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Team Hatakeyama 25

Specific functional interactions of proteins involved in DNA replication and/or DNA repair or transcription might occur in Archaea, suggesting a previously unrecognized regulatory network coupling DNA replication and translation, which might also exist in Eukarya. Comparative analysis of genomes is valuable to explore evolution of genomes, deduce gene functions, or predict functional linking between proteins. Here, we have systematically analyzed the genomic environment of all known DNA replication genes in 27 archaeal genomes to infer new connections for DNA replication proteins from conserved genomic associations.Two distinct sets of DNA replication genes frequently co-localize in archaeal genomes: the first includes the genes for PCNA, the small subunit of the DNA primase (PriS), and Gins15; the second comprises the genes for MCM and Gins23. Other genomic associations of genes encoding proteins involved in informational processes that may be functionally relevant at the cellular level have also been noted; in particular, the association between the genes for PCNA, transcription factor S, and NudF. Surprisingly, a conserved cluster of genes coding for proteins involved in translation or ribosome biogenesis is almost systematically contiguous to the group of genes coding for PCNA, PriS, and Gins15. The functional relevance of this cluster encoding proteins conserved in Archaea and Eukarya is strongly supported by statistical analysis. Interestingly, the gene encoding the S27E protein, also known as metallopanstimulin 1 (MPS-1) in human, is overexpressed in multiple cancer cell lines.Our genome context analysis suggests specific functional interactions for proteins involved in DNA replication between each other or with proteins involved in DNA repair or transcription. Furthermore, it suggests a previously unrecognized regulatory network coupling DNA replication and translation in Archaea that may also exist in Eukarya. A fewr system , are pror system . Comparar system ,15). Forr system ,17 and or system ,19.in vitro . Most oiewed in ). Severaiewed in ), suppor genomes -24; in oHere, we have performed a systematic genome context analysis of genes encoding DNA replication proteins in 27 completely sequenced archaeal genomes. Our results show that a subset of genes encoding DNA replication proteins often co-localize, that is, these genes are arranged in operon-like structures that are preserved between distant lineages (as for the majority of the cases discussed here), or they lie in a common chromosomal region less than 5 kilobases away from each other. Some of these associations are conserved between distant lineages, indicating that they reflect a functional and possibly a physical interaction between the gene products. In particular, we identified two conserved genomic associations of DNA replication genes that suggest a functional connection between the PCNA, the DNA primase and the MCM helicase via the GINS complex. We also observed that the gene for PCNA is linked to the gene coding for the transcription factor S (TFS) in 12 out of the 27 analyzed genomes, as well as to a gene encoding the ADP-ribose pyrophosphatase NudF in 8 genomes, pointing toward the existence of cross-talk between DNA replication, DNA repair, and transcription. In addition, we noticed that the gene encoding the initiator protein Cdc6 is usually adjacent to a predicted origin of replication, sometimes together with or close to the gene coding for the small subunit of DNA polymerase (Pol)D (DP1) in euryarchaeal genomes, suggesting that PolD may be recruited by Cdc6 at the origin of replication. Moreover, some proteins without clear functional assignments (an oligonucleotide/oligosaccharide-binding (OB)-fold containing protein, a recently described new GTPase, DnaG) are encoded by genes that co-localize with DNA replication genes, suggesting that they may be involved in DNA transaction processes. Surprisingly, our analysis also reveals a widely conserved clustering of a particular set of genes coding for DNA replication proteins ) with a special set of genes encoding proteins related to the ribosome . This cluster is strongly supported by a statistical analysis based on the actual distribution of gene clusters in the set of genomes analyzed in this study, suggesting the existence of a previously unrecognized regulatory network coupling DNA replication and translation in Archaea.Halobacterium sp. NRC-1 survival according to recent genetic data [We have performed an exhaustive search of all known putative DNA replication genes in the 27 archaeal genomes available at the NCBI as of 10tic data . We havePyrobaculum aerophilum [Methanopyrus kandleri , whilst the gene encoding the large catalytic subunit (DP2) is present. It would be of particular interest to get insight into the functional properties of the M. burtonii PolD to unravel whether or not a core version of PolD exhibits the expected features, given that the interaction between the two subunits has been shown to be essential for full enzymatic activities of the canonical form [DNA replication proteins are encoded by a set of genes that is present in all archaeal genomes , with the exception of PolD, which is absent in hyperthermophilic Crenarchaea; Gins23, which has only been detected in Crenarchaea and Thermococcales; RPA, which is absent in hyperthermophilic Crenarchaea; and the crenarchaeal SSB, which is currently restricted to Crenarchaea and Thermoplasmatales. We noticed a few interesting instances of missing DNA replication genes. In particular, we and others failed to detect a RPA or a SSB homolog in rophilum ,29 and tandleri . Interestingly, the genes encoding the two subunits of Topo VI are contiguous to the genes encoding the two subunits of DNA gyrase in all halophilic Archaea and in Methanosarcinales, suggesting a co-regulation of the two type II DNA topoisomerases that was selected after the transfer of the bacterial enzyme into its archaeal host. The genes encoding the two subunits of PolD are adjacent in Thermococcales only, and those for the two subunits of DNA primase co-localize in Thermococcales and Methanobacteriales; the primase genes are fused in N. equitans as previously mentioned . The genes encoding the three subunits of the heterotrimeric RPA found in Thermococcales are clustered in the four completely sequenced genomes presently known, whereas the genes encoding RPA homologs present in other euryarchaeal genomes never associate. Finally, the genes encoding the two Gins proteins in Crenarchaea and Thermococcales are never adjacent. The tendency for genes encoding different subunits of DNA replication factors to co-localize is, therefore, very different from one gene to the other, a first indication that the observed gene associations are not random.Several DNA replication factors are formed by the association of two or more different protein subunits , including RFC (RFC-s and RFC-l), primase (PriS and PriL), the PolD holoenzyme (DP1 and DP2), and Topo VI (A and B subunits). We did not detect any obvious trend of association for the genes encoding different subunits of heteromultimeric proteins among archaeal genomes, except for the genes encoding the Topo VI subunits and the genes for the RFC subunits. The genes encoding the two subunits of Topo VI are contiguous in all Archaea, except for N. equitans since all the gene strings that are conserved in all other archaeal genomes are disrupted in this archaeon. It is likely that these disruptions are due to extensive genome rearrangements that occurred in this species because N. equitans is a parasitic organism that has adapted to its lifestyle by extensive genome reduction, including the split of several genes [In the course of this work, we noticed that co-localization of DNA replication genes - encoding different subunits of heteromultimeric proteins (see above) or encoding different proteins (see below) - are more frequent in some genomes than in others. They are especially rare in al genes ,34. At tPyrococcus abyssi chromosomal replication origin (oriC), where the gene encoding Cdc6 lies together with those encoding DP1, DP2, RFC-s, and RFC-l [cdc6-2 locus in Sulfolobus solfataricus, where the genes encoding RFC-s, RFC-l, Cdc6-2, Gins23, and MCM are situated [A. fulgidus, Haloarcula marismortui, and Halobacterium salinarum and a textracts . Finallyextracts ). TherefIn the course of this analysis, we detected many genomic associations of DNA replication genes with genes coding for archaeal homologs of DNA repair/recombination proteins from Eukarya or from Bacteria . We also found associations between genes for DNA replication proteins and specific archaeal proteins that have been characterized biochemically and predicted to be involved in the repair of stalled replication forks by recombination/repair . All these observations suggest that several DNA replication proteins are also involved in base excision repair, in nucleotide excision repair, or in the repair of stalled replication forks. They are described and discussed in Additional data file 3.P. aerophilum, Aeropyrum pernix) and euryarchaeal genomes and gene capture, whereas its RNA polymerase has evolved more rapidly than other archaeal RNA polymerases [In summary, the gene for PCNA is linked to the gene coding for TFS in 12 out of the 27 analyzed genomes. Although, this gene pairing is not supported by statistical analyses since two genes clusters are frequently conserved across genomes , it cannot be a chance occurrence . Furthermore, it is remarkable that these two genes are associated in both crenarchaeal and euryarchaeal genomes representing four different orders. In our opinion, this conservation pattern indicates that this gene pairing is not coincidental, pointing towards the existence of cross-talk between replication and transcription processes and indicating that TFS and PCNA may be part of this connection. The archaeal protein TFS is homologous to the carboxy-terminal domain of the eukaryotic transcription factor TFIIS and to one of the small subunits of the three eukaryotic RNA polymerases . TFS is on forks . One posymerases .M. jannaschii [M. kandleri, which does not contain any TFS homolog, the gene for NudF co-localizes with the PCNA gene . Nudix proteins, which are found in the three domains of life, hydrolyze a wide range of organic pyrophosphates, including nucleoside di- and triphosphates, dinucleoside polyphosphate, and nucleotide sugars; some superfamily members have the ability to degrade damaged nucleotides (reviewed in ). We notnnaschii . Therefonnaschii ). The clnnaschii , respectively. Hence, the gene encoding Cdc6 is located in the vicinity of the genes encoding RFC-s1 and RFC-l in P. aerophilum; RFC-s in H. salinarum; MCM and DP2 in M. maripaludis; and DP1 in H. salinarum, H. marismortui, Methanothermobacter thermautotrophicus, and Methanosphaera stadtmanae . Remarkably, all these proteins should be recruited at the replication origin for the initiation of DNA replication. In addition, the genes that are located in the vicinity of the cdc6 gene in the genomes of P. aerophilum, Halobacteria and methanogens correspond to those that form the replication islands of Pyrococcus or Sulfolobus . Since the gene encoding Cdc6 is frequently associated with a predicted replication origin [cdc6 gene with various DNA replication genes in the vicinity of oriC could help the recruitment of DNA replication proteins to build new DNA replication factories at the origin of replication. Among the various gene associations of cdc6 with other DNA replication genes, the most recurrent is the linkage with the gene encoding the small subunit of PolD. First noticed in M. thermautotrophicus, P. furiosus and P. horikoshii [oriC via its small subunit DP1. Interestingly, we recently noticed the presence of an origin recognition box (ORB) and mini-ORB repeats in the gene encoding the DP1 subunit of the four Thermococcales [Besides the DNA replication genes that belong to the PPsG cluster, the gene that co-localizes more frequently with other DNA replication genes is n origin ,23,47, crikoshii , this ascoccales . This suA. fulgidus, M. maripaludis, and M. hungatei) harbor a gene that encodes an OB fold-containing protein without assigned function that is distantly related to the RPA32 subunit of Thermococcales (COG3390). Interestingly, in most euryarchaeal genomes, the gene belonging to COG3390 is arranged in tandem with a gene encoding a RPA41 homolog suggesting that the two gene products functionally associate , has been shown to be a partner of two proteins: XPA involved in nucleotide excision repair [Another interesting candidate is a protein that we previously identified as PACE12 in a list of proteins from Archaea conserved in Eukarya . Interess Figure . This sun repair and MBD2n repair . Such obPicrophilus torridus. The archaeal DnaG-like protein associates with archaeal exosome components in S. solfataricus [M. thermautotrophicus [Finally, our analysis suggests that the archaeal homologs of the bacterial primase DnaG may be involved in DNA replication/repair in Archaea since the gene encoding DnaG is adjacent to the gene encoding PolB3 in the three crenarchaeal lineages investigated and is located in the vicinity of a gene encoding a RPA in almost all Methanosarcinales . Furthermore, the gene encoding the archaeal DnaG is located beside the gene encoding PACE12 in ataricus and in Mrophicus . It is uN. equitans (COG2047). Therefore, this protein may interact with Nop10, maybe as a regulator given its predicted function.Surprisingly, we found that the DNA replication genes of the PPsG cluster or its subsets are frequently contiguous to a set of genes encoding proteins involved in translation. This association forms a supercluster grouping in the same orientation as the genes of the PPsG cluster and a highly conserved cluster of four genes encoding, in order, the ribosomal proteins L44E and S27E, the alpha subunit of the initiation factor aIF-2, and the protein Nop10 (involved in rRNA processing) . The complete LSIN cluster is conserved in all Crenarchaea and nearly all Euryarchaea , in Methanococcales (PG-LS) and A. fulgidus (G-LS). Interestingly, the genes encoding L44E and S27E (LS cluster) are located close to the gene encoding PolB in Thermococcales, whereas the gene encoding Nop10 (N) is close to the gene encoding MCM in N. equitans, indicating that the translation proteins encoded by the genes of the LSIN cluster are somehow linked to DNA replication .The genes of the PPsG and LSIN clusters are always organized in the same order and all transcribed in the same direction Figure . This PPM. kandleri, M. thermautotrophicum) or forms a cluster together with the genes encoding MCM and Gins23 in the four Thermococcales are impaired in the elimination of damaged transcripts after a genotoxic stress, suggesting that S27A is involved in mRNA turnover [A. thaliana exhibits a motif in common with transcriptions factors known to have roles in DNA repair [In the course of performing literature mining regarding these proteins, we focused our attention on S27E since this protein exhibits various extra-ribosomal functions. In human, the gene for this ribosomal protein was originally isolated in a screen for growth factor-induced genes and its product called metallopanstimulin (MPS-1) because it was identified as a metalloprotein expressed in a wide spectrum of proliferating tissues . S27E ; the sary phase . Thus, uS. acidocaldarius whose size is close to the average size of archaeal genomes). As intuitively expected, we determined that the probability of finding that two neighboring genes in S. acidocaldarius are still neighbors in any of the 26 randomized S. acidocaldarius genomes is very low . For instance, the probabilities of finding that two neighboring genes are still neighbors in two or three randomized genomes is 0.23% and 0.04%, respectively. Accordingly, if two or more genes are located close to each other in the genomes of more than two different species, this cannot be by chance. Two alternatives can be proposed to explain why co-localization of some genes are conserved in several species: these genes were adjacent in the genome of the ancestor of these species and have not yet been separated by chromosome recombination; or there is a selection pressure that favors organisms in which these genes are associated, either by maintaining an association already present in the ancestor of the two genomes or favoring their recurrent association. The distribution of gene clusters in present-day genomes should be the result of a combination of these two alternatives. One can reason that gene clusters maintained only by chance (genes not yet separated by recombination) disappear, on average, more rapidly in the course of evolution than those maintained by selection pressure. In that case, clusters under positive selection pressure should be essentially those present in the highest number of genomes. To perform a quantitative analysis that could be amenable to statistical analysis, we first determined the distribution pattern of gene clusters in our dataset of 27 genomes . As shown in Figure In order to evaluate the statistical significance of the various genes associations that we have detected in this analysis, we first determined the probability of finding by chance groups of two, three, and so on contiguous genes in a set of 26 randomly shuffled genomes are not statistically supported by a 5% standard threshold. For instance, although the cluster between TFS and PCNA is present in 10 genomes of both Euryarchaea and Crenarchaea ), its frequency score is not significant (33%). However, this is also the case for gene associations whose biological relevance has been validated experimentally. For instance, the frequency score of the cluster of genes coding for MCM and Gins23 is not significant (55%) despite the functional relevance of this cluster, as indicated by the work of Bell and colleagues . This agoriC for the initiation of DNA replication. We also speculate the existence of cross-talk between DNA replication, DNA repair, and transcription in which PCNA, TFS, and the ADP-pyrophosphatase NudF may be involved. Moreover, we suggest that three proteins without clear functional assignations may take part in informational processes at the DNA level.We have identified, through our genome context analysis of archaeal DNA replication genes, several conserved gene associations that have escaped previous global screening, and from which new functional connections have been inferred. Most of these gene clusters are conserved in distantly related archaeal genomes, indicating that these clusterings are not merely coincidental but probably functionally relevant, and that they should be under selection pressure to optimize functional interactions between the encoded proteins and/or to facilitate the formation of specific protein sub-complexes. In particular, we predict that the PCNA clamp, the DNA primase, and the helicase MCM are functionally connected via the GINS complex in the replication factory and that Cdc6 may interact with DP1 at Finally, and unexpectedly, we discovered that the genes coding for a particular set of proteins are almost systematically arranged in an operon-like structure with a conserved cluster of genes coding for ribosome-related proteins , suggesting the existence of a functional coupling between DNA replication and translation in Archaea. The biological relevance of this association is strongly supported by a statistical analysis of the gene cluster distribution in the 27 archaeal genomes of our dataset. Most of the genes belonging to this particular cluster have eukaryotic homologs but are absent from bacteria; thus, we anticipate that DNA replication and translation may be co-regulated by a mechanism conserved from Archaea to human. The nature of these connections remains to be deciphered but the gene cluster highlighted in this study may be a benchmark for future experimental studies aiming to address this fundamental issue.A. pernix; P. aerophilum; the three Sulfolobales, S. acidocaldarius, S. solfataricus, and S. tokodaii; N. equitans; A. fulgidus; the three Halobacteriales H. marismortui, H. salinarum, and Natronomonas pharaonis; the two Methanobacteriales M. thermautotrophicus and M. stadtmanae; the two Methanococcales M. jannaschii and M. maripaludis; M. kandleri; the four Methanosarcinales M. burtonii, Methanosarcina acetivorans, M. barkeri, and M. mazei; Methanospirillum hungatei; the four Thermococcales P. abyssi, P. furiosus, P. horikoshii, and Thermococcus kodakaraensis; and the three Thermoplasmatales Picrophilus torridus, Thermoplasma acidophilum, and T. volcanium) by means of BLASTP or PSI-BLAST [P. abyssi and S. solfataricus homologs and, if available, sequences of biochemically characterized proteins as references. All the proteins of the above list were assigned to clusters of orthologous groups (COGs) [A list of 12 factors - corresponding to both monomeric and heteromultimeric proteins - likely to be involved in DNA replication was drawn up. This list contains: the initiation factor Cdc6/Orc1; PolB1, PolB2, and PolB3; the small and large subunits of PolD (DP1 and DP2); the helicase MCM; the sliding clamp PCNA; the small and large subunits of the clamp-loader RFC (RFC-s and RFC-l); the small and large subunits of the DNA primase (PriS and PriL); the single-stranded binding protein (RPA or SSB); DNA ligase; the two subunits of Topo VI (Topo VIA and Topo VIB); RNase HII; the flap endonuclease FEN-1; and the two Gins subunits (Gins15 and Gins23) of the GINS complex. The accession numbers of these proteins or protein subunits were retrieved from 27 complete archaeal genomes (SI-BLAST performeSI-BLAST using P.s (COGs) ,70 usings (COGs) in orderThe genomic context of DNA replication genes were visualized with Genomapper. All genomic contexts were scrutinized manually since the conserved cluster of genes encoding PCNA, PriS and Gins15 was not detected with an automated tool such as STRING , likely S. acidocaldarius genome, whose 2,329 genes approximate the average gene content in completely sequenced archaeal genomes, as reference. We generated 26 random genomes as follows: all genes of the S. acidocaldarius genome were position-exchanged for another gene chosen randomly from the genome; starting with gene number one and then sequentially applying the same process to all other genes. We then counted the number of times clusters of two to nine genes, present in the genome of S. acidocaldarius, remained together in the 26 randomized genomes. For all clusters we calculated a prevalence index based on their presence and absence. That is, every time a gene cluster was indeed present in a randomized genome the prevalence gene index gained one point, otherwise it lost one point. This approach allowed us to calculate the probabilities of having gene clusters by chance only (data not shown). These probabilities were lower than 0.01%, except for clusters of two or three genes in two genomes (see text).We chose the To establish if the conservation of the gene clusters characterized in this work was statistically significant, we decided to determine the global gene cluster conservation among the 27 archaeal genomes we used for genome context analysis. A genome was chosen randomly, and from this genome a gene was taken randomly. This gene was then BLAST searched against all other 26 genomes. The same BLAST search was performed for its two neighboring genes (the first upstream and the first downstream). Every time the gene appeared with at least one of the same flanking genes in another genome, the prevalence gene index gained one point, otherwise the index lost one point. The whole operation was repeated 10,000 times. We repeated the same process for gene clusters of three to nine genes. We thus ended up with 10,000 prevalence indexes for each size of gene cluster, from which we constructed frequency distributions . At the same time we determined the prevalence indexes of 32 representative clusters containing DNA replication genes and/or translation genes . We then performed a one-tailed test to settle the significance of our clusters; we simply located the prevalence indexes of our 32 clusters in the frequency distributions (frequency score). The indexes were considered statistically supported when they were present in the 5% or less area of the right part of the distributions ; this area of the distributions contains those very few clusters highly conserved in archaeal genomes.COG, cluster of orthologous groups; DP1, PolD small subunit; DP2, PolD large subunit; LSIN, L44E S27E aIF-2 alpha Nop10; MPS-1, metallopanstimulin 1; OB, oligonucleotide/oligosaccharide-binding; PACE, proteins of Archaea conserved in Eukarya; Pol, DNA polymerase; PPsG, PCNA PriS Gins15; PriL, DNA primase large subunit; PriS, DNA primase small subunit; RFC-l, replication factor C large subunit; RFC-s, replication factor C small subunit; TFS, transcription factor S; Topo, topoisomerase.PF initiated the study. JB performed genome context analysis. DC carried out statistical analyses and simulations, and helped to interpret numerical analysis. PF and JB interpreted the data and wrote the paper.The following additional data are available. Additional data file DNA replication factors encoded by archaeal genomes analyzed in this work.Click here for fileGenomic context of all the archaeal DNA replication genes analyzed in this study.Click here for fileDescription of and discussion about genomic associations of DNA replication genes with genes coding for archaeal homologs of DNA repair/recombination proteins.Click here for filePrevalence indexes of gene clusters and frequency distributions of clusters of two genes and clusters of more than two genes.Click here for file

Secale cereale L.) belongs to tribe Triticeae and is an important temperate cereal. It is one of the parents of man-made species Triticale and has been used as a source of agronomically important genes for wheat improvement. The short arm of rye chromosome 1 (1RS), in particular is rich in useful genes, and as it may increase yield, protein content and resistance to biotic and abiotic stress, it has been introgressed into wheat as the 1BL.1RS translocation. A better knowledge of the rye genome could facilitate rye improvement and increase the efficiency of utilizing rye genes in wheat breeding.Rye useful sequences with a cumulative length of 2,032,538 bp and an average read length of 732 bp. These sequences represent 0.5% of 1RS arm. The GC content of the sequenced fraction of 1RS is 45.9%, and at least 84% of the 1RS arm consists of repetitive DNA. We identified transposable element junctions in BESs and developed insertion site based polymorphism markers (ISBP). Out of the 64 primer pairs tested, 17 (26.6%) were specific for 1RS. We also identified BESs carrying microsatellites suitable for development of 1RS-specific SSR markers.This work demonstrates the utility of chromosome arm-specific BAC libraries for targeted analysis of large Triticeae genomes and provides new sequence data from the rye genome and molecular markers for the short arm of rye chromosome 1. Secale cereale L.) is a temperate cereal belonging to the tribe Triticeae, which is grown mainly in Europe and Northern America. Its uses include grain, hay, pasture, cover crop, green fodder, and green manure. More than 50% of the annual rye harvest is used for bread making, resulting in rich, dark bread that holds its freshness for about a week. Despite its relatively low acreage compared to other cereals, rye is of great importance due to its broad tolerance to biotic and abiotic stress, a feature generally lacking in other temperate cereals. Thus, rye remains an important grain crop species for cool temperate zones.Rye has been introgressed into several hundreds of wheat cultivars . In fact quality . Thus, iDespite the economic importance of rye, little is known about its genetic make up at the DNA sequence level. To our knowledge, there is no ongoing sequencing project in rye, and there are no plans to target gene-rich fractions of its genome. Rye is underrepresented in the sequence databases compared to wheat and barley for which 1,104,002 and 529,839 sequences respectively, are deposited in GenBank. There are only 9,807 rye sequences (about 5 Mbp) available, of which about 90% are expressed sequence tags (ESTs). Updated list of rye genes, markers and linkage data was created by Schlegel and Korzun and SccImp1RShA_0079_J11F [GenBank:FI104773], contained each one complete and one partial unit of this repeat with identity ranging from 85 to 95%. These sequences were used for multiple alignment and consensus sequence calculation against the complete BES data set revealed eight complete or partial units. Assuming that our data set represents 0.5% of 1RS, one can estimate that there is more than 1,000 COP1 units in chromosome 1RS. BLAST search against NCBI nr database revealed additional units in BAC clones from T. turgidum subsp.durum [GenBank:EF081027], T. turgidum subsp. dicoccoides [GenBank:EF067844] and H. vulgare [GenBank:DQ871219] with similarities ranging from 70 to 90%. In each sequence, 3 – 5 tandemly organized units were identified.Searching BES with masked repeats against themselves and genome survey sequences (GSSs) from Aegilops tauschii [Repeat composition of rye BESs was compared to 10.8 Mbp sequence of random BESs obtained from wheat chromosome 3B and 2.9 tauschii . The fre-30) against 960,574 plant PUTs downloaded from PlantGDB [-10) was identified for 17 of them. Eleven of them have a putative function were identified in the data set using SciRoKo 3.3 software . In total, 216 SSRs were identified with an average length of 19.85 bp. The most abundant motifs were trinucleotides, which were found 92-times Table . On averSixty-four ISBP primer pairs were tested for 1RS specificity. Twelve of them (ora001 – ora012) provided an amplification product in rye and in the wheat-rye 1RS addition line but did not show any amplification with wheat DNA and thus were considered 1RS specific Figure . An addiAfa repeat proved that the repeat is localized on the D genome chromosomes. No signal was detected after FISH with the COP1 repeat on barley metaphase chromosomes accounting for 0.5% DNA of the arm. This study provides the largest amount of genomic sequence data for rye and allows the first systematic analysis of the DNA sequence composition of the rye genome. Because the BAC clones selected for end sequencing were chosen randomly and originated from BAC libraries constructed with two different restriction enzymes, the BESs produced here are expected to be randomly distributed along the whole chromosome arm. Assuming that there is no or little difference in sequence composition among different rye chromosomes, one can consider this sequence as representative of the whole rye genome. The GC content of 1RS 45.9%) is comparable to 44.5% and 44% GC content in wheat and ric% is compThe observed content of repetitive sequences 84.2%) is lower than expected and is similar to that found in the wheat B genome (85.9%) by Paux et al. . As indi4.2% is lAegilops speltoides have the same mating system, both being outcrossers. Moreover, the B genome is largest of the three homeoelogous wheat genomes and similar in size to the rye genome. On the other hand, the similarity in repeat composition between both genomes may simply reflect similar trends in the mode of their expansion via the LTR-retrotransposon activation.A comparison of the frequency of various types of repeats in genomes of rye and wheat B and D genomes suggested a greater similarity between the rye and wheat B genome than between the rye and wheat D genome. This close relationship of rye and wheat B genome was also supported at the sequence level. It is interesting to note that rye and the putative B genome progenitor A. thaliana genome is 33,000 (TAIR8 release) [Until now, the lack of sequence data did not permit estimation of the number of genes in rye. By analyzing 2 Mbp sequence from chromosome arm 1RS, we estimate 2,000 genes being present on 1RS and thus 36,000 genes in the rye genome. This first estimate for rye is consistent with the predicted gene numbers in other plants. Most recent estimate for gene number in release) , the currelease) estimateIn addition to the analysis of the rye genome composition, we used BAC end sequences for marker development. There is still a low density of markers available for rye genome and additional markers are urgently needed. Development of a genetic linkage map of rye with 183 markers was reported by Korzun et al. . BednareThis work presents a method for targeted development of molecular markers from specific parts of the rye genome using the 1RS chromosome arm as a case study. Until now, there are only a few markers available for this critical part of the rye genome. This hampers marker assisted breeding not only in rye, but also in wheat where the markers from 1RS would permit selection of lines with introgressions of desired parts of 1RS without the harmful genes. We have developed twelve new ISBPs markers for 1RS and designed almost 200 additional primer pairs for potential ISBP markers that remain to be tested. If needed, generation of additional BAC end sequences from 1RS is possible. In addition to the development of new ISBP markers, BES containing microsatellites were used to develop new 1RS-specific SSR markers. Out of the 63 tested microsatellites that were tested, 21 were specific for 1RS . Thus, iOne of the potential problems in targeted marker development from the 1RS-specific BAC library is the contamination with clones from wheat DNA. This could explain the specificity of amplification for several ISBP markers with wheat DNA. As the 1RS arm was flow-sorted from a wheat-rye ditelosomic addition line, contamination by fragments of wheat chromosomes cannot be avoided. However, the contamination of the 1RS BAC library was estimated to be only 14% and thusOryza sativa) and wild (Zizania latifolia) rice.The discovery of five ISBP markers, which were specific only for 1RS maintained in the wheat-rye ditelosomic addition line and were not found in diploid rye, was unexpected. Although the same cultivars were used for the development of the 1RS wheat-rye addition line as well as for marker testing, these insertion sites were absent from rye and wheat. This suggests that some mobile elements were activated only after the addition of 1RS telocentric to wheat, most probably as a consequence of interspecific hybridization. Among the five activated elements three are retrotransposons and two are DNA transposons (MITE and Mutator). Liu and Wendel and ShanThis work provides the first insights into the composition of the rye genome and its chromosome arm 1RS, in particular. We demonstrate that the use of chromosome arm-specific BAC libraries facilitates the analysis of complex plant genomes by targeting particular genomic regions as well as by developing molecular markers for these regions. New molecular markers from 1RS should help in saturating the genetic map of 1RS, and aid marker assisted breeding and gene cloning.1RS: short arm of rye chromosome 1; BAC: bacterial artifical chromosome; BES: BAC end sequence; ISBP: Insertion Site Based Polymorphism; PUT: PlantGDB-assembled Unique TranscriptsJB, EP and RK participated in DNA sequence analysis. JB and MH mapped newly isolated ISBP markers. DK performed the FISH experiments. PS sorted chromosomes using flow cytometry. JŠ and HŠ made an intellectual contribution to the concept of the experiment. CT sequenced BAC ends. JB drafted the manuscript. TL and CF revised manuscript critically for important intellectual content, JD conceived and supervised the project and prepared the final version of the manuscript. All authors read and approved the final manuscript.List of 1RS-specific ISBP markersClick here for file

Saccharomyces cerevisiae, all ends of telomeric DNA contain telomeric repeats of (TG1–3), but the number and position of subtelomeric X and Y' repeat elements vary. Using chromatin immunoprecipitation and genome-wide analyses, we here demonstrate that the subtelomeric X and Y' elements have distinct structural and functional properties. Y' elements are transcriptionally active and highly enriched in nucleosomes, whereas X elements are repressed and devoid of nucleosomes. In contrast to X elements, the Y' elements also lack the classical hallmarks of heterochromatin, such as high Sir3 and Rap1 occupancy as well as low levels of histone H4 lysine 16 acetylation. Our analyses suggest that the presence of X and Y' elements govern chromatin structure and transcription activity at individual chromosome ends.In Saccharomyces cerevisiae, telomeric DNA consists of tandem repeats of (TG1–3)nIn 1–3 tract, thereby generating a telomere that lacked both X and Y' elements rolled gene in Drosophila is located in heterochromatin and its expression is essential for viability in vitroGenes placed near telomeres are transcriptionally repressed, which is a phenomenon termed the telomere position effect (TPE) In budding yeast, many proteins have been identified, which can modulate TPE SIR3 gene leads to derepression of X element transcription. Our results reveal that X and Y' elements in budding yeast have distinct chromatin structure and this variation may help to explain the variations in TPE seen at different telomeres.In this study, we compare the histone density and nucleosome distribution at X and Y' elements. Unexpectedly, we find that X elements lack histones and a defined nucleosome structure. In contrast, Y' elements display high nucleosome density. Furthermore, at telomeres only containing X elements, we fail to observe significant H4K16Ac levels. In contrast, we do observe, significant levels of H4K16Ac at some regions in Y' elements and transcription analysis reveals that Y' elements are transcriptionally active. The Sir3 protein binds to X elements, but not Y' elements, and deletion of the TEF1 gene was used as an example of euchromatin histone occupancy. Our analysis showed that the occupancy at Y' elements was even higher than the histone occupancy in the euchromatic TEF1 coding region.We first measured histone H3 and H4 occupancy at chromosome ends. We used 5 different primer pairs. Due to sequence similarities between repeat elements present at different chromosome ends, we could only find one unique primer pair, which was specific to the X element at telomere C1R. The other primer pairs amplified multiple X elements. The P1, P3, and P4 primer pairs amplified between 3 to 12 different X elements, whereas the primer pair P2 amplified 13 different Y' elements . Naked genomic DNA randomly digested with micrococcal nuclease was also hybridized to tiling arrays and used to define the background. The data were normalized with the Affymetrix Tiling Array software (TAS) and visualized with the Integrated Genome Browser (IGB). To validate nucleosome maps generated this way, we compared our findings with previously published data for the CHA1 and HIS3 promoter regions Our observations so far suggested that histones are absent or at least very scarce on X elements. To further address this possibility, we analyzed positioning of nucleosomes over telomeres using DNA microarray technology. We isolated 146-bp mononuclesomes from micrococcal nuclease digested chromatin. The purified 146-bp mononucleosomal DNA was hybridized to a We next analyzed the nucleosome occupancy at X and Y' elements. Our results therefore demonstrate that X and Y' elements differ in histone as well as nucleosome density. To further confirm that X elements lack a nuclesomal organization, we used micrococcal nuclease mapping and indirect labeling to map nucleosome positioning at the right end of chromosome V. The nuclease mapping results supported our tiling array data, since no stably positioned nucleosomes were observed on the X element region.HMR locus Since stably positioned nucleosomes are absent from X elements, we wondered if the heterochromatin associated proteins Rap1 and Sir3 could bind these regions. We first analyzed Sir3 occupancy using the same set of primer pairs as in 1–3 repeats and gradually decreasing levels of Sir3 towards the euchromatic regions of the chromosome . Interestingly, primers detecting Y' element sequences showed 5–10 fold higher transcription than primers detecting X elements or genes in a telomere proximal position (X and Y' elements contain some short open reading frames (ORFs), even if most of these correspond to pseudogenes position . In agresir3 deletion strain, in order to analyze the requirement of Sir proteins for transcription silencing of genes located in X or Y' elements. RT-QPCR results only revealed derepression of genes located in X elements, whereas the Y' element located genes remained unchanged .Our results showed that the Sir3 protein interacts with X elements. We examined changes in gene transcription in a nchanged . Genome-S. cerevisiae genome have a distinct, chromosome specific organization, which is dependent on the precise location of X and Y' elements. These differences in chromatin structure may contribute to the formation of unique telomere structures, which will enable cellular processes to distinguish between different chromosome ends. The X elements do not interact with core histones and are devoid of nucleosomes. Instead of nucleosomes, we here demonstrate that X elements are bound by Sir3 and Rap1. In support of this finding, X elements have a strong silencing effect on adjacent genes, which are derepressed upon loss of Sir proteins or Rap1, lending functional evidence for a role of these proteins in X element chromatin architecture As an integral part of the subtelomeres, X and Y' elements have in general been thought of as heterochromatin regions with high nucleosome density The MNase digest generated specific, but somewhat blurry bands, which could suggest that the specific translational positions in the subtelomeric region of chromosome V is more relaxed than what has been observed at promoters Drosophila uses retrotransposons to elongate its chromosome ends In contrast to the X elements, the Y' elements display high histone occupancy and contains positioned nucleosomes. Moreover, H4K16Ac, which is associated with transcriptional activation and the maintenance of euchromatin, cannot be observed on X elements, but is present on Y' elements. In keeping with an active chromatin conformation, we could not observe Sir3 and Rap1 binding to Y' elements. Finally, Y' elements are actively transcribed, whereas X elements are silent. These findings support the idea that Y' elements possess anti-silencing properties and limits the spreading of silent chromatin. In X-Y' chromosome ends, the Y' element is located between the TG repeats and the X element. In spite of this, genes located near the Y' element are not silenced and a foreign reporter genes inserted in a Y' element can still be transcribed 1–3 repeats could therefore indicate that they play a role in this loop formation. We would like to propose that the Rap1 and Sir proteins interacting with TG1–3 repeats, fold back and contact the Rap1/Sir protein structure on X elements. We will address this interesting possibility in future work.It has been proposed that yeast telomeres form fold back loops and that this process is dependent on the Sir proteins MATa; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0) and Y07110 , obtained from EUROSCARF. Yeast cells were cultured in YPD medium containing 10 g yeast extract, 20 g peptone and 20 g glucose per liter of distilled water.Strains used for this study were BY4741 ) and incubated at 37°C for 20 min. To obtain mononucleosomes containing DNA with an approximate length of 146 bp, the amount of nuclease needed, was determined experimentally. The digestion was halted by shifting the reactions to 4°C and addition of EDTA to a final concentration of 10 mM. Proteinase K and SDS were added to the digested material, followed by incubation at 55°C over night to reverse the crosslinking. DNA was purified by phenol/chloroform extraction followed by ethanol precipitation, and incubation with RNase A. Purified DNA was run in a 1.5% agarose gel, and 150-bp mononucleosomal DNA were extracted from the gel and used for Affymetrix tiling microarray analysis. Naked genomic control DNA was randomly digested by micrococcal nuclease and used as a genomic input control. After zymolase digestion, the spheroplasts were incubated with proteinase K and SDS at 55°C over night to reverse the crosslinking and remove the nucleosome structure. DNA was purified by phenol/chloroform extraction followed by ethanol precipitation. Purified naked DNA was completely dissolved in digestion buffer and incubated at 37°C for 20 min. The digestion was stopped by EDTA . DNA was purified by phenol/chloroform extraction again followed by ethanol precipitation, and incubation with RNase A. Purified DNA was run in a 1.5% agarose gel. The naked DNA digested with micrococcal nuclease showed a smear around 100–500 bp on the gel. The smear around 150 bp (from 100 to 200 bp) were extracted from the gel and used for Affymetrix tiling analysis.BY4741 cells (1 L) were grown to OD 0.9–1.0. Then the cells were crosslinked in 1% formaldehyde for 10 min, quenched with 125 mM glycine for 5 min, and spun down at 4°C for 5 min at 3000 rpm. Pellets were washed twice in PBS and resuspended in 5 ml ice-cold lysis buffer . The resuspended cells were incubated at 30°C, 100 rpm shaking, in 50-ml conical tubes, to digest cell walls. After 30 minutes 10 ul cells were added to 1 ml 1% SDS solution to measure the OD value. The digestion was continued until an 80% decrease of the OD value was observed compared to the cells without zymolyase digestion. Spheroplasts were then spun down at 1,500 RPM for 10 min at 4°C. The pellets were resuspended in 4 ml digestion buffer ). The amounts of micrococcal nuclease used are indicated in the figure legend and the incubation was at 37°C for 10 min. EDTA , Proteinase K and SDS were used to stop the digestion. The samples were incubated at 55°C for two hours. DNA was purified by phenol/chloroform extraction, then incubated at 37°C for 30 min, purified again by phenol/chloroform again and precipitated by ethanol. Purified DNA was digested by 50 units of ScaI overnight at 37°C. The digested DNA was separated in an 1.4% agarose gel, 150 volt, 4 hours. The DNA was transferred onto a Hyrbid N+ membrane and fragments were detected by Southern blotting. The probe was amplified by PCR using a primer pair (MR5F:TTC ATC TTC TGA CGC GGT GAG CTT MR5R:TCA AGT CCA TTG GCA GCA CCT TTG). The purified PCR product was labeled by the Stratagene random primer labeling kit. Hybridization was performed at 65°C in rapid-hyb buffer . The Naked DNA control preparation was the same as we described for preparation of mononucleosomes. The Spheroplasts were incubated with proteinase K to remove all proteins. The naked genomic DNA was purified by phenol/chloroform extraction, and ethanol precipitation. The precipitated DNA was dissolved in lysis buffer and amounts of micrococcal nuclease used, are indicated in the figure legend. After 10 minutes of nuclease digestion, the reactions were stopped by addition of EDTA and SDS followed by phenol/chloroform purification of DNA. Precipitated DNA was dissolved in water and digested with 50 units of Sca I overnight. The digested naked DNA was run in parallel with micrococcal nuclease treated samples in an agarose gel and analyzed as described The Spheroplasts was prepared as described above for Mononucleosome preparations, but without formaldehyde crosslinking. Briefly, the 1 L BY4741 cells were grown to OD 0.9–1.0. Yeast pellets were washed twice in PBS and resuspended in 5 ml sorbitol buffer . After we reached an 80% decrease in the OD value, spheroplasts were spun down at 1,500 RPM for 10 min at 4°C. The pellets were resuspended in 4 ml digestion buffer . All labeling and hybridization were carried out at the Bioinformatics and Expression Analysis Core Facility at Karolinska Institutet, Stockholm. All tilling array experiments were performed with two independent biological repeats.For Microarray data sets were analyzed using the Affymetrix tiling array software (TAS). Genome annotation data were obtained from the Affymetrix website. Mononucleosome data were selected as the treatment group. Random digested input control data were selected as the background group. Bandwidth was 70 and the test type was two sides. After intensity analyses, the log2 transformed signal file was visualized by the integrated genome browser (Affymetrix). Raw data and signal files obtained from TAS have been deposited on the NCBI GEO website (GSE13615).TEF1 (euchromatin) and HMR (hetrochromatin) genes were used as controls. Experiments were typically repeated at least 3 times, error bars in figures show standard deviations. Primer sequences are presented in supplementary Real-time quantitative PCR was carried out on a Bio-Rad CFX-96 detection system with QPCR SYBR Green reagents (Bio-Rad) and with a primer concentration of 0.5 µM. PCR conditions were standardized to 40 cycles; 95°C for 10 s, 55°C for 10 s, and 72°C for 30 s. Results were analyzed as described. Serial dilutions of total extract DNA were used to generate a standard curve for each primer and each reaction. Each ChIP experiment was normalized to total DNA input. The 7 yeast cells were harvested by centrifugation, the pellet was resuspended in sorbitol buffer and incubated for 30 minutes at 30°C. The spheroplasts were collected by centrifugations for 5 min at 1500 rpm. Lysis buffer was added and the spheroblasts were lysed by vortexing vigorously. One volume 70% ethanol was added to the homogenized lysate and mixed well by pipetting. The sample was transferred to an RNeasy column and spun down for 15 seconds at 10,000 RPM. After washing, the RNA was eluted with 50 µl elution buffer. The RNA concentration was determined using nanodrop spectrophotometer (Thermo Scientific). One ug RNA was used for first strand cDNA synthesis (Roche). Quantitative PCR was carried out on a Bio-Rad CFX-96 detection system with QPCR SYBR Green reagents (Bio-Rad) and with a primer concentration of 0.5 µM. PCR conditions were standardized to 25°C 10 minutes, 95°C 3 minutes and 40 cycles; 95°C for 10 s, 60°C for 30 s. A set of 4 genes with known mRNA abundance YHR021(8200), YDR418W(4200), YGL245W(980), and YDL218W(124) were used as abundance standards. cDNA prepared from BY4741 and a sir3 mutant strain were used for transcription analysis. Data analysis was performed with Bio-Rad CFX Manager Software. Different samples were normalized to ACT1, which was used as an internal control. Target gene primer efficiency was normalized by genomic DNA serial dilution controls. Target gene mRNA abundance was calculated based on comparison with genes with known mRNA abundance. All reported experiments represent at least three biological repeats.Yeast total RNA was extracted using the RNeasy Mini Kit (Qiagen) following the manufacturers instructions. Briefly, 2×10Figure S1Nucleosome density. High resolution profiling of nucleosome position of X and Y elements. X elements presented by red rectangle and Y elements presented by Yellow rectangle. The black arrow indicated the X element nucleosome free regions. The width of these regions showed above the black arrow. X axis scale vary between different telomeres due to different length of Y elements.(2.58 MB PDF)Click here for additional data file.Figure S2Rap1 binding. Rap1 occupancy showed high enrichment to X element. The original data were obtained from lieb JD et al,2001 Nat Genet. X and Y elements indicated as red and yellow rectangle.(0.28 MB PDF)Click here for additional data file.Figure S3H4K16 Acetylation. High resolution profiling of H4K16Ac of X and Y elements. H4K16 Ac anbitbody(abcam) enriched DNA and input DNA hybrid to tiling array respectively. H4K16ac IP data were normalized to input data in TAS and visualized in IGB as we described in (2.28 MB PDF)Click here for additional data file.Figure S4Southern blotting of X,Y and TEF1 probe. Southern blot showed the fragment size of input. Probes used here were X element(X1), Y element(Y1) and euchromatin control(TEF1). Marker lane indicated the molecular weight.(0.45 MB PDF)Click here for additional data file.Table S1The primer sequences used in this study.(0.62 MB PDF)Click here for additional data file.

Realization that dental caries is a reversible, dynamic biochemical event at a micron level has changed the way the profession recognizes the caries disease and the caries lesion. The diagnosis of dental caries poses challenges due to the complex interaction of multiple endogenous causal factors. The most appropriate diagnostic aid for this purpose is the risk model of caries risk assessment. The analyses of the biological determinants provide clues to the dominant causal factor. The detection of a carious lesion has undergone a rigorous revision and revolution in order to identify the earliest mineral change so that it can be controlled without resorting to invasive management options. Apart from detection, it became mandatory to assess the extent of the lesion (noncavitated/cavitated), assess the activity status of the lesion (active/arrested), monitor the lesion progress (progression/regression over a period of time), and finally to predict the prognosis of the lesion as well as the disease. The prognosis of the disease can be best assessed by analyzing the predictor factors in caries risk assessment. The ultimate objective of such a meticulous and methodical approach aids in devising a tailor-made treatment plan, using preventing measures precisely and restorative measures minimally. This ensures the best oral health outcome of the patient. The preceding part of this series had focused on the changeovers in dental caries definition and etiopathogenesis. They areDental caries is a disease with multiple causal factors, called as determinants and confounders, manifesting itself on the tooth structure as a carious lesion. Such manifestations may range from microscopic to macroscopic changes in the tooth.The underlying causal process has been attributed to disturbances in two oral homeostases: (a) disturbance in the mineral homeostasis between the tooth and the oral fluid and (b) disturbance in the microbial homeostasis in the biofilm.The result is alternate cycles of mineral loss and gain in the hydroxyapatite crystal. These cycles are orchestrated by a set of factors called as the demineralizing factors and the remineralizing factors.With this knowledge in the backdrop, the ensuing text shall unfold the changeovers in dental caries diagnosis and prognosis.Diagnosis is “the recognition of a disease or a condition by its outward signs and symptoms”. The diagIf dental caries is a disease and the lesion is the sign of the disease, then even a dental clinician should follow the same. But in reality, this process of elimination does not take place in the diagnosis of dental caries. A dental clinician is instantly and intuitively aware that certain unique demineralizations and destructions observed on the tooth are tell-tale signs of none other than a dental caries disease. Therefore, diagnosis is rarely about finding out what the patient has but does the tooth have caries.Generally, it is a common practice that on dental examination, if demineralizations or destructions are observed, at whatever stage, they are immediately labeled as “dental caries.” Probably, the labeling may extend a little further, to include the nature and extent of the lesion . Once the lesion is labeled, a treatment plan is devised for that lesion . Thus diagnosis and treatment of the disease shrink down to just labeling and treating the lesion. Due to the absence of important steps such as differential diagnosis and diagnosis, probably, the carious lesion takes the center stage eclipsing the importance of the caries disease. An inevitable query rises with this practice of using the “sixth sense” in caries diagnosis; has the dental profession ever bothered to diagnose and treat dental caries disease? The answer to this can be elucidated by revisiting the dental caries causal process.In the presence of the biofilm, every sucrose attack creates an acidic environ in the immediate vicinity of the tooth. This initiates a sequence of random biochemical reactions of mineral loss and mineral gain. As long as the dynamic equilibrium of the mineral content of the tooth-oral fluid and the microbial content of the biofilm is maintained, the entire sequence remains within the boundary of a physiological process, where loss and gain are equalized Figure . Given tHowever, if the factor disturbing the homeostasis is strong, intense, and long lasting, then the demineralization persists and prevails. The mineral loss is now more than the gain, which becomes evident as structural changes in the tooth structure. Along a timeline, this shift in the balance leads to more changes in the tooth structure, which cannot be accepted as healthy, but should be considered as pathological. Thus the physiological dental caries process becomes a pathological dental caries disease.The transformation of dental caries from a physiological process to pathology is not a sudden cross-over, but a continuum over a period of time, affected by numerous variables. There is a lack of a definite boundary line between health and disease. This becomes the raison d'être for the inextricable confusion in the diagnosis of dental caries and even in the detection of its manifestation. The profession is perpetually burdened by the question “when is caries, caries.” ExtensivTherefore, in order to achieve this goal of best management options for the patients, a shift in disease paradigm has been suggested. The concHowever, diagnosis has another meaning: “the analysis of the underlying physiological/biochemical cause(s) of a disease or condition.” Instead Risk assessment primarily intends to identify the individuals who are prone to the disease. But caries risk assessment can be comprehended as two models, namely, risk model and prediction model. A risk model identifies one or more causative factors of the dental caries disease. The prediction model identifies the patient who is at high risk for the disease. Generally, three approaches have been proposed for risk assessment, such as past caries experience, socioeconomic factors, and biological factors. The past caries experience and the socioeconomic factors form part of the predictor model and the biological factors, such as diet, saliva, and the microbes, are used both in risk and predictor models. In otherIn the following text, only the risk model associated with diagnosis will be discussed. The predictor model of CRA will be dealt under the section of prognosis of dental caries.The causal factors, in the Key's circle, are depicted in the form of four intersecting circles of a uniform dimension. At a glance, it might be interpreted that all the four factors have to be of equal strength and severity to cause dental caries, which is not true always. Assuming that all the causal parameters are responsible for the precipitation of the disease and then developing a treatment strategy that is all-inclusive is obviously a waste of resource. If two caries-inducing factors have different magnitudes, the end product is the same, i.e., dental caries. For instance 2 sucrose factors × 10 microbial factors = 10 sucrose factor × 2 microbial factors = dental caries. The pathThe robust literature is available on the methodology for CRA.–17 The dDietary analysis is the most subjective of all, as it involves patients' attitude, motivation, cooperation, and honesty. It is aimed at finding out the cariogenicity and the nutritive value of the diet consumed by the patient. The frequency of consumption of cariogenic diet is also of vital concern. The commonly used methods are dietary history, 24-h recall, dietary record, and food frequency questionnaires.19 A 3–7 Salivary analysis is done to assess four parameters, such as flow rate, buffering capacity, pH, and viscosity. Both stimulated and unstimulated saliva can be used for this purpose.Salivary flow rate is the most important clinical parameter affecting dental caries susceptibility. With a rThe unstimulated saliva is used to test the pH because the resting pH is the correct indicator of caries occurrence. With an increased flow, the pH can shift toward reduced acidity. The buffering capacity of the saliva is its ability to reduce the acidity. This can be measured by the buffer effect. The buffThe function of minor salivary glands is assessed by testing the hydration capacity. Hydration is directly related to the accumulation and growth of the biofilm. The examination mirror sticks onto the mucosa in a dry mouth. Viscous, ropy saliva reflects less water content and thus affects the oral clearance rate. Normally, the ability to string out for saliva, when lifted with a mouth mirror, is just 2.5 cm.Microbial analysis is done by culturing the salivary sample to assess the growth of S. mutans streptococci and the lactobacilli. The acceptable level of both organisms in the saliva is less than 1 million colony forming units (CFU). Laboratory culturing techniques are used to grow both organisms in their respective culture medium. The culture medium used for lactobacillus growth is Rogosa SL-agar and the one used for the growth of mutans streptococci is MSB-agar. Commercially available kits provide a plate, which contains culture media for the both organisms on either side. The plate is then incubated in the laboratory. Currently, chair-side assessment methods are commercially available that are based on a species-specific monoclonal antibody response, where the results are obtained within 30 min. An increased mutans streptococci level is usually evident in the presence of more incipient lesions. This is because of the ability of these organisms to adhere to the tooth by the extracellular polysaccharides, rendering them responsible for the initiation of the lesion. The lactobacilli require retentive areas to proliferate and hence, a high count is usually associated with the presence of frank cavitations.Though the above-mentioned analysis is performed individually, it should be remembered that the data obtained should be evaluated collectively . It is nIn an attempt to reduce the subjectivity of the data obtained through the risk analysis, quantification is attempted in a computerized analysis. This is called as the cariogram, where the factors are weighted and projected as a pie diagram. The weighted factors are depicted as sectors in the pie, with different color codes, and the remaining sector indicates the “chance to avoid cavities.” A traffiThe objectives of clinical examination of the oral cavity are dual:Examination for carious lesionExamination of carious lesionIn the process of examination for caries, it is appropriate to use the term, detection, which means discerning something hidden or subtle. The neceA good diagnostic aid should express most, if not the whole truth, regarding the presence or absence of the disease and its related sign. An aid that is sensitive enough to produce maximum of true positive results and specific enough to produce maximum of true negative results, keeping the false negatives and false positives to the minimum are considered valid aids/correct aids. But the correct interpretation of the results is solely dependent on certain attributes of the clinician, namely, observation, knowledge, and discipline. So a diagnostic aid should also be designed in such a way that it culminates in reliable/reproducible results, with least inter-/intraobserver variation. In addition, an ideal lesion detection aid should be able to provide results with the same accuracy, in detecting signs of varying severity on various surfaces .Such a diagnostic aid for caries detection, till today, remains an evasive one. The main factor responsible is the intangible question, “how early is early?” This question stems from the indefinable caries process continuum; therefore, the answer is inexplicit as well. Every diagnostic aid should have a scale of measurement that is based on a diagnostic threshold. A diagnostic threshold determines what is recorded as “diseased” or “sound” Figure . UnfortuFormulation of stringent diagnostic criteria that is realistic and useful for the treatment decision.Concurrent evolution of the technical gadgets, aiming at lowering the diagnostic threshold more and more, in order to detect the earliest mineral loss.Revolutionary progress in quantifying the test results, in order to reduce inter- and intraobserver variation that is related to the qualitative assessment.Shift from a dichotomous, nominal and ordinal scale, to a numerical scale that helps in monitoring the progress of the lesion over time.Seeing and touching are dual aspects of cognitive perceptions. Naturally, visual and visual–tactile examinations have been the most commonly used diagnostic aids in caries examination since many years. Visual inspection that was used previously lacked definite diagnostic criteria to detect an incipient lesion. Though it proved to be good in detecting cavitated lesions, it provided less sensitive results with regard to noncavitated fissure caries, and occult occlusal and approximal lesions.A probe or an explorer was used along with the visual aid in order to fulfill this lacuna. However, studies show that such a beneficial output was not evident. In additThe introduction of roentgen rays opened up the third eye for the dental profession. In dental caries examination, it proved to be a vital tool in detecting the occult proximal lesions. It was also widely used to assess the depth of the lesion. Bite-wing radiography has been in practice since 1925, and it is preferred to the periapical radiographs mainly because of the superior imaging geometry for visualizing caries. But the Yet another aid that has been in use for the detection of occult caries is the fiber-optic transillumination test (FOTI). This device is based on the principle of light scattering. As light is scattered more in a carious tissue than in a noncarious tissue, it is observed as a dark shadow against a light background. Initially, it was used for the detection of approximal lesions mainly in the anterior teeth. But later its use was extended to detect proximal lesions in the posterior teeth as well. The hidden dentinal caries in the occlusal aspect is well detected with a three-dimensional view indicating the volume of caries. Compared with the bite-wing, it has been reported to be equally specific, but less sensitive. However,Probe, bite-wing radiograph, and FOTI reported reduced specificity when used alone, whereas, visual examination reported poor sensitivity. Therefore, they have been used in combinations, to improve sensitivity without compromising much on specificity. Nevertheless, from raw versions, all these traditional aids have undergone some internal restructuring over these years, with a simple objective of becoming more valid and reliable.A major drawback in visual examination was the use of varied diagnostic criteria by various authors. An exhaustive review regarding this concluded emphasizing on the need for one criteria system. Thus eme35After a long debate of “to probe or not to probe,” probing has resurged now as a valuable adjunct to visual examination, but with a different purpose and well-defined criteria. Today's probing is limited to the removal of plaque from the surface of an incipient lesion, thus enhancing visibility. In addition, it is used to assess the surface texture of a lesion, an attribute that implies the activity of the lesion. The mode of usage changed too. Blunt probe, manipulated at a 20–40° angle to the surface, is being recommended against a sharp probe acting perpendicular to the tooth surface.Radiographic detection has evolved to digital radiography, with the hope of shedding all the pitfalls of the conventional radiography. But studies reported digital bite-wing radiography, in terms of the diagnostic aid parameters, to be only as accurate as the conventional ones, not any better. Despite Digitization has been adopted in FOTI also, to improve the sensitivity. This device is called the digital imaging fiber-optic transillumination (DIFOTI). The device works on the same principle of light scattering, but the human eyes are replaced by CCD intraoral camera to capture the image and instantly project in the monitor. As discussed for direct digital radiography, image enhancement, storage, and comparison between images are its main advantages. The sensitivity has been reported to be better than bite-wing radiography, but strong evidence is still lacking for this device.Novel detection aids evolved with an aim to revolutionize the caries detection at a very incipient stage, by further lowering the diagnostic threshold. These devices utilize the subtle changes that happen in the physical properties of a demineralized tooth structure. Optical, electrical, and thermal properties of a demineralized tooth differ from the sound tooth structure. Observations of the interaction of energy that is applied to the tooth structure or observations of energy that is emitted from the tooth are the basic platforms on which these hi-tech gadgets operate. Gadgets Fluorescence is a phenomenon where the wavelength of the light coming from the source loses energy to the surrounding tissue, and thus is converted to a larger wavelength of different color when it travels back toward the observer. Enamel and dentin have a certain fluorescence that is called as autofluorescence. This is attributed to some chromophores present in the dental structure. Carious lesions as well as the biofilm fluoresces on incident light. This is attributed to protoporphyrin, which is a bacterial break-down product. The difference in the fluorescing capacity of the sound tooth and the carious lesion can be recorded or observed. Two gadgets are available in this concept: quantitative light-induced fluorescence (QLF) and the Diagnodent (KaVo).44Diagnodent uses a red light at 655 nm wavelength, emitted from a laser source. Caries-induced changes fluoresce in red color due to the presence of the bacterial by-product. So, logically the Diagnodent device can detect the deep lesions with bacteria and not the superficial lesions where bacterial fluorophores are absent. The more intense the fluorescence, the more is the destruction. The intensity is displayed in a continuous numerical scale from 0 to 99. A recent modification of Diagnodent is the Diagnodent pen, which has a tip of 0.4 mm; thus it facilitates easy placement in the approximal areas for the detection of an incipient proximal lesion. Systematic reviews are available that elaborate on the performance of Diagnodent on the occlusal and proximal lesions.46 The seQuantitative laser/light-induced fluorescence (QLF) is a device that works on the same principle of fluorescence of the tooth and the carious lesion, but uses a light source emitting a blue light in a shorter wavelength of 488 nm. The fluorescence emitted back in the yellow region is observed through a yellow high-pass filter to filter out all the reflected and back-scattered light. The yellow fluorescence is attributed to the proteinic chromophores that are cross-links between the chains of structural proteins. Light scattering is more in the demineralized enamel; thus absorption, and hence fluorescence are also less. In addition, the scattered light acts as barrier for the light to reach the underlying healthy tooth and also for the fluorescent light from the tooth to reach the observer. The net result is a less penetration depth and visualization of the enamel lesion as a dark spot against a light green fluorescent background. Logically, this mechanism can work for a short depth of 400 μm. To quantify the mineral loss, the device was improvised with a CCD camera and computerization. The fluorescence loss from the lesion was subtracted from the fluorescence of the tooth, quantifying the mean and maximum fluorescence loss from the lesion as well as the area of the lesion in square mm. A furtheThe electrical conductivity of a normal tooth is less compared to a demineralized lesion. The porous enamel after demineralization is filled with an ionic fluid which increases the electrical conductivity. Alternately, the electrical resistance or impedance is reduced in the porous enamel. The device that measures electrical conductivity is the electrical caries meter (ECM) and the electrical impedance is measured with electrical impedance spectroscopy (EIS). The ECM is a fixed frequency device that is used in a site-specific manner or a surface-specific manner. The latter is used in epidemiological surveys. The ECM displays quantitative data that enables monitoring of lesion progression. The diagnostic results are more sensitive, but less specific than the visual aid.Summarizing, the lack of sensitivity in a gross visual examination has been the sole impetus for the evolution of a long trail of diagnostic aids. Systematic reviews show thaThus the evolutionary cycle in lesion detection has culminated where it started, namely, the visual–tactile examination, but a very sophisticated one with stringent diagnostic criteria and with technological gadgets to assist.The statement lesions are detected on extracted teeth is expliA noncavitated white spot lesion detected on a smooth surface may be an ongoing demineralization at the point of detection, or it may be an arrested demineralization, or it may be a scar tissue remnant after remineralization. Any of these three situations is very much possible keeping in mind the dynamic disease process. The same dynamism may also result in the transformation of these lesions for the betterment or for the worse. A regressed lesion can become an active demineralization or an active demineralization can transform into an arrested lesion. The status of the lesion at a point of time and its progression over a period of time assume tremendous significance, since the vital treatment decision process rests on them. For instance, an active, noncavitated white spot lesion in a high-risk individual has the maximum probability of aggressively progressing. The treatment then becomes rigorous and follow-up regime/monitoring becomes mandatory.The progression or regression of the lesion is directly associated with the caries disease risk status. The risk status of the disease too can shift from high risk to low risk or the vice-versa, a probability that is related to many patient-related factors, such as the dental awareness, socioeconomic status, psychosocial factors, dietary habits, and oral hygiene status. Caries monitoring implies not only the observation of a change in the lesion status, but change in the risk status too, over a period.Both the assessment of caries activity status and the assessment of caries risk prediction help to determine the prognosis of the dental caries, a mission that is as significant as diagnosis and detection, for doing the right thing, done right, at the right time for the right person.The past caries experience of the patient and the socioeconomic status of the patient have been proposed as strong predictors in caries risk assessment.55 The biThe data related to caries prediction are obtained from a detailed history and clinical examination. Comprehensive dialoguing with the patient provides clues on past dental treatments, systemic illnesses predisposing to caries, socioeconomic status, and familial tendency. A detailed history can also reveal the patient's oral hygiene habits, life-style habits and dietary habits, and fluoride exposure (exposure to fluoridated water/fluoridated tooth paste/mouth wash), all of which can have a definite impact on the future of caries .The presence of restorations and edentulous spaces should be corroborated with the past dental history, if they are the effect of a carious or noncarious disfigurement. The frequency of the past treatment directly implies the frequency of a new lesion occurrence. Systemic illnesses can alter the salivary parameters, diet, attitude, and psychomotor skills and hence can affect the caries incidence and progress. Social history conveys the economical, educational, and professional status of the patient, which also conveys the attitude of the patient toward oral health and dental awareness. Family history revealing caries in siblings indicates a strong familial tendency. The presence of multiple active lesions, especially in the anterior teeth or in proximal surfaces along with a poor oral hygiene, collectively forms vital clinical parameters for caries prediction.Caries activity is “the summation of the dynamics of the caries process resulting in the net loss, over time, of mineral from a caries lesion – i.e., there is an active lesion progression.” An active caries is a “lesion, from which, over a specified period of time, there is net mineral loss, i.e., the lesion is progressing.” An inactive caries is a “lesion which is not undergoing net mineral loss – i.e., the caries process in a specific lesion is no longer progressing.” These arThe surface characteristics, such as color and texture have been adopted as defining criteria. The pathoanatomical changes in the enamel and dentin have been clearly delineated to aid in such a registration . As the 4445859The long process of diagnosis of the disease, and the detection and prognosis of dental caries culminate in a single point of focus, i.e., treatment. The treatment is not just to target the lesions alone, as practiced since antiquity, but to manage the patient in a holistic way, to ensure the best oral health for a long time. Though preventive measures constitute a major part of the management model, given the status that the biofilm is ubiquitous in the mouth and caries is life-style-influenced disease, it remains to be answered if it can be prevented in a true sense or just controlled over lif

Ovarian cancer is particularly insidious in nature. Its ability to go undetected until late stages coupled with its non-descript signs and symptoms make it the seventh leading cause of cancer related deaths in women. Additionally, the lack of sensitive diagnostic tools and resistance to widely accepted chemotherapy regimens make ovarian cancer devastating to patients and families and frustrating to medical practitioners and researchers. Here, we provide an in-depth review of the theories describing the origin of ovarian cancer, molecular factors that influence its growth and development, and standard methods for detection and treatment. Special emphasis is focused on interactions between ovarian tumors and the innate and adaptive immune system and attempts that are currently underway to devise novel immunotherapeutic approaches for the treatment of ovarian tumors. Epithelial ovarian cancer (EOC) is the most deadly of gynecological cancers and is the seventh-leading cause of cancer deaths in women. In 2008, there were 21,650 cases reported which resulted in the deaths of 15,520 women in the United States . HoweverThere are several different types of ovarian cancers depending upon the cell type of origin. Epithelial cell ovarian cancer (EOC) constitutes 90% of ovarian cancers, while gonadal-stromal (6% occurrence), and germ cell (4% occurrence) tumors make up the rest of the incidence of ovarian cancer patients . As ovarThe majority of EOC cases are sporadic in nature and occur in women with no known predisposing factors and thus, in the general population, the overall risk of EOC is low (2-5%). Only a small percentage 5-10%) of EOC patients have a genetic predisposition to the disease. Ninety percent of these patients are carriers of mutated BRCA1 and/or BRCA2 genes, which are also implicated in hereditary breast cancer . These g% of EOC The normal ovarian surface epithelium (OSE) covers the surface of the ovary. OSE is highly responsive to environmental stimuli, including those associated with ovulation . In a noThe differentiation of OSE cells from cuboidal epithelial cells to a mesenchymal phenotype that is characteristic of Mullerian duct-derived tissues is termed epithelial- mesenchymal transition (EMT). The occurrence of EMT is postulated to aid cells in movement during embryo tissue generation, tissue regeneration after wounding, and is implicated in the development of cancer . OSE celOSE cells undergo EMT transition after ovulation to remodel the extracellular matrix and repair the post-ovulatory wound that is generated during expulsion of the oocyte. Epithelial cells are characteristically polar and are bound together with molecules (such as E-cadherin) that facilitate cell-cell junctions. Conversely, the mesenchymal phenotype is that of motility and movement, as well as reduced polarity of a cell . The train vitro cells spontaneously transformed into cancerous cells [The expression of markers that are associated with those of Mullerian-duct derived tissue are found in inclusion cysts, which are the site of many neoplasms. OSE lining inclusion cysts express higher levels of EOC markers MUC16 (CA125) and CA19-9 and is two to three times more metaplastic in women with ovarian tumors compared to OSE in normal ovaries . The hypus cells -23. Anotus cells .An alternative hypothesis related to that of incessant ovulation is known as the gonadotropin hypothesis -27. HighOthers hypothesize that ovarian tumors do not arise from OSE at all, but derive directly from the Mullerian-duct tissues and migrate to the ovarian surface. Dubeau first proposed this hypothesis in 1999 . Accordi+ mutation have also been observed [+ women who underwent prophylactic salpingo-oophorectomies. This staining correlated with mutations in the p53 gene in these same cells. Because the p53 mutations were found predominantly in the distal fimbrae of the fallopian tubes (the cells that are in contact with the OSE), the location of this staining may reveal one mechanism by which ovarian tumors arise in BRCA+ women [+ was conducted. The results revealed that p53 mutations were not present in any inclusion cysts that were examined, but were present in 38% of fimbrae of fallopian tubes from these women [In addition to histological changes found in the fimbrae of the fallopian tubes, mutations in the tumor suppressor gene p53 in the distal fimbrae of women with the BRCAobserved . ChristoA+ women . In 2008A+ women . A compase women . Anotherse women -39.Dubeau states that ovarian cancer is over-diagnosed, and many of these cancers actually arise from the fallopian tube or peritoneal cavity wall. The origin of ovarian tumors is of important consideration, not only for nomenclature reasons, but for women who have the BRCA1 or BRCA2 mutation and are undergoing prophylactic surgery and who want to preserve their fertility. If the origin of ovarian cancer is indeed not the ovary, then the ovaries need not be removed, and cryopreservation of oocytes for future use is not an issue .Attempts to find an accurate screening test for EOC have, to date, been unsuccessful. CA125 (MUC16), originally thought to be an indicator of ovarian cancer, is now known to be quite non-specific as well as to lack the sensitivity to detect stage I disease. Bast and coworkers showed in the 1980s that CA125 was expressed in the serum of the majority of patients with EOC, as well as patients with cancer of the endometrium, fallopian tube, and endocervix -44. CA12Proteomic approaches are being utilized to identify molecular markers for ovarian cancer and mathematical models are being developed to identify specific patterns that are indicative of disease . Other pOvarian cancer is a surgically staged disease, meaning that it is impossible to tell what the stage of the cancer is without examining the extent of the metastasis surgically. Metastasis of ovarian cancer spreads by direct extension to neighboring organs from the ovaries or by the sloughing of tumor cells into the peritoneal cavity. These individual or groups of exfoliated cells float in the fluid of the peritoneal cavity and can subsequently bind to the wall of the peritoneal cavity and form additional lesions. The tumor cells also commonly disseminate by lymphatic spread . Proper The stages (I-IV) of ovarian cancer are determined by the extent of metastasis. Stage I EOC is confined to the ovaries whereas stage II affects other pelvic structures. In stage III, the disease has spread beyond the pelvis into the upper abdominal cavity or into the draining nodal beds irrespective of peritoneal based disease. Stage IV is defined as disease outside of the peritoneal cavity and most commonly includes parenchymal liver lesions or malignant pleural effusions. Patients with stage I disease most commonly undergo bilateral oophorectomy, hysterectomy, and surgical staging including peritoneal biopsies, omentectomy, and pelvic and aortic lymph node dissection. In select cases of younger patients who wish to preserve fertility, only the affected ovary may be removed and a hysterectomy would not be performed . ChemothTreatment is made difficult for EOC patients because metastasis is acute and tumor cells exert immunosuppressive effects. The anatomical location of the ovaries within the peritoneal cavity facilitates metastasis because tumor cells can spread by sloughing off of the main tumor and binding to many organs in the vicinity, including the peritoneal cavity surfaces and the highly vascular omentum . This coTumorigenesis requires several genetic alterations, either somatic or inherited, that confer a selective growth advantage to the neoplastic cell population. During tumor development, initial random genetic alterations result in a tumor cell population with a proliferative advantage. These tumor cells become the progenitors of a clonal population that eventually dominates the tumor mass. Tumor progression is analogous to Darwinian selection, with repeated mutations and subsequent dominance of the daughter cell population via expression of traits that confer a survival advantage .A defining characteristic of a malignant epithelial tumor is invasion beyond the basement membrane into the surrounding stromal tissues. For example, in breast disease benign tumors such as fibrocystic lesions, sclerosis adenoma, and fibroadenoma are all characterized by disorganization of the normal epithelial architecture. However, no matter how extensive this disorganization may become, these benign lesions are always characterized by a continuous basement membrane that separates the neoplastic epithelium from the stroma . MalignaOnce a tumor is established metastasis may occur. While primary tumors are usually successfully eliminated by surgical or chemotherapeutic means, metastases are more difficult to detect and treat . MetastaEOC was originally thought to be of the linear-clonal model of metastasis, which states that a late stage clone of the tumor acquires an additional genetic change that enables metastatic progression . HoweverThe complexity of metastasis increases when one considers that each cancer type typically metastasizes to different areas in the body. This is termed the "seed vs. soil" hypothesis which was first observed by Stephen Paget in 1889 . ReferriIn ovarian cancer, the "seed vs. soil" observation holds true as the most common sites of metastasis are within the peritoneal cavity. Mesothelial cells that express mesothelin line the walls of the peritoneal cavity as well as the organs within it. We and others have shown that MUC16, present on the surface of cancer cells, binds readily to mesothelin ,94. ReceIn order to efficiently metastasize, tumor cells must first detach from the primary tumor by downregulating adhesive molecules, then later upregulate adhesive molecules to attach again to the target site epithelium. The initial step of detachment requires disruption of cell-cell adhesions, and this is facilitated by a loss of E-cadherin. E-cadherin is tethered to the actin cytoskeleton, which plays a primary role in supporting cell-to-cell adhesions. The disruption of the expression of E-cadherin can then lead to cells which can disseminate from the primary tumor. Loss of E-cadherin function is necessary but not sufficient for an epithelial to mesenchymal cell type transition . Loss ofAfter detachment from the primary tumor site, the next step of metastasis is to effectively invade into neighboring tissues. Movement of the tumor cells through solid tissues requires the acquisition of phenotypes that allow cells to degrade the ECM and subsequently acquire forward propelling movements to invade into these tissues .Next, the tumor cells migrate into the circulation, lymphatic system, or peritoneal space. In EOC, metastasis is facilitated by the clockwise flow of peritoneal fluid. The final steps of metastasis include arrest in the small blood vessels of a distant organ, extravasation into the surrounding tissue and proliferation at the secondary site .+ and 10% CD16-. The CD16+ phenotype is associated with activation and cytotoxicity, while the CD16- cells release cytokines and are not cytotoxic [+ and 40% CD16-. Therefore, there are less cytotoxic cells in the PF compared to the peripheral blood [Patients with EOC often experience several periods of remission and relapse of increasingly shortening periods until their tumors become resistant to chemotherapeutic treatment . Additioytotoxic . In the al blood .+T cell proliferation and function, as well as the cells' ability to produce IFN-γ. IL-2R subunits γ and β (but not α) were significantly suppressed as measured by flow cytometry [Other immune cell subsets can also be affected by factors within peritoneal fluid. A study published in 2001 described a factor within PF that induced the loss of the T cell receptor (TcR)-associated signal transducing zeta-chain (CD3ζ) . They isytometry . Our groytometry .+ T cells, most NKT cells, and a subset of CD4+ T cells [Tumor cells also produce ligands that can bind to activating receptors on immune cells and thus downregulate the expression of these receptors. The ligands for activating receptor NKG2D are MHC class I-chain-related proteins A and B (MICA/MICB) and the UL16-binding proteins (ULBP-3) . NKG2D l T cells . When NK T cells . Wang an T cells . Another T cells . Strong T cells .Most pre-clinical models of cancer immunotherapy indicate that such treatments work best in the setting of minimal volume, sub-clinical disease. Thus it is thought that patients with minimal residual disease who clinically appear to be in remission are ideal candidates for immunotherapeutic strategies. Immunotherapies may not be robust enough to eliminate the entire tumor when used alone, however; their use after surgery and chemotherapy may be useful to eliminate remaining sub-clinical tumor cells to prevent recurrence. The high rate of clinical response to therapy and the subsequent high rate of recurrence in EOC after primary treatment is evidence of a large number of women with sub-clinical disease at the completion of therapy. These patients may offer an excellent setting for immunotherapy.There are several immunotherapies that have been targeted to MUC16 as well as mesothelin. One such immunotherapy, oregovomab, is an immunoglobulin (Ig) I gG1k subclass murine monoclonal antibody that binds with high affinity to circulating CA125. This antibody complexes with CA125 and is taken up and processed by APCs (antigen presenting cells) ,110. BotAntibodies, designated 3A5 and 11D10, against the tandem repeat sequence of MUC16 have been conjugated to the cytotoxic auristatin analogs monomethylauristatin F and monomethylauristatin E ,114. TheAbagovomab (ACA125) is an anti-idiotypic antibody against the MUC16 antibody OC125 and mimics the antigenic epitope of MUC16. It serves as a surrogate when given to patients. In phase I and II trials, patients that received abagovomab antibody developed anti-anti-idiotypic antibodies (Ab3) and this correlated with increased survival ,116. ReiPseudomonas exotoxin that mediates cell killing. Phase I trials have been completed with SS1P and have shown anti-tumor activity in heavily treated patients [Mesothelin is normally expressed by mesothelial cells that line the pleura, peritoneum, and pericardium. It is highly expressed by tumor cells associated with pancreatic, ovarian, and lung adenocarcinomas as well as malignant mesothelioma ,119. Itspatients . Pre-clipatients .Listeria monocytogenes as the vector. Pre-clinical studies have shown this vaccine to elicit CD4+/CD8+ T cell mesothelin specific responses in mice and cynomolgus monkeys. A Phase I trial for CRS-207 is underway [MORAb-009 is a high affinity chimeric monoclonal IgG1/κ with high affinity and specificity for mesothelin . This anunderway .There are several other molecular candidates that are being investigated for immunotherapy against ovarian cancer. Incubation of immune cells with ovarian cancer cells lead to generation of antigen specific T cells against THP-1 and other peptide epitopes of ovarian cancer . Other pAutoantibodies against p53 are present in ovarian cancer patients and their presence is associated with improved survival . In a phCytoreductive surgery followed by intense chemotherapy with platinum and taxol has become a standard approach for the treatment of EOC. Therapy is especially effective if the cancer is detected at early stage of progression. Future advances in the management and cure of EOC will depend on development of novel treatment modalities and diagnostic tests that can accurately detect early stage low volume tumors. While chemotherapeutic approaches have been important in the management of EOC, there is a growing sense in the field that additional supportive therapeutic approaches will be required for effective elimination of the cancer. The polyclonal nature of EOC ensures that therapeutic approaches may not eliminate the entire spectrum of cancer cells present in a patient. Combinatorial approaches that can result in direct cytotoxicity, prevent tumor angiogenesis, inhibit cancer metastasis, and also simultaneously increase the immunologic detection of tumors may be required to eliminate the polyclonal tumors. Such a holistic approach will require delineation of the molecular mechanisms that allow tumors to metastasize, promote angiogenesis, and to circumvent any effective immunological responses.The combined treatment strategies will benefit from the development of diagnostic and screening tests. To date the "gold standard" for assessing the regression and recurrence of EOC is the serum CA125 (MUC16) assay. However, this assay is limited in its scope. Development of novel proteomics based approaches for the development of diagnostic tests hold great promise. However, even after intense research, successful development of a proteomics-based diagnostic test has remained elusive.Overall, significant hurdles still remain in the effective diagnosis and treatment of EOC. The significant advances made in the molecular understanding of EOC, development of murine models and novel proteomics-based technologies, and the use of immune-based treatment approaches are likely to provide novel opportunities for the effective management of EOC.The authors declare that they have no competing interests.JAAG, JPC, and MSP did the majority of the writing of this manuscript. NC and AK contributed by writing specific sections of this manuscript. All authors have read and approved this manuscript.

The operating system incorporates most commonly used phylogenetic software, which has been pre-compiled and pre-configured, allowing for straightforward application of phylogenetic methods and development of phyloinformatic pipelines in a stable Linux environment. The software is distributed as a live CD and can be installed directly or run from the CD without making changes to the computer. PhyLIS is available for free at Phylogenetic methods are playing a growing role in nearly all fields of biology. As researchers accumulate data, the size of phylogenetic analyses and the scope of inferences for which they are employed have both increased dramatically.The availability of cheap multi-core processors exacerbates this issue, and now many even-moderately sized labs routinely build small clusters or groups of several phylogenetic workstations. Phyloinformatic research, in particular, depends largely on processing power and highly parallel analyses that are spread across many computers and processors. As phyloinformatic pipelines become more complex and sophisticated, careful standardization of operating systems across computers also becomes more complex. While this growing computational power gives researchers the ability to be more creative in attempting complex and time-consuming analyses, the process of compiling, installing, and configuring software for these machines becomes increasingly repetitive, error-prone, and time-consuming. This problem is, in principle, easily solved. What is needed is a simple, stable platform that is specifically geared toward performing phylogenetic analyses. Perhaps the simplest (from a user perspective) solution to this need is a lightweight Linux-based operating system, geared specifically toward phylogenetic and phyloinformatic research. Existing Linux distributions such as Bio-LinuxPhyLIS v1.0 is a free GNU/Linux distribution based on the popular and user-friendly Ubuntu Linux distribution. The name PhyLIS is an acronym for Phylogenetic Linux for Informatics and Systematics. The distribution comes with most commonly used phylogenetic software pre-compiled, installed, and configured, which allows this software to be executed by simply typing the appropriate command . PhyLIS The distribution intentionally re-uses most of the system maintenance packages from Ubuntu, making the actual use of the operating system very similar to Ubuntu . Overall, the non-phylogenetic aspects of PhyLIS have been kept as close as possible to Ubuntu in order to preserve the large amount of development that the Ubuntu team has put into ensuring an easy-to-use operating system. Because of this, navigating, updating and using the operating system is largely intuitive for users that are already familiar with more widely used operating systems. The bundled phylogenetic tools are avaiPhyLIS is distributed as a live CD and can be used in two ways. First, it can be booted from the CD without making changes to the underlying operating system. This is useful, for example, for temporarily employing computers to run phylogenetic analyses when not in use for their primary purpose. At the completion of the analysis, the results can be transferred to permanent storage and the machine rebooted, restoring it to its previous configuration and operating system. The live CD mode is also useful for testing PhyLIS with little time commitment before deciding whether to install it.Second, PhyLIS can be directly installed from the live CD using a simple graphical installer that allows for a new, complete installation (erasing the previous operating system), or a partitioned installation . The distribution has been tested and can be installed on most 32- and 64-bit PCs (including Apple computers that use Intel processors).http://www.eve.ucdavis.edu/rcthomson/phylis.PhyLIS aims to simplify the process of carrying out complex phylogenetic analyses and has utility both for individual researchers and for teaching environments. The operating system presents a large suite of tools in a stable platform and should be useful for system administrators performing many installations. However, it is also simple enough to use that individual researchers with little previous Linux experience can employ it effectively. PhyLIS is under active development and undergoes periodic updates every six months to incorporate new versions of software and minor bug fixes. Users are encouraged to request additional software or features that would enhance the utility of the operating system; these will be incorporated into future releases of PhyLIS. The latest release is freely available at

Mycobacterium tuberculosis are both public health problems, the mechanisms by which they affect lung functions remain elusive.Although it is established that opioid and M. tuberculosis infection exhibited significant apoptosis in the lung in wild type mice as demonstrated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. Morphine and M. tuberculosis significantly induced the expression of Toll-like receptor 9 (TLR9), a key mediator of innate immunity and inflammation. Interestingly, deficiency in TLR9 significantly inhibited the morphine and M. tuberculosis induced apoptosis in the lung. In addition, chronic morphine treatment and M. tuberculosis infection enhanced the levels of cytokines in wild type mice, but not in TLR9 knockout (KO) mice. The bacterial load was much lower in TLR9 KO mice compared with that in wild type mice following morphine and M. tuberculosis treatment. Morphine alone did not alter the bacterial load in either wild type or TLR9 KO mice. Moreover, administration of morphine and M. tuberculosis decreased the levels of phosphorylation of Akt and GSK3β in the wild type mice, but not in TLR9 KO mice, suggesting an involvement of Akt/GSK3β in morphine and M. tuberculosis-mediated TLR9 signaling. Furthermore, administration of morphine and M. tuberculosis caused a dramatic decrease in Bcl-2 level but increase in Bax level in wild type mice, but not in TLR9 KO mice, indicating a role of Bcl-2 family in TLR9-mediated apoptosis in the lung following morphine and M. tuberculosis administration.We report here that mice subjected to chronic morphine administration and M. tuberculosis infection.These data reveal a role for TLR9 in the immune response to opioids during However, opioids as clinical therapies produce benefits as well as severe side effects. Studies showed that opiates, including morphine, cause cell death and apoptosis in various systems. We have previously reported that opioids promote lymphocyte apoptosis both M. tuberculosis remains a leading global public health problem despite the availability of chemotherapy and BCG vaccine M. tuberculosis undergo increased apoptosis, which is caspase-8 dependent but caspase-9 independent M. tuberculosis replication M. tuberculosis is still controversial M. tuberculosis infection, not only M. tuberculosis infection.Toll-like receptors (TLRs) play a critical role in both innate resistance and the initiation of adaptive immunity to infectious agents M. tuberculosis in the mouse model. In addition, our data demonstrate that mice deficient in TLR9 inhibit morphine-induced apoptosis during M. tuberculosis infection. Finally, we found that inhibition of TLR9 prevents morphine and M. tuberculosis -induced apoptosis through pro-apoptotic and anti-apoptotic pathways. Taken together, these data reveal a role for TLR9 in the immune response to opioids during M. tuberculosis infection and provide an important example of TLR9 in host resistance to infection.In this study, we show opioids through potent stimulus of TLR9-dependent proinflammatory cytokine production caused altered host resistance against M. tuberculosis infection is unknown. It has been shown that morphine increase the expression of TLR9 M. tuberculosis infection on inducing apoptosis. During the mouse infection with M. tuberculosis H37Ra, we implanted wild type mice and TLR9 knockout mice with a 25 mg sustained-release morphine pellet, while control mice received a comparable placebo pellet for different time periods and determined apoptotic cells using the TUNEL technique. We found that chronic morphine administration or H37Ra infection alone induced cell apoptosis in the lungs from wild type mice, but not in TLR9 deficient mice lower expression of IL-1β, and IL-6 was observed in the TLR9 KO mice as compared with the wild type control mice (We then examined the expression levels of proinflammatory cytokines in TLR9 KO and wild type mice following morphine administration and H37Ra infection. Administration of morphine and rol mice . These d473. The levels of phospho-Akt at Ser473 in the lungs from wild type mice were significantly lower compared with control (without treatment) wild type mice via Dr. Dennis Klinman . Wild type Balb/c mice were purchased from the Jackson Laboratory and maintained in the Division of Laboratory Animal Resources at East Tennessee State University (ETSU), a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC). All aspects of the animal care and experimental protocols were approved by the ETSU Committee on Animal Care. Male mice aged 8 weeks were used in all experiments.M. tuberculosis strain H37Ra was grown in 7H9 liquid medium (Difco) supplemented with 0.05% Tween 80 and 10% BSA-dextrose-catalase (ADC) enrichment (Difco) at 37°C for approximately 2 weeks with occasional agitation.Placebo pellets and morphine pellets were kindly provided by National Institutes of Drug Abuse (NIDA). The antibodies to total Akt, phosphor-Akt (Ser 473), total GSK3β, phospho-GSK3β (Ser9) were purchased from Cell Signaling Technology . The antibodies to Bcl-2, Bax, and GAPDH were purchased from Santa Cruz Biotechnology . In situ apoptosis detection kit was purchased from Roche Diagnostic . The Quantitative PCR kit was purchased from Invitrogen . 7 bacteria in 100 µl Wide type and TLR9 KO mice were randomly divided into four groups of 10 each respectively: control group (C), morphine group (M), H37Ra group (H37Ra), and H37Ra with morphine group (H37Ra + M). Each of the mice in H37Ra and M groups received an intraperitoneal (i.p.) injection with 2.5×10The lungs were homogenized in 1 ml PBS buffer containing 0.1% Tween 80. The suspension was diluted in serial 10-fold dilutions. Triplicate samples (50 µl) were plated on 7H11 plates supplemented with ADC, and antibiotic cocktail to prevent contamination. The CFU values were counted after 3 week incubation at 37°C.CCCTGGTGTGGAACATCAT(forward) and GTTGGACAGGTGGACGAAGT (reverse); TNF-α: AGTTCCCAAATGGCCTCCCTCTCA(forward) and TGGTTTGCTACGA CG-TGGGCTACA(reverse); IL-1β: CTGGAGAGTGTGGATCCCAAGCAA(forward) and GGGAACTC- TGCAGACTCAAACTCCAC(reverse); IL-6: AGCCAGAGTCCTTCAGAGAGATACAG(forward) and CTCCAGCTTATCTGTTAGGAGAGCA(reverse).Total RNA was isolated from lungs by the VERSA GENE™ RNA Tissue Kit and the real-time RT-PCR detection technique was performed as described previously The frozen sections from wild type and TLR9 KO mice were harvested for terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay. Nucleosomal DNA fragmentation in cells was determined by the TUNEL assay using an in situ apoptosis detection kit according to the manufacturer's instructions and our previous publications Lungs were fixed by formalin, embedded with paraffin, sectioned and then stained with hematoxylin and eosin.Western blotting was performed as described in our previous publications P<0.05 was considered to be significant.All data were represented as means ± SEM. Differences among groups were compared by one-way analysis of variance (ANOVA) followed by Tukey's multiple-comparison test. The authors wish to express their appreciation to Dr. Shizuo Akira and Dr. Dennis Klinman , for providing TLR9 knockout mice. We thank NIDA for providing us with both placebo and morphine pellets.

The provision of mechanical ventilation for the support of infants and children with respiratory failure or insufficiency is one of the most common techniques that are performed in the Pediatric Intensive Care Unit (PICU). Despite its widespread application in the PICUs of the 21st century, before the 1930s, respiratory failure was uniformly fatal due to the lack of equipment and techniques for airway management and ventilatory support. The operating rooms of the 1950s and 1960s provided the arena for the development of the manual skills and the refinement of the equipment needed for airway management, which subsequently led to the more widespread use of endotracheal intubation thereby ushering in the era of positive pressure ventilation. Although there seems to be an ever increasing complexity in the techniques of mechanical ventilation, its successful use in the PICU should be guided by the basic principles of gas exchange and the physiology of respiratory function. With an understanding of these key concepts and the use of basic concepts of mechanical ventilation, this technique can be successfully applied in both the PICU and the operating room. This article reviews the basic physiology of gas exchange, principles of pulmonary physiology, and the concepts of mechanical ventilation to provide an overview of the knowledge required for the provision of conventional mechanical ventilation in various clinical arenas. The first widespread use of mechanical support for respiratory failure began with negative pressure ventilation during the poiliomyelitis epidemic of the 1930s (see below for a discussion of negative pressure ventilation). The operating rooms of the 1950s and 1960s provided the arena for the development of the manual skills and the refinement of the equipment needed for airway management, which subsequently led to the more widespread use of endotracheal intubation thereby ushering in the era of positive pressure ventilation. Although mechanical ventilation remains a commonly used technique in both the operating room and the ICU, all of us at some point are or were filled with trepidation and uncertainty when faced with initiating mechanical ventilation. Although, the devices used to provide ventilatory support and the various clinical decisions to be made will initially seem overwhelming, a logical approach to the provision of mechanical and its initial set-up will help in the decision-making process. The approach to the provision of mechanical ventilation in the critically ill patient is supported by an understanding of the basics of pulmonary physiology and gas exchange.The support of infants and children with respiratory failure or insufficiency through mechanical ventilation is one of the most common techniques or procedures performed in the Intensive Care Unit (ICU). Despite its widespread application in the ICUs of the 21Although there are a diverse group of disease processes involving the central nervous and peripheral nervous systems, cardiovascular, and respiratory systems, which may lead to respiratory failure, there are a limited number of primary indications for the institution of endotracheal intubation and mechanical ventilation in the ICU setting . As the 2) is rare, but may be encountered while providing care at higher altitudes, if there is some mechanical issue with the hospital's oxygen supply, or if there is a mismatch of the appropriate flows of gases used either in the operating (oxygen and nitrous oxide) or in the ICU setting (oxygen and helium).[2 as the primary cause of hypoxemia should never occur in the context of modern medical care, it is imperative that the FiO2 be continuously monitored when medical gases are mixed. The other four processes leading to hypoxemia may be seen in the ICU setting. True shunt refers to patients with blood that travels from the right (venous) side of the circulation to the left side without traversing the lungs. Although there is a normal 2–3% obligatory physiologic shunt which includes both the bronchial veins that empty into the left atrium and the Thebesian veins (venous drainage of the coronary system which drain into the left ventricular cavity), this has no significant impact on the arterial oxygen saturation. What occurs in clinical practice is cyanotic congenital heart disease (CHD) such as Tetralogy of Fallot, tricuspid atresia, pulmonary atresia resulting in right-to-left intracardiac shunting of blood. This cause of hypoxemia does not generally show a significant response or increase in the partial pressure of oxygen in the blood (PaO2) when the FiO2 is increased. The failure to respond to an increasing FiO2 can be used as a diagnostic test when evaluating the cyanotic newborn as an increase in the PaO2 to some degree will occur even in patients with hypoxemia from severe pulmonary parenchymal disease. Although surgical intervention may be necessary in patients with true shunt from CHD to provide adequate pulmonary blood flow, these patients generally do not present with clinical signs and symptoms of respiratory failure and tend to tolerate oxygen saturations in the 70–80% range without manifesting dyspnea or other signs of respiratory failure. Therefore, clinically it may be possible to distinguish the newborn with cyanosis from CHD versus those with pulmonary parenchymal disease, based on the fact that signs of respiratory distress are generally less obvious in the setting of CHD provided there is no accompanying congestive heart failure.Respiratory insufficiency may result primarily in hypoxemia, hypercarbia, or a combination of the two. When confronted with the hypoxemic patient , the treatment will be tailored according to the etiology of the hypoxemia. In clinical practice, there are five basic causes of hypoxemia . In clin helium). Although2) into and carbon dioxide (CO2) out of the alveoli. This latter process is generally rare in the ICU setting, occurring most commonly in conditions that thicken these layers through which O2 and CO2 must diffuse. Causes include collagen vascular disorders, sarcoidosis, and other diseases that affect the endothelial–basement membrane–epithelial layer of the alveolus. The final of the five causes of hypoxemia is hypoventilation. Associated with the hypoxemia of hypoventilation is hypercarbia, which is relatively uncommon in other causes of hypoxemia except with severe forms of ventilation–perfusion inequalities and diffusion abnormalities.Another cause of hypoxemia, also sometimes inappropriately referred to as shunt, is ventilation–perfusion mismatch. This refers to processes such as pneumonia, adult respiratory distress syndrome, or atelectasis that disrupts the normal gas exchange occurring at the alveolar–capillary level with the exposure of pulmonary capillary blood to poorly ventilated alveoli. Ventilation–perfusion mismatch refers to perfusion of poorly ventilated alveoli. The opposite of the process, ventilation without perfusion or deadspace, results in hypercarbia generally without hypoxemia. One of the goals of mechanical ventilation is to improve the matching of ventilation and perfusion to improve oxygenation through the use of recruitment maneuvers if the cause is atelectasis, or the application of positive end expiratory pressure (PEEP) if the cause is adult respiratory distress syndrome (ARDS) or other alveolar diseases (see below). Hypoxemia may also result from diffusion abnormalities from a process, which affects the pulmonary endothelial–basement membrane–alveolar epithelial complex, thereby interfering with the diffusion of oxygen . Therefore, therapy that improves mixed venous oxygen saturation may also improve arterial oxygen saturation in patients with significant ventilation–perfusion mismatch or true shunt. Mixed venous oxygen saturation is determined by the difference between the oxygen that is delivered to the tissues and the amount of oxygen used by the tissues. Methods to improve oxygen delivery include increasing the hemoglobin concentration or augmenting cardiac output while preventing hyperthermia, shivering, or agitation, which increase tissue oxygen consumption. These strategies may be of significant clinical application in patients with severe respiratory failure and refractory hypoxemia.2 and the partial pressure of oxygen in the alveoli. This is commonly referred to as the A–a or the alveolar–arterial oxygen gradient. The normal value of 10–15 mmHg frequently exceeds 200 mmHg in the critically ill patient with respiratory failure. The alveolar partial pressure of oxygen is determined using Dalton's law which states that the gases in the closed space of the alveolus must equal barometric pressure . There are generally only four gases in the alveolus including O2, nitrogen (unless the FiO2 is 1.0), water vapor , and CO2. Although in the strictest sense, the alveolar pressure of CO2 is calculated as the PaCO2/RQ, where RQ is the respiratory quotient , for clinical purposes, the alveolar pressure of CO2 can be assumed to be equivalent to the partial pressure of CO2 in the blood (PaCO2), thereby resulting in the equation: alveolar oxygen concentration = FiO2 (760 – 47) – PaCO2. As the alveolar partial pressure of O2 is approximately linear with the FiO2, a quick estimate of the alveolar oxygen concentration can be had by multiplying the oxygen concentration (percentage) by 60–70.In the setting of hypoxemia, an evaluation of the severity of the lung disease can be determined based on the difference between the PaO2 from the body in the amount needed to maintain cellular homeostasis. Oxygenation is regulated by the FiO2 and the mean airway pressure. Mean airway pressure is determined by the peak inflating pressure (PIP), PEEP, and the inspiratory time.[The primary goals of mechanical ventilation are the maintenance of adequate oxygenation and clearance of COOne of the goals of mechanical ventilation, regardless of the mode and setting, is the restoration of FRC. Critical in the maintenance of a normal ventilation–perfusion ratio is the relationship between FRC and closing capacity (CC). CC is the lung volume at which small airway closure occurs during expiration. Conditions that decrease FRC below CC or increase CC above FRC result in a maldistribution of ventilation/perfusion and adversely affect the mechanics of breathing and ventilation . In the VT) that is adequate for CO2 removal. The PaCO2 is directly related to the body's production of CO2 during the catabolism of fats and carbohydrates and inversely related to alveolar ventilation. In most clinical circumstances, the control of PaCO2 will rely on alterations in the minute ventilation; however, some control of the body's endogenous CO2 production is possible through the increase in the use of fats versus carbohydrates for nutrition or by the control of body temperature. As the respiratory quotient (CO2 production/O2 consumption) for carbohydrates is 1 versus 0.7 for fats, an increased reliance on fats to provide caloric requirements can be used to limit endogenous CO2 production and thereby minimize ventilatory requirements. Furthermore, prevention of hypothermia and even induction of mild hypothermia (35°C) can also be used clinically to control hypercarbia and limit mechanical ventilatory requirements. It must also be stressed that in patients with severe lung disease, ventilation to normocarbia is not necessary and may in fact be harmful. Current practice includes the use of permissive hypercarbia or allowing the PaCO2 to increase provided the pH is kept above 7.25. This strategy has been shown to improve outcome in patients with ARDS.[Effective mechanical ventilation also provides minute ventilation, calculated as the respiratory rate (RR) times the tidal volume that occurs with cardiac arrest, a decrease in cardiac output, or pulmonary embolism. The measurement of physiologic deadspace can be performed using Bohr's method. This is based on the principle that all exhaled CO2 comes from alveoli that are perfused since deadspace does not receive pulmonary perfusion and is therefore devoid of CO2. The Bohr equation states: VD/VT = (PaCO2 – PECO2)/PaCO2 where VD is the deadspace ventilation, VT the tidal volume, and PECO2 the partial pressure of CO2 in mixed expired gas.Although minute ventilation is defined as RR times VT, the entire VT is not involved in the effective gas exchanged. The part of the VT that does not participate in gas exchange is referred to as physiologic deadspace. Total or physiologic deadspace is composed of anatomic deadspace (the area of the conducting areas or the trachea and bronchi that do not participate in gas exchange) and alveolar deadspace . In the healthy state, the alveolar deadspace is minimal so that anatomic and physiologic deadspaces are approximately the same. Although anatomic deadspace, representing approximately 30% of a normal tidal breath or 150 mL in an average-sized adult, does not generally change regardless of the disease process, alveolar deadspace may change significantly in patients with pulmonary parenchymal disease, pulmonary vascular disease, or with changes in cardiac output resulting in alterations in pulmonary perfusion. The latter principle is clearly demonstrated by the abrupt decline in end-tidal CO2 measurements and the following equation:Since the anatomic deadspace (VD) is relatively constant in patients with healthy lungs, increasing the VT decreases the ratio of VD to VT. In effect, the VT increases alveolar ventilation. In patients with intrinsic lung disease undergoing mechanical ventilation, there is ventilation of poorly perfused regions of the lungs . In this setting, increases in VT may not decrease VD/VT since higher alveolar pressures due to larger VT may result in a further decrease in pulmonary perfusion and an increase in alveolar VD. An estimation of the effect of VT changes on VD/VT in such clinical scenarios can be provided by estimating VD/VT using capnography with ETCO2 – PETCO2)/PaCO2 In summary, it can be determined that a change in the metabolic rate with an alteration in CO2 production, a change in minute ventilation (RR or VT), or a change in VD may affect PaCO2.VD/VT = were large tanks into which the patient's entire body was placed . The patAlthough not in common clinical use today, these devices hold a place in our medical history as the first artificial ventilators used on a widespread scale for patients with respiratory failure. However, the idea of negative pressure ventilation is not dead; cuirass or vests that fit over the patient's thorax and are sealed at the waist and neck are occasionally used for the treatment of acute or chronic respiratory failure in infants and children. These deet al. demonstrated that the switch from positive pressure to negative pressure ventilation resulted in an immediate mean increase in pulmonary blood flow of 42% and a total increase of 54%.[The advantages of negative pressure ventilation devices are that they do not require endotracheal intubation, can be applied intermittently, can be used at home without the need for tracheostomy, and the interpleural pressure decreases from the beginning to the end of inspiration . An increase in interpleural pressure can decrease venous return (preload) and cardiac output. With a decrease in interpleural pressure during negative pressure ventilation, venous return and cardiac output increase thereby matching the phasic changes that occur in these variables during normal spontaneous ventilation. While most patients tolerate the negative cardiovascular effects of positive pressure ventilation without a clinically significant effect, positive pressure ventilation may have adverse clinical effects in specific populations. One such group is children following cavopulmonary anastomoses (Fontan procedures). In these patients, the anatomical presentation of a single ventricle mandates the re-routing of blood directly from the venous circulation (superior and inferior venacavae) to the pulmonary circulation to avoid chronic long-term volume overload, which would result if the single ventricle were to pump to both the circulations. In the performance of these procedures, the (SVC) Superior Vena Cava; and subsequently, the (IVC) Inferior Vena Cava are directly anastomosed to the pulmonary artery. In the postoperative setting (both acute and long-term), positive pressure ventilation by decreasing venous return can significantly decrease pulmonary blood flow which is dependent on the passive flow of blood from the venous to the pulmonary circulation. Although current clinical practice is to attempt early tracheal extubation, negative pressure ventilation using a cuirass around the patients has been evaluated in the immediate postoperative period following cavopulmonary (Fontan) anastamosis. Shekerdemian e of 54%. The imprThe era of positive pressure mechanical ventilation began with controlled mandatory ventilation (CMV) which provided intermittent positive pressure breaths to the patient without the ability to sense the patient's own respiratory efforts, with no gas flow in between the ventilator breaths, and has no means to allow the patient to breathe spontaneously thereby resulting in significant patient–ventilator asynchrony unless deep levels of sedation or neuromuscular blockade were used. An additional issue with CMV was the recognition that controlled ventilation in the absence of spontaneous breathing rapidly leads to atrophy of respiratory muscles. CMV was 2 O) within the breathing circuit of the ventilator system. Setting this value at too large a value (more than –3 cm H2O) may lead to failure to sense the patient's own spontaneous breath thereby leading to breathing without assistance resulting in an increased WOB (see below). Setting this value too low or making it too sensitive can lead to autocycling of the ventilator due to pressure changes caused by cardiac oscillations or a leak around an uncuffed ETT. In the latter scenario, as there is a leak around the tube, there is a loss of the PEEP and therefore a drop in the airway pressure, which may be interpreted by the ventilator as the patient's inspiratory effort. Given these issues, some adjustments of the sensitivity, based on the patient's characteristics, may be necessary.Given these issues, strict IMV is not used in clinical practice and is having been replaced by modes such as assist control (AC) and synchronized intermittent mandatory ventilation (SIMV) which attempt to deliver a ventilator breath that is coordinated with the patient's own inspiratory effort. Alternatively, a breath is delivered if the patient does not initiate a breath within a preset period of time. To accomplish this, the ventilator must have a way of sensing the patient's own inspiratory effort. In most clinical circumstances, this is accomplished by sensing a pressure change in airway pressure the PEEP. The limit variable (pressure or volume) will give the name to the type of ventilation chosen: pressure-limited or volume-limited.When the decision has been made to initiate mechanical ventilation, the clinician will initially have to take the following decisions: (a) the mode of ventilation such as AC, SIMV, or pressure-regulated volume-controlled (PRVC); (b) the limit variable (pressure or volume) which will control the tidal breath and its magnitude (set as either a pressure above PEEP or a specific VT); (c) the inspiratory time; (d) the ventilator rate (breaths/minute); (e) the FiOBreathing during mechanical ventilation can be controlled, assisted, supported, or spontaneous. Controlled breaths are provided regardless of the patient's respiratory efforts , whereas assisted and supported breaths are synchronized with the patient's own respiratory effort. Spontaneous breaths occur without ventilator assistance and since they impose the WOB, they are generally not allowed in the modern era of mechanical ventilation. Instead, when the SIMV mode is used, spontaneous breaths are frequently supported with either pressure or volume support (see below).2 should be maintained within the normal range; however, there are many factors other than central control of ventilation which may control the RR so that tachypnea resulting in hypocarbia may occur in sepsis, central nervous system disorders, pain, and agitation. Additionally, although AC ventilation ensures support with every breath thereby limiting the WOB, when the decision is made to wean the ventilatory support, the volume or pressure of the tidal breath must be weaned and not necessarily the rate. With the AC mode of ventilation, if the rate is set at 20 breaths/minute and the patient is breathing at 30 breaths/minute, decreasing the rate to 15 breaths/minute will have an impact neither on the amount of support provided to the patient nor on the minute ventilation.When considering assisted ventilation, the basic modes of ventilation include AC, SIMV, and PRCV. With AC, the ventilator delivers full support (either pressure or volume) every time the patient initiates a breath. If the patient fails to breathe, the ventilator will deliver a fixed number of breaths per minute according to the preset rate. The theoretical advantage of AC ventilation is that every patient initiated breath is supported and the patient determines the RR. With an intact central control of respiration, the PaCOWith SIMV, a set number of breaths per minute are synchronized with the patient's respiratory effort and the full support (pressure or volume) is delivered. If the patient breathes above the preset number of breaths each minute, there will be additional minute ventilation from this spontaneous ventilation, but there will be no added support with these breaths if SIMV is used alone. As the WOB during mechanical ventilation may be related to the resistance of the ETT , the circuit, and the ventilator, pressure or volume support may be added to augment the spontaneous breaths. Therefore, SIMV with pressure support (PS) is a frequently used mode of ventilation.2 O from the previous breath to deliver the selected VT. Thus, the ventilator is continuously adapting the inspiratory pressure to changes in the resistance and compliance of the patient's respiratory system. The theory behind this mode of ventilation is that it delivers a fixed VT (as opposed to the variable VT that occurs with pressure-limited modes of ventilation) (see below). Since it works within the confines of a preset pressure limit, PRVC may limit the incidence of barotrauma that may occur when volume-limited modes of ventilation are used. To date, there are limited data demonstrating the superiority of AC, SIMV, or PRVC modes of ventilation.PRVC is a more recently introduced mode of ventilation, which can be provided by the newest generation of mechanical ventilators. It combines the features of both volume- and pressure-limited ventilation. Like AC modes, this mode will deliver the entire tidal breath every time the patient initiates a breath. This mode utilizes a decelerating inspiratory flow waveform (like pressure-limited ventilation on the Servo 300 and 900C ventilators) to deliver a preset VT over the inspiratory time in a pressure-limited manner. During the delivery of the tidal breath, respiratory system compliance and resistance are monitored and an algorithm is devised to deliver the VT while limiting the PIP. The ventilator regulates the inspiratory pressure up or down by as much as 3 cm HThe limit variable (pressure or volume) is that parameter which is set to determine the magnitude of the tidal breath. The type of ventilation we have chosen is named according to the limit variable: pressure-limited (pressure-control by some) or volume-limited (volume-control). In the early days of IMV, volume was generally the limit variable. A fixed VT was delivered over a fixed interval (inspiratory time) regardless of the PIP, provided that the high pressure limit was not exceeded. This in turn could lead to barotrauma.With volume-limited ventilation, a specific VT is set by the clinician and an inspiratory time is chosen. The flow provided is then integrated based on the tidal volume and inspiratory time. For example, if a VT of 500 mL with an inspiratory time of 1 second is chosen, 500 mL will be delivered over 1 second using a gas flow of 30 L/minute (500 mL/second = 30 L/minute). In general, flow is constant during the inspiratory cycle (square-wave flow pattern). When mechanical ventilation was first applied, a VT of 10–15 mL/kg was frequently used. However, more recent evidence has demonstrated that such a large VT may result in repetitive overdistention of alveoli with endothelial, epithelial, and basement membrane damage with increased microvascular permeability otherwise referred to as volutrauma.10 This volVolume-limited ventilation (in either the AC or SIMV modes) is best used in patients with normal resistance and compliance. For example, volume-limited ventilation is frequently used intraoperatively when patients with relatively normal pulmonary function receive endotracheal intubation and mechanical ventilation during surgical procedures. With poor compliance or high resistance, the higher PIP may lead to barotrauma and increased mortality in patients with ARDS and other types of pulmonary parenchymal disease. The adva2O) require an investigation which should start at the ventilator and work toward the patient including a check for kinking of the circuit or ETT, obstruction to the ETT or major airways by mucus (passing a suction catheter can frequently be used as both a diagnostic and therapeutic maneuver), auscultation to assess if there are bilateral breath sounds (to rule out mainstem intubation) and to rule out bronchospasm, a radiograph to evaluate increasing alveolar space disease (pneumonia or ARDS), or external factors impeding respiratory excursion . With volume-limited ventilation, manipulation of the inspiratory time can be used to decrease the PIP as increasing the inspiratory time decreases the inspiratory gas flow rate, thereby decreasing the PIP. However, longer inspiratory times (approaching I:E ratios of 1:1) may be relatively uncomfortable for the patient who is awake since the normal I:E ratio is 1:3 or 1:4. Additionally, reversing the I:E ratio may result in air trapping and auto-PEEP. If the peak airway pressure is unacceptably high, the pressure-limited mode may be chosen (see below for a full discussion of setting the inspiratory time).When volume-limited ventilation is used, the PIP should be monitored as changes in the PIP reflect changes in resistance and compliance of the respiratory system. High pressures . Because inspiratory pressure is the limiting variable, changes in respiratory system mechanics will result in changes in the delivered VT and minute ventilation. However, given that the PIP is controlled, the risk of barotraumas is lesser than with volume-limited ventilation. With specific types of the commonly used ICU ventilators , PCV is pressure-limited and time-cycled so that when the preset level of pressure is achieved, it is held for a preset time (inspiratory time), after which exhalation begins. With the newer generation of ventilators, regardless of whether pressure or volume ventilation is chosen, the type of flow waveform (square-wave or decelerating) can be chosen. Pressure-limited ventilation may be particularly beneficial in patients with decreased compliance related to high airway resistance or alveolar space disease such as pneumonia or ARDS.2 O and the PIP at 20 cm H2 O, the ∆P is 15 cm H2O. If the PEEP is increased to 7 cm H2O, the PIP will remain at 20 cm H2O, and therefore, the ∆P decreases to 13 cm H2O. If the PEEP and ∆P are set, increasing the PEEP by 2 cm H2O will also increase the PIP by 2 cm H2O since the ∆P remains at 15 cm H2O.During pressure-limited ventilation, the delivered VT is determined by the pressure level above PEEP (sometimes referred to as the delta or ∆P), the inspiratory time, loss of VT from a leak around an uncuffed ETT, and the patient's resistance and compliance. The delta PEEP is adjusted to deliver the desired exhaled VT . Depending on the type and manufacturer of the ventilator, the ∆P is set by either setting a PIP (VIP Bird ventilator) or pressure above PEEP (Servo 900C or 300 ventilator). In the former circumstance, it should be noted that adjusting the PEEP will affect the pressure above PEEP (the ∆P), and therefore, the VT. For example, if the PEEP is set at 5 cm HApart from patients with decreased respiratory system compliance or high resistance, pressure-limited ventilation is also frequently used in neonates and infants. The delivery of a small ViT may be somewhat inaccurate based on the working parameters of the ventilator. A discrepancy of 10–20 mL in the delivered VT is not an issue when the set VT is 500 mL, but can be a significant issue when the set VT is 30–40 mL. An additional advantage of pressure-limited ventilation is the use of a decelerating flow pattern to deliver the tidal breath. Although newer ventilators allow the independent adjustment of the inspiratory flow pattern, the Servo 300 ventilator, a commonly used ventilator in the ICU population, uses a constant flow for volume-limited ventilation (square-wave pattern) and a decelerating flow for pressure-limited ventilation. The decelerating flow pattern may help in the recruitment of alveoli with long time constants (high resistance and low compliance), and thereby, over time improves the compliance. As such, it may be difficult to determine if the improved outcome of patients with ARDS who receive pressure- versus volume-limited ventilation is the result of the type of ventilation or the differences in the inspiratory flow patterns.As with volume-limited ventilation, an inspiratory time is set with pressure-limited ventilation. As most pressure ventilators actually time-cycle (end inspiration based on the inspiratory time) and do not pressure-cycle (end inspiratory when the preset pressure is achieved), increasing the inspiratory time will increase the mean airway pressure, and hence, the exhaled VT. This is in contrast to what occurs with volume-limited ventilation where extending the inspiratory time decreases the PIP, but does not affect VT.2O. Using the plateau pressure eliminates the resistance imposed by the ETT and the major conducting airways, thereby approximating the pressures that occur within the alveoli. The plateau pressure is measured by holding a breath at the end of inspiration . With a pause at the end of inspiration, the pressure within the circuit declines from the high level that occurs at the start of the breath to a baseline or plateau level.During pressure-limited ventilation, the exhaled VT should be monitored to assess the ongoing changes in the resistance and compliance of the respiratory system as opposed to monitoring the PIP during volume-limited ventilation. A decrease in the exhaled VT should prompt a thorough investigation into its cause that includes the same steps as outlined above for investigating an increase in PIP during volume-limited ventilation. In patients with severe lung disease, the goal of pressure-limited ventilation is to achieve a plateau pressure of less than 35 cm HSo far, we have discussed five basic types of ventilation including AC-pressure limited, AC-volume limited, SIMV-pressure limited, SIMV-volume limited, and PRVC ventilation. These are the five basic modes of mechanical ventilation used most commonly in the ICU today. Although most modern day ICU ventilators can provide all of these modes and options, older ventilators such as the Servo 900C cannot provide SIMV-pressure limited or PRVC ventilations. With the 900C, if pressure-limited ventilation is used, it can only be performed in the AC mode.The inspiratory time may be the most overlooked and underappreciated ventilator setting. Depending on the type of ventilation (pressure-limited or volume-limited), the effect of changing the inspiratory time has dramatically different effects. With pressure-limited ventilation, the delivery of the tidal breath is in actuality pressure-limited and time-cycled (the preset pressure or ∆P is held until the inspiratory time is completed). Extending the inspiration time will increase the VT. More importantly, the inspiratory time along with PEEP and PIP determines the mean airway pressure. Extending the inspiratory time increases the mean airway pressure and will thereby increase oxygenation. With volume-limited ventilation, extending the inspiratory time serves to decrease the inspiratory flow rate and thereby reduces the PIP. It can also be used as a therapeutic maneuver to help recruit alveoli with long time constants and to help the resolution of atelectasis.2.[With normal spontaneous ventilation, the I:E ratio is 1:3 or 1:4. The use of longer inspiratory times may be uncomfortable during spontaneous ventilation in the patient who is awake. With AC, SIMV, or PRVC ventilation, the ventilator determines the inspiratory time as opposed to supported breaths (pressure or volume support) where the patient sets the inspiratory time. In our practice, we frequently use inspiratory times of 0.3–0.5 second in infants and up to 0.7–1 second in adolescents. The use of inspiratory times that are somewhat longer than those that occur during normal tidal breathing may be helpful during acute illnesses when patients are prone to develop atelectasis while on mechanical ventilation due to lack of sighing (see below). The inspiratory time should also be adjusted based on the underlying disease process. Patients with bronchospasm and air trapping generally benefit from a shorter inspiratory time to allow for as much exhalation time as possible. Patients with alveolar space disease and poor compliance generally do better with longer inspiratory times to increase mean airway pressure and improve oxygenation. The inspiratory time can be increased up to 1.2–1.5 seconds as needed to increase the mean airway pressure and recruit alveoli, but our practice generally restricts the inspiratory time to limit the I:E at 1:1. Reversal of the I:E ratio has been used in the management of patients with severe ARDS in an attempt to augment oxygenation and allow weaning of the FiO2. Longer iSubtleties in adjusting the inspiratory time vary from one ventilator to the other. The inspiratory time may be set as a fixed time (seconds) by adjusting the inspiratory flow rate as an I:E ratio or as a percentage of the respiratory cycle. “Even when the same ventilator is used, there may even be differences in how the inspiratory time is set dependent on whether pressure or volume ventilation is used.” The inspiratory time of the VIP Bird ventilator is set by adjusting the flow rate during volume-limited ventilation and is set in seconds during pressure-limited ventilation. If the inspiratory time is set as an I:E ratio or as a percentage of the respiratory cycle, adjusting the rate will affect the actual inspiratory. For example, if the RR is set at 15 breaths/minute with an inspiratory time of 25%, it results in an inspiratory time of 1 second. Changing the rate to 20 breaths/minute with the same inspiratory time of 25% results in an inspiratory time of 0.75 seconds. Such changes can result in changes in the peak airway pressure during volume-limited ventilation or the VT during pressure-limited ventilation.Some ventilators allow the addition of an inspiratory pause. This time is added to the end of inspiration, so its contribution to the total inspiratory time must be realized in avoiding reversal of the I:E ratio. The inspiratory pause holds the inspiratory effort at the end of inspiration without the ongoing gas flow. This maneuver serves many of the same purposes as extending the inspiratory time during pressure-limited ventilation including the recruitment of alveoli with long time constants (high resistance and low compliance), promotion of collateral ventilation via pores of Cohn and canals of Lambert, reversal of atelectasis, and improved matching of ventilation and perfusion.2O) to avoid ventilation–perfusion inequalities. This mechanical inefficiency is avoided under dynamic conditions because ELV is greater than FRC secondary to a rapid RR with short expiratory times , laryngeal muscle contraction during exhalation impedes expiratory airflow (this does not occur with an ETT in place), and increased intercostal muscle tone that stabilizes the chest wall thereby increasing elastic recoil. Thus, sedated or intubated infants generally require delivery of physiologic levels of PEEP to overcome the loss of these dynamic compensatory mechanisms. Higher levels of PEEP may be required in patients with alveolar space disease, increased abdominal distention, and other pathologic conditions that increase CC and decrease FRC. PEEP increases the lung volume, restores FRC, and when applied in the proper amount, improves lung compliance so that a given change in pressure results in a greater VT.PEEP refers to positive end expiratory pressure applied during the provision of mechanical ventilation with an ETT. PEEP maintains the patency of injured lung units which may collapse during exhalation. Although physiologically accomplishing the same thing, it should be differentiated from continuous positive airway pressure (CPAP), which is applied during spontaneous ventilation. In normal adults, FRC and expiratory lung volume are equal and exceed the CC. Thus, healthy adolescents and adults require little or no PEEP to prevent atelectasis and its associated hypoxemia from occurring. In contrast, newborns with their highly compliant chest wall will have an FRC that approaches and in some cases may be less than CC under passive conditions, thereby leading to the concept of physiologic PEEP , the curve will be sigmoidal in shape with a marked increase in the volume achieved with small changes in pressure initially noted at a lower plateau pressure (lower inflection point) and then a decrease in these volumes at a higher plateau pressure (upper inflection point).[Several different methods of determining the optimal PEEP have been suggested including obtaining a chest radiograph with an evaluation of the expansion of the lung fields, increasing the PEEP to allow for an FiOn point).15 Some cIn most instances, a change in PEEP is the first step used for regulating mean airway pressure in patients with lung disease thereby moving to the steep portion of the pressure–volume curve and restoring normal compliance of the respiratory system. The application of PEEP prevents airway pressure from dropping below critical closing pressure , redistributes pulmonary edema fluid from alveoli to the interstitium, maintains alveolar surfactant activity, and improves ventilation to low V/Q lung units.17 Excess2 of 1.0. Depending on the severity of the underlying lung injury, this can generally be rapidly weaned according to the oxygen saturation on the pulse oximeter without the need for arterial blood gas analysis. The risk of toxic effects of oxygen is minimized by using the lowest FiO2 which results in an oxygen saturation of 90% or a PaO2 of 60 mmHg. In patients who cannot be weaned to an FiO2 less than 0.5–0.6, other maneuvers to increase oxygenation (increasing the mean airway pressure by increasing PEEP or inspiratory time) should be attempted. Additionally, accepting lower oxygen saturations (80–90%) may be acceptable in patients with severe lung injury. With the use of sedatives, judicious transfusion to increase hemoglobin levels, and control of peripheral oxygen consumption, adequate oxygen delivery to meet tissue can usually be maintained with an oxygen saturation of 80%.The final two decisions regarding ventilatory parameters are somewhat self-explanatory. Immediately before and for a brief period following endotracheal intubation, most patients are ventilated with an FiO2, and the VT that is delivered. In patients with severe lung injury, higher rates are used to compensate for the lower VT thereby limiting ventilator-induced lung injury. Guidelines for starting ranges of RRs include 10–12 breaths/minute for adults or adolescents, 12–16 breaths/minute for older children (6–10 years of age), 16–24 breaths/minute for toddlers, and 24–30 breaths/minute for neonates and infants. Higher rates may be needed in patients with more severe degrees of lung injury, when hyperventilation is used to treat increased intracranial pressure or pulmonary hypertension, or if endogenous CO2 production is elevated. However, ventilation to normocapnia is not always required, as modern therapy for ARDS and other types of acute lung injuries employs permissive hypercapnia where hypercarbia is allowed provided the pH is greater than 7.25.The ventilator rate is set primarily based on the patient's age, the desired PaCOThe pressure–volume (compliance) and pressure-flow (resistance) characteristics of the respiratory system determine the WOB. Physiologic factors including the impendence from the elastic recoil of the lung and chest wall and the frictional resistance to gas flow in the airways combined with mechanical factors can both further contribute to the WOB. Various disease processes through either a decrease in respiratory compliance and/or an increase in respiratory resistance (bronchospasm or upper airway lesions) can also increase the WOB.Supported ventilation is defined as a breath that is triggered by the patient, limited by the ventilator (volume or pressure), and cycled by the patient (the patient determines the inspiratory time). It is used with SIMV to support spontaneous breaths that occur between ventilator breaths and thereby limits WOB. It can also be used with controlled ventilation as a means of weaning patients from mechanical ventilation. Ventilation is spontaneous in nature because the patient determines the ventilatory pattern by initiating and terminating each breath. Therefore, supported ventilation is only used in patients with an intact ventilatory drive. With this form of ventilation, the patient provides the work to trigger the breath and then interacts with the ventilator to perform a variable amount of the remaining work with each breath.Pressure support ventilation (PSV) is a mode in which the patient triggers the ventilator to deliver a flow of gas sufficient to provide a preset pressure level. The breath is terminated when inspiratory flow decreases to a percentage of its peak value rather than by volume, pressure, or time. At that point, the exhalation valve closes to pressurize the circuit to the predetermined expiratory limit (PEEP). Therefore, the patient retains control of the cycle length and flow characteristics. The VT is determined by the patient's inspiratory effort, the preset pressure limit, and respiratory system impedance (resistance and compliance). PSV is used most commonly to compensate for the inspiratory work imposed by the ETT.19 PSV ca20Two different methods of weaning ventilation with PSV have been advocated. One approach involves setting the pressure limit high enough to achieve delivery of the typical mechanical tidal breaths (6–8 mL/kg) with no back-up SIMV rate and then gradually decreasing the pressure down to the minimum value needed to overcome the imposed work of the ETT and ventilator circuit prior to extubation. The second method involves the combined use of SIMV and PSV in which the pressure limit during PSV breaths is set to minimize the imposed work of the ETT and circuit only. The SIMV rate is then gradually decreased to 0–4, at which time the ETT is removed. Controlled studies have suggested that PSV weaning is more effective than SIMV weaning in adult patients who are difficult to liberate from mechanical ventilation.24 SimilaVolume support ventilation (VSV) is a newer mode of ventilation in which supported breaths are volume-limited while using a decelerating inspiratory flow that is flow-cycled as with PSV. With this mode of ventilation, the pressure assist is regulated to deliver the preset volume, provided a maximum pressure limit is not exceeded, with each supported breath. This mode has all of the theoretical benefits of PSV with the unique capability of providing a guaranteed minimum minute volume. To date, the experience with this mode of supported ventilation in children is limited.Appropriate weaning techniques are mandatory as complications related to mechanical ventilation and endotracheal intubation are generally dependent on the duration of support. For the 2 and transcutaneous CO2 monitoring). Once the rate has been weaned to 0–4 breaths/minute, the levels of PS and PEEP are also weaned. PS is weaned to a level that is thought to provide only enough support to overcome the WOB imposed by the ETT, the circuit, and the ventilator. No formal studies exist to demonstrate the exact level of PS required to achieve this goal; however, levels of 6–10 cm H2 0 are generally accepted as providing minimal extra support in the pediatric age group. Higher levels may provide additional support thereby suggesting that the patient can be removed from mechanical ventilation when in fact there is still a significant amount of support provided by the ventilator. When the patient has reached some arbitrary minimum SIMV rate , they are ready for a trial of extubation.[With a lack of formal studies to demonstrate the superiority of one technique over another, there are several methods of weaning patients from mechanical ventilation. In the pediatric population, SIMV remains a commonly used method for weaning of mechanical ventilatory support. This mode of ventilation allows for a gradual decrease in the amount of minute ventilation delivered by the ventilator by slowly decreasing the set RR as tolerated by the patient. In general, the frequency of positive pressure breaths is decreased in 2–4 breaths/minute increments followed by an assessment of the patient's status. This is frequently done by clinical evaluation by following the patient's RR, oxygen requirement, and evaluating signs of respiratory distress. The clinical assessment is frequently supplemented by the use of noninvasive measures of oxygenation and ventilatory status must be weaned. The AC mode may be beneficial in neonates and infants who have difficulty triggering the PS mode of some ventilators. The baseline amount of support that is acceptable prior to attempted extubation should be an amount estimated to provide only enough support to overcome the WOB imposed by the ETT and ventilator. As with PS ventilation, there are limited data in children to determine the exact level of support needed to compensate for the WOB added by the ETT and the ventilator.The ability to implement and provide mechanical support to patients with respiratory failure may be the single most important maneuver ever added to the ICU armamentarium. Although this technology started with the introduction of endotracheal intubation in the operating room, the more recent advances have centered on the design and function of modern day mechanical ventilators as well as an improved understanding of ventilator-induced lung injury. Prospective clinical studies have delineated important techniques that may improve survival and limit morbidity in patients with acute lung injury. Despite these advances, patients may fail conventional mechanical ventilation and require other modalities of support including high-frequency ventilation or extracorporeal support.

Tumours in hamsters, induced by the chicken-embryo-lethal-orphan (CELO) virus, by tumour tissue transplants, or by tumour cells grown in culture, were well circumscribed solid tumours and covered by a thin capsule-like structure. All were fibrosarcomata. However, tumours produced by the 3 inocula exhibited the following histological differences. Neoplasms induced by CELO virus were generally less differentiated and were composed of cells with polygonal or oval nuclei and indistinct cytoplasmic boundaries. Numerous multinucleated bizarre giant cells were found. Those produced by tumour tissue transplants were more differentiated and were composed of spindle shaped cells with abundant collagen fibre formation. Neoplasms induced by tumour cells grown in culture were generally undifferentiated with many mitotic figures and contained numerous giant cells.in vitro. The cell cultures consisted of large cells with oval or rounded large nuclei and prominent nucleoli. Multinucleated giant cells, cells in mitosis, and a disorganized growth pattern were also characteristic of the cell cultures. However, mitosis and a piling-up of cells occurred more frequently with cell cultures derived from the CELO virus-induced tumour.Cells from tumours induced by CELO virus or tumour transplants produced similar morphologies when cultured

Mild knee pain is a common symptom in later life. Despite this fact, there are few data on the impact of it worsening or how individuals alter their appraisals and behavior when it becomes severe. We sought to describe the changes that accompany a substantial deterioration in characteristic knee pain. A nested case-control analysis of existing cohort data identified 57 adults aged over 50 years experiencing progression from mild to severe characteristic pain intensity 18 months later and compared them, before and after this transition, with 228 controls whose knee pain did not progress. Worsening knee pain was accompanied by a marked increase in pain frequency and extent, functional limitation, depressive symptoms, catastrophising, praying and hoping, and use of oral and topical analgesia. Most individuals consulted a general practitioner either during or after this episode. Although relatively rare, substantial deterioration in knee pain has a major impact on those affected. Timely presentation to primary care, addressing potentially unhelpful appraisals and coping strategies, reinforcing core nonpharmacological management, and future research to identify triggering events for substantial deterioration and loss of adequate pain control should be part of an agenda to improve care for this important minority of older adults with knee pain.This article describes what happens when the common symptom of mild knee pain in later life becomes significantly worse. The results may help clinicians understand the health impact, changes in patient appraisal and coping, and treatments that typically accompany this change in symptoms. Knee pain is the most common pain complaint presented to the general practitioner by older adults.Early work identified the importance of self-treatment and “no-action decisions”To investigate this question, we conducted nested case-control analyses using data from a 3-year prospective, population-based observational cohort study of older adults with knee pain. We were interested in a particular transition: The change from mild to severe characteristic pain. Our hypothesis was that substantial worsening of knee pain would trigger the higher use of all coping strategies, pharmacological and nonpharmacological treatments, and general practice consultation compared with controls. Furthermore, prior to substantial worsening, when both cases and controls had comparable levels of mild knee pain, no differences in the above variables would be observed between cases and controls.We conducted a nested case-control analysis, sampling cases and controls from an existing population-based prospective observational cohort study; the Clinical Assessment Study of the Knee, abbreviated as CAS(K).The CAS(K) cohort comprises 819 individuals with knee pain, aged 50 years and over, registered with 3 general practices . Cohort members were recruited between August 2002 and September 2003 from a 2-phase postal survey. Respondents providing written informed consent to further contact attended a research clinic that included a standardized clinical interview, examination, and plain radiographs. Participants were sent a postal follow-up questionnaire 18 months and 3 years after study entry. All participants provided written informed consent to take part in the study. 742 (90.6%) participants provided additional written informed consent specifically to review their general practice medical records. The study was approved by North Staffordshire Local Research Ethics Committee. Full details of the study design and methods have been previously presented.Information was gathered by self-complete questionnaires at study entry, 18-month follow-up, and 3-year follow-up.Measures that were repeated at each time point included: Characteristic pain intensity measured using items from the Chronic Pain GradeCoping and appraisal was gathered using the 1-item Coping Strategies QuestionnaireTo identify all knee-related GP consultations, a review of the general practice consultation records from baseline to 3 years was undertaken for all participants who specifically provided written informed consent to accessing their medical records. Doctors at the practices routinely code and enter details of all patient consultations on computer. Individual problems are coded separately during each consultation. The participating practices are members of the Keele GP Research Partnership and the completeness of their coding of consultations is subject to annual quality review.We were interested in the transition from mild pain to severe pain and attempted to omit very short-term fluctuations in pain intensity. Substantial deterioration of knee pain was therefore defined as a change in characteristic pain intensity from <50/100 (‘mild’) to ≥70/100 (‘severe’) between 2 consecutive reports 18 months apart. The cut-offs for characteristic pain intensity were selected to be consistent with those suggested for 11-point pain numerical-rating scales who had mild knee pain at study entry which had become severe at 18-month follow-up and a second (Set 2) who had mild knee pain at 18-month follow-up which had become severe by 36-month follow-up. These 2 groups together formed the total “case” population for this analysis.We identified a control group from the cohort population for each case set (control: cases = 4:1). Controls for case Set 1 were a random sample of participants who had mild knee pain at study entry which was not severe at 18-month follow-up (characteristic pain intensity <70 out of 100). Controls for case Set 2 were a random sample of participants who had mild knee pain at 18 months which was not severe at 36 months. The second set of controls was selected after removal of the first set of controls (ie selection without replacement). Each set of cases and controls was mutually exclusive (ie no participant could appear in more than 1 group).0 represented the time at which both cases and controls had mild pain. T1 was the time 18 months later when cases were reporting severe pain and controls were reporting nonsevere pain. We refer to the period between T0 and T1 hereafter as the “index period”. The selection of cases and controls and timing of measures is represented in For the main analyses the 2 sets of cases were combined and compared to the combined control groups. TThe sociodemographic and clinical-history characteristics of cases and controls were described. Between-group differences between cases and controls were calculated at the beginning and at the end of the index period. Results for dichotomous data were expressed as percentage differences with 95% confidence intervals (95%CI). For numerical data, ie number of pain areas, WOMAC-PF, HAD anxiety and depression scores, the mean and standard deviation at each time point was calculated and between-group differences expressed as mean differences with 95%CI. The 95%CI expresses the precision of the estimate of mean between-group difference obtained from the sample and is more informative than a simple p-value.We conducted 2 additional separate subgroup analyses based on only 1 set of cases and controls. Set 1 (in which cases and controls were selected from baseline and 18-month data) provided information on pain experience 18 months after the index period. Set 2 (in which cases and controls were selected from 18- and 36-month data) provided information on pain experience 18 months before the index period.After removing 16 participants with an existing diagnosis of inflammatory disease at study entry (n = 16), 433 participants were eligible for Set 1, having reported mild knee pain at study entry and provided pain-intensity data at 18-month follow-up. Of these, 28 (6.2%) had severe knee pain at follow-up and qualified as cases. To act as controls for these cases, 112 participants were randomly selected from the remaining 405 participants. For Set 2, 361 participants reported mild knee pain at 18-month follow-up and provided pain-intensity data at 36-month follow-up. Of these, 29 (7.8%) had severe knee pain at follow-up. From the remaining 332 participants, 116 were randomly selected to act as controls for this second set of cases.Cases and controls were similar with respect to age, total time since onset of knee problem, and previous knee surgery . There wSubstantial deterioration in knee-pain intensity was associated with an increase in pain persistence, night pain, pain extent, functional limitation, and depressive symptoms during the index period .1) were also apparent at the start of the index period (T0) when both cases and controls had mild characteristic-pain intensity .Between-group differences in catastrophising, and praying and hoping, were present at the start of the index period although, owing to the noncollection of CSQ data at cohort-study entry, this is based on 1 set of cases and controls only and was statistically nonsignificant.There were no between-group differences either at the start or the end of the index period in the reported use of distraction, reinterpreting pain sensation, ignoring pain, or increased behavioral activities.Overall, the presence of any of the 7 coping strategies was similar at the start of the index period but was significantly higher in cases compared to controls at the end of the index period.Substantial deterioration in knee pain was associated with a marked increase in the use of oral and topical analgesia, particularly oral opioids . There wForty-six percent of cases had a record of visiting their GP about their knee problem during the time when substantial deterioration had taken place. Only 1 in 5 controls had visited their GP. This difference between cases and controls persisted after the index period. In total, 82% of cases had a record of consulting their GP about their knee problem either during the index period or in the 18-month period following substantial deterioration. This compares with 24% of controls.0) , had significantly higher pain intensity in the 18 months prior to the index period, and were already engaged in more efforts to control their pain (eg opioid intake). Together these findings suggest that substantial worsening may often be 1 phase in a history of fluctuating pain. Mild characteristic pain for controls may represent the true underlying severity of their problem: For cases, it may indicate a period where pain, usually more severe, was successfully, but temporarily, under control. Substantial deterioration may represent the loss of effective pain control.The proportion of individuals reporting substantial worsening was consistent with previous surveys of chronic-pain populations using similar repeated measures of pain intensity.Our findings extend previous cross-sectional studies that have described differences in coping and treatments between individuals with mild and severe pain by demonstrating that the transition from mild to severe knee pain is accompanied by certain changes in coping and treatment within those affected. Catastrophising was not the focus of our study but our findings appear to confirm its importance in older adults with knee pain and OAOur findings that neither anxiety nor self-rated health appeared to be affected by a substantial deterioration in knee-pain severity were unexpected. There is strong evidence for a cross-sectional association between anxiety disorders and arthritis and other painful conditions,There is continuing interest in defining, describing, and explaining clinical important change. Most previous research focuses on improvement following therapeutic intervention.The number of cases was relatively small and our findings, particularly those from the subgroup analyses, should be interpreted with caution and regarded principally as exploratory rather than hypothesis-generating. For this reason, we have confined ourselves to calculating unadjusted mean between-group differences and an estimate of the precision of these rather than p values. If we accept that the transition from mild to severe pain is an important one to investigate, our study suggests that much larger sample sizes would be required given the relative infrequency of this large change in symptoms in members of the general population. Caution is needed also given the number of comparisons made in the present analyses raising the prospect of Type I errors and the fact that our unadjusted analyses could not control for any baseline differences.One disadvantage of attempting to cover a very broad range of domains within the same study was the need to use brief questionnaires or single items for some domains to minimize respondent burden. Coping and appraisal, for example, was based on the 1-item CSQ, with items dichotomized. Such data reduction was felt to be necessary given the highly-skewed distribution although the effect of this is that increases or decreases in the frequency of use of different coping strategies could not be directly quantified. The 2-item CSQ has recently been validated in older adultsThe precise temporal sequence of changes in pain intensity and changes in coping observed in this study cannot be fully disentangled as our primary analyses are based on 2 time points. We have referred to many of the changes accompanying substantial deterioration in pain intensity as “effects”. This assumption of temporal sequence is reasonable for treatment use but may not be true of other observed changes (eg worsening pain may be an effect of increased catastrophising).Our study used between-group differences to represent the “average effects” of a substantial worsening in characteristic knee-pain intensity. Average effects can still mask differences between individuals in how they respond to substantial worsening of pain. Our study indicates what tends to happen when mild knee pain becomes severe. Idiographic within-person daily process studies have investigated sequential day-to-day relationships between pain, mood, and coping at the level of individuals,Our study is descriptive but serves to illustrate the process of substantial deterioration that may precede presentation to the general practitioner. Low mood, catastrophising, and praying and hoping for the pain to go away may be important features of the presenting problem in primary care and greater efforts are needed in translating principles of cognitive-behavioral approaches into practical interventions in this setting. Understanding why some individuals experience relatively rapid and significant deterioration in pain is a priority for future research, although the notion of a loss of adequate pain control must be considered alongside the search for distinct triggering events.Improving the uptake of core nonpharmacological treatments is a priority for clinical practice. Current guidelines and recommendations emphasize the importance of weight loss and exercise therapy for all patients with knee OA.

Considering the fact that for most photosensitizers only monotonic dose-response relationships have been reported, this result was unexpected. The present studies were, therefore, undertaken to further investigate the concentration dependent photodynamic effects of AlPcS2.Photodynamic therapy (PDT) involves excitation of sensitizer molecules by visible light in the presence of molecular oxygen, thereby generating reactive oxygen species (ROS) through electron/energy transfer processes. The ROS, thus produced can cause damage to both the structure and the function of the cellular constituents resulting in cell death. Our preliminary investigations of dose-response relationships in a human glioma cell line (BMG-1) showed that disulphonated aluminum phthalocyanine (AlPcS2 were investigated in BMG-1 cells by absorbance and fluorescence measurements, image analysis, cell counting and colony forming assays, flow cytometry and micronuclei formation respectively.Concentration-dependent cellular uptake, sub-cellular localization, proliferation and photodynamic effects of AlPcS2 concentrations was observed to be biphasic. AlPcS2 was distributed throughout the cytoplasm with intense fluorescence in the perinuclear regions at a concentration of 1 μM, while a weak diffuse fluorescence was observed at higher concentrations. A concentration-dependent decrease in cell proliferation with accumulation of cells in G2+M phase was observed after PDT. The response of clonogenic survival after AlPcS2-PDT was non-monotonic with respect to AlPcS2 concentration.The cellular uptake as a function of extra-cellular AlPcSBased on the results we conclude that concentration-dependent changes in physico-chemical properties of sensitizer such as aggregation may influence intracellular transport and localization of photosensitizer. Consequent modifications in the photodynamic induction of lesions and their repair leading to different modes of cell death may contribute to the observed non-linear effects. Considering the fact that for most photosensitizers only monotonic dose-response relationships have been reported [2 and to gain insight into the mechanisms underlying these effects.Photodynamic therapy (PDT) involves excitation of sensitizer molecules by visible light in the presence of molecular oxygen, thereby generating reactive oxygen species (ROS) through electron/energy transfer processes. The reactive oxygen species, such as singlet oxygen and hydroxyl radicals thus produced can cause damage to both the structure and the function of the cellular constituents resulting in cell death. Photodynamic effects resulting either in apoptotic, mitotic and/or necrotic cell death depend on the nature of the photosensitizer, cell type and the cellular targets for photosensitization, concentration and intracellular localization of the sensitizer ,2, the itoxicity ,9,10,12.reported , this reHuman cerebral glioma cell line , established from a mixed glioma was used3 cells/cm2 in tissue culture flasks to maintain the cells in the exponential phase. All experiments were carried out with exponentially growing cells.Monolayer BMG-1 cells were grown in DMEM with 5% fetal calf serum (FCS), penicillin (100 units/mL), streptomycin (50 μg/mL) and nystatin (2 μg/mL). Stock cultures were passaged every third day after harvesting the cells with 0.05% trypsin and seeding 8 × 102) was prepared and characterized in INMAS, Delhi and consisted of a mixture of isomers with sulphonic groups in both adjacent and opposite positions [Disulphonated aluminum phthalocyanine , SRL, and E-Merck, India.2 (1-10 μM) in HBSS at 37°C. After incubation of cells in HBSS, both the absorption and fluorescence spectra of cells and supernatant before and after washing were obtained independently . Cellular uptake was calculated using standard calibration curves of photosensitizer in HBSS.Cells were trypsinized, counted and incubated in dark for various time intervals with various concentrations of AlPcS2 was studied by fluorescence microscopy using image analysis system equipped with a monochrome CCD camera .Intracellular localization of AlPcS2, cover-slips were washed in PBS, mounted on slides and examined under the fluorescence microscope using UV excitation filter (300-400 nm) and emission recorded in 400-800 nm region of the spectrum. Images were acquired and stored in digital computer (166 MHz) and analyzed using the software provided by Optimas Corporation, USA.Cells were grown on cover-slips for these studies. After incubation with AlPcSCytoplasmic and nuclear localization of the sensitizer was estimated by analyzing the images using area morphometry by marking the appropriate regions of interest (ROI). For uptake measurements also, area morphometry that provides the average amount of the photosensitizer in the whole selected area, was used .2 (0.25-10 μM). Post-incubation, cells were washed with HBSS and exposed to red light (Power = 3 W/cm2) from a high power (1000 W) Xenon arc lamp , using an optical filter (cut off at 610 nm) with the petridishes placed on ice. Optical power at the cell surface was measured using radiometer having a detector head (SL021/FQ) with a flat response between spectral range 400-1000 nm. The cells were euoxic with oxygen levels provided by dissolved oxygen in the media. Cells were incubated for further 2 h at 37°C in HBSS before assay of cell response to treatment.Cells growing as adherent monolayer cultures were incubated in HBSS at 37°C for 2 h with varying concentrations of AlPcS2 (5%) atmosphere at 37°C for 8-10 days to allow colony formation. Colonies were fixed with methanol and stained with 1% crystal violet. Colonies having more than 50 cells were counted and plating efficiency (PE) and surviving fraction (S.F.) were calculated.Nearly 150 cells were plated in growth medium (DMEM + 10% FCS) after the treatment (as described above) and incubated in dark under humidified COAfter photodynamic treatment, attached monolayer cells were incubated in growth medium, harvested and counted (using hemocytometer) after varying intervals of time. Floating cells were collected separately before harvesting attached cells by trypsinization. Flow-cytometric measurements of cellular DNA contents were performed with the ethanol (70%) fixed cells using the intercalating DNA fluorochrome, propidium iodide (PI) as described earlier . MeasureAir-dried slides containing acetic acid-methanol (1:3 V/V) fixed cells were stained with 2-aminophenylindoledihydrochloride (DAPI) (10 μg/mL in citric acid (0.01 M), disodium phosphate (0.45 M) buffer containing 0.05% Tween-20 detergent) as described earlier . Slides m is the number of cells with micronuclei and Nt is the total number of cells analyzed. Since, micronuclei formation is linked to cell proliferation, the micronuclei frequencies were normalized with respect to the cell numbers [where N numbers .Detection and analysis of photodynamically induced apoptosis was performed by studying the morphological features, DNA content and changes in cell size, cytoskeleton structure associated with cells undergoing apoptosis.Morphologically, marked condensation and margination of chromatin, fragmentation of nuclei and cell shrinkage characterize apoptotic cells and a good correlation between these morphological changes and DNA ladder has been demonstrated . Slides 0/G1) population was taken to be indicative of the apoptotic cell population.Flow-cytometric measurements of cellular DNA content were performed with ethanol fixed cells. The presence of hypodiploid . Analysis of light scatter was performed by off-line gating using appropriate windows created with untreated cells.Relationship between surviving fraction and energy (KJ) was quantified by modeling the data with a univariate linear regression analysis with energy being an independent variable and surviving fraction as dependent variable. Overall differences of mean relative proliferation among different treatment groups as well as at each pre-specified hours were tested by using one-way analysis of variance (one-way ANOVA) with Bonferroni correction for pairwise group comparisons. For all the analysis, type-I error rate was set to 5% but multiple comparison was handled by using Bonferroni correction in which type-I error rate for pairwise group comparisons was set to 1.66%. A p-value of < 0.05 was considered statistically significant, if not stated otherwise due to Bonferroni correction for multiple comparisons. SAS v9.2 for windows was used for statistical analysis of the data.2 following incubation of cells with AlPcS2 in HBSS for different time intervals showed rapid and linear increase in the accumulation of AlPcS2 in the first 2 h, prolonged incubation (up to 24 h), however, did not result in any further increase in the uptake at higher concentrations as reported earlier in studies of cellular uptake in a human nasopharyngeal cancer cell line [Estimation of cellular content of AlPcSM Figure . This paell line .2 localized in the perinuclear region and no significant changes in the localization patterns were observed up to 4 h of incubation time (data not shown). Changes in localization as a function of concentration (1-10 μM) showed that AlPcS2 was distributed throughout the cytoplasm with intense fluorescence in the perinuclear regions up to a concentration of 2 μM, while a weak diffuse fluorescence was observed at higher concentrations did not compromise the survival.Responses to different doses of AlPcS2 during pre-incubation.Survival of glioma cells after damage induced by photo-irradiation in the presence of phthalocyanine was studied by the macrocolony assay, both as a function of light dose and concentration of AlPcS2 for 2 h in HBSS after PDT with increasing light doses up to 1800 J/cm2 . The relationship between surviving fraction and energy can be quantified by the following regression equation "Surviving Fraction = 1-0.0538*Energy". Based on the analysis, a linear decrease was observed in the clonogenic cell survival of cells pre-incubated at 1 μM AlPcS2 Figure .2 concentration dependent dose-response (performed at the light dose of 450 J/cm2) showed a linear decrease in survival up to a concentration of 1 μM , the surviving fraction did not decrease; instead a gradual increase was observed. At 10 μM, the survival was almost equal to the untreated cells , while at 5 μM even one population doubling could not be observed after 42 h post treatment at each pre-specified times i.e. 19, 30, 42 hours. By taking into account the Bonferroni correction for multiple comparison with pairwise type-I error rate as 1.66%, there were no differences between 1 μM vs. control as well as between 5 μM vs. 1 μM at 19 hours and between 1 μM vs. control at 42 hours . All other pairwise treatment differences were significant at 1.66% , while no significant change was observed at 1 μM AlPcS2 (Table Frequency of non adherent and floating cells in the culture increased after AlPcS2 accumulated at both the concentrations (1 and 5 μM), the PDT-induced differences in the proliferation kinetics observed here, must arise from the concentration dependent differences in the patterns of sub-cellular distribution of the photosensitizer.It is pertinent to note that since almost equal amounts of cellular AlPcS0 fraction of cells indicating considerable DNA fragmentation (apoptotic and necrotic death) after PDT at higher concentration of AlPcS2, while at 1 μM AlPcS2, little differences as compared to untreated controls were observed. In contrast, a reduction in forward angle light scatter implying a reduction in the cell size (measured from 20-42 h after PDT) could be observed to a significant extent even at 1 μM AlPcS2 indicating the aggregation of AlPcS2 at higher concentrations. Present observations are in agreement with studies in V-79 cells where it has been shown that intracellular fluorescence intensity of various phthalocyanine derivatives vary with their aggregation capacity [AlPcS behaves like a typical amphiphile with charged substituents located at the membrane/buffer interface and the non-polar portion of the molecule in contact with the hydrophobic lipid chains . Such a r Figure . Many ofcapacity .2 was observed to be dependent on its extracellular concentrations. It was localized in a granular fashion throughout the cytoplasm with intense fluorescence in the perinuclear region at lower concentrations while at higher concentrations AlPcS2 fluorescence was weak and diffused could also be due to the intracellular presence of photodynamically inactive species like aggregates [2, the RFI monitored by whole cell spectroscopy at 10 μM AlPcS2 was many folds less than the RFI in HBSS and methanol indicating the aggregation of AlPcS2 at higher concentrations. These observations suggest that AlPcS2 was not aggregated in HBSS before uptake but was aggregated once it was taken up by the cells. The present results are similar to the observations made earlier in V79 cells, where cells incubated with 1 μM and 3 μM of AlPcS1 were more sensitive per quantum of fluorescence than the cells incubated with 10 μM indicating that all the sulphonated AlPc derivatives inside the cells are partly aggregated, the degree of aggregation being dependent on lipophilicity [44- and pAlPcS44- (isolated by HPLC fractionation) has also been reported to reduce with increasing concentrations of the sensitizer due to aggregation at the higher concentrations in a human nasopharyngeal cancer cell line (KB) [2 fluoresce and have a detectable triplet state and also involved in the production of singlet oxygen [The decrease in photodynamic cytotoxicity induced by AlPcSgregates . Althoughilicity . The phoine (KB) . It has t oxygen .2 at different concentrations could result in varying amounts of photobleaching leading to reduced production of ROS at high concentrations.High intracellular concentrations of the sensitizer may also result in an inner filtering of light contributing to the reduced photodynamic efficiency. Phthalocyanines have been shown to be highly efficient quenchers of singlet oxygen . It is p2-PDT on the macrocolony assay , and would, thus, include modifications induced by the late repair and death processes. Also stress-induced premature senescence (SIPS), after sub-lethal oxidative damage [It is intriguing that the effects of AlPcS Figures and 3c ae damage ,48, coul1-S transition) or mitotic division (G2 block) has been shown to enhance cell survival following damage caused by many physical and chemical agents [2+M block, observed at 5 μM Figure , while t2-PDT resulted in classical features of apoptosis viz. induction of sub G0/G1 population only at 5 μM AlPcS2 [2 distributed diffusely in the cytoplasm with intense perinuclear fluorescence, damage to cytoskeletal elements could be one of the factors triggering apoptosis. This could also contribute to reduction in initial rate of proliferation of cells pre-incubated at higher concentrations of AlPcS2. However, induction of G2-block to a greater extent may allow the remaining cells to recover from the potentially lethal lesions under these conditions and contribute to a higher clonogenic survival.Although, AlPcSith ZnPC . This moith ZnPC ,51. DeponPC [2+) . Role ofnPC [2+) ,53,54. S2-PDT efficacy under certain circumstances may not increase monotonically with the increase in photodynamic dose varied by changing the concentration of the photosensitizer. Based on the present results, we hypothesize that the non-monotonic photodynamic effects could arise due to multiple reasons including (a) concentration dependent changes in physico-chemical properties of AlPcS2 due to varying degrees of aggregation leading to different patterns of cellular transport and intracellular localization, (b) complex interactions between photobleaching and singlet oxygen quenching at high intracellular densities of AlPcS2 and its aggregates and (c) competitions between cellular proliferation, cellular repair/misrepair and cell death pathways following induction of photodynamic lesions. Detailed further studies are warranted to verify this hypothesis and to elucidate precise mechanisms underlying the phenomena observed in the present studies. Most importantly, these results strongly suggest that the therapeutic efficacy of PDT need not always be higher with higher PDT doses achieved either by large sensitizer and/or light doses. Further the in vivo responses are likely to be confounded by other factors related to tumor physiology as well as systemic effects. Therefore, predictive assays using appropriate in vitro models that better represent environmental factors prevailing in tumors will be helpful in designing most effective therapy for a given tumor.Results of the present investigations imply that the AlPcS2: disulphonated aluminum phthalocyanine; BMG-1: human glioma cell line; DAPI: 2-aminophenylindoledihydrochloride; HpD: hematoporphyrin derivative; PDT: photodynamic treatment; PI: propidium iodide; RFI: relative fluorescence intensity; ROI: region of interest; ROS: reactive oxygen species.AlPcSThe authors declare that they have no competing interests.SG carried out all the experiments, acquired, analyzed and interpreted the data and drafted the manuscript. BSD participated in the design of the study, made contributions to acquisition, analysis and interpretation of data and helped to draft the manuscript. KM helped in the interpretation of the data on uptake and localization of phthalocyanine and drafting of the manuscript. VJ conceived the study and participated in its design and coordination and helped in interpretation of data and drafting the manuscript. TKS made contribution to statistical analysis and interpretation of data and helped to draft the revised manuscript.The final manuscript is read and approved by all the authors.

Although patients with cleft lip and palate (CLP) are not seen regularly in general dental practice, this is a frequent congenital anomaly; approximately one in every 800 live births results in a CLP. The cause of CLP is unknown, but possible causes are malnutrition and irradiation during pregnancy, psychological stress, teratogenic agents, infectious agents (viruses), and inheritance. Most clefts are likely caused by multiple genetic and nongenetic factors. Prosthetic reconstruction of the anterior maxilla is important for these patients. This paper describes the prosthetic rehabilitation of two patients with CLP, 19-year-old and 21-year-old women, both with surgically treated CLP. In both, an examination revealed a residual palatal defect of 2 × 3 mm and missing maxillary lateral incisors. The 19-year-old was treated with a fiber-reinforced composite resin-bonded fixed partial denture. The 21-year-old was treated with a removable partial denture with an extracoronal attachment system. The prosthetic rehabilitation of the two patients with CLP was evaluated clinically. In both, well-planned prosthetic, periodontal, and surgical therapy resulted in satisfactory function and esthetics, alleviating their deformities. With education and appropriate recall, the patients should be able to maintain their oral health. Providing maxillofacial prosthetic treatment for patients with congenital and craniofacial defects not only should address physical and functional deficiencies but also should ideally evaluate the possible psychological effects of these deformities .Over the years, we have observed that patients with partial anodontia, cleft lip and palate, amelogenesis imperfecta, dentinogenesis imperfecta, ectodermal dysplasia, and neurological defects frequently have physical anomalies. These anomalies include, but are not limited to, decreased vertical dimensions of occlusion, decreased facial support, temporomandibular joint symptoms, lack of functional occlusion, altered speech, poor esthetics, teeth sensitivity due to abnormal wear and abrasion, lack of a normal smile line, and altered anatomy in the lower third of the face. These patients often require a combination of dental and medical specialists to improve these functional and esthetic problems. Maxillofacial prosthodontic treatment offers improvement in the appearance, function, and health of patients with congenital and craniofacial defects . Although patients with cleft palate may not be seen regularly in general dental practice, this is a frequent congenital anomaly; approximately one in every 800 live births results in a cleft lip and palate –4. The cCongenitally missing anterior teeth are common in cleft palate patients. In unilateral or bilateral clefts, the lateral incisors are the most frequently missing teeth, although the canines and central incisors may also be affected . When prA conventional fixed dental prosthesis can be used in the prosthetic treatment of a unilateral cleft and palate (UCLP) patient. This requires preparing at least one tooth on each side of the edentulous space and placing complete or partial metal-ceramic restorations . ConsequThis clinical report describes two alternative prosthetic treatments for two UCLP patients.We treated a 19-year-old woman and a 21-year-old woman with surgically treated UCLP in the Department of Prosthodontics, Dicle University. An examination revealed a residual palatal defect of 2 × 3 mm and missing maxillary lateral incisors in both. The 19-year-old woman was treated with a fiber-reinforced composite resin-bonded fixed partial denture (RBFPD). The 21-year-old woman was treated with a removable partial denture (RPD) with an extracoronal attachment system.The radiographic and clinical analyses showed no bone loss around the abutment teeth . The rigFirst, proximal cavities were prepared for the inlays that would facilitate a well-aligned path of insertion Figures and 6. AlAfter cavity preparation, a piece of reinforcing fiber, which had been coated with bonding agent, was packed into the inlay cavity of one abutment tooth and the free ends of the fiber were extended to the inlay cavity of the other abutment tooth .The bulk of the crown of the pontic and the inlay cavity restoration of the abutment teeth were formed using a layer of stronger hybrid resin . The resin restoration was cured for at least 2 minutes with a resin composite-curing unit. Then, the restoration was given a final shaping and polishing Figures and 9.Cleft lip and cleft palate are among the most common congenital anomalies. The reported incidence of cleft lip and palate is 2 per 1000 live births in Japan and from 1.25 to 1.43 per 1000 in the United States , 8. WhenMaxillofacial prosthetic treatment, a combination of fixed, implant-supported, and removable prostheses in conjunction with other dental and medical treatment, may be necessary to obtain the maximum ideal outcome for the patient.The use of a fixed partial denture may create a number of problems such as the removal of sound tooth structure and difficulty in oral hygiene with reduced gingival and periodontal health. It has been recommended that two abutment teeth be used on each side of the cleft .Well-planned prosthetic, periodontal, and surgical therapy may result in satisfactory function and esthetics, alleviating the deformities. With education and appropriate recall, the patients should be able to maintain their oral health.When replacing a tooth, the following solutions may be considered: (1) an implant-supported single crown; (2) a conventional fixed partial denture (FPD); and (3) a resin-bonded fixed partial denture (RBFPD). Removable partial dentures should ameliorate the health of the remaining dentition and surrounding oral tissue . With caA removable dental prosthesis may be used as a provisional form of tooth replacement. Although it can provide good esthetics, a portion of the prosthesis must rest on the soft tissues of the palate and may cause irritation. The removable nature of the prosthesis is a common patient objection. It is used only as a definitive means of tooth replacement when multiple teeth are missing and the edentulous space is too extensive to be spanned by a fixed restoration , 7. For For cases in which the abutment teeth require no restoration, a resin-bonded fixed dental prosthesis can be used , 16. ThiOn completion of the prosthesis, routine maintenance was performed during two or three patient recalls over the next year. Probing depths varied between 1 and 1.5 mm, and there was no gingival recession or inflammation in the region of the prosthesis. The patients were satisfied and reported no functional or esthetic problems.

The tolerability, anti-tumour activity and pharmacokinetic interaction of high-dose intravenous cyclosporin combined with intravenous etoposide was evaluated in children. Eighteen patients with recurrent or refractory tumours, all of whom had previously received etoposide, were treated with a combination of high-dose cyclosporin and etoposide. In 13, cyclosporin was given as a continuous infusion (15 mg kg(-1) per 24 h for 60 h) and in five a short 3-hour infusion of 30 mg kg(-1) day(-1) on three consecutive days. Pharmacokinetic profiles of etoposide were determined with and without cyclosporin. Cyclosporin levels ranged from 1359 to 4835 ng ml(-1) and cyclosporin increased the median area under the concentration time for etoposide curve from 7.2 to 12.5 mg ml(-1) min. The major toxicity was acute with varying forms of hypersensitivity reactions. In four cases this was severe. Hyperbilirubinaemia was present in 25 of 32 courses but was of short duration. In 14 courses, creatinine and/or urea was elevated, but was also transient. Significant hypertension was seen in six courses. Four of 17 patients evaluable for response obtained a partial response and one showed stable disease. It is concluded that in children given the combination of high-dose cyclosporin and etoposide, the etoposide dose should be halved in order to achieve an area under the drug concentration-time curve similar to that with etoposide alone. A continuous infusion schedule of cyclosporin is better tolerated during the period of administration but is associated with similar hepatic and renal dysfunction to a short schedule. The 24% response rate in children who had previously received etoposide suggests that this may be an effective method of enhancing drug sensitivity and further phase II evaluation is justified.

The neurotrophin BDNF has been implicated in the regulation of neuroplasticity, gene expression, and synaptic function in the adult brain, as well as in the pathophysiology of neuropsychiatric disorders and the mechanism of action of antidepressants. Antidepressant treatments have been shown to increase the expression of BDNF mRNA, although the changes measured were found to be different depending on various factors. A few studies only have measured levels of BDNF protein after antidepressant treatments, and poor correlation was found between mRNA and protein changes. We studied the time course of expression of BDNF mRNA and protein during drug treatments, in order to elucidate the temporal profile of regulation of this effector and whether mRNA and protein levels correlate. Rat groups were treated for 1, 2 or 3 weeks with fluoxetine or reboxetine; in additional groups drug treatment was followed by a washout week (3+1). Total BDNF mRNA was measured by Real Time PCR, pro- and mature BDNF proteins were measured by Western blot.We found that mature BDNF protein is induced more rapidly than mRNA, by both drugs in hippocampus (weeks 1–2) and by reboxetine in prefrontal/frontal cortex (week 1). The temporal profile of BDNF protein expression was largely inconsistent with that of mRNA, which followed the protein induction and reached a peak at week 3.These results suggest that BDNF protein is rapidly elevated by antidepressant treatments by posttranscriptional mechanisms, and that induction of BDNF mRNA is a slower process. Brain-Derived Neurotrophic Factor (BDNF) is an abundant neurotrophin regulating neuroplasticity, gene expression, synaptic function and cognition in the adult brain ,2, that However, several studies have shown that the changes in the expression of BDNF can be quite different depending on various factors, such as type of drug, dosage, route of administration, length of treatment , a selective serotonin reuptake inhibitor (SSRI), and reboxetine (RBX), a selective norepinephrine reuptake inhibitor (NRI). The drug treatments were carried out for 1, 2 or 3 weeks and were followed by an additional washout week (3+1), that was added in order to study the fate of BDNF expression when antidepressant treatment is discontinued. In all these rat groups we assessed the expression of total BDNF at both mRNA and protein levels, measuring both the pro- and mature forms of BDNF ,22. We f4,79 = 147.31; p < 0.0001), drug and time/drug interaction . Similarly, in P/FC there was an effect of time , drug and time-drug interaction . As shown in Fig. We measured the changes in BDNF expression induced by the two drug treatments at the level of both mRNA and protein, in HPC and P/FC. Total BDNF mRNA was detected by means of quantitative Real Time PCR, using primers designed on the sequence of the coding exon (exon IX). 2-Way ANOVA showed in HPC an effect of time and mature BDNF and of time/drug interaction for pro-BDNF . In HPC, FLX induced a trend toward increase of pro-BDNF at weeks 1–2, which became significant at week 3, while RBX up-regulated pro-BDNF at weeks 2–3. Both FLX and RBX consistently up-regulated the expression of mature BDNF at all times in HPC, with maximum at weeks 2–3 for FLX and 1 to 3 for RBX and time/drug interaction for pro-BDNF and an effect of time and drug for mature BDNF. Indeed, both FLX and RBX up-regulated pro-BDNF, with the only exception of week 3 for FLX. Differently from the HPC, in P/FC the up-regulation of pro-BDNF by FLX sharply contrasted with the absence of changes in mature BDNF before the actual peak of mRNA (week 3), suggesting early translation and processing of the protein during antidepressant treatment. The same was observed for RBX in P/FC.The main findings of this work were that BDNF mRNA and protein levels during antidepressant treatments did not correlate, and that induction of mature protein preceded that of mRNA. The measurement of BDNF mRNA expression showed an overall similar temporal profile of induction with the two drugs. RBX acted somewhat faster, inducing BDNF mRNA already at week 1 in both areas, while FLX induced BDNF from week 2. The shape of this induction was also somewhat different; FLX progressively increased the expression up to week 3, while RBX induced BDNF in two distinct waves. After a precocious induction at week 1, the expression of BDNF was not further increased (P/FC) or was reduced (HPC) by RBX between week 1 and 2. A second, more pronounced, wave of BDNF induction by RBX was observed between week 2 and 3. Overall, RBX acted faster on BDNF transcription but FLX outsized RBX with regard to the maximum extent of mRNA induction. Interestingly, the washout week also differently affected the outcome of the two drug treatments in HPC, because it sharply reduced the effect of FLX in both areas and of RBX in P/FC, while in HPC of RBX-treated rats BDNF mRNA level was unchanged at week 3+1. In a companion study , we prevSurprisingly, the temporal profile of BDNF protein expression in HPC was largely inconsistent with that of mRNA expression . It has already been suggested that BDNF synthesis could be regulated at posttranscriptional level , as founFinally, the finding that BDNF, a major readout effector, is rapidly elevated in HPC by antidepressant treatment may suggest that later downstream events are required to elicit systemic and behavioral effects of antidepressants. If BDNF production is faster than thus far envisaged, perhaps the correlates of the therapeutic effects should be searched in the actual cellular/molecular changes induced by the action of BDNF, such as the modifications in synaptic plasticity and the increment of neurogenesis. Further work is required to characterize these events.The results of the present study show that BDNF protein is rapidly elevated by antidepressant treatments whereas the induction of BDNF mRNA is a slower process, suggesting that posttranscriptional mechanisms are involved in the mechanism of antidepressants.2PO4, 1 mM Na2VO4, 2 ml/ml of protease inhibitor cocktail to obtain the total extract, that was aliquoted and stored at -80°C.Experiments complied with guidelines for use of experimental animals of European Community Council Directive 86/609/EEC. All efforts were made to minimize animal distress and to reduce the numbers of animals used in this study. Groups of 12 male Sprague-Dawley rats (6 week-old at beginning of treatment) were treated with vehicle (water), FLX or RBX at 10 mg/kg rat weight/day, delivered in drinking water; the average water intake per day for each cage (two rats) was recorded for 4 days before starting and throughout the treatment, and the drug solutions were changed every two days according to animals' weight, as reported in previous studies . Rats weTotal RNA was isolated with Trizol . 2 μg of RNA were reverse transcribed using SuperScript™ II Reverse Transcriptase (Invitrogen) and 0.5 μg random hexamers in a 50 μl final volume. Real-time PCR was carried out using a LightCycler rapid thermal cycler System and SYBR Green was used for the detection of double-strand DNA. The PCR reaction was set up into microcapillary tubes in a volume of 20 μl with 2 μl of cDNA and 2 μl of LightCycler DNA Master SYBR Green I (Roche Diagnostics). For BDNF quantitation, fwd AGCTGAGCGTGTGTGACAGT and rew ACCCATGGGATTACACTTGG primers were used, while for β-actin quantitation, used as internal control ,33, fwd Western blot analysis was carried out as described previously , by incuAll data were analyzed by using two-way analysis of variance (2-Way ANOVA) for the variable treatment (FLX/RBX) and the variable time . If the analysis of variance revealed significant group differences, post-hoc Bonferroni tests were carried out to elucidate the pattern of group differences. Significance was assumed at p < 0.05. Statistical analysis of the data was carried out using GraphPad Prism 4 .LM carried out the animal treatments, the western analysis and performed the statistical analysis. AC carried out the molecular biology work. DT performed the statistical analysis, and drafted the manuscript. AB carried out the molecular biology work. MG planned the experiment for BDNF RNA transcription. SB helped to draft the manuscript and provided useful discussion. GR helped to draft the manuscript and provided useful discussion. MP designed the study, and drafted the manuscript. All authors contributed to and have approved the final manuscript.

The identification of virus replication is an important step towards the understanding of the pathogenesis process of viruses in their respective hosts. In the present study, we developed a strand-specific RT-PCR-based method for analysis of Deformed Wing Virus (DWV) replication in honey bees and in honey bee parasitic mites, Varroa Destructor.For years, the understanding of the pathogenetic mechanisms that underlie honey bee viral diseases has been severely hindered because of the lack of a cell culture system for virus propagation. As a result, it is very imperative to develop new methods that would permit the The results shows that the method developed in our study allows reliable identification of the virus replication and solves the problem of falsely-primed cDNA amplifications that commonly exists in the current system. Using TaqMan real-time quantitative RT-PCR incorporated with biotinylated primers and magnetic beads purification step, we characterized the replication and tissue tropism of DWV infection in honey bees. We provide evidence for DWV replication in the tissues of wings, head, thorax, legs, hemolymph, and gut of honey bees and also in Varroa mites.The strategy reported in the present study forms a model system for studying bee virus replication, pathogenesis and immunity. This study should be a significant contribution to the goal of achieving a better understanding of virus pathogenesis in honey bees and to the design of appropriate control measures for bee populations at risk to virus infections. Apis mellifera, the most economically valuable pollinator of agricultural and horticultural crops worldwide. In the U.S. alone, the honey bee has an annual market value exceeding 14.6 billion dollars producing honey and other hive products [in vitro.The viruses pose a serious threat to the health and well-being of the honey bee, products ,3. Recenproducts , investiVarroa destructor), a potential vector of DWV, was also investigated.Recent advances in molecular technology have greatly expanded our ability to detect and elucidate the molecular events associated with virus infections and pathogenesis. With the current molecular technology, complete genomes of several honey bee viruses have been sequenced and analyzed -14. UsinThe strand specificity of conventional RT-PCR was evaluated. As shown in Figure In this study, Tagged RT-PCR was evaluated for its specificity for amplification of both positive and negative-strand RNA from bees with deformed wings using four combinations of primers. Tagged RT-PCR assay was based on the generation of cDNAs by the primer containing a tag and further amplification of cDNA by a tag-primer and a primer complementary to the synthesized cDNA. The result showed that cDNAs generated by either tag-forward or tag-reverse primers were consistently amplified by subsequent PCR amplification, regardless of whether a pair of primers or only a single primer was used for PCR amplification. As shown in Figure In order to achieve highly strand-specific detection of RNA for DWV, strand-specific RT-PCR was conducted using a biotinylated primer for cDNA generation and magnetic separation to purify synthesized cDNA prior to PCR amplification. As shown in Figure in vitro transcribed from the pCR2.1 TA cloning vector against corresponding CT value. As shown in Figure 3 pg to 1 fg and the corresponding CT values within the concentration range. The detection limit of positive and negative-strand DWV RNAs were the same. The lowest limit of detection with qRT-PCR for DWV was 1 fg per reaction for both positive and negative-strand RNA. The RNA concentration below 1 fg could not be amplified in the reaction.Further, the strand-specific TaqMan real-time qRT-PCR incorporated with biotinylated primer and magnetic separation was carried out for quantification of DWV replication in host tissues and parasitic mites of honey bees. To ensure an accurate quantification as well as the highest sensitivity of the assay, a standard curve was first established by plotting seven 10-fold dilutions of DWV-specific RNA 4 times higher in the wings (p < 0.0001), 27.8 times higher in the legs (p < 0.0001), 19.8 times higher in the head (p = 0.0015), 107.1 times higher in the thorax (p = 0.0008) and descended down in order of wings, hemolymph, legs, gut, head, abdomen, and thorax Figure . Meanwhi) Figure .3 times more abundant than negative-strand DWV RNA in the virus-infected bees (p = 0.007). Positive-strand DWV RNA was detected in all tissues of bees with symptomatic or asymptomatic infections even though the average concentration of positive-strand viral RNA in tissues of bees with wing deformity was significantly higher than tissues of bees with normal wings; 8 × 102 times more in the hemolymph, 103 times more in the wings, 90 times more in the legs, 29.8 times more in the head, 1.8 × 103 times more in the thorax, 15.2 times more in the gut (p < 0.0001) and 29.8 times more in the abdomen (p < 0.0001) Figure .Using the same methodology, negative-strand RNA of DWV was detected in 81% (17/21) while the positive-strand RNA of DWV was found in 95.2% (20/21) of the Varroa mites tested. Quantification of positive and negative-strand in the Varroa mites showed no significant difference (p = 0.07) between the titers of the negative-strand and positive-strand RNAs as seen in the honey bees (p < 0.05).Replication is a key step in successful virus infections. The replication strategies of positive-strand RNA viruses share remarkable similarities: all replicate and express their genomes through negative-strand RNA intermediates that are used as templates for the production of positive-strand progeny RNAs packaged in new virion particles . Therefoin vitro RNA replication assay for honey bee viruses, we first evaluated the existing methods, including conventional RT-PCR and Tagged RT-PCR for specificity of strand-specific detection of DWV RNA. The results showed that DWV RNA could be amplified by conventional RT-PCR without any primer for reverse transcription. The reason for nonspecific cDNA synthesis by conventional RT-PCR could be attributed to different events, including false-priming by antigenomic viral RNA or cellular RNAs, as well as self primering due to the secondary structure at the 5'UTR of viral RNA during reverse transcription, as earlier reports suggested [In an attempt to develop an uggested ,25-27. Tuggested ,29. Howeuggested . The eviIn order to circumvent the problems of false priming and contamination of residue primers from RT, it is worthwhile to develop improved procedures for analysis of virus replication. We report here the development of a novel strand-specific RT-PCR coupled with a biotinylated primer for reverse transcription and purification of biotinylated cDNA with magnetic beads. Using biotinylated oligonucleotide primers during the reverse transcription can lead to subsequent synthesis of biotinylated cDNAs, which have a high binding affinity to the streptavidin-coated magnetic beads. The purification step of streptavidin magnetic beads-cDNA complex, ensures the capture of only biotinylated cDNAs. The disappearance of a positive signal for non-strand-specific amplification of magnetic-bead purified biotinylated cDNA in our study suggests that the assay developed is a significant modification of conventional or Tagged RT-PCR. The purification of biotinylated cDNA with magnetic beads is a key step in assuring the strand-specific detection and elimination of the residues of RNA, non-incorporated RT primers as well as enzymes and salts that would interfere with subsequent PCR amplification.While quantitative detection of DWV infection and tissue tropism of DWV in the host were previously reported ,31, therThe observation that different tissues showed distinct kinetics of DWV replication, together with the fact that differences in the titers of positive-strand DWV RNAs were not significant among tissues examined, suggested that DWV had a tropism to certain host tissues in the replication. Among seven tissues examined, the most abundant amount of negative-strand DWV RNA was detected in the wings, which suggests that the wings are likely the predominant tissue site of DWV replication. Our earlier studies ,31 demonCompared to the titer of positive-strand DWV RNA, the relatively low amount of negative-strand DWV RNA implies that a regulatory mechanism may exist to facilitate the viral replication. These findings would stimulate further investigation to unveil the regulatory mechanisms that honey bees use to control the pattern of replication. Both positive and negative-strand RNAs of DWV were also detected in Varroa mites (data not shown) using the magnetic beads methodology. This finding is in agreement with preliminary works ,23 and sIn sum, strand-specific detection and quantification of DWV RNA were achieved using qRT-PCR incorporated with biotinylated primers and purification of biotinylated cDNA with magnetic beads. The elucidation of DWV replication profiles in honey bees would have broad implications for future development of therapeutic strategies for viral diseases. The assay developed in this study represents a useful tool to study not only replication of honey bee viruses but also other single-stranded RNA viruses.in vivo using the honey bee as a model. This permitted the specific identification of an important honey bee virus (DWV) replication sites in the honey bee body and it's quantification. The screening between symptomatic and asymptomatic bees using this technology had permitted the identification of the digestive tract and abdomen as a critical replication site in the course of symptomatic infection.We conclude that qRT-PCR incorporated with biotinylated primers and purification of biotinylated cDNAs with magnetic beads is a strong approach to specific detection and quantification of a virus genome and anti-genome Varroa mites and maintained in the USDA-ARS Bee Research Laboratory backyard apiary, Beltsville, MD. Bees intended for tissue dissection were kept alive inside of containers with the cap loosened to allow airflow before dissection. Otherwise, bees were immediately stored in -80°C freezer for subsequent RNA extraction.Individual adult worker bees, with and without deformed wings, and individual Varroa mites, were collected from honey bee colonies that were left untreated for Each live bee (N = 18) was fixed on the wax top of a dissecting dish with insect pins. Under a dissecting microscope, hemolymph was collected with a micropipette by making a small hole at the joint area between the wing and body with a needle to make it bleed. Following hemolymph collection, the gut was carefully pulled out with forceps. The remaining body including wings, legs, head, thorax and abdomen were cut apart with scissors and collected individually. To prevent possible contamination with hemolymph, all tissues were thoroughly rinsed once with 1× PBS and twice with nuclease-free water. All the tissue samples were subjected to RNA extraction.pro, Amersham Biosciences). RNA samples were stored at -80°C prior to molecular detection for viruses.Collected tissues from bees and Varroa mites (n = 21) were individually homogenized in TRIzol Reagent, a solution of guanidine isothiocyanate and phenol, for RNA extraction . After the addition of chloroform to remove proteins, lipids and DNA, the upper aqueous phase containing RNA was removed to a new microcentrifuge tube, precipitated with isopropanol, and the resulting pellet was dissolved in diethyl-pyrocarbonate (DEPC)-treated water with the addition of 1 μl of RNaseOUT a ribonuclease inhibitor . The concentration of total RNA was determined by measuring the absorption at 260 nm and the purity of RNA was estimated by the absorbance ratio of 260 nm/280 nm using a spectrophotometer with a 50 μl ultramicrovolume cell holder was used for RT-PCR following the manufacturer's instructions. Primers used in the study were a pair of DWV specific primers as reported before . In ordeTagged RT-PCRs were performed under the same thermal cycling conditions as the conventional RT-PCR described above, except that the primers used in the study were modified by adding a 15-bp long sequence tag . The seqIn order to increase strand specificity, the regular primers or tagged-primers were replaced by biotinylated-primers for reverse transcription. The biotinylated-primers were synthesized by Invitrogen. After the reverse transcription reaction, cDNAs generated by either biotinylated forward or biotinylated reverse primers were magnetically purified by the Dynabeads kilobaseBINDER M-280 kit following the manufacturer's recommendations. Magnetic beads coated with a monolayer of streptavidin were added to the RT reaction mixture containing the biotinylated cDNAs. The reaction mixture was incubated at room temperature for 3 hours in a roller incubator to allow immobilization of the biotinylated cDNA onto the magnetic beads. The Dynabeads/DNA-complex were washed twice in washing solution and once in distilled water and resuspended in PBS buffer. After releasing immobilized Biotinylated cDNAs from magnetic beads, PCR amplification was conducted for each sample under the same conditions as described above. The cDNAs that were generated by biotinylated primers and directly subjected to PCR amplification without magnetic bead purification were used as a control.TaqMan real-time quantitative RT-PCR (qRT-PCR) incorporated with biotinylated-primers and magnetic bead purification was performed for quantification of negative and positive-strands of DWV using the Stratagene Mx3000P spectrofluorometric thermal cycler operated by MxPro qPCR software. The virus levels were quantified based on the value of the cycle threshold (Ct), which represents the number of cycles needed to generate a fluorescent signal above a predefined threshold and is inversely proportional to the concentration of the initial target that has been amplified. The house keeping gene, β-actin, was employed as an endogenous control for normalization of the quantification. The sequence information of primers and probes for both DWV and β-actin are the same as described before [in vitro transcription using the Megascript T7 kit in order to generate positive and negative DWV-specific ssRNAs and to construct a new standard curve for sensitive analysis and absolute virus quantification. Seven 10-fold serial dilutions of positive or negative in vitro transcribed RNA were subjected to reverse transcription with biotinylated primers followed by magnetic bead purification and qPCR amplification. Each sample was run in triplicate for statistical purposes. The standard curve was established by plotting the initial quantities of the 10-fold serial diluted RNA against the corresponding threshold value (CT).Purified DWV specific fragments were incorporated into a pCR2.1 TA cloning vector following the manufacturer's protocol. Recombinant plasmid DNA, containing DWV fragment in both directions, was purified using the Plasmid Mini Prep Kit and used as a template for The specificity of the RT-PCR assay was confirmed by sequencing analysis. RT-PCR bands were excised from a low-melting-temperature agarose gel and purified using the Wizard PCR Prep DNA purification system . The nucleotide sequences of the RT-PCR fragments were determined from both forward and reverse directions to confirm the specificity of the DWV amplification. The sequence data of each virus fragment were analyzed using the BLAST server at the National Center for Biotechnology Information, NIH.The Fisher's Least Significant Difference (LSD) Comparison of Means Test was used to analyze for significant differences of positive and negative-strand DWV titers among different tissues of the bees. The results are expressed as mean ± SD. Differences were considered statistically significant if p value < 0.05.DWV: Deformed Wing Virus; RT-PCR: Retro transcription-Polymerase chain reaction; CCD; Colony Collapse Disorder; CPE: Cytopathic effect.The authors declare that they have no competing interests.HFB conceived the research, performed the experiments, and wrote the manuscript. YPC developed the conceptual aspects of the work and wrote and edited the manuscript. All authors participated in data collection and read and approved the final manuscript.Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

Research on the vascular microenvironment has been hindered by challenges in studying this compartment in metastasis under conditions that reproduce We present a microfluidic vasculature system to model interactions between circulating breast cancer cells with microvascular endothelium at potential sites of metastasis. The microfluidic vasculature produces spatially-restricted stimulation from the basal side of the endothelium that models both organ-specific localization and polarization of chemokines and many other signaling molecules under variable flow conditions. We used this microfluidic system to produce site-specific stimulation of microvascular endothelium with CXCL12, a chemokine strongly implicated in metastasis.When added from the basal side, CXCL12 acts through receptor CXCR4 on endothelium to promote adhesion of circulating breast cancer cells, independent of CXCL12 receptors CXCR4 or CXCR7 on tumor cells. These studies suggest that targeting CXCL12-CXCR4 signaling in endothelium may limit metastases in breast and other cancers and highlight the unique capabilities of our microfluidic device to advance studies of the intravascular microenvironment in metastasis. Trafficking of cells through the vasculature and arrest of circulating cells on endothelium at specific sites are critical steps in a wide variety of physiologic and disease processes, including immune surveillance In vitro assays of cancer cells and endothelium typically are performed under static, biochemically homogeneous conditions. These static assays do not accurately model intravascular events in metastasis because blood flow alters gene expression in vivo. Animal models of metastasis also are time-consuming, labor-intensive, and expensive.Identifying molecular determinants of trafficking and arrest of circulating cancer cells on endothelium at characteristic sites of metastatic disease has been limited in large part by challenges of studying the intravascular microenvironment under physiologic conditions. We have engineered a new microfluidic model of the vasculature to overcome limitations of existing systems and advance studies of the intravascular compartment in cancer metastasis and other diseases. This microfluidic device produces defined flow rates within a range of physiologic levels and enables real-time bright field and fluorescence imaging of circulating cancer cells and microvascular endothelium. The device also allows region-specific treatment of endothelium from the basal side, recreating organ-specific localization of signaling molecules produced by stromal cells in the extravascular space. This also mimics serial interaction of circulating tumor cells with endothelia of differing potentials to promote cell adhesion and formation of metastases. To establish the utility of this microfluidic device for studying intravascular steps in metastasis, we investigated effects of chemokine CXCL12 on adhesion of circulating breast cancer cells to endothelium. High levels of CXCL12 are expressed by parenchymal cells in organs commonly affected by metastatic breast cancer, such as liver, bone, and brain Using the microfluidic vasculature to selectively manipulate cancer cells and endothelium, we demonstrate that circulating breast cancer cells preferentially adhere to endothelium stimulated from the basal side with CXCL12. CXCL12 functions through CXCR4 on endothelial cells to significantly enhance adhesion of circulating breast cancer cells, independent of CXCR4 or CXCR7 on cancer cells. These results provide new insights into pro-metastatic effects of CXCL12 in the intravascular microenvironment, focusing attention on CXCL12-CXCR4 in vascular endothelium as a potential therapeutic target to block metastasis. Moreover, the research establishes this microfluidic vasculature technology as a versatile, physiologic model for future studies of the intravascular microenvironment in metastasis and other disease processes.The microfluidic vasculature is comprised of two poly(dimethysiloxane) (PDMS) layers sandwiching a thin, porous, and optically clear polyester membrane . The topThis setup enables simultaneous and distinct localization of chemokines on the basal face and flow on the apical face of the endothelium . This ca−2 dyn cm−2 .−2 (Previous reports have shown that CXCR4 expression in cancer cells promotes metastasis to distant organs such as the lung −2 (p<0.05)−2 (p<0.05)−2 , CXCL12-−2 prolonged, extravascular stimulation of vascular endothelium with CXCL12, modeling both the high levels and directionality of this chemokine in target organs for metastatic breast and other cancers in vitro system and enables our system to evaluate the factors that mediate intravascular adhesion of circulating cancer cells in the context of physiologically defined chemical environments and physical forces.We have described a compact, easily-fabricated microfluidic system to model serial interactions of circulating cancer cells with a vascular endothelium at defined sites stimulated extravascularly from the basal side by chemokines. The microfluidic vasculature reproduces preferential adhesion of circulating cancer cells to endothelia in organs and tissues with high levels of CXCL12 in vitro flow-based systems. Our design also enables simultaneous basal chemokine and apical fluid mechanical stimulation of endothelia in a reagent efficient manner. Microliter amounts of expensive chemokine solution are localized to the bottom compartment while flow substantive enough to deliver shear stress levels known to alter gene expression Given its attributes, our system would also be useful for studies involving immune cell trafficking to endothelium either in response to chemokines such as CXCL12 Using our microfluidic endothelium, we confirmed that CXCR4 or CXCR7 on breast cancer cells promotes intravascular adhesion throughout the channel , supportin vivo studies. Furthermore, inhibiting CXCL12-CXCR4 signaling on endothelium significantly reduced adhesion of circulating breast cancer cells . Prior to cell seeding, the device was sterilized by placing under UV light for ∼30 min.The microfluidic device consiste7 cells ml−1 EGM-2 MV) was loaded through the funnel-shaped inlet reservoir, allowed to attach along the entire length of the top channel. Cells were re-fed 1–2 times per day by gravity flow from the funnel-shaped inlet and through the top channel and grown to confluence (24–48 h after seeding). We note that the presence of the bottom channels and all of these treatments were in EBM-2+Single®Quots with no serum.Human dermal microvascular cells passage numbers 6–8 were cultured in EBM-2+5% FBS+SingleQuot® kit or EGM-2 MV (Lonza) as described previously channels , which h5 cells per dish. One day after plating, cells were changed to DMEM medium with 0.2% bovine serum albumin (BSA) . Supernatants containing secreted CXCL12-GL or GL were collected from cells 18 hours later, and levels of CXCL12-GL were measured by ELISA (R&D Systems). Supernatants from CXCL12-GL secreting cells contained ≈40 ng ml−1 CXCL12, while this chemokine was undetectable in supernatants from GL secreting cells. CXCL12-GL has bioluminescence properties comparable to unfused GL and CXCR4-dependent signaling to a comparable extent as unfused secreted or synthetic CXCL12. Full characterization of CXCL12-GL will be described in a separate manuscript.293T cells stably transduced with lentiviral vectors for a fusion protein of CXCL12 and Gaussia luciferase (CXCL12-GL) or unfused Gaussia luciferase (GL) were plated in 6 cm dishes at 1×10−1 CXCL12-GL or a comparable amount of GL based on luminescence either in the top or bottom compartments of the Transwell insert. Cells and Transwell inserts were washed twice with PBS before adding 1 µg ml−1 coelenterazine (Fluka) in PBS and measuring bioluminescence in intact cells on an IVIS 100 system . Images were obtained for 20–30 seconds using high resolution acquisition. Bioluminescence was quantified by region of interest analysis using Living Image Software HDMEC cultured on 24-well Transwell inserts (Corning) were incubated for 5 hours with ≈40 ng mlHuman MDA-MB-231 breast cancer cells Total RNA from HDMECs was prep5′-ACGGACAAGTACAGGCTGCAC-3′ and 5′-CCCAGAAGGGAAGCGTGA-3′CXCR4: 5′-AAGAAGATGGTACGCCGTGTCGTCTC-3′ and 5′-CTGCTGTGCTTCTCCTGGTCACTGGA-3′CXCR7: 5′-GAAGGTGAAGGTCGGAGT-3′ and 5′-GAAGATGGTGATGGGATTTC-3′GAPDH: −1 CXCL12 for 10 minutes or 5 hours. Control cells were incubated with BSA alone. 231 breast cancer cells were cultured overnight in DMEM medium containing 0.5% serum. Breast cancer cells then were treated with 100 ng ml−1 CXCL12 (R&D Systems) for 3 or 10 minutes, respectively , 2-DQ is the flow rate per width in the system and h is the height of the channel. This equation is valid for cases where the width is much larger than the height. In our system, the height of the channels was 60 µm and the width was 800 µm.The shear stress levels on the cells within the channels were modeled with the following equation −1) for neutralizing CXCR4 or 11G8 antibody (10 µg ml−1) for neutralizing CXCR7 is added to the EBM-2+0.5% FBS starve media.Prior to each experiment, the EGM-2 MV culture in the funnel-inlet of the microfluidic device was removed, washed with PBS, and replaced with EBM-2+Single®Quots. Subsequently, the top channel was washed with EBM-2+Single®Quots by gravity-driven flow. Concurrently, the bottom channels were triple washed with PBS and replaced with either EBM-2+Single®Quots (untreated) or EBM-2+Single®Quots+cytokine or chemokine of interest. The flow in the top channel was stopped and the device was left to be treated for 5 h. Concurrently, culture flasks of the 231 breast cancer cells were washed with PBS and serum starved in EBM-2+0.5% FBS for 5 h as well. For experiments involving pre-treatment of 231 cells with chemokine receptor inhibitor , either 6 cells ml−1. The cancer cell suspension was loaded into the funnel-shaped inlet. Flow was controlled using a programmable syringe pump (KD Scientific) that withdraws fluid away from the funnel-shaped inlet at flow rates corresponding with shear stress levels of 0.50 dyn cm−2 or 2.50 dyn cm−2. We note that in our system, the shear stress levels of 0.50 dyn cm−2 and 2.50 dyn cm−2 correspond with flow velocities of 0.2 mm s−1 and 1.0 mm s−1 respectively which are in line with the in vivo blood flow velocity range of 0.1–1.5 mm s−1 reported in the microcirculation of potential sites of breast cancer metastasis −2 with the avidity decreasing rapidly with higher shear stress levels −2 compared to 0.50 dyn cm−2.After serum starvation, the breast cancer cells were collected using citric saline, a calcium ion chelator, instead of trypsin to ensure cell surface proteins remain intact. The cancer cells were centrifuged and washed twice and resuspended in EBM-2+0.5% FBS at a concentration of 2×10The microfluidic device was placed on the stage of an epi-fluorescence, inverted microscope . The duration of each experiment was 30 min followed by a controlled 1 min wash of the top channel with PBS to remove loosely attached cells. Images were recorded with a digital CCD camera (Hamamatsu). To determine cell counts, fluorescent cells in an entire region were counted blindly by a different person other than the experimentalist to negate observer bias using a semi-automatic counting protocol with NIH ImageJ and treated as one data point.−1) introduced either apical or basal to the endothelium for 5 hours. Also, the same amount of cancer cells and volume were allowed to adhere since the surface area of the Transwell insert is the same as an individual well in a 96-well plate (0.32–0.33 cm2). In these experiments, 10 minutes instead of 30 minutes were allowed for adhesion prior to triplicate PBS washing.The conditions for the static adhesion experiments were the same as the flow-based experiments described above in terms of cells, pre-treatment, and duration of experiments. The difference was the static experiments were performed in tissue culture 96-well plates (Corning) instead of microfluidic devices. Into each well, ≈10,000 cancer cells in 100 µl were added and allowed to adhere for 30 minutes. Following the adhesion experiments, each well was triple washed with PBS to remove loosely attached cells. For static adhesion in Transwells , HDMECs Sample populations were compared either pair wise using the Mann-Whitney U test or in groups of more than two using the Kruskal-Wallis test. p<0.05 was the threshold for statistical significance. Data sets that were statistically significant with the Mann-Whitney U test were indicated with a ‘*’ symbol. For data evaluated with the Kruskal-Wallis test, results that were statistically significant were indicated with a ‘**’ symbol; results that were not statistically significant were indicated with a ‘#’ symbol.Figure S1−2 flow conditions. TNF-α was applied to either the upstream (black bars) or downstream compartment (white bars) with the other compartments in the same device left untreated. Cancer cell adhesion onto the TNF-α treated endothelial region was significantly greater than the untreated region regardless of if TNF-α was applied upstream or downstream (p<0.01). (A) 0.50 dyn cm−2. (B) 2.50 dyn cm−2. ‘+’ denotes TNF-α stimulation. n = 3 each for upstream or downstream treated conditions. Data are expressed as the mean+s.e.m. (C) Boxplots representing adhesion selectivity of 231-control cells towards TNF-α treated endothelium at 0, 0.50, and 2.50 dyn cm−2 shear stress levels. The adhesion selectivity was statistically different for the different flow conditions with the selectivity increasing with increasing flow. n = 6 for each condition.231-control cells preferentially adhere onto TNF-α stimulated endothelial region at 0.50 and 2.50 dyn cm(0.26 MB TIF)Click here for additional data file.

The great epidemiological significance and costs associated with chronic heart failure pose a challenge to health systems in Western industrial countries. In the past few years, controlled randomised studies have shown that patients with chronic heart failure benefit from telemedical monitoring; specifically, telemonitoring of various vital parameters combined with a review of the symptoms, drug compliance and patient education. In Germany, various telemedical monitoring projects for patients with chronic heart failure have been initiated in the past few years; seven of them are presented here. Currently 7220 patients are being monitored in the seven selected projects. Most patients (51.1%) are in NYHA stage II, 26.3% in NYHA stage III, 14.5% in NYHA stage I and only 6.6% in NYHA stage IV respectively. Most projects are primarily regional. Their structure of telemedical monitoring tends to be modular and uses stratification according to the NYHA stages. All projects include medical or health economics assessments. The future of telemedical monitoring projects for patients with chronic heart failure will depend on the outcome of these assessments. Only of there is statistical evidence for medical benefit to the individual patient as well as cost savings will these projects continue. In the Western industrial nations, chronic heart failure has great epidemiological significance due to the rising number of patients, particularly elderly ones . This de1-receptor blockers, aldosterone antagonists, or beta-receptor blockers) and medical devices , the prognosis for chronic heart failure has improved in the past few years [Due to both, drug therapy , the most important partner in a complex telemonitoring system is the telemedical service centre. All data is collected here and integrated into the electronic patient records and constitutes the central core of case management. The patients transmit various vital parameters, either at intervals or continuously, to the telemedical service centre. The transmission of the vital parameters can be conducted via telephone or mobile phone. In Germany, one limiting factor seems to be the missing internet access, particularly in primary care practices.The relevant monitoring parameters are heart rate, ECG (arrhythmias), blood pressure, and body weight. For instance, it was shown in a clinical study that atrial fibrillation worsens the prognosis for patients with chronic heart failure . Body weIn the past few years, much international experience has been gathered in this regard on telemonitoring in patients with chronic heart failure, also in the context of randomised controlled studies. In the following, we report on selected projects in Germany.The German health system has traditionally been structured according to sector, namely, the outpatient and inpatient sectors. Government policy has made various attempts in the last few years to overcome this division to some extent. One important mean to achieve this is contracts for integrated care .In the past few years, a number of, mostly regional, model projects on telemonitoring patients with chronic heart failure have been conducted in Germany. These projects brought forth signs of cooperation between the hospitals and practising physicians, as the providers of medical care, and various health insurance organisations. In addition, providers of telemedical services and industrial partners were included in the cooperation.Currently 7220 patients are being monitored in seven selected telemonitoring projects for patients with chronic heart failure. Most patients (51.1%) are in NYHA stage II, 26.3% in NYHA stage III, 14.5% in NYHA stage I, only 6.6% of patients were in NYHA stage IV, and in 1.6% of the Patients the NYHA stage was unknown.The selected projects are presented in greater detail in the following sections.The telemedical care of patients with heart failure in the context of the HeiTel project makes it possible, through monitoring, to institute and adjust therapy optimised for the individual patient. The patient transmits by telephone certain prescribed vital parameters via modem to the telemedical centre in an automated manner. If individually defined threshold values are not reached or exceeded, an alarm is immediately triggered in the monitoring centre so that therapeutic measures can be started immediately. Independent of any alarm responses, a patient in NYHA stages III-IV is contacted proactively at least once a week, while a stage II patient is contacted at least twice a month; they are asked questions according to a standardised form. The goal of these contacts is to promote medication adherence and to recognize telltale changes in a patient's health status as early as possible. Patients can reach the telemedical centre around the clock every day of the year to report cardiopulmonary symptoms and serious complaints. Training sessions on nutrition, movement and pharmacotherapy complete the programme and reinforce the patient's self-reliance in dealing with himself and his illness. After hospitalisation or after the conclusion of the individual titration phase during the initiation of drug therapy, which generally lasts about 6 months, there is a de-escalation of the device-based home monitoring, as part of a modular plan, which then becomes care provision by means of training sessions on nutrition, movement and monitoring of pharmacotherapy using a nurse call system. The purpose of this is to reinforce the patient's self-reliance in coping with himself and his illness and to perpetuate the success of treatment.This modular plan offers the opportunity of establishing telemedical care as a cost-efficient module that is integrated into a chain of medical services. More than 200 general practitioners and cardiologists are currently actively participating in this plan.n = 210) in comparison to a control group (n = 12 000) over a period of 2 years. This justifies the assumption that the intervention has a lasting effect. Although the plan was only designed for 12 months, these results led to an extension of the contract for integrated care.The accompanying evaluation of the project documented a high-level of satisfaction among the participants. It moreover, also demonstrated the plan's efficiency in terms of health economics by means of a significant reduction both in the overall costs and in the number of days of hospitalisation for the HeiTel patients was established in 2004 by the Municipal Hospital Brandenburg in Brandenburg/Havel. Its aim is to improve the care for chronic cardiac patients in the federal state of Brandenburg. In cooperation with getemed, a medical technology company, and with funding by the European Union and by the Ministry of Economics of the state of Brandenburg a telemonitoring unit was developed which daily records and transmits all noninvasively parameters and information regarding clinical symptoms, drug compliance, and desired contact. The promising results of a self-financed pilot study led to a contract with the AOK Brandenburg for integrated medical care for patients with chronic heart failure.Cooperations with various hospitals were established, including the German Heart Centre in Berlin, and numerous hospitals in the state of Brandenburg, as well as consulting cardiologists and general practitioners. The telemedical monitoring allows surveillance of patients from far distance, which is advantageous in a large, sparsely populated state with a low density of physicians.Inclusion criteria are chronic heart failure NYHA stages III-IV, an ejection fraction ≤40%, and a history of hospitalization for acute decompensated heart failure. At the moment, 300 patients of NYHA stages III and IV participate in the program.The patients receive a telemonitoring device, developed by getemed, Teltow, a scale, ECG electrodes, and a blood pressure gauge. The patient daily measures weight, blood pressure, ECG, thorax impedance and breathing rate, and for certain indications also oxygen saturation. Patients also provide information regarding the severity of dyspnea, their subjective well-being, medication intake, and preference regarding desired personal contact. The data is encoded and transmitted by internet to the TMBZ, where it is analysed weekdays with regard to newly manifested arrhythmias, heart rate, body weight, and blood pressure using individually determined threshold values, breathing rate, and, if indicated, the oxygen saturation. The patient's subjective comments regarding dyspnea, peripheral edema, well-being, and drug compliance are evaluated to detect worsening symptoms. If deterioration has been detected, the patient's physician is informed by fax or, in urgent cases, via telephone by the tmzb. A summary of the reported results is included. This objective data enables better adjustment of the patient's medication according to individually changing health status.The patient is contacted by the tmzb if: (i) suspicious changes of parameters are recorded, (ii) the patient requested contact, (iii) no data was transmitted, or (iv) the preceding intervention was not successful. The closer collaboration between the individual patients and their individual care givers allows for an optimal care. The acceptance of telemonitoring among patients is very high, as indicated by the fact that the compliance with data transfer is 95%. Patients indicate that they feel secure. No serious technical problems have been encountered. Preliminary analyses of the data show a significant reduction both in number and days of hospitalisations for acutely decompensated heart failure .The “CorBene” project was developed as a contract for providing integrated care between practicing cardiologists, the Ford and Rhineland company health insurance programme, and the companies Vitaphone (Mannheim) and Medtronic (Düsseldorf). The goal is a transsectoral, stage-specific therapy of patients with chronic heart failure that is based on telemonitoring and is conducted according to guidelines. The participating doctors profit from a uniform documentation, improved exchange of information, and integrated quality management. This prevents, for example, redundant examinations. The telemedical concept of monitoring consists of four modules. This guarantees that the telemonitoring is appropriate to the NYHA stage and is individual. In module 1, the patient is given an ECG monitoring card to record his heart frequency and detect cardiac arrhythmias. Module 2 consists of weight monitoring. ECG and weight monitoring are linked together in modules 3 and 4. All reported data is integrated into an electronic patient record by the telemedical service centre Vitaphone, which is certified according to ISO and the Association for Engineering/Electronics/Information Technology (VDE) rules of application for telemonitoring , 24. In The “CorBene” project is being evaluated in terms of health economics. Initial data from 306 patients show that 81.2% of the patients included in the project are being treated according to the guidelines. 10.8% of these patients show clear improvement of clinical symptoms since they have been included in the project . The finIn the meantime, “CorBene”, which initially was a regional project, has been extended to cover all of North Rhine Westphalia (NRW) and Saarland. Currently more than 2900 patients with chronic heart failure are monitored by the project.Integrated concepts of providing medical care that utilize telemedicine have been proven to be appropriate instruments for improving current deficits for patients with cardiac failure. For this reason the Techniker Krankenkasse health insurance company and the German Foundation for the Chronically Ill developed the integrated programme of medical care called “Telemedicine for the Heart”. Patients from throughout Germany with NYHA stages II-IV who are insured with the Techniker Krankenkasse have profited from this programme since January 1st, 2006.The modularly structured programme for providing care, collecting data, interpreting data and training patients allows the participants to deal with their illness in a safer more self-empowered manner. It is also an important adjuvant to the treatment provided by their physicians. The goal of the patient-centred programme is to help the participating patients gain patient empowerment, to understand their illness better, to make the influencing factors between chronic heart failure, the prescribed medicine and their own life style more transparent. It also increases patient awareness of warning signs and symptoms, that indicate imminent decompensation. The analysis of life style with reference to the patient's own health situation encourages participants to reduce risk factors and expand health-promoting factors. By means of close coordination with the locally responsible physician, the programme aims to reduce the frequency and length of inpatient hospitalisations, at decreasing mortality, and at offering participants coordinated and high-quality medical care . The programme is set to run for 27 months, which are divided into three phases. The first 6-month is the training phase. It familiarises the patients with the procedures of the programme and, by means of telephone-based training, provides specific knowledge about the complex topics associated with chronic heart failure. The second phase is the 3-month stabilisation phase. It promotes a consolidation of the behavioural patterns taught during the first phase and attempts to teach how to deal with the illness independently on a daily basis. The third phase consists of an 18-month refresher phase, aiming to anchor the behavioural patterns that promote better health in daily routines of participating patients, through constant repetition. This should allow, the results that have been achieved to be maintained after the participants have left the programme. The care and training given to patients is provided by a telemedical service centre . An electronic record is maintained for each patient at the telemedical service centre. For patients in NYHA stages III and IV, telemedical monitoring of body weight, blood pressure, and the pulse is also planned. During the programme, necessary interventions are made by the telemedical service centre team in coordination with the general practitioners and/or the cardiologists if a threshold is crossed.P = .03, t-test). This means that it was possible to avoid approximately every fifth hospitalisation as the result of this intervention (i) an improved level of knowledge among the participating patients, (ii) an early warning function of the telemedical monitoring, and (iii) close coordination between the telemedical centre and the responsible physicians. In the context of a health economics evaluation conducted by the chair of health management at the University of Erlangen-Nuremberg, the data of 281 participants in the programme “Telemedicine for the Heart” (treatment group) were compared to the data of a matched control group that was three times larger in size. Examination of the number of hospitalisations per patient and year, showed that for the participants in the programme there were 21.5% fewer hospitalisations reduction in mortality within the first year, that is, that with the most intensive training and support phases. Even if the analysis is extended to the maximum period of time (including the last phase of the programme with a low intensity of care provision), the mortality was reduced by 35.1% , a value more than one third lower than the mortality in the comparison group.The evaluation also provides a clearly positive result with regard to mortality. In the group of programme participants, there was a 51.7% as well as over the maximum period of time observed normalised per year . These data make it clear that the complete 27-month programme fully paid for itself in its first year of work, while the effects of the care provision—especially the effect of the training sessions—were maintained over a much longer period of time.The medical effects reported above were achieved in a cost-efficient manner, as is shown by the overall costs. There are substantial differences in favour of the treatment group, both after 1 year of programme membership and follows patients for a period of at least 12 months. In the telemedical group, there is daily monitoring of blood pressure, ECG, oxygen saturation, and body weight. Furthermore the patients' physical activity is evaluated by an activity sensor and the patient himself evaluates his own health status. The study is financed and supported logistically by the Barmer and Bosch health insurance companies. The primary end point of the study is the period of survival. Secondary end points of the study are overall mortality, cardiovascular mortality, frequency of any kind of nonelective hospitalisation, frequency of hospitalisations for cardiovascular reasons, the plasma level of NT-proBNP over time and the quality of life over time, An evaluation from the perspective of health economics is planned , 28.The project “HeartAs” was established in combination with an integrated-care contract. Until now, 516 patients have been included in the project, 96 of which have already concluded the program. The majority of patients are in NYHA stage II and III. In this project, body weight, ECG (12-channel or 1-channel), pulse, and blood pressure are transmitted. The project consists of a modular character. In addition to compliance management and emergency management modules, regular telephoneadvice calls are given. A medical, academic, and economic evaluation will follow.Some initial experience regarding the telemedical monitoring of patients with chronic heart failure has been gathered in a project organised by the health insurance company “Kaufmännische Krankenkasse” and the Almeda company in Munich. The idea behind this project was similar to that of the ProHeart project. Participants were contacted regularly by telephone by specially trained medical personnel. On the one hand, the use of specially developed software meant that the telephone conversations were very standardized, while on the other, there was always an opportunity to discuss the patient's situation in detail in an individual and problem-oriented manner. The telephone contacts were supplemented by written training material . There wDuring the observation period, 14.7% of the participating patients died, compared to 27.1% in the control group. This difference was statistically significant .The project “ProHeart” is currently conducted by Almeda company and the Allianz health insurance. Up to now, 1545 patients in NYHA stages I–IV have been included in the projectThe German projects on telemedical monitoring of patients with chronic heart failure that are described individually above exhibit different approaches and plans for improving the situation of these patients. Nonetheless, they share some fundamental strategies. The goals are, besides monitoring the patients' status , to remind patients of their medication, to monitor symptoms indicative of imminent cardiac decompensation, and to train patients on topics relevant to heart failure. A significant concern of all the programmes is increasing the ability of the patients themselves to manage their own illness. Most programmes are planned for a limited time period. The timely limitations of this program have to be discussed critically. Most of the projects described were established and conducted within the confines of integrated-care-contracts over a predefined frame of time. Undisputed is the fact that chronic heart failure can improve only with consistent surveillance, for which reason patients need further long-term monitoring. This poses the question of which patients (NYHA stage) derive the most benefit from telemonitoring and which intensity should be adjusted to individual patient needs. As a foundation, consistent definition and measures of goals needs to be defined . The discussed projects demonstrate the heterogeneous of patients included according to differences in NYHA stages. In a meta-analysis of 14 randomised controlled studies on telemonitoring of patients with heart failure, Clark et al. demonstrated there was a 20% reduction in overall mortality . The effRoth and assoThe results clearly show that telemonitoring and telephone support programmes are beneficial with regard to mortality and the rehospitalisation rates for patients with chronic heart failure –17. The The reasons discussed for this include the high level of standard treatment for heart failure and intermittent data transmission (once a week) . The telThe electronic patient record is the central component of telemedical patient monitoring. Recognising deviations from established alarm thresholds allows both patients and the treating physician to be alerted promptly. This permits close coordination between the partners and early intervention in the patient. In addition, personal telephone contact to the patient is established through the telemedical service centre. By means of this, enquiries can be made about symptoms and complaints, it can be checked whether medication is being taken, and patient education can be coordinated. The quality of the telemedical care systems depends significantly on the work of the employees in the telemedical service centre. The aim here must be to standardise procedures and certification of the telemedical service centres in the context of quality assurance . To thisAnother essential concern in telemedical monitoring is intercommunication between general practitioners, cardiologists in private practice, hospitals and rehabilitation facilities. This aspect is of great importance in Germany since here there is traditionally a great division between outpatient and inpatient sectors. Most telemedical monitoring programmes were conducted for this reason in the context of contracts for integrated care. In doing this, in addition to physicians, medical insurance companies and telemedical providers would be directly included in the projects.The greatest challenge at the moment is the reimbursement of telemedical services. This applies both to the service providers and the telemedical providers. At the moment in Germany, telemedicine for monitoring patients with chronic heart failure is not a component of standard treatment. Thus it can also be explained why most projects are regional in character. In the next few years, it will be necessary to more strongly develop the evidence of the benefits of telemedical monitoring in patients with heart failure. For this, in addition to medical and economic evaluations , health-economic evaluations are also necessary. The programmes presented here have this health-economic evaluation integrated into them as a fixed component of the programmes. Proof of the cost efficiency of the programmes will be a crucial point for the future of telemedical monitoring programmes. Currently, none of the described projects have been completed, so that final results are not yet available.In Germany, various projects on telemedical monitoring of patients with chronic heart failure could have been set up in the past few years. Most of these projects were initiated in the context of contracts of integrated care and are mostly regional in nature. The telemedical monitoring programmes differ in terms of their choice of monitored vital parameters, sequence and frequency of data transmission, and duration. This makes a comparison only possible to a limited extent. In addition, most of the programmes are not yet completed, so that no final assessment of the medical-economic or health-economic evaluation is yet possible. The further acceptance of patients and sponsors of projects on telemedical monitoring of patients with chronic heart failure will depend in the future on the evidence for improvement of the medical variables as well as evidence of cost efficiency. Standardization is required of the programmes for telemedical monitoring of patients with chronic heart failure according to defined quality criteria. For the context of further studies some basic questions, such as which patients (NYHA stage) derive the most benefit at which degree of intensity of telemonitoring, need to be considered, in order to achieve lasting improvements for these patients. In addition, attention need to be given to the affects of the physical separation of telemonitoring on the doctor-patient relationship in order to increase the physician's and patient's acceptance of telemonitoring.

Research using the zebrafish model has experienced a rapid growth in recent years. Although real-time reverse transcription PCR (QPCR), normalized to an internal reference ("housekeeping") gene, is a frequently used method for quantifying gene expression changes in zebrafish, many commonly used housekeeping genes are known to vary with experimental conditions. To identify housekeeping genes that are stably expressed under different experimental conditions, and thus suitable as normalizers for QPCR in zebrafish, the present study evaluated the expression of eight commonly used housekeeping genes as a function of stage and hormone/toxicant exposure during development, and by tissue type and sex in adult fish.bactin1, tubulin alpha 1(tuba1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), glucose-6-phosphate dehydrogenase (g6pd), TATA-box binding protein (tbp), beta-2-microglobulin (b2m), elongation factor 1 alpha (elfa), and 18s ribosomal RNA (18s) during development ; in different tissue types of adult males and females; and after treatment of embryos/larvae (24 – 96 hpf) with commonly used vehicles for administration and agents that represent known environmental endocrine disruptors. All genes were found to have some degree of variability under the conditions tested here. Rank ordering of expression stability using geNorm analysis identified 18s, b2m, and elfa as most stable during development and across tissue types, while gapdh, tuba1, and tpb were the most variable. Following chemical treatment, tuba1, bactin1, and elfa were the most stably expressed whereas tbp, 18s, and b2m were the least stable. Data also revealed sex differences that are gene- and tissue-specific, and treatment effects that are gene-, vehicle- and ligand-specific. When the accuracy of QPCR analysis was tested using different reference genes to measure suppression of cyp19a1b by an estrogen receptor antagonist and induction of cyp1a by an arylhydrocarbon receptor agonist, the direction and magnitude of effects with stable and unstable genes differed.QPCR analysis was used to quantify mRNA levels of This study provides data that can be expected to aid zebrafish researchers in their initial choice of housekeeping genes for future studies, but underlines the importance of further validating housekeeping genes for each new experimental paradigm and fish species. To illnot 18s) . A similof gapdh . In zebranalysis ,21.cyp19a1b expression following ICI treatment demonstrates the impact on a modest gene response. When housekeeping genes affected by ICI are used for normalization the expected down-regulation is negated. If this situation were to occur when testing a novel chemical, or when using QPCR to verify results of microarray analysis, a real effect could be overlooked. In the case of cyp1a, a gene that is robustly induced by AhR ligands, a difference in housekeeping gene expression results in a more than 3-fold exaggeration in upregulation. This kind of overstatement could have real implications for data interpretation, for example, when comparing dose-response characteristics of different chemical agents, or when screening environmental samples for bioactivity. To avoid unforeseen errors in normalization, for example, by the presence of unknown agents in complex mixtures that affect reference gene expression, the stability of the chosen housekeeping gene can be routinely monitored by recording changes in Ct values.Although changes in housekeeping gene expression in response to EtOH, DMSO or low doses of chemicals appear small, even small differences can add significant error to target gene expression during normalization. The example of Given that many of the classical reference genes have proven unreliable -28, alteAnother promising method for normalization is the use of statistical software to determine the most stably expressed gene . By usingapdh clearly has large variability in its expression under all experimental conditions tested in our study and so would not be recommended for normalization. Studies by Tang et al in zebrafish [gapdh to be unsuitable for data normalization. The gene with the least variability across all the conditions assessed in this study was elfa and so may be an appropriate initial selection for normalization.The intent of this study was to provide a database that helps zebrafish researchers to identify a shortlist of candidate housekeeping genes for specific experiments. For example, although it has been a relatively popular housekeeping gene for zebrafish research, ebrafish and Filbebrafish also fouelfa, whereas gapdh was unstable under all conditions. Results of this study are intended to guide zebrafish researchers with initial selection of a reference gene, but underline the importance of accurate housekeeping gene validation for each new experimental paradigm.All eight housekeeping genes tested were found to have some degree of variability under the conditions tested here, but genes most suitable as normalizers during development, across tissue types, and in chemical treatment experiments were identified. The gene with a low degree of variability across all conditions was Danio rerio, were obtained from a commercial supplier and maintained in 30 gal aquaria at 28°C on a 14:10 light-dark cycle. Fertilized eggs were collected after natural spawning, washed, and distributed into 20 × 100 mm culture plates (Fisher Scientific). Embryos (150 embryos/50 ml egg water) were allowed to develop at 28°C on a 14L:10D cycle [Wild type adult male and female zebrafish, 0D cycle . For dev18s, reverse transcription was carried out using random primers which results in a lower reaction efficiency. A minimum of two RT reactions were performed for each biological replicate for technical replicate comparison.Adult tissues or whole embryos were homogenized in Tri Reagent (Sigma) and total RNA was extracted as previously described and treatuba1, tbp, and b2m were developed using Primer Express software (Applied Biosystems) and entered into the Real Time PCR Primer Databank . bactin1 primers were obtained from the Real Time PCR Primer Databank [Eight housekeeping genes were selected from commonly used reference genes Table . All oliDatabank . Primer Real time PCR was performed on an ABI Prism 7900 HT sequence detection system (Applied Biosystems) with SYBR green fluorescent label. Samples contained 1× SYBR green master mix, 2–4 pmol of each primer and 0.25 μl RT reaction for a final volume of 10 μl. Samples were run in triplicate in optically clear 384-well plates (Applied Biosystems). Cycling parameters were as follows: 50°C × 2 min, 95°C × 10 min, then 40 cycles of the following 95°C × 15 s, 60°C × 1 min. For each sample a dissociation step was performed at 95°C × 15 s, 60°C × 15 s, and 95°C × 15 s at the end of the amplication phase to identify a single, specific melting temperature for each primer set. PCR was performed twice on each sample for a minimum of 36 data sets generated for each sample/gene combination .QGene to determine the PCR amplification efficiency (E) for each primer pair where E = 10(-1/slope) as determined by linear regression analysis of a dilution series of reactions [[E), together with the Ct values, was used to calculate a relative gene expression value for each transcript using the equation E ΔCt(Min Ct-Ct sample) where Min Ct is the lowest Ct value for the primer pair and Ct sample is the Ct value for each amplification [t-test was used to compare differences in mean Ct values between male and female tissues. Student's t-test was also used to determine significant differences in expression following chemical treatment. Vehicle was compared to untreated and all other chemicals were compared to the vehicle of preparation (DMSO). Significance was set at P < 0.05.Data generated by real-time PCR were compiled and collected using SDS 2.2 software (Applied Biosystems). Data were exported to actions [; see Resfication . The Ct fication . The relfication . This prPCR: polymerase chain reaction; RNA: ribonucleic acid; Ct: cycle threshold; ANOVA: analysis of variance; S.E.M: standard error of the mean; EtOH: ethanolATM conceived of the study, has been responsible for all the experimental work, and has been involved in drafting and revising the manuscript. GVC participated as a supervisor in study design and analyses. She has been involved in drafting the manuscript, revising it critically for important intellectual content, and given final approval for the version to be published

The development of small molecule inhibitors of hepatitis C virus (HCV) core protein as antiviral agents has been intensively pursued as a viable strategy to eradicate HCV infection. However, lack of a robust and convenient small animal model has hampered the assessment of in vivo efficacy of any antiviral compound.The objective of this work was to develop a novel method to screen anti-core protein siRNA in the mouse liver by bioluminescence imaging. The inhibitory effect of two shRNAs targeting the highly conserved core region of the HCV genome, shRNA452 and shRNA523, was examined using this method. In the transient mouse model, the effect of shRNA-523 was detectable at as early as 24 h and became even more pronounced at later time points. The effect of shRNA-452 was not detectable until 48 h post-transduction. In a stable mouse model, shRNA523 reduced luciferase levels by up to 76.4±26.0% and 91.8±8.0% at 6 h and 12 h after injection respectively, and the inhibitory effect persisted for 1 day after a single injection while shRNA-Scramble did not seem to have an effect on the luciferase activity in vivo.Thus, we developed a simple and quantitative assay for real-time monitoring of HCV core protein inhibitors in mice. HCV infection is a major cause of chronic liver diseases, which often progresses to liver cirrhosis and hepatocellular carcinoma (up to 20%) HCV is an enveloped RNA virus that belongs to the family Flaviviridae In the present work, we established a transient mouse model and stable mouse model by hydrodynamics methods to screen of HCV core protein inhibitors. The inhibitory effect of hairpin shRNAs targeting the core region of the HCV genome was monitored in the mouse liver by bioluminescence imaging. Finally, we found that the expression level of core protein could be reflected by the activity of Fluc in the mouse model, and shRNA targeting HCV core protein could effectively downregulate core gene and Fluc gene expression in vivo. These models could be used for screening anti-HCV compounds.C57BL/6 mice were obtained from and fed in National Beijing Center for Drug Safety Evaluation and Research (NBCDSER).This study was approved by the ethics committee of the NBCDSER (Permit No.09-1425).attB, as were previously described attB-Fluc gene was fused in-frame to the downstream of HCV core gene by a short linker gene (GGTGGTGGTTCCGGTGGTGGT). Plasmid pGL3-attB-Core was generated by deleting the Fluc gene from the pGL3-attB-CoreFluc between NcoI and XbaI sites. The plasmids expressing shRNAs against the following regions of the HCV core-protein sequence: 452–472 nt, 479–499 nt, and 503–523 nt, were also constructed pCMVInt and pT-ttB-Fluc was gene5 cells/well in 24-well dishes with 0.6 µg of a plasmid DNA mixture following the manufacturer's instructions. Plasmids were purified with the Qiagen plasmid purification kits . In each case, 1 ng of a plasmid that had the Renilla luciferase gene driven by the herpes simplex virus thymidine kinase (HSV-TK) promoter was included in the assay to monitor transfection efficiency. After 48 h, cells were washed with PBS and harvested in 100 µl of Passive Lysis Buffer . Firefly and Renilla luciferase activity was measured in a GloMax™ 96 luminometer from 20 µl of lysate using the Dual-Luciferase Reporter Assay System (Promega).Huh7 cells were cultured in DMEM with 10% FBS at 37°C in 5% CO2/air. Transient transfections were performed by using Lipofectamine 2000 , starting from approximately 1×10For the long-term study, plasmids were purified with the Endotoxin Free Maxi Kit and administrated to C57BL/6 mice by the hydrodynamics method attB-1. The cycling conditions were 94°C for 30 s, 55°C for 30 s and 72°C for 30 s. The products were used as templates in the second round PCR with primers mspL1rev and attB-2 under similar conditions to those for the first round PCR. The second-round PCR products were cloned into pGEM-T (Promega) and sequenced. The primers were showed as follows 5′-TGAGGAGGAGCCTTAGCAAC-3′; attB-1: 5′-GTAGGTCACGGTCTCGAAGC-3′; attB-2: 5′-CGAAGCCGCGGTGCGGGTGCCA- 3′.Animals were sacrificed after 2weeks (control group) and 3 months (test group).The livers were removed and genomic DNA isolated using the Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer's instructions. To detect site specific integration at mpsL1 (mice pseudo-site from liver ), a nested PCR approach was followed. Mice liver genome DNA was used as template for the first round PCR with primers mspL1rev and Proteins were extracted from liver tissues or transfected cells following the manufacturer's instructions. 10 µg of proteins was loaded on a 10% SDS–polyacrylamide gel in Laemmli sample buffer. Proteins were separated by electrophoresis and transferred onto a Hybond-P membrane . The membrane was blocked overnight at 4°C in 5% nonfat milk and incubated with anti-HCV-core or anti-luciferase antibody followed by secondary antibody. Immunoreactive proteins were visualized using SuperSignalWest Pico Chemiluminescent Substrate (Pierce).For routine histological analysis, formalin-fixed paraffin-embedded liver samples were cut into sections 4 µm thick, deparaffinized in xylene, and dehydrated through a series of decreasing concentrations of ethanol. Sections were stained with hematoxylin and eosin.Serum alanine aminotransferase was measured in 25 µl samples using a microtiter plate assay IL-6 and IL-1βwere measured by immunoreactivity in a double-sandwich enzyme-linked immunosorbent assay (ELISA) format using commercially available kits for mouse cytokines according to the manufacturer's recommendations.P<0.05.All data were reported as mean±SD. For statistical comparisons, parametrical data were compared using the Student's t-test. Differences were considered significant at attB-Core, pGL3-attB-Fluc and pGL3-attB-CoreFluc were transfected into Huh7 cells respectively. Forty-eight hours after transfection, Fluc activities were measured. The Fluc activity was significantly higher in cells transfected with pGL3-attB-Fluc than in those transfected with pGL3-attB-CoreFluc. No Fluc activity was measured in cells transfected with pGL3-attB-Core were designed to target the highly conserved core region of the HCV genome. pGL3-shRNA523 . The inhshRNA523 . The resattB-CoreFluc and 10 µg of shRNA-Scramble, shRNA-452, shRNA-523 or shRNA-Fluc expression vectors respectively. Bioluminescence imaging was performed to examine luciferase expression in the liver at the indicated time after DNA injection. As illustrated in We proceeded to investigate whether two of these shRNAs employed in cell culture could similarly mediate a gene-silencing effect in adult mice by transient transfection, using real-time bioluminescence imaging. Four groups of mice were injected via the tail vein with 10 µg of pGL3-attP” sites to achieve long-term gene expression attB-CoreFluc was injected with either 10 µg of carrier plasmid pCS (control group) or the integrase expression vector pCMV-Int (test group) into the tail vein of mice. The luciferase activity was measured at different time points using the bioluminescence method. There was a high level of luciferase expression in the livers of all the mice 24 h after injection. When pCMV-Int was included, transgene expression decreased ∼30-fold within two weeks and lasted until day 420 and 91.8±8.0% (P = 0.00001) compared to before injection, at 6 hr and 12 hr after injection respectively , or shRNA523 expression vectors. Serum levels of alanine aminotransferase (ALT), a marker of liver function, were evaluated . ALT levHCV genome is an ssRNA that functions as both an mRNA and a replication template so that destruction of HCV RNA could eliminate not only virally directed protein synthesis, but also viral replication. In cell culture, siRNA, as well as vector-encoded shRNA directed against the HCV viral genome, specifically targeting the HCV-5′-NTR, core, NS3, NS4b, NS5a and NS5b inhibits HCV gene expression We described here a novel method to screen anti-core protein siRNA in the mouse liver. By using the reporter gene, anti-core protein compounds can be screened by simply bioluminescence imaging the Fluc activity in whole animals under true physiological conditions.attB-CoreFluc, which encoded the fly luciferase (reporter gene) fusing to the downstream of HCV core protein as a silencing target, were co-transfected into Huh7 cells and the mouse liver. In cell culture, all the three shRNAs caused significant reduction in the level of HCV core protein while the sramble shRNA had no inhibitory effect on core protein expression. This observation had been previously reported by other groups. But Suzuki et al considered that shRNA452 construct mediated more effective inhibition of HCV replication than the other core-shRNAs (shRNA479 and shRNA523) In this study, three shRNAs targeting the highly conserved core region of the HCV genome and the plasmid pGL3-There are some special requirements for assays used in drug discovery that are related to the nature of the information needed to understand drug action. Besides, advanced characterization of compounds typically requires answers to questions such as the relationship between duration of action and pharmacokinetics or the maintenance of efficacy after repeated dosing. So a stable mouse model can help to identify and evaluate specific compounds for their potential efficacy.attB site into endogenous positions in the genome of mouse liver cells Phage ΦC31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. Several studies document that phage ΦC31 integrase can site-specifically integrate plasmid DNA bearing an Short hairpin RNAs (shRNAs) have emerged as a novel therapeutic modality, but there is increasing concern over nonspecific effects in vivo In conclusion, a simple and quantitative method of real-time monitoring of HCV core protein inhibitors in mice has been successfully developed. Moreover, the method clearly demonstrates that shRNA targeting HCV core protein can effectively downregulate core gene and reporter gene expression in the liver of mice. This luminescence-based method allows continuous monitoring of the kinetics of HCV core protein inhibitors in live animals. This novel and simple method can be used for screening anti-HCV compounds.

The dysregulation of the mTOR pathway has been found to be a contributing factor of a variety of different cancer. To investigate the role of mTOR signal pathway in the stepwise development of gastric carcinomas, we analyzed the correlations between the mTOR and P70S6K expression and clinic pathological factors and studied its prognostic role in gastric carcinomas.mTOR and phospho-p70S6K proteins were examined by immunohistochemistry on tissue microarray containing gastric carcinomas (n = 412), adenomas (n = 47) and non-neoplastic mucosa with a comparison of their expression with clinicopathological parameters of carcinomas.There was no difference of mTOR expression between these three tissues (p > 0.05). Cytoplasmic phospho(p)-P706SK was highly expressed in adenoma, compared with ANNMs (p < 0.05), whereas its nuclear expression was lower in gastric carcinomas than gastric adenoma and ANNMs (p < 0.05). These three markers were preferably expressed in the older patients with gastric cancer and intestinal-type carcinoma (p < 0.05). mTOR expression was positively correlated with the cytoplasmic and nuclear expression of p-P70S6K(p < 0.05). Nuclear P70S6K was inversely linked to tumor size, depth of invasion, lymph node metastasis and UICC staging (p < 0.05). Univariate analysis indicated that expression of mTOR and nuclear p-P70S6K was closely linked to favorable prognosis of the carcinoma patients (p < 0.05). Multivariate analysis showed that age, depth of invasion, lymphatic invasion, lymph node metastasis, Lauren's classification and mTOR expression were independent prognostic factors for overall gastric carcinomas (p < 0.05).Aberrant expression of p-P70S6K possibly contributes to pathogenesis, growth, invasion and metastasis of gastric carcinomas. It was considered as a promising marker to indicate the aggressive behaviors and prognosis of gastric carcinomas. Gastric carcinoma ranks as the world's second leading cause of cancer mortality behind lung cancer despite a sharp worldwide decline in both its incidence and mortality since the second half of the 20th century. It continues to be a major health problem because of the slow decrease in incidence in Asia and high mortality of diagnosed gastric carcinoma in West . TherefoThe mammalian target of rapamycin (mTOR) also known as FK506 binding protein 12-rapamycin associated protein 1 is a serine/threonine protein kinase that supports cell growth, cellular metabolism, cell proliferation, cell motility, cell survival, protein synthesis, and transcription such as angiogenesis and autophagy. mTOR that is an evolutionarily conserved serine-threonine kinase of a 289-kDa in length belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family. mTOR is composed of an N-term; 20 tandem repeats-HEAT which are implicated in protein-protein interactions; and a C-term which includes a FAT domain, a FBR domain, a kinase domain, a NDR domain and a FATC domain. The FATC domain is essential to mTOR activity and the deletion of a single amino acid from this domain abrogates the activity. mTOR can be autophosphorylated via its intrinsic serine/threonine kinase activity. mTOR exerts its multiple functions in the context of two different multiprotein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 is composed of mTOR, Raptor, mLST8, and PRAS40, and importantly activates p70 ribosomal protein S6 kinase and inactivates eIF4E binding protein 1, which promotes protein translation and cell growth. Conversely, mTORC2 is composed of mTOR, Rictor, Sin1, and mLST8, phosphorylates and activates another member of the AGC kinase family, Akt. Current research indicates that mTOR integrates the input from multiple upstream pathways, including insulin, growth factors (such as IGF-1 and IGF-2), and mitogens. mTOR also functions as a sensor of cellular nutrient and energy levels and redox status [P70 S6 kinase (p70S6K) is activated in a signaling pathway that includes mTOR. P70S6K is a mitogen-activated Ser/Thr protein kinase that is required for cell growth and G1 cell cycle progression. This kinase is controlled by multiple phosphorylation events located within the catalytic, linker and pseudosubstrate domains and subsequently phosphorylates specifically ribosomal protein S6. Activation occurs via phosphorylation at ser411, Thr421 and Ser424 within the pseudosubstrate region. Phosphorylation of Thr229 in the catalytic domain and Thr389 in the linker domain are most critical for kinase function. Stimulation of mammalian cells by a variety of mitogenic stimuli results in a rapid, biphasic activation of p70S6K. Inhibition of p70 activity inhibits the entry into S phase of the cell cycle and exhibits cell cycle arrest at G0/G1 phase, suggesting that the activation of p70S6k plays an obligatory role in mediating mitogenic signals during cell activation [mTOR signaling pathway and its downstream serine/threonine kinase p70S6k were frequently activated in human cancers and the dysregulation of the mTOR pathway is implicated as a contributing factor to various human disease processes, especially various types of cancer,6,8-11. Gastric carcinomas (n = 421) were collected from the surgical resection, adenoma (n = 45) from endoscopic biopsy or polypectomy, and gastritis (n = 49) from the endoscopic biopsy in Shengjing Hospital of China Medical University and the First Affiliated Hospital of China Medical University between 1993 and 2006. All carcinomas were adenocarcinomas and the adenoma group was free from non-neoplastic polyp types, leiomyomas and benign GIST's. The patients with gastric carcinoma were 293 men and 126 women . Among them, 156 cases have carcinomas accompanied with lymph node metastasis. None of the patients underwent chemotherapy or radiotherapy before surgery. They all provided consent for use of tumour tissue for clinical research and our University Ethical Committee approved the research protocol. We followed up all patients by consulting their case documents or through telephone.All tissues were fixed in 4% neutralised formaldehyde, embedded in paraffin and incised into 4 mm sections. These sections were stained by haematoxylin-and-eosin (HE) to confirm their histological diagnosis and other microscopic characteristics. The staging for each gastric carcinoma was evaluated according to the Union Internationale Contre le Cancer (UICC) system for the extent of tumour spread . HistoloPrior to TMA construction, all tissue slides were histopathologically re-evaluated by one pathologist and. Two 2.0-mm tissue cores were taken from representative areas of gastric samples using a manual arraying device and mounted in a new recipient block. Four-μm-thick sections were consecutively incised from the recipient block and transferred to poly-lysine-coated glass slides. HE staining was performed on TMA for confirmation of tumor tissue.For the immunohistochemical procedure, 4-μm-thick sections were deparaffinized with xylene and rehydrated through an alcohol gradient. The sections were quenched with 3% hydrogen peroxide in absolute methanol for 20 min to block endogenous peroxidase activity, and heated in a microwave for 15 min in citrate buffer to retrieve the antigen. The sections were incubated with rabbit anti-mTOR antibody and anti-phospho-p70 s6 kinase for 60 min, followed by exposure to the anti-rabbit Envison-PO antibody for 60 min. Binding sites were visualized with 3, 3'-diaminobenzidine (DAB) with the 5-min reaction. After each treatment, the slides were washed with TBST three times for 1 min. After counterstained with Mayer's haematoxylin, the sections were dehydrated, cleared and mounted. Omission of the primary antibody was used as a negative control.As indicated in Figure Statistical evaluation was performed using Spearman correlation test to analyze the rank data. Kaplan-Meier survival plots were generated and comparisons between survival curves were made with the log-rank statistic. The Cox's proportional hazards model was employed for multivariate analysis. p < 0.05 was considered as statistically significant. SPSS 10.0 software was employed to analyze all data.As showed in Figure These three markers were preferably expressed in the older patients with gastric cancer and intestinal-type carcinoma . The 122 patients died from carcinoma and several cases dying from other disease has been excluded. Figure Mammalian target of rapamycin (mTOR) is also known as FKBP-rapamycin-associated protein or rapamycin and FKBP target and functions as a serine/threonine protein kinase to sense adenosine triphosphate and amino acids to balance nutrient availability and cell growth. When sufficient nutrients are available, mTOR is phosphorylated via the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway, transmits a positive signal to p70 S6 kinase (p70S6K), and participates in the inactivation of the eukaryotic translation initiation factor 4E inhibitor, 4EBP1. Therefore, mTOR plays a key role in cellular growth and homeostasis, and its regulation is frequently altered in tumors ,15.Although mTOR protein can shuttle between the nucleus and cytoplasm ,17, we oThe 70-kDa S6 kinase (p70S6K) is a cytoplasmic Ser/Thr kinase that is mainly known to regulate protein translation through phosphorylation of the 40S ribosomal protein S6. Activation of p70S6K is achieved through phosphorylation on multiple Ser/Thr residues by stimulation with growth factors such as epidermal growth factor (EGF), thrombin, and lysophosphatidic acid (LPA),24. To tAlthough gastric cancer is malignant tumor originating from the same gastric epithelium, its morphological features vary substantially with the individual patients . AccordiTo clarify the prognostic significance of mTOR, cytoplasmic or nuclear P70S6K expression, we here analyzed their relation with the survival of 412 patients with gastric carcinoma and found a close relationship link between the positivity of mTOR and nuclear phospho-P70S6K expression and favorable survival. Multivariate analysis demonstrated six independent prognostic factors such as age, depth of invasion, lymphatic invasion, lymph node metastasis, Lauren's classification and mTOR expression were independent prognostic factors for overall gastric carcinomas. However, several evidences indicated that phosphor-mTOR expression was closely linked to the poor prognosis of the patients with cervical adenocarcioma or hepatocellular carcinoma ,25. PS6KAberrant expression of p-P70S6K might play an important role of malignant transformation of gastric epithelial cells and was closely related to growth, invasion, metastasis and prognosis of gastric carcinomas and was considered as a promising marker to indicate the pathobiological behaviors. The distinct expression of mTOR and p-P70S6K could be employed to differentiate the intestinal- and diffuse-type carcinomas and underlay the molecular mechanism about the differentiation of both carcinomas. Nuclear p-p70S6K was a good marker to indicate the favorable prognosis of gastric carcinoma patients, albeit dependent on other parameters, but mTOR expression was an independent factor for the prognosis.The authors declare that they have no competing interests.LX designed the research and wrote the paper. WSL and YCW carried out the the immunoassays and collected the gastric cancer tissues. LX and WSL carried out the pathological diagnosis and data analysis. YD prepared the Tissue microarray. All authors have read and approved the manuscript.

Shortly after weaning, a complex multi-step process that leads to massive epithelial apoptosis is triggered by tissue local factors in the mouse mammary gland. Several reports have demonstrated the relevance of mechanical stress to induce adaptive responses in different cell types. Interestingly, these signaling pathways also participate in mammary gland involution. Then, it has been suggested that cell stretching caused by milk accumulation after weaning might be the first stimulus that initiates the complete remodeling of the mammary gland. However, no previous report has demonstrated the impact of mechanical stress on mammary cell physiology. To address this issue, we have designed a new practical device that allowed us to evaluate the effects of radial stretching on mammary epithelial cells in culture.We have designed and built a new device to analyze the biological consequences of applying mechanical stress to cells cultured on flexible silicone membranes. Subsequently, a geometrical model that predicted the percentage of radial strain applied to the elastic substrate was developed. By microscopic image analysis, the adjustment of these calculations to the actual strain exerted on the attached cells was verified. The studies described herein were all performed in the HC11 non-tumorigenic mammary epithelial cell line, which was originated from a pregnant BALB/c mouse. In these cells, as previously observed in other tissue types, mechanical stress induced ERK1/2 phosphorylation and c-Fos mRNA and protein expression. In addition, we found that mammary cell stretching triggered involution associated cellular events as Leukemia Inhibitory Factor (LIF) expression induction, STAT3 activation and AKT phosphorylation inhibition.Here, we show for the first time, that mechanical strain is able to induce weaning-associated events in cultured mammary epithelial cells. These results were obtained using a new practical and affordable device specifically designed for such a purpose. We believe that our results indicate the relevance of mechanical stress among the early post-lactation events that lead to mammary gland involution. Cells are able to act in response to multiple biochemical and biophysical stimuli, depending on their type and function. Particularly, it has been shown that mechanical forces trigger specific events in a variety of cell types under different physiological and pathological situations -5. For eper se, the earliest stimulus to trigger expression and release of local factors that would initiate mammary gland involution [In the mouse mammary gland, post-lactational involution is divided in two distinct phases. The first one starts only hours after weaning, when milk stasis induces the expression of local factors that trigger apoptosis of the alveolar epithelium ,9. Then,volution . To addrvolution , which aThe device designed to apply controlled equibiaxial strain to cells attached to stretchable membranes, consist in 5 different pieces Figure :® made cylinder (item #1) that constitutes the bottom of the assembled device. This piece has a protruding ring in the inner face, where the silicone membrane (43 mm diameter) is placed.1) Delrin® made ring (item #2) with an O-ring that fits the inner membrane holder. This piece is placed inside item #1 and attaches the membrane to the inner holder.2) Delrin® made), which fits in item #2.3) Indenter ring that pushes down the indenter ring (item #3).4) Flange Aluminum screw-top (item #5) that, when turned down, pushes the indenter ring (item #3) that stretches the silicone membrane.The assembly process is described in Figure Selection of materials for building this device and the stretchable membranes was made according to previous reported apparatus ,13. Sinc® made indenter (item #3). Perfluorooctanoic acid (PFOA) is a chemical compound that was detected in trace amounts in finished Teflon products. The Agency's Science Advisory Board of the Environmental Protection Agency (EPA) suggested that PFOA is "likely to be carcinogenic to humans". However, EPA is still in the process of evaluating this information and has not made any definitive conclusions at this time . This should be considered for apparatus manipulation and if the device were used for evaluating effects that could be altered or imitated by traces of this compound.It must be taken into consideration that in this device, cell medium is in close contact with the TeflonElastic silicone membranes were made by vulcanizing liquid silicone rubber with Platinum as catalyst. Preformed matrices were filled with this material that polymerized at room temperature (25°C) during 3–7 days. Later, membranes were treated with 5.7% KOH in methanol for 5 min to neutralize the polymerization-derived HCl. After being washed with double-distilled water, silicone membranes were sterilized by autoclaving. Cell attachment was facilitated by incubating membranes with 50 μg/ml of rat collagen type I in 0.02N acetic acid for 1 h at room temperature. Then, membranes were rinsed with phosphate-buffered saline solution and cells were seeded.5 cells/dish. After attachment, they were grown for approximately 48 h, until the cell culture became confluent. Then, culture medium was removed and cells were washed and held for 12 h under serum-free conditions.The HC11 cell line, derived from pregnant BALB/c mouse mammary glands, was maintained as previously described . To perfMembranes with the attached cells were removed from the culture dishes and carefully placed inside the stretching device Figure , step 1.a is pushed down an h distance, the membrane adopts an "inverted hat" shape of radius a, at the top, and b, at the bottom could be calculated by Equation 2 equipped with a Canon A520 or a Canon Rebel 350 XT digital camera . The obtained phase contrast video images could be then analyzed by the Image J 1.37v software .c-fos detection, the primers used were: sense 5'-TCCCTGGATTTGACTGGAGGTCTG-3' and antisense 5'-CGACTGAGGAAGGGTTCG-3'. The thermal cycling conditions were: 95°C for 3 min, followed by 34 cycles of 30 sec at 95°C, 45 sec at 68.7°C for annealing, and 45 sec at 72°C for extension. Levels of c-fos expression were normalized by gapdh expression that was determined by using the following primers: sense 5'-AAGAAGGTGGTGAAGCAGGCATC-3', antisense 5'-CGAAGGTGGAAGAGTGGGAGTTG-3'. The cycling conditions used were: 95°C for 3 min, followed by 35 cycles of 40 sec at 95°C, 40 sec at 65°C and 40 sec at 72°C. For lif and actin expression analysis, primers and protocols were previously described [Total RNA extraction from HC11 cells was performed using Trizol reagent . For each assay, 1 μg of total RNA was reversed-transcribed as previously described . Real-tiescribed . To dete). For band quantification, the obtained images were converted to grayscale and equal areas encompassing each band were drawn. Then, the integrated density in each rectangle was obtained and the background noise was subtracted for each band. In each case, the obtained value was normalized as indicated in each experiment.Total or nuclear proteins were extracted from HC11 cells as previously described . ProteinAfter carrying out stretching protocols, conditioned medium from stretched and control cells was collected and the Leukemia Inhibitory Factor (LIF) content was determined using a mouse LIF-enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions.Data are expressed as means ± SE. Data were analyzedby one-way ANOVA (following Bartlett's test of homogeneity of variance) followed by Tukey-Kramer as post-hoc test correction for multiple comparisons between means. Statistical comparisons were performed using Statistica (version6.0) software package . Differences were regarded as significant at p < 0.05. Each experiment was performed independently at least three times.The goal of this study was to determine whether applying controlled strain to cultured mammary epithelial cells would induce involution-associated cellular events. The device described herein is capable of delivering up to 30% of two-dimensional homogeneous strain to silicone membranes that were either purchased or made in the laboratory. The device complete description and assembly process are described in "Methods" and in Figure . The homogeneity of the equibiaxial strain was verified by determining the distance between 10 pairs of cells in each orientation in four different microscope fields. Figure Equation 1) relative to the flange displacement (or screw-top thread turns). These observations indicated there was an efficient strain transmission from the silicone substrate to the mammary epithelial cells. Once this was verified, we carried out the studies described below in confluent HC11 cells grown on the flexible membranes.As the flexible membrane remains in the same horizontal plane during strain application, the silicon support and the attached cells can be observed under an inverted microscope before and after stretching. In order to predict the percentage of radial strain applied (RSA) to the elastic substrate, we have developed a theoretical model based on the device geometry (see "Methods"). To test the resultant formula c-fos mRNA expression was induced in a dose-dependent manner under rising single-step RSAs (from 0 to 15%) for 1 h. This induction reached a plateau from 15% to 20% RSA and started to decrease at 30% RSA. Then, time course of c-fos mRNA expression in HC11 cells subjected to 20% linear strain was analyzed. We found that the highest expression levels were reached after 30 min of sustained strain .The mammary gland epithelium undergoes dramatic changes during the successive stages of pregnancy, lactation and involution. In the fully lactating gland, the layer of ductal and alveolar mammary cells form coherent sheets in which cell junctions provide a compact permeability barrier. It has been indicated that mechanical stress imposed by milk secretion on these tightly attached cells after parturition is one of the main factors that determine initiation of the ejection reflex . On the In an effort to understand mechanical strain contribution to mammary gland involution, we have designed, built and validated a new cell-stretching device. The apparatus presented in this study is inspired in the system previously described by Lee and coworkers , but it et al. [Different devices that use elastic membranes substrates to support cell adhesion and transmit strain have been previously described ,19-22. Set al. . In thise.g. strain levels, intervals of static stress, pharmacological treatments, etc.).The device described herein shows the following properties: 1) easy real time optical monitoring of cell geometry, function, and deformation upon strain application; 2) a small size that allows long-term stretching protocols to be performed in a cell incubator; 3) easy sterilization by autoclaving and 4) low cost, which permits the construction of multiple units that can be used to test simultaneously different experimental conditions families have been well characterized: the extracellular-regulated protein kinase (ERK1/2), the c-Jun NH2-terminal protein kinase (JNK), and the p38 . Upon activation through tyrosine and threonine phosphorylation, these proteins translocate to the nucleus and phosphorylate transcription factors, such as AP-1 family members and the serum response factor (SRF) . MAPK acin vivo . Our resin vivo ,45,46.fos gene transcription, it has been determined that both STAT3 and the ERK-mediated pathway co-operate in its induction. In fact, Kunisada et al. [fos mRNA expression in cardiac myocites. Therefore, the same signaling pathways might be interacting in mechanically stressed mammary epithelial cells.Not only ERK1/2, but also STAT3 might be implicated in c-Fos expression induction and activation in epithelial cells . In the a et al. showed tOne of the most critical molecular changes associated with apoptosis induction during mammary gland involution is STAT3 activation via the Janus kinase (JAK) pathway in response to cytokines and growth factors ,49. It hLevels of p-STAT3 significantly decreased after 1 h and were recovered after 6 h Figure . We beliSeveral studies regarding the impact of mechanical stimuli on protein kinase B/AKT (PKB/AKT) activation have been described in endothelial cells , in vascin vivo active (cleaved) caspase-3 was observed only in the shed cells at 12 h and 24 h involution and not in the alveolar wall until 72 h [In spite of p-AKT down-regulation, we have not detected apoptosis induction in the stretched HC11 cells. This observation has different possible explanations. First, it has been reported that in smooth muscle cells, mechanical stress induced apoptosis is p53-dependent . Therefotil 72 h . TherefoIt is important to point out that the experiments showed herein were performed in confluent, but not differentiated, HC11 cells. We observed that grown on silicon as a substrate, these cells show similar features to competent HC11 cells grown on plastic. We observed high expression of STAT5A and low levels of p-STAT5 and β-casein compared to cells treated with lactogenic hormones. Expression levels of these proteins did not significantly change upon stretching (data not shown). However, we do not know whether mechanical stress would be able to block the action of lactogenic hormones and/or would be able to trigger cell death in fully differentiated cells. More experiments are being performed to answer these questions.We have previously reported that tumor cell secreted LIF was able to decrease HC11 cell viability . Here, wThe results showed herein provide, for the first time, experimental evidence that mechanical strain applied to mammary epithelial cells induces molecular events involved in the initiation of post-lactational involution. A big advance was made when it was determined that this biological process was not only regulated by circulating hormones, but also -and primordially- by tissue local factors. Similarly, determining that mechanical forces play a relevant role in the initiation of such a complex process might reveal significant mechanisms underlying cell fate decision in the mammary gland. However, studying this scenario requires new experimental approaches involving the development and/or adaptation of methods and apparatus. Therefore, here we have made a special effort to carefully describe the device and the geometrical model we built to such a purpose. Using them we were able to demonstrate that mechanical stress can trigger intracellular pathways that facilitate epithelial apoptosis and secretion of specific cytokines that may induce death in neighboring non-stretched cells.All authors helped in the design of the cell stretching apparatus. In addition, each of them made the following specific contributions: AQ designed the protocol to prepare the silicone membranes, set up the cell culture conditions, performed the real rime RT-PCR and western blot techniques, made the statistical analysis and graphics of the obtained results and wrote the manuscript draft; MS and JP designed the geometrical model to predict stretching intensity, designed and performed visual analysis of cells and substrates and helped to improve the quality of the lab-made silicone membranes; NR performed the ELISA for LIF and ECK proposed the initial idea of testing the relevance of mechanical stress in inducing mammary involution-associated events; designed and coordinated the biological experiments and wrote the final manuscript. All authors read and approved the last submitted version.

The ethyl­eneglycol ligand forms a non-planar metallacyclic ring by chelating the Ru atom via the O atoms. The O⋯O distances of 2.554 (2) and 2.568 (2) Å are indicative of hydrogen bonding between coordinated ethyl­eneglycol and outer-sphere trifluoro­methane­sulfonate fragments. The crystal packing is stabilized by ionic forces and several CH3⋯·F (2.585 and 2.640 Å) and CH3⋯O inter­actions between the penta­methyl­cyclo­penta­dienyl ligand and trifluoro­methane­sulfonate anion. There is noticeable short inter­molecular contact [2.9039 (16) Å], between an O atom of the SO3 group and a C atom of the penta­methyl­cyclo­penta­dienyl ligand.The title compound, [Ru(C DOI: 10.1107/S1600536807067426/bg2160Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:

Recently our group reported a new class of cyclic Cα-tetrasubstituted tetrahydrofuran α-amino acids prepared from methionine and aromatic aldehydes. We now report the extension of this methodology to aliphatic aldehydes. Although such aldehydes are prone to give aldol products under the reaction conditions used, we were able to obtain the target cyclic amino acids in low to moderate yields and in some cases with good diastereoselectivity.C The product was purified with a 85:15 mixture of PE:diethyl ether (Rf = 0.2) to give 12 as a colourless oil in 2% yield . The product was obtained as an inseparable mixture of the cis and trans product with a cis:trans ratio of 1:9. 1H NMR (CDCl3): δ = 0.90–0.92 ; 1.41–1.52 ; 1.69–1.80 ; 2.42 ; 2.62–2.79 ; 3.45–3.55 ; 3.88–4.11 ; 5.02 ; 5.28 . 13C NMR (CDCl3): δ 16.7 ; 17.2 ; 18.5 ; 19.1 ; 20.4 ; 20.8 ; 27.9 ; 28.4 ; 32.6 ; 66.5 ; 66.6 ; 67.6 ; 82.3 ; 87.5 ; 154.0 ; 154.4 ; 171.2 ; 171.3 . IR (neat) [cm−1]: = 2977, 2877, 2361, 1708, 1492, 1392, 1366, 1250, 1157, 1085, 1052, 940, 848. CI-MS (NH3): m/z 218.2 (9) [MH+ − 2 C4H8], 274.2 (51) [MH+ − C4H8], 330.2 (100) [MH+]. HR-MS [MH+] calcd. for C17H32NO5 330.2280; found 330.2288. MF C17H31NO5. MW 329.43.The synthesis followed GP 1 using [3-(tert-butoxycarbonylamino)-4-tert-butoxy-4-oxobutyl]dimethylsulfonium iodide , caesium hydroxide and 3-methylbutanal . The product was purified with a 85:15 mixture of PE:diethyl ether (Rf = 0.16) to give 13 as a colourless oil in 17% yield . The product was obtained as an inseparable mixture of the cis and trans product with a cis:trans ratio of 1:5. 1H NMR (CDCl3): δ 0.87–0.90 ; 1.30–1.41 ; 1.44–1.47 ; 1.71–1.80 ; 2.37 ; 2.65–2.85 ; 3.76–4.08 ; 4.93 ; 5.12 . 13C NMR (CDCl3): δ 21.6 ; 21.9 ; 23.6 ; 23.7 ; 25.2 ; 25.7 ; 27.9 ; 28.3 ; 38.0 ; 39.3 ; 66.3 ; 66.9 ; 67.6 ; 68.2 ; 79.8 ; 81.7 ; 82.0 ; 82.1 ; 82.8 ; 154.7 ; 155.1 ; 170.6 ; 170.7 . IR (neat) [cm−1]: = 3326, 2957, 2359, 1708, 1497, 1366, 1250,1160, 1116, 1098, 1055, 885, 849, 781. CI-MS (NH3): m/z (%) = 244.2 (25) [MH+ − Boc], 288.2 (44) [MH+ − C4H8], 305.2 (19) [MNH4+ − C4H8], 344.3 (100) [MH+]. HR-MS : [M+] calcd. for C18H33NO5 343.2359; found 343.2358. MF C18H33NO5. MW 343.46.The synthesis followed GP 1 using [3-(tert-butoxycarbonylamino)-4-tert-butoxy-4-oxobutyl]dimethylsulfonium iodide , caesium hydroxide and butyraldehyde . The product was purified with a 85:15 mixture of PE:diethyl ether (Rf = 0.21) to give 14 as a colourless oil in 36% yield . The product was obtained as an inseparable mixture of the cis and trans product with a cis:trans ratio of 1:3. 1H NMR (CDCl3): δ 0.86 ; 1.21–1.55 ; 2.29 ; 2.61–2.79 ; 3.62–4.15 ; 4.91 ; 5.01 . 13C NMR (CDCl3): δ 14.0 ; 14.1 ; 19.8 ; 20.0 ; 27.9 ; 28.3 ; 31.3 ; 32.7 ; 35.6 ; 37.2 ; 66.2 ; 66.8 ; 67.3 ; 68.2 ; 79.8 ; 81.7 ; 82.0 ; 84.1 ; 84.5 ; 154.8 ; 155.1 ; 170.6 ; 170.7 . IR (neat) [cm−1]: = 3334, 2975, 2357, 1709, 1490, 1366, 1250, 1157, 1077, 949, 848. CI-MS (NH3): m/z (%) = 230.2 (18) [MH+ − Boc], 274.2 (30) [MH+ − C4H8], 291.2 (19) [MNH4+ − C4H8], 330.2 (100) [MH+], 676.6 (9) [2 M + NH4+]. HR-MS : [M+] calcd. for C17H31NO5 329.2202; found 329.2210. MF C17H31NO5. MW 329.43.The synthesis followed GP 1 using [3-(tert-butoxycarbonylamino)-4-tert-butoxy-4-oxobutyl]dimethylsulfonium iodide , caesium hydroxide and acetaldehyde . The product was purified with a 80:20 mixture of PE:diethyl ether (Rf = 0.1) to give 15 as a colourless oil in 28% yield . The product was obtained as an inseparable mixture of the cis and trans product with a cis:trans ratio of 1:2. 1H NMR (CDCl3): δ 1.16 ; 1.23 ; 1.44 ; 1.47 ; 2.21–2.40 ; 2.67–2.82 ; 3.78–3.99 ; 4.06 ; 4.94 ; 5.13 . 13C NMR (CDCl3): δ 13.3 ; 15.2 ; 26.9 ; 26.9 ; 27.3 ; 27.3 ; 34.1 ; 36.1 ; 65.1 ; 65.8 ; 66.2 ; 67.5 ; 78.9 ; 79.9 ; 80.7 ; 81.0 ; 153.8 ; 154.1 ; 169.4 ; 169.6 . IR (neat) [cm−1]: = 2973, 2361, 1705, 1489, 1369, 1247, 1158, 1073, 955, 849. CI-MS (NH3): m/z (%) = 202.1 (25) [MH+ − Boc], 246.1 (40) [MH+ − C4H8], 263.1 (34) [MNH4+ − C4H8], 302.1 (100) [MH+], 319.1 (4) [MNH4+]. HR-MS : [MH+] calcd. for C15H28NO5 302.1967; found 302.1966. MF C15H27NO5. MW 301.38.The synthesis followed GP 1 using [3-(tert-butoxycarbonylamino)-4-tert-butoxy-4-oxobutyl]dimethylsulfonium iodide , caesium hydroxide and methacrylaldehyde . The product was purified with a 80:20 mixture of PE:diethyl ether (Rf = 0.22) to give 16 as a colourless oil in 18% yield . 1H NMR (CDCl3): δ 1.37 ; 1.40 ; 1.69 ; 2.35–2.53 ; 2.57–2.71 ; 4.00 ; 4.12 ; 4.31 ; 4.86 ; 5.04 ; 5.32 . 13C NMR (CDCl3): δ 18.5 ; 26.8 ; 27.3 ; 34.8 ; 66.4 ; 67.5 ; 81.2 ; 85.5 ; 112.2 ; 139.7 ; 153.4 ; 169.4 . IR (neat) [cm−1]: = 3409, 3059, 2063, 1614, 1483, 1335, 1242, 1133, 1098, 1055, 887, 823, 708. CI-MS : m/z (%) = 228.1 (28) [MH+ − Boc], 272.1 (41) [MH+ − C4H8], 289.1 (50) [MNH4+ − C4H8], 328.1 (100) [MH+], 345.1 (8) [MNH4+]. HR-MS : [M+] calcd. for C17H29NO5 327.2046; found 327.2043. MF C17H29NO5. MW 327.42.The synthesis followed GP 1 using [3-(File 112–16NMR spectra of compounds File 215CIF file of compound

Purpose: Histological grading is currently one of the best predictors of tumor behavior and outcome in soft tissue sarcoma.However, occasionally there is significant disagreement even among expert pathologists. An alternative method that givesmore reliable and non-subjective diagnostic information is needed. The potential use of proton magnetic resonance spectroscopyin combination with an appropriate statistical classification strategy was tested here in differentiating normalmesenchymal tissue from soft tissue sarcoma. Methods: Fifty-four normal and soft tissue sarcoma specimens of various histological types were obtained from 15 patients.One-dimensional proton magnetic resonance spectra were acquired at 360 MHz. Spectral data were analyzed by using boththe conventional peak area ratios and a specific statistical classification strategy. Results: The statistical classification strategy gave much better results than the conventional analysis. The overall classificationaccuracy (based on the histopathology of the MRS specimens) in differentiating normal mesenchymal from soft tissuesarcoma was 93%, with a sensitivity of 100% and specificity of 88%.The results in the test set were 83, 92 and 76%, respectively.Our optimal region selection algorithm identified six spectral regions with discriminating potential, including thoseassigned to choline, creatine, glutamine, glutamic acid and lipid. Conclusion: Proton magnetic resonance spectroscopy combined with a statistical classification strategy gave good results indifferentiating normal mesenchymal tissue from soft tissue sarcoma specimens ex vivo. Such an approach may also differentiatebenign tumors from malignant ones and this will be explored in future studies.

Perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) are man-made, persistent organic pollutants widely spread throughout the environment and human populations. They have been found to interfere with fetal growth in some animal models, but whether a similar effect is seen in humans is uncertain.We investigated the association between plasma levels of PFOS and PFOA in pregnant women and their infants’ birth weight and length of gestation.We randomly selected 1,400 women and their infants from the Danish National Birth Cohort among those who completed all four computer-assisted telephone interviews, provided the first blood samples between gestational weeks 4 and 14, and who gave birth to a single live-born child without congenital malformation. PFOS and PFOA were measured by high performance liquid chromatography–tandem mass spectrometer.PFOS and PFOA levels in maternal plasma were on average 35.3 and 5.6 ng/mL, respectively. Only PFOA levels were inversely associated with birth weight . Neither maternal PFOS nor PFOA levels were consistently associated with the risk for preterm birth or low birth weight. We observed no adverse effects for maternal PFOS or PFOA levels on small for gestational age.Our nationwide cohort data suggest an inverse association between maternal plasma PFOA levels and birth weight. Because of widespread exposure to these chemicals, our findings may be of potential public health concern. Perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) are persistent organic pollutants (POPs), which are produced synthetically or from the metabolism of other perfluorinated chemicals (PFCs) . These cPFOA and PFOS are slowly metabolized and have half-lives in humans of around 4 years and 5 years, respectively . They arExposure of rodent dams during pregnancy to both PFOS and PFOA has been associated with decrements in birth weight and length of gestation at birth . AlthougIn this study we examined the relation between maternal plasma PFOS and PFOA levels during pregnancy and birth weight as well as length of gestation using data from the Danish National Birth Cohort .The Danish National Birth Cohort (DNBC) includes data on 91,827 pregnant women from March 1996 to November 2002 . Women wn = 87,752), who provided the first blood sample between gestational weeks 4 and 14 , and who had responded to all four telephone interviews , we randomly selected 1,400 mothers. Of the 1,400 selected, 1,102 had a second blood sample, and 200 were randomly selected among these. Of these 200, 146 had a cord blood sample from the child, and 50 were randomly selected among these. We used the second maternal and cord blood samples to examine whether PFOS and PFOA levels were stable during pregnancy and whether the mother–offspring correlation in blood levels was high. Sample sizes were determined in large part by the capacity of the laboratory to perform PFOS and PFOA assays and by what would be acceptable for the Steering Committee that gives permission for use of cohort data.Among all participants who gave birth to a single live-born child without a reported congenital malformation (http://www.bsmb.dk) (n = 10), we used the expected date of delivery provided by the pregnant women at the second interview (after their ultrasound examinations). Low birth weight (LBW) was defined as birth weight < 2,500 g. Preterm birth was defined as the birth of an infant before 37 completed weeks of gestation (259 days), post-term birth as after 42 completed weeks of gestation (294 days), and term birth as between 37 and 42 completed weeks of gestation. Small for gestational age (SGA) was defined as an infant with birth weight below the 10th percentile at a specific gestational age in weeks, based on all singleton live births of same sex and parity who fulfilled our sampling criteria.Information on potential confounders—such as infant sex, maternal age, parity, socio-occupational status, prepregnancy body mass index (BMI), and smoking during the pregnancy—was collected by highly structured questionnaires (available at bsmb.dk) . Socio-obsmb.dk) . If estiThis research was approved by the UCLA Office for Protection of Research Subjects (Reference No. 06-08-023-01) and the Danish Data Protection Agency (Reference No. J.Nr 2006-41-6324).18O2 PFOS) and PFOA (13C2 PFOA) were used in all extracted procedures. All extractions were performed using solid phase extraction techniques and based on 100 μL of plasma. Details of the analytical methods used are available elsewhere and PFOA . Only two PFOA values and no PFOS values were reported at the lower limit of quantitation (LLOQ) of 1.0 ng/mL. Values below the LLOQ (n = 2) were assigned a value of half the LLOQ. PFOS and PFOA measurements were determined on the residual plasma component from 15 frozen whole blood samples . These were multiplied by a factor of two to make them comparable to measurements made on plasma and 95% confidence interval (CI) for low birth weight, SGA, and preterm birth. The PFOS and PFOA levels were entered into the analyses as both continuous and categorical variables . Plasma levels below the 25th percentile were used as the reference group when using categorical variables. Both log-transformed and untransformed blood PFC concentrations were tested in the statistical models, and the findings were similar. For this article, we used only the results of untransformed concentrations. We also conducted regression diagnostics for influential points using Cook’s distance, but no substantial changes in estimates were observed before or after the influential points were excluded, and our results are therefore all based on the whole data set.We adjusted for risk factors that could influence fetal growth or length of gestation at birth. These factors included maternal age, parity, socio-occupational status, prepregnancy BMI, smoking during pregnancy, infant sex, and gestational week at blood drawing. For birth weight, we also adjusted for gestational age at birth, measured in days and entered as linear and quadratic terms into the model to capture the reduced weight gain during the last weeks. All covariates but gestational age at birth and gestational week at blood drawing were introduced into models as indicator variables. Stratified analyses by parity, prepregnancy BMI, term of birth and sex were also performed to evaluate the consistency of the associations across strata.2. The mean birth weight was 3,623 g, and the incidence of LBW was 1.7%; the mean gestational age at birth was 280 days, and the incidence of preterm birth and post-term birth was 3.8% and 9.9%, respectively , and lowest in the age group of ≥ 35 years (5.1 ng/mL), but after adjustment for parity the difference was reduced to 0.4 ng/mL. High levels were also observed in overweight and obese women .r = 0.62), and after adjustment for PFOS levels in the first maternal blood, the regression coefficient of PFOA became slightly stronger . Analyses stratified by term of birth revealed that the effects of PFOA on birth weight were more pronounced in preterm and post-term babies (r = 0.72 for PFOS and 0.84 for PFOA if two outliers were not included) , but thencluded) , but corTo our knowledge, this is the first nationwide population-based study on birth weight and length of gestation with prospectively collected data on levels of perfluorinated chemicals in maternal sera drawn early in pregnancy. We found only maternal plasma PFOA levels to be inversely associated with birth weight. Risk estimates for preterm birth did not increase monotonically across quartiles of PFOS or PFOA exposure. For all four outcomes evaluated, the outcome in the lowest exposure quartile differed from outcomes in the upper three quartiles, but there was no evidence of a dose–response association above the 25th percentile.Maternal plasma levels in the present study are similar to most levels reported for U.S. populations, but much lower than those found in fluorochemical workers or residents living near the fluoropolymer production facilities . We founThis study has several strengths. We used state-of-the-art laboratory facilities, rigorous blinding, and hospital records for obtaining data on birth weight and gestational age. We found a high correlation between the concentrations of PFOS and PFOA taken over pregnancy time, but the levels declined over pregnancy time, possibly related to blood volume expansion and decreased serum albumin concentration during pregnancy , changesOur nationwide study has the advantage of being selected from a well-described cohort of > 90,000 pregnant women and their infants . SelectiAdjusting for parity and prepregnancy BMI substantially changed the crude measures of association. For instance, adjustment for only parity in the regression model led to attenuation of the regression coefficient between PFOA and birth weight from −20.5 to −7.2. We did find substantially higher PFOA levels in nulliparous women than in multiparous women, which has been shown before in cord blood , perhapsUnlike the more lipophilic POPs, PFOS and PFOA do not typically accumulate in the lipid tissues because of their oil-repellent properties . BecausePFOS and PFOA can cross the placental barrier, but cord concentrations are usually lower than those observed in maternal serum or plasma . In a reThe association between PFOA and birth weight may or may not have public health importance, even if it is causal. LBW has been associated with higher mortality and morbidity that is not restricted to early years of life , althougThe incidence of LBW and preterm birth in our study population is relatively low due to the selection criteria used for sampling the sub-cohort. This should be taken into consideration when trying to extrapolate our results to other populations. We used clinical recordings of gestational age that were often ultrasound-based. Ultrasound-based estimates are biased if the exposure of interest reduces very early fetal growth. This may explain to some extent the higher incidence of preterm birth we see among the exposed, but analysis of gestational age by exposure category did not provide evidence for such an effect, and such a bias would not explain differences in mean birth weight.We observed an inverse association between PFOA levels and birth weight, although the maternal concentrations were orders of magnitude lower than those at which developmental effects have been observed in the animal toxicity studies. A risk characterization of PFOA shows average sera concentrations associated with the lower 95% confidence limit of the benchmark dose for 10% response levels for various developmental outcomes were at least 29,000 ng/mL in rat . The appIn conclusion, our nationwide cohort study suggests an inverse association between maternal levels of PFOA and birth weight. Although PFOS and PFOA have been phased out by some manufacturers in the United States and in some European countries, they are still produced elsewhere, and other similar compounds are believed to break down to PFOS or PFOA in the environment . If thes

In recent years lung cancer specialists have complained that due to stigma resulting from the association of the disease with smoking, theirs is a neglected field. This paper demonstrates that in the 1950s and 1960s, when the British Medical Research Council (MRC) started to organize clinical trials for various forms of cancer, this was not the case. Rather, the organizers of these trials saw lung cancer as a particularly promising object of research, for much was known about the disease. The cancer trials were part of a strategy to use the Randomized Controlled Trial (RCT) technology to cement the role of the MRC as the dominant body overseeing medical research in Britain. The organization of the trials, however, turned out to be very difficult, due to ethical problems and the dominance of one form of therapy, surgery. The trial results were deeply disappointing. I argue that these frustrating results contributed to the notion of hopelessness that has come to surround lung cancer, and to the shift of focus from cure to prevention that was triggered by epidemiologic studies identifying tobacco smoke as the main cause of the disease. The paper deals with an important episode in the history of clinical cancer research in postwar Britain, illustrating the ethical and practical problems faced by the organizers. It has been a common complaint in recent years among lung cancer specialists that theirs is a neglected field, and that the main reason for this is a stigma resulting from the association of the disease with smoking. Claudia Henschke and Peggy McCarthy, for example, in a recent book have argued that “in part because of pervasive negative feelings about smokers (and even ex-smokers), many lung cancer patients aren’t offered the aggressive treatments routinely provided for those with other types of cancer.” Looking at a series of clinical studies of lung cancer funded by the British Medical Research Council (MRC) between the mid-1950s and the mid-1970s, I will argue that when planning for these trials started, the expectations in lung cancer treatment were not significantly different from those for other cancers. The lung cancer trials, as we will see, were part of an attempt to introduce the Randomized Controlled Trial (RCT) technology, which had been used successfully to evaluate the effectiveness of streptomycin in the treatment of tuberculosis, to clinical cancer research. While only a minority of lung cancer patients in the 1950s could expect to be cured, and there was little hope of long-term survival for the majority, there was also no general notion that lung cancer would remain incurable. Neither, as work by Charles Webster, Virginia Berridge, Paolo Palladino, and others indicates, was there much of a stigma attached to smoking in the 1940s and 1950s. In the absence of stigma, how do we explain the apparent neglect of lung cancer research that current-day authors point to? I will argue that an explanation can be found in the history of treatment trials, whose organization was made complicated by the fact that there was a well-established treatment, surgery. This, along with the new ideal of randomization, led to ethical problems for the organizers of the trials. Could researchers withhold the option of surgery from patients if there was only a slight chance that these patients might benefit from an operation? A trial that was not ethical was also not feasible, even if it might provide interesting results. Moreover, the main groups involved with the preparation of the trials at this stage, radiotherapists and surgeons, had different opinions as to what was good practice. While the organizers of the MRC cancer trials initially considered lung cancer as a particularly suitable target for therapeutic trials, these problems, as we will see, led to trials about which hardly anybody was enthusiastic. References to the link with smoking and the perception that individuals brought the cancer upon themselves provided the clinical researchers conducting these trials with a way of dealing with the disappointing outcomes by shifting the focus of attention from cure to prevention. Lung cancer therapy today certainly seems less innovative, with fewer headlines about advances than in other cancers. Hopes for new cures and research progress, which dominate debates around other forms of cancer, are often absent in discourses centering on lung cancer. However, this was not the case in the 1950s. On 31 January 1957, five months before the publication of its important “Statement on Tobacco Smoking and Cancer of the Lung,” the Medical Research Council held a Conference on the Evaluation of Different Methods of Cancer Therapy. The notion that lung cancer was particularly well understood had its origins in the intense interest generated by the mysterious increase in the incidence of this once rare and obscure disease, which clinicians and pathologists observed to have occurred since the beginning of the twentieth century. Debates over whether this increase was real, and its possible causes, grew more intense toward the 1950s; the suspects, besides cigarette smoking, were the tarring of the roads, exposure at work, or car fumes and smog. These well-documented debates provided the background for the epidemiologic studies by Richard Doll and Austin Bradford Hill in Britain and by Evart Graham and Ernst Wynder in the United States, and for the reports by the MRC, the Royal College of Physicians (see the The standard treatment for most lung cancers was (and still is) surgery. A typical lung cancer patient in the 1950s would see his GP about chest problems: difficulties with breathing, or even blood in the sputum. The task of the GP was then to decide whether or not the patient was suffering from one of the “conventional” chest problems, such as TB or bronchitis. Usually, the patient would be referred to the local chest X-ray service. The agenda set by the recommendations of the 1957 conference was heavily geared toward the evaluation of new approaches in radiotherapy, which was the form of therapy from which British cancer specialists most expected innovative impulses—in spite of disappointments with the treatment of lung cancer. In June 1957 the Council appointed a Steering Committee, also chaired by Windeyer, to prepare the appropriate trials. favourable consideration should be given, if possible, to the support of any suitable clinical trials in the field of cancer therapy which can be carried out without too elaborate an organisation and with reasonable promise of yielding useful information. The research program drawn up by the committee was, it seems, at least as much about the development of new methods of clinical research as about finding new cancer therapies. The committee was a vehicle for applying the new RCT approach to the evaluation of well-established and new therapeutic methods, especially in radiotherapy. The use of the new technology for establishing the effectiveness of streptomycin in the treatment of tuberculosis had provided the MRC with a much-publicized success. The members of the ad hoc Working Party appointeThorax. Blair, Mr. A. L. d’Abreu, Professor J. Gough, Professor Austin Bradford Hill, and Professor Windeyer. The chairman, Scadding, was consultant chest physician at the Brompton and Hammersmith Hospitals and professor at the Institute of Diseases of the Chest, the specialist medical school associated with the Brompton Hospital. He is hailed as one of the founding fathers of respiratory medicine in Britain, and was one of the founders and later president of the Thoracic Society and the first editor of the journal The discussions among both the Steering Committee and the Working Party centered predominantly on what kinds of studies were (a) technically and (b) ethically doable. However, as it turns out, the two realms, the technical and the ethical, were difficult to keep separate. Ethical concerns, for example, were frequently raised by the prospect of randomization—an issue that, as we have heard, was central to the committee’s work. One of its members, Professor Robert W. Scarff, wondered “if strict randomisation was necessary since so many clinicians had a clear-cut impression of what was best for the patient and might feel random selection to be a little unethical.” How were such problems to be overcome and appropriate trials organized? And why did they choose to look at lung cancer therapy? At the Steering Committee meeting on 13 January 1958, Professor Mitchell proposed to look at a trial he was undertaking in Cambridge as a model, and his statement points to one of the reasons for including lung cancer in the recommendations. It had to be made sure, Mitchell suggested, that each patient received the best possible treatment appropriate to his particular case, and there should be the most careful clinical observation of each individual. Diagnosis, pathology and histology must be exact and unquestionable; a common type of tumour with a short natural history should preferably be studied to allow adequate numbers to be investigated in a reasonable time, a quantitative result without bias should be aimed at and criteria should be as objective as possible. His experience also showed that two forms of treatment, one new and one conventional, could be successfully compared and that randomisation was necessary for an accurate result. Another member of the committee, Professor Hunter, was concerned that “there were many forms of cancer which could not be suitably used in such an investigation because the pattern of treatment was so well established and so widely accepted that any deviation would cause ethical difficulty, and that this left free for investigation only the fringe of cases of hopeless prognosis.” Contrary to current notions, the members of the committee did not view carcinoma of the bronchus as exceptionally hopeless, but as “a representative problem.” They agreed that retrospective surveys could not supply the answers they were looking for. Long-term studies were too expensive , but at least five years of follow-up were necessary. However, not only survival should be recorded: other parameters should also be taken into account, which might allow conclusions concerning quality of life—such as time spent out of hospital, time spent out of work, the degree of pain and disability, dyspnea, and hemoptysis. For lung cancer it was especially important, Mitchell suggested, “to evaluate the ordeal of treatment against possible benefit, and to try to decide if, in the late cases, X-ray treatment was worth while as opposed to simple palliation.” Soon after the constitution of the ad hoc working parties in 1957 it became clear that it was not easy to find a suitable, well-contained question that The discussions in the committee seemed to go in circles, and progress was frustratingly slow. Since so far only a minority of “some ten percent” of patients was considered for therapy at all, Windeyer asked, would it not be possible to study the remaining 90 percent, maybe by comparing different The Working Party continued to pursue the idea of a trial comparing the effects of super- and orthovoltage irradiation. Despite a distinct lack of enthusiasm on his part, Bignall volunteered to draft a provisional protocol for the trial. It was decided to approach Philip D’Arcy Hart of the MRC’s Tuberculosis Research Unit about coordinating the work in collaboration with the Statistical Unit, due to these units’ previous experience with controlled trials. It was important for the Working Party to get a sufficient number of radiotherapists on board, so it made sense to involve them in the preparation. A meeting with twenty-nine consultant radiotherapists took place on 21 January 1961 in the Council Room of the Royal College of Surgeons in London. They did not. The radiotherapists, too, were unenthusiastic about the protocol. Most thought that, clearly, supervoltage was to be preferred to orthovoltage therapy. Four years after the decision to organize lung cancer treatment trials, the at times frustratingly slow negotiations over the details of these trials appeared finally to draw to a conclusion. Another meeting was scheduled with both consultant surgeons and radiotherapists on 25 July 1961. However, while the agenda was set, there were still difficulties. A central problem was the eligibility of patients for the study. After the consultation with the radiotherapists, the Working Party had returned to an option for a trial design that its members had dismissed at an earlier stage of Lancet, The results of the trial were not encouraging: after two years, only 3 of the original 71 surgical patients and 10 of the 73 radiotherapy patients were still alive. According to the report in the the number of survivors at 24 months is so small that further statistically significant differences between the series in this respect cannot now arise. Despite the The working party suggested that radiotherapy might be the slightly better choice, since postoperative complications would be avoided. prevention of the disease. However, because the results of the treatment are so poor whether by surgery or radical radiotherapy there is an urgent need for further research to improve the treatment of this condition. There is also an urgent need to apply the knowledge already available, in particular that of the role of cigarette smoking . . . to the We can see how in the light of widening acceptance of the tobacco hypothesis the focus is shifting toward prevention, in line with what Berridge observes for policy formation. A note in the administrative file dealing with the study states: “It seems to me that there is nothing at all controversial about this report, which is a straightforward account of a difficult but well organized clinical trial, the outcome of which has been as depressing as it was predictable.”Lancet after the publication of the first report indicates that this was not achieved. The study was criticized by leading specialists such as Roger Abbey Smith of the Thoracic Unit at the King Edward VII One official goal of this and other clinical trials was to provide evidence that would lead to closure in a controversy. The debate unfolding on the letter pages of the Scadding defended the study that his Working Party had organized, arguing that, even taking these criticisms into account, the results were not significantly different and the outlook remained bleak: The facts should be publicised: the incidence of a disease which has assumed epidemic proportions, which has a high mortality, and for which no current method of treatment can be regarded as satisfactory, would be reduced to a small fraction of its present level if men and women as responsible individuals chose to give up, or never to take up, cigarette smoking. It seems that the frustrating outcomes of a trial about which nobody was very enthusiastic in the first place reinforced an ongoing shift of focus from therapy to the prevention of lung cancer. However, by presenting experiences with the treatment of small-cell carcinoma, an especially malignant type of cancer, as representative of lung cancer more generally, it may be argued that Scadding made the outlook for lung cancer patients seem even bleaker than it may have been anyway. The second trial overseen by the Working Party was a study of chemotherapy as an adjuvant to surgery. The trial started in 1964, after having been discussed at a Working Party meeting in 1963. Like the first trial, this second one was also coordinated by the MRC Tuberculosis Research Unit. In charge of both trials at the Unit was Anthony Bernard Miller, replacing Joan Heffernan. The preparation of the chemotherapy trial, it seems, was much smoother than that of the first trial: there were no extensive debates in the Working Party, and no big meetings with consultants. One explanation for this lack of controversy may be that chemotherapy was tested only as a secondary therapy, an adjuvant to surgery, to prevent the growth of secondary tumors. It may also be due to the fact that in chemotherapy (unlike radiotherapy) there were few entrenched positions. It was perceived as something new, an approach that promised new channels for intervention . Patients were randomly assigned, in a double-blind set-up, to groups that were prescribed either a placebo or one of two chemotherapeutic agents, busulphan or cyclophosphamide. The only other difficulty the organizers encountered, besides the unexpectedly high incidence of hazardous toxicity with busulphan in the early stages of the study, was one that is very common to treatment trials: the problem of ensuring that patients took the right number of tablets, not more and not fewer than they had been prescribed. The organizers suggested that patients should occasionally receive home visits by health visitors, who on these occasions could count the patient’s remaining tablets. While the Working Group had shown that it was able to organize a clinical study in lung cancer that conformed to the new standards of a cooperative, double-blind, randomized controlled trial, the results did not fulfil its expectations: “The therapeutic results at two years are disappointing, for there is no evidence that either of the two cytotoxic drugs in the dosage used improved survival.” Let us disregard the disappointing results of these trials for a moment. Judging from the attention that lung cancer received from the Medical Research Council in the 1950s and 1960s, it appears that this was not a particularly neglected form of malignant disease. Indeed, the remarkable increase in incidence also triggered interest among researchers, with a view to not only lung cancer etiology, leading to the work that linked this disease firmly with cigarette smoking, but also its treatment. Nor did this interest disappear quickly. In 1979, still, about 10% of cancer treatment studies then under way in Britain were dealing with lung cancer, 21 studies out of 211 for which questionnaires were returned. What makes the treatment of one form of malignant disease a more interesting and rewarding subject for research than another? As Ilana Löwy, Jean Paul Gaudillière, and others have shown, for blood and lymph cancers it was partly the convergence of interest between cell biologists and cancer researchers.Lancet editorial in 1975 suggested that the outlook for sufferers was dire: the proportion of all patients “diagnosed and treated with the best means available” who could expect to survive for five or more years was estimated to be one in twenty in the late 1950s, and this had not changed significantly in the meantime. Bronchial carcinoma turned out to be a particularly “recalcitrant” form of malignant disease. By the time Wallace Fox and Scadding published the ten-year follow-up results of the MRC trial of surgery and radiotherapy, carcinoma of the bronchi was by far the commonest malignant tumor in Britain and most other developed countries, and was still increasing in frequency. Its mortality, moreover, was little affected by treatment. A In the light of these results, the authors of the editorial concluded that “the overwhelming importance of a preventive approach to this disease must always be emphasised. It would be a cruel deception to allow smokers to think that any improvement in treatment, or any procedure to which they might submit themselves to attain early diagnosis, is likely to diminish appreciably their risk of dying of lung cancer.” The apparent neglect of bronchial carcinoma in clinical research was not caused by stigma, as I have shown. Rather, the notion of hopelessness in lung cancer therapy and the stigma of the self-infiicted disease emerged around the same time. The tide may be turning, though. Books like that by Henschke and McCarthy, charities dedicated specifically to lung cancer (like the Roy Castle Foundation in Britain), and the mere fact that medical oncologists are specializing in lung cancer research may all be indicators of change. In a climate where smoking is increasingly medicalized and viewed as an addiction rather than a matter of choice, depicting lung cancer as a neglected field may be a good strategy for generating attention and increasing its visibility.

Mycoplasma pneumoniae deserves renewed attention.Currently, broad empiric antimicrobial treatment including atypical coverage is recommended for patients with mild to moderate community-acquired pneumonia (CAP). Therefore, the relative impact of each atypical pathogen, particularly Mycoplasma pneumoniae pneumonia (MPP). The diagnosis of MPP was based on a positive PCR from respiratory samples and/or a positive IgM-titer from an acute phase serum sample.Based on prospective data from 4532 patients with CAP included in the German CAP-Competence Network (CAPNETZ), we studied the incidence, clinical characteristics, and outcome of patients with 307 patients (6.8%) had definite MPP . Compared to patients with other definite and unknown etiologies, patients with MPP were significantly younger (41 ± 16 versus 62 ± 17 and 61 ± 18 years), had fewer co-morbidities, presented with a less severe disease, showed a lower inflammatory response in terms of leukocyte counts (median 8850 versus 13200 and 11000 μL) and CRP values (60 versus 173 and 73 mg/L), and had better outcomes, including a shorter length of hospitalization (9 ± 5 versus 14 ± 11 and 12 ± 9 days), fewer patients requiring mechanical ventilation (0.3 versus 4.5 and 2.1%), and a minimal mortality (0.7 versus 8.7 and 6.5%).In this large series of patients with definite MPP according to very strict criteria, MPP appears as a condition with a high incidence, quite specific clinical presentation, and a largely benign course. In view of a widely favorable clinical outcome, recent recommendations including regular coverage of atypical pathogens in patients with mild to moderate CAP might be reconsidered for patients in Germany as well as in other countries with comparable epidemiological settings. Mycoplasma pneumoniae as an important pathogen of community-acquired pneumonia (CAP) is only rarely diagnosed in routine practice. This is explained by the many limitations of paired serology which still is the applied diagnostic tool in most cases. In several epidemiologic studies of CAP, largely relying on paired serology, Mycoplasma pneumoniae was identified in 5–15% of cases, resulting in a second or third rank pathogen causing CAP in most series [Legionella spp. and Chlamydophila pneumoniae, these three pathogens are usually addressed as "atypical bacterial pathogens", and considered as prominent targets for broad spectrum antimicrobial treatment including atypical coverage [t series . Togethecoverage .This approach is questionable for several reasons. First, it may discourage clinical services to include microbial investigations in their diagnostic work-up and thereby promote the decline of any effort to design targeted antimicrobial treatment. Second, relying on paired serology for the diagnosis of MPP necessarily misses acute deaths from pneumonia, and may thereby provide misleading clinical descriptions of the disease and underestimate the prognostic implications of this pathogen. Finally, lumping together all three atypical pathogens throughout all pneumonia severities at admission may heavily bias the potential prognostic implications of the pathogens included in this group, and thereby lead to recommendations of initial empiric antimicrobial treatment implying frank overtreatment.Mycoplasma pneumoniae using a very strict methodology, in order to revisit the epidemiology, clinical characteristics, and the outcome of these patients. In addition, we thought to reconsider the recommended diagnostic approach to atypical pathogens on the background of our findings.For these reasons, based on the large CAPNETZ database, we aimed at identifying patients with CAP due to A detailed description of the CAPNETZ methodology is given elsewhere . In shorThe study was approved by the ethical review board, and all patients included gave informed consent.® Ulm, Germany. The study period comprised 55 months starting on 1st June 2002 and ending 31st December 2006, thus including almost five autumn-winter seasons.In this prospective study, all demographic, clinical and diagnostic data of the patients were recorded using standardized web-based data sheets created by 2 mtPhysicians were asked to provide a sputum sample from all study patients. However, if the patient was not able to produce a sputum sample, it was the physician's decision to perform more invasive procedures. Methods applied were described previously . In shorRespiratory specimens and acute-phase serum were collected, stored frozen for a maximum time period of 3 months and then sent to the central service unit in Ulm on dry ice. After arrival at the central service unit completeness of the number and kind of specimens was checked and specimens were stored at -80°C. Both respiratory specimen as well as sera were analysed retrospectively. Physicians were not aware of the test results in time to change therapy. The DNA from the clinical samples was extracted by using the QIAamp DNA Mini Kit according to the manufacturer's instructions .Mycoplasma pneumoniae by using a real-time PCR targeting the inter-repetitive region of the P1 gene. We used an antigen enriched with P1 (160 KD) mature adhesin. The real-time PCR readily detects all subtypes and variants of Mycoplasma pneumoniae with a detection limit of approximately 10 genomic equivalents [Detection of Mycoplasma DNA from respiratory samples was performed in the Institute for Medical Microbiology and Hygiene, Dresden, the German Consulting Laboratory for ivalents . To exclMycoplasma pneumoniae-specific IgG, IgA or IgM antibodies the Virotech EIA was used according to the manufacturer's instructions. The EIA is based on a defined antigen mix that includes the P1, P100, and P30 proteins.For the detection of Mycoplasma pneumonia was defined as: 1) a positive PCR based detection of Mycoplasma pneumoniae DNA in respiratory samples or 2) a positive IgM test in the acute phase serum sample.In the present study, definite Comparisons between groups were performed by means of the Chi square test for categorical variables or analysis of variances (ANOVA) for continuous variables including multiple comparisons. All analyses were performed with SPSS software . All tests of significance were 2-tailed, and alpha was set at 0.05. To correct for multiple testing the Bonferroni correction was applied to the variables presented in table Overall, 4532 patients with CAP from twelve clinical centers throughout Germany were included in our analysis. The 2492 male and 2040 female patients had a mean age of 60 ± 19 years. Sixty-five percent (n = 2922) of the patients were hospitalized when first contacted for participation in CAPNETZ. Co-morbidities were present in 2565 patients (57%). Thirty-one percent of the patients were smokers. Fifty-six percent of all patients presented with fever, 92% coughed, 73% had dyspnoea, and 8% showed signs of confusion. 106 patients (2.3%) required mechanical ventilation and 290 patients (6.4%) died . The demographic and clinical data of the patients are given in Table Mycoplasma pneumoniae PCR.Respiratory samples were available from 1538 (33.6%) of the patients Table . 148 patients had Mycoplasma pneumonia were considered as having definite ia Table .In support of our working definition, patients with a positive PCR from respiratory samples had almost identical characteristics if compared to patients with a positive IgM serum test, whilst patients with a positive IgA or IgG serum test were older, predominantly men, more often hospitalized, had higher CRB-65 scores and a fatality rate of 7.5% and 6.3%, respectively , Streptococcus pneumoniae (n = 7), Haemophilus influenzae (n = 3), and Staphylococcus aureus (MSSA) (n = 2). None of the patients with mixed infection died.Mixed infections of Mycoplasma pneumonia, 171 were treated as outpatients and 136 were hospitalized. The former group was even younger, had very mild pneumonia, had fewer diabetic patients and a milder inflammatory response. No outpatient died or length of hospitalization (p = .057) between patients with Mycoplasma pneumonia who received appropriate or inappropriate antibiotic therapy, respectively.Detailed data concerning antimicrobial treatment were available for 97% of all patients. The predominant classes of antimicrobial agents administered were macrolides/ketolides (37%) and fluoroquinolones (29%). Overall, 65% of patients with A 71-years old patient diagnosed by PCR died several hours after he had been admitted to the hospital. The cause of death was not established. An additional female patient, aged 69 years, died after 10 days of hospitalization. She suffered from COPD, was a heavy smoker and had to be mechanically ventilated. Both patients had not received antibiotics active against atypical bacteria.Mycoplasma pneumoniae is an important pathogen causing CAP 2) patients with MPP are characterized by a quite specific clinical pattern, including younger age, absent or limited co-morbidity, limited inflammatory response, and usually presented with a mild to moderate pneumonia; 3) as a consequence, the majority of patients were treated as outpatients, hospitalized patients had a shorter length of stay, and mortality was minimal; 4) patients with Mycoplasma pneumonia treated as outpatients had even milder pneumonia than those hospitalized; 5) although there was a trend for patients with Mycoplasma pneumonia receiving antimicrobial drugs active against atypical bacterial pathogens more frequently than those with other or unknown etiologies, the rate of discordant treatment remained high.The main findings of our study, based on very strict diagnostic criteria in a large population of patients with CAP, can be summarized as follows: 1) Mycoplasma pneumonia and the large number of patients identified by this diagnostic approach. It is also unique for offering an identical extensive microbiological workup for hospitalized as well as outpatients. The incidence of Mycoplasma pneumoniae pneumonia was 6.7%, which is at the lower range of previous figures reported in recent large etiologic studies.This study is unique for the strict criteria for the diagnosis of Mycoplasma pneumoniae have largely relied on serologic testing, either using paired serum samples or including IgM and/or acute IgG or IgA [Previous series of patients with CAP due to G or IgA -14. WhenM.pneumoniae pneumonia we felt safe to designate patients with a positive PCR result from respiratory samples as a proven case of M.pneumoniae infection. Due to the fact that in many cases acute phase sera were available and serological tests had been performed, we then had a closer look at our serological test results to answer the question whether patients with a positive IgM for M.pneumoniae in their acute phase serum might be considered as proven cases of M.pneumoniae infection as well. In fact, the detection of specific IgM antibodies is generally accepted as an indication of a recent infection. Two aspects persuaded us to follow this hypothesis:When considering our study patients with M.pneumoniae are only very rarely detected in the sera of healthy subjects. When evaluating the Virotech kit used in our study in blood donors and orthopaedic patients only 2 out of 602 patient samples were IgM positive (0.3%), whereas IgA and IgG antibodies were detected in a significant number of healthy persons [[(i) IgM antibodies to persons [, C. Lück(ii) Patients with a positive PCR result had very similar demographic and clinical characteristics if compared to patients with IgM antibodies only. Especially if looking at the initial CRB65 score, the proportion of patients requiring mechanical ventilation and the outcome Table , we assuMycoplasma pneumoniae proteins as antigens. Therefore, a high specificity might be assumed and was demonstrated in a recent publication [Mycoplasma pneumoniae DNA has a high positive predictive value. Albeit a persistence of Mycoplasma pneumoniae DNA after infection or within the incubation time has been reported it is generally accepted that such events are very rare [We used a commercially available test that uses specific lication . The det ,17.Mycoplasma pneumoniae after the initiation of antimicrobial treatment. In this context it should be noted that 27% of our patients received antibiotic agents at the time of inclusion into our study. The affection of antimicrobial treatment on clinical presentation and PCR or IgM responses in CAP caused by M.pneumoniae has never been studied to our knowledge. Therefore, we ignore the true effect of this confounder.In one third of our MPP patients we found concordant positive PCR and positive IgM test results. The IgM EIA used showed a moderate sensitivity in sera collected in the acute phase . Thus, iMycoplasma pneumoniae.In contrast, clinical characteristics of patients with positive IgA titers were similar to that of patients with other bacterial and unknown etiologies but clearly different from the population with positive PCR and/or IgM, indicating that positive IgA titers represent persisting titers after infection occurring at any time. Moreover, the high prevalence of IgA antibodies (5–8%) found in blood donors and patients without respiratory symptoms strongly indicates a poor specificity. Therefore, IgA antibody detection is of very limited use as a diagnostic tool of pneumonia due to Chlamydophila pneumoniae, Mycoplasma pneumoniae is the only bacterial respiratory pathogen clearly occurring more frequently in younger adults. In a large study on the etiology of CAP, Mycoplasma pneumoniae was the only age-associated pathogen [Mycoplasma pneumoniae can result as the most frequent pathogen even prior to Streptococcus pneumoniae [Mycoplasma pneumoniae have considerably fewer co-morbidities. The only concomitant disease occurring with a frequency of more than 10% in our MPP population was COPD.Despite our different and strict diagnostic approach based exclusively on real-time PCR in respiratory samples and acute phase IgM, our data confirm previous findings of MPP being associated with several peculiar clinical characteristics. Apart from pathogen . It appeeumoniae ,19. ProbMycoplasma pneumoniae reported in the literature could be observed in our series [Mycoplasma pneumonia. In accordance with the regularly mild presentation of MPP, and in line with several previous reports, more than half of our patients were treated as outpatients, hospitalized patients had a shorter length of hospitalization, and mortality was very low [Another important feature of MPP is its usually less severe presentation, both in terms of clinical CRB-65 severity scores as well as inflammatory response. None of the severe complications of r series , althougvery low -14. MoreMycoplasma pneumonia, the rate of discordant treatment was high. Discordant treatment had no discernable effect on outcomes such as length of stay and mortality, indicating that MPP is usually a mild and self-limiting disease. Of note, however, both patients who died with MPP had received discordant treatment initially.Despite a trend for patients with MPP to receive more frequently antimicrobial treatment covering atypical bacterial pathogens, indicating that clinicians may have been aware of a probable Legionella spp., Mycoplasma pneumoniae and Chlamydophila pneumoniae as the three atypical bacterial pathogens treatable by antibacterial agents, it appears that only Legionella spp. are associated with a relevant mortality. In a recent series from our group, we could show that Legionella spp. was found with equal frequency in both ambulatory and hospitalized patients. However, severity was low and mortality was zero in ambulatory patients [Our findings may have significant implications for future recommendations of empiric antimicrobial treatment in patients with CAP, particularly with respect to the need for covering atypical pathogens. If we consider patients . As a repatients . FollowiLegionella spp. was 5%, of Chlamydophila pneumoniae 7%, and of Mycoplasma pneumoniae 12%. However, the rate of patients discharged alive at 14 and 30 days was not different but appeared only significantly different when the total number of patients discharged alive was considered, hinting at non-pneumonia-related reasons for different outcomes. Several other reports also do not support the conclusion of Arnold et al. [Legionella spp. Another concern relates to mixed infections. In our series, the rate of mixed infections was low (n = 20), with no associated mortality. Most co-infections were caused by pneumococci and Legionella spp. which may be detected using routine diagnostic methods according to current ATS/IDSA recommendations [Legionella spp. or, in cases of more severe pneumonia, also for patients with Mycoplasma pneumoniae and Chlamydophila pneumoniae should obviate the need of regular atypical coverage in all hospitalized patients.Only recently, in a large study across four important world regions, Arnold et al. found a lower mortality in hospitalized patients receiving atypical coverage. As a result, and referring to several other studies with similar findings, they strongly recommended such coverage in all hospitalized patients ,21,22. Id et al. ,23-25. Tndations . Thus, aChlamydophila pneumoniae pneumonia and those with probable MPP, i.e. younger patients (age < 40 years) with absent or mild co-morbidity and a mild clinical presentation (CRB-65 < 2).At present, we suggest that in hospitalized patients with CAP coverage of atypical bacteria can be limited to patients with severe CAP, those with confirmed legionellosis, Mycoplasma pneumoniae. However, realizing the minimal risk of adverse outcomes associated with this condition, and having in mind recent findings of our group on legionellosis, atypical coverage of all patients presenting with mild CAP as defined in our study seems questionable. Instead, our data provide important hints for strategies aimed at a more judicious use of broad spectrum antimicrobial treatment with atypical coverage.In conclusion, our study confirms previous reports about characteristic patterns of CAP through The authors declare that they have no competing interests.HvB carried out analysis and interpretation of the data, performed the statistical analysis, participated in the coordination of the study and drafted the manuscript. TW participated in the design, coordination and supervision of the study as well as analysis and interpretation of the data, RM participated in the design, coordination and supervision of the study as well as analysis and interpretation of the data. NS participated in the design, coordination and supervision of the study. CL carried out all molecular and serological studies and participated in the analysis and interpretation of the data. SE participated in the analysis and interpretation of the data and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/62/prepub

Similar effects were observed using conventional whole-cell and gramicidin perforated-patch techniques. The serotonin effects persisted in the presence of tetrodotoxin and were mediated by 5-HT2C receptors, as they were reversed by the 5-HT2 receptor antagonist ketanserin and by the selective 5-HT2C receptor antagonist RS 102221. Serotonin-induced depolarizations were not accompanied by a significant change in FSI input resistance. Serotonin caused the appearance of spontaneous firing in a minority (5/35) of responsive FSIs, whereas it strongly increased FSI excitability in each of the remaining responsive FSIs, significantly decreasing the latency of the first spike evoked by a current step and increasing spike frequency. Voltage-clamp experiments revealed that serotonin suppressed a current that reversed around −100 mV and displayed a marked inward rectification, a finding that explains the lack of effects of serotonin on input resistance. Consistently, the effects of serotonin were completely occluded by low concentrations of extracellular barium, which selectively blocks Kir2 channels. We concluded that the excitatory effects of serotonin on FSIs were mediated by 5-HT2C receptors and involved suppression of an inwardly rectifying K+ current.Fast-spiking interneurons (FSIs) control the output of the striatum by mediating feed-forward GABAergic inhibition of projection neurons. Their neuromodulation can therefore critically affect the operation of the basal ganglia. We studied the effects of 5-hydroxytryptamine , a neurotransmitter released in the striatum by fibres originating in the raphe nuclei, on FSIs recorded with whole-cell techniques in rat brain slices. Bath application of serotonin (30 μ Coronal brain slices (300 μm thick) were obtained using a vibroslicer and maintained at 25°C in oxygenated artificial cerebrospinal fluid . For recordings, slices were submerged, superfused (2–3 mL/min) at 25°C and visualized with infrared/differential interference contrast microscopy. Drugs were bath-applied. Current-clamp recordings were performed in bridge mode using an Axoclamp-2B amplifier (Axon Instruments) or a BA-1S bridge amplifier (NPI electronic GmbH). Voltage-clamp recordings were performed using the AxoClamp-2B in continuous single-electrode mode, with uncompensated series resistance. Whole-cell recordings were obtained with patch pipettes (2–5 MΩ) filled with a solution containing (in mm): 125 potassium gluconate, 10 NaCl, 1 CaCl2, 2 MgCl2, 1 BAPTA, 19 HEPES, 0.4 Mg-GTP, 4 Mg-ATP, and adjusted to pH 7.3 with KOH.Wistar rats were used for the experiments. Overall, 57 animals were used; the number of animals used for each experiment corresponds to Input resistance was measured in current-clamp experiments by applying small negative current steps (0.5–1 s long), eliciting 2–10 mV deflections in FSIs. If the FSI was depolarized as a result of a pharmacological treatment, it was manually repolarized to control level for a short period while the current steps were applied; in this way, input resistance measurements were obtained at the same membrane potential. The input resistance was calculated by dividing the steady-state voltage deflection (measured at the end of the step) by the amplitude of the current step. For each pharmacological condition tested, 5–25 current steps were applied at 10 s intervals; the input resistance measurements for each condition were then grouped for statistical analysis.For perforated-patch recordings, gramicidin (10–20 μg/mL) was added to the intrapipette solution and perforation was monitored as described in I = g(Vm) × (Vm − Ek), where g(Vm) was described by a Boltzmann’s equation: g(Vm) = gmax((1 + exp(Vm − Ek − ΔVh))ν−1)−1. The parameters gmax, ΔVh and ν yielding the curve of best fit were obtained using the method of non-linear least squares provided by matlab software (The MathWorks). The slope conductance associated with the serotonin-suppressed current was estimated using the curve of best fit for I(Vm); the voltage domain was divided into 1 mV intervals and the slope conductance was calculated as the ratio ΔI : ΔV for each of these intervals.The curve of best fit for the average current suppressed by serotonin was obtained using the equation: For action potential half-width measurements, the spike threshold was defined as the point where the rate of depolarization exceeded 75 mV/ms; spike amplitude was measured as the difference between the threshold level and the peak of the spike. For each FSI, 10–30 suprathreshold depolarizing current steps were applied every 10 s in each pharmacological condition, and the half-width of the first spike induced by each step was measured and used for statistical comparison.U-tests (originpro 8 software). Drugs were obtained from Tocris Bioscience UK, apart from 5-hydroxytryptamine (5-HT) hydrochloride and gramicidin, which were obtained from Sigma-Aldrich UK.Experimental values are expressed as mean ± SD and all statistical comparisons were carried out using non-directional Mann–Whitney The first two postnatal weeks are critical for FSI development . FSIs wem). Serotonin induced reversible depolarizing effects (8.4 ± 3.6 mV) in 4/5 FSIs tested in the presence of TTX using conventional whole-cell recordings failed to elicit significant effects. In most cases, serotonin-induced depolarizations per se did not elicit action potentials. However, in five FSIs serotonin did cause the appearance of spontaneous action potentials (at an average frequency of 3.6 ± 2.1 Hz), as shown in the examples of In another series of experiments, we applied serotonin in the absence of TTX to investigate its effects on FSI excitability. Under these conditions, serotonin induced reversible membrane depolarizations (8.9 ± 2.8 mV) in 27/33 FSIs recorded with conventional whole-cell techniques . In the 6/33 cells that did not respond to 30 μ2 receptors (2 receptor antagonist ketanserin (10 μm) in nine experiments in which serotonin had depolarized FSIs by > 5 mV (four of these experiments were carried out in the presence of TTX). In 9/9 cases, ketanserin (applied in the presence of serotonin) fully reversed the serotonin effects, causing the membrane potential to return either to the level observed in control solution or to more hyperpolarized levels . Two cases in which ketanserin caused the membrane potential to attain levels more negative than in control solution are presented in In other basal ganglia neurons, serotonin depolarizing effects are mediated by 5-HT2 receptors, we also investigated the effects of the 5-HT2 receptor agonist α-methyl-5-HT (30 μm) on FSIs. α-methyl-5-HT produced depolarizing effects (8.0 ± 1.7 mV) similar to those of serotonin in 6/7 FSIs, as shown in the example of To further test the hypothesis that the effects of serotonin were mediated by 5-HT2 receptors. The 5-HT2A and 5-HT2C receptor subtypes are abundantly expressed in the striatum , still in the presence of serotonin, fully reversed these effects, causing the membrane potential to return to levels slightly more negative (on average by 1.1 ± 1.4 mV) than that observed in control solution. In four of these five FSIs, RS 102221 was then washed out, still in the presence of serotonin, and subsequently reapplied (after > 10 min). In all cases, removal of RS 102221 caused the FSI membrane potential to return to depolarized levels similar to those observed during the first application of serotonin, whereas reapplication of RS 102221 caused repolarizations to levels slightly more negative than that observed in control solution. An example of these effects is illustrated in 2C receptors.These data clearly indicated that serotonin depolarized FSIs acting on 5-HTP < 0.001). A Mann–Whitney U test was carried out on the number of spikes elicited by 10 consecutive steps in control solution and 10 in the presence of α-methyl-5-HT; in each case the steps were applied every 10 s; when ketanserin was added, the number of spikes decreased back to 2 ± 1 (10 steps). Similar results were observed in 6/6 FSIs, in which either serotonin or α-methyl-5-HT had induced, per se, subthreshold depolarizations; in all cases current steps elicited significantly more spikes in the presence of serotonin or α-methyl-5-HT than in control solution .In control solution, the effects of serotonin or α-methyl-5-HT on FSI excitability were tested by repeatedly depolarizing these cells with current injections before and after drug application. An example of these experiments is illustrated in P < 0.01; 10–20 measurements per cell per condition) decreased the latency of the first spike (on average by 53 ± 10%) and the first inter-spike intervals (on average by 63 ± 8%). An example of the effects of serotonin on spike latency, and their reversal after serotonin washout, is shown in per se subthreshold, they strongly facilitated spike generation induced by concomitant excitatory stimuli.In another series of experiments, we used current steps that elicited at least two spikes in control solution. At least 10 identical steps were applied (every 10 s), in control solution and in the presence of serotonin, to seven FSIs, which were recorded without steady current injections. In control solution, the latency of the first spike was 134 ± 35 ms and the first inter-spike interval was 245 ± 88 ms. In each of these seven FSIs, serotonin induced depolarizations of > 5 mV and significantly . We concluded that the use of perforated patch was not critical to study serotonin effects on FSIs.In a previous study on cholinergic interneurons, the effects of serotonin could only be observed using perforated-patch techniques . The obsP > 0.07; at least 10 spikes per condition) affect action potential half-width in any of the 22 FSIs tested, as shown in the example of Vh = −80 mV, serotonin induced an inward current in each of these seven FSIs . The time-course of serotonin responses was similar to that observed in current-clamp recordings. In four FSIs, a series of voltage steps (1 s long) were applied in control solution and in the presence of serotonin (after > 30 min from the onset of the application). These steps were delivered from a holding potential of −80 mV to levels between −110 and −40 mV, in 10 mV increments. A representative example of these experiments is shown in Ek (−104.3 mV). In order to quantify the inward rectification, we measured the slope conductance of the serotonin-suppressed current at −105 and −65 mV . This slope was significantly larger at −105 mV (on average 5.3 ± 0.2 pA/mV) than at −65 mV (on average −0.4 ± 0.1 pA/mV), indicating the presence of strong inward rectification.The observation that the effects of serotonin persisted in the presence of TTX suggests that its action did not involve the voltage-activated sodium currents responsible for spike generation. Consistent with this notion, serotonin did not significantly (The average current suppressed by serotonin for four FSIs is shown in m) of extracellular barium (m) in five experiments in which a previous application of serotonin had caused depolarizing effects (8.6 ± 1.8 mV) in FSIs. Barium was applied after serotonin effects had completely washed out. Barium per se induced membrane depolarizations (12.0 ± 4.8 mV) in 5/5 FSIs. The input resistance was not significantly affected by barium in 5/5 cases . In the continuous presence of barium, the FSIs were manually repolarized at control level; under these conditions, reapplication of serotonin failed to affect the FSI membrane potential in all five cases. A representative example of these experiments is illustrated in n = 4, not shown); thus, FSIs fully retained their ability to respond to serotonin after a first application.Inwardly rectifying currents mediated by Kir2 channels are selectively blocked by low concentrations = 5.3%; the reciprocal relationship between resistance and conductance (R = 1/C) implies that ΔR/R = ΔC/C. Therefore, 5.3% is also an upper limit for the percent change in resistance caused by the serotonin-suppressed current. Whole-cell recordings are inevitably contaminated by thermal and synaptic noise, as well as by small variations in the electrode access resistance; it is therefore not surprising that such small changes in input resistance did not give rise to statistically significant effects. Consistently, 100 μm barium also did not induce significant changes in FSI input resistance, despite its action as an inward rectifier potassium current blocker. It should be noted, however, that suppression of an inwardly rectifying potassium current will still produce robust depolarizations and increases in excitability. These phenomena were indeed observed in our experiments. In a minority of serotonin-responsive FSIs, serotonin induced spontaneous firing, which was never present in these neurons in control solution; in the remaining ones, in the presence of serotonin, the ability of a depolarizing input to generate action potentials was strongly increased, even when the cells were repolarized to control levels. Furthermore, when stimuli that were suprathreshold in control solution were used, in the presence of serotonin, the latency of the first spike strongly decreased and the initial firing frequency strongly increased. This suggests that FSIs will be much more responsive to cortical glutamatergic inputs . In this voltage region (−73 to −61 mV), the slope conductance of the current suppressed by serotonin was extremely small in absolute value, i.e. < 0.3 nS. Classic studies on ve slope . In the m); the electrophysiological properties of these FSIs did not differ significantly from those of serotonin-responsive FSIs. In these cells, increasing the serotonin concentration up to 120 μm was also ineffective, suggesting that they belonged to a subpopulation that was unresponsive to serotonin. Previous histological studies have shown that the 5-HT2C are not uniformly distributed in the striatum, being more densely expressed in the matrix or striosome compartments, respectively (2C receptors (or that of the potassium channels targeted by it) was low or absent in the FSIs that did not respond to serotonin, as a result of their compartmental location. Further studies will be required to clarify whether the FSIs that do not respond to serotonin belong to a specific striatal compartment.Approximately 22% of FSIs did not respond to the standard serotonin concentration used in this study (30 μectively . Further2 receptors. Cholinergic interneurons and FSIs control projection neurons in different ways but their actions on these cells may converge at a functional level. We have shown using paired recordings that cholinergic interneurons inhibit cortical glutamatergic input to projection neurons through a presynaptic mechanism (A receptors (The present observations on FSI extend our previous finding that serotonin excites striatal cholinergic interneurons . It is wechanism . Project2 receptors in patients exposed to selective serotonin reuptake inhibitors, may cause excessive suppression of the GABAergic striatal output and this could be an important causal factor in the genesis of motor side effects (From a clinical perspective, it is interesting that selective serotonin reuptake inhibitors, which are widely used for the treatment of depression, can produce extrapyramidal motor effects, including akathisia and dystonia . The pre

One-sentence summary for table of contents: These countries should consider using inexpensive generic agents to confront the next pandemic. Developing countries face unique difficulties preparing for an influenza pandemic. Our current top-down approach will not provide these countries with adequate supplies of vaccines and antiviral agents. Consequently, they will have to use a bottom-up approach based on inexpensive generic agents that either modify the host response to influenza virus or act as antiviral agents. Several of these agents have shown promise, and many are currently produced in developing countries. Investigators must primarily identify agents for managing infection in populations and not simply seek explanations for how they work. They must determine in which countries these agents are produced and define patterns of distribution and costs. Because prepandemic research cannot establish whether these agents will be effective in a pandemic, randomized controlled trials must begin immediately after a new pandemic virus has emerged. Without this research, industrialized and developing countries could face an unprecedented health crisis. More than a decade ago, the first human cases of disease caused by avian influenza virus A (H5N1) appeared in Hong Kong Special Administrative Region, People’s Republic of China. Five years ago, influenza virus A (H5N1) reemerged to cause highly lethal human disease in Southeast Asia. Health officials are concerned that these cases could be the harbinger of the next influenza pandemic. As a result, virtually all industrialized countries and many developing countries have mounted extensive pandemic preparedness efforts. However, as pointed out recently by Oshitani et al., industrialized countries face “unique and difficult issues, which make preparing for a pandemic more challenging” (If a pandemic form of influenza virus A (H5N1) emerges within the next few years, all countries will have to depend almost entirely on egg-derived inactivated adjuvanted influenza vaccines. For developing countries, this approach will not succeed. Estimates show that within the first 6–9 months of a pandemic outbreak, vaccine companies will be only able to produce enough doses to vaccinate ≈700 million persons Global Programme on Influenza concluded that “most developing countries will have no access to a vaccine during the first wave of a pandemic and perhaps throughout its duration” that might be used for prepandemic vaccination, but Oshitani et al. note that “both pandemic and prepandemic vaccines would not be available in developing countries unless an international mechanism exists to share such vaccine with them at low cost” have acquired molecular characteristics suggesting they might become more easily transmissible among humans , (7). GivThe current approach to pandemic planning for all countries involves small groups of health officials, influenza scientists, and company executives, most of whom come from industrialized countries. For the foreseeable future, this top-down approach will be incapable of providing developing countries with timely supplies of affordable vaccines and antiviral agents. The Indonesian Health Minister, for one, understands this. With little prospect that people in her country will be able to obtain vaccines against pandemic viruses, she precipitated a standoff with WHO by announcing in February 2007 that unless Indonesia is able to gain access to supplies of vaccines against pandemic viruses, her country will no longer share its influenza viruses A (H5N1) with WHO’s laboratory-based surveillance system .Other generic agents, some with direct activity against influenza virus, should also be considered was not nearly as effective as treating the mice with an antiviral agent and 2 immunomodulatory agents, mesalazine, a PPARγ agonist, and celecoxib, a cyclooxygenase (COX)–2 inhibitor or 1918-like viruses . The age–,After demonstrating the effectiveness of 1 or more treatment regimens in animals, influenza virologists should then use in vitro systems to define the molecular mechanisms responsible for their protective activity. However, some of these agents will have broader effects on the host response. For example, although administering a COX-2 inhibitor along with a PPARγ agonist improved survival rates and times in mice infected with influenza virus A (H5N1) , patterns of distribution to other developing countries, and costs for public markets . SpecialWhere feasible, clinical trials of promising treatment regimens might be undertaken in patients with severe seasonal influenza. In a few instances, clinicians might choose to treat patients infected with influenza virus A (H5N1) on a compassionate basis . Within Planning for clinical trials during the prepandemic period must start with identifying clinical investigators who will conduct these trials and institutions that will sponsor their work. Supplies of the agents to be tested must be set aside, study protocols written, and ethical approval obtained. A mechanism for rapid regulatory approval must be developed to enable trials to be conducted wherever the pandemic virus first emerges. A financing mechanism must be established that enables immediate access to funds necessary to support the trials. Finally, an internet-based communication strategy must be devised that ensures prompt dissemination of study results to physicians and health officials worldwide.None of this research on generic agents will be possible without international coordination. Thus far, the top-down approach that has characterized vaccine and antiviral research and development has lacked an international system for coordination and management to ensure rapid progress (Experience with the severe acute respiratory syndrome (SARS) in 2003 shows us how we could do much better. When SARS first came to international attention, WHO quickly established 3 virtual networks of experienced virologists, clinicians, and epidemiologists (Oshitani et al. correctly emphasize that preparing for the next pandemic requires a global perspective, but this does not necessarily mean that the measures used to confront the pandemic in developing countries must be supplied through an internationally organized top-down process. An international process will surely be required for distributing vaccines and antiviral agents, but experience indicates that the process will be slow and cumbersome and supplies of these agents will remain scarce (It is too soon to know whether generic agents could be used to confront the next influenza pandemic, yet developing countries lack realistic alternatives. For this reason, their leaders must convince scientists and international organizations, including WHO, of the urgent need for research to determine whether these inexpensive agents could mitigate the effects of a pandemic. Otherwise, developing and industrialized countries alike could be faced with an unprecedented global health crisis.

Zoonotic disease transmission systems constitute sets of interacting species, ranging from pathogens in wildlife reservoirs and transmitted directly to humans (–Disease studies that lack careful biodiversity documentation are numerous, even in the recent literature. In the August 2009 issue of this journal, I found at least 4 articles that report sampling of hosts or vectors, yet make no mention of vouchers (Mastomys rodents present (The reality, however, is quite different. First, technologies for diagnosis and testing have evolved considerably and will continue to evolve, with each iteration providing more complete information and insight into the pathogens present. The failure to preserve voucher specimens, however, makes such retesting and improved learning impossible. For example, in early studies of filoviruses, thousands of specimens were tested serologically for evidence of infection (Henipavirus distributions, in contrast to the time and effort it took to assemble other samples (Finally, and perhaps most urgent, treating biodiversity samples as disposable ignores opportunities to assemble archives of diagnostic samples for future studies. Host samples accumulated for 1 purpose could be recycled to form a strong basis for future studies of pathogens not yet known. Consider, for example, that those same samples of mammals from Africa from the early filovirus studies could have enabled quick and detailed study of Of course, biologic material that is potentially infected with dangerous pathogens carries with it some degree of responsibility, to ensure that unfortunate accidents do not occur. Two general paths are possible: ,My suggestion is not an empty dream but rather an open door. The biodiversity science community is fully prepared and willing to partner with the disease community in this effort. On the most proximate level, biodiversity specialists are eager to build scientific reference collections and are willing to curate and catalog voucher specimens. Vouchers provide permanent specimen identifiers that can be reported in publications and used to reference the voucher in genomic data bases. Furthermore, biodiversity specialists are interested in many of the same geographic regions as disease specialists and would welcome opportunities to obtain new specimen material from these regions. Finally, the role of pathogens in constraining host evolution, distribution, and ecology is of increasing interest in the biodiversity community (

People living with HIV (PLHIV) sometimes experience discrimination. There is little understanding of the causes, forms and consequences of this stigma in Islamic countries. This qualitative study explored perceptions and experiences of PLHIV regarding both the quality of healthcare and the attitudes and behaviours of their healthcare providers in the Islamic Republic of Iran.In-depth, semi-structured interviews were held with a purposively selected group of 69 PLHIV recruited from two HIV care clinics in Tehran. Data were analyzed using the content analysis approach.Nearly all participants reported experiencing stigma and discrimination by their healthcare providers in a variety of contexts. Participants perceived that their healthcare providers' fear of being infected with HIV, coupled with religious and negative value-based assumptions about PLHIV, led to high levels of stigma. Participants mentioned at least four major forms of stigma: (1) refusal of care; (2) sub-optimal care; (3) excessive precautions and physical distancing; and (4) humiliation and blaming. The participants' healthcare-seeking behavioural reactions to perceived stigma and discrimination included avoiding or delaying seeking care, not disclosing HIV status when seeking healthcare, and using spiritual healing. In addition, emotional responses to perceived acts of stigma included feeling undeserving of care, diminished motivation to stay healthy, feeling angry and vengeful, and experiencing emotional stress.While previous studies demonstrate that most Iranian healthcare providers report fairly positive attitudes towards PLHIV, our participants' experiences tell a different story. Therefore, it is imperative to engage both healthcare providers and PLHIV in designing interventions targeting stigma in healthcare settings. Additionally, specialized training programmes in universal precautions for health providers will lead to stigma reduction. National policies to strengthen medical training and to provide funding for stigma-reduction programming are strongly recommended. Investigating Islamic literature and instruction, as well as requesting official public statements from religious leaders regarding stigma and discrimination in healthcare settings, should be used in educational intervention programmes targeting healthcare providers. Finally, further studies are needed to investigate the role of the physician and religion in the local context. Stigma affects the lives of individuals infected with HIV . ParticuMore than four decades ago, Goffman 1963) defined stigma as "an attribute that is deeply discrediting", and proposed that the stigmatized person is reduced "from a whole and usual person to a tainted, discounted one" (p. 3). 3 definedDeacon (2006) constructed a sustainable theory of health-related stigma that brought together both the individual and social dimensions of this complex phenomenon that may facilitate interventions against health-related stigma. Deacon argues that stigma comes about in a social process during which the following occurs: illness is perceived as controllable or preventable, and caused by identifiable "immoral" behaviours. These behaviours are associated with certain groups that "carry" the illness, which draws on existing social constructs of the "other", who are consequently blamed for becoming infected.These "others" experience status loss from the projection of blame, and may become disadvantaged as a result . The intNumerous studies have documented the attitudes of healthcare providers toward PLHIV -15. AlthThe effect of perceived discrimination or internalized HIV stigma on access to care, regular HIV care, and adherence to treatment need further attention . ResearcAn increasing number of countries in the Middle East, North Africa and Asia, including those with Muslim majorities, have experienced or are at risk for HIV epidemics . HIV traReligious constraints on sexuality may have consequences for the transmission of sexually transmitted infections . It has In a recent comprehensive examination of literature, Nyblade and her colleagues (2009) provide a review of the various forms and causes of stigma documented in healthcare setting across regions . StudiesIn Iran, the first case of HIV was reported in 1987, and was followed by a rapid increase in the number of cases. Currently, the HIV prevalence rate in Iran is estimated at 0.2%. In 2007, there were officially 15,587 Iranians living with HIV, 14,702 (94.3%) of whom were male and 885 (5.7%) of whom were female. Over the 20-year surveillance period, the rate of HIV infections diagnosed annually among Iranian citizens gradually increased, and over the period 1997 to 2004, went from 1.38 to 4.6 cases per 100,000 people per year .While only 10% of PLHIV in Iran have been infected as a result of sexual contact ,26, sexuIn Iran, a limited number of studies have been conducted to assess the knowledge and perspectives of healthcare providers towards HIV infection -35. ThesAssessing how PLHIV perceive and experience the behaviours and attitudes of providers is important; the subjective beliefs that PLHIV hold about the situations in which they find themselves may or may not correspond to objective reality, but are nonetheless powerful forces with real consequences for their health-seeking behaviours. To address these questions and the gap in HIV and AIDS research in Iran, this qualitative study explored the perceptions and experiences of PLHIV regarding the attitudes and behaviours of healthcare providers and quality of healthcare.In this qualitative study, participants were recruited from two HIV care clinics affiliated with the Medical School of Tehran University and University of Medical Sciences, as well as the Department of HIV Infection of Imam Khomeini Hospital. Currently, there are three large HIV care clinics in the greater metropolitan area of Tehran that provide comprehensive care, including medical and psychological care. PLHIV receive referrals for drug rehabilitation, ophthalmological, gynaecological, gastroenterological, dental and other specialty care. However, the specialty care facilities are not specific to HIV patients. The study sites record serving approximately 4500 PLHIV annually.The sampling method involved the first author of this study approaching potential participants waiting in line to receive their clinical care. Seventy-six potential participants were invited to participate in the study. Of these, seven were not enrolled into the study due to lack of interest and other clinic-related reasons. A total of 69 participants then consented and were enrolled into the study.A convenience sampling method was used to choose potential eligible participants, and then a purposeful sampling method was used to achieve data saturation. Purposive samples are the most commonly used form of non-probabilistic sampling for conducting qualitative research. In order for analytic generalizations to be richer, qualitative researchers typically rely on the concept of "saturation", or the point at which no new information or themes are observed in the data ,38.The first author of this study systematically reviewed the degree of data saturation over the course of thematic analysis. We found that saturation occurred within the first 60 interviews; however, to be on the safe side, we collected data from an additional 10 participants. This study reports data from 69 semi-structured interviews with PLHIV who provided informed consent. The interviews were conducted between March 2008 and January 2009.The main author conducted all the one-on-one interviews. Semi-structured and in-depth interviews were conducted in private rooms, recorded on audiotapes, and transcribed verbatim. Prior to conducting the interviews, three qualitative research experts and five non-participants reviewed the interview items to assess the survey instrument. The interview consisted of open-ended questions, including the following: How do you feel about your healthcare providers? Do you have any problems with them? How did your disease influence the care you received from healthcare providers? What do you think about your interaction with them?At the end of each interview, we collected the participants' demographic characteristics. Each interview lasted for between 20 and 90 minutes. The transcripts were manually coded and grouped into categories to explore the initial themes. Data collection and analysis were carried out simultaneously. The analysis of the data was conducted using transcripts in Farsi (the common language of the Iranian people) by the first author. The data were further explored, using content analysis, for the identification of recurring themes. Transcripts were read several times and coded, and emergent themes were identified.The co-researchers checked the plausibility of the data interpretation and ensured that the qualitative data analysis was systematic and verifiable. To ensure the validity of the analyst interpretations, data source triangulation both across participants and across investigators were employed. More than 25% of the transcripts, codes and categories were rechecked by the study team and a high level of agreements was noted. Disagreements between the researchers were resolved by group discussions.In addition to using semi-structured interviews, we also employed in-depth interviews to provide further opportunities for the participants to share their untold stories about HIV-related healthcare-seeking experiences. The in-depth and semi-structured interviews used for the collection of data were complementary in method. However, the in-depth interviews shed more light on the consequences of stigma. Some of the participants were more eager to talk about their behavioural and emotional reactions to the HIV-related stigma to the extent that they requested to be scheduled for follow-up interviews.The Ethics Committee of Tarbiat Modares University approved the study proposal. Participants were informed about the objectives of the research and its confidentiality. Those who agreed to participate in the study were each asked to sign an informed consent document.In all, 69 PLHIV, including 42 61%) men and 27 (39%) women, took part in this study. Of the total, 49 participants (71%) reported diagnoses by their primary physician as being HIV positive and 20 (29%) reported their health status as full development of AIDS. The median age of patients was 28 and they ranged in age from 21 to 47 years. Most participants were educated at the high school level (from 10 to 12 years of education), while one participant reported no formal education, and four participants had received college educations. Table % men andWhile many participants in our study indicated they received quality care and were happy with their healthcare experience, nearly all participants, especially women, reported that they experienced stigma in the healthcare system. Table Participants perceived that healthcare providers were fearful of becoming infected with HIV and tended to hold negative views and attitudes regarding homosexuality, drug abuse, prostitution and adultery. These views may contribute to HIV-related stigma among healthcare providers.Participants perceived that healthcare providers' fear of becoming infected was a major cause of stigma against them. For example, a 36-year-old female receptionist spoke of her mistreatment by a provider:I was hospitalized due to pneumonia. At the time I reported to my doctor that when I menstruated, my bleeding was excessive and did not stop for several days, so my doctor referred me to a gynaecologist for further evaluation. When I arrived at the gynaecology service, the staffs read my medical record and were informed about my history of HIV infection; they hesitated to examine me. I heard my nurse saying to herself that I came here to infect them too.A 27-year-old unemployed male said:I have no doubt that one of the nurses in this ward is scared of taking my blood ... One of our main problems among providers, even among physicians, is that they really do not know how HIV is transmitted.Other participants described their experiences with healthcare providers who would refuse to shake their hands or touch them for fear of becoming infected. A 31-year-old supermarket salesman reported:When I told my physician about my HIV infection, he quickly sterilized his hands with disinfectant because he had shaken hands with me when I arrived. It immediately affected my spirit. I know my provider has studied this disease and he knows that HIV cannot be transmitted by shaking hands. So why did he act this way?A 32-year-old married housewife said:Most providers want to know how I was infected. Sometimes they ask this question and even before I answer, they blame me for doing something sinful. I am married, I have never had any relationships out of wedlock. However, a female doctor told me, "Are you sure you have not had a sexual relationship with someone else?" Indeed, she was interrogating me while three other individuals were in the room.A 25-year-old housewife indicated that "some of the providers think that whoever is infected with the HIV virus is a prostitute or has committed adultery".In another instance, a 34-year-old restaurant manager remarked:They look at you as if you are a lunatic who deserves to get this horrible disease. I think they assume that you abuse drugs or you are a homosexual.Similar perceptions were described by other participants who indicated that they felt as though their healthcare providers seemed to believe that whoever was infected with HIV had committed a sin, and as a result, more or less deserved to suffer.Participants in this study indicated at least four major forms of stigma, including: (a) denial of care; (b) sub-optimal care; (c) excessive precautions and physical distancing; and (d) humiliation or psychological abuse and blaming.The majority of participants stated that healthcare providers sometimes refused to provide needed services, and perceived that healthcare providers did so because of their fear of becoming infected with HIV. A 29-year-old female patient, a hairdresser, reported:Yes, it happened to me when my ovarian cyst ruptured and I needed an immediate operation. The surgeons did not operate on me. They never wanted to. They just came and went. I was examined by several surgeons.A 25-year-old patient, a housewife, reported that she was denied post-delivery care when the emergency room (ER) doctor became aware of her HIV status. She indicated:I had excessive bleeding for a few days after I had delivered my baby. I had to go to the ER. I told them that I am HIV positive. The ER doctor told me, "You have excessive bleeding. I cannot examine you. I could be easily infected with the HIV. Look, you have HIV .... Sorry, go somewhere else."A 27-year-old used car dealer spoke of a similar circumstance in which a nurse had refused to provide services:I knew a nurse [at the infection ward] who was scared of us. No nurse really wants to take care of PLHIV admitted to this ward. Some of the nurses in particular refused to take blood or give injections to the patients.With no exception, all patients who needed dental care and had revealed their HIV status to their dentists were directly or indirectly denied dental care. A 31-year-old unemployed male patient reported that on one occasion when he revealed his HIV status to his provider, the dentist immediately asked him to leave the office. A 25-year-old housewife reported visiting a dentist who claimed that his equipment had broken down and he would therefore not be able to provide treatment for her decayed teeth. She perceived his refusal to provide care as a clear demonstration of his unwillingness to serve patients with HIV.A number of patients reported that they had received sub-optimal care because of their HIV status. A 36 year old housewife said:I was preparing dinner at home when I cut my index finger; the cut was deep and I knew from experience that it would require stitches of some sort, so I went to the local urgent care clinic. I told the doctor that I was HIV positive and then the doctor informed me that I really did not need stitches after all and he wrapped the finger in gauze and tape and sent me home. After a few days the cut became infected and I had to see [the] doctor. This time, [I] went to a different one. This time the doctor had to clean the cut and stitched up my finger."A few participants described experiences of healthcare providers taking excessive precautions and keeping a physical distance, including providers who refused to shake their hands or touch them. Participants expressed frustration with their healthcare providers' excessive precautionary measures; many participants remarked that this was confusing to them: they understood that healthcare providers were medical professionals who were supposed to be aware of the HIV disease process, including how the disease was transmitted from one person to another.Some participants reacted emotionally to the perceived hostility of healthcare providers. A 25-year-old housewife related:How would you feel about talking about your disease with your doctor when you know that what you have to say will not be considered or even matter to anyone? This hurts your spirit. When the doctor does not care about me, when she/he doesn't communicate with me effectively, what should I expect from other people?Similarly, a 26-year-old patient, a housewife, said:Believe [it] or not, there is one nurse in this ward who actually ran away from me. On one occasion, when I approached the nurse's station to ask for something, she told a non-HIV-positive patient, "Cover your mouth, be careful, you do not want to get infected with the HIV." Oh my God, I really do not understand this.Many participants from different ages and backgrounds claimed that they had been humiliated, constantly blamed, and verbally abused by their healthcare providers. A 28-year-old seamstress related her experiences in the hospital during the delivery of her child:I was admitted to a hospital for delivery. When the nurse was informed about my HIV infection, she said, "Why did you become pregnant? Why are you here? There are many children in the orphanage that you could have adopted, instead of becoming pregnant." You see, they give themselves the right to decide for us.A 31-year-old factory worker reported getting negative non-verbal message from his healthcare provider. He said:Once I went to the doctor and talked about my CD4 count, he looked at me in a very nasty way. I deeply felt as though he actually blamed me for getting HIV, by the way he looked at me, even though he did not say anything.In addition, participants reported that they felt as though they were blamed for their infection because of disrespectful behaviours and negative attitudes expressed toward them by their healthcare providers. As mentioned earlier, most participants reported that healthcare providers assumed the right to ask participants how they became infected, even when they were hospitalized for reasons not directly related to their disease. Participants also reported feeling disrespected by healthcare providers who learned of their seropositive status during hospital visits.In-depth interviews with the participants in this study led us to identify the consequences of stigma as: (a) avoiding or delaying seeking healthcare; (b) refusing disclosure of HIV status when seeking care; (c) low self-esteem accompanied by a diminished motivation to stay healthy; (d) using violence and feeling vengeful; (e) using alternative medicine as a substitute for conventional care; and (f) experiencing emotional stress.A 25-year-old housewife who needed dental treatment refused to seek care, saying that:Several of my teeth needed immediate attention. Indeed, occasionally I have pain. However, I am not seeking dental care. I know as soon as I let them know that I am HIV positive, they will tell me, sorry our equipment is broken, come back later ... My husband has stopped taking his medication. His CD4 is less than 800 now. He says, "I cannot handle any more humiliation ... I just want to die."In a few cases, participants reported that they did not immediately seek treatment upon being diagnosed as seropositive for fear of being mistreated or misjudged by their healthcare providers, especially when their offices were located in the same community. One 28-year-old housewife, who lived in a small town, did not seek care at a local medical centre because she was worried about how health workers would view her and her family:My husband forced me to go to a medical centre in Tehran, in a big city. My husband did not come with me in the last four or five times that I went to meet with my doctor. I would have liked to have had a consultation in our own town, but my husband and I are not sure how the local doctors will react.Perhaps most troublesome to many of the participants was the perception that they could not communicate effectively and honestly with healthcare providers for fear of negative retribution based on previous negative experiences. Participants, as discussed, felt that healthcare providers feared becoming infected from their patients and therefore verbally and emotionally mistreated or denied treatment to patients in their care. In some cases, patients did not disclose their infection at all, while a few patients revealed their HIV status after receiving treatment.Indeed, a patient indicated that to make sure that his dentist took precautionary measures, he usually told his dentist that he has been diagnosed with hepatitis B or C. A 29-year-old housewife said:I begged them (dentists) a couple of times to take care of my bad tooth. However, they did not do anything but instead asked me to leave their clinic. After that experience, I did not tell them, dentists or others, any more about my HIV status.Several patients claimed that because they had been mistreated by their providers, they had lost their motivation to stay healthy. A 31-year-old male street worker said:You know, after my doctor mistreated me, I left his office angry. I told myself that my life is over; I really do not have any reason to take care of myself. This is the last page of my life; my life no longer has any meaning.A 22-year-old unemployed male indicated:My own doctor has no respect for me. This makes me very upset. I felt as though I do not deserve any care. I am worthless. I do not know ... maybe I should go somewhere else, another country.Several patients, particularly female patients who claimed that they had been inadvertently infected through their husbands, expressed strong vengeful feelings toward healthcare providers who openly blamed or mistreated them. A 25-year-old married patient, a housewife, said:Oh my God, their (providers') behaviours and attitudes toward me made me so upset ... I really hate them. I do not understand. They are educated ... they should know better. Sometimes I feel that I should take revenge.A 35-year-old male factory worker said:I really wanted to take revenge by using the same syringe that they had used to take blood from me.Even though none of our participants reported explicitly using alternative medicine as a substitute for conventional care, a few participants admitted that they had been using spiritual healing by travelling to Islamic holy land and to the tombs of Islamic leaders to get cured, partly because of the stigma they had experienced in the healthcare system.Participants in our study indicated that they had had numerous interactions with their healthcare providers during the course of their disease, and that these interactions caused them tremendous emotional stress. Participants reported avoiding the use of healthcare in general, including non-HIV-related care within their communities, and especially in their neighbourhoods. Also, participants reported that they did not expect to receive support if they chose to disclose their HIV status; this ultimately led to emotional stress among our participants, who reported experiencing HIV-related stigma.Several patients in our study indicated that they had felt deeply depressed when they were denied care or received sub-optimal care by their health care providers. Indeed, one of the patients indicated that her doctors' and other healthcare providers' attitudes and behaviours toward her made her feel "paranoid"; she claimed that she had every right to be paranoid.It is important to note that a vast majority of participants in this study who reported being stigmatized clearly indicated that discrimination by some healthcare providers should not be generalized to all of them. In addition to receiving sub-optimal care or being denied care, as well as being blamed for their HIV status, many participants believed that they had received strong supportive care from many other healthcare providers. A 39-year-old female airport custodian said:I was so scared when I learned that I was infected with the HIV, I had lost all hope. I was devastated. However, my doctor changed my life. He helped me to think differently. I really felt calm after talking to him.A 22-year-old housewife talked about the supportive role of physicians during her pregnancy:I was a pregnant woman when I was diagnosed as HIV positive, so I became disappointed. I thought both my baby and I would die at any moment. When I came to [my] doctor [at the] prenatal clinic, they encouraged me and said that I would not die and said I could live well. I said again, I am going to die. In response, different health workers encouraged me and said if I took precautionary measures, I would not die. Now I have become somewhat hopeful about living.Finally, a few participants in this study mentioned that when they were referred by the HIV clinic for a certain type of care, they felt less discrimination and experienced more quality in their care. For example, a 32-year-old housewife described her experience visiting a dentist to whom she had been referred:I had a toothache two years ago. I should have gotten my tooth treated, but [whenever] I called a dentist office, they did not admit me. But after a while, the same dentist whom I visited a long time ago accepted to treat my tooth. This was a change in their attitudes, because the HIV clinic had introduced me to the dentist.Prior to elaborating on our findings, it is important to note that participants for this study were selected from two HIV clinics in central Tehran, and therefore the results of this study could not be generalized to all Iranian PLHIV. Patients who had not visited these treatment sites were not included in the present research.Our data shows that nearly all participants in this study reported experiencing stigma and discrimination by their healthcare providers in a variety of contexts. While previous studies demonstrate that most Iranian healthcare providers report fairly positive attitudes towards PLHIV , our parParticipants in this study perceived that healthcare providers' fear of becoming infected with HIV, coupled with religion- and value-based assumptions about PLHIV, led to high levels of stigma. The perceived negative attitudes of healthcare providers demonstrated in this study are supported by previous studies in different countries -43.gonah) as they relate to HIV transmission and stigma. Participants felt that providers linked HIV-positive status to drug addiction, prostitution, adultery, alcoholism and homosexuality, all of which are seen as religious sins. Participants clearly perceived that religious-based values led to negative attitudes toward PLHIV. However, it is important to note that none of the participants indicated that healthcare providers who seem more religious had exercised more discrimination against them. Further studies are needed to investigate the impact of religiosity on attitudes toward providing healthcare to PLHIV in Muslim communities, or what it means to be an HIV-positive Muslim, and to live in an Islamic country where practically all healthcare providers are Muslim.PLHIV who participated in this study often pointed to the concept of religious sins regarding stigma and discrimination in healthcare settings, should be used in educational intervention programmes targeting healthcare providers.It is important to consider what should be done in future research, programming and/or policy to change this situation, and to examine how efforts made in western countries to reduce stigma may be applied in Iran. Extensive qualitative research is needed to answer these questions. Some solutions may be suggested within Islamic law itself, which equally condemns discrimination against patients. Investigating Islamic literature and instruction, as well as requesting official public statements from spiritual leaders participants experienced higher quality care when visiting healthcare providers to whom they were referred directly by an HIV clinic; and (2) participants consistently experienced poor quality care from dentists. Participants may have more positive experiences with providers to whom they are referred by HIV clinics because these healthcare providers presumably receive a large number of HIV patients through the referral process, and therefore may be more likely to be informed, educated and prepared to provide care to PLHIV.Additionally, official referrals by HIV clinics to healthcare providers may carry more formal accountability than patient-initiated requests for care. Regardless of the reasons, we suggest that HIV clinics develop an official referral office for PLHIV, and further, that PLHIV should be encouraged to request all types of referrals from HIV clinics, particularly for non-HIV-related care.et al (2006) [The second finding, that dentists have greater fear of occupation-related HIV risk than physicians and other healthcare providers, has been supported by Jovic-Vranes l (2006) . Anotherl (2006) . One plal (2006) .Previous studies have emphasized education of clinical staff through: role modelling, discussions and counselling about modes of HIV transmission; universal precautions; decreasing discrimination; and consideration of human rights . EducatiStrategies to address healthcare providers' concerns, such as the development of interventions to promote occupational safety, are likely to ameliorate the discrimination experienced by PLHIV when accessing healthcare services . In partPLHIV reported being refused care or receiving sub-optimal care; this is consistent with previous study findings ,53,54. Pmahram. In this context, mahram means that one is allowed to expose his or her body to someone who is not his or her spouse, which is otherwise condemned by Islamic law. Iranian Islamic culture traditionally considers the most prestigious professions to be the study of religion and/or the study of the body. Indeed, clergy and physicians provide care for an individual's mind and body, respectively.However, it is important to examine this rejection and blaming within the context of the patient-provider relationship in Iranian culture. This relationship is traditionally based on compassion, friendliness, altruism and mutual trust. In Iranian Shi'a-Islamic culture, the physician is mahram, rejects or mistreats his or her patient, the patient faces an extremely humiliating situation that is far more degrading than being rejected by any other service. This rejection likely causes psychological distress with negative health consequences. In our study, such consequences included participants avoiding or delaying seeking healthcare, refusing disclosure of HIV status when seeking healthcare, and using spiritual healing as a substitute for conventional care.Given this context, when a healthcare provider, who is culturally recognized as a patients' confidante and trustee under Avoiding and delaying seeking healthcare in local settings by participants who had commuted from smaller cities to the two HIV clinics in Tehran was particularly alarming. Several participants clearly reported that they had not sought healthcare in their own towns because they did not want to be mistreated or misjudged by local healthcare providers. This suggests heavier stigma and discrimination in small and local healthcare settings. Therefore, it is imperative that the Iranian healthcare system combat stigma by focusing on educational training and policy at the national level that particularly targets both HIV clinics and regular healthcare facilities.In addition to behavioural reactions to perceived acts of stigma, emotional reactions included developing low self-esteem, accompanied by a diminished motivation to stay healthy, feeling angry and vengeful, and being emotional stressed. Other researchers have reported similar results. A recent study examined the experiences of individuals living with HIV who resided in an area with low HIV prevalence, and reported that the reactions to perceived acts of stigma and discrimination by PLHIV from remote areas included anger, shame, social isolation and self-advocacy .In a recent investigation of Iranian healthcare performance on HIV and AIDS, NBC: Around the World reported, "In a region where other Muslim governments ignore the AIDS epidemic, quarantine HIV-infected people or preach abstinence as the only solution, Iran's approach is fairly progressive. Iran's AIDS programme melds up-to-date programmes and research with deep-rooted religious values" . HoweverEven though the Islamic Republic of Iran has been a leading country in HIV prevention and treatment in the Middle East ,56, the Iranian healthcare authorities must show support and designate resources to stigma-reduction activities nationally . An inteThe authors declare that they have no competing interests.FR conceived the study and participated in the design, data collection and analysis. SN conceived the study, took part in coordination and helped to draft the manuscript. MB participated in the design of the study and preparation of the manuscript. FAm participated in the design of the study and interpretation of data. FAa participated in the design of the study and interpretation of data. EH participated in the interpretation of data. SST participated in the design of the study and interpretation of data. All authors read and approved the final manuscript.

Computational modeling and experimentation in a model system for network dynamics reveal how network phase relationships are temperature-compensated in terms of their underlying synaptic and intrinsic membrane currents. Cancer borealis. The pyloric rhythm is a triphasic motor pattern in which the Pyloric Dilator (PD), Lateral Pyloric (LP), and Pyloric (PY) neurons fire in a repeating sequence. While the frequency of the pyloric rhythm increased about 4-fold (Q10∼2.3) as the temperature was shifted from 7°C to 23°C, the phase relationships of the PD, LP, and PY neurons showed almost perfect temperature compensation. The Q10's of the input conductance, synaptic currents, transient outward current (IA), and the hyperpolarization-activated inward current (Ih), all of which help determine the phase of LP neuron activity, ranged from 1.8 to 4. We studied the effects of temperature in >1,000 computational models of a bursting neuron and the LP neuron. Many bursting models failed to monotonically increase in frequency as temperature increased. Temperature compensation of LP neuron phase was facilitated when model neurons' currents had Q10's close to 2. Together, these data indicate that although diverse sets of maximal conductances may be found in identified neurons across animals, there may be strong evolutionary pressure to restrict the Q10's of the processes that contribute to temperature compensation of neuronal circuits.Most animal species are cold-blooded, and their neuronal circuits must maintain function despite environmental temperature fluctuations. The central pattern generating circuits that produce rhythmic motor patterns depend on the orderly activation of circuit neurons. We describe the effects of temperature on the pyloric rhythm of the stomatogastric ganglion of the crab, 10's) of synapses and membrane currents. We used computational models to show that the experimentally measured Q10's can promote phase maintenance. We also showed that many model bursting neurons fail to burst over the entire temperature range and that phase maintenance is promoted by closely restricting the model neurons' Q10's. These results imply that although ion channel numbers can vary between individuals, there may be strong evolutionary pressure that restricts the temperature dependence of the processes that contribute to temperature compensation of neuronal circuits.The neural circuits that produce behaviors such as walking, chewing, and swimming must be both robust and flexible to changing internal and environmental demands. How then do cold-blooded animals cope with temperature fluctuations when the underlying processes that give rise to circuit performance are themselves temperature-dependent? We exploit the crab stomatogastric ganglion to understand the extent to which circuit features are robust to temperature perturbations. We subjected these circuits to temperature ranges they normally encounter in the wild. Interestingly, while the frequency of activity in the network increased 4-fold over these temperature ranges, the relative timing between neurons in the network—termed phase relationships—remained constant. To understand how temperature compensation of phase might occur, we characterized the temperature dependence (Q The nervous systems of cold-blooded animals must function across significant ranges of temperature despite the fact that the signal transduction pathways and synaptic and intrinsic membrane currents are all temperature-dependent. This is particularly intriguing because one would not expect all cellular processes to have the same temperature dependence, and therefore it is hard to imagine that functional circuit integrity would necessarily be easily maintained when temperature is altered. Nonetheless, temperature compensation, that is maintenance of constant function as temperature is altered, is an important property of circadian rhythms 10's that describe the changes in their rates as a function of temperature. Although the Q10 for many biological processes is ∼2, Q10's for ion channels can vary from 1.5 to almost 100. For example, the Q10's for activation and inactivation of some K+ channels are 1.8–4.6 10's higher than 10 2+ channels have inactivation rates with Q10's as high as 19 10's that govern the intrinsic and synaptic conductances within a circuit be and still allow appropriate function to be maintained despite environmental temperature change? Which attributes of circuit performance are temperature dependent and which, if any, are temperature compensated?All biological processes have characteristic QCancer borealis, the crab used for this study, lives in the northern Atlantic Ocean, and can be found from Newfoundland to Florida C. borealis inhabit deeper waters (at depths of 800 m), they also are frequently found in both intertidal and subtidal ecosystems foraging for food C. borealis can experience temperature fluctuations ranging from 8°C to 24°C within a single day C. borealis is found at ocean temperatures ranging from 3°C to 16°C + current (IA) and the hyperpolarization-activated inward current (Ih) The stomatogastric ganglion (STG) of marine crustaceans generates two motor patterns that are responsible for the chewing and filtering of food The phase relationships of the network neurons are maintained relatively constant as a function of frequency 10's, we varied temperature in two different computational models, one of a bursting pacemaker neuron Although the pyloric rhythm is a “simple” neuronal circuit, its dynamics involve the activation and inactivation of many intrinsic and synaptic currents. To determine whether the biological results “automatically” arise from the effects of temperature on membrane currents with similar QThe triphasic pyloric rhythm of the STG is shown in the extracellular recordings from the motor nerves exiting the STG in p = 0.35, Mann-Whitney rank sum test, n = 45 for the non-acclimated population) p<0.05, Levene test).All of the animals used in this study were acclimated to 11°C for at least 3 wk before use. The frequency of the pyloric rhythms from 23 animals at 11°C varied from 1.0 Hz to 1.5 Hz and exhibited a mean frequency of 1.20±0.11 Hz (S.D.). The median pyloric frequency of these acclimated animals was not significantly different from that of a non-acclimated population described previously . Remarkably, although the frequency varied considerably as a function of temperature, the phase of firing of the pyloric neurons was virtually unaltered by temperature (n = 7), as indicated by Q10's not significantly different from 1 .To quantify the effects of temperature on frequency and phase of the pyloric rhythm, we recorded from the motor nerves in seven preparations that were gradually warmed from 7°C to 23°C . Figure 2.32±0.2 . Temperaperature , n = 7, t from 1 . We did To gain further insight into how phase relationships might remain stable despite the increase in frequency as a function of temperature, we examined the intracellular waveforms of the pyloric neurons as a function of temperature. 10 = 0.92±0.1, p = 0.256, one-way repeated measures ANOVA; p<0.002, one-way repeated measures ANOVA), with a Q10 of 1.56±0.17.Although the total IPSPs in LP are temperature compensated, this does not necessarily mean that the synaptic currents are also constant, as the IPSP waveform and amplitude depend on the other membrane conductances and time constants as well as the synaptic current. We measured the LP neuron input conductance in voltage clamp as a function of temperature by applying tetrodotoxin (TTX) and picrotoxin (PTX) to remove action potentials and block most of the synaptic inputs to the LP neuron. We then stepped the voltage from −60 mV to −80 mV in 5 mV steps, measured the resulting currents, and calculated the input conductance. The apparent lack of change in IPSP amplitude from 7°C to 23°C despite a nearly 2-fold increase in input conductance over that range was intriguing. Therefore, we voltage-clamped the LP neuron during the ongoing pyloric rhythm and directly measured the total Inhibitory Postsynaptic Currents (IPSCs) over the same range of temperatures. 10 of 2.29±0.25 (n = 7). 10 = 1.56±0.17; The synaptic currents recorded at −60 mV are most relevant to understanding the membrane potential trajectories seen during the ongoing rhythm . AccordiA, and the hyperpolarization-activated inward current, Ih, on the phase at which the LP and PY neurons recover from inhibition Previous work demonstrated the importance of both the transient outward current, IA. IA was measured as the difference current between the outward currents evoked from a holding potential of −100 mV and those evoked from −40 mV. The currents measured at voltages from −40 mV to +30 mV in 10 mV steps are shown at five temperatures from 7°C to 23°C the Q10's of IA and Ih were replaced by the experimentally measured values , but the biologically measured Q10's decreased this effect and increased the extent to which the LP maintained constant phase as a function of temperature.Taylor et al. d values and 5. T10's to 1.5, 2.0, and 3.0 (10's for IA and Ih (blue) to the condition in which they had the experimentally determined values (red). These plots show that in the “default” (blue) condition, temperature affected the LP onset phase for all Q10's . For the blue condition, LP onset phase came later at high temperatures for Q10 = 1.5, became approximately temperature compensated for Q10 = 2.0, and became considerably “overcompensated” for Q10 = 3.0 . Note that the differences between the red and blue conditions were smaller for larger Q10 values .We then examined the effects of changing all of the model QPoikilotherms often face considerable temperature fluctuations during a given day or over extended periods of time. This poses a series of interesting questions for the nervous system, as it needs to maintain its important functions despite these environmental perturbations. Many previous studies have documented the effects of temperature on a variety of behaviors and physiological processes 10's less than 2 are considered to be relatively temperature insensitive, and behaviors with emergent system Q10's less than 2 are conventionally termed “temperature compensated.” The best known example of temperature compensation is that shown by the circadian rhythm, and its temperature compensation is considered one of its salient features. Although the molecular basis underlying circadian rhythms have been well-elucidated NeurosporaBy convention, processes with QAplysia neuromuscular system. In this preparation, the strength of neuromodulatory effects on muscle contraction was maintained over a temperature range from 15°C to 25°C. Paradoxically, the release of neuromodulators that modulate neuromuscular contraction decreased 20-fold when temperature was increased. However, the decrease in neuromodulator release was partially compensated by increased neuromodulator efficacy. Thus in this particular example, physiological temperature compensation is achieved by opposing temperature dependencies of related functions.Zhurov and Brezina In this study, we describe a form of temperature compensation that occurs in a rhythmically active neural circuit. When we rapidly varied the temperature of the in vitro stomatogastric nervous system, the pyloric network frequency varied about 4-fold, from ∼0.7 Hz at low temperatures to ∼2.9 Hz at 23°C . At the A and Ih—have opposing functional roles, temperature compensation of the phase relationships of the pyloric neurons appears to emerge as a consequence of similar temperature dependencies of opposing cellular mechanisms. h activation and delayed by IA activation h and also favor the voltage-dependent deinactivation of IA. Consequently, as temperature increases, these opposing time and voltage-dependent processes, as a first approximation, may remain temporally scaled with each other. The LP off-phase is determined by the phase of PY onset, which is itself maintained constant, presumably by similar mechanisms.All of the component processes we measured increased in amplitude and/or rate with temperature. However, given that some of these component processes—e.g., IA, and Ihh and deinactivation of IAAt constant temperature, the pyloric network phase relationships are also maintained despite changes in frequency Thus, the mechanisms that allow for phase maintenance at constant temperature seem to employ some of the same cellular mechanisms that promote temperature compensation. This is not necessarily surprising, because the strong evolutionary pressure that gave rise to robust phase maintenance may have also forced a set of solutions that would be robust enough to compensate for the frequency changes that arise from changes in temperature.The ability to maintain phase independent of frequency has been well-described in the neuronal control of swimming in lamprey, leech, and crayfish Our commonsense knowledge of the world may lead us to believe that achieving robust behavior in cold-blooded animals over a range of temperature is simpler than it is in fact. The non-trivial nature of this problem is revealed in the computational studies reported here. In both cases, we employed families of more than 1,000 model neurons that varied in terms of the maximal conductances of their underlying currents but were otherwise identical. These modeling studies illustrate the extent to which robust physiological behavior requires that the temperature dependence of the biological processes must be closely regulated.Many of the 1,270 pacemaker models failed tA and Ih in determining phase in the pyloric network, without the computational studies shown in 10's of IA and Ih would favor temperature compensation of phase. The first result from these studies and maintained in tanks containing artificial seawater at 11°C for 3 wk before use.C. borealis physiological saline was composed of 440 mM NaCl, 11 mM KCl, 13 mM CaCl2, 26 mM MgCl2, 11 mM Trizma base, and 5 mM Maleic acid, pH 7.4–7.5. All reagents were purchased from Sigma Aldrich.The stomatogastric nervous system was dissected out of the animals and pinned out in a Sylgard (Dow Corning) coated plastic Petri dish containing chilled saline (11°C). In all cases, we worked only with fully intact stomatogastric nervous system preparations that included the commissural and esophageal ganglia with two intact superior esophageal nerves and two intact inferior esophageal nerves.The preparations were continuously superfused with physiological saline. The temperature of the superfusing saline was controlled using a Peltier device purchased from Warner Instruments. The temperature was changed at about 1 degree/min when only extracellular recordings were made and about 1 degree/2–3 min when intracellular recordings were also made. As the temperature was increased we noticed that the ganglia swelled and/or moved. This often necessitated small movements of the intracellular electrodes to maintain the intracellular recordings. When the electrodes were repositioned carefully, return to 11°C showed no changes in resting potential, firing properties, or input resistance. If the electrodes were not repositioned slightly, penetrations were usually lost with large temperature changes. The preparations were maintained at their target temperature for 10 min before data were taken.2SO4 and 20 mM KCl. Data were acquired using a Digidata 1200 data acquisition board (Axon Instruments) and analyzed using Clampfit 9.0 (Axon Instruments) Spike2 v 6.04 (Cambridge Electronic Design) and/or MATLAB 7.1 (Mathworks). Statistical analyses were performed using the SigmaPlot 10 and SigmaStat software packages (Jandel Scientific).Vaseline wells were placed around motor nerves and extracellular recordings were obtained using stainless steel pin electrodes placed in the wells and amplified using an A-M Systems differential amplifier. Intracellular recordings were obtained from cell bodies in the STG using 10–30 MΩ glass microelectrodes pulled with a Flaming/Brown micropipette puller The microelectrode solution contained 0.6 M KInput conductance was measured in two electrode voltage clamp by stepping the membrane from −60 mV to −80 mV in 5 mV steps, measuring the resulting currents, and then calculating the conductance.IPSCs were measured using two electrode voltage clamp during the ongoing rhythm. IPSCs were measured in the LP neuron at membrane potentials from −60 mV to −120 mV in 5 mV steps. Each holding potential was maintained for 8–12 s, and the membrane potential was returned to −60 mV in between each voltage step for at least 8–12 s.h and IA were both measured in the presence of 10−7 M TTX, 10−5 M PTX, and 10−2 M TEA. Ih was measured at membrane potentials from −50 mV to −120 mV in 5 mV steps using 12 s pulses with 12 s at −50 mV between each pulse. IA was measured as the difference current between steps taken from a holding potential of −100 mV and those taken from a holding potential of −40 mV. Currents were recorded from −40 mV to +30 mV at 10 mV intervals using pulses of 1.5 s following a 1.5 s prepulse.I10. The Q10 is a conventional measure used to describe how much a given rate process changes over a 10°C temperature change and defined as [rate(T+10°)/rate(T)] p was plotted against temperature T in a semilog plot and the Q10 value was then extracted from the slope of the linear regression (m) 10 (seQ10) was calculated by propagation error from the slope and the standard error of the slope (sem) of the linear regression To characterize and compare the temperature dependencies of various parameters of the pyloric network, we used a modified Arrhenius equation to determine the temperature coefficient, Q10) of all channel activation and inactivation dynamics. For simplicity we set the Q10 of all the activation and inactivation rates equal to 2. All values for ∞m, ∞h, mτ, hτ, and p are as found in Liu et al. 10.The model used to simulate bursting neurons has been previously described To create a population of model bursting neurons, we initiated 1,500 simulations with random initial values for all seven active conductances. Each model was subject to 1 h of simulated activity-dependent homeostatic regulation at the model's default temperature of 23.5°C. Models that showed poor bursting behavior after this period were excluded, resulting in a population of 1,270 model neurons that were bursting at 23.5°C and which had a variety of different values of the maximal conductances for the voltage-dependent currents. We then turned off the self-tuning function in these neurons and used them as a set of models with different underlying conductances to probe the extent to which they maintained functional bursting activity over a wide range of temperatures.Each model was run at 7, 11, 15, and 19°C—the same temperatures used in the biological studies. Voltage traces from each of these simulations were analyzed to determine how the bursting behavior varied with temperature. Bursting behavior—as measured by slow wave frequency—was extracted from each simulation using auto-correlation of a 10 s sample of the voltage trace. Here we define slow wave frequency as low-frequency membrane oscillations. We identified the frequency of these oscillations as the lowest frequency peak in the autocorrelogram of the voltage waveform. If this peak had an autocorrelation less than 0.05, we instead used the frequency corresponding to the greatest autocorrelation (this is uncommon and corresponds to very irregular activity).To determine whether the bursting neurons maintained duty cycle as a function of temperature, we calculated the coefficient of variation (CV) of each model's duty cycle. The bounds used to define the population of model bursting neurons that exhibited “constant duty cycle” were chosen to contain the central ∼85% of the experimental data (PD off phase). Models exhibiting CVs of duty cycle across temperature within this bound were classified as having constant duty cycle, while those with CVs above this value were classified as having a “non-constant duty cycle.”10's of IA and Ih affect LP phase onset were performed using the LP model described in Taylor et al. 10's could be associated with all voltage-gated and synaptic conductances. For each voltage-gated conductance, there was a Q10 for the maximal conductance, another for the activation rate, and another for the inactivation rate. The reference temperature was 10°C, as the model was originally designed to mimic LP's behavior at this temperature. Reversal potentials were not changed with temperature, as these effects are small over the temperature range used.Simulations to determine how the Q

The dihedral angle between the phenyl and pyrrolidine rings is 48.48 (6)°.The reaction of aniline with norcantharidin produced the imide title compound, C Å b = 8.4345 (3) Å c = 14.4101 (6) Å β = 93.468 (3)°V = 1163.62 (8) Å3 Z = 4 Kα radiationMo −1 μ = 0.10 mmT = 296 (2) K 0.32 × 0.25 × 0.04 mm Bruker APEXII CCD area-detector diffractometerSADABS; Sheldrick, 1996T min = 0.971, T max = 0.996Absorption correction: multi-scan (18085 measured reflections2699 independent reflectionsI > 2σ(I)1898 reflections with R int = 0.041 R[F 2 > 2σ(F 2)] = 0.041 wR(F 2) = 0.177 S = 0.61 2699 reflections163 parametersH-atom parameters constrainedmax = 0.16 e Å−3 Δρmin = −0.17 e Å−3 Δρ APEX2 used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008SHELXTL (Sheldrick, 2008SHELXL97.Data collection: 10.1107/S1600536809000282/at2701sup1.cif Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536809000282/at2701Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:

It was synthesized from the elements at 873 K in an evacuated fused-silica tube. Single crystals were grown by vapor transport with I2. NpCuSe2 crystallizes in the LaCuS2 structure type and can be viewed as a stacking of layers of CuSe4 tetra­hedra and of double layers of NpSe7 monocapped trigonal prisms along [100]. Because there are no Se—Se bonds in the structure, the formal oxidation states of Np/Cu/Se may be assigned as +III/+I/−II, respectively.The title compound, NpCuSe Å b = 7.4384 (6) Å c = 7.1066 (5) Å β = 97.156 (1)°V = 350.34 (5) Å3 Z = 4 Kα radiationMo −1 μ = 56.06 mmT = 100 (2) K 0.08 × 0.05 × 0.04 mm Bruker APEXII CCD diffractometerSADABS; Sheldrick, 2006T min = 0.045, T max = 0.212Absorption correction: numerical 1309 reflections with R int = 0.036 R[F 2 > 2σ(F 2)] = 0.028 wR(F 2) = 0.068 S = 1.35 1376 reflections37 parametersmax = 2.43 e Å−3 Δρmin = −4.48 e Å−3 Δρ APEX2 used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008CrystalMaker (Palmer, 2008SHELXTL (Sheldrick, 2008Data collection: 10.1107/S160053680900395X/wm2219sup1.cif Crystal structure: contains datablocks I, global. DOI: 10.1107/S160053680900395X/wm2219Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:

Our results suggest that even though IR expression was normally occurring, IR β-subunit tyrosine kinase activity in muscle was down-regulated leading to alterations in insulin post receptor signaling. Consistent with normal insulin receptor and post receptor signaling, our results were compatible with decreased insulin binding to IR probably due to neutralization by anti-insulin antibodies. In conclusion, this patient has immunologic insulin resistance and treatment should be based on immunosuppressive drugs as tolerated.Insulin signalling pathways were investigated in a 33 year-old woman with immunologic insulin resistance. Her past medical history was remarkable for intermittent use of insulin and allergic reactions to several drugs, and measure of plasma anti-insulin antibodies level corroborated the clinical suspicion of immune mediated insulin resistance . Treatment with several immunosuppressive regimens was tried, however the results were disappointing. Possible subcellular mechanisms of insulin resistance were investigated by performing analysis of insulin receptor and post receptor signaling in skeletal muscle biopsy. The expression of insulin receptor (IR), insulin receptor substrate 1 (IRS-1) and glucose transporter 4 (GLUT-4) was evaluated in total extract from muscle tissue by Western blotting. Basal IR, IRS-1 and GLUT-4 expression was detected, however receptor autophosphorylation was not observed. A study of translocation of GLUT-4 to plasma membrane showed that tissue presented low levels of membrane-associated GLUT-4. When Immunologic insulin resistance is a disorder recognized in the clinical practice for many years ,2. Its pAfter excluding the possibility of resistance to the subcutaneous route of administration by using the parenteral route and makiWe hereby present a case of immunologic insulin resistance that has not responded to many therapeutic immunosuppressive interventions resulting in great anxiety for assistant physicians and leading us to investigate the possibility of a subcellular mechanism of disease.A 33 year-old woman with diabetes for 25 years was treated with NPH insulin (0.77 U/kg/day) when she first came to our attention. She had a positive history of allergy to penicillin and no other morbidities. During her adolescence, she had been switched to oral hypoglycemiants for approximately 4 years, which made her diabetes difficult to classify.During follow-up we were able to gradually decrease her insulin dose until she was successfully switched back to oral agents. For 11 months she was on a regimen of glyburide (10 mg bid), metformin (850 mg tid) and acarbose (50 mg tid), before being admitted with hyperosmolar non-ketotic coma.After a prolonged hospital stay she was discharged home using NPH insulin. Over the following months, worsening glycemic control indicated progressive insulin dose increments (from 0.6 to 5.7 U/Kg/day). When 2.7 U/Kg/day was reached, we discarded all causes of secondary diabetes and plasma anti-insulin antibodies were determined . We concluded she developed immunologic insulin resistance.Several immunosuppressive regimens were tried such as glucocorticoids , cyclophSubcutaneous resistance to insulin was ruled out by intravenous administration during the inpatient period . Thereafin vitro stimulation with insulin. We found that IR and IRS-1 tyrosine phosphorylation was highly stimulated by insulin and prednisone (40 mg/d) in an outpatient basis. After one year, insulin dose was reduced from 5.8 to 3.48 U/Kg/d and glycemic control, even if not optimum, has shown some improvement (average glycemia: 390 to < 200 md/dL). It is important to note that patient did not experience fasting hypoglycemia since severe insulin resistance developed.We conclude that this patient has immunologic insulin resistance of difficult management. The best immunosuppressive regimen for her, in terms of improvement in glycemic control, was the combination of prednisone and micophenolate mophetil, however the results are slow as a result of the type of response to these drugs. The muscular biopsy helped to rule out a subcellular mechanism of insulin resistance that would be more difficult to treat.The authors declare that they have no competing interests.GFT, FHSC and CRMAJ drafted the manuscript and were responsible for patient's medical assistance, MSF carried out the molecular genetic studies, GFT, MSF and MBG participated in the design of the study. MBG coordinated the study. All authors read and approved the final manuscript.

The human genome displays extensive copy-number variation (CNV). Recent discoveries have shown that large segments of DNA, ranging in size from hundreds to thousands of nucleotides, are either deleted or duplicated. This CNV may encompass genes, leading to a change in phenotype, including drug response phenotypes. Gemcitabine and 1-β-D-arabinofuranosylcytosine (AraC) are cytidine analogues used to treat a variety of cancers. Previous studies have shown that genetic variation may influence response to these drugs. In the present study, we set out to test the hypothesis that variation in copy number might contribute to variation in cytidine analogue response phenotypes.50 values identified 11 regions with permutation p-values < 0.05. Multiplex ligation-dependent probe amplification assays were performed to verify the 11 CNV regions that were associated with this phenotype; with false positive and false negative rates for the in-silico findings of 1.3% and 0.04%, respectively. We also had basal mRNA expression array data for these same 197 cell lines, which allowed us to quantify mRNA expression for 41 probesets in or near the CNV regions identified. We found that 7 of those 41 genes were highly expressed in our lymphoblastoid cell lines, and one of the seven genes (SMYD3) that was significant in the CNV association study was selected for further functional experiments. Those studies showed that knockdown of SMYD3, in pancreatic cancer cell lines increased gemcitabine and AraC resistance during cytotoxicity assay, consistent with the results of the association analysis.We used a cell-based model system consisting of 197 ethnically-defined lymphoblastoid cell lines for which genome-wide SNP data were obtained using Illumina 550 and 650 K SNP arrays to study cytidine analogue cytotoxicity. 775 CNVs with allele frequencies > 1% were identified in 102 regions across the genome. 87/102 of these loci overlapped with previously identified regions of CNV. Association of CNVs with gemcitabine and AraC ICThese results suggest that CNVs may play a role in variation in cytidine analogue effect. Therefore, association studies of CNVs with drug response phenotypes in cell-based model systems, when paired with functional characterization, might help to identify CNV that contributes to variation in drug response. CYP2D6 deletions ("poor" metabolizers), and these genotypes are associated with variation in response to a large number of drugs , PLEKHA5 and GPR133 (G protein-coupled receptor 133), respectively. NPHP1 and PLEKHA5 overlapped the CNV regions, whereas GPR133 was 179 Kb away from the CNV region associated with AraC. The region located on chromosome 1 overlapped the KIF26B gene and was 295 Kb away from a gene encoding a histone methyltransferase, SMYD3.In the case of AraC, one region in our cell lines and the expression of this gene was also associated with AraC IC50, with a p-value of 0.0027. The Chr1CNV7 association did not pass Bonferroni correction. However, the association study was a "discovery" study - to be followed by functional genomics validation. Hence, we selected the SMYD3 candidate gene based on expression and possible biological relevance to cancer.Since we had Affymetrix U133 Plus 2.0 mRNA expression array data for the same lymphoblastoid cell lines in which we had assayed CNV ; we deteSMYD3 inhibits cervical carcinoma cell growth and invasion [SMYD3 may represent a risk factor for human cancer [SMYD3 plays crucial roles in HeLa cell proliferation and migration/invasion, so it has been suggested that it may be a useful therapeutic target in human cervical carcinomas [SMYD3 gene (chr1CNV7) is associated with AraC IC50 value, with a permutation p-value of 0.035. The chr1CNV7 deletion occurred in 3 samples, all from Caucasian subjects, so we also tested the association in only this ethnic group. Likelihood ratio testing of linear regression of AraC log IC50 values against gender and storage time, with or without the relevant CNV values, gave a p-value of 0.019. In addition, analysis of IC50 values with the chr1CNV7 region showed that deletion of this CNV region was associated with an increase in the IC50 value for AraC when compared with cells transfected with negative control siRNA as shown in Figure SMYD3, followed by cytotoxicity studies with both drugs, showed significant deviations, but the deviation was small for AraC when compared to that after gemcitabine treatment were found in other publications, and 51/102 were represented in multiple studies. Although we did find agreement for many of our CNVs with previously reported variants, there were other CNV regions previously reported that were not identified in our study. This could be due to our stringent criteria for CNVs. It also could be due to the different platforms, methods and study populations used in different studies. In addition rare events are usually not reported.In this study, we have used 550,000 SNP markers (Illumina 550 K Bead chip) to discover CNV in 197 human lymphoblastoid cell lines obtained from ethnically diverse populations. The average distance between markers on the Illumina 550 K chip is 5.8 kb, and the average size of CNVs identified in our cell lines was more than 76,000 bp, indicating that smaller CNV may be underrepresented in our study. The Illumina 550 K SNP chip was designed, in part, to interrogate gene rich regions ; which i50) for gemcitabine and AraC using a 197 lymphoblastoid cell line-based model system designed to make it possible to study common human genetic variation. Although tumor genome is critical for understanding response to therapy and disease pathophysiology, the germline genome is also critical, especially for drug response phenotypes. Obviously, we understand that these lymphoblastoid cell lines were EBV transformed from normal individuals, and that they were neither collected from cancer patients nor tumor tissues. Hence, we might miss some candidate genes that may be specific to cancer. However, lymphoblastoid cell lines have been shown by several groups, including ours, to be useful for identifying candidate genes or genetic variation associated with drug-induced cytotoxicity [50 values in 197 lymphoblastoid cell lines. 11/102 regions were associated with gemcitabine and AraC IC50 values had a gene (SMYD3) in close proximity that displayed high expression in the lymphoblastoid cell lines.Since we had Affymetrix U133 Plus 2.0 expression array data for the same lymphoblastoid cell lines ; we deteSMYD3 gene in pancreatic cancer cell lines. Knockdown made the cells more resistant to AraC, confirming the association study results, and also made them resistant to gemcitabine. Our results suggest that joining association studies with functional validation experiments may help to identify biomarkers for disease or response to therapy.SMYD3, found on the q arm of chromosome 1, encodes an alternatively spliced transcript for 369 or 428 amino acids protein. Hamamoto et. al. first described SMYD3's histone methyltransferase activity with specificity for di- and tri- methylation of lys4 on Histone 3. SMYD3's histone methyltransferase activity results in transcription induction for at least 60 targets across the genome . EnhanceB3GAT1, LRP5L, PLEKHA5, KIF26B, TYRP1, FAM5C, ADAM6, FLJ40330, NPHP1, OR52E4, GPR133 and SMYD3) with drug response phenotypes. Analysis in lymphoblastoid cell lines and functional validation in cancer cell lines suggest the probable role of SMYD3 to AraC and gemcitabine drug response phenotype. The current study provides additional information with regard to the contribution of CNVs to variation in drug response for two important antineoplastic drugs and indicates that the assay of CNV should be included in pharmacogenomic studies.We took the advantage of genome-wide SNP data obtained with 550 K Illumina Bead Chips to identify CNV regions across the genome in 197 lymphoblastoid cell lines. Association studies with gemcitabine and AraC cytotoxicity phenotypes identified CNV regions that might be associated with cytotoxicity for these two drugs. In this study we investigated the role of CNVs together with expression of neighboring genes (The conception of the study and interpretation of the analysis was performed conjointly by SH, KRK, CH, JPK, LL, LW and RW. Writing of the manuscript was performed by KRK, LW and SH and RW. KRK and CH performed the computational and statistical analysis, SH and LL performed the laboratory-based experiments. All of the authors read, corrected and approved the final manuscript.Additional file 1, Methods Section, Table S1, Table S2Click here for file

Weak inter­molecular C—H⋯O hydrogen bonding is present in the crystal structure.The two naphthyl fused-ring systems in the title compound, C Å b = 29.439 (1) Å c = 8.3518 (3) Å V = 4211.5 (3) Å3 Z = 8 Kα radiationMo −1 μ = 0.10 mmT = 123 K 0.27 × 0.12 × 0.05 mm Bruker SMART APEX diffractometerAbsorption correction: none17329 measured reflections3700 independent reflectionsI > 2σ(I)2613 reflections with R int = 0.069 R[F 2 > 2σ(F 2)] = 0.085 wR(F 2) = 0.195 S = 1.23 3700 reflections244 parametersH-atom parameters constrainedmax = 0.57 e Å−3 Δρmin = −0.50 e Å−3 Δρ APEX2 used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008X-SEED (Barbour, 2001publCIF (Westrip, 2009Data collection: 10.1107/S1600536809010824/xu2498sup1.cif Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536809010824/xu2498Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:

We introduce and describe the Breast Cancer and the Environment Research Centers (BCERC), a research network with a transdisciplinary approach to elucidating the role of environmental factors in pubertal development as a window on breast cancer etiology. We describe the organization of four national centers integrated into the BCERC network.Investigators use a common conceptual framework based on multiple levels of biologic, behavioral, and social organization across the life span. The approach connects basic biologic studies with rodent models and tissue culture systems, a coordinated multicenter epidemiologic cohort study of prepubertal girls, and the integration of community members of breast cancer advocates as key members of the research team to comprise the network.Relevant literature is reviewed that describes current knowledge across levels of organization. Individual research questions and hypotheses in BCERC are driven by gaps in our knowledge that are presented at genetic, metabolic, cellular, individual, and environmental levels.As data collection on the cohort, animal experiments, and analyses proceed, results will be synthesized through a transdisciplinary approach.Center investigators are addressing a large number of specific research questions related to early pubertal onset, which is an established risk factor for breast cancer. BCERC research findings aimed at the primary prevention of breast cancer will be disseminated to the scientific community and to the public by breast cancer advocates, who have been integral members of the research process from its inception. The Breast Cancer and the Environment Research Centers (BCERC) sponsored by the National Institute for Environmental Health Sciences (NIEHS) and the National Cancer Institute (NCI) were established to better understand how environmental factors may influence pubertal development and to enable primary breast cancer prevention strategies. In 2003, after a period of focused and thoughtful advocacy integrated with scientific consultation, BCERCs were awarded to The Fox Chase Comprehensive Cancer Center in Philadelphia, Pennsylvania; Michigan State University in East Lansing, Michigan; the University of Cincinnati, Ohio; and the University of California San Francisco Helen Diller Family Comprehensive Cancer Center.In this review, we define the BCERC research questions and present our common conceptual framework for the etiology of breast cancer and the rationale for a focus on puberty. Then, across multiple levels of biologic, behavioral, and social organization, we highlight our current scientific understanding of the development of the normal mammary gland, the nature and action of breast cancer risk factors, and our understanding of the mechanisms of breast carcinogenesis relevant to early development. Our BCERC experimental approach is driven by the knowledge gaps in this understanding. We conclude with a description of the organization of the BCERC.We explore whether exposures to environmental factors before and during puberty might set the stage for increased breast cancer risk in adulthood. Multiple hypotheses are being tested by developing and interrogating rodent models and rodent and human cell culture systems to characterize the molecular basis of mammary gland development over the life span. We seek to determine how environmental agents may affect this development and to better understand the process of breast carcinogenesis. In simultaneously conducted epidemiologic cohort studies of prepubertal girls, conceptually integrated with the studies in animal and human tissue models, additional hypotheses posit roles for the environmental, psychological, behavioral, metabolic, and genetic factors as determinants of puberty. This transdisciplinary approach will generate information about relevant exposures from human studies that can be applied to the animal studies. In turn, the animal studies will provide a mechanistic understanding of how environmental exposures may affect pubertal breast development and adversely influence breast cancer risk in adult life.This research adds to the findings of other similar research relevant to breast cancer, early development, and the environment, including the National Growth and Health Study , the ChiThe BCERC initiative is multidisciplinary in its approach to research questions and in the integrated nature of the science with community and advocacy participation. Although not prescribed by the National Institutes of Health’s request for applications, investigators are invested in making our approach truly transdisciplinary . Transdiin utero exposures through puberty and maturity to the postmenopausal years. Our framework permits multiple investigators to locate their particular hypotheses within the framework, taking into account our best understanding of pubertal development and the environment from both a socioecological and life span perspective that are participants in the research studies and whose members work to translate the current scientific knowledge, as well as findings from new research, into educational messages for the community. Simultaneously, they provide input from the community perspective into the ongoing research of the BCERCs.in utero hormonal milieu may influence breast cancer risk. Trichopoulos and others have hypothesized that higher levels of hormones during pregnancy favor the generation of a higher number of susceptible stem cells with compromised genomic stability , and possibly alpha-fetoprotein might be associated with increased breast cancer risk axis, or thelarche, and the activation of the hypothalamic– pituitary–adrenal (HPA) axis, or adrenarche, which are independent events. A surge of pituitary follicle-stimulating hormone (FSH), associated with HP axis activation, stimulates primordial ovarian follicles to secrete estrogen. Circulating estrogen in turn induces mammary duct, mammary stromal connective tissue, pituitary luteinizing hormone (LH), and vascular proliferation and growth . The ratThe manifestation of breast development that we are studying in the epidemiologic project is assumed to reflect the underlying mammary biology studied in rodent models in the biology project. In rodent models, we are characterizing the rate of development, extent, and character of the mammary epithelium. With improved animal models and biomarkers to study the impact of environmental stressors on breast cancer, we can elucidate the effects of the timing of these exposures during critical windows of vulnerability.Focusing now on the phase of pubertal development, we describe factors of influence at multiple levels of organization, starting with genes and concluding with the social environment.Relatively little is known about how genetic makeup influences breast cancer risk at the time of puberty. It is unclear, for example whether BRCA mutations interact with environmental factor to increase risk . MutatioCYP3A4 (cytochrome P450 3A4) alleles, which primarily metabolize testosterone, are associated with early puberty and are more common in African-American than Hispanic or white girls topographic location within the mammary gland tree, b) age at exposure to a known or putative genotoxic agent, and c) reproductive history of the host. Further, this structure has the highest proliferative activity, estrogen receptor (ER) content, and rate of carcinogen binding to DNA the physical environment—exposure to environmental pollutants or toxicants; b) the built environment, which influences whether children have a healthy place to live, play, and go to school; and c) the social environment—the associations with socioeconomic status (SES) and race/ethnicity, social norms of behavior, and culture. These are contextual aspects of place as opposed to constitutive aspects, which are the sum of the individual characteristics of persons living in any particular geographically defined area.Factors at the environmental level can influence pubertal development and ultimately breast cancer in three major ways: The impact of putative environmental toxicants on breast development and carcinogenesis is at the heart of BCERC studies. A broad array of environmental carcinogens is being evaluated in animal models and/or in the epidemiologic studies.in vitro tests have demonstrated the estrogenic activity of BBP , another xenoestrogen, is polymerized in the manufacture of polycarbonate plastic and epoxy resins. Human exposure occurs by the leaching of BPA from plastic-lined food and beverage containers and from some dental sealants , and theCYP1A1 such that high levels of PCB exposure and expression of CYP1A1 confer a higher risk of breast cancer are a class of chemicals that are lipophilic and resistant to degradation and thus persist in the food chain and individual fat stores. POPs include chemicals such as aldrin, chlordane, DDT (dichlorodiphenyltrichloroethane), dieldrin, heptachlor, and polychlorinated biphenyls (PCBs), which have become highly prevalent in the environment of industrialized countries since World War II. A study of brominated flame retardants (polybrominated biphenyls) among accidentally exposed farm workers in Michigan revealed an association with earlier pubic hair, but not breast development, in the daughters of exposed mothers . Polybrot cancer . In anotechanism NAT2 slow acetylator and GSTMI null genotypes that may be associated with breast cancer in adults are a large class of chemicals formed by the incomplete combustion of coal, oil, and gas, as well as tobacco smoke and other substances to which humans are exposed in ambient air. These substances are genotoxic and known to be potential breast carcinogens , perhapsOne of the most commonly voiced cancer-related public concerns is the possible impact of hormones in food, because U.S. animal food sources are frequently exposed to growth hormones to boost production of meat, dairy products, and eggs. Because higher levels of estradiol may induce the hypothalamic burst of gonadotropin that initiates puberty, the impact of hormone-treated cattle deserves further study . In the Finally, among environmental factors, ionizing radiation exposure has long been recognized as a risk factor for breast cancer in humans, probably as a result of the induction of DNA double-strand breaks. An increased risk of breast cancer has been consistently reported for radiation exposure from various sources, including the atomic weapon explosions in Nagasaki-Hiroshima , and medFor even the partial list of putative environmental agents presented above, much remains to be elucidated, not only in terms of their biologic effects in animal models, but also the risks for adverse outcomes as assessed by human epidemiology. Furthermore, it is likely that chemicals interact with each other and that their individual effects are modified by the presence of genetic polymorphisms and by the social context in which the exposure plays out. It is only with a prospective, longitudinal study with input from multiple disciplinary perspectives, such as in the BCERC, that these effects and interactions can be illuminated.In addition to chemical toxicants in the physical environment, there are also aspects of the built environment that can lead to childhood obesity and may contribute to earlier puberty and breast cancer incidence later in life.For example, it is known that people who live in socioeconomically deprived neighborhoods are more likely to be physically inactive , to haveChildhood obesity is more prevalent in nonwhite and low-income children, and thus factors associated with SES, race, and ethnicity could be contributing to observed disparities in the onset of puberty. However, the relationship of SES and race/ethnicity to breast cancer is complex. Breast cancer is one of the few cancers related directly to higher SES and to being self-identified as white compared with black (above the age of about 40 years), Hispanic, and Asian women. Within race/ethnic groups, there is also a direct relationship with SES . The mosUntil recently, the age of menarche has been lower in higher-SES populations and in more industrialized countries. In the last half-century, in the United States at least, this relationship may have changed. Epidemiologic evidence now suggests that lower SES is related to earlier puberty, as determined by entry into stage 2 breast development among girls in the United States . In one Although pathways linking SES and menarche activation are not well understood, it appears that body composition and nutrition are essential parts of the puzzle . These nhttp://www.bcerc.org), an intranet web site, and national meetings. A publications committee has developed and monitors procedures for the analysis of pooled data, publication tracking, authorship protocols, ancillary studies, and other matters pertaining to pooled data and publications from the network.In addition to the four BCERCs described above, a coordinating center at the University of California, San Francisco (UCSF) site exists to coordinate questionnaire development, data entry, centralized data management, and pooled analyses. Bioinformatics support is being coordinated at the Fox Chase Cancer Center . Other aspects being coordinated between centers include an Internet web site , University of Cincinnati, and Fox Chase (Mount Sinai School of Medicine), there is a high degree of collaboration and standardization in the epidemiologic studies across centers to maximize opportunities for pooled analyses and cross-site comparisons.Integrated into the structure of the BCERCs are four COTCs. Members of the COTC include breast cancer advocates as well as academicians with expertise in communication. The primary role of the COTCs is the translation and dissemination of major research findings to the public through their member organizations. Specifically, the COTCs conduct their own research activities in communication and dissemination, develop fact sheets and other educational materials based on BCERC research, hold town hall meetings and other community activities, and assist with recruitment and retention strategies for the epidemiologic study. Members participate on both the epidemiology and biology projects, disseminate educational information and scientific findings of the BCERC and ensure the inclusion of the community perspective in BCERC projects and facilitating the ongoing flow of information from the study team to the community and from the community back to the study team.www.bcerc.org) for a listing of these publications to date (Specific results of constituent studies are being published from individual centers and collaborative efforts across centers. The reader is referred to the study Web site ( to date .The transdisciplinary nature of our approach derives first from the highly varied areas of expertise of the scientific team and the community partners. The disciplines represented include genetics, molecular and cell biology, immunology, anatomy, radiation biology, pediatrics, endocrinology, toxicology, nutritional sciences, communication science, and epidemiology. The BCERC investigators within the network are addressing specific research questions that focus on the common shared goal of the overall project: to understand the role of the environment in pubertal development as a window on breast cancer etiology. The ongoing and integrated involvement of community members and advocates in the research process and in communication with the public is a unique and effective way to advance the transdisciplinary science approach we have adopted.

The thia­diazole ring adopts a flattened envelope conformation, with the flap sp 3-hybridized C atom lying 0.259 (1) Å out of the plane of the other four atoms. The screw-related mol­ecules are linked into chains along the b axis by inter­molecular N—H⋯O hydrogen bonds.The title heterocyclic compound, C Å b = 11.0510 (2) Å c = 16.6045 (3) Å β = 90.442 (10)°V = 1724.52 (6) Å3 Z = 4 Kα radiationMo −1 μ = 0.20 mmT = 298 (2) K 0.5 × 0.4 × 0.3 mm Bruker X8 APEX CCD area-detector diffractometerAbsorption correction: none52162 measured reflections8286 independent reflectionsI > 2σ(I)7182 reflections with R int = 0.019 R[F 2 > 2σ(F 2)] = 0.035 wR(F 2) = 0.108 S = 1.03 8286 reflections221 parametersH atoms treated by a mixture of independent and constrained refinementmax = 0.51 e Å−3 Δρmin = −0.19 e Å−3 Δρ APEX2 used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008ORTEP-3 (Farrugia,1997PLATON (Spek, 2003WinGX (Farrugia, 1999Data collection: 10.1107/S1600536809000191/ci2746sup1.cif Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536809000191/ci2746Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:

Youth Check Your Risk, on chlamydia testing rates among young people attending general practices. The secondary aim was to test the acceptability of the tool among general practitioners and young people.Targeted chlamydia screening has been advocated to reduce chlamydia associated reproductive sequelae. General practitioners are well positioned to play a major role in chlamydia control. The primary aim of this pilot study was to measure the effect of offering an online sexual health assessment tool, Youth Check Your Risk http://www.checkyourrisk.org.au for use post-consultation between March to October 2007. The proportion of young people tested for chlamydia before and during the implementation of the tool was compared. Acceptability was assessed through a structured interviewer-administered questionnaire with general practitioners, and anonymous online data provided by Youth Check Your Risk users.General practitioners at three practices in Melbourne, Australia, referred patients aged 16 to 24 years to Youth Check Your Risk and 120 used it (13.8%). Major reasons for low referral rates reported by practitioners included lack of time, discomfort with raising the issue of testing, and difficulty in remembering to refer patients.The intervention did not result in any significant increases in the proportion of 16 to 24 year old males (2.7% to 3.0%) or females (6.3% to 6.4%) tested for chlamydia. A small increase in the proportion of 16 to 19 year old females tested was seen (4.1% to 7.2%). Of the 2997 patients seen during the intervention phase, 871 (29.1%) were referred to Youth Check Your Risk. Future interventions aimed at increasing chlamydia screening in general practice with the aid of an online risk assessment tool need to identify and overcome barriers to testing.Offering an online sexual risk assessment tool in general practice did not significantly increase the proportion of young people tested for chlamydia, with GPs identifying a number of barriers to referring young people to Chlamydia trachomatis is the most common bacterial sexually transmitted infection (STI) worldwide but in the end we've got 15 minutes and I spend longer and I'm always running late, but still it's that time issue.2) Discomfort raising the issue of sexual health, particularly where patients had not presented for sexual health reasons.3) Difficulty in remembering to refer patients in the target age group.To bypass these issues, the majority of GPs at one clinic reported leaving referral to YCYR to reception staff.On the whole, GPs reported that they did not hand out the referral cards or offer chlamydia screening opportunistically to all young patients, but did so when young people presented with high risk behaviours or issues related to sexual health, such as cervical screening, screening for sexually transmitted infections, contraception or genital symptoms. The majority of GPs reported that they did not feel that their testing practices had changed as a result of YCYR; however, participation in the study did raise their awareness and reminded them of the need to opportunistically screen young people for chlamydia.really negative' and 'indifferent' to 'fairly responsive' and 'interested'. Of the GPs interviewed, only two encountered patients who provided feedback on YCYR. One patient provided general feedback on the service to their GP at a later consultation, while another patient returned to their GP with the printed recommendations. Practices indicated that patients seemed reluctant to use YCYR on site, particularly in the practice in which the computer was set up in the 'staff only' area.GPs reported mixed reactions from patients they referred to YCYR, from 'We found that opportunistically offering an online sexual risk assessment tool in general practice to young people did not significantly increase the proportion of young people tested for chlamydia, although a small increase in the proportion of 16 to 19 year old women tested was seen. While GPs generally had a favourable view of the tool, a number of barriers prevented them from referring young people to YCYR and consequently, less than a third of young patients were referred to the site, and of these, few accessed it.The provision of onsite computers by practices did not seem to improve access by young people, and while no young people provided feedback about this, the location of the computers could have been a deterrent along with the fact that the only reason patients would access them was to use YCYR, hence potentially leaving young people feeling exposed or embarrassed. At a system level, practices may not be equipped to provide patients with a private area in which they can confidentially source online resources.This study has some limitations. First, we do not know if the small increase in chlamydia testing among younger women was as a direct result of offering YCYR through general practice. In order to protect the confidentiality of patients accessing the website, it was not possible to track health service use of individual patients accessing the site, therefore we do not know how many of the patients who accessed the website actually returned to a GP for chlamydia testing. Nor can we determine which chlamydia tests were the result of the patient accessing the website and being exposed to the recommendations for chlamydia screening. It is possible that testing increased because of a greater awareness of chlamydia screening due to the practices' involvement in the study or because of exposure to the educational package provided to GPs as part of the intervention, rather than as a direct result of referral to YCYR. Secondly, although their views were sought, only a small number of YCYR users provided online feedback and none provided detailed interview feedback despite the incentive offered, so we do not know how most young patients felt about being referred to the website, its on-site access, use of the site or whether they followed up on the advice. Thirdly, as two of the practices were affiliated with universities and serviced high numbers of young people and international students, it is uncertain how generalizable the results are to other practice populations.Reviews of interventions aimed at change in primary care show that while no intervention is effective in all circumstances, systematically developed, multi faceted interventions tailored to and engaging the target group and addressing barriers and facilitators to change are more likely to be effective -17. A reIt is possible that a more rigorous methodological approach in this study may have better addressed the existing barriers. In saying this, in a study by Merritt et al , despiteWhile engaging and increasing GPs knowledge around chlamydia testing is likely to go some way towards facilitating chlamydia screening, it is unlikely to increase opportunistic screening to the levels required to have an impact on chlamydia transmission in the population. Enhancing GPs communication skills, particularly around sexual history taking, may also help, however, as previous research has shown, GPs often do not feel comfortable raising the issue of sexual health during unrelated consultations ,9 and arFurthermore, GPs' perceptions that patient's are embarrassed discussing their sexual health is associated with a reduced likelihood of taking a sexual history and thus assessing a patient's risk . Recent Offering an online sexual risk assessment tool in general practice did not significantly increase the proportion of young people tested for chlamydia, with GPs identifying a number of barriers in referring young people to YCYR.It is possible that the intervention required more rigorous development to overcome these barriers. However, before the intervention can be considered ineffective, the way in which it was implemented must also be considered . The lowThe use of YCYR in general practice therefore should not be discounted altogether. However, given the issues and barriers evident in this study and reflected in other current research, the development of any future interventions, including YCYR, must first address these barriers if they are to effectively increase screening in general practice.CYR: Check Your Risk; GP: General Practitioner; HPV: Human papillomavirus; MSHC: Melbourne Sexual Health Centre; STI: Sexually transmitted infection; YCYR: Youth Check Your Risk.The authors declare that they have no competing interests.JEB was responsible for data collection, analysis and interpretation, and prepared the first draft of the manuscript. LAS contributed to the conception and design of the study, interpretation of data and writing of manuscript. CKF contributed to the conception and design of the study, interpretation of data and writing of manuscript. JSH was involved in study design, assisted in the statistical analysis and critically reviewed the manuscript. DM was involved in study design and critically reviewed the manuscript. DJH was involved in study design, assisted in youth focus group recruitment and critically reviewed the manuscript. SMS was involved in study design and critically reviewed the manuscript. MJW was involved in study design and critically reviewed the manuscript. DAW was involved in study design, assisted in the GP reference group recruitment and critically reviewed the manuscript. MYC contributed to the conception and design of the study, interpretation of data and writing of manuscriptAll authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/29/prepub

SwaminathHaving said so, I wish to introduce a caveat. Few are artists or sculptors from the moment they begin to practice their art; most will need much instruction, and many will continue to fail with their brush or chisel no matter how long they train. Applying this metaphor, not every psychiatrist will have the personality or the talent to successfully use humour as a tool with his clients; the skill will necessarily need to be developed for use by the right person and in the right context, lest the attempts end on a negative note.Moving on to a matter not addressed by Swaminath: that of the need for humour in academia. Clinicians, researchers, and editors who read, write in, or prepare journals have one attribute in common: they are all humans. Assuming that humour is a continuous variable that is normally distributed in the general population, and assuming that an interest in journals is not a confounding factor, a quick reference to statistical tables will show that more than four-fifths of journal readers will have humour levels that are within one standard deviation of the mean, or greater.So, should there be more humour in scientific literature? I believe that there should. Journals would then become more interesting and more likely to be read, and readers would realize that even researchers and journal editors have a human side.Alice's adventures in wonderland and Through the looking glass are, after the Bible and the works of Shakespeare, among the most quoted works in the world; these were written by the 19th century Oxford mathematician Charles Lutwidge Dodgson under the pen name Lewis Carroll.Scientists and dons have for long exhibited their wit; for example The physical sciences have their humorists. For example, George GamowBritish Medical Journal often publishes offbeat articles, especially in its Christmas issue, each year. Two studies that were particularly interesting were those of Leibovici4General medical journals have their humorists, too. For example, the BMJ also carries fillers, anecdotes and other interesting articles that are frequently humorous in content.Lancet used to carry a regular humorous column titled Jabs and Jibes; regrettably, this column has not appeared for long. Other medical and specialty journals such as JAMA and Neurology include poetry, real-life story, and other columns, which, unfortunately, are sometimes tragicomic.In every issue, the Biological Psychiatry, for example, once carried an April Fool editorial;BMJ, itself, has often carried April Fool articles and responses thereto.1012Psychiatry journals have not lagged behind. Indian Journal of Psychiatry will pass a new milestone in its history, and further improve its ratings and readability.In conclusion, I believe that, with the recognition of the importance of the views of Swaminath,

The repercussion of the heated dispute on cyclooxygenase-2 (COX-2) selective nonsteroidal anti-inflammatory drugs (NSAIDs) led to the national and international withdrawal of several of the recently introduced coxibs. Further debate and research have highlighted risks of the classical NSAIDs too. There is much controversy about the cardiovascular safety of a nonselective NSAID naproxen (NAP) and its possible cardioprotective effect.The study was undertaken to determine the cardiovascular effects of NAP on doxorubicin-induced cardiomyopathy in rats.Male albino rats received a single i.p. injection of normal saline and doxorubicin (DOX) 15 mg/kg (toxic control group). Naproxen was administered alone and in combination with DOX and DOX + trimetazidine (TMZ) for 5 days after 24 h of DOX treatment. DOX-induced cardiomyopathy was assessed in terms of increased activities of serum lactate dehydrogenase (LDH), tissue thiobarbituric acid reactive substances (TBARS) and decreased activities of myocardial glutathione, superoxide dismutase and catalase, followed by transmission electron microscopy of the cardiac tissue.Doxorubicin significantly increased oxidative stress as evidenced by increased levels of LDH and TBARS and decreased antioxidant enzymes levels. Both biochemical and electron microscopic studies revealed that NAP itself was cardiotoxic and aggravated DOX-induced cardiomyopathy and abolished the protective effect of TMZ in rats.This study indicates that NAP has the potential to worsen the situation in patients with cardiovascular disease. Therefore, it should be used cautiously in patients with compromised cardiac function. Cyclo-oxygenase-2 selective nonsteroidal anti-inflammatory drugs (NSAIDs), prescribed for the treatment of arthritis and other musculoskeletal complaints are associated with reduced occurrence of gastrointestinal (GI) toxic effects compared with nonselective NSAIDs. However, the VIoxx Gastrointestinal Outcomes Research (VIGOR) and adenEvidence that rofecoxib (Vioxx) increases the risk of myocardial infarction has led to intensive research to assess the risks associated with other coxibs and conventional NSAIDs. At that time many researchers suggested and aggressively pursued the hypothesis that the increased frequency of events was not due to any prothrombic effects of rofecoxib. Non-aspirin, nonsteroidal anti-inflammatory drugs (NANSAIDs) have complex effects that could either prevent or promote coronary heart disease. This research has confirmed that some of these drugs can also increase the risk of cardiovascular events, but the mechanisms and clinical significance are still under intense debate. Results indicating an association between cardiovascular risk and the use of various conventional NSAIDs have recently emerged from some observational studies.4Naproxen (NAP) is a NSAID advocated for use in painful and inflammatory rheumatic and certain nonrheumatic conditions. There is no evidence that it is actually cardioprotective. The cardiovascular safety of nonselective NSAIDs has never been systematically studied. Case–control studies have found no cardiovascular effects of NSAID, while a few studies have shown a specific cardioprotective effect for NAP.6 These rDoxorubicin, an anthracycline antibiotic, is widely used as effective antineoplastic agent in the treatment of a variety of malignancies, including lymphoma, leukemia, and solid tumors. Unfortunately, the clinical use of this drug is limited by cumulative dose-related cardiotoxicity which may lead to a severe and irreversible form of cardiomyopathy. There arin vivo model in rats.[Trimetazidine is an anti-ischemic drug that restores the ability of the ischemic cells to produce energy and reduces the generation of oxygen-derived free radicals. Various in rats. Trimetaz in rats. In a cas in rats.In this study, DOX treatment was taken as a cardiomyopathy model to investigate the cardiovascular effects of NAP and to compare its effect with TMZ treatment.Doxorubicin HCl , naproxen , trimetazidine , and LDH diagnostic kit were obtained for the study. All chemicals were of analytical grade and chemicals required for sensitive biochemical assay were purchased from Sigma Chemical Co., USA, Hi Media, and SD Fine Chemicals. Double distilled water was used for all biochemical assays.The study was approved by the Institutional Animals Ethics Committee (IAEC), Hamdard University, New Delhi, India. Fifty-six male albino Wistar rats (250–300 g) were used. They were acclimatized at 25 ± 2 °C under standard laboratory conditions (12 h light and 12 h dark: day and night cycle) and had free access to food and water.Rats were divided into seven groups, containing eight rats per group. In the normal control group (group 1) (CTR), rats received water for injection. The toxic control group (group 2) received DOX (15 mg/kg single dose) intraperitoneally. The third naproxen per se (NAP PS) group and fourth trimetazidine per se (TMZ PS) group received NAP , and TMZ for 5 days. The fifth (DOX + NAP) and sixth (DOX + TMZ) groups received, respectively, NAP and TMZ for 5 days, 24 h after administration of DOX. In the last group, i.e., DOX + TMZ + NAP group, rats received both the TMZ and NAP for 5 days after 24 h of DOX treatment. After 24 h of last treatment blood samples were withdrawn from the tail vein of rats under light ether anesthesia for biochemical estimation of serum lactate dehydrogenase (LDH).19All the animals were then sacrificed by decapitation under light ether anesthesia and hearts were dissected out. Cardiac tissues were washed with ice-cold saline for biochemical estimation of thiobarbituric acid reactive substance (TBARS), glutathi21P values <0.05 were considered statistically significant.The results were subjected to analysis of variance followed by Bonferroni's test. In the DOX and DOX + NAP groups, the fur of animals became scruffy and developed a light yellow tinge, and there were red exudates around the eyes except in the DOX + TMZ group. All groups of animals except CTR group were suffering from diarrhoea, although more severe diarrhoea was observed in DOX + NAP group. Animals in the DOX-treated group also appeared to be sicker, weaker, and lethargic. The most predominant features in the DOX treatment groups were the development of a grossly enlarged abdomen and ascites.During the post-treatment period, 37.5% mortality was observed in the DOX + NAP group and 25% mortality was observed in the DOX group. There were no deaths in the CTR group, DOX + TMZ group, NAP PS group, and TMZ PS group. DOX + TMZ + NAP group showed 12.5% mortality.P < 0.01) decrease in the heart weight:body weight ratio in the group DOX compared to group CTR . There was no significant fall in the heart weight:body weight ratio in TMZ PS group , whereas a significant decrease in NAP PS group was observed as compared to CTR group. As compared to DOX group, a significant decrease in the heart weight:body weight ratio was found in DOX + NAP group but a significant increase was found in DOX + TMZ group and DOX + TMZ + NAP group [There was a significant ( × 10−3) .Serum LDH: There was a significant increase in serum LDH level in DOX group and DOX + NAP group as compared to normal control (CTR) group (P < 0.001) and DOX group (P < 0.001), respectively. There was a significant decrease in serum LDH level in the DOX + TMZ group as compared to DOX group (P < 0.001). There was a significant increase in LDH levels in NAP PS group (P < 0.001) whereas no significant difference was found between TMZ PS group (P > 0.05) and normal CTR group. A significant decrease in LDH levels was found in DOX + TMZ + NAP group as compared to DOX group (P < 0.05) [ < 0.05) .Myocardial TBARS: Tissue lipid peroxides estimated as the level of TBARS were significantly elevated in DOX group as compared to corresponding normal CTR group. There was a significant increase in TBARS levels in DOX + NAP group as compared to DOX group (P < 0.001), whereas a significant decrease was found in the DOX + TMZ group as compared to DOX group (P < 0.001). There was also a significant increase in TBARS levels in NAP PS as compared to control DOX group (P < 0.01) whereas no significant increase in TBARS levels in TMZ PS group was found as compared to control group (group 1) (P > 0.05). There was significant decrease in TBARS levels in DOX +TMZ + NAP group as compared to DOX group (P < 0.01) [ < 0.01) .Myocardial GSH: Tissue GSH level was reduced significantly in DOX group as compared to normal control (CTR) group (P < 0.001) [P < 0.05), whereas a significant increase was found in the DOX + TMZ group as compared to DOX group (P < 0.001). There was also a significant decrease in GSH levels in NAP PS as compared to control DOX group (P < 0.01), whereas no significant difference in GSH levels in TMZ PS group was found as compared to control group (group 1) (P > 0.05). There was no significant difference in GSH levels between DOX + TMZ + NAP group and DOX group (P > 0.05) [< 0.001) . Signifi > 0.05) .Myocardial CAT: In the DOX group, there was a significant decrease in CAT level compared to normal CTR group (P < 0.001). In the DOX + NAP group, there was a significant decrease (P < 0.05). However, a significant increase in DOX + TMZ group (P < 0.001) was noted as compared to DOX group. There was also a significant decrease in CAT levels in NAP PS group (P < 0.001), but no significant difference in cardiac tissue CAT levels was observed in TMZ PS group (P > 0.05) as compared to normal CTR group. There was a significant decrease in CAT levels in DOX + TMZ + NAP group as compared to DOX group (P < 0.05) [ < 0.05) .Myocardial SOD: Significant reduction of SOD activity was observed in DOX group when compared to normal CTR group (P < 0.001). Significant decrease in the SOD level was observed in DOX + NAP group (P < 0.01), whereas a significant increase in SOD level was observed in DOX + TMZ group (P<0.01). There was also a significant decrease in SOD levels in NAP PS group compared to normal CTR group (P < 0.001). However, no significant decrease in TMZ PS group was observed as compared to normal CTR group (P > 0.05). There was no significant difference in SOD levels between DOX + TMZ + NAP group and DOX group (P > 0.05) [ > 0.05) .Histopathological examination of cardiac tissue of normal control group, CTR group, revealed a normal architecture with regular morphology of myocardial cell membrane and well-preserved cytoplasm . Marked Transmission electron microscopical results: Dramatic morphological changes [Figure s Figure and B, is Figure and D. HThe NAP PS group Figure and F reThis study has shown that DOX produced significant cardiomyopathy, as evidenced by increased levels of serum marker enzyme (LDH) and tissue TBARS; decreased levels of myocardial endogenous antioxidants . Doxorubicin also caused a significant loss of myofibrils and cytoplasmic vacuolization in myocytes.2+) free radical complex.[·) radical. ROS reacts with lipids, protein, and other cellular constituents to cause damage to mitochondria and cell membranes of the heart muscle.Doxorubicin-induced cardiomyopathy is related to cumulative dosage. Repeated administration of DOX beyond a certain dose has been shown to cause cardiomyopathic changes in patients and as w10 complex. The lattBoth NAP PS and DOX + NAP groups elevated the levels of serum LDH and cardiac tissue TBARS, whereas it decreased the levels of cardiac tissue superoxide dismutase, catalase, and glutathione as compared to normal and toxic control groups, respectively.When TMZ was given along with DOX, it decreased the levels of serum LDH, and cardiac tissue TBARS, while increased the levels of cardiac tissue superoxide dismutase, catalase, and glutathione as compared to toxic control group. It showed the protective effect produced by this drug. This may be due to free radical scavenging effect of the drug in the cardiac muscle. Trimetazidine has been shown to protect DOX-induced acute cardiotoxicity by preservation of endogenous antioxidant and reduction of lipid peroxidation.Trimetazidine when given in combination with NAP and DOX decreased the levels of serum LDH, and cardiac tissue TBARS, whereas increased the levels of cardiac tissue superoxide dismutase, catalase, and glutathione, less significantly as compared to toxic control group thus indicating that NAP interfered with the protective effect of TMZ in DOX model.Doxorubicin-induced morphological changes in myocardium were observed by electron microscopy. In this study, DOX treatment caused significant histological changes including marked myofibril loss, cytoplasmic vacuolization, chromatin condensation and margination, and membrane blebbing, but maintained the mitochondrial and sarcolemmal integrity.The NAP PS group revealed condensed chromatin as compared to normal control group. Treatment of NAP along with DOX revealed more condensed chromatin at the margins of the nuclear membrane and extensive cytoplasmic vacuolization as compared to DOX-treated toxic control group indicating aggravation of DOX-induced cardiotoxicity by this group. Treatment of TMZ 10 mg/kg along with DOX exhibited less extensive vacuolization, with smaller and more sparsely distributed vacuoles compared to DOX-treated toxic control group.Treatment of NAP along with TMZ and DOX revealed condensed chromatin at the margin of the nuclear membrane with smaller and more sparsely distributed vacuoles as compared to DOX-treated toxic control group.Thus, biochemical and electron microscopic studies together revealed that NAP aggravated DOX-induced cardiomyopathy in rats. In addition, NAP itself was found to be cardiotoxic as revealed by biochemical and confirmed by pathological studies.et al. did an observational study to measure the effects of NANSAIDs, including NAP, on risk of serious coronary heart disease and reported no cardiac protection among long-term NANSAIDs users with uninterrupted use.[et al. reported that the use of NAP does not protect against serious coronary heart disease.[et al. reported that patients with pre-existing medical conditions appeared to have a significantly higher risk for cardiovascular events associated with the use of NSAIDs and celecoxib compared with patients without these conditions.[Earlier studies believed that NAP was cardioprotective as observed by an unexpected fivefold increase in the risk of acute myocardial infarction (AMI) with rofecoxib when compared with NAP. However,2pted use. Absence disease. Huang etnditions. Rahme annditions.Our findings support some of the abovementioned observational studies that NAP itself is cardiotoxic and it is not safe to use this drug in cardiovascular compromised patients.On the basis of the currently available data, FDA has concluded that the potential for increased risk of serious cardiovascular adverse events is a class effect of NSAIDs. Additional data from long-term, controlled clinical trials are needed to more definitively determine the magnitude of increased risk with NSAIDs, if any.According to the FDA, all NSAIDs may have similar risks that increase with duration of use and in the presence of existing cardiovascular disease and/or related risk factors. Therefore, clinicians are advised to remain alert for the development of cardiovascular events, even in the absence of previous symptoms and other treatment options should be considered in patients at increased risk for cardiovascular effects.In conclusion, DOX increased lipid peroxidation and reduced the levels of catalase and GSH in rat heart and caused morphological changes in myocardium, characteristic of apoptosis as shown by TEM and histopathology examination. NAP aggravated DOX-induced cardiomyopathy and itself was found to be cardiotoxic in rats. Trimetazidine showed good protective effect along with DOX but when given along with NAP, its protective effect was abolished. These results suggest that NAP should be used cautiously in patients with cardiovascular disease.

Formal checks of participant understanding are now widely recommended to improve informed consent processes. However, the views of the participants these assessments are designed to protect are rarely considered. In this paper the findings of a qualitative study aimed at documenting community reactions to a semi-structured questionnaire ('quiz') are reported. The quiz was administered to 189 mothers after consenting for their children to participate in a malaria vaccine trial on the Kenyan Coast.Once the malaria vaccine trial was underway, focus group discussions were held with some of these mothers , and with community-based field staff attached to the malaria vaccine trial (two groups of five workers). Individual interviews with other trial staff were also held.The quiz prompted community members to voice concerns about blood sampling and vaccine side-effects, thereby encouraging additional discussions and interactions between the research team and potential study participants. However, it also caused significant upset and concern. Some of the quiz questions, or the way in which they were asked, appeared to fuel misconceptions and fears, with potentially negative consequences for both the study and community members.Formal approaches to checking study understanding should be employed with sensitivity and caution. They are influenced by and impact upon complex social relationships between and among researchers and community members. Adequate consideration of these contexts in assessments of understanding, and in responding to the issues raised, requires strong social science capacity. Individual informed consent is a key ethical obligation for clinical studies. However, empirical studies show that the key requirements are often not met -5. Suggevaluable addition to many trials conducted in developed countries, where participants may have an incomplete understanding of the implications of their participation' [There is some evidence that a formal assessment, administered during the informed consent process, improves understanding. Checks ipation' . The neeIn this paper, community reactions to a semi-structured questionnaire aimed at exploring participant perceptions and understanding of a malaria vaccine trial are described. The trial was conducted at the KEMRI-Wellcome Trust Collaborative Research Programme on the Kenyan Coast. The trial, information giving processes, and community perceptions and understanding of the trial itself are described in detail elsewhere,16. BrieThe Social and Behavioural Research (SBR) group coordinate community liaison activities at the KEMRI-Wellcome Trust Research Programme. SBR members adapted a quiz passed by the local and national ethics review committees into a semi-structured questionnaire (hereafter 'quiz') administered to 189 caretakers, primarily mothers ; that children will fit and die immediately after the vaccine is administered; that children will slowly die off after the end of the vaccine trial; and that biscuits and juice are given to children to increase their blood for further blood taking for devils.et al [Recognition of the huge sensitivity around blood sampling led to public meetings at which the PI showed and answered questions about how blood is processed and used in the laboratory; including demonstrations of microscope slides and assay wells. These meetings were an opportunity to focus on an area that had not been covered in depth in previous information giving sessions. More importantly, they were an additional opportunity for community members to test the research teams' openness and consistency in messages. These are behaviours supportive of trust-building, and appeared essential to the successful conduct of the trial .Over the course of public meetings and in other informal interactions, concerns about the quiz itself began to emerge. Questions about the quiz were, therefore, included in FGDs with fieldworkers and community members. In almost all FGDs, participants mentioned at least one disadvantage of the quiz. An important view expressed in community groups was that the questions asked, and the way they were asked, heightened concerns about rumours circulating in the community; and that raising concerns in this way was unnecessary , and assisted in decision-making about study participation (Figure it helped some; those who decided to continue coming. But then those that misunderstood, it didn't help them because they ran away! So, did they finish the journey?Nevertheless, the quiz was clearly an unnerving experience for some participants Figure ; Quote 6Formal approaches to checking study understanding are increasingly popular for clinical research. The findings presented in this paper suggest that any such checks should be employed with sensitivity and caution. In the absence of other studies that have formally explored this issue, the experiences in Kenya suggest the following key issues influencing community reactions and the validity of the quiz itself:The interviewer. The value of quizzes is dependent on the interpersonal skills of the people administering it, and their ability to ask questions in an open and non-threatening way. Difficulties were faced in Kenya even with fieldworkers fluent in the local language who were carefully trained in qualitative methodologies and in communication skills. Interviewers from within the team may have been more appropriate in this case.Qualitative versus quantitative data. The findings of quantitative data acquired can be misleading without complementary qualitative data. Using quiz data alone to make definitive comments on individual levels of understanding or eligibility to access a trial is not therefore recommended. Imbalances in resources and research knowledge between researchers and community members can also make individual interviews intimidating for participants. In Kenya, a qualitative, group based, or less formal quiz may well have achieved the same positive impacts for all with fewer concerns raised for community members.The follow up provided after a quiz. A formal check of understanding can lead to difficulties for individuals, for the community and for researchers in the short term. Nevertheless, this is an opportunity for the issues raised to be dealt with, leading to better understanding on both sides, and a stronger relationship between participants and researchers. This is particularly the case if the assessment is incorporated into and informs a broader strategy for involving communities in aspects of the research.Expertise is required. Developing, administering and responding to assessments of understanding for clinical trials requires significant social science capacity. Strengthening the role of good quality social science in clinical trials is an important strategy for improving ethical practice.The findings may be specific to the study population and to the socio-cultural and institutional context of the study. Use of a similar quiz in phase 1 trials, on a much smaller sample size of well-educated parents, provoked less controversy. However this was not formally explored. Further studies on quizzes would indicate the generalizability of the findings and identify important contextual influences. A greater understanding of community perspectives on quizzes, and of other ethical requirements, will help give research participants and communities a voice in national and international debates on how to strengthen ethical practice.All authors were involved in the design and conduct of the study, data analysis, and the preparation and review of the manuscript. Caroline Gikonyo was responsible for data collection. Sassy Molyneux had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.Appendix 1. The introduction to and questions covered in the quizClick here for file

The sulphonamide group of drugs is implicated in bilateral acute angle closure (AAC) due to an idiosyncratic response. We report a series of three cases with bilateral AAC caused by different sulphonamide derivatives, their presentation and management. Bilateral simultaneous secondary acute angle closure (AAC) is rare and has been reported secondary to drugs, general A 60-year-old male was referred to us with a history of having undergone cataract surgery in left eye (L/E) two days earlier and in right eye (R/E) one month ago. He complained of severe pain and decreased vision in both eyes for two days. He had undergone phacoemulsification under topical anesthesia in both the eyes one month apart and the postoperative period after the first eye surgery was uneventful. He presented to the treating physician with bilateral flat anterior chambers and high intraocular pressure (IOP) four hours after his cataract surgery and was diagnosed and treated as malignant glaucoma with intravenous (IV) mannitol 20% (1-2 g/kg body weight), tab acetazolamide 250 mg thrice daily and timolol maleate 0.5% eye drops twice daily in both eyes (B/E), peripheral iridotomy in R/E and YAG hyloidotomy in R/E and posterior capsulotomy in L/E. On examination here, visual acuity was finger counting at half meter in R/E and one meter in L/E. There was bilateral lid edema, conjunctival chemosis, corneal edema, shallow anterior chambers (ACs) Fig. and b, mA 53-year-old woman presented with sudden painful decrease in vision in B/E and severe headache for 12 h. She was a known diabetic and hypertensive on insulin, amlodepine 5 mg for the past two years and indapamide 2.5 mg for one month. Her random blood sugar was 180 mg/dl. On examination, her vision was 20/60p, N36 R/E and 20/60p, N24 in L/E with a myopic shift of one diopter (D) in B/E. In B/E there was lid edema, conjunctival chemosis, shallow ACs Fig. and 3b, A 28-year-old woman presented with sudden decrease in vision and pain in B/E for one day. She had been recently diagnosed to have migraine and was started on tab. topiramate 50 mg for four days. On examination her vision was 20/200 both eyes, improving to 20/30 with -5.0 D sphere correction, both eyes showed congested conjunctivae, clear corneas, shallow ACs, clear lens and round reacting pupils. IOP was 34 mm Hg in B/E. Gonioscopy showed closed angles and an undilated fundus examination showed 0.4 cup with healthy rim in B/E. With sudden decrease in vision, myopic shift in refraction and bilateral AAC, a diagnosis of topiramate-induced secondary angle closure was made. She was advised to discontinue topiramate and was treated with IV mannitol and timolol maleate 0.5% drops. Three days later her unaided vision was 20/20 in B/E with IOP 14 mm Hg and wide open angles on gonioscopy. Timolol was discontinued.Drug-induced uveal effusions have been reported with sulphonamides. All threThe fact that the condition resolved after stopping the suspected medication indicates a causal relationship. The best method to prove causality is to re-challenge. However, re-challenge may not be ethical and might sometimes fail to reproduce the event.It is rather paradoxical that acetazolamide, an antiglaucoma medication can cause secondary angle closure and rise in IOP. Since acTo our knowledge there is no report of bilateral drug-induced angle closure caused by indapamide, an antihypertensive which is a sulphonamide derivative. However, there is a report of acute transient myopia caused by indapamide. Topirama9This case series highlights the fact that drug-induced angle closure should be suspected in any patient with features of bilateral AAC, myopic shift of refractive error and annular ciliochoroidal detachment. UBM is helpful in the diagnosis of this condition. Apart from IOP-lowering measures, a review of systemic medications and discontinuation of the most probable causative agent should be done. A high index of suspicion will help manage drug-induced bilateral acute angle closures appropriately with quick and complete visual recovery.

Many refugees arrive in Australia with complex health needs. In South Australia (SA), providing initial health care to refugees is the responsibility of General Practitioners (GPs) in private practice. Their capacity to perform this work effectively for current newly arrived refugees is uncertain. The aim of this study was to document the challenges faced by GPs in private practice in SA when providing initial care to refugees and to discuss the implications of this for policy relating to optimising health care services for refugees.Semi-structured interviews with twelve GPs in private practice and three Medical Directors of Divisions of General Practice. Using a template analysis approach the interviews were coded and analysed thematically.Multiple challenges providing care to refugees were found including those related to: (1) refugee health issues; (2) the GP-refugee interaction; and (3) the structure of general practice. The Divisions also reported challenges assisting GPs to provide effective care related to a lack of funding and awareness of which GPs required support. Although respondents suggested a number of ways that GPs could be assisted to provide better initial care to refugees, strong support was voiced for the initial care of refugees to be provided via a specialist refugee health service.GPs in this study were under-resourced, at both an individual GP level as well as a structural level, to provide effective initial care for refugees. In SA, there are likely to be a number of challenges attempting to increase the capacity of GPs in private practice to provide initial care. An alternative model is for refugees with multiple and complex health care needs as well as those with significant resettlement challenges to receive initial health care via the existing specialist refugee health service in Adelaide. Under its Humanitarian program Australia accepts approximately 13,000 refugees each year ,3. Many Despite the potential for high levels of morbidity, refugees undergo only a limited health assessment prior to their arrival in Australia. For the majority this includes a medical examination, a Chest X-ray for those 11 years and over and an HIV test for those 15 years and over . AlthougUntil recently in SA, the State funded refugee health service along with two community health centres (CHCs) with specific refugee health expertise, performed comprehensive health assessments on a large proportion of newly arrived refugees to SA. With changes to the Department of Immigration and Citizenship (DIAC) funded Integrated Humanitarian Settlement Service (IHSS) contract in 2005, the responsibility of providing this initial care in SA was passed to General Practitioners (GPs) in private practice. Whilst this is the path taken in SA, each Australian state and territory has a different model for the provision of initial health care services with varying levels of involvement of specialist refugee and community health services , althougThere has been surprisingly little written documenting the experiences of GPs who provide initial care to refugees, both in Australia and overseas, including the challenges they might face and hence their capacity to do this work is uncertain. Some general review articles have been written by health practitioners based on their own experiences providing health care to refugees and challenges listed include those related to language, ,22 the tThere are limitations, however, in the applicability of these studies to the Australian context given differing national health systems and special service entitlements for refugees and the fact that it was unclear to what extent these studies related to the experiences of GPs providing care to refugees from similar backgrounds to those currently arriving in Australia. A review of the Australian literature found only one published study addressing this issue. In a report to the Victorian Department of Human Services, the Victorian Foundation for Survivors of Torture (VFST), drawing on their experiences as well as interviews with 19 GPs performing initial health assessments for newly arrived refugees in Melbourne, identified a number of similar challenges for GPs to those already listed . AdditioWhilst the VFST study provides valuable insight into the experiences of GPs in the Australian context, much of the initial health care for refugees in Victoria is performed by GPs working in State funded community health centres where they are more likely to be salaried and have better access to supports to assist them to manage patients with multiple and complex health needs. By contrast, in SA all newly arrived refugees are referred directly to GPs in private practice for initial health care and there has been no research to date assessing their experiences providing care to refugees in SA.Given that current refugee arrivals are likely to carry a greater disease burden combined with the recent increased responsibility of GPs in private practice in SA to provide initial care, the aims of this study were: (i) to document the existence and nature of challenges for GPs who do this work in SA, (ii) to explore the ways in which these challenges could be reduced and (iii) to discuss the policy implications of this in relation to optimising the initial health care for refugees.Given that the nature of this study was exploratory, a qualitative approach was taken in order to gain a deeper understanding of the challenges faced by GPs in private practice when providing care to refugees. Semi-structured interviews were conducted with 12 GPs providing care to refugees in private practice as well as the Medical Directors of three of the Divisions of General Practice in metropolitan Adelaide with high levels of current or proposed refugee settlement. The study was approved by the University of Adelaide Human Research Ethics Committee.To recruit GPs, potential participants were identified via a database of GPs who were either currently accepting or had accepted refugee referrals in the past. One of the authors (DJ) also used his personal knowledge of GPs known to provide care to refugees through his previous work at the SA specialist refugee health service and through formal and informal networks in the refugee health sector. Additional GPs were also identified following the Division interviews. An introductory letter with a fax-back reply was sent to 77 potential GP participants. After the initial mail out, six GPs agreed to participate. Follow-up phone calls were subsequently made to another ten GPs in order to recruit the remaining six GP participants. GPs were recruited from most regions of the Adelaide metropolitan area although there were none from the southern Adelaide region (resettlement of refugees to this region had only occurred relatively recently). The twelve GPs represented eight separate practices – two groups of three GPs were recruited from the same practices. Two thirds of participants had longstanding involvement in providing care to refugees whereas the remainder had become more recently involved with increasing numbers of African refugees resettling close to their practices. Whilst African refugees made up a large proportion of newly arrived refugees seen in the past twelve months, GP participants also reported providing care to large numbers of refugees from the Middle East as well as a small number of refugees from the Former Yugoslavia.To recruit the Medical Directors of Divisions, five were contacted by email with two agreeing to participate. Follow-up telephone calls led to the recruitment of one further Division.Given that a number of potential challenges were identified from the literature as well as there being much anecdotal evidence of challenges for GPs to do this work, a semi-structured interview format was chosen to examine these specifically whilst at the same time allowing any previously unknown challenges to emerge. Different interview schedules were used for the GPs and the Divisions respectively. Although the questions for each group focused on the broad aims of the study they addressed slightly different aspects of the issue. The GP interviews generally explored the challenges GPs face when working with refugees whilst the Division interviews focused on the current or potential role of Divisions to support GPs in private to work with refugees and well as the identification of potential structural impediments for GPs doing this work.The interviews were conducted between April 2006 and July 2006. Each participant was interviewed once with interviews ranging from 40 to 70 minutes. Individual interviews were conducted with nine of the GP participants and a small group interview was conducted with the remaining three GPs. The three Divisions were each interviewed individually. The interviews were tape recorded and transcribed verbatim. In both the GP and Division interviews data saturation was reached.a priori themes in addition to new themes identified from initial reading and analysis of the transcripts. Final thematic templates for both the GP and Division transcripts were agreed upon by the Project Team and then all data was coded according to these themes, with DJ undertaking the bulk of the coding. Two transcripts were also independently coded by the other members of the Project Team. Following this, comparisons were made and a consensus reached on how the remaining data was to be coded. Coding numbers were randomly assigned to protect the confidentiality of the participants where direct quotes were reported in the results.A template analysis approach was adopted ,29 whereGPs in this study reported a range of challenges when providing care to refugees. In many cases these challenges were explicitly linked to performing initial assessments whilst at other times GPs spoke of challenges in the broader context of providing care to refugees – but which can be assumed to be operating when providing initial care. The challenges fell into three main categories: (i) refugee health issues; (ii) GP/refugee interaction; and (iii) the structure of general practice. There was a great deal of overlap, however, between these categories and a very strong theme to emerge was not having enough time to do the work required which related to any issue that made a consultation with a refugee longer. Further, these challenges did not appear to be related to how long GPs had been providing care to refugees or to the intensity of their involvement other than that more experienced GPs had a greater awareness of available interpreter services. The Divisions also reported challenges assisting GPs to provide care relating to a limited awareness of refugee numbers settling in their divisions and which GPs needed extra support as well as a lack of specific Commonwealth funding to do this work. Finally, whilst participants suggested ways these challenges could be reduced, overall strong support was provided for initial health care to be provided via a specialist health service.Challenges for GPs providing health care to refugees that related to refugee health issues included GP knowledge of previous health assessments, GP awareness of and experience managing health conditions unique to refugees, and the multiple and complex nature of refugee health conditions.A number of GPs and Divisions expressed uncertainty regarding what health assessments refugees had received prior to arrival in Australia:I don't know if there is some sort of system that they go through, or some sort of protocol that they, medically, have to go through before they are granted visas... (Dr 1)There was also uncertainty regarding what health assessments were carried out after arrival in Australia with some GPs assuming that the MHS still performed this work. Uncertainty regarding previous assessments did not relate to how long GPs had been providing care to refugees.For some GPs this resulted in confusion over their role in detecting and managing health conditions unique to refugees:So we have got the clinical exotica; we have got very little understanding of what has happened to these people before and where the responsibility stops and starts for who should be following up all these things. (Dr 7)Only one GP reported using guidelines to assist screening for exotic conditions. It was likely that many conditions unique to refugees were not being detected as indicated by one GP:I haven't personally come across anything unusual that would be something that was quite rare... I'm sure I will. I'm sure I have probably missed heaps, too. Slipped through that I haven't seen or recognised. (Dr 1)Concern was expressed, however, that refugee screening guidelines would just be another one of many such guidelines that GPs were expected to know about and follow.Even if GPs were detecting conditions unique to refugees, there was concern that they did not necessarily have the experience to manage them:I guess it is out of our comfort zone, because our medical experience doesn't include the exotic illnesses that they front up with... (Dr 7)Again, these challenges were not dependent on how long GPs had been providing care to refugees as many of these challenges related to more recently arrived African refugees.The expectation that GPs develop this expertise was also questioned as this was seen to compete with what was thought to be the important broader generalist role of general practice:...we are supposed to be highly trained now in mental health and refugee health...when actually we are general practice. We are not specialty people...I think it is important that we stay that way, because you start going down into specialty areas too much and you start to miss the bigger picture... (Dr 1)In terms of mental health problems, most GPs were aware of previous traumatic experiences of refugees and that combined with the stresses of resettlement meant that psychological problems often resulted. This also meant, however, that the time it took to build rapport and trust when seeing a refugee was far greater than with a non-refugee patient and this affected the ability to gather information about their background and past medical history.Most GPs described providing care to refugees as demanding, with refugees more likely to have multiple and complex health needs:...usually the refugees or migrants that I have seen have got multiple needs. It is usually not just one simple thing. (Dr 10)This often meant the nature of the work was time consuming:Often at the beginning there are so many issues to get through that I think it takes quite a number of long consultations before you really even have a clear idea about who this person is and what their experiences are. To get all the health issues on the table, I think, takes a really long time. (Dr 11)A number of challenges relating to the interaction between GP and refugee were raised including issues related to culture and language as well as refugee knowledge of the Australian health care system.A number of GPs observed that refugees often had a different understanding of disease causation when compared to a Western model:...the way people behave around their health and their illness is very culturally determined. To try and understand what is going on, I can't just impose my framework, because the way they will express themselves is really different about what they are feeling. (Dr 11)As a result, GPs reported having to spend much longer than normal explaining Western health concepts to refugees. This included screening activities such as Pap smears and organising referrals for mammograms, when giving a diagnosis of an illness such as Hepatitis B or when referring refugees for pathology tests. Other challenges attributed to cultural differences included uncertainty over cultural appropriateness of examination, gender related issues such as decision-making over birth control and gaining consent for invasive procedures:...one woman came in and consented to a Pap-smear and thankfully, we managed to get around it that she actually had one before and was okay to do it, but I thought if she had never had one, how was I going to explain to this woman what I was going to do. (Dr 8)Different naming practices also sometimes presented challenges in locating the correct patient file. Although GPs with longer standing involvement in refugee care may have been more aware of cultural differences, all GPs reported challenges related to this issue.A number of challenges relating to language resulted from the need to use interpreters. These included not being able to adequately provide explanations via an interpreter, difficulties dealing with mental health problems and the extra time required to both conduct a consultation with an interpreter as well as organise an interpreter when one was not pre-booked. Communication challenges were also experienced when contacting refugee patients for follow up of test results or to book an appointment. Although most GPs were aware of the need to use an interpreter when refugees were not fluent in English, those GPs with more recent involvement in refugee care were more likely to be unfamiliar with all the services offered by the Commonwealth Translating Interpreting Service (TIS), such as the doctor's priority phone line :The times that I have needed it they have been – appointments have been booked well in advance. How do you book an interpreter when someone rings up at lunchtime and sees you two hours later for something that is minor or insignificant? (Dr 1)One GP was confused about who paid for TIS believing that the practice was billed for an interpreting service when it was booked and the patient did not attend the appointment. A number of GPs also talked about difficulties contacting refugee patients for follow up of test results or to book an appointment:Communication, when you know they can't speak English, so you can't phone them, and when you know that they are quite a mobile group of people, so that when you send a letter to their address, they might have moved on. (Dr 6)Almost all GPs mentioned difficulties resulting from refugees' lack of familiarity with the Australian health care system. This related to missed appointments, which meant no remuneration for GPs, or refugees arriving late:That is a difficult problem with them... they will turn up really late for an appointment with no sort of seeming reference to the timeslot that they were given... it does sometimes make it difficult for us if we are then on the back foot for the rest of the session. (Dr 1)GPs also reported that refugees' lack of understanding of the Australian health system resulted in challenges for GPs when they were referring refugees to other agencies or specialists and also when writing prescriptions. As a result GPs reported spending more time providing explanations about how the health system worked:I take more time with them...Because the health care system here is much different... I try to make it easier for them to understand the system and how it works here. (Dr 5)A number of challenges relating to the structure of general practice were identified including GP workforce shortages, a lack of organised referral pathways for refugees to general practice as well as a lack of remuneration and infrastructure support required to perform initial assessments.As a result of the demand for GP services outstripping supply in some regions of Adelaide, providing appointments for any new patients, whether they were refugees or not, was often difficult. Three GPs in this study, with high loads of patients with complex health care needs including refugees, had closed their books to new patients and another GP described potential difficulties accepting new patients:We are having trouble accepting new patients full stop... freely accepting new patients irrespective of whether they are a refugee or not, it is difficult to actually accommodate everybody. (Dr 1)Further, as highlighted by one Division, with GP shortages most acute in socioeconomically disadvantaged areas, refugees were more likely to be affected given that they are often settled in areas where housing was cheaper:The cheaper areas for housing [are] where the workforce is the worst, so you can end up in this vicious circle where the practices go "Ah, we are closing our books, we are just not seeing anyone new". (Div 2)Overall GPs reported that there was usually no clear referral pathway for refugees to private general practice. This was perceived as a problem because it meant that GPs were not able to control the numbers of refugee patients they saw when there were limits to the amount of work they could do with patients with multiple and complex needs such as refugees:... because it is primary care you are expected to just take everybody that walks through the door. That doesn't work... lots and lots of agencies would like to refer here. We have to somehow prevent ourselves from drowning. (Dr 11)Complicating this was the fact that when a GP took on one refugee then it was most likely that the rest of the family would then come to see the same GP which could dramatically increase their caseload of patients with high health care needs. Related to this was a fear for one GP clinic of being inundated with refugees if their clinic was promoted as a formalised referral centre for refugees because of difficulties already meeting the needs of their current patient load. A situation where there was no system in place to manage referrals was described by one GP as 'a recipe for burnout'.Not having a formal referral pathway to GPs also led to problems with the transfer of health information including results from pre-departure health checks and any health services refugees had accessed in Australia. It was also noted that poor transfer of health information could also result in duplication of services such as immunisation.Half of the GPs identified remuneration as a challenge when working with refugees. This was because refugees were mostly bulk billed and many needed longer consultations which were felt to be inadequately remunerated under the current Medicare billing system:...it's just not financially viable because, as we know, long complex consultations are not a way which assists you to run your practice in a way that is financially viable... (Dr 11)Remuneration was also a challenge because of the fact that refugees often missed appointments, which meant no remuneration, and work with refugees often involved time consuming extra-consultation activities that could not be charged to Medicare:Missed appointments are fairly common...So you miss the remuneration if they don't come. The time factor; the complexity of the consult is more than what is remunerated for the time involved, because your follow-up is often phone calls to various agencies or organising things, writing letters, becoming an advocate, coordinating allied health. Very little of that is remunerated... (Dr 10)Despite the strong indication that remuneration was a challenge for GPs, the majority of GPs said that they were either not going to use or were unlikely to use the new Medicare item number for performing an initial health assessment on a refugee although there was moderate support for it in principle. This was either because of a lack of familiarity with already existing Enhanced Primary Care (EPC) item numbers or because of the high administrative burden associated with their use:It is not something I am likely to use personally. I think it is a great idea ... but I am not very good at using the specific numbers ... and the EPC items and so on. I just don't have time to sort out all the paperwork for that sort of thing. (Dr 6)Another GP was disappointed that the Divisions of General Practice had not produced a template to assist using the item number in the same way that they had with previous EPC item numbers. Finally, a number of GPs were critical that the item number did not go far enough in that it failed to recognise that the greater health care needs of refugees were ongoing.There was strong evidence provided by the majority of GPs in this study that they did not have the necessary infrastructure support, i.e. the systems and support staff, to perform initial refugee health assessments:We are not well enough equipped. We are not resourced, we do not have the supporting background structure. (Dr 9)It was particularly overwhelming for GPs when groups or families all came at once and they had not had a previous health assessment:...we were having whole families of recent arrivals come to the surgery and need all their history taken; immunisations brought up-to-date and that was just overwhelming... We do have a practice nurse, but she is usually quite busy doing other things. That was too much for us to handle. (Dr 6)The Divisions also believed that a lack of infrastructure support was a reason it would be difficult for GPs to perform initial health assessments and was a reason uptake of the new item number would be limited:If you just basically say "Here is a new item number doctors" it won't be taken up, because it is going to be all too hard. From an infrastructure perspective most practices lack the infrastructure to really make this work. (Div 3)The Divisions identified a number of challenges in assisting GPs to provide care to refugees. They expressed concerns that they did not know how many refugees were being resettled as well as precisely where these resettlements were occurring within their Divisions. The Divisions reported also having limited awareness of which GPs in their Divisions were providing care to refugees and, as a result, were not able to determine which GPs might need extra support to do this work. One Division had surveyed GPs to assess this but most had been too busy to respond. Although a number of GPs had been recruited by the IHSS provider to offer care to newly arrived refugees, the Divisions expressed frustration that they did not know who these GPs were. They were also concerned that there was no collaboration with the IHSS provider which might avoid resettling refugees in areas where they might have difficulty accessing GP services:... there is no point putting refugees in to an area where there are no GPs. There might be GPs there but they might not want to see refugees or they might have closed their books. We would have that intelligence; they would have no idea about that... (Div 1)All Divisions mentioned a lack of funding as a major reason their ability to help GPs was limited. Because refugee health was not a priority area for the Commonwealth, Divisions received no direct funding for refugee health initiatives:We are funded by the Department of Health and Ageing; we have got a whole lot of quality indicators that we have got to actually achieve in. Refugee health does not even appear in there. (Div 3)The Divisions explained that it was also a lack of funding that limited their ability to assist GPs to utilise the new item number. Despite this, two Divisions had diverted core funding to better support GPs in the area of refugee health but no Divisions had funding for specific services or programs. One Division felt their case to argue for increased funding was limited given the lack of data related to refugee numbers settling in the Division and that there was no empirical evidence that GPs weren't coping even though it was recognised that GPs were too busy to answer surveys that might provide this data.Despite GPs questioning how realistic it was for them to manage many of the exotic health conditions in newly refugees, there was some support for the provision of screening guidelines for use by GPs in private practice provided they were in a simple and readily accessible format – such as linked to the general practice software program Medical Director (MD).GPs reported they could be assisted to overcome some of the challenges related to culture if they were provided with more background information about refugee groups either through the provision of information sheets or talks from different community members regarding different cultural practices. It was also suggested that these challenges could be reduced through better provision by settlement services of health information to refugees on arrival including information about common conditions in refugees such as hepatitis B, early intervention and illness prevention activities and better initial orientation to the Australian health care system. Many GPs also believed that settlement services could provide more assistance to refugees to attend appointments. Other ways of improving refugee navigation of the health system included a greater role for voluntary organisations, practice nurses or community health care workers who could be health advocates for refugees:At times you feel like you're running around doing a lot of the work that could be partly done through either a practice nurse or another allied health worker or somebody who can, on the ground, advocate for that refugee individual. (Dr 10)In relation to referral pathways of refugees to general practice, it was strongly stated by a number of GPs that this should involve a consent process which assessed the ability of a GP to take on the care of a new refugee or refugee family:It is not a kind of fair system to plonk someone onto a practice. I think agencies should actually liaise with either one of the senior doctors or, if there is a nurse... so a referral can actually be properly organised and assessed as to whether the practice can take someone on. I think the ways it has happened in the past have been really unsatisfactory. (Dr 11)Despite limited enthusiasm for the new Medicare item number, a small number of GPs were still interested in learning more about how to use it through their Division and there was also some support for the provision of a template by the Divisions which could also be incorporated into MD.The Divisions suggested a number of ways they could assist GPs to perform initial assessments if they were provided with funding. This included improving the utilisation of the item number by educating practice nurses and developing a template as well as Division funded 'clinical attachments' at the state funded specialist refugee service to provide GPs with greater expertise in managing health conditions unique to refuges. It was also suggested that Division funded refugee health infrastructure grants could provide assistance to GPs to better set up their clinics to care for refugees via IT support and the provision of business cases for employing practice nurses. One Division, however, expressed uncertainty about the likelihood that GPs would be willing to build such systems into their practices if they were only seeing small numbers of refugees, particularly given that there was limited enthusiasm to build similar systems for high prevalence chronic diseases in the general population.Despite indicating ways they could be assisted, a number of GPs believed that the responsibility of providing initial care to refugees should not lie with GPs in private practice:There should be a front line somewhere. I don't think general practice should be the front line. (Dr 8)Instead, a system where refugees received an initial assessment via a specialist refugee or community health service was strongly supported by both GPs and the Divisions. One GP described the advantages of a community health service where a range of services could be provided to refugees in one location:I think the community health service is the best stop for an initial assessment because of the complexity of the presentations, usually, and the need for accessing a lot of different services that is really beyond most private centre GPs to be able to do that adequately. If you have got access within one building, for example, to workers who can do some of the chasing up, some of the phoning and some of the coordination, then you are much more likely to give people a good service. (Dr 10)A number of GPs indicated that they would be much happier to accept referrals of refugees if they had had an initial assessment where they could then focus on their more day-to-day health needs.This study highlights the many and diverse challenges faced by GPs in private practice when providing health care to refugees in their initial resettlement period. These challenges and their policy implications are discussed below.The extent of the challenges faced by GPs providing initial care to refugees in this study is not surprising given that it is during their early resettlement period that refugees are most likely to experience multiple medical problems, many of an exotic nature, when language and cultural barriers are likely to be greatest and when refugee knowledge of the Australian health care system and general health literacy are likely to be most limited.Whilst many of the challenges identified support previous research in this area, the most striking feature of this study is the strong evidence that GPs in private practice are not sufficiently resourced to provide initial care effectively to newly arrived refugees with multiple and complex health needs. For GPs in this study, the lack of resources existed both at an individual GP level, with GPs lacking comprehensive knowledge of the health conditions unique to refugees, as well as at a more structural health system level, where GPs lacked both the time and the infrastructure support to do this work effectively. Further, the lack of resources was not related to the length of time GPs had been providing care to refugees or the intensity of their involvement other than that GPs with less experience were less familiar with the use of interpreter services.Given that the first point of contact with the Australian healthcare system for refugees currently arriving in SA is with a GP in private practice, there are a number of health consequences for refugees if GPs do not have the necessary resources to provide them with effective care in their initial resettlement period. These include GPs missing or inadequately treating physical and mental health problems unique to refugees, refugees not following through with treatments or referrals and refugees under engaging with illness prevention activities because of poor health literacy . This caAlthough GPs mentioned a number of ways they could be assisted to provide more effective initial care to refugees, they also indicated that there would be major limitations attempting to build this capacity.Firstly, GPs questioned the expectation that they develop the specific 'refugee health' expertise needed for performing initial assessments which competed with their role as 'generalists'. It is likely that, at best, developing the required expertise to perform initial assessments will only appeal to a small number of GPs.Secondly, even those GPs who have an interest in doing this work may not want to identify themselves as a 'refugee doctor' for fears, as stated by one GP, that they will become inundated with referrals. GPs in this study indicated that there were limits to the amount of work they could do with refugee patients given the often multiple and complex needs on initial presentation. GPs, however, operate in a primary health care (PHC) system where they have little control over how patients are referred to them. Further, GPs performing initial health assessments are most likely to be the GPs who provide the ongoing care . As a result, GPs providing initial health care can quickly end up with very high numbers of patients with multiple and complex health needs. A number of GPs in this study indicated that this had contributed to them closing their books to new patients. Refugee health service providers in Adelaide as well as interstate have alsThirdly, GPs indicated that providing initial care to refugees was time consuming but the fee-for-service structure of general practice combined with GP workforce shortages limited the time GPs could offer to refugees to manage their multiple and complex health needs – a problem shared with other groups who have greater health care needs . Under tAn alternative to providing initial care to refugees in private general practice is for this to be provided in a specialist refugee service or community health setting. Such a service delivery model received strong support from participants in this study. Previous studies have similarly highlighted the central importance of community health services providing initial health care to patients with complex health needs, including refugees, as a result of better access to resources and infrastructure support ,26,34.It is acknowledged that there is not a one size fits all approach when determining which model, specialist or mainstream, best meets the special service needs of refugees in their initial resettlement period and thatThis study provided a unique and detailed insight into the experience of GPs providing health care to refugees. However, given the small number of participants in this study, these results cannot be generalised to all GPs in Adelaide or GPs in other locations. To do this, a larger quantitative study would be required. The low response rate from GPs could have meant that those GPs involved were more motivated to participate because of dissatisfaction with the current system of provision of initial health care to refugees. This low response rate, however, mirrors the experience of Divisions in this study and their difficulties getting GPs to participate in research and respond to surveys. Further, it is generally believed that at the time data was collected for this study there were a limited number of GPs providing initial care to refugees in SA. It is likely, therefore, that the views of GPs in this study not only provide a reasonably comprehensive summary of the challenges of providing initial care but are also the experience of most GPs doing this work in SA at the time. Whilst GPs interstate are likely to face many similar challenges providing initial care to refugees, it is beyond the scope of this paper to comment on how well resourced they are to provide effective care.This study provides evidence that, due to a range of challenges, GPs in private practice in SA are insufficiently resourced to provide initial health care effectively to refugees and that attempting to overcome these challenges would face a number of obstacles. Whilst further evidence is required to document the extent of these challenges in SA and how they might be best addressed, it makes sense for the existing state funded refugee health service to be involved in the delivery of initial PHC services to refugees, especially those with complex health needs and significant resettlement challenges.The authors declare that they have no competing interests.DJ conceptualised the study, conducted the literature review, undertook data collection and analysis, and drafted the paper. AZ and TB advised on all stages of the work including analysis of the data as well as reviewing and contributing to drafts of the paper. All authors read and approved the final manuscript.

PWScr) for neonatal lethality, failure to thrive, and growth retardation was narrowed to the locus containing a cluster of neuronally expressed MBII-85 small nucleolar RNA (snoRNA) genes. Here, we report the deletion of PWScr. Mice carrying the maternally inherited allele (m−/p+PWScr) are indistinguishable from wild-type littermates. All those with the paternally inherited allele (m+/p−PWScr) consistently display postnatal growth retardation, with about 15% postnatal lethality in C57BL/6, but not FVB/N crosses. This is the first example in a multicellular organism of genetic deletion of a C/D box snoRNA gene resulting in a pronounced phenotype.Prader-Willi syndrome (PWS [MIM 176270]) is a neurogenetic disorder characterized by decreased fetal activity, muscular hypotonia, failure to thrive, short stature, obesity, mental retardation, and hypogonadotropic hypogonadism. It is caused by the loss of function of one or more imprinted, paternally expressed genes on the proximal long arm of chromosome 15. Several potential PWS mouse models involving the orthologous region on chromosome 7C exist. Based on the analysis of deletions in the mouse and gene expression in PWS patients with chromosomal translocations, a critical region is missing or unexpressed. In an attempt to understand this disorder, various protein-coding genes in this region have been deleted in mice, but none of the resulting phenotypes consistently correlated with the human disease. This region also contains a cluster of genes that encode functional non-protein-coding RNAs. We deleted specifically the MBII-85 small nucleolar RNA (snoRNA) gene cluster on the parental mouse chromosome, which did not affect expression of any of the other snoRNA or protein-coding genes in the PWS region. These mice consistently displayed postnatal growth retardation starting from day 5 to 6, low postnatal lethality only in certain genetic backgrounds (<15%), and no adolescent obesity. Thus, this mouse model, with the deletion of a small, brain-specific non-protein-coding RNA, should prove useful for teasing out the various molecular pathologies of PWS. Ube3A gene . . Mkrn3 ahenotype –23. The e models and genee models ,24,25. Tins open ,18,24. Wins open to deletPWScr is the most probable candidate region for neonatal lethality, failure to thrive and postnatal growth retardation, we devised the following strategy for producing m+/p−PWScr mice (HPRT)-deficient ES cells, AB2.2, were modified through homologous recombination (HR) using targeting constructs 5′HPRT/PWScr_targ and 3′HPRT/PWScr_targ to place loxP sites proximal to the 5′ flanking region of the MBII-85 snoRNA gene cluster and distal to the Ipw exon C, respectively Resistance of ES cells to HAT media requires a functional HPRT gene. Restoring the intact HPRT gene through deletion of the PWScr leads to resistance of ES cells to HAT media. 2) Southern blot analysis of all HAT-resistant ES colonies (data not shown), as well as all PWScr-deleted mice, using 5′HR and 3′HR probes . 3) We PCR amplified and sequenced flanking regions of the PWScr locus together with the inserted HPRT gene derived from 5′HPRT/PWScr_targ-targeted ES cells were crossed with C57BL/6 females. As expected, we did not observe growth retardation in the m−/p+5′HPRT mice (p=0.275) B, and S2termates C. In addp=0.275) D.m+/p−PWScr pups as is the case with postnatal PWS patients. Insufficient milk intake resulting in growth retardation is consistent with the Holland hypothesis and observations in TgPWS mice The dictions postnatang SNRPN ,30. Bothm+/p−PWScr and m−/p+PWScr mice and observed that m+/p−PWScr males and females transmitted the PWScr deleted allele to offspring. To further extend our observations, we established 10 breeding pairs from each of the m+/p−PWScr and m−/p+PWScr males with 80 wild-type females. All matings resulted in pregnancies leading to successful live births with litter sizes around eight , and always included wild-type mice and mice carrying the PWScr-deleted allele in a ratio of 1:1 by complementarity to defined sites within these RNA targets . HoweverHPRT/PWScr_targ and 3′HPRT/PWScr_targ targeting vectors, we isolated clones RPCIP711K19517Q6 and RPCIP711J18414Q6, respectively, from the RPCI21 mouse PAC library (RZPD German Resource Center for Genome Investigation) using the MBII-85 oligonucleotide . The thymidine kinase (TK) gene was placed outside of the homologous arm , passage 17, were expanded in HEPES-buffered, Dulbecco's modified Eagle's medium supplemented with 15% fetal bovine serum (HyClone), nonessential amino acids, L-glutamine, β-mercaptoethanol, 1000 U/ml recombinant LIF (Chemicon) and antibiotics (penicillin 100 U/ml and streptomycin 100 μg/ml) on a γ-irradiated monolayer of SNL6.7 cells (from A. Bradley) or mouse primary fibroblasts. For electroporation, 2 × 107 ES cells were resuspended in 20 mM HEPES pH 7.4, 173 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM dextrose, and 0.1 mM β-mercaptoethanol [NotI linearized replacement targeting vectors 5′HPRT/PWScr_targ, 3′HPRT/PWScr_targ, and intact CRE expressing cassette pOG231 (55 μg DNA of each) were electroporated at 25 μF and 400V . After electroporation, cells were plated onto 100 mm culture dishes containing a γ-irradiated monolayer of primary, G-418-resistant, or SNL6.7 fibroblast feeder cells. Thirty-eight hours later, 350 μg/ml G418 (Invitrogen) and 0.2 μM 2′-deoxy-2′-fluoro-β-D-arabinofuranosyl-5-iodouracil (FIAU) or 1.0 - 0.5 μg/ml puromycin (Sigma) and 0.2 μM FIAU, or HAT (Sigma) were added to the culture medium. The medium was replaced every day and colonies were picked and analyzed eight days after plating. 5′HPRT loxP-targeted ES cells were analyzed using oligonucleotide pairs 85–5′screen2d/85–5′screen2r and 85–5′screen3d/85–5′screen3r for a nested PCR approach. For analysis of 3′HPRT loxP-targeted cells we employed the 85–3′screen1d/85–3′screen1r and 85–3′screen2d/85–3′screen2r nested PCR primer pair combinations. DNA blot analysis was performed as described [32p−labeled 5′HR and 3′HR probes (PWScr-deletion were injected into 3.5-day-old B6D2F1 (C57BL/6 x DBA) blastocysts, and the resulting embryos were transferred to CD-1 foster mice. Chimeras were identified by their agouti coat color.oethanol . The Notescribed . MembranR probes A. SeveraPWScr together with the inserted HPRT gene was amplified and sequenced using PCR primers MB85seqD1 and MB85seqR1. Sequencing reactions were performed using the BigDye terminator cycle sequencing reaction kit (PE Applied Biosystems) and resolved on an ABI Prism 3100 (Perkin Elmer) capillary sequencing machine.The 5.7 kb DNA fragment containing flanking regions of oC – 20 sec, touch down −1 °C, extension 67 oC – 1 min 40 sec); 35 cycles . The final extension was performed at 67 °C for 5 min.To genotype mice we performed PCR analysis of genomic DNA from tail biopsies using the primer pair MB85deld1/MB85delr1 . PCR cyc32P-labeled cDNA probes. Northern blot analysis of snoRNAs was performed with specific oligonucleotides according to the manufacturer's instructions. RNA samples, 20 μg each, were treated with RNase-free DNase I (Roche). First strand cDNA synthesis was performed using Transcriptor reverse transcriptase (Roche) and hexamer oligonucleotides, followed by PCR amplification with gene specific oligonucleotides . The cDNleotides as descrPWScr-deficient mouse line was established by breeding male chimeras nos. 2 and 5 from one mutant ES cell line with female C57BL/6 mice to produce heterozygous mice. Subsequently, heterozygous mice were interbred or bred to C57BL/6 mice. All breeding occurred at the ZMBE animal facility of the University Clinics, Münster in a controlled room with a 12:12 hour light-dark cycle, and mice were housed under non-enriched, standard conditions in individually ventilated (36 (l) x 20 (w) x 20 (h) cm) cages for up to five littermates. Pups were weaned 19 – 23 days after birth and females were kept separately from males.A t-test, or ANOVA for each day of postnatal age. Weights of placentas and embryos were analyzed using Mann-Whitney nonparametric statistics. Postnatal lethality was analyzed with the chi-square test.Statistical analysis was performed using the StatView software package. Body weight was analyzed using Student's Figure S1HPRT cassette (Genbank accession number EU233428). The sequence of Ipw exon H is underlined. The sequence positions correspond to the UCSC Genome Browser . The compiled deleted region is estimated to span ∼193 kb; however, because the gap size is only estimated , the region deleted in our mouse model could span from 143 to ≥193 kb.The primary sequence of the 5′- and 3′- regions flanking the inserted (82 KB PDF)Click here for additional data file.Figure S2 Growth dynamics of mice in 126SV x C57BL/6 x FVB/N genetic crosses (∼50% FVB/N contribution) beginning at postnatal day 1.m+/p−PWScr males. The black line corresponds to the weight gain of 3 wild-type males.(A) Growth dynamics of 11 investigated males. The yellow line corresponds to the weight gain of 8 m+/p−PWScr females. The black line corresponds to the weight gain of 8 wild-type females.(B) Growth dynamics of 21 investigated female mice. The yellow line corresponds to the weight gain of 13 Growth dynamics of mice in 126SV x C57BL/6 x BALB/c genetic crosses (∼50% BALB/c contribution) beginning at postnatal week 6.m+/p−PWScr males. The black line corresponds to 53 m+/p+PWScr males.(C) Growth dynamics of 100 analyzed male mice. The yellow line corresponds to weight gain of 47 m+/p−PWScr females. The black line corresponds to the weight gain of 51 m+/p+PWScr females. In all cases, black error bars exhibit statistically significant intervals .(D) Growth dynamics of 102 female mice. The yellow line corresponds to the weight gain of 51 (109 KB PDF)Click here for additional data file.Table S1(27 KB DOC)Click here for additional data file.Table S2P1 – P8 indicate the corresponding postnatal days. N is the number of mice investigated from each genotype. Mean is the average mouse weight per category in grams. Statistically significant differences (p < 0.05) are indicated in bold.(76 KB DOC)Click here for additional data file.Table S3m+/p−PWScr males and wild type BALB/c females resulted in 230 embryos/pups. Among those 106 were identified as m+/p+PWScr, 121 as m+/p−PWScr and 3 were not genotyped. We have performed 12 crosses between m−/p+PWScr males and wild type BALB/c females and obtained 100 embryos/pups. Fifty were identified as m+/p+PWScr, 48 as m+/p−PWScr and 2 were not genotyped.Twenty-nine crosses between (63 KB DOC)Click here for additional data file.

These viruses have resulted in >100 cases of human infection since 2002,and their pandemic potential should not be underestimated. Influenza A subtype H7 viruses have resulted in >100 cases of humaninfection since 2002 in the Netherlands, Italy, Canada, the United States, andthe United Kingdom. Clinical illness from subtype H7 infection ranges fromconjunctivitis to mild upper respiratory illness to pneumonia. Although subtypeH7 infections have resulted in a smaller proportion of hospitalizations anddeaths in humans than those caused by subtype H5N1, some subtype H7 strainsappear more adapted for human infection on the basis of their virus-bindingproperties and illness rates among exposed persons. Moreover, increasedisolation of subtype H7 influenza viruses from poultry and the ability of thissubtype to cause severe human disease underscore the need for continuedsurveillance and characterization of these viruses. We review the history ofhuman infection caused by subtype H7. In addition, we discuss recentlyidentified molecular correlates of subtype H7 virus pathogenesis and assesscurrent measures to prevent future subtype H7 virus infection. Orthomyxoviridae and possess 8negative-sense RNA segments encoding 11 known proteins. Of these, the 2 viral surfaceglycoproteins, hemagglutinin (HA) and neuraminidase (NA), form the basis of multipleserologically distinct virus subtypes. Currently, 16 HA and 9 NA subtypes have beenidentified in wild water birds, the natural host for all influenza A viruses and thereservoir from which viruses emerge to infect domestic poultry and occasionally mammals.Most influenza viruses that infect wild or domestic birds cause no or limited illnessesand deaths and are characterized as being low pathogenicity avian influenza (LPAI)viruses. However, viruses within the H5 and H7 subtypes have the capacity to acquiregenetic properties that confer high virulence and a high proportion of deaths inchickens and other fowl after their introduction into domestic poultry; these virusesare characterized as highly pathogenic avian influenza (HPAI) viruses according to theintravenous pathogenicity index method described by the World Organization for AnimalHealth ,1957 (H2N2), and 1968 (H3N2). However, none of these pandemic strains possessed the HAcleavage site mutation characteristic of HPAI viruses resulted in the infection of a laboratory workerwhen an experimentally infected seal sneezed into the face and the right eye of theworker from a human, which occurred in the United States in 1959, from the bloodof a man with clinically diagnosed infectious hepatitis outbreak in the Netherlands, LPAI (H7N3) virusescaused outbreaks in poultry in northern Italy during 2002–03.Retrospective serologic analysis of workers involved in the outbreak responseidentified 7 of 185 persons who had close direct physical contact with poultry andwere seropositive by microneutralization assay and Western blot analysis for subtypeH7 influenza have circulated in the northeastern UnitedStates live bird markets for over a decade and were the cause of a devastatingoutbreak predominantly on domestic turkey farms in 2002. One of 80 tested workersinvolved in the culling operations during this outbreak reported a temporallyrelated respiratory illness and exhibited serum-neutralizing antibody responsesconsistent with a subtype H7N2 virus infection, providing the first evidence ofpossible human infection with a North American lineage LPAI virus (H7N2) first detected in a poultry flock ineastern England was isolated from a poultry worker with conjunctivitis in 2003 , which circulated in the live bird markets of the northeastern UnitedStates for over a decade. These viruses possess a 24-nt deletion found in the HA anda 51-nt stalk deletion in the NA, which distinguishes them from other subtype H7viruses found in domestic poultry in North America. Since these viruses wereintroduced in 1994, the HA cleavage site of circulating viruses acquired additionalbasic amino acids, a known correlate of pathogenicity for avian influenza viruses European lineage virusesisolated from humans resemble subtype H5N1 viruses in their capacity for highvirulence in mammalian models has greatly improved ourunderstanding of avian viruses. Applying this knowledge toward the assessment ofother HPAI and LPAI viruses with pandemic potential, such as those within the H7subtype, will further improve our ability to respond to and reduce the severity offuture pandemics, regardless of virus subtype.

Anopheles gambiae s.s. Giles is a major malaria vector in Sub-Saharan Africa. Studies of the basic biology of this mosquito, including oviposition, provide a background for assessing which attributes might be exploited for suppressing A. gambiae populations. Here, we report on when during the diel cycle A. gambiae individuals deposit eggs as compared to the ovipositional patterns of groups.A. gambiae was determined by presenting hypergravid females with an ovipositional substrate exclusively between 1200 and 1600 h.Battery-powered wall clocks were modified so as to present a unique section of dark and wet ovipositional substrate at hourly intervals over two consecutive 12 h periods. Ovipositional periodicity of mosquito groups and individuals was determined by counting the number of eggs present on each section of the ovipositional substrate. Capacity for mid-afternoon oviposition by groups of Kisumu laboratory strain A. gambiae groups deposited 65% of their total eggs between 1800 and 0 h, and the remaining 35% were spread between 0 and 1000 h. Caged house-collected A. gambiae groups deposited 74% of their total eggs between 1800 and 200 h, ceased oviposition for 3 h, and then spread the remaining 26% of their eggs near or after dawn. Ninety-six percent of individual A. gambiae females spread their eggs over a continuous 2–4 h period without interruption. In tests of capacity for mid-afternoon oviposition, females given evening access to an ovipositional resource deposited 2% of their total eggs between 1200 and 1700 h. A. gambiae females given only access to an ovipositional resource between 1200 and 1700 h deposited 3 times more eggs during that time period than did females previously given evening access.On equatorial time, caged laboratory strain A. gambiae oviposit in a single ca. 2–4 h continuous bout per 24 h. Oviposition is most probable in early scotophase, mid scotophase, or early photophase. However, some oviposition can occur at any hour during 24 h, especially if females were previously deprived of ovipositional substrate.Confined individual Drosophila melanogaster [D. pseudoobscura [Delia antiqua [Chrysomya bezziana [Aedes aegypti [Anopheles albimanus [A. freeborni [A. albitarsis [A. gambiae [A. albimanus [D. melanogaster [D. melanogaster oviposition [D. melanogaster individuals. Chadee et al. [A. albimanus laid their entire complement of eggs at once rather than splitting them between two different periods.Diel ovipositional patterns have been studied in various Diptera, including nogaster -4, D. psoobscura , Delia a antiqua , Chrysombezziana , Aedes a aegypti , Anophellbimanus , A. freereeborni , A. albibitarsis and A. g gambiae -14. All lbimanus and D. mposition . Fluegele et al. reportedA. gambiae held in a common cage [A. gambiae was capable of oviposition at any time during the night. However, they observed that five of a total of nine batches of eggs were laid between 2000 and 2300 h. Under equatorial conditions, McCrae [A. gambiae oviposition. He postulated that the time of peak oviposition during a night was related to the time at which the blood meal was taken. However, Sumba et al. [A. gambiae commenced at scotophase (1800 h) and peaked between 1800–2100 h. A second but smaller ovipositional peak was documented by both research teams, but at inconsistent times. In all cases, some oviposition occurred throughout scotophase. In one case [Outcomes of research on the ovipositional periodicity of groups of mon cage -14 have mon cage suggeste, McCrae reporteda et al. and SumbA. gambiae s.s. originated from the Kenya Medical Research Institute (KEMRI) of Kisumu, Kenya. It was reared at Michigan State University according to Huang et al. [A. gambiae s.s. and the remainder were A. arabiensis as determined by PCR [Two sources of mosquitoes were used: 1) The Kisumu laboratory strain of g et al. . Cages od by PCR .D. melanogaster [Agrotis segetum [A. gambiae individuals vs. groups.The method of egg collection may influence the oviposition rhythm of some Diptera . Use of nogaster ,16 and A segetum . In the ® high-capacity brown paper towelling . This paper towelling appears light when dry, but dark when wet. Thus, it provided the two key stimuli (dark and wet) necessary and sufficient to strongly stimulate A. gambiae to oviposit [® flaps to prevent mosquitoes from depositing eggs on any unexposed section of the substrate. The clock face was mounted on the hour-hand driver, so that the opening in the face plate made one revolution every 12 h. The clock face was covered with white paper so as to maximize contrast between background versus the actual ovipositional site [Battery-powered wall clocks measuring 31 cm in diameter were modified to progressively present a unique section of dark and wet substrate onto which mosquitoes could oviposit over 12 h measuring 60 × 60 × 60 cm and containing approximately 500 laboratory-reared females of the Kisumu strain varying in reproductive stages. The light cycle in the environmental chamber was set at 12:12 LD, to approximate the natural light cycle found in Kisumu, Kenya. A small tungsten bulb continued to burn in the laboratory at night so as to provide the equivalent light from the night sky. Light levels during scotophase were slightly less than full moonlight . OviposThe bottom of the enclosure for these tests was the clock apparatus over which sat a 12 cm high cylindrical wire frame. Nylon netting (18 intersections/cm) was placed over the frame and secured by a drawstring. Six 2 cm diameter wet cotton balls were placed on the roof of the cage as a source of moisture. Blood meals were offered to females between 1200 and 1700 h three to four days prior to use. Three or four days after a blood meal, an individual female was gently transferred to the clock cage by aspirator before scotophase. After the female had been exposed to the ovipositional resource for 4–5 h, the female was removed from the cage and fresh paper towelling was substituted for the previously exposed paper towelling within the clock. Then, the female was carefully reinserted. The exchange of the ovipositional resource was repeated 11 h later. The numbers of eggs laid within each hourly period were counted and incorporated into a histogram. Correlation analysis (SAS software version 9.1) was used to test for a correlation between the length of the preoviposition interval and the length of the oviposition interval . It was also used to test for a correlation between the length of the preoviposition interval and the total number of eggs deposited per female. The ovipositional periodicity was measured for a total of 56 individual females, all of the Kisumu laboratory strain.The terms gravid and hypergravid as used by Sumba et al. refer thEngorged females were randomly selected from newly blood-fed cages of mosquitoes and placed in groups of 20 into 8 cages made from 15 cm high and 19 cm in diameter white cardboard cartons. The top of the cage was covered with white netting (8 intersections/cm) and a sleeve of the same netting was fitted to a 10.5 cm hole cut in the side for mosquito and ovipositional resource insertion. Females were provided with a constant source of 10% honey solution and six wet cotton balls were placed on the top of the cage to provide extra moisture. Two days after blood-feeding, an ovipositional resource was provided to half of the cages approximately 2 h before the lights were turned off to record egg deposition during scotophase. The ovipositional resource was a 100 × 35 mm clear plastic Petri dish containing 20 mL of distilled water, placed over a circular piece of black paper. At 1200 h the following day, the loaded ovipositional resources were replaced with new Petri dishes containing fresh filtered water. Four ovipositional resources were also introduced into the 4 cages from which an ovipositional resource had been withheld. These resources, identical to those previously mentioned, were used to record oviposition by gravid females during photophase. After the initial introduction, ovipositional resources were changed hourly from 1200–1600 h in the latter half of the cages and all exposed ovipositional resources were examined for the presence of eggs. Using a small brush, eggs present were brushed into lines on a piece of white paper and counted.A. gambiae of the Kisumu laboratory strain revealed two ovipositional pulses revealed no significant difference between gravid and hypergravid treatments (p = 0.61). However, correlation analysis revealed a significant positive correlation between the length of the preoviposition interval and the total number of eggs deposited (p = 0.03).Ovipositional periodicity of gravid and hypergravid females held individually was similar, although more gravid females contributed to the second ovipositional pulse than did hypergravids. A paired t-test of the total mean number of eggs oviposited per The time at which individual females initiated oviposition was highly variable Figure . The meaSeven out of the 12 cages from which an ovipositional resource had been withheld until mid-afternoon produced eggs than there was in the amount of time devoted to oviposition. In the case of the single female who split her eggs between two ovipositional periods, an interruption caused by the exchange of the paper towelling could explain this single aberration.While the rates of egg deposition by gravid and hypergravid females were not found to be different, a statistically significant correlation existed between the length of the preoviposition interval and the total number of eggs deposited per female. Individuals with longer preoviposition intervals tended to deposit slightly more eggs. However, this correlation likely has little biological significance due to the considerable scatter in the data. This is demonstrated by the width of the 95% confidence intervals surrounding the mean total numbers of eggs per preoviposition interval, which ranged from a mean of 64.4 to a mean of 19.9 .A. gambiae does have the capacity for afternoon oviposition in full light. Females denied an ovipositional resource for 18 h oviposited between 1200 and 1600 h, when the ovipositional resource was introduced. In some cases, eggs were found on the ovipositional resources between 1200 and 1600 h even when the mosquitoes had a resource beginning at 1700 h on the previous night.-3 w m-2, which is equivalent to late dusk or early dawn [A. gambiae to forage for ovipositional sites before full darkness and at or after dawn, when useful visual contrast would be more detectable.Visual contrast of the ovipositional substrate is an important stimulus for oviposition and egg placement. Huang et al. reportedrly dawn . When ovA. gambiae is not restricted only to one specific time of day, and oviposition is not fully inhibited by high light levels. Gravid females can initiate oviposition as soon as an ovipositional resource becomes available. Thus, ovitraps, a tool to monitor A. gambiae population growth and help predict malaria epidemics, should remain available throughout the full 24 hr diel to be maximally effective. Further study of abiotic factors like daily temperature and relative humidity fluctuations and their contribution to patterns in flight activity in the field may be of interest. Excess temperature and low RH may limit mid-afternoon oviposition in the field. During the daytime in the tropics, air and soil temperatures typically exceed the optimum temperature for oviposition .Our overall results establish that oviposition by A. gambiae populations are ovipositionally flexible. Rather than confining oviposition to a specific brief period during 24 h, as is true for many insects, A. gambiae can oviposit at any time after their eggs have fully developed and they have access to an ovipositional resource. But, they most commonly begin oviposition and deposit the majority of eggs shortly after lights off. Once oviposition commences, individual females deposit their eggs over a continuous 2 to 3 hr period without interruption.The author(s) declare that they have no competing interests.MLF conducted experimental work on individuals, and primarily developed the manuscript. JH, MLF and JRM collectively planned the study and conducted experimental work with groups. JRM initiated the study and designed the clock apparatus. JRM and JH developed the manuscript with MLF. MLF, JRM, JH and EDW analyzed and displayed the data. EDW secured the NIH grant under which this research was funded, arranged Kenya travel and secured the lab facilities for the study. MNB provided logistical support, research site security and interpretational analysis. JV provided research site security and secured personnel for collection of feral mosquitoes. All authors read and approved the final manuscript.

A classical O—H⋯O hydrogen bond exists between the methanol solvent mol­ecule and the perchlorate anion. Magnetic susceptibility measurements indicated the complex to be in the low-spin state in the temperature range 5–400 K.In the title complex, [Fe(C DOI: 10.1107/S1600536809040549/si2207Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:

Although human embryonic stem cells (hESCs) hold great promise as a source of differentiated cells to treat several human diseases, many obstacles still need to be surmounted before this can become a reality. First among these, a robust chemically-defined system to expand hESCs in culture is still unavailable despite recent advances in the understanding of factors controlling hESC self-renewal.In this study, we attempted to find new molecules that stimulate long term hESC self-renewal. In order to do this, we started from the observation that a commercially available serum replacement product has a strong positive effect on the expansion of undifferentiated hESCs when added to a previously reported chemically-defined medium. Subsequent experiments demonstrated that the active ingredient within the serum replacement is lipid-rich albumin. Furthermore, we show that this activity is trypsin-resistant, strongly suggesting that lipids and not albumin are responsible for the effect. Consistent with this, lipid-poor albumin shows no detectable activity. Finally, we identified the major lipids bound to the lipid-rich albumin and tested several lipid candidates for the effect.Our discovery of the role played by albumin-associated lipids in stimulating hESC self-renewal constitutes a significant advance in the knowledge of how hESC pluripotency is maintained by extracellular factors and has important applications in the development of increasingly chemically defined hESC culture systems. Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of human blastocysts. The ability of hESCs to differentiate into any cell type of the adult human body, i.e. their pluripotency, has generated hopes that in the future these cells will be used as a source of differentiated cells to treat a number of human ailments, including type I diabetes, Parkinson's and heart disease In order to develop chemically-defined media that robustly sustain hESC self-renewal, it is of critical importance that the signals and mechanisms controlling hESC fate choices (such as choosing to differentiate into a particular lineage as opposed to remaining undifferentiated) be understood in detail. Over the last few years, a number of extracellular factors have been identified that affect hESC self-renewal. Amongst these, a major role is played by members of the TGFβ superfamily of signaling molecules In this study, we find that albumin-associated lipids have strong positive effects on the self-renewal of two different hESC lines. Our results demonstrate that these lipids constitute an essential ingredient within knockout serum replacement, a supplement which is widely used to prevent hESC differentiation and stimulate hESC self-renewal.2. Plates were incubated for 30 minutes at 37°C for matrigel to solidify and then washed once in D-PBS (Invitrogen) before being used. To maintain cell lines, these were cultured in mouse embryonic fibroblast-conditioned medium (MEFCM), which was prepared as previously described Human embryonic stem cell lines HUES7 and HUES9 were cultured on matrigel-coated plates. For plate preparation, BD Matrigel (BD Biosciences) was diluted 15 times in KO-DMEM (Invitrogen) and used to coat plates at 0.11 ml/cmEmbryoid bodies (EBs) were prepared by treating cells with 2 mg/ml dispase for 15 minutes at 37°C, as done when passaging them (see above). Cells were then fully detached by pipetting with a 5 ml tip, centrifuged, resuspended in DMEM+10% FBS and plated onto non-coated, non-cell culture-treated petri dishes. Despite this, some EBs still tended to attach to the plate, so every day the attached EBs were detached by gentle scraping with a sterile cell scraper. After 6 days of growth in suspension with media changes on days 2 and 4, EBs were transferred to gelatin-coated plates (0.1% gelatin for 2 hours at 37°C) in the same medium (DMEM+10% FBS). Under these conditions, cells attached to the bottom of the plates, where they were left to differentiate for 20 additional days, changing the medium as required.2.5 ml of 50 mg/ml AlbuMAX II (Invitrogen) dissolved in PBS were extracted using a 1∶1 chloroform∶methanol mix (5 ml) and twice again with chloroform (2 ml each). The extracted lipids were then diluted to appropriate concentrations using chloroform∶methanol (1∶1) and used for HPLC analysis. HPLC standards consisted of 1 mg/ml each of triolein, cholesterol, ceramide, oleic acid, dioleoyl-phosphatidylglycerol, dioleoyl-phosphatidylcholine, dioleoyl-phosphatidylethanolamine, dioleoyl-phosphatidylserine, dioleoyl-phosphatidic acid, soy phosphatidylinositol, sphingomyelin and 0.5 mg/ml each of myristoyl-ceramide, oleoyl-lysophosphatidylcholine, oleoyl-lysophosphatidylethanolamine, oleoyl-lysophosphatidylglycerol and oleoyl-lysophosphatidylserine. The standards and the samples were injected into a normal phase HPLC column and analyzed using an evaporative light scattering detector (HPLC-ELSD).For flow cytometric analysis, cells were washed once in D-PBS, incubated for 20 minutes at 37°C in cell dissociation buffer (Invitrogen) and individualized by pipetting. Cells were then centrifuged for 5 minutes at 400 g, resuspended in D-PBS, recentrifuged and resuspended again in blocking solution , in which they were incubated for 5 minutes at room temperature. Next, cells were centrifuged once more and resuspended in BS with the desired antibodies ). After incubating cells for 30 minutes on ice with the primary antibodies, cells were washed three times in BS, incubated with secondary antibodies (Jackson Labs) as previously done for primaries, washed three more times in BS, filtered to remove any remaining cell aggregates and analyzed using a MoFlo flow cytometer (Cytomation). In case an analysis could not be performed immediately, cells were fixed in PBS+3% paraformaldehyde for 15 minutes at 4°C, washed twice in PBS, stored at 4°C in the dark and analyzed the next day.Exponentially growing hESCs were treated overnight with 200 ng/ml KaryoMAX colcemid solution (Invitrogen) to arrest cells in metaphase. Next, cell medium was aspirated and cells were detached by a 20 minute incubation with cell dissociation buffer (Invitrogen). After two washes in PBS, cells were gently resuspended in a 0.56% KCl solution and incubated at room temperature for 15 minutes. Cells were then centrifuged and gently resuspended in cold methanol∶acetic acid 3∶1 (V∶V) for fixation. After incubating for 10 minutes on ice, cells were centrifuged again and resuspended in a small volume (about 200 µl per million cells) of the same cold methanol∶acetic acid solution. At this point, cells were dropped onto slides (approx. 10 µl/slide), slides were allowed to dry and then stained with KaryoMAX Giemsa stain (Invitrogen) for at least 2 hours. After washing the slides a few times with water, they were dried and visualized in a microscope for chromosome counting.Total RNA was purified from cells using Qiagen's RNeasy kit. The RNA concentration in the samples was determined spectrophotometrically and 1 µg of RNA per sample was converted to cDNA using Superscript II reverse transcriptase from Invitrogen. PCR reactions were prepared in microamp optical 96-well reaction plates (Applied Biosystems) by mixing 0.5 µl cDNA , 5 µl SYBR green PCR master mix 2X (Applied Biosystems) and 10 picomoles of each primer in a total volume of 10 µl. Reactions were run in an ABI Prism 7900 Sequence Detector (Applied Biosystems) and results analyzed with SDS2.3 software (Applied Biosystems) using the ΔΔCt method. Primers used were hGAPDH-fwd (5′-GGA CTC ATG ACC ACA GTC CAT GCC-3′), hGAPDH-rev (5′-TCA GGG ATG ACC TTG CCC ACA G-3′), hOct4-fwd (5′-GAG AAG GAT GTG GTC CGA GTG TG-3′), hOct4-rev (5′-CAG AGG AAA GGA CAC TGG TCC C-3′), hNanog-fwd (5′-TGA ACC TCA GCT ACA AAC AGG TG-3′), hNanog-rev (5′-AAC TGC ATG CAG GAC TGC AGA G-3′), h-cTnT-fwd (5′-AGG CGC TGA TTG AGG CTC AC-3′), h-cTnT-rev (5′-ATA GAT GCT CTG CCA CAG C-3′), hGFAP-fwd (5′-CGG CTG CTT TTC CCT AAG C-3′), hGFAP-rev (5′-GGG TAC ATT TTG TGT GTG AGT AAG AAG-3′), hSox1-fwd (5′-GCC CTG AGC CGA CTG TGA-3′), hSox1-rev (5′-CCG TGA ATA CGA TGA GTG TTA CCT-3′), hSox17-fwd (5′-TGA ATG TGT CCC AAA ACA GCT T-3′), hSox17-rev (5′-CAC ACC CAG GAC AAC ATT TCT TT-3′).For immunofluorescence staining, cells grown on glass coverslips were fixed for 20 minutes at room temperature in PBS+4% paraformaldehyde followed by three washes in PBS. Next, cells were blocked and permeabilized by a 20-minute incubation with PBS-BS (PBS+0.5% BSA+0.05% saponin) and then incubated with mouse anti-TRA-1-60 antibody ) for 1 hour at 30–37°C. After three washes with PBS-BS, cells were incubated for an additional hour at room temperature and in darkness with PBS-B containing 10 µg/ml DAPI or propidium iodide (Molecular Probes) and 0.5 µg/ml FITC-conjugated donkey anti-mouse antibody (Jackson Labs). Then, cells were washed five times with PBS and coverslips mounted on slides using Vectashield (Vector Laboratories). Slides were left overnight at 4°C and analyzed the next day by fluorescence microscopy.2 and 0.1% Tween-20). After this, cells were stained by incubating them at room temperature for 15–30 minutes in the dark in staining solution containing NTMT, 0.34 mg/ml NBT and 0.18 mg/ml BCIP . Finally, the reaction was stopped by washing cells once in PBS and, after counterstaining with propidium iodide, cells were visualized in a microscope.To stain cells positive for alkaline phosphatase, cells were first fixed by incubating them for 20 minutes at room temperature in 4% paraformaldehyde in PBS. Next, cells were washed three times with PBS and then three more times in NTMT buffer that robustly sustains long term hESC self-renewal, we started by testing those media that had already been reported to achieve this goal ed cells s. On theed cells s. Analysed cells .Since KOSR is not a chemically-defined product, the next question we attempted to answer concerned the identity of the molecule/s that are responsible for the effect of KOSR on the maintenance of hESC stemness. KOSR was originally designed and patented in 1998 by Life Technologies Inc. to substitute for fetal bovine serum (FBS) in applications, such as the derivation and culture of mouse embryonic stem cells, where the use of FBS was problematic due to its high lot-to-lot variability. Unlike FBS, KOSR has a very constant composition from one lot to another, thus eliminating the need to test every batch before being able to use it. The composition of KOSR can be found in its patent Since N2/B27-CDM already contains a fairly large amount of transferrin www.expasy.ch/tools), the 607 amino acid residue sequence of BSA is predicted to have 45 cleavage sites for trypsin with a 100% probability of cleavage, and 26 additional sites with a probability higher than 80%). After culturing HUES7 or HUES9 cells for five passages in the presence of N2/B27-CDM alone or supplemented with either 15% Trypsin-KOSR or 1% Trypsin-AlbuMAX, it became evident that the trypsinized samples keep at least part of the activity of the original untreated KOSR or AlbuMAX samples, as measured by maintenance of the expression of ALP (In order to establish whether the effects of lipid-rich BSA (AlbuMAX) on hESC self-renewal are dependent on its protein or its lipid components, or both, we followed two different approaches. First, we compared the effects of AlbuMAX with those of a lipid-poor albumin . As shown in n of ALP , TRA-1-6n of ALP , Oct4 ann of ALP , indicatAlthough it has been shown that hESCs cultured in N2/B27-CDM+AlbuMAX for several passages retain expression of previously described pluripotency markers such as ALP, TRA-1-60, Oct4 and Nanog, it remained to be proven that these cells are really pluripotent in the sense that they are capable of differentiating to all three germ layers of the embryo, i.e. ectoderm, endoderm and mesoderm. To address this issue, hESCs that had been cultured for seven passages in N2/B27-CDM plus AlbuMAX, KOSR or Trypsin-AlbuMAX (or MEFCM as a control) were induced to differentiate by formation of embryoid bodies (EBs) in suspension culture, as explained in the In addition to keeping the pluripotency of hESCs intact, it is important that media used to cultivate hESCs do not induce genomic alterations in these cells. To test whether culturing in the presence of N2/B27-CDM+AlbuMAX causes any changes in chromosome number, HUES9 cells were cultured for ten passages in this medium after which the number of chromosomes in the cells was counted as described in Since lipid molecules have an essential role in mediating the self-renewal enhancement observed with AlbuMAX, our next goal was to find out what lipid or lipids are responsible for this effect. In order to do this, we decided to take two different approaches. On the one hand, we carried out a lipid analysis aimed at identifying the major lipid species present in AlbuMAX. On the other hand, given that the active molecules in AlbuMAX need not be those that are more abundant and therefore appear more prominently in the lipid analysis, we also decided to test several lipids which we thought were good candidates to mediate the observed effect. This second approach will be dealt with in the next section. To identify lipids present in AlbuMAX, we extracted them using organic solvents and analyzed them by high performance liquid chromatography see section.2) and a chemically defined mix of unsaturated and saturated free fatty acids (FFA) known commercially as chemically defined lipid concentrate (Invitrogen). To test these molecules, HUES9 cells adapted to grow on N2/B27-CDM+1% AlbuMAX were either maintained with this same medium or transferred to N2/B27-CDM alone or N2/B27-CDM supplemented with FFA (1/250 dilution), LPC (100 µM), LPA (10 µM), S1P (10 µM) or PGE2 (20 µM). Supplementation with 10 µM S1P led to massive cell death, so the S1P concentration was reduced to 3 µM, which was much less toxic for the cells. After six passages in the presence of the aforementioned lipids, cells were either fixed and stained for ALP seem to have a modest effect on hESC self-renewal, even though these effects, if they exist, are clearly weaker than the effect observed with AlbuMAX.Based on previous reports see and on t for ALP or used for ALP . As expeIn this study, we have found that knockout serum replacement (KOSR) strongly stimulates self-renewal of human embryonic stem cells cultured with a previously reported chemically-defined medium (N2/B27-CDM) 2). LPA and S1P are very active lysophospholipids that act by binding GPCRs on the cell surface, and many of these GPCRs have already been shown to be expressed by hESCs 2 was recently shown to stimulate self-renewal of hematopoietic stem cells 2 not stimulate hESC self-renewal, it actually caused cell differentiation and a reduction in cell proliferation, thus ruling out PGE2 as a mediator of AlbuMAX's effects. Regarding S1P, high concentrations of this lipid (10 µM) induced a considerable amount of cell death, which is in stark contrast with a previous report showing that 10–20 µM S1P prevents hESC apoptosis The next question we have addressed concerns the identity of the active lipids in AlbuMAX. To this end, albumin-bound lipids were extracted with organic solvents and analyzed by high performance liquid chromatography (HPLC) . Among tIn conclusion, it is still unclear what lipid/s are responsible for the effect of AlbuMAX on hESC self-renewal. One possibility is that the active lipid is not among the ones tested here. Also possible is that the effect of AlbuMAX is due to a combination of lipids that must be added together. In this respect, the most promising combination on the basis of our results would be LPA+S1P, but this has not been tested yet. Likewise, it cannot be ruled out that some of the lipids tested here are active only at concentrations different from those used in our experiments .In spite of not having been able to identify the lipid/s responsible for the effect of AlbuMAX, we think that the discovery of the role played by albumin-associated lipids in stimulating hESC self-renewal, together with the other results shown in this study, represent a significant advance in our knowledge of how hESC pluripotency is maintained by extracellular factors and has applications in the development of increasingly chemically defined hESC culture systems.

The dihedral angle formed by the benzene ring with the tetra­zole rings are 51.86 (15) and 3.76 (11)°. In the crystal structure, centrosymmetrically related mol­ecules are linked into dimers by inter­molecular C—H⋯N hydrogen-bonding inter­actions.In the title compound, [Fe DOI: 10.1107/S1600536809022442/rz2333Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:

Mammographic breast density is one of the strongest known risk factors for breast cancer. We present a novel technique for estimating breast density based on 3D T1-weighted Magnetic Resonance Imaging (MRI) and evaluate its performance, including for breast cancer risk prediction, relative to two standard mammographic density-estimation methods.The analyses were based on MRI (n = 655) and mammography (n = 607) images obtained in the course of the UK multicentre magnetic resonance imaging breast screening (MARIBS) study of asymptomatic women aged 31 to 49 years who were at high genetic risk of breast cancer. The MRI percent and absolute dense volumes were estimated using our novel algorithm (MRIBview) while mammographic percent and absolute dense area were estimated using the Cumulus thresholding algorithm and also using a 21-point Visual Assessment scale for one medio-lateral oblique image per woman. We assessed the relationships of the MRI and mammographic measures to one another, to standard anthropometric and hormonal factors, to BRCA1/2 genetic status, and to breast cancer risk (60 cases) using linear and Poisson regression.P < 0.0001). Both show strong associations with established anthropometric and hormonal factors. Mammographic percent dense area, and to a lesser extent MRI percent dense volume were lower in BRCA1 carriers but there was no association with BRCA2 carrier status. The study was underpowered to detect expected associations between percent density and breast cancer, but women with absolute MRI dense volume in the upper half of the distribution had double the risk of those in the lower half (P = 0.009).MRI percent dense volume is well correlated with mammographic percent dense area (R = 0.76) but overall gives estimates 8.1 percentage points lower (The MRIBview estimates of volumetric breast density are highly correlated with mammographic dense area but are not equivalent measures; the MRI absolute dense volume shows potential as a predictor of breast cancer risk that merits further investigation. BRCA1 or BRCA2 genes .The details of the remaining women are given in table S1 in Additional data file program . The senAt the time of recruitment 576 of the 749 women (76.9%) completed a questionnaire containing questions relating to lifestyle risk factors, reproductive and hormonal history and various standard anthropometric measures Table .The primary analyses were based on three measures of proportional density and two measures of absolute density (MRI dense volume and Cumulus mammographic dense area), as described above. All analyses of mammographic density refer to the MLO projection unless otherwise specified.The relationships between the density measures for the left and right breasts were assessed using Pearson linear correlation coefficients and t-tests. The mean unsigned difference between breasts (i.e. not taking into account which of a woman's breasts is the denser) is also presented. The relationship between the MRI percent dense volume and Cumulus percent dense area was assessed using linear regression. Correlation coefficients and weighted kappa statistics were used to describe inter-reader (MRI) and intra-reader ) consistencies.vs never smokers; women who had ever been pregnant vs never pregnant; women who had ever used hormone replacement therapy vs those who had not; women who had previously taken an oral contraceptive vs those who had not; women who had ever taken tamoxifen as part of a prevention trial vs those who had not. Women who reported that their menstrual periods had stopped due either to a natural menopause, a hysterectomy or an oophorectomy were considered as being postmenopausal.Linear regression was used to examine associations between the density measures and a range of standard variables, with the two MRI measures log-transformed to closer approximate a normal distribution. Continuous variables were divided into quartiles; age at study registration, weight, body mass index , hip, waist and chest circumferences and waist:hip ratio . Parity was defined as the number of full-term pregnancies; women with four or more were considered as a single group. The following binary variables were also considered: current/previous smokers BRCA1/2 genetic groups were adjusted for age at registration ≥ 45 years and associations between genetic status and density were tested using linear regression. The analyses were repeated additionally adjusting for BMI and parity.The mean values of each density measure for the BRCA1 mutation carriers, BRCA2 mutation carriers and uninformative/non-carriers. The power to detect a doubling of risk between the upper and lower halves of the density distribution was 72% for the MRI percent dense volume and 59% for the Cumulus percent dense area, rising to 92% and 82% for a 2.5-fold increase in risk.Finally, Poisson regression was used to test whether each of the density measures was associated with cancer risk in this population by quartile of density, and also grouping the two lower and two upper quartiles in order to improve the stability of the estimates. Each individual's at-risk period was defined as starting at their study registration and continuing until the earliest of breast cancer diagnosis, death, prophylactic bilateral mastectomy, other loss to the ONS flagging system or 31 July 2007. The analyses were performed adjusting only for age group, adjusting for genetic status and/or BMI and parity, and also separately for All statistical tests are two-sided and were conducted using Stata version 10.0 .P = 1.0 for MRI, P = 0.32 for Cumulus, P = 0.31 for VA) , compared with women with a Cumulus-density < 30% [slope = 0.43 ] (P = 0.0036 for the difference between the two slopes). The 30% cut-off was based on visual inspection of the scatter-plot. On average the MRI percent dense volume was 14.9 percentage points lower (95% CI 13.5 to 16.4) than the Cumulus percent dense area (P < 0.0001) for the women with mammographically denser breasts, but there was no significant difference for the less dense breasts (P = 0.20). Despite the strong correlation there were a few women for whom the MRI and Cumulus estimates were highly divergent. The correlation between the MRI absolute dense volume and the Cumulus absolute dense area was clearly present but was weaker than for the corresponding percent densities (R = 0.61) [Additional data file The percent dense volume measured by MRI was positively correlated with the percent dense area estimated using the Cumulus program , in order to establish whether the relationships seen in unselected populations are also true in this high-risk group of women, and to see whether breast density as measured by our novel MRI-density estimation technique is related to the same factors as mammographic breast density Tables and 2.P < 0.001 in each case) and with WHR (P = 0.003), as expected. Although there was a significant negative trend in Cumulus percent density with increasing age, a significant decrease was seen only for women in the oldest age quartile . Post-menopausal women, women who had ever been pregnant and women who had ever used tamoxifen also had lower percent dense areas .The Cumulus percent dense area was negatively associated with BMI, weight, hip, waist and chest circumferences and with parity (trend P = 0.023).Lower MRI percent dense volume was significantly associated with all of the same variables, and additionally showed a possible negative association with previous oral contraceptive use (P = 0.021) but not with any of the other tested variables.The Cumulus measure of absolute dense area was significantly associated with the same variables as the Cumulus percent density, apart from age, tamoxifen use and ever-pregnancy, but the associations were in each case less significant than for percent dense area. The MRI measure of absolute dense volume showed a modestly significant association with chest circumference and chest circumference (trend by quartiles). However, chest circumference was only available for 491 women and so, to avoid excessive reduction in sample sizes, subsequent analyses were conducted under two models: 1) adjusted for age ≥ 45 years only; 2) adjusted for age ≥ 45 years, BMI quartiles and parity.BRCA1/BRCA2 genotype. TP53 carriers and others with a Li-Fraumeni syndrome family history were not included in this part of the analysis because of their small numbers. For each of the percent and absolute density measures, women with an uninformative genetic test (the baseline group) tended to have denser breasts than either the BRCA1 or BRCA2 carriers or non-carriers. The comparison with non-carriers was only significant for the MRI percent density, and then only when BRCA1 and BRCA2 non-carriers were combined .Figures BRCA1 mutation carriers had significantly lower MRI percent dense volume (P = 0.010), Cumulus percent dense area (P = 0.001), VA percent dense area (P < 0.001) and Cumulus absolute dense volume (P = 0.002) than the uninformative group; these effects remained significant after adjustment for BMI and parity , and this result was non-significant after adjustment for BMI and parity (P = 0.31). P = 0.00 than the Figures , but theBRCA1 and BRCA2 carrier probabilities using multivariate regression. BRCA1 carrier probability was negatively associated with Cumulus percent density but not with MRI percent dense volume . BRCA2 carrier probability was not associated with MRI or Cumulus percent densities .The data were reanalysed in terms of the Boadicea-estimated Thirty-eight of the 749 women developed breast cancer during the course of the MARIBS study; a further 22 developed breast cancer between their last MARIBS screening round and the cut-off date of 31/07/2007. Unfortunately we were unable to obtain any mammogram for 12 of the women with a breast cancer during the study, so the comparison of the MRI and Cumulus/Visual density measures are not based on equal numbers.BRCA1 and BRCA2 genotype/carrier probability (Upper two quartiles vs lower two quartiles: IRR (incidence rate ratio) = 2.04 (1.16 to 3.59) P = 0.013 for MRI; IRR = 2.24 (1.18 to 4.26) P = 0.014 for VA). These incidence rate ratios ceased to be significant when further adjusted for parity and BMI, although this may be due to the smaller numbers of women for whom these data were available P = 0.021; IRR = 1.35 (1.01 to 1.80) P = 0.042 for trend in quartiles) (Table BRCA1 and BRCA2 carriers (BRCA1 IRR = 3.62 (1.32 to 9.90) P = 0.012; BRCA2 IRR = 3.06 (1.14 to 8.19) P = 0.026) but was not apparent in the non-carriers/uninformative women (P = 0.66) . Specifically, among the set of 176 BRCA1 mutation carriers analysed by Mitchell et al, 36% developed breast cancer, compared with only 17% of our 94 BRCA1 carriers, hence it is possible that their group of carriers were biased towards women with denser breasts . This would have obscured any more general association between BRCA1 mutation status and low density. Conversely, the opposite may well be true, in that we specifically excluded women who had developed breast cancer prior to recruitment (mean age at entry = 40 years). According to the same logic, this could have biased our set of BRCA1 carriers towards those with a lower than average density. The same would not necessarily have been seen for BRCA2 carriers, in whom a greater proportion would be expected to be asymptomatic at the age of entry regardless of the presence or absence of potential modifiers such as density. The observation of lower density in BRCA1 mutation carriers would seem to be at odds with a report that only BRCA1/p53-deficient mice developed lateral branches and alveoli without pregnancy [BRCA1/2 carriers and non-carriers from a Chicago study [We found carrying a regnancy . To our go study .BRCA1/2 mutation carriers.The chief goal of breast density estimation is for the study and prediction of breast cancer risk. High breast density is well established as one of the strongest known risk factors for breast cancer, with clearer associations for quantitative measures of percent density (such as Cumulus) than for qualitative measures . HoweverBRCA1/2 mutation carriers, comparing women with percent density ≥ 50% with those with density < 50%, but found the comparison to be non-significant when densities between 25 to 50% were compared with < 25% [BRCA1 and BRCA2 carriers. Only 17% of the women in our study had a Cumulus percent density ≥ 50% , giving us a 30% power to detect a difference of the same magnitude as that reported by Mitchell et al. This difference in the distribution of densities between the studies may be due to differences in ascertainment criteria, as discussed above, or may simply reflect the use of different digitizers.Mitchell et al reported a breast cancer odds ratio of 2.29 (95% CI 1.23 to 4.26) for a combined group of Our results also appear at first glance to be different from those seen in the general population; a recent meta-analysis of 42 studies reported an almost five-fold increase in risk for the densest category (≥ 75%) versus the least dense (< 5%) . HoweverTP53 mutation carriers for whom x-ray mammography is not considered safe.The MARIBS study was originally designed to have suitable power to detect differences in sensitivity between MRI and XRM breast screening , rather We also examined the value of the craniocaudal view in our data set in the light of a recent report that the predictive value of visually-assessed mammographic percent density based on a mediolateral oblique view alone was substantially improved when combined with density estimates from the craniocaudal view . We founBRCA1 and BRCA2 carriers. It is possible that this is a false positive result. If it is true, it is curious that MRI dense volume showed almost no significant associations with any of the tested anthropometric or hormonal factors. However, given that breast cancer is believed to be initiated in the stromal or epithelial cells, it is plausible that the total quantity of target tissue is a more relevant predictor of breast cancer risk than the proportional density. In this context it is interesting that the absolute MRI breast volume was the only density measure not found to be lower in BRCA1 mutation carriers. While clearly related to percent dense volume, absolute dense volume is not capturing the same information; we found that 37% of women were in a different half of the percent MRI dense volume distribution than the absolute dense volume distribution, explaining why the results for the two measures were so different. No such breast cancer association was seen for the Cumulus absolute dense area, suggesting that the differences between mammographic and MRI density are more marked when not adjusted for total breast area/volume, and that the three-dimensional MRI dense volume provides a better estimate of the amount of tissue at risk of carcinogenesis. These findings are necessarily preliminary and will need to be confirmed in further studies.Interestingly, the one density outcome for which we did see a consistent association with breast cancer risk, even after adjusting for genetic status, BMI and parity, was the absolute MRI dense volume, a measure which has not been studied before. There appeared to be an approximate doubling of risk between the upper and lower halves of the distribution, rising to an over three-fold increase in The MRIBview algorithm for the MRI-based estimation of the volume of dense tissue in the breast provides a viable alternative to quantitative mammographic density estimation and may be of particular value in women at high genetic risk of breast cancer for whom MRI breast screening is already recommended. Although the nature of the available dataset limited our power to detect associations between density and breast cancer risk, MRI percent dense volume did not appear to perform markedly differently from Cumulus percent dense area. However, the association between absolute MRI dense volume and breast cancer risk is a novel, and potentially important finding that requires replication in a specifically designed case-control study.BMI: body mass index; CC: cranio-caudal; CI: confidence interval; IQR: inter quartile range; IRR: incidence rate ratio; MARIBS: magnetic resonance imaging breast screening; MLO: medial-lateral oblique; MLPA: multiple ligation dependant probe amplification; MRI: magnetic resonance imaging; ONS: office of national statistics; RR: relative risk; UCL: University College London; VA: visual assessment; WHR: waist-hip ratio; XRM: X-ray mammographyRE has received an educational grant from VISTA diagnostics. ML has received salary as a Director of a company developing dedicated MRI scanners for breast cancer. The company also receives grant funding from the UK Technology Strategy Board. This company does not fund this research or manuscript in any way. ML is employed by the Institute of Cancer Research (University of London) which holds patents on density measurement using MRI and holds the contracts of staff contributing to this research. His department has research agreements with Philips, Siemens and General Electric, major imaging companies that might benefit from this work; these research agreements are not related to the research in this manuscript. ML has also performed paid consultancy for Roche for work unrelated to this manuscript. The other authors declare that they have no competing interests.DT performed all statistical analyses, prepared the figures and drafted the manuscript. ML, RE, DE and DGE conceived of the study and participated in its design and co-ordination. GK-L coordinated the data management. FL obtained, entered and managed the pedigree data. MK created the MRIBview program. SR and EB performed the MRI density estimates. IW traced and data-managed the mammograms. CB performed the mammographic visual density assessments. SG and SJR performed the BRCA1/2 genetic analyses. RW conceived of the study, participated in its design and coordination, performed the Cumulus density estimation and helped to draft the manuscript. All authors read and approved the final manuscript.table S1 (genetic status of participants and probabilities of carrying BRCA1 or BRCA2 mutations for untested women and women with uninformative BRCA1/BRCA2 screening tests as predicted by the BOADICEA program), Table S2 and Table S3 .Click here for fileFigure S1 .Click here for fileFigure S2 (a scatter plot showing the relationship between MRI absolute dense volume and Cumulus absolute dense area).Click here for fileThe full MARIBS authorship list.Click here for file

The hydr­oxy groups are involved in intra­molecular O—H⋯N hydrogen bonds. The crystal packing exhibits weak inter­molecular O—H⋯O and C—H⋯O hydrogen bonds.The asymmetric unit of the title compound, C Å b = 17.729 (4) Å c = 11.013 (2) Å β = 97.62 (3)°V = 6722 (2) Å3 Z = 12 Kα radiationMo −1 μ = 0.08 mmT = 173 K 0.50 × 0.33 × 0.29 mm Rigaku Saturn724+ CCD diffractometerABSCOR 7677 reflections with R int = 0.050 R[F 2 > 2σ(F 2)] = 0.059 wR(F 2) = 0.145 S = 1.17 7960 reflections784 parameters1 restraintH-atom parameters constrainedmax = 0.41 e Å−3 Δρmin = −0.24 e Å−3 Δρ CrystalClear used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008XP in SHELXTL (Sheldrick, 2008SHELXL97.Data collection: 10.1107/S1600536809035296/cv2590sup1.cif Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536809035296/cv2590Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:

Insulin resistance is a risk factor for type 2 diabetes and cardiovascular disease progression. Current diagnostic tests, such as glycemic indicators, have limitations in the early detection of insulin resistant individuals. We searched for novel biomarkers identifying these at-risk subjects.Using mass spectrometry, non-targeted biochemical profiling was conducted in a cohort of 399 nondiabetic subjects representing a broad spectrum of insulin sensitivity and glucose tolerance .FFM = 33 −1·kgFFM−1, median [interquartile range], n = 140) from insulin sensitive subjects (MFFM = 66 −1·kgFFM−1) with a 76% accuracy. By targeted isotope dilution assay, plasma α–HB concentrations were reciprocally related to MFFM; and by partition analysis, an α–HB value of 5 µg/ml was found to best separate insulin resistant from insulin sensitive subjects. α–HB also separated subjects with normal glucose tolerance from those with impaired fasting glycemia or impaired glucose tolerance independently of, and in an additive fashion to, insulin resistance. These associations were also independent of sex, age and BMI. Other metabolites from this global analysis that significantly correlated to insulin sensitivity included certain organic acid, amino acid, lysophospholipid, acylcarnitine and fatty acid species. Several metabolites are intermediates related to α-HB metabolism and biosynthesis.Random forest statistical analysis selected α-hydroxybutyrate (α–HB) as the top-ranked biochemical for separating insulin resistant (lower third of the clamp-derived Mα–hydroxybutyrate is an early marker for both insulin resistance and impaired glucose regulation. The underlying biochemical mechanisms may involve increased lipid oxidation and oxidative stress. Insulin resistance (IR) has been established as a precursor of type 2 diabetes (T2D) Traditional clinical tests do not measure IR directly and, as a result, a variety of methods have been developed: the gold standard hyperinsulinemic euglycemic clamp (HI clamp); insulin tolerance test; steady state plasma glucose (SSPG) following fixed somatostatin/glucose/insulin infusions; and modeling analysis of the oral glucose tolerance test (OGTT) or frequently sampled intravenous glucose tolerance test (FSIVGTT) The RISC study (Relationship of Insulin Sensitivity to Cardiovascular Risk), comprising a nondiabetic cohort, was initiated to address how IR may contribute to T2D and CVD progression. We report here on a global biochemical profiling technology developed for the discovery of new biochemical biomarkers. This technology has been successfully applied to identify biochemicals associated with disease, toxicity and aging FFM in the 399 RISC subjects analyzed is shown in i.e., MFFM≤45 µmol·min−1·kgFFM−1) was defined as IR. By this criterion, MFFM was 33 −1·kgFFM−1, median [interquartile range], in the IR group (n = 140) and 66 −1·kgFFM−1 in the more insulin sensitive (IS) subjects. The demographic and metabolic characteristics of the 399 subjects under analysis are described in Fasting plasma samples from the RISC cohort were analyzed in a non-targeted fashion on three separate mass spectrometry platforms, UHPLC-MS/MS (+/- ESI) and GC-MS (+EI), with 485 biochemicals measured, as illustrated in To assess the ability to classify subjects as IS or IR, Random Forest (RF) analysis was performed. As shown in p-value 1.40E-21, FFM and MWBM . Summarized in FFM) with overlap observed with the initial RF analysis in IR subjects compared to IS subjects, whether measured by the screening platform or by the targeted isotopic dilution assay.Since the initial analyses were based upon relative quantification data obtained from the non-targeted biochemical profiling technology, a targeted assay was developed to provide absolute quantitative results. As shown in −2. The 210 NGT subjects who were more insulin sensitive (NGT-IS) were 44 −2. The 61 IFG subjects had an age of 49 −2, while the 82 IGT subjects had an age of 45 −2.Subjects were classified as normoglycemic or dysglycemic based upon the results of fasting plasma glucose (FPG) and the oral glucose tolerance test (OGTT) as illustrated in Shown in FFM was significantly lower in each of the IFG, IGT, and NGT-IR groups in comparison with the NGT-IS group (p<0.0001 for each), as illustrated in FFM. Using the targeted assay, the measured levels of α–HB were significantly (p<0.0001) higher in the NGT-IR, IFG and IGT groups as compared to the NGT-IS group. Relatedly, by partition analysis, an α–HB concentration of 5 µg/ml was found to best separate IR from IS subjects. Furthermore, based upon multiple logistic regression analysis, α–HB was significantly associated with IR independently of center (collection site), sex, age, and BMI, with an odds ratio of 2.84 for each SD ( = 1.7 µg/ml) of plasma α–HB.Consistent with previous reports Interestingly, RF analysis ranked α-HB as the most important metabolite to classify NGT and IGT subjects, with a >70% classification accuracy (data not shown). Consistent with these observations, α–HB levels were significantly higher in IGT than NGT subjects (p<0.0001), as shown in r2 = 0.25, p<0.0001) even after adjusting for center, sex, age, and BMI (data not shown).In addition to measuring α-HB by absolute quantitation, targeted assays were also developed for candidate IR biomarkers identified by RF and correlation analyses, with examples of representative biochemical classes highlighted in FFM value and their fold changes in concentration from the bottom tertile to the top two-thirds of insulin sensitivity . Consistent with the screening data, α–HB is highly correlated to the glucose disposal rate .Summarized in Using a non-targeted biochemical screening approach in a large and well characterized cohort of nondiabetic subjects representing a wide spectrum of insulin sensitivity, we identified α–hydroxybutyrate (α–HB) as a biomarker segregating with clamp-derived IR in subjects with normal glucose tolerance. Furthermore, α–HB segregated with dysglycemia (IFG+IGT) independently of, and in addition to, IR. Importantly, these associations were independent of sex, age, and BMI. Thus, together with other biomarkers, α–HB may provide a diagnostic tool to identify IR and/or IGT earlier than currently used clinical tests.via a reaction catalyzed by lactate dehydrogenase (LDH) or α–hydroxybutyrate dehydrogenase (α–HBDH) (in vivo when either (a) the formation of α–KB exceeds the rate of its catabolism, which leads to substrate accumulation, or (b) there is product inhibition of the dehydrogenase that catalyzes the conversion of α–KB to propionyl-CoA α–HB is an organic acid derived from α-ketobutyrate (α–KB) Figure 7α–KB is also produced as a result of the conversion of cystathionine to cysteine. Under conditions of increased oxidative stress, a higher flux of cysteine into production of glutathione, the primary antioxidant in cells, occurs from a shift in homocysteine production from transmethylation of methionine to transsulfuration of homocysteine to produce cystathionine + ratio due to increased lipid oxidation. The first mechanism likely contributes to increased α–HB formation by supplying more α–KB substrate from increased cysteine anabolism elevation of hepatic glutathione stress resulting in an increased demand for glutathione production, and (2) elevation of the NADH/NADnabolism Figure 5+ redox balance by glutathione infusion therapy resulted in improvement of insulin sensitivity and β-cell function in normal subjects and in T2D patients Changes in other important IR-associated metabolites within metabolic pathways leading to the formation of α-KB and α-HB are highlighted in et al. identified a metabolic signature for the accumulation of branched-chain amino acids, the glutamine/glutamate couple, several acylcarnitines, and some aromatic amino acids using principal component analysis In a recent study comparing the urinary profiles of 98 intermediary metabolites measured by targeted MS in 74 obese and 67 lean individuals, Newgard With an unmet need for a practical clinical test that accurately measures IR in individuals, identification of α–HB as a significant biomarker for separating IR from IS subjects using a fasting plasma sample could lead to development of such a diagnostic test. α–HB in combination with other biochemical and clinical parameters may also prove to be useful as a clinical indicator of subclinical abnormalities of glucose metabolism.RISC is a prospective, observational cohort study whose rationale and methodology have been published previously −2 (range 16.9–42.9) - were selected for non-targeted biochemical profiling analysis. Based on the OGTT, 256 subjects had normal glucose tolerance , 82 subjects had impaired glucose tolerance , and 61 subjects had impaired fasting glycemia .Of 1293 clamped RISC subjects, 194 males and 205 females – median age 45 years and median body mass index (BMI) 25.0 kg mEGIR-RISC study had undergone appropriate review by the European Commission research program and its ethics committee. Written consent was given by the patients for their information to be stored in the hospital database and used for research purposes, aligned with the analysis described herein. The current retrospective analysis described herein did not require additional review by said ethics committee due to prior approval of future biomedical analyses when EGIR-RISC study was initiated.FFM, in units of µmol per min per kg of fat-free mass. Plasma free fatty acids (FFA) were measured in the fasting state and at timed intervals during the clamp; the values during the last 40 min of the clamp were averaged to express insulin inhibition of circulating FFA.Electrical bioimpedance (to measure fat-free mass), routine clinical chemistry, OGTT, and HI clamp were performed as described e.g., amino acids, lipids, carbohydrates), was examined to measure biochemical changes within plasma samples collected after an overnight (10–12 hours) fast. The non-targeted process used single sample extraction followed by protein precipitation to recover a diverse range of molecules .Biochemical profiling was performed using multiple platform (UHPLC and GC) mass spectrometry technology, as described Metabolites were identified by automated comparison and spectra fitting to a chemical standard library of experimentally derived spectra as previously described Upon receipt of fasted, baseline plasma samples from HI clamps, aliquots were prepared and immediately frozen at −80°C until time of analysis. At time of analysis, samples were thawed on ice and 100 µl was extracted using an automated MicroLab STAR® system . The samples were extracted using a single extraction with 400 µl of methanol, containing the recovery standards: tridecanoic acid, fluorophenylglycine, chlorophenylalanine and d6-cholesterol. The solvent extraction step was performed by shaking for two minutes using a Geno/Grinder 2000 . After extraction, the sample was centrifuged and supernatant removed using the MicroLab STAR® robotics system. The extract supernatant was split into four equal aliquots: two for UHPLC/MS, one for GC/MS and one reserve aliquot. Aliquots were placed on a TurboVap® (Zymark) to remove solvent, and dried under vacuum overnight. Samples were maintained at 4°C throughout the extraction process. For UHPLC/MS analysis, extract aliquots were reconstituted in either 0.1% formic acid for positive ion UHPLC/MS, or 6.5 mM ammonium bicarbonate pH 8.0 for negative ion UHPLC/MS. For GC/MS analysis, aliquots were derivatized using equal parts N,O-bistrimethylsilyl-trifluoroacetamide and a solvent mixture of acetonitrile:dichloromethane:cyclohexane (5∶4∶1) with 5% triethylamine at 60°C for 1 hour. The derivatization mixture also contained a series of alkyl benzenes for use as retention time markers.2O (solvent A) and 0.1% formic acid in methanol (solvent B), while the mobile phase for negative ion analysis consisted of 6.5 mM ammonium bicarbonate, pH 8.0 (solvent A) and 6.5 mM ammonium bicarbonate in 95% methanol (solvent B). The acidic extracts were monitored for positive ions and the basic extracts were monitored for negative ions in independent injections using separate acid/base dedicated 2.1×100 mm Waters BEH C18 1.7 µm particle columns heated to 40°C. The extracts were loaded via a Waters Acquity autosampler and gradient eluted directly into the mass spectrometer at a flow rate of 350 µl/min. The LTQ alternated between full scan mass spectra (99–1000 m/z) and data dependent MS/MS scans, which used dynamic exclusion.UHPLC/MS was carried out using a Waters Acquity UHPLC coupled to an LTQ mass spectrometer equipped with an electrospray ionization source. Two separate UHPLC/MS injections were performed on each sample: one optimized for positive ions and one for negative ions. The positive ion analyses were performed first, followed by negative ion analyses. The mobile phase for positive ion analysis consisted of 0.1% formic acid in HThe derivatized samples for GC/MS were analyzed on a Thermo-Finnigan Trace DSQ fast-scanning single-quadrupole MS operated at unit mass resolving power. The GC column was 20 m×0.18 mm with 0.18 µm film phase consisting of 5% phenyldimethyl silicone. The temperature program started with an initial oven temperature of 60°C and was ramped to 340°C, with helium as the carrier gas. The MS was operated using electron impact ionization with a 50–750 amu scan range and was tuned and calibrated daily for mass resolution and mass accuracy.FFM). Within each day run, samples were completely randomized to avoid group block effects. The raw area counts for each metabolite in each sample were normalized to correct for variation resulting from instrument inter-day tuning differences. For each metabolite, the raw area counts were divided by its median value for each run-day, therefore setting the medians equal to 1 for each day's run. This correctly preserves all variation between samples, yet allows metabolites of widely different raw peak areas to be compared directly on a similar graphical scale. Missing values were assumed to result from areas falling below limits of detection. For each metabolite, missing values were imputed with its observed minimum after the normalization step.Samples were analyzed over the course of two weeks. Each run day was balanced for age, BMI, gender, OGTT, and insulin-mediated total glucose disposal, MThe data extraction of raw mass spectra data files yielded information that was loaded into a relational database and manipulated without resorting to BLOB manipulation. Once in the database the information was examined and appropriate QC limits were imposed. Peaks were identified using Metabolon's proprietary peak integration software, and component parts were stored in a separate and specifically designed complex data structure.i.e., extraction, recovery, resuspension, and instrument performance) for endogenous biochemicals within technical replicate plasma samples was calculated to be 15% MRSD. These SD values reflected acceptable levels of variability for overall process and instrumentation of the analytical platform.The median relative standard deviation (MRSD), a quality assurance metric of quantification and measure of instrument variability, was determined to be 8% for a panel of 30 internal standards. Overall process variability was verified for each biochemical.For QA/QC purposes a number of additional samples were included with each day's analysis. Briefly, a selection of internal standards was added to every sample, immediately prior to injection into the instrument. These compounds were carefully chosen in order to not interfere with measurement of endogenous compounds. These QC samples were primarily used to evaluate process control for each study. Additionally, a small aliquot of each experimental sample was pooled together to serve as a technical replicate for duration of the run. This technical replicate sample was injected throughout the platform run day and across all run days, allowing variability in quantitation of all consistently detected biochemicals in the experimental samples to be monitored. With this monitoring, a metric on overall process variability was assigned for the platform's performance based on quantitation of metabolites in actual experimental samples see section.t-testing. When data from NGT, IGT, or IFG categories were used in comparisons for classification by RF, the number of in-bag samples was set to 50% of smallest sub-group to account for unbalanced samples sizes. For platform screening data and targeted analytical data, we used 50,000 and 1,000 trees, respectively. Random forest analysis was performed using the R-package “randomForest” FFM value into two groups. Multiple logistic regression tested the independent association of metabolites with lower tertile of insulin resistance; results are given as the odds ratio and 95% confidence interval (C.I.). Statistical analyses were performed using JMP , and “R” (http://cran.r-project.org/).Data are given as median and [interquartile range]. Classification and Regression Trees (CART), Random Forest (RF) For absolute quantitation, metabolites were analyzed by isotope dilution UHPLC-MS-MS . 50 µl of EDTA plasma samples were spiked with internal standard solution and subsequently subjected to protein precipitation by mixing with 250 µl of methanol. Following centrifugation, aliquots of clear supernatant were injected onto an UHPLC-MS-MS system, consisting of a Thermo TSQ Quantum Ultra Mass Spectrometer and a Waters Acquity UHPLC system equipped with a column manager module and three different columns. Each sample was analyzed using three different chromatographic systems to cover the various analytes.18 column at a mobile phase flow rate of 0.4 ml/min at 40°C. Ionization was achieved by negative HESI mode. Creatine, octanoyl carnitine, decanoyl carnitine, glutamic acid, glycine, serine, threonine, palmitoyl-lyso-PC, oleoyl-lyso-PC and linoleoyl-lyso-PC were eluted with a 0.01% formic acid in water/acetonitrile-water-ammonium formate (700∶300∶2.7) gradient on a Thermo, BioBasic SCX column at a mobile phase flow rate of 0.5 ml/min at 40°C. Ionization was achieved by positive HESI mode. Palmitic acid, palmitoleic acid, margaric acid, stearic acid, oleic acid, and linoleic acid, were eluted isocratically with 15% 5 mM ammonium bicarbonate in water and 85% acetonitrile-methanol (1∶1) on a Waters, Acquity BEH C18 column at a mobile phase flow rate of 0.4 ml/min at 40°C. Ionization was achieved by negative HESI mode. Quantitation was performed based on the area ratios of analyte and internal standard peaks using a weighted linear least squares regression analysis generated from fortified calibration standards in an artificial matrix, prepared immediately prior to each run. The following corresponding stable labeled compounds were used as internal standards: α-HB-D3, β-HB-D3, 3-methyl-2-oxobutyric acid-D7, palmitic acid-13C16, margaric acid-D3, oleic acid-13C18, stearic acid-D3, linoleic acid-13C18, linolenic acid-13C18, , creatine-D3, octanoyl carnitine-D3, decanoyl carnitine-D3, glutamic acid-D5, glycine-13C2-15N, serine-D3, threonine-13C4-15N, tryptophan-D5, linoleoyl-lyso-PC-D9 .α-Hydroxybutyric acid (α-HB), β-hydroxybutyric acid and 3-methyl-2-oxo-butyric acid were eluted with a 0.01% formic acid in water/acetonitrile-methanol (1∶1) gradient on a Waters, Acquity BEH C3 internal standard solution (30.0 µg/mL) and subjected to protein precipitation by vigorously mixing with 0.250 mL of methanol. Following centrifugation, the supernatant was removed and 2.00 µL were injected onto a Waters Acquity/Thermo Quantum Ultra LC-MS-MS system. Calibration range included 0.500 to 20.0 µg/mL α-HB. Calibration standard samples were prepared in 2% BSA or water. Chromatographic conditions included the following: Waters, Acquity C 18 BEH column, 1.7 micron 2.1×100 mm; mobile phase A: 0.01% formic acid in water; mobile phase B: acetonitrile-methanol (1∶1); flow rate: 0.400 mL/min; gradient: initial 99% phase A, 1.0 min 60% phase A, linear, 1.4 min 60% phase A, 1.5 min 99% phase A; and linear α-HB retention time was 1.22 min. Mass spectrometer settings included selective reaction monitoring, negative ionization mode; HESI source; Spray voltage: −2500 V; vaporizer temperature: 300°C, Capillary temperature: 350°C; sheath/auxillary/sweep gas: N2; collision gas: Ar, 0.5 mTorr; monitored transitions: α-HB: m/z 103.1->57.1, α-HB-D3: m/z 106.1->59.1, collision energy: 13 V, each.For extraction, 0.0500 mL of human EDTA plasma was spiked with 0.0200 mL α-HB-DAppendix S1List of EGIR-RISC Investigators and Centers.(0.03 MB DOC)Click here for additional data file.

The concentrations of TNF α were significantly elevated in patients with malignant tumors of the adrenal cortex and in patients with Conn’s syndrome compared to control. In patients with non-functioning adenomas and pheochromocytomas, TNF α levels were similar to those detected in the control. In subjects with myelolipomas, the serum concentration of TNF α was lower compared to the control. After adrenalectomy, the levels of TNF α were decreased in patients with malignant tumors and in patients with Conn’s syndrome, nonfunctioniong adenomas and pheochromocytomas compared to the concentration before surgery. The serum concentrations of soluble receptors of TNF α did not differ among different patient groups and compared to the control. After adrenalectomy, the blood concentrations of TNF α R1 and TNF α R2 were decreased in patients with Conn’s syndrome. However, to confirm practicality of the evaluation of TNF α and its soluble receptors in differential diagnosis in patients with adrenal tumors, a larger study group is needed.The peripheral blood levels of TNF Most of them are incidental findings. Although the majority of adrenocortical and adrenomedullary tumors are benign, there are no clinical and laboratory markers to distinguish most of them from malignant tumors ,2. The mα is a pleiotropic cytokine that plays a central role in inflammation and apoptosis. It modifies the inflammatory reactions and immune reactions in response to injury and infection . Seven patients with malignant cortical tumors, seven patients with Conn’s syndrome, 10 patients with non-functioning adrenal adenomas, 12 patients with pheochromocytomas and three patients with myelolipomas (according to pathology data) were studied. The clinical diagnosis of the malignant tumors was suspected in patients with weight loss, high serum concentration of dehydroepiandrosterone sulfate and irregular, large mass found by computer tomography imaging. The diagnosis of the Conn’s syndrome was based on elevated level of plasma aldosterone, low level of plasma renin and an aldosterone/renin ratio of greater than 5.4. The diagnosis of pheochromocytoma was suspected in patients with poorly controlled hypertension associated with bouts of sweating, headache, palpitations, a high serum concentration of chromogranin A and a high level of metanephrins in urine. In patients with nonfunctioning adenomas and myelolipomas, the tumors were incidentally found during computer tomography imaging.The study group was composed of 39 subjects [28 females aged 25–82 aged 36–60 (49.5 ± 8.38). The presence of adrenal tumor was excluded on the basis of physical examination, laboratory tests and ultrasound examination of the abdomen. All patients and control group were included in the study only after giving informed consent. The study was approved by the Local Ethical Committee.α and its soluble receptors were measured by enzyme immunoassay (ELISA) . The intra-assay precision for TNF α was 4.67%, for TNF α R1 – 4.43%, and for TNF α R2 was 3.53%. The inter - assay precision for TNF α was 5.8%, for TNF α R1 – 6.1%, and for TNF α R2 was 4.07%. The statistical analysis was performed using the non - parametric Kruskal-Wallis test and Wilxocon test. A p-value of less than 0.05 was considered statistically significant.The concentration of TNF 3.α, TNF α R1 and TNF α R2 before surgery and after surgery are presented in x ± SD) concentration of TNF α in the control group was 12.09 ± 4.31 pg/mL, the mean concentration of TNF α R1 was 1,479.2 ± 388.23 pg/mL, and the mean concentration of TNF α R2 was 2,456.7 ± 521.12 pg/mL. The concentration of TNF α was significantly elevated in patients with malignant tumors of the adrenal cortex, compared to the control , and in patients with Conn’s syndrome, compared to the control . The highest levels of TNF α in patients with malignant tumors of the cortex were noted. In patients with non-functioning adenomas and pheochromocytomas, TNF α levels were not significantly different from those of the control, (p > 0.05) and were respectively 11.0 ± 2.03 pg/mL; 9.14 ± 4.58 pg/mL. In patients with myelolipomas, the serum concentration of TNF α was lower, compared to control (The concentrations of TNF < 0.05) .α were lower in patients with malignant tumors , and in patients with Conn’s syndrome , non-functioning adenomas and pheochromocytomas than before surgery and 6.α and its receptors were found.No significant correlations between the size of the tumors and the serum level of TNF 4.α is a transmembrane protein described originally as a protein which causes the haemorrhagic necrosis of experimental tumors [α possesses proinflammatory activity and plays a key role in apoptosis [α and its receptors may increase susceptibility to infections [α is also probably involved in development of lymphoid tissue [TNF l tumors . Furtherpoptosis –12. A lofections . TNF α id tissue .α in pathogenesis of malignant tumors is not clearly understood. Mocellin and Nitti [α may represent one of the molecular links between chronic inflammation and further development of malignant tumors. Leibovich et al. [α may promote angiogenesis but others [α. Schweigerer et al. [α on the proliferation of endothelial cells derived from blood vessels of the brain and adrenal cortex. In both types of these cells, TNF α decreased the proliferative activity of endothelial cells.The role of TNF nd Nitti suggest h et al. revealedt others did not r et al. assessedα acts also as a paracrinal and autocrinal regulator of the growth and development of the adrenocortical cells. Call et al. [α and IL-6. They also proved, using immunoassay techniques, that the glomerular zone of the adrenal cortex is the site of the greatest synthesis of TNF α. The increased level of TNF α in our study in patients with Conn’s syndrome may confirm that the glomerular zone of the adrenal cortex mainly secretes TNF α. The secretion of TNF α by human adrenal glands confirmed the studies of González-Hernández et al. [et al. [α has an influence on synthesis and secretion of steroid hormones in adrenal cortex [α occur in the blood of patients with malignant tumors. Cimino et al. [α in the blood of patients with acute leukemia. Elevated levels of TNF α were also confirmed in the peripheral blood of patients with melanoma [TNF l et al. revealedz et al. ,20 and P [et al. . It is al cortex . Moreoveo et al. observedmelanoma , renal cmelanoma and pancmelanoma .α in patients with malignant tumors . It may be due to activation of the immunological system and cytokine net in patients with malignant tumors. The elevated levels of TNF α in patients with malignant tumors may be also due to activation of angiogenesis and overexpression of TNF α in the transformed cells. Interferon γ is known as the most important factor to increase the expression of TNF α. The regulation of the expression is at the transcriptional level [α may lead to overexpression of integrins and adhesive molecules [et al. [α on the expression of ZC3H10 (zinc finger protein) and GRHL-3 (grainyhead-like 3) in breast cancer cell lines (MCF-7). Expression of these proteins was increased after addition of TNF α to the incubation medium of the cells in vitro. However, ZC3H10 acts as an antagonist of cell growth in vitro, but GRHL-3 stimulates the migration of the endothelial cells.The results of our study showed the highest level of TNF al level . It has olecules . Guardio [et al. assessedα was decreased after adrenalectomy. These results may be due to elimination of the tumor, which was the main local source of TNF α.In our patients with malignant tumors of the adrenal cortex, in patients with Conn’s syndrome and in patients with non-functioning adenomas and pheochromocytomas the concentration of TNF α in the pathogenesis of adrenal tumors is unknown. Elevated levels of these receptors were found in the amniotic fluid of pregnant women [α receptors may bind TNF α and inactivate this molecule. On the other hand, these forms of TNF α receptors can stabilize the TNF α molecule at low concentrations of TNF α [The role of the soluble receptors of TNF nt women ,31, in tnt women and patint women . Some auof TNF α .α receptors did not differ, either among study groups or compared to the control. After adrenalectomy, the levels of the soluble forms of TNF α decreased in patients with Conn’s syndrome. It could be argued that the soluble forms of TNF α receptors are also involved in the process of tumorigenesis. The low level of TNF α and high level of soluble forms of TNF α receptors in patients with myelolipoma may confirm the hypothesis that soluble forms of these receptors inactivate this molecule. However, such a small group (3 patients) is not sufficient to draw any significant conclusions.The results of our study revealed that the concentration of soluble forms of TNF α and its soluble receptors in differential diagnosis in patients with adrenal tumors, a large study group is needed. The evaluation of the concentration of numerous cytokines in the blood has been used only in the clinical studies so far. To take advantage of these molecules in clinical practice, further studies are needed and clinical examinations, laboratory tests and imaging techniques will still be the most important means of diagnosing patients with adrenal tumors.To confirm practicality of the evaluation of TNF 5.α and its soluble receptors appear to be involved in adrenal gland oncogenesis. The peripheral blood concentrations of this cytokines before surgery may be useful for the discrimination between malignant and benign adrenal tumors.TNF

The phenyl ring exhibits a dihedral angle of 81.16 (4)° with the plane of the benzofuran fragment. The crystal structure is stabilized by π–π inter­actions between the furan and benzene rings of neighbouring mol­ecules [centroid–centroid distance = 3.874 (2) Å] and by C—H⋯π inter­actions between a phenyl H atom of the phenyl­sulfonyl substituent and the furan ring of adjacent mol­ecules. In addition, the crystal structure exhibits intra- and inter­molecular C—H⋯O inter­actions.The title compound, C Å b = 8.4238 (7) Å c = 18.963 (2) Å β = 91.535 (2)°V = 1476.6 (2) Å3 Z = 4 Kα radiationMo −1 μ = 0.23 mmT = 173 (2) K 0.40 × 0.40 × 0.10 mm Bruker SMART CCD diffractometerAbsorption correction: none8704 measured reflections3212 independent reflectionsI > 2σ(I)2544 reflections with R int = 0.041 R[F 2 > 2σ(F 2)] = 0.037 wR(F 2) = 0.104 S = 1.03 3212 reflections193 parametersH-atom parameters constrainedmax = 0.30 e Å−3 Δρmin = −0.38 e Å−3 Δρ SMART used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008ORTEP-3 (Farrugia, 1997DIAMOND (Brandenburg, 1998SHELXL97.Data collection: 10.1107/S1600536808008477/zl2106sup1.cif Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808008477/zl2106Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:

The crystal packing is stabilized by inter­molecular O—H⋯N hydrogen bonds, which link the mol­ecules into inversion dimers.In the title mol­ecule, C Å b = 8.1790 (16) Å c = 8.4978 (17) Å α = 101.56 (3)°β = 100.39 (3)°γ = 105.47 (3)°V = 351.75 (16) Å3 Z = 2 Kα radiationMo −1 μ = 0.39 mmT = 293 K 0.22 × 0.17 × 0.13 mm Rigaku R-AXIS RAPID IP area-detector diffractometerABSCOR; Higashi, 1995T min = 0.919, T max = 0.951Absorption correction: multi-scan (2778 measured reflections1234 independent reflectionsI > 2σ(I)1027 reflections with R int = 0.014 R[F 2 > 2σ(F 2)] = 0.032 wR(F 2) = 0.119 S = 1.19 1234 reflections96 parametersH atoms treated by a mixture of independent and constrained refinementmax = 0.33 e Å−3 Δρmin = −0.25 e Å−3 Δρ RAPID-AUTO used to solve structure: SHELXTL (Sheldrick, 2008SHELXTL; molecular graphics: SHELXTL; software used to prepare material for publication: SHELXTL.Data collection: 10.1107/S1600536809023095/hg2526sup1.cif Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536809023095/hg2526Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:

Current tools for the diagnosis of tuberculosis pleural effusions are sub-optimal. Data about the value of new diagnostic technologies are limited, particularly, in high burden settings. Preliminary case control studies have identified IFN-γ-inducible-10kDa protein (IP-10) as a promising diagnostic marker; however, its diagnostic utility in a day-to-day clinical setting is unclear. Detection of LAM antigen has not previously been evaluated in pleural fluid.M. tuberculosis or histology suggestive of tuberculosis.We investigated the comparative diagnostic utility of established (adenosine deaminase [ADA]), more recent (standardized nucleic-acid-amplification-test [NAAT]) and newer technologies for the evaluation of pleural effusions in 78 consecutively recruited South African tuberculosis suspects. All consenting participants underwent pleural biopsy unless contra-indicated or refused. The reference standard comprised culture positivity for Of 74 evaluable subjects 48, 7 and 19 had definite, probable and non-TB, respectively. IP-10 levels were significantly higher in TB vs non-TB participants (p<0.0001). The respective outcomes for the different diagnostic modalities were: ADA at the 30 IU/L cut-point , NAAT , IP-10 at the 28,170 pg/ml ROC-derived cut-point , and IP-10 at the 4035 pg/ml cut-point . Thus IP-10, using the ROC-derived cut-point, missed ∼20% of TB cases and mis-diagnosed ∼20% of non-TB cases. By contrast, when a lower cut-point was used a negative test excluded TB. The NAAT had a poor sensitivity but high specificity. LAM antigen-detection was not diagnostically useful.Although IP-10, like ADA, has sub-optimal specificity, it may be a clinically useful rule-out test for tuberculous pleural effusions. Larger multi-centric studies are now required to confirm our findings. Annually, over half a million pleural effusions are diagnosed world-wide and it is one of the commonest forms of extra-pulmonary tuberculosis TB; . In AfriELISA) has not previously been evaluated in other body compartments including pleural fluid.An alternative promising, but poorly studied, biomarker is IFN-γ inducible protein of 10 kDa (IP-10). IP-10, a Th1-associated chemokine, was found to be a useful discriminatory tool in three case-controlled studies M. tuberculosis and/ or histology in keeping with tuberculosis.In this study we prospectively evaluated the comparative diagnostic utility of established (adenosine deaminase [ADA]), more recent (standardized nucleic-acid-amplification-test [NAAT]) and newer technologies for the evaluation of pleural effusions in 78 South African tuberculosis suspects. The gold standard for tuberculosis was culture positivity for Seventy-eight consecutive patients with suspected TB pleural effusion were prospectively recruited at the Groote Schuur, Somerset and Victoria hospitals in Cape Town, South Africa, after informed consent see . Study aM. tuberculosis using the MGIT 960 system) evaluation.All patients had a history taken, detailed physical examination performed, routine haemtological investigations, including testing for HIV infection, chest x-ray, sputum examination when possible , and aspiration of approximately 20 ml of pleural fluid (or closest obtainable volume) for biochemical (protein and glucose), cytological , and microbiological . Patients were thus characterized as (i) definite TB: either positive M.tb culture and/ or histology in keeping with tuberculosis, and a clinico-radiological picture consistent with TB with clinical response to anti-TB treatment; (ii) non-TB: alternative diagnosis made on histology or pleural fluid aspiration, and not treated for TB, and on 3 to 6 month follow-up there were no features to suggest TB, and (iii) probable TB: clinical picture of TB but not satisfying the definite TB criteria and treated for TB by the attending physician.For accurate characterization of disease multiple closed pleural biopsies (approximately four) were undertaken using an Abraham's needle under local anesthesia, by a trained internal medicine resident . In 16 patients biopsies were not performed because of patient refusal, a contra-indication or a positive culture of fluid, or histology from another site, prior to attempted pleural biopsy. The reference standard for tuberculosis was culture positivity for ELISA, ME, USA; see http://www.clearview.com/tb_elisa.aspx). An interim analysis was done in the 1st 24 recruited patients (14 definite or probable TB cases and 10 non-TB cases) to use a go/ no-go decision point for further LAM testing. For nucleic-acid-amplification the Amplified Tuberculosis Direct Test was performed in duplicate according to the manufacturer's instructions (H37Rv served as a positive control and M. intracellulare served as the negative control) and readouts were obtained using a Leader 50 Luminometer .Pleural fluid protein and ADA levels were derived using the Biuret and colorimetric methods, respectively. LAM antigen concentration in the pleural fluid was measured in duplicate, after a heating step to dissociate antigen and antibody, according to the manufacturer's instructions . All assays were performed by an experienced laboratory technician who was blinded to patient and clinical details.To ascertain the relative value of newer tests in a high burden setting, regression models were fitted to identify variables independently associated with risk of tuberculosis, taking into account findings from the history, physical examination and pleural fluid biochemical data. The final bio-clinical scoring rule, incorporating age and protein levels, was developed by assigning a relative score or points to each of the variables included in the final multivariate model. Here we use the model to evaluate the relative incremental value of the different pleural diagnostic tests.2 test or Fisher exact test and continuous variables were compared using t-student test, whenever appropriate. Non-parametric tests (Mann-Whitney) were used for non-normally distributed variables. Concordance between tests was measured using the kappa co-efficient. Diagnostic accuracy, including 95% confidence intervals, was assessed using sensitivity, specificity, predictive values and area under the ROC in the TB and non-TB sub-groups. The study report was prepared using the Standards for Reporting of Diagnostic Accuracy (STARD initiative) format (19).Categorical variables were compared using the χA summary of the study plan is shown in th; 75th percentiles) pleural fluid cell count was 1.75×106 cells/ml and the median volume of fluid obtained was 20 ml .Of those tested 20/51 (49%) were HIV positive. In the TB vs non-TB group the mean age (years) was significantly lower though there were more people of Black African and mixed race . There was no significant inter-group difference for sex, HIV status, BCG vaccination or employment status. The mean (SD) pleural fluid protein levels were significantly higher in the TB vs non-TB group . In the final multivariate logistic regression model, age (<42 years), and protein levels (>53 g/L) [OR = 3.59, 95% CI 1.02–12.56, p = 0.04) were independently associated with the risk of tuberculosis. These variables, when incorporated into a bio-clinical score, had a maximal sensitivity and specificity of 54 and 89%. The median were positive in 1, 27 and 41 of the 48 definite TB cases, respectively, and by definition, in none of the non-TB cases. None of the probable TB cases were culture or biopsy positive but all were treated empirically for TB based on clinical suspicion. Twenty one percent (16/ 74) of patients did not have a pleural biopsy . The diagnostic outcomes of protein levels at different cut-points is shown in The IP-10 scatter-plot (n = 73) and area under the ROC is shown in By contrast the ADA (30 iu/l cut-point) had a sensitivity of 96 , specificity 69 , NPV 85 , +LR 17.4 , and −ve LR 0.09 , respectively. At the cut-point used for clinical decision making in Cape Town (30 iu/l) ADA had a sub-optimal specificity of 64% compared to a higher AUC-derived cut-point 47 iu/l; , table 2ELISA, ME, USA) in the first 22 TB suspects recruited . The sensitivity of LAM antigen was 8% (1 of 12 patients) and specificity was 100%.We measured LAM antigen levels had a better sensitivity, PPV and positive LR, though the specificity and NPV was sub-optimal implying that ∼3 in 10 non-TB subjects would be erroneously treated for TB and ∼1 in 7 patients with a negative test would in fact have TB. At a higher cut-point (47 iu/l) the former misdiagnosis would be reduced, but not eliminated, and at the expense of missing ∼1 in 10 TB cases. At the lower cut-point ADA would be an excellent rule-out test but specificity would be poor. Nevertheless, ADA is cheaper and more widely available than IP-10. Thus IP-10, which can be measured with several commercially available ELISA assays, cannot replace ADA. Rather, it could useful in a specific clinical context where ruling out TB would be useful.In the parent study using the same cohort of patients we show that unstimulated IFN-γ levels very accurately distinguishes TB from non-TB effusions in African patients (data not shown). This preliminary analysis showed a modest correlation between IP-10 and IFN-γ levels (Spearman r = 0.3836). Why does IP-10, an IFN-γ inducible chemokine, not correlate highly with IFN-γ levels, as it does in peripheral blood http://www.clearview.com/tb_elisa.aspx), which we evaluated in pleural fluid. For cost-containment purposes further testing of LAM was discontinued, after an interim analysis of the first 24 patient results, because of its poor diagnostic utility (only one out of 14 definite or probable TB cases tested positive for LAM antigen). Why LAM antigen virtually undetectable in pleural fluid? Preliminary experiments excluded technical reasons and batch variability. To exclude the lack of antigen-protein dissociation a heating step was incorporated into the test protocol to ensure dissociation. The lack of LAM antigen detection in the majority of TB pleural effusions probably reflects the pauci-bacillary nature of pleural disease, though high affinity antigen-antibody binding cannot entirely be excluded.TB antigen detection has previously been investigated for its diagnostic utility in pleural effusions. However, tuberculostearic acid was found to have limited diagnostic utility The variability of NAAT performance outcomes are highly dependant on laboratory protocols and we therefore evaluated a standardized NAAT, hitherto not undertaken in an African setting, in pleural TB suspects. The sensitivity of commercial NAATs for pleural TB are highly variable (20 to 100%; summarized in detail in We took several steps to minimize bias and ensure study validity, including consecutive recruitment with universally applied and pre-specified inclusion criteria, an experienced technician blinded to clinical details, invasive procedures to ensure accurate classification of patient and control sub-groups, and use of a pre-specified reference standard. We also provide, through comparison with clinical assessment and incremental test value over existing tools, information on clinical utility rather than test performance outcomes only (sensitivity, specificity etc; In conclusion, like ADA, IP-10 levels at the AUC-derived cut-point are not specific for tuberculosis, though at the lower cut-point they appear to be promising rule-out tests for TB in a high burden setting. Larger studies are required in other settings to confirm these findings.

Ovarian cancer is the most lethal gynecological malignancy, and the ovarian clear cell carcinoma subtype (OCCA) demonstrates a particularly poor response to standard treatment. Improvements in ovarian cancer outcomes, especially for OCCA, could be expected from a clearer understanding of the molecular pathology that might guide strategies for earlier diagnosis and more effective treatment.d's to ovarian cancer cells in the pico- to nanomolar range were obtained. Preliminary investigation of the targets of these aptamers and their binding characteristics was also performed.Cell-SELEX technology was employed to develop new molecular probes for ovarian cancer cell surface markers. A total of thirteen aptamers with KWe have selected a series of aptamers that bind to different types of ovarian cancer, but not cervical cancer. Though binding to other cancer cell lines was observed, these aptamers could lead to identification of biomarkers that are related to cancer. Ovarian cancer is the fifth most common cancer in women The most commonly used serum biomarker for clinical diagnosis and prognosis is ovarian cancer antigen 125 (CA-125). The CA-125 value is elevated in approximately 90% of late-stage cases of epithelial ovarian cancer (stages 3 and 4). However, it is only elevated in 50-60% of women with early stage disease and is also elevated in a number of benign conditions The utilization of aptamers has great potential for the identification of new biomarkers. Aptamers, which are probes capable of specifically binding to cell surface markers expressed by targeted tumor cells The target specificity and affinity of aptamers are similar to those of antibodies, but with several advantages over antibodies for clinical use. Aptamers may be chemically synthesized in a short time at relatively low cost, allowing better batch-to-batch reproducibility and easier incorporation of chemical modifications. Since aptamers for cells are selected without prior knowledge of the target molecules, selected aptamers can be used to identify new surface markers on cancer cells In this work, a total of 13 aptamers was selected for two model ovarian cancer cell lines: the OCCA line TOV-21G Two model ovarian cancer cell lines were chosen for the selection of ovarian cancer aptamers: the OCCA cell line TOV-21G and the ovarian serous adenocarcinoma cell line CAOV-3. In order to identify aptamers that specifically bind to ovarian cancer cells, the cervical cancer cell line HeLa was used for counter-selection. The SELEX procedure for TOV-21G is described briefly below. A detailed description is provided in the experimental section.To start the selection process, 20 pmol of naive library was enriched by sequential binding to TOV-21G cell monolayers. Sequences showing non-specific binding to general cell surface markers were removed by incubating the enriched pool with HeLa cells . The eluted pool for each round of SELEX was amplified through PCR, after which the ssDNA pools of interest were recovered and monitored for enrichment toward TOV-21G by flow cytometry. As the selected pools were enriched with sequences that recognize and bind to the target cell line, an increase in fluorescence signal was observed . But attFollowing completion of the selection process, three pools were chosen and submitted for sequencing: the final pool (round 22), the previous pool (round 21) and a pool showing minimal enrichment (round 13). Pool sequencing was used to help identify aptamer candidates by generating large quantities of sequences. This number of sequences (here a minimum of 2000 per pool) is large enough to allow identification of aptamers that only have a small representation in the pool (i.e. less then 1%). As can be seen in Sequences were aligned into families according to sequence homology. The number of homologues was compared across the different sequenced pools to validate their enrichment through the selection procedure using basic bio- informatics. Ten sequences showing the best homology throughout the pools were selected as aptamer candidates, synthesized and tested for binding to the model ovarian cancer cell lines. All the candidates showed binding to TOV-21G, with binding affinities in the pico- to nano-molar range . This ded  = 0.25±0.08 nM) and aptTOV2 (0.90±0.25 nM) bind very tightly to TOV-21G cells. As shown in As shown in The binding of the selected aptamers was tested with different adenocarcinoma cell lines, as well as other types of cancer cell lines, as shown in Since both selections took place at 4°C, the selected aptamers were tested at physiological conditions. The aptamers were incubated with the target cell line at 37°C and 4°C and their binding was measured. All aptamers showed similar binding at 4°C and 37°C and serous adenocarcinoma (CAOV-3). By counter selection against HeLa cells, aptamers that can distinguish ovarian cancer from cervical cancer were selected. In particular, AptTOV 1 showed very high affinity towards TOV-21G, with a Kper se. However, since the aptTOV apamers do not bind to a cancer of similar etiology (CAOV3) and also not to HeLa, they still have the potential to provide more insight into the pathology of ovarian cancer. It has been observed that there are significant differences in the proteome of serous and clear cell ovarian cancer Given the limited number of biomarkers for ovarian cancer currently available, the aptamers obtained from these selections have potential for improving diagnosis and treatment of this deadly disease. Because the aptamers also bind benign cysts , the aptThe discovery of two aptamers that were insensitive to protease digestion is intriguing. Additionally, their binding to all tested adenocarcinoma cells, but not to any of the leukemia cell lines, suggests the potential to further elucidate the underlying molecular differences between these cancer types. Further investigation is warranted to identify the targets of these aptamers and assess their performance in clinical samples.2, dNTPs (each at 2.5 mM), 0.5 µM of each primer, and Hot start Taq DNA polymerase (5units/µL). PCR was performed on a Biorad Thermocycler and all reagents were purchased from Takara. Monitoring of pool enrichment, characterization of the selected aptamers, and identification of the target protein assays were performed by flow cytometric analysis using a FACScan cytometer (BD Immunocytometry Systems). Trypsin and Proteinase K were purchased from Fisher Biotech. The imaging of cells was performed with an Olympus FV500-IX81 confocal microscope . The DNA sequences were determined by the Genome Sequencing Services Laboratory at the University of Florida with the use of 454 sequencing (Roche).All oligonucleotides were synthesized by standard phosphoramidite chemistry using a 3400 DNA synthesizer (Applied Biosystems) and were purified by reversed-phase HPLC (Varian Prostar). All PCR mixtures contained 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.0 mM MgCl2 atmosphere.The CAOV-3, HeLa, Hs832(C)T and TOV-21G cell lines where obtained from the American Type Cell Culture (ATCC). The CAOV-3 and TOV-21G ovarian cancer cell lines where maintained in culture with MCBD 105: Medium 199 (1∶1); the HeLa cell line was cultured in RPMI-1640; and the Hs832(C)T cell line was cultured in Dulbecco's Modified Eagle's Medium (DMEM). All media where supplemented with 10% FBS and 100 UI/mL Penicillin-Streptomycin. Other cell lines used for selectivity assays included CEM (T cell leukemia), Ramos (Burkitt's Lymphoma), HCT-116, DLD-1, HT-29 , NCI_H23 and A172 (glioblastoma), all of which were cultured according to ATCC specifications. All cell lines where incubated at 37°C in a 5% CO2 in Dulbecco's phosphate buffered saline with calcium chloride and magnesium chloride (Sigma). Binding buffer (BB) used for selection was prepared by adding yeast tRNA (0.1 mg/mL) (Sigma) and BSA (1 mg/mL) (Fisher) to the wash buffer to reduce background binding.During the selection, cells were washed before and after incubation with wash buffer (WB), containing 4.5 g/L glucose and 5 mM MgClThe HPLC-purified library contained a segment of randomized sequence of 40 nucleotides (nt) flanked by 20-nt primer hybridization sites:5′- ATC CAG AGT GAC GCA GCA (N)40TGG ACA CGG TGG CTT AGT-3′) and (5′-ACT ACC AAC GAG CGA CCA CT (N)40AGA GTT CAG GAG AGG CAG GT-3′). The forward primers were labeled with 5′-FITC and the reverse primers were labeled with 5′-biotin. with WB and incubated with the DNA pool on ice in an orbital shaker for 30 min. In later selection rounds, the cells were washed with increased stringency to remove weakly binding sequences . The bound sequences were eluted in 500 µL BB by heating at 95°C for 15 min, cooled on ice for 5 min and centrifuged at 14,000 rpm for 2 min.The supernatant containing the DNA sequences was then incubated with a negative cell line to perform a subtraction of general sequences, as described above. The remaining sequences were amplified by PCR using the FITC- and biotin-labeled primers. Amplifications were carried out at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, followed by final extension for 3 min at 72°C. The selected sense ssDNA was separated from the biotinylated antisense ssDNA by streptavidin-coated sepharose beads (Amersham Bioscience). The ssDNA was eluted from the sepharose beads by melting in a 0.2M NaOH solution.The enrichment of specific sequences was assayed using flow cytometry as explained below. When the level of enrichment reached a plateau, pools of interest were submitted for sequencing. The aptamer selection for the CAOV3 cell line was performed using the same protocol, however a 1 minute trypsinization step was used to suspend the cells before adding the pools. Supplemental 5) were incubated with various concentrations of 5′-biotin labeled aptamers on ice for 20 min in 100 µL of BB. Cells were then washed twice with 500 µL of BB, and suspended in 100 µL of BB containing streptavidin-PE-Cy5.5. Cells were then washed twice with 500 µL of WB, and were suspended in 200 µL of BB for flow cytometric analysis, using a 5′-biotin labeled random sequence as the negative control. All the experiments for binding assays were repeated at least 2 times. The specific binding intensity was calculated by subtraction of the mean fluorescence intensity of the background binding from the mean fluorescence intensity of the aptamers. The equilibrium dissociation constant (Kd) of the fluorescent ligand was obtained by fitting a plot of the specific binding intensity versus (Y) the aptamer concentration (X) to the equation Y = BmaxX/(Kd+X) using SigmaPlot. . Supplemental To determine the binding affinities of the aptamers, the target cells were detached using non-enzymatic cell dissociation solution. After resuspension, the cells were washed with 3 mL of PBS and then incubated with 1 mL of 0.05% trypsin/0.53 mM EDTA in HBSS or 0.1 mg/mL proteinase K in PBS at 37°C for 1, 5, 15, 30 and 60 minutes. Pure FBS was added to quench the proteinases. After washing with 2 mL of BB, the treated cells were used for binding assays as described above.Target cells Click here for additional data file.Data S2Allignment of the final CAOV3 pool.(0.20 MB TXT)Click here for additional data file.Data S3This file contains the flow data from the figures presented in this article.(3.69 MB ZIP)Click here for additional data file.Data S4Legend to the amount of pluses. This figure was our guideline to determine the amount of pluses for (0.04 MB PDF)Click here for additional data file.

Pattern recognition methods have become increasingly popular in fMRI data analysis, which are powerful in discriminating between multi-voxel patterns of brain activities associated with different mental states. However, when they are used in functional brain mapping, the location of discriminative voxels varies significantly, raising difficulties in interpreting the locus of the effect. Here we proposed a hierarchical framework of multivariate approach that maps informative clusters rather than voxels to achieve reliable functional brain mapping without compromising the discriminative power. In particular, we first searched for local homogeneous clusters that consisted of voxels with similar response profiles. Then, a multi-voxel classifier was built for each cluster to extract discriminative information from the multi-voxel patterns. Finally, through multivariate ranking, outputs from the classifiers were served as a multi-cluster pattern to identify informative clusters by examining interactions among clusters. Results from both simulated and real fMRI data demonstrated that this hierarchical approach showed better performance in the robustness of functional brain mapping than traditional voxel-based multivariate methods. In addition, the mapped clusters were highly overlapped for two perceptually equivalent object categories, further confirming the validity of our approach. In short, the hierarchical framework of multivariate approach is suitable for both pattern classification and brain mapping in fMRI studies. Multi-voxel pattern analysis (MVPA) methods have been widely used in fMRI studies to characterize the relationship between fMRI responses and cognitive functions. In a typical MVPA framework, a multivariate classifier is trained on multi-voxel patterns of brain activities with known labels, and the trained classifier is then used to classify untrained data or clusters (MIC) and constructing a linear SVM classifier for classifying experimental conditions from the selected clusters. The test data set was then used to evaluate the predictive accuracy of the classifier constructed from the training data set.v and w , similar to the regions of interest approach in a univariate analysis. The assumption was that the fMRI responses of voxels within a homogeneous cluster are sampled from an independent and identical distribution. Thus, a multi-voxel pattern In contrast to the univariate summation, multivariate methods can capture fine-scale information embedded in the multi-voxel pattern by relaxing the independent and identical distribution assumption. Here we used Gaussian Naïve Bayesian (GNB) d is the number of clusters) and d-dimensional discriminant w to minimizeTo identify informative clusters, discriminative weights were derived from linear Support Vector Machine (SVM) jth cluster to the classification can be measured and ranked by the absolute value of its corresponding discriminative weight To classify a new pattern Simulated fMRI dataThe simulated fMRI data sets were created by following the procedure described in Kriegeskorte et al. ST), performance metrics of the MIC were evaluated at different values of ST .Partition of homogeneous regions and multivariate analyses were done with in-house code. To evaluate the influence of minimal cluster size to account for the hemodynamic delay and then by averaging the signal intensity within blocks for each voxel. Thus there were 8 (runs) ×3 (blocks) samples for each condition. The extracted multi-voxel patterns with their corresponding experimental condition labels were fed into a four-fold cross-validation of both the MIC and MIV mappings and classification. This process was repeated 10 times to achieve stable results. In each fold, the mapping of informative regions and the training of classifier were carried out with 3/4 of the data, whereas the predictive accuracy of the trained classifier was tested with the rest 1/4 of the data.For each of 4×4 combinations of the parameters, an ROC curve was produced by plotting sensitivity against 1-specificity in detecting the preset informative voxels at different thresholds of discriminative weights. Area under the ROC curve was used to evaluate the overall performance of these two mapping methods so as to examine to what extent both sensitivity and specificity were achieved S. In evaluating the robustness of functional mapping, we focused on within-subject variance because the selection of informative voxels or clusters in different folds varied significantly. Thus the robustness of mapping was defined as an averaged overlap rate among voxels selected in the four cross-validation folds. The overlap rate between voxels was quantified with a set-wise similarity metric Real fMRI dataThe real fMRI data were collected from an object recognition experiment consisting of stimuli from six object categories: male faces, female faces, houses, scenes, cars, and chairs. Ten subjects participated in the study. All subjects were right-handed, and had normal or correct-to-normal vision. Written informed consent was obtained from each subject before participation. The study was approved by the IRB of Beijing Normal University.Each subject participated in a single session consisting of twelve blocked-designed runs. Each run contained a total of six 32-second stimulus blocks, one stimulus category each, interleaved with seven 16-second fixation blocks, which gave 12 blocks for each category. Each stimulus block contained 40 trials, with a 300 ms stimulus display and a 500 ms blank display per trial. The stimuli were presented near fixation, and their positions were jittered across trials to prevent repetitive suppression of BOLD signals. Each of 20 stimulus exemplars from a category was presented twice in a block. The sequence of exemplars was pseudo-randomized, and four immediate repetitions were added to each block. The subjects were instructed to pay attention to each stimulus and to press a button when a repetition was detected .Data were acquired on a Siemens 3T Trio scanner with a 12-channel phased-array head coil at BNU Imaging Center for Brain Research, Beijing. T2*-weighted gradient-echo, echo-planar images (EPI) of the whole brain were collected . Structural images were acquired with MPRAGE, an inversion prepared gradient echo sequence . The structural images were used in registering the functional data to cortical surfaces and generating a mask of gray matter with Freesurfer http://surfer.nmr.mgh.harvard.edu). Preprocessing of the fMRI data involved motion correction and grand-mean intensity normalization.Preprocessing and univariate analyses of the fMRI data were conducted with the Freesurfer functional analysis stream .For multivariate analyses, only the voxels within the anatomical mask of gray matter generated by Freesufer with the structural images were included. Linear detrending within runs was conducted for each voxel on the preprocessed fMRI data with no spatial smoothing. The remaining analyses were the same as those performed in the simulated data.ps<0.05), and the area under ROC in the MIV was significantly higher than that in the uMIC (ps<0.01) at all levels of the CNR and We first evaluated the performance of the mapping methods by calculating area under ROC at various CNR. Because the size of the preset informative regions did not affect the results across different methods , the resps<0.05) . The preps<0.05) , with thps<0.05) . More imCNR and , whereasCNR and .Thus, the mMIC outperformed the MIV in robustness of localizing the informative voxels with slightly better or at least identical predictive accuracy, whereas uMIC showed the worst performance in both predictive accuracy and the robustness of mapping. In the next step, we used real fMRI data to further test the feasibility and validity of the mMIC. Because the performance of the uMIC was lower in both classification and mapping, we did not include it in the further analysis. In addition, because the mMIC was relatively tolerant of the change of the preset cluster size , only one Ts was chosen in analyzing the real fMRI data. The ted data .Information on the partition of homogeneous clusters in the real fMRI data was summarized in Because there were six object categories, the classification between any two categories gave rise to fifteen contrasts in total. Predictive accuracy and the robustness of mapping were calculated for each contrast respectively. The pattern of predictive accuracy was similar to that in simulated data: the mMIC achieved higher or comparable predictive accuracy compared to the MIV . The reaps<0.05). Similar results were found for each of the 15 contrasts at each of the 10 feature levels. More importantly, the mMIC outperformed MIV in all the contrasts in mapping and female faces (versus houses) for the mMIC and the MIV was calculated respectively. The overlap rate was significantly larger in the mMIC than that in the MIV at almost all feature levels ( voxels) . The ove voxels) , which cMVPA has been successful in discriminating mental states by investigating subtle information embedded in multi-voxel pattern of fMRI activities. Here we proposed a new method to improve the validity and reliability of MVPA in localizing the discriminative patterns by taking the local homogenous clusters as the basic unit in the mapping . The results from both the simulated data and the fMRI data showed that the MIC via multivariate within-cluster summation (mMIC) exceeded the voxel-based MVPA methods (MIV) in functional brain mapping.The advantage of the mMIC over the MIV is non-trivial. First, mapping informative regions at the level of local homogeneous clusters not only agreed with the spatiotemporal nature of fMRI data, but also echoed the findings from neurophysiological and neuroimaging studies. That is, neurons with similar response profiles are usually clustered together, forming relatively homogeneous cortical regions Our method is in line with previous studies on mapping information regions with MVPA. To address the spatiotemporal correlation of fMRI data, Carroll et al. Although our method is better than voxel-based MVPA methods, several issues remain. First, although the mMIC was better in discriminating some experimental conditions (e.g. houses versus scenes) than the MIV, the predictive accuracy of the mMIC in general was not significantly better, possibly because the accuracy in other experimental contrasts was at ceiling. Future experiments are needed to systematically compare these two methods in predictive accuracy when it is not at ceiling . Second, the preset size of homogeneous clusters with two smallest region sizes (15 and 30)), and the performance of the MIV was better than that of the uMIC . For the predictive accuracy, the mMIC was comparable to or slightly better than the MIV, and the MIV was significantly better than the uMIC . For robustness of mapping, there was no significant difference between the MIV and uMIC, and the mMIC was significantly better than either the MIV or uMIC .(TIF)Click here for additional data file.Figure S2Within-cluster temporal correlations in real fMRI data. To examine the within-cluster homogeneity of clusters partitioned by the region growing algorithm, we calculated the mean vale of inter-voxel temporal correlation coefficients between fMRI time courses of all pairs of voxels within the same cluster. The histogram shows the distribution of the within-cluster correlations from all clusters and from all ten subjects.(TIF)Click here for additional data file.Figure S3Slice view of homogeneous clusters. Homogeneous clusters of a representative subject are shown in a slice view. The homogeneous clusters were partitioned by the iterative region growing method from all gray matter voxels of this subject. Colors were used to mark different homogeneous clusters.(TIF)Click here for additional data file.Figure S4Performance of the mMIC on homogeneous clusters versus non-homogeneous clusters. To examine whether the homogeneous clusters help improve the performance of the mMIC, we partitioned the brain into non-overlapped cubic-shaped clusters, irrespectively to the embedded correlations among the BOLD signals of voxels. The rest procedure, including the sum-up of the within-cluster patterns and the classification of multivariate GNB discriminants, was the same as the mMIC. The modified method is referred as the mMICc for simplicity. Three levels of cluster size in the mMICc were chosen to match the mean size of the homogeneous clusters with Ts of 15, 40, and 60 in the mMIC. To simplify the comparison, the performance on each experimental contrast was pooled together, and the averaged performance was then submitted to a two-way ANOVA with factors as homogeneity (homogeneous clusters versus cubic-shaped clusters) and cluster size. We found that 1) for the predictive accuracy, the performance was similar at all cluster size tested, with a slight gain in using homogeneous clusters (Left); 2) for the robustness of functional mapping, the mMIC based on homogeneous clusters was significantly better in overall  = 56.7, p<0.001), especially at larger cluster sizes  = 4.5, p = 0.001; cluster size III, t(9) = 2.3, p = 0.04) (Right). In addition, because neighboring voxels usually share similar response characteristics, the cubic-shaped clusters likely contained large amount of homogeneous voxels. Thus, it is not surprising that its performance was significant better than that of the MIV (gray dotted line) that completely ignored correlations in BOLD signals among voxels at all sizes . Taken together, our result suggests that the homogeneous information embedded in BOLD signals among voxels helps improve the robustness of functional mapping without compromising the predictive accuracy. Error bars indicate standard error of mean across subjects.(TIF)Click here for additional data file.Figure S5The effect of spatial smoothing on MVPA analyses. Recently, it has been shown that spatial smoothing is beneficial for MVPA analyses ts<1) (Left). For the contrast of male versus female faces, the MIVs was better than the MIV in predictive accuracy (t(9) = 2.06, p = 0.07), whereas there was no difference between mMIC and MIVs (t<1). For the contrast of houses versus scenes, the MIVs was inferior to the mMIC (t(9) = 2.11, p = 0.06), and there was no difference between the MIVs and the MIV (t<1). The robustness of functional mapping of the MIVs showed a similar pattern (Right). The MIVs outperformed MIV in all the contrasts . More critically, the MIVs was inferior to the mMIC in all the contrasts . B) Overlap between informative clusters mapped for female and male faces (versus houses). The functional validity of the MIVs, MIV, and MIC was examined by calculating the overlap rate between informative regions mapped with male faces (versus houses) and female faces (versus houses). Similarly, the overlap rate in the MIVs was significantly higher than that in the MIV , and was significantly smaller than that in the mMIC . Error bars indicate standard error of mean across subjects.(TIF)Click here for additional data file.Table S1Predictive accuracy in the simulated data by uMIC, mMIC, and MIV, under various settings of CNR and Ts.(DOC)Click here for additional data file.Table S2Robustness of mapping in the simulated data by uMIC, mMIC, and MIV, under various settings of CNR and Ts.(DOC)Click here for additional data file.

The two phenyl rings form dihedral angles of 64.6 (1) and 87.8 (1)° with the best plane through the piperidine ring. The crystal packing is governed by inter­molecular C—H⋯O inter­actions.In the title compound, C Å b = 10.6369 (5) Å c = 11.1497 (7) Å β = 100.373 (3)°V = 882.21 (8) Å3 Z = 2 Kα radiationMo −1 μ = 0.08 mmT = 293 K 0.30 × 0.25 × 0.20 mm Bruker Kappa APEXII area-detector diffractometerSADABS; Sheldrick, 2001T min = 0.977, T max = 0.985Absorption correction: multi-scan (12819 measured reflections3457 independent reflectionsI > 2σ(I)2400 reflections with R int = 0.042 R[F 2 > 2σ(F 2)] = 0.046 wR(F 2) = 0.145 S = 1.02 3457 reflections220 parameters1 restraintH-atom parameters constrainedmax = 0.23 e Å−3 Δρmin = −0.17 e Å−3 Δρ APEX2 used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008ORTEP-3 (Farrugia, 1997SHELXL97 and PLATON (Spek, 2009Data collection: 10.1107/S1600536809028049/bt2989sup1.cif Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536809028049/bt2989Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:

Progression of solid tumors including lung cancer is angiogenesis-dependent. We previously introduced a bioinformatics-based methodology to identify endogenous anti-angiogenic peptide sequences, and validated these predictions in vitro screening, we have evaluated the ability of a 20 amino acid peptide derived from the α5 fibril of type IV collagen, pentastatin-1, to suppress vessel growth in an angioreactor-based directed in vivo angiogenesis assay (DIVAA). In addition, pentastatin-1 suppressed tumor growth with intraperitoneal peptide administration in a small cell lung cancer (SCLC) xenograft model in nude mice using the NCI-H82 human cancer cell line.One family of peptides with high activity is derived from the α-fibrils of type IV collagen. Based on the results from the in vivo in a dose dependent manner. The peptide also decreased the rate of tumor growth and microvascular density in vivo in a small cell lung cancer xenograft model.Pentastatin-1 decreased the invasion of vessels into angioreactors The peptide treatment significantly decreased the invasion of microvessels in angioreactors and the rate of tumor growth in the xenograft model, indicating potential treatment for angiogenesis-dependent disease, and for translational development as a therapeutic agent for lung cancer. Judah Folkman pioneered the field of tumor angiogenesis by demonstrating that solid tumors are dependent on their blood supply to grow and metastasize, thus placing the field of tumor angiogenesis at the center of cancer biology and therapeutics . Lung cain vitro in cell proliferation and migration assays on human umbilical vein endothelial cells (HUVECs) [in vivo angiogenesis assay (DIVAA), and to an in vivo NCI-H82 SCLC xenograft model. We demonstrate high activity in each of these assays, in addition to directly inhibiting proliferation of NCI-H82 SCLC cells and 3T3 fibroblasts in vitro, indicating strong potential for pentastatin-1 as a therapeutic agent for lung cancer.We recently developed a bioinformatics-based approach to predict over 100 novel endogenous anti-angiogenic peptides [(HUVECs) . Most peThe peptide pentastatin-1 (LRRFSTMPFMFCNINNVCNF) was synthesized using a solid-phase synthesis technique by a commercial provider . The endogenous human and mouse sequences are identical for this peptide. The manufacturer provided HPLC and mass spectrometry analysis to guarantee >95% purity. The peptides were stored at -80°C in lyophilized form. Since pentastatin-1 is hydrophobic, it was solubilized using 10% dimethyl sulfoxide (DMSO) and water without any demonstrated effect on cell viability.In vitro viability assays were completed with pentastatin-1 on NCI-H82 small cell lung cancer and mouse 3T3 fibroblast cell lines. NCI-H82 human SCLC cells were obtained from the laboratory of Dr. D. Neil Watkins . The cells were propagated in RPMI 1640 cell medium supplemented with 10% v/v fetal bovine serum, 10 mM of HEPES, 2 mM of L-glutamine, 1% v/v of pen/strep, 1.5 g/l of sodium bicarbonate, and 1 mM of sodium pyruvate. The cells do not attach to the flask, but grow in small floating colonies and are passaged by centrifugation without trypsinization and subsequently resuspended in fresh media. 3T3 mouse fibroblast cells were acquired from ATCC and grown under standard conditions in Dulbecco's Modified Eagle Medium (DMEM) (ATCC) with 10% FBS and 1% pen/strep.3 cells were seeded per well in a 96-well microplate, centrifuged at 1,000 RPM for 5 minutes, and exposed for 3 days to peptide concentrations of: 3.2, 6.3, 12.5, 25, 50, and 100 μg/mL. Cells were tested in triplicate for each concentration. Microplates containing NCI-H82s were centrifuged at 1,000 RPM for 5 minutes to minimize loss of non-adherent cells before application of WST-1. As an experimental control, equivalent to normal cell viability, the cells were cultured without any agent in complete medium, containing growth factors and serum without any exposure to peptide.The effects of the peptide on the NCI-H82 and 3T3 cell viability were measured using the colorimetric cell proliferation reagent WST-1 . Approximately 2 × 10Cell proliferation was measured by BrdU (5-bromo-2'-deoxyuridine) incorporation assay on both NCI-H82 cells and 3T3 fibroblasts according to manufacturer's recommendations. Briefly, 10,000 cells/well were plated into 96 well plates in the presence of pentastatin-1 at 3.8, 15, and 60 μg/mL, and BrdU label (1:2000 dilution) for 24 hours. Plates were then washed, fixed with anti-BrdU antibody, and peroxidase goat anti-mouse IgG conjugate. Immunocomplex formation was measured using tetra-methylbenzidine solution, and the reaction terminated using 2.5 N sulfuric acid. The measured intensity is proportional to the amount of incorporated BrdU in the cells. Absorbance was measured at 450 nm using a Victor 3 V plate reader . Cells were tested at five wells per concentration.in vivo method of assaying angiogenesis. Silicone cylinders of 20 μl volume and 100 μm of inner diameter (angioreactors) were closed on one side, and filled with an extract of extracellular matrix containing VEGF and fibroblast growth factor (FGF) with or without premixed peptides as a control. These angioreactors were then implanted subcutaneously in the abdominal region of C57BL/6 mice, two angioreactors per animal, ventrally one per each side of the peritoneal cavity. Capillary sprouts originating from the host vessels invaded the extracellular matrix and formed vessels in the angioreactor. 12 days after the implantation, the mice were euthanized and angioreactors were removed. The extracellular matrix, containing the developed vasculature, was removed from the cylinder and endothelial cells were stained using FITC-Lectin, quantified by fluorescence at 510 nm emission with 485 nm excitation using a fluorescence plate reader . The intensity of the signal is proportional to the number of endothelial cells contained in each of the angioreactors. The results were normalized to the mean of the experimental controls. Statistical significance was tested by the Student's t-test at p < 0.05 *.DIVAA is a quantitative Animal protocols were approved by the Animal Care and Use Committee at the Johns Hopkins Medical Institutions. Athymic nude mice were obtained from Harlan . The mice were 4-6 weeks of age and housed on a 12 hour light dark cycle with food and water provided ad lib. All mice were fed with a standard caloric diet for their age.in vitro cell viability experiments. Prior to inoculation, the cells were prepared in a solution containing 1:1 mixture of PBS and Matrigel . 1 × 106 cells in 200 μl volume were injected subcutaneously in the right flank area of the mice. Tumors were inspected 5 to 6 days after inoculation. The tumor inoculation efficiency was approximately 75%.Mice were allowed to acclimate for one week prior to inoculation with NCI-H82 cells. NCI-H82 cells were grown under the same conditions for 3 to 200 mm3. Equivalent volumes of 10% DMSO in water were injected as an experimental control. Separate controls of scrambled peptide sequences were also made to test that the anti-angiogenic efficacy of pentastatin-1 was sequence-dependent. The injections were continued for 12 days, with a total of 8 animals per group used for the experiments per peptide per concentration. The location and side of the injection were alternated every day. The higher dose of 10 mg/kg was repeated twice with similar tumor suppression and without a statistically significant change.Following growth incubation of 6-7 days, tumor size volume was estimated by measurement of tumor dimensions with digital calipers, and peptides were administered once per day for 12 days, intraperitoneally (i.p), at 5 mg/kg and 10 mg/kg. In most cases the initial tumor volume ranged from 150 mm(4/3)πab2, where a is the measurement for the long semi-axis and b is the measurement for the short semi-axis.The tumor dimensions were measured every three days. We measured two dimensions for each tumor, a long and short axis. In some cases the tumors developed as two lobes, in which we considered the whole tumor as a single lobe with two associated axes. In order to estimate the volume we considered the tumors as prolate spheroids with volume equal to t-test with significance defined at p < 0.05 * and p < 0.01 **. Statistical significance was tested among the peptide condition and the experimental control in addition to the scrambled peptide.The average tumor size per condition and the standard error of the mean are reported over time. The statistical significance measured using the Student's Immediately following the sacrifice of the mice, tumors were excised and stored in a zinc-based fixative for 10-14 days, and sent to the JHMI Immunohistochemistry Core Facility for paraffin wax-embedding and processing. After deparaffinization and rehydration, 5 μm cross sections were treated overnight at room temperature with a monoclonal rat anti-mouse CD31 (PECAM-1) IgG antibody (1:100) , which specifically recognizes an epitope on the surface of endothelial cells. Secondary antibody incubation was made using biotinylated rabbit anti-rat IgG antibody (1:200) for one hour, and lightly counterstained with hematoxylin.Similar analysis was made for the apoptotic marker cleaved (activated) caspase-3. After deparaffinization and rehydration of slides, primary antibodies were applied at dilutions of 1:400 for cleaved caspase-3 , diluted in antibody dilution buffer and incubated at 14 hours at 4°C. Primary antibodies were detected using the Power Vision Plus HRP-polymer detection system per manufacturer's instructions. DAB chromogen was applied to develop the secondary detection reagent. Slides were counterstained with hematoxylin and mounted on cover slips.t-test at p < 0.05 * and p < 0.01 **.All histological samples were digitized, then processed using Aperio Image Scope Software , and quantified using FRiDA software . For the CD31 antibody staining 100% represents the mean of the experimental controls. Apoptotic cell counts were made per frame at 20× magnification using FriDA. Each peptide concentration was compared to the control and scrambled peptide equivalent using the Student's In vitro cell viability experiments show pentastatin-1 decreases NCI-H82 SCLC and 3T3 fibroblast viability at increasing concentrations . The percent inhibition for each condition over the 12 days for the experiment is summarized in Fig. In the tumor xenograft experiment we follow the mean tumor volume of pentastatin-1 treated xenografts Fig. , along w 12 Fig. . BeginniFig. in vivo half-lives and degradation by proteases, they are also advantageous due to low toxicities, high selectivity, and potential to enhance existing cytotoxic therapies [Currently, there is a high unmet need for treatment of small cell lung cancer as upwards of 95% of patients diagnosed with the disease will die within five years , e.g. tyherapies .Collagen IV forms the bulk of the structure of the vascular basement membrane, and is known to contain several cryptic fragments with anti-angiogenic activity . Severalin vitro. Similarly, we show pentastatin-1 effectively inhibits viability of NCI-H82-SCLC cells and 3T3 fibroblasts by WST-1 colorimetric agent, and inhibits synthesis of DNA by BrdU incorporation indicating the peptide targets multiple cell types in addition to being anti-angiogenic. Previously published xenograft work shows pentastatin-1 has low toxicity measured by H&E staining of vital organs in severe combined immunodeficient (SCID) mice [1 and β3 integrins, which are expressed on endothelial cells [We have previously shown pentastatin-1 decreases HUVEC migration and viability ID) mice . We haveal cells , and areal cells includinal cells , indicat1 and β3 integrin receptors on endothelial cells is consistent with the decrease in microvascular density, as the peptide targets these receptors, while a lower microvascular density is correlated with a decrease in tumor development and growth.In our xenograft model we show pentastatin-1 has a monotonic dose response and suppresses tumor growth at both 5 mg/kg and 10 mg/kg, while decreasing the microvascular density and increasing apoptotic cell count in response to the peptide application. At the higher dose the peptide suppresses tumor growth significantly with an inhibition of 53%. Although the tumors grow and reach a size larger than the initial size when peptide was administered, the peptide still effectively suppresses the tumor growth when used as monotherapy. The scrambled peptide equivalent shows that the peptide's activity is sequence-dependent, as it is not statistically different from the control. The presence of βin vitro HUVEC proliferation and migration assays. Small cell lung cancer is very resistant to chemotherapy, and has a high mortality rate indicating the need for comprehensive treatment. The disease has been shown to be angiogenesis-dependent. Based on previous in vitro experiments, we selected pentastatin-1 for application to the angioreactor-based DIVAA, and a small cell lung cancer xenograft model using nude mice. The peptide has shown to be effective in a pre-clinical model for limiting tumor growth, and has the potential for translational research in lung cancer.Type IV collagen contains several cryptic fragments with anti-angiogenic properties. We previously identified several anti-angiogenic fragments through a bioinformatics-based methodology, including pentastatin-1, which showed high potency to The authors declare that they have no competing interests.in vitro and in vivo experimental data. JK prepared the manuscript for submission. BT, HH and RP supervised xenograft inoculation and provided experimental and technical assistance regarding data analysis. DNW provided NCI-H82 small cell lung cancer cells, and experimental guidance. AP conceived design of the study, supervised in vitro and in vivo assays, and provided useful discussion for data interpretation. All authors read and approved the final manuscript.JK and MK conducted and analyzed all The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/29/prepub

Cellular processes such as metabolism, decision making in development and differentiation, signalling, etc., can be modeled as large networks of biochemical reactions. In order to understand the functioning of these systems, there is a strong need for general model reduction techniques allowing to simplify models without loosing their main properties. In systems biology we also need to compare models or to couple them as parts of larger models. In these situations reduction to a common level of complexity is needed.κB pathway.We propose a systematic treatment of model reduction of multiscale biochemical networks. First, we consider linear kinetic models, which appear as "pseudo-monomolecular" subsystems of multiscale nonlinear reaction networks. For such linear models, we propose a reduction algorithm which is based on a generalized theory of the limiting step that we have developed in . Second,Our approach allows critical parameter identification and produces hierarchies of models. Hierarchical modeling is important in "middle-out" approaches when there is need to zoom in and out several levels of complexity. Critical parameter identification is an important issue in systems biology with potential applications to biological control and therapeutics. Our approach also deals naturally with the presence of multiple time scales, which is a general property of systems biology models. Model reduction techniques are used to reduce the dimensionality of complex dynamics. Applications of model reduction techniques in chemical engineering , in eTrajectory based techniques use the integration of the dynamical equations and look for a small number of reduced variables and zero elsewhere.for κn ≪ κi, i ≠ n) then this expression in the first order is simplified toIf we have a system with well separated constants .which means that most of substance is concentrated just before the "bottleneck" κn ≪ κi, i ≠ n, it is sufficient to remove the slowest step of the cycle An → An-τ+1. After removing, we will have acyclic non-branching system of reactions with eigenvalues and eigenvectors that can be computed from the formulas in the previous section. These formulas give n - 1 eigenvector sets corresponding to n - 1 non-zero eigenvalues λi = -κi, i = 1..n - 1. For example, removing A8 → A6 step at Fig. To approximate the dynamics of the reaction network for Ai, let us define κi as the maximal kinetic constant for reactions Ai → Aj: κi = maxj{kji}. For correspondent j we use notation ϕ(i): ϕ(i) = arg maxj{kji}. The function ϕ(i) is defined under condition that for Ai outgoing reactions Ai → Aj exist. If there exist no such outgoing reactions then let us define ϕ(i) = i.Now let us consider an arbitrary linear reaction network with well-separated constants. For each Ai → Aϕ(i) with kinetic constants κi. The correspondent kinetic equation isAn auxiliary reaction network is the set of reactions i → Φ (i) that is used to compute the eigenvectors of the kinetic matrix. The auxiliary network can have several connected components. In each connected component the minimal sink is an attractor of the auxiliary dynamical system, hence it is either a node, or a cycle.The auxiliary network also defines a auxiliary discrete dynamical system 1) Let us consider a reaction network κi are known) or to obtain the 0–1 asymptotics (if only the ordering of κi is known).2) If the auxiliary network does not contain cycles then the auxiliary network kinetics (16) approximates relaxation of the initial network C1, C2, ... with periods τ1, τ2, ... > 1.3) In general case, let the system Ci, we introduce a new vertex Ai. The new set of vertices is Ci and add vertices Ai).By "gluing" cycles into points, we transform the reaction network A → B do not change.A → Ai (to the whole glued cycle) with the same constant.2. Reactions from the second group ("entering to cycles") transform into Ai → B with the rate constant renormalization: let the cycle Ci be the following sequence of reactions A1 → A2 → ... A1, and the reaction rate constant for Ai → Ai+1 is ki (A1). For the limiting reaction of the cycle Ci we use notation kilim . If A = Aj and k is the rate reaction for A → B, then the new reaction Ai → B has the rate constant kkilim /kj. This corresponds to a quasistationary distribution on the cycle (15). It is obvious that the new rate constant is smaller than the initial one: kkilim /kj <k, because ki lim <kj due to definition of limiting constant. If after gluing, several reactions Ai → B appear, then only the one with the maximal constant should be kept.3. Reactions of the third type ("exiting from cycles") change into Ai → Aj.4. The same constant renormalization is necessary for reactions of the fourth type ("between cycles"). These reactions transform into 5. Reactions of the fifth type ("inside cycles") are discarded.4) After the new network Ci can be neglected when they are dominated by a stronger flux of Ci are minimal sinks in A → B in A ∈ Ci, B ∉ Ci. Nevertheless, there may be such reactions in the initial network The algorithm produces an hierarchy of cycles. Notice that the algorithm is based on an asymmetry between entering reactions and outgoing reactions from cycles in the hierarchy. Indeed, some fluxes of ified in .Ai ∈ i = 1..n which is called dominant kinetic system. Dynamics of this acyclic system can be computed from . To construct the dominant kinetic system, the following algorithm is applied:Now we show how to find an approximation of the dynamics of the reaction network Let 1. For 2. For each glued cycle node τi,a) Recall its nodes b) Let us assume that the limiting step in c) Remove τi vertices d) Add e) Add to B in B with the same constant, i.e. outgoing reactions f) If there exists an outgoing reaction B → g) If there exists an incoming reaction in the form A → B, and substitute the reaction by B with the same constant, as for 3. If in the initial m ← m - 1, and repeat steps 1–4 until no glued cycles left.4. Let One has to notice that in the process of network preprocessing some reaction rates are substituted by monomials of the initial reaction constants, i.e. expressions in the form K and does not depend on the production reactions K0. In particular, relaxation times do not depend on the system being closed or not. However, the stationary distribution cs and the sequence of relaxation events depends on production reactions (see Eq.(2)).The dominant kinetic system fully describes the relaxation modes of the network. The construction of this system depends only on the matrix Kc = 0, therefore they are determined up to multiplication by positive constants. They form a k-dimensional cone where k is the multiplicity of the zero eigenvalue of the matrix K, also the number of minimal sinks of the network.For closed systems, steady states are solutions of the linear homogeneous equation k sink vertices of the auxiliary network Ai, i = 1..n be vertices in the initial network Let ith sink vertex 1. Let us take the x = b = 1, and a null vector bi ∈ Rn.2. Define x is not a glued cycle then it corresponds to a vertex Aj ∈ bi has components δij; stop.3. If x is a glued cycle then recall all its vertices x1, ..., xτ.4. If κlim = mins=1..τ {smin = arg mins=1..τ {5. Determine the limiting rate constant xj of the cycle repeat:6. For each vertex j ≠ smin and a) Let xj corresponds to a simple vertex Ak then cj,b) if xj corresponds to a glued cycle then do recursively steps 4–6 with x = xj and b = cj.c) if Any possible stationary distribution has form In brief, the distribution of the concentrations on any cycle is approximated by the first order expression (15), and this procedure is applied recursively for the vertices that represent glued cycles. The state thus obtained is equally a good approximation of the steady state of the dominant kinetic system.c0 = 1. The corresponding reaction are ∅ → Ai and the constants are the production rates ki0. The normalization c0 = 1 is possible for all bounded steady states, because these are determined up to multiplication by a constant. Furthermore, all steady states are bounded, provided that the following topological condition holds: if there exists an oriented path from ∅ to Ai, then there exists an oriented path from Ai to ∅. We suppose this condition to be always fulfilled. Applying the algorithm to the closed system we obtain Open systems can be reduced to closed ones by considering that all production reactions originate from the node ∅ that has concentration Example. Let us consider an example of the network 2) An auxiliary reaction network A3 → A4 → A5 → A3 in the auxiliary reaction network is glued in one vertex A4 → A2, renormalize its rate to 3) The cycle A3 → A2 → A3 in the auxiliary reaction network 4) The cycle A3 → A2 → A3 and try to determine the limiting step in it, we have two possibilies: k32 and k32. Let us consider them separately:Now if we restore the cycle Case k32k32, then we remove the limiting step A2 and obtain 3.1.2).3.1.1) Since A2 → A2 → A3 in 3.1.3) We restore the glued cycle corresponding to A3 → A4 → A5 → A3 (it is A5 → A3) and as a result we obtain acyclic dominant kinetic system shown at Fig. 3.1.4) We remove the limiting step reaction in the cycle Case k32k32, then we remove the limiting step A2 → 3.2.1) Since A2 to the head of the limiting step in the cycle (it is A5 vertex); the rate of A5 → A2 is 3.2.3) We restore the glued cycle corresponding to A3 → A4 → A5 → A3 (it is A5 → A3) and as a result we obtain acyclic dominant kinetic system shown at Fig. 3.2.4) We remove the limiting step reaction in the cycle Dominant approximations of hierarchical linear reaction network allow us to introduce some new concepts important for the dynamics of multiscale systems.Piecewise affine dynamics has been widely used to approximate dynamics of gene regulatory networks -37 as a j0, ci (0) ~ λk an exponential vanishes in Eq. (2) and the state has a "jump" -rk -cs). Let us consider that eigenvalues are ordered λ1 >> λ2 >> ... >> λn-1. Then, the sequence of right eigenvectors rk such that -cs) ≠ 0 codes the dynamics starting in c(0). In other words, there is a sequence of well separated times τ1 = 1/λ1 <<λ2 = 1/λ2 << ... <<τn-1 = 1/λn-1 such that something happens (a state transition) between each one of these times. Left eigenvectors provides the lumping and right eigenvectors provide the sequence of state transitions. On timescales τk <t <τk+1 one can observe a jump -rk in state space provided that - cs) ≠ 0. On this timescale the dynamics is equivalent to the degradation of pseudospecies , λk .Indeed, zero-one approximation of eigenvectors in hierarchical linear systems justifies a discrete coding of dynamics. Suppose that initial state is concentrated in Our approach to dominant subsystems emphasizes some simple but important principles. First of all, dynamics of a hierarchical linear network can be specified if a) the topology of the network is given and if b) to each reaction we associate a positive integer representing order ; c) for cyclic topologies, some monomials grouping constants of several reactions have also to be ordered in the same manner .second slowest constant is the reaction number. Unless reactants and products belong to compartments of different volumes αji, βjk are nonnegative integers (stoichiometric coefficients). Reactions involving components from different compartments have non-integer stoichiometry. For instance, a reaction translocating a molecule from nucleus to the cytosol has stoichiometry where kv is the volume ratio of cytosol to nucleus.where Dynamics of nonlinear networks is described by a system of differential equations:νj = βj - αj is the global stoichiometric vector. S is the stoichiometric matrix whose columns are the vectors νj. The reaction rates c) are non-linear functions of the concentrations. For instance the mass action law reads There are no simple rules to relate timescales to reaction constants of nonlinear models. The units of the inverse constants of bimolecular reactions are concentration multiplied by time and one needs at least one concentration value in order to construct a timescale. Generally, timescales are functions of many reaction constants and concentration variables. These functions are not necessarily smooth. Near bifurcations , at least one timescale of the system diverges for finite changes of the reaction constants. However, nonlinear biochemical networks have wide distributions of time-scales, as can be shown by simple (Jacobian based) analysis of models.Various reduction methods of nonlinear models are based on projection of the dynamics on a lower dimensional invariant manifold -8. The rA major improvement in calculating dominant subsystems can be obtained by combining quasi-stationarity and averaging. Averaging techniques are widely used in physics and chemistry to simplify models by eliminating fast, oscillating (microscopic) variables -24. Our I be the set of indices of intermediate components, that will be eliminated. I. Rates of T be the set of indices of terminal species. Terminal species represent the frontier between the rest of the system and the subsystems made of intermediate species. Although instead of terminal the name boundary species could be more appropriate, the latter term has already been employed in systems biology with a different meaning, which is species whose concentrations are fixed in a simulation.Let f Temkin are callS the columns corresponding to the reactions SI and the terminal stoichiometric matrix ST, respectively.Extracting from the matrix In multiscale biochemical systems, some components react much more rapidly to changes in the environment than others. The reasons for the existence of such fast species can be multiple. Thus, rapidly transformed or rapidly consumed molecules , or promoter sites submitted to rapid binding/unbinding processes are examples of fast species. Fast species are good candidates for intermediate species. Indeed, it is easy to prove that they can be eliminated by quasistationarity. When production rates are not weak, fast species are those whose concentrations are small and well separated from the concentrations of other species. Though straightforward, the precise condition connecting quasi-stationarity and smallness of concentrations can not be easily found in literature, hence we briefly discuss it below.ϵ be a small parameter, representing concentrations. Suppose for simplicity that reactions SI RI = KI (cT) cI + cT), where RI is the restriction of the vector R to the intermediate species. An important assumption is cT) = Let Suppose that among the reactions cI = ϵ), it means that KI (cT) = ϵ). This leads to the following asymptotic:Because cI/ϵ, ϵKI = Ic is the complement of I designating species other that I. Intermediate species are fast and the system (19) can be reduced using Tikhonov's , but most of the properties of this probability distribution will not be used here. The only property that we will use is the following: if ki > kj, then ki/kj ≫ 1 (with probability close to one). It means that we can assume that ki/kj ≫ a for any preassigned positive value of a that does not depend on k values. One can interpret this property as an asymptotic one for α → -∞, β → ∞. This property allows us to simplify algebraic formulas. For example, ki + kjcan be substituted by max{ki, kj} , orIn linear hierarchical models, ensembles with well separated constants appear . We coua, b, c, d.for nonzero k1 + k2) - k1, if k1 ≫ k2? If we first simplify the expression in brackets, it is zero, but if we open brackets without simplification, it is k2. This is a standard difficulty in use of relative errors for round-off. If we estimate the error in the final answer, and then simplify, we shall avoid this difficulty. Use of o and k1 ≫ k2, then we can write (k1 + k2) = k1(1 + o(1)), and k1(1 + o(1) - k1 = k1o(1). The last expression is neither zero, nor absolutely small – it is just relatively small with respect to k1.Of course, some ambiguity can be introduced, for example, what is 1/kB <<kA. Both cases mean that degradation of A, B is weak and/or the propensity of complex formation is high. Case a) means also that B is in excess, the opposite being true in case b).Supposing A, B quasi-stationary we have to find the positive solutions of the equations kA - kB = kB and from the second nonlinear equation we obtain Rc = kcAB. Actually, the two solutions can be summarized by:Let us consider the case a). We consider that the order of A from the linear equation we remain with a quadratic equation for B) we can show that the min-funnel approximation (29) is valid under less restrictive conditions. The only separation condition that we need is min >> kdeg, A kdeg,B/kc. We can easily identify the critical parameters kA, kB and the non-critical ones kdeg,A, kdeg,B, kc. The validity of the expression (29) depends on order relations involving monomials of critical and non-critical parameters.Using the exact solution of the system .Averaging is an useful model reduction technique for high-dimensional clocks or for other types of oscillating molecular systems has an attractive hyperbolic limit cycle x = ψ , of period T(y): ψ (τ + T(y), y) = ψ (τ = t/ϵ). Then, after a short transient, the slow variable satisfies the averaged equation:It is supposed that for any x = ψ describes damped oscillations, with damping time much larger than ϵ, i.e. when the fast dynamics (30) has a stable focus and the eigenvalues of the Jacobian ∂f/∂x calculated at the focus are of the form -λ ± iμ, 0 <λ <<μ = ϵ).The result can be extended to the case when y:The following averaged steady state equation allows to eliminate the slow species y are constant in time and can be considered to be conserved, which has two significant consequences.If (32) has a stable steady state, we always reach this situation. In this case, the slow non-oscillating variables First, Eq.(33) restores conservation. Slow variables are often the result of broken conservation laws. In fact, in biological open systems, nothing is conserved. Conservation laws result from balancing production and degradation either passively (slow processes) or actively (feed-back). Thus, we can ignore production and degradation of molecules whose level is rigorously controlled. Eq.(33) describes such a case.y reach stationarity, the only variables that change in time are the oscillating variables x. Eq.(30) describes the dynamics of x, considering that y satisfies (33). For oscillators, averaging provides a way to eliminate slow non-oscillating variables, which is formally equivalent to quasi-stationarity and represents a new case of applicability of Clarke's method. The difference between the two cases is that we eliminate fast variables by solving quasi-stationarity equations and we eliminate slow variables by solving averaged stationarity equations. Thus, intermediate non-oscillating variables are expressed in terms of only non-oscillating terminal variables. If there are no non-oscillating terminal variables, then non-oscillating intermediates become conserved quantities.Second, (33) are averaged steady state equations for the slow variables. If slow variables In this section, we demonstrate hierarchical model reduction, model comparison and critical parameter identification. Critical parameters are identified during the reduction procedure.n species, r reactions and p parameters is designated by M. Our conception about systems biology models is summarized by the following idea. Instead of providing a single model, it is better to provide a hierarchy of models, and the relations between them. Depending on the application, we can choose the most appropriate model in the hierarchy or couple several simple models into a larger model.Model reduction starts with a complex model, from which we obtain a hierarchy of reduced models by eliminating various intermediate species. The intermediate species are either quasi-stationary species , or non-oscillating species (for oscillators). The complexity of a model is quantified by three integers. A model with nk = 2nr + ni kinetic constants, where nr, ni are the numbers of reversible and irreversible reactions. Reactions with kinetic constants zero are not considered in the counting. Each one of the nc conservation laws adds an extra parameter, the value of the conserved quantity. These values follow from initial data and are important parameters for the dynamics. For multi-compartment models, the ratios of the compartments volumes are extra parameters. Thus p = nk + nc + 1.The number of parameters in a model are obtained as follows. If the elementary reactions follow mass action law kinetics, there are Model comparison has a similar flowchart. By model comparison we understand a) mapping one model to another one by model reduction or mapping each model to a third one, closest in some sense to both; b) compare predictions of the models for sets of parameters related one to the other by the mapping at a). In this case, the choice of intermediate species is dictated by the differences between the models to be compared.κB is involved in a wide diversity of domains such as the immune and inflammatory responses, cell survival and apoptosis, cellular stress and neuro-degenerative diseases, cancer and development. NF-κB is sequestered in the cytoplasm by inactivating proteins named IκB. Upon signalling, IκB molecules are phosphorylated by a kinase complex, then ubiquitinylated, and finally degraded by the proteasomal complex. NF-κB bound to IκB molecules is then transported to the nucleus to activate its target genes. There are known five members of the NF-κB family in mammals, Rel (c-rel), RelA (p65), RelB, NF-κB1 (p50 and its precursor p105) and NF-κB2 (p52 and its precursor p100). This generates a large combinatorial complexity of dimers, affinities and transcriptional capabilities. IκB family comprises seven members in mammals , x8 = [A20], x13 = [IkBa : p50 : p65@csl], kp1 21= k3k9/k4, k21p2 = k15p2 = k15/k13, k15p2 = (k15k3k9)/(k4k13), k21p3 = k15p3 = k5/k4.where After reduction of the first module we obtain the model ℳ.The second module is situated in the nucleus and contains IkBa@ncl and IKBnp50:p65@ncl. Three intermediate reactions (translocations of inhibitor and of the complex and complex formation) are replaced by one simple submechanism describing the nuclear complex formation and translocation (IkBa@csl + p50:p65@ncl → IkBa:p50:p65@csl) whose dominant rate is:x7 = [p50 : p65@ncl], kp1 14= k23, where This reduction step leads to the model ℳ. The dynamics . Both period and phase changes result from the reduced delay on the negative feed-back loop containing A20. Reducing the species mRNAIkB has destabilizing effect on the oscillations. It is no longer possible to obtain self-sustained oscillations and damping times are generally smaller than for the non-reduced model. It is well known that delayed negative feed-back favors stable oscillations and that reducing the delay destabilizes oscillations. Our findings suggest that the delay along the IkBa negative feed-back loop is more important for the stability of the oscillations than the delay along the A20 loop.We have tested reduction of two more species that have small concentration but are not quasi-stationary. Reducing the species mRNAA20 leads to the model ℳ Intermediate reactions (representing the transcription/translation module) are replaced by a single one (production of protein), of parameter Model reduction allows to identify critical and non-critical parameters. Parameters of reduced models are monomials of parameters of the non-reduced models (see Eqs.(34),(35),(36)). Some parameters of the non-reduced model may not occur in these monomials; these are non-critical parameters. Among monomials, only some are critical. Critical monomials are detected by sensitivity studies performeτ be the studied quantity and k the parameter . We say that k is critical if A > 0 is some fixed constant and k0 some central value of the parameter. The sensitivity study is presented in Fig. kp114, k18, kp20, kp121, k22, k26, C0. By changing these parameters, the oscillations can be modified from damped to self-sustained. The above parameters are the critical monomials from which we get the critical parameters (with respect to damping time) of the unreduced model: k23, k18, k16, k20, k17, k3, k9, k4, k22, k26, C0. The degrees of the critical monomials represent logarithmic sensitivities, therefore they provide both sign an strength of the influence of the critical parameters on the studied property. For instance, from kp1 21= k3k9(k4)-1 we can say that damping time can be increased (produce sustained oscillations) by reducing k3, or by reducing k9, or by increasing k4), see also Fig. As an example, we detect critical monomials in the simplest reduced model ℳ5, 8, 15), first with respect to damping time and then with respect to the period of the oscillations. Deciding rigorously what large sensitivity means is not easy. In we propo, 8, 15, kp 20stands for increasing the NF-κB dependent A20 production . Increasing k26, k22 stands for increasing the NF-κB dependent IκB production. The latter effect has been exploited in , x11 = [IkBa@ncl], Where p50 : p65@csl.The fourth step is a min funnel simplification of the production of the complex R40, R47, R52 are replaced by ∅ → p50:p65@csl, of rate:This leads to the model ℳ.R53–54, R71–72, that produce and consume p50 : p65, the total amount of p50 : p65 (free or in complexes with other species) would be conserved. Considering that the degradation reactions R53–54, R71–72, R72 have the same constant k, the total amount of NF-κB represented by the variableThe fifth step, justified by averaging, introduces a new conservation law has no conservation law). Without the reactions C = [p50 : p65@csl] + [p50 : p65@ncl]/kv + [p50 : p65 : IkBa@csl] + ... satisfies the equation :k and a rapid timescale, the period of the oscillations has two time scales, a slow timescale 1/where the average is over a period of the oscillations.R52, R53, R54, R71, R72 are eliminated. Initial conditions of the system are chosen such that initial total NF-κB is C0 .In the fifth reduction step, reactions κB transcription rate κB concentration x11 . This phenomenon is not taken into account in [R26 = k26x7.We obtain the model ℳ that has the same species and reactions as Lipniacki's model ℳ, but slightly more parameters. The difference in the number of parameters comes from the more complex expressions of the mRNA Icount in where thC0 and k26 which are obtained by averaging (C0 is given by Eq.(42) and k26 is calculated in order to have equal average production rates of mRNAIκB in the two models, see Fig. κBa. Without signal, the quantity of inhibitor in ℳ is small (it is largely in excess in the other two models). With signal, the amplitude of the oscillations is higher in ℳ. These differences follow from the different kinetic laws for the transcription of IκBa. Basically, in M and ℳ, IκBa has some negative influence on its own production (see Eq.(39)).One important objective of model comparison is to obtain the parameter mapping. This allows to calculate the parameters of one model if the parameters of the other model are known. Then, dynamical properties of the models can be compared. In our example, all the parameters of ℳ should be equal to the corresponding parameters of ℳ except for κB total quantity. Although this is not important in normal situations (when C is conserved), it could become important if one wants to cope with strong perturbations of NF-κB activity.Our most complex model can account for phenomena that can not be studied by any of the conservative models ℳ, ℳ, namely it can take into account variations of the NF-Thus, using a more complex model depends on the experimental situation . The role of mathematics and modeling in quantitative biology is to predict the behavior of a system. Depending on which behavior, the simplest theory can change, and we want a hierarchy of models and model mapping methods. The process can go in both directions: reducing, or increasing the number of details.Model mapping also allows to identify non-critical and critical parameters. Let us give only two examples. The constants or reactions 13,14 (formation of the complex) are not critical and one does not need to know them with precision. Actually, variations by a factor 100 in these constants do not change the dynamics.C0 is a critical parameter. Reference [μm3. This means C0 = 0.06 μM. Nevertheless, cell volume estimate is not really precise and errors can easily shift the model from a damped oscillatory to a sustained oscillatory dynamics. In this case, it is the comparison between model prediction and theoretical observation that can fix the value of the critical parameter.The values that we use come from , who citeference proposes with the following ids: MODEL7743656488, MODEL7743631122, MODEL7743608569, MODEL7743576806, MODEL7743528808, MODEL7743444866.The sequence of reduction steps described in this section is illustrated on Fig. 2S1–2S7 in Additional File We have presented a methodology for reducing and comparing systems biology models. We show how to produce a hierarchy of coarse grained models that can be used for understanding functioning of the biological systems. We show how models in the hierarchy can be mapped one onto another, thus allowing to decrease or to increase the number of details that are needed for the description of the system. Our method identifies the set of critical parameters of the system. This can be particularly useful for robustness studies (when robustness is understood as stability against parameter variability ) or for We did not approach aspects of multi-scale modeling that occur in multi-organ physiology, or spatial aspects. Relation with stochastic modelling has been only briefly discussed and will be presented in detail elsewhere . The reduction methods presented here can be applied to systems of biochemical reactions modeling cell physiology and can A central idea in our treatment is the hypothesis that biological systems are hierarchial, involving many separated time scales. The reduction methods were adapted to exploit this situation . The hierarchical nature of the systems is not sufficiently exploited by more traditional approaches. For instance, singular perturbation copes with two time scales and eliminates the fastest. In biology, we are often interested in a "middle" time-scale, corresponding to a particular process that we study. We have shown how to eliminate both faster and slower variables. Another specificity of systems biology is the quest for critical parameters. Our approach offers naturally a solution: critical parameters are detected in the reduction process. It also extends the theory of limiting step to complex networks . ShowingAs future work we will improve our algorithms in order to propose fully automated reduction tools. At present, the automated sections of our methods are the calculation of dominant subsystems of pseudo-monomolecular subsystems and the calculation of simple sub-mechanisms stoichiometries and rates. The detection of quasi-stationary and non-oscillating species is semi-automated. The solutions of quasi-stationarity and averaged stationarity equations are not yet fully automated (except for the pseudo-monomolecular case).We also plan to consider other applications such as high dimensional switches .Concerning our model comparison methods, we would like to study hierarchies of kinetic models coming from various organisms, for which the conserved and the specific parts are the result of evolution.κB model. All authors drafted, read and approved the final manuscript.OR proposed the methodology to reduce nonlinear models. AG developed the general theory of multiscale linear system, together with OR and AZ. AZ and OR designed and implemented the algorithms. AL designed the NF-κB models. This file shows the hierarchy of models using Systems Biology Graphical Notation (SBGN) and the results of a study of the truly non-linear reactions.Hierarchy of NFClick here for file

Neck lymph node status is the most important factor for prognosis in head and neck squamous cell carcinoma. Sentinel node detection reliably predicts the lymph node status in melanoma and breast cancer patients. This study evaluates the predictive value of sentinel node detection in 50 patients suffering from pharyngeal and laryngeal carcinomas with a N0 neck as assessed by ultrasound imaging. Following 99m-Technetium nanocolloid injection in the perimeter of the tumour intraoperative sentinel node detection was performed during lymph node dissection. Postoperatively the histological results of the sentinel nodes were compared with the excised neck dissection specimen. Identification of sentinel nodes was successful in all 50 patients with a sensitivity of 89%. In eight cases the sentinel node showed nodal disease (pN1). In 41 patients the sentinel node was tumour negative reflecting the correct neck lymph node status (pN0). We observed one false-negative result. In this case the sentinel node was free of tumour, whereas a neighbouring lymph node contained a lymph node metastasis (pN1). Although we have shown, that skipping of nodal basins can occur, this technique still reliably identifies the sentinel nodes of patients with squamous cell carcinoma of the pharynx and larynx. Future studies must show, if sentinel node detection is suitable to limit the extent of lymph node dissection in clinically N0 necks of patients suffering from pharyngeal and laryngeal squamous cell carcinoma.British Journal of Cancer (2002) 87, 711–715. doi:10.1038/sj.bjc.6600445www.bjcancer.comCancer Research UK© 2002 Paralell to other tumour entities there were intensified efforts within the last two decades, which are still discussed controversially, to limit the extent of lymph node dissection in the clinically staged N0 situations also for head and neck cancer patients. The aim of reducing a potential excess of surgical therapy for the patient is currently achieved quite successfully in other tumour entities by applying the so-called sentinel node (SN) concept (This study was approved by an ethics committee and written informed consent was obtained from the patients at the beginning of therapy.n=33), the larynx (n=14) and the hypopharynx (n=3) an intraoperative SN detection was performed. B-mode-ultrasound assessed a N0 neck in all 50 patients. Lymph nodes >1 cm in diameter, lymph nodes with a spherical appearance or diffuse borders to the surrounding soft tissue were defined as criteria for malignancy in neck sonography. These cases required fine needle aspiration cytology and if they were preoperatively classified as N1-neck these cases were not enrolled in this study. Further details on the location and TNM-classification can be withdrawn from Table 1In 50 previously untreated patients suffering from squamous cell carcinomas of the oropharynx (At the beginning of the surgery (tumour resection and neck dissection) the application of a total amount of 1.2 mCi Tc99m-nanocolloid, which was dissolved in 0.2–0.35 ml normal saline, was performed by intraoperative injections. This amount was chosen under the consideration of the special anatomical challenges in the lymphatic system of the upper aerodigestive tract, which is characterised by regional variation, but high density of lymphatics. An excessive amount of tracer substance leads to drainage into several neighbouring lymph node levels due to an unphysiological increase of the interstitial pressure. Before this background, it has to be pointed out, that more than 100 years ago Mascagni could show, that lymphatic fluids usually pass up to eight lymph nodes until they enter the blood vessels.The tracer substance, which had been aspirated into a 1 ml syringe was injected into four spots at the perimeter of the tumour under microscopic control. In cases of easily accessible SCCs of the oropharynx a hypodermic 24 Gauge needle of 25 mm length was used. Laryngeal and hypopharyngeal carcinomas were exposed via rigid endoscopy in general anaesthesia. The tracer substance was then injected under microscopic control into the perimeter of the tumour, using a 23 Gauge needle of 80 mm length , ensuring that no considerable amounts of the tracer were spilled.Based on the results of previous studies (n=20) with identification of radiolabelled lymph nodes on both sides of the neck had to be performed. Indications for bilateral neck dissection were carcinomas, which extended to the midline or trespassed it. With one exception all supraglottic carcinomas (5/6) were also treated by bilateral neck dissection. The exception to the rule was a patient suffering from a small T2 carcinoma of the supraglottis which was located on the free edge of the epiglottis with a wide clear margin to the midline. The extent of neck dissection depended on the size and likelihood of occult metastatic spread.Using a 14-mm diameter straight collimated probe the intraoperative identification of the sentinel nodes was performed within a maximum delay of 6 h (time range 2–6 h). This time span which may seem extended is based on the fact that in certain cases a bilateral neck dissection (n=30) or bilateral (n=20) neck dissection was then performed. Following uni- or bilateral neck dissection, the primary tumour was excised either by conventional surgery (n=3) or laser surgery (n=47), depending on the size and location of the lesion. In the remaining eight patients, in whom the carcinoma had been removed transorally by laser microsurgery, the operation had been performed directly prior to neck dissection, which facilitated intraoperative SN detection. The main cause for improved detection of the sentinel nodes was the elimination of scattered radiation from the peritumoural tissue. When performing this type of procedure, which is currently favoured by us, a time space of 15–20 min passes between tracer injection and laser microsurgical resection of the primary.Subsequent intraoperative readings with the gamma probe were performed to identify the sentinel nodes. Readings were usually performed after elevation of the cutaneous flaps. In the following, the lymph node containing tissue of the predominant metastatic region was mobilized, until the operation specimen could be rotated carefully. It was necessary to increase the distance between neck dissection specimen and primary injection site of the tracer substance to reduce the influence of radioactive scattering on identification of the SN with the gamma probe. The identified hot nodes were excised and extra-corporal control readings were performed. Due to the fact that especially carcinomas of the upper aerodigestive tract may initially drain into more than one lymph node, the three hottest nodes, containing more than 10 times the background level of radioactivity, were defined as sentinel nodes (SN1-3). Unilateral .In all 50 patients one or more sentinel nodes could be detected intraoperatively. A total number of 90 sentinel nodes could be identified. Sixteen out of ninety sentinel nodes were found on the contralateral side in carcinomas, which were either situated in the midline or expanded over the midline (Table 2n=70).During pathohistologic examination a total of 2538 lymph nodes were analysed. On an average 36 lymph nodes (range 15–67) were examined histologically per neck dissection specimen , while in seven patients a solitary macrometastasis (H&E) and in one patient a micrometastasis (MNF 116) was proven during histopathological examination (pN1(mi)). In the remaining patient a pitfall occurred (Table 3n=8) and 0% (0/41) in cases of tumour free sentinel nodes (n=41). The number of isolated tumour cells was 21.9% (9/41) in cases of tumourfree sentinel nodes. In the patient with a false negative sentinel node the neck dissection specimens (pN1) contained one further micrometastases.In this clinically N0-neck patient group the rate of occult nodal disease was found to be 18%. The rate of micrometastases (<2 mm) with a size between 0.5–1 mm within the neck dissection specimen was 87.5% (7/8) in cases of histologically proven macrometastases within the sentinel nodes – with respect to the known direct influence of the prognosis – in 41 of 50 investigated pharyngeal and laryngeal cancer patients. At this point it should not be omitted, that, despite a rate of 0% for micrometastases, the rate of isolated tumour cells in the remaining neck dissection specimen was found to be 21.9%. But it has to be pointed out, that this finding was determined throughout an advanced histopathological examination, which does not seem to be feasible for the clinical routine due to high costs and expenditure of labour. The status of such an advanced work-up of the neck dissection specimen can not be estimated at present. We would like to agree with The detection of solitary macrometastasis (pN1) in seven patients and of a micrometastasis (pN1(mi)) in one patient with clinically staged N0 neck seemed to be very interesting. The detailed investigation of each lymph node of the neck dissection specimen revealed in 7/8 patients 1–2 further micrometastases within the lymph nodes of the neck dissection specimen. Among 41 patients without macrometastases were 0/41 patients suffering from micrometastases and 9/41 patients in whom isolated tumour cells could be detected. Thus, the rate of occult metastatic spread in the presented investigation was 18% for nodal disease, which is in accordance with the literature for SCC of the head and neck which we not included in the present study for obvious reasons. For these situations we actually developed an injection via a butterfly cannula , which can be inserted in an oblique angle.Some surgeons perform a selective neck dissection in T3 glottic carcinomas because of the important influence of lymphogenic metastatic spread on prognosis of patients with carcinomas of the head and neck, although the likelihood of occult lymphogenic metastatic spread in T3 glottic carcinoma is only estimated to be 10–15% therapy is necessary or whether a wait and see policy is justified.The radioguided dissection of the 2–3 hottest nodes and the surrounding tissue may be a further step in reduction of radicality in surgery of the clinically staged N0 neck. However, the value of such an approach has to be examined in multicentric trials. As future perspective it may be possible to direct the efforts on a development of an endoscopic neck dissection (

Searching the Web for documents using information retrieval systems plays an important part in clinicians’ practice of evidence-based medicine. While much research focuses on the design of methods to retrieve documents, there has been little examination of the way different search engine capabilities influence clinician search behaviors.Previous studies have shown that use of task-based search engines allows for faster searches with no loss of decision accuracy compared with resource-based engines. We hypothesized that changes in search behaviors may explain these differences.In all, 75 clinicians were randomized to use either a resource-based or a task-based version of a clinical information retrieval system to answer questions about 8 clinical scenarios in a controlled setting in a university computer laboratory. Clinicians using the resource-based system could select 1 of 6 resources, such as PubMed; clinicians using the task-based system could select 1 of 6 clinical tasks, such as diagnosis. Clinicians in both systems could reformulate search queries. System logs unobtrusively capturing clinicians’ interactions with the systems were coded and analyzed for clinicians’ search actions and query reformulation strategies. The most frequent search action of clinicians using the resource-based system was to explore a new resource with the same query, that is, these clinicians exhibited a “breadth-first” search behaviour. Of 1398 search actions, clinicians using the resource-based system conducted 401 in this way. In contrast, the majority of clinicians using the task-based system exhibited a “depth-first” search behavior in which they reformulated query keywords while keeping to the same task profiles. Of 585 search actions conducted by clinicians using the task-based system, 379 were conducted in this way.This study provides evidence that different search engine designs are associated with different user search behaviors. Searching for information on the Web to support decision making is now an important part of clinician practice . While mRecent studies of clinical search strategies have concentrated on methods of optimizing queries sent to information retrieval systems that enhance the performance of the retrieval. Hoogendam and colleagues conducted a prospective observational study of how physicians at a hospital used PubMed to search for information during their daily clinical activities . They foFew studies have looked at how clinicians reformulate queries and select sources to retrieve information during a search session to answer clinical questions. In previous studies, we have shown that a task-based search engine design allows for faster clinical decision making compared with purely resource-based engines at no cost in correctness of answers . SimilarIn all, 75 clinicians practicing in the state of New South Wales, Australia, were recruited to use an online information retrieval system to answer questions on 8 clinical scenarios within 80 minutes in a controlled setting in a university computer laboratory 9]. Par. Par9]. Participants were randomly allocated to use either a resource-based or a task-based version of an online information retrieval system to answer the 8 questions. All participants were given a brief written orientation tutorial regarding their allocated system. Questions were presented in random order. Each participant was asked to use the allocated system to locate documentary evidence to help answer each question. Participants were asked to work through the questions as they would in a real clinical setting and not spend more than 10 minutes on any one question.The search systems used by participants were essentially identical in that both systems allowed users to first select a profile to delimit their search and then to enter keywords to specify the focus of their search. The resource-based system first required clinicians to select a profile by specifying one of six online resources. These included PubMed, MIMS , Therapeutic Guidelines , the Merck Manual, Harrison’s Principles of Internal Medicine, and HealthInsite . Of the six resources, five presented evidence in a predigested, summarized form with references available for follow-up.The task-based system first required the clinicians to select a profile by selecting one of six clinical tasks: diagnosis, drug information, etiology, patient education, treatment, and other . Four keSystem logs unobtrusively capturing participants’ interactions with the systems were coded and analyzed for their search actions and query reformulation strategies. For each clinical scenario question, participants were able to reformulate queries and conduct a sequence of searches as they explored information to assist in answering the question. We first coded these query reformulations by the change in profile selection (task or resource) between consecutive searches in a session as “new profile,” “same profile,” or “previously used profile.” We next coded the keyword changes, as indicating a syntactic and/or a semantic reformulation . ExampleChi-square analyses and the test for difference between proportions were conducted to detect statistically significant differences in profile and query search actions between clinicians using the resource-based and task-based systems.Of 75 clinicians, 39 were randomly allocated to use of the resource-based system and 36 to use of the task-based system. Two resource-based scenarios were not completed, giving a total of 310 search sessions, 1708 searches, and 1455 document accesses using the resource-based system. The task-based system generated 288 search sessions, 873 searches, and 1136 document accesses.2 2 = 103.45, P < .001) , (2) ref < .001) , and (3) < .001) .P < .001), and 5.9% more likely to select a profile that was previously visited and apply no changes to keywords (P < .001), 7.5% to keep the same profile and apply both syntactic and semantic changes to the query , and 6.5% to keep the same profile and apply semantic changes to the query . Also, c < .001) . Further < .001) .P < .001) (P < .001) (P < .001) (P = .006) , and cli < .001) . At the < .001) , and cli = .006) .P < .001) (P < .001) ( < .001) . Among c < .001) .Clinicians using the resource-based system appeared to favor a “breadth-first” search strategy, exploring different resources with the same keywords in the query before searching in a specific resource with query reformulations. Clinicians using the task-based system were provided with results from multiple resources in each search and so appeared to favor a “depth-first” search strategy, searching in the same task profile exhaustively with different keyword reformulations in the query before moving to other profiles.We have previously shown that changes in search engine design and interface were associated with changes in clinical decision velocity, number of search actions undertaken, and ultimate decision outcome . To undeFurther study is needed to understand how clinicians assess the results of a search and formulate the next step in their strategy. We have discussed elsewhere that the process of searching can be thought of as a conversation where inAccording to Grice’s conversational maxims , (which One can hypothesize, when clinicians are faced with a choice of several resources with no clear indication of which is the best, they scan multiple resources to gauge the "competence" of each before committing to a detailed conversation with the resource they feel best qualified to help. In contrast, clinicians with a task-based system are simultaneously receiving answers from multiple resources and so should be able to quickly form a view of the overall capabilities of the group of resources being simultaneously searched. Not faced with concerns about the competence of the system they are interacting with, clinicians focus on improving the dialogue with the system. This is done by finding different ways to ask the same question or by changing the question focus if there has been a “misunderstanding.” As a result, this could explain why users of task-based systems conduct fewer searches and consult fewer documents , that isOverall, given the clear differences in the styles of user-system dialogue demonstrated in this study, and the impact of such behavior on the clinical utility of information retrieval systems, discovering ways of optimizing the dialogue between knowledge sources and users seems a productive line of further enquiry.

Mycobacterium avium subsp paratuberculosis (M. paratuberculosis) using an intratonsillar infection model. In addition, we have recently developed a partial protein array representing 92 M. paratuberculosis coding sequences. These combined tools have enabled a unique look at the temporal analysis of M. paratuberculosis antigens within the native host. The primary objective of this study was to identify M. paratuberculosis antigens detected by cattle early during infection. A secondary objective was to evaluate the humoral immune response in cattle during the initial year of infection.Our laboratories have previously reported on the experimental infection of cattle with M. paratuberculosis specific protein, encoded by MAP0862, was strongly detected initially, but the antibody response became weaker with time. The most reactive protein was a putative surface antigen encoded by MAP1087. A second protein, MAP1204, implicated in virulence, was also strongly detected by day 70 in both cattle. Subsequent experiments showed that these two proteins were detected with sera from 5 of 9 naturally infected cattle in the subclinical stage of Johne's disease.Sera from two experimentally infected cattle, taken pre-inoculation and at day 70, 194 and 321 post infection, identified dynamic antibody reactivity among antigens with some showing an increased response over time and others showing declining levels of reactivity over the same time period. A M. paratuberculosis proteins are detected by sera from experimentally infected cattle as early as 70 days after exposure. These data further suggest at least two antigens may be useful in the early diagnosis of M. paratuberculosis infections. Finally, the construction and use of a protein array in this pilot study has led to a novel approach for discovery of M. paratuberculosis antigens.Collectively these results demonstrate that Mycobacterium avium subsp paratuberculosis (M. paratuberculosis). A recent survey estimated that 20%–40% of dairy herds in the United States are infected with M. paratuberculosis and producers lose $227 USD annually for each infected animal [Johne's disease is an economically significant intestinal disease caused by d animal . These cM. paratuberculosis infection by ingestion of contaminated milk or grass containing fecal material from a shedding cow, there is a lengthy subclinical phase that can last for several years. During this stage the cows may appear healthy, but can intermittently shed low numbers of mycobacteria in the feces, enabling transmission to other animals including wildlife species. A major challenge in controlling Johne's disease is the ability to detect infected cattle prior to appearance of disease signs, such as diarrhea and heavy fecal shedding of M. paratuberculosis. An unknown trigger, possibly stress during lactation or parturition, advances the disease from subclinical to clinical where disease signs such as weight loss and diarrhea become evident [M. paratuberculosis if a subclinically infected animal is not shedding bacilli at the time the fecal or milk sample is collected. M. paratuberculosis antigen induced interferon (IFN)-γ has been shown to be elevated in subclinical animals, but this cytokine declines in the clinical stage concomitant with an increase in M. paratuberculosis specific IL-10 production [M. paratuberculosis-infected cattle [After evident ,3. This evident . Currentoduction ,6. A comd cattle .M. paratuberculosis at early stages of infection in cattle. There are several reasons for this, but one in particular is that cattle that appear healthy are not routinely evaluated using serial test bleeds and analysis. Furthermore, there are numerous studies that show the cell-mediated immune response in cattle predominates during the early stages of infection and is responsible for the initial control of this infection [With a few notable exceptions -10, thernfection ,6,11. HoM. paratuberculosis in neonatal calves [M. paratuberculosis was detected in all calves by 271 days post challenge, no clinical signs were observed in any animal throughout the study [The recent literature has revealed an emphasis on developing consensus animal models for Johne's disease study. One publication is a resl calves . Serial he study . Howeverhe study .M. paratuberculosis gene products [The study described herein combines the intratonsillar infection model with newproducts . That arM. paratuberculosis recombinant fusion proteins including hypothetical proteins (20), known antigens (6), membrane proteins (10), and proteins with no similarity in public sequence databases (25). The affinity tag present within these recombinant fusion proteins is the maltose binding protein (MBP). MBP was also expressed as a fusion with the E. coli LacZ alpha peptide and used as a control to determine antibody reactivity to this tag in each experiment. In addition, there are two spots that contain a sonicated whole cell extract of M. paratuberculosis and one spot with only spotting buffer as a negative control. A complete listing of the proteins present on the array along with their sizes, concentration and predicted function are listed in Table A 96-spot protein array was constructed on nitrocellulose filters using a dot blot apparatus to imprint the array. Represented among these spots are 92 proteins 0, and prThe performance of the array was tested using a monoclonal antibody developed to the MBP tag as shown previously . These eM. paratuberculosis proteins. In general, the antibody response against the set of 92 proteins was remarkably similar between the two animals. One notable difference was that fewer proteins were recognized at day 70 in animal 5904 compared to 5902, as only 11 proteins compared to 27 proteins were detected, respectively need to be included along with MAP1204 and MAP1087 in order to detect all nine subclinical cows.A panel of sera collected from 9 cows in the subclinical stage of infection was analyzed by immunoblot against the two strongest antigens identified from the protein array experiments. These animals are housed at the National Animal Disease Center and are monitored four times a year for shedding of bacilli and immunological status as measured by ELISA assays for antibody and IFN-γ Table . MAP1087M. paratuberculosis early during infection. Antigen-based diagnostic tests for Johne's disease currently use a complex mixture of antigens, such as PPD or mycobacterial membrane fractions, that can exhibit cross reactivity with other mycobacteria [M. paratuberculosis as this information now enables researchers to select and characterize M. paratuberculosis sequences of interest. Our interest is in characterizing those sequences that are novel and specific to M. paratuberculosis in order to develop the best available diagnostic tools. This study represents the most comprehensive M. paratuberculosis antigen analysis on a temporal scale. Data from this study provide a first look at how specific antibody reactivity changes over time and in direct comparison to other proteins.Diagnosis of Johne's disease is difficult because as many as 90% of infected animals may show no signs of disease during sample collection and there is a lack of sensitive and specific assays that detect bacteria . ResultsM. paratuberculosis. The current study extends these initial findings by using a recently developed protein array to probe sera from these experimentally infected calves. The two proteins that elicited the strongest antibody reactivity were MAP1087, a probable peptide transport system permease and MAP1204, a p60 protein homolog. These proteins have a calculated size of 15.4 kDa and 25.4 kDa, respectively. Therefore, it is unlikely that the proteins encoded by MAP1087 and MAP1204 are the same 2 antigens detected in the initial study by Waters et al [M. smegmatis and M. avium strain 104. This raises the potential for cross-reactivity if these antigens were incorporated into a diagnostic test for Johne's disease.When a temporal analysis of serial bleeds from experimentally infected calves was undertaken, we discovered a 50-kDa protein that was detected by day 14 post-challenge and a 60-kDa protein was evident by day 42 . Howeverrs et al . AlthougM. paratuberculosis protein array was initially constructed from 43 recombinant proteins and tested with sera from a variety of host species [M. paratuberculosis. Performing a test to detect M. paratuberculosis antigens when clinical signs of the disease are already evident in the host, make the control of this disease by test and cull strategies futile. An optimized serological test that incorporates antigens detected at early times post-infection is a critical tool that animal producers/farmers need to control Johne's disease. In this way infected animals can be identified prior to shedding and exposure of herd mates to the disease causing bacterium. Regarding uninfected cows, one key advantage of the experimental model used is that each cow had its own animal-specific negative control (the preinfection bleed). This type of control is not available in most Johne's disease studies. In summary, a highly specific test that could identify infected animals within herds may have a greater and more immediate impact on cattle industries than any other current management approach, including vaccination.A species . The proM. paratuberculosis protein antigen likely remains undiscovered simply because all the proteins produced by M. paratuberculosis are not represented on the current protein array. In this study, promising candidates have been found, but the genome sequence annotation suggests that M. paratuberculosis produces a total of 4,530 proteins [The ideal proteins . TherefoM. paratuberculosis. Those studies have revealed less than 40 coding sequences that are uniquely present in M. paratuberculosis [M. paratuberculosis-specific sequences for immunoreactivity with sera from animals exposed to M. paratuberculosis [M. paratuberculosis protein array has many of these same coding sequences represented. Both MAP0862 and MAP2963c were detected in each animal in this study, but MAP3732c is not present on the array used in the current study. The antigens identified in this study should be further evaluated with naturally infected animals in field trials before incorporation into a diagnostic test for Johne's disease.Our laboratory, in collaboration with others, has successfully completed the genome sequence of this significant veterinary pathogen and haverculosis ,25. Thisrculosis . Among trculosis . Furtherrculosis . Those sM. paratuberculosis proteins analyzed in this study, 2 emerged as potential antigens for the early detection of Johne's disease in cattle. These proteins are encoded by MAP1087 and MAP1204. The combination of MAP1087 and MAP1204 detected more subclinical cows than either protein alone, suggesting that the ideal detection antigen will comprise a mixture of more than one protein. Finally, this is a pilot scale study that demonstrates the method is effective for use in a large-scale antigen discovery project.Of the 92 recombinant M. paratuberculosis by instillation of four weekly doses of approximately 4 × 106 CFU (in 0.2 ml PBS) into both tonsillar crypts weekly from 2 to 5 weeks of age. The subclinical cows used in this study comprised Holsteins, Brown Swiss and Guernsey with ages that ranged from 2–7 years.All cattle used in this study were housed at the National Animal Disease Center (NADC) and handled using institutional care guidelines set by the animal care and use committee. Experimentally infected calves were put in biosafety level 2 containment immediately following birth to prevent exposure to environmental mycobacteria. Naturally infected subclinical cattle are contained on a field pasture-barn set up on site at NADC. The experimentally infected animals consisted of male Holstein calves less than 1 year of age . The heaM. paratuberculosis proteins recombinantly produced in E. coli with 89 of these using the pMAL-c2 expression vector (New England Biolabs), and 3 using the pET vector. All expression clones were sequenced to confirm that the cloned insert matched the native M. paratuberculosis gene and was in-frame with expression signals built into the expression vector. One spot on the array contains the MBP/LacZ protein used as a control to assess antibody reactivity to the MBP affinity tag. This control protein comprises the 42-kDa maltose binding protein along with the 8-kDa LacZ alpha peptide. Two spots contain a sonicated whole cell extract of M. paratuberculosis prepared as described previously [The protein array consists of a 96-dot format of proteins spotted onto nitrocellulose. There are 92 total M. paratuberculosis proteins. All protein arrays were sequentially produced and no single array was used for more than one experiment. To print each array, the nitrocellulose membrane, pre-soaked in PBS, was placed over a rubber gasket such that it covered all wells of the Bio-dot 96-well manifold apparatus . The top of the Bio-dot apparatus was then positioned over the membrane and fastened in each corner. Each well was pre-washed with 200 μl of PBS followed by a brief vacuum to pull the buffer through the membrane. Diluted working stocks of purified proteins were stored in deep 96-well plates with a 1-ml capacity per round bottom well . Transfer of these proteins from the deep well plate to the assembled Bio-dot apparatus was performed using an 8-channel multi-pipette. Each well of the assembled 96-well dot blot apparatus was loaded with the appropriate protein at a final concentration of 3 μg/well as measured in a NanoDrop spectrophotometer (Thermo Fisher Scientific) at 280nm. The PBS-diluted samples were allowed to flow through the membrane by gravity flow. Each well was rinsed with 200 μl of PBS, 0.1% Tween-20 (TPBS). The vacuum was engaged to pull the rinse solution through and the nitrocellulose membrane was removed from the apparatus and placed in a Petri dish containing a blocking solution . After 1 hour in the blocking solution, the immunoblot assay was performed.PBS was used as the spotting buffer and diluent for all M. paratuberculosis infected calves were diluted in the blocking buffer and exposed to spotted arrays for two hours at room temperature on a rocker platform. All cattle sera were diluted 1:500. After three washes in TPBS, the nitrocellulose array was incubated for 1.5 h with Horse Radish Peroxidase-conjugated anti-bovine IgG antibody (Pierce) diluted at 1:20,000 in TPBS-BSA. This was followed by a final three washes in TPBS. Assay development was with SuperSignal detection reagents (Pierce) and Kodak BioMax MR film.Serum from Spot intensity was measured using the Adobe Photoshop CS3 extended application. This version has the ability to record pixel gray values using the measurement scale of 1 pixel equals 1 pixel. Each spot was measured identically using a window area of 1804 pixels. Values were exported into a spreadsheet for further analysis. The background statistics were calculated by determining the mean and standard deviation of the 24 spots within each array that had the least signal intensity. Each intensity score was compared to the calculated background intensity. Values that were within two standard deviations of the background mean were set to zero and all other intensity values were subtracted from the background mean to give positive intensity values that were adjusted for the array background. To determine the reactivity distribution of all proteins in the complete set, the highest intensity score for each protein was examined as a way to determine if there was distinct stratification of maximal antibody response. Kernel Density Estimation performed on the log-transformed maximum intensity scores indicated there were three distinct normally distributed peaks. These distributions define the proteins belonging to the low, medium, or high reactivity groups presented in Figure JPB conceived of the study, printed the protein arrays, participated in study design and coordination and drafted the manuscript. DOB provided bioinformatics support, statistical analysis and assisted with densitometric analyses of protein arrays. WRW and MVP conceived the experimental calf challenge model and performed the immunological characterizations. JRS carried out the immunoassays and determined immunological status for the subclinical cattle. MLP participated in the design of the study and performed bioinformatics analyses. All authors read and approved the final manuscript.

Commercial samples with high and low zinc levels were analysed by the proposed method and the results were compared with those obtained with the standard ASTM method. The t test for mean values showed no significant differences at the 95% confidence level. Precision (RSD%) was better than 5% for the high concentrations system. The carryover between successive injections was found to benegligible.A flow-injection system is proposed for the determination of metal-based additives in lubricating oils. The system, operating under computer control uses a motorised syringe for measuring and injecting the oil sample (200 μL) in a kerosene stream, where it is dispersed by means of a packed mixing reactor and carried to an atomic absorption spectrometer which is used as detector. Zinc was used as model analyte. Two different systems were evaluated, one for low concentrations (range 0–10 ppm) and the second capable of providing higher dilution rates for high concentrations (range 0.02%–0.2% w/w). The sampling frequency was about 30 samples/h. Calibration curves fitted a second-degree regression model (

Liver-selective thyromimetics have been reported to efficiently reduce plasma cholesterol through the hepatic induction of both, the low-density lipoprotein receptor (LDLr) and the high-density lipoprotein (HDL) receptor; the scavenger receptor class B type I (SR-BI). Here, we investigated the effect of the thyromimetic T-0681 on reverse cholesterol transport (RCT) and atherosclerosis, and studied the underlying mechanisms using different mouse models, including mice lacking LDLr, SR-BI, and apoE, as well as CETP transgenic mice.T-0681 treatment promoted bile acid production and biliary sterol secretion consistently in the majority of the studied mouse models, which was associated with a marked reduction of plasma cholesterol. Using an assay of macrophage RCT in mice, we found T-0681 to significantly increase fecal excretion of macrophage-derived neutral and acidic sterols. No positive effect on RCT was found in CETP transgenic mice, most likely due to the observed decrease in plasma CETP mass. Studies in SR-BI KO and LDLr KO mice suggested hepatic LDLr to be necessary for the action of T-0681 on lipid metabolism, as the compound did not have any influence on plasma cholesterol levels in mice lacking this receptor. Finally, prolonged treatment with T-0681 reduced the development of atherosclerosis by 60% in apoE KOs on Western type diet. In contrast, at an earlier time-point T-0681 slightly increased small fatty streak lesions, in part due to an impaired macrophage cholesterol efflux capacity, when compared to controls.The present results show that liver-selective thyromimetics can promote RCT and that such compounds may protect from atherosclerosis partly through induction of bile acid metabolism and biliary sterol secretion. On-going clinical trials will show whether selective thyromimetics do prevent atherosclerosis also in humans. We further studied the impact of T-0681 on the development of atherosclerosis in mice, and analyzed the underlying mechanisms.In preliminary dose-titration studies in wild-type (WT) mice we observed a marked increase of hepatic SR-BI expression at 36 nmol/kg/d T-0681, and a concomitant 50% decrease of plasma cholesterol. Higher doses than 36 nmol/kg/d showed no further lipid-lowering effect (data not shown). Accordingly, Parini and coworkers recently presented data on SR-BI-inducing properties of the thyromimetic GC-1 in liver of WT mice In subsequent experiments in WT mice, 36 nmol/kg/d of T-0681 was found to reproducibly increase hepatic SR-BI expression and to decrease both LDL-C and high-density lipoprotein cholesterol . Plasma [3H]-cholesterol levels tended to be reduced . In addition, in contrast to WT mice, no induction of hepatic CYP7A1 and ABCG5/G8 was observed , via apoB-containing lipoproteins Interestingly, we observed no effect of T-0681 on hepatic LDLr expression of WT mice (data not shown). This finding is in agreement with data by Parini and coworkers P<0.01), along with a marked decrease in plasma cholesterol . No difference was observed in the hepatic expression of SR-BI, LDLr, ABCG5/G8 and CYP7A1 . There was no difference in both hepatic SR-BI and LDLr protein expression. However, we found a 3-fold upregulation of ABCG5, and a 2-fold increase of CYP7A1 in livers of T-0681 treated apoE KOs All animals were handled in strict accordance with good animal practice as defined by the Austrian Authorities, and all animal work was approved by the Austrian Animal Care and Use Committee. Male C57/B6 (WT) mice, obtained from Charles River Laboratories , were fed a standard chow diet . After 2 weeks of acclimatization, mice were divided into two groups and subcutaneously implanted with Alzet micro-osmotic pumps carrying T-0681 in PBS (36 nmol/kg/d) or PBS alone as control for 14 days. After 14 days of treatment, animals were fasted for 5 h and anesthetized. Blood samples were taken, mice sacrificed by cervical dislocation, and organ biopsies were snap-frozen.Male SR-BI KO and LDLr KO mice, obtained from Jackson Laboratories were fed a standard chow diet , and subcutaneously implanted with Alzet micro-osmotic pumps carrying T-0681 in PBS (36 nmol/kg/d) or PBS alone as control. Mice were then fasted for 5 h, blood samples were taken, mice were sacrificed by cervical dislocation, and liver biopsies were snap-frozen.2. Macrophage in vivo RCT studies were performed as described 3H]-cholesterol and 40 µg/ml acetylated LDL (AcLDL) 3H]-cholesterol–labeled and AcLDL-loaded J774 cells were injected intraperitoneally. Feces were collected continuously from 0 to 48 h and stored at 4°C before extraction of cholesterol and bile acids. At study termination (48 h after injection), mice were exsanguinated, perfused with cold PBS, and portions of the liver were removed and snap-frozen. Liver lipid, fecal cholesterol as well as bile acid extractions were performed as described 6 J774 macrophages containing 0.65×106 cpm in 0.6 ml PBS.J774 macrophages were obtained from the American Type Culture Collection and cultured in supplemented DMEM medium at 37°C and 5% CO6 cells containing 0.6×106 cpm in 0.6 ml PBS) were performed as described elsewhere Macrophage in vivo RCT studies in male WT mice with primary murine bone marrow-derived macrophages for 48 h. Then cells were washed and equilibrated overnight in either the presence or absence of T-0681 (100 nM) in serum-free medium. For the cholesterol efflux, medium containing 2.5% mouse plasma from the 4-weeks and the 8-weeks studies was added to cells. After 4 hours, supernatant was collected and centrifuged . Cells were washed with PBS and lyzed in 1 ml 0.1 N NaOH. 300 µl of supernatant and cell lysate were measured in a liquid scintillation counter. Percentages of cholesterol efflux were calculated using following formula: J774 macrophages were labeled with [Male apoE KO mice were obtained from Jackson Laboratories After 2 weeks of acclimatization, mice were set on a Western type diet , divided into two groups and subcutaneously implanted with Alzet micro-osmotic pumps carrying T-0681 in PBS (36 nmol/kg/d) or PBS alone as control. After 4 and 8 weeks, mice were fasted for 5 h, blood samples were taken, mice sacrificed by cervical dislocation, and liver biopsies were snap-frozen. Hearts were prepared and atherosclerotic lesions quantified as described Total cholesterol was measured in whole plasma of each animal employing an ABX Diagnostics commercial kit . Additionally, pooled plasma of each group was subjected to FPLC fractionation analysis with two tandem Superose 6 columns as described previously 50 mg of dried feces were boiled in 1 ml alkaline methanol at 80°C for 2 h after addition of 50 nmol 5α-Cholestane as internal standard for neutral sterol analysis. After cooling down to room temperature, neutral sterols were extracted using three times 3 ml of petroleum ether, boiling range 60–80°C. The residual sample was diluted 1∶9 with distilled water. 100 µl of the solution were subjected to an enzymatic total bile acid measurement Sitosterol and campesterol were extracted from 10 µl plasma from each animal in duplicate samples. Samples were derivatized with trimethylsilane reagent prior to gas-chromatography-mass spectrometry analysis Preparation of proteins from murine liver samples as well as from T-0681-treated J774 macrophages and subsequent Western blot analysis were performed as described Total RNA was extracted using RNA bee according to the manufacturer's protocol and reverse transcribed with Omniscript-RT Kit . Primers and probes for murine ABCA1, ABCG5, ABCG8, CYP7A1 were described previously t-test for unpaired data. A difference was considered statistically significant when P<0.05.Results are presented as mean ± s.e.m. The statistical significance of the differences between the means of the experimental groups was tested by the Student's

Borrelia burgdorferi species, and act as dilution hosts in parts of North America. Whether European lizards significantly reduce the ability of B. burgdorferi to maintain itself in enzootic cycles, and consequently decrease the infection rate of Ixodes ricinus ticks for B. burgdorferi and other tick-borne pathogens in Western Europe is not clear.Lizards are considered zooprophylactic for almost all B. burgdorferi sensu lato, compared to questing ticks in heath (24%) or forest (19%). The prevalence of Rickettsia helvetica was significantly higher in ticks from lizards (19%) than those from woodland (10%) whereas neither was significantly different from the prevalence in ticks from heather (15%). The prevalence of Anaplasma and Ehrlichia spp in heather (12%) and forest (14%) were comparable, but significantly lower in ticks from sand lizards (5.4%). The prevalence of Babesia spp in ticks varied between 0 and 5.3%. Tick load of lizards ranged from 1 - 16. Tick densities were ~ 5-fold lower in the heather areas than in woodlands at all four sites.Ticks were collected from sand lizards, their habitat (heath) and from the adjacent forest. DNA of tick-borne pathogens was detected by PCR followed by reverse line blotting. Tick densities were measured at all four locations by blanket dragging. Nymphs and adult ticks collected from lizards had a significantly lower (1.4%) prevalence of B. burgdorferi s.l. infection rate of questing ticks. In contrast, sand lizards might act as reservoir hosts for R. helvetica. Remarkably, the public health risk from tick-borne diseases is approximately five times lower in heather than in woodland, due to the low tick densities in heather.Despite their apparent low reservoir competence, the presence of sand lizards had insignificant impact on the Rickettsia species have been identified as causative agents of tick-borne rickettsioses in Europe [B. burgdorferi in Europe is Ixodes ricinus. This tick species feeds on a wide variety of warm- and cold-blooded vertebrates, including humans. Some of these tick hosts are incompetent hosts for various B. burgdorferi genospecies. The hypothesized origin of such specific associations is that different groups of vertebrates exhibit distinct types of innate immunity, which may either tolerate or destroy certain B. burgdorferi genospecies [Several tick-borne diseases are emerging in Europe as shown by the example of a dramatic increase of Lyme disease cases over the last decade ,2. Furthn Europe ,4. The un Europe -8. They ospecies -11. IncoLacerta agilis (sand lizards), have been identified as incompetent hosts for all B. burgdorferi genospecies, except B. burgdorferi lusitaniae [B. burgdorferi to maintain itself in an enzootic cycle [B. burgdorferi in ticks and diverting tick bites away from competent hosts [At least eight lizard species, including sitaniae -21. The sitaniae -24. The ic cycle ,26 and mnt hosts ,27. Thisnt hosts . Lizardsnt hosts ,30. In tnt hosts ,32.I. ricinus on sand lizards. The mean number of ticks per lizard varied from 0.2 to 23 and maximal numbers of ranged from 42 to 61 in different study areas [I. ricinus. Small rodents, probably the most relevant B. burgdorferi reservoir in the Netherlands, serve mainly as hosts for the larval stage of I. ricinus while sand lizards feed often a much higher proportion of nymphs [B. burgdorferi s.l species except B. burgdorferi lusitaniae [B. burgdorferi prevalence in questing ticks have not been investigated.Various studies in the Netherlands and other European countries evaluated the prevalence of dy areas -35. Thesrodents) ,36. Due sitaniae ,20, but Rickettsia helvetica was identified the most prevalent potentially pathogenic Rickettsia in Dutch ticks [R. helvetica can be transmitted vertically from one tick generation to the next by trans-stadial and transovarial transmission it is less dependent on the presence of competent vertebrate hosts and therefore a dilution effect is less likely to occur [Rickettsia in tick populations [R. helvetica whereas no incompetent hosts have been found so far [R. helvetica is unclear, but infection with R. helvetica has been suspected in acute perimyocarditis, unexplained febrile illness and sarcoidosis [Recently, ch ticks ,38. As Rto occur ,39. Neveulations ,40. A fed so far ,41. To dcoidosis -49.Ehrlichia and Anaplasma species are intracellular bacteria that can be transmitted by tick bites. The causative agent for human monocytic anaplasmosis is Ehrlichia chaffeensis [Anaplasma phagocytophilum [Anaplasma and Ehrlichia in Dutch ticks found mainly Ehrlichia schotti variant, which is of unknown pathogenicity [ffeensis and an otophilum . A recengenicity .Babesia species are known to cause disease in humans, cattle and companion animals and are usually transmitted by tick bites. In Europe, Babesia divergens is thought to be the most important species to cause human disease but other species have recently been identified as human pathogens in Europe as well [B. microti and a Babesia EU1 (proposed name Babesia venatorum) have been repeatedly been identified in ticks in the Netherlands [Various as well . The pro as well ,54. B. mherlands .B. burgdorferi s.l. and R. helvetica. A non-invasive field study was set up which includes the collection of ticks from lizards and vegetation in their habitats as well as from vegetation in geographically nearby control areas that did not serve as a lizard habitat. Tick and lizard densities as well as the infection rate of the different habitats were determined and compared.Here, we investigated the role of sand lizards in the ecology of tick-borne pathogens, in particular et al. in four different geographical upcountry areas from both heathland (sand lizard habitat) and forest areas (unsuitable lizard habitat), which were directly adjacent to each other [Field sampling was conducted between May and October in the years 2007 till 2009. Ticks were collected by flagging vegetation as described by Wielinga ch other . Sand liTick densities were estimated at the different sampling locations by dragging a 100 cm wide flannel cloth over a distance of 100 m with checking for ticks every 25 m. Nymphs and adult ticks were collected from the cloth and counted. This was repeated four times at each location and the counts were averaged. Tick densities of adjacent heath and forest areas were determined on the same day and under equal weather conditions. Lizard densities were estimated by expert judgment, based on the yearly monitoring data from all study areas .Rickettsia spp., B. burgdorferi s.l., Ehrlichia/Anaplasma spp. and Babesia spp.) was determined by polymerase chain reaction (PCR) followed by reverse line blotting (RLB) as described before [Total DNA was extracted from the ticks by alkaline lysis as described elsewhere . DNA extd before ,57,58. TB. burgdorferi s. l. was amplified by PCR with the HotStarTaq master mix with the following conditions: 15 min 94°C, then cycles of 20 s 94°C, 30 s 70°C, 30 s 72°C lowering the annealing temperature 1°C each cycle till reaching 60°C, then 40 cycles at this annealing temperature and ending by 7 min 72°C.The 23S-5S intergenic spacer region of Rickettsia species was amplified by PCR with the HotStarTaq master mix with the following conditions: 15 min 94°C, then cycles of 20 s 94°C, 30 s 72°C, 30 s 72°C lowering the annealing temperature 1°C each cycle till reaching 62°C, then 40 cycles at this annealing temperature followed by a final elongation step for 7 min at 72°C. For this study we designed two new RLB probes that are able to hybridize to DNA of most Rickettsia species except for R. helvetica and closely related species. These probes were designed with the purpose to establish the occurrences of possible multiple infections of ticks with Rickettsia species. Newly designed probes were tested with a series of samples and positive controls before incorporating them in the assay (not shown). In a second PCR on a small subset of samples a longer fragment of the 16S rRNA gene was amplified and sequenced to be able to compare the sequences in more detail.The 16S rRNA gene of Ehrlichia and Anaplasma species was amplified by PCR with the HotStarTaq master mix with the following conditions: 15 min 94°C, then cycles of 20 s 94°C, 30 s 65°C, 30 s 72°C lowering the annealing temperature 1°C each cycle till reaching 55°C, then 20 cycles at this annealing temperature and an additional 20 cycles with an annealing temperature of 63°C followed by a final elongation step for 7 min at 72°C.The 16S rRNA gene of Babesia was amplified by PCR using a reaction mix containing 1× buffer, 1.75 mM magnesium chloride, 200 μM dNTPs, 0.25 μM of each primer and Taq polymerase. The PCR reaction was run under following conditions: 10 min 94°C, then cycles of 20 s 94°C, 30 s 67°C, 30 s 72°C lowering the annealing temperature 1°C each cycle till reaching 57°C, then 40 cycles at this annealing temperature followed by a final elongation step for 7 min at 72°C.The 18S rRNA gene of http://www.ncbi.nlm.nih.gov/ using BLAST. The sequences were aligned and analyzed using BioNumerics 5.1 .To minimize cross contamination and false-positive results, positive and negative controls were included in each batch tested by the PCR and RLB assays. Furthermore DNA extraction, PCR mix preparation, sample addition, and PCR analysis were performed in assigned separate labs. PCR products of some samples were sequenced by dideoxy-dye termination sequencing of both strands, and compared with sequences in Genbank Data were analyzed using tools provided by OpenEpi and QuanIn total 713 ticks were collected: 491 from vegetation and 222 from lizards Table , with anB. burgdorferi s.l., Rickettsia and Ehrlichia/Anaplasma species were present in ticks collected from all geographical areas to 26% . From the 58 ticks collected from lizards from the Hullenberg area, only three were positive for B. burgdorferi genospecies while ticks from forest (woodland) and heather in this area had significantly higher infection rates of 21% and 44% , respectively. The only B. burgdorferi species associated with lizards, B. lusitaniae, was not detected in any tick tested during this study. The infection rates were not significantly different between questing ticks in forest (woodland) and heathers areas. The genospecies of B. burgdorferi isolates were determined by RLB. B. afzelii was the most prevalent B. burgdorferi species in ticks from all areas and vegetation types. Other species identified were B. burgdorferi garinii in a total of 5 ticks, all collected from woodlands, B. burgdorferi sensu stricto (n = 6) and B. burgdorferi valaisiana . Eight samples hybridized with the catch-all probe for B. burgdorferi s.l. but could not be identified to the species level by RLB. The 5S-23S rRNA intergenic spacer regions of these samples were later sequenced and identified as B. afzelii (not shown).PCR and RLB showed that as Table . The oveRickettsia spp. were found in all four investigated areas in ticks collected from heather, woodland and lizards with high spatial variations and infection rates ranging from 5-33% (Table Rickettsia spp. were identified as R. helvetica by RLB. The remaining positive samples hybridized with a Rickettsia typhi probe (n = 3) or the catch-all probe for Rickettsia spp. only. The 16S rRNA sequences of these samples were later identified as Rickettsia bellii-like sequences with 1 to 6 of about 356 nucleotides difference to R. bellii (Genbank accession No U11014). No infections with more than one Rickettsia species were found.3% Table . Compari3% Table . Further3% Table . Eighty-Ehrlichia/Anaplasma spp. were found with infection rates between 0 and 26% with highly variable infection rates between the three areas and within the different tick groups. From all ticks collected from Heumensoord only one (0.8%) was positive for Ehrlichia/Anaplasma while in Hullenberg 15% were positive. All but nine positive ticks were identified as Ehrlichia schotti. Two were identified by RLB as Anaplasma phagocytophilum. The remaining seven samples could not be determined to the species level. Ehrlichia/Anaplasma prevalence was significantly higher in ticks collected from forest (p = 0.002) and heather (p = 0.02) than in ticks collected from lizards . This study indicates that sand lizards are probably incompetent hosts for B. burgdorferi species commonly present. To confirm the incompetence of sand lizards to acquire or transmit B. burgdorferi further studies and direct sampling of lizard tissue will be necessary.The current study confirms earlier observations ticks taken from lizards, including sand lizards, are almost free of eri s.l. ,21,29,63is study . Only veB. burgdorferi prevalence in questing ticks collected from forest and heather vegetation were similar. How high the B. burgdorferi prevalence would be in the same habitat without lizards is unknown. Our data corroborate the previous notion that an incompetent host does not automatically act as a dilution host [B. burgdorferi infestation rate in questing and attached ticks would further deepen the knowledge on the specific characteristics of lizards as possible dilution hosts for B. burgdorferi. A high passive mobility of ticks would also lead to an attenuation of a dilution effect [B. burgdorferi s.l., like a decrease of its overall prevalence in questing ticks, but also on the emergence of adapted species like B. burgdorferi lusitaniae and other tick-borne pathogens.We did not find evidence of a dilution effect of sand lizards since the ion host ,28. Studn effect that wouR. helvetica was the most prevalent Rickettsia species found in The Netherlands [R. helvetica is of uncertain pathogenicity but in recent years increasing numbers of publications confirm the pathogenic status of R. helvetica [R. typhi was found in three ticks. The DNA of R. typhi has been detected in tick lysates before, but its relevance is unclear [R. bellii.As found previously, herlands . R. helvelvetica -49. Scan unclear . SeveralR. helvetica prevalences in larvae, nymphs and adult ticks during a longitudinal study and concluded that ticks are a major reservoir for R. helvetica in the Netherlands [et al. reported much lower infection rates in nymphs than in adult ticks which might indicate the necessity of reservoir hosts to sustain rickettsial infection of a tick population on a long term [R. helvetica. Blood and tissue samples of lizards could have delivered additional information on the ability of sand lizards to maintain and transmit rickettsial infection but obtaining these would have been invasive and was therefore not possible.High spatial variations of Rickettsia infection rates as found during this study had been observed in earlier studies at our laboratory and have also been reported by other groups . Variatiherlands . Contrarong term . ProbablFor Anaplasma species the opposite picture was observed than for Rickettsia. Prevalence of Anaplasma was significantly lower in ticks collected from lizards compared to those from forest and heather vegetation indicating that lizards might be incompetent hosts for these pathogens.R. helvetica. Low tick density in heather had been reported previously [A dilution effect for Anaplasma was not observed in the ticks collected from heather. In order to assess health risks for humans we attempted to calculate the infected-tick-density. The density of infected ticks is an approximation of the exposure risk and is therefore of importance when evaluating infection risks. Tick densities and density of infected ticks in forests were much higher (p < 0.0001) than in the corresponding heather areas the risk of exposure to a tick bite is higher as well. So even though the presence of lizards in heathland did not lead to a dilution effect, the risk of contracting Lyme disease is much lower in heathlands. The same is true for eviously ,67 and cB. burgdorferi prevalence in ticks. However, as lizard density data are only rough estimates, the positive association could not be tested with statistical methods. This study has shown that a single vertebrate species correlates with different tick-borne pathogens in opposing directions. While sand lizards are probably incompetent hosts for the B. burgdorferi species present in the Netherlands they seem to be reservoir hosts for Rickettsia spp. The different impacts of any single vertebrate host on tick-borne diseases will make it difficult to reduce the overall tick-borne disease risks for man and pets by manipulating vertebrate host communities. Reduction of tick-densities is a more immediate measure to reduce exposure and would evenly decrease the risk of all tick-borne diseases. A reduction of tick densities achieved by altering vegetation or reducing the abundance of vertebrate hosts for adult ticks is unpredictable and not ecologically acceptable. The low tick numbers in heather mean a decreased risk for Lyme disease or rickettsioses and with regard to tick-borne diseases heathland therefore seems a safer recreational area than woodland.The four areas were classified as having very high (Leusderheide and Hullenberg), high (Bergherbos) and medium (Heumensoord) lizard densities. Surprisingly, the lizard density seems to be positively associated with The authors declare that they have no competing interests.ET and MF collected data, performed lab tests and developed new methodology. ET analyzed data and wrote initial draft. HS and JR designed the study and wrote the final manuscript. AS designed the study, collected data, provided data on lizard density, and co-refined the intellectual content of the manuscript.

To the Editor:Acinetobacter species are ubiquitous in the environment. In recent years, some species, particularly A. baumannii, have emerged as important nosocomial pathogens because of their persistence in the hospital environment and broad antimicrobial drug resistance patterns is a commensal organism of human skin, oropharynx, and perineum that shows tropism for urinary tract mucosa .All 10 patients were immunocompromised; 8 had used an intravascular catheter and 2 had used a urinary catheter. Blood cultures of the patients were analyzed with the BacT/ALERT 3D system . Isolates were identified as A. lwoffii isolates to 18 antimicrobial drugs were determined by the broth microdilution method, according to Clinical and Laboratory Standards Institute guidelines (A. lwoffii resistant to >4 classes of drugs were defined as multidrug-resistant (MDR) isolates.Susceptibilities of 10 A. lwoffii isolates were genotyped by pulsed-field gel electrophoresis (PFGE) to determine their epidemiologic relatedness. Chromosomal DNA was digested with SmaI (>4 bands) and aztreonam. The other 4 isolates were MDR: 3 were susceptible only to imipenem (MICs 1–4 μg/mL), meropenem (MICs 1–2 μg/mL), and amikacin (MICs 2–4 μg/mL). The fourth MDR strain was susceptible to imipenem (MIC 2 μg/mL), meropenem (MIC 2 μg/mL), amikacin (MIC 4 μg/mL), and ciprofloxacin (MIC 1 μg/mL). Seven antimicrobial drug resistance profiles were detected (Among the 10 detected .A. lwoffii isolates identified 8 distinct PFGE types. Two MDR strains (strains 2 and 3 in the Table), which were isolated from patients in different wards, and 2 non-MDR strains (strains 8 and 9), which were isolated from patients in the same ward, had similar PFGE patterns and identical resistance phenotypes. These findings suggest nosocomial transmission. Nine of the 10 patients survived after catheter removal or treatment with appropriate antimicrobial drugs. These results confirm that catheter-related A. lwoffii bacteremia in immunocompromised hosts is associated with a low risk for death (Macrorestriction analysis of the A. lwoffii MDR strains that cause bacteremia in immunocompromised catheterized patients. Our data are consistent with those of previous reports on the role of catheters as the principal source of A. lwoffii infections.This study identified

For the newly introduced class of integrase inhibitors, a huge discrepancy between these two measures of efficacy was observed. Hence, a thorough understanding of the relation between these two measures of drug efficacy is imperative for guiding future drug discovery and development activities in HIV. In this article, we developed a novel viral dynamics model, which allows for a mechanistic integration of the mode of action of all approved drugs and drugs in late clinical trials. Subsequently, we established a link between in vivo and in vitro measures of drug efficacy, and extract important determinants of drug efficacy in vivo. The analysis is based on a new quantity—the reproductive capacity—that represents in mathematical terms the in vivo analog of the read-out of a phenotypic assay. Our results suggest a drug-class specific impact of antivirals on the total amount of viral replication. Moreover, we showed that the (drug-)target half life, dominated by immune-system related clearance processes, is a key characteristic that affects both the emergence of resistance as well as the in vitro–in vivo correlation of efficacy measures in HIV treatment. We found that protease- and maturation inhibitors, due to their target half-life, decrease the total amount of viral replication and the emergence of resistance most efficiently.Predictive markers linking drug efficacy to clinical outcome are a key component in the drug discovery and development process. In HIV infection, two different measures, viral load decay and phenotypic assays, are used to assess drug efficacy in vivo measure of drug efficacy, whereas phenotypic assays are used to assess drug efficacy in vitro. The recent development of novel HIV drugs resulted in a huge discrepancy between viral load decay and in vitro predictions of drug efficacy. We used a mathematical modelling approach to resolve this discrepancy by introducing a new quantity, the reproductive capacity, that allows a transfer of the in vitro drug efficacy measure into the in vivo context, enabling a direct comparison. We developed a novel model of viral dynamics that incorporates the mechanism of action of all established and novel antivirals. Based on the model, we analyzed the ability of the viral infection to replicate under different drug treatments, and estimated class-specific times until virological failure. We conclude that the half life of the targeted viral stage is an important class-specific attribute that impacts on the overall success of a drug in vivo. Our findings have direct implication for the drug discovery and development process.To guide drug discovery and development, measures of drug efficacy that are linked to clinical outcome are of key importance. In HIV treatment, decay of plasma viral load is typically used as an Since 1996, human immunodeficiency virus (HIV) infection is treated with a combination therapy, known as highly active anti-retroviral therapy (HAART) in vivo potency of novel antivirals is usually assessed by viral load decline in small clinical trials of monotherapy, e.g., in vitro potency of antivirals is typically assessed by using phenotypic/single-round infectivity assays During therapy, plasma viral load (HIV RNA per mL blood plasma) is recommended by the National Institute of Health as a marker of therapy success in vitro, using phenotypic/single-round infectivity assays, and in vivo, using viral load decline, was observed in vitro efficacy is amongst the lowest Investigation of novel drug targets for the treatment of HIV infection resulted in the development of new drug classes. In 2003 and 2007, the fusion inhibitor (FI) enfuvirtide in vivo dynamics of HIV. Sedaghat et al. in vivo potency.Mathematical modelling of viral dynamics has lead to many insights into the pathogenesis and treatment of HIV. It is a valuable tool to interpret the time course of virological markers during HIV treatment Consequently, viral load decay may be misleading for assessing the potency of integrase inhibitors (and other novel inhibitors) in comparison to existing drug classes. However, an alternative, more appropriate measure of drug efficacy, which allows to directly compare drugs from different drug classes is still missing.mechanistic way; and (iii) to analyze determinants of drug efficacy critical for drug discovery and development. The proposed measure of drug efficacy, termed reproductive capacity, extends the established in vivo marker, plasma viral load, by incorporating additional infectious viral stages, and the in vitro phenotypic/single-round infectivity assays by taking into account host specific defense mechanisms. This enables us to understand the observed discrepancies between in vitro and in vivo efficacy for integrase inhibitors, and to elucidate and quantify the role of immune-system related clearance mechanisms in drug action. The results presented herein are of particular value to categorize different molecular targets in the HIV life cycle and are expected to be of significance for guiding future HIV drug discovery and development.The objectives of this article are (i) to develop a novel, generic measure of drug potency that facilitates comparison across different drug classes; (ii) to develop a novel mathematical model of the viral replication cycle that incorporates the action of established and novel drugs in a We derived a detailed virus-target cell interaction model as depicted in gag-pol-polyprotein contained) enzymes. During the final step, the viral protease, which is packed into the correctly assembled, immature virions Target cells are produced by the immune system with some constant rate Depending on the stage of the life cycle, the host organism has different abilities to clear the virus. It was assumed that infectious, immature and defective virions The system of ordinary differential equations (ODEs) describing the rate of change of the different viral species and target cells in the detailed model (depicted in Gag-polyprotein) The actions of the different drug classes within the viral life cycle are shown in ed model , PIs theWe used the detailed virus-target cell interaction model to predict the effect of the different drug classes on the distinct stages of the viral life cycle. In order to enable a direct comparison between the different drug classes, we artificially eliminated the feedback by keeping the uninfected target cell hown in , while dwn in and finally incorporated the viral mutation process (resulting from erroneous reverse transcription) into the overall model. Whether the longer lived cell population consists solely of macrophages The model enabled us to mechanistically incorporate the action of all drugs that are approved or in late clinical trial. The impact of a compound on a corresponding (lumped) parameter in the model is specified by process , denotegiven in . Since sThe production of infectious offspring is crucial for the survival of a viral population. The phenotypic single-round infectivity assay measures the amount of infectious offspring after one round of replication. For a given drug, the assay quantifies the drug's efficacy by measuring the reduction in viral offspring relative to the drug-free situation. We defined a new quantity—termed the reproductive capacity The basic reproductive number Based on the two-stage virus dynamics model, the basic reproductive number in vitro reproductive capacity, corresponding to the read-out of the phenotypic assay in vivo measure, the in vitro measure would not take into account: (i) the clearance of any infective stage by the immune system is therefore delayed by the viral life cycle. MIs and PIs do not interfere with the total amount of virus that is being released, but rather shift the ratio of infective to total virus, see , inset, load see , when asload see , in agreload see yield siIn contrast to other inhibitor classes, InIs decrease the amount of late infected cells see , which hnds see . Sedaghands see , which din vivo efficacy of an antiviral drug depends on many different factors, particularly the ability of the virus to adapt to the pharmacological challenge by developing resistance mutations. The ability to develop drug resistance is strongly dependent on the induced pattern of resistance mutations against a particular drug, but might also be influenced by the velocity at which replication competent compartments are removed from the body. However, viral load decay focusses on only one single variable, namely the total output of virus, whereas other infectious stages and (S31), Supplementary The mechanistic mode of action of a compound at its target site can be elucidated by cell free assays that use purified viral protein, e.g. reverse transcriptase for RTIs. The influence of viral mutation, the immune system and pharmacokinetics are absent in this type of assay. However, it is possible to deduce the pharmacodynamic mode (e.g. Eq. (1), see also in vivo pharmacokinetic data is available . We did not consider them in this study, since they are expected to contribute little to the dynamics analyzed herein (the first and the second decay phase).in silico. In its current form, the reproductive capacity requires detailed knowledge about (i) the composition of the viral population, and (ii) the fitness of the different viral strains under a given treatment and (16)–(19)).The reproductive capacity is a useful concept to analyze and monitor drug efficacy in vitro, e.g., by phenotypic assays. We model strain specific fitness The fitness of certain viral strains can be assessed ce e.g., , usuallyAcquisition of detailed knowledge about the composition of the viral population might, due to recent advances in sequencing technology t = 0), the effects of the drug treatment were simulated until the viral population size reached 90% of its pre-treatment value, i.e., virological rebound occurred. During a simulation, the stochastic partitioning of the reaction system was dynamically updated and stochastic reaction events were realized accordingly. Every numerical calculation was computed with a relative error tolerance of The overall virus dynamics in our model comprise different viral strains with copy numbers that can vary over several orders of magnitude. For this reason we have chosen a hybrid (stochastic deterministic) setting for numerical simulation. This approach (i) takes stochastic fluctuations in the slow reaction processes into account; and (ii) reduces the computational costs for the simulation of the fast (deterministic) system dynamics. We used the direct hybrid method proposed in Text S1This file contains the derivation of the simplified model from the(0.30 MB PDF)Click here for additional data file.Figure S1Delay in the onset of viral load decay, exemplified for PI treatment. Simulation results (red line) using the novel two stage virus dynamics model and simulating 100% effective PI treatment are shown together with median clinical data (black diamonds) from PI (RTV) monotherapy.(0.91 MB EPS)Click here for additional data file.

Background: Septic abortion caused by transplacental salmonella infection is extremely rare; thereare no reported cases of serotype oranienburg as an etiology. Case: We describe a patient with non-typhoidal Salmonella enteritidis serotype oranienburg as acause of first-trimester pregnancy loss. The rapid progression of this patient's septicemia and adverseoutcome is described. The epidemiology and natural history of salmonella infections are also discussed. Conclusion: Non-typhoidal salmonella is still a cause of morbidity in Western countries. Thisinfection can result in rapid-onset fetal demise and septic abortion.

Previous studies suggest that p27kip1, a kind of cyclin-dependent kinase inhibitor, play an important role in the smooth muscle cell proliferation. However, the mechanism of hypoxia and the subcellular interactions between p27kip1 and prostacyclin analogues in human pulmonary arterial smooth muscle cell (HPASMC) are not fully understood.Hypoxia induces the proliferation of pulmonary arterial smooth muscle cell (PASMC) kip1 in the ability of Beraprost sodium to inhibit the proliferation of HPASMC during hypoxia. To clarify the biological effects of hypoxic air exposure and BPS on HPASMC, the cells were cultured in a hypoxic chamber under various oxygen concentrations (0.1–21%). Thereafter, DNA synthesis was measured as bromodeoxyuridine (BrdU) incorporation, the cell cycle was analyzed by flow cytometry with propidium iodide staining. The p27kip1 mRNA and protein expression and it's stability was measured by real-time RT-PCR and Western blotting. Further, we assessed the role of p27kip1 in HPASMC proliferation using p27kip1 gene knockdown using small interfering RNA (siRNA) transfection.We investigated the role of p27kip1 protein degradation, whereas BPS suppressed HPASMC proliferation under both hypoxic and normoxic conditions by suppressing p27kip1 degradation with intracellular cAMP-elevation. The 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), a cAMP analogue, had similar action as BPS in the regulation of p27kip1. Moderate hypoxia did not affect the stability of p27kip1 protein expression, but PDGF, known as major hypoxia-induced growth factors, significantly decreased p27kip1 protein stability. We also demonstrated that BPS and 8-Br-cAMP suppressed HPASMC proliferation under both hypoxic and normoxic conditions by blocking p27kip1 mRNA degradation. Furthermore, p27kip1 gene silencing partially attenuated the effects of BPS and partially restored hypoxia-induced proliferation.Although severe hypoxia (0.1% oxygen) suppressed the proliferation of serum-stimulated HPASMC, moderate hypoxia (2% oxygen) enhanced proliferation in accordance with enhanced p27kip1 down-regulation probably via the induction of growth factors such as PDGF, and BPS inhibits both the cell proliferation and p27kip1 mRNA degradation through cAMP pathway.Our study suggests that moderate hypoxia induces HPASMC proliferation, which is partially dependent of p27 Many modeling -12.2) is thought to improve exercise tolerance and survival in patients with either primary or secondary PH through its ability to inhibit the growth of PASMC [2 analogue to increase intracellular cAMP levels via adenylate cyclase activation [2, such as activating adenylate cyclase and increasing intracellular cAMP levels, through activation of the PGI2 receptor. Owing to its chemical characteristics, BPS is more stable and persistent than natural PGI2 and has higher affinity for the PGI2 receptor [Prostacyclin , as well as interactions of the CDK inhibitor p27kip1. We assessed the effects of BPS and 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), a cAMP analogue, on cell proliferation and p27kip1 expression, and examined the role of p27kip1 in HPASMC proliferation using p27kip1 gene silencing.The proliferation of PASMC, which causes pulmonary vascular remodeling, requires the cells to enter the cell cycle. The most important molecular process for cell cycle progression is retinoblastoma protein phosphorylation by cyclin-dependent kinase (CDK)-cyclin complexes, and CDK activities are mainly regulated by CDK inhibitors such as le cells ,21-23. Opression . On the hypoxia -27. Theskip1 polyclonal antibody, mouse anti-β-actin monoclonal antibody, horseradish peroxidase-conjugated goat anti-mouse and rabbit antibody, p27kip1 and control small interfering RNA (siRNA) and BPS was a gift from Toray Industries Inc. . All other chemicals were purchased from Sigma .We obtained reagents and materials from various sources as follows: Humedia SG medium, recombinant human EGF and FGF, gentamycin, streptomycin, and amphotericin B ; bromodeoxyuridine (BrdU) proliferation assay kits ; low-pH cAMP ELISA kits ; ECL detection system ; Moloney murine leukemia virus reverse transcriptase , Quantitech™ SYBR Green PCR kits ; Lipofectamine 2000, 4 – 12% Bis-Tris Nupage gels, and MES-SDS running buffer ; DC protein assay kit and polyvinylidene difluoride (PVDF) membranes in a cell-culture incubator and used at the seventh passage after trypsinization in all experiments. Oxygen concentrations (0.1% ~10%) were modified using N2-CO2 incubators .HPASMC supplied by Kurabo Ltd. were cultured in Humedia SG medium containing 5% fetal bovine serum, with 50 μg/ml of gentamycin, 50 ng/ml of amphotericin B, 1 ng/ml of recombinant human EGF, and 1 ng/ml of recombinant human FGF. The cells were incubated in 75-cm2) seeded in 96-well culture plates for 48 h in serum-free DMEM, then changed the medium to DMEM containing 10% FBS and antibiotics. Thereafter, the cells were incubated for 24 h in various oxygen concentrations with or without 10 μM BPS. We measured BrdU incorporation using BrdU proliferation assay kits according to the manufacturer's protocol. Briefly, the cells were labeled with 10 ng/ml of BrdU during the incubation, washed 3 times with cold PBS, fixed, air dried and incubated with mouse anti-BrdU monoclonal antibody . The antibody was aspirated, the cells were washed 3 times and then incubated with peroxidase goat anti-mouse IgG at room temperature for 30 minutes. The cells were washed 3 times, and 100 μM substrate was added to each well and incubated for 10 minutes in darkness. Thereafter, we measured absorbance at dual-wave lengths of 450 to 540 nm.We incubated HPASMC seeded in 6-well culture plates for 48 h in serum free DMEM, then changed the medium to DMEM containing 10% FBS and antibiotics. The cells were further incubated for 24 h under normal or hypoxic conditions with or without 10 μM BPS. The cells were harvested with trypsin-EDTA and fixed using 70% ethanol. The ethanol was removed and the cells were incubated in PBS containing RNase (172 k units/ml) at 37°C for 30 minutes, stained with propidium iodide (50 μg/ml) and suspended in PBS for 30 minutes on ice. DNA fluorescence was measured and flow cytometry proceeded using an EPICS XL .We examined whether the cell cycle was influenced by the oxygen concentration using flow cytometry with propidium iodide staining. We incubated HPASMC seeded in 24-well culture plates for 48 h in serum-free DMEM then changed the medium to DMEM containing 10% FBS. The plates were incubated for various periods under normal or hypoxic conditions in the presence of 10 μM BPS. The medium was aspirated and adherent cells were solubilized with 200 μl of 0.1 N HCl and 0.1% Triton X. Thereafter, cAMP concentrations in the cell lysates were measured using a low-pH cAMP ELISA kit according to the manufacturer's protocol.We incubated HPASMC seeded in 6-cm dishes for 48 h in serum-free DMEM. The cells were washed twice with PBS, and then placed in DMEM containing 10% FBS and antibiotics under normal or hypoxic oxygen concentrations for various periods with or without 10 μM BPS or 1 mM of 8-Br-cAMP. The cells were then harvested by trypsinization, washed 3 times, and pelleted by centrifugation. Total cellular RNA was obtained by one acid guanidinium thiocyanate-phenol-chloroform extraction [2+ in a total volume of 20 μl. Annealing proceeded at room temperature for 5 minutes, extension at 44°C for 40 minutes, and chain termination at 99°C for 5 minutes.We cultured HPASMC using DNA-binding SYBR green dye to detect PCR products. The cycling conditions were as follows: initial denaturation at 95°C for 15 minutes, 50 cycles of denaturation at 94°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 15 seconds. The β-actin gene served as the reference. The PCR products were isolated from the LightCycler™ glass capillaries, resolved by electrophoresis on 1.5% agarose gels and confirmed by ethidium bromide (EB) staining. Each assay was repeated in 6 independent experiments.We then performed PCR using the RT products and specific oligonucleotide primers for p272) seeded in 6-cm dishes for 48 h in serum-free DMEM. The cells were washed twice with PBS, placed in DMEM containing 10% FBS and antibiotics and then cultured under normal or hypoxic oxygen conditions for various periods with or without 10 μM BPS or 1 mM 8-Br-cAMP. The cells were then harvested and resuspended in protein lysis buffer and incubated for 30 min on ice. Cell lysates were clarified by centrifugation at 10,000 g for 15 minutes at 4°C, then the protein content in the supernatants was quantified using DC protein assay kits. Thereafter, 25 μg of protein per lane was loaded onto 4 – 12% Bis-Tris Nupage gels with MES SDS running buffer, according to the manufacturer's protocol. The gels were transferred to PVDF membranes by electrophoresis at 100 V for 1 h, then non-specific binding was blocked in PBS containing 0.2% Tween 20 (PBS-T) and 5% nonfat milk (blocking buffer) at room temperature for 1 h. All antibodies were diluted in blocking buffer. The membrane was then probed with rabbit anti-p27kip1 polyclonal antibody or mouse anti-β-actin monoclonal antibody (diluted 1:5000), and incubated for 1 h at room temperature. Membranes were washed with PBS-T and incubated with horseradish peroxidase-conjugated goat anti-rabbit or mouse IgG for 2 h at room temperature. After washing with PBS-T, proteins were detected using the ECL system. Each assay was repeated in 4 independent experiments.We cultured HPASMC seeded in 6-cm dishes for 48 h in serum-free DMEM. The cells were washed twice with PBS, then placed in DMEM containing 10% FBS and cultured under normal or hypoxic conditions for the indicated periods in the presence of the transcription inhibitor actinomycin D (Act D) (400 nM), or the protein synthesis inhibitor cycloheximide (CHX) (25 μg/ml), and with or without 10 μM BPS, 1 mM 8-Br-cAMP or 25 ng/ml of platelet-derived growth factor (PDGF). The cells were then counted and mRNA and protein stability was examined per 50,000 cells incubated with Act D and CHX using RT-PCR and Western blotting, respectively. Each assay was repeated in 4 independent experiments.We cultured HPASMC into 6-cm dishes and 96-well culture plates, then incubated in DMEM supplemented with 10% FBS and antibiotics for 24 h under normal or hypoxic conditions with or without 10 μM BPS. We then measured BrdU incorporation into the transfected cells and confirmed target gene silencing by p27kip1 siRNA using Western blotting.We incubated HPASMC in 10-cm dishes in DMEM containing 10% FBS for 24 h, until they reached about 60% confluence. After rinsing, the cells were incubated for 6 h with serum-free Opti-MEM medium, 5 μl/ml of Lipofectamine 2000, and 50 nM control or p27The results are expressed as means ± SE. Statistical analysis was performed using ANOVA with Bonferroni correction for multiple comparisons. Comparisons were considered statistically significant at p < 0.05.Moderate hypoxia (2% oxygen) promoted, whereas severe hypoxia (0.1% oxygen) suppressed DNA synthesis in serum-stimulated HPASMC Fig. . Under n0/1 phase and that the cell cycle was arrested (quiescent state). Moderate hypoxia significantly promoted cell cycle progression and forced the cells to enter the S and G2/M phases compared with the control under normal oxygen conditions and BPS significantly suppressed the cell cycle progression of cells that were serum-stimulated under hypoxic conditions , p27xia Fig. .kip1 mRNA expression, we assessed p27kip1 mRNA stability using Act D. Both BPS and 8-Br-cAMP significantly suppressed p27kip1 mRNA degradation in cells incubated with Act D under both normoxic and moderately hypoxic conditions. Although moderate hypoxia did not change p27kip1 mRNA expression, mRNA stability was slightly decreased under moderate hypoxia enhanced the proliferation of serum-stimulated HPASMC in accordance with promoted p27Increased levels of growth factors derived from the accumulation of hypoxia-inducible factor 1α (HIF-1α) are thought to regulate PASMC proliferation under hypoxic conditions since a partial HIF-1α deficiency decreases muscularizartion of pulmonary arterioles in animals exposed to chronic hypoxia . Althougkip1 expression has been demonstrated in mice with pulmonary hypertension induced by hypoxia [kip1, which blocks the cell cycle at the G0/1 phase, is regulated via several mechanisms including transcription, protein degradation and translation [kip1 mRNA degradation. Our data also suggested that the hypoxia-induced down-regulation of p27kip1 was not apparently mediated by hypoxia per se, but rather mitogenic factors such as PDGF derived via hypoxia enhanced p27kip1 protein degradation. We demonstrated that the decrease of p27kip1 expression during hypoxia was post-transcriptional regulation from the results of RT-PCR and western blot analysis. We hypothesized that the discrepancy between the results of p27kip1 protein expression and protein stability during hypoxia may be explained by the effect of CHX which could suppress the protein expression of the hypoxic signal transduction including hypoxia-induced growth factors, such as PDGF. Our results that PDGF decreased the stability of p27kip1 is consistent with our hypothesis, and we believe that these results are consistent with conclusion that p27kip1 down-regulation mediates hypoxia-induced HPASMC proliferation. The suppressive effect of PDGF on p27kip1 expression has been demonstrated using rat aortic vascular smooth muscle cells [kip1 protein [The suppressive effect of hypoxia on p27 hypoxia . Howevernslation -36. The le cells and in hle cells , and onc protein . Since b0/1 phase even under hypoxia, with p27kip1 elevation being associated with increased intracellular cAMP expression, which was not affected by the oxygen concentration. These results indicated that the BPS-cAMP pathway functioned even under hypoxic conditions and that p27kip1 elevation might be a consequence of BPS-induced intracellular cAMP elevation. To confirm this hypothesis, we investigated the effects of the cAMP analogue 8-Br-cAMP on p27kip1 expression and of BPS on DNA synthesis in p27kip1 gene knockdown HPASMC. The effects of 8-Br-cAMP and BPS on p27kip1 expression were similar and p27kip1-dependent regulation of proliferation was confirmed in the p27kip1 knockdown cells. Overexpression of p27kip1 in rat PASMC decreased thymidine uptake and cellular proliferation while p27kip1 knock-out PASMC from mouse had increased cellular proliferation compared with p27kip1 wild-type PASMC [kip1 expression in the lung and the anti-proliferative effects of heparin during hypoxia were absent in p27kip1 knock-out mouse compared with p27kip1 wild-type mouse [kip1 siRNA, we demonstrated that the anti-proliferative effects of BPS during hypoxia were lessened in the decrease of p27kip1. Therefore, we consider that our results from 27kip1 siRNA experiments are consistent with the published results, and we believe that our results demonstrate the importance of p27kip1 in the hypoxic regulation of PASMC proliferation and hypoxia-induced pulmonary hypertension and remodeling, which would add an important additional advancement in this field.Our results showed that HPASMC incubated with BPS were arrested at the Gpe PASMC . As wellpe mouse . Using pkip1 mRNA degradation under both normoxic and hypoxic conditions. Although cAMP regulates the expression of several genes, and the control of the mRNA degradation rate by cAMP is also an important regulatory mechanism of gene expression [kip1 mRNA stability is controlled by interactions between MAPK-dependent regulation [kip1 mRNA stability during exposure to agents that elevate cAMP and hypoxia. Therefore, to clarify the detailed mechanisms of hypoxia and cAMP with respect to p27kip1 expression, additional studies are required to explain the relationship between cAMP and Rho.We also found that BPS and 8-Br-cAMP suppressed p27pression -42, the gulation and Rho-gulation . In addigulation ,46, whicgulation ,48. Theskip1-cAMP and hypoxia-induced pathways. We believe that clarification of the precise mechanisms of pulmonary smooth muscle proliferation will lead to improved therapeutic strategies that targets hypoxic pulmonary hypertension and remodelling of the pulmonary circulation.In summary, we found that BPS and hypoxia play critical roles in HPASMC growth through p27The author(s) declare that they have no competing interests.MK carried out the laboratory measurement and data analysis.SM conceived the study idea and participated in the laboratory measurement and drafted the manuscript.YD, SA, and IM participated in the design of the study.TI supervised the study and was involved in the manuscript writing.All authors read and approved the final manuscript.

Both epidemiologic and strain-related factors may contribute to large clusters of tuberculosis patients. Mycobacterium tuberculosis are described as clustered. Cluster size may depend on patient or strain characteristics. In a 7-year population-based study of tuberculosis in Karonga District, Malawi, clusters were defined by using IS6110 restriction fragment length polymorphism, excluding patterns with <5 bands. Spoligotyping was used to compare strains with an international database. Among 682 clustered patients, cluster size ranged from 2 to 37. Male patients, young adults, and town residents were over-represented in large clusters. Cluster size was not associated with HIV status or death from tuberculosis. Spoligotypes from 9 (90%) of 10 large cluster strains were identical or very similar (1 spacer different) to common spoligotypes found elsewhere, compared with 37 (66%) of 56 of those from nonclustered patients (p = 0.3). Large clusters were associated with factors likely to be related to social mixing, but spoligotypes of common strains in this setting were also common types elsewhere, consistent with strain differences in transmissibility.Tuberculosis patients with identical strains of Many studies have investigated risk factors for clustering, but relatively little is known about the determinants of cluster size , or they are more virulent and are therefore more likely to give rise to secondary cases within the period studied based on the ISEpidemiologic differences can be explored by examining risk factors for cluster size. Giordano et al. strains from patients with smear-positive tuberculosis in 1998 or 1999, as examples of strains that had apparently not spread in the population; and from all positive cultures from 2002. Previously identified spoligotypes were defined as widespread if the international database described them as both “ubiquitous” and “recurrent,” “common,” or “epidemic.”Spoligotyping . Cluster size was divided into 4 groups , and assOver the study period, 1,248 cases of culture-positive tuberculosis were diagnosed in patients in Karonga District. RFLP results were available on 1,194 isolates from 1,044 patients. After we excluded 25 isolates because laboratory error was suspected (Cluster size varied from 2 to 37. The determinants of cluster size are shown in >15 people) were found in at least 4 of the 6 geographic areas of the district, and most were found throughout the district. The distributions of the 4 largest clusters are shown in the All of the large cluster strains (>15 people) were compared with the international database , respectively. The spoligotype for strain kps121, spoligotype129, was not similar to any widespread types.The spoligotypes from the RFLP-defined large cluster strains were compared with spoligotypes from patients with positive cultures in 2002, and from patients with smear-positive tuberculosis and unique RFLP patterns in 1998 through 1999. Overall, 9 (90%) of 10 of the large cluster strains had spoligotypes that were identical to, or only 1 spacer different from, previously described widespread spoligotypes. For the patients from 2002, this proportion was 90 (71%) of 126 (p = 0.3 when compared to the large cluster strains), and for the smear-positive unique strains, it was 37 (66%) of 56 (p = 0.3 compared to the large cluster strains).All the spoligotypes that were found in the RFLP-defined large cluster strains were also found among (RFLP-defined) unique strains. Seventeen of the unique strains had spoligotype 59, and 2 others had closely related patterns ; 1 had spoligotype 21, and 1 had a closely related pattern; 4 had spoligotype 53, and 2 had closely related patterns; and 6 had spoligotype 129. Of the 56 patients from 1998 to 1999, none had Beijing spoligotypes, but we have previously described strains with Beijing spoligotypes and unique RFLP patterns in this population had spoligotype 59, and 10 more had closely related patterns; 11 (9%) had spoligotype 21; 8 (6%) had spoligotype 53, and 2 had closely related patterns; 7 (6%) had the Beijing spoligotype; and 8 (6%) had spoligotype 129. The 36 isolates with spoligotype 59 had 23 different RFLP patterns with a similarity coefficient of 63%.This study suggests that both epidemiologic and strain-related factors may contribute to large cluster size. In large clusters young adults, male patients, and those living in the town were over-represented, all factors likely to be associated with increased social mixing. Similar associations with age and sex have been found previously, in the United States and Denmark. In Denmark the largest cluster was particularly predominant in the capital city and cluster size, but most patients had sputum smear–positive disease. There was also no statistically significant association with degree of smear positivity (not shown). An overall association with infectiousness would not necessarily be expected: the infectiousness of the first cases of a cluster may be important in determining size, but the first cases for the large clusters, which were found throughout the period of study, are not identifiable. There was no significant association with isoniazid resistance, but only 39 (6%) patients had resistant strains. Isoniazid resistance has been associated with reduced clustering and reduced generation of secondary cases . Virulent strains could lead to large clusters if virulence were associated with increased transmission rates or increased rates of disease after infection (Evidence that strain characteristics may have contributed to cluster size comes from the finding that the spoligotypes of most of the common RFLP-defined strains in this study were identical to, or only 1 spacer different from, widespread spoligotypes already described. Unique RFLP-defined strains from smear-positive patients in the early part of the study were used as a comparison group. Smear-positive case-patients were chosen to maximize the likelihood of transmission occurring; early cases were used to allow time for secondary cases to have been identified if they had occurred. These unique strains were less likely than the large cluster strains to have spoligotypes that were closely related to widespread types, but this difference was not statistically significant, and the spoligotypes that were found in the large cluster strains were also found among the unique strains. Interestingly, strain kps121, which was the only large cluster strain with a spoligotype not closely related to a widespread previously described type, was also the 1 large cluster strain that was clearly decreasing in the Karonga population.The finding of large cluster strains with previously described widespread spoligotypes may suggest that these strains are particularly transmissible or particularly likely to cause disease. Other possibilities are that they are older in evolutionary terms, and thus have had more time to become widespread, or that we are seeing a founder effect in some populations with subsequent spread following human migration patterns. Spoligotype 59 was common in the Malawi population in all groups of patients, clustered and unique, and was associated with a wide diversity of RFLP patterns, which suggests that it may be a longstanding strain in this area. It was also the most common spoligotype found in studies in Zimbabwe and Zambia (

The 2004 International Conference on Improving Use of Medicines recommended that emerging and expanding health insurances in low-income countries focus on improving access to and use of medicines. In recent years, Community-based Health Insurance (CHI) schemes have multiplied, with mounting evidence of their positive effects on financial protection and resource mobilization for healthcare in poor settings. Using literature review and qualitative interviews, this paper investigates whether and how CHI expands access to medicines in low-income countries.We used three complementary data collection approaches: (1) analysis of WHO National Health Accounts (NHA) and available results from the World Health Survey (WHS); (2) review of peer-reviewed articles published since 2002 and documents posted online by national insurance programs and international organizations; (3) structured interviews of CHI managers about key issues related to medicines benefit packages in Lao PDR and Rwanda.In low-income countries, only two percent of WHS respondents with voluntary insurance belong to the lowest income quintile, suggesting very low CHI penetration among the poor. Yet according to the WHS, medicines are the largest reported component of out-of-pocket payments for healthcare in these countries (median 41.7%) and this proportion is inversely associated with income quintile. Publications have mentioned over a thousand CHI schemes in 19 low-income countries, usually without in-depth description of the type, extent, or adequacy of medicines coverage. Evidence from the literature is scarce about how coverage affects medicines utilization or how schemes use cost-containment tools like co-payments and formularies. On the other hand, interviews found that medicines may represent up to 80% of CHI expenditures.This paper highlights the paucity of evidence about medicines coverage in CHI. Given the policy commitment to expand CHI in several countries and the potential of CHI to improve medicines access and use, systematic research is needed on medicine benefits and their performance, including the impacts of CHI on access to, affordability, and use of medicines at the household level. Low-income countries face considerable challenges in financing health care for their populations: limited tax revenues and complex management of social health insurance translate into enormously inadequate health expenditures per capita with individuals directly absorbing the financial burden of diseases.any scheme managed and operated by an organization, other than a government or private for-profit company that provides risk pooling to cover all or part of the costs of health care services' [Over the past decade, policy makers have presented community financing as an alternative to user fees strategies and a viable option to improving health care systems in low-income countries ,2. Severervices' . The actervices' ,6, but tervices' ,7-10. Obervices' -13. In cervices' , Senegalervices' , or Lao ervices' , CHI expEssential medicines are critical to treating infectious diseases and chronic conditions. Improving access to medicines in developing countries is one key to achieving the Millennium Development Goals ,17,18. IWe believe that CHI has the potential to improve access to and quality use of medicines, and that members may be more likely to voluntarily participate in CHI with medicines coverage. To begin to examine this hypothesis, we sought to describe the prevalence of CHI among poor populations; the characteristics of medicine benefits offered by CHI plans; and how medicines coverage may affect the performance of CHI and the satisfaction of members with their plans.low-income countries as defined by the World Bank, e.g. countries with a Gross National Income of US$875 or less per capita in 2005 [To control for the diversity of CHI, we focused our investigation on in 2005 . We used in 2005 . Because in 2005 . Informa in 2005 .Agence Française de Développement. Peer-reviewed articles in English and published reports in English or French that identified a CHI plan were selected and classified by country. For each low-income country, we extracted data on the number of schemes, total membership, as well as existence and role of CHI networks. At the individual plan level, we collected information about technical design features related to medicine benefits , organizational incentives related to medicines , or any mention of members' attitudes about how CHI affects access to medicines. When two articles provided the same level of technical information on a scheme, we chose the more recent one.We performed a literature review looking for data on characteristics of medicines coverage offered by CHI plans in low-income countries. Publications were identified with EMBASE and MEDLINE from January 2002 through December 2006, using the search terms: community-based health insurance, community health planning, cost sharing, developing countries, drug benefit, fees and charges, health expenditures, health insurance, health resources, health services accessibility, insurance, and medicines coverage. We also searched websites of major international organizations such as the International Labor Organization, World Bank, World Health Organization, and websites of international development agencies such as the US Agency of International Development and the .Finally, to illustrate how individual plans operate, we conducted interviews using a structured questionnaire in two countries where governments have taken the lead in using CHI to expand health insurance coverage to poor populations: one country in Africa (Rwanda) and one in Asia (Lao PDR). This questionnaire is available at Over a third of the world's population resides in low-income countries, where about 70% of people live in rural areas.the outlays of private social insurance schemes, commercial and nonprofit insurance schemes, health maintenance organizations, and other agents managing prepaid medical and paramedical benefits including the operating costs of these schemes". Overall, private health care expenditures adjusted for purchasing power parity (ppp) are similar between low-income countries with and without private prepaid plans . However, in the former, out-of-pocket expenditures constitute a smaller percentage of private health expenditures (78.4% vs. 89.6%), likely due in part to the 5.3% average contribution by private prepaid plans.According to NHA data Table , out-of-NHA do not report on categories of population covered by prepaid health insurance plans, but WHS data give some indication about the economic status of privately insured people. WHS data provide information on the overall prevalence of health insurance, and of "voluntary insurance" which includes CHI. Among the 84,135 households interviewed by the WHS in the 17 low-income countries that reported data on the WHS insurance section, a total of 785 respondents reported having some form of health insurance Table . Of thosWHS data from low-income countries provide some insight about the importance of medicines in household expenditures, and on how economic factors affect access to medicines Table . MedicinWe identified fifty-three publications containing information about 1066 CHI plans in 19 low-income countries ,16,27-75Gicumbi and Centre Universitaire de Santé Publique (CUSP) illustrate how CHI functions in Rwanda. These two schemes were selected because they represent two different geographic areas (Northern and Southern Provinces) and two levels of activity . Both plans had been in operation for over three years at the time of interviews.In Rwanda, a comprehensive political and legal framework supports the systematic expansion of CHI as the instrument of progression towards universal health insurance coverage ,76. ConsCUSP plan operates at the local level. Created in 2003 in a mixed rural/urban environment, it now insures between 5,000 and 10,000 persons. Membership is voluntary and individual. Yearly premiums are 1,000 Frw (US$1–2) on average. Members pay a fixed co-payment of 100 Frw (US$ 0.20) for every medicine obtained at health centers, and a coinsurance of 10% of retail price for medicines received in the hospital. Medicine costs represented 80% of the total expenditures of CUSP in 2006. The Gicumbi plan, created in 1999, operates at the district level coordinating 21 plans linked to health centers and 1 plan linked to a hospital, and insuring 85% of the target population. Membership is by family and voluntary, except for the very poor chosen by the community and automatically enrolled without paying premiums. Annual premiums average 3,500 Frw (US$ 6–7). Members pay a fixed 10% coinsurance for all medicines. Outpatient benefits only include medicines on the national EML and prescribed by health centers; some medicines not on the EML are covered by the inpatient benefit. At the district level, the Gicumbi district/regional authority buys medicines from the Rwanda central pharmacy (CAMERWA) at average wholesale price plus 5% margin and sets retail prices. Medicines expenditures of the Gicumbi plan increased by a factor of five after implementation of the Ministry of Health decision to cover the very poor, suggesting a broader access to both care and medicines by these populations. Most frequently used medicines are amoxycillin, paracetamol, quinine, cotrimozaxole, and penicillin V.The Gicumbi's policy of familial membership and free-of-charge enrollment of some of the poorest in the community indicates an emphasis on equity in access of care. Its outpatient drug coverage restrictions suggest an effort to control the quantity and appropriateness of medicines used that may be beneficial to its long-term financial sustainability. On the other hand, CUSP's policy of individual membership for everyone without subsidy may be less oriented towards equity. Its strategy of fixed co-payment for every medicine obtained at the health center without coverage restrictions may target over utilization without trying to influence the choice of medicines.According to the interviews, Like Rwanda authorities, those of Lao PDR have adopted a national strategy to increase health resource mobilization for the poor. In April 2005, regulations for a nationwide implementation of community based health insurance were issued after three pilot CHI projects showed promising results . HoweverWe combined three complementary sources of data to explore CHI medicines coverage in low-income countries. Our findings highlight the paucity of evidence about medicines coverage and medicines utilization in community-based health insurance programs, as well as the need for understanding better the role of medicines coverage in the overall health care financing strategy of low-income countries. Several factors make systematic analysis of data about the structure and processes of CHI plans particularly challenging: small size, diversity of communities where they function, and lack of infrastructure or technical capacity. Indeed, most available data come from schemes currently receiving assistance from governments, large micro-insurance networks, or international organizations,77 A plaThe paucity of data may also reflect the fact that many CHI plans operate in environments where drug supply systems are so weak that covering medicines is not a realistic option. Nevertheless, as WHS data demonstrate, payments for medicines constitute about half of out-of-pocket expenditures in low-income countries, and this proportion increases with poverty. Even in settings where medicines supply may not be adequate, poor people still use a substantial portion of their meager resources to buy medicines.Overall, our findings confirm that voluntary insurance, which includes CHI, has a very low penetration in low-income countries where private payments, representing over half of health care expenditures, are mostly made of out-of-pocket. Our literature review identified CHI plans in only one-third of low-income countries (19/54); in only four countries did the plans cover more than 1% of the population. Reasons may vary with national contexts. For instance in Vietnam, the recent emphasis by the Social Security Agency on extending social health insurance coverage to poor populations is consistent with the relatively high percentage of voluntarily insured Vietnamese in the lowest income bracket (11.1%), and may explain why CHI has not been the focus of attention in this country in recent years -81. WHS The raison d'être of CHI is to improve access to care for the poorest of the society. With low penetration among the poor, CHI does not yet achieve this goal. We believe that a focus on inpatient and outpatient medicines coverage, adjusted to local circumstances, may be one strategy to encourage higher rates of voluntary enrollment in CHI by the poor. CHI plans which cover medicines can devise strategies to ensure that quality medicines are consistently available at health centers, provide incentives (such as lower copayments) to use generics, and encourage appropriate prescribing through education and administrative systems. A key challenge will be to ensure financial sustainability of the CHI plans as enrollment and demand for medicines increase.To be successful, CHI must perform well in two areas: revenue collection and strategic purchasing . MedicinAssociation d'Entraide des Femmes scheme preferentially contracts with religious health care providers, because these providers receive donations of brand name medicines that patients prefer, and sell them at discounted prices [Financial access to medicines can be expanded by integrating evidence about effectiveness, preferences of providers, and medical needs of members in decisions about which medicines to cover, negotiating affordable prices with providers, and working with local medicines outlets to ensure availability of medicines. The potential of CHI to influence prescription behavior and to control medicine costs is real. Even if they do not purchase medicines directly, CHI plans can negotiate payments with those who purchase and supply medicines, and they can design incentives to use recommended medicines. In Benin, the d prices . While tFrom our review, it is clear that appropriate tools to assist in designing and managing medicines benefit packages adapted to low-income environments do not exist yet, despite the multiplicity of workshops and manuals targeting CHI administrators ,84. TraiThe paucity of medicines coverage data prevented us from examining the role of CHI on improving access to medicines. Yet, interviews suggest that medicines represent between 65% and 80% of costs incurred by some plans, highlighting the importance of monitoring medicines utilization and expenditures in CHI. Medicines coverage policies can be used to control medicine expenditures and to improve quality use by rewarding adequate prescription behavior and by encouraging the use of effective and safe medicines at lowest possible prices. In poor communities, CHI provides a critical institutional link between patients, providers, and suppliers of medicines: it can play a key role by negotiating with medicine suppliers, by adjusting medicines coverage to local health care priorities, by disseminating education materials tailored to the community about quality use of medicines, by linking medicines coverage to treatment adherence, and by rewarding providers and community-based workers who follow treatment guidelines. In this context, the large-scale social experiment of CHI development in Rwanda is of significance, since it may bring important progress to the understanding of challenges faced by CHI in low-income settings. Interviews with CHI administrators in Rwanda illustrate the interplay of government support and community involvement needed to design medicine benefits for the poor. Much more could be learned in this setting by comparing membership and utilization among plans with different medicine benefits, and by exploring the relationship between medicines coverage and enrollment, cost recovery, and financial stability.In conclusion, our results show that the extent and nature of medicines coverage offered by CHI in low-income countries are not well-reported at this time. Lack of access to medicines is a crucial issue in the developing world. We believe that better medicines coverage and well-designed medicine benefits are part of a strategy to address this problem. Further research is needed to characterize current coverage and utilization of essential medicines in CHI, to identify effective medicines benefit packages in low-income settings, and to study the impacts of these benefits on patterns of service utilization and clinical outcomes. A better understanding of medicine policies in CHI can help national policymakers and insurers who develop strategies to improve health care financing systems, prevent catastrophic health expenditures, and achieve the Millennium Development Goals. To that effect, the Rwanda experience may constitute a unique opportunity to evaluate the contribution of CHI in improving access of medicines, medicines utilization, and ultimately health outcomes in low-income environments.The authors declare that they have no competing interests.CVV carried out the literature review, designed the structured interviews, and drafted the manuscript. DRD conceived of the study, participated in its design and reviewed the manuscript. JN provided input during the initial phase of the project, carried out interviews in Rwanda, and reviewed the manuscript. AW participated in the design of the study, carried out an interview with three officials from the National Institute of Public Health, the Ministry of Health, and the WHO office in Lao PDR, and reviewed the manuscript.

Eucalyptus with cavities that are relatively large and rich in essential oils.The biosynthesis of plant natural products in sub-dermal secretory cavities is poorly understood at the molecular level, largely due to the difficulty of physically isolating these structures for study. Our aim was to develop a protocol for isolating live and intact sub-dermal secretory cavities, and to do this, we used leaves from three species of Aspergillus niger was found to allow isolation of intact cavities after a relatively short incubation (12 h), with no visible artifacts from digestion and no loss of cellular integrity or cavity contents. Several measurements indicated the potential of the isolated cavities for further functional studies. First, the cavities were found to consume oxygen at a rate that is comparable to that estimated from leaf respiratory rates. Second, mRNA was extracted from cavities, and it was used to amplify a cDNA fragment with high similarity to that of a monoterpene synthase. Third, the contents of the cavity lumen were extracted, showing an unexpectedly low abundance of volatile essential oils and a sizeable amount of non-volatile material, which is contrary to the widely accepted role of secretory cavities as predominantly essential oil repositories.Leaves were digested using a variety of commercially available enzymes. A pectinase from Eucalyptus species with sub-dermal secretory cavities, and should find wide application in studies of the developmental and functional biology of these structures, and the biosynthesis of the plant natural products they contain.The protocol described herein is likely to be adaptable to a range of The application to plants of systems biology technologies, such as metabolomics and transcriptomics, has generally followed a non-targeted approach using bulk material composed of numerous tissue types . AttemptEucalyptus and Citrus species [The specialised secretory structures in which essential oils (mono- and sequiterpenes) are synthesised and stored are ideal candidates for fine scale application of systems biology techniques . The oil species ,9. Such species .Recent advances in elucidating the pathways for essential oil biosynthesis have come with the application of transcriptomics to isolated glandular trichomes from species such as peppermint , sweet bRosa spp.), Rutaceae (Citrus spp.) or Myrtaceae . The multicellular secretory structures in these families are generally ellipsoidal in shape and consist of a sub-epidermal lumen delimited by an internal layer of flattened, thin-walled secretory cells, and an external layer of thicker-walled parenchymatous cells [The internally embedded secretory structures have proven much more difficult to isolate from the surrounding tissues, and consequently have not been as well studied at the molecular level. The most advanced work has been on the monoterpenes and diterpene resin acids (collectively oleoresin) housed in the resin ducts of conifers ,20. Thisus cells .Eucalyptus species have long been of economic value as pharmaceuticals and as fragrance additives [The essential oils extracted from the secretory cavities of a number of dditives , but thedditives and othedditives ,25. Manydditives ), and giEucalyptus sub-dermal secretory cavities free from all surrounding leaf mesophyll tissues, without compromising the integrity of the cells bounding the lumen and without the loss of any lumen contents. The relatively large sub-dermal secretory cavities of Eucalyptus species not only house economically important essential oils, but these structures have recently been shown to contain other, non-volatile natural products [Eucalyptus globulus Labill. ssp. globulus (Tasmanian blue gum), the world's major source of the pharmaceutical monoterpene 1,8-cineole [E. polybractea R.T. Baker , Australia's key commercial source of 1,8-cineole [E. froggattii Blakely the secretory cavities of which contain substantially lower levels of monoterpenes relative to sesquiterpenes [We aimed to develop a protocol to isolate live products , making -cineole , E. poly-cineole , and a nterpenes .-1 buffer. Two enzymes were from Aspergillus niger: pectinase PASE and pectinase in glycerol P-4716 ; two were from Rhizopus: pectinase P-2401 (Sigma) and Calbiochem macerase-pectinase ; and two were from Trichoderma viride: cellulase C-1794 (Sigma) and cellulase 'Onozuka' RS . Fresh leaves were cut into 2 mm strips and placed in 'Standard' buffer containing each enzyme and incubated for between 12 and 24 h at temperatures as per manufacturer's instructions. To maximize the chance of isolating cavities with live cells, 24 h was chosen as the maximum allowable incubation time.Secretory cavities were isolated from surrounding leaf tissues following partial enzymatic digestion of leaves. Six commercially available enzymes were trialed at amounts ranging from 50 to 250 units mlAspergillus at 250 units ml-1 standard buffer produced numerous isolated secretory cavities (free from mesophyll cells) from each leaf strip within minutes of teasing leaf tissues apart . In addition to lower cavity yields, incubation with Worthington PASE resulted in browning of the mesophyll cells in the leaf strips during incubation, which subsequent analysis following the method of Vernon (1960) [Rhizopus pectinases produced semi-isolated cavities with mesophyll cells still attached showed whed Fig. and thesity Fig. . Incubat-1 standard buffer; 25°C), a pipette was used to collect each individual cavity from within the milieu of mesophyll cells and vasculature . Intact cavities were collected, washed with standard buffer and concentrated (100 g for 10 min).After successful enzymatic digestion with Sigma P-4714 .Photosynthetic pigments were extracted and quantified as indicators of mesophyll cell contamination in triplicate collections of 50 isolated cavities. The results were compared to those for sections of digested leaf dissected to encompass 50 cavities. Total chlorophyll (mean ± s.e.) was 173.42 ± 20.28 ng cavityE. polybractea secretory cavities i.e. 1 ng RNA cavity-1. Nanodrop spectrophotometer readings gave a 260/280 nm ratio of 2.1, indicating high purity RNA was obtained. A monoterpene synthase gene and a ubiquitous actin gene (control) were successfully amplified from secretory cavity cDNA and sequenced using an AB3730xl 96-capillary sequencer . Specific monoterpene synthase primers amplified a 289 bp region, which Blastx (NCBI) showed to have 99% identity at the amino acid level with two monoterpene synthase sequences from Eucalyptus globulus and 85% identity with a putative monoterpene synthase sequence from Melaleuca alternifolia with r2 = 0.86, a significant slope (± 1 s.e.) of 0.42 ± 0.04 , but a non-significant intercept of 0.05 ± 0.06. Similarly, the linear regression for E. globulus was significant with r2 = 0.95, a significant slope (± 1 s.e.) of 0.59 ± 0.03 , and a non-significant intercept of 0.16 ± 0.09. The linear regression for E. froggattii was also significant with r2 = 0.80, a significant slope (± 1 s.e.) of 0.41 ± 0.05 , and a non-significant intercept of 0.46 ± 0.26. The significant slope values for each species are relatively similar and indicate that the essential oil contents of the cavities were only 42%, 59% and 41% of the cavity lumen volume for E. polybractea, E. globulus and E. froggattii, respectively.We next quantified the relative abundances of the volatile essential oils and non-volatile resinous components within the lumena of secretory cavities by applying the enzymatic isolation protocol to ing Fig. . The lin-1 and for those hand-dissected in tissue blocks was 0.23 ± 0.02 nL nL-1. A one-way ANOVA detected no statistical difference between the means , thus it appears that no essential oils are lost during the isolation process.To test if the regression slopes were artificially low due to a loss of essential oils from the cavities during the isolation process, ten blocks of leaf tissue each encompassing a single cavity were hand-dissected from fully expanded leaves, imaged using a dissecting microscope with transmitted lighting and the oil extracted as for the isolated cavities. Only outer cavity diameters could be accurately estimated in the hand dissected tissue blocks therefore total cavity volume (lumen and cavity cells) was calculated, rather than lumen volume. The mean (± s.e.) extracted oil volume per unit total cavity volume for enzymatically isolated cavities was 0.22 ± 0.01 nL nLEucalyptus secretory cavities showed the dominant constituent in E. polybractea and E. globulus cavity lumena was the oxygenated monoterpene 1,8-cineole , whereas E. froggattii cavities contained on average only 5% of this key monoterpene has previously been reported, but structural integrity was compromised during isolation resulting in a loss of between 83 and 96% of lumen contents [Eucalyptus secretory cavities [The protocol described herein is able to successfully isolate intact, live secretory cavities from three contents . Our procavities . We havecavities , rather cavities ,32, and Eucalyptus has demonstrated the potential importance of these poorly understood constituents. In a number of studies of isolated glandular trichomes, essential oils have been found to co-occur with less volatile compounds. For example, the trichomes of species in the Lamiaceae can contain monoterpenes and non-biosynthetically related compounds such as diterpenoids in white horehound [Eucalyptus globulus is the world's major source of eucalyptus oil and E. polybractea is Australia's key commercial source, the high estimates of 41 and 58% non-volatile components in the cavity lumena, respectively, suggest that future research on the biosynthesis of the resinous component may have implications for increasing commercial essential oil yields.The use of the protocol to estimate high abundances of non-volatile resinous components within the secretory cavities of orehound , phenylporehound , and flaorehound and mintorehound . Neverthorehound . In eucaEucalyptus secretory cavity lumena is composed of the monoterpenoid glucose esters cuniloside B and froggattiside A [Eucalyptus secretory cavity lumena described here . This cEucalyptus. The protocol is likely to be adaptable to a broad range of Eucalyptus species with sub-dermal, foliar secretory cavities and should find application in biosynthetic studies of the numerous commercially important natural products found in eucalypt leaves.Surprisingly little is known about the physiology of plant secretory structures, and in particular, those of an embedded nature. This paucity of knowledge has been ascribed to the low abundance of the cells that compose secretory structures, and the difficulty of physically isolating these cells for study . The proEucalyptus polybractea and E. froggattii, but are highly abundant in seedling leaves of E. globulus. Therefore E. polybractea leaves were sampled from plantation-grown adult trees , whereas seedlings of glasshouse-grown E. globulus were sampled and oxygen depletion in the buffer measured and logged every 2 min for 60 min using a MI-730 micro-oxygen electrode connected to an Orion 5-star meter . A solution of 'Standard' buffer (125 μl) without secretory cavities was used as the control and control measurements were logged for 1 h before and after the isolated cavity measurements.2) and also on triplicate batches of 50 isolated cavities. The leaf sections were excised from leaf strips post enzymatic digestion with Sigma P-4716 pectinase. In each case, tissue was ground in a microcentrifuge tube with a microtube pestle and extracted with 300 μl acetone . Extracts were centrifuged, 250 μl of the supernatant collected and its absorbance at 647 and 664 nm measured using a Beckman DU640 spectrophotometer . Total chlorophyll per cavity was determined using the equations of Jeffrey & Humphrey (1975) [Chlorophyll concentrations were determined on a per cavity basis for three leaf sections, each encompassing 50 cavities (~16 mm4 (0.5% w/v), and dehydrated in a graded ethanol series. Cavities in 100% ethanol were then dried using a Baltec CPD 030 critical point dryer, gold-coated using an Edwards S150B sputter coater and observed with a Philips XL30 FEG Field Emission Scanning Electron Microscope in 'Standard' buffer for 20 min was used to visualize intact nuclei under a UV filter for 10 min was used to visualize lipids under a UV filter after isolated cavities were punctured with a 1 μm microprobe for 5 min. The cavities were then mounted in ethanol (100%) and examined by confocal fluorescence microscopy with a Leica DMIRB laser scanning microscope and a Leica TCS SP2 imaging system Fig. . For scaope Fig. . For fluope Fig. &2b. Stater Fig. . Staininobe Fig. .-1 tridecane as an internal standard, and ground using a Retsch MM300 mixer mill for 20 s. The hexane extract was collected and analysed using a Perkin Elmer Autosystem XC GC-FID fitted with a Zebron-5 column and with He as the carrier gas at a flow rate of 1 ml min-1. The column temperature was held at 70°C for 4 min following injection of a 2.5 μL aliquot, then ramped at 10°C min-1 to 250°C and held for a further 4 min. Oil constituents were identified by retention time comparison with known standards or by GC-MS using a 7890A GC coupled to a 5975C mass spectrometer operated with the same column and conditions as for the GC-FID. Oil constituents were quantified by comparison with the known standards or by using the average response ratio of all compounds.Isolated cavities were imaged on a micrometer slide under a dissecting microscope with transmitted lighting to enable visualization of the lumen through the translucent cavity epithelial cells. The lumen volume for each cavity was estimated assuming an elipsoid shape and averaging two measurements for each of the equatorial radii and the polar radius of the lumen made using ImageJ software . Each cavity was then transferred by pipette to a microtube containing two tungsten balls and 40 μl of hexane containing 100 μg mlE. polybractea cavities were transferred via pipette to a microfuge tube containing 100 μL of a 50:50 v/v solution of sorbital buffer and RNAlater and frozen in liquid N2 before being ground with a plastic microfuge pestle. Total RNA was extracted using an RNeasy Plant Mini Kit . RLC buffer (500 μL) with β-mercaptoethanol (5 μL) was added to the ground secretory cavity tissue and the sample was then vortexed and transferred to a QIAshredder tube. Total RNA quantity and purity (260/280 nm) was measured in a 1 μL aliquot using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). Genomic DNA was removed using DNaseI and cDNA was synthesised from 30 ng of RNA using SuperScript III First-Strand Synthesis SuperMix (Invitrogen) with oligo(dT)20 primers according to manufacturer's instructions.Leaf strips were incubated for 12 h with pectinase in glycerol P-4716 in a solution comprised of 50% standard buffer containing 5 mM DTT and 50% RNAlater . One hundred isolated Eucalyptus globulus, sage, rosemary and Arabidopsis thaliana . PCR conditions were as follows: 60 s at 94°C; 40 cycles of 30 s at 94°C, 60 s at 61°C, 60 s at 72°C; 300 s at 72°C. Primers specific to E. polybractea 5'-GGTATGACTTGTGCAAAGCCT and 5'-CACCGTATTGAATTCGTGGTCT were designed using sequences obtained from PCR with degenerate primers. Actin primers 5'-ACGGCCTGGATGGCGACGTACATG and 5'-GCAGAAGGACGCCTACGTTGGTGAC for the sorghum ac1 actin gene (GenBank accession no. X79378) were used as a control [Ex Taq DNA polymerase with specific monoterpene synthase and actin primers. PCR conditions were as follows: 60 s at 94°C; 40 cycles of 30 s at 94°C, 30 s at 61°C, 60 s at 72°C; 300 s at 72°C.Degenerate primers 5'-TTGGAAGAGCTRSARCTATTCAC and 5'-GTTCCATCTTYTTCCATGYTKKGTC were designed based on an alignment of the published sequences of monoterpene synthase genes from One-way ANOVA and regression analyses were performed using SPSS version 18.0 .The authors declare that they have no competing interests.JG conceived the study, developed the protocol and drafted the manuscript. AH performed the microscopy and helped to draft the manuscript. MM & EN carried out the molecular studies and helped to draft the manuscript. DK helped develop the protocol and performed the gas chromatography analyses. IW helped to design the study and draft the manuscript. All authors read and approved the final manuscript.

In a patient undergoing regular hemodialysis through an arteriovenous fistula access, pleural effusion is a known long term complication. However, a unilateral hemothorax is relatively uncommon. Here we report a 46 year old male, end-stage renal disease patient, on maintenance hemodialysis, who presented with a giant brachiocephalic AV fistula in his left arm and progressive breathlessness. Radiological imaging revealed a left sided pleural effusion. Ultrasound guided aspiration revealed a hemorrhagic pleural fluid. A Doppler study of the fistula revealed a high velocity blood flow through the fistula, thereby establishing the cause of the unilateral hemothorax. Ligation of the fistula resulted in complete resolution of the hemothorax. The other possible causes for hemothorax in a dialysis patient are also discussed in this case report. AV (arteriovenous) fistulas are recognized as the preferred access method for hemodialysis in patients with end stage renal disease. However, they are associated with a separate subset of complications, in addition to those that can potentially occur with hemodialysis. Pleural effusion, with or without hemorrhage, is one among these complications. High flow through the arteriovenous fistula is recognized as an uncommon cause of unilateral hemothorax in such patients. Here we report a patient with a giant AV fistula and a same sided hemorrhagic pleural effusion.A 46 year old male, a known hypertensive on treatment for the past 10 years, was diagnosed to have chronic renal failure 3 years ago. Consequently he progressed to ESRD and hemodialysis was initiated in December 2006 for the same, with the creation of a brachiocephalic arteriovenous fistula over the left arm. He has been undergoing regular thrice weekly hemodialysis since then. He is a school teacher by occupation. He had no past history of diabetes mellitus, hypothyroidism, coronary artery disease, or hepatic disease. No past history of Tuberculosis or TB contact. He is married and has two sons. He was a non-smoker and a non-alcoholic.rd June 2008, he presented to the emergency department of our tertiary care hospital with complaints of progressive breathlessness over the past 15 days. There was no history of associated chest pain, palpitations, syncope, cough, expectoration, or orthopnea.Over the past year, the artificial AV fistula enlarged in size to attain its current dimensions Figure . On 3rd On examination, he had pallor and bilateral pitting pedal edema. He was afebrile, with a pulse rate of 108/min, BP of 140/80 mmHg, respiratory rate of 28/min. A giant arteriovenous fistula was seen extending from the cubital fossa of the left arm up to the left supraclavicular region (Figure th day after admission. Following ligation of the fistula, the patient's left sided pleural effusion decreased. The chest drain was removed after 7 days and patient was discharged after 3 more days. On follow up 6 weeks later, the patient did not have a recurrence of pleural effusion (Figure His blood investigations showed hemoglobin of 4.5 g/dl, BUN – 13 mg/dl, creatinine- 2.6 mg/dl, with other parameters being within normal limits. His coagulation profile was slightly deranged with a raised INR (Table The incidence of pleural effusion in hospitalized patients receiving long-term hemodialysis is 20.2%; of them 48% have unilateral effusion . UnilateUremia causes uremic pleuritis. This evolves into a fibrinous pleuritis which, if untreated, progresses to become a fibrothorax. The pleural effusion takes the form of exudates, which at times is hemorrhagic, and often unilateral. It usually disappears with dialysis and adequate renal replacement therapy, or develops into a fibrothorax. An ultrasonogram may depict septae, fibrinous bands, and debris -4. ConsiIn patients on dialysis, hemothorax may occur due to coagulopathy. This can arise secondary to platelet dysfunction of uremia and/or anticoagulant use ,5,7,8. AHemothorax could arise as a complication of central venous catheterization. This is usually secondary to malpositioning or malfunctioning of the vascular catheter ,8. In ouAs a result of the increased venous pressure due to the high venous flow through the arteriovenous fistula, stenosis and/or thrombosis of the brachiocephalic and/or subclavian veins can occur. A stenosis of the brachiocephalic vein, in association with high venous flow rates, considerably increase venous pressures in the intercostal and bronchial veins of the left side of the chest. Consequently, local hemodynamics would be altered such that pleural fluid resorption would not occur normally, and excess pleural fluid formation would occur. This leads to unilateral pleural effusion over the same side as the vein stenosis/thrombosis . DopplerThe other possibility of pulmonary embolism due to a thrombus arising from the AV fistula also seemed unlikely, as there was no evidence of thrombosis in the fistula and the patient's D-dimer levels were within normal limits. Pulmonary embolism usually produces minor pleural effusions unlike the massive effusion in our patient ,9.The possibility of the hemothorax occurring secondary to tuberculosis or other infective etiology seems improbable as the patient improved significantly following ligation of the fistula, and had no evidence of recurrence even after 6 weeks. During this entire period he was not given antibiotics or antituberculous therapy. A tuberculous hemothorax resolving without antituberculous therapy is not possible.A malignant etiology for hemothorax is again unlikely in view of rapid resolution and absence of recurrence of the hemothorax. In addition, the pleural fluid showed no evidence of malignant cells.There have been reports of hemothorax occurring as a consequence of excessive flow in an AVF access . As a reHigh flow state of an arteriovenous fistula access must be thought of as a differential diagnosis for ipsilateral hemothorax, in a patient on hemodialysis through the same. Dramatic improvement results with ligation of the fistula.People used to give me odd stares because of the snake-like structure over my arm. When I was admitted, the sight of blood coming out of my chest when the tube was inserted really scared me. I feel a lot more content now that this fistula has been removed. I feel like I have been cured of this problem.AV: arteriovenous; AVF: arteriovenous fistula; ESRD: end stage renal disease; TB: tuberculosis; Hb: hemoglobin; TC: total count; RBC: red blood cells; WBC: white blood cells; BUN: blood urea nitrogen; PT: prothrombin time; PTT: partial thromboplastin time; INR: international normalized ratio; USG: ultrasonogram; PCR: polymerase chain reaction.The authors declare that they have no competing interests.SS, PG, ADP, AAK, DV, VJ, DR, GPSS were involved in the patient care, acquisition of data, analysis and interpretation of data, review of literature, drafting and revising the manuscript.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor – in – Chief of this journal.

The mol­ecular structure features short intra­molecular C—H⋯O contacts and the crystal packing exhibits weak inter­molecular C—H⋯S and π–π inter­actions [centroid-to-centroid distances 3.734 (2)–3.888 (2) Å].In the title compound, C Å b = 10.1269 (7) Å c = 28.233 (2) Å V = 4400.2 (6) Å3 Z = 8 Kα radiationMo −1 μ = 0.37 mmT = 295 (2) K 0.25 × 0.20 × 0.20 mm Bruker Kappa APEX2 diffractometerSADABS; Sheldrick, 1996T min = 0.914, T max = 0.931Absorption correction: multi-scan (25628 measured reflections5212 independent reflectionsI > 2σ(I)3570 reflections with R int = 0.040 R[F 2 > 2σ(F 2)] = 0.053 wR(F 2) = 0.175 S = 1.04 5212 reflections290 parameters2 restraintsH-atom parameters constrainedmax = 0.55 e Å−3 Δρmin = −0.55 e Å−3 Δρ APEX2 used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008PLATON (Spek, 2003SHELXL97.Data collection: 10.1107/S1600536809003493/bt2862sup1.cif Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536809003493/bt2862Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:

The aim of the present clinical study was to determine the local bone density in dental implant recipient sites using computerized tomography (CT) and to investigate the influence of local bone density on implant stability parameters and implant success.A total of 300 implants were placed in 111 patients between 2003 and 2005. The bone density in each implant recipient site was determined using CT. Insertion torque and resonance frequency analysis were used as implant stability parameters. The peak insertion torque values were recorded with OsseoCare machine. The resonance frequency analysis measurements were performed with Osstell instrument immediately after implant placement, 6, and 12 months later.Of 300 implants placed, 20 were lost, meaning a survival rate of %. 93.3 after three years (average 3.7 ± 0.7 years). The mean bone density, insertion torque and RFA recordings of all 300 implants were 620 ± 251 HU, 36.1 ± 8 Ncm, and 65.7 ± 9 ISQ at implant placement respectively; which indicated statistically significant correlations between bone density and insertion torque values (p < 0.001), bone density and ISQ values (p < 0.001), and insertion torque and ISQ values (p < 0.001). The mean bone density, insertion torque and RFA values were 645 ± 240 HU, 37.2 ± 7 Ncm, and 67.1 ± 7 ISQ for 280 successful implants at implant placement, while corresponding values were 267 ± 47 HU, 21.8 ± 4 Ncm, and 46.5 ± 4 ISQ for 20 failed implants; which indicated statistically significant differences for each parameter (p < 0.001).CT is a useful tool to determine the bone density in the implant recipient sites, and the local bone density has a prevailing influence on primary implant stability, which is an important determinant for implant success. The use of dental implants to restore missing teeth has become increasingly widespread over the past two decades. Numerous clinical studies with dental implants have revealed encouraging outcomes -4. The sClinical reports suggest that dental implants for the mandible have higher survival rates than those for the maxilla, especially for the posterior maxilla ,8. CompaSeveral factors, such as implant geometry, preparation technique, and quality and quantity of local bone influence primary stability, and primary implant stability is one of the main factors influencing implant survival rates . It is aThe purpose of the present study was to examine the correlations between the local bone density from CT, and the implant stability parameters including insertion torque and resonance frequency analysis and the implant survival rates.A total of 111 patient files were analysed. The mean age of the patients was 55 ± 11. All patients have been provided with a total of 300 implants in two clinics from 2003 to 2005. Details of the diameter and length of implants are presented in Table The presurgical evaluation consisted of clinical and radiographic examinations including computerized tomography scans. All patients were thoroughly informed about the procedure and signed a written consent. Also, local ethic approval was obtained for the main study.15, and the mean bone density of the implant recipient area was measured using software incorporated in the CT machine machine was utilized. Prior to CT scan, previously fabricated surgical acrylic templates including 1 mm-diamater indicator metal rods, which were located in the center of the missing teeth, or the existing removable complete dentures attached with the same indicator rods for edentulous patients were placed in the mouth. The same scanning conditions were provided for each CT scan. The cross-sectional, coronal and axial images for each maxilla/mandible were obtained from the CT machine. The suitable implant for each previously designated implant recipient site was selected by using the cross-sectional images. The rectangular area of each implant selected was plotted on the cross-sectional images with a tool incorporated in the CT machineOne hour prior to implant surgery, the patients were given 2 grams of amoxicillin. Standard one-stage surgical technique was utilized to prepare the surgical sites. Full-thickness mucoperiosteal flaps were raised while the patients were under local anesthesia. 300 Brånemark Mk III TiUnite implants were placed under sterile saline irrigation. All drilling and implant insertion procedures were carried out with the Osseocare motor . Immediately following implant placement, resonance frequency analysis meaurements with an Osstell instrument were performed.Conventional (3 months or 6 months after implant placement) and early loading protocols were used for the implants. The prostheses delivered to the patients were comprised of single-implant crowns, implant-supported fixed partial/full prostheses, and implant-supported overdentures.During the implant insertion, the maximum insertion torque value was recorded by means of the same OsseoCare motor . Starting from 20 Ncm, the placement torque was increased in steps of 5 Ncm, when the rotation stopped because of friction before the implant was fully inserted. The OsseoCare motor was developed to provide a well-controlled insertion torque to avoid mechanical overload of the equipment or bone tissue. The final maximum insertion torque value of each implant was recorded in 20, 32, and 45 Ncm.The resonance frequency analysis (RFA) measurements were performed using the Osstell instrument . All RFA measurements were performed at implant level immediately after implant placement, and at 6- and 12-month follow-up visits. Therefore, the prostheses and abutments were removed in order to perform RFA measurements. In essence, the 8.5 mm-height transducer was mounted on the implants orthoradially with the upright part on the oral side. The resonance frequency analysis transducer was designed as an offset cantilever beam with attached two piezoceramic elements. Exciting these elements vibrates the beam. The excitation signal is a sine wave typically varying in frequency from 5 to 15 Hz with a peak amplitude of 1 V. The captured data are recorded in Implant Stability Quotient (ISQ) ranging from 1 to 100. ISQ values are derived from the stiffness (N/μm) of the implant/bone system and the calibration parameters of the transducer. High ISQ value indicates high stability, whereas low value indicates a low implant stability.Implants had to meet the following criteria, which are a modification of the proposal by Albrektsson and Zarb , to be rP < 0.05 was considered statistically significant.SPSS statistical software was used for all statistical analysis. The distribution of data was non-parametric, which was determined by the Kolmogorov-Smirnov test. Mann Whitney U test was used to verify possible differences between groups in terms of the bone density, insertion torque, and resonance frequency values. Correlations between the bone density, insertion torque, and implant stability values were determined by using Spearman's rho test. One hundred and eleven patients receiving 300 dental implants were included in this study. Twenty implants were lost in 15 patients, resulting in a failure rate of 6.7% after three years (average 3.7 ± 0.7 years). Only the implants failed before prosthesis delivery were included in the present study and no implant was lost after prosthesis delivery. The distribution of the failed implants was presented in Table The total of 300 implant sites consisted of 100 anterior mandibular sites (846 ± 234 HU), 60 posterior mandibular sites (526 ± 107 HU), 70 anterior maxillary sites (591 ± 176 HU), and 70 posterior maxillary sites (403 ± 95 HU). It was found that the bone density in all patients ranged from 199 HU to 1231 HU. The mean bone density, insertion torque and RFA recordings of all 300 implants were 620 ± 251 HU, 36.1 ± 8 Ncm, and 65.7 ± 9 ISQ at implant placement respectively; which indicated statistically significant correlations between bone density and insertion torque values , bone density and ISQ values , and insertion torque and ISQ values .The mean bone density, insertion torque and RFA values were 645 ± 240 HU, 37.2 ± 7 Ncm, and 67.1 ± 7 ISQ for 280 successful implants at implant placement, while corresponding values were 267 ± 47 HU, 21.8 ± 4 Ncm, and 46.5 ± 4 ISQ for 20 failed implants, and the differences between succesful and failed implants were statistically significant for each parameter (p < 0.001). When 280 successful implants were considered, the mean ISQ values slightly decrease from implant surgery (67.1 ± 7 ISQ) to 6- month follow-up visit (66.9 ± 6 ISQ) (p > 0.05), and increased from 6-month to 12-month follow-up visit (68.6 ± 7 ISQ) (p < 0.001). The detailed analysis of the changes in ISQ values during the 1-year observation period and statistical analysis were presented in Figure The mean bone density, insertion torque and RFA values were 542 ± 20 HU, 34.5 ± 8 Ncm, and 64 ± 9 ISQ for 145 implants placed in females, while corresponding values were 692 ± 271 HU, 37.6 ± 8 Ncm, and 67.3 ± 8 ISQ for 155 implants placed in males, and the differences between females and males were statistically significant for each parameter (p < 0.001) while 179 implants were placed in non-smokers and 9 implants were lost (5.02%). When compared to the non-smokers, the higher percentage of implant failures in the smokers observed in the present study is in agreement with the earlier studies ,37. StriAs an alternative to CT scanning, laser Doppler flowmetry has recently been introduced as a valid method of determining bone vascularity and, as a derivation, bone quality . VerdoncUnder the guidelines of this study, the following conclusions can be drawn:1. CT scanning, which is a non-invasive method may be used to determine the regional bone quality before implant surgery.2. Significant correlations found between bone quality and implant stability parameters indicate that clinicians may predict primary stability before implant insertion, and they may modify their treatment plans before implant surgery, where the bone quality is poor.IT gathered and analyzed all retrospective data, and did statistics. Also wrote the main version of the article. EG wrote the final version of the article and did corrections. The authors declare that they have no competing interests.The pre-publication history for this paper can be accessed here:

Sarcoptes scabiei. It is common worldwide and spreads rapidly under crowded conditions, such as those found in socially disadvantaged communities of Indigenous populations and in developing countries. Pruritic scabies lesions facilitate opportunistic bacterial infections, particularly Group A streptococci. Streptococcal infections cause significant sequelae and the increased community streptococcal burden has led to extreme levels of acute rheumatic fever and rheumatic heart disease in Australia's Indigenous communities. In addition, emerging resistance to currently available therapeutics emphasizes the need to identify potential targets for novel chemotherapeutic and/or immunological intervention. Scabies research has been severely limited by the availability of parasites, and scabies remains a truly neglected infectious disease. We report development of a tractable model for scabies in the pig, Sus domestica.Scabies is a parasitic skin infestation caused by the burrowing mite Sarcoptes scabiei var. suis. To increase intensity and duration of infestation without generating animal welfare issues we have optimised an immunosuppression regimen utilising daily oral treatment with 0.2mg/kg dexamethasone. Only mild, controlled side effects are observed, and mange infection can be maintained indefinitely providing large mite numbers (>6000 mites/g skin) for molecular-based research on scabies. In pilot experiments we explore whether any adaptation of the mite population is reflected in genetic changes. Phylogenetic analysis was performed comparing sets of genetic data obtained from pig mites collected from naturally infected pigs with data from pig mites collected from the most recent cohort.Over five years and involving ten independent cohorts, we have developed a protocol for continuous passage of in vivo studies on host immune responses to scabies including the relations to the associated bacterial pathogenesis and more detailed studies of molecular evolution and host adaption. It is a most needed tool for the further investigation of this important and widespread parasitic disease.A reliable pig/scabies animal model will facilitate Sarcoptes scabiei, is a major driving force behind bacterial skin infections in tropical settings. Aboriginal and Torres Strait Islander peoples are nearly twenty times more likely to die from acute rheumatic fever and rheumatic heart disease than individuals from the wider Australian community. These conditions are caused by bacterial pathogens such as Group A streptococci, which have been linked to underlying scabies infestations. Community based initiatives to reduce scabies and associated disease have expanded, but have been threatened in recent years by emerging drug resistance. Critical biological questions surrounding scabies remain unanswered due to a lack of biomedical research. This has been due in part to a lack of either a suitable animal model or an in vitro culture system for scabies mites. The pig/mite model reported here will be a much needed resource for parasite material and will facilitate in vivo studies on host immune responses to scabies, including relations to associated bacterial pathogenesis, and more detailed studies of molecular evolution and host adaptation. It represents the missing tool to extrapolate emerging molecular data into an in vivo setting and may well allow the development of clinical interventions.Scabies, a neglected parasitic disease caused by the microscopic mite Sarcoptes scabiei. Human scabies is a widespread disease in developing regions of the world, and remains a significant problem amongst indigenous populations in developed countries Streptococcus or Staphylococcus aureusScabies, or sarcoptic mange, is an infectious skin disease caused by the mite Sarcoptes infest up to 40 different mammalian hosts across 17 families S. scabiei infestation in both human and animal populations, the pathogenesis and immune responses to this disease is not well understood Parasitic mites of the genus Scabies has historically been a difficult disease to study. Scabies mites cannot be maintained or propagated away from their animal host, and it is difficult to collect mites in large quantities, as a typical infestation of human scabies involves fewer than twenty mites. Access to hyper-infested hosts has enabled the construction of cDNA libraries from human in vitro. To overcome these barriers, the availability of a tractable animal model for scabies would be of enormous benefit. Despite being morphologically very similar S. scabiei variants appear to be predominantly host specific, and investigations whether they are genetically distinct are ongoing Despite these recent advances, access to hyper-infested hosts remains opportunistic and sporadic. Moreover, there has been an almost complete failure in efforts to maintain viable mites in the laboratory for longer than 24–48 hours, and no established methods are available to propagate mites S. scabiei. Herein we report progress in developing a sustainable experimental model of chronic porcine mange, which now consistently provides large numbers of mites, facilitating the less restrictive conduct of research on human and animal scabies.When infested with mange, pigs show similar epidermal, morphological, and immunological changes to humans All animals were handled in strict accordance with good animal practice as defined by the Australian code of practice for the care and use of animals for scientific purposes and the NHMRC's Animal Code of Practice, and all animal work conducted with ethical approval from both DEEDI and QIMR Animal Ethics Committees .Samples were initially sought from pigs for the purpose of obtaining sufficient mites to attempt transmission of infection to mice. Six ears from slaughtered pigs with mangy appearance were supplied to the laboratory in a weekly basis by a Southeast Queensland abattoir from June 2004 to November 2007. Skin pieces were dissected from the inner ear and incubated in glass petri dishes at 27°C, which encourages mites to crawl out towards the heat source. Dishes were examined and mites picked under a dissecting microscope using microscopic needles.2). Several crusts were inserted into both ear canals of naïve piglets. Pigs were temporarily restrained to prevent dislodgement of the crust by agitation, thereby allowing successful infestation.Pigs were housed at the DEEDI Animal Research Institute, Yeerongpilly, QLD, and at the Centre for Advanced Animal Studies, Gatton, QLD. It was ensured that the care and the experimental practices conformed to the Australian animal ethics guidelines. Pigs with suspected natural mange infections (Skin samples were collected by gently scraping and lifting off encrusted areas from the inner ear area of the pig with a sharpened teaspoon and subsequently examined for mites.S. scabiei var. suis DNA from 10 mites collected from one pig in group 4 in 2006 and from another pig in a later group 10 in 2010 was prepared as described previously S. scabiei var. hominis cDNA used as outgroups (data not shown). Sequence alignments and subsequent phylogenetic analysis was performed using MEGA4 The quantity of mites obtained from abattoir samples was variable, exceeding 3,000 mites per ear on some occasions, whereas at other times no mites were observed for several months . AlthougDespite having clinical features of mange, such as scratching behaviour, hair loss, reddened and flaky skin, the untreated pigs in groups 1 and 2 either had no or very few mites and symptoms resolved within 6 weeks of acquisition . These oTo increase mite numbers and maintain a prolonged infestation by counteracting natural immunity, it was proposed to treat mange infected pigs with corticosteroids. This concept is supported by the observation that corticosteroid therapy often results in the development of crusted scabies in humans Because dexamethasone had not been tested previously in scabies infected pigs, and to give appropriate consideration to animal welfare issues, a gradual, conservative treatment program was initiated.As naturally infected pigs self cured over time , Group 3Since 0.1mg/kg appeared to be inadequate to achieve sufficient immunosuppression, moderate increments in dosage were commenced, whereby a sentinal pig was exposed to the higher dose for six weeks prior to the rest of the group, allowing for observation of detrimental effects. At 0.3mg/kg pigs started to develop much larger crusted lesions which also spread to regions beyond the ears . At thisAt the maximal dose of 0.3mg/kg dexamethasone mite infestation dramatically increased, but noticeable side effects were observed. The most obvious of these was a tendency of growth retardation and a change of body shape. Retardation in weight gain in dexamethasone treated pigs has been reported Most these side effects parallel that of iatrogenic Cushing's syndrome seen in humans, with glucocorticoid excess resulting in symptoms such as centralised adiposity, bone osteoporosis, hirsutism, depression and anxiety. It should be emphasised that the side effects observed in this study were mild, and aside from the physical effects, pigs appeared normal in spite of their mange infestation. Pigs were closely monitored by skilled veterinary staff for side-effects including increased susceptibility to disease, but other than mange, no additional infections were observed. Good husbandry and infection control measures were paramount to this process.Since the optimal treatment regimen has now been established, the dosage of dexamethasone can be adjusted accordingly to maintain required mite burden, while minimising side effects. Continued tailoring of dexamethasone is also important since it became evident through the trial that pigs showed great variation in inherent immunologic responses to both steroid treatment and scabies. For example, one pig on low dose dexamethasone still developed chronic mange and the side effects described above. Similarly, other pigs had relatively low mite burdens despite receiving higher doses of dexamethasone. Variation in pig mange responses are common Our data with data from pig mites collected from one pig in group 10 (2009). Thus, we were investigating genetic changes in a closed mite population maintained isolated for over 3 years.We focussed on the SMIPP-S-B2 gene, belonging to a multigene family of at least 32 closely related gut proteases homologous to the group 3 major allergens of astigmatid house dust mites The pig mite derived SMIPP-S-B2 sequences form a distinct cluster from the corresponding human mite sequence. We also observed a considerable degree of intra-species heterogeneity. In the 2006 mite population, three distinct B2 isoforms (B2-1 to 3) were observed, with polymorphisms at 7 amino acid sites with isoin vivo in human and pig populations, it is difficult to undertake such studies in human settings. In previous genetic analysis on mites from human crusted scabies, patients had been infected over many years and had undergone multiple treatments over several episodes. In microsatellite data reported by Walton et al.While heterogeneity within the SMIPP family most likely happens Although this SMIPP-S analysis was limited in its scope, it nevertheless highlights the potential of this model for investigations of the molecular evolution of scabies mite genes. Representative isolates collected over the continuing passage of mites through successive generations may provide fascinating insights into genetic diversity, evolution and host-adaption of scabies mites, particularly within the novel SMIPP family.In conclusion, over a prolonged timeframe of five years, involving ten independent cohorts , we have successfully developed a sustainable experimental infestation of scabies mite on immunosuppressed pigs. Encrustment on the ears occurs after 6–12 weeks and can be maintained for at least 12 months, depending on drug dosage and individual pig response. This animal model now consistently provides large mite numbers (>6000 mites/g skin) for molecular-based research on scabies. Projects that have benefitted from this to date include detailed studies of gene expression in scabies mites in vivo studies, including investigation of pig immune responses to scabies and dexamethasone pathways of immunosuppression, in addition to more detailed studies of molecular evolution and host adaption. Furthermore, our pig model is now at a stage to be optimised as pig/scabies/GAS model. Such research should result in innovative tools for the prevention, monitoring and further investigation of this important and widespread parasitic disease.Most importantly, further research may utilise the full features of this animal model which facilitates

With the completion of the whole genome sequence for many organisms, investigations into genomic structure have revealed that gene distribution is variable, and that genes with similar function or expression are located within clusters. This clustering suggests that there are evolutionary constraints that determine genome architecture. However, as most of the evidence for constraints on genome evolution comes from studies on yeast, it is unclear how much of this prior work can be extrapolated to mammalian genomes. Therefore, in this work we wished to examine the constraints on regions of the mammalian genome containing conserved gene clusters.We first identified regions of the mouse genome with microsynteny conservation by comparing gene arrangement in the mouse genome to the human, rat, and dog genomes. We then asked if any particular gene types were found preferentially in conserved regions. We found a significant correlation between conserved microsynteny and the density of mouse orthologs of human disease genes, suggesting that disease genes are clustered in genomic regions of increased microsynteny conservation.The correlation between microsynteny conservation and disease gene locations indicates that regions of the mouse genome with microsynteny conservation may contain undiscovered human disease genes. This study not only demonstrates that gene function constrains mammalian genome organization, but also identifies regions of the mouse genome that can be experimentally examined to produce mouse models of human disease. The availability of several mammalian genome sequences has enabled comparative genomic studies to identify regions of conserved linkage among different organisms (reviewed in -4). ThesC. elegans, essential genes are located in clusters in regions with low recombination [Although it is becoming increasingly apparent that genomes display a large degree of structural plasticity, there are nevertheless significant evolutionary constraints on genome structure. Previous studies have provided evidence for functional constraints on genome organization in prokaryotic and eukaryotic genomes . Studiesbination .Clustering of genes with similar functions has also been observed in mammalian genomes. In the human genome, genes that are in the same pathway are in closer proximity than would be expected by chance . SimilarMany of the previous studies to detect gene clustering were based on bioinformatic analysis of genome annotation. However, there is also support for functional constraints on mammalian genome organization from experimental data. Analysis of saturation levels of mouse mutagenesis screens for lethal phenotypes directed at specific genomic regions demonstrated that mouse essential genes are disproportionately found in regions of conserved microsynteny , at leasWe evaluated the level of microsynteny conservation between the mouse genome and those of human, dog and rat. First, we obtained all protein-coding genes and their genomic locations on all mouse autosomes as annotated in the Ensembl mouse genome browser ,31 should not be rejected. However, the use of non-overlapping 20 Mb windows restricted the resolution of the study. For example, chromosome 19 has only two observations from non-overlapping windows. To improve the resolution of our study, we next examined 20 Mb intervals staggered by 5 Mb. This sliding window analysis allowed more observations on each chromosome.As the data conformed to a normal distribution, we therefore calculated Z-scores (number of standard deviations above or below the mean) for each 20 Mb sliding window. Windows with Z>1 were considered to have increased conservation, those with Z<-1 were considered to have decreased conservation, and windows with 1>Z>-1 had intermediate conservation. There were 51 sliding windows of the mouse genome found to have Z>1, indicative of higher microsynteny conservation, and 91 intervals found to have Z<-1, indicative of lower microsynteny conservation. Three hundred twenty-two genomic regions demonstrate intermediate microsynteny conservation with scores of 1>Z>-1. On individual chromosomes there is variation in the conservation of microsynteny, with most chromosomes containing both windows of increased microsynteny conservation and windows of decreased microsynteny conservation . Such a representation over-estimates the true correlation between the two sets, since gene density varies considerably in different windows. This confounds the analysis as regions with high gene density would be expected to have both high numbers of disease orthologs and high numbers of genes with conserved microsynteny regardless of whether there is an additional correlation between microsynteny conservation and the presence of disease gene orthologs. When corrected for gene density, a significant correlation between microsynteny conservation and disease gene ortholog density is still observed .We next assessed whether the orthologs of disease genes were located in regions of the mouse genome with increased microsynteny conservation. We detect a correlation between regions of the genome with conserved microsynteny and the distribution of disease gene orthologs over the whole genome . Orthologs of these genes in the human, rat, and dog genomes, along with their genomic positions, were retrieved using Ensembl BioMart homology filters. Any mouse genes that did not have an ortholog in another genome identified in BioMart were then subjected to a BLAT search http://genome.ucsc.edu/cgi-bin/hgBlat?command=start on the other genome using the mouse protein sequence retrieved with the BioMart sequences filter. The genomic location of the best match to the mouse protein sequence from the second genome was retrieved. Locally developed software was used to identify genes with conserved microsynteny. Each mouse gene is analyzed in turn. For each mouse gene m, its neighbors in the mouse genome (m-1 and m+1) are identified. The orientation of m-1, m and m+1 is determined from the start and stop positions. In the dog, human and rat genomes, the homologue of m is identified from both Ensembl BioMart homology filters and BLAT searches. We term these homologues d, h and r. For each of these genes their neighbors in their respective genomes are identified, and their orientation determined. The gene m is defined as having conserved microsynteny only if all of the following are true: (i) m, d, h, and r comprise a homologous set of genes (ii) m-1, d-1, h-1 and r-1 comprise a homologous set (iii) m+1, d+1, h+1 and r+1 comprise a homologous set (iv) the m, d, h, and r have the same orientation (v) m-1, d-1, h-1 and r-1 have the same orientation and (iv) m+1, d+1, h+1 and r+1 have the same orientation. Results are robust whether or not we limit the definition of conservation only to those genes close to each other in the genome (within 200 Kb). Results presented have conservation limited in this manner.All protein coding genes on mouse autosomes were retrieved using a BioMart search on the Ensembl Genome Browser database. Genomic positions of synteny blocks were retrieved from the database and tabulated.Each highly conserved mouse genomic region was compared to synteny maps based on sequence alignment. Maps of the mouse compared to dog, rat, and human were retrieved from the Ensembl genome browser found in mouse genomic regions with microsyteny conservation of Z>1.Click here for fileData on conserved and disease genes by window for mouse genome. An excel file listing for each window in the mouse genome the number of conserved or disease genes, with the Z-score of each window. Each window size is presented as a separate worksheet.Click here for file

A new method for more timely monitoring of cancer patient survival was employed to assess progress in 5-year survival of breast cancer patients in Saarland, Germany, between 1980-84 and 1990-94. Five-year relative survival gradually increased from 68.8% to 73.5%. Improvements were most pronounced among age groups 50-59 and 60-69. The latter had the highest 5-year relative survival (77.1%) in 1990-94.

Dementia is one of the most disabling disorders afflicting the elderly, with a staggering emotional and economic impact. Antidementia agents have been used for delaying cognitive decline. Antipsychotics are commonly prescribed for behavioral symptoms associated with dementia.To explore the use of anti-dementing agents and antipsychotics used in patients with a diagnosis of dementiaA retrospective chart review method; geriatric clinic of tertiary care setting.The study sample included 51 consecutive patients with a diagnosis of dementia. The commonest subtype of dementia that was diagnosed was Alzheimer's disease (45%), followed by Frontotemporal dementia (25%).The commonest antidementia drug that was used was donepezil, which alone was prescribed in 27 patients (52%). The commonest antipsychotic used was quetiapine, which was used in 24 patients (47%).The study found donepezil to be the most commonly prescribed antidementia drug and quetiapine to be the most commonly used antipsychotic in a tertiary care geriatric clinic, in a developing country. There is a need to study the cost-effectiveness of antidementia and antipsychotic drugs in patients with dementia, in developing countries. Dementia is a silent epidemic afflicting the elderly with a staggering emotional and economic impact, throughout the world, including India. The Delphi consensus study estimateThe treatment of dementia varies through the course of the illness, because symptoms evolve over time. Cholinesterase inhibitors that include donepezil, rivastigmine, and galantamine have been recommended for use in mild-to-moderate Alzheimer's dementia. Memantine, a noncompetitive N-methyl d-aspartate (NMDA) antagonist, is the only drug that has been recommended for use in severe dementia, with evidence supporting its use in moderate dementia. AppropriBehavioral and psychological symptoms of dementia (BPSD) are an integral part of the dementia syndrome. Psychosis, agitation, and other behavioral symptoms are reported in patients with dementia causing significant caregiver distress and leading to early institutionalization. The geneThe prescription pattern of antidementia drugs and antipsychotics in dementia patients has not been studied in developing countries where cost may be the most important factor in determining their choice. We therefore studied retrospectively, patients with cognitive complaints presenting to the tertiary care geriatric clinic of our hospital with an aim to investigate the use of antidementia and antipsychotics drugs prescribed to dementia patients.The study reported in this article was conducted at the Geriatric Clinic Outpatient Department of a tertiary center . This study is a clinical audit based on a retrospective chart review of 51 individuals with an ICD-10 diagnosiIn all patients, the diagnosis was made by a detailed, face-to-face, semi-structured clinical interview by resident doctors in psychiatry utilizing the ICD - 10 criteria. After a The bulk of this study focused on the detailed reading of individual case files of patients. Initially, lists of individuals with ICD-10 diagnosis of dementia were identified from the outpatient services of the geriatric clinic. After identification, these case notes were reviewed, and relevant information was incorporated in the study, mainly focusing on the antidementia and antipsychotic drugs prescribed. The data were suitably coded and extracted on a custom Microsoft excel sheet and analyzed. As this was a chart review of patients already in treatment with voluntary consent, individual informed consent was not obtained.Fifty-one consecutive patients with dementia, who registered at the Geriatric Clinic Outpatient Department of a tertiary center, met the study criteria for the study sample. The mean age of the sample was 65.72 years. The sample included 23 males (45.1%) and 28 females (54.9%). A majority of the patients belonged to the middle-socioeconomic status (57%).The commonest subtype of dementia that was diagnosed was Alzheimer's disease (45%), followed by fronto-temporal dementia (25%), followed by others,. The HMSThe commonest antidementia drug that was used was donepezil, which alone was prescribed in 27 patients (52%). It was prescribed in a dose range of 5 to 10 mg/day. Memantine alone was prescribed in nine patients (18%). A combination of choline-esterase inhibitor and memantine was used in five patients (10%). Galantamine alone was used in three patients only, whereas, Rivastigmine alone was used in a single patient. Six patients (12%) were not prescribed any antidementia drugs. The findings are depicted in The commonest antipsychotic used was Quetiapine, which was used in 24 patients (47%). It was used in a dose range of 25 to 300 mg/day. The duration of follow-up was up to 11 months . A similar number of patients did not receive any antipsychotics. A minority of the patients received the other antipsychotics . The findings are depicted in Dementias are common among elderly patients, but they are poorly recognized and treated in developing countries, including India. To the bIn terms of comparative effectiveness, there is very little to choose from among the three choline-esterase inhibitors. A systemFood and Drug Administration (FDA), based on a meta-analysis of 17 double-blind randomized placebo-controlled trials among elderly people with dementia, commented that atypical antipsychotics were associated with a significantly (1.6-1.7 times) greater mortality risk when compared with placebos. However, there is a paucity of any other evidence-based treatment alternatives to antipsychotics for this population.[The study found that quetiapine was the most common antipsychotic used for behavioral and psychotic symptoms. None of the patients were prescribed typical antipsychotics. The lesser propensity of quetiapine to cause extra pyramidal symptoms among the atypical antipsychotics may be the most plausible reason for prescribing it. The sedation that is commonly seen with quetiapine may be the other probable reason for the choice of the drug. The greater metabolic side effect profile of olanzapine and risperidone may also have deterred the use of these drugs in spite of them being cheaper. Antipsychotic drugs should be considered for BPSD only if there is a specific need, or if other treatments have failed; decision-making should be individualized and documented after a risk-benefit analysis. The results of the phase I outcomes from the Clinical Antipsychotic Trial of Intervention Effectiveness study for Alzheimer's disease (CATIE-AD) suggest that antipsychotics may be more effective for particular symptoms, such as anger, aggression, and paranoid ideas. They do not appear to improve the functioning, care needs, or quality of life. The Foodpulation. A watchfpulation.SSRIs were prescribed in a small minority of the patients. The patients who received SSRIs were those with fronto-temporal dementia. Only two patients received benzodiazepines, which were avoided probably due to risks such as falls and amnesia.In summary, the study found donepezil and quetiapine to be the most commonly prescribed antidementia drug and antipsychotic, respectively. There are reasons to exercise caution against our study results as this was a small sample, and has to be taken with all the caveats of a retrospective chart-review design. Notwithstanding the above shortcomings, this investigation has indeed thrown up valuable results that will surely help fine tune the clinical prescription approach to dementia. The audit highlights the need to study the cost-effectiveness of antidementia and antipsychotic drugs for patients with dementia, in developing countries.

The present study examined income-related household food purchases among a sample of 90 households from the community.Annotated food purchase receipts were collected for a four-week period by the primary household shopper. Receipt food source and foods items were classified into specific categories, and food quantities in ounces were recorded by research staff. For home sources, a limited number of food/beverage categories were recorded. For eating out sources, all food/beverage items were recorded. Median monthly per person dollars spent and per person ounces purchased were computed. Food sources and food categories were examined by household income tertile.A community-based sample of 90 households.Higher income households spent significantly more dollars per person per month from both home and eating out sources compared with lower income households . Compared with lower income households, higher income households spent significantly more home source dollars on both fruits/vegetables and sweets/snacks , but did not differ on home dollars spent on sugar sweetened beverages . The proportion of home beverages that were sugar sweetened beverages was significantly higher among lower income households . Within eating out sources, lower income households spent a significantly greater percent of dollars per person at carry out places . No income differences were observed for dollars spent at discount grocery stores, small grocery stores or convenience stores.Higher income households spent more money on both healthy and less healthy foods from a wide range of sources. Lower income households spent a larger proportion of their eating out dollars at carry out places, and a larger proportion of their home beverage purchases were sugar sweetened beverages. Population-based surveys of individual intake show that lower income is associated with a poorer quality diet . IndividExamining food purchase patterns at the household level may provide insight into potential reasons for less healthful individual food intake. Household food purchases may influence individual intake through simple availability and from social influences such as behavioral modeling . Lower iTwo possible reasons for these differences in foods purchased and food sources selected are related to food cost and food availability. When food cost constraints are high, as they are among lower income households, shoppers may choose more energy dense foods to maximize the calories obtained per dollar expended ,3,7,8. TEating out is another possible means by which household income may influence individual dietary quality ,14. AbouThe household food purchase receipt or record is a method that has been used to examine household food purchases . The purpose of the present research was to explore income-related differences in household level food purchases that might be influenced by access to food sources and by food costs. The present paper reports associations between household income, food sources and food purchases among a community-based sample of 90 households. It was hypothesized that lower income households would be more likely than higher income households to spend their food dollars on home sources, such as grocery stores, rather than eating out sources. However, among eating out sources, it was hypothesized that lower income households would be more likely to spend food dollars at carry out places compared to full-service restaurants. Lower income households were hypothesized to spend more of their home source dollars at small grocery stores, convenience stores and gas stations, compared with higher income households. Lower income households were hypothesized to purchase fewer fruits and vegetables, more sweets and snacks and more sugar sweetened beverages compared to higher income households.et al. Household obesity prevention intervention targeting television, soft drinks, fast food and physical activity (under review). The study was a group-randomized trial, with households the unit of randomization, intervention and analysis. Data presented here are cross-sectional baseline data only. Households were recruited from the community over an eight-month period. Recruitment sources included community libraries, worksites, schools, daycare centers, health clinics, religious institutions, park and recreation centers, grocery stores and food co-ops. Fliers and brochures were distributed in local community centers, libraries, grocery stores and schools. In-person recruitment was conducted at community events, health fairs, and after-school programs. Interested households contacted study staff and were screened for eligibility. Study eligibility criteria were related to the weight gain prevention intervention aims. Eligibility criteria included: 1) at least one child ages ≥5 yrs and two household members ages ≥12 yrs; 2) residence in a private house or apartment within 20 miles of the university; 3) household TV viewing weekly average of ≥10 hrs per person; 4) no household members with dietary, medical, psychological, or physical limitations that would prevent their participation in intervention activities; and 5) willingness to be randomized to active intervention or control group. Eligibility was assessed during an initial telephone call conducted by a trained staff member and confirmed during an in-person baseline clinic visit. Overall, 732 inquiries were received, 289 families were eligible, 106 households completed the baseline clinic visit, and 90 households completed the baseline home visit and 4 week receipt data collection and thus were enrolled in the study and randomized. The University of Minnesota IRB approved the study.Data for the present study were collected as part of a community-based household weight gain prevention intervention conducted in Minneapolis, Minnesota, USA in 2007-2008 . Primary shoppers were instructed to query other household members about any food purchases they made during the week. The primary shopper was instructed to complete a receipt annotation sheet regardless of whether a receipt was available . For simplicity, we use the term receipt to refer to receipt annotation sheet hereafter.Due to the complexity and time required for receipt annotation, a decision was made to limit grocery store annotation to foods and beverages targeted by the intervention because of their potential link to excess weight gain. The specific targeted food categories were: fruits, vegetables, prepackaged snacks and sweets, prepackaged entrees, and beverages. For eating out receipts, primary shoppers were instructed to record all food and beverage items on the annotation sheet, including entrees, side orders, and beverages such as alcohol and specialty drinks . Details about the receipt data collection and coding protocol are available elsewhere .Statistical analyses were conducted using the Statistical Analysis System Release 8.2. . Food anTo examine purchases by household income, tertiles of household income were created based on self-reported household income from the clinic data collection visit. Personal pre-tax income was reported only on the adult survey, and all adult income was summed within the household to generate the household income. Median values for each study variable are presented by household income and statistical tests of increasing or decreasing trends across income were performed using the Jonckheere-Terpstra test, which is a non-parametric test for trend . The houOne hundred six households completed the initial baseline clinic visit. Of these, 90 households completed four weeks of receipt collection and were enrolled in the study. Households were enrolled and randomized only if the baseline clinic visit, the home visit, and the four-week receipt collection activity were completed. Thus, the drop out rate from clinic visit to receipt collection was 15% (90/106 = 85% completers). A total of 2,483 receipt annotation sheets were returned by the 90 households. Of these, 1,892 (75.2%) included receipts plus annotation sheets, and 591 included annotation sheets only (no receipt). A total of 9,498 food line items were included in the receipt annotation sheets.2 versus 29.0 kg/m2).Households differed in the number of receipts returned to research staff. The number of receipts returned by a household depends on many factors, including household income and the number of adults and children who reside in the household. The median number of receipts returned by high, medium and low income households was 35, 21, and 19, respectively. Only 10 households returned 10 or fewer total receipts during the four week receipt collection period. Of these 10 households, 1, 2, and 7 were high, medium and low income, respectively. Compared with households that turned in more than 10 receipts, households that turned in 10 or fewer receipts reported lower income , had primary shoppers who were less educated (30% advanced education versus 64%), and had a higher body mass index (34.7 kg/m2 (SD = 7.2 kg/m2). Household primary shoppers reported eating from a carry-out restaurant 2.3 times and from a sit down restaurant 1.1 times during the preceding week. On average, household primary shoppers reported that the household ate six meals together during the preceding week.Ninety-three percent of the primary shoppers were female, and 78% were white. Households on average were comprised of four people (1.8 adults (range 1-4) and 2.0 children (range 1-5)). The most common configuration was two adults and two children (51%). The primary shopper was on average 40.8 years (SD = 7.3 yrs), 64% were married or living with their significant other; 25.8% had more than a college degree. Reported household income was distributed as follows: 35% ≤ US $45,000 per year; 30% between US $50,000 and $95,000; and 35% ≥ US $100,000 per year. The average body mass index for the primary shopper was 29.7 kg/mP = .09). As a percent of eating out dollars, lower income households spent more at carry out restaurants (54%) compared with higher income households (37%) (P = .01).Table Household per person monthly dollars spent was significantly higher among middle and high income households for premium chain grocery stores, and among high income households for wholesale stores and specialty food stores. No significant income differences were observed for per person dollars spent at discount chain grocery stores, small grocery stores, convenience stores, gas stations or nonfood stores.Table No income-related differences were observed for sugar sweetened beverage purchases from home or eating out sources. However, the percent of home beverage dollars spent for sugar sweetened beverages was significantly higher among lower income households (45%) compared with higher income households (26%). Higher income households spent significantly more dollars per person on non-caloric beverages from both home and eating out sources. The relative proportion of per person dollars spent on sweets and snacks versus fruits and vegetables did not significantly differ by household income.The total ounces purchased for each food and beverage category is shown in Table It was initially hypothesized that higher income households would spend more on healthful foods and less on sugar-sweetened beverages and snack foods -3,5. TheFood purchase sources were also hypothesized to differ by household income, such that lower income households would purchase a larger proportion of their food purchases from sources such as convenience stores and small markets, which typically have more expensive and less healthful food choices. The results of the present study did not support this hypothesis. Lower income households did not differ from higher income households in the per person dollars spent at discount chain grocery stores, small grocery stores, convenience stores, gas stations or nonfood stores. The majority of food purchases were from discount chain grocery stores, regardless of household income level. Higher income households spent significantly more dollars per person from premium chain discount stores, wholesale stores and specialty food stores. Thus, in this sample of households, access to large chain retail food outlets does not seem to be a barrier specific to lower income households.Higher income households spend a larger proportion of their food dollars eating out . US natiThese results suggest that the overall amount of money available for food purchases may be the main characteristic related to food purchases by households of different income levels, more than a lack of access to food outlets. Although lower-income households may shop at similar food sources, the types and quality of the foods they purchase may be different from higher income households. For example, in the present study, lower income households may have economized by purchasing less expensive versions of food such as fruits and vegetables. However, it does appear that when they do purchase beverages from stores, a large percent of the beverages are sugar beverages. They also chose carry out restaurants more often than full-service restaurants. Thus, the lower income households appear to have choices about where to shop and what to buy. Higher income households may have more choices, given the greater amount of money they can afford to spend on food of all types from all sources. Food price is less of a barrier to purchase choices for high income households.The present study had several strengths. Detailed data were available on the frequency of food purchases from a wide range of retail food sources over a lengthy time period. The sample was a diverse community sample of households with a range of configurations of adults and children. Eating out sources were included in the receipt collection and annotation. Limitations of the study included the lack of coding of every grocery store food and beverage item purchased and the study's focus on certain targeted food and beverage categories. It is not known whether the receipt collection captured all food and beverage purchases during the four week period, nor whether the purchases of all household members were captured. The number of eating-out receipts declined over the four-week period, suggesting that frequent small purchases from coffee shops or fast food places may not have been completely captured . HoweverThe households that comprised the sample were not representative of the community and self-selected to participate in a weight gain prevention intervention involving eating, physical activity and television viewing behaviors. Compared to national census statistics, the sample was comprised of more married , well educated and higher income people . Thus, tFuture research is needed to examine household income-related food and beverage purchasing patterns in a representative sample of households. Documentation of food sources other than retail food stores is needed, particularly among lower income households that may receive food from sources such as food shelves or federal food programs. Examination of the full range of foods purchased will also provide more complete data about household income differences in food purchases, both sources and types of foods and beverages. A more detailed collection and analysis of specific food types and prices paid will help provide a more clear understanding of how lower income households spend less money for food but perhaps purchase more calories and thus increase risk of excess weight gain and obesity. These data will also help clarify how higher income households can spend more money on food and beverages but remain at lower risk of excess weight gain.Higher income households spent more money on all types of foods from a wide range of sources. Lower income households spent a larger proportion of their eating out dollars at carry out places, and a larger proportion of their home beverage purchases were sugar sweetened beverages. More detailed research on household food spending can promote a better understanding of how retail food sources, food prices and specific foods purchased contribute to household energy purchased, energy balance and risk for excess weight gain.The authors declare that they have no competing interests.SAF conceptualized the research idea and drafted the manuscript. NRM and MW conducted data analysis, provided scientific input and edited the manuscript. All authors read and approved the final manuscript.

The second author's name was incorrectly listed. The correct name is: M. A. Sokrates Stein. The correct citation is: Stöbe P, Stein MAS, Habring-Müller A, Bezdan D, Fuchs AL, et al. (2009) Multifactorial Regulation of a Hox Target Gene. PLoS Genet 5(3): e1000412. doi:10.1371/journal.pgen.1000412

The protonated N atom and one of the two 2-amino groups are hydrogen bonded to the 4-nitro­benzoate anion through a pair of N—H⋯O hydrogen bonds, forming an R 2 2(8) ring motif. The carboxyl­ate mean plane of the 4-nitro­benzoate anion is twisted by 3.77 (5)° from the attached ring and the nitro group is similarly twisted by 2.28 (10)°. In the crystal, the mol­ecules are linked by N—H⋯O and C—H⋯O inter­actions into sheets parallel to (100).In the title salt, C Å b = 6.7365 (1) Å c = 11.4489 (3) Å β = 101.967 (1)°V = 609.83 (2) Å3 Z = 2 Kα radiationMo −1 μ = 0.12 mmT = 100 K 0.25 × 0.17 × 0.10 mm Bruker SMART APEXII CCD area-detector diffractometerSADABS; Bruker, 2005T min = 0.972, T max = 0.988Absorption correction: multi-scan (11659 measured reflections2808 independent reflectionsI > 2σ(I)2155 reflections with R int = 0.045 R[F 2 > 2σ(F 2)] = 0.052 wR(F 2) = 0.116 S = 1.04 2808 reflections229 parameters1 restraintH atoms treated by a mixture of independent and constrained refinementmax = 0.43 e Å−3 Δρmin = −0.30 e Å−3 Δρ APEX2 used to solve structure: SHELXTL (Sheldrick, 2008SHELXTL; molecular graphics: SHELXTLsoftware used to prepare material for publication: SHELXTL and PLATON (Spek, 2009Data collection: 10.1107/S160053680902100X/tk2462sup1.cif Crystal structure: contains datablocks global, I. DOI: 10.1107/S160053680902100X/tk2462Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:

Hantavirus pulmonary syndrome (HPS) occurs in most infections with Sin Nombre virus and other North American hantaviruses. We report five cases of acute hantavirus infection that did not fit the HPS case definition. The patients had characteristic prodromal symptoms without severe pulmonary involvement. These cases suggest that surveillance for HPS may need to be expanded.

The scientific community is showing increasing interest in characterizing the function, evolution and language of these sequences. Despite this, there remains little in the way of user-friendly access to a large dataset of such elements in conjunction with the analysis and the visualization tools needed to study them.Comparative genomics is currently one of the most popular approaches to study the regulatory architecture of vertebrate genomes. Fish-mammal genomic comparisons have proved powerful in identifying conserved non-coding elements likely to be distal . In an interactive and intuitive way the website displays data on > 6800 non-coding elements associated with over 120 early developmental genes and conserved across vertebrates. The database regularly incorporates results of ongoing in vivo zebrafish enhancer assays of the CNEs carried out in-house, which currently number ~100. Included and highlighted within this set are elements derived from duplication events both at the origin of vertebrates and more recently in the teleost lineage, thus providing valuable data for studying the divergence of regulatory roles between paralogs. CONDOR therefore provides a number of tools and facilities to allow scientists to progress in their own studies on the function and evolution of developmental cis-regulation.Here we present CONDOR (COnserved Non-coDing Orthologous Regions) available at: By providing access to data with an approachable graphics interface, the CONDOR database presents a rich resource for further studies into the regulation and evolution of genes involved in early development. This is predominantly because we know so little about their language, mode of action and origin, and traditional ways of identifying them are difficult and laborious. The burgeoning field of evolutionary developmental biology (termed evo-devo) is increasingly shifting its emphasis towards the importance of gene regulatory networks in the evolution of developmental pathways [In recent years, although great advances have been made in our ability to annotate gene sequences, the annotation of sequence elements that control complex gene expression is lagging far behind. With exons accounting for only a tiny proportion of the genome < 1.5%) there remains a vast amount of unannotated sequence, containing an unknown number of functional elements. In particular, the annotation of the complex network of .5% therepathways -5). ThesThe confidence with which putative functional non-coding elements can be identified depends on the evolutionary distance between the species selected, and a balance must be found between eliminating background noise and retaining sufficient sequence similarity for detection . The incFugu rubripes, with its highly compact non-coding regions, has been commonly used as a model genome to increase the efficacy of detecting putative functional sequences. The similarities between mammals and Fugu are recognisable despite their large evolutionary divergence. Both vertebrates, they share not only the defining characteristic of a backbone, but also many basic anatomical and physiological similarities and patterns of development. The strong parallels in early developmental patterning are likely to be under control of a common core set of genes and regulatory elements. Significantly, a strong spatial association between highly conserved non-coding elements (CNEs) and genes involved in transcriptional regulation and/or development (termed trans-dev genes) has been described in many studies [in-vivo function reproducibly to up-regulate tissue-specific expression of reporter genes during development.The large evolutionary distance between fish and mammals means that even the slowest evolving DNA has diverged sufficiently to significantly improve the signal to noise ratio in genomic alignments. In particular the genome of the pufferfish, studies ,4,7-9. F studies ) or usin studies are cons studies and zebrcis-regulation in early developmental genes. Here, such elements are rigorously defined by optimal alignment strategies for sequences as divergent as fish and tetrapods, ensuring that sequences are derived from orthologous regions. In addition, we cater for the growing number of researchers in evo-devo interested in sets of experimentally verified developmental enhancers. Thus we couple both bioinformatics and functional data in a visual and searchable form.With an increasing supply of functional data, we have created CONDOR (Conserved Non-coDing Orthologous Regions), a database resource for the study of evolutionary conserved Fugu-mammal multiple alignments, in particular the CNEs identified from them. The second stores functional (lab-based) data from in vivo enhancer assays carried out in-house. While these two datasets are integrated in the database their derivation will be outlined separately.The relational database structure can be divided conceptually into two main parts, representing the two main sources of data stored within it. The first relates to the computationally derived dataset from Fugu genome and four mammalian genomes including human, mouse, rat and dog. The full methodology used to identify CNEs can be seen in Figure Fugu scaffolds using additional assembly data (such as BAC ends and sequence from older assemblies) from the Fugu Information Network [Fugu and human genomes [trans-dev genes. The CONDOR database is therefore designed around these distinct regions. Multiple alignments are more sensitive and better suited for identifying conservation between highly diverged sequences such as fish and mammals than more commonly used pairwise alignments [Fugu) encompassing 129 trans-dev genes conserved in synteny with these clusters in both fish and mammals. The chromosomal locations of developmental gene regions currently in the database can be seen in Figure The core of the CONDOR database is made up of a set of more than 6800 CNEs identified through a combination of sensitive multiple and multi-pairwise alignments of orthologous regions between a baseline teleost Network . The ali genomes . These cignments and wereignments . Currentcis-regulatory elements can exert their regulatory influence large distances from their target gene [trans-dev gene(s) but rather by the initial whole-genome CNE synteny map resulting in the identification of CNEs that may be located large distances from the trans-dev gene or within the introns of neighbouring genes they arvergence . CNEs thvergence . The empvergence . The firvergence . The higvergence means in targets .PAX8 region to 342 in the IRX3/5/6-SALL1 region, possibly reflecting differences in regulatory complexity and the extent to which regulation has been conserved between fish and mammals as well as the possible presence of overlapping regulatory domains. CNEs in CONDOR range in length from 21–864 bp (mean = 117 bp) and cover some 750 Kb of human sequence, or around 0.05% of the non-repetitive genome. Therefore, although this set represents a tiny fraction of the ~5% of the human genome thought to be under selective constraint [Numbers of CNEs in each region in CONDOR vary considerably, ranging from two in the nstraint it is prnstraint ,11.Fugu). Specific identifiers are only assigned to sequences in these species (as opposed to any of the other vertebrate genome in which a CNE is present), as they are only sequences that can be strictly guaranteed to derive from orthologous regions.It is important for future analyses that published sets of tested CRMs identified through species comparisons can in some way be traced back to a sequence repository and thus properly defined, similar to the definition of sequences in protein or transcript databases. Commonly in currently published work, functionally characterized conserved non-coding sequences are referred to by some arbitrary name and by their location in the genome or the distance from the start of a gene. This often changes with the release of updated genome assemblies and makes it difficult for other researchers to track the sequence back from the literature. In response, CONDOR is the first database to use specific identifiers for conserved non-coding elements in each genome, independent of their current location in a genome assembly. Unique identifiers are used both to refer to a CNE orthologous unit (akin to a cluster of orthologous genes), e.g. CRCNE00000054, which is useful in defining a CNE in any vertebrate genome to which it is conserved, as well as individual sequences within the CNE, e.g. CRCNEAC00000172 (in human), from sequences used to define the element in the original multiple alignment .The central search page allows CNE-containing gene regions to be located from a pull-down list or searched more generally through a text search by gene name, protein accession number, GO term etc for any genes contained within these regions. Once located, CNEs within a gene region can be visualized via an interactive graphical browser (termed 'CONDOR View') or in a tabular form (termed 'Text View'). Each gene region analysed extends as far as the shared synteny and gene order between fish and mammals, allowing for long-range 'Text view' displays information on the CNEs in a tabular format providing additional data to that in 'CONDOR View', giving details of conservation in other vertebrate genomes Figure and highin silico and experimental annotations can be readily viewed. A similar track in the UCSC Genome Browser is also planned in the future.The database is also available as a permanent DAS source within the most current human, mouse and rat assemblies at the Ensembl Genome Browser so that The vast majority of CNEs exhibit no sequence identity to any other sequence in the genome ,28,29. Hcis-regulatory elements complementing and adding to the growing list of enhancers tested in the mouse model [CONDOR will enable greater accessibility to ongoing functional data of se model with thetrans-dev paralogs and offer an important resource for studying cis-regulatory element evolution after duplication and how this influences paralogous gene function. It is worth noting that since CNEs can act over a long range, identifying their target gene can become complex when studying a region that contains more than one developmental gene. In the case of dCNEs, the retention of both element and gene after duplication, strongly implies association [Whilst the vast majority of CNEs conserved across vertebrates are unique in the human genome ,29 an inociation . CONDOR Fugu from alignments where both Fugu gene regions are compared independently to the same region in mammals. Recent comparative analysis of CNEs in these regions reveals assymetrical evolutionary rates and a pattern of retention and loss of CNEs between duplicated genes indicative of regulatory subfunctionalization [In addition, the CONDOR database is the first resource to make available data on CNEs identified from a number of teleost-specific duplicated regions within lization . To faciIn addition to providing data on the locations and sequence information of each CNE, CONDOR also provides a number of other features to help study the function and evolution of these sequences:in vivo or in vitro analysis, a number of automatically generated PCR primer predictions using the EPrimer3 program [• To facilitate the amplification of CNEs from a genome of choice for program are prov• Sensitive BLAST alignments of any CNE against a variety of nucleotide sequence databases can be displayed dynamically . Database searches include a variety of vertebrate genomes allowing lineage or species specific mutational events to be detected. The output produces the multiple alignment in a number of formats that can be directly viewed in the browser or downloaded for further downstream analysis.• CNEs from each of the main genomes can be downloaded in bulk as a set of sequences in FASTA format or as a tab-delimited file with substantial sequence and positional data. Bulk downloads can also be filtered by genome, gene region or CNE length and conservation.The database is run on a Solaris platform and housed using the MySQL (v1.4.0) database management system at the School of Biological and Chemical Sciences, QMUL, London. The web interface is run on an Apache web server that serves a set of CGI scripts and static web pages. The "CONDOR-View" graphical browser is created using the Bioperl Bio::Graphics module . CNEs arThe identification and characterisation of distal CRMs that control the complex gene regulatory networks in early vertebrate development remain one of the greatest challenges of the post-genomic era. It is of increasing interest to the scientific community for datasets of predicted CRMs that are well defined, easily accessible and manageable in number. Here we present CONDOR, a database resource of both predicted and experimentally verified orthologous non-coding elements conserved across the vertebrate lineage and associated with early developmental regulators. This resource is designed both for the increasing number of experimental groups interested in prioritising a set of elements for experimental verification and computational users interested in training sets with high regulatory potential.. A full tutorial can be found on the website. Please cite this paper when publishing data using computational data from CONDOR. Use of functional data in publications is only permitted with prior agreement from the corresponding author. Positions of CNEs within vertebrate genome assemblies are constantly updated as new versions are released and in vivo zebrafish enhancer annotations are added to the database automatically once results for each experiment are collated.The CONDOR database is publicly available without payment or registration at in vivo data collection. DG, JC, SS, PS, HC and GE developed and carried out the in vivo enhancer assays. DG and GM helped write parts of the manuscript. GE conceived the database, and participated in its design and coordination.AW carried out all multiple alignments, computational data extraction and post-processing, designed and implemented the database schema and web front end and wrote the first draft of the manuscript. JC designed the zebrafish expression sub-domains and colour-coding system involved in the

The dihedral angle betwen two benzene rings is 21.32 (7)°. The adamantane unit consists of three fused cyclo­hexane rings in classical chair conformations, with absolute values of C—C—C—C torsion angles in the range 57.5 (2)–60.9 (2)°. Weak inter­actions of the type C—H⋯O link mol­ecules of each enanti­omer into chains parallel to the b axis and lying about inversion centers. The crystal packing is also stabilized by inter­molecular π-π stacking inter­actions [centroid–centroid distance of 3.8566 (11) Å].In the title compound, C Å b = 6.8474 (14) Å c = 24.465 (5) Å β = 95.62 (3)°V = 4283.1 (15) Å3 Z = 8 Kα radiationMo −1 μ = 0.08 mmT = 120 K 0.50 × 0.40 × 0.40 mm Kuma KM-4-CCD diffractometerCrysAlis RED; Oxford Diffraction, 2006T min = 0.928, T max = 0.976Absorption correction: multi-scan (24394 measured reflections3768 independent reflectionsI > 2σ(I)2791 reflections with R int = 0.025 R[F 2 > 2σ(F 2)] = 0.048 wR(F 2) = 0.151 S = 1.09 3768 reflections273 parametersH-atom parameters constrainedmax = 0.74 e Å−3 Δρmin = −0.30 e Å−3 Δρ CrysAlis CCD used to solve structure: SHELXS97 (Sheldrick, 2008SHELXL97 (Sheldrick, 2008ORTEP-3 (Farrugia, 1997Mercury (Macrae et al., 2008SHELXL97.Data collection: 10.1107/S1600536809015888/pv2152sup1.cif Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536809015888/pv2152Isup2.hkl Structure factors: contains datablocks I. DOI: crystallographic information; 3D view; checkCIF report Additional supplementary materials:

Isolated isletsmaintained in tissue culture were infected withfive well- characterised strains of CBV-4. Aliquotsof the culture medium were analysed with regardto virus replication and insulin content. Infectedand uninfected islets were examined by light microscopyto determine the degree of virus-inducedcytopathic effect (CPE). The results showed thatthe islet cells were susceptible to infection by allthe strains of CBV-4 although the outcome of theinfection differed. The virus titres obtained at 48and 72 hours post infection differed significantlybetween all the CBV-4 strains (p < 0.001), indicatingdifferent ability to replicate in islet cells. Pronouncedto weak CPE, which was partly due tothe origin (donor) of the islets, was induced byfour of the five CBV-4 strains. One strain (VD2921)replicated without causing CPE despite highvirus titres. One (V89-4557) of the CBV-4 strainsalways revealed pronounced CPE. Infection by this strain also caused functional impairment thatsignificantly affected insulin response to high glucoseat 48 hours post infection (p < 0.001). Replicationof another CBV-4 strain (JVB) in the islet cellssignificantly increased the release of insulin comparedto non-infected control cells (p < 0.001) indicatingdamage of the β-cells leading to leakage ofinsulin.The aim was to study whether different strains ofCoxsackievirus B4 (CBV-4) are able to infect humanpancreatic islet cells

This paper describes the design of an event ontology being developed for application in the machine understanding of infectious disease-related events reported in natural language text. This event ontology is designed to support timely detection of disease outbreaks and rapid judgment of their alerting status by 1) bridging a gap between layman's language used in disease outbreak reports and public health experts' deep knowledge, and 2) making multi-lingual information available.This event ontology integrates a model of experts' knowledge for disease surveillance, and at the same time sets of linguistic expressions which denote disease-related events, and formal definitions of events. In this ontology, rather general event classes, which are suitable for application to language-oriented tasks such as recognition of event expressions, are placed on the upper-level, and more specific events of the experts' interest are in the lower level. Each class is related to other classes which represent participants of events, and linked with multi-lingual synonym sets and axioms.We consider that the design of the event ontology and the methodology introduced in this paper are applicable to other domains which require integration of natural language information and machine support for experts to assess them. The first version of the ontology, with about 40 concepts, will be available in March 2008. Timely detection of disease outbreak events and rapid judgment of their alerting status are the cornerstone of defence against the threat of infectious diseases. Today, it is estimated that about 65% of the initial reports on disease outbreaks come from news articles and informal information sources on the Web . HoweverThis paper presents a rationale and design of the multi-lingual event ontology. This complements the extant BioCaster Ontology for objects , which iBridging the gap between layman's languages and public health experts' knowledge. Disease outbreak reports on the Web such as news articles are often written in non-technical language, while public health experts assess outbreak events with highly technical knowledge. It is necessary to provide them with information on the Web in a form which conforms to their own knowledge.• Making information available in multiple languages. Usually the earliest information on diseases is reported in the local language. There is a need for access to foreign language information translated into the expert's first languages.• In this paper, we use the term ‘event’ as having the same meaning as perdurants, or occurrences in philosophy, or eventualities used in linguistics . AlthougBCEO can be regarded as a domain ontology for a particular purpose. It is not a domain ontology in the ordinary sense, since it does not have a detailed knowledge model. The application orientation impacts on the design in at least three ways: 1) the level of granularity, 2) scope (type of events described) and 3) multi-linguality.We identified the following events which public health experts seek through the news articles available online. These are key events in disease news reports, and we desire to recognize the relevant linguistic expressions.• Disease outbreaks (group infection)• Infecting events in individuals• Health status of patients • Control efforts to contain diseases We found that when the experts judge the seriousness of disease events in news reports, they look at the following aspects. Most of them are the properties of participants of events .• Kind of infected organism: animal infection, human infection• Number of patients: more cases are considered more relevant.• Location: outbreak in endemic area, non-endemic area, nosocomial outbreak• Transmission route: animal-to-human, human-to-human• Infectious capacity of pathogen• Virulence (morbidity) of pathogen• Potential for use in bio-terrorism• Drug resistance of pathogenCorrelations of these aspects are taken into consideration to estimate the urgency and abnormality of events. For example, a single case of a virulent disease such as smallpox is more serious than a cluster case of a less virulent disease, and the first occurrence of an animal disease in humans is more important than its re-occurrence in animals. It is desirable that the surveillance system can automatically judge the relevance of events and alert the experts once a dangerous situation occurs. We consider that this can be partly achieved by appropriate modelling of knowledge in the event ontology combined with the rules that are customized to the interests of individual public health workers.Although public health experts look at disease-related events with technical knowledge as described above, reports on the Web are usually written in non-technical language, by non-experts. Not many technical terms appear in news articles compared to more professional publication types, and sometimes their meanings are generalized, vague or inaccurate. This makes it challenging to apply regular medical ontologies using traditional indexing strategies.In many cases, disease-related events are mentioned with verbs (e.g. ‘infect’), verbal nouns (e.g. ‘infection’) and verb phrases. We also find many synonymous event expressions: e.g., infecting events can be expressed by many verbs and verb phrases such as ‘infect’, ‘transmit’, ‘contract’, ‘communicate (pathogen/disease)’, ‘catch (pathogen/disease)’, ‘get (pathogen/disease)’, verbal nouns such as ‘infection’, ‘transmission’, ‘contraction’. There are also cases such that an infecting event is not directly mentioned, but only implied, as in sentences like “A man died of bird flu”.Participants of those events, such as disease cases, pathogens, source of infection, time and location are also mentioned in various ways. Sometimes they occur as arguments of verbs or verbal nouns which denote events, but in other cases they are expressed as non-argument modifiers (adjuncts). We also observe that sometimes an event is mentioned in one sentence and some of its participants are mentioned in another. Regarding the variety and complexity of event expressions, we consider the description of relations between relations and those between expressions in an ontology to be beneficial in avoiding loss of data.It is important for any disease surveillance system to process multi-lingual information in order to detect earliest outbreak reports. In fact, some extant disease surveillance systems already provide a multi-lingual capability. Good examples are GPHIN 165], which supports the United Nation's official languages, and MedISys (An Infecting event induces a BeingInfected state)root term, which serves as an interlingual pivot. The synonym sets are different from synsets in WordNet [Each event class is linked to sets of synonymous terms via a WordNet 18] in t in troot WordNet and Word1. Make two sentences from the verbs , adding the same participants, time, and location to both.2. See if there is a situation where you can affirm one while negating the other, and vice versa. If you cannot find any, the two expressions are synonymous.Let us see an example. Suppose we would like to see if the two verbs, ‘contract’, and ‘infect’ are synonymous. Following part 1 of the test, we construct sentences “Mr. A contracted influenza virus from Mr. B on Jan 15 in Oedo-city”(S1) and “Mr. B infected Mr. A with influenza virus on Jan 15 in Oedo-city”(S2), providing each of the verbs with the same participants, time and location. Then we see if we can affirm S1 while negating S2 as in “Mr. A contracted influenza virus from Mr. B on Jan 15 in Oedo-city, but Mr. B DID NOT infect Mr. A with influenza virus on Jan 15 in Oedo-city”(S3) and vice versa as in “Mr. A DID NOT contract influenza virus from Mr. B on Jan 15 in Oedo-city, but Mr. B infected Mr. A with influenza virus on Jan 15 in Oedo-city”(S4). We can see that neither S3 nor S4 can be true in any situation, thus the two verbs are synonymous. Although this example is monolingual, this test can be applied to multi-lingual texts on condition that there is a person who can make the linguistic judgment in both languages.BCEO described so far will be applied for 1) annotating/grounding text mentions of events , 2) translation of terms which denote events, 3) modelling of public health experts' knowledge used to judge the alerting relevance of disease-related events. (1) is important in making a basis for automatic recognition of varieties of event expressions in disease outbreak reports, in addition to the traditional named entities. (2) is necessary to provide disease outbreak information in users' native languages, and grasp relationship between reports written in different languages. In BCEO, multi-lingual synonym sets will be used for these purposes. While (1) and (2) are rather language-oriented aspects, (3) is a knowledge-oriented aspect. The lower-level event classes, along with their OWL properties and formal definitions in axioms can be utilized for describing the knowledge.We now compare how disease-related event concepts and event expressions have been covered in BCEO to some of the existing ontologies.PHSkb is a codroot term, in order to bridge sets of synonyms in different languages. These lexical resources are not intended to be domain specific and covers rather general and common event expressions. We referred to them when we expand our synonym sets which is related to more specific event concepts. PropBank [Linguistic expressions which refer to disease related events in outbreak reports are mostly covered by lexical resources such as WordNet 18] and and 18] PropBank has a liTop-level classification of events is provided by upper ontologies such as SUMO , BFO 2727 and DOAs we have discussed, in order for an event ontology to be a basis for machine understanding of natural language information on disease outbreaks, it needs to include 1) a model of important disease-related events, 2) event expressions found in outbreak reports on the Web, 3) formal definitions of event concepts. Although each of the existing ontologies seems to meet a part of the requirements in the disease monitoring task, it is hard to find one which satisfies all of them. This calls for re-organizing knowledge and terminology in a way to integrate all three required aspects.We have introduced the design and construction of BCEO, which is being developed for application to multi-lingual text mining for disease surveillance. The event ontology integrates a model of experts' knowledge for disease surveillance, a structured vocabulary of linguistic expressions, and descriptions of event classes in meta-linguistic, logical representation. These features are essential to bridge between natural language texts and experts' deep knowledge. We consider that the design of the event ontology and the methodology introduced in this paper are applicable to other domains, and will be useful especially in those which treats emergency information such as chemical and nuclear incidents. Now we are developing the first version of the ontology with about 40 event concepts, linked to synonym sets in English and Japanese. The first version will be published online in March 2008.The authors declare that they have no competing interests.This work was directed by NC. AK designed the ontology by extending the basic framework made by NC. HC analyzed 1000 online news articles and examined linguistic expressions which denote disease-related events in English. Japanese expressions are complemented by AK. MS identified important events and public health experts' knowledge which should be covered by the ontology. All authors contributed during the whole length of the ontology construction and writing of the paper. All authors read and approved the final manuscript.

HD CAG repeat, whose length determines age at onset, undergoes tissue-specific somatic instability, predominant in the striatum, suggesting that tissue-specific CAG length changes could modify the disease process. Therefore, understanding the mechanisms underlying the tissue specificity of somatic instability may provide novel routes to therapies. However progress in this area has been hampered by the lack of sensitive high-throughput instability quantification methods and global approaches to identify the underlying factors.In Huntington's disease (HD), an expanded CAG repeat produces characteristic striatal neurodegeneration. Interestingly, the HD CAG repeat instability and integrated this with genome-wide bioinformatic approaches. Using tissue instability quantified in 16 tissues as a phenotype and tissue microarray gene expression as a predictor, we built a mathematical model and identified a gene expression signature that accurately predicted tissue instability. Using the predictive ability of this signature we found that somatic instability was not a consequence of pathogenesis. In support of this, genetic crosses with models of accelerated neuropathology failed to induce somatic instability. In addition, we searched for genes and pathways that correlated with tissue instability. We found that expression levels of DNA repair genes did not explain the tissue specificity of somatic instability. Instead, our data implicate other pathways, particularly cell cycle, metabolism and neurotransmitter pathways, acting in combination to generate tissue-specific patterns of instability.Here we describe a novel approach to gain insight into the factors responsible for the tissue specificity of somatic instability. Using accurate genetic knock-in mouse models of HD, we developed a reliable, high-throughput method to quantify tissue Our study clearly demonstrates that multiple tissue factors reflect the level of somatic instability in different tissues. In addition, our quantitative, genome-wide approach is readily applicable to high-throughput assays and opens the door to widespread applications with the potential to accelerate the discovery of drugs that alter tissue instability. HD CAG repeat occurs in the striatum and cortex, brain regions that are major targets of the pathogenic process. Furthermore, studies both in HD patients and in a knock-in mouse model of HD provide compelling evidence indicating that somatic expansion in these brain regions accelerates the ongoing pathogenic process [Expansions of trinucleotide repeat sequences over certain thresholds cause more than 30 human diseases including Huntington's disease (HD), a number of spinocerebellar ataxias (SCAs), myotonic dystrophy 1 (DM1), and fragile X syndrome. Interestingly, expanded trinucleotide repeat sequences undergo progressive, expansion-biased tissue-specific somatic instability . As the process -9. Therecis-factors [HD CAG instability occurs in many tissues to varying extents [Somatic instability is critically dependent on DNA repair genes and is also influenced by -factors ,8,10-16.-factors . Given t extents ,6,17, weUsing accurate genetic knock-in mouse models of HD ,18 that Previous methods for determining instability following PCR amplification of repeats from 'bulk' genomic DNA have either been qualitative, or have failed to adequately account for amplification efficiencies that differ between stable and unstable tissues. In contrast, quantitative small pool-PCR (SP-PCR) methods are extrHdhQ111 mice is expansion-biased and contraction is not highly variable between tissues . Figure We then compared instability indices using our relative peak height threshold method to somatic instability quantified using SP-PCR on genomic DNA of tissues from the same mouse increases over time [HD CAG knock-in allele) to the HdhQ111 mice in this study. While age and genetic background-related gene expression changes will increase the noise in our system, this broad, tissue-based analysis allows us to pull out major tissue-specific gene expression differences that occur over and above age- and genetic background-related effects.With the aim of investigating the tissue specificity of somatic instability in a global and unbiased manner we then took a bioinformatics approach. Using 16 different tissues from 5-month Thus, we modeled instability index as a function of gene expression using partial least square regression (PLSR) . An instHdhQ111/+ tissues and compared these with instability indices predicted from the regression model in the same tissues , indicating that instability index can be relatively precisely predicted from the gene expression signature. Furthermore, these data demonstrate that although the model was based on gene expression data and instability index data from mice that differed in age and genetic background, it nevertheless has significant predictive power. This indicates the presence of tissue-specific factors related to instability independent of age and genetic background.We then confirmed the predictive power of this instability-correlated gene expression signature by comparing measured instability indices with predicted instability indices from our regression model in new independent samples. For this, 1) we measured instability indices of four new independent s Figure , blue, as Figure , red. AsOur sensitive quantification method and instability-correlated gene expression signature/regression model is a versatile tool. One of the advantages of our regression model is that the 'propensity' for instability can be predicted when instability can not be directly measured. For example, our approach allowed a prediction of an instability index in 78 different tissues and conditions in the mouse tissue gene expression data set Table , a far gHD CAG somatic expansion, particularly, why the repeat is so unstable in the striatum. Our instability quantification/bioinformatics approach provides a novel, global and unbiased means of probing these factors. One possibility that could at least in part explain the tissue specificity is that somatic instability occurs as a result of the ongoing HD pathogenic process, as previously hypothesized [HdhQ111/111 mice that exhibit an ongoing pathogenic process and somatic instability in striatum but not in cerebellum [Hdh+/+ littermates, and predicted instability using the regression model above. Interestingly, as shown in Figure propensity for somatic expansion that is unrelated to the HD CAG pathogenic process. Although wild-type striata possesses this propensity, the normal HD CAG repeat does not actually expand because it does not present a sufficiently long target to be susceptible to the processes that mediate expansion.We are interested in understanding the factors that contribute to the tissue specificity of thesized . We firsrebellum , and on Hdh CAG repeat is both a source of a pathogenic process and a target of instability, it is very difficult to delineate the relationship between the HD pathogenic process and somatic instability. Therefore, we used genetic mouse models in which neurodegenerative processes are modulated or caused by factors independent of the HD CAG repeat. We first investigated HdhQ92 mice lacking the dopamine transporter (DAT), which show accelerated HD pathogenesis in the striatum [HdhQ92/+ DAT-/- and HdhQ92/+DAT+/+ mice were not different, indicating that HD CAG instability is not contributed by the disease process. We also tested whether inducing neurodegeneration in the cerebellum, a normally stable tissue, would cause instability in the cerebellum by crossing HdhQ111 mice to Harlequin (Hq) mice, a model of cerebellar granule cell degeneration [HdhQ111/+ Hq/Y mice and HdhQ111/+ +/Y control mice exhibited similar low cerebellar instability indices, indicating that neurodegeneration per se is insufficient to induce instability.To test the prediction that somatic instability does not occur as a consequence of ongoing pathogenesis, we performed two genetic experiments. Since the expanded striatum . As showneration . As showHD CAG disease process is not responsible for the striatal specificity of HD CAG repeat instability, arguing against the sequestration of DNA repair proteins or other factors, as a contributor to somatic instability as previously suggested [Taken together, these results support the prediction from our mathematical model, that the uggested . Our resuggested , and wituggested ,5.Msh2, Msh3, Ogg1 and Cbp), previously shown to play important roles in CAG repeat instability [Msh3, Ogg1 and Cbp did not correlate with instability index and Msh2 expression level showed a weak negative correlation with instability index , in which gene expression data is analyzed at a the level of biological pathways rather than individual genes . ConfirmIt is possible that as striatum is particularly unstable, the highly correlated pathways are simply those that are predominantly present or absent in this tissue, and that the correlation with instability is coincidental. However, pathways significantly up-regulated or down-regulated in striatum compared to cerebellum (data not shown) showed little overlap with those that correlated with instability; for example, the dopamine pathway is strongly up-regulated in striatum, but does not correlate with instability. This suggests that the instability-correlated pathways are directly related to instability rather than simply being striatal-specific.HdhQ111/+) derived from striatal primordia of HdhQ111/+ E14 embryos [-5 instability index units/week), consistent with the prediction from the negative correlation between cell cycle and the instability index. It is notable that the HD CAG repeat in STHdhQ111 cells is extremely stable over multiple passages and under numerous different experimental conditions (data not shown). Cell cycle arrest is the only condition we have identified so far that has resulted in any expansion of the repeat. These findings indicate that the negative correlation of cell cycle pathways with the instability index more likely reflects a contribution of cell proliferation to preventing instability rather than a reduction of these pathways as a consequence of instability.Instability-correlated pathways may either directly modify instability or may represent cells' secondary responses to instability. To distinguish these alternatives, we asked whether alteration of an instability-correlated pathway would influence instability. Cell cycle pathways were negatively correlated with instability index Table , and our embryos . These cAlthough cell cycle pathways may be directly involved in modifying instability, some tissues (e.g. cerebellum) with a high proportion of non-proliferating cells were relatively stable. This indicated that each correlated pathway may explain a small part of the tissue instability and that the contributions of each pathway may be different for each tissue. Therefore, to investigate further the contributions of the different instability-correlated pathways, we compared the expression levels across different tissues of genes in the two most strongly correlated pathways (positive correlation). Interestingly, although 'UDP-galactose beta-N-acetylglucosamine beta-1,3-galactosyltransferase activity' was the most significantly correlated pathway Table , liver wHD CAG repeat instability that combines a reliable, high-throughput method for quantifying somatic instability with mathematical modeling based on gene expression data. Predictions based on our modeling were confirmed using genetic, biochemical and cell culture-based experiments, indicating the validity of our bioinformatics approach.We have developed a novel approach for use in investigations of tissue-specific somatic HD CAG instability. In addition, DNA repair proteins have been found to be essential factors for somatic instability of trinucleotide repeats [HD CAG instability. In addition, Hdh expression levels did not correlate with instability index in tissues (data not shown), confirming observations that although transcription through expanded repeats may be important in somatic instability [Hdh mRNA. Alternatively, our study suggests new pathways, notably metabolism, neurotransmitter, and cell cycle that may contribute, in combination, to the level of somatic instability in different tissues, providing a starting point to identify additional factors that contribute to somatic instability. Notably, there was no predominant factor that could explain the tissue-specificity of HD CAG instability, suggesting that patterns of instability are determined by the combined effects/interactions of many genes.It has been proposed that somatic instability may be a consequence of disease pathogenesis , potenti repeats ,8,11-16.tability , tissue-trans-acting factors, but has also been shown to depend on cis-acting sequences [Somatic instability of trinucleotide repeats not only requires equences . Thus, wequences ,29. It wOur bioinformatics method based on gene expression data can only address aspects of tissue instability that are related to steady-state mRNA levels. In principle, however, a similar bioinformatic approach could be also applied to proteomics data. Irrespective of the particular method however, the strength of our approach is in its high-throughput, global and predictive nature, facilitating a number of important applications. Our GeneMapper quantification method is readily applicable to high-throughput assays such as screening small molecules that modulate instability in cells, or screening for genetic modifiers in mice. A powerful application of our bioinformatics approach is that the instability-correlated gene expression signature can be used as a surrogate marker for instability in situations where repeat instability cannot be directly measured. For example, gene expression databases can be screened to identify cell or tissue states that have the propensity for somatic instability, even in the absence of an expanded CAG repeat target as a read-out. Similarly, databases can be screened for compounds that reduce the instability propensity. Together, these approaches promise to accelerate the discovery of drugs that modulate instability and that are therefore candidate modifiers of disease.Our study demonstrates that multiple tissue factors including metabolism, neurotransmitter, and cell cycle combine to reflect the level of somatic instability in different tissues. Our findings also indicate that DNA repair proteins act largely in a non tissue-specific manner. In addition, the combination of our instability quantification method and mathematical modeling is a powerful strategy that has allowed us, in an unbiased manner, to gain critical new insights into the tissue specificity of trinucleotide repeat instability in HD. It opens the door to widespread downstream applications with the potential to make significant advances in novel avenues for therapeutic intervention in both Huntington's disease and trinucleotide expansion disorders in general.HdhQ111 knock-in mice with 109 CAGs [HdhQ111/+ (CD1) and HdhQ92/+ mice (CD1) [Hq) mutant (B6CBACa-AW-J/A) [DAT) knockout mice (C57Bl/6J) [HdhQ92 mice were crossed with DAT knock-out mice and progeny intercrossed to generate HdhQ92/Q92 DAT-/- mice and HdhQ92 DAT+/+ control littermates for comparisons of instability. HdhQ111 males were crossed with Hq/+ females, and HdhQ111/+ Hq/Y males and control HdhQ111/+ +/Y littermate males used for comparisons of instability. All animal experiments were performed to minimize pain and discomfort, under an approved Institutional Animal Care and Use Committee protocol.109 CAGs were use109 CAGs . For accce (CD1) were cro-AW-J/A) and dopa57Bl/6J) , respectHD CAG repeat-specific primers as previously described [Genomic DNA, isolated from mouse tissues and cell lines , was used for PCR amplification using escribed . The forEcoRV and diluted in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA containing 0.1 μM carrier primer (MD16) to a final concentration of approximately 10 ng/μl. The amount of input DNA equivalent to a single amplifiable mutant Hdh allele was determined empirically using Poisson analysis, and for each tissue between 32 and 117 single mutant amplifiable molecules were analyzed. A nested PCR protocol was used, in which only the mutant (knock-in) Hdh allele is amplified. Mutant Hdh alleles were amplified using 0.5 μM MD16 primer 5'-CCCATTCATTGCCTTGCTGCTAAG (forward) [2, 6.7 mM 2-mercaptoethanol, 4.4 μM EDTA, 1 mM dNTPs and 113 μg/ml BSA), 10% DMSO and 0.5 U units Taq polymerase (Fisher). Cycling conditions were 94°C 5 min, 35 cycles of 94°C 30 sec, 58°C 30 sec, 72°C 3 min, followed by 10 minutes at 72°C. PCR products were diluted 100-fold in TE and amplified in a second round using 0.8 μM Hu4 primer 5'-CCTGGAAAAGCTGATGAAGG (forward) and 0.8 μM Hu3 primer 5'-GGCGGCTGAGGAAGCTGAGGA (reverse) in a PCR buffer containing 67 mM Tris-HCl pH 8.8, 16.7 mM (NH4)2SO4, 2 mM MgCl2, 0.17 mg/mg BSA, 10 mM 2-mercaptoethanol, 10% DMSO, 200 μM dNTPs, with 0.5 U Taq polymerase (Fisher). Cycling conditions were 94°C 90 sec, 25 cycles of 94°C 30 sec, 65°C 30 sec, 72°C 90 sec, followed by 10 minutes at 72°. Hu4 was fluorescently labelled with 6-FAM (Applied Biosystems). PCR products were resolved using the ABI 3730 automated DNA analyzer (Applied Biosystems) using GeneMapper v.3.7 and GeneScan 500-LIZ as internal size standard to assign repeat size. HD CAG size was assigned as the highest peak. All PCR reactions were set up in a laminar flow hood and 20% of zero DNA control PCR reactions were included per run. To determine a small pool instability index we determined the frequency of each CAG repeat length, and multiplied each frequency by the number of repeats (+ or -) from the modal CAG length. These values were then summed.Genomic DNA was digested with forward) and 0.5 HdhQ111/+, 5 months, n = 4-6 mice for each tissue) and compared them with instability indices predicted by our model. Secondly, we additionally analyzed gene expression profiles of striatum and cerebellum and used these to predict instability indices for comparison to previously measured instability indices in these tissues. Test set RMSEP was calculated based on the difference between measured and predicted instability indices. Prediction of instability index for each of the tissues analyzed in mouse Gene Expression Atlas was based on our regression model and the instability-correlated signature.We used the mouse tissue gene expression database of Genomics Institute of the Novartis Research Foundation . All microarrays were background corrected and normalized by gcRMA. To identify an instability-correlated gene expression signature, Pearson correlation coefficients and corresponding p values between gene expression levels and instability indices of training samples were calculated for each probe, and the gene expression data was sorted by p values. We used Pearson correlation coefficients only as a ranking metric and this linear correlation information has not been used in actual modeling. Therefore, our models capture not just linear relationship but covariance between instability and expression. To identify an instability-correlated gene expression signature, we sequentially introduced the top n most highly correlated probes into the regression algorithms in a forward selection procedure, and calculated root mean squared error of prediction (RMSEP) by leave one out cross validation (LOO CV) of training samples . In addition to LOO CV of training samples, we further tested our model using 2 different test set samples. Firstly, we measured instability indices in additional tissues correlated with the instability index.Click here for file

Advances in technology and the scientific understanding of disease processes are presenting new opportunities to improve health through individualized approaches to patient management referred to as personalized medicine. Future health care strategies that deploy genomic technologies and molecular therapies will bring opportunities to prevent, predict, and pre-empt disease processes but will be dependent on knowledge management capabilities for health care providers that are not currently available. A key cornerstone to the potential application of this knowledge will be effective use of electronic health records. In particular, appropriate clinical use of genomic test results and molecularly-targeted therapies present important challenges in patient management that can be effectively addressed using electronic clinical decision support technologies.Approaches to shaping future health information needs for personalized medicine were undertaken by a work group of the American Health Information Community. A needs assessment for clinical decision support in electronic health record systems to support personalized medical practices was conducted to guide health future development activities. Further, a suggested action plan was developed for government, researchers and research institutions, developers of electronic information tools , and standards development organizations to meet the needs for personalized approaches to medical practice. In this article, we focus these activities on stakeholder organizations as an operational framework to help identify and coordinate needs and opportunities for clinical decision support tools to enable personalized medicine.This perspective addresses conceptual approaches that can be undertaken to develop and apply clinical decision support in electronic health record systems to achieve personalized medical care. In addition, to represent meaningful benefits to personalized decision-making, a comparison of current and future applications of clinical decision support to enable individualized medical treatment plans is presented. If clinical decision support tools are to impact outcomes in a clear and positive manner, their development and deployment must therefore consider the needs of the providers, including specific practice needs, information workflow, and practice environment. In the coming years, electronic health records (EHR) will have increasingly important roles in the delivery of health care, particularly in creating new opportunities to improve quality and effectiveness. Among the most important features offered by EHR are electronic exchange and interoperability of patient health information; new tools for carrying out some care delivery functions, like drug prescribing; supporting providers in the delivery of evidence-based care, including incorporation of evolving practice guidelines into point-of-care accessible information formats; measurement of quality in care delivery; and widespread access to large networks of data for research and quality purposes. While improvements in quality of care involve all of these functions, it is particularly in the area of clinical decision support (CDS) that EHR-based technology is expected to improve delivery of care in a continually evolving manner.There will be an interdependent relationship between CDS and personalized medicine in the effort to achieve improvements in the quality of health care. CDS will help health care providers cope with an increase in the number of clinical decisions and acceleration of knowledge development that would not be manageable without EHR systems. As medical knowledge increasingly steers toward variability in treatments based on individual patient factors, including genomic-related clinical variables, the need for CDS as a central part of care delivery will grow. Thus, improving quality of care is expected to involve increasing numbers of options for therapy, based on increasingly detailed amounts of quantitative data and knowledge, and resulting in increasing precision and effectiveness of care for each patient. For the most part, CDS tools today do not provide support for computing of complex quantitative determinations such as risk assessment, determination of combinatorial therapeutic interventions, and prediction of outcomes from interventions. Improvements in CDS development will be essential to achieving measurable advances in clinical outcomes in scenarios where health care providers are faced with increasing numbers of clinical variables.Today, even as the basic foundation of EHR systems is being laid, the pathways for CDS development are simultaneously being designed. This task is complex in itself, and it is made even more complex by the need for CDS design to anticipate and accommodate the expected growth of differentiation in patient care - i.e., personalized medicine. In this paper, we examine the potential and current state of CDS development; we identify roles for key stakeholders involved in the various elements of CDS design and construction; and, we recommend steps the various stakeholders can take at this stage to enable the long term goal of CDS support for personalized medicine.While there are many contexts for which the term 'personalized medicine' can be applied, we take a broad view of its future applications. Personalized medicine is defined as the delivery of health care in a manner that is informed by each person's unique clinical information; genetic, genomic, and other molecular/biological characteristics; and environmental influences. The goals of personalize medicine are to take advantage of a molecular understanding of disease, combined with other individual factors, to optimize preventive health care strategies while people are still well or at the earliest stages of disease. In 2007, the American Health Information Community (AHIC) established a work group to address standards and interoperability specifications to enable EHR systems to exchange and apply genomic information in medical decision-making. CDS was among the four priority areas targeted for workgroup activity in large part because of the complexity that individualized patient care delivery presents to health care providers' practices. [The completion of the Human Genome Project, followed by the development of a more manageable understanding of the human genome in the Hap Map project and the launch of genome-wide association studies (GWAS), marked a great accomplishment and initiated a burst in scientific discovery of the genetic underpinnings of common diseases. This is Realization of personalized medicine is dependent on the ability to collect, disseminate, and process complex information in the context of clinical care. The traditional paradigm of a provider reading current literature and submitting adverse event reports cannot support this complexity. It requires an EHR infrastructure to provide access to key clinical data with CDS capabilities that provide the right information, at the right time, at the point-of-care.CDS tools have the potential to enable personalized approaches to health care by providing health care providers, their staff, and patients with information and preferences specific to the individual, intelligently filtered and combined at appropriate times, to enhance health and health care. Primary methodologies for CDS include information retrieval; evaluation of logical conditions; probabilistic and data-driven classification or prediction; heuristic modeling systems; calculations, algorithms, and multi-step processes; and associative groupings of elements. CDS encoIn summary, CDS tools will likely become critical components of personalized medicine paradigms, which rely on EHR capabilities to assess a variety of data types, including an individual's genetic/genomic data, within evidence-based guidelines and practices. As the individualized information is accumulated and the applications of tailored treatment decisions expand, it is likely that providers and patients will need CDS tools to reap the benefits from the information influx. When combined with the provider's clinical judgment, these tools will complement non-EHR-based data sources to offer an additional resource to assist with clinical decision-making.The following commonly occurring clinical scenarios compare and contrast current and future applications of information support to facilitate individualized decision-making. In addition, these examples demonstrate the gaps in capabilities and can serve as a springboard for recommendations to close them. These scenarios represent the current knowledge base and are reflective of the types of genetically-based information used in decision-making today.Patient X commences antiretroviral treatment after testing positive for HIV. His health care provider prescribes a non-nucleoside reverse transcriptase inhibitor (NNRTI) and concurrently orders a genotype resistance test to identify HIV-reverse-transcriptase-based mutations, as certain variants may decrease the effectiveness of the prescribed inhibitor. However, despite the identification of the K103N mutation in the reverse transcriptase, which is broadly accepted to confer definitive resistance, [While it may seem difficult to imagine that a health care provider would order a resistance panel and then seemingly ignore its results, the scenario is more complex than this simple analysis. As with many rapidly-advancing fields, the consensus on genotype is constantly evolving, as is the health care providers' understanding of and currency with this knowledge. In a recent study concerning HIV genotypic resistance testing and antiretroviral prescription practices in a group of experienced HIV health care providers, researchers analyzed the frequency with which patients were prescribed antiretroviral therapies that were inconsistent with viral genotypic information. Among paPatient Y commences antiretroviral treatment after testing positive for HIV. Using e-prescription software embedded within the EHR system, his health care provider prescribes an NNRTI and is concurrently prompted to order a genotype resistance test to identify mutations that may decrease the NNRTI's effectiveness; the order is added with a single click from the alert screen. The results are returned, and the test identifies the K103N mutation in the reverse transcriptase. The automated electronic patient record system cross-references the test result with the current prescription, notifies the provider about the patient's resistance to the current medication, and suggests several alternate NNRTIs that are not contraindicated by this genotypic result. The tool provides the physician with an "information button" in the EHR that provides information that augments the decision to change medication. The physician makes thus makes medication changes (with the help of tools similar to the one that facilitated the test ordering), and the patient achieves the desirable reduction in viral load.Patient X has a family history of breast cancer and initiates a conversation with her provider to discuss risk prediction and preventive strategies. The provider utilizes a web-based tool or user-installed program on her personal digital assistant device such as the National Cancer Institute's Breast Cancer Risk Assessment Tool more efficiently.An EHR contains standardized family health history, medical history, laboratory results (including genomic information from previous testing), and other clinical information. In addition, the EHR has embedded risk-prediction tools, which can utilize more complex information sources and do not require re-entry of data that are contained within the patient's medical record. As the patient enters various life stages for which risk factors are modified or updated, the risk determination is actively revised and provided to the patient and provider, along with personalized information and guidance on interpreting and addressing the information provided. For example, on her 35As demonstrated above, CDS tools will enable health care providers and patients to more efficiently and effectively gather, interpret, process, and apply an increasing volume of complex information needed for up-to-date, best-practice care. However, for CDS to be effective in managing decisions often encountered in primary care, such as diabetes, heart disease, or mental illness, algorithms must integrate an increasing array of variables to tailor an accurate and useful risk probability assessment to a given patient. In addition, mitigating factors such as co-morbidities and non-mutation-based genomic changes occur over time and may create additional considerations when developing an intervention. When one considers that multiple gene combinations may suggest different management paradigms for a given condition, the landscape becomes quite complex.BRCA (genes associated with hereditary breast cancer) scenario reveals, the use of genetic and genomic tests, when coupled with predictive risk assessments provided by family health history information, offers tremendous potential to deliver timely, appropriate prevention and care. However, it also adds complexity to the decision-making process that will only increase as additional genomic information becomes validated.The impact of EHRs and related platforms on patient outcomes has been inconsistent and difficult to quantify. While inIn addition, interoperable tools must be founded on an agreed-upon baseline set of evidence-based best practices that exclude ad-hoc, vendor-specific, or experiential rules. Third, formal medical education typically provides minimal training in applying genetic/genomic tests and data, limiting many health care providers' abilities to interpret this information. Furthermore, constraints and demands of current clinical practice often discourage the convenient acquisition of this knowledge. To appropriately interpret results, the provider must seek information from external sources, such as the laboratory performing the tests, knowledge repositories, or genetic specialists. Currently, this process is inefficient, and the burden for improved information-handling will increase, particularly among primary care practices, as more genomic information is integrated into the health care system. If CDS tools are to impact outcomes in a clear and positive manner, their development and deployment must therefore consider the needs of the provider, including specific practice needs, information workflow, and practice environment.Compared to paper-based systems, interoperable CDS resources offer the potential to transfer information more rapidly and perhaps more reliably, however much work remains to be done before these resources become standard practice. Wide deployment of CDS tools supporting clinical medical decision making will be facilitated through standardization (agreed-upon standards for the rules and data fields that will enable the various electronic resources to utilize various data types and resources) and interoperability (the capability of an information system to exchange data with other systems). Data from different sources, including EHRs, claims databases, genomic databases, and laboratory results files, are used in various ways. Standards and interoperability enable information in each of these resources to be related to each other, creating new knowledge from their integration. Currently, this goal is hampered by the challenges of incorporating proprietary technologies that have developed over time in existing EHR systems with emerging applications and capabilities. Even widely-used predictive algorithms, such as the National Cancer Institute's Breast Cancer Risk Assessment Tool and the As CDS becomes incorporated into EHRs, additional efforts are required, both in research and application, before these tools can be optimized for personalized disease management at the point-of-care. Appropriate oversight can facilitate the integration process by promoting interoperable standards, assisting in the development of quality control metrics for information used in CDS tools, and assessing the impact of the tools on outcomes and performance measures. The recommendations discussed in this paper offer a dialogue to create avenues that will ultimately facilitate the adoption and use of CDS tools in medicine.The pathway to use CDS in support of personalized medicine requires many integrated components to be developed in parallel with the emerging EHR foundation in health care. Developing a health care system that optimizes personalized prevention and treatment requires engagement from many diverse stakeholders. Organizing the work that needs to be done by stakeholder organizations is a helpful framework to consider the actors and actions needed for the complete picture of personalized medicine CDS systems to emerge. These include the consumer/patient, molecular diagnostic (laboratory test or device) developer, providers, payor, CDS developers, and oversight and regulatory bodies, among many others.An overview of the continuum of information flow from evidence development through clinical application using EHR technology is shown in Figure To address the information needs outlined in the framework above, this section discusses the key steps and which stakeholders will likely be best suited to address them. A summary of recommendations for the various stakeholders is provided in Table Integrating CDS tools into clinical practice will require multi-layered policy interventions to overcome systemic barriers and challenges currently facing personalized medicine. ,17 In 20Numerous government agencies currently participate in initiatives that support information needs for personalized medical practices including the Department of Health and Human Services' (HHS) Personalized Health Care Initiative, ,20 the AAmong the priorities to enhance CDS development for future personalized medicine approaches is the establishment of EHR standards for a minimum data set of key variables. This minimum data set would include demographic data, physical measures, family health history, medications, health risk factors, and other parameters. CDS tools can then be built through rules and algorithms deployed through EHR systems using data supported from the patient's EHR. Other priorities include establishing pathways for integrating requirements for quality measures and standards and certification with the primary organizations responsible for them.CDS resources will accelerate the integration of new knowledge and technologies into health care practice. Standards development can orient this information towards appropriate end-users, but only if the needs and the output of those users are well understood. As such, the development of optimal CDS tools will require input from basic, clinical, and outcomes researchers. As new information that may personalize medical care emerges, researchers should consider engaging with professional societies and EHR vendors to understand the types of information that will be necessary to integrate the new tools into care. Many current clinical systems include mechanisms to provide providers with relevant, trusted information as they make clinical decisions. As new tools are integrated, however, researchers should explore methodologies to present tools to providers, assess patient-provider interfaces, evaluate the impact of electronic tools in shared decision-making, and support innovative software applications that integrate multiple variables such as test results, medical history, medication history, patient preferences, and family health history. Current research on the use of CDS tools in quality improvement measures, workflow, and shared decision-making practices should continue. For examDevelopers of electronic information tools can advance personalized medicine by creating tools to identify appropriate molecular diagnostic tests, provide additional background material or references, or produce a personalized interpretation based on existing medical data at the point-of-care. When setting priorities, vendors should build tools to support medical decision-making, including workflow integration for diagnostic test orders, integration of test results into confirmatory tests, intervention options, and patient information resources. High-impact areas needed for development include tools for detecting potential adverse events, genetic assay support for treatment selection, and reminder systems for risk detection, disease screening, and prevention.These tools may capitalize on established EHR technologies such as e-prescribing. For example, by augmenting the information provided to pharmacies, the pharmacist could become a resource providing assurances for patient safety, minimizing adverse events, and improving health outcomes. Recent initiatives advanced by the Centers for Medicare and Medicaid Services are providing financial incentives for e-prescribing, and recent legislative action is providing a stimulus incentive to physicians to use this technology. ProvidinEvidence-based practice guidelines offer the health care provider a general reference framework from which to choose interventions to address a patient's health care needs. However, no two patients are alike, and an individual's unique genomic/biological profile and personal preferences may suggest appropriate treatment avenues that differ from those applicable to other individuals who manifest the same disease. Practice guidelines should include patient-specific factors for disease management when these considerations are supported by an evidence base. Given the multi-factorial nature of chronic conditions commonly encountered in primary care , however, it is reasonable to expect increasingly nuanced treatment algorithms to emerge. To benefit patients fully, clinical practice guidelines and quality measures must incorporate differences among subsets of patients as the relationships between molecular profiles and treatment response are elucidated. As mentioned previously, CDS tools have the potential to identify diagnostic tests and other criteria to stratify patients and identify the appropriate clinical guidelines for a given subset of patients. In turn, these tools can inform quality improvement efforts at the level of the practice and quality metrics related to policy development and education. However, practice guidelines must be translated into machine-readable formats to integrate into CDS tools, and it is recommended that organizations that develop practice guidelines work with electronic tool developers to create standard data formats that can be used to structure guidelines so that they can be imported and used in CDS formats.Recent legislation, such as the Medicare Improvements for Patients and Providers Act of 2008 and AmerThe broad integration of complex, personalized information into the health care delivery process through CDS tools will require the development of standards to permit disparate entities to utilize and exchange data. Considerations may include standardized terminology, metrics, and guidelines; examination of the workflow between various entities; identification of special datasets to collect ; and identification of policy and technical issues. EHR standards development organizations are encouraged to consider standards for templates that enable the integration of best practices into EHR systems. In addition, standards developers can recognize the value and potential of coupling genetic and family history information by prioritizing standards development through the Health Information Technology Standards Panel and the Science, technology, and medical practice continue to enable innovative individualized approaches to health care that require sophisticated reasoning and decision making. The rate of development of these approaches will only accelerate, dramatically expanding patients' and providers' needs for tools to help process this information and take appropriate action. At present, electronic tools to support this approach to health care are used infrequently in EHR systems, yet these tools will be essential to support future point-of-care decision-making. As CDS development activities begin to unfold, assurances that these technologies will accommodate individualized approaches in medical decision-making will be essential for achieving more effective care through personalized medicine. Electronic information tool developers, researchers and research institutions, and standards development organizations will each have a role in bringing individualized approaches to health care, thereby effecting a transition that will benefit all health care stakeholders. It will require the efforts of numerous individuals and teams to develop the health care system that leverages individual differences for the prevention, early detection, and tailored treatment of human disease. Identifying key roles and activities across the health care enterprise can help accelerate the achievement of this goal for the benefit of patients and providers.CDS: clinical decision support, EHR: electronic health records, HITSP: health information technology standards panel, CCHIT: certification commission for healthcare information technology, AHIC: American Health Information Community, GWAS: genome-wide association studies.JAO is chief clinical informatics officer of Thomson Reuters.Conceptual framework, organization, and content of the paper were developed equally by GD and JO. All authors contributed equally to the drafting, editing, and integration of comments from advisors. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6947/9/44/prepub

Retroviruses engage the ESCRT pathway through late assembly (L) domains in Gag to promote virus release. HIV-1 uses a PTAP motif as its primary L domain, which interacts with the ESCRT-I component Tsg101. In contrast, certain other retroviruses primarily use PPxY-type L domains, which constitute ligands for NEDD4-type ubiquitin ligases. Surprisingly, although HIV-1 Gag lacks PPxY motifs, the release of HIV-1 L domain mutants is potently enhanced by ectopic NEDD4-2s, a native isoform with a naturally truncated C2 domain that appears to account for the residual titer of L domain-defective HIV-1. The reason for the unique potency of the NEDD4-2s isoform has remained unclear. We now show that the naturally truncated C2 domain of NEDD4-2s functions as an autonomous Gag-targeting module that can be functionally replaced by the unrelated Gag-binding protein cyclophilin A (CypA). The residual C2 domain of NEDD4-2s was sufficient to transfer the ability to stimulate HIV-1 budding to other NEDD4 family members, including the yeast homologue Rsp5, and even to isolated catalytic HECT domains. The isolated catalytic domain of NEDD4-2s also efficiently promoted HIV-1 budding when targeted to Gag via CypA. We conclude that the regions typically required for substrate recognition by HECT ubiquitin ligases are all dispensable to stimulate HIV-1 release, implying that the relevant target for ubiquitination is Gag itself or can be recognized by divergent isolated HECT domains. However, the mere ability to ubiquitinate Gag was not sufficient to stimulate HIV-1 budding. Rather, our results indicate that the synthesis of K63-linked ubiquitin chains is critical for ubiquitin ligase-mediated virus release. To promote its escape from cells, HIV-1 hijacks cellular budding machinery through so-called L domains in its structural Gag protein. However, HIV-1 lacks a type of L domain that recruits NEDD4 ubiquitin ligases, a family of cellular enzymes that attach one or more copies of a small protein called ubiquitin to other proteins. Surprisingly, one NEDD4 family member, which is known as NEDD4-2s and stands out because its membrane-binding domain is uniquely truncated, can nevertheless potently stimulate HIV-1 release. Our study reveals that NEDD4-2s can do this because its altered membrane-binding domain allows it to associate with HIV-1 Gag. Remarkably, when tagged with the altered membrane-binding domain of NEDD4-2s, even a distantly related yeast protein becomes capable of stimulating the release of HIV-1. We also show that only the portion of NEDD4-2s that acts as an enzyme is required when targeted to HIV-1 Gag in an alternative manner. Taken together, our findings indicate that it is not simply the ability to attach ubiquitin to Gag, but rather the ability to form a particular type of ubiquitin chain in the immediate vicinity of Gag, that is critical to stimulate virus release. Retroviruses such as HIV-1 usurp the cellular Endosomal Sorting Complex Required for Transport (ESCRT) machinery to promote the detachment of infectious progeny virions from the plasma membrane nL-type, binds to the V domain of ALIX, and has an auxiliary role Retroviruses recruit the ESCRT machinery through so-called late assembly (L) domains in Gag, the viral polyprotein that drives particle assembly and release nL-type L domain in HIV-1 p6 as expected, and also on the NC domain of Gag, which binds to a different region of ALIX ΔPTAPPThe profound release and infectivity defects of HIV-1 PTAP L domain mutants can be largely corrected by increasing the cellular expression levels of ALIX ΔPTAPP, with even full-length NEDD4-2 showing only minimal activity ΔPTAP release and infectivity NEDD4 family ubiquitin ligases have a common modular architecture, with an N-terminal C2 domain involved in membrane binding, multiple substrate-binding WW domains, and a C-terminal HECT domain with intrinsic catalytic activity In the present study, we show that the residual C2 domain of NEDD4-2s constitutes an autonomous HIV-1 Gag-targeting module that can be functionally replaced by the unrelated HIV-1 Gag-binding protein cyclophilin A (CypA). Remarkably, CypA could also substitute for the entire substrate-recognition domain of NEDD4-2s. When targeted to Gag via the C2 domain remnant of NEDD4-2s, other NEDD4-type ubiquitin ligases, including yeast Rsp5, and a subset of isolated HECT domains acquired the ability to rescue HIV-1 budding. These observations imply that divergent isolated HECT domains are either capable of recognizing a functionally relevant substrate when targeted to Gag, or can engage the ESCRT pathway themselves upon autoubiquitination. Our results also indicate that the synthesis of K63-linked ubiquitin chains by NEDD4 family members is critical for the ability to stimulate virus budding.ΔPTAPPΔ1-31 was completely inactive, again confirming earlier results Δ1-31 was nearly as active as WT NEDD4-2s, and stimulated the release of particulate CA approximately 25-fold. Furthermore, Cyp-NΔ1-31 partially corrected the Gag processing defect of the Tsg101 binding site mutant or all four of the WW domains, in effect leaving only the HECT domain in place (NHECT). As shown in Δ1-275 and Cyp-NHECT stimulated the release of particle-associated CA by 30- and 20-fold, respectively. Of note, identical NEDD4-2s truncation mutants that lack CypA at the N-terminus exhibited no activity in this assay The residual C2 domain of NEDD4-2s is separated from the catalytic HECT domain by an approximately 420-amino-acid region that harbors four WW domains involved in substrate binding . To examΔPTAPP1-245/WWP1) was at least as active as WT NEDD4-2s in rescuing particle production and Gag processing by HIV-1ΔPTAPP increased mature particle production by HIV-1ΔPTAPP almost 20-fold, despite being poorly expressed . We infer that the residual C2 domain of NEDD4-2s is sufficient to confer the ability to function in HIV-1 budding to widely divergent NEDD4 family members from different species.To determine the generality of these findings, we examined whether it was possible to convert a yeast ubiquitin ligase into a form capable of correcting HIV-1 budding defects. r levels . In contWT, which has NC and p6 replaced by a foreign dimerization domain and efficiently produces VLP in an ESCRT pathway-independent manner WT Gag exhibits less baseline ubiquitination than authentic HIV-1 Gag. These properties of ZWT Gag had allowed us to analyze the effect of NEDD4-2s on the ubiquitination of VLP-associated Gag, and the uptake of NEDD4-2s into VLP, independent of its effect on VLP production We previously showed that the residual C2 domain of NEDD4-2s is required for its ability to induce Gag ubiquitination, and for its incorporation into VLP WT Gag-based assay system as in our previous study to determine whether chimeric versions of WWP1, ITCH, or Rsp5 are capable of inducing Gag-ubiquitin conjugates. All of the parental and chimeric ubiquitin ligase constructs used in this analysis had N-terminal FLAG tags to allow a comparison of relative expression levels. As previously reported WT Gag and NEDD4-2s led to the appearance of additional Gag species in ZWT VLP that migrated slower than the ZWT Gag precursor rescued both particle production and the conversion of CA-p2 to mature CA to a similar extent as full-length NEDD4-2s, which served as a positive control induced the ubiquitination of ZWT Gag to a comparable extent as NEDD4-2s. In contrast, the constructs harboring the HECT domains of SMURF1, E6AP, or HERC6 failed to induce Gag ubiquitination. It is possible that the isolated HECT domains of E6AP and HERC6 lacked catalytic activity, since no evidence for autoubiquitination was apparent. Also, HERC6 is an interferon-induced protein that, based on similarity to human HERC5 and mouse HERC6, may be a ligase for ISG15 rather than ubiquitin 1-37/SMURF1HECT construct appeared as capable of autoubiquitination as the two HECT domain constructs that efficiently rescued HIV-1 budding were not incorporated, even though they were expressed as well or better than the constructs that were taken up into VLP. The third inactive construct (N1-37/HERC6HECT) was incorporated at about 4-fold higher levels than NEDD4-2s, normalized for expression levels and WT NEDD4-2s exhibited comparable activities in the ΔPTAPP rescue assay . The N1-245/ITCH E6AP C lobe chimera appeared to have no effect on HIV-1 budding, but was too poorly expressed to yield reliable results (data not shown). In contrast, the N1-245/ITCH-HUWE1 C lobe chimera was expressed at higher levels. However, even at expression levels similar to or higher than those obtained with the parental N1-245/ITCH construct, N1-245/ITCH-HUWE1 had at most a small effect on the release of HIV-1ΔPTAPP, whereas N1-245/ITCH potently rescued budding . Nevertheless, when the same samples were examined by Western blotting with the K63-linkage specific Apu3 antibody, only the parental N1-245/ITCH construct yielded three prominent bands (right panel). The mobility of these three bands was as expected for ZWT Gag modified with di-, tri-, or tetra-ubiquitin chains. Equivalent results were obtained with HWA4C4, another K63-linkage specific antibody (data not show). We conclude that the chain type specificities of N1-245/ITCH and N1-245/ITCH-HUWE1 in living cells differ considerably, and that the ability to catalyze K63-linked chains is of critical importance for the rescue of HIV-1 release.Interestingly, the pattern of Z245/ITCH , consistWe show here that widely divergent human and yeast ubiquitin ligases of the NEDD4 family, and even a subset of isolated HECT domains, possess the intrinsic ability to function in HIV-1 release. The truncated C2 domain of NEDD4-2s provides a natural Gag-targeting module, which accounts for the unique ability of authentic NEDD4-2s to rescue HIV-1 budding defects. However, other NEDD4 family members, including yeast Rsp5, and in some cases even their isolated catalytic HECT domains, acquire the same ability if targeted to HIV-1 Gag. A common property that is shared by widely divergent NEDD4 family members is the preferential catalysis of K63-linked ubiquitin chains, and at least in the case of yeast Rsp5, the isolated HECT domain is sufficient to synthesize such chains In our previous study, the unique potency of NEDD4-2s in the ΔPTAPP rescue assay did depend on its C2 domain being truncated, and was not shared by several other NEDD4 family members with intact C2 domains Our previous results suggested that the C2 domain remnant of NEDD4-2s is required for activity in the ΔPTAPP rescue assay, because it mediates the association of the ubiquitin ligase with HIV-1 Gag HECT E3s contain two broad functional regions: a large N-terminal region required for substrate recognition, and a C-terminal region (the HECT domain) which catalyzes the ubiquitination of bound substrates ΔPTAPP release by NEDD4-2s depends on Tsg101/ESCRT-I One potential transacting factor is ESCRT-I, because Sundquist and colleagues have demonstrated that the stimulation of HIV-1Saccharomyces cerevisiae, can strongly stimulate HIV-1ΔPTAPP release and Gag processing when its C2 domain is replaced. Thus, if an interaction with an ESCRT pathway component is required for activity in the ΔPTAPP rescue assay, such an interaction and the interfaces involved must be conserved between yeast and man. One reported interaction that potentially meets these criteria is that between NEDD4 and ALIX or their yeast homologues Rsp5 and Bro1 ΔPTAPPIt has also been suggested that NEDD4 family E3s interact through their HECT domains with as yet unknown components of the ESCRT pathway, because several NEDD4 family members, and the isolated HECT domain of WWP1, localized to aberrant endosomal class E compartments induced by dominant-negative VPS4 In principle, ESCRT pathway components could also be recruited via ubiquitinated Gag, because the upstream ESCRT complexes each possess at least one component that binds ubiquitin K63-linked ubiquitin chains are required for the transport of at least some cargo into MVB Structural studies indicate that the conformations of K63- and K48-linked chains are markedly distinct. Specifically, K63-linked di- or tetraubiquitin chains exhibit an extended conformation in which functionally important surface hydrophobic residues are constitutively exposed, whereas K48-linked chains can adopt a closed conformation in which these hydrophobic surface residues are sequestered ΔPTAPP is a mutant of HXBH10, a vpu-positive version of the infectious HXB2 proviral clone of HIV-1, with an in-frame deletion of codons 7 through 11 of p6 WT variant of HXBH10 encodes a chimeric Gag precursor that has NCp1p6 replaced by a leucine zipper dimerization domain HXBH10Δ1-31 (residues 32–834 of NEDD4-2s), or WWP1s (residues 110–922 of WWP1) with an N-terminal FLAG tag have been previously described, and are based on the mammalian expression vector pBJ5 Δ1-31, Cyp-NΔ1-275, and Cyp-NΔHECT constructs encode CypA followed by HA and FLAG epitopes, which in turn are followed by NEDD4-2s residues 32–834, 276–834, and 432–834, respectively. Vectors expressing CypA-HA/FLAG fused to the isolated HECT domains of SMURF1 (residues 366–757), E6AP (residues 462–852), or HERC6 (residues 637–1014) were made in an analogous manner using Cyp-NΔ1-31 and cDNA clones encoding SMURF1 (KIAA1625), E6AP (BC009271), or HERC6 (BC042047) as templates. Overlap extension PCR was also used to generate pBJ5-based vectors expressing FLAG-tagged residues 1–73, 1–110, or 1–245 of NEDD4-2s fused to residues 383–922 of WWP1 , using previously described vectors expressing FLAG-NEDD4-2s and FLAG-WWP1s as templates 1-37/ITCH), residues 138–809 of yeast Rsp5 (yielding N1-37/Rsp5), residues 366–757 of SMURF1 (yielding N1-37/SMURF1HECT), residues 1207–1606 of HECW1 (yielding N1-37/HECW1HECT), residues 1162–1572 of HECW2 (yielding N1-37/HECW2HECT), residues 462–852 of E6AP (yielding N1-37/E6APHECT), or residues 637–1014 of HERC6 (yielding N1-37/HERC6HECT). The templates used included previously published plasmids Plasmids expressing NEDD4-2s, N6) were seeded into T-25 tissue culture flasks 24 hrs prior to transfection. A calcium phosphate precipitation technique was used to transfect cells with HXBH10ΔPTAPP (between 0.5 and 1 µg) or ZWT proviral DNA (2 µg), along with expression vectors encoding the indicated E3 constructs (between 1 and 6 µg) or empty vector. Total DNA transfected was normalized to 8 µg by the addition of carrier DNA (pTZ18U). At 24 hrs post-transfection, cell culture supernatants were removed and clarified by low-speed centrifugation and passage through a 0.45 µm filter. Clarified supernatants were layered on to 20% sucrose, and viral particles were separated using high-speed centrifugation . Cells were lysed using 1 x RIPA buffer with protease inhibitors. Virus pellets and cell lysates were analyzed by SDS-PAGE and Western blotting. The anti-HIV CA antibody 183-H12-5C was used to detect Gag, Gag cleavage products, and Gag-ubiquitin conjugates. Ectopically expressed ubiquitin ligase constructs were detected using anti-FLAG or anti-HA antibodies . The K63-linkage specific antibodies Apu3 and HWA4C4 were purchased from Millipore. Western blots were quantitated with the ImageJ software.293T cells (1.2×10NEDD4-2s: BC000621; WWP1: BC036065; ITCH: NM_031483; SMURF1: NM_020429; HECW1: NM_015022; HECW2: NM_020760; E6AP: NM_130838; HERC6: BC042047; HUWE1: NM_031407; CypA: NM_021130.

Platelet-derived growth factor (PDGF) and insulin promote the survival of neuronal cells, including retinal ganglion cells (RGCs), via activation of phosphoinositide 3-kinase (PI 3-kinase)/Akt signaling. Of importance, recent studies have shown that imatinib inhibition of PDGF receptors induces retinal toxicity in some patients. To date, the extent of activation and the functional significance of insulin-induced PI 3-kinase/Akt signaling remain unclear in the context of dysregulated PDGF receptor signaling in retinal cells. In the present study, we tested the hypothesis that the pro-survival effect of insulin-induced PI 3-kinase/Akt signaling is compromised by imatinib inhibition of PDGF receptor signaling in RGCs.RGC-5 cells were subjected to acute and long-term treatments with imatinib, a PDGF receptor tyrosine kinase inhibitor. Afterwards, the changes in RGC phenotype and apoptotic markers were assessed by fluorescence and phase contrast microscopy and caspase-3/poly(ADP-ribose) polymerase (PARP) cleavage, respectively. In addition, imatinib regulation of PDGF- and insulin-induced PI 3-kinase/Akt survival signaling was determined by immunoblot analyses, immunoprecipitation, and in vitro PI 3-kinase assays.Treatment of RGC-5 cells with imatinib for up to 48 h resulted in apoptosis, which was not rescued by insulin supplementation. The apoptotic phenotype was associated with upregulation of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase. Time dependency experiments revealed that imatinib-mediated apoptosis was preceded by early and sustained abrogation of PDGF-induced increases in PDGF receptor tyrosine phosphorylation and phosphotyrosine-associated PI 3-kinase activity. In addition, imatinib inhibited PDGF-induced downstream phosphorylation of Akt, GSK-3β, and p70S6kinase. However, imatinib exposure did not affect insulin-induced insulin receptor substrate (IRS)-associated PI 3-kinase activity and the downstream phosphorylation of Akt, GSK-3β, and p70S6kinase.Together, these data indicate that disruption of PDGF receptor signaling compromises the pro-survival effect of insulin-induced IRS-dependent PI 3-kinase/Akt signaling in RGCs, and that the maintenance of PDGF-induced PI 3-kinase/Akt signaling is critical for the survival of retinal neuronal cells. The central nervous system consists of different neuronal cell types, including retinal, cerebellar, and cortical neuronal cells , the surPlatelet-derived growth factor (PDGF) is another important neurotrophin that promotes the survival of central nervous system neurons, including retinal ganglion cells (RGCs). In this regard, PDGF induces PI 3-kinase/Akt survival signaling to prevent apoptotic cell death -15. TranRecent studies have shown that imatinib inhibition of PDGF receptors induces retinal toxicity in patients with chronic myeloid leukemia (CML) -23. ThesA PI 3-kinase ATP was purchased from MP Biomedicals . All other chemicals were from Fisher Scientific or Sigma Chemical .Recombinant human platelet-derived growth factor-BB (PDGF-BB) was purchased from R&D Systems . Human insulin (Novolin R) and imatinib mesylate was obtained as a gift from Dr. Neeraj Agarwal . RGC-5 cells (passages up to 10) were maintained in culture using Dulbecco’s Modified Eagle Medium along with 10% fetal bovine serum and antibiotic and antimycotic solution (Sigma) in a humidified atmosphere of 95% air and 5% COescribed ,31. AfteRGC-5 cells were seeded onto 6 well plates. The subconfluent cells were then maintained in complete medium (+FBS) or serum free medium (-FBS) for 48 h, during which time the cells were exposed to increasing concentrations of imatinib (1 µM to 30 µM). In addition, serum-free medium (-FBS) was supplemented with either 30 ng/ml PDGF or 30 nM insulin during RGC-5 cell exposure to imatinib. After 48 h, the cells were washed with PBS (pH 7.4), fixed with 4% paraformaldehyde for 15 min, and then labeled with 0.5 µg/ml 4,6 diamidino-2-phenylindole (DAPI). The apoptotic phenotype was then assessed by nuclear staining with DAPI (fluorescence microscopy) and by morphological changes (phase-contrast microscopy). For each treatment condition, at least 3 different fields were chosen to verify apoptotic cell death characterized by nuclear condensation, cytoplasmic shrinkage, and rounding-up of cells. The images of RGC-5 cells obtained from fluorescence and phase-contrast microscopy are representative of 4 to 5 separate experiments.RGC-5 cell lysates (10 µg protein each) were electrophoresed using pre-cast 4%–12% NuPage mini-gels (Invitrogen), and the resolved proteins were transferred to nitrocellulose membranes . The membranes were blocked and probed with the respective primary antibodies. The membranes were washed three times with Tris-buffered saline . The components of TBS included 25 mM Tris base, 137 mM NaCl, 3 mM KCl, and 0.1% Tween-20. Subsequently, the immunoreactivity was detected using specific HRP-conjugated secondary antibodies followed by enhanced chemiluminescence (Amersham Biosciences), as described [RGC-5 lysates obtained using buffer A were sonicated (15 s×4) and centrifuged at 16,000x g (4 °C) for 10 min. The respective supernatants were then used for protein assays . The aliquots of supernatants (60 µg protein) were subjected to immunoprecipitation with 2 µg each of anti-phosphotyrosine, anti-IRS-1, anti-p85α, or anti-p110β primary antibody that was pre-conjugated (2 h at 4 °C) to Gammabind G Sepharose. Prior to the PI 3-kinase assays, the immunocomplexes were washed with buffer A and TNE buffer, which consisted of 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EGTA, and 0.1 mM sodium orthovanadate .32P-ATP. The reactions were stopped by adding 20 µl of 6N HCl and 160 µl of CHCl3/CH3OH (1:1). Subsequently, the assay tubes were vortexed for 20 s and centrifuged at 16,000 xg at room temperature for 5 min. The phospholipid-containing lower organic phase from the respective reaction tubes was recovered and spotted on to silica gel thin layer chromatography plates that had been heated to 100 °C for approximately 1 h. The thin layer chromatography plates were then subjected to ascending chromatography using a freshly prepared solvent mixture that contained (in ml) 60 CHCl3; 47 CH3OH; 11.3 H2O; 2 NH4OH. Phosphatidylinositol 3-phosphate (PI3P) spots thus resolved were visualized and quantified by autoradiography and phosphorimager analyses , respectively. As negative controls, mock immunoprecipitations were performed using lysis buffer, which revealed negligible formation of 32P-labeled PI3P.PI 3-kinase assays were performed according to the following protocol . After it-test. Values of p<0.05 were considered statistically significant.Results shown are the mean±standard error of the mean of 3 or more experiments. Statistical analyses of the data were performed by one-way repeated measures ANOVA followed by Bonferroni Previously, we and several other investigators showed that PDGF or insulin promotes the survival of neuronal cells, including RGCs ,4,12. InApoptotic cell death is induced by the upregulation of cleaved caspase-3 and cleaved PARP. Cleaved caspase-3 is the activated form of cytosolic caspase-3, whereas nuclear PARP is one of the substrates of activated caspase-3. The activation of caspase-3 may result from diminution in the phosphorylation or activation of cell survival kinases, including Akt, and so we examined if imatinib perturbs these pathways in RGC-5 cells. Subconfluent RGC-5 cells were maintained in serum-free conditions for 24 h or 48 h, during which time the cells were exposed to 10 µM and 30 µM imatinib. The cell lysates were subjected to immunoblotting using the following primary antibodies: anti-caspase-3 primary antibody that detects endogenous full length inactive caspase-3 (35 kDa) and the cleaved caspase-3 19 kDa; ; and antTo determine whether imatinib dysregulates agonist-specific receptor tyrosine phosphorylation, we subjected serum-deprived (24 h) RGC-5 cells to pretreatments without (control) or with imatinib (0.1 to 30 µM) for 30 min. Subsequently, control and imatinib-pretreated cells were stimulated with PDGF or insulin for 6 min. As shown in Previous studies have shown that imatinib inhibits PDGF-induced Akt phosphorylation in vascular smooth muscle cells and IRS-The data shown in To determine the sustained effects of imatinib that dysregulates agonist-specific receptor tyrosine phosphorylation, we subjected serum-deprived (24 h) RGC-5 cells to pretreatments without (control) or with 10 µM imatinib for 3 h and 24 h. Subsequently, control and imatinib-pretreated cells were stimulated with PDGF or insulin for 6 min. As shown in 2-ceramide inhibits insulin-induced IRS-associated PI 3-kinase activity (unpublished observations). To verify whether long-term imatinib exposure affects PDGF as well as insulin-induced PI 3-kinase activity, further studies were performed. As shown in Recent studies demonstrate that prolonged incubation of leukemia cells with imatinib results in the generation of endogenous ceramides , which mTo further support the observations shown in The present study provides evidence that the survival of RGCs, a key component of the central nervous system, is compromised by impaired PDGF receptor signaling. Imatinib inhibition of PDGF receptor signaling leads to apoptotic cell death, which is not rescued by insulin that triggers prosurvival signaling pathway . The majPI 3-kinase activation and PIP3 production are critical for neuronal cell survival and development, neurite formation and elongation, and axon specification -40. ThisSeveral studies have shown that IRS-dependent and IRS-independent PI 3-kinase/Akt signaling pathways mediate the survival of neuronal cells. In this regard, insulin and insulin-like growth factor-1 utilize IRS as the intermediary signaling component to activate PI 3-kinase/Akt survival signaling ,40. In cvs insulin-induced pro-survival signaling in the neuroretina.At this juncture, it is pertinent to note that imatinib therapy in CML patients with diabetes improves insulin sensitivity and fasting blood glucose levels ,25. On tPDGF exerts neuroprotective effects in several neuronal cell types. After optic nerve axotomy, RGCs undergo apoptosis due to diminished PDGF expression levels . In hippRGC-5 cells express several ganglion cell markers including Thy-1 and NMDA receptors, and thereby possess the characteristics of primary RGCs in culture ,54. In aPDGF receptor antagonism induces apoptotic loss of RGCs (present study), neuroprogenitor cells , and bra

Like everyone else we had been hearing about the recent Mumbai abortion case through the media. As Pediatric Surgeons and sensitive human beings we all felt quite disturbed over various aspects of the issue. The case raised a debate regarding the rights of an unborn baby and those of its parents.A 30-year-old mother, had a 24-week pregnancy. The baby was diagnosed to have a congenital heart disease. Knowing about the morbidity related to the disease, the couple decided not to have the baby; however, the pregnancy was already well beyond 20 weeks. They asked for legal permission from the court for abortion. The court denied it as it was against the law. The case ended with the twisted tale of some wrong reports from the hospital and with the miscarriage possibly because of the stress the mother had undergone. The event definitely left us with some food for our thoughts.A nationwide debate followed, with the majority of the learned politicians, professionals, lawyers and doctors arguing against the abortion. However, a significant proportion of the general public did think that parents should be allowed to decide whether to have a defective baby or not, as they are the people who will face the ground realities and not the society or the system who is forcing them to keep the baby.There was a very noticeable fact that many categories of people were being asked for their opinion . These people saw the issue in their limited perspectives. We never saw any one asking any pediatric surgeon about what is probably scientifically correct. We feel that pediatric surgeons, after gynecologists, are the people directly related to facing situations where a mother comes with an antenatal ultrasound past 20 weeks with congenital defects in the baby. It would not be wrong to say that a pediatric surgeon is the most authentic person to give his opinion regarding the morbidity associated with a congenital anomaly. We see and analyze the situation in a much wider perspective while considering a whole lot of other congenital anomalies being increasingly diagnosed antenatally, many of which prove to be incompatible with life or associated with poor quality of life. The family and the society have to bear the severe burden of these babies later on.The 1971 MTP act specifically mentions laws regarding:When a pregnancy can be terminated.By whom it can be terminated.Place where pregnancy can be terminated.The punishments of violation.The major point of discussion in relation of these laws is that no pregnancy can be terminated after 20 weeks have elapsed in any circumstance unless there is a life-threatening medical emergency to the mother, like in a situation of a threatened abortion. Hence, even if a mother is diagnosed to have a baby with anencephaly or multiple defects incompatible to life after birth, she will be forced to keep the baby once the time limit of 20 weeks has been crossed.After enactment of the law in 1972, a few amendments were made that were minor ones and changed the definition of a mentally ill person versus a lunatic, change in the places authorized for MTP, the equal liability of the owner of the place where MTP is performed even if he is not the doctor and the degree of punishment after violation.The UK Abortion Act 1967: Much of the Indian law was copied from the UK Abortion Act 1967, which included a similar time limit of 20 weeks at the time of enactment, but, with the changing scenario, the act was amended in 1990 and the time limit was increased to 24 weeks.http://www.efc.org.uk/Foryoungpeople/Factsaboutabortion/MoreonUKabortionlaw).The exact text from the law amended is quoted below Rest of the world , 13 weeks , 14 weeks , 18 weeks (Sweden), viability (Netherlands and to some extent the United States) and 24 weeks (Singapore and the United Kingdom). Here, the term viability is equivalent to 24 weeks.Liberalize the time limit to 24 weeks instead of 20.Develop a categorical list of congenital anomalies that are either:incompatible with life,carry extreme morbidity after birth orkeep the individual away from a socially productive life.Any of these listed anomalies if found at any stage of pregnancy, the parents should have the right to legally terminate.Legal provisions making advanced-level antenatal ultrasound mandatory (which is to be made available free of cost by the state if possible).There were many arguments against the liberalization of the law in such circumstances as seen in the Mumbai abortion case. Many of them were valid. It will result in killing of the unborn baby, specially the girl child. But again, there are both good and bad aspects of the situation. All these can be controlled by strict law enforcement and monitoring agencies like the PNDT Act and the government health departments.We are all aware that the health care scenario for an average citizen is not optimal in India and is in a grim situation. Even the most basic antenatal health care program does not receive priority over other health schemes. In this situation, it is almost impossible to provide good antenatal screening services for congenital anomalies. In the absence of a state-sponsored health insurance, the entire cost of treatment has to be borne by the parents. This stands in contrast to the developed countries like the UK and the USA. Perhaps the law makers, politicians and all the people who were giving their opinion in the mentioned case should keep this in mind and think with a wider perspective of the field situation in our country. Even the judge, while giving the final decision, said that “There was no law in place for the court to order termination of the pregnancy in a situation like this. The government can consider making this change in the future, but what can we do now?” This meant that while giving this decision even the judges realized that what they were giving was not fair and changes need to be made.We have a strong association in the form of IAPS, which can be verbose enough to deliver authentic opinions on such issues. We have to speak up with both the ground realities as well as the scientific facts. The only way to jump into the scene and produce significant contributions is by constituting a scientific committee that can study the situation and convey the key message to the law makers.The above text deliberately leaves several aspects of the agenda untouched, perhaps to provoke “us,” who will bring our thoughts from passive subconsciousness to active and executable decisions after reading this. In the end, we can say that change with time is a rule and even the rules have to change with time in order to remain valid in the most current context. We made a rule 38 years back and did not make any major change in it, a thing that other countries did as they progressed with time.

We investigated a possible role of antidepressants as inhibitors of P2X4 receptors and analysed their analgesic mechanism using an animal model of neuropathic pain.Neuropathic pain is characterized by pain hypersensitivity to innocuous stimuli that is nearly always resistant to known treatments such as non-steroidal anti-inflammatory drugs or even opioids. It has been reported that some antidepressants are effective for treating neuropathic pain. However, the underlying molecular mechanisms are not well understood. We have recently demonstrated that blocking P2X2+ responses in P2X4 receptor-expressing 1321N1 cells, which are known to have no endogenous ATP receptors. Paroxetine exhibited the most powerful inhibition of calcium influx via rat and human P2X4 receptors, with IC50 values of 2.45 μM and 1.87 μM, respectively. Intrathecal administration of paroxetine produced a striking antiallodynic effect in an animal model of neuropathic pain. Co-administration of WAY100635, ketanserin or ondansetron with paroxetine induced no significant change in the antiallodynic effect of paroxetine. Furthermore, the antiallodynic effect of paroxetine was observed even in rats that had received intrathecal pretreatment with 5,7-dihydroxytryptamine, which dramatically depletes spinal 5-hydroxytryptamine.Antidepressants strongly inhibited ATP-mediated Ca4 receptors may underlie the analgesic effect of paroxetine, and it is possible that some antidepressants clinically used in patients with neuropathic pain show antiallodynic effects, at least in part via their inhibitory effects on P2X4 receptors.These results suggest that paroxetine acts as a potent analgesic in the spinal cord via a mechanism independent of its inhibitory effect on serotonin transporters. Powerful inhibition on P2X Neuropathic pain is caused by lesions of the central or peripheral nervous system, mainly in patients with diabetes, post-herpetic neuralgia or cancer. Neuropathic pain is especially problematic because of its chronic, severe and intractable pain state, and is characterized by tactile allodynia, which drastically affects the quality of patients' lives. Although a number of patients suffer from neuropathic pain, its pathogenesis is not fully understood. It is widely known that neuropathic pain is nearly always resistant to general analgesics, such as non-steroidal anti-inflammatory drugs or even opioids, but some antidepressants and anticonvulsants have been successful in treating neuropathic pain.Antidepressants have been used for over 30 years to manage several intractable pain states including chronic headache, low back pain, rheumatoid arthritis and fibromyalgia ,2. AccumIt has been well known that antidepressants induce antidepressive effects via their inhibitory effects on 5-hydroxytryptamine (5-HT) and norepinephrine (NE) transporters in the central nervous system . MonoamiIn addition to their inhibitory effects on monoamine transporters, antidepressants have been reported to affect multiple neurotransmitter receptors and ion channels implicated in pain transmission such as NMDA receptors ,17 and o4 receptors in activated microglia plays a key role in the pathogenesis of neuropathic pain. Spinal nerve injury induced upregulation of P2X4 receptors on activated microglia in the spinal cord and spinal blockade of P2X4 receptors induced significant antiallodynic effects i response to ATP stimulation (data not shown). 1321N1 cells stably expressing rat P2X4 receptors displayed a reproducible [Ca2+]i response to ATP stimulation significantly inhibited ATP-evoked currents in rat P2X4 receptor-expressed 1321N1 cells Figure . The ATP) Figure . Pretreas Figure . Some ans Figure . Paroxetn Figure . Using 1s Figure . Paroxetn Figure . Both ine Figure and 2c. s Figure and 3b as Figure and 3d.4 receptors, has antiallodynic effect, because we have shown that inhibiting P2X4 receptors reversed tactile allodynia in neuropathic rats i increase in 1321N1 cells was induced by calcium influx from extracellular fluid via P2X4 receptors. Recombinant rat or human P2X4 receptors expressed in 1321N1 cells showed pharmacological properties similar to those previously described i response via rat P2X4 receptors as previously reported i increase was markedly suppressed by paroxetine, suggesting that paroxetine inhibits rat and human P2X4 receptors in a non-competitive manner. Using an electrophysiological technique, we found that similar to the results in calcium imaging, the pretreatment of cells with paroxetine strongly inhibited the ATP-induced currents on rat P2X4 receptor-expressed 1321N1 cells. Therefore, it is proposed that paroxetine directly inhibits P2X4 receptors. Furthermore, paroxetine strongly inhibited the ATP-induced currents on primary cultured microglial cells. We have previously shown that an exposure of such concentration of ATP to primary microglia selectively activates P2X4 receptors i level at high concentrations greater than 50 μM and induces apoptosis in MG63 cells [Intrathecal administration of paroxetine showed a potent antiallodynic effect at 7 days and 14 days after nerve injury. We have previously shown that intrathecal administration of TNP-ATP induces significant antiallodynic effects at higher doses (10 or 30 nmol) than par63 cells . The wea2+]i response mediated by human P2X4 receptors . These results indicate that the difference in the potency of inhibition on P2X4 receptors may explain the difference in the clinical effectiveness of antidepressants in patients of neuropathy.We also found that fluvoxamine produced a much weaker antiallodynic effect than paroxetine, and citalopram produced no antiallodynic effect, although these SSRIs have similar inhibitory action on 5-HT transporters. Citalopram has been reported to be less effective than paroxetine in patients of diabetic neuropathy ,38. Inte7 receptors as well as P2X4 receptors [2+]i response of P2X7 receptor-expressed 1321N1 cells . Therefore, it is conceivable that intrathecally administered paroxetine may also inhibit P2X7 receptors in the spinal cord. However, we have previously shown that PPADS, a non-selective antagonist for P2X receptors including P2X7, has no effect on mechanical allodynia in neuropathic pain model incorporated into pcDNA3.1+ [4 receptors were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 95% air and 5% CO2 at 37°C and split 1/6 every three days. For the measurement of [Ca2+]i, the cells were plated onto poly-L-lysine-coated glass coverslips, placed in silicon rubber walls and maintained for about 48 hr.The cDNAs encoding rat and human P2Xiew, CA) were intPrimary cultured microglia were prepared according to the method described previously . In brie2+]i in single cells was monitored by a fura-2 ratio imaging system. The cells were incubated with 2.5 μM fura-2AM for 45 min in a balanced salt solution at room temperature. Then, the cells were washed with BSS and mounted on an inverted fluorescence microscope equipped with a Xenon-lamp and band-pass filters of 340 nm and 380 nm. The emission fluorescence was measured at 510 nm. Image data were detected with Aquacosmos , and [Ca2+]i was expressed as the ratio of the fluorescence intensities at 340 nm and 380 nm. Applying 30 μM ATP for 20 sec to the 1321N1 human astrocytoma cells expressing rat or human P2X4 receptors, a first [Ca2+]i response (S1) was measured. Drugs were added to the cells for 10 min, and [Ca2+]i response (S2) was measured by a second ATP application. Inhibitory effects of the drugs were evaluated by the S2/S1 ratio. After washing out the drugs by BSS, we confirmed recovery of [Ca2+]i response by a third ATP stimulation.i response via human P2X4 receptorsEffect of citalopram on ATP-evoked [Ca. Effect of pretreatment of cells with citalopram on the ATP-evoked [Ca2+]i response via human P2X4 receptors. Citalopram has no effect on the ATP-evoked [Ca2+]i response via human P2X4 receptors. Data are means ± SEM of 164–181 cells.Click here for file2+]i response via rat P2X7 receptorsEffect of paroxetine on BzATP-evoked [Ca. Paroxetine significantly inhibited the BzATP induced [Ca2+]i response in rat P2X7-expressed 1321N1 cells (***p < 0.001 by unpaired t-test). Data are means ± SEM of 95–113 cells.Click here for file

Anopheles fauna of Timor-Leste using these techniques.The island of Timor lies at the south-eastern edge of Indonesia on the boundary of the Oriental and Australian faunal regions. The country of Timor-Leste, which occupies the eastern part of the island, is malarious but anopheline faunal surveys and malaria vector incrimination date back to the 1960 s. Over the last decade the malaria vectors of south-east Asia and the south-west Pacific have been intensely studied using molecular techniques that can confirm identification within complexes of isomorphic species. The aim of this study is to accurately identify the Anopheles sundaicus and Anopheles subpictus individuals was also assessed using DNA sequences from the ITS2 and mitochondrial cytochrome-b. All specimens, other than those from larval surveys, were processed to detect the presence of the Plasmodium parasite circumsporozoite protein by ELISA for vector incrimination.The survey was carried out over the period February to June 2001. Standard entomological techniques - human landing collections, larval collections and CO2 baited light traps - were used to collect anophelines from the main geographical regions: coastal plains, inland plains, and highlands. Specimens were processed for identification by morphology and genotyped for the ribosomal DNA ITS2 by restriction analysis and/or DNA sequencing. Phylogenetic relationship of Anopheles barbirostris, Anopheles aconitus, Anopheles annularis, Anopheles maculatus, Anopheles peditaeniatus, An. sundaicus and Anopheles vagus. These were confirmed by molecular analysis with the addition of Anopheles flavirostris and an unidentified species designated here as An. vagus genotype B. This latter species was morphologically similar to An. vagus and An. subpictus and is likely to be the An. subpictus described by other workers for Timor. However, genetically this species showed strong affinities to the An. sundaicus complex. Anopheles vagus was the most common species but was rarely collected coming to bite humans; An. barbirostris and An. vagus genotype B were the two most common species collected in human landing catches and both were found positive for CS protein.Of 2,030 specimens collected, seven species were identified by morphology: Anopheles were identified; two species: An. barbirostris and An. vagus genotype B, were incriminated as malaria vectors.The anopheline fauna of Timor-Leste is of Oriental origin with no evidence of elements from the Australian Region. The existence of species complexes will make the use of morphological markers problematic in the country. Using molecular analysis a number of issues regarding the anopheline fauna of Timor-Leste were resolved and nine putative species of Timor, of which Timor-Leste (formerly East Timor) is part, is the largest island at the eastern end of the Lesser Sunda Islands which form part of the Malayan Archipelago and the country of Indonesia from which Timor-Leste recently gained independence. Malaria is endemic in Timor-Leste, though transmission is not high, tending to be mesoendemic on the coast, hypoendemic in the inland lowland regions, and very low to absent in areas over 500 m above sea level (asl) . MalariaAnopheles fauna appears to be distinctly Oriental, although it has been reported that members of the Punctulatus Group, which includes the major malaria vectors of the south-west Pacific, may occur there [Anopheles barbirostris, Anopheles sundaicus, Anopheles subpictus and Anopheles aconitus. Of these An. barbirostris, An. subpictus and An. sundaicus are considered as the main vectors [The island of Timor lies on the border of the Oriental and Australian faunal regions, and while, like Australia, it is of Gondwanic origin the island has never been connected with Australia, even during the glacia-maxima of the Pleistocene era and the existence of the Shula Shelf . Its Anour there . Variousur there ,6. Faunaur there ,8. In 19 vectors -9.The discrepancies presented by the various authors with regards to anopheline speciation in Timor-Leste highlights the problems of using alpha taxonomy for identifying specimens especially where cryptic or isomorphic species are involved. Here the problem is exacerbated as Oriental species have, over the last 5 M years, moved down from the south-east Asian mainland through the islands of the Malayan Archipelago allowing for founder effects and island isolationism, resulting in cryptic species with subtle changes in morphology.Accurate species identification, which allows important vectorial parameters to be applied to species, is crucial in malaria transmission studies and in implementing and monitoring control strategies. Over the last ten years a considerable amount of work has been done to validate the identity of the malaria vectors of south-east Asia and the south-west Pacific using molecular techniques. Species identification is now predominantly based on a polymerase chain reaction (PCR) of the ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2) region either through restriction fragment length polymorphism analysis (RFLP) or an allele-specific PCR (AS-PCR) ,11. To dPlasmodium falciparum and 21 cases of Plasmodium vivax in-country and 212 cases of relapsing P. vivax on return to Australia [Plasmodium parasite and incriminate malaria vectors.In support of Timor-Leste's transition to independence, a United Nations Peace Keeping Force was deployed in September 1999. During the first six months the Australian Defence Force (ADF) contingent suffered 267 malaria infections consisting of 43 cases of The island of Timor lies 9°S and 125°E; Timor-Leste occupies 18,900 sq km (about two-thirds) of the eastern part of the island. The climate is tropical monsoon with distinct wet and dry seasons. Precipitation ranges between 1,000-2,000 mm p.a.; the wet season occurs from November to May and accounts for 80-85% of the annual rainfall, whereas the dry season is from June to October. The country is mountainous with a central range up to 2,900 m asl separating the north and south coasts. There are three main geographical regions: a narrow coastal plain (0-20 m asl), inland lowland plains (100-500 m asl), and highland regions (>500 m asl).2 baited EVS traps [Collections of anophelines were made from the Dili area and the Bobonaro District including coastal, lowland inland and highland areas Figure . Human lVS traps . SeventeHinf I, Hha I, Tru 9I, Hsp 92 III, Ali I, Sau 3AI, Sal I, Msp I, Dde I, and Rsa I) that recognise four nucleotide motives to produce diagnostic RFLPs of the ITS2 region.All larvae were reared to adults in the site water from which they were collected. All adult females were identified morphologically using the keys of Bonne-Wepster and Swellengrebel, Reid, and O'Connor and Soepanto ,14,15. S2, 200 pM of each dNTP, 0.4 μM of each primer, 0.5U of Taq DNA polymerase and approximately 1-20 ng of genomic DNA template (1 μl of extraction). Cycling conditions included initial denaturing at 93°C for 4 min followed by 35 cycles of 93°C for 1 min, 50°C for 1 min, 72°C for 1.5 min. The PCR product was purified using a QIAquick PCR purification kit (Qiagen) and sequenced by the Australian Genome Research Facility on an ABI3730xl.The rDNA ITS2 region was sequenced from species belonging to recognised complexes based on differential PCR-RFLP profiles . The mtDAn. subpictus specimens collected from Papua New Guinea (PNG) and Vietnam. Anopheles sundaicus and An. subpictus ITS2 and cyt-b sequences were obtained from Genbank using both species' name searches and nucleotide Blast (Blastn) searches through the National Centre for Biotechnology Information (NCBI). Sequences drawn from GenBank were limited to An. sundaicus and related An. subpictus species using a rapid dip stick method [n = 5) negative control value [All adult mosquitoes collected in human landing catches were processed for the presence of CS protein of ol value . Only thThree HLC were conducted 1900-0700 hr and indicated that while anophelines fed sporadically throughout the night, there was a peak feeding time in the first hour of the night when the majority of feeding occurred. Accordingly the remaining HLC were made from 1900-0100 hr. Thirty-eight larval collections were made throughout the survey area. Figure An. barbirostris, An. peditaeniatus, An. aconitus, An. annularis, An. maculatus, An. sundaicus and An. vagus. All species, except An. maculatus, were collected in HLC. Anopheles barbirostris, An. vagus, An. maculatus and An. annularis were also collected as larvae. The results of CO2 baited traps collections were poor and from the 17 traps set only 16 anophelines were collected: An. vagus (×12), An. aconitus (×1), An. annularis (×2) and An. peditaeniatus (×1).A total of 2030 anophelines were collected, 932 from HLC, 1082 as larvae, and 16 from trap collections. From these the following seven species were identified by morphology: An. vagus from Balibo and no anophelines were collected using this method in the vicinity of Bobonaro. Around Balibo and Bobonaro the terrain is rugged and steep with little flat ground and few potential anopheline larval habitats. Only two An. vagus larval sites were located at Balibo and one at Bobonaro. Table Collections from highland sites were limited, HLC (1900-0100 hr) resulted in one Anopheles barbirostris was common and widespread throughout the coastal and inland lowland regions. It was one of the dominant anophelines collected in HLC both on the coast and inland though only three larval sites were located. These sites were large permanent bodies of water with well-established flora and fauna. Sixty-six specimens were analysed by PCR-RFLP and there appeared to be only one ITS2 genotype.Anopheles peditaeniatus was collected in HLC, but not as larvae. This species was rarely found on the coast (1/66) but was common in the inland lowland plains (65/66). The ITS2 region was examined in 29 specimens; all were found to be the same RFLP genotype. It was determined that the restriction enzyme Dde I would produce RFLPs that would reliably separate An. sinensis, An. crawfordi and An. peditaeniatus. This enzyme identified all the Timor-Leste material as An. peditaeniatus (confirmed by sequencing: GenBank accession number AF543862). Additionally sequences from the ITS2 region from these specimens matched those of An. peditaeniatus from China [Anopheles nigerrimus and Anopheles argyropus was not always possible.om China . With maAnopheles aconitus was collected on the coast and inland (<500 m asl); all in HLC with no larval sites found. Of the 26 specimens collected 18 were subjected to molecular analyses [An. aconitus and four were An. minimus. All 18 specimens were originally identified as An. aconitus based on the pale scaling on the proboscis. Given that this is an unusual character for Anopheles minimus, the four specimens of this type were sequenced and found to be Anopheles flavirostris. Anopheles aconitus was found coastally and in the inland lowland plains; An. flavirostris was only found in the inland plains. The ITS2 sequence from An. aconitus (GQ500119) was unique although it shows high similarity (97-98%) to An. aconitus isolates from south-east Asia, whereas An. flavirostris (GU062188) showed one nucleotide difference to the same species from the Philippines.analyses . Of thesAnopheles annularis was collected as larvae and in HLC; all collections were made from the inland plains. The numbers collected in HLC were low; larval habitats were natural ground pools with established flora and fauna. All specimens showed the same ITS2-RFLP profiles and a subset were sequenced (GU062187) and showed 99% identity with the same species from south-east Asia.Anopheles maculatus was collected as larvae from six locations, all inland below 500 m asl. Anopheles maculatus appeared at the end of the wet season (from May onwards) when large numbers of larvae were commonly found in shallow pools formed in the gravel beds of receding rivers and in shallow water remaining in rice fields post harvest. Anopheles maculatus is one of several closely related species within the Maculatus Group [An. maculatus sequences from Malaysia.us Group , the memus Group . MoleculAnopheles sundaicus was found at Port Hera, with eight specimens collected in HLC. The taxon sundaicus was recently split based on mitochondrial (COI and cyt-b) and ribosomal ITS2 markers, with the mainland type designated Anopheles epiroticus and the island type An. sundaicus [An. sundaicus [An. epiroticus, An. sundaicus s.s., and An. sundaicus species E [An. sundaicus sequences from south-east Asian individuals grouped these specimens with An. sundaicus individuals from Malaysia-Borneo.undaicus -28. The undaicus . Variantpecies E , we founAnopheles vagus was the most common species in the survey region with 1315 specimens identified by morphology. It was taken in larval collections, HLC and CO2 baited light traps from 32 of the 36 collection sites. It had the widest distribution of any species being collected on the coast, inland plains, and highland regions. The presence of a small pale patch of scales on the apex of the labium - a character used to separate An. vagus var. vagus from An. vagus var. limosus [An. subpictus [An. vagus and An. subpictus [An. subpictus. limosus and fromubpictus - was foubpictus ,15, was An. vagus were amplified and digested with the restriction enzymes Msp I and Rsa I. Both enzymes generated two unique RFLP profiles, and these genotypes were designated A (901 specimens) and B (414 specimens). The size of the undigested ITS2 differed between the two genotypes with A being approximately 700 bp and B 600 bp. All 27 specimens with white scaling on the apex of the proboscis were identified as genotype A. The ITS2 sequences of these two genotypes were compared to existing sequences in GenBank. Genotype A matched an existing sequence of An. vagus (FJ654649), whereas genotype B showed highest identity (98%) to An. sundaicus carrying the ITS2 sequence Variant I (AY768540) [An. sundaicus and a detailed morphological description provided; the neotype and associated topotypic specimens were collected from Sarawak [An. sundaicus neotype in that the femur and tibia are not speckled or mottled with white scales. This characteristic is considered to be of major importance in separating An. sundaicus from An. vagus and An. subpictus [An. vagus and An. subpictus than An. sundaicus. Phylogenetic assessment using: the four ITS2 sequence variants of An. sundaicus, An. vagus genotype B, and An. subpictus from PNG, Indonesia, Sri Lanka and Vietnam, strongly supports genotype B clustering with An. sundaicus species . A neoty Sarawak . The spes Figure . Additios Figure places AAn. vagus collected only 61 were in HLC and these were all from inland sites. The larvae were taken both on the coast and inland and were found in a wide variety of water bodies including swamps (fresh and brackish), rice fields, smaller ground pools with both clay and gravel substrates, animal wallows and hoof prints, wheel ruts, concrete drains, domestic waste water polluted with detergents, truck tyres and folds in plastic wraps where it cohabited with the container breeder Aedes albopictus.Of the 901 specimens of An. vagus genotype B were commonly collected in HLC (335/414). All specimens collected by this method were from coastal sites except for one inland location . Anopheles vagus genotype B was only collected as larvae on the coast and was more conservative in its larval habits than An. vagus, being found in large, well-established bodies of water including coastal lagoons (fresh and brackish) and large pools in creek lines.Specimens of Anopheles barbirostris and An. vagus genotype B made up 82.1% of the HLC. The landing rate of An. vagus genotype B was 15.6/human/night on the coast, while the rate for An. barbirostris was 7.44/human/night on the coast and 5.52/human/night inland.The landing rate for all anophelines in coastal areas was 24.0 anophelines/human/night (based on 132 hours of collecting) and for inland areas 14.64 anophelines/human/night (based on 108 hours of collecting). For all species there appeared to be an observable peak feeding period early in the evening. On the coast, 24.6% of all feeding occurred in the first hour of the night from 1900 to 2000 hr, and inland, 49.3% of feeding occurred at this time. The anophelines/human/hr for 1900-2000 hr was 3.46 for all species while the average hourly landing rate after this was 1.2 anophelines/human/hr.An. barbirostris, 61 An. vagus, 355 An. vagus genotype B, 66 An. peditaeniatus, 20 An. aconitus, 8 An. sundaicus, and 7 An. annularis. Of these, one An. barbirostris, collected from the inland village of Marko (site 32), was found positive for CS protein for both Plasmodium falciparum and Plasmodium vivax (210 variant); and one An. vagus genotype B, collected from the village of Aidabelatan (site 8), was found positive for P. vivax (247 variant) CS protein.From the HLC 907 specimens were processed for circumsporozoite antigen, these consisted of 390 The data presented here on anopheline species' composition supports the concept that the anopheline fauna of Timor-Leste is of Oriental origin and contains no species from the Australian Region. Thus Timor-Leste is one of the most south-easterly limits of an Oriental anopheline fauna that has filtered down from south-east Asia over the last 5 M years.Anopheles in Timor-Leste, this being a compilation of the studies of various earlier workers. More comprehensive studies, conducted by Portuguese workers, collected both larvae and adults from 17 locations over a period of five months, covered most of the country, and identified nine species of Anopheles [An. barbirostris, An. aconitus, An. annularis, An. flavirostris, An. maculatus, An. sundaicus and An. vagus. In addition, we collected An. peditaeniatus and An. vagus genotype B whereas Fraga de Azevedo and others [An. subpictus and Anopheles tesselatus. Other Portuguese workers [An. nigerrimus and Anopheles barbumbrosus to the records, though for Timor-Leste this latter species is more likely to be An. barbirostris [Lien and others list 14 nopheles . The pre workers added Anirostris .Anopheles taxa undermines the effectiveness of this identification method. Still, preliminary morphological identification remains indispensable for initially assigning specimens to species groups and complexes, thus simplifying the task of subsequent molecular analysis.All previous surveys on the anopheline fauna of Timor-Leste predate the advent of molecular genetic technologies (PCR and DNA sequencing) and have relied solely on morphological identification. Morphology is still the mostly commonly used method of identifying anophelines in malarious countries throughout the world. It has the benefits of being relatively inexpensive and quick (when compared to molecular methods) and can be carried out in the field. However, the variation of morphological characteristics within and between species, the practical limitations of local keys, and the presence of cryptic species within many of the An. flavirostris from An. aconitus and differentiated An. sundaicus from An. epiroticus, supporting the concept of island and mainland species [An. sundaicus confined to the islands of south-east Asia. Earlier workers have identified An. argyropus, An. nigerrimus and An. sinensis from the Hyrcanus Group in Timor-Leste [An. argyropus, An. nigerrimus and An. peditaeniatus, however PCR and sequencing of the ITS2 confirmed all the material as An. peditaeniatus and it is possible that earlier collections of An. argyropus, An. nigerrimus and An. sinensis were misidentifications of this species. Several members of the Hyrcanus Group are found in Indonesia, but only An. peditaeniatus has been found as far east as the island of Lombok (900 km west of Timor) [Species identification is paramount to understanding malaria epidemiology and so the genetic characterisation of cryptic species and the subsequent use of molecular diagnostic tools provide vital support for studies into mosquito surveillance and malaria control initiatives. In this study, molecular genetic methods separated species with An.or-Leste . There aor-Leste ,31. It hf Timor) .Anopheles vagus genotype B morphologically resembles An. subpictus and An. vagus. At the molecular genetic level however, this mosquito was placed, with strong branch support by the ITS2 and cyt-b markers, between An. subpictus and An. sundaicus, with closer genetic affinities to An. sundaicus. Anopheles vagus genotype B is probably an undescribed new species that has in the past been misidentified as An. subpictus and thus may be an important vector of malaria in Timor-Leste [An. sundaicus, An. subpictus is also a species complex, but only a few ITS2 sequences were available for comparative analyses. Some of these were from India and Sri Lanka and appeared to be distantly related. To provide additional comparative data, sequences of An. subpictus specimens from PNG and Vietnam were included in the analysis; however, An. vagus genotype B could still not be placed confidently with either An. subpictus or An. sundaicus.or-Leste -9. Like An. sundaicus and An. vagus genotype B from Timor-Leste cannot be amplified with the An. sundaicus PCR diagnostic that separates An. epiroticus, An. sundaicus s.s, and An. sundaicus E [An. sundaicus from Timor groups with individuals from Malaysia-Borneo, which are regarded as An. sundaicus s.s., and not Sumatra and Java (Indonesia) individuals, which are geographically closer and regarded as An. sundaicus E [An. vagus genotype B at the base of the An. sundaicus complex, and as sister taxa to An. subpictus species, supports the concept that this is a new as yet undescribed malaria vector species.Interestingly, both daicus E , becausedaicus E . The plaAn. sundaicus complex and the An. subpictus complex in that they are both coastal species that have adapted to utilising brackish water for oviposition and the development of the immature stages [An. vagus genotype B was mainly found on the coast and the larvae of this species were collected from brackish water sites. Only one inland site was found and the ITS2 sequences of specimens from this site matched those of An. vagus genotype B collected from the coast; earlier workers collected specimens of An. subpictus from two inland locations up to 300 m asl [An. vagus and An. subpictus groups to validate existing morphological markers. For example, how important is the apical patch of pale scales on the proboscis in separating An. vagus and An. subpictus?A shared evolutionary trait exists within both the e stages . In this00 m asl . A furthAnopheles vagus, identified in this study by morphology and confirmed by PCR-RFLP and DNA sequencing, fits the behavioural characteristics of this species in that it is a highly adaptable species, capable of utilising a wide variety of aquatic habitats including artificial containers. Its reported zoophilic behaviour [ehaviour is suppoAn. subpictus has been identified with sporozoites whereas oocysts have been found in An. barbirostris, An. sundaicus, and An. aconitus [An. barbirostris and incriminates An. vagus genotype B, which is likely to be An. subpictus of earlier authors.In April-May 2001 the malaria prevalence rate was determined by mass blood surveys in seven villages located in the area where the vector surveys were conducted [aconitus -9. This Anopheles collected in Timor-Leste was early in the evening (1900-2000 hr). This may have implications for malaria control strategies such as indoor residual spraying or long-lasting insecticide-treated bed nets, as early night feeding vectors can avoid these measures by feeding early when most of the local population is outdoors and unprotected.The peak feeding time for all species of An. maculatus, were found to feed on humans and so could be potential vectors of malaria. How important this role might be will depend on their ability to achieve appreciable numbers, their host preference and their longevity. Anopheles vagus was very common in the survey region but appeared to be only an indifferent feeder on humans and probably plays no role in malaria transmission, confirming similar observations made by other workers [Anopheles sundaicus has been incriminated as a vector in Timor-Leste, but at the time of our surveys its numbers were very low. Higher numbers have been collected between March and November by other workers, with some of these specimens positive for oocysts [An. maculatus only appeared in large numbers at the end of the survey period when the aquatic habitats it appears to favour were plentiful and so in these surveys the peak period for this species may have been missed. This highlights the limitation of surveys conducted over one season; only longitudinal surveys over several years can help to resolve seasonal fluctuations in species densities.All species collected in this survey, except workers ,14. The oocysts . SimilarThe anopheline fauna of Timor-Leste consists of Oriental species with no species from the Australian Region. Accurate morphological identification is difficult due to the presence of cryptic taxa with overlapping morphological characters, possibly the result of genetic drift in small isolated island populations following founder events leading to morphological changes. However, the simplicity and timeliness of identification by morphology makes it invaluable in the field even if only as an initial screening tool prior to molecular analysis.Anopheles morphologically; molecular methods confirmed these and in addition identified An. flavirostris and a new species referred to here as An. vagus genotype B. Anopheles barbirostris and An. vagus genotype B were the two taxa most commonly attracted to humans and both these species were found positive for Plasmodium CS protein.This study identified seven species of The authors declare that they have no competing interests.RDC designed the study, organised the field work, participated in the field collections, performed the preliminary identification using conventional PCR-RFLP and wrote the manuscript. MDE participated in the field collections and was involved in drafting and revising the manuscript. SPF organised parts of the field work, participated in the field collections and was involved in drafting and revising the manuscript. NWB was responsible for the overall molecular analysis of the specimens and was involved in drafting and revising the manuscript. All authors read and approved the final manuscript

Thymopentin (TP5) triggers an immune response by contacting with T cells; however the molecular basis of how TP5 achieves this process remains incompletely understood. According to the main idea of immunomodulation, we suppose that it would be necessary for TP5 to form complex with human class II major histocompatibility complex DR molecules (HLA-DR) before TP5 interacts with T cells. The uptake of TP5 by EBV-transformed B cells expressing HLA-DR molecules and the histogram of fluorescence intensities were observed by using fluorescent- labeled TP5, testifying the direct binding of TP5 to HLA-DR. The binding specificity was confirmed by the inhibition with unlabeled TP5, suggesting the recognition of TP5 by HLA-DR. To confirm the interaction between TP5 and HLA-DR, the complex formation was predicted by using various modeling strategies including six groups of trials with different parameters, alanine substitutions of TP5, and the mutants of HLA-DR. The results demonstrated that TP5 and its alanine substitutions assumed distinct conformations when they bound to HLA-DR. The observation further showed that there was flexibility in how the peptide bound within the binding cleft. Also, the molecular analysis supplemented a newly important discovery to the effect of Val anchor on TP5 binding HLA-DR, and revealed the important effects of Glu11 and Asn62 on the recognition of TP5. These results demonstrated the capability of TP5 to associate with HLA-DR in living antigen presenting cells (APC), thereby providing a new and promising strategy to understand the immunomodulation mechanism induced by TP5 and to design potential immunoregulatory polypeptides. Thymopentin (TP5) is a synthetic pentapeptide, corresponding to position 32∼36 of thymopoietin MHC II molecules are proteins anchored in the cell membrane of APC, where they present antigenic peptides to CD4 positive T helper cells in situ with an apparent dissociation constant (Kd) of 7.2×10−6 M. Furthermore, the binding specificity was tested by competitive binding assay with unlabeled TP5. The molecular modeling of the interaction between ligands and receptors demonstrated that TP5 and its alanine substitutions adopted distinct conformations when they bound to HLA-DR. The observation further showed that there was flexibility in peptide binding with MHC II binding cleft. More importantly, the molecular analysis supplemented a newly important discovery to the effect of Val anchor on TP5 binding HLA-DR. Also, the molecular analysis revealed the key effects of Glu11 and Asn62 on the recognition of TP5 based on the mutants of HLA-DR. The study provides a better understanding to the mechanism of interaction between TP5 and TCRs and a rational strategy to design TP5 analogs.In the present study, we have established combined experimental and computational strategies to verify the hypothesis of the complex formation of MHC II/TP5. Taking advantage of confocal-laser scanning microscopy (CLSM) and flow cytometry (FCM) techniques, we examined the binding of fluorescent-labeled TP5 to HLA-DR in living APC To validate the ability of FITC-labeled TP5 to load on EBV-transformed B cells expressing HLA-DR, a qualitative CLSM assay was used to examine the fluorescent signal of EBV-transformed B cells. The surface fluorescence was hardly observed from the cells in the absence of FITC-labeled TP5 at the excitation of 488 nm . In sharKd of 7.2×10−6 M was obtained from the double- reciprocal plot of the equilibrium binding data is close to the reported Kd value −5 M to 10−2 M, the fluorescence intensity decreased , by incubation for 45 min on ice followed by CLSM and FCM.EBV-transformed B cells expressing HLA-DR molecules were maintained in RPMI 1640 containing 2 mM L-glutamine supplemented with 10% standard FBS at 37°C and in 5% CO5 cells, washed twice with phosphate-buffered saline (PBS) buffer (pH 7.4) supplemented with 0.1% BSA and 0.05% NaN3. Ten µM FITC- labeled TP5 was added to the treated cells in PBS (pH 7.4) containing 1% BSA (binding buffer), then incubated for 2 h at 37°C. Thereafter, the cells were washed twice and resuspended in 500 µl PBS, followed by CLSM analysis using Leica Microsystems .We harvested 3×105 per sample) were incubated with various concentrations of FITC-labeled TP5 in binding buffer for 2 h at 37°C. Thereafter, the cells were washed twice and resuspended in 500 µl PBS, followed by flow cytometric analysis using a FACSCan analyzer . A total of ten thousand events were acquired for each sample. The MFI was used to evaluate the binding ability of FITC-labeled TP5 to APC.EBV-transformed B cells (3×105 per sample) were preincubated in the presence of varying concentrations of TP5 in binding buffer for 2 h at 37°C. Then, the cells were washed and resuspended in 50 µl binding buffer, followed by incubation with 20 µl mAb-DR L243 labeled with FITC for each sample for 45 min on ice in dark. Thereafter, cells were washed twice and analyzed by FCM. In each analysis, ten thousand cells were examined. Percent inhibition was calculated as [(1–MFI in the presence of TP5/MFI in the absence of TP5)×100%].EBV-transformed B cells was used as the reference. The structure of TP5 was achieved based on the crystal nonapeptide by mutating method In the first phase, TP5 was docked into the binding site by means of the package Autodock 3.0.5 In the second phase, the lowest-energy complex predicted by molecular docking was subjected to 1000 steps of energy minimization by using the perl script of minAmber MMTSB_tool for removing clashes and refining complex docked by using an Amber-based procedure Next, we studied the dockings for alanine substitutions of TP5 through scanning the alanine on each position for the same receptor (HLA-DR). Moreover, the HLA-DR double mutant DR11/62 and two single mutants DR11, and DR62, were used by molecular modeling method for the same ligand TP5. All preparations and parameters were consistent with the contents above described.

Over the past decade, genetic analysis has shifted from linkage studies, which identify broad regions containing putative trait loci, to genome-wide association studies, which detect the association of a marker with a specific phenotype. Because linkage and association analysis provide complementary information, developing a method to combine these analyses may increase the power to detect a true association. In this paper we compare a linkage score and association score test as well as a newly proposed combination of these two scores with traditional linkage and association methods. One advantage of association analysis is it can be carried out in samples of unrelated individuals, which may be easier to recruit. On the other hand, family samples provide extra information about segregation of the phenotype, and both linkage and association analysis may be performed when genotype and phenotype data are available on family members.Improvement in genotyping technologies has led to great advances in the quest to map genes influencing complex traits. In the late 1980s came linkage studies in family samples that identified broad regions containing putative trait loci. Recently, dense single-nucleotide polymorphism (SNP) chip technology has resulted in genome-wide association analysis, where the genome is queried for association with a specific phenotype. The high number of SNPs run enables relatively thorough coverage of the genome, but also greatly increases the chance of false-positive results. A low Variance-component analysis is a comPopulation-based methods can be applied to family samples provided that familial correlation is accommodated and there is no population stratification . Linear mixed-effect models are particularly well suited for association analysis of quantitative traits in family samples. The genotype effect and additional covariates are modeled as fixed effects while familial correlation is accommodated with a random effect with the covariance structure depending on the degree of relatedness between individuals. LRTs may be applied to determine the genotype-phenotype association. Alternatively, an efficient score statistic for association analysis with the variance computed conditional on the observed phenotype may offer a robust option against departure from the normality assumption .The objectives of this paper are two-fold: first, we compare the LRT with conditional score statistics for both linkage and association analyses using the Genetic Analysis Workshop 16 simulated Framingham Heart Study dataset (Problem 3). Second, we explore whether a combination of the linkage and association score improves power to detect associated SNPs.The subset of simulated Framingham Heart Study samples analyzed in our report consists of 736 families and 381 unrelated individuals. The families range in size from 2 to 291 participants with available genotypes and phenotypes, for a total of 6372 individuals.Initial data cleaning was performed using the software PLINK ,6 based Lipid-related phenotypes were available at three exams for each participant. To evaluate methods robust to departure from normality, we concentrated on low-density lipoprotein (LDL) and triglyceride levels (TG) levels because of their skewed distributions. We analyzed each trait measured at the first exam, but also averaged each trait over the three exams.Chromosome 11 was chosen because a group of 39 polygenes influencing high-density lipoprotein (HDL) clustered in the range of 110 Mb to 134 Mb and because of the presence of two major genes, one influencing LDL and the other influencing TG. Chromosome 22 was selected for ease of computation and because it harbors a major gene explaining 1% of the LDL variance.p-value ≥ 0.05, and a r2 < 0.04. Mendelian transmission errors were detected and corrected using the program PEDCHECK [For linkage analysis, we selected markers with low linkage disequilibrium, which may bias identity-by-descent (IBD) estimates when parental information is unavailable . We usedPEDCHECK .kth family, Y, has a multivariate normal distribution with mean E(Y|X) = μX = μ + βX and covariance matrix: πtij is the proportion of alleles shared IBD by relatives i and j at genome location t; and ϕij is the kinship coefficient. The covariance matrix unconditional on the IBD (Σ) is obtained by setting πtij = ϕij in the expression for Σπ.The basic variance-component model assumes that the vector of phenotype in the t is N pedigrees. To test the null hypothesis of no linkage (H0: Aπ is the matrix of centered IBD and W = (Σ-1(Y - μX)(Y- μX)'-I)Σ-1 with elements wij. The squared of the efficient score is standardized by an estimate of the variance conditional on the observed phenotypes to make it robust to violation of the normality assumption [The log likelihood for a QTL at sumption ,8.E = μ + βX + γg, where g is the coded genotypes. The covariance unconditional on the IBD proportions, Σ, is typically used, although one could easily substitute Σπ at the expense of added computation complexity. The null hypothesis of no association H0: γ = 0 is typically tested using a LRT. Alternatively, the efficient score test may be constructed to test for association. The efficient score statistic for association reduces to The linear mixed-effects model to test for association analysis is very similar to the variance-component model, with the exception that the genotype effect is included in the mean rather than in the covariance matrix: ociation .The conditional association score and our implementation of the linkage score are uncorrelated under the null hypothesis of no linkage and no association . Therefop-value above 10-6, and a low r2 (< 0.01) with all polygenes and major genes on chromosomes 11 and 22. We calculated power as the proportion of significant major gene association detected over all 200 phenotype replicates.We computed the LRT and association-conditional score statistics using an additive genetic model and a general 2-df genetic model for each SNP. For all models we adjusted for sex and age, or average age in the case of averaged phenotypes. We compared the association statistics in terms of type I error and power. To determine type I error, we selected SNPs with a call rate above 95%, a HWE We computed multipoint IBD probabilities using the software LOKI , making Figure As seen in Table α = 5% is presented in Figure α = 5% to detect this association as shown in Figure α = 10-8 (data not shown). Power to detect association between rs901824 and LDL ranged between 60% and 80% at α = 5%, but power was 0 for all statistics at α = 10-8 (results not shown). All methods achieved 100% power to detect SNP rs2294207, an additively acting SNP on chromosome 22 explaining 1% of the LDL variation with either LDL phenotypes (data not shown).Power to detected major SNPs at Despite selecting phenotypes that violated the normality assumption, type I error rates for the additive association-conditional score and LRT statistics were not inflated, but slight type I error inflation was observed for the 2-df LRT statistic and moderate inflation for the 2-df association-conditional score. The type I error rate inflation for the 2-df statistics is most likely due to a violation of asymptotic assumptions caused by SNPs with low cell counts. Note that filtering by MAF less than 10% lowers the false-positive rate for both 2-df statistics.In our analyses, all additive statistics failed to detect the association between TG levels and rs603446, a SNP with a non-additive effect. This result suggests, as do Lettre et al. , that thα = 0.05 to accommodate the extra degree of freedom used by the combined conditional score is 0.467 for the additive model and 0.396 for the general 2-df model. The percent of iterations that reached the minimum threshold ranged from 4.0% for the additive model for visit 1 TG levels at SNP rs603446 to 31.5% for the general 2-df model for average LDL at SNP rs901824. This suggests that a larger linkage signal or a more efficient combination of the conditional association and linkage scores may ultimately increase power.Including the information from the linkage analysis using the combined conditional score did not consistently increase the power to detect an associated SNP. However, despite the lack of a large linkage signal and even though the combined conditional score statistic uses an extra degree of freedom, the power remained fairly constant when using the combined conditional score compared with the LRT and the association-only conditional score. The minimum LOD score needed at A notable difference between the LRT and the conditional score for association was computation time. Performing the analysis for a total of 35,979 SNPs on chromosomes 11 and 22 using a single processor would take more than 16 hours using the LRT and less than an hour using the association-conditional score. The computation times for the linkage analysis were more comparable. Once the IBD was computed, the LRT using SOLAR took about 45 minutes to scan chromosome 11, while the linkage-conditional score took about 3.25 hours. Despite the longer linkage computation time, the combined conditional score was computed much faster than the LRT.Although more research is needed, conditional score analysis provides an interesting possibility of a gain in power by combining linkage information using the combined conditional score. Regardless, the conditional score for association provides a fast and comparable alternative to LRTs for analysis of family data.HDL: High-density lipoprotein; IBD: Identity by descent; LDL: Low-density lipoprotein; LOD: Logarithm of the odds; LRT: Likelihood ratio test; SNP: Single-nucleotide polymorphism; TG: TriglycerideThe authors declare that they have no competing interests.AEH performed the initial data cleaning, carried out the LRT association analysis, and wrote and revised the manuscript. YZ carried out the variance-component linkage analysis and revised the manuscript. JD conceived the study, implemented the conditional score analysis, and drafted and revised the manuscript. All authors read and approved the final manuscript.