We found that upon infection, the viral RNAs from the incoming particles travel together until they reach the nucleus. The viral RNAs were then detected in distinct locations in the nucleus; they are then exported individually and initially remain separated in the cytoplasm. At later time points, the different viral RNA segments gather together in the cytoplasm in a microtubule independent manner. Viral RNAs of different identities colocalize at a high frequency when they are associated with Rab11 positive vesicles, suggesting that Rab11 positive organelles may facilitate the association of different viral RNAs. Using engineered influenza viruses lacking the expression of HA or M2 protein, we showed that these viral proteins are not essential for the colocalization of two different viral RNAs in the cytoplasm. In sum, our smFISH results reveal that the viral RNAs travel together in the cytoplasm before their arrival at the plasma membrane budding sites. This newly characterized step of the genome packaging process demonstrates the precise spatiotemporal regulation of the infection cycle.The Influenza A virus genome consists of eight negative sense, single-stranded RNA segments. Although it has been established that most virus particles contain a single copy of each of the eight viral RNAs, the packaging selection mechanism remains poorly understood. Influenza viral RNAs are synthesized in the nucleus, exported into the cytoplasm and travel to the plasma membrane where viral budding and genome packaging occurs. Due to the difficulties in analyzing associated vRNPs while preserving information about their positions within the cell, it has remained unclear how and where during cellular trafficking the viral RNAs of different segments encounter each other. Using a multicolor single-molecule sensitivity fluorescence  Influenza A viruses cause one of the major respiratory infection diseases in humans. The viruses possess a genome consists of eight different RNA segments and the incorporation of all the eight RNA segments is required for the generation of an infectious virus particle. The precise process of how these eight viral RNA segments are co-packaged into progeny virus particles remains undefined due to the limitations of methodology to determine the locations of different vRNA segments in infected cells with single-molecule resolution. In this study, we established an experimental system to examine the localization of different viral RNA segments in an infected cell with high spatial precision. We found that viral RNA belonging to different segments gather together in the cytoplasm which is facilitated by cellular recycling endosomal protein Rab11. Our results supported the idea that eight different viral RNAs likely form a super-complex as they travel to the site for virion incorporation. These findings extend our knowledge on the process of influenza virus genome packaging and suggest a mechanism by which the genome assembly of different viral RNA segments is regulated. The assembly process of different vRNA segments during virus infection can therefore be studied with high resolution. Influenza virus is one of the rare RNA viruses which has a nuclear replication phase The Influenza A virus genome consists of eight negative-sense, single-stranded RNAs. In a virus particle or an infected cell, the viral RNAs exist in the form of viral ribonucleoprotein complexes (vRNPs) with the viral RNA (vRNA) encapsidated by the nucleoproteins (NP) and associate with the polymerase complex It has been suggested that the reassortment of influenza vRNAs happens in the cytoplasm because when the cytoplasm of two different virus infected cells was fused, the segments from the two virus strains were incorporated into progeny viruses in a random manner in situ hybridization assay to visualize vRNAs of different segments in influenza virus infected cells. By using this technique, the degree of colocalization between two different vRNAs can be determined with great spatial precision. By performing smFISH and colocalization analysis on virus infected cells at different time points post infection, we found that the incoming vRNPs remain associated until they are imported into the nucleus. Newly synthesized vRNPs were detected at different locations in the nucleus and the newly exported vRNPs of different identities gather together at later stages of replication when the vRNPs are loaded onto Rab11 positive vesicles. Herein, we have provided evidence that progeny vRNPs of different identities travel together in the cytoplasm. These results likely suggest that different vRNPs are selected into a pre-formed super-complex during their trafficking to the plasma membrane, where budding and genome packaging occur.In this present study, we applied single-molecule sensitivity fluorescence in situ hybridization (smFISH) system for influenza vRNAs to determine their locations in infected cells. Single-molecule sensitivity was obtained using 48 single-fluorophore-labeled short DNA oligos targeting different regions of the same vRNA. The targeted vRNAs were bound by multiple probes at the same time giving high fluorescence intensity signals, allowing them to be distinguished as diffraction-limited spots. The number of spots and the location of their centers in 3-dimensional space can then be determined using - imaging processing programs. When 48 Cy5 labeled probes against the PB2 segment were used, fluorescence spots can be observed in influenza A/Puerto Rico/8/34 (PR8) virus infected cells at 4 hours post infection (hpi) but no spots were detected in the mock infected cells , leptomycin B (LMB) or importazole (IPZ), to disrupt different steps of influenza vRNP nuclear transport. The colocalization efficiency of PB2 and NA vRNAs was then analyzed at 20, 40 and 60 minutes post-infection. In the mock treated cells, both PB2 and NA vRNAs could be detected in the nucleus as early as 20 minutes post infection  to block nuclear import of vRNPs. IPZ has been found to interfere with the interactions between importin-β and Ran-GTP, an event critical for the release of imported cargos. Treatment with IPZ, therefore, inhibits the importin-β dependent nuclear import of cargo and retains the imported cargos at the rims of the nuclei Since the results of colocalization analysis of PB2 and NA vRNPs at different time points post infection indicated that vRNPs of different identities colocalized in the cytoplasm, cellular factors that may be involved in this process were further investigated. It has been reported that influenza vRNA trafficking depends on microtubules, and it has been observed that vRNAs accumulated at the microtubule-organization center (MTOC) after their export from the nucleus To further confirm that an intact microtubule network is not important for PB2 and NA vRNAs to colocalize, we inhibited the polymerization of microtubules with nocodazole in PR8 virus infected cells. MDCK cells infected with PR8 were treated with nocodazole at 1.5 hpi and the cells were then fixed for hybridization at 6 and 8 hpi. Cells were stained with the Alexa488-conjugated wheat germ agglutinin (WGA) to label the plasma membrane and Golgi apparatus. At 6 hpi, large numbers of vRNAs were observed to accumulate at the juxta-nuclear positions in mock treated cells while in nocodazole treated cells, the vRNAs were spread out in the cytoplasm and accumulated toward the lateral plasma membrane . This obIt has been reported that influenza vRNAs travel to the plasma membrane in a Rab11 dependent manner To further demonstrate the involvement of Rab11 in the colocalization of PB2 and NA vRNAs, A549 cells were transfected with GFP tagged Rab11 in either the wild type or dominant negative forms followed by PR8 virus infection. The degree of colocalization between PB2 and NA vRNAs was compared in cells that expressed the wild type Rab11 (WT-Rab11) and cells that expressed the dominant negative Rab11 (DN-Rab11). The viral proteins hemagglutinin and M2, specifically the cytoplasmic tail of M2, have been reported to be involved in the assembly of budding virions The role of M2 protein was assessed with a similar strategy. Two recombinant viruses in the PR8 virus background were generated In this study, we have analyzed the disassembly and the subsequent assembly of influenza vRNPs segments in virus-infected cells using smFISH. We show that vRNPs of different segments remain associated after their release from the incoming virions and they travel as a package to the nuclear membrane. Newly synthesized vRNPs of different segments do not occupy the same space in the nucleus and they are likely exported individually into the cytoplasm because colocalization of the exported vRNPs of different segments is not observed during the early stages of infection. Different viral RNPs colocalize in the cytoplasm at later stages during infection (6–8 hpi in MDCK cells) and they are often found to be associated with Rab11-recycling endosomes. These results provide evidence that vRNPs belonging to different segments follow the same trafficking route and the selection for the correct combination of the eight vRNA segments likely takes place before the vRNPs reach the cell surface .in-vitro transcribed vRNAs suggested that specific RNA/RNA interactions among vRNAs exist and that they may participate in the process of genome selection The detection of vRNPs as early as 20 minutes post infection demonstrates the high sensitivity of the smFISH methodology, which allows analysis of the spatial relationship of the vRNPs during the viral entry process. When uncoating of the incoming virus particles was blocked by increasing the pH in the endosomal compartments, the PB2 and NA vRNAs were found to colocalize with very high efficiency. These results indicate that the colocalized vRNPs originate from the same virions, in accordance to the previous report that influenza virus particles package their eight different vRNAs with high efficiency The replication of vRNAs of cytoplasmic negative-sense RNA viruses has been found to occur in virally induced compartments, such as Negri bodies for rabies virus The Rab11 recycling endosome system has been shown to be involved in the transport of vRNPs from the peri-nuclear regions to the apical plasma membrane The efficient incorporation of influenza viral genome into virions has been linked to the cytoplasmic tail of the virus transmembrane proteins: HA, NA and M2 In conclusion, we have shown with smFISH and colocalization analyses that different vRNA segments in influenza virus-infected cells colocalized in the cytoplasm before they reached the plasma membrane. These findings shed light on the selection process of influenza vRNPs packaging 2. Influenza A/Puerto Rico/8/34 (PR8) and PR8 cH9/1 virus strains were grown in 10-day old embryonic chicken eggs as previously described Madin-Darby canine kidney (MDCK) epithelial cells were maintained in Modified Eagle's Medium (MEM)  supplemented with 10% fetal bovine serum (FBS). Adenocarcinomic human alveolar basal epithelial cells (A549) were grown in Dulbecco's Modified Eagle's Medium (DMEM)  supplemented with 10% FBS. Both cell lines were incubated at 37°C with 5% COThe reverse genetics method for generating recombinant PR8-HA-GFP-HA virus was as described previously Infection of MDCK cells or A549 cells with influenza viruses was performed as previously described 4 A549 cells were transfected with 1 µg of GFP-rab11-WT or GFP-rab11-DN plasmid using lipofectamine 2000 reagent according to manufacture protocol (Invitrogen). Cover slips were coated with fibronectin (Sigma) by incubating the cover slips in 50 µg/ml of fibronectin in PBS for 45 min at RT and rinse them once with PBS. The transfected cells were plated onto the fibronectin coated coverslips and incubated at 37°C for 24 hours before virus infection.5×10in situ hybridization (FISH) was performed according to published protocols with some modifications 4 cells/well and grown overnight at 37°C. At certain time points post infection, the cells were washed once with ice-cold PBSM , followed by fixation with 4% paraformaldehyde in PBSM for 10 min at room temperature (RT). After a brief wash with ice-cold PBSM, the cells were permeabilized with 0.5% Triton X-100 in PBSM for 1 min at RT. The cells were then washed with PBSM and incubated in 2XSSC  with 10% formamide for 5 min before hybridization. To detect viral RNAs, 4 µM of labeled probes in 40 µl of hybridization buffer , 0.02% RNAse-free BSA, 50 µg E. coli tRNA, 2XSSC, 10% formamide) was used for each sample. Hybridization was carried out in humidified chambers maintained at 37°C for 16 hours. The samples were then washed twice with 10% formamide 2×SSC supplemented with 2 mM VRC for 30 min at 37°C. Nuclear staining using 0.5 µg/ml of DAPI was performed afterwards and the coverslips were mounted in ProLong Gold antifade mounting media (Invitrogen). The cured samples were subjected to microscopy examination or were stored at −20°C. For samples used for both immunofluorescence and FISH analyses, cells were first blocked with 1% BSA in PBSM for 1 hour at RT after fixation and permeabilization. The coverslips were then subjected to primary and secondary antibody staining in 1% BSA in PBS followed by another fixation step with 4% paraformaldehyde for 10 min. The cells were then washed once with PBSM and equilibrated with 10% formamide 2XSSC for 10 min before the in situ hybridization procedures.RNA fluorescence Cells were placed on a Zeiss Axioplan2IE microscope equipped with a 100×, 1.4 numerical aperture (NA) oil-immersion objective (Zeiss) and a Zeiss AxioCam MRm camera. Cells were imaged using 200 nm z-dimension axis steps across a range of approximately 4 µm.We performed subpixel localization and intensity quantification of the spots using custom-designed MatLab (Mathworks) programs which were previously described Figure S1Specificity of single-molecule sensitivity FISH analysis of influenza viral RNAs. (A) MDCK cells were infected with PR8 virus at MOI = 5. DAPI signal and Cy5 fluorescence from sm-FISH probes targeting the PB2 vRNAs in mock infected and PR8 virus infected cells at 4 hpi are shown in 2D images constructed using maximum-intensity projections. Scale bar = 10 µm. (B) MDCK cells were infected with PR8 virus at MOI = 5 and hybridized with 48 Cy5 labeled probes targeting the PB2 vRNAs. Histogram of the fluorescence spots intensity at 1 hour post infected is shown. The spot intensity distribution displays a well-defined single peak characteristic of single molecules. Black circles: data; red line: Gaussian distribution fits of the data. (C) MDCK cells were infected with either PR8 or PR8 cH9/1 virus (containing the HA ORF expressing the head region of the H9 subtype) for 6 hours before smFISH was performed using Cy3 labeled probes against the HA1 vRNAs and Cy5 labeled probes against the HA9 vRNAs. Maximum intensity merges of a pair of z-stack images taken in the Cy3 channel  and the Cy5 channel  are shown. DAPI staining (blue) stains the nuclei in the cells. Scale bar = 25 µm.(TIF)Click here for additional data file.Figure S2Empirical determination of the maximum distance threshold between the centers of spots which defines colocalization. (A) The distances between the centers of colocalized spots were empirically determined by using two probe sets targeting different regions of the same viral RNA. Two different regions of NA vRNA were bound by two probe sets, one labeled with Cy3 fluorophore and the other labeled with Cy5 fluorophore. Z-stack images were taken in both fluorescence channels and the centers for the Cy3 spots and Cy5 spots were localized in 3D space using the spot detection program. The distances between the Cy3 spots and their nearest-neighboring Cy5 spots were measured. The Distributions of the distances between the neighboring spots of different colors is shown here. The distances between the centers of the Cy3 and Cy5 spots were found mainly within 1 pixel (102 nm) and spread to approximately 2.5 pixels (255 nm). Since electron microscopy analysis suggested the packaged vRNPs range from 80 nm to 100 nm , the nearest neighbor distances measured here likely represent the accuracy of measurement of the same diffraction-limited object in Cy3 and Cy5 channels. We therefore define spots as being colocalized if their centers are within 2.5 pixels. (B) In contrast to colocalizing spots, the distribution of nearest neighbor distances between the NA vRNAs (Cy5 spots) and β-actin mRNA (Cy3 spots) in infected cells shows that the nearest-neighbor distances range from 1 pixel to 10 pixels (1020 nm), with most of the spots clustered within a range from 3.5 pixels to 6 pixels. The red dashed lines show the 2.5 pixels distance threshold to define colocalization of spots in the quantitative colocalization analysis.(TIF)Click here for additional data file.Figure S3Distribution of vRNPs in the cytoplasm. Maximum intensity merge images of MDCK cells infected with PR8 virus at 2, 3 (A) and 6 (B) hpi. The cells were probed against PB2 vRNAs (green), NA vRNAs (red) and stained with DAPI (blue) to define the nuclear regions. The white arrows show the exported vRNAs detected in the cytoplasm. The red arrows indicate the peri-nuclear accumulations of vRNAs. Scale bar = 10 µm.(TIF)Click here for additional data file.Figure S4Growth kinetics of PR8 virus in MDCK cells. MDCK cells were infected with PR8 virus at MOI = 5. Supernatant of the infected MDCK cells were collected at 0, 1, 2, 4, 7, 10 and 12 hours post infection. The virus particles released into the supernatant were titered using standard plaque assays. The titer of the released virus particles at each time point is shown.(TIFF)Click here for additional data file.Figure S5The colocalization of vRNPs of different identities can be observed at 6 hpi. MDCK cells were infected with PR8 virus at MOI = 5 and sm-FISH and colocalization analyses were performed for different pairs of vRNPs. Quantification of the colocalization efficiency between the indicated vRNA pairs is shown. Low localization efficiency is observed at 4 hpi for all the vRNA pairs tested while elevated colocalization efficiency of these vRNA pairs is detected at 6 hpi, demonstrating similar kinetics.(TIF)Click here for additional data file.Figure S6Positive control of LMB treatment on MDCK cells. As a positive control for the effect of LMB, we examined the nuclear-cytoplasmic shuttling protein MAP kinase (MEK) (TIF)Click here for additional data file.Figure S7Automatic Segmentation of Microtubules and Rab11 Particles.Top: Microtubule Segmentation Example. Left, representative plane from a deconvolved image stack of microtubules labeled with an antibody against tubulin. Center, mask image showing the output of the automated segmentation algorithm for the plane on the left. Right, Overlay of data (green) and segmentation result (red). Bottom: Rab11 Particles Segmentation Example. Left, Maximum Intensity Projection of an image stack of Rab11 particles labeled with antibody against Rab11. Center, Maximum Intensity Projection of the output of the automated segmentation algorithm for the image stack represented on the left. Right, overlay of data (green) and segmentation results (red). Scale Bar: 10 µm.(TIF)Click here for additional data file.Figure S8The colocalization of PB2 and NA vRNAs in A549 cells. A549 cells were infected with PR8 virus at MOI = 5 and sm-FISH and colocalization analyses were performed for PB2 and NA vRNAs (Cy5 labeled PB2 and Cy3 labeled NA vRNAs). Quantification of the colocalization efficiency between the two vRNAs is shown. Low localization efficiency is observed at 4 hpi and 6 hpi while elevated colocalization efficiency is detected at 8 hpi and 10 hpi, demonstrating a delayed kinetics compared to that in MDCK cells (CK cells .(TIF)Click here for additional data file.Text S1Probes sequences used to target vRNA or cellular mRNA in influenza virus infected cells. Sequences for each probe used to target influenza vRNA or cellular mRNA are listed here.(DOCX)Click here for additional data file.


ECM1 gene located on chromosome 1q21. The aim of the study was to investigate the molecular genetic defect underlying lipoid proteinosis in a consanguineous Pakistani family.Lipoid proteinosis is a rare autosomal recessive disease characterized by cutaneous and mucosal lesions and hoarseness appearing in early childhood that is caused by homozygous or compound heterozygous mutations in the ECM1 gene. To screen for mutations in the ECM1 gene, all of its exons and splice junctions were PCR amplified from genomic DNA and analyzed by SSCP and sequenced directly in an ABI 3130 genetic analyzer.Genotyping of seven members of the family was performed by amplifying microsatellite markers, tightly linked to the ECM1 locus. Sequence analysis of the coding exons and splice junctions of the ECM1 gene revealed a novel homozygous mutation (c.616C > T) in exon 6, predicted to replace glutamine with stop codon (p.Q206X) at amino acid position 206.The results revealed linkage of the LP family to the ECM1 gene for the development of lipoid proteinosis.The finding of a novel mutation in Pakistani family extends the body of evidence that supports the importance of  ECM1, which encodes for the glycoprotein extracellular matrix protein 1. To date, several mutations in the ECM1 gene have been reported in unrelated LP families from different geographical areas. In this study we report a novel nonsense mutation in a consanguineous Pakistani family affected with lipoid proteinosis; and an update of ECM1 gene mutation data base.Lipoid proteinosis LP; MIM 247100) also known as Urbach-Wiethe disease or hyalinosis cutis et mucosae, was first reported by Urbach and Wiethe, in 1929 47100 als. Skin leA consanguineous Pakistani family with autosomal recessive LP was ascertained from Rawalpindi district. Two individuals (ages 15 and 23 years) in the family were affected with the disorder  amplified. Each reaction was carried out in 10 μl volume containing 1.5 mM MgCl2, 0.6 μM of each primer, 0.2 mM each dNTPs, 1U Taq DNA polymerase and 1 × PCR buffer {16 mM (NH4) 2SO4, 67mMTris-HCI (pH 8.8), and 0.01% of the nonionic detergent Tween-20} . Amplification was performed with an initial denaturation for 5 min at 94°C, followed by 35 cycles of denaturation at 94°C for 45 sec, annealing at 55°C for 45 sec, extension at 72°C for 45 sec and a final extension at 72°C for 10 min. The PCR products were separated on 8% non-denaturing polyacrylamide gels stained with ethidium bromide and alleles were assigned by visual inspection.Genomic DNA from seven individuals of the family was genotyped using microsatellite markers tightly linked to the ECM1 gene, 8 sets of primers were used to amplify all coding exons and adjacent splice sites by PCR. PCR products were initially screened for mutations by single stranded conformational polymorphism (SSCP) analysis. For this, aliquots of 10 μl of each PCR product was mixed with 10 μl denaturing solution , heated for 7 min at 95°C in PxE thermal cycler  and chilled quickly on ice for 5 min. Denatured DNA was subjected to 8% polyacrylamide gel electrophoresis (20 × 20 × 0.1 cm) containing 7% glycerol and 1 × tris-borate EDTA (TBE) buffer at constant 30W for 3.5-4.0 hrs. The gels were stained with ethidium bromide (1 μg/ml) in 1 × TBE buffer for about 5 min and visualized under UV transilluminator gel documentation system . The PCR products with mobility shift were then purified for DNA sequencing using commercially available QIAquick PCR Purification Kit . Direct sequencing was carried out by using Big Dye® Terminator v3.1 cycle sequencing kit in an ABI 3130 genetic analyzer .For detection of mutation in the The affected individuals had hoarseness of voice, pseudo-solar elastosis of the cheeks and forehead and waxy papules along the margins of eyelids. Progressive thickening and scarring of the skin and mucous membranes, hyperkeratosis with warty papules on the palms and dorsum of the hands, elbow and knee were also observed. Mobility of the tongue was limited with yellow discoloration of the lips. The clinical features of LP were identical in both patients; however, severity was varied, which may be due to difference in age as many of the clinical features of LP only manifest fully with time. Both the affected individuals showed initial symptoms during infancy. The heterozygous parents and siblings revealed no clinical signs and symptoms of LP upon detailed skin examination.ECM1 gene. The markers were fully informative, and the results revealed that the affected individuals were homozygous for the markers suggesting linkage to the ECM1 gene.Genotyping of two affected and five normal individuals of the family Figure  was perfECM1 gene. Mutant allele was found to be co-segregating with the disease phenotype in the family. Both affected individuals  in the family were homozygous for the mutant allele, while their parents  and normal siblings  were heterozygous.The SSCP analysis revealed mobility shift bands in PCR products of exon 6 of the ECM1 gene  in exon 6 of the e Figure . To see ECM1 gene comprises of 10 exons and encodes for the extracellular matrix protein 1. There are four splice variants including ECM1a, ECM1b and ECM1c, encoding proteins of 540, 415 and 559 amino acids, respectively. The recently described fourth splice variant comprises transcription of 71 bp at the 3' end of intron 1 and part of exon 2 to give a truncated 57 amino acid protein [The  protein . It prom protein -14. The ECM1 gene have been described so far in unrelated patients affected with lipoid proteinosis  in exon 6 of the ECM1 gene in a consanguineous Pakistani family. Five ECM1 mutations have been previously reported in unrelated Pakistani LP families (Table Forty six mutations in the ECM1 gene in a Pakistani family extending the mutation spectrum of the gene. The study extends the body of evidence that supports the role of ECM1 gene in the development of lipoid proteinosis. Identification of pathogenic mutations in the ECM1 gene should be helpful to improve genetic counseling and DNA based prenatal diagnosis.We have identified a novel nonsense mutation in exon 6 of the The authors declare that they have no competing interests.1 performed experimental work, AL performed clinical study of the family, MA participated in experimental work, RQ updated mutation database and participated in manuscript preparation, MN2 analyzed data and prepared manuscript, AH designed research plan and analyzed data. All authors read and approved the final manuscript.MN


HER2 genetic assays with different characteristics that could affect the performance to analyze the human epidermal growth factor 2 (HER2) gene copy number under low and high throughput conditions. The study included 108 tissue samples from breast cancer patients with HER2 immunohistochemistry (IHC) results scored as 0/1+, 2+, and 3+. HER2 genetic status was analysed using chromogenic in situ hybridization (CISH) and fluorescence in situ hybridization (FISH). Scoring results were documented through digital image analysis. The cancer region of interest was identified from a serial H&E stained slide following tissue cores were transferred to a tissue microarrays (TMA). When using TMA in a routine flow, all patients will be tested for HER2 status with IHC followed by CISH or FISH, thereby providing individual HER2 results. In conclusion, our results show that the differences between the HER2 genetic assays do not have an effect on the analytic performance and the CISH technology is superior to high throughput HER2 genetic testing due to scanning speed, while the IQ-FISH may still be a choice for fast low throughput HER2 genetic testing.The purpose was to evaluate and compare 5 different  HER2 testing on tissues scored as borderline cases (2+) found by IHC [HER2 gene amplification assays are commercially available from a multiple vendors using either fluorescence in situ hybridization (FISH) or chromogenic in situ hybridization (CISH), where the various tests have differing characteristics  probe. The direct labeling of the different FISH probes from Dako and ZytoVision uses red (TexasRed), orange (Rhodamine), or green (FITC) fluorocrome, while the CISH-based assays give rise to either red, green, or blue chromogenic precipitation. Various strategies for blocking of nonspecific probe binding and detection systems have been implemented into the different HER2 genetic assays. ZytoVison uses repeat-free oligonucleotides and thereby does not need to block repeated sequences  while another system (Dako) has developed alu sequence blocking peptide nucleic acids (PNAs) to lower the background generated from the repeated sequences that are located in the HER2 DNA probe [HER2 IQ-FISH (Dako) reduces the assay time from two days to four hours. This is achieved by breaking the hydrophobic forces in the DNA helix used in stacking [HER2 gene copy number determination in breast cancer [HER2 genetic testing and still has a high sensitivity and specificity during routine diagnostic of breast cancer [HER2 assays in a high throughput routine setting using TMA containing breast cancer tissue and to evaluate if different characteristics between the five HER2 genetic assays could affect the perfomance when using digitalization of the HER2 stained slides before manually scoring on a monitor screen.Human Epidermal growth factor Receptor 2 (HER2) expression is investigated routinely on all breast cancer cases to make the therapeutic decisions for patients with breast cancer. The American Society of Clinical Oncology (ASCO)/College of American Pathologist (CAP) recommendations for HER2 status testing are first immunohistochemical (IHC) staining and secondary to perform genetic d by IHC . Ratio-beristics . The HERNA probe . A newlystacking  with thet cancer . CISH hat cancer . Multiplt cancer –11. The The study included 108 consecutive breast carcinomas from patients diagnosed at Herlev Hospital, Denmark, with information of HER2 status performed by IHC.TMAs were constructed from formalin-fixed and paraffin-embedded donor blocks, using a fully automated ATA-27 (Beecher Instrument). The areas of interest at the margin of tumor were marked on H&E stained slides by a pathologist. Four cores of 1 mm or one core of 2 mm in diameters was used per donor block and mounted in a new recipient block. Tissue preparation, microscopy, and subsequent laboratory analyses were carried out as part of the daily routine.HER2 genetic testing was performed using 5 assays: HER2 CISH pharmDx Kit-SK109, HER2 FISH pharmDx Kit-K5331, HER2 IQ-FISH pharmDx-K5731 , ZytoDot 2C SPEC HER2/CEN17 Probe Kit-C-3022-40 and SPEC HER2/CEN17 Dual Colour Probe Kit-Z-2020 . The tests were conducted according to the manufacturers' instructions with minor changes, which are specified below. All samples were treated with pepsin for 8 minutes at room temperature. The last step of the ZytoDot CISH dehydration (3x 30 s in 100% ethanol and incubate 2x 30 s in xylene) was changed to air drying for 30 minutes before mounting. This preserves the red signals which can be faintly stained when using xylene or ethanol and avoids trapping of bubbles underneath the coverslip caused by water or air.The HER2 genetic testing were counted without knowledge of patient outcome, IHC status and results from other HER2 genetic testing. Three separate tumor areas were selected and at least 60 signals (either red or green) from invasive tumor cells were counted [HER2 gene and centromere 17 (CEN17). Scoring criteria used for analysis were nonamplified (<1.8), equivocal (1.8–2.2), and amplified (>2.2) according to ASCO/CAP guidelines [HER2 ratios, another 60 signals were scored and the final ratio of the case was calculated from the total number of signals. The final scoring of the reanalysed equivocal HER2 ratios was reported according to the cut-off criteria nonamplified (<2.0) and amplified (≥2.0).The CISH- and FISH-stained TMAs were scanned using a bright field/fluorescent panoramic scan (3D HisTech) equipped with a 40x dry objective and using single focus layer for CISH TMAs and five focus layers separated by 0.75 microns (z-stacking) for FISH TMAs. The scanned TMA full slide was analysed using Panoramic Viewer and manually scored on a computer monitor. Scores for  counted . All oveidelines . For allHER2 genetic assays and κ statistics [The statistical analysis of the accuracy was performed by calculating the agreement between a constructed consensus assay from the five different atistics .HER2 genetic assays  were investigated, resulting in 540 scoring results. The success rate of FISH and CISH HER2 genetic testing during routine condition was 100%. The scanning success was 97,6% (527 out of 540). Thirteen samples failed; of those FISH accounted for 11 samples and CISH accounted for 2 samples. Failures of FISH scanning were missing autofocus, high background staining, and persistent autofluorescence. Failures of CISH scanning for the two samples were caused by a fingerprint and an air bobble captured underneath the coverslip. These 13 patients were excluded (n = 65 scoring results), resulting in a total cohort of evaluated patients of 95 (n = 475 scoring results). All thirteen scanning failures were located on the 2 mm core TMA's, while at least two of the four cores on the 1 mm core TMA could be scanned and analysed successfully. The mean digital imaging scanning time of CISH using a 40x objective was 29 sec per mm2, while the FISH stained slides using five extended focus layers for the HER2 and CEN17 filters and a single layer for DAPI were 764 sec/mm2.A total of 108 breast carcinomas were included in the study and 5 different HER2 ratio of CISH and FISH scores, concordance was found in 99% (94/95) of the cases , which support earlier published results [HER2 genetic assays. The concordance within the CISH assays was 97.9% and 99.0%  showed 3.0 HER2/CEN17 ratio amplification. An explanation for this difference is that it may be caused by tumour heterogeneous amplification, as a region on the 2 mm core were clearly HER2 amplified. Case 69, four assays showed amplification and one FISH assay showed nonamplification. The divergent scoring results may be due to tumour heterogeneous amplification, where the correct amplified tumour area was not identified on the 2 mm core. With cases 48 and 84, the scorings were very close to the borderline indicated by HER2/CEN17 ratio amplification close to 2.0. The IHC scores were 2+ and 1+, respectively (Discordant patients . In caseectively . This inHER2 status between dual-colour CISH and FISH analysis is reported as well as a reduction in scoring time and laboratory hands on time [HER2 copy number and the mean HER2/CEN17 ratio were lower than those estimated with FISH but did not result in discrepancy of the final result. The number of HER2 counted signals can be underestimated due to overlapping signals. This discrepancy is only a risk in low amplified cases and in our study it was only seen in heavily amplified HER2 signals.HER2 testing is required to identify of breast cancer patients that may benefit for trastuzumab adjuvant therapy. Significant correlation in  on time , 16–18. HER2 analyses is needed. By using TMA, an additional possibility emerges to analyse all patients with regard to both IHC HER2 and HER2 genetic status without considerable increase of cost [Introduction of TMA into a clinical routine laboratory can be a useful technique when a high throughput of  of cost .HER2/CEN17 is homogeneously amplified; however in approximately 10% of the carcinomas, HER2 genetic testing shows unusual signal pattern [HER2 amplification staining patterns. Homogeneously amplified tumors with identical results in scored areas, intratumour heterogeneous amplification/nonamplification areas, in which a minimum of one region was scored as amplified and another scored nonamplified and can be considered as hot spot. A third heterogeneous amplification pattern was illustrated by a single cell with extensive amplification surrounded by cells that are nonamplified. However, little is known about the clinical implication of such patterns, except for one study demonstrating intratumour heterogeneity of HER2 gene amplification to be associated with a shorter disease-free survival [In most tumours, the  pattern , 20. We The different use of DNA and PNA probes together with different blocking reagents against repeated regions did not affect the genetic test results.HER2 genetic testing aimed at identifying the best technique for preparation of digital imaging and scanning. The high level of agreement obtained between CISH and FISH genetic testing with respect to assay performance makes the two techniques equivalent, in a technical perspective. The most important advantage of CISH over FISH is the much faster digitalization of a CISH-stained slide in combination with low failure rate. The success of CISH digitalization is an advantage for a future automatic image analysis of HER2 genetic testing in a routine laboratory.To our knowledge, the first study to analyse the difference between CISH and FISH for high throughput HER2 genetic assays do not have effect on the analytic performance and the CISH technology is superior to high throughput HER2 genetic testing due to scanning speed, while the IQ-FISH may still be a choice for fast low throughput HER2 genetic testing when only analysis of a few patients is required.In conclusion, our results show that the differences between the five


The prevalence of coronary artery disease increases with age. A significant proportion of patients commonly referred for investigation of coronary artery disease are often unable to perform exercise testing because of advanced age, and can only be assessed with pharmacologic tests. Stress cardiac magnetic resonance (CMR) imaging is a non-invasive modality used for detection of myocardial ischemia, necrosis and viability.The aim of the present study is to determine the applicability and safety of stress CMR in patients older than 70 years.We reviewed the data of all patients older than 70 years who were referred for stress CMR (1.5 Tesla) from January 2006 to February 2010 to our outpatient center. Standard protocol consisted of: 1) assessment of myocardial function at rest; 2) pharmacological stress induced either by dobutamine  until achieving submaximal heart rate ([220-age] x 0.85), or by adenosine (protocol of 140 µg/kg/minute during 3 minutes followed by a bolus of 10 ml of gadolinium at 4 ml/second "first pass"); 3) assessment of myocardial scar and/or viability by delayed enhancement sequences.Among the 309 patients ≥70 years old referred for a stress CMR, the test could be performed in 297 (96%) patients . Mean test duration was 50±9 minutes. Phamacological stress was induced with dobutamine in 98 (33 %) patients and adenosine in 199 (67 %) patients). The test could not be carried out in 12 (4 %) patients because of claustrophobia (8 patients) and excessive thoracic diameter (4 patients). Among the patients who underwent stress CMR, target heart rate was not reached in 13 (4%) patients. Side effects included one case of sustained supraventricular tachycardia and one case of a transient severe hypotension. In 4 patients gadolinium contrast was not injected due to severe renal insufficiency. No other complications occurred. No ischemia or infarction was found in 170 patients (58%), while isolated ischemia was found in 20 patients (7%) and ischemia in the presence of an infarction in 34 patients (12 %). Infarction without ischemia was found in 67 patients (23%).Our data shows that stress CMR performed in elderly ambulatory patients is safe and well tolerated. Myocardial ischemia and/or infarction could be confirmed in 42% and excluded in 58% of patients in less than one hour exam.


RIFLE provides standardized criteria for defining acute kidney injury (AKI) . It is bAn observational study for 6 months in ICU patients admitted ≥48 hours. For the first 7 days we calculated daily the number of patients diagnosed with AKI-0 using the four AKI definitions.One hundred and one patients (39%) had a known premorbid sCr. Mean age and APACHE was respectively 64 13) and 22 (7). Figure 3 and 22 The early diagnosis of AKI is significantly reduced when urine output criteria are neglected in the RIFLE definition, and also when baseline sCr is estimated. This may significantly impact the assessment of biomarker performance.


S. cerevisae to produce in vivo recombinant ribosomes which have a ribonuclease tethered to specific sites. Activation of the MNase activity by addition of calcium led to specific rRNA cleavage events in proximity to the ribosomal binding sites of the fusion proteins. The dimensions of the RNP environment which could be probed by this approach varied with the size of the linker sequence between MNase and the fused protein. Advantages and disadvantages of the use of MNase fusion proteins for local tertiary structure probing of RNPs as well as alternative applications for this type of approach in RNP research are discussed.Analyses of the conformational dynamics of the numerous cellular ribonucleoprotein particles (RNP) significantly contribute to the understanding of their modes of action. Here, we tested whether ribonuclease fusion proteins incorporated into RNPs can be used as molecular probes to characterize the local RNA environment of these proteins. Fusion proteins of micrococcal nuclease (MNase) with ribosomal proteins were expressed in  Direct RNA interaction sites of the studied protein could most likely not be mapped by such an approach. However, it could detect RNA elements which are located in tertiary structure (but not necessarily in primary structure) in proximity to the MNase fusion protein and therefore reveal characteristics of the actual folding state of the analyzed RNP  to an optical density of 1 (OD600) were harvested by centrifugation at room temperature (3000 g) and were washed once in 20 ml ice cold water and twice in 1 ml ice cold buffer AG200 . 1.5 ml of cold buffer AG200 per gram of wet cell pellet were added and the resulting suspension was shaken in the presence of glass beads (1.4 g of 1 mm diameter glass beads per 0.8 ml suspension in a tube of 2 ml volume) on a vibrax mixer (IKA) at 4°C and maximum speed three times for 5 minutes. The extract was clarified twice by centrifugation for 5 and 10 minutes at 13000 g and 4°C. Protein concentrations of the clarified extracts were determined by Bradford protein assay (Bio-Rad Laboratories). MNase was activated by incubation of the extracts at 22°C in the presence of 8 mM calcium chloride. To stop the reaction, extract volumes corresponding to 200 µg of proteins were added to 500 µl ice cold buffer AE+  and samples were frozen at −20°C. Subsequently, RNA extraction by hot acidic phenol treatment was performed as described in 32P-labelled probes  equilibrated in buffer AG200 containing 0.5% Triton X-100 and 0.1% Tween20. After one hour incubation at 4°C the matrix was transferred into a poly prep chromatography column (Bio-Rad Laboratories) and washed three times with 2 ml and one time with 10 ml AG200 containing 0.5% Triton-X100 and 0.1% Tween 20. After an additional wash step with 1 ml of buffer AG200 the affinity matrix was suspended in 1 ml AG200 and split into two parts which were centrifuged for 2 minutes at 2000 g and 4°C to discard the supernatant. One half of the affinity matrix was suspended in SDS sample buffer and further analyzed by SDS polyacrylamide gel electrophoresis (16% acrylamide gels were supplemented with 4.5 M Urea) and Coomassie staining or Western blotting. Total RNA was extracted from the other half and analyzed by agarose or polyacrylamide/TBE gel electrophoresis followed by ethidium bromide staining.Figure S1Oligonucleotides used in this study.(PDF)Click here for additional data file.Figure S2Plasmids used in this study.(PDF)Click here for additional data file.Figure S3Yeast strains used in this study.(PDF)Click here for additional data file.Figure S4Possible mode of action of a nuclease fusion protein incorporated into an RNP. The figure legend in the lower panel gives a description of the symbols used to represent specific structural features of the RNP and the nuclease fusion protein. The left and right part of the figure show two different conformational states of the RNP which might be the consequence of changes in the RNP’s interaction partners. Small arrows and transparent shapes of the linker, the nuclease and nuclease sensitive sites indicate varying local conformations. In the linear RNA sequence the RNA binding sites of the tested protein can be far away from the respective RNA cleavages made by the fused nuclease.(PDF)Click here for additional data file.Figure S5Growth curves of yeast strains BY4741, Y2361, Y2369 and Y2371. Yeast strains Y206 (wildtype BY4741), Y2361 (MNase-rpS13), Y2369 (MNase-rpL5) and Y2371 (MNase-rpL35) were grown overnight in YPD medium and then diluted to an OD600 of 0.02 in 0.2 ml of fresh YPD in a covered 96 well plate. Cells were incubated at 30°C in a TECAN infinite F500 reader with measurements taken in kinetic cycle mode . Generation times in logarithmic growth phase of strains Y2369 (MNase-rpL5) and Y2371 (MNase-rpL35) were increased less than 15% compared to the one of the wildtype strain Y206 (103 and 104 minutes versus 93 minutes). Generation time of Y2361 (MNase-rpS13) was increased less than 40% (128 minutes versus 93 minutes). Growth measurements using larger culture volumes incubated at 30°C in Erlenmeyer flasks on a rotary shaker gave identical results in regard to these relative changes in generation times.(PDF)Click here for additional data file.Figure S6MNase fusion proteins of rpS13, rpL5 and rpL35 expressed in yeast strains Y2361, Y2369 and Y2371 get incorporated into ribosomal particles. Cellular extracts of yeast strains which express no MNase fusion protein  or fusions of MNase linked with rpS13 , rpL5  or rpL35  by two consecutive HA tags were used for affinity purification with an anti-HA affinity matrix as described in (PDF)Click here for additional data file.Figure S7Major cleavage events in extracts of strain Y2371 depend on the addition of exogenous calcium ions. A cellular extract of yeast strain Y2371 expressing rpL35 in fusion with MNAse was prepared as described in (PDF)Click here for additional data file.Figure S8rRNA degradation in yeast cellular extracts after addition of exogenous MNase. Cellular extracts of yeast strain Y206 (wildtype BY4741) were prepared in buffer AG200 as described in (PDF)Click here for additional data file.Figure S9Map of 25S rRNA and 5.8S rRNA including positions of oligonucleotides used in this study and the major cuts observed in yeast strains Y2371 and Y2369. Oligonucleotides are represented by red boxes, major cleavage events by blue arrows and resulting rRNA fragments by grey lines.(PDF)Click here for additional data file.


Plasmodium falciparum is still only partially understood. Regulation of var genes, the most studied gene family involved in antigenic variation, is orchestrated by a dynamic pattern of inherited chromatin states. Although recent evidence pointed to epigenetic regulation of transcribed and repressed rif loci, little is known about specific on/off associated histone modifications of individual rif genes. To investigate the chromatin marks for transcribed and repressed rif loci, we cultivated parasites and evaluated the transcriptional status of chosen rif targets by qRT-PCR and performed ChIP assays using H3K9ac and H3K9me3 antibodies. We then monitored changes in the epigenetic patterns in parasites after several reinvasions and also evaluated the “poised” mark in trophozoites and schizonts of the same erythrocytic cycle by ChIP using H3K4me2 specific antibodies. Our results show that H3K9 is acetylated in transcribed rif loci and trimethylated or even unmodified in repressed rif loci. These transcriptional and epigenetic states are inherited after several reinvasions. The poised modification H3K4me2 showed a tendency to be more present in loci in trophozoites that upon progression to schizonts strongly transcribe the respective locus. However, this effect was not consistently observed for all monitored loci. While our data show important similarities to var transcription-associated chromatin modifications, the observed swiftly occurring modifications at rif loci and the absence of H3K9 modification point to a different dynamic of recruitment of chromatin modifying enzymes.The regulation of variant gene expression in  Plasmodium falciparum maintains its persistence in the vertebrate host at least in part by expression of several variant proteins at the surface of infected erythrocytes. The most important variant antigen is PfEMP-1  which is encoded by approximately 60 members of the var multigene family var genes are expressed per single parasite The human malaria parasite rif family has approximately 160 different intact members in the 3D7 strain annotated in the PlasmoDB databank. Recently, we rif transcription switches apparently faster than transcription of var genes, at least in vitro. Similar to var genes, rif gene transcription and silencing is dependent on the histone deacetylase PfSir2A rif gene loci are associated to histone 3 lysine 9 trimethylation as are other virulence-associated multigene families var, rif, and stevor seem to be controlled by a common factor and overexpression of artificial loci containing a rif promoter suppressed the transcription of var loci var genes, recently gained further support The multigene var gene which is actively transcribed was associated to histones carrying an acetyl group at the lysine 9 of histone 3 (H3K9) and Chookajorn and colleagues var locus active in early trophozoite stage and silenced in schizont stage  was associated with a dimethylated lysine 4 in histone 3 At the molecular level, Lopez-Rubio and colleagues P. falciparum, we addressed the question if dynamic histone modifications which are decisive in var regulation are also associated with active or silent rif loci. For this, the transcription of rif genes was monitored in consecutive cultures and compared to the histone modifications found for each locus in the same parasites.Given the observation of similar histone landmarks in ChIP-on-Chip experiments Var gene loci in their active state are associated with class 3 histones (H3) carrying an acetylation mark at their lysine 9 (H3K9ac), while silenced var loci associate with H3K9me3 rif loci are associated with the same epigenetic landmarks as var loci, we first performed a qRT-PCR-based rif transcript analysis in parasites previously selected for cytoadherence in CHO-CD36, since in this phenotype a number of rif loci were found active with rapid switching after a small number of reinvasions and given the large number of rif genes the analysis was restricted to these loci rif genes after 10 and 20 reinvasions after the last panning procedure. As expected, a number of rif genes were actively transcribed while others were silenced or transcribed at background levels . Noteworthy, the overall intensity of immunoprecipitation varied between loci. In order to show if predominant H3K9-acetylation - expressed as a higher ratio H3K9ac/H3K9me3 – statistically correlated with higher transcript abundance, we divided the H3K9ac/H3K9me3 ratios after 20 reinvasions by the H3K9ac/H3K9me3 ratios after 10 reinvasions and plotted them against the coefficients of relative transcript quantity from each locus after 20 and 10 reinvasions (2 = 0.5299). The extreme outliers were not considered in the analysis (Pf080138 and PFB0040c). A significant correlation of H3K9ac/H3K9me3 and transcript quantities was also observed when the data were analyzed separately for the 10 and 20 reinvasion material  were reselected for cytoadherence on CHO-CD36 cells. After two selection rounds, trophozoites at 20–24 h post reinvasion showed most transcripts from the PFI0025c locus and minor quantities from loci PFD1240w, PFD0070c and PF100397  rif loci which are actively transcribed in trophozoites (20–24 h post reinvasion) and/or schizonts (30–34 h post reinvasion), we cultivated parasites previously cytoadherence-selected on CHO-CD36 (25 reinvasions after selection), synchronized them and harvested chromatin 20 and 30 h post reinvasion and analyzed for their modification at H3K9 and H3K4 by ChIP. In trophozoites, we detected relatively low rif transcription at the tested loci (below 0.5 relative units), while in schizonts more transcripts accumulated (rif loci showed a tendency to slightly increased acetylation over trimethylation when transcripts were detected (above 0.1 relative units) and there was a significant correlation of transcript quantity with H3K9ac/H3K9me3 ratios in schizonts but not in trophozoites (rif PFB0040c (silent in trophozoites) showed increased acetylation at H3K9 when compared to trophozoites, and the strongly transcribed PF100397 showed accentuated acetylation. The only locus which did not follow the pattern was PFD0070c, which showed very few transcripts in trophozoites and in schizonts, although its locus was strongly acetylated at H3K9. The loci with no detectable transcription in trophozoites or in schizonts followed the pattern of either stronger trimethylation or lower levels of any modification  when we plotted the transcript quantities against the H3K4me2/H3 enrichment ratio. The silent loci PFL2640, PFD1010w, PF080138 and the weakly transcribed locus PFB0040c were not significantly marked  Posttranslational modification (PTM) of histones is a well-known mechanism that is related to the control of gene expression in eukaryotes. It is believed that a “histone code” orchestrates the accessibility of transcription factors to promoter regions through modification of histone tails by methyl, acetyl and phosphate groups, and proteins as ubiquitin and SUMO as reviewed by Berger rif gene transcription activation and silencing, we performed a detailed analysis of a number of rif genes. Since the selective upregulation of rif genes by phenotypic selection is not yet possible as it is for var genes e.g. rif genes and the frequent transcriptional activity of the herein tested loci In order to elucidate mechanisms responsible for var gene locus activity seem at least partially also functional for rif loci: When a significant presence of transcripts for any tested locus was observed, H3K9 acetylation dominated. This mark has not been identified before for active rif genes. Inversely, in the absence of transcripts, loci were either associated with strong tri-methylation of H3K9, in accordance to data published by Lopez-Rubio and colleagues var loci, the modifications are dynamic var genes, one would perhaps expect higher acetylation in “on” rif genes of which oligos amplify in the 5′ region of the gene – in proximity to the promoter region which is supposed to carry most of the acetyl residues rif genes are much shorter than the sometimes 12 kb-var genes. It is possible that the short central 500 bp range in which the oligos were allocated in the different rif genes may not allow to discriminate these positional effects, taking also in account that the immunoprecipitated genomic DNA fragments generated during the ChIP procedure had around 400 bp (data not shown). In order to address the question of positional effects of priming sites in genes analyzed by ChIP, we compared signal differences in the loci PFI0025c and PF100397, using additional specific oligo pairs in the coding sequence. For this experiment, material from the experiment in rif gene  is distant from transcribed rif genes and no specific pattern of transcript quantity and genomic context  following instructions of the manufacturer. The washed RNA pellet was briefly dried at RT and dissolved in water and stored at −80°C until use. About 5 µg of total RNA was used for cDNA synthesis. Briefly, total RNA was treated 3 times with DNAse I (Fermentas) prior to synthesis to prevent genomic DNA contamination. The treated RNA was reversed transcribed using MuLV-Reverted aid transcriptase reverse (Fermentas) and random hexamer primers following the manufacturers' instructions.9 parasites were crosslinked with 1% formaldehyde and RBC were lysed in PBS supplemented with saponin 0.1%. Following saponin lysis, parasites were resuspended in lysis buffer , supplemented with 1× Protease Inhibitor Cocktail . After 30 minutes of incubation, parasites were lysed using a Dounce Homogenisator (100 strokes), centrifuged for 10 minutes at 14.000 rpm/4°C, and then sonicated using a cell disruptor (Unique Ultrasonic) until resulting DNA fragments had 200–500 bp. At this stage, 10% of the material (“input”) was retrieved and submitted directly to the reversion of crosslinking (see below). For immunoprecipitations, sonicated chromatin was diluted in ChIP buffer . Chromatin diluted in ChIP buffer was pre-cleared in 50 µL/mL of a DNA sperm salmon/protein A-sepharose slurry for 2 h/4°C. Afterwards, chromatin was incubated with 1∶250 of anti-H3K9ac, H3K9me3, H3K4me2 (Upstate) and H3 (Abcam). For negative controls (mock samples) we used an anti-IgG mouse, and for calibration of the overall chromatin quantity, similar amounts of material from the “input” fraction were employed. The chromatin-immune complexes were then washed under stringent conditions. Following this, crosslink was reverted by incubation at 65°C for 4 h and 0.2 M NaCl. DNA was extracted twice with Phenol/Chloroform/isoamylalcohol, precipitated and resuspended in 20–50 µl sterile water. The results presented are the average of two or three independent immunoprecipitations from different extracts of parasites . The significance of differences between signals was evaluated by Student's T test (two tailed) using GraphPad Prism 5.ChIP was conducted as described in rif transcript sequences and only oligos with perfect primer-to-target specificity were considered. Later, oligonucleotide pairs were selected for global target specificity by ePCR using the 3D7 genome sequence as template. Importantly, the primer performance in qPCRs differed no more than 1 Ct between oligonucleotide pairs used herein and the internal control oligonucleotide pair. PCRs were performed in triplicates and equal amounts of input DNA (0.05–0.5 µg) per imunoprecipitated sample and mock immunoprecipitations (unrelated IgG) were analyzed together with samples. Mock samples presented CTs>35, which was considered as unamplified targets. All rif oligos in each immunoprecipitation were calibrated with the input DNA signal using the formula 2−ΔCt = (CtoligoAb−Ctoligoinput), where Ab is the respective antibody used for ChIP. The plotted enrichment was calculated as the ratio of immunoprecipitated Ab oligo signal/immunoprecipitated H3 oligo signal. The internal control transcript used for calibration throughout the experiments was locus seryl t-RNA transferase (PlasmoDB No. PF07.0073), previously shown as reliable  and D, schizonts 30–36 h p.i. from the same reinvasion cycle as in D. E: Material from trophozoites 20–24 h p.i. freshly repanned over CHO-CD36.(TIF)Click here for additional data file.Figure S2Position of oligos used in qPCR and ChIP in the deduced transcript sequence of analyzed rif targets and their genomic context. A) The black bars above and the numbers below the scheme of each rif coding region indicate the initial position of the amplification of the primer. The numbers at the end of each gene indicate the size of the respective coding regions. The genes were divided in the figure according to the rif promoter classification proposed by Joannin et al 2008 C and T means telomeric and centromeric position, respectively. Rif A type genes are believed to encode IRBC-surface displayed RIFINs while type B RIFINs are probably localized to Maurer's clefts rif loci, extracted from http://plasmodb.org (6/15/2011).(PPTX)Click here for additional data file.Figure S3Relative quantities of rif transcripts and chromatin enrichment for H3K9ac, H3K9me3 and H3K4me2 (poised mark) in trophozoite and schizont stages for selected rif loci. A: relative transcript amounts of chosen rif targets in trophozoites and schizonts in 3D7 parasites of the same reinvasion, calculated as above. Chromatin enrichment of H3K9ac and H3K9me3 for trophozoites (B) and schizonts (C). The chromatin enrichment was normalized using the qPCR data from ChIP with anti-H3 and 10% of the input. Significant differences between H3K9 trimethylation and acetylation are depicted by single asterisks, and significant H3K4me2 modification (compared to the H3K9me3 control) are indicated by two asterisks .(TIF)Click here for additional data file.Figure S4Raw results from experiment in  showing the influence of primer localization on the qPCR signal after ChIP at two rif loci. Immunoprecipitated material from the experiment in (PPTX)Click here for additional data file.


However molecular mechanism underlying apoptosis as a result of thyroid hormone signaling is poorly understood. The antiapoptotic role of Senescence Marker Protein-30 (SMP30) has been characterized in response to varieties of stimuli as well as in knock out model. Our earlier data suggest that thyroid hormone 3, 3′5 Triiodo L Thyronine . From the stand point of established epigenetic concept we have categorised these two TREs as negative response elements. Our attempt of siRNA mediated silencing of TRβ, reduced the fold of repression of SMP30 gene expression. In presence of thyroid hormone, Trichostatin- A (TSA), which is a Histone deacetylase (HDAC) inhibitor further inhibited SMP30 promoter activity. The above findings are in support of categorisation of both the thyroid response element as negative response elements as usually TSA should have reversed the repressions.In highly metastatic MCF-7, human breast cancer cell line T3 treatment repressed SMP30 expression leading to enhanced apoptosis. Analysis by flow cytometry and other techniques revealed that overexpression and silencing of SMP30 in MCF-7 resulted in decelerated and accelerated apoptosis respectively. In order to identify the cis–acting elements involved in this regulation, we have analyzed hormone responsiveness of transiently transfected hSMP30 gene expression by thyroid hormone which in turn induces apoptosis in MCF-7 human breast cancer cells. We believe that our study represents a good ground for future effort to develop new therapeutic approaches to challenge the progression of breast cancer.This is the first report of novel mechanistic insights into the remarkable downregulation of  However, due to conflicting results regarding the clinical correlation between breast cancer and thyroid diseases, any precise association between thyroid status and the pathogenesis of human breast cancer remains elusive The TRs are ligand modulated transcription factors encoded by two genes, TRα and TRβ, located on human chromosomes 17 and 3 respectively The effect of thyroid hormone at the cellular level is mediated through TRs, by interacting with thyroid hormone response elements (TREs) in the promoters of the target genes to regulate transcription. Depending on their ability to bind to thyroid hormone, TRs can activate or suppress gene expression in a tissue specific manner through heterodimerisation with retinoid X receptors (RXRs) and interaction with the positive or negative response elements commonly known as TREs in the regulatory regions of target genes SMP30 gene in breast cancer cells in which programmed cell death is deregulated. Additionally, we observed downregulation of SMP30 expression in rat liver by thyroid hormone SMP30, which is an antiapoptotic gene.The role of thyroid hormones in inducing apoptosis in different systems has been well documented in literature . SMP30 was initially identified as a novel protein that is highly expressed in hepatocytes and in renal tubular epithelia, and its amount decreases with aging SMP30 gene, molecular mechanism of its regulation and consequences in response to thyroid hormone treatment in MCF-7 cells. These highly metastatic breast cancer cells are found to have endogenous expression of TRs as well as SMP30. Here, we are providing promising data in support of challenging breast cancer by targeting thyroid hormone receptors which might provide enough supplementary strength to interfere breast cancer metastasis by adjuvant therapies such as Selective estrogen receptor modulators (SERMs).The mechanism underlying thyroid hormone induced regulation of SMP30 level in breast cancer cells as well as the role of thyroid hormones against breast cancer cells has not been addressed. In our current study, we have attempted to explore the importance of SMP30 gene expression both at transcriptional and translational level. MCF-7 breast cancer cells express a substantially good amount of SMP30 which is a well established antiapoptotic gene. Here we have examined the effect of thyroid hormone on SMP30 (hSMP30) promoter, we performed an electrophoretic mobility shift assay (EMSA). We have scanned for TREs within 2 kb hSMP30 promoter from transcription start site. Transcription start site of hSMP30 gene was analysed by primer extension analysis as shown in SMP30 promoter we got two TR binding half sites i.e. at 613 bp and 1.2 kbp from hSMP30 transcription start site. The sequence of the former from −637 to −600 was found to be TGACCTTAGGATGTTGGTCAGGCTGGTCTCAAACTCCGAAGGACATTAAAGGGACAATTTCTATGACCTGGTGand that of later from −1274 to −1235 was . These two DNA fragments were 32P-labeled, incubated with nuclear extract (N.E) of MCF-7 cells - transfected with TRα, TRβ and RXR α expression vectors. The electrophoretic mobility of the radiolabelled DNA fragments was retarded in presence of nuclear extract suggesting possible interaction between the 32P-labeled DNA fragments and TRs present in the nuclear extract. This binding was later confirmed by competition with 50 and 100 fold molar excess of cold self oligos, mutated and non specific oligos as shown in SMP30 promoter has specific binding sites for TRs.To determine whether there were any TR binding sites within human SMP30 promoter in MCF-7 cells by measuring luciferase activity. We transfected reporter constructs having an oligo representative of the TREs appended to some minimal promoter along with or without expression vectors in MCF-7cells. The minimal promoter (−684 to −455) region cloned in PGL3 basic vector is known as hSMP30 TRE1 and cloned (−1290 to −1015) promoter fragment is known as hSMP30 TRE2. Luciferase activity of both the reporter vectors in MCF-7 cells having endogenous TRs did not show any significant difference in presence or absence of T3 where as luciferase activity was induced by over expressing TRs along with RXRα in absence of ligand and repressed in presence of ligand as shown in SMP30 promoter behaved similarly to those of TSHα SMP30 promoter were negatively regulated by T3.We examined the response of T3 on transcriptional activity of hSMP30 TRE2 (in the presence and absence of T3) was decreased as compared with wild type and the changes in the repression ratios were also small as compared with those noted with the wild type shown in SMP30 TRE1 did not show the similar pattern. Repression was still observed although decreased luciferase activity was detected in SMP30 gene similar to earlier observation in case of some other genes i.e CD44 gene The total luciferase expression of mutated construct of hSMP30 TREs by estradiol (E2) stimulation, we did luciferase assays of hSMP30 TREs in presence and absence of E2. We found ligand independent or dependent activation of promoter activity in case of both hSMP30 TRE1 and TRE2.However, in case of thyroid hormone treatment there was downregulation of luciferase activity as shown in To analyze the effect of TRs on hSMP30 TRE1, TRE2 in MCF-7 cells having endogenous TRs did not show any significant difference in presence or absence of T3 where as luciferase activity was induced by over expressing TRα in absence or presence of ligand. However there was significant repression found while overexpressing only TRβ in presence of ligand as presented in SMP30 promoter responds to exogenous expression of TRβ and this effect is synergized by T3 through further repressing the promoter activity.Luciferase activity of both the reporter vectors h3 is known to recruit HDACs to the thyrotropin releasing hormone (TRH) and thyroid stimulating hormone TSH β promoters during ligand dependent negative regulation. Hence, histone deacetylation may also be an important mechanism for the negative regulation of other target genes by T3 [32 and 52].T3 mediated SMP30 repression in MCF-7 cells by referring the following papers SMP30 reporter construct was abolished when TSA was added as shown in We therefore examined the effects of the histone deacetylase inhibitor TSA, on TSMP30 gene expression in MCF-7 cells was analysed both at RNA and protein level by RT-PCR and Western Blot analysis in 3 treatment resulted in repression of SMP30 gene expression by 40% and TSA further repressed SMP30 expression by 20% as here in 3 dependent histone acetylation of SMP30 promoter has an important role in regulation of SMP30 gene expression. The above results also imply that deacetylation did not affect T3 dependent silencing of transcription of the SMP30 promoter, but instead was involved in the ligand independent activation, thus reinforcing the differences in the mechanisms behind TR dependent transcriptional regulation of negative thyroid response element (nTREs) vs positive thyroid response element ( pTREs).Effect of TSA on 3-dependent histone acetylation of SMP30 promoter by ChIP analysis  to nTREs by ChIP assays  SMP30 promoter after T3 treatment involves SRC-1 recruitment.We next investigated the cofactors that participated in the Tanalysis . It was P assays .LocationSMP30 gene by T3. Cotransfection of hSMP30 TRE1 and hSMP30 TRE2 luciferase construct with TRs and RXRα expression vectors resulted in increased basal transactivation of SMP30 gene in absence of T3. However, in presence of T3 the promoter activity of SMP30 returned to basal level. siRNA mediated silencing of TRβ, completely abolished the basal transactivation of SMP30  thiazol-2-yl)hydrazone] on SMP30 promoter transcriptional activity. CPTH-2, a known modulator of Gcn5 network 3, presumably by decreasing histone acetylation as shown in  revealed histone acetylation of shown in . These rFinally, we investigated whether induction of apoptotic death in MCF-7 cells by thyroid hormone is mediated through down regulation of SMP30 expression. Compared to untreated control, thyroid hormone treatment enhanced the proportion of MCF-7 cells undergoing apoptosis by 50–60% . OverexpSMP30 gene to T3 signaling has been reported in an investigation in mouse model SMP30 gene.Recent findings indicate that epigenetic alterations of the genome such as acetylation/ deacetylation and other modifications of histones prove to be key factors in breast carcinogenesis SMP30 gene expression. In an effort to unravel the underlying mechanism, we performed primer extension analysis to find out the transcription start site of human SMP30 (hSMP30) gene, scanning of hSMP30 promoter for TRE sites followed by gel retardation assays. On the basis of these experiments we confirmed that there are two important putative TREs at 613 bp as (hSMP30 TRE1) and 1.2 kbp (hSMP30 TRE2) from hSMP30 transcription start site. The luciferase assays which were conducted by taking different deletion constructs of hSMP30 promoter validated our findings of the putative TRE sites. Looking at the distinct repression caused by liganded TRs (through T3) in SMP30 gene. Similar to our line of study, Guigon et al (2011) have identified STAT-5 as a downstream key molecule in the aberrant thyroid hormone signaling emerging out of mutated TRβ. As the consequence of this the mice is predisposed to development of mammary tumors SMP30 transcription. The above findings revealed that hSMP30 promoter responds to exogenous expression of TRβ and this effect was synergized by T3 through further repressing the promoter activity. Silencing of TRβ in MCF-7 cell line reduced the amount of repression as a result of T3 treatment both in terms of protein as well as promoter activity.In the current study our investigation by flow cytometry revealed that in response to thyroid hormone treatment, apoptosis could be induced in MCF-7 cell line as a result of down regulation of SMP30 TRE2 (in the presence and absence of T3) was decreased as compared with wild type and the changes in the repression ratios were also small as compared with those noted with the wild type. From this it is quite clear that the decrease in the repression might be due to decrease binding affinity of mutated TREs with TRs. However, mutated hSMP30 TRE1 did not show the similar pattern. Repression was still observed although decreased luciferase activity was detected. In summary, these results indicated that high affinity TR binding TREs were not necessary for T3 mediated negative regulation of SMP30 gene. In literature one hypothesis suggests that indirect involvement of TR in T3 mediated negative regulation is through the squelching of coregulators from other transcription factors The total luciferase expression of mutated construct of hSMP30 promoter.The supershift assay performed in presence of TRα, TRβ and RXRα antibodies indicated that TRβ has direct binding affinity for both TRE1 and TRE2. However, it might be possible that TRα, TRβ and RXRα protein forms a heterodimer complex then binds to thyroid response elements of both the sites present in hSMP30 promoter are negative response element in the context of liganded TRs. By observing both the TREs differentially regulated by TRs we would like to further distinguish them on the basis of different cofactors recruitment on those TREs after T3 treatment. Our specific finding of distinct recruitment of SRC-1 on TRE2 due to the enhanced association of SRC-1with TRβ in response to T3 signaling as evidenced in SMP30 gene expression. Distinct withdrawal of SRC-1 from TRE1 supports the possibility that another coregulator/s with HAT activity may participate in the negative regulation of SMP30 gene expression. Previous studies in SRC-1 knockout mice and TRβ activation function-2 domain mutant knock in mice suggest that coactivators may be important for negative regulation of genes . In our investigation another interesting but opposite result was observed in the recruitment pattern of HDAC1, HDAC2 as well as NCoR on TRE1 and TRE2. These differential recruitment patterns also make our above argument more logical. Similar kinds of observations were reported for SOD1, Necdin, and CD44 - target gene that they are under negative regulation by T3 . We believe enhanced recruitment of AcH3 locally does not speak for the complete active chromatin structure. The recent reports by Wang et alSMP30 gene by TSA (Trichostatin A) which is an inhibitor of deacetylases. The repression of SMP30 gene by liganded TRs was reversed after treatment with HAT inhibitor. From this observation it was very clear that histone acetylation played a major role in the negative regulation of SMP30 gene by liganded thyroid receptor. It also established the authenticity and importance of our characterisation of two negative thyroid response elements i.e TRE1 and TRE2 of SMP30 promoter. These observations resemble the functioning of other liganded TR mediated negative TREs (nTREs) et al discussed that TR mediated recruitment and basal activation by SMRT and NCoR in absence of T3 and reversal of basal activation by dissociation of corepressors in the presence of T3 SMP30 promoter to T3 signaling may not be analysed in isolation with regard to either TRE1 or TRE2. Rather the repression of SMP30 gene expression is the combined outcome of the response of both the above elements to T3 signaling.On the basis of our results we interpreted that thyroid response elements cannot be categorically classified as absolute positive or absolute negative elements. However by considering acetylation of histone i.e H3 as bench mark for the above classification we concluded that both TRE1 as well as TRE2 in SMP30 promoter. To ascertain the role of SMP30 in the thyroid hormone induced apoptosis of MCF-7 cells, we studied the effect of thyroid hormone after overexpressing and knocking down the SMP30 gene in MCF-7 cells respectively. Overexpression of SMP30 in our study resulted in reversal of thyroid hormone induced apoptosis of MCF-7 cells while knocking down of SMP30 made the cells increasingly susceptible to thyroid hormone induced apoptosis. These findings indicated anti-apoptotic role of SMP30 in MCF-7 cells which is in accordance with earlier reports regarding the role of SMP30 in literature SMP30 gene in MCF-7 cells. Interestingly, this repression was further enhanced as a result of T3 as well as TSA treatment. It is a well established fact that TSA which is a HDAC inhibitor induces apoptosis in different types of cancer et alet al (2011) have shown downregulation of STAT-5 as a result of mutation in TRβ which was manifested in terms of predisposal of the mice to the development of mammary tumors. Our current study and the emerging importance of thyroid hormone in breast cancer support our claim that why we need to seriously think for a combinatorial therapy by taking both T3 as well as other anticancer drugs including those related to TSA Observations in the present study revealed that thyroid hormone mediated down regulation of SMP30 was direct and accomplished through negative TREs within h2 atmosphere in 37°C until 70–80% confluent. For stimulation with T3, culture medium was removed, the cells were rinsed once with phosphate buffer saline (PBS) and medium containing10% charcoal-stripped fetal bovine serum (CS-FBS) was added and incubation continued for 3 days. T3 (1 µM), (from Sigma) was diluted in medium and charcoal-stripped 10% fetal bovine serum was added to cells for the times indicated in figure legends.MCF-7and HEK 293T were obtained from National Centre for Cell Sciences, Pune. They were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) mantained in 5% COSMP30 TRE1 reporter construct was prepared by amplifying human SMP30 promoter from MCF-7 genomic DNA by using hSMP PCR 1F2 and hSMP PCR 1R1 primer sequences are shown in pBlue TOPO TA vector (Invitrogen). Insert was taken out from pBlue TOPO TA vector by HindIII digestion and ligated in to HindIII digested PGL3 Basic vector to obtain hSMP TRE1 reporter construct. Similarly, hSMP TRE2 reporter construct was prepared by amplifying human SMP30 promoter from MCF-7 genomic DNA using hSMP PCR 2F and hSMP PCR 2R1 primer sequences shown in pTARGETtm vector. Insert was taken out from pTARGETtm vector by digesting in Kpn I and Xho I enzyme, ligated in to digested PGL3 basic vector. RT-PCR product of human SMP30 was prepared from MCF-7 cDNA by using hSMP30 EcoRI F and XhoI R primers , RXRα expression vector or pCMV vector and 100 ng of pRL-TK control vector were co transfected using Fugene HD transfection reagent (from Roche) as per manufacture's instruction. After 24 hrs of transfection 1 µM T3 hormone was added and treatment of TSA and HAT inhibitor (from Sigma) were carried out accordingly as shown in figure legend. The cell lysates were prepared, and luciferase activities were measured in duplicates in three independent experiments.Transient transfections were carried out using MCF-7 cells and similar experiments were also carried out in HEK 293T cell lines. 20 hrs before transfection, cells were plated in DMEM 10%CS media, at a density of 1×103, then cells were harvested for luciferase assays and readings were taken in duplicates in three independent experiments.24 hrs after transfection of reporter plasmid DNA and expression vectors, 200pmole of TRβ siRNA or nonspecific scrambled siRNA (syntesized from Eurogentec) per well were transfected using oligofectamine according to manufacture's instruction (Invitrogen). After 24 hrs of second round transfection, cells were incubated for an additional 24 hrs in the presence and absence of 1 µM TSMP30 TRE sites were synthesized as shown in 32P ATP using T4 poly nucleotide kinase and annealed to its complementary unlabelled strand. Nuclear extracts of MCF-7transfected with TRα, TRβ and RXRα expression vectors (10 µg) to maintain appropriate concentration of functionally active nuclear receptors were incubated with 20fmoles of radiolabelled oligonucleotide duplex and 1 µg poly (d I-d C) in 30 µl reaction mixture containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, 5% glycerol for 20 mins at room temperature. In competition experiments, 100fold molar excess of unlabeled self and mutated oligonucleotide duplexes (sequences as shown in Electrophoretic mobility shift assay was performed as described in SMP30 TRE1 (h SMP PCR 1F1 and Xho2 R), TRE2 promoter (h SMP PCR 3F1 and 2R2) and non TRE region. These primer sequences were listed in ChIP assay was performed as previously described Co-Immunoprecipitation (Co-IP) and western blot assays were performed as described SMP30 and GAPDH mRNA expression were determined by quantitative RT PCR using SYBR greensystem (SIGMA). Relative values (mean ± SD) normalized to GAPDH expression.MCF-7 cells were harvested after 15 hrs of T3 followed by 6 hrs of TSA with or without treatment. RNA was isolated and SMP30 were given in SMP30 was as follow: initial denaturation (94°C 3 min),  for 30 cycles and additional extension was per`formed at 72°C for 5 min. The PCR condition for GAPDH as follows: initial denaturation (94°C 3 min),  for 30 cycles and additional extension was performed at 72°C for 5 min. Then PCR products were electrophoresed in 1.5% agarose gel.RT-PCR was preformed as described elsewhere Whole cell extracts were prepared from cells and Western blotting was performed as described elsewhere SMP30 gene as (SMP30-FLAG) were incubated in presence or absence of 10 µM T3 for 24 hrs in culture. Knocking down of endogenous SMP30 expression or non specific scrambled siRNA was followed by incubation of MCF-7 cells with or without 10 µM T3. After 24 hrs of incubation cells were harvested and assayed for apoptosis using the Annexin V FITC apoptosis detection kit, IMGENEX according to manufacturer's instruction. Cells were analyzed in FACS Calibur Analyzer (Becton Dickinson) by using Cell Quest Pro software.To detect intact, necrotic and apoptotic cells, flow cytometry was performed. MCF-7 cells transfected with mock vector (CMV-FLAG) and vector containing Detection of apoptosis by Annexin-V-FITC detection kit was further confirmed by intracellular staining with PE-conjugated polyclonal antibody against cleaved PARP (BD Biosciences 552933) according to manufacturer's instruction. This was followed by washing with PBS twice and analysis by Flow cytometer. Percentage of population shifting right in the gate represent population of cells showing cleavage of caspase substrate PARP.Two bases of the TREs were mutated as shown in the Figure S1Determination of hSMP30 Transcription start site (TSS). Determination of transcription initiation sites by primer extension analysis. A Lane 1–4, sequencing reactions; lane 5, primer extension product of MCF-7 total RNA, lane 6, labeled DNA marker from Promega. The sequence corresponding to the transcription start site has been marked by a line and is complementary to the sequencing reactions shown in the figure. B. A schematic diagram of human SMP30 promoter showing two TREs, CAAT box and transcription start site.(DOC)Click here for additional data file.Figure S2To analyze the effect of T3 on SMP30 Promoter activity in MCF-7 cell in relation to RXRα. Transient transfections of hSMP30 TRE1, TRE2 were carried out using MCF-7 cells. 20 hrs before transfection, cells were plated in DMEM 10%CS media, at a density of 1×105 cells per well in 12 well plates. For transient transfection, 0.5 µg of reporter plasmid DNA, 0.5 µg of TRβ and TRα (TRs), RXRAαexpression vector, 100 ng of pRL-TK control vector and only vector to control cells were co transfected using Fugene HD transfection reagent (from Roche) as per manufacture's instruction. After 24 hrs of transfection, cells were subjected to overnight treatment with 1 µM T3 and vehicle to control cells in 10% CS –DMEM. Then cell lysates were prepared and luciferase activities were measured. Values are the mean of three independent experiments ± SD normalized to Renilla activity. *** P<0.0001difference from vehicle control using ANOVA.(DOC)Click here for additional data file.Figure S3Luciferase activity of SMP30 TREs in HEK293 cell line. Transient transfections of hSMP30 TRE1, TRE2 were carried out using HEK 293 cells. 20 hrs before transfection, cells were plated in DMEM 10%CS media, at a density of 1×105 cells per well in 12 well plates. For transient transfection, 0.5 µg of reporter plasmid DNA, 0.5 µg of TRβ and TRα (TRs), RXRα expression vector, 100 ng of pRL-TK control vector and only vector to control cells were co transfected using Fugene HD transfection reagent (from Roche) as per manufacture's instruction. After 24 hrs of transfection, cells were subjected to overnight treatment with 1 µM T3 and vehicle to control cells in 10% CS –DMEM. Then cell lysates were prepared and luciferase activities were measured. Values are the mean of three independent experiments ± SD normalized to Renilla activity. ***P<0.0001difference from vehicle control using ANOVA.(DOC)Click here for additional data file.


Ecology and Evolution 2013; 3(8): pages 2547–256710.1002/ece3.636doi: 4, and for the Sinking Creek form at Locality 31, which is actually polymorphic for GAPDH2 and GAPDH3. We still assert that the allozyme frequencies at Locality 36 may reflect gene exchange with LGF.Table 2 reports incorrect GAPDH allozyme frequencies for the Lemon Gap form at Locality 61, which is actually fixed for GAPDHA corrected version of “The populations at localities 36 and 73 are fixed or nearly fixed for the same variants at 16 presumptive loci, but exhibit complete differentiation at the remaining six . At each of these diagnostic loci except GAPDH, the population at Locality 36 shares a variant with LGF at Locality 61 (Table