In embryonic stem (ES) cells, developmental regulators have a characteristic bivalent chromatin signature marked by simultaneous presence of both activation (H3K4me3) and repression (H3K27me3) signals and are thought to be in a ‘poised’ state for subsequent activation or silencing during differentiation. We collected eleven pairs (H3K4me3 and H3K27me3) of ChIP sequencing datasets in human ES cells and eight pairs in murine ES cells, and predicted high-confidence (HC) bivalent promoters. Over 85% of H3K27me3 marked promoters were bivalent in human and mouse ES cells. We found that (i) HC bivalent promoters were enriched for developmental factors and were highly likely to be differentially expressed upon transcription factor perturbation; (ii) murine HC bivalent promoters were occupied by both polycomb repressive component classes (PRC1 and PRC2) and grouped into four distinct clusters with different biological functions; (iii) HC bivalent and active promoters were CpG rich while H3K27me3-only promoters lacked CpG islands. Binding enrichment of distinct sets of regulators distinguished bivalent from active promoters. Moreover, a ‘TCCCC’ sequence motif was specifically enriched in bivalent promoters. Finally, this analysis will serve as a resource for future studies to further understand transcriptional regulation during embryonic development. Embryonic stem (ES) cells have the unique ability to self-renew indefinitely as well as to differentiate in response to internal as well as external stimuli69Bivalency of chromatin has therefore become an important property to investigate the functional relevance of a gene through development, and the presence of bivalent genes in human and mouse ES cells has been validated by many studies independently9121314ChIP sequencing raw data for H3K4me3 and H3K27me3 histone marks in murine and human ESCs was obtained from Gene Expression Omnibus (GEO)The one2one orthologous regions between human and mouse  were obtained from Ensembl BioMartFor the clustering of bivalent promoters into four groups, we gathered chip sequencing data for three different forms of RNAPII: RNAPIIS5P, RNAPIIS7P and 8WG16We calculated the CpG density as the ratio of observed to expected CpG countsFor identification of factors enriched at bivalent and H3K4me3 promoters, we used data from 49 and 99 ChIP-seq experiments for several factors in human and mouse embryonic stem cells respectivelyRNA sequencing data in murine ES cellsThe sequence motif enrichment analysis was performed with the command findMotifs.pl from HOMERr) = 0.75, for H3K4me3 r = 0.84), but not across human datasets . There are other factors contributing to the variation between samples, for example ES cells were grown in diverse culture conditions, and using different cell lines as well as various antibodies across datasets  of H3K4me3 and H3K27me3 ChIP sequencing (ChIP-seq) datasets for human ES cells and 8 pairs for mouse ES cells from the Gene Expression Omnibus (GEO) database and the Roadmap Epigenomics Project . After adatasets . We therdatasets . Eight Hdatasets . Adding datasets . There wdatasets .rd of H3K27me3 top promoters in any individual dataset overlapped with HC bivalent promoters   and S7, romoters .We also checked whether the peaks of H3K27me3 and H3K4me3 modifications were present at the same genomic location within a promoter region and found that over 95% of H3K27me3 and H3K4me3 peaks overlapped in each pair of samples at HC bivalent promoters. Both chromatin modifications were indeed present at the same genomic location . We compIn summary, by integrating data from multiple studies we identified HC human and murine bivalent promoters, which could not be identified by simply selecting the top peaks from individual samples. The HC bivalent promoters were highly enriched for developmental regulators compared to non-HC bivalent promoters.Bivalent promoters are known to show variation in their levels of occupancy by RNA polymerase II−51) while cluster 4 promoters were enriched for developmental functions such as organ morphogenesis . Cluster 3 promoters contained transcription factors important for specific lineages like haematopoiesis factors Gfi1 and Meis1, whereas cluster 4 contained multiple members of transcription factor families controlling development such as winged helix/forkhead box (Fox) and Hox families.HC bivalent promoters could be classified in four distinct clusters based on the presence of PRC1 components and forms of RNAPII . The firet al. (12) suggested that PRC1 was absent in our PRC1 low bivalent promoter  and PRC1 high (cluster 3 & 4). Ku promoter . Ring1b promoter .In summary, all HC bivalent promoters are occupied by components of both PRC1 and PRC2. There exists a distinct set of metabolic genes (cluster 2) which though bivalently marked has RNAPII (S7P) and is expressed at a higher level than other bivalent genes.RNA polymerase II (PolII) may be present but stalled at the promoters of bivalent genes and short (abortive) transcripts may be detected at their promotersAs bivalent genes are thought to be poised for activation or repression, we hypothesised that these genes might be more likely to be differentially expressed upon perturbation of ES cells. We therefore used a collection of differentially expressed genes upon deletion or over-expression of 91 transcription and epigenetic factors in mouse ES cells, and found that 98% of differentially expressed gene sets by the overexpression of at least one TF significantly overlapped  with bivalent genes, and 89% differentially expressed gene sets by the down-regulation of at least one TF . To chec−71) and transcription factor activity ; whereas species-specific promoters were not enriched for the two above terms (−16) and glycoprotein  and the human-specific for plasma membrane part  and alternative splicing .To perform a systematic comparison of chromatin status between human and mouse promoters in ES cells, we used 16,639 one-to-one orthologous genes between the two speciesve terms . SpecifiTo check whether the chromatin status across species is reflected in the gene expression status, we focused on five groups of promoters : three gAs shown in the first section, the bivalent status of promoters is primarily determined by the detection of an H3K27me3 modification . CpG isl3738Over 90% of our HC bivalent promoters in ES cells in both species overlap with at least one CGI region, whereas only 8% (37 of 397) of human H3K27me3 only promoters contained a CGI and no mouse H3K27me3 only promoters (none of 152) contained a CGI . Previou41The loss of H3K27me3 in rodents (mouse and rat) compared to human ES cells at many developmental genes has been associated with depletion of CGIs; mouse CGI erosion has been characterised at MYO1G, CLEC4G and MYF6 gene loci with corresponding H3K27me3 lossIn summary, the H3K27me3-only CpG-poor promoters demonstrate that polycomb recruitment does not only depend on CpG density. Although the CpG density largely correlates with H3K4me3 and H3K27me3 profiles across promoters, the loss of CGI on a promoter does not always imply a corresponding loss of the H3K4me3 and/or H3K27me3 modification on that promoter.As both active (H3K4me3-only) and bivalent promoters are CpG-rich, we investigated possible modes of distinction between the two in ES cells. Voigt, Tee, and Reinberg−256). Moreover, the co-repressor c-terminal binding protein 2 (CTBP2), required for PcG recruitment in Drosophila4647−3). Four other epigenetic regulators, Utf1, Tet1, Rest and Setdb1 were highly enriched at mouse bivalent regions. Utf1  was recently identified as a component of bivalent chromatin by acting as a buffer against full activation of bivalent genesTo identify factors preferentially binding to bivalent promoters, we calculated the overlap between transcription and epigenetic factor binding sites (peaks) and bivalent promoters. Four out of 49 and eleven out of 99 factors characterised by ChIP-seq preferred bivalent promoters in human and mouse respectively . As expeAs expected, many TFs (33 out of 49 factors in human and 39 out of 99 factors in mouse) were enriched at active (H3K4me3-only) promoters. This included known regulators of pluripotency in ES cells such as Klf4, Esrrb, Oct4, Sox2, and Nanog . Only twde novo motif identification on bivalent promoters by providing active promoter sequences as background in HOMER softwarede novo motif discovery). The ‘TCCCC’ motif was most similar to the known binding sequence of the Mzf1 transcription factorde novo motif enrichment was performed on active human and mouse promoters using bivalent promoter sequences as background, they were enriched for a ‘CGGAA’ motif found in 40% of the active promoter sequences, which was not enriched in bivalent promoters. This motif is the most similar to the known motif for Elk1 transcription factor  promoters, but more than H3K27me3 only and latent promoters. Active promoters were preferentially occupied by pluripotency factors. On the other hand, bivalent promoters were enriched for Polycomb factors as well as other chromatin modifiers. The factors enriched at bivalent promoters show very little overlap with the ones enriched at active promoters. These findings are consistent with the observed spatial segregation of transcriptional networks in ES cells where Nanog and Polycomb proteins were shown to occupy distinct nuclear spacesBivalent chromatin domains bearing both H3K4me3 and H3K27me3 modifications have been shown to be a key feature of developmentally regulated genes in ES cells8913149149Bivalent promoters are thought to be poised for rapid activation or inactivation during differentiation13We computed binding profiles of PRC components (PRC1 and PRC2) and various forms of RNA polymerase II at bivalent promoters in murine ES cells. All HC bivalent promoters were marked by Suz12, Jarid2, Ring1b and Cbx7. To note, the PRC2-only group defined byMore than half of high-confidence bivalent promoters were conserved between human and mouse, suggesting the existence of a set of genes bivalently marked across most mammalian ES cells . These g54et al. (2014) recently suggested that bivalency is the default chromatin structure for CpG-rich, G+C-rich DNASince a high density of un-methylated CpG is sufficient for vertebrate polycomb recruitment3839On bivalent promoters, the CpG density and H3K27me3 modification are highly correlated. By performing a cross-species comparison, a small fraction (~5%) of human CpG-rich HC bivalent promoters has the corresponding CpG eroded in the mouse genome, while no CpG-rich bivalent promoters in mouse are eroded in human. This erosion of CpG density was correlated with the loss of H3K27me3 and H3K4me3et al.It is intriguing how bivalent domains are established in ES cells. Voigt De novo motif discovery at HC bivalent promoters identified a ‘TCCCC’ motif in both human and mouse ES cells which was not enriched at active promoters. This motif was present in about half of the HC bivalent promoters and is similar to the sequence motif of MZF1As active (H3K4me3-only) and bivalent promoters are both CpG rich, it is key to unravel the distinguishing factors between these two groups. In summary, this meta-analysis revealed several novel aspects of bivalency in mammalian ES cells and will serve as a resource for future studies to further understand transcriptional regulation during embryonic development. Further work will be aimed at understanding how the HC bivalent promoters identified here are resolved in different cellular lineages during differentiation.How to cite this article: Mantsoki, A. et al. CpG island erosion, polycomb occupancy and sequence motif enrichment at bivalent promoters in mammalian embryonic stem cells. Sci. Rep.5, 16791; doi: 10.1038/srep16791 (2015).


The homeodomain transcription factor Hoxa2 interacts with the RING-finger type E3 ubiquitin ligase RCHY1 and induces its proteasomal degradation. In this work, we dissected this non-transcriptional activity of Hoxa2 at the molecular level. The Hoxa2-mediated decay of RCHY1 involves both the 19S and 20S proteasome complexes. It relies on both the Hoxa2 homeodomain and C-terminal moiety although no single deletion in the Hoxa2 sequence could disrupt the RCHY1 interaction. That the Hoxa2 homeodomain alone could mediate RCHY1 binding is consistent with the shared ability all the Hox proteins we tested to interact with RCHY1. Nonetheless, the ability to induce RCHY1 degradation although critically relying on the homeodomain is not common to all Hox proteins. This identifies the homeodomain as necessary but not sufficient for what appears to be an almost generic Hox protein activity. Finally we provide evidence that the Hoxa2-induced degradation of RCHY1 is evolutionarily conserved among vertebrates. These data therefore support the hypothesis that the molecular and functional interaction between Hox proteins and RCHY1 is an ancestral Hox property. Hoxa2 belongs to the extremely well-conserved Hox gene family which includes 39 members in mammals. Mammalian Hox genes are organized in four clusters located on different chromosomes and can be classified in 13 paralogue groups based on their sequence similarities and their relative position within the clusters. Hox genes code for transcription factors which fulfill well-documented functions during embryonic development. In particular, their most spectacular activities are associated to the patterning of the main body axis, limb development and organogenesis .Kip1, polH, CHK2 and c-MYC, are known to be involved in the control of cell proliferation and cell death. Consequently, by influencing the abundance of these targets, RCHY1 has been shown to be a regulator of DNA damage response and cell cycle progression . RCHiewed in , 9. Indeiewed in –13. Moreiewed in .In a former study, we reported that Hoxa2 expression correlated with a decrease in RCHY1 protein levels. We provided evidence that the RCHY1 decay induced by Hoxa2 involved the proteasome pathway but in an ubiquitin-independent way. Finally, correlatively to the RCHY1 degradation, Hoxa2 expression was also shown to decrease the ubiquitination of p53, in turn, increasing its abundance ), both terminal domains (Hoxa2HD [139–198]), or the homeodomain (Hoxa2ΔHD [δ139–198]) were removed  that the HOXA2-RCHY1 interaction mainly takes place in the nucleus (2) that the RCHY1 decay induced by HOXA2 depends on both the 19S and the 20S proteasome particles, (3) that the interaction involves molecular determinants from at least two distinct HOXA2 regions providing contacts which are sufficient but not necessary for the binding of RCHY1 and (4) that some HOXA2 deletion derivatives, though still capable of interacting with RCHY1, have lost the ability to provoke its protesomal degradation. Finally, the results provided in the present study support that the downregulation of RCHY1 provoked by HOXA2 is conserved among orthologues from other vertebrate species and that binding to RCHY1 seems to be generic characteristic of HOX proteins yet the ability to stimulate its protesomal degradation is shared only by a subset of them.WMAA protein mutated in the hexapeptide sequence located in the N-terminal part of Hoxa2. Similarly to Hoxa2ΔN, the Hoxa2WMAA protein has been shown to induce RCHY1 decay [ΔN and Hoxa2WMAA mutants lack the hexapeptide sequence but maintain full activity with regards to RCHY1 destabilization, we propose that the Hoxa2-mediated effect on RCHY1 degradation is independent of the transcription activity of Hoxa2 and corresponds to a novel non-transcriptional activity for Hoxa2.The N-terminal part of Hoxa2 seems dispensable to the induction of RCHY1 degradation. Indeed, the Hoxa2 deletion mutant lacking the N-terminal portion of the protein destabilizes RCHY1 to the same extent as the WT Hoxa2. This result could be related to what we previously showed for the Hoxa2Y1 decay . This heY1 decay  and in tY1 decay . As bothKQN-RAA), as we previously reported [Conversely, although all the HOXA2 variants tested so far remain capable of interacting with RCHY1, the ability to induce RCHY1 destabilization is only affected upon deletion of the C-terminal part or the entire homeodomain of HOXA2, or as a consequence of point mutations in the homeodomain .COS-7 cells were transfected with vectors coding for VN(TIF)Click here for additional data file.S2 Fig173RCHY1 coding vector together with the VC155p27Kip1 coding vector. Nuclei were stained with DAPI (blue).COS-7 cells were transfected with the VN(TIF)Click here for additional data file.


Decreased ability in oxygen delivery and diffusion of the blood vessels and increased oxygen consumption in the rapidly proliferating cancer cells both contribute to reduced oxygen availability in the solid tumors, a major signature of the tumor microenvironment . The oxyHIF is a heterodimeric transcription factor, consisting of an oxygen-regulated α subunit and a constitutively expressed β subunit. The regulation of HIF-α protein levels is a critical determinant of HIF-mediated gene transcription. Our previous study showed that chronic hypoxia causes the selective decay of HIF-1α at least in part by HSP70- and CHIP-dependent ubiquitination and proteasomal degradation . The levSLC2A3, GPI, PDK3, HGF, in HeLa cells exposed to chronic hypoxia (1% O2 for 72 hr) [2 for 24 hr) [Moreover, we further found that double knockdown of PRDX2 and PRDX4 selectively increases expression of a subset of HIF target genes, including HIFs have been the attractive targets for cancer therapy. Identification of the specific HIF regulators under conditions of acute and chronic hypoxia may yield the new therapeutic strategies. Indeed, high levels of PRDX2 and PRDX4 are significantly correlated with increased survival of patients with bladder cancer (our unpublished data), suggesting that PRDX2 and PRDX4 may be the new therapeutic targets for treatment of cancer.


During 2001–2014, predominant influenza A(H1N1) and A(H3N2) strains in South America predominated in all or most subsequent influenza seasons in Central and North America. Predominant A(H1N1) and A(H3N2) strains in North America predominated in most subsequent seasons in Central and South America. Sharing data between these subregions may improve influenza season preparedness. During 2002–2008, infection with influenza viruses caused 40,880–160,270 deaths each year throughout the Americas  and North America 4,535,508 results  to the Global Influenza Surveillance and Response System  of 8 years in Central America and 2 (17%) of 12 years in South America, when there was 1 southern temperate winter epidemic and a smaller northern temperate winter epidemic, all subregions had 1 annual influenza epidemic that lasted ≈5 months.The predominant influenza A(H1N1) virus strains in South America predominated in 9 of 9 subsequent seasons in Central America and 12  of 13 subsequent seasons in North America . SimilarThe predominant A(H1N1) virus strains in North America predominated in 7  of 9 subsequent seasons in Central America and 10  of 12 subsequent seasons in South America. A(H3N2) virus strains in North America predominated in 8  of 12 subsequent seasons in Central America and 10  of 13 subsequent seasons in South America. Influenza B virus strains in North America predominated in 9  of 12 subsequent seasons in Central America and 7  of 13 subsequent seasons in South America. Virus strains that predominated in North America during 1 season were less likely to predominate in the subsequent season in North America .At least 1 component of the Southern Hemisphere vaccine composition recommendations matched a predominant antigenic characterization in South America in 13  matched the Southern Hemisphere recommendations and 24  matched the Northern Hemisphere recommendations .Our findings suggest that virus strains identified during influenza epidemics in South America typically became predominant in subsequent epidemics in Central and North America. Almost as frequently, virus strains identified during epidemics in North America became predominant in the subsequent Central and South America epidemics. Although strain selection for 1 hemisphere’s vaccine formulation typically occurs before influenza activity is widespread in the opposite hemisphere, health officials have an opportunity to anticipate which influenza virus strains may predominate by observing activity in other subregions. For example, influenza A(H1N1)pdm09 virus predominated in Brazil during 2013  and then aggregated by subregion. These samples may not be geographically representative. Additional data will be needed to determine whether the characteristics of 1 subregion reliably predict influenza epidemics in another. New viral strains that appear might be introduced from outside the Americas (In summary, health officials in North and Central America may find clues about which influenza A virus strains are likely to predominate during an upcoming season by observing which were predominant in South America and vice versa. Our findings underscore the need to share timely and representative specimens with World Health Organization Collaborating Centres. In the future, shorter vaccine production times using novel technology might facilitate matching vaccine composition more closely to circulating virus strains. edit 1 Most commonly identified antigenic characterizations of influenza strains in the Americas during 2002–2013.


Incidence of tuberculous lymphadenitis, accounting for 30 – 40% of extra-pulmonary tuberculosis, has increased in parallel with increase in the incidence of tuberculosis worldwide. The diagnosis becomes more challenging when clinical presentation is suggestive but bacteriological proof is lacking.Mycobacterium tuberculosis by Ziehl neelsen (ZN) staining, culture on a Lowenstein Jensen medium, and cytological examination for findings suggestive of tubercular lymphadenitis. DNA from all the samples was amplified by conventional PCR targeting 123 bp and 240 bp fragments of IS6110 and MPT64 genes respectively and RealTime PCR targeting MPT64 gene only.Sixty lymph node aspirates from suspected cases of tuberculous lymphadenitis were examined for MPT 64 gene in PCR could diagnose 45 cases unlike IS6110 gene which could help in diagnosis of 42 cases (7.5 vs 7%). Whereas PCR gave 13 positivities which were negative by any of the conventional methods, it was negative in 2 samples that were positive on cytology only.Histopathology, ZN staining, and culture gave positivity in 25, 20 and 16 cases respectively; out of which 6 (10%) were diagnosed by positivity in all the three conventional techniques, whereas 36 (60%) were positive by any of these three techniques. Diagnosis could be made by conventional PCR in 43 cases when compared to 45 by the RealTime PCR method (7.2 vs 7.5%) while targeting the Though conventional diagnosis remains the method of choice in tuberculous lymphadenitis alternative diagnostic method such as PCR which are more rapid and reliable looks promising and help well manage patients.


The reported study  is similChinese Journal of Cancer [“The clinical value of adjuvant radiotherapy in patients with early stage breast cancer with 1 to 3 positive lymph nodes after mastectomy,” published in the f Cancer .PLOS ONE paper.This previous work was cited as reference number 20 in the PLOS ONE paper, patients receiving treatment from July 1998 to November 2007 were included in the study (79 patients received postmastectomy radiotherapy (PMRT); 618 patients did not receive PMRT). In the Chinese Journal of Cancer paper, patients receiving treatment from January 1998 to May 2007 were included in the study .In the PLOS ONE study [The authors wish to clarify the sampling methodologies. For the earlier study , only paNE study  additionThe authors also note additional differences between the two studies. The clinical value of PMRT in patients with T1-2 breast cancer and 1–3 positive axillary lymph nodes (T1-2N1M0) remained controversial following publication of the first study [


Cilia formation starts at the mother centriole and requires the activity of the microtubule affinity regulator, MARK4, which promotes axoneme extension during the initial phase of ciliogenesis. The defective axoneme extension in MARK4 depleted cells could be in part rescued by co-depletion of the inhibitory complex CP110/Cep97. Whether MARK4 only influences CP110/Cep97 or influences additional inhibitory components is not known. Interestingly, MARK4 has been recently shown to regulate the position and movement of autophagic vesicles within the cell. Recent studies also revealed that autophagy promotes ciliogenesis by inducing the selective degradation of the centriolar satellite pool of OFD1, an inhibitor of centriole elongation and axoneme extension. Here, we investigated whether MARK4 is functionally linked to autophagy to promote cilia formation. Analysis of RPE1 cells stably expressing YFP-LC3 shows that autophagosomes exhibit a perinuclear clustering upon MARK4 depletion. Quantitative immunofluorescence analysis shows that in MARK4 depleted cells, OFD1 levels at the centriolar satellites are higher than in control cells. This could be reverted by partial knock down of OFD1. By co-depleting MARK4 and OFD1, the cilia loss phenotype of MARK4 knockdown was partially reverted. Our results suggest that MARK4 acts in ciliogenesis by regulating the movement and position of the autophagosomes that degrade OFD1 at the centriolar satelites.


We also show for the first time that a potent and selective TRPA1 antagonist GRC 17536 inhibits citric acid induced cellular Ca+2 influx in TRPA1 expressing cells and the citric acid induced cough response in guinea pigs. Hence our data provides a mechanistic link between TRPA1 receptor activation in vitro and cough response induced in vivo by citric acid. Furthermore, we also show evidence for TRPA1 activation in vitro by the TLR4, TLR7 and TLR8 ligands which are implicated in bacterial/respiratory virus pathogenesis often resulting in chronic cough. In conclusion, this study highlights the potential utility of TRPA1 antagonist such as GRC 17536 in the treatment of miscellaneous chronic cough conditions arising due to diverse causes but commonly driven via TRPA1.Cough is a protective reflex action that helps clear the respiratory tract which is continuously exposed to airborne environmental irritants. However, chronic cough presents itself as a disease in its own right and despite its global occurrence; the molecular mechanisms responsible for cough are not completely understood. Transient receptor potential ankyrin1 (TRPA1) is robustly expressed in the neuronal as well as non-neuronal cells of the respiratory tract and is a sensor of a wide range of environmental irritants. It is fast getting acceptance as a key biological sensor of a variety of pro-tussive agents often implicated in miscellaneous chronic cough conditions. In the present study, we demonstrate  Cough is a vagally mediated reflex and a primary defensive mechanism to protect the airway by forceful expulsion of irritant agents from the respiratory tract. Although a protective response, sometimes it becomes excessive and harmful to the airway mucosa leading to compromised quality of life. Cough is broadly divided into acute and chronic persistent cough in vitro and in vivo studies have recently established TRPA1 as a major chemosensory receptor of the airways Activation of sensory nerves innervating anatomical regions implicated in cough reflex, including the larynx, trachea and large bronchi, by exogenous inhaled or aspirated substances or by locally produced endogenous biochemical mediators can produce cough 2 or TRPA1 activators - 4-Hydroxynonenal (4-HNE), reactive oxygen species (ROS), reactive nitrogen species (RNS), 15d-PGJ2 in the esophagus during GERD TRPA1 on sensory nerves detects not only exogenous environmental agents that may be inhaled, but is also activated by known endogenous tussive molecules such as PGE2 and bradykinin produced during tissue inflammation. Additionally, it also senses the inflammatory environment present in inflammatory lung disease, viral/ bacterial infection or extrapulmonary diseases. Literature demonstrates TRPA1 receptor expression in the esophagus and simultaneous presence of inflammatory mediators that are known to be either TRPA1 sensitizers - histamine, bradykinin, PGE+2 influx not only in recombinant TRPA1 expressing/ CHO cells but also in human primary lung fibroblasts and airway epithelial cells that express TRPA1 endogenously. Two potent and selective TRPA1 antagonists (GRC 17536 and compound 7) was able to abrogate TRPA1 agonist (s) mediated functional response in these cells. We also further provide in vivo evidence in guinea pig model that citric acid induced cough response is inhibited by GRC 17536. Since TLR7 and 8 have been implicated in viral pathogenesis, we also evaluated effect of TLR ligands on TRPA1 receptor. We demonstrate that TLR ligands, in particular the TLR4 (LPS), TLR7 (loxoribine) and TLR8 (ssRNA) ligands upregulated the expression of TRPA1 and also enhanced calcium uptake mediated by TRPA1. This response was attenuated by TRPA1 specific antagonist, GRC 17536 and compound 7. Hence, TRPA1 receptor could be a common and crucial molecular mediator behind various chronic cough etiologies.In the present study, we investigated if citric acid, which is used for many years to provoke cough in animals as well as human studies 45Ca+2 , Microscint PS , G418  and FBS .DMEM F-12, BSA and HEPES were purchased from Sigma Aldrich Inc. , MEM from Hyclone . Other reagents were procured from vendors as indicated: 2). For Calcium fluorescence assay, Ca+2, Mg+2 free PBS were used instead of DDB for drug dilution. LPS, AITC, capsaicin, H2O2, and crotonaldehyde were procured from Sigma Aldrich Inc. . ssRNA40 and loxoribine were purchased from Invivogen , 15d-PGJ2 from Enzo life sciences , and citric acid from Merck .Glenmark proprietary TRPA1 antagonists , TRPV1 antagonist (GRC 6211) and compound 7  were synthesized in-house by the discovery chemistry group. A 10 mM stock of the above antagonists was prepared in DMSO. Subsequent dilutions from the stock solution were made in drug dilution buffer (DDB) (DMEM F-12 containing 1.8 mM CaCl2. Chinese hamster ovary (CHO) cells stably expressing human TRPA1 (hTRPA1/CHO), TRPV1 (hTRPV1/CHO), TRPV3 (hTRPV3/CHO), TRPV4  were obtained from American Type Culture Collection  and cultured in MEM supplemented with 10% FBS. Human pulmonary alveolar epithelial cell line A549 (ATCC CCL-185) was also obtained from ATCC and maintained in DMEM F-12 supplemented with 10% FBS. Both the cell types were cultured at 37°C in humidified air containing 5% COad lib standard guinea pig pellet diet and water containing dietary amounts of ascorbic acid.Male Hartley guinea pigs  were acclimatized in cages  before beginning of the experiments and were maintained on an ad lib standard guinea pig pellet diet and water containing dietary amounts of ascorbic acid. All animal experiments were approved by Institutional Animal Ethics Committee (IAEC) of Glenmark Pharmaceuticals Ltd and the study was carried out in strict accordance with recommendations of Committee for the Purpose of Control and Supervision of Experiments on Animals . All animal work was conducted as per CPCSEA guideline to minimize discomfort to animals. Guinea pigs were euthanized humanely with CO2 inhalation at the end of the study.Guinea pigs were housed in cages with a light/dark cycle of 12/12 hours and were maintained on an (-ΔΔCT) Livak method of analysis.Total RNA was extracted from hTRPA1/CHO cells using TRI reagent . 3 µg of RNA was reverse transcribed using iScript select cDNA synthesis kit  according to the manufacturer's instructions to produce single-stranded cDNA. RT-PCR was performed with an real-time thermal cycler Eppendorf Mastercycler Ep  using iTaq SYBR Green Supermix with ROX kit  with human TRPA1 specific primers: 5′-GATATTGTTAACACAACCGATGGA-3′(sense primer) and 5′-CTTTTATGTCTACTTG GGCACCTT-3′(antisense primer). RT-PCR reaction was carried out for 40 cycles at 94°C for 30 s, 60°C for 15 s and 60°C for 60 s. Data was interpreted using 2+2 influx assay was performed as described earlier 2O. Subsequent dilutions were made in Ca+2, Mg+2 free PBS (pH 7.3). No significant change in pH was observed upto 10 mM citric acid. At 30 mM concentration of citric acid, the pH of the solution dropped to 5.5. TLR ligands - LPS , loxoribine  and ssRNA40  were dissolved in media and added to the cells for 16–18 hours. TRP selectivity of GRC 17536 and compound 7 was performed for TRPV1, TRPV3, TRPV4 and TRPM8 using Capsaicin (1 µM), 2-APB (1 mM), 4α-PDD (30 µM) and Icilin (250 nM) respectively, as agonist.Fluorimetric and radiometric CaCough was detected both by pressure change and by sound and recorded with a Buxco cough analyzer  as described earlier in vitro experiments, data was analyzed using GraphPad Prism 3.0 . For in vivo experiment, data from the treatment groups were compared with the control group. Mean values were statistically analyzed by one-way analysis of variance (ANOVA) followed by the Dunnett's Multiple Comparisons Test to evaluate significant differences between groups.For +2 influx in CCD19-Lu, A549 and hTRPA1/CHO cells  at the highest concentration of citric acid. In CCD19-Lu cells maximum Ca+2 influx was observed at 10 mM citric acid concentration followed by a decrease in calcium levels at the next higher concentration (30 mM). In A549 and hTRPA1/CHO cells peak Ca+2 influx was observed at 3 mM citric acid concentration followed by a drop in calcium level at the next higher concentration. Although the citric acid response pattern and the maximum Ca+2 influx response observed in these three cell types are different, the responsiveness to citric acid stimulus in the form of increased Ca+2 influx was a clear trend displayed by these TRPA1 expressing cell types, unlike the lack of any significant response seen with the hTRPV1 expressing CHO cell line (data not shown). The differences in response to Citirc acid within these TRPA1 expressing cells could also be due to differential receptor expression level or cellular background .TRPA1 receptor has been earlier reported to be a general sensor for weak acids which can diffuse across the cell membrane in the protonated form and cause intracellular acidification HO cells . Treatme+2 influx. As shown in +2 influx in all the three cell types tested. The IC50 values for GRC 17536 and compound 7 in the above assays was 8.2 nM and 27.9 nM  +2 influx by GRC 17536 is not restricted to a particular chemical class, we tested GRC 17770, a selective TRPA1 antagonist with potency similar to GRC 17536, but belonging to a chemically diverse series in hTRPA1/CHO cell based assay. GRC 17770 also inhibited citric acid induced Ca+2 influx with an IC50 of 7.19 nM . We next evaluated whether these TLR ligands could potentiate TRPA1 agonist mediated calcium uptake. As demonstrated in 2O2 and 15d-PGJ2 induced 45Ca+2 uptake in hTRPA1/CHO cells  or ssRNA40 (1 µg/ml) resulted in a 20% and 30% increase in 45Ca+2 uptake over AITC alone  in both the cell types (50. Complete inhibition of 45Ca+2 uptake was observed at the highest concentration of the compound (1 µM). GRC 17138, another compound structurally close to GRC 17536 but devoid of any functional antagonism on TRPA1/CHO cells was used to rule out the chemical class effect and confirm TRPA1 involvement in the blocking Ca+2 influx. It failed to inhibit LPS, Loxoribine or ssRNA40 enhanced calcium flux in either hTRPA1/CHO or A549 cells up to a high concentration of 10 µM , no significant protective response was observed. Dextromethorphan at 30 mg/kg dose showed 66% inhibition of cough response.To investigate whether Citric acid mediated cough response involved TRPA1 channel, we studied effect of GRC 17536 in citric acid induced cough model in guinea pigs. Guinea pigs exposed to citric acid challenge showed significant cough response. The animals treated with GRC 17536 showed 79 and 89% inhibition of cough at 60 and 100 mg/kg respectively compared to the vehicle treated animal group . At the Cough is a reflex that helps clear the respiratory tract of environmental irritants, and is often associated with symptomatology of inflammatory airway diseases such as asthma, bronchitis and COPD in vitro evidence that TRPA1 is a direct sensor for citric acid using recombinant TRPA1 overexpressing CHO cells as well as in human primary lung fibroblast cells (CCD19-Lu) and pulmonary alveolar epithelial cell line (A549). We demonstrate functional activation of TRPA1 by citric acid, observed as a concentration dependent increase in Ca+2 influx in these cells. Similar findings demonstrating TRPA1 as a general sensor of weak acids have been published by Wang et al 2 via direct gating of the channel by intracellular protons +2 influx in recombinant hTRPV1/CHO cells with citric acid under similar experimental conditions as used for TRPA1 (data not shown). Higher concentrations of citric acid  elicited a drop in Ca+2 influx presumably due to receptor desensitization. Similar observations have been reported earlier for TRPA1 with AITC as agonist In the present study we provide +2 influx in all three cell types tested with similar potency as reported earlier +2 influx in CCD19-Lu and A549 cells. Since human lung epithelial and fibroblast cells are known to express endogenous levels of both TRPA1 and TRPV1 +2 influx in these cells by GRC 6211 further confirms TRPA1 as a mediator of citric acid induced Ca+2 influx.The specificity of TRPA1 activation by citric acid was further confirmed by using two potent and selective TRPA1 antagonists - GRC 17536 (Glenmark) and compound 7 . GRC 17536 concentration dependently inhibited citric acid induced Cain vitro validation of TRPA1 as a mediator for citric acid mediated Ca+2 influx encouraged us to explore whether the in vitro observation can be translated to in vivo animal models. We demonstrated that GRC 17536 reversed citric acid induced cough response in guinea pigs. A dose dependent decrease in tussive response was observed with GRC 17536. This is the first in vivo evidence demonstrating effectiveness of selective TRPA1 antagonist in citric acid mediated cough. Several other environmental irritants known to directly activate TRPA1 receptor e.g. acrolein, crotonaldehyde etc. have also been shown to provoke cough via TRPA1 receptor in guinea pigs and humans Citric acid has been used to provoke cough in human as well as in animal studies. Extensive 2O2, prostaglandins etc. that are known to be TRPA1 activators 45Ca+2 uptake in presence of TLR ligands and upregulation of TRPA1 gene expression.TRPA1 activation is known to induce the release of a variety of neuropeptides and neurotransmitters, e.g. calcitonin gene-regulated protein (CGRP), Substance P and neurokinin, which causes vasodilation and immune cells recruitment to the site of assault, thereby exacerbating the cellular and tissue inflammatory response. The recruited immune cells secrete a number of inflammatory mediators e.g. hypochlorite, HIn summary, TRPA1 is expressed on the pulmonary C fibers that project into airways and terminate into the mucosa and submucosa of the pharynx, larynx and trachea which are anatomical regions driving cough reflex. TRPA1 is activated by endogenous and exogenous pro-tussive agents that are amply documented in literature and some of these induce cough response in guinea pigs and humans. Asthma, COPD, GERD, PNDS and post viral cough seem to be major diseases with chronic cough manifestation which is untreatable. The causes of cough in these pathologies could be diverse but the common link between them could be the activation of TRPA1 receptor on the airway vagal sensory nerves. The implicated role of vagal TRPA1 receptor in such wide variety of cough etiologies suggests TRPA1 to be a major driver of cough reflex. Further, our work demonstrates antitussive effect of GRC 17536 -a potent selective antagonist of TRPA1 receptor in guinea pig cough model. The pulmonary and vagus afferent control of human airways is generally believed to be similar to other mammals. Thus TRPA1 seems to be a perfect target for development of novel anti-tussive agents for various chronic cough conditions.


The seasonality of influenza virus infections in temperate climates and the role of environmental conditions like temperature and humidity in the transmission of influenza virus through the air are not well understood. Using ferrets housed at four different environmental conditions, we evaluated the respiratory droplet transmission of two influenza viruses  and concurrently characterized the aerosol shedding profiles of infected animals. Comparisons were made among the different temperature and humidity conditions and between the two viruses to determine if the H3N2 variant virus exhibited enhanced capabilities that may have contributed to the infections occurring in the summer. We report here that although increased levels of H3N2 variant virus were found in ferret nasal wash and exhaled aerosol samples compared to the seasonal H3N2 virus, enhanced respiratory droplet transmission was not observed under any of the environmental settings. However, overall environmental conditions were shown to modulate the frequency of influenza virus transmission through the air. Transmission occurred most frequently at 23°C/30%RH, while the levels of infectious virus in aerosols exhaled by infected ferrets agree with these results. Improving our understanding of how environmental conditions affect influenza virus infectivity and transmission may reveal ways to better protect the public against influenza virus infections. Influenza viruses display distinct seasonal patterns in temperate climates with peak infection rates occurring in the winter months and little to no influenza virus activity detected at other times of the year . HoweverThere are three generally accepted modes of influenza virus transmission . Aerosolin vitro studies have demonstrated that influenza viruses in droplets and aerosols maintain viability better at low humidity [Temperature and humidity are inextricably linked when considering their effects on evaporation rates and on infectious particle dynamics . Althoughumidity ,17. Animhumidity . The ferhumidity . To moreAll ferret procedures were approved by Institutional Animal Care and Use Committee (IACUC) of the Centers for Disease Control and Prevention and in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility. Animal studies were performed in accordance with the IACUC guidelines under protocol #2234MAIFERC: "Transmissibility of influenza viruses with pandemic potential".Virus stocks of A/Panama/2007/1999 (H3N2) were propagated in the allantoic cavity of 10 day old embryonated hens’ eggs while A/Indiana/8/2011 (H3N2v) was grown in Madin-Darby canine kidney cells (MDCK) as previously described [3 AH); 23°C/30%RH (6.2 g/m3 AH); 23°C/50%RH (10.3 g/m3 AH) and 23°C/70%RH (14.4 g/m3 AH). Prior to inoculation, ferrets were allowed to acclimatize inside the chamber for at least four days. Baseline temperatures were measured using an implantable subcutaneous temperature transponder  and baseline serum, weight and minute volume (MV) of respiration measurements were collected as described [Male Fitch ferrets 5–11 months of age (Triple F Farms), serologically negative by hemagglutination-inhibition (HI) assay for currently circulating influenza viruses, were housed during each experiment in cages placed inside a custom environmental chamber  with HEPA filtration operating at 20 air changes per hour. Experiments were conducted at four environmental settings with consideration for the comfort of the animals: 5°C/70%RH  and was found to be negligible (<0.05 m/s) for all of the cages. One day post inoculation (dpi) a naïve ferret was placed in a cage adjacent to each inoculated animal. Nasal washes (NW) were collected every other day for up to 11 dpi or days post contact (dpc)  for viruThree inoculated ferrets from each experiment were anesthetized and exhaled aerosol samples were collected and analyzed for size distribution using an aerodynamic particle sizer . Aerosol samples were collected from ferrets immediately after exhalation on 2, 4 and 6 dpi for 15 minutes of closed-mouth, normal breathing and for 5 minutes of induced sneezing as described . Size diExhaled aerosols from inoculated animals were analyzed for infectious virus using a viable two-stage cascade impactor  on 1, 3, and 5 dpi as described with modifications . Aerosol2–103 pfu) directly on prepared impactor plates and placing the impactor assembly inside the environmental chamber at one of the four environmental conditions [The effects of the aerosol collection procedure on influenza virus recovery was evaluated in duplicate by applying a known amount of virus .3.8–105.5 pfu of PN99 or IN11 virus   and allo11 virus . One dpiRegardless of the inoculum, changes in activity levels in infected animals were minor (relative inactivity indexes ranged from 1.0–1.2) while anorexia and nasal discharge were the most common clinical signs observed in ferrets housed at any of the controlled environmental settings. Peak temperatures were detected 2–3 dpi in inoculated animals and ranged from 1.2–2.4°C above baseline for PN99 virus and 1.9–3.3°C above baseline for IN11 virus . MV of r4.2 to 104.8 pfu/mL in ferrets inoculated with PN99 virus and were 3–24 times higher (105.2 to 105.7 pfu/mL) in ferrets inoculated with IN11 virus housed under the respective environmental condition; the greatest difference was observed at the 23°C/70%RH condition  on 1, 3 and 5 dpi and then quantified for influenza virus by plaque assay as well as real-time RT-PCR. Infectious virus detected at any time point and in either size range in normal breathing samples was ≤10 pfu  and in sThe impact of the aerosol collection procedure on virus recovery was tested for each controlled environmental condition by spiking the collection medium on each impactor stage with known quantities of virus. After air was pulled through the impactor for 15 or 5 minutes, to represent normal breathing and sneezing sampling times, respectively, the collection medium was harvested and processed as described for the ferret samples. PN99 virus RNA recovery from either stage and for the 15 and 5 minute collection times ranged from 54 to 79%, similar to IN11 virus RNA recovery which was 47 to 81% . InfectiThe influence that temperature and humidity have on aerosolized influenza virus and the consequence for transmission remain poorly understood despite the potential impact on the seasonality of influenza epidemics in the temperate regions of the world. Climatic conditions in tropical regions of the world represent a different set of parameters affecting transmission, and furthermore, routes of transmission not involving airborne virus (e.g. direct or indirect contact) demonstrate the complexity of global influenza virus transmission and the multitude of potential mechanisms at play each impacted differently by environmental conditions. Here, we evaluated the transmissibility of two H3N2 influenza viruses (PN99 and IN11) using ferrets housed under diverse environmental conditions and concurrently evaluated the aerosol shedding profiles of infected animals. We compared the two virus groups to each other and then combined them to identify common differences at each environmental condition. Higher levels of IN11 virus were measured in ferret nasal washes and in exhaled aerosol samples compared to PN99 virus but overall, we found little difference in the transmission capabilities of the two viruses at any of the environmental conditions with transmission most frequently observed at 23°C/30%RH (83%) and least frequently observed at 23°C/50%RH and 5°C/70%RH (17%).The viruses chosen for this study were selected because one represents a seasonal H3N2 strain PN99 virus), the other one is a swine-origin H3N2 variant strain (IN11 virus) responsible for a summertime infection ,25 and b9 virus, Any successful transmission event through the air requires that virus pass from an infected host to a susceptible one while maintaining infectivity. As this occurs, aerosols are released from the warm, humid environment of a respiratory tract, through the ambient conditions outside of the host and eventually reach the warm, humid environment of a recipient’s respiratory tract. Depending on the ambient conditions and the solute composition of the aerosol, evaporation and condensation processes may have deleterious effects on any virus present in the aerosols . Using cDetection of influenza virus in exhaled aerosols has been largely limited to the detection of viral genetic material via RT-PCR with few reporting infectious virus detection in humans ,13 or laIn the temperate regions of the world, people spend the vast majority of their time indoors, especially during the winter months where the environment is typically maintained at a warm temperature and low RH . This sc


Congenital heart disease (CHD) is the most common congenital defect, affecting 1% of all livebirths. Initial cardiac development and the subsequent maturation of the heart are temporally and spatially regulated by several Bone Morphogenetic Proteins (BMPs). BMP signaling plays an important role in the specification and patterning of the early embryo. Animal model studies have established a strong link between BMPs and cardiac development. At least 6 BMPs  are expressed in the heart where they have both independent and redundant functions. BMP2, 4 & 7 are expressed in the AtrioVentricular cushion and Out Flow Tract (OFT) and helps in their formation. However due to functional redundancy of BMP ligands, relatively little is known about the role of individual BMPs. The association of BMPs with CHD has not been studied so far in human subjects. Here we report the genetic variants in BMP2 (NM_001200.2), BMP4 (NM_001202.3) and BMP7 (NM_001719.2) in individuals with CHD.We screened 190 unrelated probands with CHD and 100 healthy controls by PCR and DNA sequencing for 3 genes.We have identified 6 novel sequence variants in BMP2. Out of which 3  are missense variants. One nonsense variants (K241X) has also been identified in one case having Patent Ductus Arteriosus and is not found in 100 controls. In BMP7, 10 novel sequence variants have been identified, out of which 3  are missense, 3  are in 5’UTR, 1 synonymous change (c.682G>T) and 3  are intronic variants. In BMP4, 3 sequence variants are found, out of which only one novel (c.1695A>C) in 3’UTR region has been found in one individual. Other than these some already reported single nucleotide polymorphisms have also been identified in these 3 genes and some of them are more frequently found in CHD cases. In-silico analyses based on evolutionary, biochemical and structural aspect of the gene revealed the disease causing effect of these variants. Further biochemical analysis would reveal the regulatory circuits involving these cardiac transcription factors and their possible role in pathogenesis of the disease.


Pharmacogenetic effects of recombinant human growth hormone according to growth hormone receptor (GHR) exon 3 polymorphism (fl vs. d3) were controversial. We investigated growth hormone response in children with growth hormone deficiency (GHD).Total 58 prepubertal children (31 boys and 27 girls) with GHD were enrolled in this study. Subjects were divided to 2 groups according polymorphism , and compared baseline phenotypes and the first year growth response to growth hormone treatment.The distribution of GHR exon 3 isoforms in children with GHD demonstrated that the frequency of fl/fl (82.8%) is higher than that in most of European studies. There was no significant difference in baseline height SDS between 2 groups. Height velocity during the first year of growth hormone replacement therapy tended to be higher in subjects who have d3 allele (fl/d3 and d3/d3), but there was no statistical difference according to genotype.It seemed that d3 allele of GHR exon 3 had no impact on the baseline phenotype and growth hormone response in patients with GHD. Relationship between GH dose and IGF-1% to help fully elucidate the value of IGF-1 testing in GH treatment.


LAS1) in the oxidosqualene cyclase repertoire, in addition to cycloartenol synthases (CAS1) and to non-steroidal triterpene synthases that contribute to the metabolic diversity of C30H50O compounds on earth. Furthermore, plant LAS1 proteins have been unambiguously identified by peptidic signatures and by their capacity to complement the yeast lanosterol synthase deficiency. A dual pathway for the synthesis of sterols through lanosterol and cycloartenol was reported in the model Arabidopsis thaliana, though the contribution of a lanosterol pathway to the production of 24-alkyl-Δ5-sterols was quite marginal  PNAS 106, 725). To investigate further the physiological relevance of CAS1 and LAS1 genes in plants, we have silenced their expression in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences from a Nicotiana tabacum CAS1 or derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1. In this report, we show a CAS1-specific functional sterol pathway in engineered yeast, and a strict dependence on CAS1 of tobacco sterol biosynthesis.The plant sterol pathway exhibits a major biosynthetic difference as compared with that of metazoans. The committed sterol precursor is the pentacyclic cycloartenol  and not lanosterol , as it was shown in the late sixties. However, plant genome mining over the last years revealed the general presence of lanosterol synthases encoding sequences ( It is nonetheless worth noting that 2-tritio lanosterol and 2-tritio cycloartenol when fed to Sorghum bicolor leaves were converted to cholesterol and sitosterol eventhough cycloartenol was a more effective sterol precursor than lanosterol Plants, algae and some protists synthesize their sterols through a biosynthetic route that contains a pentacyclic steroidal cyclization product of 2,3-oxidosqualene, namely, cycloartenol , being able to isomerize cycloeucalenol into the tetracyclic 4α-methyl sterol, obtusifoliol Arabidopsis thaliana cpi1-1 mutant indicates that a normal life span cannot be attained with the CAS1 biosynthetic segment only The essential role of CAS1 in sterol biosynthesis and plant development was demonstrated by a series of in planta characterization of CAS1 and LAS1 was restricted to Arabidopsis thaliana. In order to further document dual cycloartenol and lanosterol pathways in higher plants we have isolated a CAS1 from Nicotiana tabacum and studied its function in yeast and in Nicotiana benthamiana. We used virus induced gene silencing (VIGS) based on gene specific sequences derived from the solgenomics initiative (http://solgenomics.net/) to challenge the respective roles of CAS1 and LAS1 in Nicotiana benthamiana. In this report, we show a strict dependence on CAS1 of tobacco sterol biosynthesis.The co-existence of functional CAS1 and LAS1 in plants requires further studies. So far, Nicotiana orthologs of CAS1 and LAS1. We then cloned a Nicotiana tabacum CAS1 cDNA by recursive PCR using degenerated primers over conserved sequences. Primers used for that cloning procedure are given AtCAS1 and consequently was named NtCAS1 (GenBank accession KM452913). We next identified in the genome of Nicotiana benthamiana (http://solgenomics.net/) two genes whose products had 70% and 58% identity with AtCAS1 and AtLAS1, respectively. Relevant sequences identified in Nicotiana and in other Solanaceae are given in Nicotiana benthamiana with the functionally identified tomato triterpene synthases Arabidopsis thaliana β-amyrin synthase  in the vicinity of the conserved DCTAE motif implicated in substrate protonation, whereas all LAS1 proteins have a strict requirement for a V481 (erg7 (lanosterol-deficient) mutant led to a current understanding of the catalytic differences in both reactions We cloned by RT-PCR a cDNA fragment from d NbLAS1 , confirmm annuum  shows fer a V481  [31], .(PDF)Click here for additional data file.Table S1Gene nomenclature and protein references used to generate .(PDF)Click here for additional data file.Table S2Primers used for NtCAS1 cloning and for qPCR measurements of CAS1 and LAS1.(PDF)Click here for additional data file.Table S3The Solanaceae OSC signatures and gene references.(PDF)Click here for additional data file.References S1(DOCX)Click here for additional data file.


To assess the effectiveness of transperineal template guided mapping biopsy (TTMB) in detecting prostate cancer in patients with previous multiple negative transrectal ultrasound guided (TRUS) biopsies of the prostate.From April 2011 to February 2013 22 patients who had previously undergone 2 or more TRUS biopsy with a continuing rising PSA were biopsied using an anatomically guided template. Clinical parameters included age, initial PSA, PSA pre TTMB biopsy and number of previous biopsies. Number of cores sampled, number of cores positive for cancer and Gleason score were assessed. Results: Average age is 61.45 years with average of 2.5 previous biopsies and pre biopsy PSA of 15.5. On average 22.3 cores were sampled in each patient. PSA rose 6.4 ng/ml between initial biopsy and PSA triggering TTMB. Average elapsed time from initial biopsy to TTMB was 3.3 years. Overall 31.8% of patients (n=7) were diagnosed with prostate cancer at TTMB. There were 4 patients found to have Gleason 3+3=6, 1 Gleason 3+4=7 and 4 Gleason 4+4=8.TRUS biopsy is known to miss clinically significant pathology of the prostate. In this single surgeon single institution analysis we examined the efficacy of the TTMB in identifying prostate cancer in patients with multiple previous negative biopsies and a rising PSA, and found over 30% of patients with previous multiple negative biopsies to have clinically significant prostate cancer. This study demonstrates that in the right hands TTMB can be an integral step in the management pathway of patients with suspected prostate cancer.


The latest guidelines of the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) to test Human Epidermal Growth Factor Receptor 2 (HER2) in breast cancer after being revised in 2008 underwent a second modification in October 2013. The modification includes changes in cut-offs: 10% strong membranous staining for score 3+ on immunohistochemistry (IHC) (previously 30%) and using the ratio of >2 or absolute gene-copy-number (6 or more) alone or in combination with each other by in-situ-hybridisation technology (previously >2.2 and average copy-number of 6 or more). Hereby we addressed the question, which impact the modified cut-offs had on overall HER2-positivity in a single institution.We prospectively analysed 617 consecutive diagnostic breast-cancer cases which underwent double HER2 testing by immunohistochemistry and fluorescent in-situ hybridisation (FISH), using the modified 2013 ASCO/CAP-guidelines for one year (October 2013–October 2014). Results were compared with HER2-test results on 1,528 consecutive diagnostic breast-cancer cases from two previous years (2011–2012), using the 2008 ASCO/CAP guidelines, also tested with IHC and FISH in each case.Between October 2013 and October 2014, overall HER2-positivity was 15.8% (98 of 617 cases were either IHC 3+ or FISH amplified). 79 of 617 cases (13%) were IHC 3+, 96 of 617 cases (15.5%) were FISH amplified. Equivocal cases were seen in 25 of 617 cases (4.1%). 22 of 25 equivocal cases (88%) in 2013–2014 were IHC 1+ or 2+. In 13 equivocal cases, there was a repeated IHC/FISH testing: 2 of 13 cases (15%) became FISH amplified, 1 of 13 cases (7.5%) became IHC 3+. In 2011–2012, overall HER2-positivity (IHC/FISH) was 13.8% . 185 of 1,528 cases (12%) were 3+ on IHC, 181 of 1,522 cases (12%) were amplified by FISH. Six of 1,528 cases were equivocal by FISH, and interpreted as non-amplified (0.3%).Applying the modified ASCO/CAP guidelines from 2013 resulted in an increase (2%) in overall HER2-positivity rate compared to overall-HER2-positivity rate using the 2008 ASCO/CAP guidelines. The increased positivity rate was mainly due to more FISH-positive cases (3.5% more than until 2013). The high rate of equivocal cases (4.1%) did not contribute to increase in overall HER2-positivity, but resulted in delay in definitive HER2-status. The assessment of HER2 status in breast cancer is a part of routine tests on primary and / or on recurring lesions –3. HER2 In this study we compared overall HER2 positivity rate prior to the introduction of the modified ASCO/CAP guidelines from October 2013 with the ones using the new definitions from this time.We prospectively analysed consecutive HER2 diagnostic cases from the period of 2011–2012 (n = 1528) and compared with HER2 test results between October 2013 and October 2014 n = 617). All breast cancer cases underwent double HER2 testing by immunohistochemistry and fluorescence in situ hybridisation according to the institutional guidelines as published earlier [17. All bConsecutive patients with the diagnosis of invasive breast carcinoma undergoing HER2 testing at the Institute of Surgical Pathology, University Hospital Zurich, Zurich, Switzerland were analysed. Two periods were evaluated, 2011–2012 (two years) and from October 2013 to October 2014 (one year). Data on HER2 status from 2011–2012 were partially included in a previous paper, cases with polysomy or equivocal HER2 status were not included in this previous study . In the During the whole period (2011–2014), the same laboratory procedure for IHC and FISH was applied as published earlier .Protein expression and gene copy number score were scored using the time current ASCO/CAP guidelines as published earlier , 6.Score 0: no staining.Score 1+: weak and incomplete membrane staining in less than 10% of the invasive tumor cells.Score 2+: weak complete staining of the membrane in more than 10% of invasive cancer cells.Score 3+: strong complete homogenous membrane staining in more than 30% of the invasive tumor cells.HER2 signals were scored as follows: the number of signal copies and the ratios (HER2/CEP17) were calculated: gene copies (> 6) or cluster formations  were counted. A ratio > 2.2 was set as amplified status; a ratio < 1.8 was negative, and a ratio of 1.8–2.2 was referred to as equivocal (according the 2008 guidelines) and using ratio > 2.2 and > 6 HER2 gene copies (in > 10% of the tumor cells) for a positive status (according to 2008 guidelines). If ratio was <2.2, the gene copy number was not considered alone as amplified based on the definitions in the recommendations in Tables Protein expression and gene copy number score were scored using the latest ASCO/CAP guidelines published in October 2013 .Score 0: no stain or faint incomplete membrane stain in not more than 10% within the cells. Score 1+: weak and incomplete membrane staining in more than 10% of the tumor surface. Score 2+: Complete intense membrane staining in not more than 10% of the invasive tumor cells or weak/moderate heterogeneous incomplete staining in more than 10% of the invasive tumor cells.Score 3+: strong complete homogenous membrane staining in more than 10% of the invasive tumor cells.HER2 gene copy signals were scored as follows:Positive status was defined either as an average HER2 gene copy numbers of 6 at cases with a HER2/CEP17 ratio of less than 2 or a HER2/CEP17 ratio of 2 or more independently of the average gene copy number.Negative status was defined as average gene copy number of less than 4 with a HER2/CEP17 ratio of less than 2.Equivocal cases were defined as average gene copy number of at least 4 and less than 6 with a HER2/CEP17 ratio of less than 2.Comparison of the overall HER2 positivity rate in two periods with at the time current guidelines (ASCO/CAP 2008 and 2013)  are showOverall HER2 positivity (IHC score 3+ or amplified by FISH) was 13.8% (211 of 1528 cases). 185 of 1528 cases (12%) were 3+ on IHC, 181 of 1528 cases (12%) were amplified by FISH.Six of 1528 cases were equivocal on FISH (0.3%). The one case with IHC score 3+ was considered as HER2 positive. The other five cases underwent repeated IHC/FISH testing on the core biopsy or on a different tumor block, and none of them became amplified or IHC positive.Data are shown in Overall HER2 positivity (IHC score 3+ or amplified by FISH) was 15.8% (98 of 617 cases were either 3+ on IHC or amplified by FISH. 79 of 617 cases (13%) were 3+ on IHC, 96 of 617 cases (15.5%) were amplified by FISH.Equivocal cases by FISH were seen in 25 of 617 cases (4.1%). 22 of 25 equivocal cases (88%) were either scores 1+ or 2+ on IHC. In 13 equivocal cases, there was a repeated IHC/FISH testing: 2 of 13 cases (15%) became amplified by FISH, 1 of 13 cases (7.5%) was 3+ on IHC. The other 10 cases remained negative or equivocal by FISH and score 1+ or score 2+ by IHC. Altogether 3 of 13 (23%) equivocal cases on FISH and IHC became HER2 positive after re-testing. Time to re-testing and to definitive diagnosis on HER2 status required up to 5 weeks .Data are shown in In our study we could demonstrate, that overall HER2 positivity rate in breast cancer increased in 2% after implementing the modified ASCO/CAP guidelines in 2013 on HER2 testing. Our data show, that this increase is mainly due to newly defined cut-offs on FISH positivity, whereas score 3+ on immunohistochemistry did not essentially influence the increase in HER2 positivity rate. Additionally, a relative high percentage of equivocal cases (up to 4%) were observed by using these guidelines. This new phenomenon in our diagnostic HER2 testing, being virtually absent prior to these guidelines, resulted in a considerable delay in the definitive pathology report on HER2 status due to re-testing.There is only limited data on long-term impact of the modified ASCO/CAP Guidelines in the literature, as time passed since the official implementation of these guidelines encompasses around one and half years first –13. NeveConsequently to increased IHC score 2+ cases, the need for reflex testing with ISH technology is also arising, resulting in higher workload in ISH, which reportedly can be up to doubling the number of ISH assays when comparing numbers from using the previous guidelines in some recent works , 15. In The question of reproducibility of modified scoring criteria was addressed by Bianchi et al in a recent multicentre study . Based oAlthough the updated guidelines address issue on analytical validity and clinical utility, there is also a recommendation on re-testing in cases with histopathological discrepancies as downgrading in histological grade on excision specimens , 17–19. In conclusion it can be stated, that implementing the modified ASCO/CAP Guidelines from 2013 resulted in an increase of 2% in overall HER2 positivity rate in comparison to overall positivity rate by applying the previous ASCO/CAP guidelines. The high rate of equivocal cases (4.1%) did not considerably contribute to the increase in overall HER2 positivity, but resulted in delay of the definitive diagnosis on HER2 status. Further data and experiences from diagnostic HER2 services are needed to be collected and prospectively analysed so that adjustments in the guidelines can be met based on evidence and experience.


GLI1 and GLI2 were highly expressed in two out of three cell lines and correlated with downstream target gene expression of PTCH1and PAX6. Treatment of canine osteosarcoma cells with GANT61 resulted in decreased expression of GLI1, GLI2, PTCH1, and PAX6. Furthermore, GANT61 inhibited proliferation and colony formation in all three canine osteosarcoma cell lines. The finding that GLI signaling activity is present and active in canine osteosarcoma cells suggests that spontaneously arising osteosarcoma in dogs might serve as a good model for future preclinical testing of GLI inhibitors.The Hedgehog-GLI signaling pathway is active in a variety of human malignancies and is known to contribute to the growth and survival of human osteosarcoma cells. In this study, we examined the expression and regulation of GLI transcription factors in multiple canine osteosarcoma cell lines and analyzed the effects of inhibiting GLI with GANT61, a GLI-specific inhibitor. Compared with normal canine osteoblasts, real-time PCR showed that  The Hedgehog (Hh) signaling pathway has long been known to play a critical role in early embryonic development While canonical Hh signaling is mainly mediated by PTCH1 and SMO, recent data has clearly demonstrated a parallel existence of non-canonical Hh signaling pathways in vitro and in vivoAs an alternative to targeting SMO, compounds such as arsenic trioxide have been found to inhibit GLI transcription factors and can inhibit growth of sarcoma or OSA cells that express GLI Spontaneously occurring OSA in the dog very closely models the human disease with striking similarities at the clinical and molecular levels GLI1 and found high expression of GLI1 mRNA (transcript) in 2 out of 3 cell lines (Abrams and D17) relative to osteoblast cells , D17 cells showed a significant reduction of ed cells . We alsoed cells . Importaanalyses .PTCH1 mRNA expression by approximately 60% in D17 cells compared to vehicle DMSO treated and untreated cells . However, GLI1 showed 10–15 times higher fold mRNA expression compared to GLI2 mRNA in canine Abrams and D17 OSA cell lines. This suggested that GLI1 might possibly be the major Hh-GLI signaling regulatory transcription factor in Canine OSA. While previous studies have identified GLI1 as the main transcription factor of Hh-GLI signaling pathway in several tumor systems, two independent studies have concluded that GLI2 appears to be of highest relative importance in human OSA Initial experiments demonstrated high constitutive expression of We found that GANT61 was capable of inhibiting cell proliferation in all three canine cell lines but was most effective inhibiting the growth of D17 cells that expressed highest levels of GLI. Importantly, the IC50 value of GANT61 (based on reporter activity) is in the range of 5 µM PTCH1 compared to osteoblast cells and this correlated with low expression of GLI1 and GLI2 expression in these cells. Importantly, PTCH1 expression was significantly reduced in D17 cells after treatment with GANT61, supporting the notion that PTCH1 mRNA can be regulated by the Hh-GLI pathway in canine OSA cells. These results are in agreement with previous studies showing that GLI1 knockdown decreases PTCH1 expression in human medulloblastoma and glioblastoma cells To further examine the effects of GANT61 on canine OSA cells, we went on to examine expression of downstream target genes before and after treatment with GANT61. The majority of studies investigating downstream target genes of GLI have focused on genes with known binding of the GLI1 consensus sequences within the promoter region. Therefore, we chose to first examine expression of constitutive GLI1 downstream target genes PTCH1 and PAX6, in canine OSA cells. We then went on to determine the effect of GANT61 on the expression of these same target genes. High expression of PTCH1 in Abrams and D17 cell lines initially supported the notion that GLI signaling was active in canine OSA. However, the Moresco OSA cell line showed low expression of PAX6, one of the putative Hh/-GLI signaling downstream target genes, showed high expression in Abrams and D17 canine OSA cell lines as would be expected from GLI1, GLI2, and PTCH1 expression analyses. GANT61 treatment reduced 40% of the PAX6 expression in canine OSA cells which is in agreement with a previous study wherein GLI1 knockdown reduced the expression of PAX6 in human medulloblastoma PAX6 expression in osteocytes suggests that PAX6 regulates sclerostin, an osteocyte marker that inhibits canonical Wnt signaling antagonist PAX6 gene can possess both proto-oncogenic and tumor suppressor characteristics and these may vary among tumor types We also found that Altogether, the results of our studies demonstrate that 1) GLI signaling is present in canine OSA cell lines, 2) GANT61 is capable of altering expression of GLI target genes in canine OSA cells, and 3) inhibition of GLI is capable of reducing proliferation of canine OSA cells. While these findings indicate that Hh-GLI signaling is quite similar between canine and human OSA, several questions remain unanswered. Specifically, our studies have not examined the individual contribution of GLI proteins in canine OSA, whereas, GLI2 appears to be the major driver of Hh/-GLI signaling in human OSA GANT61  was purchased from Calbiochem and dissolved in DMSO at a stock concentration of 5 mg/ml.2 chamber. All cell lines tested and confirmed free from mycoplasma. Canine osteoblast cells were grown in growth medium (Cn417D-500) purchased from Cell Application Inc. under identical conditions.Canine osteoblast cells (Cn406-05) were purchased from Cell Application Inc. Abrams and Moresco cell lines were gifts from Dr. Douglas Thamm, Department of Clinical Sciences College of Veterinary Medicine and Biomedical Sciences Colorado State University Canine OSA cell lines Abrams, D17 and Moresco were plated in triplicate at a density of 700 cells per well in 96-well plates. After 24 hours, culture medium was replaced with fresh 2% FBS medium containing various concentrations of GANT61 at concentrations between 0–20 µM for 4 days. Cell viability was measured by incubation with the 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt (MTS) one –solution assay reagent  at 37°C for 1 h before reading absorbance on a spectrophotometer  at 490 nm. DMSO was used as a vehicle to dissolve GANT61 and for controls.Cell survival was determined by clonogenic assay based on previous publication GLI1, GLI2, PTCH1, and PAX6 was determined by using the StepOne Plus Real-Time PCR system (Applied Biosystem). Taq-Man assays (Applied Biosystem) GLI1 (Assay ID # Cf04230663_m1) GLI2 (Assay ID#4331348), PTCH1 (Assay ID #Cf02690587_m1), and PAX6 (Assay ID#Cf02675240_m1) were used to determine the expression. All transcripts expression was equilibrated with endogenous control HPRT1 (Assay ID#Cf02626256_m1) expression , Abrams, D17 and Moresco cell lines with Tri Regent (Sigma) and chloroform-ethanol method. Then determined the quantity and quality of RNA with the help of Biophotometer (eppendorf). Total 2 µg of total RNA was converted to cDNA by using High Capacity RNA to cDNA Kit (Applied Biosystem). Transcript expression (mRNA) level of pression , S2, S3.The D17 cell line was treated with 20 µM GANT61 or vehicle DMSO for 96 hrs and cell lysate was made in lysis buffer. Protein was quantified using a BCA protein Assay Reagent (bicinchonic acid) (Pierce). A total of 40 µg of protein was loaded per sample in the 7.5% polyacrylamide gels under denaturing and reducing conditions and protein was transferred to nitrocellulose membranes. After transfer of protein, the membrane was probed and incubated overnight at 4°C with antibodies Rabbit anti-GLI1 (Abcam ab49314), Rabbit anti-GLI2 (Abcam ab26056), Rabbit anti-Patch1(Sigma-Aldrich P0088), Rabbit anti-Pax6 (Abcam ab5790), and Mouse anti-Actin (Santa Cruz sc-56459) in 5% non-fat milk. Then membranes were washed and subsequently exposed to the appropriate HRP-conjugated secondary antibodies for 1 hr. at room temperature. Bands were visualized by the enhanced chemiluminescence (Pierce) in Fluorchem E Imaging system . Constitutive expression levels of GLI1 and GLI2 were performed similarly, in the absence of treatment for Abrams, D17 and Moresco cell lines.All results shown are representative of experiments done in triplicate. Analyses were performed using one-way ANOVA and post-doc Tukey's testing (GraphPad Prism version 5.0). Differences were considered significant with a p<0.05.Figure S1Amplification plot of GLI1, GLI2, PTCH1 and PAX6 RNA expression in canine OSA cell lines: This plot was used to determine the Cycle threshold (Ct)value; PCR Cycle number as X and the mean ΔRn  with the quenching dye emission (Q) during the Real Time PCR amplification process) value as Y. The standard ΔRn (Y = 0.05) of exponential phase of amplification was selected to determine the optimal CT value. Less cycle number to reach exponential phase of amplification indicates high copy number of RNA . (A) Amplification plot of GLI1 in Canine osteoblast (CO), Moresco, Abrams and D17. (B) Amplification plot of GLI2 in Canine osteoblast (CO), Moresco, Abrams and D17. (C) Amplification plot of PTCH1 in canine CO, Moresco, Abrams and D17. (D) Amplification plot of PAX6 in canine CO, Moresco, Abrams and D17.(TIF)Click here for additional data file.Figure S2Amplification plot of GLI1, GLI2, PTCH1 and PAX6 mRNA expression in canine cell line D17 after the treatment of GANT61 and DMSO. (A) Amplification plot of GLI1 expression in Canine OSA D17 cell line compared to GANT61 and DMSO (control). (B) Amplification plot of GLI2 expression in D17 cell line compared to GANT61 and DMSO (control). (C) Amplification plot of PTCH1 in canine OSA cell line D17 compared to GANT61 and DMSO treatment. (D) Amplification plot of PAX6 in canine OSA cell line D17 compared to GANT61 and DMSO treatment. GANT61 treated cells showed increased number of cycle to reach exponential phase of amplification compare to DMSO (control) (decreased mRNA copy number in GANT61 treated cells compared to DMSO).(TIF)Click here for additional data file.Figure S3Amplification plot of GLI1 and GLI2 mRNA expression in canine cell line Moresco after the treatment of GANT61 and DMSO. (A) Amplification plot of GLI1 expression in Canine OSA Moresco cell line after the treatment GANT61 and DMSO (control). (B) Amplification plot of GLI2 expression in Canine OSA Moresco cell line after the treatment of GANT61 and DMSO (control). GANT61 treated cells showed increased number of cycle to reach exponential phase of amplification compare to DMSO (control) (decreased mRNA copy number in GANT61 treated cells compared to DMSO).(TIF)Click here for additional data file.


Heart failure secondary to cardiac siderosis is the prevalent cause of mortality in patients with primary or secondary iron overload. Myocardial iron assessment using T2* has been successfully calibrated against myocardial iron at 1.5T and the black blood (BB) technique has demonstrated high reproducibility such that it has become the clinical gold standard. T1 appears to correlate with T2* in cases of significant iron loading (T2* < 20 ms) but this relationship weakens when T2* is in the normal range. We evaluated this further.69 subjects  comprising 20 healthy volunteers (controls) and 49 patients  referred for routine iron assessment were recruited. The same mid-ventricular short axis cardiac slice was used to acquire both BB T2* images and to generate T1 MOLLI maps for each subject at 1.5T . 20 subjects underwent repeat studies on the same day to evaluate reproducibility. Regions of interest were selected in the septum using CMRtools.2 = 0.696; Figure There was a reasonable correlation between BB T2* and T1 values described by a logarithmic relationship (RIn this analysis, T1 mapping using a MOLLI sequence identified all those individuals with significant iron loading as defined by the current gold standard T2* technique and has excellent reproducibility. As expected, T1 can also identify patients with mild cardiac iron loading that is not considered clinically significant. It is possible that T1 may have a clinical role in non-cardiac conditions with very low levels of iron loading, where factors other than iron affect the T2* signal, however tissue calibration remains lacking at this time. Further work is needed to determine whether there might be clinically significant advantages of T1 over the T2* cardiac technique at 1.5T for assessment of cardiac iron, but there are none at this time.This research was supported by the NIHR Cardiovascular Biomedical Research Unit at Royal Brompton & Harefield NHS Foundation Trust and Imperial College London.


In the legend of Figure 2 of the original version of this article  the capFigure 2. Spectral characterization of BVMOAf1. Visible spectra of native BVMOAf1 (solid line) and BVMOAf1 after unfolding by 1% SDS and incubation at 80°C (dotted line). Spectral changes observed upon reduction of BVMOAf1 by excess of NADPH (dashed line).The listing of ‘(solid line)’ and ‘(dotted line)’ are incorrectly placed in the text and in fact should be located in reverse. The caption should correctly read as follows:Figure 2. Spectral characterization of BVMOAf1. Visible spectra of native BVMOAf1 (dotted line) and BVMOAf1 after unfolding by 1% SDS and incubation at 80°C (solid line). Spectral changes observed upon reduction of BVMOAf1 by excess of NADPH (dashed line).


Blood sugar control for patients with sepsis remains controversial. We aimed to test the hypothesis that the variation of blood sugar level is associated with patient outcome in this study.A retrospective cohort study on nontraumatic adult patients who visited the ED of a tertiary hospital in 2010 and had a clinical diagnosis of severe sepsis was conducted. Patients with two sets of blood culture ordered by emergency physicians and at least two blood sugar tests results available during the first 48 hours of hospitalization were included. The coefficients of variation  of the blood sugar level were analyzed with multivariate logistic regression models to test the association between in-hospital mortality.Of the 1,537 patients included, most were older than 70 years of age , male (54%), without a diagnosis of severe sepsis (63%) and had a previous diagnosis of diabetes (84%). The initial blood sugar levels of patients with and without previously diagnosed diabetes were 259 ± 9.9 and 154 ± 5.7, respectively (mean ± SEM). The CoV of the consecutively monitored blood sugar level during the first 48 hours of admission for patients with and without previously diagnosed diabetes were 29.4 ± 0.5% and 21.0 ± 0.5%, respectively. Patients with CoV lower than 10% and higher than 30% tended to have higher mortality rate, compared to patients with 10 to 30% CoV level (11% vs. 12% and 7%, respectively, Figure In this retrospectively cohort study, we found that increased blood sugar variation was associated with worse patient outcome. However, further study is merited to test the possible causal relationship between variation of blood sugar level and patient outcome.