Genes enriched in transcription regulator activity showed the common up regulation of 2 core molecular clock genes, ARNTL and CLOCK. Commonly down regulated genes enriched Rhythmic process and included: NR1D1, DBP, BHLHB2, OPN4, and HTR7, which regulate intracellular circadian rhythms. Changes in mammary, liver and adipose transcriptomes at the onset of lactation illustrate the complexity of homeorhetic adaptations and suggest that these changes are coordinated through molecular clocks.Genes associated with lactation evolved more slowly than other genes in the mammalian genome. Higher conservation of milk and mammary genes suggest that species variation in milk composition is due in part to the environment and that we must look deeper into the genome for regulation of lactation. At the onset of lactation, metabolic changes are coordinated among multiple tissues through the endocrine system to accommodate the increased demand for nutrients and energy while allowing the animal to remain in homeostasis. This process is known as homeorhesis. Homeorhetic adaptation to lactation has been extensively described; however how these adaptations are orchestrated among multiple tissues remains elusive. To develop a clearer picture of how gene expression is coordinated across multiple tissues during the pregnancy to lactation transition, total RNA was isolated from mammary, liver and adipose tissues collected from rat dams (n = 5) on day 20 of pregnancy and day 1 of lactation, and gene expression was measured using Affymetrix GeneChips. Two types of gene expression analysis were performed. Genes that were differentially expressed between days within a tissue were identified with linear regression, and univariate regression was used to identify genes commonly up-regulated and down-regulated across all tissues. Gene set enrichment analysis showed genes commonly up regulated among the three tissues enriched gene ontologies  Taxonomic variation in milk composition is extensive, and is driven by neonatal requirements as well as life history and reproductive strategies of the dam. Maternal substrate demands of lactation are either met by increased dietary intake or by mobilization of nutrients stored in tissues Lactation is part of the reproductive process in mammals and is the most metabolically stressful period of an adult female's life Previous work in our lab in-vitro rate of incorporation of 14C-labeled glucose into lipids was used as an indicator of the in-vivo metabolic capacity of mammary, liver and adipose tissue on pregnancy day 20 (P20) and lactation day 1 (L1). Rate of lipid synthesis on P20 was low in mammary tissue, when the mammary gland was not synthesizing milk. With the onset of lactation there was approximately a 10-fold increase in the rate of lipid synthesis in the mammary gland , and univariate regression was performed to identify individual genes that were commonly up-regulated and down-regulated across all the L1 tissues versus all the P20 tissues When nominal P-values were adjusted with false discovery rate, mammary tissue had by far the greatest number of statistically significant changes in gene expression during the transition from pregnancy to lactation . Only 18n = 15 across tissues versus n = 5 within tissues).There were 68 genes (P<0.001) commonly up- and down-regulated across all three tissues   . InteresIt is interesting to point out that ATRN, attractin, was one of the most significantly commonly up regulated genes  transcription factor activity GO gene set  to the external world, principally via light-dark cycles. Synchronization of circadian clocks to the external world enables organisms to anticipate and prepare for periodic and seasonal changes in their environment Daily and seasonal rhythms are coordinated in mammals by the master clock that lies in the suprachiasmatic nuclei (SCN) of the hypothalamus. Internal and external synchronizing factors affect the autoregulatory transcription–translation feedback loop of core clock genes that generate circadian rhythms Intracellular circadian rhythm generation occurs through an auto regulatory transcription–translation feedback loop During the transition from pregnancy to lactation there was a significant (P<0.05) induction of ARNTL (4 fold), CLOCK (1.4 fold), NPAS2 (5 fold) and RORA (3 fold) in the mammary gland . AlthougWhen expression statistics of core clock genes were examined for common up or down regulation during the transition from pregnancy to lactation, these data suggested that ARNTL, CLOCK and RORA genes were significantly induced, and expression of BHLHB2 and NR1D1 were significantly reduced in all three tissues  We examined the expression changes in genes involved in fatty acid synthesis during the transition from pregnancy to lactation in all three tissues in relation to changes in core clock genes . The fatBmal1 (ARNTL) as measured with qPCR every 4 hrs for 48 hrs.Although we only examined one time point across 2 days, others have shown that there are amplitude changes in core circadian clock genes in the mammary gland during the transition from pregnancy to lactation. Specifically, in mouse dams there is an increase in the amplitude of expression of Bmal1 (Arntl) and decrease in amplitude of expression of Per2 during the transition from pregnancy to lactation Clock mutant mice, which have a genetic mutation that disrupts circadian rhythms, exhibit severe alterations in energy balance, with a phenotype associated with metabolic syndrome, including obesity, hyperlipidemia, hepatic steatosis, high circulating glucose, and low circulating insulin Clock mutant mice fail to thrive, suggesting that their milk production may not be adequate enough to nourish their young Interestingly, homozygous We hypothesize that the master clock modifies peripheral clocks and hormonal levels at the onset of lactation in order to coordinate the changes needed to stimulate lactogenesis and accommodate the increased metabolic demands of milk synthesis. Following modification of the clocks there is a change in the mammal's metabolome that results in the partitioning of nutrients to the mammary which in turn are used to synthesize milk . Based oMultiple pathways and gene sets related to energy homeostasis are changed in mammary, liver and adipose tissues during the transition from pregnancy to lactation. Gene sets enriched with genes up regulated during the pregnancy to lactation transition in the mammary gland reflect the turning on of secretory processes in this tissue and the down regulation of catabolic processes. Gene sets enriched with genes up regulated in liver were related to P450 pathways which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids, and the transcriptional signature of genes down regulated in liver at the onset of lactation suggest that there is a reduction in breakdown fatty acids, so that fats can be spared for milk synthesis in the mammary gland. There was a similarity between the molecular signature of adipose tissue at the onset of lactation and adipose tissue from rats exposed to long-term caloric restriction, in particular the enrichment of up regulated genes in inflammation related genes sets and the enrichment of down regulated genes in cytoskeletal and ECM gene sets. The majority of the gene sets enriched with genes commonly up regulated among all three tissues were related to metabolic processes. Genes commonly down regulated among the three tissues were related to perception and transduction of external stimuli, light and taste as well as rhythmic processes.Molecular signatures of mammary, liver and adipose were also enriched with gene sets classically associated with central nervous systems reception, integration and response to environmental and internal stimuli. In particular we found that core clock genes were commonly changed among the three tissues at the onset of lactation. These signatures illustrate the complexity of homeorhetic adaptations as well as the role of the nervous system in orchestrating the response, and suggested that changes in multiple tissues may be coordinated by changes in molecular clocks. We envision that environmental gene interactions leading to taxonomic variation in milk composition are mediated through changes in molecular clocks, which in turn mediate changes in the animal's transcriptome, proteome and metabolome, and thus metabolic output, milk.ad libitum during the experimental period. On P20, five rats were removed from the maternity cages, anaesthetized with isoflurane, and tissues were collected for analysis. Animals were euthanized by cardiac puncture. Real time videography was used to identify the precise time of birth so that dams could be euthanized within 18–36 hrs after delivery of the pups (L1). All dams were euthanized for tissue collection between Zeitgeber time (ZT) 09:00–12:00 on P20 (mean ZT 10:45) and ZT 11:00–13:00 on L1 (mean ZT 12:17), with ZT 00:00 =  time of lights on.Ten time-bred female Sprague-Dawley (190–280 g) rats  were obtained on day 2 of pregnancy (P2) and were used according to a protocol approved by the NASA Animal Care Committee. During the period of pregnancy (P2) to post natal day 1, the dams were individually housed in maternity cages and maintained under standard colony conditions . Standard rat chow (Purina #5012 pellets) and water were available in-vitro rate of incorporation of 14C-labeled glucose into lipids was used as an indicator of the in-vivo metabolic capacity of mammary, liver and adipose tissue on P20 and L1 On day P20 and L1, mammary glands, liver and visceral adipose tissue samples were collected from anesthetized dams. Mammary and liver tissues were kept in 25 mmol/L Tris, 0.25 mol/L sucrose, 1 mmol/L EDTA (pH 7.3) on ice, and adipose tissue was kept in saline at 37°C to maintain tissue viability during the time between tissue removal and incubation for metabolic analysis. As described previously At each experimental time point, mammary gland #4, liver and visceral adipose tissues were collected from anesthetized dams and were snap frozen in liquid nitrogen and stored at −80°C. Total RNA was extracted from frozen liver and mammary tissue using Trizol® Reagent  according to manufacturer's instructions. Total RNA was extracted from visceral adipose tissue using RNeasy Lipid Tissue Mini Kit  as detailed by the manufacturer. Isolated RNA was resuspended in nuclease free water , and quantity and quality of the RNA was assessed using the Nanodrop® ND-1000 UV-Vis Spectrophotometer  and on the Nanochip using the Bioanalyzer 2100 , respectively. RNA integrity number (RIN) for all samples was ≥7.0.www.ncbi.nlm.nih.gov/geo, accession no. GSE12132).Total RNA from all 3 tissues of 5 rat dams on P20 and 5 rat dams on L1 was amplified and biotinylated using NuGEN's Ovation Biotin System  to generate products for the Rat 230 2.0 GeneChips . Gene expression data were generated following the manufacturer supplied protocols. Briefly, GeneChips were hybridized in an Affymetrix 640 Hybridization oven at 45°C for 16 hours with 60 rpm rotation. After hybridization, gene chips were washed on a Fluidics station , stained, and then scanned using an Affymetrix Genearray scanner GSC3000, with 7G upgrade (Affymetrix). The efficiency of amplification and hybridization were assessed by incorporating Affymetrix Poly-A RNA and Hybridization controls with every sample. The microarray data were deposited in the Gene Expression Omnibus . This was performed by converting human GO sets to corresponding rat GO sets using NCBI homologene. For each gene in a given GO set, the expression of the gene in each L1 sample was compared to the median expression of gene in the P20 samples. The entire set of genes in each ontology was given an enrichment score using the parametric gene set enrichment analysis method (PGSEA) to test for enrichment in up or down-regulated genes TM Gene Expression kit  specific for rat. Samples, no template controls (NTC) and no reverse transcription controls (NoRT) and TaqMan reaction mixes (20 µl) were loaded into MicroAmp Fast Optical 96-well reaction plates and sealed with MicoAmp Optical adhesive film (Applied Biosystems).We used MIQE guidelines when measuring gene expression with QPCR 2Microglobulin ; Actin, beta ; ribosomal protein L10A . Target gene TaqMan hydrolysis probes were as follows: aryl hydrocarbon receptor nuclear translocator-like ; circadian locomoter output cycles kaput ; neuronal PAS domain protein 2 ; RAR-related orphan receptor alpha ; basic helix-loop-helix domain containing, class B2 ; casein kinase 1, epsilon ; nuclear receptor subfamily 1, group D, member 1 (NR1D1 Assay ID Rn00595671_m1); sterol regulatory element binding protein-1 ; acetyl CoA carboxylase-α ; and fatty acid synthase . The qPCR data across the tissues was normalized relative to the abundance of a validated endogenous control  mRNA.Three reference genes were tested to compare efficiency of amplification with target genes: βq) across samples was compared among three reference genes  to select the most appropriate reference gene for our study. The range of Cq was calculated across all samples for each reference gene and differences of Cq among all samples were ≤1.7 for Actb and B2M, and ≤2 for Rpl10a. Thus Actb and B2M were used as the reference genes for the study. Secondly PCR amplification efficiency was compared between the Actb reference gene and each of the target genes using calibration curves. The slope of Actb calibration curve was −3.2. Slopes of target gene calibration curves ranged from −3.1 to −3.4, these data indicate that the amplification efficiency is similar enough (<0.25 difference in slope from reference gene) between target and reference genes to use the delta delta Cq (Δ Δ Cq) method for calculating differences in relative gene expression (RQ). The mean Cq of target and mean Cq of reference gene for each sample were calculated from duplicate wells. The relative amounts of target gene expression for each sample were then calculated using the formula 2−ΔΔCqhttp://www.physics.csbsju.edu/stats/t-test_NROW_form.html) and data were presented as mean +/− SEM.The variation of quantification cycle  Gene ontology and B) KEGG Pathway gene sets enriched with up regulated genes or down regulated genes in mammary during the transition from pregnancy to lactation. Each column represents data from an individual lactating (L1) rat compared to the average of the 5 pregnant rats (P20). For each L1 rat comparison, enrichment scores for each pathway were calculated and the pathways that were most consistently deregulated across the tissues were identified and the results plotted as a heat map (1.02 MB TIF)Click here for additional data file.Figure S2A) Gene ontology and B) KEGG Pathway gene sets enriched with up regulated genes or down regulated genes in liver during the transition from pregnancy to lactation. Each column represents data from an individual lactating (L1) rat compared to the average of the 5 pregnant rats (P20). For each L1 rat comparison, enrichment scores for each pathway were calculated and the pathways that were most consistently deregulated across the tissues were identified and the results plotted as a heat map (1.02 MB TIF)Click here for additional data file.Figure S3A) Gene ontology and B) KEGG Pathway gene sets enriched with up regulated genes or down regulated genes in adipose during the transition from pregnancy to lactation. Each column represents data from an individual lactating (L1) rat compared to the average of the 5 pregnant rats (P20). For each L1 rat comparison, enrichment scores for each pathway were calculated and the pathways that were most consistently deregulated across the tissues were identified and the results plotted as a heat map (0.86 MB TIF)Click here for additional data file.Table S1Expression changes in genes commonly up and down regulated across all three tissues  during the transition from pregnancy to lactation, and changes within mammary, liver and adipose tissues. Values are log base 2 fold change and corresponding adjusted and unadjusted  p-values.(0.03 MB XLS)Click here for additional data file.Table S2Changes in expression of genes that enrich the KEGG_PATHWAY:hsa03320:PPAR signaling pathway, selected based on enrichment by 22 genes that were down regulated in mammary  during transition from pregnancy to lactation. Values are log base 2 fold change, adjusted and unadjusted  p-values as calculated across all three tissues (common) and within mammary, liver and adipose tissue.(0.02 MB XLS)Click here for additional data file.Table S3Changes in expression of genes that enrich the KEGG_PATHWAY:hsa04920:Adipocytokine signaling pathway, selected based on enrichment by 8 genes that were down regulated in liver  during transition from pregnancy to lactation. Values are log base 2 fold change, adjusted and unadjusted  p-values as calculated across all three tissues (common) and within mammary, liver and adipose tissue.(0.02 MB XLS)Click here for additional data file.Table S4Changes in expression of genes that enrich the GOTERM_MF_ALL:GO:0004888∼transmembrane receptor activity, selected based on enrichment by 33 genes that were down regulated in liver  during transition from pregnancy to lactation. Values are log base 2 fold change, adjusted and unadjusted  p-values as calculated across all three tissues (common) and within mammary, liver and adipose tissue.(0.02 MB XLS)Click here for additional data file.Table S5Changes in expression of genes that enrich the GOTERM_CC_ALL:GO:0043292∼contractile fiber, selected based on enrichment by 12 genes that were down regulated in adipose  during transition from pregnancy to lactation. Values are log base 2 fold change, adjusted and unadjusted  p-values as calculated across all three tissues (common) and within mammary, liver and adipose tissue.(0.02 MB DOC)Click here for additional data file.Table S6Changes in expression of genes that enrich the GOTERM_MF_ALL:GO:0003700∼transcription factor activity, selected based on enrichment by 112 genes that were commonly up regulated in all three tissues  during transition from pregnancy to lactation. Values are log base 2 fold change, adjusted and unadjusted  p-values as calculated across all three tissues (common) and within mammary, liver and adipose tissue.(0.05 MB XLS)Click here for additional data file.Table S7Changes in expression of genes that enrich the GOTERM_MF_ALL:GO:0003700∼transcription factor activity, selected based on enrichment by 97 genes that were commonly down regulated in all three tissues  during transition from pregnancy to lactation. Values are log base 2 fold change, adjusted and unadjusted  p-values as calculated across all three tissues (common) and within mammary, liver and adipose tissue.(0.04 MB XLS)Click here for additional data file.Table S8Changes in expression of genes that enrich the GOTERM_BP_ALL:GO:0048511∼rhythmic process, selected based on enrichment by 13 genes that were commonly down regulated in all three tissues  during transition from pregnancy to lactation. Values are log base 2 fold change, adjusted and unadjusted  p-values as calculated across all three tissues (common) and within mammary, liver and adipose tissue.(0.02 MB XLS)Click here for additional data file.Table S9Changes in expression of genes that enrich the GOTERM_BP_ALL:GO:0050953∼sensory perception of light stimulus, selected based on enrichment by 24 genes that were commonly down regulated in all three tissues  during transition from pregnancy to lactation. Values are log base 2 fold change, adjusted and unadjusted  p-values as calculated across all three tissues (common) and within mammary, liver and adipose tissue.(0.02 MB XLS)Click here for additional data file.Table S10Changes in core clock gene set during the transition from pregnancy to lactation across the three tissues (common) and within mammary, liver and adipose tissue. Values are log base 2 fold change and corresponding adjusted and unadjusted  p-values.(0.02 MB XLS)Click here for additional data file.


CF1 mice received a standard laboratory diet and another group was placed on dietary p-dimethylaminoazobenzene (0.5% w w−1). After 40 days of treatment, animals of both groups received p-dimethylaminoazobenzene and two weekly doses of cimetidine  during a following period of 35 days. Cimetidine prevented and reversed δ-aminolevulinate synthetase induction and cytochrome P450 enhancement provoked by p-dimethylaminoazobenzene. However, cimetidine did not restore haem oxygenase activity decreased by p-dimethylaminoazobenzene. Enhancement in glutathione S-transferase activity provoked by p-dimethylaminoazobenzene, persisted in those animals then treated with cimetidine. This drug did not modify either increased lipid peroxidation or diminution of the natural antioxidant defence system  induced by p-dimethylaminoazobenzene. In conclusion, although cimetidine treatment partially prevented and reversed cytochrome P450 induction, and alteration on haem metabolism provoked by p-dimethylaminoazobenzene AB, it did not reverse liver damage or lipid peroxidation. These results further support our hypothesis on the necessary existence of a multiple biochemical pathway disturbance for the onset of hepatocarcinogenesis initiation.Many drugs and xenobiotics are lipophilic and they should be transformed into more polar water soluble compounds to be excreted. Cimetidine inhibits cytochrome P450. The aim of this study was to investigate the preventive and/or reversal action of cimetidine on cytochrome P450 induction and other metabolic alterations provoked by the carcinogen p-dimethylaminoazobenzene. A group of male British Journal of Cancer (2002) 86, 630–635. DOI: 10.1038/sj/bjc/6600102www.bjcancer.comCancer Research UK© 2002  Cytochrome P450 (P450) plays a key role in the oxidative metabolism of drugs and xenobiotics, many of which are lipophilic and to be excreted they should be transformed into more polar water soluble molecules by the system of hepatic mono-oxygenases .P450 can be influenced by a number of exogenous and endogenous factors  and its We have earlier proposed a mechanism for the onset of hepatocarcinogenesis involving an activating status of the whole liver which reflected an important and sustained increase in P450 levels, leading to biochemical aberrations in haem metabolism which in turn would lead to the tumorigenic process  is a H2-histamine antagonist, not inhibiting hepatic mixed-function oxidases, has not a binding site on P450, suggesting therefore that CIM would alter the oxidative metabolism of some compounds by having a direct inhibitory effect on P450. If CIM exerts a significant general effect upon haem biosynthetic and/or degradative pathway, we would expect these effects to be manifest in both P450 and in other haem containing proteins when animals are treated with CIM .CF1 mice (30 g) received a standard laboratory diet   or were placed on dietary p-dimethylaminoazobenzene  . After 40 days, animals of both groups (A and B) received DAB and two weekly doses of CIM (120 mg kg−1 i.p.)  or saline  for a following period of 35 days (n=6) received SLD along the whole period of assay and were injected with CIM under the same scheduled protocol than the DAB treated groups. Animals of control group (n=6) were fed with the SLD and received saline i.p. twice a week for the same period. The treatment protocol is shown inFigure 1ad libitum.Male All animals were inspected at least twice daily. Body weight and food consumption were measured at intervals throughout the study. Food was removed from animals 16 h before sacrifice. Mice were killed  under ether anaesthesia at the indicated times and liver samples were processed immediately as previously described .δ-Aminolevulinic acid synthetase (ALA-S) activity was measured as described by Cytochrome P450 (P450) content was determined in the microsomal fraction according to −1 protein.The lipid peroxidation (LP) index was evaluated by the formation of malondialdehyde and determined as thiobarbituric acid reactive species (TBARS) by the method of Newman-Keuls test was used to assess the degree of significance. A probability level of 0.05 was used in testing for significant differences between controls and treated animals.CIM) partially prevented the enhancement of P450 content. When animals were pre-treated with DAB and then received CIM (BCIM) induction of P450 was also partially reversed. In both cases, after CIM treatment P450 levels were still 50% above basal levels  and even partially reversed this induction in animals pre-treated with DAB (BCIM), achieving a 43% diminution respect to the BS group  in porphyric adult rats . In thisin vivo and in vitro the rate limiting enzymes of haem degradation, and it was suggested that the drug itself or a metabolite would inhibit HO   did not modify increased LP produced by DAB  (It was demonstrated that CIM could not prevent CClM: 220%) .As expected CIM produced only 25% increase in GST activity. The significant enhancement (110%) in GST activity provoked by DAB  are generated. Free radicals induced LP and the role of free radicals in carcinogenesis is also well known . LP indeWe have also shown that free radicals excess was not modified by CIM in animals subjected to the carcinogenic diet, and as a consequence catalase inhibition was not affected by co-treatment of DAB and CIM. This irreversible autocatalytic process produces then a continuous increase in ROS and RNS generation leading to a desbalance between radicals and the antioxidant defence system (It is noteworthy, that increased GST, diminished catalase, and enhanced LP reflecting initiation of carcinogenesis (These results further support our hypothesis about the necessary multiple biochemical pathway disturbances for the onset of hepatocarcinogenesis initiation (Figure 6These findings also provide evidence for the importance of animal experiments in studying the multiple action and crosstalk among different biochemical pathways provoked by the administration of xenobiotics.


To survive, bacteria must be able to adapt to environmental stresses. Small regulatory RNAs have been implicated as intermediates in a variety of stress-response pathways allowing dynamic gene regulation. The RNA binding protein Hfq facilitates this process in many cases, helping sRNAs base pair with their target mRNAs and initiate gene regulation. Although Hfq has been identified as a critical component in many RNPs, the manner by which Hfq controls these interactions is not known.fhlA system was used as a model. OxyS is induced in response to oxidative stress and down regulates the translation of fhlA, a gene encoding a transcriptional activator for formate metabolism. Biophysical characterization of this system previously used a minimal construct of the fhlA mRNA which inadvertently removed a critical element within the leader sequence of this mRNA that effected thermodynamics and kinetics for the interaction with Hfq.To test the requirement of Hfq in these mRNA-sRNA complexes, the OxyS-fhlA mRNA. Hfq binds fhlA mRNA using both the proximal and distal surfaces and stimulates association kinetics between the sRNA and mRNA but remains bound to fhlA forming a ternary complex. The upstream Hfq binding element within fhlA is similar to (ARN)x elements recently identified in other mRNAs regulated by Hfq. This work leads to a kinetic model for the dynamics of these complexes and the regulation of gene expression by bacterial sRNAs.Herein, we report thermodynamic, kinetic and structural mapping studies during binary and ternary complex formation between Hfq, OxyS and  Cis RNAs derive from the same genetic locus as the regulated message but are transcribed from the antisense strand; thus exhibiting perfect complementarity with their target. These RNAs are known to control regulatory pathways such as transcriptional attenuation, RNA processing and decay, and translation initiation Small non-coding RNAs (sRNA) mediate gene regulation in both bacteria and eukaryotes E. coli, and is highly abundant where an estimated 10,000 hexamers present in the cytoplasmic fraction, often in association with ribosomes E. coli and other organisms have shown that strains lacking Hfq exhibit pleiotropic effects such as decreased growth rates, increased stress sensitivity , ineffective tRNA maturation and mini-cell formation Hfq is a homohexamer that belongs to the Sm/LSm family of RNA binding proteins sodB, ftnA, bfr, acnA and sdhC and thus acts as a regulatory node allowing a complex and integrated response to a given growth condition, in this case low iron concentrations Hfq is known largely for its role in post-transcriptional gene regulation by facilitating pairing between sRNAs and mRNAs. A common feature in these pathways is the presence of overlapping networks of RNA interactions where one sRNA regulates multiple genes. For example the sRNA RybB has been shown to regulate fhlA system X sequence element x element by Link and co-workers who showed based on crystallographic and biochemical studies the site has broader specificity than AAYAA x to distinguish it from AREs while still retaining the necessary information about the sequence specificity. To understand whether the (ARN)x element is a commodity among regulatory networks involving Hfq, we tested this hypothesis in the OxyS-fhlA system.To further understand the requirement of Hfq during sRNA:mRNA pairing, we have studied the OxyS-A system . OxyS isfhlA leader sequence enhances the kinetics and thermodynamics of fhlA associating to Hfq stimulating ternary complex formation with OxyS. Structural probing of the leader sequence confirmed Hfq-binding to the (ARN)x element at position −76 in the upstream region of the fhlA mRNA. Finally, we show that Hfq binds to the fhlA leader sequence using both its proximal- and distal-RNA binding surfaces which helps explain the kinetic and thermodynamic properties of Hfq association with mRNAs. These data lead us to revise the model for Hfq-dependent gene regulation to be less sRNA-centric and providing more importance to the manner in which Hfq interacts with a subset of bacterial messages that are subject to regulation by sRNAs.Here we show that the extension of the fhlA facilitates the interaction with OxyS and Hfq as previously described for the rpoS-DsrA system fhlA was extended from the previously characterized position −53 relative to the start codon, to −136 and −220 respectively. A putative upstream Hfq-binding element was absent in the minimal fhlA53 construct but is encoded at position −76 and thus is present in both extended constructs. RNA transcripts are denoted according to the length of their leader sequence: fhlA220 has 220 nt upstream of its start codon. In its genomic context, fhlA is part of a polycistronic message, transcribed under the control of two promoters PhypA and PhypBTo test whether the upstream leader region of fhlA mRNA leader constructs to undergo post-transcriptional regulation, gel mobility shift assays were used to detect binding to radiolabeled OxyS. Equilibrium experiments were performed with fhlA136, but neither binary complex with OxyS nor ternary complexes with OxyS and Hfq were observable using EMSA. For fhlA220, on the other hand, both binary and ternary complexes with OxyS and Hfq were stable. The OxyS•fhlA220 (O•F220) complex formed with a KD of 360±60 nM (fhlA53 (O•F53) complex where a KD of 35±7 nM was measured ) fhlA220 was surprising, but this phenomenon was also observed for DsrA and RprA, both of which bound extended rpoS leader sequences 10–20-fold less tightly than to the truncated leader To test the potential of 60±60 nM . This afmeasured . This vafhlA220 interacts with Hfq, [5′-32P] fhlA220 mRNA was incubated with Hfq at concentrations of 0 nM to 1.67 µM Hfq (hexamer). The Hfq•fhlA220 (H•F220) complex was resolved on native gels to provide a KD of 15±3 nM Hfq hexamer  revealing that the extended structure may present extra Hfq binding elements or a more favorable Hfq-binding motif (fhlA220•Hfq ternary complex (O•F220•H) was also resolved using gel shift assays when pre-formed [5′-32P] OxyS•Hfq complex was titrated with 0.5 nM–11.2 µM fhlA220 mRNA , a distal surface binder (A18) or both simultaneously. The complexes were then visualized by native gel electrophoresis . Thus this minimal construct mimics only part of the native interaction with Hfq.Hfq binds RNAs using two distinct surfaces – a proximal surface that favors U-rich sequences like those in many Hfq-binding sRNAs (such as DsrA and RybB) and the distal surface that binds poly-A RNAs  [11], - fhlA220 mRNA was titrated with increasing concentration of Hfq in the range of 0 to 1.67 µM hexamer.(0.59 MB TIF)Click here for additional data file.Figure S3.fhlA53 mRNA interaction with Hfq. (A). Gel shift assay for Hfq•fhlA220 binary complex formation. (B) Poly-acrylamide gel showing the effect of Hfq binding on Tb(III)-mediated cleavage of 32P-fhlA53. (C) Quantitative analysis of Hfq binding based on the gel in panel B. Data are represented as a ratio of the intensity of each band in the absence and presence of 1 µM Hfq hexamer. Values greater than 1 represent protection. Data between +1 and −1 were considered to be no significant effect.(2.28 MB TIF)Click here for additional data file.Figure S4fhlA220 and fhlA53. (A) Representative SPR sensorgram for OxyS•fhlA53 interaction is shown. 5′-Biotin labeled fhlA53 mRNA was immobilized and varying concentrations of OxyS was titrated . (B) SPR sensorgram for OxyS binding to fhlA220. Here the biotin label was added to OxyS sRNA and titrated with fhlA220 to monitor the interaction . For both interactions data were fitted into a Langmuir binding model to yield kinetic constants. The model is an over-simplification of a complex system as it ignores unimolecular RNA structural rearrangements that might be required prior to association, but the model sufficient to illustrate the interactions in the absence of Hfq and their approximate rates.Kinetic analysis of OxyS interaction with (0.54 MB TIF)Click here for additional data file.Figure S5fhlA220 complex. (A) Overview of handoff experiment. The Hfq•fhlA220 complex was pre-formed and dissociation kinetics of Hfq were monitored by titrating competing RNAs DsrA , A18  or both DsrA and A18. (B) Sensorgram of Hfq dissociation from the Hfq•fhlA220 complex in the presence of 500 nM and 300 nM DsrA and A18. (C) Sensorgram of Hfq dissociation from the Hfq•fhlA220 complex in the presence of 500 nM and 300 nM DsrA. (D) Sensorgram of Hfq dissociation from the Hfq•fhlA220 complex in the presence of 500 nM and 300 nM of A18. All dissociation data were fitted in to Langmuir model.Handoff kinetics of Hfq from Hfq•(1.02 MB TIF)Click here for additional data file.


Cytology performed very well in this older cohort of women. The newer Hybrid Capture II microtitre test may be a useful adjunct, especially if the results reported here are reproducible in other studies. A combined screening test offers the possibility of greater protection and/or longer screening intervals, which could reduce the overall cost of the screening programme. © 1999 Cancer Research CampaignCertain types of the human papilloma virus (HPV) are well established as the primary cause of cervical cancer. Several studies have shown that HPV testing can improve the detection rate of high-grade cervical intraepithelial neoplasia (CIN), but these have been carried out primarily in younger women. In this study we evaluated the role of HPV testing as an adjunct to cytology in women aged 35 or over. An additional aim was to evaluate commercially available kits for HPV testing. A total of 2988 eligible women aged 34 or more attending for a routine smear in 40 general practitioner practices received HPV testing in addition to routine cytology, after having given written informed consent. Samples were assayed by polymerase chain reaction (PCR) and two versions of the Hybrid Capture test for HPV, and women were invited for colposcopy if there was any cytological abnormality (including borderline smears) or the PCR test was positive. Any apparent abnormality was biopsied and loop-excision was performed as necessary. CIN was judged by histology; 42 women had high-grade CIN, of which six were cytology negative (86% sensitivity for borderline or worse) and three had a borderline smear (79% sensitivity for mild dyskaryosis or worse). The positive predictive value of a borderline smear was only 3.1%. Eleven high-grade lesions were negative by the PCR HPV test (sensitivity 74%). The first generation Hybrid Capture II test had a similar sensitivity but an unacceptably high false positive rate (18.3%), while the newer Hybrid Capture II microtitre kit had a 95% sensitivity and a 2.3% positivity rate in normal women when used at a 2 pg ml


Sc) showed marked alteration in the PrPSc glycoform ratio propagated after transmission of classical CJD prions, consistent with the PrP point mutations directly influencing PrPSc assembly. These data indicate that P102L or E200K mutations of human PrP have differing effects on prion propagation that depend upon prion strain type and can be significantly influenced by mismatch at the polymorphic residue 129.Approximately 15 % of human prion disease is associated with autosomal-dominant pathogenic mutations in the prion protein (PrP) gene. Previous attempts to model these diseases in mice have expressed human PrP mutations in murine PrP, but this may have different structural consequences. Here, we describe transgenic mice expressing human PrP with P102L or E200K mutations and methionine (M) at the polymorphic residue 129. Although no spontaneous disease developed in aged animals, these mice were readily susceptible to prion infection from patients with the homotypic pathogenic mutation. However, while variant Creutzfeldt–Jakob disease (CJD) prions transmitted infection efficiently to both lines of mice, markedly different susceptibilities to classical (sporadic and iatrogenic) CJD prions were observed. Prions from E200K and classical CJD M129 homozygous patients, transmitted disease with equivalent efficiencies and short incubation periods in human PrP 200K, 129M transgenic mice. However, mismatch at residue 129 between inoculum and host dramatically increased the incubation period. In human PrP 102L, 129M transgenic mice, short disease incubation periods were only observed with transmissions of prions from P102L patients, whereas classical CJD prions showed prolonged and variable incubation periods irrespective of the codon 129 genotype. Analysis of disease-related PrP (PrP PRNP), and constitute about 15 % of all human prion disease (Inherited prion diseases (IPDs) are fatal neurodegenerative disorders caused by autosomal-dominant mutations in the human PrP gene ( disease . Over 30C) is the principal, and possibly the sole, constituent of the transmissible agent or prion  in PrP, is one of the most prevalent, being responsible for the high incidence of CJD amongst Libyan Jews and in areas of Slovakia and Chile, and is recognized in many other countries  of human PrP is the most common mutation associated with the GSS phenotype and was first reported in 1989 (Polymorphism at residue 129 of human PrP [where either methionine (M) or valine (V) can be encoded] not only affects susceptibility to sporadic and acquired human prion diseases 196 mice, expressing lower levels of the same mutant transgene product, was reported  spontaneously developed neurological dysfunction at 166 days of age . The profoundly dissimilar consequences of the same mutation in mouse and human PrP questions the whole approach of modelling human pathogenic mutations on non-homologous PrP sequences from other species. In particular, the destabilizing effects measured in a mouse protein cannot be assumed to be equivalent in the human protein. The present study differs from all previous reports because we have investigated the biological properties of naturally occurring mutations in human PrP itself expressed in transgenic mice.Importantly, there are examples of IPD where the amino acid change thought to be pathogenic is found as a normal variant in other mammalian species  null background . Similarly, we have generated two further transgenic lines that are both homozygous for human PrP200K,129M transgenes, again on a Prnpo/o background . Here, we report the relative susceptibilities of these transgenic mice to classical (sporadic and iatrogenic) CJD prions, homotypic IPD isolates and variant (v) CJD prions.We have now generated two transgenic mouse lines that are both homozygous for the human PrPPfu polymerase from human genomic DNA with appropriate mutations, using forward primer 5′-GTCGACCAGTCATTATGGCGAACCTT-3′ and reverse primer 5′-CTCGAGAAGACCTTCCTCATCCCACT-3′. Restriction sites SalI and XhoI (underlined) were introduced in the forward and reverse primers, respectively, for use in subsequent cloning steps. The blunt-ended PCR fragments generated by Pfu polymerase were subcloned into SmaI-digested pSP72 vector and sequenced to ensure that no spurious alterations have been introduced by the PCR other than the expected existing point mutations. The mutant human PrP ORFs with the appropriate point mutation confirmed were then isolated by using SalI and XhoI. Subsequent subcloning into the cosmid vector SHaCosTt  recipient females. Two homozygous lines were established for P102L designated Tg27 and Tg33 with mutant transgene expression levels of three and one and a half times, respectively, compared with pooled normal human brain levels. Similarly, two homozygous lines were established for E200K designated Tg23 and Tg49 with relative expression levels of three- and twofold, respectively.The purified Tail biopsies were taken from putative transgenics and screened by PCR using human PrP-specific primers (5′-GTGGCCAGATGGAGTGACCTGGGCCTC-3′ and 5′-GGCACTTCCCAGCATGTAGCCG-3′). Founders were confirmed by Southern blotting using a 900 bp 3′ UTR fragment as a radiolabelled probe. Lines were bred to homozygosity and 20 mice were set aside for long-term neurological observation.2, and intracerebrally inoculated into the right parietal lobe with 30 μl of a 1 % (w/v) brain homogenate prepared in PBS. All mice were thereafter examined daily for clinical signs of prion disease. Mice were killed if exhibiting any signs of distress or once a diagnosis of prion disease was established.All procedures were carried out in a microbiological containment level III facility with strict adherence to safety protocols. A panel of inocula comprising four inherited P102L, three sporadic and three iatrogenic CJD cases were used for the 102L transgenic mice. For the 200K transgenic mice, two inherited E200K and one iatrogenic CJD inocula were used. Inocula were prepared from the brain of neuropathologically confirmed cases of sporadic and inherited CJD with consent from relatives and with the approval from the Institute of Neurology/National Hospital for Neurology and Neurosurgery Local Research Ethics Committee. Mice were anaesthetized with a mixture of halothane and ONeuropathology and immunohistochemical analyses were done as described previously  with the2+ or Mg2+ ions (D-PBS) by serial passage through needles of decreasing diameter. Aliquots were analysed with or without proteinase K digestion  by electrophoresis and immunoblotting as described previously (Sc glycoforms was performed using ImageQuant software (Molecular Dynamics).Brain homogenates  were prepared in Dulbecco's PBS lacking Caeviously . Blots weviously . Quantif102L,129M+/+ Prnpo/o)-27 and Tg-33 that expressed human PrP 102L,129M at three and one and a half times the human endogenous PrPC levels, respectively  transgenic line. Mice that died earlier than 400 days from intercurrent illnesses showed no evidence of prion-related neuropathology. Importantly, mice in the cohort survived to advanced age without clinical or neuropathological signs of prion disease or detectable PrP200K,129M+/+ Prnpo/o)-23 and Tg-49 that expressed human PrP 200K,129M at three and two times, respectively  lines and observed them long-term. Mice that died earlier than 400 days from intercurrent illnesses showed no evidence of prion-related neuropathology. Again, mice in the cohort survived to an advanced age without clinical or neuropathological signs of prion disease or detectable PrPSc, with the oldest mouse living to more than 955 days  of samples from the brain of uninoculated mice showed equivalent proportions of di-, mono- and non-glycosylated PrP to that seen in Tg35 and Tg45 transgenic mice expressing wild-type human PrP 129M . Clinical disease with high attack rates and short incubation periods ranging from 185 to 191 days accompanied the transmission of four different IPD P102L cases to Tg27 transgenic mice . Using Tg27 as a representative line, transmissions of both classical and IPD P102L prions were characterized by generalized synaptic PrP deposits in the cerebral cortex, basal ganglia, hippocampus, thalamus  and the other homozygous for PRNP 129V (designated E200K-129VV). In addition, we transmitted one case of iatrogenic CJD to both lines in order to investigate its interactions with human PrP 200K.Groups of Tg23 and Tg49 transgenic mice were challenged with brain homogenates from two different human IPD E200K cases, one homozygous for IPD E200K-129MM inoculum (I1091) transmitted clinical disease to 8/8 of Tg23 mice, with a short mean incubation period of 184±3 days , and this pattern of neuropathology closely resembled that produced in both transgenic lines by IPD E200K-129MM inoculum (I1091)  or classical CJD prions  with a previously characterized vCJD isolate designated I336  and Tg49  CJD (C expressed in uninoculated wild-type or mutant PRNP transgenic mice, this change in PrPSc glycoform ratio is consistent with the PRNP point mutations acting to directly dictate the stoichiometry and packing order of the three PrP glycoforms into disease-related fibrils or other aggregates (Sc is probably controlled in a strain-specific manner (Importantly, our data now show that the PrPSc isoforms in a new host will also be determined by host genetic background, PRNP sequence and prion strain type (The interpretation of primary transmission data for IPD isolates is complicated by the heterogeneous composition of disease-related PrP isoforms that may be present in the primary inoculum . For exaain type . These d


Crohn’s disease and ulcerative colitis are inflammatory bowel diseases (IBD) quite common in the United States and other Western countries. Patients suffering IBD are at greater risk of developing colorectal adenocarcinoma than the general population. Both, the adenomacarcinoma and the inflammation-carcinogenesis processes are characterized by active angiogenesis. Recent studies also have shown that anti-angiogenesis might be a novel therapeutic approach for IBD. Thrombospondin 1 (TSP1) is an extracellular protein well known for its anti-angiogenic properties. TSP1 also has key functions in inflammation, which is assumed to be the primary cause for carcinogenesis in IBD. This review is focused on the role of TSP1 in colorectal carcinogenesis. The therapeutic effects of TSP derived-peptides on inhibiting the inflammation-carcinogenesis progression are also discussed. The role of inflammation in carcinogenesis has been broadly corroborated. GranulocThere are many examples in which cancer is induced and promoted by inflammation. Colorectal carcinomas are among the best demonstrations for the crucial role of inflammation in cancer. Chronic Inflammatory bowel disease (IBD) such as Crohn’s disease and ulcerative colitis (UC) affect children and adults and they are recognized as premalignant disorders.,7 SustaiUC-carcinogenesis could be a consequence of two main pathways: inflammation and hyperproliferation. PreviousThe role of inflammation in colorectal carcinoma is demonstrated by the fact that non-steroidal anti-inflammatory drugs cause a dramatic involution of adenomas, the precursors of carcinomas.Angiogenesis is a landmark of cancer development and metastasis. In additThe endothelium also secretes molecules that inhibit the immune response. As an example, factors involved in the protein C pathway are activated during inflammation and endothelial activation as well as leukocyte adhesion are both diminished. These anin vitro and in vivo.(Multiple evidence points to thrombospondin 1 (TSP1) as an inhibitor of angiogenesis and inflammation  in vivo.,14 In thThese studies strongly suggest an important role of TSP1 in the mechanisms developing IBD and cancer. The use of TSP1 derived peptides could represent an innovative strategy to attenuate chronic inflammation and delay colorectal carcinogenesis.The thrombospondins (TSP) include five calcium-binding extracellular glycoproteins.,14 TSP1 The functions of TSP1 have been extensively studied in many laboratories. TSP1 is involved in the formation and resolution of a fibrin clot and binds to many proteins and proteases. TSP1 intNumerous cell assays attribute functions to TSP1 in cell proliferation, apoptosis, angiogenesis inhibition, cell attachment and motility. Most of Of great importance is the interaction of TSP1 with the scavenger receptor CD36. CD36 is expressed in endothelium  and a variety of epithelial and stromal cells. Some of TSP1 is an inhibitor of EC proliferation.,30 HowevMore recently, TSP1 has been reported to block the pro-angiogenic effects of nitric oxide (NO) in endothelial and vascular smooth muscle cells. Low leveThe anti-angiogenic effects of TSP1 may also be CD36 independent, it suppresses the cell cycle, increasing p21 and unphosphorylated retinoblastoma (Rb) in EC. Many rec+ CD25+ T regulatory cells (Tregs). These cells help to maintain self-tolerance and induce a suppressive phenotype. induced a more severe colitis in TSP1−/−mice. Focal in−/− mice showed more angiogenesis than controls after only 2 days of DSS treatment.A robust angiogenesis was a common feature in DSS-induced colitis in both genotypes. This process was present on the acute lesions as well as in regenerative areas. TSP1Intense TSP1 expression was detected in the submucosa, among the inflammatory infiltrate in WT colons. Fibrin deposits and thrombus were also positive for TSP1. UpregulTSP1 is considered a cell adhesion protein. It has also been described that bacterial cellular adherence could be promoted by proteins such TSP1. In fact,−/− mice contained a significantly lower density of fecal coliforms than feces from control mice, exposed or not to DSS. Density of lactobacilli did not vary significantly among samples. These data suggest that the lack of TSP1 in the colonic epithelium may affect the distribution of Escherichia coli (E. coli), possibly altering interactions between the bacteria and adhesin ligands in the epithelium. The mechanisms involved and the possible replacement of commensal E. coli by pathogenic strains during chronic colitis warrant further investigation.TSP1 might promote bacterial colonization and inflammation. Studies in vivo and clinical studies have shown an inverse correlation between TSP1 expression, malignancy and poorer prognosis. develop spontaneous adenomas and carcinomas in the colon. as compared to lesions developed in ApcMin+/ TSP1−/− mice. These results indicate that higher proliferation and accelerated carcinogenesis are typical features in the lesions that arose in ApcMin+/ TSP1−/− mice whose even smaller adenomas showed elevated PCNA indexes. Importantly, apoptotic indexes were unmistakably diminished on ApcMin+/ TSP1−/− tumors, suggesting that TSP1 might also target tumor cells.Clinical studies have reported a decrease in TSP1 protein expression in human colorectal carcinomas.,61 When observed. Interesthe colon. These tuTGFβ1 decreases proliferation in colon carcinoma cells and a correlation between TSP1 and TGFβ1 expression has been found in human colon carcinomas.,65In a retrospective clinical study of more than 60 colorectal carcinomas, aberrant p53 expression was related with high expression of TSP1 and lymph node metastases. Patients−/− colons tissues and secreted fas ligand was diminished compared to the levels detected in control mice. Foci of dysplasia, p53 and βcatenin nuclear expression were more frequently seen in TSP1−/− mice. Although the grade of leukocyte infiltration was similar among the genotypes by the end of the study, higher levels of both, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were secreted by TSP1−/− colons. These data support the idea that angiogenesis is initiated by chronic inflammation but even if this condition is resolved, DNA damage and changes in the epithelium will perpetuate the angiogenesis cycle and trigger carcinogenesis.Using multiple and low doses of DSS, inflammation, apoptosis and key proteins in intestinal carcinogenesis such as p53 and βcatenin were evaluated in mice lacking TSP1. In that In recent studies, anti-angiogenic compounds have proved to be effective in IBD models. TSP1 in Min+/model, ABT-510 administration using osmotic mini-pumps significantly diminished the number of adenomas compared with mice controls.(ABT-510 has been found effective as therapy in brain tumors. In these studies, ABT-510 induced apoptosis in tumor cells and diminished angiogenesis and proliferation. In the Acontrols. Angiocidcontrols.,73In the DSS model of induced colitis, ABT-510 consistently diminished angiogenesis and bleeding in treated mice. This pepAn important mechanism could be the TSP1/NO interaction. Decreasing the levels of NO by TSP1 will also inhibit leukocyte activation and angiogenesis. In additIn conclusion, the relevance of TSP1 on colorectal inflammation and carcinogenesis has been reviewed emphasizing recent data. TSP1 seems to play an important pathological function in these processes acting by multiple pathways. The study of these signaling mechanisms could have a significant impact on future therapeutic strategies for IBD and colorectal cancer.


Cygnus spp.) has been the first indicator of the presence of H5N1 in various Asian and European countries; however their role in the geographic spread of the disease remains poorly understood. We marked 10 whooper swans (Cygnus cygnus) with GPS transmitters in northeastern Mongolia during autumn 2006 and tracked their migratory movements in relation to H5N1 outbreaks. The prevalence of H5N1 outbreaks among poultry in eastern Asia during 2003–2007 peaked during winter, concurrent with whooper swan movements into regions of high poultry density. However outbreaks involving poultry were detected year round, indicating disease perpetuation independent of migratory waterbird presence. In contrast, H5N1 outbreaks involving whooper swans, as well as other migratory waterbirds that succumbed to the disease in eastern Asia, tended to occur during seasons (late spring and summer) and in habitats  where their potential for contact with poultry is very low to nonexistent. Given what is known about the susceptibility of swans to H5N1, and on the basis of the chronology and rates of whooper swan migration movements, we conclude that although there is broad spatial overlap between whooper swan distributions and H5N1 outbreak locations in eastern Asia, the likelihood of direct transmission between these groups is extremely low. Thus, our data support the hypothesis that swans are best viewed as sentinel species, and moreover, that in eastern Asia, it is most likely that their infections occurred through contact with asymptomatic migratory hosts  at or near their breeding grounds.Evaluating the potential involvement of wild avifauna in the emergence of highly pathogenic avian influenza H5N1 (hereafter H5N1) requires detailed analyses of temporal and spatial relationships between wild bird movements and disease emergence. The death of wild swans ( Anser indicus) and whooper swans (Cygnus cygnus) at these outbreak locations have become references to determine the genetic origins of viruses isolated in subsequent outbreaks Highly pathogenic avian influenza H5N1 (hereafter H5N1) has been circulating in avian populations since 1996 after it first emerged in eastern Asia and spread to large parts of Asia, Europe, Africa, and the Middle East. Mortality events involving large numbers of migratory birds first gained attention during spring 2005 with outbreaks occurring at Qinghai Lake, People's Republic of China (hereafter China), where >6,000 wild birds are reported to have died Cygnus olor), which are not generally considered a migratory species, although whooper swans, which are migratory, also have been affected The migratory bird die-offs in the spring of 2005 at Qinghai Lake and in northern Mongolia during the summer of 2005 raised concerns about the propensity of wild birds to become infected in one location and then translocate H5N1 over large distances when they migrate Cygnus columbianus bewickii) infected with low pathogenicity avian influenza Experimental inoculation trials indicate that immunologically naïve swans are highly susceptible and die soon after exposure to H5N1 The breeding range of whooper swans in eastern Asia extends from the Russian tundra south to the desert zone wetlands of China and Mongolia A virus following standard testing protocols Tracheal and cloacal swab samples were taken from each swan and stored at 4°C before being frozen in liquid nitrogen (maximum 3 hours post collection). Initial field diagnostic tests were performed using Flu Detect Antigen Test strips . Samples were shipped to the Southeast Poultry Research Laboratory , where they were screened for influenza Transmitters were programmed to collect GPS locations at 2 -hr intervals throughout the 24 hr day and transmit signals to Argos satellites every 65 s for an 8 -hr on-cycle, followed by a 48 -hr off-cycle to facilitate solar recharging. Data were recovered from the Argos Data Collection and Location System  via receivers aboard polar-orbiting weather satellites. CLS calculated PTT location estimates that were derived from the perceived Doppler-effect shifts in transmission frequency during a satellite overpass. The accuracy of each location was rated by class. Class G locations indicated that the position was a GPS location with mean accuracy ±18.5 m. Conventional Argos location classes 0, 1, 2, and 3 indicated the location was derived from ≥4 transmissions, with accuracy >1000 m, 350–1000 m, 150–350 m, and ≤150 m, respectively. Location classes A (3 transmissions) and B (2 transmissions) are not assigned accuracy estimates by CLS, and location class Z indicates that no locations were obtained.http://alaska.usgs.gov/science/ biology/ spatial/). The filtering algorithm flags improbable locations based on user-defined distance and velocity thresholds. We used the algorithm to compile two datasets for analysis. The first dataset included one location per duty cycle, based on the highest-accuracy location class. Quality of signals was judged first on the basis of location class (G>3>2>1>0>A>B), and then by indices of residual frequency error. Our primary interest for compiling a dataset with one location per duty cycle was to evaluate broad-scale migration routes and movement chronology. For finer scale spatial analyses, we constructed a comprehensive dataset that included all GPS locations that were Class 1 or higher.We compiled and validated our Doppler-derived location data using the Argos Filter Algorithm . The MODIS/Terra Land Cover Classification contains multiple classification schemes, the primary of which identifies 17 classes defined by the International Geosphere-Biosphere Programme (IGBP). We used a University of Maryland modification of the IGBP scheme (Land Cover Type 2), which we compiled into 4 land cover categories: (1) natural vegetation, including barren or sparsely vegetated areas; (2) water; (3) cropland or cropland mosaic; and (4) urban.To relate swan movements to landscape features and potential risk factors associated with H5N1 transmission we integrated our telemetry locations with digital thematic maps using ArcGIS, Version 9.2 . For habitat features we used MODerate-resolution Imaging Spectroradiometer (MODIS)/Terra Land Cover Classification, distributed by the Land Processes Distributed Active Archive Center, located at the United States Geological Survey Center for Earth Resources Observation and Science (http://www.fao.org/geonetwork). Methodology and sources of the estimates are described in the FAO's Gridded Livestock of the World 2; Poultry density information was obtained from the United Nations Food and Agriculture Organization (FAO) via the Geonetwork  to extract the habitat type and interpolated poultry density at each H5N1 outbreak location. Similar analyses were performed to determine the proportion of PTT-marked whooper swan locations occurring in each habitat type in relation to the underlying poultry density. We then evaluated trends in the occupancy patterns of croplands and urban areas through the annual cycle of the PTT-marked swans. To evaluate temporal variation in the possibility for direct contact between whooper swans and poultry, we weighted each PTT-marked swan equally and calculated weekly average poultry exposure estimates throughout the annual cycle.FAO-OIE confirmed H5N1 outbreaks were summarized by location and date. To evaluate seasonality of outbreak events we challenged a null hypothesis of equal probability of an outbreak for wild and domestic birds for all dates by comparing likelihood ratios during the whooper swan breeding and post-breeding periods, fall migration, winter period, and spring migration. For spatial evaluation, we used the Intersect Point feature in Hawth's Tools  breeding and post-breeding period (1 May to 2 October), 2) fall or southern migration (3 October–18 November), 3) non-breeding or winter period (19 November–20 February), and 4) spring or northern migration (21 February–30 April). The dates and annual cycle stages we used were appropriate for our sample population, but we recognize differences in chronology for whooper swans migrating to other breeding areas, particularly more northerly breeding areas, as well as the possibility for interannual variation in migration timing.All ten swans were tracked during the post-breeding period . DepartuDeparture dates from post-breeding areas ranged from 24 September–8 October 2006, with a mean of 2 October . Four ofArrival dates on the wintering area ranged from 6 November–15 December and averaged 19 November . Swan s5Estimated departure dates for spring migration ranged from 20–21 February for the three swans  that still wore functioning PTTs at the end of the winter period . The lasAll seven of the whooper swans that were tracked during migration exhibited the potential for long distance dispersal in a short (<5 d) time period. The rate of travel was particularly rapid during fall. For example, s577 spent 58 d at different wetlands within 90 km of its capture site and then flew 430 km south across the Gobi Desert during an 18 hr period (23.9 km/hr: Larus ichthyaetus), brown-headed gulls , ruddy shelducks (Tadorna ferruginea), great cormorants , goosander (Mergus merganser), Eurasian wigeon (Anas penelope), black-necked crane (Grus nigricollis), and several unspecified species of grebe (Podiceps spp.), egret or heron (family Ardeidae), and “wild ducks” not identified to species (family Anatidae).We included 95 FAO-OIE confirmed H5N1 outbreak events involving wild and domestic birds in eastern Asia in our analyses. These included 79 cases involving poultry, eight in which wild waterbirds were affected, and eight that were restricted to non-waterbird species. A majority of cases involving poultry did not specify whether Galliformes  or Anseriformes (e.g. domestic ducks and geese) were the primary species affected. Therefore, we evaluated poultry as a single group rather than comparing these potentially different categories. The incidents involving waterbirds affected several species, including: whooper swans, bar-headed geese, Pallas's (great black-headed) gulls  were affected. It should be noted that detection of wild bird mortality events is more likely to occur when large numbers of individuals perish at once, when there are fewer scavengers, in habitats that are monitored as wildlife parks, protected wetlands or RAMSAR sites, in habitats frequented by tourist or birders, or in locations with greater human densities. As well, large white birds such as whooper swans are more likely to be observed dead in the environment than small passerine species. Along the migration routes of whooper swans in this study, there is some passive surveillance, annual distribution and population counts are performed, and it is more likely that a mortality event would be detected in the southern part of the migration pathway as this coincides with greater human densities and important birds and conservation habitats regularly monitored.Two of the eight outbreaks involving mostly waterbirds also affected small numbers on non-waterbird species. Also, official reports of H5N1 outbreaks involving non-waterbird species were somewhat imprecise with regard to species and indicated that crows and magpies (family Corvidae) and hawks (family Accipitridae), including one mountain hawk eagle  H5N1 outbreaks involving waterbirds occurred during the whooper swan breeding period and likelihood ratio tests indicated an over representation of occurrence during this period relative to the number of exposure days  and cropland (N = 1) habitat types, where poultry densities averaged 337 (±114 SE) head/km2 and ranged from 0–874 head/km2. The outbreaks involving poultry occurred in a wider variety of habitats, with most categorized as urban (N = 38) or cropland (N = 29), but several were associated with areas of natural vegetation (N = 12). Estimated poultry densities at the location of outbreaks in poultry averaged 1020±118 head/km2 and ranged from 0–6070 head/km2.With respect to habitats, all eight of the outbreak events involving waterbirds occurred in natural vegetation (grasslands), where poultry densities were estimated to be <1 head/kmPTT-marked whooper swans almost exclusively occupied habitats comprised of natural vegetation types or water (98.4%) during the breeding and post-breeding periods, when most location estimates were characterized as grassland (81.7%). During fall migration, predominant occupancy continued to be in natural areas (48.9%), but there was an increasing diversity of habitat occupancy, including water (26.8%) and croplands (23.9%). For the winter period as a whole, habitat categories were spread evenly among natural vegetation types (25.5%), water (29.4%), croplands (21.1%), and urban (24.0%) areas. However, there was also a pattern wherein use of croplands and urban areas tended to increase from the early to late winter, eventually constituting >90% of all locations just prior to spring migration. During the early stages of spring migration use of croplands was high suggesting that whooper swans likely moved into agricultural fields to forage before departing for breeding areas.2 during the post-breeding period , and then decreased to 0–4 head/km2 during the breeding season.Not surprisingly, potential interactions between PTT-marked whooper swans and poultry mirrored habitat use patterns. Estimates for the mean poultry density at whooper swan locations ranged from 0–5 head/kmg period . EstimatThe correlation between potential exposure to poultry and the timing of H5N1 incidents in waterbirds in eastern Asia was very low . None ofAmong poultry, most outbreaks occurred during winter when migratory waterbirds were present. However, there was a strong tendency for outbreaks affecting poultry to occur in urban and cropland areas with high poultry density, rather than in areas with abundant natural vegetation and less poultry, the more frequently used wintering habitat of whooper swans and other waterfowl. Moreover, the frequency of outbreaks involving poultry did not increase during late winter and the early spring migration period when whooper swans were observed to spend more time in urban and cropland habitats where there is a higher potential exposure to poultry.Understanding the role that migratory waterbirds birds play in the ecology and transmission of H5N1 requires the integration of habitat data, seasonal movement chronology and routes, domestic poultry production information, dates and locations of H5N1 outbreak events, and analyses at both temporal and spatial levels. For many regions in which H5N1 is endemic and migratory waterbirds are suspected of disease introduction or dispersal, such basic data are often lacking. We predicted that if highly susceptible migratory waterbirds like whooper swans contracted H5N1 directly from poultry, then an association should be apparent with respect to the timing and location of outbreaks. This was clearly not the case.Although outbreaks of H5N1 involving poultry were numerous in East Asian Flyway portions of China and the Korean Peninsula from 2003–2007, very few cases involving non-waterbird species were reported in close association with poultry outbreaks although non-waterbird outbreaks did occur amongst a background of moderate poultry density . This is2) and the timing was, in nearly all instances, several weeks to months after the initial estimated departure from wintering areas where poultry density was high. In this respect, the situation in eastern Asia differs from that documented in Europe, where numerous swan mortalities were detected in proximity to locations where poultry are present Additionally, wild bird outbreak sites were characterized by grassland habitats where poultry density was very low , mallard (Anas platyrhynchos), common teal (A. crecca), northern pintail (A. acuta), and Eurasian wigeon (A. penelope) are the numerically dominant duck species migrating between breeding areas in Siberia and Mongolia and wintering areas in Japan, China, Southeast Asia, and India In the East-Asian Australasian and Central Asian flyways, ruddy shelduck , and although less wetland associated, domestic geese populations exceed 300 million birds 10 EID50/mL) for up to 4 d before the onset of clinical signs From a disease spread and transmission standpoint, laboratory studies have demonstrated that whooper swans experimentally inoculated with H5N1 can shed significant virus (>4log


The medium plating density was found ideal in generating optimum number of progenitors and also yielded about 80% mature neurons in a serum free culture set up barring any exogenous inducers. We could also demarcate and quantify the neural stem cells/progenitors among the heterogeneous cell population of differentiating ES cells using nestin intron II driven EGFP expression as a tool. The one week post-plating was determined to be the critical time window for optimum neural progenitor generation from ES cells that helped us further in purifying these cells and in demonstrating their proliferation and multipotent differentiation potential. Seeding cells at varying densities, we could decipher an interesting paradoxical scenario that interlinked both commitment and maturation with the initial plating density having a vital influence on neuronal maturation but not specification and the secretory factors were apparently playing a key role during this process. Thus it was comprehended that, the neural specification was a default process independent of exogenous factors and cellular interaction. Conversely, a defined number of cells at the specification stage itself seemed critical to provide an auto-/paracrine means of signaling threshold for the maturation process to materialize.The neural induction has remained a debatable issue pertaining to whether it is a mere default process or it involves precise instructive cues. We have chosen the embryonic stem (ES) cell model to address this issue. In a devised monoculture strategy, the cell–cell interaction availed through optimum cell plating density could define the niche for the attainment of efficient  Neurogenesis is a complex cyto-architectural enigma that involves intricate organizational events during embryogenesis and requires precision to the minutest extent. In fact, it involves an intelligent interplay of different developmental factors regulated intrinsically by the developing embryo and/or by extrinsic influencing factors. The paradox being whether the neurogenesis represents a default state in vitro without any exogenous inducers.Cell–cell communication is an important phenomenon both during development and differentiation. In fact, cell aggregation promotes the intercellular communication during the differentiation of ES cells and thereby helps transducing the signals and sets up the milieu for lineage commitment and differentiation. The fine tuning of these event cascades is brought about by both cell intrinsic and extrinsic cues. Studies till date have focused on various factors We have carried out the neural induction in a 2D culture set up to understand its default state. Cells were differentiated successfully into neural population even in the absence of any extrinsic factors. In fact, we could demarcate the neural progenitors using the nestin enhancer mediated cell trapping approach and could quantify the same that remained identical irrespective of the plating density. However, the differentiation efficiency depended on the initial cell seeding density. We could generate about 80% neurons with a better efficiency seen at medium plating density compared to that at very low or high ones. Interestingly, the transcripts levels of certain secretory factors especially Wnt corresponded well with the cell density thus implying that, the neural induction was influenced by the concentration/dilution of factors not from the medium but through the auto-/paracrine effect of the cells themselves. Our study in fact, revealed the importance of the initial seeding density that had a direct bearing and more pronounced effect on the maturation than the initiation stage of neurogenesis; thus hinting at the maturation and hence the overall neural induction process to be an instructive event, while the commitment alone represented the default state.An adherent monolayer culture strategy was devised for neural induction from ES cells using D3 and h-nestin EGFP transfected ES cell clones (nes-EGFP). The single dispersed cells when cultured in differentiation medium (DM), generated colonies in a two-dimensional fashion due to their self renewal characteristic. On day 2 (d2), the serum containing medium was substituted with serum replacement medium (KO) and this resulted in initial proliferation and a gradual switching to differentiation. The supplementation of RA (KR) expedited this process further. The colonies in fact, grew in diameter and formed cell clusters often resembling the plated EBs . A numbe+ intricate neuronal mosaics (∼80%)  . A similal cells  and EGFPure ones . As seenure ones  followedure ones .Interestingly, migratory single cells in many areas in the periphery of the clusters also exhibited smaller processes extending from the cell body . This in+ neural rosette like structures were quite prominent within one week of differentiation  with CNTF treatment. However, the overall neurogenesis remained comparable  Map2mparable  among th+ neurons started appearing at higher densities but not in 2 K plated cells thus implying a probable modulatory role of Wnt on density dependent neuronal differentiation.The variation in the density dependent neural maturation from naïve ES cells brought forth to an interesting paradigm implicating the influence of certain intrinsic secretory regulatory molecules on neural commitment and subsequent differentiation. Accordingly, we investigated the expression status of some of the key players such as BMP4, FGF4, noggin and Wnt to assess their contributory role in modulating neurogenesis. The RT-PCR was carried out on RNA samples isolated at different time periods both prior to and during differentiation from cells plated at varying densities. As seen in + neural progenitors by flowcytometry. Our data revealed an initial increase in the EGFP expression attaining the peak during 6–9 dpp followed by its down-regulation on subsequent days  with RA exposure at the respective plating densities  on the gelatin coated culture dishes. FACS analysis was also carried out on these cells to determine the density dependency on neural progenitor generation and maturation. The number of EGFP expressing neural progenitors was comparable (M2: 20–23%) at one week irrespective of the plating densities except for 100 K (15+/−1.361%) indicating that the seeding density might have a minor influence on neural commitment . Certainensities . Moreove+). A comparable number of Map2+ cells were detected in both densities with marginal increase (5–10%) in KR medium at one week time period that became almost identical (∼80%) by two weeks post-plating with or without RA. Taken together, the medium density was found ideal for both progenitor generation and neural maturation associated with extensive neurite processes thus suggesting a vital influence of initial seeding density on neurogenic efficacy and the medium being serum free could still support this.Further to monitor the differentiation profile, concurrent investigations were carried out either by the flowcytometry quantification or by selecting 5–10 random fields and counting various neural populations relative to the DAPI positive total number of cells in the medium and high density plated cultures. While, 25–30% of mature neurons were detected by first week (9 dpp), it ranged from 70–80% or more (76.8+/−6.9%) on second week. Similarly, the GFAP population also showed an increase from 3–5% to ∼20% by second week of differentiation. Contrary to this, there was a decline in the A2B5 population from first week (∼64%) to second (8–10%). Our differentiation strategy also yielded ∼25% dopaminergic neurons . The results from the influence of density on the differentiating cells thus directly portrayed that, the intrinsic factors were probably secreted and regulated the differentiation process in an auto-/paracrine fashion. The lack of appreciable influence of exogenous growth factors on neural differentiation indeed substantiated this further.The ES cell derived neurogenesis would include both the committed neural stem cells/progenitors and the differentiated progenies. This demanded the requirement of properly demarcating and quantifying the neural progenitor population from the differentiating neurons using nestin, the neuroepithelial stem cell marker + neurons were first detected. Considering the density dependent positive influence of Wnt on neuronal differentiation A number of factors have in fact been implicated in modulating neurogenesis RA is one of the key players that exerts pleiotropic influence during development + neural committed population that resided around 4 days of initiation of differentiation while the effective time window for nestin+ neural progenitors was about 6–9 dpp suggesting the existence of developmental hierarchy during early neurogenesis. In fact, Sox1+ stage cells could be split and differentiated upon re-plating without affecting their survival (unpublished data). Earlier Conti et al, in vitro to different lineages other than the neural and the conditions opted till date might not be ideal for the same. Never-the-less, incase the counter view on FGF mediated neural specification In fact, the neural induction has remained a debatable issue pertaining to whether it is a mere default process or it involves precise instructive cues. The present investigation has highlighted a combinatorial cell intrinsic and neighboring effect that influenced the cells to undergo neural specification and differentiation from ES cells. While the neural commitment was density independent, the maturation efficiency was dictated by the initial cell plating density thus questioning how appropriate could it be to regard neurogenesis that would include both commitment and maturation; as a default transition in the absence of inhibitory signals The murine ES cells, line D3 (ATCC) were maintained routinely on STO fibroblasts and cultured without fibroblast prior to differentiation as described Following similar strategy of monoculture, the in-house established and partially characterized human ES cell line/outgrowth, RH1; derived from a discarded abnormal embryo with three pronuclei  on 35 mm dishes (9.8 cm2) and were monitored for their qualitative and quantitative differences. The growth characteristics and the differentiation were monitored under the microscope  and were characterized. Various growth factors were added on d2 to the medium density culture in KR medium at the described concentrations (see figure legend), to assess their influence on neural differentiation. The RNA was isolated from cells during different stages of differentiation using Trizol reagent (Invitrogen) and was subjected to reverse transcription PCR (RT-PCR) To discern the influence of cell density on neural differentiation, the D3 ES cells and one of the representative nes-EGFP clones (17) were plated at varying densities; [low: 1–2×10+/GFAP+differentiated neuronal population among the Dapi+ cells during ES cells differentiation from a minimum of three experiments.The cellular phenotypes attained during ES cells differentiation into neural lineage in a monolayer culture were discerned by immunocytochemistry using antibodies against neurons and glia following the standard protocol The EGFP expression in nes-EGFP clones was analyzed at various developmental stages using a FACSVantage flowcytometer (Becton Dickinson) equipped with a 488 nm argon-ion-laser (150 mW) as described To assess whether the ES cells would involve an intervening neural stem cell stage that would have self-renewing ability, we dispersed the differentiating ES cells and re-plated those on gelatin coated dishes. The dispersion was carried out either enzymatically using Trypsin-EDTA or mechanically during early time points (3 dpp and 8 dpp). While the mechanical isolation yielded cells in clumps, the enzymatic dispersion gave single cells. Subsequently, the cells were cultured in either DM or KO medium.To purify the neural progenitors, the plated cells at d6-8 during differentiation were enzymatically dispersed and were allowed to revive their membrane integrity by incubating those in DM for 2 hours in the CO2 incubator. The cells were washed subsequently with PBS and subjected to MACS to purify the neural progenitors following the specified protocol (Miltenyi Biotec). In brief, the cells were suspended in 500 l of pre-chilled MACS buffer  and were incubated with either of the following primary antibodies  for 30 minutes at 4°C. The unbound antibodies were removed by washing thoroughly and the cells were suspended in 80 l MACS buffer. The cell suspension was added with magnetic beads conjugated secondary antibody slurry (20 l) and was incubated for further 20 minutes at 4°C. The washing step was repeated and finally the cells were suspended in 500 l of MACS buffer followed by loading onto a separation column placed under the magnetic field that was pre-equilibrated with MACS buffer. The flow-through was collected (−ve fraction) and the column was removed from the magnetic field after washing with the MACS buffer. Subsequently, the bound cells (+ve fraction) were eluted with MACS buffer (1 ml) using the plunger. The cells from both the +ve and the –ve fractions were pellet down and the cell pellets were suspended in KO medium. The cells were further maintained in DM or KO medium to assess their growth and differentiation characteristics.Figure S1The neural differentiation in ES cells depended on the initial plating density of cells. The extent of cell growth seen in nes-EGFP cells at d2 with 2 K (A), 60 k (B) and 130 K (C) plating densities. The neural differentiation in cells at 2 K plating density was observed occasionally in KO (D) or KR (E) medium at second week post-plating (d2+10) and remained confined to cell dense areas only. However, cells (D3 ES) plated at higher density (60 K) exhibited extensive neural differentiation with well sprouted neural processes by second week (F–H). Single cells migrating away from the cell cluster also retained neurogenic differentiation potential in those . Scale: 30 µM(0.95 MB TIF)Click here for additional data file.Figure S2+ neural progenitors showed surface expression (7 dpp) of NCAM  and A2B5  when cultured in KO or KR medium respectively. Interestingly, both these markers were expressed as early as 5 dpp as seen in D3 ES cells . Scale: 40 µM.The ES cells during differentiation showed the neuronal and neuro-glial progenitor markers, NCAM and A2B5 respectively. The EGFP(0.77 MB TIF)Click here for additional data file.Figure S3in vitro. The D3 ES cells cultured in KR medium without (A) or with bFGF (10 ng/ml) (B), NGF (10 ng/ml) (C), BDNF (10 ng/ml) (D), CNTF (2 ng/ml) (E) and FGF8 (100 ng/ml) (F) respectively showed similar neural differentiation pattern at 2 weeks (14 dpp). The inset represents the TuJ1+ neurons in each. (G): The ES cells cultured in KR medium when dispersed on d3 and re-plated on DM followed by changing to KO on d2 of re-plating displayed extensive neural differentiation with well sprouted processes as seen on d11 of re-plating. Scale: 30 µM.Exogenous supplementation of growth factors into the medium did not have pronounced effect on neural differentiation from ES cells (0.91 MB TIF)Click here for additional data file.


Activating mutations within the epidermal growth factor (EGFR) tyrosine kinase domain identify non-small cell lung cancer patients with improved clinical response to tyrosine kinase inhibitor therapy. Recently, we identified two EGFR mutations in a cohort of 25 salivary gland carcinomas (SGCs) by screening the tumour samples for the both most common hotspot mutations in exons 19 and 21 by allele-specific PCR. Here, we present a comprehensive sequencing analysis of the entire critical EGFR tyrosine kinase domain in 65 SGC of the main histopathological types. We found EGFR mutations in the tyrosine kinase domain to be a rare event in SGCs. No additional mutations other than the two known exon 19 deletions (c.2235_2249del15) in a mucoepidermoid carcinoma and an adenoid cystic carcinoma have been detected. Other putative predictive markers for EGFR-targeted therapy in SGCs might be relevant and should be investigated. Cetuximab (erbitux) has been tested in two phase II studies in patients with recurrent and/or metastatic SGCs including mainly ACC, a cancer with generally poor outcome. In 50% of patients, clinical benefit  could be achieved , mucoepidermoid carcinoma (n=10), myoepithelial carcinoma (n=8), acinic cell carcinoma (n=12) and adenocarcinoma ex pleomorphic adenoma (n=10).Surgically removed formalin-fixed tumour samples were obtained from 65 patients  treated with the histopathological diagnosis of an SGC according to the WHO classification .Epidermal growth factor exons 18, 19, 20 and 21 were amplified by nested PCR using primers described previously  with a heterozygous exon 19 deletion mutation. The two exon 19 E746_A750 deletions (both of them were variant c.2235_2249del15) were identified in a mucoepidermoid carcinoma of the left parotid gland grade 2 , and in an ACC of solid type of the right parotid gland . The female patient with the mucoepidermoid carcinoma was treated with surgery and postoperative radiation, the male patient with ACC by surgical tumour resection alone. Both patients had a favourable course of their cancer disease with no manifestation of recurrences (follow-up time 24 months).In exon 20, an SNP c.2607 G>A (p.Q787Q) was detected in 29 out of 65 tumour samples .Somatic mutations in the EGFR kinase domain are found in a subset of lung adenocarcinomas and are associated with sensitivity to TKI . Three kinase domain mutations are associated with drug resistance: D761Y in exon 19, T790M in exon 20 and an exon 20 insertion (D770N771insNPG). The exon 19 deletions and L858R in exon 21 represent 85–90% of EGFR mutations in non-small cell lung cancer. In two recent trials in advanced non-small cell lung cancer, therapy with gefitinib administered in a genotype-directed fashion to patients harbouring EGFR mutations resulted in very favourable clinical outcomes with good tolerance (http://www.cityofhope.org/cmdl/egfr%5Fdb/).Given the comparable embryonal tissue origin between lung adenocarcinoma and SGCs and the potential for genotype-directed trials of EGFR inhibitors, we undertook a comprehensive mutational analysis of EGFR in SGCs. We found EGFR mutations in the tyrosine kinase domain to be a rare event in SGCs of Caucasian origin. Whereas approximately 10–30% of lung carcinoma contain EGFR mutations , they seBoth of the mutations detected in our cohort were found in histopathological types (mucoepidermoid carcinoma and ACC), which also occur in the lung. In a recently published study, in two out of five (40%) of mucoepidermoid carcinoma of lung , the L858R mutation was detected and all five patients well responded to TKI therapy independent of the mutation status  who achieved stable disease after cetuximab treatment in the phase II trials and the apparently low incidence of drug-sensitising EGFR mutations, it would appear that also other biological factors than EGFR tyrosine kinase mutations might influence the therapy response. The underlying mechanisms of response to targeted therapies are complex and still not fully elucidated. Investigating those mechanisms and the mutation status of proteins in the cascade downstream of EGFR might identify further predictive markers for EGFR-targeted therapy in SGCs and would allow the selection of patients for a successful treatment.