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Mental fatigue, with decreased concentration capacity, is common in neuroinflammatory and neurodegenerative diseases, often appearing prior to other major mental or physical neurological symptoms. Mental fatigue also makes rehabilitation more difficult after a stroke, brain trauma, meningitis or encephalitis. As increased levels of proinflammatory cytokines are reported in these disorders, we wanted to explore whether or not proinflammatory cytokines could induce mental fatigue, and if so, by what mechanisms.+) in humans suffering from mental fatigue. At present, this is not possible for technical reasons. Therefore, more knowledge of neuronal-glial signaling in in vitro systems and animal experiments is important.It is well known that proinflammatory cytokines are increased in major depression, "sickness behavior" and sleep deprivation, which are all disorders associated with mental fatigue. Furthermore, an influence by specific proinflammatory cytokines, such as interleukin (IL)-1, on learning and memory capacities has been observed in several experimental systems. As glutamate signaling is crucial for information intake and processing within the brain, and due to the pivotal role for glutamate in brain metabolism, dynamic alterations in glutamate transmission could be of pathophysiological importance in mental fatigue. Based on this literature and observations from our own laboratory and others on the role of astroglial cells in the fine-tuning of glutamate neurotransmission we present the hypothesis that the proinflammatory cytokines tumor necrosis factor-α, IL-1β and IL-6 could be involved in the pathophysiology of mental fatigue through their ability to attenuate the astroglial clearance of extracellular glutamate, their disintegration of the blood brain barrier, and effects on astroglial metabolism and metabolic supply for the neurons, thereby attenuating glutamate transmission. To test whether our hypothesis is valid or not, brain imaging techniques should be applied with the ability to register, over time and with increasing cognitive loading, the extracellular concentrations of glutamate and potassium (KIn summary, we provide a hypothetic explanation for a general neurobiological mechanism, at the cellular level, behind one of our most common symptoms during neuroinflammation and other long-term disorders of brain function. Understanding pathophysiological mechanisms of mental fatigue could result in better treatment. Diagnostic and Statistical Manual of Mental Disorders, 4th edition ec and neurodegeneration characteristic of excitotoxicity, while the loss of neuronal glutamate transporter does not elevate [Glu]ec smission and also neurons . In fact neurons . Even mo [Glu]ec .A large number of factors have been shown to affect the activity and expression of the glutamate transporters GLT-1 and GLAST. For example, GLT-1 is stimulated by phosphorylation by protein kinase C (PKC), while GLAST is inhibited by PKC at a non-PKC consensus site . The synProinflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 have since long been known to impair astroglial glutamate uptake even if the mechanisms are not fully understood. The inhibitory function of TNF-α was established as early as the 1990s, when TNF-α was shown to inhibit astroglial glutamate uptake reported2+) signaling between BBB endothelial cells by reducing gap junction coupling and inhibiting triggered ATP release reduced . There i reduced , with lesyncytia -33, and syncytia could insyncytia ,36. Thissyncytia and leadsyncytia .ec would also lead to astroglial cell swelling, with a resulting decrease in the extracellular space volume, and locally further increased [Glu]ec [+]ec [+]ec levels have been shown in experimental systems to inhibit glutamate release [Increased [Glu] [Glu]ec -42. The ec [+]ec ,44. Even release .Recent data indicate a dynamic and fine-tuning regulation of the glutamatergic transmission. One mechanism by which neurons regulate excitatory transmission is by altering the number and composition of glutamate receptors at the postsynaptic plasma membrane. This has been shown for the NMDA receptor in experimental systems and could have prominent importance for dynamic processes as learning and memory . Of greaFurthermore, in states of decreased astroglial glutamate uptake capacity, even astroglial glucose uptake, and consequently the supply of metabolic substrates to the neurons, has been reported to decrease -50 and tec in the basal frontal cortex could lead to a decrease in the noradrenaline and serotonin (5-HT) release in the cerebral cortex, which would also decrease glucogenolysis There is an extensive literature on inflammatory response with microglial activation and the production of proinflammatory cytokines in neuroinflammatory/infectious and neurodegenerative diseases as well as in stroke and trauma ,54. The Several groups have also described enhanced production of proinflammatory cytokines in major depression [see ] and sicIn states of anxiety and stress, often experienced as secondary to mental fatigue, increased glucocorticoid levels have been demonstrated. Interestingly, long-term increases in glucocorticoids have been demonstrated to result in the production of both TNF-α and IL-1β .In the search for pathophysiological correlates to fatigue in MS, Roelcke and co-workers demonstrec. The initial consequence would be slightly increased [Glu]ec, with less precision in glutamate transmission. This would disintegrate the "filter", which normally selects information and prevents it from reaching the cerebral cortex. We can take the sound from a low-frequency fan as an example. This sound is normally sorted out after hearing it for a while. If this sound is handled with less precision by auditory recognition systems, it will continually be recognized by brain centers as "new" information and be processed in the cerebral cortex as long as the sound is on. The "filter" that normally restrains already recognized information from reaching higher brain centers, has been "opened". From a physiological point of view, it seems appropriate that the individual, and not the brain at the synaptic level, should determine which information should reach, and be processed by, the cerebral cortex. The decreased attention, increased loudness and light sensitivity, and irritability could be physiological ways of avoiding overstimulation of higher cortical centers. In case the individuals cannot protect themselves from too much sensory stimulation, the filter's opening leads to overstimulation of the cerebral cortex. Here, the final shutdown of the glutamate transmission could be one mechanism underlying mental exhaustion could be studied in animal models. Effects of astroglial dysfunction with regard to glutamate transport capacity would be of special interest. Even clinical studies with different treatment strategies could be important in casting some light on the accuracy of the hypothesis. Of utmost importance in all such studies would be test batteries making it possible to objectify and even quantify the degree of mental fatigue.It is not possible at present to ultimately prove whether or not the altered neuronal-glial interactions in glutamatergic transmission induced by proinflammatory cytokines could serve as a model to explain cellular mechanisms underlying mental fatigue. Brain imaging techniques able to determine and follow [Glu]Normally, mental fatigue and the associated symptoms disappear when the brain dysfunction is over. In some patients, the symptoms persist. We have at present no explanation for this, but if our hypothesis is correct, there could be a genetic failure preventing astroglial glutamate transporters from upregulating. Another explanation for why the symptoms persist could be that the pathological stimulation by brain plasticity creates new neuronal networks ,36.Providing information about mental fatigue, its cause and the prognosis, is of utmost importance for breaking the vicious circle, which comes with the risk for secondary anxiety and depression. Furthermore, it is important for the patient to imagine and learn how much sensory stimulation they can tolerate prior to feeling too exhausted. Due to recent results on changes in cell signaling and neuronal plasticity ,36, it mADHD attention deficit hyperactivity disorderAMPA alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionateATP adenosine triphosphateBBB blood brain barrier2+ calciumCaEc extracellularGLAST glutamate aspartate transporterGLT-1 glutamate transporter-1ec extracellular glutamate concentration[Glu]5-HT 5-hydroxytryptamineth revisionICD-10 International Classification of Diseases, 10IL-1/-6 interleukin-1/-6+ potassiumK+]ec extracellular potassium concentration[KLC locus coeruleusLTP long term potentialMS multiple sclerosis+ sodiumNaNA noradrenalineNFκB nuclear transcription factor kappaBNMDA N-methyl-D-aspartateNO nitric oxidePACAP pituitary adenylate cyclase-activating polypeptidePI3K phosphatidylinositol-3-kinasePKC protein kinase CSiv mac simian immunodeficiency virus macaquesTNF-α tumor necrosis factor alphaThe author(s) declare that they have no competing interests.Equal contributions by both authors.

Xenopus laevis.Rho GTPases and their downstream effector proteins regulate a diverse array of cellular processes during embryonic development, including reorganization of cytoskeletal architecture, cell adhesion, and transcription. Changes in the activation state of Rho GTPases are converted into changes in cellular behavior by a diversity of effector proteins, which are activated in response to changes in the GTP binding state of Rho GTPases. In this study we characterize the expression and function of one such effector, XCEP2, that is present during gastrulation stages in Xenopus laevis, a gene encoding a Rho GTPase effector protein was isolated, and found to be highly homologous, but not identical, to a Xenopus sequence previously submitted to the Genbank database. These two gene sequences are likely pseudoalleles. XCEP2 mRNA is expressed at constant levels until mid- to late- gastrula stages, and then strongly down-regulated at late gastrula/early neurula stages. Injection of antisense morpholino oligonucleotides directed at one or both pseudoalleles resulted in a significant delay in blastopore closure and interfered with normal embryonic elongation, suggesting a role for XCEP2 in regulating gastrulation movements. The morpholino antisense effect could be rescued by co-injection with a morpholino-insensitive version of the XCEP2 mRNA. Antisense morpholino oligonucleotides were found to have no effect on mesodermal induction, suggesting that the observed effects were due to changes in the behavior of involuting cells, rather than alterations in their identity. XCEP2 antisense morpholino oligonucleotides were also observed to cause complete disaggregation of cells composing animal cap explants, suggesting a specific role of XCEP2 in maintenance or regulation of cell-cell adhesion in early embryos. This loss of cell adhesion could be rescued by co-injection with a morpholino-insensitive version of the XCEP2 mRNA.In a search for genes whose expression is regulated during early stages of embryonic development in Xenopus laevis. XCEP2 is involved in maintenance of cell-cell adhesion, and as such may constitute a regulatory component that could help to balance the need for tissue integrity and plasticity during the dynamic cellular rearrangements of gastrulation.XCEP2 appears to be an essential component in the early developmental program in Xenopus laevis coordinated changes in cell shape and motility guide the initial formation of the dorsal lip, the initial site of mesodermal involution [Vertebrate gastrulation depends upon exquisite regulation of diverse morphogenetic processes including changes in cell shape, cellular adhesion and migration. For example, in volution ,2. Latervolution . Convergvolution . The cooXenopus laevis and zebrafish, non-canonical Wnt signaling on the dorsal side of developing embryos directly initiates the cellular rearrangements and migration that contribute to convergent extension of involuting mesoderm [Xenopus, resulting in severe gastrulation defects [Drosophila [The molecular basis for initiating and regulating the complex cellular movements of vertebrate gastrulation is only beginning to be discerned. Recent work has shown Wnt proteins as key upstream regulators in gastrulation movements. In mesoderm ,6. Othermesoderm ,8. Inactosophila , vertebrosophila ,11, and osophila ,12-14.Xenopus [Experiments perturbing the activity of Rho GTPases have suggested important regulatory roles for these proteins during gastrulation in Xenopus ,10,12,14Xenopus , cell adXenopus , cell miXenopus , vesiculXenopus and signXenopus -21. ReguXenopus, when the integrity of the elongating mesodermal sheet must be maintained at the same time that major cellular rearrangements are occuring. Downregulation of C-cadherin-mediated cellular adhesion has been observed to correlate with convergent extension movements of mesodermal cells [Xenopus embryos suggests that analogous upstream regulatory mechanisms may operate during Xenopus gastrulation [Regulation of cell-cell adhesion is thought to be of particular importance during gastrulation movements in al cells . This chal cells . In addial cells -27. Recerulation .Xenopus gene encoding a member of the recently characterized CEP/BORG family of RhoGTPase effector proteins [In this study, the embryonic expression pattern and function of a proteins ,30 is exproteins ,30, and proteins ,32.Xenopus Cdc42 Effector Protein 2, hereafter referred to as XCEP2, is shown here to be developmentally regulated during early embryonic stages, with diffuse expression of mRNA in the animal region that diminishes during late gastrula to neurula stages. Experiments employing morpholino-mediated inhibition of translation suggest that expression of this protein is essential for normal gastrulation movements, and that its activity is required for maintenance of cell-cell adhesion between blastomeres of animal cap explants.Xenopus embryo. The sequence of one of these fragments was found to be highly homologous to the translational start site region of database cDNA sequences encoding Cdc42 effector protein 2. A full length cDNA corresponding to our isolated fragment was amplified using 3' RACE PCR, cloned and sequenced. An alignment of the predicted amino acid sequence for our isolated full length cDNA sequence with human [Xenopus (accession number BC045241) CEP2/BORG1 genes from Genbank revealed significant homology . These differences were found to occur almost exclusively in regions of the protein outside the three conserved domains that define the CEP family of effector proteins. Given the allotetraploid genetic ancestry of Xenopus laevis, and the degree of similarity between the sequences, it is likely that these two sequences constitute pseudoalleles of the same gene. We choose to refer to our isolated pseudoallele as XCEP2A and the database allele as XCEP2B.A differential display approach was employed to reveal genes that are developmentally regulated during the transition from blastula to neurula. This screen allowed for the isolation of numerous cDNA fragments corresponding to differentially regulated mRNAs present in the th human and XenoIn situ hybridization revealed that XCEP2A mRNA is distributed uniformly over the animal hemisphere of the embryo at blastula and early gastrula stages , maintenance of this expression level through mid-gastrula stages, with a downregulation of mRNA expression starting at stage 10.5 and becoming more marked by stage 12.5 , MO1 antisense oligonucleotides pseudoalleles elicited significant gastrulation delay as compared to an identical dose of control morpholino oligonucleotide . Given this observation, it is possible that injection of either antisense XCEP2 morpholino significantly reduces the endogenous expression of both pseudoalleles. In addition to the dosage issue in the combined condition (noted above), cross-inhibition of this type could contribute to the observed lack of additive effects in the combined MO1 + MO2 condition.The antisense morpholino oligonucleotides used in these studies were found to elicit specific translational inhibition of XCEP2. Injection of antisense morpholino against our isolated pseudoallele resulted in largescale inhibition of translation of a myc-tagged fusion protein from co-injected mRNA . The absence of effects may not be unexpected, given current models of RhoGTPase effector protein activation. RhoGTPase effector proteins are activated by direct binding to GTP-bound RhoGTPases. As such, the number of activated RhoGTPase molecules may constrain the number of effector proteins bound and activated, regardless of the heightened expression levels of the effector protein.Xenopus embryos suggests that XCEP2 may play a direct role in directing the morphogenetic movements of gastrulation. This interpretation would be consistent with the limited characterization that have thus far been conducted on functional properties of this class of proteins [The observed effects of antisense XCEP2 morpholinos in proteins ,30. HoweIn order to assess the mechanism by which XCEP2 influences gastrulation, the effects of XCEP2 morpholinos on cultured animal cap explants were evaluated. While the initial intent of these experiments was to discern whether XCEP2 morpholinos prevented normal activin-induced convergent extension of animal caps, it soon became clear that morpholino-treated explants had a more fundamental deficiency. Within a 24 hour period, animal cap explants derived from XCEP2 antisense morpholino (MO1) -injected embryos were found to completely lose their integrity . CurXenopus Cdc42 effector protein 2 (XCEP2) in gastrulation movements. XCEP2 is a member of the recently characterized CEP family of Rho GTPase effector proteins [The data presented here indicate a role for the proteins ,30, whicproteins . Effectoproteins ,30. By aproteins ). The exproteins ,30.Consistent with a potential role for this class of proteins in embryonic morphogenesis, we have shown that XCEP2 expression is temporally regulated at gastrulation stages, when major modulations of cellular morphology, cytoskeletal organization, and cellular adhesion are occurring. mRNA for XCEP2 is present prior to mid-blastula transition, persists through mid gastrulation, and is strongly down-regulated by the time the blastopore closes and neurulation begins. This pattern would suggest that XCEP2 protein would be present through the period when active gastrulation movements are occurring. The diffuse spatial pattern of mRNA and, presumably, protein expression of XCEP2 may suggest that XCEP2 functions broadly in cells of the animal and equatorial regions. However, the observed broad spatial distribution of XCEP2 mRNA does not preclude the possibility that the XCEP2 protein may be functionally activated or inactivated at discreet times and locations during early embryonic development.Xenopus gastrulation, delay the closure of the blastopore and inhibit embryonic elongation. The observed rescue with XCEP2 mRNA is strong evidence for the specificity of the antisense morpholino effect. These effects are not due to a loss of mesodermal induction, as brachyury and goosecoid expression do not change in response to antisense XCEP2 morpholinos. This is particularly relevant given recent reports demonstrating a direct link between brachyury expression and control of cellular migration [Furthermore, we show that antisense morpholino oligonucleotides capable of blocking translation of the XCEP2 message interfere with igration .The effects we report require relatively high, although not unprecedented, doses of morpholino antisense oligonucleotide. This dosage requirement may reflect the difficulty inherent in morpholino-mediated translational knockdown of maternally expressed genes. XCEP2 mRNA and (presumably) XCEP2 protein are present prior to midblastula transition. Given this situation, the timing morpholino induced protein downregulation is dependent both upon the effectiveness of translational blockade, and the half life of the protein in the cytoplasm. In this context, it may be essential to impose close to complete translational inhibition in order to reduce protein levels rapidly enough to affect early embryonic events, such as gastrulation.Clearly, specific probes to assess endogenous XCEP2 protein expression will be necessary for fuller characterization of the role of this protein during gastrulation. For this reason, antibodies are currently being raised against the XCEP2 protein. These antibodies will be important in the characterization of the developmental time course of endogenous XCEP2 protein expression, assessment of the subcellular localization of the XCEP2 protein, isolation of potential XCEP2 binding partners, and in assessing and further optimizing the extent of protein down-regulation in morpholino injected embryos.Currently, the specific mechanism by which XCEP2 exerts its role in gastrulation is unknown. However, our preliminary data suggest that XCEP2 may either contribute to a required "ground state" of cellular adhesion or play a role in modulations in the strength of cadherin-mediated cell-cell adhesion that are known to occur during gastrulation ,39,40. MIn embryos Wnt-mediated signals have been shown to activate Cdc42, a process that is required for normal gastrulation movements ,12. In fThe known functional properties of the CEP class of effector proteins, and the characteristics of CRIB domain effector proteins in general, suggest some interesting possibilities relating to the control of cell adhesion during gastrulation. Consistent with the observed functions of the XCEP2 homologs in cultured cells, XCEP2 in embryos may impinge on regulatory circuits downstream of Cdc42 that control actin filament assembly, which in turn may affect diverse cellular processes, including assembly of adherens junctions. Alternatively, XCEP2 may more directly impinge upon cadherin functional activity, perhaps by influencing the association of IQGAP or other molecules with cadherin complexes. In future studies, it will also be important to establish whether embryonic activation of whether there are links between Wnt-mediated activation of Cdc42 and functional activation of XCEP2 and to characterize the mechanism(s) by which XCEP2 contributes to cell adhesion between cells of gastrulating embryos.Xenopus gastrulation. For this reason it will be interesting and important to discern further the role of XCEP2, with regard both to its relationship to intracellular signalling pathways and its effects on cellular behavior during gastrulation.It has become clear in recent years that an integrated network of signals involving Rho GTPase proteins and their effector proteins help to control and regulate the diverse and intricate morphogenetic processes that occur during embryonic development. Less clear are the specific modes of functional interaction between the multiple Rho GTPases and the diversity of potential effector proteins. We have shown that XCEP2 is one component in the complex regulatory puzzle contributing to morphogenetic processes during Adaptor 1-5'-ATGAGTCCTGAC-3' (upper strand)4-CGGTCAGGACTCAT-3'(lower strand).5'-POAdaptor 2 (the 3' phosphate group of the lower strand inhibits DNA polymerase extension)-5'-ACTGGTCTCGTAGACTGCGTACC-3'(upper strand)4-CGGGTACGCAGTC-PO43' (lower strand).5'-PO"Universal" RFDD-PCR primer (complementary to Adaptor 2)-5'-ACTGGTCTCGTAGACTGC-3'"Selection" Primer 4 AAG-3' (3-nucleotide extension is underlined)5'-ATGAGTCCTGACCGAXCEP2 cDNA forward-ATGTCCGCCAAG-3'5'-ATTGCAAAGXCEP2* cDNA forward-CGGGATCCTAGATGTCGGCGAAAGCGCCGATATACCTAAAGAG AAG-3'5'-XCEP2 cDNA reverse-5'-AACGTATCCCCTTCCCCA-3'XCEP2A forward (complementary to 5' untranslated region of the cDNA sequence)5'-AACGTATCCCCTTCCCCA-3'XCEP2A reverse (complementary to 5' untranslated region of the cDNA sequence)5'-AAAGAGAAGTAGCCGTAAAGGA-3'XCEP2B forward5'-GCCAAGGCCCCGATATAC-3'XCEP2B reverse5'-CCAATAGCAGGTAGGGAA-3'Brachyury forward5'-GGATCGTTATCACCTCTG-3'Brachyury reverse5'-GTGTAGTCTGTAGCAGCA-3'Goosecoid forward5'-ACAACTGGAAGCACTGGA-3'Goosecoid reverse5'-TCTTATTCCAGAGGAACC-3'ODC forward-5'-GTCAATGATGGAGTGTATG-3'ODC reverse5'-TCCATTCCGCTCTCCTGA-3'Antisense morpholino oligonucleotides, which specifically block translation of targeted mRNAs were synAntisense XCEP2 MO1 -CATCTTTGCA-3'5'-GGGCCTTGGCGGAXenopus Genbank sequence accession # BC045241)-Antisense XCEP2 MO2 are ligated to restriction digested, double stranded cDNA. Amplification of this cDNA is conducted with the "universal primer", complementary to one of the adaptor sequences, and one of 64 "selection" primers that bind largely to the to the second adaptor sequence but have a 3-nucleotide 3' extension that is complementary only to a subset of cDNA inserts. An extension-inhibiting chemical modification of the lower strand Adaptor 1, consisting of a 3' phosphate group in our modified procedure, and the 3-nucleotide extension of the selection primer largely restricts amplification to cDNA fragments that have different adaptor sequences ligated on opposite ends. Furthermore, any particular selection primer will selectively amplify only a subset of cDNA fragments .Restriction Fragment Differential Display PCR (RFDD-PCR) procedures were patterned closely after the commercially available DisplayProfile kit from QBiogene , with minor modifications. In this differential display procedure, Adaptors 1 and 2 . RNA from Xenopus laevis embryonic stages 7, 8.5, 9.5, 10.5 and 12.5 were prepared. First strand cDNA was synthesized using oligo dT primer and Superscript III reverse transcriptase (Invitrogen), followed by second strand synthesis catalyzed by DNA polymerase I . Double stranded cDNA was purified using GeneClean silica resin , and then digested with TaqI restriction enzyme . An annealed mix of RFDD-PCR of Adaptor 1/Adaptor 2 oligonucleotides was added, and ligated to the TaqI digested cDNA ends using T4 DNA Ligase (Roche). Touchdown PCR using the universal RFDD-PCR primer, 35S-labelled dCTP, and "Selection" Primer 4 was conducted using Accuprime PCR mix (Invitrogen), with the following cycling parameters:Total RNA from staged, duplicate batches of Pre-dwell: 94°C 4 minutes10 cycle touchdown PCR, with a 0.5 temperature decrement each cycle:94°C 30 seconds60°C→ 55°C 30 seconds72°C 1 minute30 cycle standard amplification:94°C 30 seconds55°C 30 seconds72°C 30 secondsPost dwell: 72°C, 5 minutesXenopus homologue of the Cdc42 Effector Protein 2, which we refer to as "XCEP2". The full length cDNA was amplified using gene-specific primers and poly-dT primers using standard 3' RACE procedures [Samples were run on a standard 6% denaturing DNA sequencing gel, and the gel was dried and subjected to autoradiography. Bands showing differential expression were excised from the gel and eluted by heating at 95°C for 15 minutes. Eluted fragments were re-amplified with 30 cycles of standard PCR, using the original primers (above). Direct cycle sequencing of isolated product revealed that one of the fragments corresponded to the 5' end of a The RACE amplified full-length fragment isolated from the RFDD-PCR procedure (see above) was cloned into the pCR2.1 vector, using the pCR2.1 TA cloning kit (Invitrogen), and commercially sequenced (MWG Biotech). To construct a vector encoding XCEP2A fused to a C-terminal 6xmyc tag, PCR was conducted using primers directed to the 5' and 3' end of the XCEP2A cDNA sequence (XCEP2A cDNA forward and XCEP2A cDNA reverse) with the cloned, full length XCEP2A as template. The primers used in this PCR mutated the normal stop codon and introduced a BamHI and ClaI site at the 5' and 3' ends of the amplified product, respectively. BamHI/ClaI digested full length cDNA was directionally cloned in the sense orientation into the corresponding into BamHI/ClaI digested pCS2-myc vector, producing the plasmid pCS2-XCEP-myc. In this context, the full length X-CEP2A cDNA encodes a fusion protein with a C-terminal 6x myc tag. A second construct, containing introduced mutations in the XCEP2A antisense morpholino target region, was also derived by PCR. To construct this vector, PCR was conducted using XCEP2* forward and XCEP2 reverse primers with the full-length XCEP2 DNA as template. XCEP2* forward introduced multiple changes in the sequence of the cDNA encoding the translational start site region , producing the plasmid pCS2-XCEP2-anti. In a similar fashion, an RNA expression vector containing the XCEP2 insert in the sense orientation in pCS2+ was also constructed.For transcription of antisense probe for in vitro using the mMessage mMachine kit (SP6) from Ambion , and were dissolved in distilled water prior to injection.Capped mRNAs were synthesized RT-PCR Primers, above). 25 cycles of PCR were carried out using Accuprime PCR mix (Invitrogen) in 25 ul reactions containing 2 ul of 200 ug/ml reverse transcribed cDNA, 0.3μM of each primer, and a trace of radiolabelled nucleotide to allow for autoradiographic visualization. Parallel reactions containing primers specific to the metabolic gene, ornithine decarboxylase (ODC), were used as a loading control in all semi-quantitative RT-PCR experiments. RT-PCR products were separated electrophoretically on 5% acrylamide gels, which were dried and subsequently subjected to autoradiography. RT-PCR of the XCEP2A allele resulted in stronger amplification than with the XCEP2B primer set. XCEP2B bands required correspondingly longer exposure times (2–4 fold) for detection.Total RNA was isolated from embryos using Trizol (Invitrogen) according to manufacturer's instructions. Reverse transcription was carried out using SuperscriptIII (Invitrogen). Assessment of relative levels of gene expression over developmental time, or under different experimental conditions, was carried out using standard semi-quantitative RT-PCR methods , with thXenopus laevis were purchased from Nasco . Eggs were obtained and fertilized according to standard methods. Staging was determined according to Nieuwkoop and Faber [in vitro transcribed mRNA's were injected into two-cell stage embryos, with injected volumes of approximately 13 nl in doses as described. All injections were carried out using the Nanoject positive displacement microinjector .Adult nd Faber . AntisenMeasurement of blastopore to embryo diameter ratios was accomplished by dividing the measured blastopore diameter by the embryo diameter. Diameter measurements were obtained using digital images of individual embryos, and the "Ruler" tool of the Adobe Photoshop software.To assess the effects of morpholino antisense oligonucleotides on animal cap explants, morpholino injected and control embryos were allowed to develop until stage 8.5. At stage 8.5 animal caps were dissected, and then cultured in 1xMMR for 24 hours. Non-dissected sibling embryos were allowed to develop until post-neurula stages in order to asses the effectiveness of the morpholino treatment.Proteins from embryos were extracted in 10 volumes of isotonic buffered saline containing 1% NP40. Extracts were cleared by a brief centrifugation, and supernatants were denatured by a 5-minute incubation at 95°C after diluting samples with SDS-PAGE sample buffer. Proteins were separated on 8–16% gradient polyacrylamide gels, and transferred to nitrocellulose. After blocking, blots were probed with monoclonal antibody 9E10 , which rin vitro from linearized pCS2-XCEP2anti plasmid using a digoxygenin nucleotide labeling mix (Roche) and the mMessage mMachine in vitro transcription kit (SP6) from Ambion. In situ hybridization was carried out essentially as described [Digoxygenin-labelled antisense RNA probe was transcribed escribed , using nin situ hybridization methodology in the laboratory, and conducted in situ hybridizations. RWN conceived of the study, designed and conducted molecular biological, embryological and biochemical experiments, and wrote the manuscript. Both authors read and approved of the final manuscript.KKN played a critical role in establishing

Our obstetrics and gynaecology undergraduate teaching module allocates 40–50 final year medical students to eight teaching hospital sites in the West Midlands region. Based on student feedback and concerns relating to the impact of new curriculum changes, we wished to objectively assess whether the educational environment perceived by students varied at different teaching hospital centres, and whether the environment was at an acceptable standard.A Dundee Ready Education Environment (DREEM) Questionnaire, a measure of educational environment, was administered to 206 students immediately following completion of the teaching module.The overall mean DREEM score was 139/200 (70%). There were no differences in the education climate between the teaching centres.Further research on the use of DREEM inventory, with follow up surveys, may be useful for educators to ensure and maintain high quality educational environments despite students being placed at different teaching centres. The undergraduate curriculum at our medical school was redesigned in 1998/99 to bring it in line with recommendations suggested by the General Medical Council (GMC) in Tomorrow's Doctors [Based on previous student feedback reporting differences in educational experiences, together with our concerns relating to the impact of new curriculum changes, we wished to objectively assess whether the educational environment perceived by students varied at different teaching hospital centres, and whether the environment was at an acceptable standard. In particular, was there any potential loss of teaching experience when students were placed away from the principal Teaching Hospital. Thus, the null hypothesis we wished to test was that there was no difference in the learning environment between centres. Several questionnaire-based educational tools are available that set out to 'quantify' the educational environment -4. HowevThe DREEM questionnaire, based on a Likert scale, was administered to the full class of 206 final-year Birmingham University medical students undertaking the exam module in Obstetrics and Gynaecology in 2000. All questionnaires were distributed and returned the same day of the exam, which allowed us to achieve a 100% response rate. Students were told to only comment on their recent 8-weeks experience of Obstetrics and Gynaecology. Statistical analysis was performed using Microsoft Excel and Arcus Quickstat Biomedical Statistical software, and utilised single-sample T test and One-way analysis of variance (ANOVA).The year group comprised 42% male and 58% female. The overall mean DREEM score for the study group was 139/200 (95% CL 136.1 to 141.9), or expressed as percentage of the maximal score, 70% (95% CL 68% to 71%). There was no statistically significant difference between the mean scores for the contributory DREEM domains, which were as follows: perception of learning, 34.52/48 (72%); perception of teaching, 32.05/44 (73%); academic self-perception, 19.46/32 (61%); perception of atmosphere, 34.07/48 (71%), and for social self perceptions, 18.90/28 (68%). The DREEM scores for each hospital, with comparison of all contributory elements of the DREEM inventory, are depicted in Table When converting the raw DREEM score to percentages, two-sided P-value single-sample Student's T test showed no statistically significant difference between hospitals by each DREEM domain, or between each DREEM domain within the same hospital. Greatest variation between hospitals occurred in the Students' Perception of Atmosphere domain, where there were four hospitals beyond the 95% Confidence Limits; this compared to three hospitals beyond 95% Confidence Limits in all other DREEM domains Table s. One-WayWe have used the Dundee Ready Education Environment Measure (DREEM) in 'diagnosing' the educational environment of eight different teaching centres and making comparative analysis between these centres. The overall mean DREEM score was 139/200, or expressed as a percentage, 70% (95% CL 68–71%). The educational learning environment did not vary between centres. The two lowest scoring contributory domains, academic self-perception (61%) and social self-perceptions (68%), were not statistically significantly different from the other three DREEM domains or overall mean DREEM score.This study has benefited by using an established educational measure and obtaining a 100% response rate. No students had been previously taught at the principal teaching hospital as this was solely used for Obstetrics and Gynaecology teaching. However, some of the students had previously attended the other seven teaching hospital centres due to prior clinical teaching attachments. Thus, previous experiences may have biased the teaching assessment completed by some students. Furthermore, the DREEM questions are of such a nature that it is likely that the environment of the entire curriculum was being assessed. However, by performing the DREEM survey immediately at the end of the obstetrics and gynaecology module, and emphasising reporting only the last eight weeks experience, we believe this maximised the chance that the DREEM measure assessed only the recent hospital teaching site and minimised any recall bias. Other groups have higThe DREEM domains are unlikely to be independent variables, and may be less of an environment test but more of a measure of the overall motivation and learning attitude of the individual. The Course Valuing Inventory (CVI) score is made up of five domains: worthiness of learning experience, emotional awareness, personal development, cognitive enhancement and task drive. A recent study of first year medical students showed a correlation between higher Course Valuing Inventory (CVI) scores, female gender, stronger self-confidence as a learner, greater motivation to learn and higher DREEM scores .There is no accepted agreement on what is an acceptable DREEM inventory score from published literature. Nevertheless, our DREEM score of 139/200 was higher than other reports. A study of final year medical students in Trinidad reported an overall mean DREEM of 109.9/200 . A largeThe non-significant differences between the DREEM domains and between hospitals were significant findings. This was conveyed to our tutors based at the various teaching centres as a positive and encouraging result. In practical terms, this meant that regardless of hospital capacity or student group size, their education delivery and environment was no different to other centres in the student's curriculum. The DREEM inventory may thus be a useful tool for educators to ensure and maintain high quality educational environments and uniformity in educational delivery despite students being placed at different teaching centres.The author(s) declare they have no competing interests.RV and ET carried out the statisitcal analysis, data interpretation, and drafted the manuscript. JKG conceived and coordinated the study, acquired the results, and made revisions of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:

In clinical practice, visual gait observation is often used to determine gait disorders and to evaluate treatment. Several reliability studies on observational gait analysis have been described in the literature and generally showed moderate reliability. However, patients with orthopedic disorders have received little attention. The objective of this study is to determine the reliability levels of visual observation of gait in patients with orthopedic disorders.The gait of thirty patients referred to a physical therapist for gait treatment was videotaped. Ten raters, 4 experienced, 4 inexperienced and 2 experts, individually evaluated these videotaped gait patterns of the patients twice, by using a structured gait analysis form. Reliability levels were established by calculating the Intraclass Correlation Coefficient (ICC), using a two-way random design and based on absolute agreement.The inter-rater reliability among experienced raters was comparable to that of the inexperienced raters . The expert raters reached a higher inter-rater reliability level . The average intra-rater reliability of the experienced raters was 0.63 (ICCs ranging from 0.57 to 0.70). The inexperienced raters reached an average intra-rater reliability of 0.57 (ICCs ranging from 0.52 to 0.62). The two expert raters attained ICC values of 0.70 and 0.74 respectively.Structured visual gait observation by use of a gait analysis form as described in this study was found to be moderately reliable. Clinical experience appears to increase the reliability of visual gait analysis. Patients exhibiting gait deviations caused by orthopedic impairments are often referred to a physical therapist for treatment. In order to determine treatment goals or to evaluate the effect of a therapeutic intervention, physical therapists visually observe the patient's gait -3. This et al. [et al. [Several reliability studies on observational gait analysis have been described in the literature. These studies included patients with hemiplegia -7, amputet al. , found oIn the Netherlands a gait analysis form has been developed which focuses mainly on orthopedic disorders . Visual The purpose of this present study is to determine the inter- and intra-rater reliability of videotaped observational gait analysis with use of an orthopedic gait analysis form when applied to a cohort of patients suffering from orthopedic impairments. In addition, this study determines how well the raters perform observational gait analysis by comparing their assessments with a criterion, based on the experts' opinion. In order to gain insight into how the results may give guidance to physical therapy treatment, this study also investigates which items on the gait analysis form, that have been considered to be disturbed by visual observation, receive high priority in the physical therapy treatment program according to the physical therapist who performs the visual gait analysis.Thirty videotapes of patients' gait were selected from the archives of the department of Physical Therapy of the University Medical Center Nijmegen, the Netherlands. These videotapes involved patients who had been referred to a physical therapist for gait treatment. It is common practice at this department that prior to gait training therapy the gait of each patient is videotaped according to a standardized protocol.The criteria for inclusion of the videotapes were: (1) the presence of mild to severe gait deviations due to an orthopedic impairment; (2) patient was wearing shorts or underwear to allow for a more accurate observation of the joint movement; (3) ability of a patient to walk 15 meters at least four times, twice in a semi-circle and twice in a straight line on a gymnasium floor; (4) and patient's written informed consent. The first thirty patients who complied with these criteria were included.The group consisted of 15 male and 15 female patients with a mean age of 37.8 years (range: 15 to 62 years). The type of orthopedic impairments varied from status post hip, knee, ankle surgery (n = 8), status post hip or knee prosthesis (n = 6), status post femur, tibia or ankle fracture (n = 3) and traumatic or non-traumatic non-specific hip, knee or ankle pain (n = 13) , anterior and posterior view at each of the three sub-phases of stance and the two sub-phases of swing. All patients were recorded from a lateral view (both sides) while walking 15 meters in a semi-circle (radius approximately 10 m) at a comfortable self-selected walking speed. We used a semi-circle in order to be able to observe the patient's gait in the sagittal plane from one position. The anterior and posterior views were videotaped while the patient walked five meters toward and away from the camera.®). Manufactured videos were reduced into a one-minute film-clip in which the patient's gait could be viewed in the lateral and frontal plane. Subsequently, these videos were converted to analog format again, so that they could be played by a regular video player. Sampling frequency was 24 Hz.The collected videos were edited with use of the computer program adobe premiere 6.0 to the items they scored as disturbed, with respect to a physical therapy treatment program. In other words, which items would receive important attention in the physical therapy intervention if the rater was going to treat this patient for his or her gait disorder.In order to determine the level of performance of observational gait analysis of all experienced and inexperienced raters, we compared their ratings with a criterion. This gives us an indication about how well the raters were capable in performing visual gait analysis. The criterion was attained during a consensus session of the two expert raters: After individually assessing the 30 patients for the second time, the two expert raters jointly observed the videotaped gait of all 30 patients for the third time.Inter- and intra-rater reliability levels were assessed by using Intraclass Correlation Coefficients (ICCs), validated for use with multiple raters and calculated in a two-way random model based on absolute agreement. We used ICCs because it has been shown that with data that are rated as a dichotomy, the ICC is equivalent to measures of nominal agreement, simplifying computation in cases where more than two raters are involved . In addiThe inter-rater reliability among experienced raters was 0.42 (95%CI: 0.38–0.46). This level of reliability is comparable to the inter-rater reliability of in-experienced raters, which reached an ICC value of 0.40 (95%CI: 0.36–0.44). The expert raters reached the highest inter-rater reliability (ICC: 0.54 (95%CI: 0.48–0.60)).There were no differences in inter-rater reliability between the first and second rating session of all three groups separately, based on the overlap of 95% confidence intervals.The average intra-rater reliability of the experienced raters was 0.63 (ranging from 0.57 to 0.70). The inexperienced raters reached an average intra-rater reliability of 0.57(ranging from 0.52 to 0.62). The two expert raters attained ICC values of 0.70 and 0.74 respectively.The agreement between the outcome of the joint assessment of the expert raters (criterion) and those of the individual experienced raters ranged from 0.43 to 0.55 with an average ICC value of 0.48. The inexperienced raterrs attained agreement levels ranging from 0.41 to 0.55, with an average of 0.49. There is no difference in the level of performance of visual gait assessments of experienced or inexperienced raters, when compared to the experts' opinion.The inter-rater reliability per item on the gait analysis form between the two experts is generally moderate to substantial . Only one item, extension movement of the knee during mid stance, had an ICC value for intra-rater reliability of less than 0.6. The experienced raters were able to attain good intra-rater reliability for item 2, posture of the trunk during walking (ICC = 0.81). Three items reached substantial intra-rater reliability . Two items of the gait analysis form, pelvis rotation and ankle movement during late stance, were not intra-rater reliable (ICC < 0.40). The inexperienced raters reached the highest intra-rater reliability for the assessment of arm swing during walking (ICC = 0.81). Three items had inadequate intra-rater reliability levels; flexion of the knee in early stance (ICC = 0.36), extension of the knee in mid stance (ICC = 0.22), and ankle movement during the late stance phase (ICC = 0.37).No reliability score was obtained from item 3, which describes a trunk position behind the hips, because this item was observed only once.On average, with respect to all items, in about a quarter of the cases items were judged to be disturbed by the expert and experienced raters . Expert raters showed the least variability between the first and second session; five items showed to have a mean intra-rater reliability level that is considered good (ICC > 0.80).The results of this study suggest that a brief introduction in normal gait kinematics in inexperienced raters gives comparable reliability levels of observational gait analysis in patients with orthopedic impairments compared to experienced physical therapists, who have worked for several years with patients with gait disorders. However, expert raters – those that work significantly more intensive with patients with gait disorders – accomplish higher reliability levels.As mentioned in the methods section, the gait analysis form used in this study is also used in daily practice to guide the treatment of the patient's gait disorder. In the physical therapist's treatment program, some items on the form will obviously receive higher priority than others. The results of this study show that physical therapists mainly focus their intervention on movement disorders of the lower extremity. However, the expert raters also report to give priority to asymmetry of the stance phase and excessive lateral flexion of the trunk during gait. Of the three items in this study that achieved the highest reliability levels, only the movement of the knee received generally a high priority in the treatment program of experienced raters. This implies that experienced raters will mainly focus their treatment on items that have generally a low inter- and intrarater reliability.Structured visual observation of a patient's gait by use of a gait analysis form as described in this study is found to be only moderately reliable, but may be a useful guide to the physical therapist in setting up a gait training or exercise therapy program. Intra-rater levels have shown that visual gait analysis will supply the observer with a fair indication of changes in a person's gait. However, to evaluate the effect of an intervention on a patient's gait we recommend more objective instrumentation which has been proven reliable and valid.The author(s) declare that they have no competing interests.JB carried out the data analysis, participated in the design of the study and drafted the manuscript. CvU participated in the design and coordination of the study, assisted with statistical analysis, and helped to draft the manuscript. SvM participated in the design of the study and supplied the videotapes. JK participated in the design and coordination of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:

Salmonella enterica serovar Typhimurium aroA strain in two Raf dependent lung tumor mouse models.Serine-threonine kinases of the Raf family are central players in cellular signal transduction, and thus often causally involved in the development of cancer when mutated or over-expressed. Therefore these proteins are potential targets for immunotherapy and a possible basis for vaccine development against tumors. In this study we analyzed the functionality of a new live C-Raf vaccine based on an attenuated Escherichia coli α-hemolysin and expressed in secreted form by an attenuated aroA Salmonella enterica serovar Typhimurium strain via the α-hemolysin secretion pathway. The effect of the immunization with this recombinant C-Raf strain on wild-type C57BL/6 or lung tumor bearing transgenic BxB mice was analyzed using western blot and FACS analysis as well as specific tumor growth assays.The antigen C-Raf has been fused to the C-terminal secretion signal of Salmonella enterica serovar Typhimurium aroA strain using the E. coli hemolysin secretion system. Immunization of wild-type C57BL/6 or tumor bearing mice provoked specific C-Raf antibody and T-cell responses. Most importantly, the vaccine strain significantly reduced tumor growth in two transgenic mouse models of Raf oncogene-induced lung adenomas.C-Raf antigen was successfully expressed in secreted form by an attenuated Salmonella enterica serovar Typhimurium could form the basis for a new generation of live bacterial vaccines for the treatment of Raf dependent human malignancies.The combination of the C-Raf antigen, hemolysin secretion system and The Raf proteins are located upstream of MEK and downstream of Ras and represent an essential part of the mitogenic cascade -3. InterSalmonella enterica serovar Typhimurium aroA strain. Such recombinant live vaccines have been shown to efficiently elicit both, humoral and cellular immune responses against a variety of heterologous antigens [E. coli α-hemolysin (HlyA) secretion system which is fully active in Salmonella [s), which is recognized by the HlyB/HlyD/TolC-translocator, leading to direct secretion of the entire protein into the extracellular medium without the formation of periplasmic intermediates. In addition, fused to the C-terminus of heterologous proteins, the HlyAs leads to efficient secretion of such proteins by the recombinant bacteria. In our case, the whole C-Raf antigen fused to the C-terminal secretion signal of hemolysin was efficiently expressed and secreted by an attenuated Salmonella enterica serovar Typhimurium aroA strain. The effects of this recombinant vaccine were assessed in wild-type C57BL/6 or tumor bearing transgenic BxB mice.Here we describe the development of a C-Raf vaccine on the basis of an attenuated antigens ,8. In orlmonella . This trThe bacterial strains, plasmids, cell lines and mice used in this study are listed in Table Salmonella enterica serovar Typhimurium LB5000 at 2.5 kV, 25 microfarads (μF), and 200 Ohm in a 0.1 cm electroporation cuvette.The plasmids pMOhly1 and pMOhly-Raf were first transformed in competent E. coli ,11. SubsATGCATCTTGGAAG-3' and antisense primer 3'Raf: 5'-CAACTAGAATGCATGCAGCCTCGGGGA-3' were used to amplify by PCR a 1950 bp DNA fragment representing the entire craf gene from plasmid pUC13-c-raf-1 [NsiI restriction enzyme, the DNA fragment carrying the craf gene was inserted into the single NsiI site of the export vector pMOhly1 [E. coli DH5α (Invitrogen), analyzed and transformed in S. typhimurium SL7207 were centrifuged at 5000 × g at 4°C for 5 min. The supernatant proteins were precipitated with 10% (V/V) trichloroacetic acid (TCA) for 1 h on ice, collected by centrifugation and resuspended in SDS sample buffer. The supernatant proteins were separated on 10% gels by SDS-PAGE [SDS-PAGE . For imm9 bacteria/100 μl phosphate-bufferd saline (PBS) at 5-day intervals. At day 45 after the start of the vaccination, these mice were boosted intravenously (i.v.) with a single-dose of 5 × 105 bacteria/100 μl PBS. The BxB23 mice received a second i.v. boost of 5 × 105 bacteria at day 90 after the start of the vaccination. Induction of Raf-specific immune responses were analyzed at day 50 for C57BL/6 or at day 95 for BxB23 respectively.In order to achieve a broad immune response encompassing both, the mucosal and systemic immunity against C-Raf, we combined oral immunization (p.o.) with an intravenous (i.v.) boost. In these experiments, seven weeks old C57BL/6 or BxB23 mice were immunized, first p.o. three times with 5 × 107bacteria/30 μl phosphate-buffered saline (PBS) at 14-day intervals. The vaccine was applied using a micropipette onto the nares of mice under anesthesia.Seven weeks old BxB23 mice were immunized i.n. four times with 1 × 10Induction of Raf-specific immune responses were analyzed at day 70.8salmonellae in 10 μl PBS at 14-day intervals. The vaccine was applied using a micropipette into the nares of mice without anesthesia.Four months old BXB11 mice were immunized i.n. three times with 1 × 10b haplotype . The EL4 tumor cell line is a murine thymoma cell line of the H-2ype Tab. . 10 μg pAnimals were sacrificed and a single cell suspension of splenocytes was prepared by passage of the spleen through a sieve into RP 10 medium [RPMI medium (Life Technologies) supplemented with glutamine (1%), 50 μM β-mercaptoethanol , penicillin , streptomycin and 10% fetal calf serum .4Cl, pH 7.3) for the lysis of erythrocytes. After 2 minutes, 10 ml RP 10 medium was added to stop lysis. After centrifugation, cells were resuspended in 2 ml RP 10 medium and counted.The cell suspension was centrifuged and resuspended in 3 ml lysis buffer or 5 × 105 EL-4 cells or with the addition of 10 ng/ml of phorbol myristyl acetate and 500 ng/ml of ionomycin (Sigma) or with medium alone.In 6 ml FACS tubes and a total volume of 100 μl RP 10 medium in the presence of 30 U/ml recombinant IL-2 and 10 μg/ml Brefeldin A at 37°C, 5% COAfter restimulation, cells were washed with PBS-0.1% bovine serum albumin and incubated with 1 μl of anti-CD8-CyChrome (Pharmingen Nr. 01082A) in a volume of 100 μl PBS-0.1% BSA for 20 min on ice. Subsequently, cells were washed and fixed for 20 min at room temperature with PBS-2% paraformaldehyde (Sigma). After washing with PBS-0.1% (BSA), cells were permeabilized with PBS-0.1% BSA-0.5% saponin buffer. After incubation for 5 min at room temperature, cells were washed with P-B-S buffer and incubated in a volume of 100 μl at room temperature with polyclonal rat immunoglobulin G (IgG) antibodies (JacksonImmunoResearch) to block nonspecific binding and 1 μl anti-IFN-γ-FITC for 20 min. After incubation, cells were washed twice with P-B-S buffer and another time with P-B buffer. Cells were resuspended in 400 μl PBS-PFA 0,5% and kept at 4°C until analysis.+ cells.Cells were analyzed by flow cytometry in a FACS Calibur flow cytometer (Becton Dickinson) using CellQuest 3.0 software (Becton Dickinson). Lymphocytes were chosen according to their size and granularity in a forward/side – scatter diagram. Numbers are expressed as percent IFN-γ positive CD8t test. Findings were regarded as significant, if P values were <0.05. Survival curves were compared using a log rank test.The statistical significance of differential findings between experimental groups was determined by Student's Salmonella enterica serovar Typhimurium aroA strain SL7207 secreting the C-Raf antigen was achieved by cloning the human craf cDNA from pUC13-c-raf-1 [craf-hlyAs fused gene and the functional hlyB and hlyD genes required for its secretion and with Salmonella enterica serovar Typhimurium SL7207 as control in order to test the induction of Raf-specific immune responses. The data showed that 20% of the sera of BxB23 mice immunized with SL7207/pMOhly-Raf contained Raf-specific antibodies .To assess the induction of Raf-specific T-cell responses, mice were sacrificed and Raf specific T-cell responses were assessed using intracellular IFN-γ staining followed by FACS analysis. For this purpose, T-cells were restimulated with C-Raf overexpressing EL-4 cells. Using this technique, we could detect Raf specific CD8Salmonella strains, groups of 6 to 10 heterozygous BxB23 mice at the age of 7 weeks were immunized p.o./i.v. or i.n. with recombinant Salmonella enterica serovar Typhimurium strain (SL7207/pMOhly-Raf) or with SL7207 as control. BxB23 mice normally show an induction of lung adenomas with short latency and at 100% incidence [To test the protective capacity of the immune responses induced by the recombinant ncidence ,16. The craf transgenic strain, which develop lung adenomas after a shorter latency period compared to BxB23 mice [In order to confirm these data we repeated the protection experiments using BxB11, another B23 mice . In thisThese results suggest that the immunization with the vaccine strain SL7207/pMOhly-Raf can achieve a partial protection against tumor growth in the BxB mouse model.The major problems for vaccine development against cancer are the heterogeneity of the tumor cells and the fact that all tumor antigens are self-antigens. Therefore specific T-cells might be anergic or tolerant ,18.However, despite these problems, several cancer vaccines have already reached clinical trials . The sucSalmonella enterica serovar Typhimurium aroA strain as a "live vaccine". In general, recombinant aroA salmonellae are efficient live bacterial vectors that stimulate strong mucosal immunity, humoral and cell-mediated responses with a great potential as live vaccine carriers in both humans and animals [aroA Salmonella vaccines include their safety and easy administration [aroA Salmonella enterica serovar Typhimurium strains were already successfully used as carriers for DNA vaccines against cancer in mice [Here we describe a new strategy for achieving an anti-tumor immune response with a C-Raf vaccine on the basis of an attenuated animals ,20. Advastration ,22. In a in mice .Salmonella enterica serovar Typhimurium aroA strain using the E. coli hemolysin secretion system. This system allows an efficient antigen secretion and presentation, which are necessary for an optimal immune response against the given heterologous antigen [Salmonella enterica serovar Typhimurium aroA strain SL7207 secreting C-Raf resulted in the induction of a humoral immune response, manifested by the presence of Raf-specific antibodies in some of the vaccinated mice. In addition, C57BL/6 mice immunized with Salmonella enterica serovar Typhimurium SL7207/pMOhly-Raf developed a Raf-specific CD8+ T-cell response. The C-Raf vaccine thus is able to break the peripheral tolerance of the immune system towards C-Raf and to induce a specific immune response. Although the transgenic model is closer to the human setting in comparison to challenge models with tumor cell lines, we can not formally exclude that lung specific expression of the transgene in our BxB model is not sufficient for the induction of peripheral tolerance. Furthermore, side effects due to autoimmunity might occur in humans who have a more generalized expression pattern. However, the lung tissue does not belong to immunoprivileged sites and we have never observed any signs of lung pathology in immunized animals which strongly suggests that the data could, in principle, be translated into the human setting.In this study the C-Raf antigen was delivered in secreted form by attenuated antigen . In factSalmonella enterica serovar Typhimurium SL7207/pMOhly-Raf. However, we were not able to assess Raf specific CTL responses in these mice due to the high variability and background and therefore we cannot conclude whether protection is really due to cytotoxic T-cells or might be the result of other effects, e.g. an increased intratumoral level of IFN-γ produced by Raf specific CD4+ T-cells. Interestingly, the observed variation occurred only in treated BxB23 mice, not in naïve BxB23 mice. Therefore, the observed effect might be due to a spontaneous induction of an immune response mediated by tumor infiltrating bacteria. It is interesting to note that spontaneous Raf specific immune responses also occur in the human context, which we have recently demonstrated B-Raf V599E specific CTL and B-Raf /B-Raf V599E specific humoral responses in melanoma patients [We demonstrated also a partial tumor protection both in BxB23 and in BxB11 mice after immunization with recombinant patients ,25.+ T-cells are effective in eliminating antigen positive tumor cells. The combined data thus provide proof of concept for the development of Raf-based anti-cancer vaccines.Most importantly, the lack of B-Raf V599E mutations in metastases of melanoma patients with strong B-Raf V599E CD8 response supportsE. coli hemolysin secretion system is a versatile tool for the delivery of cancer antigens in Salmonella enterica serovar Typhimurium. Moreover, we have recently shown that this secretion system is also fully active in Salmonella enterica serovar Typhi Ty21a, the only Salmonella vaccine strain registered for human use [S. typhi Ty21a could form the basis for a new generation of live bacterial vaccines against cancer.This is the first example demonstrating that the uman use . TherefoSalmonella enterica serovar Typhimurium aroA SL7207 strain via the E. coli hemolysin secretion system. In addition, the immunization of wild-type C57BL/6 or tumor bearing transgenic mice with this C-Raf secreting Salmonella strain provoked specific C-Raf antibody and T cell responses. Most importantly, the vaccine strain induced partial protection against lung cancer in two transgenic mouse models of Raf oncogene-induced lung adenomas.Taken together we have demonstrated that the C-Raf antigen can be successfully expressed and secreted by the attenuated The approach may provide a new strategy for the rational design of cancer therapies.The author(s) declare that they have no competing interests.IG, JF and JT designed the study. IG drafted the manuscript. JF was also involved in writing the report. AS and JF did the FACS analyses. JT constructed the EL4Raf cell line. TP, AS, JF and IG carried out the immunization of mice and the Western blot analyses. WG and URR were involved in providing the conceptual framework for this study. All authors approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:Supplementary figure Click here for file

EMO syndrome, defined as a triad including exophthalmus, pretibial myxedema and osteoarthropathia, is a rare condition in patients suffering from hyperthyreosis.We here describe an interesting case of EMO syndrome associated with unilateral fibromatosis of the hand and an initial stage of generalized myxedema of the skin. To our knowledge a similar case has not yet been described in literature though reports about associated fibromatosis, e.g. located retroperitoneally, already exist. Familiar explanations include its initiation by autoimmune processes or aberrant T-cell cytokine stimulation leading to an overwhelming production of glycosaminoglycans.Interpreting our case in context with previous reports we conclude that associated fibromatosis induced by autoimmune processes may affect a variety of different localizations and therefore requires careful monitoring. A therapeutical attempt by using UVA1 irridation for pretibial myxedema remained without a satisfying regression. EMO syndrome is a rare condition seen in patients suffering from hyperthyreosis. It is defined as a triad of exophthalmus, pretibial myxedema and osteoarthropathia occurring in less than 1% of patients suffering from Graves' disease [In October 2003, a 64-year-old male Caucasian patient was admitted with aggravating pretibial myxedema. Five years ago, he was diagnosed with hyperthyreosis caused by Plummer's disease. Laboratory findings revealed a repressed TSH of 0.01 mU/L (0.4–4.0 mU/L). The patient was treated with radioiodine therapy. Today he suffers from hypothyreosis and a daily substitution of 100 μg L-Thyroxin is performed. Within the months following the radioiodine therapy, an erythema progressing into a manifest pretibial myxedema developed, followed by exophthalmus and fibromatosis of the right hand within the next three years. Surgical orbita decompression due to massive exophthalmus was performed, followed by a subsequent correction of the ocular muscles. Additionally, the fibromatosis of the right hand was excised. Retrospective histological findings revealed a regressive sclerotic fibrosis of firm kollagenous fibers embedded in soft and fat tissue were consistent with a generalized fibrosing process as known in myxedema. Within the last months, decent mucous plaques of the upper limps decreased. The patient now reported progressing congestion of lymph and decreasing flexibility of the lower legs caused by the pretibial myxedema. Clinical examination confirmed massive pretibial myxedema, lymphatic congestion of the lower legs, generalized myxedema accentuating the upper limps, a residual postsurgical nodular tumor of the ulnar aspect of the right hand, exophthalmus, and severe hypertrophic osteopathy of the distal phalanges with clubbed fingers and hippocratic nails . While the congestion of lymph clearly improved, pretibial myxedema remained without any signs of regression.Grave's disease is known to eventually develop after radioiodine therapy and thisA massive recurrence after excision of thyroid dermopathy, as described in literature, could not be observed in our patient during a follow-up of 2.5 years.Generalized fibrosing processes are thought to be based on an accumulation of glycosaminogylcans. Common explanations comprise its initiation by autoimmune processes, e.g. thyreotropin receptors on fibroblasts, or aberrant T-cell cytokine stimulation, leading to overwhelming glycosaminoglycan production Fibrosin2 UVA1 (seven treatment sessions). However, we suggest that phototherapy might also be considered as an adjunct therapeutic alternative in persistent EMO syndrome.Therefore, we conclude that concomitant fibromatosis might appear in various localizations requiring elaborated diagnostic procedures and monitoring in all patients affected. We started low-dose UVA1 irradiation of the pretibial myxedema known to be able to degrade pathologic collagenous architecture by inducing dermal matrix-metalloproteinases as well as by decreasing abnormal cytokine liberation following T-cell apoptosis . NeverthThe author(s) declare that they have no competing interests.C.A. attended to the patient, participated in the design of the case report, and drafted the manuscript. A.K. conceived the study. F.B., A.B. and P.A. participated in its design and coordination.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:

Hypertension is among the most common chronic condition in middle-aged and older adults. Approximately 50 million Americans are currently diagnosed with this condition, and more than $18.7 billion is spent on hypertension management, including $3.8 billion for medications. There are numerous pharmacological agents that can be chosen to treat hypertension by physicians in clinical practices. The purpose of this study was to assess the cost of alternative antihypertensive treatments in older adults with isolated systolic hypertension (ISH).Using the Systolic Hypertension in the Elderly Program (SHEP) and other data, a cost-minimization analysis was performed. The cost was presented as the cost of number-needed-to treat (NNT) of patients for 5 years to prevent one adverse event associated with cardiovascular disease (CVD).It was found that the cost of 5 year NNT to prevent one adverse CVD event ranged widely from $6,843 to $37,408 in older patients with ISH. The incremental cost of the 5 year NNT was lower to treat older patients in the very high CVD risk group relative to patients in the lower CVD risk group, ranging from $456 to $15,511. Compared to the cost of the 5 year NNT of other commonly prescribed antihypertensive drugs, the cost of SHEP-based therapy is the lowest. The incremental costs of the 5 year NNT would be higher if other agents were used, ranging from $6,372 to $38,667 to prevent one CVD event relative to SHEP-based drug therapy.Antihypertensive therapy that is diuretic-based and that includes either low-dose reserpine or atenolol is an effective and relatively inexpensive strategy to prevent cardiovascular events in older adults with isolated systolic hypertension. Use of the diuretic-based therapy is the most cost-effective in patients at high risk for developing cardiovascular disease. Hypertension is among the most common chronic conditions in middle-aged and older adults. Approximately 50 million Americans are currently diagnosed with this condition, and more than $18.7 billion is spent on hypertension management, including $3.8 billion for medications[Treatment of hypertension can significantly decrease the risk of developing CVD ,3. The SCurrently, primary care physicians can choose from numerous pharmacological agents to treat hypertension. The commonly used antihypertensive drug classes include diuretics, beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, alpha-blockers, and calcium channel blockers. Selection of an evidence-based therapy with demonstrated efficacy, safety, and low cost has important economic implications. The purpose of this study was to: 1) assess cost of the SHEP-based antihypertensive treatment to prevent adverse events associated with CVD, including death, stroke, myocardial infarction, and heart failure; and 2) to compare cost of the SHEP-based treatment to the costs of other commonly used antihypertensive agent treatments.The SHEP trial is a randomized, double-blind, placebo-controlled clinical trial sponsored by the National Heart, Lung, and Blood Institute and the National Institute on Aging that tested the efficacy of diuretic-based stepped-care antihypertensive drug treatment of isolated systolic hypertension (ISH) to prevent strokes .The study subjects consisted of community-dwelling men and women 60 years and older who had isolated systolic hypertension, defined as an average systolic blood pressure (SBP) ≥ 160 mm Hg and an average diastolic blood pressure (DBP) < 90 mm Hg over 2 baseline visits. The primary endpoint of the trial was combined nonfatal and fatal stroke over a 5-year period. Secondary endpoints included nonfatal myocardial infarction (MI) plus fatal coronary heart disease (CHD) and major cardiovascular disease (CVD) morbidity and mortality. A total of 2,365 and 2,371 persons were randomized into the treatment and placebo group of the study respectively.4. Fasting blood samples were analyzed at a central laboratory, including serum glucose, lipid levels, creatinine, uric acid, sodium, and potassium.Subjects who met the preliminary blood pressure (BP) eligibility criteria at the initial contact visit were referred to SHEP clinics for the baseline visits. At the baseline visits, subject's demographics, medical conditions, health behaviors, and cardiovascular risk factors were obtained. Methods of these measurements have been reportedOf the 4,736 SHEP participants, 4,189 were included in this analysis. The 547 participants were excluded either because of missing data concerning CVD risk factors (n = 283) or with previous CHD or stroke (n = 264). These 547 excluded subjects had similar age, sex, race, and other characteristics as those who were included in this analysis.<160 mm Hg and for those with SBP between 160 and 179 mm Hg to have a reduction of at least 20 mm Hg. All participants were given chlorthalidone, 12.5 mg/d, or matching placebo (step 1 and dose 1 medication). Drug dosage (step 1 and dose 2 medication) was doubled, 25 mg/d, for participants failing to achieve the SBP goal at the follow-up visits. If the SBP goal was not reached at the maximal dose of step 1 medication, atenolol, 25 mg/d, or matching placebo was added (step 2 and dose 1 medication). When atenolol was contraindicated, reserpine, 0.05 mg/d, or matching placebo could be substituted. When required to reach the blood pressure goal, the dosage of the step 2 drug could be doubled . Potassium supplements were given to all participants who had serum concentration below 3.5 mm0l/L at two consecutive visits. The SHEP participants were followed up monthly until SBP reached the goal or until the maximum level of stepped-care treatment was reached [A stepped-care treatment approach was used, with the goal for individuals with SBP >180 mm Hg to reduce to reached ,7The present analysis focused on five types of events: 1) death; 2) first-occurring major cardiovascular event, including stroke, MI, or heart failure; 3) first-occurring stroke; 4) first-occurring MI; and 5) first clinical diagnosis of congestive heart failure (CHF). The adjudication and clarification of the events was done by a panel of three physicians blinded to treatment assignment and blood pressure status. Members of the panel reviewed the documentation of new cardiovascular events over the study period and adjudicated outcome events according to predetermined criteria. [a priori global score for the risk of developing future cardiovascular events, according to the Multiple Risk Factor Assessment Equation jointly proposed by the American Heart Association and the American College of Cardiology.[Information at the baseline on age, sex, total cholesterol, high density lipid (HDL) cholesterol, systolic blood pressure, diabetes (diabetic vs. non-diabetic), and smoking (current vs. never or past smoking) were used to calculate an rdiology. The equaThe methods of economic evaluation include cost-effectiveness analysis, cost-utility analysis, and cost-benefit analysis, which can be used to assess the trade-off between costs and benefits in choices of antihypertensive treatment regimens. The primary aim of this analysis was to examine cost of the diuretic-based antihypertensive drug intervention in the SHEP trial. A cost-minimization analysis is a special type of cost-effectiveness analysis. It can be used to compare cost difference among competing alternatives of antihypertensive drug treatments when these treatments are medically equivalent. In this study, we used cost-minimization analyses to compare costs and incremental costs of NNT for 5 years to prevent one adverse event related to CVD among antihypertensive treatment regimens. The perspective of this economic evaluation was that of a national health insurance system.We used the number-needed-to-treat as an unit of common outcome measure in the analysis. The number-needed-to-treat to prevent one adverse outcome has become a widely used measure of treatment benefits derived from the results of clinical trials. The NNT is the reciprocal of the absolute risk reduction (ARR) which is the difference between the proportions with the adverse event in the treatment and placebo groups. The 95% confidence interval of NNT was calculated based on the regression-based method described by Laupacis et al. The cost specified in the analysis includes the drug acquisition cost of SHEP treatment from the perspective of a national health insurance system. According to the SHEP treatment protocol, the stepped-care was classified into four types of drug treatments: 1) the Step 1 and Dose 1: chlorthalidone 12.5 mg/d; 2) the Step and Dose 2: chlorthalidone 25 mg/d; 3) the Step 2 and Dose 1: chlorthalidone 25 mg/d plus atenolol 25 mg/d or reserpine 0.05 mg/d; and 4) the Step 2 and Dose 2: chlorthalidone 25 mg/d plus atenolol 50 mg/d or reserpine 0.1 mg/d. Direct drug acquisition costs were calculated based on the minimum average wholesale prices (AWP) within drug manufacturers in the year 2000. All drug1 × C1 + W2 × C2 + W3 × C3 + W4 × C4EC = WThe W1, W2, W3, and W4 represent proportions of the participants using the Step 1 and Dose 1, the Step 2 and Dose 2, the Step 2 and Dose 1, and the Step 2 and Dose 2 medication, respectively. C1, C2, C3, and C4 represent the drug acquisition cost of the Step 1 and Dose 1, the Step 2 and Dose 2, the Step 2 and Dose 1, and the Step 2 and Dose 2 medication, respectively. A Monte Carlo method was performed to estimate the average cost and its standard deviation.To compare the cost of the SHEP-based therapy to other antihypertensive drugs, it was assumed that all antihypertensive drugs in the comparisons have equal efficacy in terms of the NNT for 5 years to prevent one CVD related event. The NNT was calculated based on the method. All drug costs were expressed as dose-specific cost per patient in 1-year and/or 5-year. Using the approach, costs were calculated for each representative drug based on equipotent doses in terms of blood pressure reduction. The non-In this analysis, we focused on the drug acquisition cost for antihypertensive management. Therefore, the monitoring cost for antihypertensive treatment was not included. Total treatment cost includes antihypertensive drug cost and monitoring cost. The monitoring of treatment in ambulatory care settings including physician visits and laboratory tests have an estimated cost of $284 per patient per year. Total coTable Results of the 5 year NNT to prevent one adverse event and its associated cost by event type are shown in Table Table In Table The result of an economic evaluation essentially shows the cost per benefit gained from adapting a specific treatment. The effective and efficient use of resources has been increasingly emphasized from society, health plans, and health care providers. This cost-minimization analysis incorporating outcome data from the SHEP trial presents information treatment cost for older patients with ISH. We found that a long-term, low-dose and diuretic-based antihypertensive therapy is relatively inexpensive and effectively prevents adverse events associated with cardiovascular diseases, especially in older patients who had a high CVD risk profile.Our findings indicate that the total and incremental treatment costs of antihypertensive drugs in ambulatory care settings range widely among drug classes as well as within drug classes. This analysis suggests that diuretic-based antihypertensive treatments are the least expensive, whereas atenolol (beta-blocker) is less costly than enalapril (ACE inhibitor) and nifedipine , and terazosin is the most expensive drugs in terms of the 5 year NNT to prevent one CVD event. It appears that use of the SHEP-based drug therapy offers greater economic benefits for controlling isolated systolic hypertension in the elderly than other antihypertensive drug treatments. Using a decision analysis model that simulated clinical decisions and outcomes that would occur when primary care physicians follow the JNC IV hypertension management guidelines, it was found that a newer class of calcium channel blockers can slightly increase the proportion of patients who achieve and maintain hypertension control, but at a substantially higher cost than with a generic diuretic drug. For our analyses, we presumed that all drugs offer equivalent therapeutic benefits. This assumption may have introduced a conservative bias into our primary findings. In fact, randomized controlled trials directly comparing active treatments for hypertension reported that calcium antagonists and doxazosin were inferior to low-dose diuretics or other agents in preventing cardiovascular events, suggesting that the cost-effectiveness of diuretic-based treatments may be even more favorable than estimated in the present study. -17 FurthWith regard to costs projected in our study, it is noteworthy to consider that compared to the SHEP treatments, costs of treatments based on more recently developed antihypertensive agents (than reported here) are likely to be even higher than estimated in the present analyses.The results of this study are limited to men and women 60 years and older who have isolated systolic hypertension and no presumed contraindication to any one class of antihypertensive medications. One limitation to our study relates to the fact that comparisons were based on costs of monotherapies, while combination therapies are frequently needed to control blood pressure.The number-needed-to-treat to prevent one adverse outcome has become a widely used measure of treatment benefits in medical community, which is easy for physicians to understand. The shortcomings of NNT are that the outcome measure of an effect is with one dimension- survival probability and that it measures the specified outcome at a single point in time. Therefore, a measure of NNT can not capture an outcome in effectiveness of the intervention with two dimensions: time and survival probability. These limitations may not allow us to take time and discounting on cost and effect into account in this study.Based on our findings, antihypertensive therapy that is diuretic-based and that includes either low-dose reserpine or atenolol represents a cost-effective regimen in preventing or delaying cardiovascular events in older adults. Use of the diuretic-based therapy is the most cost-effective in patients at high risk for developing cardiovascular disease. These results suggest that clinicians should consider using diuretics plus low-dose reserpine or atenolol as first-line therapy in patients with isolated systolic hypertension who are greater than 60 years old when there are no contraindications among these patients.ACE: angiotensin-converting enzymeALLHAT: Antihypertensive and Lipid Lowering treatment to prevent Heart Attack TrialARR: absolute risk reductionAWP: average wholesale priceBP: blood pressureCHD: coronary heart diseaseCHF: congestive heart failureCVD: cardiovascular diseaseDBP: diastolic blood pressureHDL: high density lipidISH: isolated systolic hypertensionJNC IV The Sixth Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure.NNT: number-needed to treatSBP: systolic blood pressureSHEP: Systolic Hypertension in the Elderly ProgramThe author(s) declare that they have no competing interests.GC, LF, WM and MP participated the development of the analytic framework. GC performed all data analyses. GC, LF, WM and MP drafted and revised the manuscript. All authors approved the final manuscript.

Helicobacter pylori diagnosis and susceptibility profile directs the applicability of recommended treatment regimens in our setting. To our knowledge, there is no published data on the culture and local susceptibility pattern of Helicobacter pylori in the Philippines.52 dyspeptic adult patients undergoing endoscopy from the Outpatient Gastroenterology clinic of the University of the Philippines-Philippine General Hospital underwent multiple gastric biopsy and specimens were submitted for gram stain, culture, antimicrobial sensitivity testing, rapid urease test and histology. Antimicrobial susceptibility testing was done by Epsilometer testing (Etest) method against metronidazole, clarithromycin, amoxicillin, and tetracycline.H. pylori infection (mean age of 44 years ± 13), 70% were males. H. pylori culture showed a sensitivity of 45% (95% CI [29.5–62.1]), specificity of 98% (95%CI [81.5–100%]), positive likelihood ratio of 19.93 (95% CI [1.254–317.04]) and a negative likelihood ratio of 0.56 (95% CI [0.406–0.772]). All H. pylori strains isolated were sensitive to metronidazole, clarithromycin, amoxicillin and tetracycline.Sixty percent (60%) of the study population was positive for Knowledge of the antibiotic susceptibility patterns in our setting allows us to be more cautious in the choice of first-line agents. Information on antibiotic susceptibility profile plays an important role in empiric antibiotic treatment and management of refractive cases. Helicobacter pylori is a gram-negative bacterium that colonizes the gastric mucosa of more than half of the world's population ), positive likelihood ratio of 19.93 (95% CI [1.254–317.04]) and a negative likelihood ratio of 0.56 (95% CI [0.406–0.772]). The positive predictive value was 97% (95% CI [74.7–99.7%])) and the negative predictive value was 55% (95% CI [39.8–69.7%]).All included patients underwent the 3 diagnostic tests for the diagnosis of ) figure . FourteeH. pylori organisms were isolated from 52 clinical specimens. The mean number of incubation time was 3.8 days ± 1 day. All isolates grew on primary plates. All isolates were highly sensitive to amoxicillin (mean MIC of 0.016 ug/ml by Etest)), tetracycline (mean MIC of 0.164 ug/ml SD ± 0.16 SD by Etest), metronidazole (mean MIC of 0.061 ug/ml SD ± 0.04 by Etest) and clarithromycin (mean MIC of 0.016 SD ± 0 by Etest) . A study of the World Health Organization's Programme for appropriate Health Care Technology (ATH) has shown a correlation between the occurrence of multi-resistant bacteria and antibiotic consumption patterns. The Philippines has the highest percentage in 1983 of antibiotic utilization among countries surveyed (>25%). However, majority of the people whether rich or poor allot minimum expenses for medical care at 2.7% and 1.2%, respectively[H. pylori has been well documented in other studies, the absence of primary resistance in our results may also be an underestimate of the true prevalence of H. pylori resistance because of the smaller sample size compared with published literature[H. pylori in the Philippines.Potential reasons for the absence of resistant pectively. Such paurveyed >%. Howeveiterature. Only wiH. pylori infection approximates that in published literature abroad. In the absence of standard disk diffusion zone sizes for regimens used in H. pylori eradication regimen except for metronidazole, further establishment of the susceptibility pattern of locally occurring isolates by comparing zone size breakpoints with Etest, agar dilution method and as well as molecular genotyping of resistant strains will be the future direction of this pilot study.Knowledge of the antibiotic susceptibility patterns in our setting allows us to be more cautious in the choice of first-line agents. The use of culture technique in the diagnosis of H. pylori infection, information on antibiotic susceptibility profile plays an important role in empiric antibiotic treatment and management of refractive casesWhile the use of culture is not an ideal test for the rapid diagnosis of H. pylori, manuscript writing and editingRVD, LJB, CSA contributed in the microbiologic isolation of EDL, MOD, VD contributed in the specimen processing of biopsy samples, manuscript writing and editingRLG contributed in the research design planning, manuscript content and final editing

Prostate cancer is a leading killer of men in the industrialized world. Underlying this disease is the aberrant action of the androgen receptor (AR). AR is distinguished from other nuclear receptors in that after hormone binding, it preferentially responds to a specialized set of coactivators bearing aromatic-rich motifs, while responding poorly to coactivators bearing the leucine-rich “NR box” motifs favored by other nuclear receptors. Under normal conditions, interactions with these AR-specific coactivators through aromatic-rich motifs underlie targeted gene transcription. However, during prostate cancer, abnormal association with such coactivators, as well as with coactivators containing canonical leucine-rich motifs, promotes disease progression. To understand the paradox of this unusual selectivity, we have derived a complete set of peptide motifs that interact with AR using phage display. Binding affinities were measured for a selected set of these peptides and their interactions with AR determined by X-ray crystallography. Structures of AR in complex with FxxLF, LxxLL, FxxLW, WxxLF, WxxVW, FxxFF, and FxxYF motifs reveal a changing surface of the AR coactivator binding interface that permits accommodation of both AR-specific aromatic-rich motifs and canonical leucine-rich motifs. Induced fit provides perfect mating of the motifs representing the known family of AR coactivators and suggests a framework for the design of AR coactivator antagonists. A structural study of the androgen receptor shows how it interacts with coactivator proteins. The results also provide a possible framework for the development of new receptor antagonists The androgen receptor (AR) is the cellular mediator of the actions of the hormone 5-α dihydrotestosterone (DHT). Androgen binding to AR leads to activation of genes involved in the development and maintenance of the male reproductive system and other tissues such as bone and muscle. However, it is the pivotal role of AR in the development and progression of prostate cancer that has led to increasing interest in this nuclear receptor. Presently, hormone-dependent prostate cancer is treated with a combination of strategies that reduce circulating levels of androgens, such as the administration of antiandrogens that compete for the androgen-binding pocket in the core of the C-terminal ligand-binding domain (LBD). The benefits of these treatments are typically transient, with later tumor growth associated with increases in expression levels of AR or its cofactors, or mutations that render AR resistant to antiandrogens . AlternaThe precise details of how AR binds the dozens of coregulator proteins reported to associate with different regions of AR in vivo remain poorly understood . Many nuAR, however, utilizes multiple mechanisms to activate gene transcription. Generally, AR activity is dependent on contributions from multiple transactivation functions that lie within the N-terminal domain (NTD) collectively called AF-1. Although the AR AF-2 can bind to a restricted set of LxxLL motifs and is rPresently, how the AR AF-2 surface can accommodate residues with bulky aromatic side chains and distinguish FxxLF motifs from LxxLL motifs is not known. To understand the structural basis of this unusual coactivator recognition preference, we characterized the full repertoire of interacting sequences using phage display to define amino acids preferred at the AR coactivator binding interface. Crystal structures of the AR LBD in complex with several phage display–derived peptides reveal the structural basis of FxxLF motif specificity and an induced fit of the receptor that allows accommodation of other related hydrophobic motifs. Comparisons of the structures suggest strategies for the design of AR coactivator antagonists.10 randomized peptides against DHT-bound AR LBD. Selections identified sequences containing hydrophobic motifs that were primarily aromatic in character, consistent with another recent study (Phage display has been used to study coactivator recognition specificity and to identify coactivator motif sequence variants preferred by the estrogen receptor (ER), thyroid hormone receptor (TR) β, and most recently AR . Using pnt study . SurprisP212121 with one molecule per asymmetric unit and unit cell dimensions similar to those observed in previous AR LBD crystal structures reveals that the AF-2 cleft reorganizes to accommodate the bulky peptide side chains see B and 4. The preference of AR for motifs with aromatic groups over leucine-rich motifs was assessed with a crystal structure of the AR LBD in complex with the LxxLL peptide. The structure reveals similarities between the binding modes of the LxxLL and FxxLF motifs to AR, and other LxxLL motifs to other nuclear receptors. The LxxLL motif adopts a helical conformation, and interactions of the motif with the AF-2 cleft are predominantly hydrophobic, with the three leucine residues of the motif contributing most of the interactions. However, significant differences can be seen between the binding mode of the LxxLL motif to AR and that of p160-derived LxxLL motifs to other nuclear receptors. First, flanking residues were largely disordered, with only two N-terminal flanking residues and one C-terminal residue visible in electron density maps see and 3B. Side chains of residues flanking the first leucine of the motif make additional hydrophobic interactions with the AR surface see B. Trp+2 To understand how the AR AF-2 accommodates tryptophan residues, structures of AR in complex with peptides containing tryptophan substitutions at the +1 or +5 position, or both, were determined . SurprisFinally, effects of substitutions at the +4 position were assessed in structures of AR in complex with peptides containing FxxFF and FxxYF motifs . SurprisBy contrast, the conformation of FxxYF was the closest to FxxLF see A. Other Together, the structures described above permit an assessment of the way that individual subsites of the AR AF-2 cleft accommodate hydrophobic groups. The indole rings of tryptophan and the phenyl rings of phenylalanine fit into their pockets analogously with the +1 and +5 residues bound facedown and edgewise, respectively, into the AF-2 cleft. On the other hand, the position of the +4 residue is variable, with binding in this shallow pocket largely dictated by the position of the peptide backbone caused by the bound conformations of the +1 and +5 residues see C. Small The binding mode detected in the +1 pocket is the most conserved of the three hydrophobic subsites see C. The beBinding of tryptophans in the +5 pocket is slightly more variable see C. Trp+5 The crystal structures reported here reveal how AR binds coactivator motifs with bulky aromatic hydrophobic groups and permit construction of a profile of the AR coregulator interface see . In someThe same characteristics that make the AR AF-2 ideal for binding of longer, aromatic side chains also make it less well suited for binding of shorter, branched side chains. Although changes in the position of Met734 widen the groove towards the +5 subsite to permit binding of leucine residues, the gross features of the groove remain largely the same see B. As a rUnlike the conserved interaction modes of aromatic residues with the +1 and +5 sites, binding interactions at the +4 site are variable and characterized by nonspecific interactions. This finding agrees with the relatively high conservation of residues at the +1 and +5 positions of AR-interacting motifs and suggests that these residues drive peptide interaction with the LBD, whereas the +4 site is less critical. Indeed, the +4 pocket is shallow, surface exposed, and relatively featureless, explaining the assortment of residues selected at the +4 position. It is likely that any hydrophobic residue that does not clash with surrounding residues would be suitable at this subsite.While peptide motif recognition is governed by hydrophobic interactions, polar interactions from backbone atoms and residues outside the core motif also contribute. With the exception of FxxFF, motifs containing phenylalanines at the +1 and +5 positions present canonical main chain interactions with both charge clamp residues, Lys720 and Glu897. This finding stands in contrast to predictions of previous studies , which cOn the other hand, Glu897 interacts with the FxxLF peptide backbone, but is disengaged from the LxxLL peptide backbone. One possible explanation for the apparent requirement for Glu897 in LxxLL binding is that it might interact with residues outside of the core motif. The corresponding glutamate of GR, Glu 755, forms hydrogen bonds with the −3 asparagine of TIF2 NR box 3 , and GluSequence alignment of NR coactivator sequences shows that positively charged residues are favored N-terminal to the core hydrophobic motif while negatively charged residues are favored C-terminal to the motif . Our phaFinally, the AR AF-2 surface is an attractive target for pharmaceutical design. Selective peptide inhibitors that bind the AF-2 surface of liganded ERα, ERβ, and TRβ have been developed , and simE. coli strain BL21 (DE3) STAR in the presence of 10 μM DHT. Induction was carried out with 30 μM IPTG at 17 °C for 16–18 h. E. coli cells were lysed in buffer supplemented with 0.5 μg/ml lysozyme, 5 U/ml benzonase, 0.5% CHAPS, and 10 μM DHT. All buffers for further purification steps contained 1 μM DHT. Soluble cell lysate was adsorbed to Glutathione Sepharose 4 Fast Flow resin (Amersham Biosciences), washed with buffer containing 0.1% n-octyl β-glucoside, and eluted with 15 mM glutathione. After cleavage of the GST moiety with thrombin, final purification of the AR LBD was carried out using a HiTrap SP cation exchange column (Amersham Biosciences). Eluted AR LBD was dialyzed overnight at 4 °C against buffer containing 50 mM HEPES (pH 7.2), 10% glycerol, 0.2 mM TCEP, 20 μM DHT, 150 mM Li2SO4, and 0.1% n-octyl β-glucoside, then concentrated to greater than 4 mg/ml for crystallization.Expression and purification of the AR LBD for crystallization were performed essentially as described . The cDNE. coli biotin ligase (Avidity) into BL21 (DE3) STAR cells. Protein expression was carried out as above but with induction supplemented with 50 μM biotin to ensure quantitative biotinylation of AR LBD.Purification of AR LBD for use in phage affinity selection was carried out as above without the final dialysis and concentration steps. The expression construct contained the AR LBD as an inframe fusion with GST in a modified pGEX-2T vector containing both a flexible region and an AviTag sequence allowing in vivo biotinylation. The GST–AR LBD fusion expression plasmid was cotransformed with a plasmid-encoding 10 different random or biased amino acid sequences were added to the wells containing immobilized AR LBD and incubated for 3 h at 4 °C. After washing, bound phage were eluted using pH 2 glycine. Enrichment of phage displaying target-specific peptides was monitored after each round of affinity selection using an anti-M13 antibody conjugated to horseradish peroxidase in an ELISA–type assay.Phage affinity selections were performed essentially as described . BiotinySynthetic peptides corresponding to the deduced amino acid sequences from receptor-specific phage were tested for their ability to interact with purified AR LBD using a FRET–based assay format. Peptides were synthesized according to the deduced amino acid sequence displayed on phage with an additional C-terminal amino acid sequence consisting of SGSGK to allow the attachment of a biotin tag . Flourophor conjugates were prepared by incubating either biotinylated peptides with streptavidin-cryptate , or biotinylated AR LBD with streptavidin-XL665 (Cis Bio). Interaction between peptide and AR LBD was monitored by the ratio of energy transfer by excitation at 320 nm and emission at 625 nm and 665 nm.Affinities of peptides to the AR LBD were determined with a Biacore 2000 instrument. A peptide derived from silencing mediator for RXR and TR 2 (SMRT2) served as a negative control. 1 mM peptide stock solutions in DMSO were diluted into HBS-P buffer to generate 10 μM working solutions. HBS-P buffer was flowed through the cells to achieve a stable baseline prior to immobilization of the biotinylated peptides. To achieve the binding of approximately 250 RU of peptides to individual cells, working solutions of peptides were diluted to 100 nM in HBS-P buffer. Unbound streptavidin sites were blocked by injection of a 1 mM biotin solution at a rate of 10 μl/min.Purified AR LBD was diluted into HBS-P buffer to a concentration of 10 μM and injected into all four Flowcells using the Kinject protocol at a flow rate of 10 μl/min . Following the dissociation phase, the surface of the chip was regenerated to remove residual AR LBD by QuickInject of buffer containing 10 mM HEPES and 50% ethylene glycol (pH 11). Following the establishment of a stable baseline, the same procedure was repeated using a series of AR LBD dilutions in an iterative manner. Analysis of the data was performed using BIAevaluation 3.0 software (Biacore). The SMRT2 signals were subtracted as background from the three remaining peptide signals. Data were best fit using the two-state conformational change model , 2002.Purified, concentrated AR LBD was combined with 3x to 6x molar excess of peptide and incubated 1 h at room temperature before crystallization trials. Complexes were crystallized using the hanging drop vapor diffusion method. Protein–peptide solution was combined in a 1:1 ratio with a well solution consisting of 0.6–0.8 M sodium citrate and 100 mM Tris or HEPES buffer (pH 7–8). Crystals typically appeared after 1–2 d, with maximal size attained within 2 wk. For data collection, crystals were swiped into a cryo-protectant solution consisting of well solution plus 10% glycerol before flash freezing in liquid nitrogen. The addition of ethylene glycol to a well concentration of 10%–20% was later found to both improve crystal quality and enable the freezing of crystals directly out of the drop.A-weighted 2Fo − Fc, Fo − Fc, and simulated annealing composite omit maps. Superposition of structures was performed with LSQMAN , beamline 8.3.1, with either a ADSC Quantum 315 or Quantum 210 CCD detector. Data were processed using Denzo and Scalepack . Moleculh LSQMAN . Buried h LSQMAN . Coordinhttp://www.ebi.ac.uk/swissprot) accession numbers for the gene products discussed in this paper are AR (P10275), ARA54 (Q9UBS8), ARA55 (Q9Y2V5), ARA70 (Q13772), ER , glucocorticoid receptor-interacting protein 1 NR box 3 (Q61026 ), GR (P04150), NR box 3 of TIF2 (Q15596), and TR β (P10828).The Swiss-Prot (http://www.rcsb.org/pdb) accession numbers for the structures used in this paper are FxxFF (1T73), FxxLF (1T7R), FxxLW (1T79), FxxYF (1T7M), LxxLL (1T7F), unbound (1T7T), WxxLF (1T74), and WxxVW (1T76).The Protein Data Bank (

One of the most time-consuming tasks after performing a gene expression experiment is the biological interpretation of the results by identifying physiologically important associations between the differentially expressed genes. A large part of the relevant functional evidence can be represented in the form of graphs, e.g. metabolic and signaling pathways, protein interaction maps, shared GeneOntology annotations, or literature co-citation relations. Such graphs are easily constructed from available genome annotation data. The problem of biological interpretation can then be described as identifying the subgraphs showing the most significant patterns of gene expression. We applied a graph-based extension of our iterative Group Analysis (iGA) approach to obtain a statistically rigorous identification of the subgraphs of interest in any evidence graph.We validated the Graph-based iterative Group Analysis (GiGA) by applying it to the classic yeast diauxic shift experiment of DeRisi et al., using GeneOntology and metabolic network information. GiGA reliably identified and summarized all the biological processes discussed in the original publication. Visualization of the detected subgraphs allowed the convenient exploration of the results. The method also identified several processes that were not presented in the original paper but are of obvious relevance to the yeast starvation response.GiGA provides a fast and flexible delimitation of the most interesting areas in a microarray experiment, and leads to a considerable speed-up and improvement of the interpretation process. Microarray experiments can provide a comprehensive picture of gene expression levels in biological samples. In a typical application they compare expression of several thousand genes under two different conditions , using a small number of replicate experiments. Various techniques have been developed to rank genes according to their expression changes, e.g. based on the t-statistic or the sThe main challenge to the biologist is contained in the next step of the analysis. It consists in identifying the biologically relevant expression changes, the "big picture" of the experiment. As microarray experiments tend to generate unexpected observations in areas outside the specialized expertise of the experimentalist, this can be quite difficult and time-consuming. A principled mechanism to identify the significant higher-level features of the experimental results would therefore be very useful.The biological interpretation process consists to a large extent of finding evidence connecting certain genes that are differentially expressed. This evidence can consist, e.g., of joint participation in some physiological process, physical interaction at the protein level, reported co-expression in earlier microarray experiments, a shared functional annotation, etc. This kind of evidence can intuitively be represented as a graph, and this feature is regularly used to visualize biological data, in the form of metabolic or signaling pathways or protein interaction maps. The task can then be described as the identification of subgraphs that as a whole show a statistically significant expression change. This would allow the biologist to focus her analysis on the most promising areas, without prior bias, while at the same time presenting the relevant evidence underlying each association for critical evaluation.We have recently developed an approach, iterative Group Analysis (iGA) that identifies significantly changed functional classes of genes in a microarray experiment . In contHere we extend this approach to the analysis of "evidence graphs", which offers much larger flexibility of the annotations that can be used and allows substantially improved visualization. Evidence graphs can be represented as bigraphs with two types of nodes, one for genes and one for the associated "evidence" Fig. . For evam) and, if present, all adjacent nodes of ranks equal or smaller than m. Hence, at each step of the extension process the newly extended subgraph is not adjacent to any outside node with a rank lower than m or unchanged based on an arbitrary selected significance cut-off and thus discards most of the relative-change information (gene ranks) used by GiGA . Therefore, this method is difficult to apply to very noisy or unreplicated experiments where a reliable delimitation of the "changed" genes becomes impossible.The recently released commercial Pathway Analysis software from Ingenuity Systems To validate the GiGA approach, we used the yeast diauxic shift experiment by DeRisi et al. . In thisIn their original publication, DeRisi et al. highlight the following changes during starvation: Rechanneling of metabolites into the tricarboxylic acid (TCA) and glyoxylate cycle, increase in aerobic respiration (cytochrome c oxidase and reductase), gluconeogenesis, and carbohydrate storage . In contrast, 95% of ribosomal proteins, as well as tRNA synthetases and translation elongation/initiation factors were strongly down-regulated. Twenty hours after the initial inoculation of the sample about 20% of all genes showed at least a two-fold change in expression.Table This connectivity between genes and functional classes is provided by GiGA. Table The performance of GiGA (as well as iGA) is best appreciated when compared to the results of an extensive expert interpretation of the same data. Table It is important to be aware that each of the highlighted subgraphs has to be carefully evaluated for its biological relevance. On the one hand, the sheer number of possible subgraphs in an evidence network creates a major multiple-testing problem, which means that some of the detected associations may be due to chance. Random permutations of the expression data – which can be generated by the GiGA software – can give an idea of the expected false-discovery rate. On the other hand, functional annotations are at present notoriously unreliable and spurious edges may affect the details of the results. Also, not all genes within a detected subgraph will necessarily show a strong expression change, because sometimes less strongly affected genes may connect those genes that do change. Such a relation is for example expected for many transcription factors and their targets . Nonethem1 and m2 whenever their mass difference (Δm = |m1 - m2|) can be explained by a common biochemical transformation (e.g. dehydrogenation: Δm = 2* mass(hydrogen)). The set of relevant transformations can easily be collected from any biochemistry textbook. In addition, one can introduce edges for condensation reactions between observed masses, i.e. if m1 + m2 = m3 + mass(H2O) then edges between m1 and m3, and m2 and m3 are added to the evidence network. We are currently developing the application of GiGA to this kind of data in the Sir Henry Wellcome Functional Genomics Facility at the University of Glasgow ; data not shown).The GiGA method is not restricted to use with exhaustively annotated genomes. It can work on a wide variety of "evidence" to build the necessary network, including hypothetical predicted functions or associations. It is even possible to apply GiGA to metabolomics results, which are characterized by the absence of any significant amount of annotation – usually only exact molecular masses and their differential abundance are known. In that case, an evidence network can be built from the measured masses themselves, linking compounds The present analysis of a biologically well-understood test case demonstrates the reliable performance of GiGA. The method automatically identifies all relevant physiological processes, puts them into context, summarizes them in an intuitive format, and associates them with the underlying evidence Fig. and 3. I. GeneOntology annotations were obtained from Affymetrix . The enzyme substrate networks were built based on information contained in the annotation of the yeast proteome in the SwissProt database . The GiGA algorithm has been implemented as a Perl script and compiled as a Windows command line executable. These files are available as Yeast gene expression data for the diauxic shift experiment were obtained from the Stanford Microarray Database RB devised and implemented the GiGA technique and drafted the manuscript. AA and PH supervised the project. All authors read and approved the final manuscript.GiGA program. For use from the Windows command line.Click here for fileGiGA source code.Click here for fileGiGA manual. Describes the use of GiGA applied to the example data .Click here for fileGene expression data. Sorted list of genes, based on expression during the yeast diauxic shift.Click here for fileEvidence network. List of gene pairs connecting genes whenever their gene products are enzymes that share a common substrate. Based on annotation derived from SwissProt.Click here for fileGenenames file. Contains descriptive names of the yeast genes contained in Additional file 4.Click here for fileExample output in text format. List of significantly affected subgraphs detected in the experimental data using GiGA with default settings.Click here for file.Example output in graph-description language format. Contains the same results as Additional file 7, but in a format that can be visualized and explored using graph-layout software, e.g. aiSee, which is freely available for academics at Click here for file

The early stages consecutive to infection of sheep (e.g. primo-infection) by Bovine leukemia virus mutants are largely unknown. In order to better understand the mechanisms associated with this period, we aimed at analyzing simultaneously three parameters: B-lymphocytosis, cell proliferation and viral replication.Sheep were experimentally infected either with a wild type BLV provirus or with selected mutants among which: a virus harboring an optimalized LTR promoter with consensus cyclic AMP-responsive elements, two deletants of the R3 or the G4 accessory genes and a fusion-deficient transmembrane recombinant. Seroconversion, as revealed by the onset of an anti-viral antibody response, was detected at 3 to 11 weeks after inoculation. At seroconversion, all sheep exhibited a marked increase in the numbers of circulating B lymphocytes expressing the CD5 and CD11b cluster of differentiation markers and, interestingly, this phenomenon occurred independently of the type of virus. The net increase of the absolute number of B cells was at least partially due to accelerated proliferation as revealed, after intravenous injection of bromodeoxyuridine, by the higher proportion of circulating BrdU+ B lymphocytes. BLV proviral DNA was detected by polymerase chain reaction in the leucocytes of all sheep, as expected. However, at seroconversion, the proviral loads were lower in sheep infected by the attenuated proviruses despite similar levels of B cell lymphocytosis.We conclude that the proviral loads are not directly linked to the extent of B cell proliferation observed during primo-infection of BLV-infected sheep. We propose a model of opportunistic replication of the virus supported by a general activation process of B lymphocytes. Bovine leukemia virus (BLV) is an oncogenic retrovirus closely related to the primate T-cell leukemia viruses . These vA major advantage of the BLV system is the possibility to study viral genetic determinants in relation with infectivity and pathogenicity in vivo. A strategy, which we previously described, is based on the use of a cloned BLV provirus whose sequence can be mutagenized in vitro. Well characterized mutants can subsequently be injected into sheep and compared to the wild type virus (WT) . This ex(i) the R3 and G4 accessory genes are required for efficient viral spread in vivo, although their deletion or mutation does not hamper infectivity .(ii) restoring a CRE consensus in the triplicate motif of the imperfectly conserved Tax-responsive sequence increases LTR promoter activity, as expected, but restricts the proviral loads in vivo, suggesting that repression of expression is required for immune escape .(iii) formation of multinucleated syncytia by envelope dependent cell fusion in vitro is paradoxically not required for infectivity or efficient viral spread in vivo .Importantly, only the A60V envelope mutant behaves as wild type in terms of infectivity and pathogenesis, in contrast to the others therefore referred to as attenuated. The two categories of viruses, either wild type (WT and A60V) or attenuated thus permit to characterize and compare the processes occurring during primo-infection.+ (triangles) and CD8+ (crosses) T cell populations remained remarkably constant over extended periods of time, as expected and mutants deleted in the R3 and G4 genes (CRX3 and IG4 proviruses) are impaired in their ability to propagate efficiently within their host ,19,21. T3/ml of blood to 4,858 103/ml, respectively, at days 21 and day 28; see squares), while the absolute counts of CD4+ (triangles) and CD8+ (crosses) T cells remained relatively constant . Furthermore, maximal B cell accumulation corresponded to the day of seroconversion characterized by the onset of an anti-BLV humoral response , the latter behaving as wild type in terms of pathogenesis and infectivity in vivo. Interestingly, B cell lymphocytosis occurred in all sheep independently of the type of provirus Figure was indeExpression of the CD5 and CD11b cluster of differentiation markers has been associated with BLV-infection, although B lymphocytes negative for these receptors are also less efficient targets for the virus ,24. TherIn terms of absolute cell counts , it appeared that B+CD5+ or B+CD11b+ lymphocytes accumulated at the seroconversion day in wild type infected sheep 4535 and 4536 . Figure Since BLV mutants also induce a transient lymphocytosis, a kinetics of BrdU incorporation was performed in 4 additional sheep infected with proviruses IG4, CRX3, CRE and GP30 yielded a 1 kb fragment as expected. Under similar conditions, a weaker signal was generated by amplification of genomic DNA isolated from animals infected by mutant proviruses or so-called attenuated mutants . Experimental infection of sheep with these two types of viruses led to a surprising observation, namely, a similar extent of transient lymphocytosis independently of the proviral loads . AnswerTrypanosoma evansi and Pasteurella haemolytica [With the aim to correlate lymphocytosis with a defined B cell sub-population, we have demonstrated that two surface molecules, CD5 and CD11b, whose expression has been previously associated with late stages of BLV infection -15,23,24molytica ,36. In tDifferences in the infectious potential of the attenuated viruses are the consequence of mutations in accessory genes (R3 and G4) or in the LTR promoter (CRE) ,21. AlthIn conclusion, we have characterized here the initial steps consecutive to BLV infection of sheep. We show that this period is characterized by a transient accumulation of CD5+ / CD11b+ B lymphocytes resulting, at least in part, from increased proliferation. Furthermore, the extent of B cell lymphocytosis is not directly linked to the proviral loads reached by the wild type and mutant viruses. On basis of a comparative leukemia approach, these results could be informative for the related human T-lymphotropic viruses.Roche Diagnostics) in 1 ml of HBS and injected intradermally into the back of each sheep. Plasmids containing the mutant proviruses pBLVIG4 techniques [Twelve sheep of one year old were kept under controlled conditions at the Veterinary and Agrochemical Research Centre . Two animals (n° 4533 and 4534) were used as uninfected controls whereas sheep n° 4535 and 4536 were experimentally infected with a BLV wild type cloned provirus (strain 344) . Brieflyg frame; , pBLVCRXg frame; ) and pBLg frame; ) were inchniques .VMRD Inc. Cells were then labeled with a rat anti-mouse IgG1 phycoerythrin (PE)-antibody (Becton Dickinson Immunocytometry Systems) or with a goat anti-mouse IgG2b fluorescein isothiocyanate (FITC)-conjugate . Finally, PBMCs were analyzed by flow cytometry on a Becton Dickinson FACScan flow cytometer. Ten thousand events were collected for each sample and data were analyzed with the Cellquest software (Becton Dickinson Immunocytometry Systems).Peripheral blood mononuclear cells (PBMCs) were isolated by Percoll gradient centrifugation and their viability was estimated by trypan blue dye exclusion ). PBMCs Sigma Aldrich) resuspended in physiologic serum (NaCl 0.9%). To evaluate BrdU-incorporation, blood was collected at three and seven days after each BrdU injection. The red blood cells were lysed with 1× FACS Lysing Solution (Becton Dickinson Immunocytometry Systems), the leucocytes were washed twice with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich) and incubated in the presence of biotinylated 1H4 monoclonal antibody for 30 min at 4°C. Next, the cells were labeled with streptavidin-phycoerythrin (Becton Dickinson Immunocytometry Systems) and incubated with 1× FACS Permeabilizing Solution (Becton Dickinson Immunocytometry Systems). Finally, leucocytes were stained with anti-BrdU FITC antibody in the presence of DNase (Becton Dickinson Immunocytometry Systems) and analyzed by flow cytometry.Each week during two months, sheep were injected intravenously with 500 mg of 5-bromo-2'-deoxyuridine (Wizard®Genomic DNA Purification Kit (Promega). An aliquot of 300 μl of blood were mixed with 900 μl of Cell Lysis Solution and incubated for 10 minutes at room temperature. After two washes with the same buffer, the cells were resuspended in 300 μl of Nuclei Lysis Solution and incubated for one hour at 37°C. Then, the samples were digested during 15 minutes at 37°C in the presence of 1.5 μl of RNase Solution. Proteins were precipitated by adding 100 μl of Protein Precipitation Solution to the nuclear lysates. After centrifugation at 13,000 g, the supernatant was mixed with an equal volume of isopropanol, centrifuged and ethanol precipitated. Five hundred nanograms of the purified DNAs were amplified in the presence of 200 μM of deoxynucleotides, 2.5 U of Taq DNA polymerase, and 200 ng of primers. The primers used (PCRTA 5'-CTCTTCGGGATCCATTACCTGA-3' and PCRTC 5'-CCTGCATGATCTTTCATACAAAT-3') encompass the region from position 7999 to 6990 of the BLV tax gene [gapdh gene. The samples were denatured for 5 min at 94°C, and amplified by 25 cycles of PCR . After a final elongation step of 10 min at 72°C, 20 μl of the amplification products were resolved on a 1% agarose gel, transferred to a Hybond N+ membrane (Amersham Pharmacia Biosciences), and hybridized either with a BLV tax (a 1-kb ClaI insert from plasmid pGEM7zfLOR1) or with a gapdh probe labeled with α-32P dCTP. Quantification of 32P signal was performed using a PhosphorImager .DNA isolations were performed directly on blood using the tax gene . In parapol gene and the 18S ribosomal DNA sequences essentially as described in reference [Real-time PCR was performed using 6FAM-labeled MGB probes specific for the BLV eference .BLV: Bovine Leukemia Virus; BrdU: 5-bromo-2'-deoxyuridine; CRE: Cyclic-AMP Response Element; PBMCs: Peripheral Blood Mononuclear Cells; WT: Wild Type.The author(s) declare that they have no competing interests.CD carried out the most experimental work and drafted the manuscript. MS and FM performed the sample collections and the determination of the proviral loads. PK was responsible for the sheep studies. RK participated to experimental design and interpretation of data. LW conceived the study, its design and coordination. All authors read and approved the final manuscript

Diatoms are unicellular algae with ornate silica shells. Their dazzling ability to build tiny structures could inspire applications in the semiconductor industry, drug delivery, and engineering Thalassiosira pseudonana (http://genome.jgi-psf.org/diatom/).Diatoms, unicellular algae with ornate silica shells, have fascinated amateur and professional biologists ever since the invention of the microscope. But these days, diatoms and their exquisite shells are also attracting the attention of nanotechnologists who hope that diatoms will teach them how to make minute structures currently beyond the capabilities of materials scientists. And now these nanotechnologists, together with ecologists interested in the global carbon cycle—in which diatoms play a central role—have a genomic blueprint to help them in their studies: the annotated genome sequence of Diatoms, microalgae that are found in all aquatic and moist environments, first appeared more than 180 million years ago. Since then, diatom diversity has literally exploded; no one is sure how many living species there are—probably about 100,000—or why there are so many different types. Plant molecular biologist Chris Bowler explains that molecular phylogeny and morphological studies suggest that diatoms originated ‘probably as the result of a eukaryote being invaded or engulfed by a photosynthetic eukaryote, most probably a red alga’.The basic structure of all diatoms is similar: a single cell, often with a large vacuole, contained within a silica shell or frustule made of two overlapping halves or valves joined by girdle bands, which are also made of silica. The girdle bands form the rims of the two valves and allow unidirectional growth of the diatom during vegetative division. ‘The shell is rather like a Camembert cheese box or a petri dish’, explains marine ecologist Christian Hamm .There are only two main types of diatom: centric diatoms, which often have a circular symmetry, and pennate diatoms, which are usually bilaterally symmetrical. Nevertheless, diatom shells come in a dazzling array of forms and sizes ; Box 1. Richard Gordon, Professor of Radiology at the University of Manitoba in Winnipeg, Canada, somewhat accidentally laid the foundations of ‘diatom nanotechnology’ in 1988 when he was invited to give a lecture at an engineering conference. ‘I'm not an engineer’, explains Gordon, ‘but I knew engineers were interested in what was then called microfabrication so I told them about diatoms because they are so good at making small things’. Gordon, a keen diatom hobbyist, explained to his audience how diatoms could make a three-dimensional micro- or nanoscale structure for them without them lifting a finger. By contrast, says Gordon, ‘nanotechnology techniques then and now are tedious, involving painstakingly building three-dimensional structures up layer by layer’.Such tedious techniques are currently used in the semiconductor industry. At present, explains Michael Sussman, Director of the Biotechnology Center at the University of Wisconsin-Madison , ‘features are etched onto circuit boards using light. However, the wavelength of light limits the smallest size that can be achieved, and for the next generation of faster computers, engineers need to get denser features onto computer chips than is possible with light etching’. Diatoms, says Sussman, ‘are natural-born lithographers in the nanometre range. If we could work out how diatoms lay down micro lines of silica, then we may be able to simulate it’. The proteins that diatoms use to direct silica deposition could be very useful to the semiconductor industry, says Sussman.There are other ways in which diatoms could help us clumsy humans build nanoscale ‘widgets’. Molecular biologist Mark Hildebrand is a member of a collaborative project trying to develop genetically engineered micro/nanodevices . Already, engineers are using diatoms to help them build extremely sensitive sensors based on microfluidic devices, he explains. Hildebrand is also interested in the optical properties of diatoms. ‘Information processing technology is moving from electronically to optically based hardware, which allows more information to be carried and stored. Optical systems need materials with regularly repeating structures with features below the micrometre size range. These are very difficult to make by standard manufacturing techniques, but diatoms make structures like this all the time’.It might also be possible to use diatom shells as delivery vehicles for drugs, suggests chemical engineer Tony Rogers, an assistant professor at Michigan Technological University . ‘They have a uniform nanoscale pore structure and are chemically inert and biocompatible’. Rogers envisages loading diatoms with a drug that would then leach out into the blood stream at a rate dependent on the diatom species used. By incorporating ferromagnetic particles within the diatom structure, it might be possible to use a magnet to guide the drug to the right organ, he suggests.Diatom structures are not just of interest to people interested in tiny objects. As Hamm comments, ‘in diatoms, Nature has solved many of the problems that engineers want to solve. For example, diatoms are particularly good at making lightweight but strong structures. Because it is possible to scale static structures like shells, diatoms can teach us how to make lightweight constructions for the aerospace and car industry’.Some of the potential applications of diatoms can be investigated right now, using naturally occurring diatoms. In addition, subtle but important changes can be induced in diatoms by varying the amount of silica in their environment or changing the water flow. Gordon also envisages a device he calls a compustat, which would be used to select diatoms for a specific purpose. Diatoms taken from the sea, for example, would be individually examined using a computer-controlled microscope. ‘We would tell the computer what characteristics we were looking for, and it would go through the culture, zapping those diatoms furthest from the ideal with a laser beam. The culture would then be allowed to grow up again and the process repeated until we got the sort of diatoms we wanted’, says Gordon.Gordon has not built a compustat yet—it may not work, he says, because we don't know how far we can push diatoms by forced evolution. And even if the compustat does work, to make the most of the nanotechnological potential of diatoms, we need to know exactly how diatoms make their shells. At present, all we know is that silicon transporters and a group of long-chain, polyamine-containing proteins called silaffins, which act as nucleation points for silica deposition, are involved. This is where the diatom sequencing project at the United States Department of the Environment's Joint Genome Initiative (JGI) at Walnut Creek, California, comes in.T. pseudonana, a marine centric diatom ‘We believe that knowing this genome will help us to figure out how to mimic the processes that diatoms use to construct their very precise structures, and that we can then learn how to create similarly precise structures ourselves’. Also, he adds, diatoms are extremely important on an ecological level.Daniel Rokhsar, Department Head for Computational Genomics at JGI, explains why his institute undertook the sequencing and computer annotation of the genome of T. pseudonana, explains further. Diatoms are responsible for between 25% and 40% of all the primary productivity of the oceans, she says. ‘They also keep the biological pump going. By fixing carbon dioxide and then sinking, diatoms draw carbon dioxide out of the atmosphere and take it into the deeper waters of the ocean, where it is retained for longer than it would be if the diatoms stayed near the surface’.Oceanographer Ginger Armbrust , Principal Investigator on the sequencing project for T. pseudonana, she continues, was chosen as the first diatom to sequence in part because it has a small genome, but mainly because it represents a cosmopolitan genus of diatoms and its physiology has been well studied. Once the primary sequence of the genome had been determined, molecular biologists, oceanographers, and ecologists from around the world gathered at JGI for a ‘genome jamboree’. ‘The first of these was in October 2002, a massive brainstorming session at which we all dug around in the genome for our favourite genes and tried to get a feel for what was there’, explains Bowler. ‘It was really refreshing to get the insights of oceanographers and ecologists into what this genome was telling us’.T. pseudonana genome can tell them about the difference between photosynthesis on land and in the sea. They also want to investigate how these organisms adapt to their environment. ‘Now that we have the genome’, says Armbrust, ‘we can investigate how gene expression varies at different places in the water column, for example. This will be the first time a eukaryotic genome has been interpreted in this ecological sort of way’.Among other things, Armbrust and her collaborators are interested in finding out what the T. pseudonana genome is that we can figure out quite a bit from it about how this diatom deals with organic materials, but it is hard to figure out what it is doing with silicon’, admits Rokhsar. ‘The only way we can really figure out what a gene is doing is by comparing it with known genes in other organisms, but because diatoms are so unique in their use of silicon, we don't have that option. We literally just have the parts list’.‘One of the striking things about the T. pseudonana genes is important in silicon metabolism, Sussman is using microarrays to investigate how silicon concentrations affect gene expression patterns in the diatom. ‘There may be a few hundred genes whose expression changes in response to silicon stress’, he predicts, ‘and we can then focus on the role that these genes play in silicon metabolism’. In another approach, Hildebrand is purifying the proteins present in diatom shells. ‘Once we have isolated these proteins, we can get a little bit of protein sequence, and from there go back to the genome to pull the gene out’, he explains.To get a hook on which of the 10,000 or so T. pseudonana. Unfortunately, this can't currently be done. ‘The only diatom we can genetically manipulate is Phaeodactylum tricornutum, a pennate diatom’, explains Bowler. P. tricornutum, he says, is the ‘lab rat’ of the diatom world but is much less important ecologically than T. pseudonana by oceanographer David Thomas. ‘What we saw down the microscopes just blew our socks off’, says Parker-Eaton. ‘We could see how the plankton moved, the forms, the incredible different layers within the diatoms. What we particularly liked about diatoms was their complexity—they are totally unlike any other life form’.Navicula sp. The main body is sycamore, the ‘blobs’ are resin, and the object is coloured with inks. The piece is held together by magnets but splits in half to reveal two silver inserts at its centre. More examples of these artists' work can be seen at http://www.louiseandsarah.com.Parker-Eaton and Hibbert translate what they see down the microscope into objects made of wood and silver, usually small enough to hold in the hand.

Macaca nemestrina rhadinovirus-2 (MneRV2). These primers showed little similarity to the corresponding sequences of the macaque RV1 rhadinoviruses, retroperitoneal fibromatosis herpesvirus Macaca nemestrina (RFHVMn) and Macaca mulatta (RFHVMm). To determine viral loads per cell, an additional TaqMan QPCR assay was developed to detect the single copy cellular oncostatin M gene.Two distinct lineages of rhadinoviruses related to Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) have been identified in macaques and other Old World non-human primates. We have developed a real-time quantitative PCR (QPCR) assay using a TaqMan probe to differentially detect and quantitate members of the rhadinovirus-2 (RV2) lineage. PCR primers were derived from sequences within ORF 60 and the adjacent ORF 59/60 intergenic region which were highly conserved between the macaque RV2 rhadinoviruses, rhesus rhadinovirus (RRV) and 6 cells were detected in PBMC from randomly sampled macaques from the Washington National Primate Research Center. Screening tissue from other primate species, including another macaque, Macaca fascicularis, and a baboon, Papio cynocephalus, revealed the presence of novel rhadinoviruses, MfaRV2 and PcyRV2, respectively. Sequence comparison and phylogenetic analysis confirmed their inclusion within the RV2 lineage of KSHV-like rhadinoviruses.We show that the RV2 QPCR assay is linear from less than 2 to more than 300,000 copies using MneRV2 DNA, and is non-reactive with RFHVMn DNA up to 1 billion DNA templates per reaction. RV2 loads ranging from 6 to 2,300 viral genome equivalent copies per 10We describe a QPCR assay which provides a quick and sensitive method for quantitating rhadinoviruses belonging to the RV2 lineage of KSHV-like rhadinoviruses found in a variety of macaque species commonly used for biomedical research. While this assay broadly detects different RV2 rhadinovirus species, it is unreactive with RV1 rhadinovirus species which commonly co-infect the same primate hosts. We also show that this QPCR assay can be used to identify novel RV2 rhadinoviruses in different primate species. Rhadinovirus genus of the gammaherpesviruses are lymphotrophic and are associated with a variety of lymphoproliferative diseases. Herpesvirus saimiri (HVS), the prototype rhadinovirus isolated from the South American squirrel monkey, causes fulminant T-cell lymphomas in closely related host species [Members of the species . Kaposi' species . Other r species ,4.M. mulatta) in the New England National Primate Research Center (NENPRC) [M. nemestrina RV2 (MneRV2) [We and others have demonstrated the existence of two distinct lineages of KSHV-like rhadinoviruses in Old World non-human primates ,6. The r(NENPRC) and pig-(MneRV2) ,10,11. W(MneRV2) ,13, phyl(MneRV2) . Althoug(MneRV2) -17.While complete genomic sequences have been obtained for two closely related strains of the RV2 lineage rhadinovirus of rhesus macaques, RRV strain H26-95 from the NENPRC and RRV M. nemestrina, which has been previously shown to contain a co-infection of both MneRV-2 and RFHVMn rhadinoviruses [The ORF 59 and ORF 60 genes show high levels of homology between the related rhadinoviruses, KSHV and RRV, with 52% and 70% identity at the amino acid level, respectively . Using tMultiple alignment of the RRV and MneRV2 nucleotide sequences revealed large regions of identical sequences within both the ORF 59 and ORF 60 coding regions and the ORF 59/60 intergenic region. As shown in Figure M. nemestrina OSM gene (data not shown). Multiple alignment of the human, monkey and macaque OSM sequences revealed a region within exon 3 which was highly conserved. Using Primer Express software, a set of primers and a probe (OSM-FAM) were identified . We had ied Fig. which coThe RV2 and OSM QPCR assays were optimized using DNA obtained from the spleen of a rhesus macaque, MmuA01111, which we have previously determined to contain RRV DNA in a background of macaque genomic DNA . Initial5 copies of MneRV2, with a slope of -3.320 (100% efficiency) and r2 = 0.997. For the OSM assay, MmuA01111 genomic DNA was assayed in duplicate using 4-fold dilutions, with the amount of DNA tested ranging from 0.06 ng up to 1 μg . The assay was linear across this range with a slope of -3.322 (100% efficiency) and r2 = 0.999 was assayed in duplicate using 4-fold dilutions. As seen in Figure 999 Fig. .4 RRV copies per μg genomic DNA (MmuA01111 DNA undiluted) with a slope of -3.318 (100% efficiency) and r2 = 0.988 to more than 1.7 × 106 copies of the KSHV genome and a sample containing 109 copies of a PCR product of the ORF59/60 junctional region from RFHVMn were used as templates in the RV2 assay. The RV-2 QPCR assay was negative for these templates under the standard reaction conditions.To ensure that the RV2 assay does not detect RV1 viruses, the assay was performed using DNA from the human and macaque RV1 rhadinoviruses. A DNA sample from the KSHV infected BCBL-1 cell line containiM. mulatta and M. nemestrina, RRV and MneRV2, respectively, we tested to see if this assay could be used to identify a novel RV2 rhadinovirus in M. fascicularis. DNA was obtained from spleen tissue of Mfa95044, an M. fascicularis from the Tissue Distribution Program at the WaNPRC. Approximately 250 ng of spleen DNA produced a positive result in the RV2 QPCR assay with an average cycle threshold (CT) of 31.9 cycles. In order to prove that the assay detected a novel rhadinovirus, CODEHOP primers were used in a PCR amplification reaction with the Mfa95044 spleen DNA to obtain the ORF59/60 intergenic region of this rhadinovirus as described in the Materials and Methods. An 832 bp PCR product was obtained and sequenced. A comparison of this sequence with the corresponding region from RRV and MneRV2 showed 94% and 86% nucleotide identity, respectively. The nucleotide identity with the corresponding region in RFHV and KSHV was only 59% and 60%, respectively. Phylogenetic analysis showed a close clustering of the M. fascicularis sequence with the RRV sequence and a more distant relationship with the MneRV2 sequence, confirming its origin from an RV2 rhadinovirus of M. fascicularis, herein termed MfaRV2 , herein termed PcyRV2. Phylogenetic analysis demonstrated that while PcyRV2 clustered within the RV2 rhadinovirus lineage, it branched off separately from the macaque RV2 rhadinoviruses as expected for a baboon rhadinovirus [Previously, an RV2 rhadinovirus, PapRV2, was detected in a baboon ( anubis) , and a p anubis) to ampliT values . The cumulative fluorescence curve for the MneRV2 and RRV samples were superimposable with slopes typical of those seen in the assays performed in Figures M. fascicularis and baboon templates produced fluorescence curves with significantly decreased slopes, indicating lower amplification efficiencies. The efficiencies of these PCR reactions were calculated to be approximately 81% (r2 = 0.900) for the MfaRV2 and 72% (r2 = 0.929) for the PcyRV2, however, the low levels of virus in these samples made it difficult to accurately determine the efficiencies, as indicated by the correlation coefficients.In order to compare the ability of the RV2 QPCR assay to detect different rhadinovirus templates, test samples containing roughly equivalent viral copy numbers in a background of genomic DNA were prepared. DNA from purified MneRV2, DNA from MmuA01111 spleen which contains RRV, and DNA from Mfa95044 spleen which contains MfaRV2 were diluted in DNA from a virus negative macaque to have approximately the same virus load as that found in the baboon lymphocyte DNA containing PcyRV2. As shown in Figure The novel ORF 59/60 intergenic regions of MfaRV2 and PcyRV2 were aligned with the corresponding sequences of RRV, MneRV2, RFHVMn, and KSHV. Also aligned was a partial sequence of the ORF 59/60 region obtained from RFHVMm . As shown in Figure 6 cells and RV2 (71 bp), viral loads can even be determined in formalin-fixed paraffin embedded tissue in which significant degradation of the DNA has occurred. Due to the similarities in sequence of the human, macaque and African green monkey OSM genes, the OSM QPCR assay may be suitable for quantitation of DNA in tissue from a number of other Old World primate species.M. nemestrina, 1 of 7 M. fascicularis and 1 of 2 macaques whose species is not known. In these macaques, the viral copy number was determined to range from 6–2300 per 106 cells. Although the copy number in the single positive M. fascicularis was calculated to be 250 viruses per 106 cells, this would be a low estimate due to the 81% efficiency of the amplification of that template, as discussed above. Our results for RV2 rhadinoviruses in the macaque species tested at the WaNPRC were similar to those determined for RRV in rhesus macaques at the Tulane National Primate Research Center [6 cells. In the other 28 animals, the RRV load was below the level of detection. While RRV was detected more frequently in SIV-infected macaques in this study, the virus load was similar to that seen in healthy macaques.We have screened DNA from a number of random PBMC samples from macaques at the WaNPRC for the presence of an RV2 rhadinovirus. We detected RV2 rhadinovirus DNA in 6 of 30 macaques; 4 of 20 h Center . In the Macaca fascicularis) and the lymphocytes of a baboon . The standard RV2 assay had an amplification efficiency less than 100% with the M. fascicularis and P. cynocephalus templates which cautions against its use for accurate quantitation of the MfaRV2 and PcyRV2 rhadinoviruses. The primer and probe binding regions of these two rhadinoviruses showed nucleotide mismatches which correlate with the decrease amplification efficiency of the assay.The Tulane RRV assay had a similar sensitivity to our RV2 assay, with a lower limit of one RRV genome per 10,000 cell equivalents however, it was designed to specifically target only RRV while our RV2 assay is capable of detecting RRV, MneRV2 and other macaque and baboon rhadinoviruses. In this report, we have used the RV2 assay to detect novel RV2 rhadinovirus homologs in both the spleen of a crab-eating macaque 442N were provided by R. Shibata while at the National Institutes of Health, Bethesda, MD. This pig-tailed macaque had been experimentally infected with a pathogenic SHIV strain [Macaca mulatta (Mmu) YN91-224, an SIV-infected rhesus macaque diagnosed with RF, was kindly provided by H. McClure, Yerkes National Primate Research Center. Fresh frozen spleen tissue samples were also obtained from Macaca mulatta (Mmu) A01111 at the WaNPRC, a rhesus macaque that had been experimentally infected with SIV which we have shown to be co-infected with the RV1 and RV2 macaque rhadinoviruses, RFHVMm and RRV, respectively (unpublished observations). Fresh frozen spleen tissue from a Macaca fascicularis (Mfa) 95044 and lymphocytes from a baboon (Pcy78404) were kindly provided by H. Bielefeldt-Ohmann and C.-C. Tsai, respectively, from the WaNPRC. DNA from the PBMC of thirty random healthy colony macaques was also obtained from the virus screening program at the WaNPRC.Fresh frozen spleen tissue samples from V strain . We haveV strain . Fresh fThe KSHV-infected pleural effusion lymphoma cell line, BCBL-1, was obtained from D. Ganem (Howard Hughes Institute – UCSF), and was carried in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin, streptomycin, glutamine, and β-mercaptoethanol. Rhesus primary fetal fibroblasts (RPFF) were kindly provided by Dr. Michael Axthelm (ONPRC).M. nemestrina, MneJ97167, at the WaNPRC. The MneRV2 was used to infect cultures of RPFF and viral particles were harvested from culture supernatent by high speed centrifugation. Viral DNA used as positive controls in the PCR assays was obtained by disruption of the viral particles using phenol/chloroform and ethanol precipitation.An isolate of MneRV2, was obtained from an DNA was extracted from frozen tissues using standard proteinase K-phenol/chloroform extractions and concentrated by ethanol precipitation.The protein sequences of the ORF 59 and ORF 60 genes from KSHV and RRV were aligned using ClustalW. The consensus-degenerate hybrid oligonucleotide primer (CODEHOP) technique ,21 was uTo obtain the ORF 59/60 junctional regions between the RDEL motif of ORF 60 and the PQFV motif of ORF 59 of MneRV2, PcyRV2, RFHVMn, and RFHVMm, DNA was obtained from different sources and used in PCR amplification with different CODEHOP PCR primers. Reactions were performed in 1 μM forward and reverse primers, 200 μM each dNTP, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, and 2.5 units Platinum Taq polymerase (Invitrogen) using a 55–70°C annealing temperature gradient (BioRad Icycler). For MneRV2, PCR amplification was performed on Mne442N spleen DNA using primers RDELa and PQFVb. For PcyRV2, PCR amplification was performed on lymphocyte DNA from baboon Pcy78404, using SRDEa and QFVRb. In both cases an ~830 bp PCR fragment was obtained and sequenced. To obtain the sequence of RFHVMn which had a low copy number, it was necessary to amplify the RDEL-PQFV region in two fragments. A CODEHOP primer NFFEa . Phylogenetic tree output was produced using TreeView.The RV2 assay was designed to amplify a 71-bp amplicon from the ORF 59/60 junctional region of macaque viruses belonging to the RV2 rhadinovirus lineage using consensus primers "RV2a" (forward primer 5'-TCTGAATATGTCACATCCGTTCATA-3') and "RV2b" (reverse primer 5'-GGCCCGGAAAATGAGTAACA-3') with a TaqMan probe "RV2" 5'-(6-FAM)-TGATCTGTAGTCCCCATGTGTCC-(BHQ-1)-3' (Table T) determined using the Bio-Rad software. The viral load was calculated as a cellular genome copy equivalent by using the formula:Reactions (50 μl) contained approximately 250–1000 ng of template DNA, 1 μM forward and reverse primers, 100 nM probe, 200 μM each dNTP, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, and 2.5 units Platinum Taq polymerase (Invitrogen). Magnesium chloride concentrations were 4.0 mM for the RV2 assay and 2.0 mM for the OSM assay. After activation of the polymerase by incubation for 1 minute at 95°C, amplification was performed on a Bio-Rad iCycler equipped with an optical module for 45 cycles of 95°C for 30 s, 62°C for 30 s and 72°C for 30 s. The copy number for each assay was calculated from the cycle threshold = Viral copy number/diploid OSM copy numberSamples were assayed in duplicate and the means were determined. Standard deviations were calculated using the sum of the errors of the viral and OSM copy number determinations.T, cycle threshold; KSHV/HHV8, Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8; Mfa, Macaca fascicularis; MfaRV2, Macaca fascicularis rhadinovirus-2; Mm/Mmu, Macaca mulatta; Mn/Mne, Macaca nemestrina; MneRV2, Macaca nemestrina rhadinovirus-2; ORF, open-reading frame; OSM, oncostatin M; Pcy, Papio cynocephalus; PcyRV2, Papio cynocephalus rhadinovirus-2; PCR, polymerase chain reaction; QPCR, quantitative PCR; RFHV, retroperitoneal fibromatosis herpesvirus; RRV, rhesus rhadinovirus; RV1, rhadinovirus-1; RV2, rhadinovirus-2;AGM, African green monkey; CODEHOP, consensus-degenerate hybrid oligonucleotide primer; CThe author(s) declare that they have no competing interests.Design and conception of the study ; development of the methods for amplification of the ORF59/60 regions ; Development of the QPCR assays and quantitative analysis ; Virus isolation and preparation (MET); Sequence analysis, alignment and phylogeny ; Manuscript preparation . All authors read and approved the final manuscript.

Feeding normal rats with high dietary levels of saturated fat leads to pathological conditions, which are quite similar to syndrome X in humans. These conditions such as hypertriglyceridemia, hypercholesterolemia, obesity, and hyperglycemia might induce hypertension through various mechanisms. Metabolic syndrome and the resulting NIDDM represent a major clinical challenge because implementation of treatment strategies is difficult. Vascular abnormalities probably contribute to the etiology of many diabetic complications including nephropathy, neuropathy, retinopathy, and cardiomyopathy. It has been shown that in Streptozotocin induced diabetic animals there is an increase in maximal responses to 5-Hydroxytryptamine and Angiotensin II. The purpose of this study was to evaluate High fat diet fed rats for the development of hypertriglyceridemia, hypercholesterolemia, hyperinsulinemia and hyperglycemia and to assess their vascular responses to 5-Hydroxytryptamine and Angiotensin II.Male Sprague Dawley rats were used for this study and were divided into two equal groups. One of the groups was fed with normal pellet diet and they served as the control group, whereas the other group was on a high fat diet for 4 weeks. Body weight, plasma triglycerides, plasma cholesterol, and plasma glucose were measured every week. Intraperitoneal glucose tolerance test was performed after 4 weeks of feeding. At the end of fourth week of high fat diet feeding, thoracic aortae were removed, and cut into helical strips for vascular reactivity studies. Dose-response curves of 5-Hydroxytryptamine and Angiotensin II were obtained.2, with 5-Hydroxytryptamine and Angiotensin II in both groups but Emax was increased.There was no significant difference in pDThese results suggest that hypertension in high fat diet rats is associated with increased in vitro vascular reactivity to 5-HT and Ang II. Syndrome X comprises a plethora of conditions such as obesity, dyslipidemia, impaired glucose tolerance, insulin resistance and hypertension . It placThe aim of this study was to elucidate the contractile responses to 5-HT and Ang II in HFD fed rat thoracic aorta which will provide an avenue for further exploratory studies. We have selected HFD fed rat model for our study because it is a useful model of the putative effects of excess fat intake in humans and it represents the major sub type of diabetes mellitus, non insulin dependent diabetes mellitus (NIDDM).HFD fed rats showed significant increase in body weight as compared to NPD fed rats Table . In addimax . The order of potency of agonists in both groups was Ang II>5-HT>KCl.Cumulative concentration response curves of 5-HT and Ang II for both HFD fed rat thoracic aortae showed an increase in Emax Fig and 3 wiObesity is a major risk factor for several metabolic diseases, frequently clustering to form the metabolic syndrome or syndrome X . Obese pHyperglycemia is observed in insulin resistance where glucose utilization is reduced. We saw significant elevations in blood glucose levels. Intraperitoneal glucose tolerance tests confirm severe glucose intolerance. Oversupply of dietary lipids causes insulin resistance in rats . Randle Insulin resistance with compensatory hyperinsulinemia is a prominent feature of metabolic syndrome. The most common reason for the development of hyperinsulinemia in insulin resistance is obesity. It stands as one of the major cardiovascular risk factors in patients with obesity. The present study on HFD rats demonstrated higher plasma insulin levels than control values. This marked hyperinsulinemia could be due to a combination of increased β-cell mass and decreased insulin clearance, as well as failure of insulin to suppress hepatic gluconeogenesis .Elevated cholesterol is also observed in insulin resistant individuals. For this reason we measured plasma cholesterol levels which were found to be more than normal values. Previous studies have reported a down regulation of LDL receptors and associated decrease in LDL clearance, increased total cholesterol levels .According to National Cholesterol Education Program's Adult Treatment Panel III (Third report) easily measured clinical findings for syndrome X includes increased abdominal circumference, elevated triglycerides, low high-density lipoprotein-cholesterol, and elevated fasting blood glucose and/or elevated blood pressure. Three of these five are required for diagnosis. Our study demonstrated three of the clinical parameters indicating conditions of syndrome X in HFD fed rats . InsulinIn order to elaborate the pathways that connect syndrome X to hypertension we have studied the contractile responses to 5-HT and Ang II in both HFD and NPD fed rat thoracic aortae. Previously we have demonstrated increased contractile responses with synthetic alpha adrenoceptor agonist, phenylephrine, in HFD fed rat thoracic aorta . This en2 value. Endothelial denudation obviates any related mechanisms such as impairment of NO release, increased destruction of EDRF and substrate availability for the production of EDRF. Hence, the probable reasons for these enhanced 5-HT and Ang II responses may be due to receptor mediated or non-receptor mediated pathways.The present vascular studies demonstrated that the magnitude of responses to 5-HT and Ang II was significantly enhanced in HFD fed animals without change in pD2A upregulation as observed in spontaneously hypertensive rats or due to serotonin acting through alpha adrenoceptors [The role of non-receptor mediated contraction can be ruled out for there was no change in contractile response to KCl. Vascular studies have also shown functional evidence that hypertension developed in HFD fed rats may be associated with enhanced vasoreactivity to various vasoconstrictor agents. Further the enhanced responses to 5-HT in HFD fed rats could be due to increased PKC as previously reported with STZ and alloxan (ALL) induced diabetic animals . Increasoceptors .Increased Ang II responses in HFD fed rats are may be due to upregulation of Ang II receptors as observed in hyperinsulinemia or via amplified secondary messenger systems . A propoIn summary, the present study has shown that HFD feeding in rats produces conditions similar to syndrome X. Increased vasocontractile responses observed in the model are not only mediated via alpha adrenoceptors but also due to 5-HT and Ang II , India), 160–200 g, were kept in controlled environmental conditions with room temperature 22 ± 2°C, humidity 55 ± 5% and 12-h light/dark cycles. All the animals had free access to food and water. The rats were divided into two dietary groups and fed with standard rat normal pellet diet (NPD) and HFD . Composition of HFD is described in Table Blood samples from the retro orbital plexus of anaesthetized rats were collected into the heparinized tubes and immediately centrifuged at 5000 rpm for the separation of plasma. Plasma was stored at -20°C until assayed. The plasma was used for the estimation of glucose , triglycerides, and cholesterol by commercial kits. Plasma insulin was determined by radioimmuno assay using rat insulin as standard Glucose tolerance tests were carried out after four weeks of feeding of both NPD and HFD. After an overnight fast, blood samples were collected from the retro orbital plexus. Glucose levels were measured at time zero (0 min) and glucose was injected into the rats . Additional blood samples were taken at 15, 30, 60 and 120 min. following the glucose load. Plasma glucose levels were measured by the glucose oxidase reaction (GOD/POD) using commercial kit . Area under the curve (AUC) was calculated for both NPD and HFD fed rats.3, 2.6 CaCl2 2H2O 1.2 NaH2PO4, 1.2 MgCl2 6H2O, 5.5 glucose). With the help of a steel rod, aortic endothelium was deliberately denuded. The aorta was cut into helical strips 3 mm wide, 20 mm long and then placed in a well-oxygenated (95% O2-5% CO2) bath of 10-ml KHS with one end connected to a tissue holder and the other to an isotonic transducer . The tissue was equilibrated for 60 min under a resting tension of 1.0 g. At the beginning of each experiment, aortic strips were primed with depolarizing concentration (90 mM) potassium chloride (KCl). After the equilibration period, contractile responses to various concentrations of 5-HT (10 nM-30 μM) and Ang II (1 nM-300 nM) were recorded.After 4 weeks of feeding, rats were sacrificed by cervical dislocation. The section of the aorta from between the aortic arch and the diaphragm was removed from the euthanized rats and placed in oxygenated, modified Krebs-Henseleit solution and the concentration necessary to produce 50% of its maximal response (EC50) were determined. The EC50 values were converted to the negative logarithms and expressed as pD2. Results were shown as mean ± SEM; n refers to the number of animals from which vessels were taken. Agonist potencies and maximal effects were compared by student's t test by using statistical software (GraphPad Prism 3.01). Values were considered significantly different at p < 0.05.Contraction responses are expressed as percentage. For each contractile agent, both the maximal contraction , Ang II , All drugs were dissolved in KHS. Drugs were added to the organ chambers in volumes not greater than 0.2 ml.HFD – High fat dietNPD – Normal pellet dietNIDDM – Non insulin dependent diabetes mellitusSTZ – Streptozotocin5-HT – 5-HydroxytryptamineAng II – Angiotensin IIKHS – Krebs-Henseleit solutionGOD/POD – Glucose oxidase/peroxidaseAUC – area under the curvemax – Maximal responseE50 – Concentration required producing 50% of maximal responseEC2 – -log EC50pDSG carried out the all experimentations. PR has conceived the study and participated in its design and coordination. Authors read and approved the final manuscript.

Spirituality has become a subject of interest in health care as it is was recognized to have the potential to prevent, heal or cope with illness. There is less doubt that values and goals are important contributors to life satisfaction, physical and psychological health, and that goals are what gives meaning and purpose to people's lives. However, there is as yet but limited understanding of how patients themselves view the impact of spirituality on their health and well-being, and whether they are convinced that their illness may have "meaning" to them. To raise these questions and to more precisely survey the basic attitudes of patients with severe diseases towards spirituality/religiosity (SpR) and their adjustment to their illness, we developed the SpREUK questionnaire.In order to re-validate our previously described SpREUK instrument, reliability and factor analysis of the new inventory (Version 1.1) were performed according to the standard procedures. The test sample contained 257 German subjects (53.3 ± 13.4 years) with cancer (51%), multiple sclerosis (24%), other chronic diseases (16%) and patients with acute diseases (7%).As some items of the SpREUK construct require a positive attitude towards SpR, these items (item pool 2) were separated from the others (item pool 1). The reliability of the 15-item the construct derived from the item pool 1 respectively the 14-item construct which refers to the item pool 2 both had a good quality . Factor analysis of item pool 1 resulted in a 3-factor solution which explains 53.8% of variance. Factor analysis of item pool 2 pointed to a 2-factor solution which explains 58.8% of variance. Generally, women had significantly higher SpREUK scores than male patients. Univariate variance analyses revealed significant associations between the sub-scales and SpR attitude and the educational level.The current re-evaluation of the SpREUK 1.1 questionnaire indicates that it is a reliable, valid measure of distinct topics of SpR that may be especially useful of assessing the role of SpR in health related research. The instrument appears to be a good choice for assessing a patients interest in spiritual concerns which is not biased for or against a particular religious commitment. Moreover it addresses the topic of "positive reinterpretation of disease" which seems to be of outstanding importance for patients with life-changing diseases. Spirituality has become a subject of interest in health care, and an increasing number of studies, commentaries and reviews examine the connection between religiosity/spirituality and health, its potential to prevent, heal or cope with diseases -10. MoreHowever, although religiosity and spirituality were interchangeable words, these constructs may not be identical. It is well established to divide Religiosity into three sub-constructs: Intrinsic, Extrinsic, and Quest Religiosity -20, whilSpiritual Well-Being Scale [Daily Spiritual Experience Scale [Santa Clara Strength of Religious Faith Questionnaire [Functional Assessment of Chronic Illness Therapy – Spiritual Well-Being (FACIT-Sp) scale has a much more open design [The measurability and operability of spirituality and religiosity remains a problem and thus several questionnaires address this topic. Most of them measure beliefs of specific religious groups, and ask about the relationship with God and their adjustment to their illness, we developed the SpREUK questionnaire ,29,36-39In this article we report the re-validation of the SpREUK 1.1 questionnaire , an instrument designed to examine attitudes of patients with life-threatening and chronic diseases towards spirituality/religiosity.All individuals were informed of the purpose of the study, were assured of confidentiality, and gave informed consent to participate. The patients were recruited consecutively in the cancer service, the multiple sclerosis service, and two internal medical units of the Communal Hospital in Herdecke (Germany). All subjects completed the questionnaire by themselves. Demographic information is provided in Table The sample contained 257 subjects of whom 70% were women. The mean age was 53.3 ± 13.4 years. The majority had a Christian nomination (80%), 17% had no religious orientation, and 3% other nominations. Cancer was diagnosed in 51%, multiple sclerosis in 24%, and other chronic diseases in 16% ; 7% of the individuals were patients with acute diseases . Patients in final stages of their disease were not enrolled.The items of the SpREUK 1.0 were developed with the patients' input and experts' statements ,36, rathAccording to a previously conducted reliability and factor analysis ,37 the SIn order to more precisely differentiate the three topics guidance, control and message of disease in scale 2 of the version 1.0, for the current version of the questionnaire, six new items were added . All items were scored on a 5-point scale from disagreement to agreement . The SpREUK scores are referred to a 100% level (4 "applied very much" = 100%).Reliability and factor analysis of the new inventory were performed according to the standard procedures. Next, to combine several items with similar content, we relied on the technique of factor analysis which examines the correlations among a set of variables, and to achieve a set of more general "factors." Factor analyses were repeated rotating different numbers of items in order to arrive at the solution which demonstrated both the best simple structure and the most coherence.Differences in the SpREUK scores were tested using the Kruskal-Wallis-Test for asymptomatic significance. We judged p < 0.05 significant, and 0,05 < p < 0.10 as a trend.To tested the impact of several variables on the SpREUK sub-scales, we performed analysis of univariate variance (ANOVA). As in several cases Levene's test for equality of variances was significant, and we judged p < 0.01 as significant.All statistical analyses were performed with SPSS for Windows 10.0.In order to eliminate items from the item pool that were not contributing to the questionnaire reliability, the reliability of the scale and distinct sub-scales was evaluated with internal consistency coefficients, which reflect the degree to which all items on a particular scale measure a single concept.Our item pool consisted of the previously established set of items ,36,37 anReliability analysis revealed that 6 items from the new item pool 1 had a poor corrected item-total correlation and thus were eliminated : F1.2 , F1.3 , F2.1 , F2.2 . F2.3 , and F3.1 . One item (F3.6 "The "true being" ("inner core") can not be affected by illness") was omitted because of a weak reliability (0.2997) and – even more important – it points to a distinct "field of meaning" that would need more items in the questionnaire, and thus will be used as marker item until the construct will be revised for this topic.As shown in Table Thus, the internal consistency of the 29-item SpREUK 1.1 construct was sufficiently high. The level of difficulty is 0.6205 for item pool 1 resp. 0.6014 for item pool 2. With the exception of item F3.7 , all values are in the acceptable range from 0.2 to 0.8.To combine several items with similar content, we relied on the technique of factor analysis which examines the correlations among a set of variables, and to achieve a set of more general "factors." Factor analyses were repeated rotating different numbers of items in order to arrive at the solution which demonstrated both the best simple structure and the most coherence.With a Kaiser-Mayer-Olkin value of 0.850 (item pool 1) resp. 0.939 (item pool 2), which measures the degree of common variance, the 15 resp. 14-item-pool seems to be suitable. Barlett's test for non-sphericity was highly significant .Primary factor analysis of item pool 1 pointed to a 5-factor solution. However, due to a low item number in the tentative subscales 2–5 (with 2 or 3 items each), we favoured the more appropriate 3-factor solution which explains 53.8% of variance reveal that items F1.8 ("looking for purpose and meaning in life") load good on sub-scale 2 (0.414). Analysis of the side-loadings of item pool 2 revealed that several items load also on the other sub-scale. Moreover, sub-scale 4 showed a strong but negative inter-correlation with sub-scale 5 , the lowest for sub-scale 1 ("Search for meaningful support"). Means and standard deviations for study variables are provided in Table Women had significantly higher SpREUK scores than male patients. With respect to age, the lowest SpREUK scores were found in the group of < 30 years of age. With increasing age, the trust in a higher supporting presence and the beneficial effects of resp. support through SpR increased.With respect to the marriage status, widowed patients obviously has to rely on external guidance but not the patients living with a partner not married with. Widowed and divorced patients find support in external relations through their SpR engagement , while – in contrast to married patients which may find hold in their partnership – especially divorced patients are in search for meaningful support .Search for meaningful support and positive interpretation of diseases were depending on the educational level, as patients with lower educational level had significantly lower scores than those with a higher level. A higher educational level was associated with higher scores in the sub-scales 4 and 5 which deals with the beneficial effects of SpR.Illness itself (but not the duration of disease) has a significant impact on the SpREUK scores, as MS patients had the lowest scores in all 5 sub-scales. The SpREUK scores of cancer patients revealed slight differences when compared to patients with other chronic diseases.With the exception of sub-scale 2, patients without confessional affiliations had the lowest scores for all sub-scales, indicating that the "message of disease" was not depending on a denomination. Surprisingly, the few patients with other than a Christian orientation had the highest scores for sub-scales 1, 3, 4, 5.(R+S+); 35% as religious, but not spiritual (R+S-); 23% as neither religious nor spiritual (R-S-); 10% claimed that they were spiritual, but not religious (R-S+). Thus, the numbers of patients with denominational affiliation and self-reported spiritual/religious attitudes is somewhat similar. A spiritual attitude (R+S+ and R-S+) was associated with "search for meaningful support" and "positive interpretation of disease", while a religious attitude (R+S+ and R+S-) was associated with the highest scores for the "trust in external guidance" sub-scale 3.Since nominational affiliation is not necessarily identical with religiosity or spirituality, we asked whether the patients would describe themselves as religious or spiritual ,34,35. TThe living area and the duration of diseases had no significant impact on the SpREUK scores.As shown in Table In detail Table , prayingNext we tested the impact of several variables on the SpREUK sub-scales, such as sex and marital status, educational level and confession, age and SpR attitude, and disease and duration of disease. Using the method of univariate analyses of variance we identified several sources of variability Table :SpR attitude is an important covariate for the "Search for meaningful support", "Positive interpretation of disease", "Trust in external guidance", and both "Support through SpR" sub-scales.• The educational level is an important covariate for "Search for meaningful support", "Trust in external guidance" and to a minor content for "Support in relations with the External life through SpR" – but not for the "Positive interpretation of disease".• The Age is an important covariate only for "Trust in external guidance".• Confession is an important covariate for "Trust in external guidance".• Disease itself has an impact on the "Search for meaningful support• Data from the current analysis demonstrate the reliability and validity of the SpREUK construct. Moreover, the sub-scales 1 and A (= 4) and B (= 5) of the preliminary version 1.0 were confirmed in the new version 1.1. In order to more precisely differentiate the three topics guidance, control and message of disease from the SpREUK version 1.0, six new items were added. Due to this fact, some items from the original item pool decreased the reliability of the construct and thus, two items from the sub-scale 1 had to be deleted of disease" has a good correlation with an existentialistic practice, which seems to be of outstanding importance for patients with life-changing diseases. It may be desirable to use such a measure that allows to assess attitudes which are independent of any religion or specific belief.A third strength is that the validation was performed in a sample with at least two different types of life-changing diseases and a healthy control group.Beyond conceptual boundaries, our instrument differentiates the self-addressed "religious" and "spiritual" attitudes of the patients with life-threatening diseases and heeds their search for support and meaning, and integrates the topic of "meaning in illness". We cannot exclude the possibility that these topics are not relevant for healthy individuals.In future studies we have to correlate our scales with other relevant instruments which measure aspects of SpR. Nevertheless, evaluation of the SpREUK questionnaire indicates that it is a reliable, valid measure of distinct topics of SpR that may be especially useful of assessing the role of non-religious spirituality in health related research. The focus of a larger study is to enrol patients from the highly secular Eastern Europe, and to run longitudinal studies with cancer, multiple sclerosis patients, but also cardiac failure and spinal cord damage.The SpREUK with its additional SpREUK-P manual to measure a patient's engagement in distinct forms of SpR practice is currently available in English and German language.AB conceived the study, designed and developed the questionnaire, performed statistical analysis and drafted the manuscript. TO participated to conceive and design the study, performed additional statistical analysis and helped to draft the manuscript. PFM conceived the study and participated in the design and development of the questionnaire. All authors read and approved the final manuscript.

The principles of Virchov's triad appear to be operational in atherothrombosis or arterial thrombosis: local flow changes and particularly vacular wall damage are the main pathophysiological elements. Furthermore, alterations in arterial blood composition are also involved although the specific role and importance of blood coagulation is an ongoing matter of debate. In this review we provide support for the hypothesis that activated blood coagulation is an essential determinant of the risk of atherothrombotic complications. We distinguish two phases in atherosclerosis: In the first phase, atherosclerosis develops under influence of "classical" risk factors, i.e. both genetic and acquired forces. While fibrinogen/fibrin molecules participate in early plaque lesions, increased activity of systemic coagulation is of no major influence on the risk of arterial thrombosis, except in rare cases where a number of specific procoagulant forces collide. Despite the presence of tissue factor – factor VII complex it is unlikely that all fibrin in the atherosclerotic plaque is the direct result from local clotting activity. The dominant effect of coagulation in this phase is anticoagulant, i.e. thrombin enhances protein C activation through its binding to endothelial thrombomodulin.The second phase is characterized by advancing atherosclerosis, with greater impact of inflammation as indicated by an elevated level of plasma C-reactive protein, the result of increased production influenced by interleukin-6. Inflammation overwhelms protective anticoagulant forces, which in itself may have become less efficient due to down regulation of thrombomodulin and endothelial cell protein C receptor (EPCR) expression. In this phase, the inflammatory drive leads to recurrent induction of tissue factor and assembly of catalytic complexes on aggregated cells and on microparticles, maintaining a certain level of thrombin production and fibrin formation. In advanced atherosclerosis systemic and vascular wall driven coagulation becomes more important and elevated levels of D-dimer fragments should be interpreted as markers of this hypercoagulability. The blood coagulation system comprises three basic elements: platelet adhesion, activation and aggregation, fibrin formation, and fibrinolysis. These elements interact with each other and with the blood vessel wall and under physiological conditions blood flow to tissues is unimpaired by clotting . Under pvenous thrombosis of the lower limbs, stasis, local inflammation on activated vascular endothelial cells induced by adhering leukocytes and platelets and in some cases direct vascular damage, promotes local thrombus formation. In a first episode of venous thrombosis the pre-existing composition of the blood is particularly important where congenital and acquired hypercoagulable factors such as factor V Leiden mutation and oral contraceptives, respectively, act in concert to accelerate clotting . Un. Un30]. The involvement of coagulation in the pathological substrate of atherosclerosis is beyond dispute. For many years pathologists have noted the abundant presence of fibrin in advanced atherosclerosis and this finding has fueled part of the debate on the relevance of fibrin or fibrinogen for vessel wall lesions. Rokitansky and later Duguid proposed the encrustation theory as concept for the role of fibrin in atherosclerosis (reviewed in ). In thiearly event in atherosclerosis, i.e. a small amount of fibrinogen in a thickened intima was demonstrated in a 4 year old boy [-/- background did not have fewer arterial lesions ranging from early lesions to complex fibrous plaques, suggesting that fibrinogen is not an essential molecule for atherosclerosis [-/- x apo(a) crossbred animals [Autopsy data have indicated that fibrinogen accumulation in the vessel wall may be an old boy . The depclerosis . However animals .The specific effect of fibrin and its split products in the vessel wall has also been studied. In general it appears that with increasing complexity of lesions there is an increase in the presence of intimal fibrinogen/fibrin and threads of fibrin, as well as an accumulation of various split products that may be involved in atherogenesis. The effect of fibrin and its split products on smooth muscle cells may be such that fibrin stimulates proliferation, while split products inhibit this process. Fibrin cleavage products may be detrimental for endothelial cell function, increasing permeability and promoting endothelial cell migration ,41,42. D-/-) results in markedly discrepant effects on atherosclerosis. While Plg-/- mice with an apoE-/- background showed an accelerated development and progression of intimal lesions, Plg-/- mice were protected against atherosclerosis in association with transplantation . The orAccumulating fibrin that polymerizes in the vessel wall triggers fibrinolysis. Fibrinolytic enzymes tissue plasminogen activating factor and urokinase plasminogen activating factor are present in intima and are secreted by endothelial cells and likely play an important role in vascular remodeling -44. Howe-/- mice give important clues regarding the range of mechanisms that are influenced. Effective fibrinolysis may be important in limiting fibrin accumulation and atherosclerosis in the initial phases. However, upon stronger inflammatory stimulation the effect on cell trafficking into the vessel wall becomes more dominant and the outcome may reverse such that impaired fibrinolysis may limit atherosclerosis. The latter would imply that high levels of PAI-1 may even be protective against atherosclerosis under certain conditions. Consequently, high concentrations of D-dimers, reflecting active fibrinolysis, may indeed be regarded as a sign of progressive atherosclerosis under inflammatory conditions.The net effect on fibrin cleavage and progression of atherosclerosis cannot be estimated. The above mentioned experiments with PlgAll of these issues may have therapeutic consequences since several drugs that are routinely used in patients with atherosclerosis including statins, angiotensin converting enzyme inhibitors as well as angiotensin receptor blockers appear to influence the balance of coagulation and fibrinolysis, which may influence atherosclerosis on the long term by altering vascular properties .As mentioned above, a large number of clinical studies in different groups of patients with atherosclerotic disease have generally shown that increased levels of D-dimer fragments in plasma are associated with an increased risk of severe atherosclerosis and an increased risk of vascular complications. In population based studies the contributable risk of an increased D-dimer level is quite small but statistically significant. In specific cohorts of patients the risk association is more outspoken, but of course here selection bias may produce slightly stronger associations than may be found in "real life".Before addressing specific study findings a few general observations deserve attention.First, strong associations between age and sex on the one hand, and D-dimer levels on the other hand, are noted ,11,42. DSecond, D-dimer levels are oftentimes associated with markers of inflammation, i.e. CRP and Il-6 ,25,42,46Before addressing the mechanisms we will consider D-dimer as an independent entity, i.e. a marker of disease severity. The most striking associations with clinical disease come from patients with peripheral artery disease (PAD), a reflection of systemic and advanced atherosclerosis in the majority of individuals. The total risk of clinical complications or mortality reaches figures of up to 25% annually in patients with PAD 48). In patients with PAD, elevated D-dimer levels are independent predictors of complications and are associated with severity of atherosclerosis -14,16,25. In patiElevated levels of D-dimers are usually considered as a marker of increased clotting activity. This assumption is one of the key elements of the controversy regarding cause and consequence of hypercoagulability. Indeed, Herren et al observed increased levels of D-dimer in patients with PAD, correlating with severity of disease. They also noted an association between hypercoagulability (higher F1+2 and TAT) and occurrence of myocardial ischemia during exercise testing , suggestsystemic clotting activity then a specific anticoagulant intervention may theoretically be the preferred intervention. Clinical studies with anticoagulants in patients randomized or stratified on the basis of D-dimer levels have, however, not been carried out yet.In spite of the substantial observational data, application of D-dimer assays or other risk factor measurements such as for CRP have not gained acceptance in individual patients with PAD or other cardiovascular disease yet. Thus, secondary prevention of complications is not guided by any laboratory assay, but limited to general recommendations such as the advice to stop smoking and the prescription of a platelet inhibiting drug . Three rseverity of atherosclerosis such an intervention may be inappropriate and potentially harmful because of the avoidable risk of bleeding and calcification of the arterial vessel wall upon long-term administration (at least with vitamin K antagonists). Alternatively, if D-dimer levels merely reflect inflammation, than therapy should preferably consist of anti-inflammatory agents including higher doses of aspirin, statins or ACE inhibitors. Thus, the interpretation of elevated D-dimer levels is quite important in order to guide decisions about individual therapy.If however, D-dimers are a reflection of Atherosclerosis is a chronic inflammatory disease ,28. ThisStudies from the sepsis field including models of endotoxemia and sepsis in humans and primates, respectively, have shown that inflammatory stimulation leads to activation of blood coagulation ,53. TissIn this concept, there is an increased generation of thrombin and fibrin in the blood circulation due to increased presence of inflammatory cytokines and proteins, but this does not necessarily lead to increased free thrombin in plasma. One should realize that coagulation enzymes that are generated associate with any available "scavenger", which can be an inhibitor such as antithrombin, but could also be a protease activated receptor (PAR) on platelets or endothelial cells -56. ThusThe exact contribution of subendothelial fibrin formation and cleavage to D-dimer levels in blood cannot be estimated. While locally deposited tissue factor acts as a trigger of thrombin generation, it has not been shown that this is a source of ongoing subendothelial coagulation activity. The recent discovery of factor VII in plaque contents and the in vitro evidence for production of this Gla-protein by smooth muscle cells might form a basis for local thrombin production, but there is no indication yet that this might be quantitatively important as compared to hepatic production of factor VII .systemic hypercoagulability, a conclusion based on the above arguments and the experimental evidence indicating the intimate relationship between inflammation and coagulation [The point to make is that high D-dimer levels in blood from patients with atherosclerosis should be primarily viewed as an indication of gulation .Early studies from Rosendaal et al, suggested that thrombophilic traits including the prothrombin 20210 gene variant would be a risk factor for myocardial infarction in specific individuals such as heavy smoking young women . These dIn people of advanced age this situation may change considerably and the weight of risk factors may change over time. A study that specifically addressed this point is the Bruneck community study . In thisIn advanced atherosclerosis the influence of coagulation may indeed be more prominent than in early stages, but it should be realized that acquired rather than genetically determined forces are involved. In this regard, the similarities between arterial thrombosis and the risk of recurrent venous thrombosis was used by Reitsma to make the point that inflammation is a key player under such conditions, reducing the influence that genetic thrombophilic background might inflict. On the other hand the same argument could be used to illustrate that indeed inflammation plays a more prominent role in advanced atherosclerosis where it more strongly drives the risk of thrombosis.Let us consider this situation from the scope of venous thrombosis; this is not far edged because a recent study suggested similarities in risk factors between patients with previous venous thrombosis and atherosclerosis . Recent The influence of blood coagulation on atherosclerosis follows a two stage model in which variants may occur under exceptional conditions. In general in the first phase, roughly covering the first four decades of life, atherosclerosis develops under influence of "classical" risk factors, including hypercholesterolemia and smoking, i.e. both genetic and acquired forces. While fibrinogen/fibrin molecules participate in early plaque lesions, increased activity of systemic coagulation is of no major influence on the risk of arterial thrombosis, except in rare cases where a number of specific procoagulant forces collide. The dominant effect of coagulation is anticoagulant, i.e. thrombin enhances protein C activation through its binding to endothelial thrombomodulin. Defects in the protein C mechanism may indeed precipitate arterial thrombosis, but only under highly thrombogenic conditions. Fibrinolysis limits fibrin accumulation in the intima and herewith progression of plaque lesions. At this stage elevated PAI-1 levels may diminish fibrinolysis and may stimulate plaque progression, which may explain that in a large Japanese study the PAI 4G/5G polymorphisms appeared to be a risk factor for myocardial infarction in women .The second phase is characterized by advancing atherosclerosis, with greater impact of inflammation and increased infiltration of fibrin in the arterial vessel wall, enforcing pro-inflammatory effects. The extensive interactions between inflammation and coagulation enzymes and inhibitors (in much greater detail than discussed here) amplify the chain of events that determine the risk of atherothrombosis. Inflammation overwhelms protective anticoagulant forces, which in itself may have become less efficient due to down regulation of thrombomodulin (TM) and endothelial cell protein C receptor (EPCR) expression. In this phase, evidence of activated coagulation is measurable in peripheral blood reflecting both the extent of atherosclerotic burden and the systemic clotting tendency, which poses a direct risk of thrombotic complications. This point of view deviates from Tracy's viewpoint and provides a more constructive model for integrating coagulation in arterial disease.We would also propose that D-dimer and other, novel assays such as for endogenous thrombin generation or for a

Recently, intracardiac echocardiography emerged as a useful tool in the electrophysiology laboratories for guiding transseptal left heart catheterizations, for avoiding thromboembolic and mechanical complications and assessing the ablation lesions characteristics. Although the value of ICE is well known, it is not a universal tool for achieving uncomplicated access to the left atrium. We present a case in which ICE led to interruption of a transseptal procedure because several risk factors for mechanical complications were revealed.A case of a patient with paroxysmal atrial fibrillation and atrial flutter, and distorted intracardiac anatomy is presented. Intracardiac echocardiography showed a small oval fossa abouting to an enlarged aorta anteriorly. A very small distance from the interatrial septum to the left atrial free wall was seen. The latter two conditions were predisposing to a complicated transseptal puncture. According to fluoroscopy the transseptal needle had a correct position, but the intracardiac echo image showed that it was actually pointing towards the aortic root and most importantly, that it was virtually impossible to stabilize it in the fossa itself. Based on intracardiac echo findings a decision was made to limit the procedure only to ablation of the cavotricuspid isthmus and not to proceed further so as to avoid complications.This case report illustrates the usefulness of the intracardiac echocardiography in preventing serious or even fatal complications in transseptal procedures when the cardiac anatomy is unusual or distorted. It also helps to understand the possible mechanisms of mechanical complications in cases where fluoroscopic images are apparently normal. Since the advent of ICE in the electrophysiology practice it proved its value in guiding transseptal procedures with providing an extra safety margin for the patients. The possibility to visualize the oval fossa, the LA free wall and the aortic root helps in preventing mechanical complications. ICE can visualize also intracardiac thrombus and spontaneous echocontrast, which is helpful in avoiding thromboembolic complications. Although the value of ICE is well known, it is rather hard to admit that it is a universal tool for achieving uncomplicated access to the left atrium. The aim of this case presentation is to show that ICE can lead to interruption of a transseptal procedure due to the presence of risk factors for mechanical complications when the fluoroscopic image is seemingly satisfying.A seventy-year-old male patient with paroxysmal atrial fibrillation and atrial flutter, and concomitant arterial hypertension was referred to our institution for LA circumferential ablation. The preprocedural TEE described an aneurysm of the IAS See . During In the last years ICE emerged as a useful adjunctive tool in the field of interventional electrophysiology. It serves not only scientific purposes but practical issues as well. Its value for achieving successful and uncomplicated transseptal access to the LA cavity is well known -4. HowevIn this case ICE showed that the aneurysm of the IAS observed during the TEE was actually the angulated continuity between the enlarged aortic root and the IAS. The echocardiographic orientations of ICE are sometimes clearly off axis in comparison to standard transesophageal echocardiographic views. Nevertheless, to our opinion ICE is superior in providing more detailed picture of the neighboring cardiac structures. Although useful in guiding transseptal catheterizations, TEE does not always provide complete avoidance of complications even in patients with normal hearts ,6. FurthEP procedures are relatively safe procedures and have low complication rate. One of the most frequent complications is related to cardiac wall perforation with consequent pericardial effusion and tamponade. ICE, especially the one with phased-array transducer (deeper penetration) allows continuous monitoring of the pericardial space during EP procedures. This permits prompt detection of a pericardial effusion and an immediate guidance of a therapeutic puncture. Phased-array transducers are equipped with Doppler capabilities allowing assessment of the pulmonary venous flow pattern after pulmonary vein ablation on top of diameter measurements to exclude pulmonary vein stenosis, which is the most important complication of this procedure. Monitoring microbubble formation during RF energy application allows prevention of pulmonary vein stenosis . ICE alsWe strongly believe that this case is a good illustration of the usefulness of the ICE in the electrophysiology field and further enhances its value as a tool for avoiding complications when the intracardiac anatomy is unusual or distorted. As the number of transseptal procedures in the electrophysiology laboratories all over the world is steeply growing ICE definitely has the potential to become a routine at least in those institutions with large volume of left atrial procedures.EP – electrophysiologicIAS – interatrial septumICE – intracardiac echocardiographyLA – left atrium; left atrialTEE – transesophageal echocardiographyPreprocedural transesophageal echocardiography. The irregular oval-shaped structure at the center of the screen is the aortic root. At a certain moment one can see at its upper part the ostium and the most proximal part of the right coronary artery. Below is situated the left atrium and to the left – the right atrium. The oval fossa is in between.Click here for fileIntracardiac echocardiography, showing distorted intracardiac anatomy. The oval shape in the center of the screen is the non-coronary sinus of Valsalva. Below is the right atrium at the bottom of which the transseptal needle is clearly visible. The prominent muscular structure in the left-hand part of the image is the terminal crest. The membrane of the oval fossa, adjacent to the right-hand part of the non-coronary aortic sinus shows bidirectional flapping motion. During the atrial contraction the cavity of the left atrium virtually disappears.Click here for fileIntracardiac echocardiography showing sliding of the needle towards the aorta. The transseptal needle is already in the oval fossa with its tip pointing to the aorta. This is especially clearly visible after a premature beat.Click here for file

Background to the debate: The US and Canadian task forces on preventive health recently declared that there is not enough evidence to recommend for or against routine universal screening of women for domestic violence. Yet some experts argue that routine enquiry is justified. Experts from both sides of the Atlantic debate whether screening can be justified based on the available clinical evidence Domestic violence is a misunderstood topic. The context of a trusted health professional talking to a woman is one that provides an important opportunity for providing information to counter misconceptions.asking all women about domestic violence and not in terms of screening women for domestic violence. It is not appropriate or helpful to regard enquiry about being abused as a form of screening. Domestic violence is not a disease present in the body of the person who experiences it—rather it is a health-related risk factor.I deliberately talk about this in terms of As such, knowledge of abuse puts health professionals in a position to respond better to the needs of women affected by it. Professionals can respond by providing information on specialist services—usually provided outside the health service—that women may access if they wish. By giving information to affected women, health professionals can also help to reduce women's sense of isolation and stigmatisation. Asking about experience of domestic violence can be seen as a routine part of history taking, just as health professionals regularly and repeatedly ask patients about their smoking behaviour, alcohol use, weight, and exercise.The prevalence of domestic violence among women is such that, even if it is not a personal issue for the woman concerned, it most likely will be for one or more of her relatives, friends, and neighbours . Since mMost women experiencing domestic violence report that the specialised services that exist to respond to their needs were difficult to find out about . The proStudies have examined women's views on being asked about domestic violence. These studies have shown that once they have experienced being asked, they are usually in favour of being asked. This is true both for those who have experienced or are experiencing abuse, and those who have not . It is oAsking about abuse should be done in a flexible fashion—the particular questions used should respond to the circumstances of the consultation. For example, it is appropriate to ask women about domestic violence as part of a health check in a Well Woman Clinic, but it would be completely inappropriate in a consultation where another adult or a child was present. By being flexible, health professionals can integrate their questioning within a variety of different encounters. Integrating questions about abuse into routine encounters provides for the maintenance of confidentiality and safety. In order to do this, health professionals require training on raising the issue and knowledge about local advice and support services.Committees on both sides of the Atlantic have rejected the notion of screening women for domestic violence, arguing that there is insufficient evidence of the effectiveness of interventions ,5. Part In one example of an excluded study, researchers used a randomised design to evaluate an advocacy service for women experiencing domestic violence ,8. WomenSystematic reviews have also excluded, or devalued, evidence from qualitative studies. For example, a study of 200 women who had used domestic violence outreach services found that about half were living in situations of domestic violence when they first contacted the service. All of these women reported that the outreach services had helped them to leave the abusive relationship—a valued outcome for them .Given the health impacts on women who experience domestic violence (not to mention their children) and the prevalence of the problem, routinely asking women about abuse should be seen as an important form of primary and secondary prevention for a wide range of health problems.Screening tools for domestic violence are abundant, and many are effective at identifying women experiencing abuse ,10. HoweGiven the morbidity and mortality associated with domestic violence, it is tempting to suggest that universal screening for abuse should be integrated into routine clinical care, such that all women, regardless of their reason for presenting to a clinical setting, should be “asked the question.” Some argue that this approach is justified by the need to increase awareness of domestic violence as a significant problem with serious health and social consequences, and to make abused women aware that they are not alone in their experience. These are important considerations.Certainly all women who disclose that they have been exposed to violence should be provided with options regarding seeking help . Good diwithout obvious signs and symptoms of domestic violence—such as a woman who comes to the clinic for assessment of an upper respiratory tract infection? Should such women be prompted to disclose whether they are being abused? The woman who is not being abused will answer to that effect, and the appointment can carry on. But for the woman who is experiencing violence, who has not volunteered this information, several factors must be considered. An important issue is whether she is ready—both psychologically and in terms of taking specific actions—to confront the issue. A number of excellent qualitative studies have examined the process that women undertake in acknowledging that they are “victims” of “abuse” and embarking on the often long and difficult journey to avoid, reduce, and ultimately stop the violence in their lives [However, what about women presenting ir lives ,17. GiveAny potential benefits of screening must then be weighed against its potential harms, including labelling women, prompting potentially premature disclosure, and triggering possible reprisal violence from the abuser if he discovers she has sought help. The last of these might be particularly exacerbated for the woman with the respiratory tract infection who was unprepared to disclose and did not take necessary precautions. Other potential harms include exposure to the ramifications of laws on mandatory child protection reporting, whereby health providers must report such disclosures to child protection authorities. This can lead to an investigation that potentially increases a woman's risk of exposure to violence, and in some cases of having her children placed in foster care. Research has shown that many of these potential harms are of concern to women when mandatory universal screening and/or reporting protocols are in place . FinallyGiven the lack of clear data on the benefits of screening and of the interventions to which women are referred, and the lack of data on potential harms, we and others have concluded the following ,19,20. UI agree entirely with Nadine Wathen and Harriet MacMillan that practice should be based on evidence. There are further areas of agreement. We agree that there is a lack of knowledge on effective interventions for abusers and on harm occurring as a result of enquiry, and that targeted case finding is important.all women about domestic violence. Underlying this difference is the issue about how much evidence we need, and of what type. My position is that the evidence that already exists is sufficient to justify the promotion of routine enquiry, aiming to ask all women about their experience of abuse. There is evidence of actual benefits to women—and their children—from interventions provided by specialised services for domestic violence and from brief discussions with health professionals [The key difference that exists between my viewpoint and theirs is the conclusion about whether health professionals should aim to ask ssionals .Aiming to ask all women has several advantages over targeted case finding . It contThe twin issues of women's safety and harm minimisation are extremely important, for both routine enquiry and targeted case finding. These issues are important reasons why training and protocols for enquiry are necessary. Standard principles of confidentiality should be reinforced in training and protocols, which need to be tailored to relevant legal requirements, such as when child protection issues are involved. Training and protocols also need to emphasise that the role of routine enquiry is to facilitate, and not force, disclosure. It must remain the woman's choice as to if, when, and to whom, she discloses.all women or only in situations where asking about it is part of a specific diagnostic assessment. As with screening for a disease, universal screening for domestic violence should not be implemented unless we are sure that interventions are available to help those identified via screening and that screening plus appropriate treatment will do more good than harm.We agree with Ann Taket that domestic violence is not a disease, and that the paradigm of “screening for disease” is problematic in this context. At issue, however, is the question of whether domestic violence should be “talked about” with Professor Taket outlines the importance of integrating discussions about abuse in consultations to raise community awareness. Unfortunately, there is no evidence that this type of consciousness-raising occurs, or if it does, what benefit it might have. Given the lack of effectiveness of educational campaigns in general, it is difficult to be optimistic about this approach.We disagree with her conclusion that existing systematic reviews have “excluded studies done outside the health service setting….” Our review included interventions such as the post-shelter advocacy counselling approach to which Professor Taket refers . This inFinally, we concur that qualitative studies are invaluable in understanding domestic violence. Such research has provided insight into the complex process that women undertake to address the violence in their lives. Until there is evidence that universal screening actually helps with this process, the focus should be on developing evidence-based approaches to assist women when they do disclose abuse and on training health professionals to respond appropriately to such disclosures.

The cellular signaling pathway (network) is one of the main topics of organismic investigations. The intracellular interactions between genes in a signaling pathway are considered as the foundation of functional genomics. Thus, what genes and how much they influence each other through transcriptional binding or physical interactions are essential problems. Under the synchronous measures of gene expression via a microarray chip, an amount of dynamic information is embedded and remains to be discovered. Using a systematically dynamic modeling approach, we explore the causal relationship among genes in cellular signaling pathways from the system biology approach.In this study, a second-order dynamic model is developed to describe the regulatory mechanism of a target gene from the upstream causality point of view. From the expression profile and dynamic model of a target gene, we can estimate its upstream regulatory function. According to this upstream regulatory function, we would deduce the upstream regulatory genes with their regulatory abilities and activation delays, and then link up a regulatory pathway. Iteratively, these regulatory genes are considered as target genes to trace back their upstream regulatory genes. Then we could construct the regulatory pathway (or network) to the genome wide. In short, we can infer the genetic regulatory pathways from gene-expression profiles quantitatively, which can confirm some doubted paths or seek some unknown paths in a regulatory pathway (network). Finally, the proposed approach is validated by randomly reshuffling the time order of microarray data.Arabidopsis thaliana and metabolic shift pathway from fermentation to respiration in yeast Saccharomyces cerevisiae, are reconstructed using microarray data to evaluate the performance of our proposed method. In the circadian regulatory pathway, we identified mainly the interactions between the biological clock and the photoperiodic genes consistent with the known regulatory mechanisms. We also discovered the now less-known regulations between crytochrome and phytochrome. In the metabolic shift pathway, the casual relationship of enzymatic genes could be detected properly.We focus our algorithm on the inference of regulatory abilities of the identified causal genes, and how much delay before they regulate the downstream genes. With this information, a regulatory pathway would be built up using microarray data. In the present study, two signaling pathways, i.e. circadian regulatory pathway in Biological phenomena at different organismic levels have revealed some sophisticated systematic architectures of cellular and physiological activities implicitly. These architectures were built upon the biochemical processes before the emergence of proteome and transcriptome -3. Underin situ synthesized oligonucletide and Cry2 [X10] are commonly regulated by Lhy [X3] (LATE ELONGATED HYPOCOTYL) in reciprocal ways with significant values (0.7569 in Eq.(6) and -1.8773, Eq.(10) of Table Lhy on crytochrome genes. In addition, from Eq.(10) in Table Ccal [X4] (CIRCADIAN CLOCK ASSOCIATED 1) has the greatest positive regulation (2.3465) on Cry2, meaning that Cry2 is jointly regulated by Lhy and Ccal. Because the binding sites of Lhy and Ccal found in the promoter regions of Cry2 and PhyA [X7] in Eq.(6) and Eq.(7) of Table Cry2 [X10] is also positively regulated by PhyA [X7] with 0.5-hr activation delay similar to that in Cry1 (Eq.(6) in Table PhyA is considered as a post-transcriptional regulator of phosphorylation to crytochrome within 1.0-hr after transcription. On the other hand, PhyB [X11] down-regulates Cry2 with a significant effect (-0.7141) while Cry2 [X10] up-regulates PhyB (0.0511) weakly by feedback (see Eqs.(10), (11) in Table Cry2 and PhyB in nuclear speckles that are formed in a light-dependent fashion are also confirmed by Mas et al. [Cry1 and Cry2 are both negatively co-regulated by PhyD [X8] and PhyE [X12] significantly (see Eqs.(6), (10) in Table PhyA has apparently different behavior from PhyB, PhyD, and PhyE in activating crytochrome. This might suggest the mechanism that PhyA mediates the blue light by up-regulating Cry1 and Cry2, whilst PhyB, PhyD, and PhyE would mediate the red light by inhibiting blue photoreceptors and Ccal [X4], well-known biological clock genes in the circadian system [Gi [X15] (GIGANTEA) in Figure Gi sequence lacks any motifs suggesting that it is a transcription factor of phytochromes (EARLY FLOWERING 3) opposite to Gi on phytochrome, especially for PhyA, PhyB and PhyE (Eqs.(7), (11) and (12) in Table Lhy or Ccal, Elf3 might play the same role as Lhy and Ccal. Just as expected, Elf3 contains glutamine-rich motif suggesting that it is a transcription factor (0.1223) (see Eq.(9) in Table Toc1 [X13] is transcriptionally regulated by Lhy [X3] (0.7009) and Ccal [X4] (-1.4704) whilst Pif3 [X16] (-0.1698) is presumably considered as the bridge between Toc1 and phytochrome (Eq.(13), Table Lhy and Ccal point of view, they are both positively affected simultaneously by Pif3 implying the regulation on the transcriptional level Figure is mediaal level . In addiChs [X5] (CHALCONE SYNTHASE), Pap1 [X1], and Co [X2] (CONSTANS) in Figure Chs is known as correlated with UV-B protection. It seems that Ccal and Lhy have greater effect on Chs than Pap1 (-0.0455) as a transcription factor (Eq.(5) in Table Chs is regulated by Pap1 in a small scale with amplifying effect on the cis-regulatory level. Co is recognized as a pivotal gene of photoperiodic regulation of flowering. Indeed, strong regulations from Ccal and Lhy are identified to show that Co is regulated with a large-scale attenuation effect on the cis-regulatory level (Eq.(2) in Table We also infer some downstream pathways of Chs X , represented by black lines with more linking to upstream genes, the photo-transduction pathways are stabilized to provide oscillation. On the other hand, more blue lines of inhibitive interactions are revealed in the biological-clock regulatory pathways relevant to Co, Pap1, and Chs underlying the anti-phase functional regulation between these output pathways and the oscillator. In addition, the essential signal transduction factors of Fkf1, Gi, Elf3, and Pif3 make some critical links between the functional blocks mentioned above in the circadian system encoding an enzyme, which catalyzes PEP (Phosphoenolpyruvate) to pyruvate, is negatively regulated (-5.8763) by Pck1 [Y26] (Eq.(4) in Table Pck1 could be intepreted here as an indirect upstream transcription factor or regulatory gene for Pyk1 due to its function of decarboxylation and phosphorylation of oxalacetat in the presence of a nucleoside triphosphate and a divalent metal ion to yield PEP. Another Gcrl [Y15] gene is also identified as the strongest positive regulation (5.9829) to Pyk1 in Table Gcrl of Pdc1 [Y6] (PYRUVATE DECARBOXYLASE ISOZYME 1) plays a more essential role in Table Rap1 [Y17] (0.1164), and Pyk1 [Y4] is an upstream regulatory factor coding an enzyme with the most positive effect (3.1295) on Pdc1 according to the production of acetaldehyde from pyruvate. In the last kernel of the fermentation, Adh1 [Y7] and Adh2 [Y3] are involved in the ethanol metabolism of carbohydrate storage. Adh2 is implicated to up-regulate Adh1 in Table Adh1 in Table Gcr2 [Y16] and Sfp1 [Y30] with consistently dominant negative influences on Adh2 and Adh1 respectively would be at the transcriptional level presumably (see Eqs.(3), (7) in Table In the fermentation direction, by Pck1 Y6 (Eq.(4)by Pck1 Y6 (Eq.(4)by Pck1 Y6 (Eq.(4)by Pck1 Y6 (Eq.(4)by Pck1 Y6 (Eq.(4)by Pck1 Y6 (Eq.(4)Eno2 [Y2] (ENOLASE ISOZYME 2) is regulated by Pck1 [Y26] (-0.7195) in the same way as Pyk1 while the main transcription factor is Stp2 [Y31] with significantly positive regulation in Table Eno2, Gpm1 [Y8] (PHOSPHOGLYCERATE MUTASE l), Tpi1 [Y10] (TRIOSE-P ISOMERASE 1), Fba1 [Y11] (ALDOLASE 1), and Pgi1 [Y9] (PHOSPHOGLUCOSE ISOMERASE 1) indicates the construction of a trunk of the glyconeogenesis (see Eqs.(8), (9), (10), and (11) in Table Rap1 [Y17] and Gcrl [Y15] are the common regulators of Gpm1, Pgi1, and Tpi1. This means that Rap1 and Gcrl might be the most important regulators in the glyconeogenesis pathway by the transcriptional binding. Finally, Pgm2 [Y5] (PHOSPHOGLUCOMUTASE 2) co-regulated by Glk1 [Y27] (GLUCOKINASE 1), Hxk1 [Y28], and Hxk2 [Y29] significantly confers another pathway leading to the synthesis of UDP-GLU from Glucose-6-P (see Eq.(5) in Table In the sub-pathway of glyconeogenesis, Adh1 and Adh2 are apparent when compared with the obscure relationships in glyconeogenesis . Interestingly, three transcription factors Gcr1, Gcr2 and Rap1 appear to have very significant effects on the metabolic shift pathway. Finally, in order to validate the proposed method, an independent validation is also given by randomly reshuffling the time order of microarray experiment but with same choices of target gene and regulatory gene, as shown in Figure In the overview of the metabolic shift pathway in Figure Microarray expression analysis by the dynamic system approach offers an opportunity to generate functional regulation interpretation on the genome-wide scale. The crucial ontology behind using dynamic system techniques is that the causality between gene expression profiles could be identified according to the differential equation underlying a dynamic system. Therefore, because the microarray data were harvested with time progression, the simultaneously varied gene expressions implicated in a genetic regulatory system would be detected to infer the regulatory pathways in spite of the versatile interactions such as transcriptional control, protein phosphorylation, or specific enzyme regulation.The clustering method answers the problem of what is the functional catalogue of a specific gene by the identification of resembling patterns of gene expressions. Similarly, the co-regulations of upstream genes in our method also imply their concurrent functions. In contrast to the clustering algorithm, the causality of time-course data has been smoothly drawn by our dynamic method. The Bayesian networks were used merely for forward probabilistic estimation with time transition lacking in the feedback linkages. This unidirectional problem would not happen in our algorithm. Owing to the quantitative regulatory abilities of our model, we have a greater diversity of regulatory influence than the Boolean networks, which are deterministic with merely two states.In our dynamic system approach, we not only can link qualitatively the upstream genes to the downstream ones iteratively, but also indicate quantitatively their regulatory relationships, including the regulatory abilities and the activation delays. In terms of the regulatory abilities, the comparison between the upstream regulatory genes of a target gene can inspire us to ask which one is significant biologically and whether it is a positive or negative influence on the investigated gene. Moreover, the speculation of activation delays benefits the empirical reference by providing us when the upstream regulatory genes might interact with their target genes. Since any gene can be considered as a target gene to trace back its upstream regulators, these regulators are then considered as target genes to trace back their upstream regulators. Iteratively, the genetic regulatory pathway (or network) can be constructed to the genome-wide. According to the qualitative and quantitative features imbedded, two regulatory pathway examples are characterized as in Figure In the two pathways under investigation, we have a more detailed understanding about the regulatory interactions among phytochrome, crytochrome and biological clock in the circadian regulatory system. On the other hand, the sophisticated knowledge of the metabolic pathway after the diauxic shift can be unfolded properly in our analysis. Furthermore, the independent validation of our approach is also given by randomly reshuffling the time order of microarray experiment. We found that the proposed pathways in Figures There are some shortcomings in our study. First, although the time-course microarray data are available, its lower samplings will distort the real changes of gene expressions, especially for quick dynamic evolution. A more sampling experiment with respect to the intrinsic turnover rate is expected to have more precise analysis. Secondly, a regulatory gene with larger activation delay would not be recognized because the less activation delay criterion is used, but this might be overcome by properly relaxing the criterion. Thirdly, activation profiles under the proteome should be highly correlated with the transcriptional profiles to elevate the interpretation of our system model. In general, the synchronous time-course microarray assay is more suitable to underlie the transcriptional binding among causal genes, but an inference of physical interactions in the post-transcriptional level also has sufficient feasibility in our study.In the near future, the most pressing task is to investigate our presumed paths in the laboratory. As the pathway construction algorithms are further developed, we expect this system approach to have immense impact in elucidating the underlying molecular mechanisms of pathways in a variety of organisms, especially after the maturation of the protein chips. Ultimately, we envision that biologists will perform routine pathway inference to seek some novel regulations and to identify the evolutionarily conserved links.Gi(t) in equation (1.2), we substitute equation (1.2) into equation (1.1) to obtain the following dynamic equation for the expression profile of the i-th gene,After the decomposition of ai, bi, αn, and βn should be estimated by the time profile of microarray data of the i-th gene, i.e. these parameters should be specified so that the simulating output Xi(t) of the dynamic model in equation (3.1) should meet the empirical expression profile of the i-th gene. The least-squares estimation method is employed to solve this parameter estimation problem.In the above dynamic equation, parameters t = t1,…, tm and is then arranged in a vector differential form. Consequently, the vector differential form underlined in this equation is applied to m time points in order.To make the dynamic model effective, the dynamic equation in (3.1) should meet the expression profile at all time points , , and m denotes the number of time points.where In the next step, formula (3.2) can be translated into a differential matrix equation as follows,Yi = AiΦi + Ei     (3.3), Φi = [ai bi α0 β0 … αN βN]T, and are in vector forms, while is a matrix.where i, the least-squares method below is used to derive the optimal parameters estimation of ,To estimate the relevant unknown parameters in ΦEi as the noise of the gene-expression profile or of the microarray chips. So the consideration of modeling error makes equation (3.3) approach more the reality. By the way, in order to get accurate data of and from the expression profile of the target gene, the cubic spline should be employed to interpolate the time profile of the target gene. Furthermore, the choice of N is based on the tradeoff between the accuracy of approximation in (1.2) and the complexity of parameter estimation in (3.4). In this study N = 6 is chosen because these harmonics are enough to approximate regulation functions.Actually, the modeling error could be concluded into i in equation (2.4) is given as follows:Maximum likelihood method for Ωm data points is then described byThe log-likelihood function for given σ2 is , by which equation (2.5) is obtained.The necessary condition for the maximum likelihood estimation of variance Substituting equation (2.5) into equation (4.2) yields,i by minimizing the value of σ2 in equation(2.5). Then, the maximum likelihood estimate in equation (2.6) is obtained by in equation(2.5).meaning that we can find the maximum likelihood estimation of ΩW.C. Chang and Chang-Wei Li carried out the computational studies and analysis. B.S. Chen gave the topic and suggestions. All authors read and approved the final manuscript.The raw data has been transform to .mat file, which is one of the Matlab files. This file has been divided into three parts: Name, Time, and Profile. see Name denClick here for fileThe 'Shuffled_data' is the Shuffled raw data. see Click here for file

As anorexia and hypermetabolism are common in cirrhosis, leptin levels may be increased in this disease. In this study, we investigated the relation between the severity of disease and serum leptin levels in post-hepatitis cirrhosis and the role of body composition, gender and viral aetiology of cirrhosis in this association.Thirty-five cases with post-hepatitis cirrhosis and 15 healthy controls were enrolled in this study. Body composition including body mass index, body fat percentage and body fat mass were determined. Serum leptin levels were assayed.Leptin levels were significantly higher among cirrhotic patients independent of sex compared to controls (p = 0.001). Female patients in both groups have had higher leptin levels than males .Cirrhotic patients in each of A, B and C subgroups according to the Child- Pugh classification revealed significantly different levels compared to controls . Male cirrhotics in Child-Pugh Class B and C subgroups had significantly higher leptin levels compared to male controls . On the other hand, female patients only in Child Pugh class C subgroup have had higher levels of serum leptin compared to controls (p = 0.022).Child-Pugh classification has been found to be the sole discriminator in determination of leptin levels in cirrhotics by linear regression (beta: 0.435 p = 0.015).Serum leptin levels increase in advanced liver disease independently of gender, body composition in posthepatitic cirrhosis. The increase is more abundant among patients that belong to C subgroup according to the Child- Pugh classification. Leptin, a 16-kilodalton protein, is involved in the regulation of food intake and body composition and has In normal humans, circulating level of leptin is higher in women than in men ,5. BesidMalnutrition is a common feature of cirrhotic patients . A negatIn this study, we investigated the relation between the severity of disease and serum leptin levels in post-hepatitis cirrhosis and the role of body composition, gender and viral aetiology of cirrhosis in this association.Thirty-five cases with post-hepatitis cirrhosis which were diagnosed on the basis of the clinical, laboratory, radiological, and/or histopathological findings, and 15 healthy controls were enrolled in this study. Cirrhotic cases were assigned into 3 groups on the basis of the Child-Pugh classification as folloControl group consisted of healthy individuals with normal medical history, physical examination and blood biochemistry. None of them have had a restriction of diet for loosing weight during the last three months. Subjects who receive any medication have not been included into control group. The local human institutional review committee approved the study and written consents were received from all participants.th percentile compared to controls .Gender based serum leptin levels of controls and cirrhotic cases that were grouped according to Child Pugh Classification were as shown in Figure When age, gender, BFM, hepatitis B and C virus as etiologic factors of cirrhosis and child A, B and C as Child-Pugh classification were tested as independent variables for determination of serum leptin levels as dependent variable by linear logistic regression analysis in cirrhotic group, analysis result showed that Child-Pugh classification was the sole discriminator in determination of serum leptin levels in cirrhotic cases Table .Leptin regulates body weight by suppressing appetite and increasing energy expenditure ,3,4. AnoLeptin levels are higher in woman than in men ,6. McCulSince BMI and BFM values do not differ according to the sex and the presence or absence of cirrhosis, increased serum leptin levels could not be simply dedicated to BFM or malnutrition status in cirrhosis. In addition, linear regression test in the present study has shown that disease severity, which was determined by Child-Pugh classification, was the sole significant determinant of serum leptin levels in cirrhosis. In previous studies, association between the severity of cirrhosis and serum leptin levels is controversial -13. HenrIn an animal study, it has been shown that chronic ethanol consumption leads to increased serum concentrations of tumor necrosis factor and related cytokines such as leptin by inducing over production of these factors in the liver and peripheral adipose tissues . Leptin Serum leptin levels increase in advanced liver disease independently of gender, body composition and viral etiologic factor in post-hepatitis cirrhosis. The increase is more abundant among patients that belong to C subgroup according to the Child- Pugh classification.BMI, body mass index; BFP, body fat percentage; BFM, body fat massThe authors declare that they have no competing interests.Bolukbas FF conceived of the study, and participated in its design and coordination. Bolukbas FF, Bolukbas C, Erdogan M and Zeyrek F collected the samples and carried out the laboratory analysis. Bolukbas C conceived of the study and participated in the sequence alignment and drafted the manuscript. Horoz M participated in the design of the study, participated in the sequence alignment and drafted the manuscript. Gumus M collected the clinical data and performed the statistical analysis. Yayla A drafted the manuscript and revised it critically for important intellectual content. Ovunc O participated in study design and coordination and revised the manuscript critically for important intellectual content.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:

When publishing large-scale microarray datasets, it is of great value to create supplemental websites where either the full data, or selected subsets corresponding to figures within the paper, can be browsed. We set out to create a CGI application containing many of the features of some of the existing standalone software for the visualization of clustered microarray data.We present GeneXplorer, a web application for interactive microarray data visualization and analysis in a web environment. GeneXplorer allows users to browse a microarray dataset in an intuitive fashion. It provides simple access to microarray data over the Internet and uses only HTML and JavaScript to display graphic and annotation information. It provides radar and zoom views of the data, allows display of the nearest neighbors to a gene expression vector based on their Pearson correlations and provides the ability to search gene annotation fields..The software is released under the permissive MIT Open Source license, and the complete documentation and the entire source code are freely available for download from CPAN Microarray experiments produce vast amounts of data. The resulting datasets are highly complex and contain large matrices of expression measurements as well as sequence and experiment annotations that provide biological context to the data. To organize these different types of data in a way that allows intuitive exploration of the data, and provides the ability to gain important insights into relationships within a given dataset requires sophisticated visualization tools. Such visualization tools are of benefit not only to researchers analyzing and presenting or publishing their own data, but also to Model Organism Databases (MODs) for compiling and displaying microarray data for a given model organism.There are several excellent free tools available that allow an individual user to analyze their own data. These tools are either accessible on the web, or can be downloaded and used on a desktop machine. Examples include the EPCLUST , GEPAS ,3 and FGThe application was written using object oriented Perl following the Model-View-Controller (MVC) design paradigm . GeneXplThe Microarray::CdtDataset has two essential functions: during dataset creation (see below) it decomposes the data file into its constituent data parts and creates the files needed during data viewing (see below). During data viewing it provides the API for the viewer, and allows searching and retrieval of the data. Under the current model the dataset object itself is immutable. Microarray::CdtDataset was implemented as a client of the Microarray::DataMatrix module, which provides an API for accessing matrices of expression data. In the design of the classes certain compromises had to be made to accommodate the stateless client server environment in which the program operates. Specifically, to allow rapid responses, pre-generated images and correlation data are cached in a compact format on the web-server.There are two stages required to publish a microarray dataset on the web using GeneXplorer. The first stage (executed only once per dataset) involves creation of all the necessary files for GeneXplorer to use. The second stage uses these files to produce the display using the GeneXplorer web front-end.Dataset creation requires a file in the Clustered Data Table (CDT) format: a simple tab delimited text file format that would allow us to do the updates via web services.The GeneXplorer package is provided as a typical Perl distribution on the Comprehensive Perl Archive Network (CPAN), and adheres to the usual installation mantra of perl modules. After unpacking the software, a user with administrative privileges merely needs to type:perl Makefile.PLmakemake testmake installThis will install the libraries and the executable files that are needed for dataset creation by GeneXplorer into the regular system locations, unless otherwise specified during the first step above. The example in Figure In addition to its use within SMD, GeneXplorer has been used by many publications to provide access to microarray datasets through their web supplements, that can be accessed through SMD's publication page , and wasWe are planning to further develop GeneXplorer to enable it to handle other data formats. Specifically, we would like it to be able to accept data files in MAGE-ML format , which iWe have developed a web-application, GeneXplorer, which allows the visualization of microarray datasets over the Internet using only a web browser. This application has been extremely useful in our experience, where it serves both SMD users during analysis of their data and the public while browsing published datasets.GeneXplorer is available at under thSMD: Stanford Microarray Database.CAR designed and wrote the initial version of the GeneXplorer. This was extensively re-factored and modularized by JCM (library modules for dataset) and JD (for explorer). DB was involved in the guidance of the early stages of this project. GS wrote the correlations software and the DataMatrix classes, and guided the development of this project. All authors read and approved the final version of the manuscript.

Silene which have an X-Y sex-determining system . We used the X-linked copies to build a genetic map of the X chromosomes, with a marker in the pseudoautosomal region (PAR) to orient the map. The map covers a large part of the X chromosomes—at least 50 centimorgans. Except for a recent rearrangement in S. dioica, the gene order is the same in the X chromosomes of all three species. Silent site divergence between the DNA sequences of the X and Y copies of the different genes increases with the genes' distances from the PAR, suggesting progressive restriction of recombination between the X and Y chromosomes. This was confirmed by phylogenetic analyses of the four genes, which also revealed that the least-diverged X-Y pair could have ceased recombining independently in the dioecious species after their split. Analysis of amino acid replacements vs. synonymous changes showed that, with one possible exception, the Y-linked copies appear to be functional in all three species, but there are nevertheless some signs of degenerative processes affecting the genes that have been Y-linked for the longest times. Although the X-Y system evolved quite recently in Silene (less than 10 million years ago) compared to mammals (about 320 million years ago), our results suggest that similar processes have been at work in the evolution of sex chromosomes in plants and mammals, and shed some light on the molecular mechanisms suppressing recombination between X and Y chromosomes.To help understand the evolution of suppressed recombination between sex chromosomes, and its consequences for evolution of the sequences of Y-linked genes, we have studied four X-Y gene pairs, including one gene not previously characterized, in plants in a group of closely related dioecious species of Similar processes have been at work in the evolution of sex chromosomes in plants and mammals. A recently evolved plant X-Y system is helping to shed light on these processes Drosophila sex chromosomes and some plants [Silene [Drosophila miranda [Drosophila are excellent for studying the rate and causes of degeneration, but cannot shed light on question (i).Newly evolved sex chromosome systems, such as those in plants and fishe plants , but note plants ? Second,e plants , and emp [Silene ,7 and fr miranda . Recent Silene. This genus is a model for the study of plant sex chromosome evolution, since the sex chromosomes evolved recently [Studies of the evolutionary divergence of gene pairs on mammalian X and Y chromosomes suggest that recombination between the X and nonrecombining parts of the Y was successively suppressed. In many X-Y systems, including that in mammals, there is a “pseudoautosomal” region (PAR) where the X and Y recombine, and it has been found that DNA sequence divergence between homologous X- and Y-linked genes increases with distance from this region. This pattern has been termed “evolutionary strata” ,10. Partrecently ,13.Silene species includes S. latifolia, S. dioica, and S. diclinis, which have an X-Y sex-determination system with a male-determining Y [Silene species are hermaphroditic or gynodioecious . Dioecy and sex chromosomes thus probably evolved within this genus [Silene species have n = 12 chromosomes [One group of closely related dioecious mining Y ,14, whilis genus . All dipomosomes , so theromosomes .S. latifolia have recently been identified and sequenced ceased recombining long before the three dioecious species split, whereas the X and Y copies of SlX1-SlY1 (termed locus 1) continued to recombine until recently. We discuss the implications of these results for the mechanism of recombination arrest between the sex chromosomes.Several sex-linked genes from equenced , allowinS. latifolia cDNA. The SlX3 open reading frame of 575 amino acids encodes a protein sequence similar to calcium-dependent protein kinases (CDPKs) from tobacco, rice, and Arabidopsis thaliana . CDPKs are associated with various kinds of stress responses [S. latifolia [Locus 3 was identified from esponses . Thus, latifolia ,19.S. latifolia and S. dioica [latifolia-dioica, latifolia-diclinis, and dioica-diclinis , but for gene 1 no such sites were found. If, on the other hand, X1 and Y1 diverged after the species split, some sites should differ between the species, but not between X and Y of the same species. This is found for mammalian and bird sex chromosomes, and phylogenetic analysis suggests that some X and Y genes ceased recombining independently in different taxa [Silene species are very closely related [X1 sequences. However, some Y variants are exclusive to each species; we found five nucleotide variants fixed only in the S. latifolia Y (plus nine indel variants), and ten fixed only in S. dioica Y (plus one indel). Since only 11 S. dioica Y sequences were analyzed, the number of fixed Y variants is probably overestimated, however . Furthermore, in a tree estimated excluding these sites with fixed differences in the Y-linked sequences (as is appropriate for such closely related species), the Y sequences are nested within those of the X of each species between the X and Y sequences of S. latifolia and S. diclinis (dSX-Y) correlates with the gene's distance from the PAR in the X chromosome genetic map . X-Y synonymous divergence also differs significantly between genes 1 and DD44 (p = 0.01). These results suggest progressive suppression of the recombination between X- and Y-linked alleles of different genes. In S. dioica, the same correlation exists, using the S. latifolia or S. diclinis gene order; thus, the rearrangement probably arose recently in S. dioica, consistent with its absence in the other dioecious species. A recent rearrangement, such as an inversion, after the DD44-X and -Y sequences had diverged for some time, would not affect this gene's X-Y divergence relative to that of gene 1. In mouse species, where rearrangements have occurred, evolutionary strata corresponding to those on other mammalian X chromosomes are still plainly discernible [Synonymous divergence etic map B. X-Y sycernible .(dN) was less than dS for divergence between X and Y sequences tests for these two genes, the dN/dS difference between Y and X is highly significant . Our observation of similar dS values contrasts with previous analyses [S. diclinis Y3 gene also seems to evolve faster than the other Y3 genes and moving toward the current PAR. This pattern resembles the “evolutionary strata” for mammalian X-Y gene pairs based on Ks values, a measure of divergence per site similar to dS [dSX-Y values among our four gene pairs is 26% for locus 3 in S. diclinis. This overlaps the values for the mammalian stratum 4 and 3 genes ; these strata are inferred to have ceased recombining between the X and Y 30–50 million years ago (MYA) for stratum 4, and 80–130 MYA for stratum 3, whereas strata 1 and 2 diverged 130–320 MYA [The correlation of ar to dS ,10. Howe–320 MYA ,11.S. latifolia, S. dioica, and S. diclinis X-Y sequence divergence data show that X-Y differentiation was already advanced in the common ancestor of these species, except for locus 1. The maximum synonymous X-Y divergence observed for our genes is approximately 25%, including SlAp3, which probably transposed from an autosome onto the Y soon after the sex chromosomes evolved [MROS3-X/Y, whose Y-linked copy is degenerated [Silene species, the Silene sex chromosomes must have evolved much more recently than mammalian sex chromosomes.The evolved ; all theenerated . Unless Silene. For the nuclear genes Chs and Adh in the family Brassicaceae, estimated rates are, respectively, 1.4 × 10–8 to 2.2 × 10–8 substitutions per synonymous site per year [Ipomoea [S. latifolia group of species. However, substitution rates for some plant Adh genes are almost ten times slower, particularly for plants with long generation times [DD44 differentiated before the S. dioica–S. latifolia–S. diclinis speciation, whereas gene 1 may have ceased recombining after this, perhaps independently in S. latifolia and S. dioica; no analysis can be done in S. diclinis without diversity data for this species, but suppression of X-Y recombination within this species after its split from the other dioecious species is also possible suggest that the S. latifolia Y may carry genes suppressing recombination, and should help test whether mechanisms other than inversions contributed to reduction of the PAR.The mechanism suppressing X-Y recombination is unknown. Recombination could be reduced either by inversions (or other major recombination rate changes), and/or by modifiers reducing local crossover rates. The “strata” of different divergence in mammalian sex chromosomes may have resulted from a series of Y inversions disrupting X-Y recombination . Inversinversion . Thus, gn Silene ,33, requSilene. Recombination suppression may be selectively favored to preserve advantageous Y-linked combinations of alleles at different loci, such as genes that are advantageous in males but not in females [The mechanism of recombination reduction between X and Y chromosomes is important for understanding the diversity in loci that recently ceased recombining, such as gene 1 in females , althoug females ,22.Silene sex chromosomes. Degeneration is likely, since genotypes with a Y but no X chromosome are inviable [MROS3-Y in S. latifolia [Silene Y is uncertain, because most currently known sex-linked genes in these plants were ascertained from a cDNA-based search for Y-linked genes. Bacterial artificial chromosome clone sequencing may provide unbiased comparisons of homologous X- and Y-linked regions, and this has been started in papaya [Silene can also be inferred when dN values in the Y are elevated compared with X lineages. This is seen for the two “old” Silene Y-linked genes, locus 3 and locus 4 . Thus the higher dN in the Y-linked alleles is not due to a higher mutation rate (higher dS) in the Y than the X. Moreover, the Y-linked copy of locus 3 fails to amplify in RT-PCR experiments, and may be degenerated.Our analyses suggest that both reduction of recombination and Y degeneration may be in progress for inviable ,35, but atifolia . The extn papaya . Some de locus 4 . DiffereDD44, and no diversity data have yet been obtained for gene 3. The result of this test was nonsignificant; there is thus no evidence that Y1 and Y4 evolution is driven by selection. There is, however, very low polymorphism in the Y copies, so the test has low power [These observations, plus those for gene 1 , suggestow power .Genetic degeneration is supported by low levels of polymorphism of Y- compared with X-linked genes, taking into account the lower Y effective population size ,22. ThisSilene Y-linked genes? Our analyses suggest that degeneration of the genes studied here is partial, at most, consistent with a recent origin of the Silene sex chromosomes. However, there has probably been enough time for degeneration, since this occurred rapidly for genes on the neo-Y chromosomes of D. miranda [Silene Y. Silene sex chromosomes are more advanced in sex chromosome evolution than in some other plants. The papaya sex-determining region is just a small nonrecombining part of one chromosome, yet there is evidence for considerable differentiation, including addition of repeat sequences and some evidence for gene loss [D. miranda neo-Y chromosome [Buchnera [D. miranda neo-Y [Why is degeneration so slight for our miranda , which aene loss . More liene loss ) may thuromosome , the birromosome , and in Buchnera . In all da neo-Y ), suggesS. latifolia plants were from Edinburgh and from Fontainebleau forest (France). S. dioica plants were collected in Corrèze (France). S. dioica plants from the Sherringham population , used for isolation of the ScOpa09 marker, were kindly provided by D.L. Mulcahy . S. noctiflora and S. vulgaris were obtained from the seed collection of the Lyon Botanical Garden . Seeds of S. diclinis were obtained from the seed collection of the Institute of Biophysics in Brno (Czech Republic). Interspecific hybrid Silene diclinis × latifolia plants were generated by pollination of a S. diclinis female with pollen of an MAV line male (S. latifolia) kindly provided by S. Matsunaga . The S. latifolia U9 line, which was used for pollination of the interspecific hybrid, was kindly provided by S. Grant .Genomic DNA was extracted from leaves as described . For RT-S. latifolia by the approach that yielded loci 1 and 4 (lX4/SlY4 ). From alX4/SlY4 , and the walking , using t walking , SoutherS. dioica, S. diclinis, and S. noctiflora or S. vulgaris using primers designed from S. latifolia sequences , were as follows. One incubation at 94 °C for 5 min; 35 cycles of: denaturation at 94 °C for 30 s, annealing at a temperature that depended on the primers for 30 s, and elongation at 72 °C for a time depending on the length of the amplicon being the probable ortholog of SlY/X1. In S. latifolia, some of the copies have been shown not to be sex-linked . Southern blots were not done in S. dioica or S. diclinis, but RT-PCR reactions in all the dioecious species always amplified a single sequence in the females plus another very similar sequence in males, clearly representing the expected X and Y copies. Thus, for the analyses presented later, this gene can be treated as a single-copy gene, as was also the case in previous mapping work [All genes, except gene 1, are single-copy see . In S. lrn blots , and theing work .S. dioica, S. diclinis, and S. vulgaris were amplified, either by RT-PCR (X copies) or from genomic DNA (Y copies). PCR products were cloned into pGEM-T Easy vector , and multiple clones were sequenced for each gene. Sequencing reactions were carried out with ABI Big Dye Terminator V1.1 DNA sequencing kit, on an Applied Biosystems 3100 sequencer .For locus 3, the 3′ two-thirds of the coding sequences of S. dioica or S. latifolia. We have now confirmed sex linkage of all four loci by segregation analysis in all three dioecious species were designed from this sequence. In plants grown from seeds of this family, presence or absence of the expected amplified fragment accords exactly with results using the original pseudoautosomal RAPD marker primer OPA09. With our primers, S. dioica plants from the Corrèze population were genotyped by digesting PCR products with the restriction enzyme TaqI; the recombination frequency between the marker locus and sex was approximately 2.5%, confirming the pseudoautosomal location. In S. latifolia, genotyping was done using the same primers and an AluI site polymorphism. Genotype data were analyzed by both three-point and multipoint mapping, using JoinMap version 1.4 [To orient the X genetic map, we used a pseudoautosomal marker. For this, we cloned and sequenced a RAPD fragment incompletely linked to the X chromosome of the pollen donor of the eveloped . The seqsion 1.4 . Thus thhttp://pbil.univ-lyon1.fr/) [The primer sequences were removed before sequence analyses. For each gene, the nucleotide sequences were aligned using the corresponding amino acid sequences as a guide, using ClustalW with the Seaview interface (on1.fr/) . Alignmehttp://pbil.univ-lyon1.fr/) [http://pbil.univ-lyon1.fr/) [http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) [Phylogenetic trees were estimated including all sites except those with gaps by neighbor joining (NJ), maximum parsimony, and maximum likelihood (ML), using Phylo_Win (on1.fr/) . For NJ on1.fr/) and Treeew.html) .dS and dN site divergences were estimated using PAML 3.13 (http://abacus.gene.ucl.ac.uk/software/paml.html) [http://pbil.univ-lyon1.fr/) [dS and dN are similar under various substitution models and JaDion1.fr/) . Estimatang 1994 , Yang anang 1994 , and Neiang 1994 , implemeang 1994 using Jaang 1994 .dS or dN of X and Y sequences were compared using HyPhy 0.99 expresses dS values for Y lineages as multiples of values for X lineages: Rsyn = dSY/dSX. In model 2 , Rsyn was constrained to be equal to 1 (dSY = dSX). We compared the ML values by a LR test with model 2 as the null hypothesis. We used the same approach to compare dN values (with dN/dS replacing Rsyn). To compare dN/dS using LR tests, we again defined two models. Model 1 assumed two global variables (dN/dS)X and (dN/dS)Y so that the nonsynonymous rates of branches of the X lineage were expressed in terms of the synonymous rate (dN/dS)X, and similarly using (dN/dS)Y for Y branches (“shared dN/dS”), while model 2 assumed (dN/dS)X = (dN/dS)Y.Values for species to polarS. diclinis Y3 evolves faster than other Y3 sequences, we assumed a common Rsyn for S. dioica and S. latifolia, as in model 1 above, but added a further parameter, the dSY/dSX ratio for S. diclinis (model 1*). We compared models 1 and 1* using a LR test as above; we tested dN and dN/dS differences similarly.To test whether DD44, and no diversity data are yet available for gene 3.McDonald-Kreitman tests were done using DNAsp software, version 3.95 . The divS. latifolia by contingency tests, using DNAsp. To infer fixed differences rigorously, we used diversity data within S. latifolia for genes 1, 4, and DD44. For gene 3 no diversity data are yet available; however, because this gene pair has high X-Y divergence, raw divergence values should suffice, so for this gene we estimated numbers of differences from single X and Y sequences.To test for differences in divergence between the X and Y sequences of different genes, we compared numbers of fixed X-Y differences in S. latifolia and S. dioica Y and X sequences, plus one sequence from each of two outgroup species, S. vulgaris and S. conica; this enables us to identify whether the changes were in the X or Y lineages, using parsimony. With global gap-removal, the program unambiguously distinguishes fixed differences, including insertions and deletions, from polymorphisms within species. The outgroup sequences are shorter than the other sequences, so some fixed differences in the S. dioica Y could not be analyzed.To test whether X and Y sequences of gene 1 continued recombining and started to diverge after the dioecious species split, a C program was written to find fixed differences in a set of multiple http://www.lirmm.fr/guindon/phyml.html) [As this dataset includes the first approximately 2,000 sites of gene 1, including coding and intron sequences , a more ml.html) , excludiTable S1(36 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/) accession number for AtMSI4 is AF028711; the UniProt/TrEMBL (http://www.ebi.ac.uk/trembl/) accession number the ScOpa09 marker is Q9FW98.The GenBank (

Natural killer (NK) cells represent a first line of defence against a developing cancer; however, their exact role in colorectal cancer remains undetermined. The aim of the present study was to evaluate the expression of CD16 and CD57 [immunohistochemical markers of natural NK cells] in colorectal adenocarcinoma.Presence of NK cells was investigated in 82 colorectal adenocarcinomas. Immunohistochemical analysis was performed, using 2 monoclonal antibodies . The number of immunopositive cells (%) was evaluated by image analysis. The cases were characterized according to: patient gender and age, tumor location, size, grade, bowel wall invasion, lymph node metastases and Dukes' stage.NK cells were detected in 79/82 cases at the primary tumor site, 27/33 metastatic lymph nodes and 3/4 hepatic metastases; they were detected in levels similar to those reported in the literature, but their presence was not correlated to the clinical or pathological characteristics of the series, except for a negative association with the patients' age (p = 0.031).Our data do not support an association of NK cell tissue presence with clinical or pathological variables of colorectal adenocarcinoma, except for a negative association with the patients' age; this might possibly be attributed to decreased adhesion molecule expression in older ages. Colorectal adenocarcinoma is a neoplasm in which prognosis is mainly determined by the histological stage. However, the prognosis of certain patient groups, especially those of intermediate stages, remains vague . The maiNatural killer (NK) cells play a pivotal role in innate immunity and immunological surveillance. Their cytotoxic effects may be initiated without prior immunization and thus NK cells have been considered as a first line of defence of the host against a developing cancer ,3. In spThe aim of this study was to evaluate tissue presence of NK cells in a series of colorectal adenocarcinomas and to attempt to correlate their presence with clinical and pathological variables and prognostic markers of colorectal cancer.From June 1997 to May 2000, 82 patients from our Department underwent colectomy, due to a diagnosis of colorectal adenocarcinoma of conventional histologic type. NK cell presence was examined via standard 3-step immunohistochemical analysis (ABComplex), which was performed on formalin-fixed, paraffin-embedded tissue sections. Briefly, two appropriate monoclonal antibodies were used at dilutions of 1/200 with overnight incubation. Antigen retrieval was necessary and was performed by usual microwave treatment. In the examined samples, tissue identification of NK cells was based on strong CD16 immunostaining, which was frequently accompanied by CD57 immunoreactivity. Tissue sections from hypertrophic tonsils were used as positive markers. Diaminobenzidine tetrahydrochloride 0.06% in phosphate-buffered saline buffer containing 0.03% hydrogen peroxide was used as a chromogen. Images were acquired using a Zeiss Axiolab microscope with a mechanical stage, fitted in a Sony-iris CCD video camera . The video camera was connected to a Pentium II PC, loaded with the appropriate image analysis software . Slides were examined at a ×200 magnification. The ratio, expressed in percentile proportion (%), between the number of immunohistochemically positive-stained cells and the total number (stained and unstained) of lymphocytes was calculated. All tumors had been characterized according to the following classical clinical and pathological variables: patient gender and age , tumor location , size , grade , bowel wall invasion , lymph node metastases and Dukes' stage . All "D" Dukes' stage cases involved hepatic metastases. The tumors were categorized according to their location into two larger groups: those involving the rectum as well as the left colon and those involving the right colon . This was performed for reasons of statistical analysis.Moreover, positive cases were divided into two groups: those in which immunopositive cells ranged from 0–9% of the total number of lymphocytes ("weak" tissue presence of NK cells) and those in which they were ≥10% ("strong" NK cell presence). This was performed somewhat arbitrarily, as we considered that the cutoff level of 10% is in accordance to what a pathologist would consider to be the cutoff level between "negative" and "positive" cases in a qualitative examination, and in order to perform statistical analysis. Associations of NK cell presence with the patients' sex, tumor location, grade, and presence of bowel wall invasion, as well as lymph node metastases and Dukes' stage were examined using chi-square statistics; associations with the patients' age and tumor size were determined using the Mann-Whintey U test.NK cells were detectable in the primary tumor site of 79 cases (96.3%); their percentages were up to 32% of the primary site lymphocytes . Of the 33 cases with metastatic lymph nodes, NK cells were found in 27 cases (81.8%); moreover, NK cells were found among liver lymphocytes in 3 of the total 4 cases with hepatic metastases (75% of stage "D" cases).Forty-nine of the 82 primary tumors (59.8%) had a "weak" tissue presence of NK cells (0–9%), whereas the remaining 33 primary tumors (40.2%) had a "strong" NK cell presence (≥10%). Of the 33 cases with metastatic lymph nodes, 18 cases (54.5%) had "weak" and 15 "strong" NK cell presence.NK cell presence in the primary tumor site was not associated with the patients' sex , tumor size , tumor location , tumor differentiation , wall invasion , the presence of metastatic lymph nodes , or Dukes' stage . However, a negative association between the presence of NK cells at the primary tumor site with the patients' age was noticed .Moreover, the presence of NK cells in the metastatic lymph nodes was not associated with the patients' sex , age , tumor size , tumor location , tumor differentiation , wall invasion , or Dukes' stage (C or D) .Although colorectal adenocarcinoma has been characterized as a model neoplasm in which clinical prognosis is mainly determined by the histological stage, pathologic markers often fail to predict the prognosis of a great number of patients. This has necessitated the evaluation of several alternative prognostic markers . ImmunohCD16 and CD57 were chosen in the present study, as they are markers of the presence of NK cells, a cellular line which could reasonably play a significant role in the pathophysiology of colorectal adenocarcinoma. NK cells were considerably present in the primary tumor site, as well as the metastatic lymph nodes of many cases in our series. This observation is in line with data deriving from the literature -14; thisHowever, what seems to be pivotal for rapid and efficient migration of NK cells from the circulation to the tumor stroma, is the expression of appropriate adhesion molecules . As exprIn conclusion, our findings suggest that less NK cells are found in the stroma surrounding the primary tumor site in older patients with colorectal adenocarcinoma. This could possibly be attributed to decreased adhesion molecule-mediated migration; however, this hypothesis needs to be further investigated through more studies.The pre-publication history for this paper can be accessed here:

Ras induces expression of p19Arf, which, in turn, activates p53 and growth arrest. Here, we used a multistage model of squamous cell carcinoma development to investigate the functional interactions between Ras, p19Arf, and p53 during tumor progression in the mouse. Skin tumors were induced in wild-type, Arfp19-deficient, and p53-deficient mice using the DMBA/TPA two-step protocol. Activating mutations in Hras were detected in all papillomas and carcinomas examined, regardless of genotype. Relative to wild-type mice, the growth rate of papillomas was greater in Arf-p19deficient mice, and reduced in p53-deficient mice. Malignant conversion of papillomas to squamous cell carcinomas, as well as metastasis to lymph nodes and lungs, was markedly accelerated in both Arf-p19  and p53-deficient mice. Thus, p19Arf inhibits the growth rate of tumors in a p53-independent manner. Through its regulation of p53, p19Arf also suppresses malignant conversion and metastasis. p53 expression was upregulated in papillomas from wild-type but not  Arfp19-null mice, and p53 mutations were more frequently seen in wild-type than in  Arfp19-null carcinomas. This indicates that selection for p53 mutations is a direct result of signaling from the initiating oncogenic lesion, Hras, acting through p19Arf.Ectopic expression of oncogenes such as A squamous cell carcinoma model shows Ras mutation not only initiates tumor development but, through Arf and p53, directly influences the subsequent evolutionary trajectory of the tumors Ras, and two tumor suppressors, Arf,p19  and p53, using a multistage epithelial tumor model.Tumor development and metastasis is a multistep process of somatic cell evolution that includes uncontrolled proliferation, impaired apoptosis, loss of differentiation, immortalization, neovascularization, invasion, and metastatic spread . This evRas is among the most frequently mutated oncogenes in human cancer, with approximately 30% of tumors carrying an activating mutation in one of three family members, Hras, Kras, or Nras seen in human tumors ; p = 0.010) and  Arf+/−p19 mice had an average of 2.60 more papillomas ; p = 0.045) in weeks 18–30 after DMBA administration. Average papilloma size was also greater in both  Arf−/−p19 and  Arf+/−p19 mice compared to wild-type mice ( Arf−/−p19 mice were greater than 8 mm in diameter versus 14% (38/267) from wild-type mice (p < 0.0001). Papillomas from  Arfp19-deficient mice measured up to 16 mm in diameter while very few papillomas on wild-type mice measured more than 9 mm. Thus,  Arfp19 deficiency resulted in faster growing papillomas, indicating a role for  Arfp19 in regulating the early stages of benign tumor growth.Both in vitro and in vivo studies have demonstrated that p19ppressor . However5 wk see . Papilloype mice A. Relatiype mice B. This eHras are found in more than 95% of DMBA/TPA-induced skin tumors ,  Arf+/−p19 (4/4), and  Arf−/−p19 (5/5) mice contained the identical A→T transversion at codon 61 of Hras, resulting in an amino acid change from glutamine to leucine and a constitutively activated Ras protein ; p = 0.005) and 3.11 times higher for  Arf+/−p19 mice ; p = 0.073) compared to wild-type mice. The reduced carcinoma latency and increased conversion frequency in the  Arfp19-null mice implicate loss of p19Arf as a critical rate-limiting step in malignant SCC progression.The rate of malignant conversion of papillomas to carcinomas is greatly increased in the absence of p53 function . To deteer 22 wk C. By 28  Arf+/−p19 and  Arf−/−p19 mice also showed a range of grades but a significant number (9/12) were characterized as spindle cell carcinomas. These were characterized by packed and spindle-shaped cells with elongated pleiomorphic nuclei and abundant abnormal mitotic figures. These cells grew in a homogenous pattern with very little evidence of the cellular organization typical of low-grade tumors. These tumors showed focal areas of squamous differentiation, indicating that they were derived from squamous epithelium.Histologic analysis revealed that the carcinomas from control mice ranged in grade from well-differentiated to poorly differentiated SCCs. Carcinomas from  Arfp19 deficiency also increased dissemination and establishment of metastatic SCCs. Carcinoma-bearing p19Arf-deficient mice frequently presented with enlarged lymph nodes, and in several cases tumors were noted on the lungs. should be seen in the remaining wild-type  Arfp19 allele in carcinomas. Alternatively,  Arfp19 could be recessive, in which case LOH or reduction to a homozygous null state would be expected. The fate of the wild-type allele of  Arfp19 in tumors from heterozygous mice was assessed by semiquantitative PCR analysis of genomic DNA. Three of 15 (20%) papillomas examined showed evidence of loss of the wild-type  Arfp19 allele, compared to ten of 15 (67%) carcinomas (p = 0.0027) A, indicamal skin B. IncreaArf and p53 were upregulated in papillomas, that p53 expression was reduced in  Arp19f-null papillomas, and that loss of p19Arf had a similar effect on tumor progression as that of loss of p53, provide strong in vivo support of the model whereby p19Arf regulates p53 in response to mutational activation of Hras. However, enhanced tumor growth in  Arfp19-null mice, in contrast to reduced tumor growth in p53-null mice ; p = 0.0001) 10–16 wk after the DMBA administration, while  Arfp19-null mice averaged 2.68 more papillomas ; p = 0.015) 18–40 wk after the DMBA administration (unpublished data). Thus, p53-regulated apoptosis does not appear to play a major role in SCC development, at least at the papilloma stage.To determine whether tumor growth in mice lacking p19ng index C. The p1Arf and p53 cooperate during tumor progression, papilloma to carcinoma conversion was evaluated in Arf−/−, p53−/−,p19 and Arf−/−p53−/−p19 littermates. Tumor progression was accelerated in all  Arf-p19 and p53-deficient genotypes compared to wild-type littermates p53 A, indicaints see B. This c+/−p53 mice there is strong selective pressure to lose the wild-type allele during conversion to malignancy p19 were assessed for LOH of p53 by semiquantitative PCR analysis of genomic DNA. Two out of seven papillomas from  Arf+/+p53+/−p19 mice show loss of the remaining p53 allele, while all papillomas examined from  Arf+/−p53+/−p19 (n = 8) and  Arf−/−p53+/−p19 (n = 8) mice show retention of wild-type p53 from Arf+/−p53+/−,p19  but only one of 12 (8%) from  Arf−/−p53+/−p19 (p = 0.036) showed loss of p53. Thus, deletion of p19Arf disrupts the activation of p53 and thereby reduces selection for mutations in p53 during malignant progression.In lignancy . As p19Atype p53 B. Seven Hras and the tumor suppressors Arfp19 and p53. Somatic mutation of Ras is an early and frequent event in this model of tumor development. Against this backdrop, p19Arf has at least two distinct tumor suppressor properties, which act at different stages of tumor development and which show a range of gene dosage effects. Loss of one or both  Arfp19 alleles leads to accelerated growth of benign tumors, indicating  Arfp19 is partially haploinsufficient for suppression of this early growth phenotype. Although p19Arf regulates p53 at this stage, suppression of tumor growth per se by p19Arf does not appear to be mediated through p53. p19Arf also inhibits the benign to malignant transition and subsequent tumor cell dissemination and metastasis, and this effect of p19Arf is, in contrast, mediated through p53. LOH of Arfp19  occurs preferentially in malignant tumors, indicating complete loss of p19Arf is favored during progression. Thus, p19Arf inhibits several stages in Ras-driven tumor progression. Furthermore, Ras is connected to both p19Arf and p53 through a signaling pathway, indicating that selection for mutations in the p19Arf / p53 pathway are a direct consequence of the initial Ras mutation.Using a multistage model of tumor progression, we have examined the functional interactions between the oncogene Hras mutations are found at high frequency in papillomas from wild-type and both p19Arf- and p53-deficient mice indicates that squamous epithelial cells harboring Hras mutations have a strong selective advantage, with or without the presence of p19Arf or p53. This permits analysis of the effects of Ras on defined genetic backgrounds in the natural setting of tumor cell evolution. The expression levels of both p19Arf and p53 were increased in wild-type papillomas but not in  Arf−/−p19 papillomas, indicating that p19Arf regulates p53 in response to activated Ras in vivo. However, other signals to induce p53, such as those stemming from DNA damage, remain intact in the absence of p19Arf, as shown by the rapid increase in p53 in irradiated  Arfp19-null tumors. Thus, of the many stimuli that have been shown to activate p53 using a variety of experimental systems or exogenous stimuli . However, it remains possible that these other modes of p53 activation might predominate in other tumor types or in other circumstances.The observation that systems , the RasArf and p53 were induced in papillomas, loss of p19Arf or p53 had opposite effects on early tumor growth. p19Arf deficiency resulted in increased tumor cell proliferation and tumor growth while p53-deficient mice had reduced tumor cell proliferation, tumor numbers, and tumor size. The observation that tumors harboring mutant Ras grew faster in the presence of p53 than in the absence of p53 differs from in vitro studies, in which ectopically expressed Ras induces p53-dependent growth arrest or senescence were provided by Martine Roussel and Charles Sherr , and carcinogenesis studies were performed on the F3 littermates of this cross. 20 mice of each genotype,  Arf−/−, p19 Arf+/−,p19 and  Arf+/+p19 were treated. The backs of 8-wk-old male and female mice were shaved and treated with a single application of DMBA followed a week later by twice weekly applications of TPA (200 μl of 10−4 M solution in acetone) for 15 wk. The number and size of papillomas on each mouse were recorded every 2 wk. Mice were sacrificed if moribund or following detection of carcinomas. Tumors were frozen for DNA extraction and/or fixed in formalin to be processed and stained with hematoxylin/eosin (H&E) for histological examination.p19es Sherr . To incrp53 (7 backcross to NIH) were crossed to  Arfp19-deficient (F4 backcross to NIH) mice to generate   Arf+/− p53+/−p19 mice. These mice were intercrossed to generate all nine possible  Arf /p53p19 genotypes. Some 20–30 mice of each genotype were subjected to the same DMBA/TPA protocol and monitored as described above.Mice deficient for p53 and p53 .In order to remove abundant keratin present in papillomas and carcinomas, nuclear extracts were prepared as described with modHras was amplified with standard PCR , with 5′-GACTCCTACCGGAAACAGGT-3′ and 5′-CTGTACTGATGGATGTCCTC-3′ primers. We used the internal primer 5′-TGGTCATTGATGGGGAGACA-3′ to sequence exon 2, using PE Biosystems Dye-Terminator and Big-Dye cycle sequencing.Genomic DNA was prepared from tumor and normal tissue by QIAamp DNA Mini Kit . A 400-bp PCR fragment containing exon 2 of Sections of normal skin, papillomas, carcinomas, and other organs were removed and fixed in 10% normal buffered formalin for 4 h. After fixation, tissues were processed and then embedded in paraffin. From the tissues, 4-μm sections were cut and stained for either p53 or pan-keratin using high-temperature antigen retrieval in 10 mM citrate buffer (pH 6), or for BrdU after treating with 2N HCl followed by 0.1% trypsin. After staining with the primary antibody, the sections were stained with a biotin-conjugated secondary followed by StreptABComplex/HRP (Dako). Slides were developed with DAB/NiCl and counterstained with methyl green. Control sections with no primary antibody were run concurrently. Other sections were cut and stained using a standard H&E method. Proliferation index was determined by counting the number of BrdU-stained cells per 40× field. The apoptotic index was determined by counting the H&E slides for the number of apoptotic figures per 40× field. All counts were done on a Nikon Labophot-2 microscope.Arfp19 wild-type allele in Arf+p19/− tumors, wild-type and knockout alleles were amplified by PCR separately then combined for electrophoresis. Primers 5′-AGTACAGCAGCGGGAGCATGG-3′ (Arf1), 5′-TTGAGGAGGACCGTGAAGCCG-3′ (Arf2), and 5′-ACCACACTGCTCGACATTGGG-3′ (ArfN) were used to amplify wild-type (Arf1 and Arf2) and knockout alleles (Arf2 and ArfN) from 100 ng of genomic DNA using 68 °C for annealing and extension at 90 s for 30 cycles. Equal amounts of each PCR product were then combined for electrophoresis on a 2% TAE agarose gel. Wild-type and knockout alleles of p53+/− tumors were amplified in a separate reaction as described . To measure LOH of the escribed for 30 cArfp19 (or Arf /p53p19) genotypes on the development of papillomas, longitudinal profiles of papilloma counts were analyzed using the generalized estimating equation (GEE) approach (Arfp19 (or Arf /p53p19) genotypes. Estimates of relative odds were adjusted for sex effects. Fisher's exact test was used for comparing two proportions such as comparing LOH proportions between papillomas and carcinomas. All statistical tests were two-sided.In order to assess the impact of approach . GEE is approach was used

Low adherence is a key factor in explaining impaired effectiveness and efficiency in the pharmacological treatment of hypertension. However, little is known about which factors determine low adherence in actual practice.The purpose of this study is to examine whether low social participation is associated with low adherence with antihypertensive medication, and if this association is modified by the municipality of residence.1288 users of antihypertensive medication were identified from The Health Survey in Scania 2000, Sweden. The outcome was low adherence with antihypertensives during the last two weeks. Multilevel logistic regression with participants at the first level and municipalities at the second level was used for analyses of the data.Low social participation was associated with low adherence with antihypertensives during the last two weeks , independently of low educational level. However, after additional adjustment for poor self-rated health and poor psychological health, the association between low social participation and low adherence with antihypertensives during the last two weeks remained but was not conclusive . Furthermore, the association between low social participation and low adherence with antihypertensives during the last two weeks varied among municipalities in Scania .Low social participation seems to be associated with low adherence with antihypertensives during the last two weeks, and this association may be modified by the municipality of residence. Future studies aimed at investigating health-related behaviours in general and low adherence with medication in particular might benefit if they consider area of residence. The effectiveness ,2 and efLow adherence is one important cause of uncontrolled hypertension ,10. Yet,Social participation is an important concept for understanding the influence of social factors on individual health and behaviour, and can be viewed as a feature of individual social networks . Good soMultilevel analysis handles both the micro-scale of people and the macro-scale of context simultaneously within one model . This anThe first aim of this study is to examine whether low social participation is associated with low adherence with antihypertensives during the last two weeks, independently of low educational level and health status . The second aim is to analyse whether the hypothesised association between low social participation and low adherence with antihypertensives during the last two weeks varies between municipalities in Scania.The Health Survey in Scania 2000 (HSS-2000) was a postal self-administered questionnaire sent out to a random sample of 23 437 individuals born from 1919 to 1981 living in Scania. The purpose of the HSS-2000 was to obtain information about health conditions and different types of health hazards among the inhabitants of Scania . The proThe Ethical Committee at the Medical Faculty of Lund University approved the study proposal of The HSS-2000, and all of the participants received written information about the survey.Use of antihypertensives was based on an affirmative answer to the question "Have you during the last year used medicine, which was bought at the pharmacy...?" and indicating "Medication for high blood pressure"Low adherence with antihypertensives during the last two weeks (dichotomised) was based on the question "Have you used (this) medicine during the last year, but not during the last 2 weeks?" Those participants who answered yes were considered to have low adherence.Age was categorised in five groups: <35 (reference), 35–44, 45–54, 55–64 and ≥ 65 years.Low social participation (dichotomised) was assessed after the respondent stated involvement in three or fewer activities (lowest quartile) of 13 formal or informal activities , which the respondent might have participated in during the previous 12 months [2 months .Low educational level (dichotomised) was defined as having nine years of education or less.Poor self-rated health (dichotomised) was defined as a value of ≤ 3 on an ordinal self-rated health scale ranging from 1 ("Very bad") to 7 ("Very good") [y good") .Poor psychological health (dichotomised) was determined by giving three or more affirmative answers to the 12 items composing the Standardised General Health Questionnaire (GHQ-12) [(GHQ-12) .Because of the hierarchy in the data, with individuals nested in municipalities, we used multilevel logistic regression with indi was created to study the influence of low social participation on low adherence with antihypertensives during the last two weeks, adjusting for age and sex. The second model ii was extended to also include low educational level, because low educational level could be a confounder in the association between low social participation and low adherence with antihypertensives during the last two weeks. The third model iii additionally contained poor self-rated health and poor psychological health. Impaired health may affect both low social participation and low adherence with antihypertensives.The first model The results are shown as odds ratios (OR) with 95% confidence intervals (CI)We calculated the second level variance regarding prevalence of low adherence with antihypertensives during the last two weeks , and the second level variance regarding the association between low social participation and low adherence with antihypertensives during the last two weeks . We also calculated the covariance between intercept and slope residuals. The covariance gives information about whether the association between low social participation and low adherence with antihypertensives during the last two weeks depends on the prevalence of low adherence in the different municipalities .Parameters were estimated using the Restricted Iterative Generalized Least Squares (RIGLS) and penalised quasilikelihood (PQL). Extra-binomial variation was explored systematically in all models and we found no evidence for under- or over-dispersion. The MLwiN, Version 1.1 software package was usedLow adherence with antihypertensives during the last two weeks was found among 11% (145/1 288) of the participants and 49% (635/1 288) were classified as having low social participation. The participants mean age was 63 years. Those participants classified as having low social participation more often reported low adherence with antihypertensives during the last two weeks, low educational level, poor self-rated health and poor psychological health than those who were not classified as having low social participation and in the association between low social participation and low adherence with antihypertensives during the last two weeks (slope variance) Table . The negOur results suggest that low social participation is associated with low adherence with antihypertensives during the last two weeks, independently of low educational level. In other words, the association between low social participation and low adherence withstood adjustment for socio-economic position in our analyses. Social participation might therefore be considered as a real construct, and not only a proxy for socio-economic position. However, the association between low social participation and low adherence with antihypertensives during the last two weeks was weakened after additional adjustment for poor self-rated health and poor psychological health. Furthermore, the association between low social participation and low adherence with antihypertensives during the last two weeks may vary between municipalities in Scania.The weakening of the association between low social participation and low adherence with antihypertensives during the last two weeks after we adjusted for poor self-rated health and poor psychological health may be an expression for confounding. Impaired health may negatively affect both social participation and adherence with antihypertensives. On the other hand, the observed reduction of the association may instead be telling us that physical and mental health are in the pathway between low social participation and low adherence with antihypertensives during the last two weeks . Social Our present finding that the association between low social participation and low adherence with antihypertensives during the last two weeks may vary between municipalities in Scania gives empirical support to the existence of cross-level interactions associated with health-related behaviours, such as low adherence with medication. These behaviours may be a result of the interaction between a person and his or her area of residence . In a prThe present study shows that multilevel regression analysis can be used for investigation of geographical disparities in health and health-related behaviour , without analysing any specific area characteristic . In multThe rather low participation rate (59%) may increase the risk of selection bias and reduce the ability to generalise the results and compare them to other populations. Nevertheless, the participation rate for participants aged 51–80 was about 65% and the participants using antihypertensives had a mean age of 63 years.The low adherence question we used was stated in a non-threatening manner, which might facilitate for participants to give an honest response and not underreport low adherence . Self-rePeople with low social participation and low adherence with antihypertensives during the last two weeks may have been less inclined to respond to the HSS-2000 questionnaire. This possible selection bias could lead to an underestimation of the association between low social participation and low adherence with antihypertensives during the last two weeks.Our results suggest that low social participation is associated with low adherence with antihypertensives during the last two weeks, independently of low educational level. In addition, the association between low social participation and low adherence with antihypertensives during the last two weeks seems to vary between the municipalities in Scania, which gives empirical support to the existence of cross-level interactions associated with health-related behaviours, such as low adherence with medication. We have recently showed that factors related to the area of residence influence the individual blood pressure level, especially in people using antihypertensive medication , which iFuture studies aimed at investigating health-related behaviours in general and low adherence with medication in particular might benefit if they consider that area of residence may modify associations between individual variables.The author(s) declare that they have no competing interests.JM and KJ developed the original idea, participated in the design of the study, performed the statistical analyses and drafted the manuscript. LR and JS participated in the design of the study and revised the manuscript. TL participated in the design of the study, helped to collect the data and revised 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/1471-2458/5/17/prepub

Identifying regions of the human genome that have been targets of natural selection will provide important insights into human evolutionary history and may facilitate the identification of complex disease genes. Although the signature that natural selection imparts on DNA sequence variation is difficult to disentangle from the effects of neutral processes such as population demographic history, selective and demographic forces can be distinguished by analyzing multiple loci dispersed throughout the genome. We studied the molecular evolution of 132 genes by comprehensively resequencing them in 24 African-Americans and 23 European-Americans. We developed a rigorous computational approach for taking into account multiple hypothesis tests and demographic history and found that while many apparent selective events can instead be explained by demography, there is also strong evidence for positive or balancing selection at eight genes in the European-American population, but none in the African-American population. Our results suggest that the migration of modern humans out of Africa into new environments was accompanied by genetic adaptations to emergent selective forces. In addition, a region containing four contiguous genes on Chromosome 7 showed striking evidence of a recent selective sweep in European-Americans. More generally, our results have important implications for mapping genes underlying complex human diseases. An analysis of 132 human genes suggests that the migration of modern humans out of Africa into new environments was accompanied by genetic adaptations to emergent selective forces Despite intense study and interest, a detailed understanding of the evolutionary and demographic forces that have shaped extant patterns of human genomic variation remains elusive. An important goal in studies of DNA sequence variation is to identify loci that have been targets of natural selection and thus contribute to differences in fitness between individuals in a population. Identifying regions of the human genome that have been subject to natural selection will provide important insights into recent human history , the funThe neutral theory of molecular evolution , which pOne way out of this conundrum is to recognize that population demographic history affects patterns of variation at all loci in a genome in a similar manner, whereas natural selection acts upon specific loci . Thus, bHere, we describe an extensive analysis of the molecular evolution of 132 genes that were comprehensively resequenced in 24 African-Americans and 23 European-Americans. In total, over 2.5 Mb of baseline reference DNA was sequenced, spanning 20 autosomal chromosomes and the X chromosome. The sampling of a large number of loci dispersed throughout the genome has allowed us to clarify the relative contributions of demography and selection to patterns of genetic variation at individual genes. Specifically, we developed a rigorous computational approach for taking into account multiple hypothesis tests and demographic history, and we found that while many apparent selective events can instead be explained by demography, there is also strong evidence for positive or balancing selection at eight genes in the European-derived population. In addition, we describe a striking example of a previously unreported recent selective sweep in European-Americans that spans four contiguous genes on Chromosome 7. More generally, our data provide insight into the demographic histories of African-American and European-American populations and have important implications for genetic association studies of complex diseases, as several of the genes showing evidence of selection have been implicated in susceptibility to complex human diseases.p < 0.05) in one or more tests of the allele frequency distribution .We resequenced 132 genes primarily involved in inflammation, blood clotting, and blood pressure regulation and discovered a total of 12,890 SNPs . We firsribution . Thus, tThe direction of Tajima's D, Fu and Li's D*, and Fu and Li's F* is potentially informative about the evolutionary and demographic forces that a population has experienced. For example, negative values reflect an excess of rare polymorphisms in a population, which is consistent with either positive selection or an increase in population size. Positive values indicate an excess of intermediate-frequency alleles in a population and can result from either balancing selection or population bottlenecks. In the European-American sample, we observed eleven significantly positive and five significantly negative values for one or more of these three test statistics . In the The observations of both significantly positive and significantly negative values of Tajima's D, Fu and Li's D*, and Fu and Li's F*, combined with the largely nonoverlapping set of significant genes, could reflect selective pressures unique to one population , different demographic histories, spurious results, or most likely some complex combination of all of these factors. Although these results are intriguing, their interpretation is confounded by two issues: (1) We have not corrected for multiple hypothesis tests, and (2) rejection of the standard neutral model can result from either selective or demographic forces. In the subsequent sections, we develop approaches to address these issues with the dual goals of identifying genes that possess strong evidence of natural selection and of inferring population demographic history.p values (–8) distribution for one or more tests of the allele frequency distribution and S2. Although neutrality tests of the allele frequency distribution reveal many significant deviations, it is impossible to unambiguously interpret these data as evidence for natural selection, because the null model used to assess significance makes unrealistic assumptions about population demographic history. In principle, it is possible to distinguish between demography and selection, because demography affects all loci in the genome, whereas selection acts upon specific loci. Thus, by sampling a large number of loci dispersed throughout the genome, we can begin to construct a more realistic null hypothesis by which to evaluate the evidence for or against selection .To this end, we used the empirical data to explore four different demographic models A, which We reestimated the significance of Tajima's D, Fu and Li's D*, Fu and Li's F*, and Fay and Wu's H in each population for each of the four demographic models using the best-fit parameter values. All simulations included recombination and correction for multiple tests using the FDR method (with a FDR of 5%) as described above. Population history can clearly have a profound effect on tests of natural selection , and givEPHB6, TRPV6, TRPV5, and KEL;EPHB6, TRPV6, TRPV5, and/or KEL possess specific alleles that have conferred local adaptation to a unique environmental pressure in European-derived populations. Consistent with this hypothesis, we observed strong levels of population subdivision , which An alternative explanation for why we observed fewer significant results in African-Americans than in European-Americans is that African-Americans are an admixed population , and theABO in African-Americans of accelerated evolution in either the human (TRPV6 and EPHB6) or chimp (DCN) lineage. In addition, Recently, EPHB6, TRPV6, TRPV5, and KEL. To our knowledge, this is the largest footprint of selection that has been described in the human genome, and likely reflects the combination of strong and recent selective pressures and reduced recombination in this region . Based on our current data it is impossible to identify which gene (or perhaps genes) has been the target (or targets) of selection. However, TRPV6 is a particularly interesting candidate, as it possesses three nonsynonymous amino acid substitutions that are each nearly fixed for the derived allele in European-Americans, show significant frequency differences between European-Americans and African-Americans, and are located in the most significant regions of both Tajima's D and reduced nucleotide diversity to determine the derived allele for Fay and Wu's H test. These data were generated under the auspices of the SeattleSNPs Program for Genomic Applications, which resequences candidate genes involved in inflammatory processes in humans. In general, we resequenced the complete genomic region for each gene, including introns and approximately 2 kb 5′ of the gene and 1 kb 3′ of the gene using Big-Dye terminator chemistry on an ABI 3700 or ABI 3730XL . For several exceptionally large genes, such as F13A1, less than complete coverage was obtained (see http://pga.gs.washington.edu/).Human DNAs were obtained from the Coriell Institute . We analyzed DNA from 24 African-Americans from the Human Variation Panel, African-American Panel of 50 (HD50AA) and DNA from 23 European-Americans derived from various CEPH pedigrees. We also sequenced each gene in a common chimpanzee . In order to correct for multiple tests, we repeated the coalescent simulations as described above, but included recombination. Following 4 coalescent realizations, we sampled the recombination rate from a Gamma distribution whose expectation equals the average genome-wide recombination rate of 10–8/generation .We initially assessed the significance of these statistics by comparing the observed values to 10ulations , conditineration . The posST as described in We quantified the allele frequency differences between the European- and African-American samples by the statistic FTRPV6 is the target of selection and the selected haplotype is defined by the C157R, M378V, and M681T polymorphisms. If mutations are Poisson-distributed, the expected number of segregating sites in a genealogy is E[S] = μE[T], where S, μ, and T denote segregating sites, neutral mutation rate of the locus, and total branch length of the genealogy, respectively. Assuming a star-shaped genealogy, E[T] = n × t, where n is the number of selected haplotypes. Thus, the time back to the selective sweep, t, can be estimated by S/(nμ). For TRPV6 in European-Americans, n = 45 , S = 11, and μ = 2.5 × 10–5.We estimated the time since the selective sweep for the Chromosome 7q region in European-Americans by analyzing the amount of nucleotide diversity that has accumulated on the selected haplotype as described in Wfor each population. The bottleneck model is specified by the parameters F (the inbreeding coefficient) and t (the time in years measured from the present) at which the bottleneck occurred. Values of F and t considered were F = and t = . The exponential expansion model is determined by the parameters α (the growth rate/generation) and t . Values considered for α and t were: α = and t = . The population structure under an island model is specified by the population migration rate between demes, M = 4om,N where oN and m are the effective subpopulation size and fraction of migrants in each subpopulation per generation, respectively. Values of M considered were M = . The structure model assuming population splitting with no subsequent migration is determined by the parameter t (the time in years since the populations diverged). Values of t considered were t = . In all simulations we assumed an effective population size of 10,000 and a generation time of 25 y in order to facilitate comparisons to a previous study .Click here for additional data file.Table S2(534 KB DOC).Click here for additional data file.Table S3(87 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/LocusLink/) for the genes discussed in this paper are ABO (28), ACE2 (59272), APOH (350), BDKRB2 (624), BF (629), C2 (717), CCR2 (1231), CD36 (948), CEBPB (1051), CRF (10882), CRP (1401), CSF2 (1437), CSF3 (1440), CSF3R (1441), CYP4A11 (1579), CYP4F2 (8529), DCN (1634), EPHB6 (2051), F10 (2159), F11 (2160), F12 (2161), F13A1 (2162), F2 (2147), F2R (2149), F2RL1 (2150), F2RL2 (2151), F2RL3 (9002), F3 (2152), F5 (2153), F7 (2155), F9 (2158), FGA (2243), FGB (2244), FGG (2266), FGL2 (10875), FSBP (10646), GP1BA (2811), ICAM1 (3383), IFNG (3458), IGF2 (3481), IGF2AS (51214), IL10 (3586), IL10RA (3587), IL10RB (3588), IL11 (3589), IL12A (3592), IL12B (3593), IL13 (3596), IL15RA (3601), IL17B (27190), IL19 (29949), IL1A (3552), IL1B (3553), IL1R1 (3554), IL1R2 (7850), IL1RN (3557), IL2 (3558), IL20 (50604), IL21R (50615), IL22 (50616), IL24 (11009), IL2RB (3560), IL3 (3562), IL4 (3565), IL4R (3566), IL5 (3567), IL6 (3569), IL8 (3576), IL9 (3578), IL9R (3581), IRAK4 (51135), ITGA2 (3673), ITGA8 (8516), JAK3 (3718), KEL (3792), KLK1 (3816), KLKB1 (3818), KNG (3827), LTA (4049), LTB (4050), MAP3K8 (1326), MC1R (4157), MMP3 (4314), MMP9 (4318), NOS3 (4846), PFC (5199), PLAT (5327), PLAU (5328), PLAUR (5329), PLG (5340), PON1 (5444), PON2 (5445), PPARA (5465), PPARG (5468), PROC (5624), PROCR (10544), PROS1 (5627), PROZ (8858), PTGS2 (5743), SCYA2 (6347), SELE (6401), SELL (6402), SELP (6403), SELPLG (6404), SERPINA5 (5104), SERPINC1 (462), SERPINE1 (5054), SFTPA1 (6435), SFTPA2 (6436), SFTPB (6439), SFTPC (6440), SFTPD (6441), SMP1 (23585), STAT4 (6775), STAT6 (6778), TF (7018), TFPI (7035), TGFB3 (7043), THBD (7056), TIRAP (114609), TNF (7124), TNFAIP1 (7126), TNFAIP2 (7127), TNFAIP3 (7126), TNFRSF1A (7132), TNFRSF1B (7133), TRAF6 (7189), TRPV5 (56302), TRPV6 (55503), VCAM1 (7412), VEGF (7422), and VTN (7448).LocusLink ID numbers (http://coriell.undmj.edu/) repository numbers for human genomic DNAs sequenced for this study are as follows. DNAs from African-Americans were NA17101–NA17116 and NA17133–NA17140. DNAs from European-Americans were NA06990, NA07019, NA07348, NA07349, NA10830, NA10831, NA10842–NA10845, NA10848, NA10850–NA10854, NA10857, NA10858, NA10860, NA10861, NA12547, NA12548, and NA12560.Coriell (

Subcutaneous leiomyosarcoma is a rare condition that accounts for 1% to 2% of all superficial soft tissue malignancies. Approximately 10% of cases arise in the trunk, although the extremities are the most commonly affected.We report herein the case of a 31-year-old man with a subcutaneous leiomyosarcoma, measuring 124 × 105 mm, arising in the left inguinal region. A wide local excision (with a resection margin ≥ 20 mm) was performed. Histological examination of the resected specimen revealed a leiomyosarcoma with high cellularity and two mitoses per 10 high-power fields. The patient remains well with no evidence of disease 5 years and 8 months after the operation.This is the first reported case of subcutaneous leiomyosarcoma arising in the inguinal region and also one of the largest tumors reported. The experience of this case and a review of the English-language literature suggest that a resection margin of ≥ 10 mm is recommended when excising this rare tumor. Subcutaneous leiomyosarcoma arises from smooth muscle in the walls of arterioles and veins. It is a rare tumor accounting for 1% to 2% of all superficial soft tissue malignancies . It usuaA 31-year-old man presented with a painless left inguinal tumor, which had gradually grown during the past six months. Physical examination on admission revealed a fist-sized subcutaneous tumor in the inguinal region. The overlying skin appeared normal without ulceration Figure . With a The resected tumor, measuring 124 × 105 mm, was solid, encapsulated, and a homogeneous yellowish white in color, without central necrosis and hemorrhage on its cut surface. Routine histological examination with hematoxylin-and-eosin revealed that the tumor comprised spindle-shaped cells with high cellularity in parts Figure and two The patient had an uneventful recovery and was discharged on the 9th postoperative day. As the resection margin was negative, no adjuvant treatment was given. He remains well with no evidence of disease 5 years and 8 months after excision.Although subcutaneous leiomyosarcoma commonly arises in the lower extremities, it occasionally affects the trunk ,2,4. A ret al., [As subcutaneous leiomyosarcoma is resistant to radiotherapy and chemotherapy ,3,9, suret al., demonstrThe author(s) declare that they have no competing interests.KY, YS, NF, and DS took part in the operation, performed the literature search and drafted the manuscript for submission. HU performed histological examination. KH supervised the preparation of the manuscript and edited the final version for publication. All authors read and approved the final manuscript.

To investigate the genetic underpinnings of a particular biological process, geneticists screen large collections of mutant organisms to characterize their physical defects. By comparing the genetic makeup of nonmutant organisms to mutants, it's possible to tease out the genes responsible for a defective appearance, or phenotype. In a classic study in the fruitfly, Christiane Nüsslein-Volhard and Eric Weischaus bred many lines of flies with mutations that were lethal: the fly embryos died, but not before displaying a wide range of developmental defects. Since it was known that the fruitfly needed only a single wild-type copy of these genes to survive, the mutations in these “embryonic lethals” had to be recessive, meaning that both copies, or alleles, of the gene had to be mutated for the lethal defect to appear. Nüsslein-Volhard and Weischaus's work revealed many such recessive genes crucial to early development and earned them a Nobel Prize.Among the model systems for studying development, the zebrafish has become prized because its transparent embryo develops outside the mother's body. The zebrafish has helped biologists identify many genes involved in embryogenesis and, because it's a vertebrate animal, has become a valuable resource for identifying genes involved in human disease. Now, a team led by Nancy Hopkins of the Massachusetts Institute of Technology, has created over 500 lines of zebrafish with lesions in key embryogenic genes and used them to identify a group of genes that predispose the fish to cancer, with some surprising results.(rp)—that is, each line carried one healthy version and one defective version of a different rp gene. These proteins are components of ribosomes—the massive molecular complexes within cells that mediate protein synthesis—and are essential for embryonic development.All of the 500 lines created by the researchers carried a recessive embryonic lethal mutation; for about 400 of the lines, mutations in 300 distinct genes were identified as the cause of the embryonic phenotype. During the process of cultivating some of these mutant lines, the Hopkins team noticed that an abnormally large percentage of fish experienced early mortality , while the surviving fish in these lines developed large, highly invasive malignant tumors; both phenotypes persisted over successive generations. The tumors resembled malignant peripheral nerve sheath tumors (MPNSTs) that have been found in other fish species as well as in mammals. Suspecting that these mutant lines had elevated rates of cancer, the researchers investigated the genetic makeup of the fish and discovered to their surprise that each line was heterozygous for a mutation in a different ribosomal protein gene rp mutations, the researchers report, either reduced or eliminated expression of the corresponding rp gene. In the case of “classic” tumor suppressor genes, the wild-type allele must be lost for the defective allele to set the stage for cancer. Here, the wild-type allele appeared to remain intact in the tumor cells, implicating the proteins as “haploinsufficient” tumor suppressors—a reduction from two gene copies to one functional copy seems to be enough to increase the risk of cancer. Apart from the mutations in rp genes, the authors also found a loss-of-function mutation in a gene that acts as a tumor suppressor in mammals—establishing the soundness of this approach for identifying mammalian cancer genes.All of the rp genes whose mutations caused cancer from the five other rp genes whose mutations did not, the authors raise a number of possibilities for future study. And given the high degree of conservation of genes and pathways among vertebrates, it's likely that rp mutations also raise cancer risk in humans. Together, these results demonstrate that the tiny freshwater workhorse of developmental biology has a promising future as a model system for human cancer.While these experiments do not explore how these mutations lead to cancer, the results suggest that some shared, ribosome-associated function allows these genes to act as tumor suppressors and that disrupting this function somehow leads to tumor formation. Though it's not clear what distinguishes the 11

With billions of cells in the adult human body, all replicating and dividing in an environment laden with toxins, radiation, and free radicals, a certain amount of DNA damage is guaranteed to occur. Fortunately, all organisms have built-in checkpoints throughout the cell cycle that prevent such mistakes from propagating. At the G1 checkpoint during cell division, for example, molecules survey nuclear DNA for errors and breaks before the cell is deemed fit to undergo S phase, the DNA replication stage. If damage is found, enzymes either work to repair it or, in some cases, trigger programmed cell death, or apoptosis. But when checkpoints fail, and DNA damage is left unrepaired, disease such as cancer can result. A better understanding of these events, as provided, for example, by Vincenzo Costanzo and colleagues in this issue, will consequently lead to a better understanding of the mechanisms that give rise to cancer.A serious form of DNA damage, called a double strand break (DSB), cuts the helix clean through—a far worse scenario than if just one strand slips free. In response to a DSB, the cell recruits a signaling protein called ATM and a three-protein complex called MRN, whose components selectively bind to broken DNA ends. A malfunction of this signal and repair pathway is dire. People who suffer from the genetic disease ataxia-telangiectasia (A-T) lack a functioning ATM molecule and therefore cannot properly handle DSBs or successfully navigate the G1 checkpoint. This condition leads to a host of problems, including abnormal chromosomes, deficient immune function, and a predisposition to cancer. A-T-like disease (ATLD), another rare genetic condition, has very similar symptoms. The only difference is that the protein missing is Mre11, a subunit of MRN. While recent work on the cellular level has indicated that MRN activates ATM, the biochemical relationship between these proteins has yet to be fully understood.Studying these two molecules using traditional biochemical assays is difficult because knocking out the activity of these proteins is lethal to many cells. Costanzo and colleagues used a novel test system of cell-free frog extracts and found that Mre11 is necessary for both ATM activation and for the formation of large protein–DNA complexes apparently responsible for triggering the cascade of signaling molecules underlying the DNA damage response at the G1 checkpoint.The frog extract system allowed the team to manipulate the presence or absence of Mre11 and accurately measure the response triggered by the addition of fragmented bits of DNA . As predicted, without a functional Mre11 protein, ATM was not activated and there was no response. By simply adding the protein Mre11 back to the mixture, the damage response was restored. But when the researchers added a mutant form of Mre11, still capable of performing its essential tasks in another stage of the cell cycle—DNA replication—the G1 damage response remained suppressed. This mutant form of the Mre11 protein lacks the C-terminal, or DNA-binding, end.Costanzo and colleagues also found that this DNA-binding end is required for the assembly of DNA–ATM–MRN complexes in the presence of fragmented DNA and seems to direct the entire damage response. This work helps to explain the similarity between patients with A-T and those with ATLD, and hints at the formation of a large “signaling” complex that helps to orchestrate the crucial response to DBSs in DNA.

Endothelial cell dysfunction may be implicated in the development of multiple organ failure (MOF) by a number of mechanisms. Among these, altered fibrinolysis promotes fibrin deposition, which may create microvascular alterations during inflammation. Elevated concentrations of C-reactive protein (CRP), especially when these persist over time, are correlated with an increased risk of MOF and death. CRP may inhibit fibrinolysis by inducing plasminogen activator inhibitor-1 (PAI-1) release from human aortic endothelial cells. Moreover, the administration of recombinant CRP in volunteers may increase circulating PAI-1 levels.In this study, we tested the hypothesis that CRP is associated with hypofibrinolysis in intensive care patients with and without sepsis.We studied the association of inflammation and abnormal fibrinolysis in intensive care unit (ICU) patients with (n = 11) and without (n = 21) sepsis. The inflammatory response was assessed by serum concentration of C-reactive protein (CRP), a marker of the acute phase reaction, which increase rapidly in the inflammatory response, and the plasma fibrinolytic capacity was evaluated by the Euglobulin Clot Lysis Time (ECLT), determined by a new semi-automatic method.2 = 0.45; p < 0.001). In a multivariate analysis, CRP was the strongest predictor of ECLT . In addition, the overall ICU length of stay was significantly correlated with CRP and ECLT .ECLT was significantly higher in septic than non-septic patients and was significantly correlated with CRP concentration (RIn critically ill patients a significant correlation thus exists between plasma fibrinolytic capacity and serum CRP levels. Our data were obtained in the first 24 hours of ICU admission or of sepsis, thus, the relation between CRP and hypofibrinolysis appeared very quickly. This finding is compatible with a link between inflammation and abnormal fibrinolysis, and may explain the negative prognostic value of CRP in critically ill patients. Endothelial cells have a key role in the control of vascular permeability and vessel tone, coagulation and fibrinolysis, and inflammatory response . There iEndothelial dysfunction/or activation is associated with an imbalance in hemostatic functions. Endothelial cells are responsible for the release of tissue plasminogen activator (t-PA) and contribute to the release of plasminogen activator inhibitor-1 (PAI-1). Inhibition of the fibrinolytic system amplifies the pathogenic role of fibrin deposition during severe inflammation . MultiplIn infected patients, elevated concentrations of serum CRP are correlated with a risk of MOF and death , especiaCRP can act directly on endothelial cells, inducing, for example, the expression of intercellular adhesion molecule (ICAM)-1 and the In this study, we tested the hypothesis that CRP is associated with hypofibrinolysis as measured by ECLT in intensive care patients with and without sepsis.After approval by the A. Vésale hospital ethics committee, we studied 32 ICU patients with severe sepsis n = 11) or other diagnoses (n = 21). Infection definition required isolation of a microorganism from a normally sterile body site, concurrent with accompanying signs and symptoms of sepsis and decision of antibiotic therapy. Criteria for severe sepsis included signs of at least one organ dysfunction attributed to sepsis . All pat or other®. Fibrinogen was determined by thrombin time on a STA® automate (STAGO). Leukocyte and platelet counts were determined on a hemocytometer . All tests were performed on blood obtained from the same venipuncture.Blood samples were obtained during the first 24 hours of sepsis or on the first day of admission for non-septic patients. Serum samples were collected in vacuum tubes without anticoagulant. Plasma samples were harvested in citrated vacuum tubes and put in melting ice. Whole blood was collected on EDTA-treated tubes. CRP was evaluated by antibody-binding and turbidity measurement on SYNCHRON LXThe Euglobulin Clot Lysis Time (ECLT), which is the most common test used to estimate the plasma fibrinolytic capacity, represents the balance between t-PA and PAI-1 activities . ECLT wa® software package (Jandle Scientific). The data are presented as mean ± SD. Correlation between variables was analyzed using a Pearson correlation test. A multivariate analysis was used with stepwise backward selection of the explicative variables. Sepsis was considered as a dichotomous variable while all other data were considered as continuous . ECLT was the dependent variable. A probability level of p < 0.05 was considered as statistically significant.We used SigmaStatThe major cause of severe sepsis (9 patients) or septic shock (2 patients) was pneumonia (8 patients); angiocholitis was the cause in 1 patient, and in 2 patients the cause was not identified. In 8 patients (7 with pneumonia and 1 with angiocholitis), the infection was due to a Gram negative bacteria. Only 1 patient had documented bacteremia. Non-septic patients were admitted for postoperative surveillance (7 patients), intracerebral hemorrhage (3 patients), heart failure (3 patients), drug intoxication (3 patients), or aggravated chronic obstructive pulmonary disease (5 patients).2 = 0.45; p < 0.001) with no perceptible threshold . In multivariate analysis, ECLT was best predicted by the CRP level and not significantly by sepsis or the fibrinogen concentration. Interestingly, the ICU length of stay was significantly correlated with CRP and ECLT in all patients, and in the survivors .As expected, inflammatory parameters such as white blood cells and CRP levels, the SAPS II score and ECLT were higher in the septic than the non-septic population Table . In an uMany studies have demonstrated that sepsis is associated with endothelial cell dysfunction and promotes coagulation activation . Althougin vivo. Our data suggest that CRP could itself be involved in the processes leading to endothelium dysfunction. The observed relationship does not prove a direct biological link between increasing CRP and hypofibrinolysis; however, indirect arguments exist in support of the concept.Inhibition of the fibrinolytic system may contribute to MOF, as fibrin can activate endothelial cells leading to a disorganization of the monolayer and the release of inflammatory cytokines . Increasin vitro studies have reported the direct effects of CRP on endothelial cells [In vivo, Cleland et al. [G-monomethyl-L-arginine (L-NMMA), reflecting endothelial dysfunction. Bisoendial et al. reported that the administration of CRP in volunteers impairs the fibrinolytic balance [Several al cells -8. In vid et al. reported balance . In addi balance . In a no balance .in-vitro and in-vivo. This mechanism seems to be important in sepsis, as high plasma levels of PAI-1 are associated with poor outcome [CRP could also act indirectly on endothelial cells via the action of monocytes and the release of tumor necrosis factor-α (TNF-α). TNF-α is a strong inducer of PAI-1 production outcome . Moreove outcome . CRP can outcome .CRP also has essential biological functions. No polymorphism of either the gene coding sequence or of the protein itself has been described in humans . Also, hThis work is a pilot study. We have chosen to include patients in the first day of ICU admission to limit the possible rapid effect of inflammatory response on fibrinolysis, especially in septic patients. Indeed, this particular patient population has inflammatory reaction before signs of severe sepsis and thus before their admission to ICU. In fact, we have studied ECLT test at the onset of the organ dysfunction and the inflammatory reaction. Other studies with serial measurement of ECLT in patients who developed nosocomial ICU infections are needed to study the time course of these events. Moreover, we could not definitively exclude that all patients in the non-septic groups were non infected. For example, some non-septic patients with decompensated COPD may have minor infections, despite the negative microbiology cultures and the absence of antibiotic therapy. Viral infections were also possible in some patients.Moreover, it would be of great interest to determine the interactions between CRP and ECLT with IL-6, TNF-α and endothelium dysfunction markers such as soluble thrombomodulin and soluble von Willebrand factor.Despite accumulating evidence that the inflammatory and coagulation systems are activated in sepsis, little is known about the mechanisms that ultimately lead to organ dysfunction and death. Our data were obtained in the first 24 hours of ICU admission or of sepsis, thus, the relation between CRP and hypofibrinolysis appeared very quickly. Prospective studies including the time course of CRP and hypofibrinolysis would provide additional information about this relationship.KZB: Laboratory analysis, writing of the manuscript and design of the study.MP: patients recruitment and design of the study.DB: coordination and design analysis of the results.MG: design of the study.PC: laboratory analysis.JLV: design of the study and analysis of the results.CR: design of the study and analysis of the results.YB: patients recruitment.MV: statistical analysis and coordination.

The aim of this analysis was, however, to identify the influencing factors on high catecholamine levels Various potential risk factors that could impact the humoral stress response before induction of anaesthesia were recorded in 84 males undergoing coronary aortic bypass surgery, and were entered into a stepwise linear regression analysis. The plasma level of norepinephrine measured immediately after radial artery canulation was chosen as a surrogate marker for the humoral stress response, and it was used as the dependent variable in the regression model. Accordingly, the mean arterial blood pressure, heart rate and the calculated pressure-rate product were taken as parameters of the hemodynamic situation.-1) on the morning of surgery was the only significant predictor (p = 0.004) of the high variation in preoperative norepinephrine plasma levels. This intervention decreased norepinephrine levels by more than 40% compared to no clonidine administration, from 1.26 to 0.75 nmol·l-1. There was no evidence for dose-responsiveness of clonidine. All other potential predictors were removed from the model as insignificant (p > 0.05). The use of beta-blocker, ace-inhibitors, ejection fraction, and body mass index were significant determinants for the hemodynamic situation of the patient during the pre-induction period.Stepwise regression analysis revealed that the oral administration of low-dose clonidine (mean dose 1.75 μg·kgThe oral administration of clonidine is the only significant predictor for the observed variation of norepinephrine levels during the preoperative period. Lack of significant dose responsiveness suggests that even a low dose of the drug can attenuate the preoperative stress response and thus is recommended in cardiovascular high risk patients. Even ve drugs ,6-19.However, little attention has been paid to the stress response of cardiac high risk patients when entering the operating area, during initiation of routine monitoring, and finally during awake venous and arterial canulation. Especially the latter procedure can cause significant discomfort for patients even when performed under local anaesthesia . In seveAfter Ethics Committee approval was obtained, patients gave their written and informed consent. Eighty-four consecutive male patients undergoing CABG-surgery were enrolled into this observational study. The only exclusion criterion was emergency operation.-1 and the same-day coefficient of variation for norepinephrine measurements determined by repeated measures of a standardized probe was 3%.Due to the observational character of the study, drugs administered preoperatively (including clonidine) were given at the discretion of the anaesthetist performing the preoperative examination. Each patient received an oral premedication with clorazepate 20 mg in the evening before surgery and in the morning of surgery. In about half of the patients, benzodiazepine premedication was combined with clonidine 75–300 μg. Patients were maintained on their regular cardiac and antihypertensive medication up to the day of surgery but all inhibitors of platelet aggregation were discontinued 3–7 day preoperatively. After arrival at the operating theatre an i.v. line was initiated and 500 ml hydroxyethyl-starch was infused. A 12-lead channel ECG with an automatic ST-segment analysis, oxygen saturation and invasive blood pressure monitoring were connected to the patients. A radial artery catheter was then inserted after local anaesthesia with 1 ml mepivacaine 1%. Heart rate (HR) and mean arterial blood pressure (MAP) were recorded every 15 seconds online using a Laptop computer connected to a Solar 9500 monitor . HR and MAP were multiplied to receive the pressure-rate product (PRP). These variables were used as a measure of the hemodynamic stress response. To determine humoral stress response, an arterial blood sample for the measurement of norepinephrine plasma level was taken immediately after placing the radial artery catheter (this measurement was the main outcome of the study) and 5, 15, and 60 minutes after endotracheal intubation was performed and sufentanil (10 μg.kg-1.h-1). After loss of consciousness propofol was reduced to 3 mg·kg-1.h-1 and sufentanil to 1.5 μg·kg-1.h-1. Endotracheal intubation was performed after administration of pancuronium bromide 0.1 mg·kg-1.General anaesthesia was standardized. After administration of midazolam 0.05 mg·kgTo allow comprehensive analysis of potential factors associated with a reduced stress response, the following data were recorded prospectively:• Age• Bodyweight• Height• Body mass index (BMI)• Clorazepate dose per kilogram bodyweight• Clonidine (yes – no)• Clonidine per kilogram bodyweight• Time from morning premedication until observational period• Inhibitors of the angiotensine converting enzyme system (ace-inhibitors)• Beta-blocking drugs• Calcium antagonists• Angiotensin-2 receptor inhibitors• Left ventricular ejection fraction (EF)• Number of affected vessels2 of 0.3 and higher, attributed to 14 independent variables using an F-test with a significance level of 0.05. All potential relevant factors were subjected to a stepwise linear regression analysis using a backward technique. In each step the least significant factor was eliminated when F was lower than 3.96. The quality of the regression model was judged using the Durbin-Watson statistic , and by checking if the standardized residuals are normally distributed. All calculations were performed using SPSS 11.0 for Windows. All continuous data are presented as mean and standard deviation when normally distributed and as median (25th–75th percentile) when normal distribution had to be rejected using the Kolmogorov-Smirnov-test.A power analysis had revealed that 80 patients provide a power of more than 95% to detect an R-1) on the morning of surgery was the only significant predictor (p = 0.004) of the high variation in preoperative norepinephrine plasma levels. This intervention decreased norepinephrine levels by 40% compared to no clonidine administration (from 1.26 to 0.75 nmol·l-1). In this analysis, the dichotomous variable (clonidine administration: yes-no) was a better predictor for the norepinephrine levels than variables including the clonidine dose (absolute dose or dose per body weight), indicating that our data provide no evidence for a strong dose-responsiveness of clonidine in this context.Stepwise regression analysis revealed that the single administration of low-dose clonidine (mean dose 1.75 μg·kgAll other of the investigated factors (see methods) were removed from the regression model as not significant. The two factors that were eliminated with a p < 0.1 during the last but one and during the final step were body mass index (removed in step 11 with a p = 0.064) and age (removed in step 12 with a p = 0.076). Both factors were associated with increased norepinephrine levels.The overall quality of the regression model was excellent. The Durbin-Watson coefficient was 2.04 (very near to the optimum of 2.0) and the standardized residuals were normally distributed.For mean arterial blood pressure, heart rate and the calculated pressure rate product, however, preoperative clonidine administration was not an influencing factor.For the mean arterial pressure (MAP), a higher ejection fraction (EF) was a statistically significant predictor (p = 0.024). Each 10% increase of EF was associated with a 2.7 mmHg higher MAP. Administration of an ace-inhibitor was the second predictor in the final model of MAP (p = 0.03). These patients had a 7.5 mmHg lower MAP than patients without ace-inhibitors.-2 (p = 0.001).For heart rate (HR) there were three significant predictors that remained in the model. Administration of beta-blockers and ace-inhibitors were both associated with a decreased HR (p = 0.004). Each of them decreased HR between 6–7 beats per minute (bpm). Additionally, a higher BMI was associated with a 1.3 bpm higher HR per kg·mSince the PRP is the product of HR and MAP, it is not surprising that similar variables contributed to its prediction. These were the administration of beta-blockers (p = 0.017) and ace-inhibitors (p = 0.004), each of them reducing the PRP, whereas the EF was associated with an increase in PRP (p = 0.014).No patient had signs of cardiac ischemia on arrival at the operating theatre until induction of anaesthesia (defined as ST-T change > 0.1 mV in any ECG lead). There were no major adverse events during the entire induction period and surgery.2-adrenergic receptor agonist clonidine acts by decreasing central sympathetic nervous system activity in all hyperadrenergic situations. In addition to its sedative, anxiolytic, analgesic and antihypertensive properties [The αoperties ,22 it haoperties ,24 and toperties ,26.In many investigations attention has been drawn to the stressful stimulus of endotracheal intubation ,4,6-19, Thus, it was the major aim of this observational study to identify factors that might contribute either to increased humoral stress or that might help to attenuate this response.Our results show, that a single application of low dose oral clonidine was the only factor that was associated with significantly decreased norepinephrine levels on arrival at the pre-induction area. The question that arises from this observation is, if this is simply an association or if clonidine premedication is the cause for lower norepinephrine levels. In our opinion the latter is the case. Firstly, there were no differences considering any other variables between those patients who had received clonidine and those who had not (see table -1) was low compared to all other trials. Data concerning the appropriate dose of clonidine to attenuate the stress response to intubation vary considerably between 0.625 and 10 μg·kg-1. For example, one trial demonstrated that clonidine 0.625 and 1.25 μg·kg-1 i.v. were sufficient to attenuate pressure response to laryngoscopy and intubation [-1 clonidine i.v. was equally effective as placebo, and only 4 and 6 μg·kg-1 significantly attenuated hemodynamic and adrenergic reactions in an equal manner. It could also be shown that 4 or 6 μg·kg-1 were necessary to reduce norepinephrine levels before induction of anaesthesia, however 2 μg·kg-1 where not sufficient in this setting [However, it is interesting to notice that the mean dose administered to our patients . Furthermore, a post-hoc comparison between the patients receiving either 75 or 150μg clonidine did not show relevant differences (p = 0.91 using the Mann-Whitney U-test).Higher age and higher body mass index showed a non-significant tendency to increase the catecholamine concentration. No other of the investigated factors had statistically significant impact on norepinephrine levels.An explorative post-hoc analysis of the impact of clonidine premedication (none versus any dose) and clonidine dose on norepinephrine levels during the entire induction period proves the results of the main analysis. There was a pronounced reduction of norepinephrine plasma levels after induction of general anaesthesia with lower values in the clonidine-group. However, a statistically significant interaction term p = 0.012) suggests that the fall of norepinephrine levels are more marked in the untreated group and thus mainly caused by induction of general anaesthesia rather than effects of clonidine (figure 2 suggestThis observational trial demonstrates that patients undergoing coronary artery bypass graft surgery have a great variation of norepinephrine levels when entering the operating theatre. We could identify oral clonidine premedication as the only predictor for increased humoral stress response. There was no strong evidence for a dose dependency, indicating that even small doses, like 75–150 μg attenuate the humoral stress response before coronary artery bypass graft surgery. Clonidine did not have a negative impact on hemodynamic parameters.None declared.AMM processed the data and wrote the manuscript.GG conceived the study, collected the clinical data and participated in its design.US collected the clinical data.MK designed the study and collected the clinical data.HAA performed the laboratory investigations.HW participated in the conception of the study.LHJE designed the study, performed the statistical analysis and extensively revised the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:

While previous authors have emphasized the importance of integrating and reinforcing evidence-based medicine (EBM) skills in residency, there are few published examples of such curricula. We designed an EBM curriculum to train family practice interns in essential EBM skills for information mastery using clinical questions generated by the family practice inpatient service. We sought to evaluate the impact of this curriculum on interns, residents, and faculty.Interns (n = 13) were asked to self-assess their level of confidence in basic EBM skills before and after their 2-week EBM rotation. Residents (n = 21) and faculty (n = 12) were asked to assess how often the answers provided by the EBM intern to the inpatient service changed medical care. In addition, residents were asked to report how often they used their EBM skills and how often EBM concepts and tools were used in teaching by senior residents and faculty. Faculty were asked if the EBM curriculum had increased their use of EBM in practice and in teaching.Interns significantly increased their confidence over the course of the rotation. Residents and faculty felt that the answers provided by the EBM intern provided useful information and led to changes in patient care. Faculty reported incorporating EBM into their teaching (92%) and practice (75%). Residents reported applying the EBM skills they learned to patient care (86%) and that these skills were reinforced in the teaching they received outside of the rotation (81%). All residents and 11 of 12 faculty felt that the EBM curriculum had improved patient care.To our knowledge, this is the first published EBM curriculum using an individual block rotation format. As such, it may provide an alternative model for teaching and incorporating EBM into a residency program. Evidence-based medicine (EBM) strives to provide a systematic approach to integrating the best research evidence with clinician expertise and patient preferences to provide better patient care . While tWhile there have been several studies of residency EBM curricula ,9-11, noThe UCSF family practice residency program is based at San Francisco General Hospital (SFGH), a large county hospital serving the urban poor. The program runs a busy family practice inpatient service (at SFGH) as well as the Family Health Center, an outpatient clinic that includes both continuity practices and acute care services. Within this setting, we formulated 3 primary goals for our EBM curriculum: (1) to teach interns basic EBM concepts and skills; (2) to disseminate and reinforce EBM skills to second and third year residents and faculty; and (3) to apply EBM to the care of patients. This study was approved by the UCSF Committee on Human Research.The EBM curriculum for UCSF family practice residents began, in its current form, summer of 2001. The core of the curriculum is a 2-week, individual EBM rotation for interns. In contrast to the usual format of multiple lectures or learning modules scattered throughout residency, the two-week individual block format provides for a concentrated time in which to learn EBM. It also allows tailoring of the rotation to fit the residents' backgrounds and interests. During each of the two weeks, residents have 3 half-day clinics, with the remainder of the time available for EBM. The EBM portion of the rotation fulfills the ACGME requirements for resident research and scholarly activity.patient/problem, intervention, comparison, and outcome). The entire process generally takes 5 to 10 minutes and includes modeling of how to formulate an appropriate 'answerable' question by senior residents and the inpatient attending faculty. The question(s) generated are then used by the librarian Co-director as material for a tutorial on developing search strategies and using high-quality web-based EBM resources. The emphasis of the tutorial is to introduce the intern to the concept of information mastery [The rotation begins with a meeting with the EBM faculty Director (DT) during which the structure and goals of the rotation are communicated, the intern's knowledge and experience related to EBM are assessed by review of his or her experience and by a pre-rotation test of EBM skills and knowledge (described below). Interns receive reference materials ,12 and a mastery , and lea mastery .During the first week, the intern completes a web-based EBM tutorial which coDuring the initial 3 months of the curriculum, interns completed a written test of their EBM skills and knowledge before and immediately following the rotation. The tests were adapted, with permission, from a similar instrument developed by Sean Schafer, MD and Katie Ramos, PhD at the University of California Fresno Family Practice Residency Program . Scores As others have noted, progress in the use of EBM depends on the availability of information support services to resident and faculty at the point of patient care . In conjWe estimate that training 13 interns per year requires approximately 70 hours of librarian time and 200 hours of the faculty Director's time. In the event that the medical librarian Co-Director (JH) is not available, the faculty Director (DT) provides coverage for this portion of the rotation. Absence of the faculty Director for one or two days can usually be covered by schedule modifications and communication by telephone and e-mail. An extended absence of the faculty Director requires another faculty member assuming supervisory responsibility.We evaluated our EBM curriculum with respect to our 3 primary goals using pre- and post-rotation questionnaires (completed by each EBM intern) and by a survey of family practice residents and faculty.The EBM intern questionnaires consisted of pre- and post-rotation self-assessments of confidence in EBM knowledge and skills . Self-confidence in EBM knowledge and skills was assessed by asking the intern to rate his or her level of comfort from 1 = very uncomfortable to 5 = very comfortable for (1) MEDLINE searching to answer a clinical question; (2) use of Web-based EBM resources to answer a clinical question; and (3) use of EBM principles to critically evaluate articles. At the end of the rotation interns were asked to identify the most useful and least useful aspects of the rotation and what could make the rotation better. In addition, residents completed and returned to the Residency Coordinator a standard evaluation of the rotation.We evaluated the dissemination and reinforcement of EBM skills and knowledge to residents and faculty and the application of EBM to patient care by surveying residents and faculty. These surveys were distributed and collected by a program assistant. Surveys were labeled with a code number for each resident and faculty and results were reported in aggregate to provide anonymity. The resident survey asked "How frequently have you continued to apply EBM concepts and tools from the rotation to answer clinical questions?" and "How frequently have EBM concepts and tools been reinforced via teaching by faculty or senior residents?" For both questions, response options were 1 = never, 2 = seldom (less than once per month on average); 3 = occasionally (1 to 3 times per month on average); 4 = often (1 or 2 times a week on average); and 5 = frequently (3 or more times per week on average). For faculty and residents who had had a clinical question answered by the EBM intern on the inpatient service were asked. "How often did the EBM answer provide useful information?" and "How often did the answer change your management of a patient?" For both questions, response options were 1 = less than 25% of the time; 2 = 25% to 75% of the time; and 3 = more than 75% of the time. Finally, all faculty and residents were asked if they believed that "the presence of the EBM rotation improves the quality of patient care within the family practice residency program."During the period from July 1, 2001 to September 30, 2003, 30 EBM interns presented 30 journal clubs and generated 74 CATs in response to questions from the inpatient service. Journal club presentations and CATS are electronically archived and make accessible via the residency website . InternsSurveys to evaluate goals 2 and 3 were completed by 21 of 25 residents (response rate of 84%) and 12 of 13 faculty (response rate or 92%). As seen in Table As shown in Table While the above findings support the acceptance and perceived utility of our EBM program, they do not provide a formal measure of its effectiveness. Changes in resident knowledge, skills and confident were measured over a short period of time. The frequency of reinforcement of EBM and the impact of EBM on clinical care was by resident and faculty report and may have been biased. No attempt was made to observe changes in physician behaviors or patient outcomes. There were no measures of EBM use prior to the introduction of the EBM rotation and no comparison group was available. It is therefore not possible to objectively determine to what degree current levels of awareness and utilization of EBM are the result of the rotation. It is also not possible to separate out the effects of the 2-week EBM rotation from the adjunct changes of establishing a medical information website or promoting the use of PDAs, except to the extent that the questions asked specifically about the 2-week EBM rotation.Our block EBM rotation differs substantially from the approaches reported in previous studies ,11,16 inInterns reported greater confidence in their search skills after the two week rotation. Two randomized controlled trials and a controlled before-after study have demonstrated benefits of training in electronic search skills -19. InteOne study that evaluated the impact of a 1-month pilot program to use EBM methods on an inpatient service, reported results roughly similar to ours . Our curPrevious studies have found only small increases in residents' knowledge and skills from journal clubs alone, leading to the suggestion that journal clubs should be used as a component of EBM training rather then being 'stand alone' activities -8. WhileOther investigators have reported educational interventions aimed at improving faculty knowledge and skills in medical informatics and EBM. One study reported increases in faculty self-rating of EBM skills following an intervention consisting of 2 half-day workshops and substantial amount of individual mentoring . We founOur EBM curriculum, based on an individual 2-week EBM rotation in the first year, appears to be successful in increasing resident's EBM skills and confidence. In addition, resident and faculty both perceive EBM as being incorporated and reinforced beyond the rotation, and that the presence of EBM in the residency improves the quality of patient care. We hope that our experience provides a useful model for teaching and integrating EBM into a busy, resource-limited, family practice residency.The authors declare that they have no competing interests.All authors participated in the development of the curriculum. DT conceived of and conducted the evaluation. JH and PSS reviewed the survey instrument. JH and PSS reviewed and suggested changes to the paper. JH drafted the subsection describing the teaching the use of electronic. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:

A new study adds to the mounting evidence implicating T cells as an important component of the inflammation in chronic obstructive pulmonary disease Chronic obstructive pulmonary disease (COPD) is a global epidemic of major proportions that is predicted to become the third most common cause of death and fifth most frequent cause of chronic disability by 2020. In developed countries it is mainly caused by cigarette smoking, but the reasons why only a proportion 10%–20%) of smokers develop progressive airflow limitation is currently unknown. The disease is characterized by a chronic inflammatory process predominantly in the small airways and lung parenchyma, with increased numbers of macrophages, neutrophils, and T lymphocytes [0%–20% of3 of lung and the extent of emphysema [+ (T helper) and CD8+ (suppressor/cytotoxic) T cells were increased in the airways and lung parenchyma of patients with COPD, with a predominance of CD8+ cells [+ cells, which are predominantly of the T helper 2 (Th2) pattern, with increased expression of interleukin (IL)-4, IL-5, and IL-13 (see Glossary), and which are associated with an increased number of eosinophils. In smokers who develop COPD there appears to be activation of adaptive immunity, with the infiltration of CD8+ and CD4+ cells in the alveolar walls and small airways and—in patients with the most severe disease—the presence of lymphoid follicles that contain a core of B lymphocytes surrounded by T cells [+ and, to an even greater extent, CD8+ cells.T lymphocytes were first reported to be increased in patients with COPD by Finkelstein and colleagues, who showed a correlation between the number of T lymphocytes/mm8+ cells ,5. This PLoS Medicine takes the story forward [+ and CD8+ cells appear to be fully activated, as they would be after being presented with antigens, and they show predominantly a T helper 1 (Th1)/cytotoxic T 1 (Tc1) pattern, with increased expression of interferon-γ (IFN-γ) and Th1 chemokines. This is consistent with the recent demonstration of increased expression of IL-12 in bronchial biopsies of patients with COPD and activation of the transcription factor STAT-4 in T cells, subsequent STAT-4 nuclear translocation, and IFN-γ gene induction, and thus a Th1 commitment in the T cells [The study by Grumelli et al. (2004) published in this issue of forward . The CD4 T cells .+ cells [As well as producing the cytokines IL-2 and IFN-γ, Th1 and Tc1 cells also express the chemokine receptor CXCR3 and the ligands that activate this receptor, IFN-γ inducible protein 10 , monokine induced by IFN-γ (CXCL9), and IFN-inducible T cell α chemoattractant (CXCL11). There is an increase in the expression of IP-10 in the airways of patients with COPD and an increase in the number of CXCR3+ cells . CXCR3 i+ cells and the severity of emphysema [It is likely that Th1 cells are the major source of IFN-γ in the lungs of patients with COPD and therefore drive and maintain the T cell response and promote an “immune inflammation” with neutrophils and macrophages. However, it is the role of Tc1 cells that is of particular interest, as these cells are cytotoxic to epithelial cells through the release of granzymes and perforins, which induce apoptosis. Increased concentrations of perforins have recently been reported in the sputum of patients with COPD . In suppmphysema .The T cell inflammatory response appears in mild COPD but increases markedly with disease severity. It is possible that the initial immune response becomes self-perpetuating because of endogenous autoantigens resulting from inflammatory and oxidative lung injury. There are also antigens in tobacco, but the inflammatory response appears to become independent of smoking status, and there is intense inflammation even in patients who stopped smoking many years previously , as seenCOPD is characterized by destruction of the lung parenchyma and loss of elastin due to elastolytic enzymes, such as neutrophil elastase and certain matrix metalloproteinases (MMPs). The predominant MMP in COPD appears to be MMP9, which is released in much larger amounts from alveolar macrophages of patients with COPD than from those of smokers without the disease . The stuThere are currently no treatments that reduce the relentless progression of COPD, and none that have significant anti-inflammatory effects. However the recognition that an adaptive immune T cell response, most likely driven by antigens, may play an important pathophysiological role in the pathogenesis of COPD has important therapeutic implications. It is possible that T cell inhibitory strategies, such as the use of immunosuppressants, might be effective, although side effects may be a problem, and there is particular concern about increasing the risk of bacterial infection. Another approach might be to block the trafficking of Th1 and Tc1 cells to the lungs by blocking CXCR3, and there is now a search for small-molecule inhibitors of these receptors. Inhibition of IFN-γ signaling might be another approach.The mounting evidence implicating T cells, and thus an adaptive immune response, as an important component of the inflammation in COPD is overwhelming. A better understanding of the immune mechanisms involved in COPD is important, since it might lead us to new and more effective therapeutic approaches to this important disease.+ (helper) T cell:CD4 T lymphocyte that enhances the inflammatory response+ (cytotoxic/suppressor) T cell:CD8 T lymphocyte that suppresses the inflammatory responseCXCR3: Chemokine receptor that is selectively activated by IP-10, monokine induced by IFN-γ, and IFN-inducible T cell chemoattractantCytotoxic (Tc1) cell: T cell that is characterized by secretion of INF-γGranzyme: Enzyme released by cytotoxic T cellsInterferon-γ inducible protein 10 : Chemokine of 10 kDa that selectively activates CXCR3Interferon-inducible T cell γ chemoattractant : Chemokine that selectively activates CXCR3Interferon-γ (IFN-γ): Protein secreted by Th1 and Tc1 cellsInterleukin-4 (IL-4): Protein secreted by Th2 cells that is important in increasing IgE secretionInterleukin-5 (IL-5): Protein secreted by Th2 cells that is important for eosinophiliaInterleukin-12 (IL-12): Protein secreted by antigen-presenting cells that promotes differentiation of Th1 cellsInterleukin-13 (IL-13): Protein secreted by Th2 cells that is important for IgE secretionMatrix metalloproteinase (MMP): Proteolytic enzyme that degrades connective tissueMMP9, MMP12: MMPs that destroy elastin fibersMonokine induced by interferon-γ : Chemokine that selectively activates CXCR3Neutrophil elastase: Enzyme released from neutrophils that destroys elastin fibersPerforin: Protein released by cytotoxic T cells that induces apoptosisSTAT-4: Transcription factor specifically activated by IL-1T helper 1 (Th1) cell: T lymphocyte that is characterized by secretion of INF-γT helper (Th2) cell: T lymphocyte that is characterized by increased secretion of the cytokines IL-4, IL-5, and IL-13; characteristically increased in allergic inflammation

Chronic food restriction augments the rewarding effect of centrally administered psychostimulant drugs and this effect may involve a previously documented upregulation of D-1 dopamine receptor-mediated MAP kinase signaling in nucleus accumbens (NAc) and caudate-putamen (CPu). Psychostimulants are known to induce striatal glutamate release, and group I metabotropic glutamate receptors (mGluR) have been implicated in the cellular and behavioral responses to amphetamine. The purpose of the present study was to evaluate whether chronic food restriction increases striatal MAP kinase signaling in response to the group I mGluR agonist, DHPG.Western immunoblotting was used to demonstrate that intracerebroventricular (i.c.v.) injection of DHPG (500 nmol) produces greater activation of ERK1/2 and CREB in CPu and NAc of food-restricted as compared to ad libitum fed rats. Fos-immunostaining induced by DHPG was also stronger in CPu and NAc core of food-restricted relative to ad libitum fed rats. However, i.c.v. injection of saline-vehicle produced greater activation of ERK1/2 and CREB in CPu and NAc of food-restricted relative to ad libitum fed rats, and this difference was not seen when subjects received no i.c.v. injection prior to sacrifice. In addition, although DHPG activated Akt, there was no difference in Akt activation between feeding groups. To probe whether the augmented ERK1/2 and CREB activation in vehicle-injected food-restricted rats are mediated by one or more GluR types, effects of an NMDA antagonist , AMPA antagonist , and group I mGluR antagonist were compared to saline-vehicle. Antagonist injections did not diminish activation of ERK1/2 or CREB.These results indicate that a group I mGluR agonist induces phosphorylation of Akt, ERK1/2 and CREB in both CPu and NAc. However, group I mGluR-mediated signaling may not be upregulated in food-restricted rats. Rather, a physiological response to "i.c.v. injection stress" is augmented by food restriction and appears to summate with effects of the group I mGluR agonist in activating ERK1/2 and CREB. While the augmented cellular response of food-restricted rats to i.c.v. injection treatment represents additional evidence of enhanced CNS responsiveness in these subjects, the functional significance and underlying mechanism(s) of this effect remain to be elucidated. The twelve unoperated/uninjected rats were simply removed from home cages and exposed to CO2 followed by decapitation. Brains were rapidly removed and immediately frozen in powdered dry ice. Five hundred-micrometer sections were cut using an IEC Minotome cryostat, and CPu and NAc were micropunched, under an Olympus dissecting microscope, from a series of 8 consecutive frozen sections. The tissue was then homogenized in 10 volumes of 50 mM Tris-HCl, pH 7.5 containing 50 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 40 mM β-glycerophosphate, 50 mM NaF and 5 mM Na4P2O7, 1% Tx-100, 0.5 μM okadaic acid, 0.5% sodium deoxycholate and 0.1% SDS, followed by centrifugation and protein determination using BCA reagent kit as described by the manufacturer (Pierce) Supernatants were mixed with 5 × SDS-PAGE sample buffer, boiled for 5 min, cooled on ice and kept at -80°C until future use.Prior studies have indicated that MAP kinase activation is transient and the optimal time-point to study phosphorylation of ERK 1/2 after physiological or pharmacological treatment is 15–20 min . TherefoProtein (10–30 μg per lane) was separated by electrophoresis on precast 10% polyacrylamide gels . Precision Plus protein standard molecular weight markers were also loaded to assure complete electrophoretic transfer and to estimate the size of bands of interest. The gels were transferred to nitrocellulose membrane (Osmonics) for 2 h, with a constant voltage of 100 volts. Membranes were blocked for 1 hr at room temperature with blocking buffer, 5% non fat dry milk in 50 mM Tris-HCl, pH 7.5 containing 150 mM NaCl and 0.1% Tween 20 (TBS-T), then probed overnight at 4°C using primary monoclonal antibodies for phospho-(Thr202/Tyr204)-p44/42 ERK1/2 , or polyclonal antibodies for phospho-Akt (Ser 473) , and phospho (Ser 133) CREB . Total levels of ERK1/2, Akt and CREB were detected on the same blots using anti-rabbit p42/44 ERK1/2 antibody 1:2000, , anti-rabbit total Akt , or anti-rabbit CREB antibody . After detection of phosphorylated ERK1/2, phospho-Akt and CREB blots were stripped with 25 mM Glycine, pH 2.0 containing 1% SDS for 30 min at room temperature, washed six times in TBS-T buffer, blocked in blocking buffer for 1 h and then incubated overnight at 4°C in total ERK1/2, total Akt or CREB antibody. After probing with primary antibodies and washing with TBS-T buffer (3 × 5 min), membranes were incubated with 1:2000 dilution horseradish peroxidase conjugated anti-mouse or 1: 2000 dilution anti-rabbit IgG . Proteins were visualized using a chemiluminescense ECL kit (Pierce). Densitometric analysis of the bands was performed using NIH image software. Phospho-p42/44 MAPK, phospho Akt and phospho CREB values were normalized to total p42/44 MAPK, Akt and CREB values respectively.Ninety min after DHPG injection rats were anesthetized with sodium pentobarbital and transcardially perfused with isotonic phosphate buffered saline (PBS) followed by 4% paraformaldehyde in PBS. Brains were then removed and maintained in 20% sucrose at 4°C for 48 h. Forty μm sections were cut on an IEC Minotome cryostat and collected in a cryoprotective solution. Fos immunostaining was carried out using a rabbit polyclonal c-Fos antiserum and the avidin-biotin peroxidase complex .Sections were washed in 1% sodium borohydride followed by PBS and incubated for 2 hrs in 4% normal goat serum plus 1% BSA in PBS containing 0.2% Triton X-100 (Sigma-Aldrich) to block nonspecific binding. This was followed by incubation, overnight, with rabbit polyclonal c-fos antiserum (1:5000 dilution). Following several PBS washes, sections were incubated with a secondary antiserum for 60 min and subsequently reacted with avidin-biotin complex (ABC) (Vector). The peroxidase reaction was visualized with a chromogen solution containing 100 mM nickel sulfate, 125 mM sodium acetate, 10 mM imidazole, 0.03% diaminobenzidine (DAB), and 0.01% hydrogen peroxide at pH 6.5. Sections were then mounted on chrome-alum coated slides, dehydrated, and coverslipped.Objective counting of c-Fos positive cells in CPu at coronal levels +1.7 and -0.3 mm, and NAc core and shell at coronal level +1.5 mm in relation to bregma was accoFor each Western blot, film exposure time was set as needed to visualize distinct bands in the control samples of each experiment. Immunoblots were analyzed using NIH imaging software. For each blot, relative phospho-protein levels were calculated from the ratio of optical density of the phosphorylated protein/total protein to correct for small differences in protein loading. In addition, tubulin levels were analyzed in several representative gels and no differences were observed between treatment groups. Results were expressed by comparison to the normalized control, which in Experiment 1 was defined as the ad libitum fed group injected with vehicle. In the first part of Experiment 2, unoperated/uninjected ad libitum fed rats served as control. In the second part of Experiment 2, food-restricted rats injected with i.c.v. saline vehicle served as control. Differences between treatment conditions in Experiment 1 were analyzed by two-way analysis of variance . Differences between treatment conditions in the first and second parts of Experiment 2 were analyzed by student's t-test and one-way ANOVA, respectively.YP conducted the majority of immunoblotting experiments plus the immunohistochemistry experiment, contributed to experimental design and assisted in manuscript preparation. YB provided technical supervision of immunoblotting experiments and assisted in manuscript preparation. KC contributed to design of the study, assisted in all experiments, and wrote the final draft of the manuscript.

The role of amino acids as substrates for protein synthesis is well documented. However, a function for amino acids in modulating the signal transduction pathways that regulate mRNA translation has only recently been described. Interesting, some of the signaling pathways regulated by amino acids overlap with those classically associated with the cellular response to hormones such as insulin and insulin-like growth factors. The focus of this review is on the signaling pathways regulated by amino acids, with a particular emphasis on the branched-chain amino acid leucine, and the steps in mRNA translation controlled by the signaling pathways. The response of translation initiation to a change in amino acid and/or hormone availability can be general, i.e., affecting the translation of most if not all mRNAs, and/or specific, i.e., affecting the translation of a single class or subset of mRNAs. Both the general and specific responses can be mediated through regulation of either the met-tRNAi and/or mRNA binding steps. The specific response may also involve an additional regulatory site, i.e., the phosphorylation status of ribosomal protein rpS6, one of the proteins composing the 40S ribosomal subunit. Learning how the cell recognizes a sufficiency of amino acids is presently the objective of intense research. Present evidence, however, suggests multiple recognition sites and multiple signaling pathways. Below, we summarize our current knowledge of the signaling pathways known to respond to changes in amino acid availability. In addition, the translation initiation factors and mRNA structural elements that are involved in changes in both global and specific modulation of mRNA translation are discussed.Recent advances in biomedical research reveal a key role for amino acids as nutritional signals in the regulation of a number of cellular processes. Studies employing a variety of cell types and different tissues demonstrate that one such process affected is the regulation of gene expression through modulation of the translation of messenger RNA (mRNA). The studies show that cells recognize changes in amino acid availability and generate alterations in signal transduction pathways that are also regulated by hormones and growth factors. The cells then respond to the integrated signaling input by either upregulating or downregulating translation initiation, i.e., the process during which initiator methionyl-tRNA and internal ribosome entry sites (IRESs). Phosphorylation of eIF2α is mediated by any of four known eIF2α kinases in mammalian cells: the mammalian ortholog of the yeast general control non-derepressing kinase-2 (mGCN2), the heme-regulated inhibitor (HRI), the protein kinase dsRNA-activated (PKR), and the PKR-like endoplasmic reticulum kinase and livresponse . Howeverresponse . A recenresponse . Thus, iHRI was first identified in rabbit reticulocytes and shown to be activated in response to hyperoxia and iron and heme deficiency . SubsequPKR is a ubiquitously expressed serine-threonine protein kinase that is activated by double-stranded RNA and is induced by interferon [reviewed in ]. PKR isi essentially halts cell growth and triggers apoptosis . Morrsk . TheAlthough eIF2α phosphorylation is one mechanism for enhancing the translation of mRNAs containing an IRES element(s), it is not unique. For example, during apoptosis or infection by certain types of viruses, eIF4G is cleaved. The normal function of eIF4G is to assemble the translation initiation factors eIF4A and eIF4E and the poly(A) binding protein into a complex that mediates the binding of mRNA to the 40S ribosomal subunit. Cleavage of eIF4G during apoptosis or viral infection separates the binding domain for PABP and the mRNA cap binding protein, eIF4E, from the domains that bind eIF4A and allow ribosome attachment (referred to as the middle fragment of eIF4G or M-FAG). A recent study reported that M-FAG generated in etoposide-treated cells M-FAG promotes the preferential translation of certain, but not all, IRES-containing mRNAs including Apaf-1 and death-associated protein (DAP)-5 . MoreoveAnother structural element within the 5'-UTR of some mRNAs that is involved in selective mRNA translation is an oligopyrimidine tract, referred to as a TOP sequence, immediately downstream of the 5'-cap structure ,69. MessMost mRNAs that are efficiently translated, e.g. GAPDH and β-actin, have 5'-UTRs that are short (<200 nt), have a low content of G and C residues, and are relatively unstructured . In contSecondary structure may also function as a cis-acting regulatory element through the binding of specific trans-acting factors. A well-characterized example of such regulation is the modulation of ferritin and δ-aminolevulinate (ALA) synthase mRNA translation in response to changes in iron availability . Both thNutrients, and in particular certain amino acids, play important roles in the control of gene expression through their ability to modulate the initiation phase of mRNA translation. All essential amino acids have the potential to globally regulate mRNA translation through the eIF2α kinase mGCN2. In addition, changes in eIF2α phosphorylation can selectively modulate the translation of mRNAs encoding particular proteins if the 5'UTR of the mRNA contains uORFs and/or IRES elements. Selective control of mRNA translation can also occur through changes in signaling through mTOR. Activation of S6K1 by mTOR leads to phosphorylation of rpS6 and eIF4B which is thought to promote preferential translation of TOP mRNAs and mRNAs with highly structured 5'-UTRs, respectively. In addition, mTOR phosphorylates the eIF4E binding proteins leading to enhanced assembly of the eIF4F complex. In combination with eIF4B phosphorylation, enhanced eIF4F assembly leads to preferential translation of mRNAs with highly structured 5'-UTRs. Although other amino acids have been shown to increase signaling through mTOR, leucine is arguably the most potent of the amino acids in activating the pathway.Both authors contributed equally to the writing of this manuscript.None declared.

In this paper I will describe some of the sentinel events in Aboriginal and Torres Strait Islander health policy and strategy during 2003 and the early part of 2004. This will involve discussion on the:• National Strategic Framework in Aboriginal and Torres Strait Islander Health• National Strategic Framework for Aboriginal and Torres Strait Islander Peoples Mental Health and Social and Emotional Well Being 2004–2009• National Aboriginal and Torres Strait Islander Health Performance Framework• The roll-out of the Primary Health Care Access Program• The National Aboriginal and Torres Strait Islander Social Survey and the National Indigenous Health SurveyThese developments are consistent with a policy agenda that has evolved, in general terms, since the release of the National Aboriginal Health Strategy in 1989. However, I will also consider significant developments in the broader context for Aboriginal and Torres Strait Islander affairs, particularly the decision made in early 2004 by the Howard government to abolish the Aboriginal and Torres Strait Islander Commission (ATSIC). While the key events and developments that are reported in this paper elaborate on an agenda that has been developing for more than a decade, the decision to abolish ATSIC is likely to have a revolutionary impact on the future development of Aboriginal health strategy. Following the lead of the National Aboriginal Health Strategy (NAHS) , nationa• Framework Agreements in Aboriginal and Torres Strait Islander Health ;• Joint Planning Forums .st of July 2003, the "National Strategic Framework for Aboriginal and Torres Strait Islander Health" . Agreement has also been recently brokered that details strategies for Indigenous social and emotional well-being, which is one of the Key Result Areas for the "National Framework". Significant progress has also been made in the development of a national performance management framework for Aboriginal and Torres Strait Islander health that aligns with the "National Framework".The NAHS has been the guiding framework for action in this field since it was endorsed in 1989. Consequently, it was significant that the Australian Health Ministers Conference endorsed its successor on the 31The agenda in Aboriginal and Torres Strait Islander health strategy that was adopted by the health portfolio post 1995 had focussed on reform priorities focussed on the development of :• The capacity of primary health services to respond to Aboriginal and Torres Strait Islander health need (with a particular focus on financing and workforce);• disease and risk strategies that aimed to improve Aboriginal and Torres Strait Islander health outcomes;• the evidence base for policy and practice in this sector .With respect to primary care capacity, the roll-out of the Primary Health Care Access Program (PHCAP) continues to be one of the central planks of this agenda and I will provide an overview of recent progress. Significant progress has also been made over the last couple of years in the development of the Australian Bureau of Statistics Indigenous Survey program, which promises to enhance the information available to assist decision-making within the sector. I will provide a report on the recent developments in the roll-out of this program.While the recent developments in Aboriginal and Torres Strait Islander health policy and strategy represent an evolution of a health reform agenda that has been developing for more than a decade, the abolition of ATSIC points to a much more revolutionary change in the broader institutional and programmatic context for Aboriginal affairs. ATSIC had play a critical role in integrating Australian government programs in Indigenous affairs and providing an institutional structure that facilitated Aboriginal and Torres Strait Islander input into policy and program development. ATSIC, for instance, continued to play a role in health strategy following the transfer of specific health program responsibilities in 1995. It retained, for instance, responsibility for the delivery of environmental health service. A memorandum of understanding was developed between the Department of Health and Human Services and ATSIC to support collaboration between the sectors . ConsequThe National Aboriginal and Torres Strait Islander Health Council (NATSIHC) oversaw the development of the "National Framework". However, this process was stalled by political conflict between the key stakeholders. In December 2000, Council members representing the National Aboriginal Community Controlled Health Organisation (NACCHO) resigned in protest over a consultation draft of the 'National Framework'. NACCHO, which is the peak body representing the Aboriginal community controlled health sector, was concerned with :The way the Draft is written distances Aboriginal and Torres Strait Islander people, undermines the concept of Aboriginal community control of primary health care service delivery and diminished structures which NACCHO believe are still useful. The document's tone and language is wrong in a number of ways...Following further negotiation, NACCHO withdrew its resignation, and the NATISHC was reconstituted with revised membership and Terms of Reference . DespiteThe agreed "National Framework" consists of two documents:• The "National Strategic Framework for Aboriginal and Torres Strait Islander Health – Framework for action by Governments", which sets out a five- to ten-year reform agenda in 9 key result areas .• The "National Strategic Framework for Aboriginal and Torres Strait Islander Health – Context", which outlines the rationale for the Framework and its context .There are nine Key Result Areas set out in the Framework including :• Community controlled primary health care: building community capacity so that individuals and communities can better address and manage their own health needs.• Health system delivery framework: improving the responsiveness of the mainstream health system to Indigenous Australians and developing stronger partnerships between mainstream and Indigenous-specific services.• A competent health workforce: improving the training, supply, recruitment and retention of appropriately skilled health professionals, health service managers and policy officers in both mainstream and Indigenous-specific health services.• Emotional and social well-being: improving outcomes with respect to mental health, suicide, family violence, substance misuse and male health .• Environmental health: improving the delivery of safe housing, water, sewerage and waste disposal.• Wider strategies that impact on health: undertaking action in portfolios outside the health sector and implementing health gain strategies in the areas of education, employment transport, food and nutrition, custodial health, aged and disability services, recreation and exercise.• Data, research and evidence: aiming to improve the quality of information about how well the health sector is meeting the needs of Indigenous Australians.• Resources and finance: aiming to provide an optimal level of resources for Indigenous health commensurate with levels of need, costs of delivering services and community capacity to deliver health outcomes.• Accountability: both to communities and to governments for the delivery and effectiveness of health services.The "National Framework" was endorsed as a plan to guide all Australian governments in a coordinated, collaborative and multi-sectoral approach to achieving Aboriginal and Torres Strait Islander health gain over the next decade. It does not have a specific funding program attached to its implementation, although arguably, the roll-out of the Primary Health Care Access Program (described later) will provide additional capacity to the implementation of the "National Framework". It is also possible that the National Framework will guide the allocation of any new resources made available through the joint planning processes established under the Framework Agreements.To further these ends, it is significant that the "National Framework" was endorsed through each government's cabinet process, providing a whole-of-government commitment to its implementation in each State and Territory and at the Commonwealth level. Each jurisdiction is developing its implementation plan against which it will report annually on progress and outcomes in health portfolios and biennially on whole of government progress. The plans will identify the specific strategies and timeframes for each action area. The National Aboriginal and Torres Strait Islander Health Council will develop a plan for an independent mid-term and final evaluation.The "Social and Emotional Well Being Framework (SEWB Framework)" is basedThe nine guiding principles for the "SEWB Framework" were been extracted from "Ways Forward" , an earlIn 2003 the Social Health Reference Group (SHRG) (established to oversee its development) conducted extensive consultations on a draft framework document. Since then the 'SEWB Framework' has endorsed by the NATSIHC and the National Mental Health Working Group in November 2003, and the Standing Committee on Aboriginal and Torres Strait Islander Health in December 2003. It was anticipated that the final 'SEWB Framework' document would be endorsed out of session by the Australian Health Ministers Advisory Council by the middle of 2004.The development of the Aboriginal and Torres Strait Islander Health Performance Framework has built on the foundations of earlier work which has established the key elements of this framework, including the:• national performance indicators in Aboriginal and Torres Strait Islander health for the Australian Health Ministers Advisory Council ;• service activity reporting for Aboriginal community controlled health services ;• Australian government health portfolio indicators ; and• the reporting against key indicators of Aboriginal and Torres Strait Islander disadvantage for the Council of Australian Governments .It is intended that the Aboriginal and Torres Strait Islander Health Performance Framework will both integrate those government performance reporting processes that have already been developed; streamline reporting processes in Indigenous health and, ensure the strategic management of policy relevant and quality information in published reports . As star• mapping the relationship between the Key Result areas of the 'National Framework' and key domains of health performance ; and• identifying priorities for data development and improvement based on priorities of the 'National Framework'.The Primary Health Care Access Program (PHCAP) was introduced in the 1999–2000 Federal Budget to improve access to primary health care for Aboriginal and Torres Strait Islander people. PHCAP achieves this by funding increased primary health care provision, such as additional general practitioners, nurses, Aboriginal Health Workers, and through preventive and health promotional activities, such as diabetes education and management. Funds are also used for supports to service provision such as capital works and equipment. The program also aims to work with existing health services to ensure they are responsive to the needs of Aboriginal and Torres Strait Islander people.On average across Australia, PHCAP aims to bring the level of Commonwealth funding for Indigenous primary health care to three times the average MBS usage for all Australians. The key objectives of PHCAP are :• Increased availability of appropriate primary health care services where they are currently inadequate;• Local health systems that better meet the needs of Aboriginal and Torres Strait Islander people; and• Individuals and communities that are empowered to take greater responsibility for their own health.Services can be provided through a mix of arrangements, including Indigenous specific, mainstream or a combination of these. Funding can also be used to support mechanisms to assist service providers to deliver better services and enable individuals and communities to become more involved in improving their health.Up until March 2004, new and additional services have been funded in Central Australia (5 regions), Queensland (3 regions) and South Australia (4 regions) through PHCAP, as well as continued funding of services provided through the former Aboriginal Coordinated Care Trials .During 2003 a more streamlined approach to the management of PHCAP rollout was developed ,16, resuFrom these proposals, $11.8 million in funding was approved on 14 March 2004 for:• additional health professional and support staff, for example, over 20 more health professional positions in the Kimberley region of WA;• capital works for health clinic upgrades and the construction of staff housing in remote communities;• minor capital purchases such as medical equipment; and• one-off health promotional activities and health board support and training.Longer term strategies around enhancement of local service systems, to ensure they are more accessible for Aboriginal and Torres Strait Islander people, and ensuring the commitment by state/territory governments to at least maintain their funding commitments, will continue to be pursued. While the PHCAP program has provided a significant injection of resources into what is generally considered an inadequately funded primary heath care system, the amount made available through this program still does not meet its programmatic benchmarks and targets .One of the development priorities established by the heath portfolio when it took responsibility for the administration of the Aboriginal health program was to develop the evidence base to support policy reform and the development of health service capacity .The National Health Survey, undertaken by the Australian Bureau of Statistics with funding support from the Australian Department of Health and Ageing, collects information about the health status, use of health services and facilities, socio-economic status and health-related aspects of the lifestyle of Australians. The Indigenous component of this survey aims to benchmark information on a range of health issues and enable comparisons between the health characteristics of Indigenous and non-Indigenous Australians and to allow trends in the health of Indigenous Australians to be monitored over time.The Indigenous Health Survey that was run in 2001, collected data from approximately 3,500 individuals which was reportable at the national level . In 2004In parallel with the health survey program the Australian Bureau of Statistics collected data for the 2002 National Aboriginal and Torres Strait Islander Social Survey from August 2002 to April 2003 . It is pOn 20 April 2004, the Prime Minister, John Howard and the Minister for Aboriginal Affairs, Senator Amanda Vanstone, announced the intention of the Australian government to abolish the ATSIC.ATSIC had been established in 1989 when the program responsibilities of the Commonwealth Department of Aboriginal Affairs and the Aboriginal Development Corporation were merged into a structure that enable the regional allocation of resources through elected regional councils. The board of Commissioners, elected by ATSIC regional councils, was responsible for national policy development and the oversight of national programs. At the Commonwealth level, ATSIC had the lead agency responsible for the administration of a range of programs such as: community development and employment (CDEP); housing and infrastructure; cultural heritage, broadcasting services; legal services; native title, land rights and the Indigenous land fund, etc.The agency also played a critical role in co-ordinating and integrating the Aboriginal strategy across the different government program areas. ATSIC and the health portfolio had collaborated in the implementation of health infrastructure priority projects . The MemATSIC played a pivotal institutional role in the development of 'whole of government' strategies across the Australian government. It was in effect the only institutional mechanism that enabled this. This was until the Council of Australian Governments (COAG) resolved (in 2000 and 2002) to trial, in up to 10 regions across the country, innovative administrative arrangements, developed in partnership with Indigenous communities, which aimed to provide "more flexible programs and services based on priorities agreed with communities" [From its first term in 1996, the Howard Coalition government had a conflictual relationship with the Commission. However, government confidence in the ATSIC Board deteriorated significantly under the chairmanship of Geoff Clark (first elected chairperson in 1999) to the extent that the Minister for Indigenous Affairs suspended him on the ground of misbehaviour (under section 40 of the ATSIC Act 1989) . A revieThe Federal cabinet, nevertheless, resolved to a more radical agenda than outlined in the review findings, and announced its intention to abolish ATSIC, its regional councils and the mainstreaming of the administration of all the programs for which ATSIC had been responsible. It is proposed that the elected advisory structures will be replaced by a government appointed national council. It is also proposed that Indigenous specific program dollars will be quarantined and a whole of government approach is to be developed for the delivery of Indigenous specific funding. The key elements of this reform package have been positioned within the broader context of a government commitment to reforms aimed at producing "'joined up' government and the 'seamless' delivery of programmes" . This ne• Ministerial Taskforce: which would operate as a cabinet committee, provide collaborative leadership at a government level and set strategic directions.• Secretaries group: which would support Ministerial decision-making, coordinate across government agencies, and oversight annual reporting.• National Indigenous Council: in which the Minister would appointment Indigenous leaders in health, education, employment, law and justice to provide advice and monitor performance.The proposed mechanisms and structures that would be established to deliver this 'joined-up' framework including regional partnership agreements and community shared responsibility agreements . It is also proposed to establish Indigenous co-ordination centres which will provide a single shopfront in regional and remote Australia for indigenous specific programs, lead the negotiation of regional partnerships an shared responsibility agreements but maintain line responsibility to mainstream departments.The impact of this radical reform agenda to national Aboriginal health strategy is difficult to predict. One the one hand the actual changes to the administration of the health program is insignificant . A mainstream department has administered this program since 1995. One the other hand, the implementation of this reform agenda could have potentially very significant consequences for the development of inter-sectoral strategies in Indigenous health. This depends on the success in implementing the new mechanisms, and on their effectiveness. Furthermore, the political consequences of this radical agenda on the relationship between the Australian government and Aboriginal and Torres Strait Islander peoples have yet to really become clear. Specific partnership arrangements that have been developed within the health sector are tenuous – as is evident by the politics in the development of the "National Framework". These partnerships are critical to successful implementation of strategy in Indigenous health. Consequently, deterioration in the broader relationship between Indigenous Australians and the Australian government may have significant negative consequences for the partnership processes specific to the health sector. Even though 2003 was a year in which policy and strategy in Indigenous health made no or new radical departures, it was a year of considerable tumult in relations between the Australian government and Indigenous peoples. The ramifications of this are only now beginning to unfold.ATSIC Aboriginal and Torres Strait Islander CommissionNAHS National Aboriginal Health StrategyNATSICH National Aboriginal and Torres Strait Islander Health CouncilNACCHO National Aboriginal Community Controlled Health OrganisationPHCAP Primary Health Care Access ProgramThe authors declare that they have no competing interests.

This paper reports on a study investigating the epidemiology of sporadic cryptosporidiosis in the North West of England and Wales using a Geographical Information System (GIS) to map location of residence of cases. Some 747 reports of cases were made to CDSC North West of which 649 reports were suitable for analysis. Cases were plotted on the maps of water supply zone and water quality area boundaries, provided by the two main water utilities.It was notable that there were major spatial variations in attack rate across the North West and Wales. The most dramatic example was the large difference between the Greater Manchester conurbation with many reports and Liverpool with none. Given the distribution of previously detected waterborne outbreaks in the region it was initially thought that drinking water source may be an explanation. However, an analysis of the distribution of cases in the Greater Manchester area showed no correlation with any of five water supplies that serve the conurbation.Our study has shown a dramatic variation in the incidence of laboratory confirmed cryptosporidiosis within two regions of the United Kingdom. Further analysis has not been able to prove drinking water as a likely explanation of this variation which so far remains unexplained. Cryptosporidium. Most infections in the UK are due either to C. parvum (previously known as C. parvum genotype 2 or bovine strain) or C. hominis (previously known as C. parvum genotype 1 or human strain) [Cryptosporidium spp. are protozoan parasites. In otherwise healthy individuals they tend to cause a self-limiting form of gastroenteritis which can last for several days and, sometimes, weeks. In patients with certain forms of immune deficiency, most notable the Acquired Immune Deficiency Syndrome (AIDS), the infection can cause a severe and prolonged diarrhoeal illness which, prior to the widespread use of at highly effective antiretroviral therapy was often fatal [Cryptosporidiosis is infection with species of the genus Cryptosporidiosis has now become the most commonly identified protozoal cause of gastroenteritis in the United Kingdom. Most of the epidemiological data to-date has been related to reports of outbreaks. Between the years 1983 to 1997 there were 80 outbreaks cryptosporidiosis in England and Wales affecting 4649 individuals . OutbreaThe epidemiology of sporadic (non-outbreak-related) cases is largely unknown. Of three large case-control studies reported in the past few years only one found an association with drinking mains water -9. The sThe question remains what proportion of sporadic cryptosporidiosis infections may be due to the consumption of mains drinking water. This paper reports a study using GIS to investigate the epidemiology of sporadic cryptosporidiosis and particularly address the issues of whether sporadic cases are also associated with consumption of drinking water.Table It can be seen that there is substantial spatial variation in the distribution of reported cases. In part, this variation can be explained by variation in population density. However, much of the variation is unexplained. For example, reports from Liverpool are very uncommon, whilst reports from Greater Manchester are very common.It was decided to investigate the excess case reporting from Greater Manchester in further detail to look for any possible association with water supplies. Water to the Greater Manchester area comes from five main water treatment works; Lostock (derived from Thirlmere in the Lake District and chlorinated but not filtered), Woodgate Hill , Arnfield-Godley , Buckton Castle and Wybersley .In order to determine whether there was any relationship between attack rate and water supply, all water supply zones in the North West that received any water from one or more of these five supplies were identified Figure . For eacAs already mentioned, care should be taken in the interpretation of this analysis. It is notable that the proportion of reports that could not be allocated a correct postcode varied from one health authority to another to some extent. Also variation in attack rate between water supply zones or water quality areas was as likely to be due to differences in population size as to differences in reported cases. This was most obvious in zones/areas with relatively small population sizes where random effects could have a particularly important affect. However, there are a number of obvious features.The most obvious is the large number of cases from the Greater Manchester conurbation. This covered the Bury and Rochdale, Manchester, Salford, Stockport, West Pennine, and Wigan and Bolton Health Authorities. This excess of cases in Manchester is even more remarkable when compared with the virtual absence of cases from the Liverpool conurbation . The reason for the excess of cases in Greater Manchester is unclear. Although different reporting habits could play a part, we doubt that it could explain more than a small part of the difference. Reporting practices are not that greatly different across the North West . A sero-Cryptosporidium [An alternate explanation could be that the increase represents different water supplies. Salford, and Wigan and Bolton Health Authorities get much of their water supply from Thirlmere, a supply known to be prone to contamination by poridium , none ofporidium . NeverthA further explanation could be that the Manchester population experience other risk factors more commonly than the Liverpool population. Possible explanations include contact with contact with animals, visiting swimming pools and overseas travel. We do not have access to data to show whether or not people from Manchester are more exposed to these factors than people from Liverpool. However, Manchester is closer to a major National Park, The Peak District. If people from Manchester use their proximity to the Peak District to spend more time in the countryside and so are more likely to come into contact with farm animals, this could explain the difference. This would be an interesting hypothesis to test in a further study.In addition to Greater Manchester, there are also areas of increased reporting from North Wales and from North West Lancashire. These hotspots also remain unexplained. North West Lancashire, however, receives much of its water from Thirlmere and a water source cannot be excluded. However, many cases were reported from the Fylde peninsula which only receives a small proportion of its water from Thirlmere.et al. [The use of GIS to study the spatial distribution of cases has been useful in identifying geographical variation, but not necessarily for identifying the reasons for this variation. However, initial analysis does not support the hypothesis that differences in drinking water source is the major reason for this variation. We agree with Dangendorf et al. that GISConsultants in Communicable Disease Control in the North West Region of England and in Wales were asked to forward details of cryptosporidium cases upon notification from the laboratory. A data collection form was completed for each case, giving the following details: name, address, postcode, date of birth, GP name, GP address and date of notification. The form was faxed or e-mailed to Communicable Disease Surveillance Centre (CDSC) – North West as soon as possible.Enhanced surveillance for the North West of England and Wales were set up separately, North West England in mid December 2000 and Wales in February 2001. Both ran until February 2002. To check for accuracy, the data were audited every 2 months. Each CCDC was sent a list of the cases they had notified to CDSC North West in the preceding 2 months. Any cases that had not been notified were forwarded to CDSC.The first stage in the geographical analysis was to check the 747 records for possible duplicate records. These were selected on the criteria of 2 individuals with identical names, dates of birth and postcodes being present in the database. Given that a postcode contains on average only 15 addresses the chances of these being legitimate is highly unlikely. Through this procedure 10 records were deleted from the database. Consequently 737 cases of cryptosporidiosis were identified during the period of enhanced surveillance.The next step was to assign a grid reference to each postcode and this was achieved using the Royal Mail Postcode Address File. Eighty eight records were excluded as either, an incomplete postcode was entered into the cryptosporidiosis database or a match could not be found in the postcode address file. Therefore, in total the database was reduced to 649 cryptosporidiosis cases. These were plotted as points against a backdrop of the water supply zones for the two main water utilities. The water supply zone and water quality area boundaries were provided by the two main water utilities (United Utilities and Welsh Water). A "water supply zone" is an area designated by a water undertaker providing water to the residences of not more than 50,000 people. In general the source is consistent across a particular zone.Using the GIS each case was also assigned its corresponding water supply zone and the number of cases in each WSZ was divided by the population, based upon data supplied by the two water utilities, to produce the attack rate maps. The analysis was undertaken in ArcGIS 8.1 using point in polygon techniques .PRH was lead on study design, did the statistical analyses and co-wrote the paper.SH co-designed the study and co-wrote the paper and undertook most of the data collection.QS co-designed the study and co-wrote the paper.SW co-designed the study and co-wrote the paper.IL undertook the geographical analyses and co-wrote the paper.KO co-designed the study, co-wrote the paper and obtained data on the water distribution.RC co-designed the study and co-wrote the paper.

ST estimates, Nei's DA distance and phylogenetic relationships.Approximately 800,000 primarily feral dogs live on the small island of Bali. To analyze the genetic diversity in this population, forty samples were collected at random from dogs in the Denpasar, Bali region and tested using 31 polymorphic microsatellites. Australian dingoes and 28 American Kennel Club breeds were compared to the Bali Street Dog (BSD) for allelic diversity, heterozygosities, F-statistics, GST of 0.088 and also with high bootstrap support to the Australian dingo and Akita in the phylogenetic analysis.The BSD proved to be the most heterogeneous, exhibiting 239 of the 366 total alleles observed across all groups and breeds and had an observed heterozygosity of 0.692. Thirteen private alleles were observed in the BSD with an additional three alleles observed only in the BSD and the Australian dingo. The BSD was related most closely to the Chow Chow with a FThis preliminary study into the diversity and relationship of the BSD to other domestic and feral dog populations shows the BSD to be highly heterogeneous and related to populations of East Asian origin. These results indicate that a viable and diverse population of dogs existed on the island of Bali prior to its geographic isolation approximately 12,000 years ago and has been little influenced by domesticated European dogs since that time. Bali, a province of the Republic of Indonesia, is an island just 87 km from north to south and 142 km from east to west and home to more than 2.9 million people [More than 90% of the residents of Bali are Hindu with mytAs a result of overpopulation, many BSDs suffer from chronic skin diseases, internal parasites, parvo- and distemper-virus infections, and malnutrition. In an effort to reduce the dog population and to care for their medical needs, the Bali Street Dog Foundation (Yayasan Yudisthira Swarga) was founded in 1998 . They prThe BSD population is of interest for both its genetic diversity and historical relationships. It is also a population that has bred more or less randomly for thousands of years with limited genetic influx, due mainly to geographic barriers and a strict rabies control program in effect since 1926. The present study is concerned with the genetic diversity of this unique canine population and its relationship to other canine subpopulations in Asia and throughout the world. Data presented herein was derived from the DNA testing of 40 BSD samples from the Denpasar city region of Bali with 31 polymorphic microsatellite loci. The genetic diversity of the BSD was compared to that of the Australian dingo and 28 American Kennel Club (AKC) breeds.T values greater than 0.700. Average HS was 0.577 for the 30 subpopulations, with all but three loci having HS values greater than 0.500. The HS and HT values were closest for C23.123 and farthest for C22.279 and C10.404. HWE analysis revealed that all but one locus had at least one population out of equilibrium for the 30 populations sampled. C01.424, C31.646 and CPH16 had 7 populations out of HWE and AHT130 did not have any populations with p values below 0.05. The level of locus diversity attributable to subpopulation structure was evaluated with two statistics – RST and FST. Both statistics gave similar average values at 0.230 and 0.236 respectively. However, RST ranged from 0.098 to 0.486 while FST ranged from 0.179 to 0.328.Analysis of locus diversity across all 30 subpopulations revealed that the number of observed alleles ranged from six to 20 with a total of 366 for all loci Table . OverallE) and observed (HO) heterozygosities and a 28.4% higher HE than the average AKC breed (0.573). HO was also highest in the BSD at 0.692, versus 0.426 in the Australian dingo and 0.563 in the average AKC breed.Overall, the BSD was the most genetically diverse population surveyed here, displaying 239 total alleles out of the 366 seen in all 30 subpopulations or 65.3% of the total observed alleles .IS estimates were calculated to assess the level of inbreeding for each subpopulation . Appearing at the highest frequency was allele 201 of locus CPH08 in the BSD, Australian dingo and Chow Chow at frequencies of 21.3%, 10% and 20%, respectively. Further, the BSD, Australian dingo, Chow Chow and Akita share allele 113 of locus C22.279 at frequencies of 23.8%, 67.5%, 15% and 5%, respectively. Results for locus PEZ08 demonstrate a lack of influence of European alleles where a high frequency of deviations from n+4 alleles were observed in the AKC breeds sampled yet no deviation from n+4 alleles was observed in the BSD or the Australian dingo.ST analysis was performed for each locus between the BSD and the two closest subpopulations: the Australian dingo and Chow Chow and the Australian dingo , and least closely with the Airedale Terrier .Further distance analysis was performed for all 31 loci between all 30 subpopulations using both Nei's DA distance and pairwise Fes Table . Across Genetic distance relationships amongst the five Asian subpopulations were further explored using neighbor-joining dendograms with four non-Asian subpopulations for comparison Fig. . The BSDO was high in three feral dog subpopulations of Korea, Sakhalin and Taiwan, ranging from 0.539 in the Taiwanese to 0.717 in the Korean dogs. Given that the loci used in that study had an average allele number of 7.75, these values are similar to the HO of 0.692 observed in the BSD. Wilton et al. [O of 0.387 using microsatellites with an average allele number of 6.93, similar to the HO of 0.426 for the Australian dingoes reported herein with an average allele number of 11.8.Microsatellites have been previously used to assess genetic diversity and relationships in feral dog subpopulations ,7. Kim eIS. Even after adjusting for sample size, the BSD maintains their status as the most heterogeneous population in the study. Unlike the Australian dingo which exhibits a much lower level of diversity, the BSD findings suggest either a large founding population on Bali and/or a consistent genetic influx since the geographic isolation of ~12,000 years ago. This data also supports that the BSD appears to approximate a randomly breeding population with little selection pressure.Given the size of the island of Bali, it is extraordinary that 800,000 feral dogs can thrive and maintain such high levels of genetic diversity. Of all the subpopulations surveyed here, the BSD has the highest number of observed alleles, the highest heterozygosity, the fewest number of loci out of Hardy-Weinberg equilibrium and the lowest FE, such as the Boxer with a HE of 0.320, breeds like the Jack Russell Terrier have a high HE of 0.713 and overall their HE is higher than that of the dingo. Of first note, the selection of the dogs that contribute to a breed composition mostly occurs prior to official breed recognition primarily by genetic drift due to geographic isolation and selection for particular working or physical characteristics. After official breed recognition future breeding choices are based primarily on the availability of sires and dams that approximate the breed standard. As a result, there is a founding population that proceeds to breed mostly by convenience. Also, many breed standards have changed considerably over the years resulting in retention of a certain level of diversity within each breed, some breeds retaining much more than others. Finally, dogs comprising the comparison AKC breeds were sampled from across the United States, removing any geographical bias of the genotypes observed and slightly elevating the heterozygosities.When comparing the heterogeneity of the BSD to that observed within the AKC breeds some caveats should be addressed. One may initially expect long established, well-defined dog breeds to be much less heterogeneous than reported here. While some breeds do have a low HT is 0.577. The average values for the 11 subpopulations surveyed in the Kim et al [The average allelic diversity of the loci used in the present study was 11.8 alleles per locus, versus 7.75 in the Kim et al work . Howeverim et al work werST and FST values were nearly identical across all subpopulations and all loci, indicating that approximately 23% of the differences observed in allele frequencies can be attributed to differences between subpopulations. FST provides an unbiased estimate of genetic drift between subpopulations by comparing alleles identical by state. RST takes advantage of the stepwise mutation model, which assumes that mutations most often occur as whole repeat unit losses or gains from the original allele size. As a result, the number of mutations provides an estimate of time from divergence. It is interesting, therefore, to compare RST and FST values by locus. Eighteen of the 31 loci studied have an RST to FST ratio greater than 1.1 over that of other study groups. Blood samples from the Australian dingo were taken from captive animals in Australia. Australian dingoes were known to be unrelated by at least one generation.Dogs from 28 American Kennel Club (AKC) breeds, equally representing the AKC group designations, were sampled with buccal swabs for a previous study . Twenty Thirty-one of the 100 microsatellites multiplexed into 12 PCRs by the Veterinary Genetics Laboratory had beenForward primers were synthesized and dye labeled with either Fam, Hex or Vic, or Tamra or Ned , Foster City, CA). Reverse primers were synthesized by Operon . Primer sequences and concentrations for all markers are available as BSD and AKC breed DNA was derived from buccal cells harvested from the inside of the cheek with nylon bristle cytology brushes . Samples were collected by owners or field volunteers and submitted directly to the laboratory. DNA was extracted by heating a single swab for 10 min at 95°C in 400 μl 50 mM NaOH and then neutralized with 140 μl 1 M Tris-HCl, pH 8.0. Australian dingo DNA was extracted from blood using a standard sodium hydroxide digest.2, 200 μM of each dNTP , 0.6 unit AmpliTaq (ABI), and 2% DMSO (Sigma) then covered with 15 ul Chill-out™ Liquid Wax to prevent evaporation. One of five thermal cycler programs was used for each primer mix ranging from 56° to 64° degrees for the annealing temperature. All PCR work was done in polycarbonate 96-well v-bottom microtiter plates on MJ Research PTC-100 thermal cyclers . Protocols are also available in A 2 μl aliquot of extract was used in each PCR which equates to approximately 50 ng DNA. All markers and DNAs were amplified with a PCR reagent mix of 1X PCR buffer (ABI), 4.17 mM MgClOne μl aliquots of PCR product were mixed with 2 μl Fluorescent Ladder (CXR) 60–400 (Promega 400) or Internal Lane Standard 600 (Promega 600) fluorescent size standard, denatured on MJ Research PTC-100 thermal cyclers for three minutes at 95°C, then held at 5°C or placed on ice for at least one minute before gel loading. Two μl aliquots were then loaded onto a 6% denaturing polyacrylamide gel and run on an ABI 377 Automated Sequencer using ABI 10" × 7 1/8" short plates (12 cm). Gels were run at 1.10 kV (constant) voltage, 60.0 mA current, 200 W power, 51°C and 40.0 mW (constant) laser power for up to 2 hours when using Promega 400, and up to 3 hours using Promega 600. DNA fragment analysis was performed with in-house designed STRand software , which rO) were determined by direct counting for each of the 30 subpopulations. Hardy-Weinberg equilibrium (HWE) tests were performed using Genepop version 3.4 [ST estimates and subpopulation expected heterozygosities (HE) for the 30 breeds or dog groups were performed using Genepop version 3.4 [IS estimates (inbreeding coefficient of each subpopulation) for each allele following Weir and Cockerham [Allelic diversity and observed heterozygosities and its associated parameters, HS (average heterozygosity among subpopulations) and GST (coefficient of genetic differentiation), were calculated across all loci using the public domain software, DISPAN [ST [ST [Gene diversity or total population heterozygosity (H, DISPAN . Two addSPAN [ST and RST [ST [ST ,28 were Allele frequencies were used to compute a matrix of genetic distances , which wBSD: Bali Street DogIS, FST RST, GST: F-statistics indicesFS, HT, HE, HO: Heterozygosity indicesHHWE: Hardy-Weinberg equilibriumA: Number of allelesNAES: American Eskimo DogAS: Australian ShepherdAT: Airedale TerrierBCO: Border CollieBLT: Bull TerrierBMD: Bernese Mountain DogBS: Brittany SpanielBT: Belgian TervurenBU: BulldogBX: BoxerBZ: BorzoiChow: Chow ChowDingo: Australian dingoDP: Doberman PinscherGH: GreyhoundMBT: Miniature Bull TerrierPG: PugRR: Rhodesian RidgebackGR: Golden RetrieverJRT: Jack Russell TerrierKE: KeeshondLR: Labrador RetrieverNE: Norwegian ElkhoundPG: PugPM: PomeranianPPN: PapillonPWC: Pembroke Welsh CorgiRR: Rhodesian RidgebackWM: WeimaranerYT: Yorkshire TerrierDNI performed the majority of data acquisition and analysis, wrote first draft of the manuscript and prepared the final draft for submission. ALS performed the majority of sample processing, assisted in data acquisition and the writing of the subsequent drafts of the manuscript as well as final draft preparation. SG sampled the dogs tested, provided background for the manuscript and assisted in the final draft preparation. ANW provided the Australian dingo data for comparison and assisted in the subsequent drafts of the manuscript. NCP directed the research and assisted in the writing of the manuscript. All authors read and approved the final manuscript."MS 2784776144318916 Supplement1.xls" and contains the primer sequences, expected size range, primer concentration used and annealing temperatures used. In a separate sheet within the same file the protocols for each PCR reaction are listed.Click here for file"MS 2784776144318916 Supplement2.xls" and contains the individual genotype data for each animal used to derive the statistics and phylogenetic results presented herein.Click here for file

Many different things combine to cause epidemics of disease. Among these factors are the characteristics of the infecting organism, the resistance of the host, and, as is increasingly realized, climatic conditions.El Niño, the best known climatic disturbance, is caused by a warming of the Pacific Ocean, which then affects the climate globally. Previous work has suggested that this recurring phenomenon can have a profound effect on the incidence of many diseases, including dengue, malaria, and diarrheal diseases.PLoS Medicine, Sultan and colleagues from a climate research institute and an infectious diseases center in France looked at the relation between climate and meningitis outbreaks in Mali in West Africa, a region that every year between February and May sees devastating epidemics of meningococcal meningitis affecting up to 200,000 people. The most important recurring climatic event in this region is a dry wind, known as the Harmattan, that blows throughout the winter, causing a drop in humidity and the production of vast quantities of dust.In a paper in this month's What the authors found was that over the years 1994–2002, the week of the onset of the yearly meningitis epidemic came at around the same time as the peak of one measure of the wind—the sixth week of the year. As Pascual and Dobson say in their Perspective article on this study, “Sultan and colleagues' study is exceptional in that it illustrates a clear relationship between an external environmental variable and the initiation of disease outbreaks.”How do climatic changes influence disease? In some cases, such as the role of flooding in spreading a waterborne disease, the causes are perhaps obvious, but why should a dry wind affect disease incidence? Previous works have suggested that the climate can work in a number of ways, by influencing the life cycle of both disease vectors and the disease-causing organism, and, as here perhaps, by affecting the resistance of the host. Sultan and colleagues speculate that the drying effects of the wind on the mucous membranes could increase the chances of the organism getting established in the human host. Whatever the causes, one very useful feature of climate is that, once the patterns are understood, they can often be predicted.A way of predicting these meningitis epidemics could be enormously useful. Sultan and colleagues looked at only a few years, but if these findings are confirmed over a longer time period, they could make preparing for an epidemic much more efficient.

Nutrition & Metabolism (N&M) will publish articles that integrate nutrition with biochemistry and molecular biology. The open access process is chosen to provide rapid and accessible dissemination of new results and perspectives in a field that is of great current interest. Manuscripts in all areas of nutritional biochemistry will be considered but three areas of particular interest are lipoprotein metabolism, amino acids as metabolic signals, and the effect of macronutrient composition of diet on health. The need for the journal is identified in the epidemic of obesity, diabetes, dyslipidemias and related diseases, and a sudden increase in popular diets, as well as renewed interest in intermediary metabolism.A new Open Access journal, Nutrition & Metabolism (N&M) include 1) an awareness of an epidemic of obesity, diabetes, dyslipidemias and related diseases, 2) a sudden increase in the popularity of diets, such as low carbohydrate diets, to achieve weight loss and combat diabetes, and 3) a renewed interest in intermediary metabolism accompanied by the development of new tools and techniques for genomic and metabolic analysis.Recent events that provide the rationale for a new Open Access journal, ) and similar motions exist in the US congress [N&M feel that, at this point, the burden of proof is on proponents of perpetuating the current system. We are, however, not doctrinaire on this point and believe one should pay for a service if it is valuable. Beyond information, printed collections provide convenience and we intend to offer bound copies of articles on individual topics as the journal proceeds.With the considerable activity shown in these areas, rapid and easily accessible dissemination of new information is clearly valuable. Whereas articles in existing journals do discuss intermediary metabolism in a nutritional context, there is a need for a unique and explicit focus for this discipline. In addition, it is precisely because publications in nutritional biochemistry are spread over such a large number of existing journals, few libraries and almost no individual can subscribe to all. It is in areas like this that free, open access becomes important. There is a large published debate on open access . Most rcongress ,3. The eN&M has its own strengths and interests as indicated by the board of editors . Three areas of particular interest are lipoprotein metabolism, amino acids as metabolic signals, and the effect of macronutrient composition of diet on health. This is reflected in our opening research articles by Darimont, et al. on the control of obesity and lipid structure by adrenergic systems, and by Volek, et al. on the effectiveness of low carbohydrate diets, and differential effects on fat and lean mass.Nutrition and metabolism is a broad field and we welcome submissions from all areas of nutrition and related biochemistry. Like any journal, however, , while not recommending any particular diet, highlighted many of the relevant issues in macronutrient control of metabolism. In our initial publications, contributors to the conference will provide reviews of the various topics covered. In the first posting, Klaas Westerterp summarizes the importance of macronutrient composition in thermogenesis, and Stephen Phinney discusses the impact of ketogenic diets on physical performance. Kimball and Jefferson review the regulation of mRNA translation in general. The article provides a nice overview of various mechanisms involved in the control of protein synthesis when amino acids become limiting. Perhaps the most important from a practical standpoint, Nuttall and Gannon summarize potential benefits of higher protein diets in diabetes.The sudden popularity of low carbohydrate diets is one of the most remarkable phenomena in nutrition today. A recent editorial by Walter Willett points out how important it is that we understand them . SimilarN&M is free. Articles are included in PubMed and archived in PubMed Central. Online submissions can be made at .Nutrition & Metabolism welcomes contributions in all areas of research in which nutrition interacts with biochemistry and molecular biology. Emphasis will be on the molecular, biochemical, and physiologic understanding of various metabolic pathways. The journal will publish Original Research, Reviews, Commentaries and Perspectives, Brief Communications, Methods and Book Reviews. Access to all articles in

E. Coli genes is affected by changes in the level of chromosome supercoiling, suggesting that supercoiling transmits regulatory signals from the environment to many cellular pathways.Microarray analysis shows that transcription of 306 Escherichia coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. In turn, supercoiling influences local DNA structure and can affect gene expression. We used microarrays representing nearly the entire genome of Escherichia coli MG1655 to examine the dynamics of chromosome structure.The chromosome of We measured the transcriptional response to a loss of supercoiling caused either by genetic impairment of a topoisomerase or addition of specific topoisomerase inhibitors during log-phase growth and identified genes whose changes are statistically significant. Transcription of 7% of the genome (306 genes) was rapidly and reproducibly affected by changes in the level of supercoiling; the expression of 106 genes increased upon chromosome relaxation and the expression of 200 decreased. These changes are most likely to be direct effects, as the kinetics of their induction or repression closely follow the kinetics of DNA relaxation in the cells. Unexpectedly, the genes induced by relaxation have a significantly enriched AT content in both upstream and coding regions.The 306 supercoiling-sensitive genes are functionally diverse and widely dispersed throughout the chromosome. We propose that supercoiling acts as a second messenger that transmits information about the environment to many regulatory networks in the cell. Escherichia coli is a circular double-stranded DNA molecule that is maintained in a negatively supercoiled state. Supercoiling induces torsional tension in the DNA, and thus can influence processes that involve the opening of the double helix, such as replication initiation [The chromosome of itiation , DNA looitiation and tranitiation . A numbeitiation ), suggesE. coli, supercoiling is maintained at a precise range during log phase growth by the topoisomerases DNA gyrase, topoisomerase I (topo I), and topoisomerase IV (topo IV) [gyrA and gyrB), and for topo I (topA). Relaxation of the chromosome upregulates gyrA and gyrB and downregulates topA as a form of feedback control [fis , ilvG (an amino-acid synthase subunit) and cydAB (an oxidase involved in aerobic respiration), has been found to be sensitive to supercoiling [In topo IV) -7. DNA gtopo IV) -10, whertopo IV) . Togethetopo IV) ,13. The control -16. Thisrcoiling -19, suggrcoiling . If supercoiling ,17,18.E. coli K-12 genome to systematically identify those genes that respond to relaxation of the chromosome during log-phase growth. We used antibiotics and mutations in the topoisomerase genes to change supercoiling levels by independent mechanisms and thus discerned the general effects of chromosome relaxation. We classify supercoiling-sensitive genes, or SSGs, according to their response to DNA relaxation. Therefore, we call 'relaxation-induced genes' those genes whose expression is increased upon DNA relaxation, and 'relaxation-repressed genes' those whose expression is repressed by DNA relaxation.In this study, we used cDNA microarrays ,22 repregyrA or topA, and their expression patterns correlated with the supercoiling level of a reporter plasmid in the cells. SSG transcripts have the same rates of RNA decay as non-SSG transcripts, and thus the changes in expression were due to a change in the rate of RNA synthesis, rather than RNA decay.An extensive statistical analysis of our experimental results revealed 200 relaxation-repressed genes and 106 relaxation-induced genes; in total, around 7% of all genes in the genome were found to be significantly affected by supercoiling changes. Many of these genes are more sensitive to supercoiling than p < 0.0001) AT-rich in their upstream sequences, and also have AT-rich coding regions. Relaxation-repressed genes had a corresponding preference for GC-rich sequences. The SSGs are dispersed throughout the chromosome, and fall into many different functional classes. We propose that the large number and functional diversity of the SSGs reflects the role of supercoiling as a second messenger that responds to environmental changes and feeds into different regulatory networks.We discovered that the sequences of the relaxation-induced genes are significantly (tetA gene in pBR322 anchors this plasmid to the membrane [We sought to determine the genes that are activated or repressed by relaxation of the (-) supercoils in the chromosome. To isolate the expression changes due to the loss of supercoiling from those due to the experimental approach, we used three different methods to relax the chromosome. In two of the methods we inhibited gyrase and topoIV with either quinolone or coumarin antibiotics, and in the third we used a temperature-sensitive strain in which gyrase is inhibited at 42°C. Because it is technically difficult to quantify the supercoiling state of the bacterial chromosome, we used a plasmid, pBR322, in the strains as a reference. Co-transcriptional translation of the membrane , and thumembrane . The supgyrA and gyrB) or topo IV (parC and parE) as controls, to separate expression changes due to undiscovered drug targets from those directly due to changes in supercoiling. When we inhibited gyrase by treating gyrA+ parCR cells with 15 μg/ml norfloxacin, the reporter plasmid in the cells was rapidly relaxed (Table gyrAR parCR strain remained (-) supercoiled. After 30 minutes, there was a 103-fold drop in viability in the sensitive strain, but only a 17% drop in the resistant strain. A norfloxacin concentration of 50 μg/ml fully inhibited both gyrase and topoisomerase IV in the wild-type strain (data not shown), while the resistant strain retained wild type levels of (-) supercoiling and showed only a slight drop (15%) in viability, indicating that we did not overcome the drug resistance. At bacteriocidal concentrations similar to these, quinolones cause a decrease in the sedimentation coefficient of the nucleoid, indicating relaxation of the chromosomal supercoils [t = 0) and was labeled with Cy3 (green). RNA samples taken 2, 5, 10, 20 and 30 minutes after drug addition were labeled with Cy5 (red). The labeled experimental and reference samples were mixed in equal amounts before hybridization to a microarray.The quinolone antibiotic norfloxacin selectively and immediately inhibits gyrase and topo IV -26. We upercoils . The refInhibition of topoisomerases by quinolones leads to double-strand breaks in the chromosome . Thus, nWe also relaxed the chromosome using novobiocin, a coumarin antibiotic that inhibits gyrase, and at a higher concentration, topo IV ,31. NovogyrB234 cells upon shift to the restrictive temperature and subsequent relaxation of the DNA to determine which genes change significantly during an experiment can bias expression analysis towards genes with very low or variable expression levels . We usedet al. . Brieflyrocedure . The corp-value represents the robustness of the response to relaxation, whereas correlation with plasmid supercoiling may represent sensitivity to changes in supercoiling levels. For example, a gene that is always completely repressed in response to relaxation will have a low p-value, but may show little correlation with intermediate levels of supercoiling. Similarly, a gene with more variable expression may have a higher p-value, but may also have a higher sensitivity to intermediate supercoiling levels. Taken together, these metrics provide a detailed account of supercoiling sensitivity.As an independent metric of supercoiling sensitivity, we measured how closely gene expression followed the level of DNA supercoiling, by calculating the correlation of the expression of each gene across all of the experiments with the level of supercoiling in the reporter plasmid. Relaxation-induced genes showed a positive correlation with plasmid linking number supercoiling is lost, both linking number and gene expression increase), up to a maximum observed Pearson correlation coefficient of 0.91. Relaxation-repressed genes showed a negative correlation with plasmid linking number to a minimum Pearson coefficient of -0.88. The majority of genes showed no strong correlation with plasmid supercoiling, resulting in Pearson coefficients between 0.5 and -0.5. The p-values for all of the genes versus their correlation to plasmid supercoiling are plotted in Figure p-values and little correlation with plasmid supercoiling. Those genes with the lowest p-values tended to be more strongly correlated (or anticorrelated) to plasmid supercoiling. The data for all genes can be found in Additional data file 2. Among all genes there is a continuous variation in both p-value and correlation to plasmid supercoiling. We found a total of 306 genes at p < 0.05, which we define as SSGs. Of these, 106 genes were induced by DNA relaxation and have a positive correlation with plasmid linking number, while 200 genes were repressed by relaxation and these have a negative correlation with plasmid linking number. The correlations of the SSGs with plasmid supercoiling are shown in Figure p-value. Just over half of the SSGs have high significance, p < 0.005. The high redundancy of our data minimized the influence of any single array measurement. Thus we can be confident that the genes we classed as SSGs have a reproducible response to supercoiling changes.The p-value (from the top). Each colored entry in the diagram corresponds to one spot on one array . Conversely, these relaxation-repressed genes should have low ratios (and black squares) in the control experiments 1 to 14. The significant difference in SSG expression between the controls and relaxation experiments is reflected by the sharp contrast between the mostly black controls and the bright green relaxation experiments. At the top we have shown a model expression profile representing the supercoiling of the reporter plasmid in each experiment are listed, along with their correlations to plasmid supercoiling levels.Figure p-values, correlations to supercoiling, and expression levels in each experiment can be found in Additional data file 3.The 106 genes that are induced by relaxation are similarly shown in Figure gyrA+parC+ cells, plasmid supercoiling levels changed dramatically within the first minute should respond quickly to relaxation. We used a finer time-course experiment to determine which genes had the fastest response to chromosomal relaxation. When 15 μg/ml norfloxacin was added to e Figure . Signifie Figure . We rankE. coli mRNAs have a mean half-life of 5.2 ± 0.3 minutes in LB media [The speed of the transcriptional response to relaxation, combined with the strong correlations to supercoiling of the reporter plasmid in the cells, is strong evidence that the SSGs are directly regulated by supercoiling changes. Furthermore, given that LB media , RNA synp-value with the published values of RNA half-life [We found no correlation of alf-life and in gp = 3E-4).We searched for a basis of supercoiling sensitivity at the nucleotide sequence level by examining the AT content in and around the SSGs. We considered only the first genes in an operon. Whereas relaxation-repressed genes have a slightly depleted AT content both upstream of their promoters and within the coding sequence, relaxation-induced genes have an emphatic elevation of AT content in similar regions. The AT content of relaxation-induced genes from 800 nucleotides before to 200 nucleotides after the start codon is 54.6%, compared with 51.7% for non-SSGs. To illustrate the very low probability of selecting by chance a set of genes with such an elevated AT content, we randomly selected groups of first-in-operon non-SSGs 50,000 times and calculated AT content within the same window. We never found a set with an AT content as high as the relaxation-induced genes .This is not the only region in which the AT content of SSGs deviates from the rest of the genome. Figure Striking as these differences in AT content are for SSGs as a group, they are not sufficient to distinguish an individual SSG from a non-SSG. That is, not all genes with high or low AT content were supercoiling sensitive in our experiments. Although such genes are rare in the non-SSG population, the greater size of the pool of non-SSGs results in many genes with wide variations in AT content. Also, supercoiling sensitivity cannot solely be due to differences in AT content, as a few SSGs were highly sensitive to supercoiling changes in spite of having an AT content similar to the rest of the genome.topA [gyrA [p-values. Accordingly, although the topoisomerase genes topA and gyrA both clearly respond to supercoiling . The omission of these topoisomerase genes from our list of SSGs reflects the conservative statistical approach we used to define the list. There are probably other genes that respond to supercoiling changes in different conditions from those we tested (log-phase growth in rich media). Also, we defined SSGs by focusing on the immediate effects of relaxation, and thus considered only primary transcriptional changes, rather than downstream effects mediated by other gene products . When downstream effects are considered, changes in supercoiling are likely to affect transcription of an even greater proportion of the genome.In this analysis of supercoiling effects on transcription, we identified 306 genes that quickly and reproducibly respond to chromosomal relaxation. The comprehensive nature of our investigation, with responses of 93% of the genome in 21 different relaxation experiments and 14 control experiments, allowed us to be more stringent than previous studies in our definition of SSGs, and to identify those genes that had statistically significant changes after the chromosome was relaxed by different methods. Genes that are sensitive to relaxation but are also affected by temperature shifts (including topA and gyrApA [gyrA ) showed E. coli. Two early studies used either nylon membranes or two-dimensional protein gels to compare topoisomerase mutants with slightly different homeostatic levels of supercoiling, and neither study found a large number of genes [There have been several previous attempts to measure the effects of supercoiling on gene expression in of genes ,44. ThisE. coli [ynhG, yrbL, otsB and yifE). Seven others had p < 0.1 in our relaxation experiments, and the rest had still higher p-values in this study. It is possible that these genes are only responsive to supercoiling changes in the context of osmotic stress.A more recent analysis by Church and colleagues used microarrays to gauge the osmotic stress response of E. coli . SurveyiJust as supercoiling is affected by many environmental changes, such as osmotic shock, oxygen tension, nutrient upshift and temperature change, so too do changes in supercoiling affect genes in a large number of classes. Not surprisingly, a substantial fraction (6.9%) of the SSGs encode genes involved in DNA replication, recombination, modification and repair. However, the SSGs span many other classes, and thus are well positioned to feed into many different regulatory networks. Thus, supercoiling can act as a second messenger that quickly translates environmental changes to transcriptional programs, inducing and repressing specific genes independently of protein synthesis.smtAmukBEF operon on loss of supercoiling is intriguing, given the importance of mukB, mukE and mukF in chromosome supercoiling and segregation [E. coli [Several of the SSGs warrant further inspection. For example, the repression of the regation ,46. Cons[E. coli , these gyrdD, a 'putative topoisomerase'. yrdD encodes a 19 kilodalton (kDa) protein 30-40% identical to the carboxy-terminal domain of topoisomerase I from Bacillus subtilis, Helicobacter pylori and Methanococcus jannaschii. The function of YrdD is unknown, but the repression by chromosomal relaxation provides an intriguing lead.Another relaxation-repressed gene that may be involved in chromosomal maintenance is cls (cardiolipin synthase) and ileS (isoleucine tRNA synthetase), which is consistent with the earlier discovery that these genes were involved in sensitivity to gyrase inhibitors [deoA and deoC are induced on relaxation. For these genes, DNA relaxation may be a signal of DNA damage, and their induction would allow the cell to recycle nucleotides necessary for DNA repair. Finally, the induction of rpoD, the σ70 subunit of RNA polymerase, may help the cell compensate for the increased difficulty of melting the relaxed DNA template.Chromosomal relaxation leads to the repression of hibitors . Also, wlacps and ilvGP [in vitro. The more relevant issue is promoter regulation on chromosomes in vivo, where other factors may dominate. The CRP protein increases lac operon transcription at the low to moderate superhelicities found in vivo, and the nucleoid-associated protein IHF is implicated in the supercoiling sensitivity of the ilvGMEDA operon [Salmonella leu-500 promoter by supercoiling requires that the promoter is either on the chromosome or on a plasmid anchored to the cell membrane by transcription and translation of a gene such as tetA [What is the basis of supercoiling sensitivity? Most of the well controlled analyses of supercoiling-sensitive promoters, notably of the nd ilvGP ,49-51, wA operon . Also, tA operon -55. We f as tetA . FurthergyrA and gyrB promoters, Menzel and Gellert [gyrA and gyrB. Promoter clearance has also been invoked in the mechanism of supercoiling sensitivity of some promoters in vitro [in vivo, and propose that promoter clearance is generally a key regulatory step for supercoiling sensitive transcription.It is striking that there are so many relaxation-induced genes that are relatively repressed when the chromosome is (-) supercoiled. This is surprising because (-) supercoiling should favor formation of an open promoter complex. The promoter regions of many of the genes induced by relaxation are AT rich, which will make it easier to form an open promoter complex even when the DNA is relaxed and the energy required is greater. Alternatively, the difference in AT content could reflect structural features such as curvature or flexibility. Curved sequences of DNA can influence the position of plectonemic supercoils, and thus could serve to localize a promoter sequence to the apex of a superhelical loop . We note Gellert found thin vitro . As our The AT-rich regions of our relaxation-induced genes extend downstream of the translational start site, and thus may involve transcription elongation in addition to promoter clearance. There is growing appreciation of the regulation of transcription elongation -61. The E. coli chromosome [The SSGs are useful as topological probes of the chromosome in living cells. While the SSGs are scattered throughout the chromosome, they are not evenly spread, but rather have regions of high and low density. The SSGs are plotted on a chromosomal map in Figure romosome . The disromosome . Relaxatromosome . Here weWe have shown that supercoiling acts as a transcription factor, with positive and negative effects on specific genes while leaving the majority of the genome unchanged. Like other transcription factors such as TrpR and ArcAE. coli K-12, strain MG1655 (Sigma-Genosys), were generously supplied by Fred Blattner (University of Wisconsin) and Carol Gross . ORFs were amplified from MG1655 genomic DNA using ExTaq polymerase (PanVera) and failed PCR reactions were attempted again using Platinum Taq (Invitrogen) or previous successful reactions as the DNA template. Ninety-six percent of the ORFs were successfully amplified. PCR conditions were set according to those supplied with the primers. DNA was precipitated with isopropanol and prepared for microarray printing as described in [tyrT, have been shown to respond to changes in supercoiling [Amino- and carboxy-terminal primers for protein-coding open reading frames (ORFs) of ribed in . We did rcoiling .Detailed instructions on slide preparation, microarray printing and processing microarrays can be found online . 384-welE. coli cells were grown with shaking in LB media to an OD600 = 0.45-0.55 at 37°C, or at 30°C for temperature-sensitive strains. Samples of cells were withdrawn at intervals and added to a 1/10 volume of either 95% ethanol plus 5% phenol or 2 M NaN3 to stop transcription. Cells were then quickly harvested by centrifugation in a microcentrifuge. The supernatant was aspirated and the pellets frozen in liquid N2. Total RNA was prepared using the Qiagen RNeasy mini kit, except that 4 mg/ml lysozyme and a 30 sec incubation was used in the first step. For each microarray, 20 μg total RNA was primed with 1-2 μg of random hexamers and labeled by reverse transcription in the presence of Cy3- and Cy5-conjugated dUTP (Amersham Biosciences). For each experiment or condition, a Cy5-labeled experimental sample was combined with a Cy3-labeled reference sample and hybridized to a processed microarray as described [escribed . After 5Scanned array images were visually inspected, and non-uniform spots were excluded from further analysis. The background was subtracted from the images that were then (median) normalized using the Scanalyze 2.0 program, v. 1.44 such that the total fluorescence in each channel was equal.E. coli genes, that could be considered for further study. Because we were interested in changes in expression levels due to variations in supercoiling rather than to drug or genetic effects, we used a two-sample comparison approach rather than a factorial analysis approach. We tested two commonly used methods to determine differentially expressed genes in the comparison of two samples. We found that the method of Dudoit et al [et al [We tested several methods of imputation to estimate the values of spots missing due to hybridization defects (described in ), and afit et al , which cl [et al .32P-labeled DNA probes for gyrB and asnB (as a loading control) were synthesized from their respective PCR products, and radioactivity was quantified by a phosphorimager.Samples were run on formaldehyde-MOPS 1% agarose gels and blotted onto a nylon membrane . 32P-labgyrB234 mutant are in a C600 strain background, but all strains used have been described in greater detail elsewhere [ΔacrA strain that greatly slows drug efflux [0, where Lk0 for pBR322 = 4,361 bp/10.5 bp/turn = 415. Samples were run on parallel 20-cm gels containing 0, 2.8 or 10 μg/ml chloroquine for 18-26 h at 48-52 V with constant buffer recirculation, which allowed visualization of the entire distribution of topoisomers. Gels were southern blotted [32P-labeled probe made from random priming of pBR322. Radioactive blots were quantified using a phosphorimager.Plasmid DNA was extracted from cells by the alkaline lysis method or the Qlsewhere ,35,73. Tg efflux . The supg efflux from the blotted , and hybgyrB by both methods 5 min after addition of the gyrase inhibitor novobiocin to ΔacrA cells at 5, 20, 50 and 200 μg/ml. The microarray ratios for these concentrations were 2.3, 4.8, 4.9 and 6.3, respectively, while the ratios from northern hybridizations were 2.8, 4.7, 4.7 and 5.1. Second, as an internal control we compared the transcription of genes in 153 known polycistronic operons. We found no operons with genes that changed expression more than 1.5-fold in opposite directions (data not shown). Third, we compared two identically grown cultures with the same microarray . We used two strains that were isogenic, except that one had point mutations conferring norfloxacin resistance on gyrase and topo IV. The correlation coefficient of the gene-expression levels was 0.982, indicating the negligible variation between the cultures. In contrast, when we treated cells with the gyrase inhibitor norfloxacin , the correlation coefficient with respect to the untreated cells was only 0.391 and hundreds of genes showed large differences in expression. We conclude that gene-expression changes resulting from slight genotypic changes or experimental repeats were negligible compared with the changes resulting from topoisomerase inhibition, and that the E. coli microarrays are a reliable method for quantifying these changes.We tested the validity of our microarrays in three ways. First, we compared gene expression ratios measured with microarrays to values obtained by northern hybridization. We measured induction ratios for p-values < 0.05) over a total of 35 experiments, in which DNA gyrase was inhibited with novobiocin, norfloxacin or by a mutation that rendered gyrase temperature-sensitive. Next we determined the correlation of gene expression with the σ of a reference plasmid in the same cells. To calculate the correlation of gene expression to plasmid supercoiling, we created a model profile made up of the ratio of plasmid σ in each experiment to plasmid σ in the (supercoiled) reference for that experiment (Table We limited the list of SSGs to those whose expression difference between treatments and controls was statistically significant (nt Table . The maxThe following additional data files are available with the online version of this paper: Additional data file Data on the induction of the SOS response to DNA damageClick here for additional data fileGene-expression ratios for all genes across all experimentsClick here for additional data fileGene-expression ratios for supercoiling-sensitive genes across all experimentsClick here for additional data fileData on the reproducibility of microarray measurement of RNA levelsClick here for additional data file

Popular methods to reconstruct molecular phylogenies are based on multiple sequence alignments, in which addition or removal of data may change the resulting tree topology. We have sought a representation of homologous proteins that would conserve the information of pair-wise sequence alignments, respect probabilistic properties of Z-scores (Monte Carlo methods applied to pair-wise comparisons) and be the basis for a novel method of consistent and stable phylogenetic reconstruction.We have built up a spatial representation of protein sequences using concepts from particle physics (configuration space) and respecting a frame of constraints deduced from pair-wise alignment score properties in information theory. The obtained configuration space of homologous proteins (CSHP) allows the representation of real and shuffled sequences, and thereupon an expression of the TULIP theorem for Z-score probabilities. Based on the CSHP, we propose a phylogeny reconstruction using Z-scores. Deduced trees, called TULIP trees, are consistent with multiple-alignment based trees. Furthermore, the TULIP tree reconstruction method provides a solution for some previously reported incongruent results, such as the apicomplexan enolase phylogeny.The CSHP is a unified model that conserves mutual information between proteins in the way physical models conserve energy. Applications include the reconstruction of evolutionary consistent and robust trees, the topology of which is based on a spatial representation that is not reordered after addition or removal of sequences. The CSHP and its assigned phylogenetic topology, provide a powerful and easily updated representation for massive pair-wise genome comparisons based on Z-score computations. We based our model on mathematical properties that alignment scores should respect; i) information theory     (22)a* and b* the random variables corresponding to the shuffled sequences of a and b respectively. The sum of the logarithms corresponds to the product of the two probabilities, an expression of the hypothesis of independence of lineage. Interestingly, equation (22) provides an expression of the symmetric effect of time on the variations that independently affected a and b.with t appears as a function of Z-score ratios. For any set of homologous proteins, it is therefore possible to measure a table of pair-wise divergence times and build phylogenetic trees using distance methods.From relation (20), , where numbrand is the number of randomizations. In the case studies presented here, we set numrand = 2000 (see Methods). By contrast, stability of MAB trees is sought by bootstrapping approaches and consensus tree reconstruction. MAB trees appear as the result of a complex learning process including possible re-adjustment of the multiple alignments after eye inspection pragmatically applied to assist the reconstruction. Alternatively, Bayesian analyses have been recently proposed for phylogenetic inference μ,+∞μ + ψ ,+∞[.In consequence, s0 is not trivial because it depends on the knowledge of the cumulative distribution function. Extensive studies provided experimental and theoretical supports for an extreme value distribution of alignment scores [i.e. the Gumbel distribution [The estimation of t scores ,43,44. Uribution , the cumθ and β (β > 0) the location and scale parameters. The probability density function g(s) is defined by dG(s) = g(s)ds. We observe with that . Using the Taylor's polynomial formula, i.e. :with In consequence, for a Gumbel score probability distribution:ψ from a Gumbel distribution is illustrated in Figure A graphical determination of a and b is relatively high, that is s ≥ μ + ψ, then the trivial inequality s ≥ s impliesIf a pair-wise alignment score of two sequences s - μ)2)) ≥ - μ)2))     (h), we deduce that a and b, alignment was achieved with the Smith-Waterman method and the BLOSUM 62 scoring matrices, using the BIOFACET package from Gene-IT, France [z, z, z, z, with 2000 sequence shuffling. For all computations, an estimation of the Gumbel parameters θ and β was made using the computed μ and σ of any S and the formula and θ = μ - βΓ'(1), where Γ'(1) ≈ 0.577216 is the Euler constant. In all computations, both Gumbel parameters were very close = 35.04, SD(θ) = 0.12, mean(β) = 3.92, SD(β) = 0.08). As a consequence, the assumption Q ≈ Q was verified for any pair of sequences. We used the parameters to estimate μ = θ + βΓ '(1) , and μ + ψ ≈ μ + 10.5178 ≈ 47.85. As any pairs of computed scores are higher than this critical threshold, we used relation [To build PHYLIP trees, multiple sequence alignments were created with ClustalW . PHYLIP , France . We comprelation . Trees wCSHP, configuration space of homologous proteins, TULIP, theorem of the upper limit of a score probabilityOB conceived the main theoretical model, designed and developed the method to build phylogenetic trees and drafted the manuscript. SR and PO participated in the theoretical model refinement and in the design and development of computational methods to build TULIP trees. EM contributed to the conception of this study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.

Should we be considering the social and economic ramifications of a society where life-span could be limitless? Sinclair suggested, as have other experts, including his mentor Lenny Guarente and the National Institute on Aging advisory council member Elizabeth Blackburn, that Kass and other bioconservatives are creating a false alarm about life extension, because only a modest increase in human life span is achievable by biomedical intervention, whereas Kass's apprehensions concern extreme or indefinite life extension. Kass retorted that science isn't like that: modest success tends to place the bit between our teeth and can often result in advances far exceeding our expectations.The biogerontologist David Sinclair and the bioethicist Leon Kass recently locked horns in a radio debate of age-related molecular and cellular damage—consisting of just seven major categories of ‘rejuvenation therapy’ (I agree with these predictions in two respects: that the degree of life extension achieved by first-generation drugs of this sort may well approach the (currently unknown) amount elicitable by caloric restriction itself in humans, and that it is unlikely to be much exceeded by later drugs that work the same way. In two other ways, however, I claim they are incorrect. The first error is the assumption of proportionality: I have recently argued , from evescribed , 2004 a therapy’ —that appCoping with Methuselah and by other commentators is demographic. Life expectancy is typically defined in terms of what demographers call a period survival curve, which is a purely artificial construction derived from the proportions of those of each age at the start of a given year who die during that year. The ‘life expectancy’ of the ‘population’ thus described is that of a hypothetical population whose members live all their lives with the mortality risk at each age that the real people of that age experienced in the year of interest. The remaining life expectancy of someone aged N in that year is more than this life expectancy minus N for two reasons: one mathematical and one biomedical . My spirits briefly rose on reading Aaron and Harris's explicit statement (p. 69) of the latter reason. Unfortunately, they didn't discuss what would happen if age-specific mortality rates fell by more than 2% per year. An interesting scenario was thus unexplored: that in which mortality rates fall so fast that people's remaining life expectancy increases with time. Is this unimaginably fast? Not at all: it is simply the ratio of the mortality rates at consecutive ages (in the same year) in the age range where most people die, which is only about 10% per year. I term this rate of reduction of age-specific mortality risk ‘actuarial escape velocity’ (AEV), because an individual's remaining life expectancy is affected by aging and by improvements in life-extending therapy in a way qualitatively very similar to how the remaining life expectancy of someone jumping off a cliff is affected by, respectively, gravity and upward jet propulsion . The escape velocity cusp is closer than you might guess. Since we are already so long lived, even a 30% increase in healthy life span will give the first beneficiaries of rejuvenation therapies another 20 years—an eternity in science—to benefit from second-generation therapies that would give another 30%, and so on ad infinitum. Thus, if first-generation rejuvenation therapies were universally available and this progress in developing rejuvenation therapy could be indefinitely maintained, these advances would put us beyond AEV. Universal availability might be thought economically and sociopolitically implausible , so it's worth considering the same question in terms of life-span Coping with Methuselah is representative, is the most significant because of its urgency. First-generation rejuvenation therapies, whenever they arrive, will surely build on a string of prior laboratory achievements. Those achievements, it seems to me, will have progressively worn down humanity's evidently desperate determination to close its eyes to the prospect of defeating its foremost remaining scourge anytime soon. The problem is that this wearing-down may have been completed long before the rejuvenation therapies arrive. There will come an advance—probably a single laboratory result—that breaks the camel's back and forces society to abandon that denial: to accept that the risk of getting one's hopes up and seeing them dashed is now outweighed by the risk of missing the AEV boat by inaction. What will that result be? I think a conservative guess is a trebling of the remaining life span of mice of a long-lived strain that have reached two-thirds of their normal life span before treatment begins. This would possess what I claim are the key necessary features: a big life extension, in something furry and not congenitally sick, from treatment begun in middle age.The third oversight that I observe in contemporary commentaries on life extension, among which ten years, given adequate and focused funding (perhaps $100 million per year). And nobody in Coping with Methuselah said so. This timeframe could be way off, of course, but as Wade notes (p. 57), big advances often occur much sooner than most experts expect. Even the most obvious of these lifestyle changes—greater expenditure on traditional medical care, avoidance of socially vital but risky professions—could severely destabilise the global economy; those better versed in economics and sociology than I would doubtless be even more pessimistic about our ability to negotiate this period smoothly. Overpopulation, probably the most frequently cited drawback of curing aging, could not result for many decades, but the same cannot be said for breadth of access irrespective of ability to pay: in a post-9/11 world, restricted availability of rejuvenation therapies resembling that seen today with AIDS drugs would invite violence on a scale that, shall we say, might be worth trying to avoid.It is the prospect of AEV, of course, that makes this juncture so pivotal. It seems quite certain to me that the announcement of such mice will cause huge, essentially immediate, society-wide changes in lifestyle and expenditure choices—in a word, pandemonium—resulting from the anticipation that extreme human life extension might arrive soon enough to benefit people already alive. We will probably not have effective rejuvenation therapies for humans for at least 25 years, and it could certainly be 100 years. But given the present status of the therapies listed in Coping with Methuselah (p. 143). Once AEV is achieved, there will be no going back: rejuvenation research will be intense forever thereafter and will anticipate and remedy the life-threatening degenerative changes appearing at newly achieved ages with ever-increasing efficacy and lead time. This will bring about the greatest economic change of all in society: the elimination of retirement benefits. Retirement benefits are for frail people, and there won't be any frail people. The graph just mentioned amply illustrates how much wealth will be released by this. My hope, therefore, is that once policy makers begin to realise what's coming they will factor in this eventual windfall and allocate sufficient short-term resources to make the period of limited availability of rejuvenation therapies brief enough to prevent mayhem. This will, however, be possible only if such resources begin to be set aside long enough in advance—and we don't know how long we have.Am I, then, resigned to a future in which countless millions are denied many decades of life by our studied reluctance to plan ahead today? Not quite. The way out is pointed to in

The initiation of re-entrant cardiac arrhythmias is associated with increased dispersion of repolarisation, but the details are difficult to investigate either experimentally or clinically. We used a computational model of cardiac tissue to study systematically the association between action potential duration (APD) dispersion and susceptibility to re-entry.+ channel gKmax set to 0.004 mS mm-2. Within the central 40 × 40 mm region we introduced square regions with prolonged APD by reducing gKmax to between 0.001 and 0.003 mS mm-2. We varied (i) the spatial scale of these regions, (ii) the magnitude of gKmax in these regions, and (iii) cell-to-cell coupling.We simulated a 60 × 60 mm 2 D sheet of cardiac ventricular tissue using the Luo-Rudy phase 1 model, with maximal conductance of the KgKmax in regions with prolonged APD from 0.003 to 0.001 mS mm-2 increased APD dispersion from 22 to 70 ms, and the susceptible window from <1 to 56 ms. Decreasing cell-to-cell coupling by changing the diffusion coefficient from 0.2 to 0.05 mm2 ms-1 increased APD dispersion from 57 to 88 ms, and increased the susceptible window from 41 to 74 ms.Changing spatial scale from 5 to 20 mm increased APD dispersion from 49 to 102 ms, and the susceptible window from 31 to 86 ms. Decreasing We found a close association between increased APD dispersion and susceptibility to re-entrant arrhythmias, when APD dispersion is increased by larger spatial scale of heterogeneity, greater electrophysiological heterogeneity, and weaker cell-to-cell coupling. Cardiac disease remains an important cause of sudden death in the industrialised world, and in many cases the lethal events are the cardiac arrhythmias called ventricular tachycardia (VT) and ventricular fibrillation (VF). Spontaneous episodes of VT and VF occur in patients where cardiac disease or congenital abnormality has remodelled either the structure or function of cardiac cells and tissue. There is abundant experimental evidence to support the idea that VT and VF are sustained by re-entry ,2, but tSlow conduction and unidirectional block have long been known to facilitate re-entry , and exp-1 were associated with block and re-entry [Regional differences in repolarisation are often described as action potential duration (APD) dispersion. The difference between the longest and shortest observed APD is a conceptually simple and easily obtained quantity and has widely been used to measure APD dispersion, although other indices have been proposed . Some exre-entry ,19,20. Sre-entry ,21,22. T+ channel conductance between regions with short and long APD, and (iii) strength of cell-to-cell coupling.Computational models offer a powerful research tool for addressing these questions, because the properties of a virtual tissue can be controlled precisely and independently in a way that would be extremely difficult to achieve experimentally. The purpose of this study was therefore to investigate systematically how measured APD dispersion and vulnerability to re-entry in a computational model of ventricular tissue are related to: (i) the spatial scale of heterogeneity, (ii) the magnitude of differences in KWe simulated electrical activation in a 2 D isotropic monodomain virtual tissue Vm is membrane voltage, Cm specific membrane capacitance, D a diffusion coefficient and Iion current flow through the cell membrane per unit area. We used the Luo-Rudy phase 1 (LR1) model [Iion,where 1) model to give INa, ICa and IK are time and voltage dependent currents flowing through Na+, Ca2+, and K+ channels, IK1 a time-independent K+ current, IKp a plateau K+ current, and Ib a background current. We changed two parameters from the original Luo and Rudy paper [+ conductance from 0.23 mS mm-2 to 0.16 mS mm-2 as in the later version of the model [-2 to 0.0005 mS mm-2 to produce an APD comparable to that in the canine ventricle. We controlled repolarisation by varying maximum K+ conductance (gKmax) from the default value of 0.00282 mS mm-2 to a value between 0.001 mS mm-2 and 0.004 mS mm-2 (see below).where dy paper . We reduhe model , and we Cm to 0.001 μF mm-2, and set D to between 0.05 and 0.2 mm2 ms-1. We used an adaptive timestep of either 0.02 or 0.1 ms depending on the magnitude of dVm/dt at each grid point [2 ms-1 we obtained a conduction velocity (CV) for a stable plane wave of 0.56 m s-1, with a speedup of about two times and an error in CV of 2.5 % compared to computations with a fixed timestep of 0.01 ms. Simulations with smaller fixed and adaptive timesteps yielded plane waves with a comparable CV. Changing the space step to 0.25 mm and 0.15 mm resulted in a change of CV for a plane wave of <5 % compared to the CV computed with a space step of 0.2 mm. These findings indicated the stability of our numerical method.We solved equation 1 and the LR1 equations using an explicit Euler method, with both a lookup table of the voltage dependent parameters in the LR1 model, and an adaptive operator splitting technique . We applid point . With a gKmax. In each case a propagating action potential could be elicited with a minimum diastolic interval of about 10 ms, indicating that the refractory period was very close to the APD.Figure gKmax set to 0.004 mS mm-2. The central 40 × 40 mm region was heterogeneous, and was subdivided into squares. Within alternate squares gKmax was set to either 0.004 mS mm-2 or to a specific lower value (see below). Thus the virtual tissues had a border region with short APD, and a central heterogeneous region divided into a chequerboard with alternating squares of either short or long APD of 0.003 mS mm-2. The diffusion coefficient in the whole virtual tissue was set to 0.1 mm2 ms-1.In our reference virtual tissue, the 40 × 40 mm heterogeneous region was divided into 16 squares giving heterogeneity with a spatial scale of 10 mm. In half of the 16 squares spatial scale of heterogeneity by changing the size of heterogeneity in the reference virtual tissue from 10 mm to 20 mm and 5 mm at every grid point. We estimated APD dispersion during steady pacing with three measured that have been used in experimental studies [APDdiff). Second we measured the standard deviation of APD (APDSD) across the whole virtual tissue. Finally we measured the APD difference between each grid point and its neighbours 1 mm above, below, left and right, and determined the maximum value of measurements throughout the whole virtual tissue (maxLD) [Action potentials were initiated in each virtual tissue by holding the membrane voltage along one edge at 0 mV for 2 ms. We measured APD to 90% recovery (APD studies . First w (maxLD) .gKmax supported propagating plane waves following S1 stimulation along one edge. These propagating plane waves had a depolarising wavefront and a repolarising wave back both aligned parallel to the edge that was stimulated. A premature S2 stimulus delivered to the same edge as the S1 stimulus therefore resulted either in block or in a propagating plane wave.A spatially homogenous control virtual tissue with uniform block if the S2 stimulus failed to propagate, re-entry if the S2 stimulus elicited re-entry that completed more than one cycle, wavebreak if the S2 stimulus elicited a wave that broke but did not re-enter, or propagation if the S2 stimulus elicited a wave that propagated without wavebreak.In the heterogeneous virtual tissues the wave back was not a plane wave because some regions repolarised more quickly than others. A premature S2 stimulus could produce a wavefront that would encounter a mixture of recovered and refractory tissue, and hence elicit wavebreak and re-entry. We therefore assessed vulnerability to re-entry in the heterogeneous virtual tissues by delivering two S1 stimuli to one edge at 500 ms intervals, and then a premature S2 stimulus to the same edge. We varied the timing of the S2 stimulus in steps of 1 ms. The virtual tissue response was characterised as either susceptibility to re-entry, and to the range of S2 intervals as the width of the susceptible window.Vulnerability to re-entry is typically estimated by applying a local premature stimulus that interacts with repolarising tissue, and the vulnerable window is the range of stimulus strength and timing that elicits re-entry. In this study, we investigated the initiation of re-entry by S1 S2 stimulation from the same stimulus site, and we estimated the vulnerability of each virtual tissue to re-entry from the range of S2 intervals that resulted in either wavebreak or re-entry. To avoid confusion we refer to our estimate of vulnerability as + channel and conductance of the time independent K+ channel gK1.We made a preliminary investigation into two candidate mechanisms for reducing susceptibility, inactivation of the Na+ current to support a propagating wavefront [+ channels have not recovered from inactivation, then a propagating wave blocks and dissipates [+ channel inactivation is controlled by the j-gate in the LR1 model [+ channels from inactivation throughout the virtual tissue by multiplying the time constant of Na+ channel inactivation τj by 10 [Block of an action potential occurs if there is insufficient Naavefront ; when Nassipates . Na+ chaR1 model . We prolτj by 10 .+ current iK1 is a voltage dependent current that holds the membrane at its resting potential. It is activated during repolarisation and at rest, and is also activated close to the core of re-entrant waves [iK1 throughout the virtual tissue.The time independent Knt waves ,31. We i90 in variants of the virtual tissue during pacing at a cycle length of 500 ms from the bottom edge. The three columns show the effect of changing spatial scales or re-entry (orange), and hence an increase in the width of the susceptible window The lower bound of the susceptible window where S2 was blocked depended on the refractory period of the border tissue with short APD, and was not greatly affected by changes in spatial scale, Δgkmax or strength of cell-to-cell coupling. The upper bound showed a similar trend to the measures of APD dispersion shown in Figure gkmax and weak cell-to-cell coupling associated with a wide susceptible window.Figure The virtual tissue with a spatial scale of 20 mm showed a different pattern of susceptibility compared to the others, with two ranges of S2 that initiated wavebreak and two ranges of S2 that initiated re-entry. This behaviour is illustrated in Figure APDdiff and reveals an approximately linear relationship. The correlation coefficient R2 = 0.99, which indicates a strong association between APDdiff and susceptibility. The interception of the line with the APDdiff axis also suggests that for the S1 S2 configuration used in this study, the susceptible window falls to zero for APD dispersion less than 20 ms. The association between the other measures of APD dispersion effect on maximum and minimum APD, the width of the susceptible window was decreased from 56 ms to 39 ms. The lower bound of the susceptible window moved from 198 to 216 ms, reflecting an increase in the refractory period of the virtual tissue as well as more prominent conduction velocity restitution.Although prolonging τgK1 increased current flow across the membrane during repolarisation, shortening APD by about 10% and decreasing APDdiff from 70 ms to 49 ms. The width of the susceptible window was also reduced from 56 ms to 42 ms when gK1 was doubled. The lower bound of the susceptible window moved from 198 ms to 175 ms, as a result of the shorter APD.Doubling gkmax, and strength of cell-to-cell coupling on APD dispersion and susceptibility to re-entry. Wavebreaks and re-entry can be created in cardiac tissue when an activation wavefront encounters a gradient of recovery. Experimental studies have therefore found an association between increased APD dispersion and greater susceptibility to re-entry because a premature stimulus is more likely to be blocked in tissue with regions of prolonged APD. In this study we found that large spatial scale heterogeneity, large Δgkmax, and reduced strength of cell-to-cell coupling all increased both APD dispersion and susceptibility to re-entry. Tissue heterogeneities produce APD dispersion, and APD dispersion is modulated by electrotonic current flow [gkmax affected APD dispersion directly, whereas changing the strength of cell-to-cell coupling affected electrotonic current flow. This study indicates that each of these factors could be an important component in arrhythmogenesis, and that the susceptibility of a heterogenous tissue to re-entry can be estimated from simple measures of APD dispersion.In this study we have used a computational model of cardiac tissue to dissect out the effects of spatial scale, Δent flow . In thisgkmax and strength of cell-to-cell coupling in the experimental study are difficult to establish. Nevertheless the initiation of re-entry by block and retrograde activation in the region with prolonged APD followed a broadly similar pattern to our simulations, although the activation pathways in the experimental study were more complex, presumably due to anisotropic conduction in the rabbit ventricle. A decrease in strength of cell-to-cell coupling results in slowed conduction, and this is a common finding in tissue damaged by ischaemia, infarction [Although the spatial scale of heterogeneity has been identified as potentially important in other studies , there ifarction , and othfarction . Severalfarction ,35-37.Recent computational studies have addressed the influence of heterogeneous acetylcholine distribution on the vulnerability to and stability of re-entry in the atria ,32. The The dynamical behaviour of APD is recognised as important not only for the stability of re-entry but alsoThe effect of heterogeneity on the stability of spiral waves has been investigated by Xie et al . This stNormal ventricular tissue is remarkably resistant to the initiation of re-entry, but this robustness is greatly reduced by actions that increase the spatial dispersion of refractoriness. In this computational study we have shown that regional differences in repolarisation have an interlinked effect not only on the initiation of re-entry but also on measures of APD dispersion. Measures of APD dispersion are valuable in clinical practice because they could provide an estimate of arrhythmia risk, and various indices have been developed in experimental studies . In our + channels from inactivation can be prolonged, or if the conductance of the iK1 channel can be increased. The effect of this kind of intervention in the intact heart may however be more complex. Other computational studies have shown that modifying the kinetics of the Na+ channel can have a pro-arrhythmic effect. Delaying recovery of Na+ channels from inactivation can increase the slope of the APD restitution curve and hence the likelihood of alternans and re-entry [+ channel conductance increases the vulnerable window [iK1 channel in experimental studies [iK1 channel conductance on susceptibility to re-entry investigated in the present study, it does highlight the potential importance of this channel for the mechanisms of re-entry.These preliminary investigations suggest that, in our model, susceptibility to re-entry could be reduced if recovery of Nare-entry , and rede window . Differe studies . AlthougThe influence of individual ion channel currents on the initiation and subsequent behaviour of re-entry is an important direction for future research, but will require more biophysically detailed cell models than the LR1 model used in this study.The electrical behaviour of cardiac tissue is complex, and depends on processes that act at tissue, cell, sub-cellular, and molecular levels. Computational models of electrical activation and conduction in the heart aim to simulate processes that are relevant to the research question, and simplifications are made accordingly. This study involved a large number of computations to establish susceptibility to re-entry, and so we chose to use a model that was a compromise between fidelity to real cardiac tissue and computational requirements.2+ storage and release have been developed [2+ handling may become heterogeneous in addition to APD, and so it is possible that susceptibility to re-entry could also be modified if this additional feature is taken into account.More detailed versions of the LR model and others incorporating a fuller description of ion channels, pumps, exchangers, as well as Caeveloped ,25,45,46In the present study we chose to use an idealised geometrical heterogeneity based on square regions because this approach allowed us to assess the initiation of re-entry under well controlled conditions. In real cardiac tissue we would expect the heterogeneities to be much more irregular in shape and gradient, and the conditions that favour re-entry to be dependent on the relative location of the heterogenous region and the stimulus site.The behaviour of re-entry in 3 D tissue is more complex than in 2 D, especially when the effects of rotational anisotropy and transmural differences in action potential shape and duration are taken into account . StudiesRHC conceived and designed the study, wrote the simulation code, and ran the simulations. AVH participated in the study design, and helped draft the manuscript. Both authors read and approved the final manuscript.Movie relating to Figure 5aClick here for fileMovie relating to Figure 5bClick here for fileMovie relating to Figure 5cClick here for fileMovie relating to Figure 7aClick here for fileMovie relating to Figure 7bClick here for fileMovie relating to Figure 7cClick here for file

Vif functions to counteract an anti-retroviral cellular factor in non-permissive cells named APOBEC3G. The current mechanism proposed for protection of the virus by HIV-1 Vif is to induce APOBEC3G degradation through a ubiquitination-dependent proteasomal pathway. However, a new study published in Retrovirology by Strebel and colleagues suggests that Vif-induced APOBEC3G destruction may not be required for Vif's virus-protective effect. Strebel and co-workers show that Vif and APOBEC3G can stably co-exist, and yet viruses produced under such conditions are fully infectious. This new result highlights the notion that depletion of APOBEC3G is not the sole protective mechanism of Vif and that additional mechanisms exerted by this protein can be envisioned which counteract APOBEC3G and enhance HIV infectivity.The viral infectivity factor, Vif, of human immunodeficiency virus type 1, HIV-1, has long been shown to promote viral replication Retrovirology article [In contrast to most animal viruses, infection with the human and simian immunodeficiency viruses results in prolonged, continuous viral replication in the infected host. Remarkably, viral persistence is not thwarted by the presence of apparently vigorous, virus-specific immune responses. Several factors, including the evasion of an innate cellular anti-viral defense by HIV-1 as discussed in a recent article . Interes article -5. Earli article ,6.APOBEC3G is a virion-encapsidated cellular protein that deaminates dC to dU in minus-strand viral cDNA during reverse transcription -10. The vif+ virus are not known; but, current observations are that APOBEC3G confers a major deleterious effect to the HIV-1 genome when the Vif protein is absent. Historically, Vif has been known to play a dramatically important role in HIV-1 infectivity [vif-defective virus can replicate in some permissive cells such as Jurkat and SupT1 cells, but cannot replicate in other non-permissive cells such as macrophages, primary human T cells, and some restrictive T cell lines [The effects of APOBEC3G and its G-to-A deaminase activity on the survival of wild type HIV-1 ectivity ,13. Vif ectivity -17. HIV-ll lines -20. For ll lines .Following on the heels of that initial observation, an enormous amount of effort emerged from several laboratories directed at elucidating how Vif mechanistically counteracts APOBEC3G in order to protect HIV-1 . These results strongly support a new mechanistic function of HIV-1 Vif protein, complementing the model where Vif counteracts the inhibitory effects of APOBEC3G by enhancing its degradation via ubiquitin-proteasome pathway may indicate an alternative protective mechanism used by HIV-1 to eliminate innate cellular immunity. Nevertheless, we cannot exclude that HIV-1 uses Vif to exert multiple mechanisms to synergistically and more effectively inhibit the anti-viral activity of APOBEC3G.m pNL-A1 . Even thytoplasm ,46. If AIn conclusion, the existence of two different mechanisms may represent two faces of the same coin, with the common goal of inhibiting APOBEC3G's anti-viral activity. As is often the case with new findings, new questions are posed. The link between APOBEC3G's enzymatic function, its degradation pathway, and its incorporation into virions in the presence of Vif is certainly to require additional attention. Answers to these questions are likely to keep many of us busy for the foreseeable future.None declared.The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; Vif, Viral Infectivity Factor; SIV, simian immunodeficiency virus; NC, nucleocapsid protein; APOBEC3G, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G; APOBEC3F, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3F PBMC, peripheral blood mononuclear cells; Cul5, Cullin type 5; SCF, skp1-cullin-F-box protein ligase.

PLoS Medicine, Ben Lopman and colleagues argue that although it is right to criticize the lack of evidence on unsafe medical injection, field data are hard to collect. They note that in the only published study addressing this issue, Kiwanuka and colleagues found no link between unsafe injections and HIV spread in rural Uganda. In an effort to “inform the debate” further, Lopman and colleagues looked at the association between HIV and unsafe injection practices in rural Zimbabwe.Of the 40 million people worldwide with HIV, 30 million live in the developing world. By far the worst hit region is sub-Saharan Africa, where nearly four million children have lost one or both parents to HIV/AIDS since 2000. Is heterosexual transmission the driving force behind the HIV epidemic in sub-Saharan Africa? In a controversial debate, some researchers have suggested that other factors such as unsafe medical injection practices may also be to blame, and that by overlooking, and even suppressing, analysis of this possible route of transmission, the current focus on preventing sexual transmission may be misguided. In this month&apos;s The team analyzed data from adults in Manicaland, a rural part of Zimbabwe, who were taking part in the Manicaland HIV/STD Prevention Study. In 1999 and 2000, eligible patients were tested for HIV and surveyed (86.7% were HIV negative at the start of the study), and were followed up three years later. The team collected survey data on injections in the patients, who were male and female adults aged 15 to 54 years old, and tested for an association between injection exposure and HIV infection. In 2002 and 2003, 505 of the men and 1,342 of the women, representing a 69.7% follow-up, were again interviewed and tested for HIV infection. Of these, 40% reported having had an injection or needle prick during the study period. A total of 67 patients developed HIV during the study; of these 13 (19%) said they had not had sex during the study period and 40 (60%) said they had not had an injection. The statistical analysis found no significant association between injections and HIV infection in men or women.Patients who had HIV when the study began did not have higher rates of injections. Instead, injections were highly associated with childbirth and pregnancy. But since HIV–positive women have reduced fertility, a reduction in the use of maternal services may partially explain why injections were not more common in these HIV-positive patients. In this study, the strongest predictor of HIV infection was symptoms of sexually transmitted disease.Despite problems of recall bias and under-reporting of sexual activity—a particularly difficult problem in studies in Africa—sexual behavior is consistently linked with HIV incidence. Where does this leave the debate over injections in Africa? Certainly, for this community, they do not seem to be a major source of HIV infection, and local policy-makers would therefore do best to concentrate on the prevention of sexually transmitted infections.

A 42 year-old male former semi-professional soccer player sustained a right lower extremity popliteal contusion during a soccer game. He was clinically diagnosed with a possible traumatic deep vein thrombosis (DVT), and sent for confirmatory tests. A duplex doppler ultrasound was positive for DVT, and the patient was admitted to hospital for anticoagulation . Upon discharge from hospital the patient continued oral warfarin anticoagulation (six months), and the use of compression stockings (nine months). He followed up with his family doctor at regular intervals for serial coagulation measurements, and ultrasound examinations. The patient's only identified major thrombotic risk factor was the traumatic injury. One year after the initial deep vein thrombosis (DVT) the patient returned to contact sport, however he continued to have intermittent symptoms of right lower leg pain and right knee effusion.Athletes can develop vascular injuries in a variety of contact and non-contact sports. Trauma is one of the most common causes of lower extremity deep vein thrombosis (DVT), however athletic injuries involving lower extremity traumatic DVT are seldom reported. This diagnosis and the associated risk factors must be considered during the initial physical examination. The primary method of radiological diagnosis of lower extremity DVT is a complete bilateral duplex sonography, which can be augmented by other methods such as evidence-based risk factor analysis. Antithrombotic medication is the current standard of treatment for DVT. Acute thrombolytic treatment has demonstrated an improved therapeutic efficacy, and a decrease in post-DVT symptoms.There is a lack of scientific literature concerning the return to sport protocol following a DVT event. Athletic individuals who desire to return to sport after a DVT need to be fully informed about their treatment and risk of reoccurrence, so that appropriate decisions can be made. Athletes are susceptible to a variety of vascular injuries, secondary to either repetitive motion, or high-speed collisions . The difth, 2003 in the emergency department, with the chief complaint of right leg pain. The patient had been playing soccer 10 days prior to this visit, and recalled a traumatic "tackle" injury to the posterior area of his right lower extremity. He denied experiencing any sensation of tearing or popping in the right knee during the index trauma, and was able to complete the game with only minor discomfort. On day 3 post-injury the patient noted severe pain in his knee and calf with ambulation. The patient visited his primary doctor on post-injury day 8 and was diagnosed with a right lower extremity soft tissue injury. A right lower extremity echo-doppler ultrasound (US), and a semi-quantitative D-dimer automated latex procedure were ordered to rule out a vascular disorder. The US investigation demonstrated a DVT in the distal femoral, popliteal, and distal calf veins, with a heterogenous mass without a doppler signal in the right popliteal fossa. The D-dimer result was also positive for a suspected thrombosis . The patient was instructed by his physician to proceed immediately to the emergency department for further evaluation and treatment.A 42 year old Polish born male former semi-professional soccer player was seen on May 16The past medical and family history of the patient was non-contributory for a history of thrombophilia or other thrombotic major risk factors. The patient had a remote (11 years old) surgical history of a right-sided inguinal hernia that could have created scar tissue contributing to vascular obstruction and stasis. The initial emergency department examination demonstrated an exquisitely tender right calf with a 3 cm difference in mid-calf girth ; a 1+ right knee supra patellar effusion; and a palpable popliteal mass with visible ecchymosis. Laboratory tests were negative for metabolic, hematological or familial abnormalities. A repeat US investigation confirmed the results of the previous outpatient results. The patient was anticoagulated simultaneously with unfractionated heparin and Warfarin sulfate. A multiview plain film x-ray examination of the right lower extremity demonstrated no fracture, dislocation, or bony mass.A magnetic resonance image (MRI) of the right knee was done several days after admission, to verify a torn right knee meniscal cartilage that had been previously diagnosed. The official MRI radiological report included a small free-edge tear of the posterior horn root junction of the lateral meniscus, chondromalasia , and a moderate joint effusion with a bursal cyst or dilated semimembranous-gastronemius bursa.Anticoagulation was achieved on day 6 of the patient's hospitalization. He was discharged on 5 mg of warfarin per day, with instructions to continue the use of compression stockings. The patient was also advised to follow up with his primary physician for regular monitoring, and to avoid contact or collision activities during anticoagulation.The patient was maintained on warfarin for six months, with weekly physician monitoring for the first three months post-injury. The monitoring interval was changed to once per month for the remainder of the treatment period. Hematologic investigations were obtained three months post injury. There were no contributory thrombophilic factors found in these investigations. Laboratory levels of Protein C activity 22% (range = 70–140%), Protein S activity 48% (range = 75–140%), INR 2.57 (range = 0.88–1.12), and PT 27.5 sec (range = 9.6–12.0 sec); APTT 38.5 sec (range = 23.4–35.4 sec) were found to be appropriately reactive to the anticoagulant therapy.The patient underwent two arthrocentesis procedures to remove small amounts of serous fluid from the joint, and each time was injected with a lidocaine/corticosteroid combination. US examinations after the hospitalization period failed to demonstrate a recurrence or new onset of DVT, however residual echogenic material characteristic of a chronic thrombus was demonstrated in the popliteal vein. Compression stocking use was maintained after hospital discharge, and was discontinued after nine months. The patient returned to soccer after anticoagulation, with a full understanding of his increased risk of DVT recurrence.One-year post injury the patient continued to suffer from intermittent right lower extremity discomfort and swelling often unrelated to activity. An elective arthroscopy was recently performed on the patient's right knee to investigate his long-standing meniscal disruption and effusion. The arthroscopy demonstrated several areas of arthrosis , and a torn lateral meniscus. Appropriate partial lateral menisectomy and debridement, and chondroplasty of the areas of arthrosis were preformed. An arthroscopic examination of the posterior compartment demonstrated a small cleft-like area just medial to the semimembranosis where the Baker's cyst likely originated. The patient returned to the orthopedist one week post-op with a large (150 cc's) hemarthrosis that was aspirated from the knee. He was requested to follow-up in one month for re-evaluation.This case study illustrates the importance of considering deep vein thrombosis in the diagnosis of sport-related extremity trauma. DVT is classically related to venous stasis, intimal injury, and coagulation diathesis (Virchow's triad). The estimated incidence of DVT from all causes is 0.5 to 1.6 per 1000 persons per year, and may be an underestimation due to the number of DVT that are asymptomatic .Standard risk factors for DVT are immobilization, pregnancy, recent surgery (particularly orthopedic), malignancy, older age, smoking, coagulation deficits or hypercoagulable states, connective tissue disorders, sex steroid administration, severe dehydration, and major trauma. Bates et al. presenteCoagulation diathesis through congenital or acquired thrombophilia may promote coagulation . CoagulaThe testing for hypercoagulable states in an individual after a single episode of thrombosis is a costly, yet routine procedure in many centers. The common assumption that an identified presence of a thrombophilic abnormality increases the risk of recurrence, and justifies prolonged therapy is without clear supportive evidence. A review of the current literature concerning the treatment of individuals with coagulation deficits concludes that there is no clear evidence that modifying treatment because of an identified hypercoaguable state alters the outcome, or that more intensive therapy is required in patients with laboratory evidence of thrombophillia .Exercise is thought to act as a protective mechanism against thrombosis, due to the controlled balance between the exercise activated coagulation and fibrinolytic pathways . Upper eLower extremity DVT with a traumatic sporting injury in otherwise healthy active adults is seldom mentioned in the medical literature -29. ThisThe popliteal, posterior tibial and peroneal veins are susceptible to intimal trauma by the sudden hyperextension and torsion that the lower extremity experiences in a soccer "kick" or "tackle" motion. The popliteal arteries and veins are susceptible to direct, sheering, and muscular compressive forces due to their anatomical position, especially with rapid knee hyperextension or anterior dislocation ,22.The literature demonstrates the importance and efficacy of a complete bilateral duplex sonography as the primary method of DVT diagnostic investigation . US findAnticoagulation is effective in preventing DVT propagation and PE, but has no chemical fibrinolytic activity. This type of therapy allows for intrinsic fibrinolysis to occur. Radiographically demonstrable clot lysis occurs in only 50% of anticoagulated patients, and the incidence of complete resolution is less than 5%. Intrinsic fibrinolysis that occurs slowly does not preserve the function of the venous valves, which become fibrotic and fixed after a few weeks of being trapped in clot .The symptoms experienced by individuals without complete clot resolution include heavy or achy legs, edema, throbbing paresthesia, purities, numbness, stiffness, and difficulty standing or ambulating. Postthrombotic syndrome (PTS) is characterized by brawny edema of the leg, stasis dermatitis, hyperpigmentation, induration, ulceration and chronic leg pain. This syndrome is associated with an extraordinary level of chronic pain and disability, and approximately 40% of the total cost of treating DVT is spent on PTS .Zeigler et al. investigThere is growing evidence that the early lysis provided by thrombolytic therapy is more likely to preserve valve function, decreasing the likelihood of DVT recurrence, and the occurrence of PTS ,39. ReceThe general knowledge concerning quality of life and burden of illness in patients with persistent post-DVT symptoms is limited. This issue is especially important to the athletic patient, as participation in sport is usually an extremely important component of quality of life. For routine monitoring of outcomes in chronic venous disorders there are questionnaires that are available ,41. HednThe athlete's primary concern upon the initial DVT diagnosis is return to play. The issue of return to sport after a lower extremity DVTs has only been addressed only once in the literature concerning return to non-contact sport . GeneralAn athlete who wants to return to a contact or collision sport should be informed of the possible increased risk of recurrent DVT that he or she may face, above the current estimates derived from the general population. There is no current evidence in the literature that investigates the specific risk factor of a traumatic collision, and the recurrence of a DVT. This lack of evidence suggests that the patient and physician should work together to make an informed return to play decision involving the patient's current individual risk profile, the likelihood of DVT recurrence, athletic goals, and the perceived importance of the particular sport to quality of life.The potential limitations of this case study include the lack of testing for prothrobin mutation, and fibrynolitic parameters .The author(s) declare that they have no competing interests.PE developed, researched, wrote and revised the case study; RU assisted in study development and manuscript revision; DM assisted in manuscript development and revision; HJ assisted in manuscript development and revision.

The objective of this study was to develop simultaneously a new questionnaire, the Pediatric GERD Caregiver Impact Questionnaire (PGCIQ), in American English and American Spanish in order to elucidate the impact of caring for a child with GERD.Two focus group discussions were conducted in American English and American Spanish to develop a relevant conceptual model. Focus group participants were the primary caregivers of children with GERD (newborn through 12 years of age). Participant responses were qualitatively analyzed to identify potential differences in caregiver perspectives by the caregiver's language, socio-economic status and demographic profile as well as the child's age and disease severity level. Items in the PGCIQ were generated simultaneously in English and Spanish by reviewing results of qualitative analysis from focus groups in each language. The PGCIQ was finalized in both languages after testing content validity and conducting an in-depth translatability assessment.Analysis of focus group comments resulted in the development of a first draft questionnaire consisting of 58 items in 10 domains. Content validity testing and an in-depth translatability assessment resulted in wording modification of 37 items, deletion of 14 items and the addition of a domain with five items. Feedback from the content validity testing interviews indicated that the instrument is conceptually relevant in both American English and American Spanish, clear, comprehensive and easy to complete within 10 minutes. The final version of the PGCIQ contains 49 items assessing ten domains. An optional module with nine items is available for investigative research purposes and for use only at baseline.The PGCIQ was developed using simultaneous item generation, a process that allows for consideration of concept relevance in all stages of development and in all languages being developed. The PGCIQ is the first questionnaire to document the multidimensional impact of caring for an infant or young child with GERD. Linguistic adaptation of the PGCIQ in multiple languages is ongoing. A validation study of the PGCIQ is needed to examine its psychometric properties, further refine the items and develop an appropriate scoring model. Gastric reflux of short duration is a normal physiological event for all infants less than six to seven weeks old. When gastric reflux occurs more frequently and past the age of seven weeks, it becomes a clinically significant problem that is diagnosed as pediatric gastroesophageal reflux disease (GERD). Symptoms of pediatric GERD include pain, irritability, frequent spitting-up or vomiting, constant or sudden crying, poor sleep habits and frequent waking. At present, these symptoms represent a clinically significant problem for one of every 500 infants between the ages of six weeks and 18 months .The prevalence of symptoms consistent with pediatric GERD varies with age and depends upon the type of symptoms. When referring solely to regurgitation symptoms, over 80% of children experience spontaneous remission by age 18 months . In compMany health care professionals (HCPs) specializing in the treatment of GERD feel that published prevalence rates underestimate the extent of the condition ,7. InterIn addition to having serious consequences for infants and young children, reports from parent advocacy groups suggest that untreated or ineffectively treated pediatric GERD exerts a substantial negative impact on the life of the child's primary caregiver . CaregivThe current study had two objectives related to the assessment of the impact of pediatric GERD on the caregiver's daily life. The first objective was to determine if there was an existing instrument suitable for measuring the impact of caring for an infant or child with GERD. If no instrument could be identified, the second objective was to develop an instrument suitable to quantify the impact of pediatric GERD on caregivers, thereby providing a means to improve public awareness of the issue.A focused literature review was conducted to determine if a caregiver-reported outcome measure that assesses the impact of caring for an infant or child with GERD had been developed. This review included a search of commercial and Mapi Values in-house medical databases of published literature from 1990 to the present in order to identify available instruments and studies relevant to caregiver burden in pediatric GERD. Additionally, the review examined whether generic quality of life measures had been previously applied to the assessment of the impact of caring for a pediatric GERD patient. The literature review uncovered numerous instruments developed for caregivers of adult, elderly or terminally ill patients , evaluatThe "Pediatric GERD Caregiver Impact Questionnaire" (PGCIQ) was developed in American English and American Spanish to address the need for an instrument to assess the impact of caring for a child with GERD. This instrument was developed for use in observational studies and multi-national clinical trials to systematically assess and document the physical, psychosocial, psychological and financial impact of caring for pediatric GERD patients. In addition, items in the PGCIQ were specifically developed to capture changes in caregiver burden in response to successful treatment. This instrument will allow documentation of the impact of caring for a child with GERD and provide evidence to increase public and physician awareness of the condition.The PGCIQ was developed simultaneously in American English and American Spanish to accommodate the rapidly changing population composition of the United States (U.S.). The U.S. Hispanic population grew by 61% from 1970 to 1980 and by another 53% in the following 10 years . BetweenAlthough the questionnaire was initially developed in American English and American Spanish, it was designed to be suitable for cross-cultural and linguistic adaptation into multiple languages. Thus, the PGCIQ was developed to provide valuable information in multi-national clinical evaluations regarding the value of different GERD treatments from the caregiver's perspective. Information obtained from the PGCIQ can be used to inform and educate health care providers and payers about the needs of caregivers of pediatric GERD patients.The development of the PGCIQ followed the methodology of simultaneous questionnaire development. This process was selected to reduce the risk of systematic measurement error at the item level and ensure that construct bias, which occurs when the construct is measured, was identical across all developed language versions. When combining this methodology with a translatability assessment conducted by linguistic experts, the procedure was intended to yield an "optimal" measure for adaptation of the American English version into different cultures. Additionally, the process aimed to produce a measure that was less susceptible to cultural differences than a questionnaire developed in one language and followed by translation into other languages .Participants were recruited by pediatric gastroenterologists and pediatricians. The primary caregivers included in the discussions were 18 years or older and were caring for children (newborn to 12 years of age) who were either newly diagnosed with GERD or seeking treatment for a new episode of GERD after a period without treatment. Diagnoses of GERD required that children present with common clinical manifestations of GERD. Neonates and young babies were required to have chronic regurgitation, most commonly demonstrated by vomiting. In young children, diagnosis was confirmed by physical discomfort that manifested as prolonged crying, fussing, arching, or refusal of feedings. As well, all children three months or older were required to have symptoms of GERD requiring acid suppressive therapy.Caregivers were excluded if their child had a history of acute life-threatening events due to manifestations of GERD or a severe unstable illness that could exacerbate caregiver burden. In order to optimize the relevance of the instrument across a wide range of age groups, the study authors attempted to achieve an equal distribution of children within three groups: premature infants to three months, four to 11 months and 12 months to 12 years. Across all three groups, participant eligibility was confirmed by case report forms completed by the treating physician.Four focus group discussions, two in American English and two in American Spanish, were conducted to elicit issues relevant to caring for a child with GERD. For each language group, discussions were conducted on the East and West Coasts to ensure adequate representation of Mexicans, Puerto Ricans and Cubans, the three largest groups of Spanish-speaking Americans currently residing in the US .Each two-hour focus group was conducted using a structured focus group discussion guide that was developed in American English and linguistically adapted into American Spanish. American English and American Spanish focus groups were facilitated by two female researchers both living in the U.S. The native Spanish speaker was from Ecuador. Focus group moderators received identical, in-depth training from Mapi Values, and all focus groups were videotaped to ensure adherence to the discussion guide. Caregivers were asked to discuss how caring for their child has impacted their lives in the following areas: daily activities, social and family life, emotional and physical functioning and financial well-being. At the start of each focus group discussion, caregivers completed a 48-item GERD Caregiver Informational Survey, developed for this study, which contained questions about the caregiver's and child's demographic, socioeconomic and clinical profile.Qualitative analysis of the focus group discussions was conducted separately for each language group. This analysis included a comprehensive review of verbatim transcripts by native speaking researchers who also conducted the focus group discussions. Participants' comments were coded to highlight key concepts and psychosocial correlates. Coded comments were subsequently grouped together to elicit the domains and issues important to caregivers. These domains and correlates provided the framework of the conceptual model for questionnaire development Figure .Following creation of a conceptual model, global concepts within each language group were compared to evaluate similarities and differences in caregiver perspectives. In addition, responses were evaluated to identify potential differences by the child's developmental stage, severity of disease, socio-economic status and demographic profile. Participants' verbatim quotes from each language group were then consolidated within the common domains.Items were simultaneously generated in American English and American Spanish after consolidating caregivers' verbatim comments. For each item, American English and American Spanish speaking interviewers discussed relevant concepts identified from the focus group discussions and agreed upon conceptually equivalent wording. This process ensured that each item in the questionnaire was pertinent to caregivers in both language groups and formed the basis for Version 1 of the questionnaire.A panel of linguistic experts conducted a translatability assessment of the PGCIQ to finalize Version 1 of the PGCIQ. The questionnaire was evaluated for its cultural adaptability and translatability in all potential target languages to ensure the cultural equivalence of items. Additionally, this process was used to limit the threat of bias in all current and potential target languages. Items identified as irrelevant for future target languages were considered for deletion or modification in the English and Spanish versions.Content validity interviews were conducted by the native speaking researchers who also conducted the focus group discussions. Researchers interviewed caregivers to assess the ease of comprehension, clarity, cultural equivalence and relevance of the first version of the PGCIQ. Participants were recruited following the same methods and criteria as for the focus group discussions. The interviews aimed to assess the clarity, comprehension and appropriateness of all instructions, questionnaire items and response scales.Interviews were transcribed verbatim in the participants' native language. Each transcript was comprehensively reviewed, analyzed and coded to highlight caregiver impressions of Version 1 of the PGCIQ. Coded comments were subsequently grouped together to elicit the caregivers' feedback by item. Items were considered for modification if more than one caregiver in either language had difficulties with or suggestions for the item.The interview results from each language group were analyzed separately and later compared for potential differences. In addition, caregiver responses were evaluated for potential differences by the child's developmental stage, severity of disease, socio-economic status and demographic profile. If an item was identified during content validity testing as potentially problematic, the item was reviewed and simultaneously modified by interviewers in both languages. If an item was problematic in at least one of the languages, the interviewers reviewed the caregivers' verbatim suggestions and attempted to agree upon a conceptually equivalent modification. If no suitable wording could capture the same concept in both languages, the item was deleted from the questionnaire.The final version of the questionnaire aimed to be relevant to caregivers of pediatric GERD patients, conceptually equivalent in American English and American Spanish, free from bias or jargon and easy to understand, interpret and complete within 10–15 minutes. The final version of the questionnaire was also developed to be appropriate for administration for a fifth grade literacy level, as confirmed by a Flesch-Kincaid Grade level test and lingTwenty-seven caregivers, 12 American English-speaking and 15 American Spanish-speaking, participated in the focus group discussions. The final distribution of interview participants by geographic region was East Coast English (n = 7), West Coast English (n = 6), East Coast Spanish (n = 5) and West Coast Spanish (n = 9). The average age of focus group participants was 37 years of age with a range of 18 to 64 years of age. The participants were predominantly female (89%), married (81%), living with their spouse and children (89%) and recipients of a high school diploma (81%). The fact that participants were primarily female reflects the composition of the population of primary caregivers seen by the investigators (referring physicians). Moreover, the study investigators attributed the fewer numbers of single and divorced caregivers in the discussion groups to difficulties in taking time off work. Analysis of the caregiver characteristics also revealed differences by language group. Relative to Spanish-speaking participants, a greater proportion of English-speaking respondents were female (100% vs. 80%) and married (100% vs. 67%). Table The children in this study were primarily female (52%) and their average age was 2.8 years, with a range of six months to eleven years. The average age was determined after excluding an outlier (12 year-olds) from the calculation. In terms of the target age groups, 8% of the sample population was premature up to three month-old infants; 33% were four month to 12 month-old infants; and 55% were 12 month to 12 year-old children. In total, 81% of the children were three years-old or younger, with only five of the children over the age of three. The majority of this sample consisted of children under the age of three years. The age range of this population was consistent with the literature which indicated that the prevalence of GERD symptoms significantly declines with age, with less than 30% of GERD cases persisting past three years of age .According to caregivers' ratings, their children were primarily in very good (26%), good (26%) or fair health (26%). Analysis of additional clinical measures revealed significant differences in the children's health by language group, with Spanish-speakers reporting more hours of fussiness per day, emergency room visits and hospitalizations. Table Qualitative analysis of the focus group discussion results revealed nine key concepts relevant to caregivers of pediatric GERD patients: experiences related to GERD diagnosis, taking care of the child, daily activities, emotional well-being, household expenses, physical health, social life, relationships, and employment prior to and after GERD diagnosis. Each key concept is discussed in the following sections.Caregivers recalled feelings of fear, helplessness and guilt associated with the onset of their children's GERD symptoms. Many of these feelings were discussed in the context of negative interactions with HCPs. Caregivers shared stories about their struggle to get doctor appointments or referrals and being told that their children's symptoms were "normal" or that they were "overreacting" or "paranoid." These experiences appeared to be particularly stressful for the Spanish-speaking caregivers. In several cases, Spanish-speaking caregivers stayed with their physicians even though they received inadequate medical attention due to their lack of comfort with the U.S. health system. Thus, their children tended to be diagnosed at an older age than children of English-speakers resulting in elevated feelings of fear, helplessness and guilt. Although caregivers stressed the trauma they experienced in order to obtain their child's diagnosis, this experience did not appear to be change once the child had received an accurate GERD diagnosis. Therefore, this domain will most likely not be affected by treatment and therefore, has been included as optional.Caregivers discussed four unique parenting issues related to taking care of a child with GERD: using special feeding techniques, putting the child to bed safely, disciplining the child and finding a reliable childcare provider. First, caregivers of children under four years of age discussed the need to use special feeding techniques. These caregivers commented that they needed to feed their children "small portions," feed them "constantly" and prepare special meals or formulas. Second, caregivers of infants reported adopting unique bedtime routines. These caregivers used equipment such as wedges, car seats, pillows and monitors to put their children to bed safely. Third, caregivers of children over the age of four cited discipline as a challenging issue. Several caregivers recalled emotional tales of disciplining their children for throwing up "on purpose," while others feared that their children would spit up every time they were "yelled at" or "punished." Finally, virtually all of the caregivers cited childcare as a major concern. Most caregivers were unwilling or unable to find childcare providers capable of handling their children's special needs. Even those caregivers who found suitable care providers experienced a sense of distress due to concerns that others would not care for their child adequately. For example, one caregiver stated "No one else is going to give your child the attention [he/she] needs".The caregivers cited three daily activities that were significantly impaired by their children's GERD: mealtimes, housework and bathing. Concerning mealtimes, the caregivers shared stories about spending time "preparing multiple meals" for the family , lacking time to "sit down and eat" and having to eat cereal, frozen meals or take-out for dinner multiple evenings in a row. With regard to housework, the caregivers shared anecdotes about having to do laundry "every day," "constantly cleaning the carpet" and putting plastic covers over the furniture to protect it from their children's spit-up or vomit. Finally, the caregivers recalled stories about not having time to bathe, struggling to shower while holding the child and feeling too tired to get dressed. Limitations in these three activities appeared to be most pertinent among caregivers of infants and caregivers of children with more severe GERD symptoms.Caring for a child with GERD had a major impact on the caregivers' emotional well-being. The caregivers mentioned a myriad of feelings including fear, worry, grief and depression. The lingering feelings of fear experienced by caregivers most frequently pertained to the possibility that their children might choke and stop breathing. These feelings of fear were reported primarily by caregivers of infants. Feelings of worry were experienced by caregivers of all age groups and predominantly focused on the child's "failure to thrive." These feelings of worry were particularly burdensome for caregivers of children with severe GERD who experienced substantial weight loss and significant developmental delays.Two specific differences between language groups were found in this domain. English-speaking caregivers mentioned feelings of guilt for "disliking" or "not wanting" their children and feelings of envy upon seeing other "healthy babies." In contrast, Spanish-speakers explained that it was a "given" that caregivers would be close to their children and that they did not experience the feeling of "disliking" their child.Caregivers discussed incurring additional expenses for a variety of products and services related to caring for their children including doctor/hospital bills, special infant formula(s), medicine, laundry, childcare, new or replaced furniture, new or replaced carpets, cleaning supplies and special equipment designed for managing GERD . During the focus group discussions, the majority of caregivers spontaneously provided information related to their employment and income level when discussing the financial impact of caring for their children. Where possible, this information was noted and recorded by the moderators for analysis. Additionally, the caregivers were asked information about their insurance coverage and typical expenses in the Caregiver Informational Survey. Analysis of these data indicated that the caregivers' expenses did not differ by their socio-economic status or language group. However, caregivers from lower income households appeared to be more greatly impacted by their additional household expenses and reported greater financial strain.The majority of caregivers indicated that their physical health had declined since their children first exhibited symptoms. Common terms used by the caregivers to describe the state of their physical health were "tired," "exhausted," "stressed out" and "distracted." In addition, the caregivers shared numerous stories about carrying their infants all day, sleeping in shifts with their spouses and developing physical ailments such as migraines and skin conditions due to the strain of caring for a child with GERD. These physical health challenges seemed to be particularly burdensome for caregivers of children with more severe cases of GERD.Caregivers reported reduced social interactions outside of the home and increased social isolation due to their children's GERD. Numerous caregivers commented that they preferred to "stay at home" due to the embarrassment of dealing with their children's constant "vomiting" and "screaming" in public and the emotional strain of seeing healthy babies of the same age as their children. For several caregivers, social isolation was exacerbated by difficulties experienced while talking on the telephone due to their children's "vomiting", "screaming" and need for attention. Issues of social isolation appeared to be most relevant for caregivers of younger infants and caregivers of children with more severe symptoms.Caregivers described how caring for their child's GERD affected their relationships with their spouse/partner, relatives, other child(ren) in the family, and their child with GERD. Discussions about the caregiver's relationship with their spouse/partner focused upon the tension experienced between the couple jointly caring for a child with GERD. Tension was often attributed to disagreements over the child's medical care, reduced personal time, reduced desire for physical intimacy and decreased communication. Comments about their relationships with relatives emphasized feelings of frustration with family members who "just don't get it," "complain so much" and "always judge." Remarks about their relationships with other children in the family reflected reduced time to dedicate to the siblings of the GERD patient. Some caregivers confessed that due to the attention required by their child with GERD, they were "less patient," more "demanding" and more "neglecting" of their other children. Finally, stories about the impact on the bond between the child with GERD and the primary caregiver reflected a dual-directional relationship. In some cases, the child's illness seemed to weaken the bond with the primary caregiver, whereas the illness appeared to strengthen the bond in other instances. No major differences were revealed in the impact on these four relationships by language, age, socio-economic or symptom severity group.The impact of caring for a child with GERD on an individual's employment was primarily dependent on the caregiver's employment situation prior to the child's diagnosis. The caregivers who were previously employed mentioned a number of ways that caring for a child with GERD affected their paid employment including: changes in work schedules, ability to take enough time off to care for their child, reduced productivity and changes in long-term career goals. In order to provide sufficient care for their children, many of the caregivers changed their work schedule by leaving their jobs altogether, switching jobs or cutting back their hours. Moreover, several caregivers took more paid or unpaid vacation time in order to take their children to doctors' appointments, share the burden of care with their spouses or provide more attention to their children. Employed caregivers also described indirect costs in terms of reduced productivity and inability to concentrate at work. Many of those caregivers who were not employed prior to the child's diagnosis reported altering or sacrificing their career plans to ensure they had enough time to devote to the child with GERD. The aforementioned employment issues appeared to be relevant to caregivers across language groups, age groups and symptom severity levels. Overall, however, the employment issues appeared to be especially pertinent for individuals from low-income households, many of whom worked in hourly rather than salaried positions.Item generation based on nine identified areas and the caregivers' verbatim responses resulted in 10 domains with a total of 58 items that were simultaneously generated in American English and American Spanish. The first draft assessed the following areas: experiences related to diagnosis, caring for the child, daily activities, emotional well-being, physical health, social life, relationships, household expenses and employment prior to onset of GERD and current employment.The domain assessing the caregivers experiences related to diagnosis was developed for baseline use only. This domain contains six items with "yes" and "no" response options and has a recall period of present time. Issues about employment were divided into two domains pertaining to the time before the child's diagnosis as well as the current time period in order to quantify how the direct and indirect costs associated with caring for a child with GERD change in response to treatment.With the exception of four domains , all questions in the remaining domains have ordinal scales evaluating intensity and frequency. These questions are phrased in the first person, for example: "During the last two weeks, I needed to prepare special meals or formulas for my child." The response format, a 5-point Likert scale was chosen, as the upper limit of an individual's capacity for discriminate judgments has been shown to be near seven (plus or minus two) . PreviouThe nine core domains and one optional domain in the first version of the PGCIQ were selected to correspond with the key themes identified from the focus group discussions. To ensure cultural equivalence of items, only those concepts that had been identified in both language groups served as the basis for generating items. Considering that 80% of caregivers had children under the age of three, item generation was predominantly based upon verbatim comments elicited by caregivers of infants and young children.In the item generation focus group discussions, over 80% of the caregivers had children aged three years or younger. Specific concepts that were noted in the focus group discussions as relevant to caregivers of older children and adolescents, but not to caregivers of infants or toddlers, included discipline issues, concern about the child's emotional well-being and concern about the child's academic development and self-esteem. In contrast, concepts that were relevant to caregivers of infants and toddlers, but not to caregivers of older children or adolescents, included the need to feed the child frequently, feed the child small portions and spend a significant amount of time feeding the child. In general, infants and toddlers also seemed to require more constant one-on-one attention, thereby exerting a greater impact on the caregivers' daily activities, social functioning and family lives.The PGCIQ was formulated in caregivers with a range of socio-economic and educational backgrounds. In general, all domains were relevant to caregivers across socio-demographic groups. However, the "employment" and "household expenses" domains appeared more relevant to caregivers of lower economic and educational status due to a higher prevalence of hourly wage earners. Employees working for hourly wages reported greater difficulties getting time off from work to care for their child, less coworker understanding, more financial strain and more pressure to remain in positions they did not enjoy than employees who were salaried. Those in lower income households also struggled more with additional household expenses due to the child's GERD and were more likely to report they could not afford products or services to improve their children's health.The focus group discussions were conducted in American English and American Spanish. All domains appeared to be relevant to both language groups. Specific issues that were more relevant to the English-speaking groups included experiencing feelings of "envy," feelings of guilt for "disliking the child" and feeling "bonded with the child with GERD." In contrast, employment challenges and issues of tension, reduced intimacy and communication in the partner relationship appeared to be more relevant to the Spanish-speaking groups. Relative to the English-speakers, the Spanish-speakers also voiced the following unique challenges: more financial strain, more pressure to be an "ideal" parent, more difficulty getting attention from English-speaking doctors, less understanding from co-workers and less access to information about GERD.The results of the in-depth translatability assessment revealed that 16 of 58 items were potentially problematic for future adaptation of the PGCIQ into other languages. These 16 items were reviewed by native speakers. Four of the items were deleted and 12 of the items were modified to improve the cultural adaptability of the PGCIQ. The modified items were flagged for further testing in content validity interviews.Version 1 of PGCIQ was tested in twenty caregivers, 10 English-speaking and 10 Spanish-speaking, who agreed to participate in content validity testing interviews. The final distribution of interview participants by geographic region was East Coast English (n = 4), West Coast English (n = 6), East Coast Spanish (n = 3) and West Coast Spanish (n = 7). The characteristics of the interview population were similar to those of the focus group population. The average age of the participants was 38 years with a range from 24 to 79 years. The majority of the participants were female (70%), married (75%), living with their spouse and children (70%) and recipients of a high school degree (75%). These characteristics resemble those of the focus group population and favor the experiences of married females. Table The distribution of children was 45% female and 40% male. An additional 15% of the gender data was missing and is not included in these figures. The average age of the population was 2.3 years, with a range from 0.33 to 10.5 years. As noted, the majority of comments used for item generation of Version 1 pertained to infants and toddlers under the age of three years. Thus, an attempt was made to test the relevance of the PGCIQ in caregivers of older children during the content validity interviews. Despite attempts to recruit caregivers of older children, the final age distribution was skewed towards children 12 months-old and younger. Premature through three month-old infants composed 17%, four month-old to 12 month-old infants composed 56% and 12 month-old through 12 year-old children composed 28% of the final distribution. Similar to the age distribution of the focus group discussions, 85% of the interview participants cared for children through the age of three, with only three of the 20 caregivers having children over three years. Table Overall, the respondents had positive impressions of the PGCIQ and described it as "easy to understand" and "easy to answer." The respondents were also satisfied with the recall period, length and the format of the questionnaire. The average time of completion was eight minutes, with a range from five to 15 minutes.The caregivers suggested a number of revisions to improve clarity. These suggestions, combined with the results of the in-depth translatability assessment, resulted in wording modification of 25 items and deletion of 10 items. The majority of wording modifications were slight changes to enhance the clarity of the items. For example, one item "During the last two weeks, I was limited in preparing meals" was modified to "During the last two weeks, I was limited in preparing meals for my family."Additionally, several caregivers suggested including a new domain to capture their relationships with family members. In response to this feedback, a new domain, "Your Relationship with Your Family Members," was added. Items in this section used the same wording as the items in the "Your Relationship with Your Partner" section, with the word "partner" replaced by "family member(s)."The version of the PGCIQ created after content validity testing contains 49 items assessing 10 core domains: "Taking Care of Your Child," "Your Daily Activities," "Your Emotional Well-Being," "Your Household Expenses," "Your Physical Health," "Your Social Life," "Your Relationship with Your Partner," "Your Relationship with Your Family Members," "Your Employment Prior to Caring for Your Child with GERD," and "Your Current Employment." An additional, optional module with nine items, "Your Experiences Related to Diagnosis," is available for investigative research purposes and for use only at baseline.The following sections present considerations for the PGCIQ's use and potential limitations of the study. The following topics are addressed: "Your Relationship with Your Family Members"; symptom severity level; child's age and caregiver characteristics.The new domain, "Your Relationship with Your Family Members," was added following content validity testing interviews based on feedback from participants that additional questions about relationships with family members would provide valuable information. This new domain has not been tested with the wording "family members." As this is a significant change that has not been tested, this domain must be scrutinized during the linguistic validation and psychometric testing process.The PGCIQ was generated utilizing data from caregivers of children with a range of severity levels. Children with more severe GERD or significant co-morbidities tended to increase the impact upon the primary caregiver across all nine domains. Conversely, a longer time period since diagnosis tended to reduce the impact on the caregiver. The reduction in impact over time appeared to be the result of two factors. First, many children eventually received successful treatment, which alleviated the GERD symptoms and reduced the strain on the caregiver. Second, the caregivers learned to accept and adapt to their children's condition with time.Given the age distribution of the study population, the PGCIQ is currently recommended for use in caregivers of pediatric GERD patients, newborn through three years old. Despite attempts to recruit across three age groups, ranging from newborn to 12 years of age, 80% of the focus group participants and 85% of the interview participants cared for a child aged newborn through three years of age. Although these age distributions were consistent with findings in the literature that approximately 70% of GERD cases spontaneously remit after age three, we felt that the majority of data favored infants and toddlers. Further testing of this instrument is therefore recommended in order to consider its use in populations of caregivers of children over the age of three. Researchers interested in extending the PGCIQ to children over the age of three are encouraged to consider the issues relevant to a child's age presented in the results section.The focus group discussion and content validity participants were primarily married women. From interviews with physicians, it was found that the majority of caregivers who seek treatment for pediatric GERD patients are women. Even though this instrument has been developed in a primarily female population, the identified caregiver issues appear to span gender roles. The men who participated in the focus group discussions were a small, but vocal minority, who elicited the same core domains as the female participants. Particular items of concern when using the questionnaire in men may be those related to domestic daily activities, given that these items tap more stereotypically female responsibilities. However, if the man is the primary caregiver, then it would stand to reason that he would be responsible for domestic daily activities. Future studies should seek to test the validity of the PGCIQ in larger populations of men and women to ensure that the items are equally relevant across gender roles.The PGCIQ has been developed in American English and American Spanish following a rigorous methodology of simultaneous item development for use in observational studies and multi-national clinical trials. The instrument is being linguistically validated from the American English version into several other languages following the appropriate methodology. After linguistic validation and psychometric testing is complete, a theoretical scoring model can be developed, and the PGCIQ may be used in international studies.Considering the age composition of the focus group and content validity interview population, the PGCIQ is currently recommended for use only in caregivers of pediatric GERD patients, newborn through three years old. Further testing of this instrument is recommended in order to consider its use in populations of caregivers of children over the age of three. Additionally, the study population was predominantly female, married and of moderate to high educational status. However, analysis of the caregivers' verbatim comments suggested that the nine domains were equally relevant to men and women, married and unmarried participants and caregivers of different educational levels. Future tests of the PGCIQ should include large samples of men and women of different marital and educational status to confirm the relevance of these domains.The PGCIQ is the first questionnaire to document the multi-dimensional impact of caring for an infant or young child with GERD, making it a valuable tool to gather information and quantify the relevant issues and impacts experienced by caregivers of pediatric GERD patients. Information gathered from this tool can be used to inform and educate physicians, managed health care organizations, pharmacists and other health care providers about the needs of caregivers. In addition, items in the PGCIQ were specifically developed to capture changes in caregiver impact in response to successful treatment. Because caregivers mentioned all nine domains as affected by their child's GERD, we conjecture that intervention effects would be associated with changed scores in all nine domains. Thus, the PGCIQ may provide valuable information in multi-national clinical evaluations regarding the value of different GERD treatments from the caregivers' perspective.This simultaneously generated patient-reported outcome instrument is one of the few examples of simultaneous development identified in the literature. This methodology results in a measure with a reduced risk of factors that might otherwise threaten the successful cross-cultural adaptation of the instrument, making the PGCIQ an appropriate instrument for adaptation into multiple languages.It is important for the PGCIQ to undergo psychometric testing to develop a final scoring model before the instrument can be used to assess the impact of pediatric GERD on caregivers. Academics and researchers interested in obtaining a copy of the PGCIQ should contact the lead author.JK Participated in the study design and coordinationDLK participated in the design, coordination and analysis and its interpretationSB participated in the field work and coordination and analysis and interpretationJAC conceived of the study and participated in its design

Virology 77:179–190, 2003). Here we evaluate the impact of these vaccines on oral transmission and evolution of SIV envelope variants.Oral infection of infant macaques with simian immunodeficiency virus (SIV) is a useful animal model to test interventions to reduce postnatal HIV transmission via breast-feeding. We previously demonstrated that immunization of infant rhesus macaques with either modified vaccinia virus Ankara (MVA) expressing SIV Gag, Pol and Env, or live-attenuated SIVmac1A11 resulted in lower viremia and longer survival compared to unimmunized controls after oral challenge with virulent SIVmac251 (Van Rompay env) region revealed two major env variants in the uncloned SIVmac251 inoculum. Plasma sampled from all infants 1 week after challenge contained heterogeneous SIV env populations including one or both of the most common env variants in the virus inoculum; no consistent differences in patterns of env variants were found between vaccinated and unvaccinated infants. However, SIV env variant populations diverged in most vaccinated monkeys 3 to 5 months after challenge, in association with the development of neutralizing antibodies.Limiting dilution analysis of SIV RNA followed by heteroduplex mobility assays of the V1–V2 envelope (These patterns of viral envelope diversity, immune responses and disease course in SIV-infected infant macaques are similar to observations in HIV-infected children, and underscore the relevance of this pediatric animal model. The results also support the concept that neonatal immunization with HIV vaccines might modulate disease progression in infants infected with HIV by breast-feeding. The continued need for breast-feeding in developing countries due to nutritional or socio-economic reasons poses a considerable risk for postnatal mother-to-child transmission of HIV, and breastfeeding is estimated to account for 33–50% of infant HIV infections worldwide -5. This Advances in the understanding of the mechanisms of oral transmission of HIV variants may aid the development of an effective infant HIV-1 vaccine. Recent studies have demonstrated that infants of HIV-infected women can be infected with single or multiple HIV variants ,7 shortlLongitudinal studies of HIV-infected adults have shown that the rate of disease progression is inversely related to the rate of evolution of HIV envelope quasispecies ,9. Also,env) region of SIV variants present in the SIVmac251 virus inoculum and compare the transmission and evolution of the SIV env quasispecies in plasma following oral inoculation of these vaccinated and unvaccinated infant macaques. Three major questions were addressed: (i) Compared to the SIVmac251 virus inoculum, are few SIV envelope variants transmitted orally?, (ii) Is the lower viremia and better clinical outcome of vaccinated infants related to the initial genetic diversity of SIV env quasispecies?, and, (iii) Is the evolution of SIV envelope quasispecies during the course of infection associated with the development of SIV neutralizing antibody? We demonstrate that while the vaccines did not modulate oral transmission of viral variants, an association was found between vaccine-induced enhanced antiviral immune responses, increased env diversity, and a slower disease course. These findings in vaccinated infant macaques are similar to observations in HIV-infected children with slow disease progression and support the relevance of the SIV infant macaque model for developing neonatal vaccine strategies to prevent pediatric HIV infection and AIDS.Simian immunodeficiency virus (SIV) infection of infant macaques is a useful and relevant animal model of pediatric HIV infection for rapidly testing the efficacy of pediatric HIV vaccine and drug interventions -20. Thisenv variants. To determine the most common variant(s) in the virus stock, six independent serial dilution experiments were conducted. Viral RNA was isolated from 1 ml of virus stock and 10-fold dilution series (undiluted to 10-9) of the RNA were prepared from 6 separate aliquots of virus stock. The resulting RNA was analyzed by RT-PCR and HMA. Figure -5 or 10-6 dilutions of viral RNA indicates that the undiluted SIVmac251-5/98 stock contains multiple env variants at high frequency. An RT-PCR endpoint (i.e. dilution to a single variant) was not reached in 3 of the 6 dilution experiments. An example of this is shown in dilution series A that migrated to two different positions on the HMA gels. Based on these positions, the homoduplex bands that migrated furthest were referred to as VSEV-1 and the variants that migrated a shorter distance were designated VSEV-2 had 10- to 100-fold lower virus levels one week after SIVmac251 challenge than all but one infant (31378) infected with more heterogeneous populations of SIV variants to VSEV-1 and VSEV-2, respectively (data not shown). The most common variants by end-point dilution in the 11 monkeys with transmission pattern A and A/C were similar to VSEV-1 , or VSEV-2 or both .env populations in plasma of the monkeys during the course of infection Fig. . Resultsenv quasispecies in plasma varied among animals at the first sample as indicated by the gel banding pattern for all unvaccinated animals (Group 1) and for 7 of the 12 vaccinated animals was observed by 3 to 5 months of infection in 4 of the 8 vaccinated monkeys that were still relatively healthy at 28 weeks , we also observed little change or a decrease of genetic divergence of plasma env quasispecies was evaluated by measuring levels of plasma antibodies that neutralized the homologous challenge virus, SIVmac251-5/98, during the course of infection of initial infection were not potent enough to modulate the variant transmission patterns.Neither of the SIV vaccines used in this experiment (MVA-SIVgpe and SIVmac1A11), nor the presence of maternal antibodies in one of the MVA-SIVgpe immunized groups altered which envelope variants were transmitted because in each group, some monkeys became infected with more heterogeneous and others with more homogeneous virus populations. It is possible that neither MVA-SIVgpe nor SIVmac1A11 elicited immune responses that effectively targeted the predominant SIV env variants tended to be lower one week after oral inoculation with SIVmac251-5/98. The higher initial virus levels in infants infected with multiple variants may reflect higher replication capacities of diverse variant populations compared to those comprised of one main variant, especially in the initial target cells during the first days of infection. We have observed this previously for adult macaques inoculated intravaginally , where N is the number of partitions in a lane, and P(i) is the fraction of the total signal in partition i. The maximum possible entropy is ln(N), and we defined the normalized entropy as S/ln(N). The normalized entropy has a range of 0 to 1, where 0 reflects no diversity , and 1 reflects maximum entropy, in which the signal is evenly distributed throughout all partitions in the lane. Thus, entropy is large for lanes with many, closely spaced or overlapping bands and small for lanes with only one band or a few, narrow bands.The quasispecies diversity for each sample was estimated by calculating a normalized Shannon entropy, a measure of the breadth or spread of the signal distribution in each HMA gel lane, using the HDent program for each HMA gel lane using the HDent program. The MMS is a measure of the midpoint of the total signal in an HMA gel lane that has values between 0 and 1, where 0 corresponds to the bottom of an HMA gel lane (i.e. nearest homoduplex bands) and 1 corresponds to the top of the lane. Thus, a MMS score of 1 reflects maximum sequence diversity ; a MMS value of 0 reflects maximum sequence similarity (> 98%) where all signal for a lane is in homoduplex bands and there are no visible heteroduplexes.Shannon entropy estimates quasispecies genetic diversity by measuring the pattern of SIV V1–V2 strands ,48,49. T® DNA mini kit, QIAgen, Valencia, CA) was used to screen for the presence of the rhesus macaque major histocompatibility complex (MHC) class I alleles Mamu A*01 and Mamu B*01, using a PCR-based technique [DNA extracted from lymphoid cells . For all statistical comparisons, a P value less than 0.05 was considered significant.Fisher's exact test, performed with Instat v. 2.03 , was used to evaluate possible association of SIV The author(s) declare they have no competing interests.JG carried out the HMA studies and drafted the manuscript; KVR participated in the design and coordination of the study, acquisition and analysis of data, and helped draft the manuscript; DM analyzed and interpreted all neutralizing antibody assays; PE and BM designed and provided the MVA constructs, participated in the experimental design and manuscript writing; MM designed and coordinated the study, assisted in the data analyses and helped draft the manuscript.

Although soy protein and its isoflavones have been reported to reduce the risk of osteoporosis in peri- and post-menopausal women, most of these studies are of short duration (i.e. six months). The objective of this study was to examine if one year consumption of soy-containing foods (providing 25 g protein and 60 mg isoflavones) exerts beneficial effects on bone in postmenopausal women.Eighty-seven eligible postmenopausal women were randomly assigned to consume soy or control foods daily for one year. Bone mineral density (BMD) and bone mineral content (BMC) of the whole body, lumbar (L1-L4), and total hip were measured using dual energy x-ray absorptiometry at baseline and after one year. Blood and urine markers of bone metabolism were also assessed.Sixty-two subjects completed the one-year long study. Whole body and lumbar BMD and BMC were significantly decreased in both the soy and control groups. However, there were no significant changes in total hip BMD and BMC irrespective of treatment. Both treatments positively affected markers of bone formation as indicated by increased serum bone-specific alkaline phosphatase (BSAP) activity, insulin-like growth factor-I (IGF-I), and osteocalcin . Neither of the protein supplements had any effect on urinary deoxypyridinoline excretion, a marker of bone resorption.Our findings suggest that although one year supplementation of 25 g protein per se positively modulated markers of bone formation, this amount of protein was unable to prevent lumbar and whole body bone loss in postmenopausal women. It is estimated by the year 2010, 35 million women in the United States either will have osteoporosis or be at risk of developing the disease if appropriate preventive measures are not taken -13. Soy Epidemiological data suggest that populations with high intakes of soy, i.e. Asians, have a lower incidence of osteoporotic fractures ,17. AsiaFrom the research point of view, there are a number of animal studies which have shown that soy protein and/or its isoflavones positively influence bone mineral density (BMD) ,20-26. IThe purpose of the present study was to examine the effects of one-year supplementation of soy-based products containing 25 g protein and 60 mg isoflavones on BMD, bone mineral content (BMC), serum and urinary markers of bone turnover in postmenopausal women. The rationale for choosing this amount of soy protein was based on the average intake of Asians ,19 and tPostmenopausal women younger than 65 years who were not on HRT or any prescription medications or herbal supplements, including soy isoflavones, known to positively influence bone were recruited. Women with cancer, liver disease, hypo- or hyperthyroidism, gastrointestinal disorders, insulin-dependent diabetes mellitus, pelvic inflammatory disease, and endometrial polyps were excluded from the study. The study protocol was approved by the Institutional Review Board at Oklahoma State University. Subjects signed a consent form after being provided with oral and written descriptions of the study. A complete medical history was obtained from all subjects before initiating the treatments. Subjects were also given routine physical and gynecological examinations. Subjects were independent living and were advised to maintain their usual physical activity.Eighty-seven eligible postmenopausal women were randomly assigned to one of two dietary treatments in a double-blind parallel study. The dietary treatments consisted of 25 g protein from soy products or comparative control. The test foods were in the form of a snack bar, drink mix or cereal and were consumed daily for a period of one year. The soy products were soy protein-based and delivered 60 mg isoflavones per day whereas the control regimen was devoid of soy protein and isoflavones.To ensure double-blinding, the study participants were randomly assigned to one of the two treatments and the study supplies were provided to the study participants in unlabeled packages. Additionally, the identity of each treatment was revealed to the investigators and research personnel involved in the collection and analyses of the data only after all analyses were completed. For this study compliance was measured in two forms. First, study participants were provided with customized calendars for subjects to record how much of each of the cereal, the snack bar, or the drink mix they consumed, if any. Second, study participants were asked to return any unconsumed foods to the study site so they could be tallied. The study participants were advised by a registered dietitian to make appropriate adjustments in their daily food consumption to account for the additional energy and nutrients supplied by the treatment regimen.For each subject, medical and nutrition histories were obtained at the beginning of the study. One-week food frequency questionnaires were completed via interview by a registered dietitian at the beginning and at the end of the study. Nutrient analysis was performed using food analysis software . Anthropometric data were collected at the beginning, six months, and at the end of the study by a single trained staff member, as described elsewhere . Height Bone density was assessed at the beginning and at the end of treatment using dual energy x-ray absorptiometry equipped with appropriate software for whole body BMD and BMC. Additionally, select regional sites, i.e. total hip and lumbar spine (L1-L4) were analyzed using high resolution software. The intra- and inter- assay coefficients of variations were 3.4% and 5.1% and 2.5% and 4.7% for BMC and BMD, respectively.Study participants were provided with routine physical and gynecological exams including a pap smear at baseline and at the end of the study. A venous blood sample was obtained after an overnight fast from each subject at the beginning, six months, and at the end of the study for various analyses. Blood samples were centrifuged at 2500 × g for 15 min at 4°C, serum samples were separated and stored at -20°C until analyses. Each study participant collected a 24-h urine specimen, excluding the first void, at the beginning, after six months, and at the end of the study. Urine volume was recorded and aliquots were stored at -20°C for later analyses.2), estrone (E1), estrone sulfate, follicle stimulating hormone (FSH), and sex hormone-binding globulin (SHBG) were assessed as we have previously described [To assess whether soy isoflavones modulate sex steroids and their availability, serum levels of 17β-estradiol using a Cobas Fara II clinical analyzer. Urinary deoxypyridinoline (Dpd), a specific marker of bone resorption , was meaP < 0.05 was regarded as significant.Data were analyzed using analysis of variance methods with PROC MIXED in PC SAS analyzing the main and interaction effects of the two factors, treatment (soy protein or control) and time (baseline or after treatment), using the SLICE option. Since each subject was measured at baseline and after treatment, a split plot (repeated measures) model was utilized. The mean changes in endpoints for the soy protein and control treatment groups were compared by analyzing interaction effects of the two factors, treatment and time, using the SLICE option. Data are reported as least square mean ± standard error (SE); unless otherwise indicated, Sixty-two of the 87 women completed the one year study resulting in an attrition rate of approximately 29%. Reasons for dropping from the study included medical conditions preventing continuation in the study (2 women in the soy group and 1 in the control group), starting HRT (1 woman in the soy group and 3 in the control group), noncompliance (2 women in the soy group), dislike of the volume or flavor of the food (3 women in the control group), gastrointestinal side effects (2 women in the control group), food was causing headaches (1 woman in the control group), and personal reasons (3 women in the soy group and 2 in the control group). Five additional women in the soy group decided to discontinue the study without citing a particular reason.Women in both treatment groups had similar baseline characteristics Table . Body wedata not shown).Subjects in both the soy and control groups lost whole body and lumbar BMD and BMC after one year, but no change was observed in the total hip Table . Whole bBoth dietary protein supplements significantly increased serum markers of bone formation, i.e. osteocalcin, BSAP, IGF-I and ALP Table . However2, E1, and estrone sulfate. None of the treatment significantly influenced these sex hormones isoflavones independent of soy protein can prevent ovarian hormone deficiency-associated bone loss; 2) consumption of soy containing food or intake of isoflavones on a daily basis is necessary to observe the expected beneficial effects on bone or simply intermittent use will produce the same results; 3) the effect of soy protein or its isoflavones on bone is transitory; and 4) the combination of soy isoflavones and lower doses of antiresorptive agents can prevent postmenopausal bone mineral loss. As these and other questions are answered, the efficacy of soy protein and its isoflavones as alternative and/or adjunctive treatments for postmenopausal osteoporosis can be determined.The author(s) declare that they have no competing interests.

Published October 19, 2004PLoS Medicine, volume 1, issue 1:In Nevirapine and Efavirenz Elicit Different Changes in Lipid Profiles in Antiretroviral-Therapy-Naive Patients Infected with HIV-1Frank van Leth, Prahpan Phanuphak, Erik Stroes, Brian Gazzard, Pedro Cahn, et al.DOI: 10.1371/journal.pmed.0010019The following information was missing from the legend for There was also a line with open circles in

Previous research indicates that many patients with fractures indicative of underlying osteoporosis are not receiving appropriate diagnostic follow-up and therapy. We assessed osteoporosis treatment coverage in older home care clients with a diagnosis of osteoporosis and/or prevalent fracture.Subjects included 330 home care clients, aged 65+, participating in a longitudinal study of medication adherence and health-related outcomes. Data on clients' demographic, health and functional status and service utilization patterns were collected using the Minimum Data Set for Home Care (MDS-HC). A medication review included prescribed and over-the-counter medications taken in the past 7 days. Criteria for indications for osteoporosis therapy included diagnosis of osteoporosis or a recent fracture. Coverage for treatment was examined for anti-osteoporotic therapies approved for use in 2000.Of the 330 home care clients, 78 (24%) had a diagnosis of osteoporosis (n = 47) and/or had sustained a recent fracture (n = 34). Drug data were available for 77/78 subjects. Among the subjects with osteoporosis or a recent fracture, 45.5% were receiving treatment for osteoporosis; 14% were receiving only calcium and vitamin D, and an additional 31% were receiving drug therapy (bisphosphonate or hormone replacement therapy). The remaining 54.5% of subjects were not receiving any approved osteoporosis therapy.The high prevalence of undertreatment among a population of older adults with relatively high access to health care services raises concern regarding the adequacy of diagnosis and treatment of osteoporosis in the community. Osteoporosis is defined as ' . . .a skeletal disorder characterized by compromised bone strength predisposing a person to an increased risk of fracture. Bone strength reflects the integration of two main features; bone density and bone quality' . The preThe purpose of this study was to examine osteoporosis treatment among older home care clients with a diagnosis of osteoporosis and/or prevalent fracture. As this population has relatively frequent contact with health care providers, we expected that osteoporosis treatment rates should be higher than previously reported.Participants were 330 older home care clients participating in a longitudinal study of medication adherence and related health outcomes. Details of the primary study can be found elsewhere . BrieflyData on demographics, health and functional status and service utilization were collected with a standardized assessment tool, the Minimum Data Set for Home Care (MDS-HC) ,18, and Fracture prevalence and diagnosis of osteoporosis were determined from the disease diagnoses section of the MDS-HC (Section J). The MDS-HC includes specific categories for charting of: 'hip fracture', 'other fracture', and 'osteoporosis'. Prevalent fractures were defined as those that had resulted in a hospitalization (in last 90 days), currently required treatment and/or symptom management or were being monitored by a home care professional. Coverage for treatment was examined for anti-osteoporotic therapies approved for use in 2000 . These iPrevalence estimates of treatment for osteoporosis among subjects with a prevalent fracture and/or diagnosis of osteoporosis were reported. Descriptive bivariate comparisons were also conducted to examine potential variations between osteoporotic subjects who received treatment and those who did not. Fisher's exact test was reported for the bivariate comparisons. Due to the limited number of subjects with osteoporosis or fracture, multivariable analyses were not feasible.Demographic and health status variables are summarized in Table The prevalence of osteoporosis diagnoses and fractures are summarized in Table at least minimal treatment for osteoporosis . Only 30 subjects (39%) were receiving therapy with an approved prescription drug (bisphosphonate and/or hormone replacement therapy), with 5 subjects (6%) being treated only with calcium and vitamin D. Six subjects receiving prescription drug therapy were also taking calcium and vitamin D . Of the 42 subjects not receiving minimal treatment , 3 were taking calcium only, and 10 were taking a multivitamin that may have contained calcium and/or vitamin D. Bivariate comparisons of potential factors that may influence receipt of treatment are summarized in Table versus 31%). The proportion of subjects with lower education levels was also relatively higher among the non-treated group. None of the 5 men among the osteoporotic group were receiving treatment.Specific therapeutic interventions for osteoporosis among subjects with a diagnosis of osteoporosis and/or fracture are shown in Figure versus <39%, respectively) [Home care clients are expected to have relatively frequent contact with health care providers. Among those with a diagnosis of osteoporosis or prevalent fracture n = 78), 30% had weekly contact with nursing staff, 42% had at least one emergency or emergent care visit or hospitalization in the past 3 months, and the majority (67%) had a recent (<6 months) review of their total medication regimen. Thus, more opportunity for intervention following fracture, and higher rates of treatment would be expected among this population. Although the proportion of home care clients receiving treatment was somewhat higher than reported in most previous studies -13, the ctively) , we alsoOur data also indicate that subjects who fracture at sites other than the hip may be less likely to receive treatment. While many of the fractures observed in this study Table were typWe also examined several factors potentially associated with undertreatment. However, the small numbers of subjects with osteoporosis or prevalent fracture limit the interpretation of our findings. Although some trends are apparent, such as undertreatment of men and those with lower education, we cannot comment conclusively on these observations due to the limited sample size and cross-sectional nature of the study. Further evaluation of these factors utilizing a larger sample and appropriate multivariable analysis may provide further insight regarding the potential impact of specific determinants.Speculation regarding reasons for the apparent undertreatment of osteoporosis has focused on physician oversight. However, several factors may play a role in the choice to initiate treatment. A recent study indicated that some physicians have concerns about the proven efficacy of osteoporosis treatment among older populations and may be exercising judgment with respect to minimizing polypharmacy in this population . FurtherDespite limitations, our findings highlight the growing concern that many patients with osteoporosis are not receiving appropriate therapeutic interventions. The lower treatment rate among subjects with fractures at sites other than the hip suggests that physicians may not be recognizing the probability of underlying osteoporosis. While the diagnosis of osteoporosis by BMD measurement has been widely publicized, recent clinical practice guidelines have emphasized the importance of a history of fragility fracture in the identification of patients with osteoporosis . Our finFour previous studies conducted in Canada have examined osteoporosis diagnosis and treatment interventions following fracture. Our data confirm low treatment rates among patients with fracture, and also indicate that even patients with a documented diagnosis may not be receiving therapy. The reasons for lack of treatment of osteoporosis are not yet clear. The reports of patient and physician concerns regarding side effects and polypharmacy warrant further investigation, and suggest that nonpharmacologic interventions may be more acceptable in certain patient populations. Considering the complexity of issues involved in decisions to initiate and continue with treatment, future studies focusing more on evaluation of physician and patient awareness of osteoporosis and factors influencing treatment decisions are needed.Evaluation of other patient-related factors, such as adherence and persistence with osteoporosis therapy, and their impact on health outcomes (e.g. fracture), are also relevant. Such studies may provide insight regarding specific interventions needed to reduce risk of morbidity and mortality associated with osteoporosis.Shelly Vik and Colleen Maxwell have no competing interests. David Hanley's competing interests include consultancies with, honoraria for speaking from, or involvement in research with, the following companies or organizations: Amgen, Astra-Zeneca, Aventis, the Dairy Farmers of Canada, Eli Lilly, Merck, Novartis, NPS Pharmaceuticals, Pfizer, Procter and Gamble, Roche, and Wyeth.Analysis of data, interpretation and the original draft were completed by Shelly Vik. Colleen Maxwell and David Hanley contributed to conception and interpretation, and provided critical evaluation of clinical and methodological content, and subsequent revisions.The pre-publication history for this paper can be accessed here:

The antibacterial activity of host defense peptides (HDP) is largely mediated by permeabilization of bacterial membranes. The lipid membrane of enveloped viruses might also be a target of antimicrobial peptides. Therefore, we screened a panel of naturally occurring HDPs representing different classes for inhibition of early, Env-independent steps in the HIV replication cycle. A lentiviral vector-based screening assay was used to determine the inhibitory effect of HDPs on early steps in the replication cycle and on cell metabolism.Human LL37 and porcine Protegrin-1 specifically reduced lentiviral vector infectivity, whereas the reduction of luciferase activities observed at high concentrations of the other HDPs is primarily due to modulation of cellular activity and/ or cytotoxicity rather than antiviral activity. A retroviral vector was inhibited by LL37 and Protegrin-1 to similar extent, while no specific inhibition of adenoviral vector mediated gene transfer was observed. Specific inhibitory effects of Protegrin-1 were confirmed for wild type HIV-1.Although Protegrin-1 apparently inhibits an early step in the HIV-replication cycle, cytotoxic effects might limit its use as an antiviral agent unless the specificity for the virus can be improved. The lowrn model ,22.Some of these peptides are induced at epithelial surfaces in response to invading organisms -25. ManyIn addition, several antiviral activities were reported. Recently it has been demonstrated that rabbit neutrophil peptide alpha-defensin NP1 protects cells from infection with HSV-1 and 2 . Other sThe antibacterial activity of HDPs is largely mediated by pore formation leading to permeablization of the bacterial membrane. Although some selectivity for bacterial membranes has been described, the lipid membrane of enveloped viruses might also be a target of antimicrobial peptides ,40. ThisGiven the potential cytotoxicity of HDPs, it was important to discriminate between modulation of cell metabolism and direct antiviral effects. We were concerned that HDPs could modulate host cell metabolism without affecting cell viability as assayed for example by standard MTT assays. We therefore decided to use immunodeficiency virus-based vectors transferring the luciferase gene to determine both, the HDP antiviral activity and modulation of cell metabolism. The luciferase activity of target cells, stably transduced with the lentiviral vector prior to HDP treatment should reveal any effect of HDPs on cellular transcriptional and translational efficacy. Treating cells with HDPs during infection with the lentiviral vector and comparison with results obtained with the stably transduced cells should then allow identifying HDPs that inhibit early steps in the viral replication cycle. To validate the luciferase-based assay for modulation of cell metabolism, 293 cells were stably transduced with a lentiviral vector transferring the luciferase gene. Incubation of transduced cells with increasing concentrations of Protegrin-1 and LL37 led to a dose-dependent inhibition of luciferase activity , fungal Plectasin and Novispirin G10, inhibited gram positive and gram negative bacteria revealing antimicrobial activity in the expected range (Table 50) of LL37 and PG-1, resulting in IC50s of approximately 30 μg/ml and 16.8 μg/ml, respectively . To evaluate whether LL37 and PG-1 directly target VSV-G or a lentiviral protein, the inhibitory effect of these HDP against the lentiviral vector was compared side by side to their effect on a retroviral vector containing the amphotropic MLV Env for entry into target cells. The dose dependent reduction in the luciferase activity in the target cells was very similar in cells transduced with the lentiviral or the MLV vector Fig. .50s of 25 μg/ml and 14 μg/ml were observed for LL37 and PG-1, respectively [syn, 2 μg of pHIT-G and 5 μg of VLΔBH were transiently cotransfected by the CaPO4coprecipitation method into 293T cells as previously described [syn, pcTat [To generate lentiviral vector particles transferring the luciferase gene, a codon-optimized HIV-1 (Hgpsyn) and a VS(Hgpsyn) were use(Hgpsyn) .5 μg of escribed . An HIV escribed was alson, pcTat , pcRev [n, pcTat and pHITn, pcTat . The MLVn, pcTat . Two dayin vitro ligation [dl327 , utilizing the unique Bst1107 I restriction site. The vector was propagated in 293 cells and purified by two rounds of CsCl density centrifugation [2 and 10% glycerol four-times (1 hour each) at 4°C, and stored at -80°C until use. The concentration of the vector was determined by measuring absorbency at 260 nm [Beginning with a first generation E1- and E3-deleted adenoviral vector, we generated a replication-competent adenoviral vector Ad.OW126, which harbors in the E1 region the firefly luciferase cDNA ), a IRES element ,51, and ligation to H5dl3fugation , dialyzet 260 nm , and thet 260 nm . The ratsyn and pHIT-G. 293A cells were plated in 24 well plates at a density of 50.000 cells / well and transduced with 200 μl of VLΔBH-SIN vector for two hours. Two days after plating cells were transferred to one well of a six well plate and transduced again with 1 ml of VLΔBH-SIN vector. Cells were subsequently expanded resulting in 293-Luc cells.To stably transduce the luciferase gene into 293A cells, a self-inactivating version of VLΔBH, VLΔBH-SIN similar to VGΔBH-SIN was packThe supernatant of infected 293A or 293-Luc cells, cultured in 96 well plates was removed and cells were lysed in 50 μl of cell lysis buffer . 20 μl of the cell lysates were used in the firefly luciferase assay system of Promega as described by the manufacturer. Each single value of the triplicates was expressed as percent of the mean of triplicates of control cultures infected with the same vector in the absence of HDPs and the mean and the standard deviation of the percent values was calculated for each triplicate.in vitro and in vivo studies. Potential endotoxin contamination was monitored with the chromogenic Limulus amoebocyte lysate assay using Escherichia coli endotoxin (supplied with the kit) as the standard. Endotoxin levels for the peptides were not detectable.The antimicrobial peptides used in this study were prepared by solid phase synthesis and purified by RP-HPLC. Recombinant human β-Defensin-2 was produced by a molecular farming approach in transgenic potato tubers and purified by perfusion chromatography (data not shown). The peptides (≥ 98% pure) were dissolved in 0.01% acetic acid and used for all Acinetobacter baumannii (ATCC 19606), Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) Gram-positive strains: Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228) and Enterococcus faecalis (ATCC 29212).The following strains were used in this study: Gram-negative strains: 620 × 2.5 × 108.All bacterial strains were analyzed with API test strips to confirm identity and aliquots were stored frozen in 50 % skim milk at -80°C. Bacteria were grown overnight in trypticase soy broth at 275 rpm and 37°C. An aliquot of the resulting stationary phase cultures was then transferred to 20 ml of trypticase soy broth and incubated at 37°C for 2.5 hours to reach log phase. This subculture was transferred to a 50 ml conical polystyrene tube and centrifuged for 10 min at 4°C at 880 g. The bacterial pellet was washed once with chilled phosphate buffered saline, pH 7.4, and resuspended in 5 ml of the same cold buffer. One milliliter was removed to measure its optical density at 620 nm. The bacterial concentration was calculated from the following formula: CFU/ml = ODin vitro a radial diffusion assay was performed as previously described [6 CFU) were mixed with 10 ml of underlay gel (43°C) and immediately poured into square 9 × 9 cm petri dishes. A series of 3 mm wells was punched after the agarose solidified. After appropriate serial dilutions were done, 5 μl of HDP, vancomycin , gentamicin, ciprofloxacin, or fluconazole were added to the designated wells. Plates were incubated at 37°C for 3 hours. The bacteria-containing layer was covered with a 10 ml overlay of the nutrient rich agar. The overlay agar consisted of 6% (w/v) TSB and 1% agarose in PBS for all assays. After 18 h of incubation at 37°C, the plates were stained with 0.001% Coomassie blue for 10 h. The clear zones around the punched wells indicated antibacterial activity. The diameters of the clear zones were converted into units by subtracting the well diameter and multiplying the difference by 10. Results were plotted using a semi log scale and correlation coefficients and X-intercepts obtained from linear regression analysis. The minimal effective concentration (MEC) corresponded to the X-intercept value. All assays were performed in triplicates and repeated at least once.To monitor bacterial growth inhibition escribed . Briefly3 cells / well. After 48 hours, 50 μl of MTT-solution (3 mg/ml) was added and incubated at 37° C under 5% CO2 for 1 hour. After this time medium was removed and 100 μl 0.04 N HCL + 10% SDS was used to dissolve the resulting blue formazan crystals in living cells. The optical density was determined at 550 nm. Each single value of the triplicates was expressed as percent of the mean of triplicates of control cultures infected with the same vector in the absence of HDPs and the mean and the standard deviation of the percent values was calculated for each triplicate. In addition the BrdU Cell proliferation ELISA with chemiluminescence detection was performed. After 293-Luc cells or P4CCR5 cells were plated in 96 well plates at a density of 2 × 103 cells/well BrdU (5-bromo-2'-deoxyuridine) was added to the cells with a resulting concentration of 10 μM for the last 22 h of the incubation period. After removing the culture medium, the cells were fixed and DNA was denatured in one step with Fixdenat. Thereafter the cells were incubated with Anti-BrdU-POD for 1 h at room temperature. The chemiluminescence detection was measured after automatic injection of substrate solution with a microplate-luminometer .293-Luc cells were plated in 96 well plates at a density of 2 × 103 cells / well. After overnight incubation, the supernatant of the wells were removed and replaced by 25 μl vector preparation and 25 μl HDP at twice the final concentration indicated. 50 μl fresh medium with HDP at the final concentration indicated was added after two hours. One or two days after infection, the supernatant was removed and 50 μl of cell lysis buffer (Promega) was added. Lysates were stored at -80°C until determination of the luciferase activity of the extracts. The affect of HDPs on cell metabolism was determined in parallel by plating 293-Luc cells exactly the same way as 293A cells and the luciferase activity was determined after one or two days of incubation with HDPs. For GFP-expressing vectors, vector titers were calculated from the number of GFP positive cells per well as previously described [To determine the effect of HDPs on vector infectivity and cell metabolism, 293A target cells were plated in triplicates into 96-well plates at 2 × 10escribed and the 3 cells / well of 96 well plate. 50 μl fresh medium with HDP at the final concentration indicated was added after two hours. Two days after infection, the supernatant was removed and cells were stained by X-Gal. The number of infected cells per well were counted in the microscope.Stocks of HIV-1 were generated by transient transfection of 293T cells with the molecular clone pNL4-3. 25 μl of the virus stock were incubated for 30 minutes at room temperature with 25 μl of LL37 or PG-1 adjusted to twice the final concentration indicated. The mixture was added to P4CCR5 cells plated the day before at a density of 2 × 10The author(s) declare that they have no competing interests.LS, BT and JM performed most of the experiments. LS, OW, ML, EL, HH, HS and KU participated in the experimental design, data interpretation and writing of the manuscript.

Elevated low-density lipoprotein (LDL)-cholesterol is associated with a significantly increased risk of coronary heart disease. Ezetimibe is the first member of a new class of selective cholesterol absorption inhibitors. It impairs the intestinal reabsorption of both dietary and hepatically excreted biliary cholesterol. Ezetimibe is an effective and safe agent for lowering LDL-C and non HDL-C. Short term clinical trials have established the role of ezetimibe monotherapy and its use in combination with statins. Furthermore, ezetimibe and statin combination therapy increased the percentage of patients who achieved their LDL-C treatment goal.Studies using surrogate markers of atherosclerosis have suggested a possible role of ezetimibe in combating atherosclerosis. Ezetimibe provides an effective therapeutic strategy for the management of homozygous familial hypercholesterolemia (HoFH) and sitosterolemia. The lack of outcomes and long term safety data is attributed to the relatively recent introduction of this medication. Over 60 million Americans suffer from cardiovascular disease (CHD). The incidence of CHD and stroke has been on the rise partly because of the increase in life expectancy and the explosive epidemic of diabetes and the metabolic syndrome . CHD is Animal and human studies have established the role of cholesterol in the development and progression of atherosclerosis. LDL-cholesterol (LDL-C) constitutes approximately 60–70 % of total serum cholesterol. Epidemiological studies directly implicated LDL-C to the development of atherosclerosis and CHD. Furthermore, LDL-C level appears to be directly related to the development and recurrence of CHD .Animal studies suggested a protective effect of low LDL-C against atherosclerosis . MultiplMost of the landmark CHD prevention trials involved the use of statin medications. LDL-C remains the primary target of treatment in most instances, and statins are the mainstay of LDL-C lowering treatment .The National Cholesterol Education Program/ Adult Treatment Panel III (NCEP/ATP III) updated guidelines table for deteEzetimibe, a cholesterol absorption inhibitor, is the first agent of a new class of lipid-lowering compounds that selectively inhibits the intestinal absorption of cholesterol and related phytosterols. Ezetimibe undergoes extensive glucuronidation to an active metabolite in the intestinal mucosa . EzetimiAfter oral administration, ezetimibe is absorbed and extensively conjugated to a pharmacologically active phenolic glucuronide (ezetimibe-glucuronide) , the druPlasma concentrations for total ezetimibe were about 2-fold higher in older individuals (>65 years), levels were similar in adolescents to healthy adults and may be higher in women than in men . In patiAdverse experiences were reported in approximately 2% of patients treated with ezetimibe and included fatigue, arthralgia, diarrhea, abdominal pain and back pain. Angioedema and rash were reported after general clinical use of this medication . With co1. Primary Hypercholesterolemia , Ezetimibe is indicated in this case for use as both mono and combination therapy.2. The reduction of elevated total-C and LDL-C levels in patients with homozygous familial hypercholesterolemia (HoFH) either as primary or as an adjunct to other lipid-lowering treatments.3. In patients suffering from Homozygous Sitosterolemia, as adjunctive therapy to diet for the reduction of elevated sitosterol and campesterol levels in patients with homozygous familial sitosterolemia.Multiple studies conducted to examine the effects of ezetimibe monotherapy have concluded that this drug was effective in lowering LDL-C versus placebo.Analysis of multicenter, double-blind, placebo-controlled trials demonstrated that ezetimibe at the 10 mg once daily clinically approved dose significantly modified cholesterol and cholesterol subtypes in patients with hypercholesterolemia when compared to placebo. Ezetimibe significantly lowered total-Cholesterol (TC) (12 %), LDL-C (18 %), apolipoprotein B (Apo B) (15 %), and triglycerides (TG) (7%) and increased high density lipoprotein (HDL-C) (3.5%) -24. LipoIn a case series report, we analyzed the effects of Ezetimibe on cholesterol particle size and number using NMR technology . We found that Ezetimibe lowered cholesterol particle number by a mean 26 % and had no significant effect on cholesterol particle size [The effects of ezetimibe on cholesterol and its subtypes were not influenced by risk-factor status, gender, age, race, time of administration, or baseline lipid profile . The oveThe struggle to achieve the NCEP/ATP III guidelines LDL-C goals through primary utilization of statins is often frustrating for the clinician. In a study to examine the efficacy of statin titration on attainment of LDL-C goal, the authors concluded that for high risk patients, approximately half were able to achieve their LDL-C goal at the appropriate statin starting dose, and only one third of the titration group were able to achieve the NCEP/ATP III cholesterol goal . Now witHMG-CoA reductase inhibitors (statins) act on the rate-limiting step to inhibit HMG-CoA conversion to mevalonate, effectively decreasing LDL-C synthesis. They result in a decrease of LDL-C ranging between 30–60 %, depending on the individual statin and the dose administered. Statin induced LDL-C lowering appears to be effective in reducing CHD and CHD related mortality and morbidity. The extent of CHD and CHD related events reduction is proportionate to the extent of LDL-C reduction ,28. LDL-Reasons to initiate combination therapy to treat hyperlipidemia include: further LDL-C lowering, reducing side effects related to higher doses of statins, modifying other risk factors besides LDL-C such as HDL-C and TG.Increasing the dose of statins has a limited effect on reducing LDL-C, as it is well established that doubling the dose of a statins leads to a 5 % more reduction in total TC and 7 % more reduction in LDL-C with each doubling .Although statins have demonstrated similarity in CHD related events, they are heterogeneous not only in LDL-C lowering efficacy but also in their safety profiles. The bulk of the statins effect on LDL-C occurs at the initial recommended dose and they are safer when used at doses below the maximal recommended dose.Statins are the most effective drugs known to modify LDL-C, but in terms of HDL-C and TG modifying capacity, other classes of lipid lowering medications used alone or in combination with statins offer higher efficacy.The metabolism of cholesterol is an intricate process that involves both produced and ingested cholesterol. The mechanism of action of HMG-CoA reductase inhibitors affects the production of cholesterol, whereas that of cholesterol absorption inhibitors affects absorbed cholesterol, thereby offering potential synergism of action when the medication are used in combination. Trials examining the efficacy have demonstrated synergism and consistency in LDL-C lowering in the absence pharmacokinetic interaction between the statins and ezetimibe.In a relatively large, multicenter study, involving patients with primary hypercholesterolemia already receiving statin monotherapy , patients were randomized to receive either ezetimibe or placebo in addition to their current statin therapy. At the conclusion of this 8 week study, the ezetimibe and statin groups were found to have a significantly lower total-C, LDL-C, Apo B, and TG, and increased HDL-C when compared to the statin only and placebo groups. Furthermore, LDL-C reductions induced by ezetimibe were generally consistent across all statins groups .Another multicenter, double-blind, randomized trial examined the effects of ezetimibe on patients suffering from (HoFH). At the initiation of the trial patients were receiving either atorvastatin or simvastatin. The addition of ezetimibe reduced LDL-C by an additional 20.5 % in contrast to only 6.7 % reduction that resulted from doubling the statin dose . SimilarThe addition of ezetimibe to statins is superior to treatment with statins alone in lowering non-HDL-C, ezetimibe co-administered with simvastatin lowered non-HDL-C by 47.1% whereas, simvastatin monotherapy lowered non-HDL-C by 33.6% when results were pooled across different doses [In terms of modifying risk factors other than LDL-C, the co-administration of ezetimibe with statins had a more favorable effect on HDL-C and TG when compared to statins therapy alone . In concEzetimibe co-administration with fibric acid derivatives was examined in a randomized, evaluator-blind, placebo-controlled, parallel-group study of 32 healthy hypereholesterolemics. Ezetimibe co-administration with fenofibrate was found to produce clinically significant reductions in LDL-C (36.3%) compared to the fenofibrate group (22.3%) with a more favorable TG and HDL-C profile .Sitosterolemia is a rare inherited disorder caused by mutation in either the ABCG5 or ABCG8 genes located on chromosome (2p21) . First dClinical manifestations include: tendon and tuberous xanthomas, episodes of hemolysis, accelerated atherosclerosis, and premature coronary artery disease. It is important to note that close to 50 % of these patients have normal cholesterol levels -41.A recently reported trial demonstrated that treatment with ezetimibe reduces plant sterol levels in patients with sitosterolemia. The authors reported a decrease in sitosterol concentrations by 21% and campesterol by 24 % .It is generally accepted that atherosclerosis is an inflammatory disorder. It is believed that the atherosclerotic process begins with endothelial cell activation, which is triggered by multiple factors such as oxidized lipoproteins. Cholesterol lowering agents as a group have demonstrated great efficacy in prevention and cessation of the progression of atherosclerosis. The efficacy of ezetimibe in monotherapy or in combination on CHD morbidity and mortality has not been well established.One of the unique features of cholesterol absorption inhibitors is their ability to modify post-prandial hyperlipidemia. There is increasing evidence that post-prandial lipoproteins (particularly cholesterol-rich chylomicron remnant) are atherogenic. Ezetimibe has the potential to reduce the cholesterol content of chylomicrons by up to 60% , which mHigh-sensitivity C-reactive protein (hs-CRP) is an inflammatory mediator whose levels correlate with increased coronary risk. Ezetimibe co-administered with simvastatin resulted in significant incremental decreases in hs-CRP in patients with primary hypercholesterolemia. Changes in individual lipid parameters did not explain the observed decreases in hs-CRP and were possibly consistent with an additional anti-inflammatory effect compared with simvastatin monotherapy .In a prospective trial to study effects of ezetimibe co-administered with atorvastatin in patients with primary hypercholesterolemia, ezetimibe plus atorvastatin significantly provided an additional (10%) lowering of hs-CRP versus atorvastatin alone .Ezetimibe and its class of cholesterol absorption inhibitors are new, and there is a lack of outcomes data to explore whether its cholesterol modifying effects will translate to lower CHD mortality and morbidity. The safety of this medication has not yet been established with long term trials data as most of the studies conducted were short term. With the advent and increased utilization of combination therapy in the management of dyslipidemia, further trials are needed to explore the efficacy, indications and safety profile of ezetimibe use in combination with Peroxisome proliferator-activated receptors (PPARs), niacin and bile acid resins.The increased popularity of special weight loss diets such as the high protein diet, poses questions of whether such diets will alter the efficacy or safety of cholesterol absorption inhibitors.Finally, the efficacy of statins in reducing CHD related events has lead to the controversial hypothesis regarding whether or not statins poses a pleiotropic (non lipoprotein) effect. If a pleiotropic effect exists, one might argue that a statin at a higher dose might be more beneficial than combination therapy producing the same effect.Ezetimibe is the first clinically approved cholesterol absorption inhibitor. It is effective in lowering LDL-C as monotherapy or in combination with statins. The use of combination LDL-C lowering medication is expected to become a much more common modality of treatment, especially after the recent NCEP/ATP III update. Ezetimibe offers further lowering of LDL-C and non HDL-C that is consistent and probably safer than increasing the dose of the individual statin. It also provides another effective treatment option for HoFH and sitosterolemia patients. Because of its recent introduction, we still lack both outcomes and long term safety data.

After reading the Learning Forum by Fleming and Lynn (1) Morphology: the essential point of dermatological diagnosis is morphology, a low tech, but hard to master, skill. Dermatological diagnosis, as any other medical diagnosis, starts by collecting adequate information from the patient, and follows by its elaboration. Many doctors consider that dermatological diagnosis can be made on a quick recognition basis, but an ordered and syndromic approach is essential to get to an adequate diagnosis. I think that most dermatologists would agree that a good description of a patient by an experienced colleague is a better starting point for diagnosis than many pictures. I would describe the lesions seen in Figure 1 of [1] not simply as shallow ulcers, but as clearly polycyclic erosions (a finding highly suggestive of herpetic infection).PLoS Medicine, with many readers in less developed countries, this test should not be forgotten.(2) Indicated investigations: Tzanck test is the microscopic evaluation of cell morphology on a cutaneous smear. It can be done in about 15 minutes, requiring a microscope and a trained doctor. Access to this test is probably much easier than to viral cultures or polymerase chain reaction tests. In this setting, a positive Tzanck test would be enough to confirm the clinical diagnosis at a minimum cost. Considering the widespread audience of (3) This case, and the suspicion about systemic manifestations of skin disease, is a wonderful opportunity to disseminate an old concept, very frequently forgotten in medical literature: the skin is an organ, in fact, the biggest one in the body. Its main functions are to act as a barrier, to control temperature, to serve immunological and hormonal roles, and, physiologically less important but very important for patient well-being, to participate in personal relationships. When these functions are not adequately performed, skin failure appears, exactly as is the case with heart or renal failure. Skin failure can have many manifestations, including noninfectious fever, bacteremia, or sepsis. As is the case with renal or cardiac failure, it is easier and more practical to learn about this syndrome than to discuss the systemic manifestations of the many diseases that can cause it. I would highly recommend the following references for doctors interested in the subject: ,3.

Direct payments to local communities to conserve wildlife could prove effective but is biodiversity a commodity that can be bought and sold? The language of conservation is changing: protecting biodiversity is no longer just about ethics and aesthetics; the latest buzzwords are commodities and consumers. Traditionally, conservation initiatives have talked up the benefits they will bring to the global community—saving species, habitats, ecosystems, and ultimately the planet. But conservation also has its costs, and these are usually borne by local people prevented from exploiting the resources around them in other ways. It is unfair to expect a localised minority to pick up costs that ultimately benefit a dispersed majority, argue conservation biologists. There has to be more money made available by concerned individuals, non-governmental organisations, national governments, and international bodies, and there need to be better ways to spend this money if conservation is to be effective, they say. Biodiversity is a commodity that can be bought and sold. We are consumers and must pay.2 of the country , and is using direct payments to help reduce the slaughter of turtles by local fishermen. The Watamu Turtle Watch Program is currently paying fishermen just over $3 a turtle to release the animals from their nets rather than kill them. Before the scheme started in 2000, only around 50 turtles were being released from nets each year. By 2003, more than 500 a year were making it back into the sea. Elsewhere along the Kenyan coast, where fishermen do not get these payments, turtles continue to be killed, says Trott. However, the financial incentives are only part of a grander program of education and support to sensitise people to the conservation message, he says. Eventually, the plan is to stop payments altogether. ‘Payment will be reduced as education and awareness is increased to the point where it's phased out,’ he says.Marine biologist Steve Trott agrees. He is project coordinator for the Local Ocean Trust, a charity-based conservation organisation operating in the Watamu and Malindi Marine Parks and Reserve in Kenya in Costa Rica . In the

Physicians often recommend to older adults that they should engage in mentally stimulating activity to reduce the risk of dementia. But is this recommendation based on sound evidence? Physicians now commonly advise older adults to engage in mentally stimulating activity as a way of reducing their risk of dementia. Indeed, the recommendation is often followed by the acknowledgment that evidence of benefit is still lacking, but “it can't hurt.” What could possibly be the problem with older adults spending their time doing crossword puzzles and anagrams, completing figural logic puzzles, or testing their reaction time on a computer? In certain respects, there is no problem. Patients will probably improve at the targeted skills, and may feel good—particularly if the activity is both challenging and successfully completed.But can it hurt? Possibly. There are two ways that encouraging mental activity programs might do more harm than good. First, they may offer false hope. Second, individuals who do develop dementia might be blamed for their condition. When heavy smokers get lung cancer, they are sometimes seen as having contributed to their own fates. People with Alzheimer disease might similarly be viewed as having brought it on themselves through failure to exercise their brains.Three types of evidence are cited to support the idea that mental exercise can improve one's chances of escaping Alzheimer disease.Having more years of education has been shown to be related to a lower prevalence of Alzheimer disease in cross-sectional, population-based studies In epidemiological studies, people cannot be randomly assigned to different levels of education, or to different kinds and levels of participation in leisure activities. Consequently, researchers must try to identify confounders and take them into account analytically. However, uncertainties remain. Both education and leisure activities are imperfect measures of mental exercise. For instance, leisure activities represent a combination of influences. Not only is there mental activation, but there may also be broader health effects, including stress reduction and improved vascular health—both of which may contribute to reducing dementia risk Another problem with these epidemiological studies is that reverse causation could be involved—in other words, that incipient dementia could be causing reduced engagement in leisure activities, although some prospective studies have been particularly attentive to controlling for this possibility Many studies support the possibility of enhancing memory and other cognitive performance, or of slowing cognitive decline in older adults without dementia These limitations are evident in one of the largest randomized controlled trials of cognitive training with older adults, a large, multisite study named ACTIVE The third type of evidence suggesting that mental exercise may help to prevent Alzheimer disease comes from neurobiology studies that show greater brain complexity in those with higher levels of mental activity. Many such studies, done with animals, show greater neural complexity after having been exposed to an enriched environment that provides lots of stimulation, for example by including wheels, tunnels, toys, and gnawing sticks The concept of cognitive reserve is often used to explain why education and mental stimulation are beneficial. The term cognitive reserve is sometimes taken to refer directly to brain size or to synaptic density in the cortex. At other times, cognitive reserve is defined as the ability to compensate for acquired brain pathology. This definition encompasses coping skills as well as recruitment of other brain areas, with cognitive reserve thus accounting for individual differences in severity of cognitive dysfunction when there are pathological neural changes. People with a higher level of education have greater cognitive reserve. In some studies, education or occupation are even used as proxy measures of cognitive reserve, while others are beginning to measure neural substrates that correspond to reserve Taken together, the evidence is very suggestive that having greater cognitive reserve is related to a reduced risk of Alzheimer disease. But the evidence that mental exercise per se can increase cognitive reserve and stave off dementia is weaker. Epidemiological studies suggest that individual differences in cognitive reserve may actually be lifelong. In addition, people with greater cognitive reserve may choose mentally stimulating leisure activities and jobs, leading to a chicken-and-egg dilemma for the interpretation of the relationship between mentally stimulating activities in adulthood and dementia risk. Cognitive training has demonstrable effects on performance, on views of self, and on brain function—but the results are very specific to the skills that are trained, and it is as yet entirely unknown whether there is any effect on when or whether an individual develops Alzheimer disease. Further, the types of skills taught by practicing mental puzzles may be less helpful in everyday life than more prosaic “tricks,” such as concentrating, or taking notes, or putting objects in the same place each time so that they won't be lost.So far, we have little evidence that mental practice will help prevent the development of dementia. We have better evidence that good brain health is multiply determined, that brain development early in life matters, and that genetic influences are of great importance in accounting for individual differences in cognitive reserve and in explaining who develops Alzheimer disease and who does not. At least half of the explanation for individual differences in susceptibility to Alzheimer disease is genetic, although the genes involved have not yet been completely discovered For older adults, health practices that could influence the brain include sound nutrition, sufficient sleep, stress management, treatment of mood or anxiety disorders, good vascular health, physical exercise, and avoidance of head trauma. But there is no convincing evidence that memory practice and other cognitively stimulating activities are sufficient to prevent Alzheimer disease; it is not just a case of “use it or lose it.”

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes—for the measurement of population activity, in particular—have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2. Winfred Denk and colleagues succeed in generating transgenic mice that express one of two calcium indicators in their cells, creating a valuable tool to study neuronal activity Two classes of genetic Ca2+ indicators have been designed that use different mechanisms of action. The first class, called “cameleons” regulation system , G-CaMP, camgaroo-2 (Cg2), and the cameleons YC 2.12 and YC 3.12 (n = 5 cells) and IP (n = 2 cells), respectively were used for analysis of expression patterns by immunohistochemistry . In high- and moderate-expression lines mice, but there it had very different, much broader emission spectra and one moderate-expression line MTH-Cg2-14), using confocal imaging spectrometry with excitation at 488 nm. The emission spectra of smooth and punctate fluorescence were similar to each other and to Cg2-expressing HEK cells . Imaging up to and occasionally beyond a depth of 500 μm was possible . Densely2+ response properties of FCIPs using a variety of different preparations and stimulation methods.Next we tested the CaMainly to test temporal response characteristics, we performed a series of somatic electrical recording and synaptic stimulation experiments on pyramidal cells in brain slices. Targeted whole-cell tight-seal recordings of FCIP-expressing layer-2/3 cortical cells showed that FCIP-expressing cells have normal electrophysiological properties A. SomatiFirst, cortical slices from a Cg2 animal (MTH-Cg2-14) were imaged using a CCD camera. Stimulation effectiveness was monitored in a distantly located soma (≈200 μm) by whole-cell tight-seal recording C. Short 2+ responses were seen (eight cells in two retinas tested), consistent with YC3.12 results in other tissues. In lines expressing Cg2 (MTH-Cg2-14), fluorescence became too weak and bleached too quickly for optophysiological measurements . In one of two IP-expressing mice tested , a subset of ganglion cells was strongly labeled see K but no endrites C and 7D.FCIPs are expressed in the olfactory bulb in afferent sensory axons and granule cells, with relative expression levels varying somewhat with FCIP type and across lines (data not shown). Substantial changes in fluorescence were observed for both IP and Cg2 (MTH-IP-12 and MTH-Cg2-19) in response to odor stimulation . We do n2+] dynamics in sensory afferents rather than [Ca2+]. It is, however, unlikely that the changes we saw are dominated by pH effects for the following reasons. In the case of Cg2, stimulation-induced pH changes certainly does not apply to IP and Cg2 since we found large responses even in mice aged 8–12 wks, which had been expressing indicators since the onset of αCamKII expression before birth. However, even in strongly expressing smooth-fluorescence lines, a substantial fraction of the indicator protein was found to be immobile and potentially nonfunctional at various ages. It is surprising that punctate fluorescence occurs predominantly in weak lines since precipitation typically occurs at high concentrations. It could, however, be that a limited number of binding and sequestration sites for FCIPs exist in the cell and that only after these sites are saturated does the accumulation of mobile see J, cytosog motifs that lacromoters but not romoters . This isromoters . SimilarOne of the remaining problems hampering imaging of population activity with FCIPs is that expression, while cell-type specific, is not complete, i.e., not in every cell of one type, even in lines that express FCIPs at high levels. One possible explanation is position effect variegation (PEV), which occurs when a transgene integrates adjacent to a heterochromatin domain in the genome. In such a situation, expression of the locus variegates, being active in some cells and silent in others . If this2+ indicators in mice, albeit for an unexpected reason. Unlike other promoter systems, such as the CMV promoter, which also appears to resist gene silencing, the TET system allows cell specificity via the expression of the transactivator, which appears to be controllable by cellular promoters without gene silencing.In any case, it appears that the use of the TET system allows the expression of genetically encoded Ca2+ indicators opens the way for the measurement of neural activity patterns in mammals in vivo and in vitro. While the sensitivity of genetic indicators does not (yet) quite reach those of synthetic compounds, it is sufficient for single-trial measurements at least in some applications and FL were cloned into a Ptetbi vector . The resulting plasmids were sequenced and transfected into HeLa cells that stably express tTA were fixed in 4% paraformaldehyde in PBS for 4 h and washed twice with PBS. Brain slices were cut to a thickness of approximately 70 μm using a vibratome . Distribution of Calontech) and the http://rsb.info.nih.gov/ij/) and IgorPro .All two-photon measurements described in the following sections were done using custom-built two-photon microscopes. Fluorescence was two-photon-excited by a mode-locked Ti-sapphire laser coupled into a custom-built imaging system. The objective used was a 40X/0.8 NA water immersion lens . A photomultiplier-based whole-field detector detectedRegions rich in neurites were repeatedly scanned in the two-photon microscope. After a first bleaching run from mice were prepared according to published procedures for hippocampal experiments and for Acute brain slices (see above) were maintained in ACSF. Whole-cell tight-seal recordings were made using pipettes made from borosilicate glass (5–10 MΩ) containing 135 mM K gluconate, 4 mM KCl, 10 mM HEPES, 10 mM Na2-phosphocreatine, 4 mM Mg-ATP, and 0.3 mM Na-GTP. Recording pipettes also contained 0.2% biocytin and 1–5 μM Alexa 568, which is spectrally distinct from the FCIPs and can be used to unambiguously identify the recorded cell. For synaptic stimulation we used saline-filled glass electrodes or tungsten microelectrodes (1 MΩ) . Synaptic transmission was blocked in some experiments by the addition of 40 μM 6-cyano-7-nitroquinoxaline-2,3-dione and 100 μM 2-amino-5-phosphovaleric acid.Wide-field images were taken with a MicroMax, 512 × 512 back-illuminated CCD camera binned 5 by 5. For experiments involving extracellular stimuli we calculated the change in fluorescence relative to the prestimulus period (ΔF/F) for each binned pixel frame by frame. The prestimulus period of regions of interest was fitted with an exponential curve, which was then subtracted from the entire fluorescence time course to correct for bleaching. No corrections were made for autofluorescence, so that the relative FCIP fluorescence changes are likely to be larger. When analyzing action-potential-induced signals from individual neurons, we calculated the fluorescence changes by subtracting the average of two nearby areas from the total fluorescence to account for background fluorescence to record baseline fluorescence (100 pulses at 100 Hz or 20 pulses at 100 Hz). Image sequences were 6 or 12 s long. The background level (average intensity with the laser off) was subtracted from every frame in the image sequence. The average of the five prestimulus (rest) images was subtracted from the average of five “response” images (response minus rest). The built-in smoothing function of ImageJ was used to reduce the noise further. The brightness of the difference image was enhanced for better display. Localized small fluorescent structures or hot spots in neurites were identified visually. For IP, traces with high time resolution were acquired using 64-pixel line scans at 500 Hz. Fluorescence was averaged over the width of the soma. The fluorescence from a neighboring region was subtracted to account for background. Fluorescence changes (percent ΔF/F) were calculated relative to the resting fluorescence.Mice were dark-adapted for several hours before the experiments and all subsequent procedures were carried out under dim red illumination to minimize photobleaching. Animals were anesthetized with halothane and subsequently killed by cerebral dislocation or by intraperitoneal injection of pentobarbital. Immediately afterward, both eyes were removed and dissected free in Ames medium (Sigma-Aldrich). A piece was cut from a retina and placed photoreceptor side down into the recording chamber, and maintained at 35 °C in Ames medium continuously perfused with oxygen. The remaining retina was kept for further use.2+-mediated fluorescence (emission 520 BP 30 nm) changes in retinal ganglion cells using a custom-built two-photon microscope. The laser (Coherent Mira 900F) was tuned to 925 nm and body temperature was maintained at 37 °C. For two-photon imaging, a custom-built headplate with an imaging window (4 mm × 3 mm) was glued to the top of the skull using cyano-acrylate and attached to a fixed metal bar before thinning of the skull. The combination of rigid headplate and thinned-skull reduces respiration and cardiac-pulsation-induced brain motion. The microscope objective was positioned at an angle so that the optical axis was perpendicular to the surface of the cortex.

Accidental poisoning is one of the leading causes of injury in the United States, second only to motor vehicle accidents. According to the Centers for Disease Control and Prevention, the rates of accidental poisoning mortality have been increasing in the past fourteen years nationally. In Texas, mortality rates from accidental poisoning have mirrored national trends, increasing linearly from 1981 to 2001. The purpose of this study was to determine if there are spatiotemporal clusters of accidental poisoning mortality among Texas counties, and if so, whether there are variations in clustering and risk according to gender and race/ethnicity. The Spatial Scan Statistic in combination with GIS software was used to identify potential clusters between 1980 and 2001 among Texas counties, and Poisson regression was used to evaluate risk differences.Several significant (p < 0.05) accidental poisoning mortality clusters were identified in different regions of Texas. The geographic and temporal persistence of clusters was found to vary by racial group, gender, and race/gender combinations, and most of the clusters persisted into the present decade. Poisson regression revealed significant differences in risk according to race and gender. The Black population was found to be at greatest risk of accidental poisoning mortality relative to other race/ethnic groups (Relative Risk (RR) = 1.25, 95% Confidence Interval (CI) = 1.24 – 1.27), and the male population was found to be at elevated risk when the female population was used as a reference.The findings of the present study provide evidence for the existence of accidental poisoning mortality clusters in Texas, demonstrate the persistence of these clusters into the present decade, and show the spatiotemporal variations in risk and clustering of accidental poisoning deaths by gender and race/ethnicity. By quantifying disparities in accidental poisoning mortality by place, time and person, this study demonstrates the utility of the spatial scan statistic combined with GIS and regression methods in identifying priority areas for public health planning and resource allocation. The accidental poisoning mortality rate has been increasing in the United States. In the 21-year period spanning 1981 to 2001, mortality rates due to accidental poisoning more than doubled from 2.0 per 100,000 in 1981 to 4.9 per 100,000 in 2001 / [D - n])D - d)(I     (1) the purely spatial scan statistic and (2) the space-time scan statistic adjusting for time.ETN was responsible for the design and implementation of the project and the final paper. ETN also contributed to data analysis. CEH contributed to database design, visual basic application programming and data analysis. CEH also assisted in the preparation and editing of the final paper. VIH contributed to the preparation and editing of the final paper, including reviewing the relevant literature. AMH was responsible for the layout and design of the illustrative maps and also assisted in preparing and editing the final paper. All authors read and approved the final manuscript.

Although a rare disease, uveal melanoma is the most common primary intraocular malignancy in adults, with an incidence rate of up to 1.0 per 100,000 persons per year in Europe. Only a few consistent risk factors have been identified for this disease. We present the study design of an ongoing incident case-control study on uveal melanoma (acronym: RIFA study) that focuses on radiofrequency radiation as transmitted by radio sets and wireless telephones, occupational risk factors, phenotypical characteristics, and UV radiation.We conduct a case-control study to identify the role of different exposures in the development of uveal melanoma. The cases of uveal melanoma were identified at the Division of Ophthalmology, University of Essen, a referral centre for tumours of the eye. We recruit three control groups: population controls, controls sampled from those ophthalmologists who referred cases to the Division of Ophthalmology, University of Duisburg-Essen, and sibling controls. For each case the controls are matched on sex and age (five year groups), except for sibling controls. The data are collected from the study participants by short self-administered questionnaire and by telephone interview. During and at the end of the field phase, the data are quality-checked.To estimate the effect of exposures on uveal melanoma risk, we will use conditional logistic regression that accounts for the matching factors and allows to control for potential confounding. Although a rare disease, uveal melanoma of the eye is the most common primary intraocular malignancy in adults, with an incidence rate of up to 1.0 per 100,000 person years in Europe ,3. AnothSome uveal melanoma are associated with neurofibromatosis. However, the vast majority of familial cases reported are non-syndromic . Some reOccupation may be also relevant, and may include chemical work ,12, arc The population-wide introduction of analog and digital mobile phone techniques in the recent years, which has been coined as the mobile revolution , has resUntil now, the majority of epidemiological cancer studies focussed on brain cancer because the brain may be exposed to RFR -20. WithThe RIFA study is planned to answer several etiologic questions with a special focus on electromagnetic radiation especially radio-frequency radiation as emitted by mobile phones and radio sets. First, is the finding of an increased risk of uveal melanoma among subjects with frequent use of RFR devices reproducible? Second, if there is an association, is this association site-specific in terms of laterality of the uveal melanoma and major site of mobile phone use? Third, if there is an association, is there a dose-response relationship between RFR and uveal melanoma risk? Another set of etiologic question relates to pigmentation characteristics including iris colour, hair colour, tendency of the skin to burn and to tan, freckling, number of cutaneous nevi. A further study questions relates to exposure to work and leisure time related ambient ultraviolet radiation and uveal melanoma risk. In addition, our study focuses on several occupational exposures or jobs that are suspected to be associated with an increased risk of uveal melanoma ,15 incluth, 2002 to September 24th, 2004 are eligible, if they are referred to the Division of Ophthalmology, University of Duisburg-Essen during the recruitment period, are aged 20–74 years at diagnosis, are living in Germany, and are capable to complete the interview in German. The majority (about 70–80%) of uveal melanoma treated at the University Hospital Essen receive episcleral plaque therapy without histological verification. For this reason we did not include a reference pathologist who reviews the diagnostic certainty of the cases. Experiences from our previous case-control studies showed that there is a nearly perfect agreement between the local eye doctors in the reference centre and the international reference pathologist [The case recruitment is hospital-based and takes place in the Division of Ophthalmology, University of Essen which is a the referral centre for eye cancer in Germany, currently treating about 400–500 eye cancer patients per year. Eligible uveal melanoma cases have to fulfil several criteria. Patients with newly diagnosed first uveal melanoma located in the choroid, iris, and/or ciliary body during t London) .We considered a diagnosis of uveal melanoma as definite if the results of the clinical examination of the eye and ultrasound were unambiguous. The inclusion and exclusion criteria of the cases are listed in table th, 2002 through March 31th, 2004. Assuming comparable incidences of uveal melanoma in the federal states of Germany, the crude rate (referred cases divided by population at risk aged 20–74 years) may be considered as an indicator of the referral effect.Interim analyses showed that the majority of cases comes from the territory of former West Germany. Figure Obviously, the referral effect varies by federal states. However, it is difficult to judge whether the case referral to the Division of Ophthalmology, University of Duisburg-Essen is a random sample of all newly diagnosed uveal melanoma cases in Germany. We therefore decided to recruit three different control groups. First, if we assume that cases treated in Essen are a random sample of all cases in Germany, a population-based control group would be the most appropriate control group. For this approach, we randomly select controls from mandatory lists of residence that cover the total population of the city or local district. These lists are regarded as the most complete sampling frame for population-based studies in Germany.Second, if the referred cases are not a random sample of all newly diagnosed cases of uveal melanoma in Germany, a control group sampled from those ophthalmologists who referred cases to the Division of Ophthalmology, University of Duisburg-Essen, would be most appropriate ,26. To iIn addition, we recruit sibling controls of cases (matching ratio 1:1) in order to assess whether genetic factors may confound the effect of exposure. The sibling controls are matched in genetic background. The inclusion criteria of the three control groups are presented in table Based on our former uveal melanoma case-control studies ,22 we esPopulation-based prevalence estimates of mobile phone use in the general population are scant. A recent telephone survey from 2001 showed that 82% of male and 74% of female participants aged 14–44 years use mobile phones; within the age group 45–59 years, 63% of male and 58% of female participants use mobile phones. The oldest age group (> = 60 years) shows considerable lower prevalences of mobile phone use . To deteTable The questionnaire on mobile phone use is the same instrument which has been used by the international case-control study on brain cancer and mobile phone use sponsored by the International Agency for Research on Cancer, called Interphone study . In contCompared to other studies on the aetiology of uveal melanoma, the RIFA study uses a detailed assessment of pigmentary characteristics. The self-administered questionnaire contains a eye and hair colour card that allows the participants to choose the most appropriate colour of their eyes and hairs at age 20 years. During the telephone interview, the skin reaction to sun exposure is asked according the concept of Fitzpatrick . FitzpatFor the assessment of nevi of the upper arms and the dorsum of the feet with a diameter of at least 3 mm, participants receive a template with a 3 mm hole that enables them to count all nevi of this minimum size. In addition, the CATÍ (computer assisted telephone interview) includes a detailed history of sunburns in the recent 15 years before interview and tendency to freckle as a child. The CATI contains a question on eye colour with the typical categorical answers as has been used by several others , we use 16 job-specific supplementary questionnaires to obtain details of the job tasks and materials used.The quality control program includes several procedures. The study is designed to fulfill the recommendations of the German Good Epidemiologic Practises .Eight month before the main study started, a manual of standard operating procedures (MOP) was written and a pilot study of four weeks was conducted to test the field work and exposure questionnaires. After some minor revisions of the MOP, report forms, and questionnaire instruments, the principal investigator and participating epidemiologists had to sign that they fully agree with the final version of the MOP.Interviewers of the study were introduced into the field work and were blinded against our study hypotheses. After an initial interviewer training course, interviewers are regularly monitored and receive regular training courses.The recruitment progress, given as number of registered cases and controls, distribution of inclusion and exclusion criteria, response proportions, is monitored monthly. The analysis of nonresponse reasons is supplemented by an short questionnaire for subjects not willing to participate. This questionnaire includes few demographic and exposure items that help us to assess potential selection effects due to nonresponse.A plausibility control of the interview data is done quarterly and is the basis for the regular training courses of the interviewers.The completeness of case registration is checked by regular comparison of the list of registered cases with lists of admissions to the referral centre. In addition we compare our list of cases with data of the hospital information system that includes information on diagnoses.The self-administered short questionnaires are visually edited by the study personnel before the telephone interview starts. The visual editing includes a completeness check and coding of the life-long job history. For each job period, the occupation and branch of industry is coded according to ISCO-68 and NACEThe CATI contains internal quality checks that prevent data entry errors. For example, interviewers are not able to fill in the detailed questions on mobile phones, if the entry question on ever having used mobile phone has been answered with no.At the end of the field phase, the data are quality-checked. To estimate the effect of exposures on uveal melanoma risk, we will use conditional logistic regression that accounts for the matching factors and allows to control for potential confounding. We will classify people exposed to an occupational category if they ever worked within this category for at least six months. The quantification of mobile phone use will be based on average number of phone calls and average duration of phone calls per time unit. The association between pigmentary characteristics and uveal melanoma risk will be assessed by detailed matrix containing information on hair colour at age 20 years, eye colour, freckling tendency, and skin colour. Final results of these analyses are scheduled to be published in summer 2005.None declared.The pre-publication history for this paper can be accessed here:

The MidSouth Computational Biology and Bioinformatics Society (MCBIOS) describes its efforts to provide local opportunities for researchers to learn and connect with colleagues The rich get richer while the poor get poorer. This is as true for research in the life sciences as it is for society in general. Why is this? To put it simply: because existing resources can be leveraged to generate new ones. In life science research, these resources are often expressed in terms of people, equipment, and funding. Those who have more in these areas—large research organizations, support staff, extensive facilities, and pipelines of funding—can be more productive, and thereby more successful in maintaining and even expanding their infrastructure. In the United States, this “scientific wealth” is concentrated in certain geographic areas, particularly along the east and west coasts. While there are many highly competent individuals in other, less wealthy, regions, they often struggle to attract funding because they lack this infrastructure.Fortunately, scientific progress is also made through collaboration and cooperation—activities fostered through the exchange of viewpoints and the exploration of ideas. Given a framework in which this networking can occur, some of the problems that result from a lack of infrastructure can be overcome. By definition, collaboration involves the sharing of ideas, capabilities, and resources. While it may not be possible to access all necessary resources locally, finding partners—who have their own local capabilities and resources—can enhance one's own research environment. Through these kinds of efforts, “virtual infrastructures” grow. And through collaborative efforts, those of us in some of the “poorer” regions of the United States have opportunities to successfully compete for national sources of funding by becoming better able to address the “existing research environment” consideration on most grant applications.www.MCBIOS.org), created to serve a geographical area that includes Arkansas, Louisiana, western Tennessee, Missouri, Mississippi, Oklahoma, and east Texas. By its very nature, bioinformatics involves people from many different backgrounds and is therefore ideal for collaborative efforts. Like many other regional societies, our primary goal is to provide a framework in which collaboration and cooperation can occur. By sponsoring regional activities we hope to bring educators, researchers, and especially students together with others who have similar and/or complementary interests. Through the society, not only can medical scientists make contact with computational experts, but researchers in Arkansas can connect with researchers in Oklahoma, educators from Missouri can interact with educators from Louisiana, and students from Mississippi can find others from Tennessee who are working in the same area. Communication across specialties and areas of expertise is especially important for fostering interdisciplinary efforts and exposing students to a broader range of topics than might be available at their own institution. Nowhere is this truer than in the application of informatics to a variety of disciplines.Building a virtual infrastructure is the primary purpose behind the formation, in the United States, of the MidSouth Computational Biology and Bioinformatics Society , for example, has recognized MCBIOS under their “regional affiliate” program. Likewise, MCBIOS is encouraging its members to form “local chapters” . These local chapters are eligible to host the annual MCBIOS conference and are able to support even closer interactions among their local participants. The hierarchy of affiliations provides an abundance of opportunities for members to participate based upon their interests, financial resources, and tolerance for travel.Regional societies need not be in competition with national or international societies. In fact, many of these larger organizations are finding that it is to their advantage to encourage close relationships with their regional counterparts. The International Society of Computational Biology , we have deliberately chosen locations for the MCBIOS annual conference that would be within a day's drive for all members. At the inaugural 2003 MCBIOS conference, for example, with a theme of “Building Networks,” a number of computer scientists attended who were able to find out more about bioinformatics and their possible contribution to life science research; they would probably not have been able to make the investment to attend a national or international meeting on bioinformatics.While still maintaining high standards, regional events also increase the number of venues for the presentation of research results and creative efforts in the educational arena. While the ISCB program, which is already highly self-selecting, accepts less than 15% of their submissions, almost all submissions at the first annual MCBIOS conference could be accommodated, at least in poster form. These regional events can also attract well-respected keynote speakers and provide training opportunities that might not otherwise be available to the membership. For example, Dr. Alan Leshner, CEO of the American Association for the Advancement of Science, gave the keynote address of the 2004 MCBIOS conference. In 2003, we were also able to host a special training session by the National Center for Biotechnology Information on their GenBank and molecular biology tools.While MCBIOS is still new, we have plans to extend our activities—and thus our impact—beyond our annual conference. We are dedicated to supporting our local chapters, and have plans to develop a speakers' bureau to serve them. Plans to collaborate on regional technology efforts and on multi-institutional educational programs are also in the works. Through the auspices of the society, we hope to increase regional credibility and attract national funding in support of these infrastructure improvements.In the long term—and in addition to supporting the intellectual efforts of our members through a vibrant organizational community—our goal is to increase extramural funding for our represented institutions. We hope to achieve this by fostering competitive research. And this competitive research will be possible on a larger scale through our development of a virtual infrastructure—one that comes about through regional collaboration and a pooling of resources. In conjunction with other infrastructure-building efforts , we hope to see externally funded research in the life sciences significantly increase within our region, the American “Midsouth.”http://BRIN.UAMS.edu] of the National Center for Research Resources through a grant to underwrite student participation and awards at the conferences. The 2004 MCBIOS conference was also supported in part by a grant from the National Center for Toxicological Research [www.FDA.gov/NCTR].)(The 2003 and 2004 MCBIOS conferences have been supported in part by National Institutes of Health grant P20 RR-16460 from the Arkansas Biomedical Research Infrastructure Network Program [

We evaluated the impact of ibpAB, which is innocuous under physiological conditions, impaired culture growth during α-glucosidase production. At higher temperatures, accumulation of stress proteins including disaggregation chaperones (DnaK and ClpB) and components of the RNA degradosome, enolase and PNP, was intensified. Overexpression of ibpAB, conversely, suppressed the heat-shock response under these conditions. Inclusion bodies of α-glucosidase started to disaggregate after arrest of protein synthesis in a ClpB and DnaK dependent manner, followed by degradation or reactivation. IbpA/IbpB decelerated disaggregation and degradation at higher temperatures, but did hardly influence the disaggregation kinetics at 15°C. Overexpression of ibpAB concomitant to production at 42°C increased the yield of α-glucosidase activity during reactivation.Deletion of ibpAB, inclusion body removal is faster, but cells encounter more intense stress and growth impairment. IbpA/IbpB thus exert a major function in cell protection during stressful situations.IbpA/IbpB attenuate the accumulation of stress proteins, and – at high temperatures – save disaggregated proteins from degradation, at the cost, however, of delayed removal of aggregates. Without Escherichia coli can induce several stress responses [Escherichia coli [Production of recombinant proteins as inclusion bodies in esponses -3 and maesponses -6. The mesponses ,8 may reesponses ,9,10. Moesponses ,12. Whilhia coli , the highia coli ,15. The Analogously, incubation at extreme temperatures is characterised by near exclusive heat-shock protein synthesis as a consequence of extensive aggregation of cellular proteins ,19. To dE. coli sHsps, IbpA and IbpB, reversibly dissociate upon prolonged incubation at 50°C from multimers of 2 – 3 MDa to monomers in the case of IbpA and to oligomers of 650 – 700 kDa in the case of IbpB, exposing hydrophobic sites [in vitro and facilitates their subsequent refolding by DnaK [in vitro and in vivo is mediated by ClpB together with DnaK [ibpAB as well as in some cases in strains lacking ibpAB [The small heat-shock proteins (sHsps) are intrinsic holding chaperones of multimeric structure. The multimers dissociate at high temperatures into smaller entities for exposure of client binding sites. After binding of misfolded client proteins, the sHsps form large globular assemblies . Therebyic sites ,25. IbpAic sites , but sho by DnaK -25, a ro by DnaK ,27. IbpA by DnaK ,28. The ith DnaK , can be ng ibpAB ,32,33. Ing ibpAB ,35.ibpAB confers resistance to heat and other stresses [ibpAB operon does not affect growth even at high temperatures. Only recovery from prolonged exposure to extreme temperatures such as 50°C are impaired in an ibpAB mutant, and growth of ibpAB dnaK double mutants at high temperatures [Saccharomyces cerevisiae produced in recombinant E. coli is a suitable model system. It accumulates as inclusion bodies and induces the heat-shock response including prolonged expression of the ibpAB operon [E. coli if produced at low temperature [The role of IbpA/IbpB in cell protection is less clear. Overexpression of stresses , but deleratures ,36. In tB operon . It is, perature , enablinibpAB operon was induced already during α-glucosidase production at 30°C, with moderately higher levels at elevated temperatures or concomitant to production at lower temperature.Post-induction growth of the res Fig. , althougThus, the sHsp are effective only under conditions that favour inclusion body formation.ibpAB deletion mutant at all temperatures, in addition to the induction at elevated temperatures . While the concentration of α-glucosidase was 10–25% higher in dnaK and clpB deletion mutants than in the wildtype strain, the specific activities obtained during the production phase were less than 0.1 Umg-1, compared to about 0.3 Umg-1 in the other strains (data not shown). Importantly, neither disaggregation, nor reactivation nor degradation took place in these mutants within two hours, and the concentration of α-glucosidase inclusion bodies remained unchanged.The impact of chaperones on the metabolism of α-glucosidase inclusion bodies was examined in wildtype proteins . In the ibpAB, were investigated as a function of temperature. After production of α-glucosidase at 37°C, the cultures were transferred to various temperatures. As protein synthesis was arrested before the transfer, the initial IbpA/IbpB level did not depend on the disaggregation temperature. The α-glucosidase concentration in the insoluble cell fractions of all cultures decreased with time, but with different kinetics. In the ibpAB deletion strain, the disaggregation kinetics were not influenced by the incubation temperature .Disaggregation of α-glucosidase inclusion bodies was accompanied by an increase of the specific α-glucosidase activities in the cell extracts. The reactivation showed a clear temperature optimum in the range of 24 to 30°C, most evident with wildtype levels of IbpA/IbpB Fig. . The spened Fig. . Both, iibpAB deletion mutant at all temperatures . About one third of the α-glucosidase produced concomitant to ins Fig. . Also thins Fig. , resemblins Fig. . Thus, tibpAB overexpression U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR and JGT17 MC4100 Δ ibpAB [argU tRNA and carrying the lacI repressor gene [HindIII restriction sides, coding for the ibpA and ibpB genes including their native promoter, was cloned into the HindIII site of the plasmid pKK177-3/GLUCP1 [GLUCPI gene and the 5ST1T2 terminator, to give the new plasmid named pKK177-3/GLUCP1_ibpAB, in which the ibpAB operon is under the control of its native promoter and of the tac-promoter upstream of the α-glucosidase gene. Proper orientation of the insert was tested by EarI digestion. Δ ibpAB were use Δ ibpAB . Plasmidsor gene was cotrsor gene . The seqsor gene between 3/GLUCP1 , located-1, chloramphenicol 50 μg mL-1), on a rotary shaker at 37°C to an OD600 of 0.5, induced with 1 mM IPTG and transferred to different temperatures as indicated in the results section. For reactivation experiments, tetracycline was added to a final concentration of 25 μgmL-1 two hours after induction to arrest protein synthesis, and the cultures were transferred to the indicated temperatures.The cultures were incubated on Luria-Bertani (LB) medium, supplemented with appropriate antibiotics .α-glucosidase activity was measured as described . For the600 mL were dissolved in 200 μL lysis buffer containing 8 M urea, 4% CHAPS, 60 mM DDT, 2% Pharmalyte 3–10 and 0.002% bromophenol blue and incubated for one hour at room temperature. The first dimension was carried out on an IPGphor using 24 cm pH gradient 3–10 strips (amersham pharmacia biotech), with the voltage profile recommended by the manufacturer. The second dimension was run in an Ettan Dalt six (amersham pharmacia biotech) on 12.5% polyacrylamide gels. Gels were stained with Coomassie brilliant blue for quantification or by silver staining for visualisation. Gels for cultures incubated at 42°C were performed in quadruplicate, for other temperatures once. Protein spots were identified by comparison with the E. coli 2D database . Spot identities were verified by N-terminal sequencing for ClpB (RLDRLTN) and enolase (SKIVKII). α-glucosidase was not dissolved from the inclusion bodies and is not visible in the gels.Cell pellets from a culture volume of 8/OD

How genomics and related health biotechnologies can improve the health of the poor and contribute towards meeting the Millenium Development Goals Making New Technologies Work for Human Development, identified technical progress as the largest factor in reducing mortality rates and improving life expectancy from 1960 to 1990 Development experts and policy makers agree that investment in science and technology is important for economic growth and development. The 2001 United Nations (UN) Development Programme report, Genomics and Global Health, presented recently to Task Force 10, we addressed how genomics and related health biotechnologies can improve global health and contribute towards meeting the MDGs This capacity is essential if the world is to achieve the UN Millennium Development Goals (MDGs), which were adopted by all UN members in 2000 in a commitment to promote sustainable development and eliminate poverty in the world. As part of the Millennium Project, the UN established task forces to come up with strategies to help developing countries achieve the MDGs. One of these is the Task Force on Science, Technology, and Innovation (Task Force 10), created in recognition of the fact that many of the goals, especially those related to health and the environment, cannot be realized without a strong contribution from science and technology Genomics refers to the powerful new wave of health-related life sciences energized by the human genome project and the knowledge and tools it is spawning. It is a relatively new science that has tremendous potential to address health problems in developing countries.http://www.malariatest.com/]). Recombinant vaccines, a result of genetic engineering, promise to be safer, cheaper, and possibly easier to store and transport than traditional vaccines The role of genomics in global health has been highlighted previously in the World Health Organization's 2002 report Genomics and Global HealthIn The GGI could also facilitate the sharing of good practices. For example, the Canadian Prime Minister, Paul Martin, in his February 2004 reply to the Speech from the Throne, set a long-term target for Canada to devote 5% of its research and development spending to the challenges of developing countries. If successfully implemented and replicated by other industrialized countries, this target could make a real difference to global health.Our report concludes that the key actors are developing countries themselves. We explore how to put genomics and related technologies to work in developing countries within the next Today there are examples of strategies that some developing countries, including China, Cuba, Brazil, India, and South Africa, have followed to institute learning processes that are helping them to build NSIs in biotechnology. China seized the opportunity to take part in the Human Genome Project and quickly set up major institutions in genomics, such as the Beijing Genomics Institute, equipped with state-of-the-art sequencing facilities and computers. It has also followed a strategy of private-sector development in line with the NSI framework. Because of a government policy encouraging their return, Chinese expatriates are increasingly active in setting up small health biotechnology firms, bringing to the country knowledge from the world's major centers for genomics and related technologies.The government in Cuba became interested in biotechnology in the early 1980s when the field was still in its infancy and created an interdisciplinary group, the Biological Front, to explore the possibilities of the technology for Cuba. It has continued to support biotechnology even during periods of economic hardship, set up institutions with research, development, and manufacturing facilities, and encouraged linkages between these institutions by setting up a biotechnology cluster, the West Havana Scientific Pole. Encouraging linkages has been a core policy of the government in Cuba, and its health biotechnology development has benefited from the ties with a strong public health system.Brazil has a relatively long history of supporting science and technology, and the country is increasingly focusing on genomics and related technologies. The lack of commercialization of its cutting edge science and technology has been a weakness of the system in Brazil, but the country is now trying to overcome this weakness by proposing an “Innovation Law” that encourages cooperation between universities and the private sector.Since its independence in 1947, India has followed a vision to improve the quality of life of its people by emphasizing science and technology. Limited resources and a patenting system that did not allow patenting of pharmaceutical products but only patenting of processes encouraged firms to come up with low-cost process innovation. This has resulted in health products such as the Shanvac-B hepatitis B vaccine, which is produced in India for a fraction of the cost in developed countries.The South African government's Biotechnology Advisory Committee recognizes that successful commercialization of public-sector-supported research and development requires strong linkages within the NSI. The committee has recommended the creation of several Regional Innovation Centres to act as nuclei for the development of biotechnology platforms that can effectively launch new products and services. These strategies will provide important lessons for many other developing nations as they begin participating in the genomics revolution.First, the development gap between developing countries and the industrialized world continues to grow. The international community is beginning to promote science and technology to reduce this gap. The genomics revolution holds tremendous potential to improve health in developing countries and, if harnessed appropriately, could help to reduce the development divide.Second, genomics and related biotechnologies can help to achieve the UN MDGs. Fast, accurate molecular diagnostic devices, safer recombinant vaccines, female-controlled vaginal microbicides, and low-cost bioremediation tools are some examples of biotechnologies that can have an impact.Third, genomics knowledge has the characteristics of a global public good. In order to harness the benefits of genomics for development, the developing world needs, above all, access to genomics knowledge. Fourth, the promotion of the science of genomics as a global public good and the encouragement of global knowledge flows could best be achieved through international partnerships. A GGI involving an international partnership of public and private entities from both developed and developing countries could catalyze genomics knowledge and learning worldwide.Fifth, countries that have genomics capacity are best positioned to take advantage of the genomics revolution to meet their health needs. For the transfer of technologies to be effective and sustainable, it must be accompanied by transfer of science and knowledge. As well, receiving countries must have the capacity to absorb and use the technology.And sixth, learning is important for building genomics capacity, and is central to the creation of NSIs in biotechnology in developing countries. These countries can strengthen the building blocks of the NSI framework by doing the following: re-energizing academic institutions and public-sector research to strengthen their science base; training people and building human capital to use, adapt, and innovate biotechnologies; encouraging regional and international cooperation to create new channels for knowledge exchange and trade; improving the policy environment to encourage the building of capacity; and fostering the growth of the private sector, encouraging it to address local health needs, and strengthening linkages between public and private sectors to create new biotechnology goods and services.

From a biochemical perspective, a living cell is a collection of molecules jampacked into a confined space by a flexible barrier, called the plasma membrane. A diverse array of proteins embedded in the plasma membrane act as conduits between the cell interior and its external environment, conveying nutrients, metabolites, and information. The life of a cell—as well as that of any multicellular organism—depends on a cell's ability to communicate with its neighbors, both near and far. One way cells do this is with transmembrane receptors outfitted with both extracellular and intracellular domains that mediate information flow between the cell's external and internal environment. One class of transmembrane receptors, called integrin receptors, specializes in interacting with and binding to other cells and the extracellular matrix, a complex of molecules surrounding cells that provides structural support. By integrating various components of the extracellular matrix, integrins , play an important role in such diverse processes as cell differentiation, programmed cell death, wound healing, and metastasis.Integrins can be regulated by signals within the cell to bind to their ligands with either low or high affinity. While a multitude of integrin ligands have been identified and the general mechanics of both the extracellular and intracellular domains of these receptors are known, exactly how a signal crosses the receptor's transmembrane segment to regulate affinity has remained obscure. Now, Bing-Hao Luo, Timothy Springer, and Junichi Takagi have taken a mutational approach to shed light on the inner workings of the transmembrane segment and to explain how it transmits information.Much of what we know about the function of integrins has come from studying the crystal structures and models obtained from structural analysis. These analyses have generated information not only about the structure and composition of the extracellular and intracellular domains of integrins, but also about the conformational changes that accompany signaling events. Integrins contain a large extracellular domain, a transmembrane segment, and a relatively short intracellular “tail.” Integrins are heterodimers—molecules that contain two subunits composed of different amino acids—made up of an α chain and a β chain. Tight association of the two subunits is associated with an inactive, or low-affinity, state of the extracellular ligand-binding domain. Separation of the intracellular subunits is associated with a dramatic conformational change and activation of the extracellular domain, changing a bent structure with a downward-pointing ligand-binding site into an extended one with an outwardly stretched ligand-binding site. This mechanism differs from most transmembrane signaling molecules, which usually achieve activation through association with their target molecules.To investigate how the transmembrane segment mediates these changes, Luo, Springer, and Takagi systematically replaced amino acids in both the α and β transmembrane domains of the heterodimer with cysteines, creating the potential for binding interactions through a chemical reaction, disulfide bond formation, between the two subunits. By analyzing 120 possible cysteine pairs, the researchers not only confirmed the structure of the transmembrane region as helical but also mapped the proximal amino acid residues between the helices. To understand how the helical transmembrane domains transmit signals, the team introduced activating mutations in the amino acids of the α subunit cytoplasmic tail. Using this approach, they observed the loss of the contact between the subunits, indicating a separation of the transmembrane helices. Furthermore, when disulfide bond formation occurred, linking the transmembrane segments together, activation was suppressed. While previous models had proposed various modes of subunit movements, including hinge- and piston-like models, these results strongly support the notion that lateral separation of the subunits is the driving force behind the signal. As many diseases arise from defects in integrin adhesion, understanding the conformation and mechanism of integrin activation could suggest promising avenues for drug development aimed at correcting such defects.

In a series of 11 microsatellite stable (MSS) and 9 microsatellite unstable (MSI) colon cancer cell lines and primary colon carcinomas (25 MSS and 28 MSI) with known ploidy stem line and APC, KRAS, and TP53 mutation status, we analyzed the promoter methylation of the following genes: hMLH1, MGMT, p16INK4a (CDKN2A α-transcript), p14ARF (CDKN2A β-transcript), APC, and E-cadherin (CDH1). We compared the DNA methylation profiles of the cell lines with those of the primary tumors. Finally, we examined if the epigenetic changes were associated with known genetic markers and/or clinicopathological variables.Tumor cell lines are commonly used as experimental tools in cancer research, but their relevance for the hMLH1, MGMT, p16INK4a, p14ARF, APC, and E-cadherin, respectively, whereas 21%, 40%, 32%, 38%, 32%, and 40% of the primary tumors were methylated for the same genes. hMLH1 and p14ARF were significantly more often methylated in MSI than in MSS primary tumors, whereas the remaining four genes showed similar methylation frequencies in the two groups. Methylation of p14ARF, which indirectly inactivates TP53, was seen more frequently in tumors with normal TP53 than in mutated samples, but the difference was not statistically significant. Methylation of p14ARF and p16INK4a was often present in the same primary tumors, but association to diploidy, MSI, right-sided location and female gender was only significant for p14ARF. E-cadherin was methylated in 14/34 tumors with altered APC further stimulating WNT signaling.The cell lines and primary tumors generally showed similar overall distribution and frequencies of gene methylation. Among the cell lines, 15%, 50%, 75%, 65%, 20% and 15% showed promoter methylation for in vitro models, comparable with the in vivo situation, as the cell lines display many of the same molecular alterations as do the primary carcinomas. The combined pattern of epigenetic and genetic aberrations in the primary carcinomas reveals associations between them as well as to clinicopathological variables, and may aid in the future molecular assisted classification of clinically distinct stages.The present study shows that colon cancer cell lines are in general relevant RB, p15INK4b, p16INK4a), the TP53 pathway (p14ARF), the WNT signaling pathway , DNA repair , apoptosis (DAPK), and the metastasizing process .Development of colorectal cancer through various morphological stages has been linked to several genetic and epigenetic changes. The majority of carcinomas have several chromosomal aberrations, a phenotype often referred to as chromosomal instability. Approximately 15% of the tumors are near diploid but exhibit microsatellite instability (MSI), seen as genome-wide short nucleotide insertions and deletions . This phin vitro studies. Although many of the known genetic aberrations in colon cancer cell lines have been comprehensively described [Human cancer cell lines are important tools in cancer research. Their commercial availability and unrestrained growth make them well suited for escribed , severalThe frequencies of both methylation and gene mutation differ among various studies of cell lines and primary tumors. The genome characteristics, profiles of gene mutations, and methylation status are rarely reported in the same samples, let alone in large series. In the present report we address these potentially connected pathogenetic mechanisms by presenting methylation profiles of a set of genes in a series of MSI and microsatellite stable (MSS) colon cancer cell lines and primary colorectal carcinomas. The methylation profiles are compared with various known genetic and clinicopathological features of the same series.hMLH1, MGMT, p16INK4a, p14ARF, APC, and E-cadherin, respectively, whereas 0/11, 5/11, 8/11, 5/11, 2/11, and 1/11 of the MSS cell lines were hypermethylated for the same genes (Table The colon cancer cell line methylation-specific PCR (MSP) results are summarized in Table es Table , 2a. In Methylation status was assessable in more than 99% of the total number of analyses .hMLH1 promoter to 61% among the MSI tumors for the p14ARF gene are shown in Table P = 0.231, Mann-Whitney test).Several of the primary tumor samples displayed widespread CpG island methylation Figure . Eighteep14ARF (P < 0.001) and hMLH1 (P = 0.015). Sixteen of 49 primary tumors harbored TP53 mutations, and all of the tumors with TP53 mutations also harbored unmethylated hMLH1 (P = 0.009). p14ARF hypermethylation was less common in tumors with mutated TP53 than in tumors with wild type TP53, although this was not statistically significant (P = 0.127). Four tumors displayed a G:C to A:T TP53 mutation and three of them simultaneously harbored a methylated MGMT gene. Four of 11 tumors with G:C to A:T KRAS (KRAS2) mutations were methylated at the MGMT promoter. Overall, the presence of KRAS mutations was not associated with the methylation status of the genes analyzed. Among the 20 tumors with p14ARF methylation, 10 were also methylated at the adjacent p16INK4a gene (P = 0.067). Finally, the APC promoter was methylated in 17/53 (32%) tumors, and 8/17 (47%) tumors displayed both APC mutation and methylation.The methylation status of the primary tumors was compared with genetic characteristics of the same tumors Table . In geneP = 0.080). We found no statistically significant associations between tumors with widespread methylation and presence of TP53, KRAS, or APC mutations.Among the tumors with widespread methylation (3 or more methylated genes), 13/18 (72%) tumors demonstrated MSI, whereas 5/24 (21%) were MSS (hMLH1 (P = 0.043) and p14ARF (P = 0.050). Tumors from patients younger than the mean age (68 years) had a lower methylation frequency for p16INK4a than did tumors from older patients, although this was not statistically significant (P = 0.074). There was a strong association between methylation and right-sided tumor location as 10/11 (91%) tumors methylated in hMLH1 and 12/19 (63%) of the tumors methylated in p14ARF were located in the right side of the colon . There was no statistically significant association between methylation and histological grade. Most of the tumors with APC methylation belonged to the Dukes' B group, but the differences were not statistically significant (P = 0.068).The clinicopathological features and methylation status of the primary tumors are summarized in Table P = 0.035). We saw no statistically significant associations between presence of widespread methylation and the remaining clinicopathological variables included in the present study.Tumors with widespread methylation (≥ 3 loci) are associated with right-sided localization; 10/17 (59%), versus 5/17 (29%) left-sided have also shown similar frequencies of TP53, KRAS and APC mutations [in vitro system is a suitable experimental tool that closely reflect the in vivo situation.It has been debated for some time whether cell lines are more frequently methylated than primary tumors . Regardiutations and ploiutations , which fhMLH1 primers we designed amplify a region of the promoter, in which methylation invariably correlates with the lack of hMLH1 expression [Previously reported variations in promoter hypermethylation frequencies of different tumor suppressor genes in colorectal cancer can be explained by various ratios of MSI versus MSS samples in the series analyzed, different methods for analyzing methylation, the inter-individual variation in scoring of methylated samples, incomplete bisulphite modification, tumor heterogeneity, and the fact that different parts of the gene promoter region in question have been analyzed. In the present study, we used primer sets known to only detect methylation in tumor cells, never in normal tissues from the same patients ,31,38-42pression ,43,44. Mpression ,43,44.hMLH1 and p14ARF genes, whereas the four additional genes analyzed revealed similar methylation frequencies in the MSS and MSI groups. Promoter methylation of the hMLH1 gene was, not surprisingly, found only in tumors and cell lines with MSI, not in the MSS samples. The MSS tumors and cell lines per definition contain functional hMLH1 protein, and transcriptional silencing of hMLH1 by hypermethylation is known to be the main cause of MSI in sporadic CRC [p14ARF methylation may have a specific role in MSI tumors, since it seems to be most often inactivated in tumors with wild type TP53 (see below). However, the relatively high methylation frequencies of the remaining analyzed genes, and also their overall similar frequency in MSI and MSS samples, imply that they are important in colorectal carcinogenesis independently of tumor site and MSI status.As expected, the MSI primary tumors showed more methylation overall than did the MSS group. However, this was only significant for the adic CRC ,28,45. Ap16INK4a, have been reported to be methylated consistently in most tumor types analyzed [p16INK4a methylation frequencies vary from 18% [p16INK4a and p14ARF are more commonly methylated in tumors with MSI than in MSS [p14ARF is higher than that for p16INK4a in MSI colorectal carcinomas.Inactivation of tumor suppressor genes by promoter hypermethylation has been recognized to be at least as common as gene disruption by mutation in tumorigenesis . Indeed,analyzed . In colofrom 18% to 50 % from 18% with mosfrom 18% ,46,49-51n in MSS ,11,51-536-guanine by an irreversible transfer to an internal cysteine residue [6-guanine has a tendency to base pair with thymine during replication, thereby introducing a G:C to A:T transition mutation in the DNA [MGMT gene has previously been reported to be associated with G:C to A:T mutations in the tumor suppressor gene TP53 [KRAS [TP53 but seemingly not for KRAS, although no certain conclusions can be drawn from the limited number of samples with G:C to A:T mutations.The DNA repair protein MGMT is able to remove promutagenic alkyl groups from O residue . Left un the DNA . Inactivene TP53 and the 53 [KRAS ,58. Our ARF protein interacts in vivo with the MDM2 protein, neutralizing MDM2's inhibition of TP53 [p14ARF in tumors with mutated TP53 than in tumors with wild type TP53 has been reported previously [TP53 mutation in colorectal carcinomas [p14ARF gene in MSI tumors with few TP53 mutations is in agreement with a recent study [The p14 of TP53 . Less hyeviously . Additiorcinomas -62. The APC gene is frequent in colorectal and other gastrointestinal carcinomas, usually by truncating mutations [APC methylation in the upper range of what has been seen in previous studies [APC are common in colorectal cancer [APC mutations in our study were also methylated. We have not looked at allele-specific mutation, but methylation and mutation in the same tumor might reflect one mutated allele and methylation of the other, in accordance with Knudson's two hit hypothesis. This has previously been demonstrated for APC in colorectal cancer samples by Esteller et. al [MYC and CCND1 (cyclin D1) further stimulating cell proliferation [APC caused by methylation (n = 17) and/or gene mutation (n = 26). The E-cadherin gene was also methylated in 14/34 tumors with altered APC, presumably further stimulating WNT signaling [APC methylation seemed to be more common in Dukes B stage tumors.Inactivation of the utations ,64. An a studies ,65,66. Sl cancer ,68 and, l cancer ,22,69, aferation . Among tignaling . IntereshMLH1 in sporadic carcinomas is associated with proximal tumor location in the large bowel [hMLH1 promoter were taken from the right side of the colon. An association between sporadic proximal colon carcinomas and methylation has also been reported for p16INK4a and p14ARF [p14ARF. However, p16INK4a demonstrated the same tendency. Both hMLH1 and p14ARF are strongly associated with MSI and MSI is in turn strongly associated with proximal tumor location [The present study confirms that methylation of ge bowel ,21,45,70location ,72, hencp14ARF does not exclude p16INK4a methylation, is in accordance with previous studies [p16INK4a or p14ARF methylation with female gender and increased age has been described in some studies [p14ARF and hMLH1, but not of p16INK4a. We also found a weak association between p16INK4a methylation and increasing age. This potential age-specific methylation was not confirmed for any of the other genes studied. The gender-associated methylation of hMLH1 has previously been described [When it comes to gene methylation and its association with other clinicopathological features, contradictory results have been reported. Our observation that methylation of studies ,24. Corr studies but not studies ,21,24. Wescribed ,74 and met. al [Like Toyota et. al , we founin vivo situation. The methylation profile of specific genes, in particular hMLH1 and p14ARF, has strong associations with genetic and clinicopathological features and might be related to biologically distinct subsets of colorectal tumors.The data presented here demonstrate that multiple genes are methylated in colorectal carcinomas. This underlines the important role epigenetic inactivation of tumor suppressor genes plays during the process of tumor development. Epigenetic changes in colon cancer cell lines are overall comparable with those of primary carcinomas of the large bowel, which make the cell lines relevant models for the TP53, KRAS and APC [et. al [Fifty-three primary colorectal carcinomas from 52 patients, including 25 MSS tumors and 28 MSI tumors, were submitted to methylation analyses. One of the tumors was from a patient with hereditary non-polyposis colorectal cancer (HNPCC), whereas the rest of the cases were sporadic . The tum and APC ,64,77. T and APC ,78-80, 3 and APC , and tot [et. al . The pri [et. al . The DNAhMLH1, MGMT, p16INK4a, p14ARF, APC and E-cadherin by MSP, a method that distinguishes unmethylated from methylated alleles of a given gene [Promoter methylation was studied in ven gene . After bMGMT [p16INK4a [p14ARF [APC [E-cadherin fragments [hMLH1 fragments were designed in accordance with hMLH1 promoter methylation and gene expression studies [One or two μg DNA from each sample was modified as described . PreviouMGMT ,82, p16Ip16INK4a ,82, p14A [p14ARF , APC [39ARF [APC and E-caragments , 200 μM dNTP , and 0.625 U HotStarTaq DNA Polymerase (QIAGEN). PCR products were loaded onto 7.5% polyacrylamide gels, stained with ethidium bromide, and visualized by UV illumination. An independent second "methylated reaction" of the MSP was performed for all the samples included in the present study. In cases with diverging results from the two rounds of MSP, we did a third independent MSP round.All the PCRs were carried out in a total volume of 25 μl containing 1 × PCR Buffer was used as a positive control for MSP of methylated alleles, whereas DNA from normal lymphocytes was used as a control for unmethylated alleles. Water was used as a negative PCR control in both reactions.Human placental DNA treated 2 test. Two of the statistically significant cross-tables analyzed by the Pearson χ2 had cells with expected count less than 5, with a minimum count of 2.96 (Table P values are derived from two tailed statistical tests using the SPSS 11.5 software.All 2 × 2 contingency tables were analyzed using Fisher's exact test. Three × 2 tables were analyzed by the Pearson χ96 Table . The ManAPC mutation status in the cohort. GIM and TOR have collected the series of human primary tumors and provided all clinical and pathological information. RH provided all cell lines and information about them. ME contributed with scientific discussions of the results and participated in the writing of the manuscript. RAL conceived the study, was responsible for its design and coordination, and contributed in the evaluation of the results and in preparation of the manuscript. All authors have read and approved of the final manuscript.GEL cultured and isolated DNA from all cell lines and carried out the MSP analyses of these and of the patient samples. GEL interpreted the results, performed the statistics and drafted the manuscript. LT participated in the study design, scored the MSP results independently of author 1, and contributed to manuscript preparation. TL was responsible for the update of the Additional file 1 lists the MSP primers used in the present study.Click here for file

Totally implantable central venous access devices (intraports) are commonly used in cancer patients to administer chemotherapy or parenteral nutrition. Rupture of intraport is a rare complication.During 3 years period, a total of 245 intraports were placed in cancer patients for chemotherapy. Four of these cases (two colon cancer and one each of pancreas and breast cancer) had rupture of the intraport catheter, these forms the basis of present report.insitu for intraports was 164∀35 days. Median follow-up time was 290 days and total port time in situ was 40180 days. The incidence of port rupture was 1 per 10,000 port days.Mean time Three of the 4 cases were managed by successful removal of catheters. In two of these the catheter was removed under fluoroscopic control using femoral route, while in the third patient the catheter was removed surgically. One of the catheters could not be removed and migrated to right ventricle on manipulations.Port catheter rupture is a rare but dreaded complication associated with subcutaneous port catheter device placement for chemotherapy. In case of such an event the patient should be managed by an experienced vascular surgeon and interventional radiologist, as in most cases the ruptured catheter can be retrieved by non operative interventional measures. Totally implantable central venous access devices (intraports) are commonly used in patients with cancer to administer chemotherapy, blood and blood products, antibiotics, parenteral nutrition and to obtain blood samples for laboratory analysis. The catheter is usually placed in the subclavian vein under local anesthesia. There are many complications associated with this technique like hemothorax, pneumothorax, pocket infection, infection of the tunnel or the port, bleeding, hematoma and thrombosis of the catheter or the vein. A very rare complication of the intraport catheter is rupture inside the subclavian vein .The literature regarding common complications is abound, however there is very little information on port ruptures. Some attempts has been made to correlate these with type of port used, site of placement, type of chemotherapy, duration of catheter use etc ,3. Howev® with titanium portal and detachable silicon rubber catheter were used in all cases. All patients requiring removal or replacement of the device, prior to completion of the intended treatment were identified from the operative room and hospital records.Between June 1995 and May 1998, 245 patients underwent intraports insertions at the "Agii Anargiri" Anticancer Hospital of Kifissia, Athens, Greece, and were followed up for possible device-related morbidity. Baseline demographic information and the indication for the placement were obtained from the retrospective review of patients' medical and operative reports. Follow-up continued till the device was removed or the patient died. Follow-up time ranged from 1 month to over 3 years. Port-A-CathAll intraports were inserted by percutaneous access technique to the subclavian vein. The tip of the catheter was positioned in the superior vena cava or the right atrium. The ports were then placed within a subcutaneous pocket created on the anterior chest wall. The position was documented by immediate intraoperative chest roentgenogram. Four of these patients had port rupture of which three were complete and one partial, one of these ruptures was accidental.Routine removal of the subcutanous intraport was carried out in the operating room under local anesthesia with xylocaine 1% (10 cc). An incision was placed on the skin in the area over the drum of the device. Then the drum was prepared by cutting off the tissues that surround the port. The port and the catheter were caught with Kocher forceps. The tissues around the catheter were dissected and the catheter was slowly pulled out. The catheter was cut into three pieces and was sent for culture. Patients were discharged after two hours of observation.in situ was 40180 days, while mean (SD) port time in situ was 164 (35) days.Over a three year period between 1995 and 1998 a total of 245 port devices were fitted in cancer patients . Mean age of the patients was 58 ± 6.3 years. Total port time After a median follow-up of 290 days (range 30–690 days) four ports ruptured, thus bringing the number of fractures per 1000 port days to 0.01% or one rupture every 10,000 port days. Two patients had cancer of the colon and one each had cancer of breast and pancreas. Both patients with colonic cancer received 5 flurouracil and leukovorin, patient with breast cancer received epirubicin and paclitaxal and patient with pancreatic cancer received gemcitabine, paclitaxal and cisplatinum. No correlation was observed with type of chemotherapy or site of disease. Three of the port ruptures were full and one partial one of the ruptures was accidental (case 1). The detail of the cases has been provided in next section. Three of the patients survived for two years after the catheter removal, while the fourth patient with accidental port rupture and whose catheter was left behind in the right ventricle died of progressive disease two month later.enoxaparin sodium, 1 mg/kg/12 h for 5 days and 40 mg/day for further 14 days in order to prevent thromboembolic event. The patient died two months later due to progressive disease without obvious complications related to the retained catheter.A 65-years-old female with colonic cancer, received intraport for chemotherapy administration. After 6 months due to the poor response to chemotherapy it was decided to remove the catheter. During the manipulations for removal, the catheter, accidentally ruptured at the point of its entrance in to subclavian vein. The peripheral part of the catheter remained in the vein, while only the central part could be removed. Another attempt to uncover the subclavian vein till superior vena cava failed. Patient underwent thoracotomy for removal of remaining catheter two days later. Superior vena cava was opened and catheter removal was attempted, however, during this process the catheter slipped into right atrium and further attempts to retrieve it were abandoned. Patient was started on anticoagulant treatment using et al [A 68-year-old female with colonic cancer received intraport for administration of chemotherapy. After one and a half year of treatment it was decided to remove the catheter as the catheter has thrombosed due to non heparinization. At the time of its removal the catheter ruptured at the point of its entry to subclavian vein. The peripheral part of the catheter remained in the vein. An unsuccessful attempt was made to expose subclavian vein till superior vena cava. Later this catheter migrated to right ventricle. The catheter was removed using the technique of Yedlicka et al , throughA 74-years-old female suffering from breast cancer underwent intraport insertion for administration of chemotherapy. After fourteen months of treatment it was decided to remove the catheter as the catheter had thrombosed. On attempted removal the catheter was found to be ruptured at its entry to subclavian vein (Figure Next day, the broken part of the catheter was removed successfully under fluoroscopic control using the technique described above for case 2 (Figure n Figure . Next da2 Figure .In a 56-year-old female patient with pancreatic carcinoma underwent an intraport placement for chemotherapy. Eight months later, the patient complained of pain in the back during the administration of chemotherapy. A fluoroscopic examination showed partially broken catheter in the vein while the other part was lying in the subcutaneous tissue. The catheter was removed from the subclavian vein carefully to avoid complete rupture of the catheter. Similar to case 3 above, the biomedical examination showed a significant reduction in the elasticity of the catheter material.Since Aubaniac first described the percutaneous central venous catheterization in 1952, insertion of central venous access devices for fluid administration has increased rapidly. The total complication rate associated with central venous catheter devices ranges from 0.4 to 29% ,4,6.et al, in 1997 [Spontaneous rupture of the port catheters appears to be a very rare and dreaded event. Biffi in 1997 .The biomechanical analysis, of ruptured catheters in our series showed a significant decrease in the elasticity of the material. No correlation between changes of mechanical properties of the material and specific chemotherapy administrated through the port has been established so far ,8. Even In case 1 the port catheter rupture was due to wrong manipulations during catheter removal and accidental cutting of peripheral part without first holding it in clamp. The further catheter movement was induced by negative intra-thoracic pressure during respiration . Other pet al [Ballarini et al , suggestet al ,10-12. IThere is no controversy that the foreign bodies inserted in the systematic circulation must be removed. This is best achieved under fluoroscopy with specific catheters and snare loops. Removal of intravascular material by means of minimally invasive techniques presents excellent results, while at the same time it minimizes morbidity and mortality ,14. If sIntravascular rupture of subcutaneous port catheters is a rare complication. Etiology still remains elusive, however wrong placement has been advocated as the most important cause. Other causes include catheter material faults and alterations of the material's mechanical properties, probably due to the administered substances; however, there is no data to support the effect of administered substances. Ruptured catheters are best removed by minimally invasive radiological techniques.The authors declare that they have no competing interests.DF participated in the operations, and drafted the manuscript.CT participated in the operations, patients follow-up search of literature and preparation of draft.GF conducted the follow-up and helped to draft the manuscript.AN and SR participated in the operations, design of the study and preparation of manuscript for publication.All authors read and approved the final manuscript.

As part of a long-term initiative to improve cancer surveillance in New York State, small area maps of relative risk, expressed as standardized incidence ratios (SIRs), were produced for the most common cancers. This includes prostate cancer, the focus of this paper, since it is the most common non-dermatologic malignancy diagnosed among men and the second leading cause of cancer deaths for men in the United States.ZIP codes were chosen as mapping units for several reasons, including the need to balance between protecting personal privacy and public demand for fine geographic resolution. Since the population size varies greatly among such small mapping units, hierarchical Bayes spatial modelling was applied in this paper to produce a map of smoothed SIRs. It is further demonstrated how other characteristics of the large sample from the stationary posterior distribution of SIRs can be mapped to investigate various aspects of the statewide spatial pattern of prostate cancer incidence.Thematic mapping of the median and 95 percentile range of SIRs provided, respectively, a map of spatially smoothed values and the uncertainty associated with these smoothed values. Maps were also produced to identify ZIP codes expressing a 95% probability, in the Bayesian paradigm, of being less than or greater than the null value of 1.The model behaved as expected since areas that were statistically elevated coincided with areas identified by the spatial scan statistic, plus the relative uncertainty increased as a ZIP code's population decreased, with an exaggerated effect for low population ZIP codes on the edge of the state border.The overall smoothed pattern, along with identified high and low areas, may reflect difference across the state with respect to socio-demographics and risk factors; however, this is confounded by potential differences in screening and diagnostic follow-up. Nevertheless, the Bayes modelling approach is shown to provide not only smoothed results, but also considerable other information from a large empirical distribution of outcomes associated with each mapping unit. Geographic surveillance of chronic disease is central to understanding spatial or spatial-temporal patterns that may help to identify discrepancies in disease burden among different regions or communities. As part of ongoing efforts in New York State to understand spatial patterns of cancer and to help implement cancer prevention and control programs, small area maps of cancer relative risk, expressed as standardized incidence ratios (SIRs), have been produced and shared with the public for the Prostate cancer, the focus of this paper, was included because it is the most common non-dermatologic malignancy diagnosed among men and the second leading cause of cancer deaths for men in the United States (US) . AlthougResults for prostate cancer are reproduced in Figure It is well recognized that the stability of population-based statistics like the SIRs in Figure y, as a random variable that has arisen from a probability distribution with expectation θ. This expectation is modeled, via an appropriate link g(·), as a linear function g(θ) = α + β x'+ ε, for a common value α, explanatory covariates β x'and a random effect ε that captures unexplained variation. If the random effect is associated with exchangeable spatial heterogeneity, estimates are smoothed towards a global mean, whereas if the random effect is associated with local spatial autocorrelation, estimates are smoothed towards a local neighborhood mean, which is typically more meaningful in geographic epidemiology. There are different approaches to modelling local spatial dependence, and section 6.3 of Cressie , where l is the mean maximum achievable log likelihood, obtained for a saturated model where a parameter is assigned to each datum, and l is the mean log likelihood obtained for the model in question. This takes the conventional assessment of deviance for generalized linear models [Variations of the model defined above were compared by evaluating the mean deviance of 1000 iterations chosen from the three independent Markov Chains after burn-in. This was done by obtaining the mean of -2(log likelihood) for each iteration, as provided by the r models and applr models .Incorporating a random effect associated with local spatial structure (CAR term) provides much stronger prior information than the exchangeable random effect alone Table , which a

The insect exoskeleton or cuticle is a bi-partite composite of proteins and chitin that provides protective, skeletal and structural functions. Little information is available about the molecular structure of this important complex that exhibits a helicoidal architecture. Scores of sequences of cuticular proteins have been obtained from direct protein sequencing, from cDNAs, and from genomic analyses.Most of these cuticular protein sequences contain motifs found only in arthropod proteins.Drosophila melanogaster and the Anopheles gambiae genomes, that have been predicted to be cuticular proteins, based on a Pfam motif (PF00379) responsible for chitin binding in Arthropod cuticle. The total number of the database entries is 445: 370 derive from insects, 60 from Crustacea and 15 from Chelicerata. The database can be accessed from our web server at .cuticleDB is a relational database containing all structural proteins of Arthropod cuticle identified to date. Many come from direct sequencing of proteins isolated from cuticle and from sequences from cDNAs that share common features with these authentic cuticular proteins. It also includes proteins from the CuticleDB was primarily designed to contain correct and full annotation of cuticular protein data. The database will be of help to future genome annotators. Users will be able to test hypotheses for the existence of known and also of yet unknown motifs in cuticular proteins. An analysis of motifs may contribute to understanding how proteins contribute to the physical properties of cuticle as well as to the precise nature of their interaction with chitin. One particular family of cuticular proteins constitutes one of the largest multigene families known in insects . UnrelatThe database nomenclature is based either on the names given by those who deposited the sequences or on codes assigned by genome projects. Thus, we have retained the existing names/codes for the convenience of the users.cuticle, exoskeletal, carapace) to the Protein databases of Entrez and Uniprot (release 1.8) [Anopheles gambiae and Drosophila melanogaster, from Ensembl [The data collection has been basically done in two ways. First, by submitting appropriate keywords ase 1.8) we colle Ensembl and EBI, Ensembl . This mo Ensembl and most we coll Ensembl . A short Ensembl , and, as Ensembl , was wid Ensembl ,3and refIn order to ensure that our data collection is complete, we scanned all protein sequences of Uniprot (release 1.9) for PF00379. Again manual filtration was required. In addition to PF00379, other motifs have been described in cuticular proteins, some are found along with PF00379 while others define other families of cuticular proteins ,3. All rThe data for our database was obtained by parsing the fields Definition, Accession, GI from Version, Organism and Origin from the Entrez entries. From the Uniprot entries we used Primary accession number, Protein name, Origin of the protein, Cross-references and Sequence information. This retrieval was done with Perl scripts. Additional information, concerning temporal and local expression of the proteins or corresponding mRNAs, was drawn from literature.The data have been organised based on a relational model and is stored in a PostgreSQL database system. The user has supervisory access through our Apache web-server. The database is managed by an interferential software, written in Java, which tends to settle any web-server's query. Also, it implements a homemade computational tool that performs motif search as described below.The main page of cuticleDB includes the following interfaces: Introduction, Data Retrieval, User manual and Contact. On clicking the Data Retrieval icon, users are presented with the search interface of the database. The query can be done in two ways: either by searching in fields or by gathering a set of proteins Figure .The separate fields in which the user may search are Protein name, Taxonomy, references in other databases and the protein sequence. The protein sequence can be searched against any pattern according to the user's imagination and, therefore, hypotheses for novel motifs can be tested. This is performed by a separate, homemade tool that has been integrated in cuticleDB and which gives the user the opportunity to detect new motifs in cuticular proteins. The integration of this tool is of importance especially in a database such as this, given the significance of motifs not only in cuticular proteins, but in structural proteins in general.Users can gather all protein entries from a single species (35 species are included in cuticleDB) or all protein entries whose protein sequence contains one of a series of motifs. However, this series of motifs has been pre-selected by the constructors of the database and cannot be modified by the user. The selection criterion was the frequency of appearance of these motifs in the literature. The most commonly found motifs were searched against all protein sequences of the database and have properly been assigned to each entry.A typical cuticleDB entry contains the following fields: Protein Name, References to other databases , Taxonomy, Expression Details, Protein Sequence and its Length, Database-Source of the sequence and the method by which the sequence was obtained Figure . The fieD. melanogaster, A. gambiae). The only verified cuticular proteins are those where the complete protein sequence or a unique N-terminal region was determined from a protein extracted from a cleaned cuticle or where a specific antibody reacted with proteins in cuticle or extracted from it. Finding mRNA in the epidermis is presumptive evidence that a protein is cuticular. The majority of cuticular proteins in this database were designated as cuticular proteins based on their sequence similarity to authentic cuticular proteins. Such proteins where sequence is the sole criterion for assignment are marked as "putative" in the database. Furthermore, at present, the annotation of the proteins of A. gambiae is preliminary. Many proteins are missing signal peptides, other clearly have been incorrectly assembled. Such sequences are marked as preliminary as well as putative. This database will be continuously updated at regular intervals to accommodate annotation.Taxonomic data are taken from Entrez. The total number of entries in cuticleDB is 445. These proteins are distributed in the three large taxa: Insecta (370 entries), Crustacea (60 entries) and Chelicerata (15 entries). The database includes entries from 6 orders of the class Insecta: Diptera (258 entries), Lepidoptera (39 entries), Orthoptera (37 entries), Hemiptera (6 entries), Coleoptera (22 entries), Dictyoptera (8 entries). The large number of proteins in Diptera is due to the inclusion of cuticular proteins from the two genomes . Both oSubsequently, we used these Profile Hidden Markov Models as a prediction tool for classifying the cuticular proteins into two groups RR1, RR2. The prediction was in agreement with the literature as far as the known RR1 and RR2 proteins are concerned. The total number of RR1 and RR2 proteins in cuticleDB are 132 and 148, respectively. The start and end positions of the two motif-types are shown in the corresponding entry of each protein. A smaller class, RR-3, with 75 conserved residues was also identified by Andersen [We have also studied the appearance of another motif: AAP(A/V). This small, hydrophobic tetrapeptide has been found to occur mainly in proteins of hard cuticles ,3, whereThe most severe problem of genome projects to date is that of correct annotation. So, accurate and specialized databases as cuticleDB with its description of highly conserved motifs will be of help to genome annotators. Therefore, cuticleDB can be used as a basis for annotating new cuticular proteins by similarity in future Arthropod genome projects.cuticleDB can also be utilized in molecular research as well, due to its focus on motif appearance. Cuticular proteins, as is the case with all structural proteins are marked by the presence of characteristic motifs. Some motifs are repeated within a protein sequence, whereas others appear only once. cuticleDB has been designed in such a way that the user can have a complete view of motif occurrence in the sequence of each protein entry. First, each entry shows the exact position of the most common cuticle motifs in the protein sequence. Secondly, the user is given the opportunity to search the sequence for novel motifs and therefore, test hypotheses for the existence of new patterns. Subsequently, hypotheses for possible interactions between cuticle macromolecules (either proteins with chitin or proteins with proteins) can be tested. Moreover, our results of the RR1 and RR2 predictions can be used as a guide for identifying a certain protein as coming from either soft or from hard regions of the cuticle. Most importantly, the information about the RR1 and RR2 distinction can be used for studies of cuticle's mechanical properties. As RR1 and RR2 proteins appear in soft and hard cuticles respectively, which means that the former interact with chitin more loosely than the latter, one can gain an insight in cuticle's molecular construction combining our data on the sequences of RR1 and RR2 proteins with some experimental work. Moreover, one could use the Expression Details, namely where and when each protein is expressed, when studying the differential construction of the cuticle among different developmental stages or among different regions of a single cuticle.The goal of cuticleDB constructors was the collection of all cuticular protein sequences that have appeared to date and their detailed and correct annotation. The better the organisation of the data, the easier the work will be for researchers dealing with cuticle and structural proteins in general. cuticleDB will help them to answer questions like : 'What kind of proteins appear in hard cuticles?' 'Why do RR2 proteins interact with chitin more tightly than RR1 proteins?' 'Which motifs contribute to protein-protein interaction in the cuticle?' 'From which stage can a certain protein be extracted?' Furthermore, it is hoped that, detection of common properties of these proteins, as well as recognition of important differences that are responsible for cuticle's complexity and important functions will be facilitated by its existence.Last but not least, it is hoped that this database will be of help to genome annotators in the near future as more arthropod genomes become available.. An e.mail biodb@biol.uoa.gr may also be used for comments, corrections and further data (sequence) submission.cuticleDB was created and is maintained in the Department of Cell Biology and Biophysics, Faculty of Biology of the National and Kapodistrian University of Athens. It is freely available at the URL: RR1: The extended Rebers and Riddiford Consensus, type IRR2: The extended Rebers and Riddiford Consensus, type IIHMM: Hidden Markov ModelCKM performed the data collection, and test procedures, and also participated in the design and the implementation of the databaseICS carried out the design of the algorithms and the database,. implemented all the algorithms, and also created the web interfaceVAI supervised the data collection and the testsJHW compiled the first draft of known cuticular proteins, provided a critique of the data base during its constructionSJH coordinated and supervised the whole project, suggesting the general directions and innovative features of the databaseAll authors have read and accepted the final manuscript.

Coronary artery bypass grafting surgery is an effective treatment modality for patients with severe coronary artery disease. The conduits used during the surgery include both the arterial and venous conduits. Long- term graft patency rate for the internal mammary arterial graft is superior, but the same is not true for the saphenous vein grafts. At 10 years, more than 50% of the vein grafts would have occluded and many of them are diseased. Why do the saphenous vein grafts fail the test of time? Many causes have been proposed for saphenous graft failure. Some are non-modifiable and the rest are modifiable. Non-modifiable causes include different histological structure of the vein compared to artery, size disparity between coronary artery and saphenous vein. However, researches are more interested in the modifiable causes, such as graft flow dynamics and wall shear stress distribution at the anastomotic sites. Formation of intimal hyperplasia at the anastomotic junction has been implicated as the root cause of long- term graft failure.Many researchers have analyzed the complex flow patterns in the distal sapheno-coronary anastomotic region, using various simulated model in an attempt to explain the site of preferential intimal hyperplasia based on the flow disturbances and differential wall stress distribution. In this paper, the geometrical bypass models (aorto-left coronary bypass graft model and aorto-right coronary bypass graft model) are based on real-life situations. In our models, the dimensions of the aorta, saphenous vein and the coronary artery simulate the actual dimensions at surgery. Both the proximal and distal anastomoses are considered at the same time, and we also take into the consideration the cross-sectional shape change of the venous conduit from circular to elliptical. Contrary to previous works, we have carried out computational fluid dynamics (CFD) study in the entire aorta-graft-perfused artery domain. The results reported here focus on (i) the complex flow patterns both at the proximal and distal anastomotic sites, and (ii) the wall shear stress distribution, which is an important factor that contributes to graft patency.The three-dimensional coronary bypass models of the aorto-right coronary bypass and the aorto-left coronary bypass systems are constructed using computational fluid-dynamics software (Fluent 6.0.1). To have a better understanding of the flow dynamics at specific time instants of the cardiac cycle, quasi-steady flow simulations are performed, using a finite-volume approach. The data input to the models are the physiological measurements of flow-rates at (i) the aortic entrance, (ii) the ascending aorta, (iii) the left coronary artery, and (iv) the right coronary artery.The flow field and the wall shear stress are calculated throughout the cycle, but reported in this paper at two different instants of the cardiac cycle, one at the onset of ejection and the other during mid-diastole for both the right and left aorto-coronary bypass graft models. Plots of velocity-vector and the wall shear stress distributions are displayed in the aorto-graft-coronary arterial flow-field domain. We have shown (i) how the blocked coronary artery is being perfused in systole and diastole, (ii) the flow patterns at the two anastomotic junctions, proximal and distal anastomotic sites, and (iii) the shear stress distributions and their associations with arterial disease.The computed results have revealed that (i) maximum perfusion of the occluded artery occurs during mid-diastole, and (ii) the maximum wall shear-stress variation is observed around the distal anastomotic region. These results can enable the clinicians to have a better understanding of vein graft disease, and hopefully we can offer a solution to alleviate or delay the occurrence of vein graft disease. The complex anatomy of coronary vessels has made the investigation of coronary flow and hemodynamics one of the most difficult and challenging studies until today. Leonardo Da Vinci , developed insightful sketches (some 550 years ago) of the anatomy of the heart and its blood vessels, as shown in Figure The major cause of death in both the developed and developing countries is cardiovascular disease. The study of coronary arterial circulation is important, because it is crucial in maintaining heart perfusion and function. The process of arteriosclerosis involves the formation of atherosclerotic plaques in the coronary tree. With time, this results in stenosis of the blood vessel and in turn decreases coronary flow. When it cannot match the oxygen demand of the myocardium, it results in ischemia and thereafter in an infarct. In order to overcome this problem of diminished perfusion of the affected myocardium, CABG is one of the treatment options that are being used to improve coronary perfusion. In this procedure, new routes around narrowed and blocked arteries are constructed with both arterial and venous conduits, allowing sufficient blood flow to be delivered to the ischemic heart muscles.In spite of CABG being an effective surgical technique to revascularize the myocardium, 20–50% of bypass grafts fail due to the formation of intimal hyperplasia -3. PreviFlow regimes in end-to-side anastomosis, provide pertinent information concerning wall shear stress gradient which affects intimal hyperplasia thickening. The three- dimensional experimental studies of flow in an end-to-side anastomosis by Ojha et al have revAlthough wall shear stress distribution is a major factor in the onset of coronary diseases it cannot be measured directly and is hence calculated from velocity profiles. Even though these velocity profiles can be obtained in vivo using magnetic resonance (MR) or color-flow Doppler ultrasound (CDU), there are some limitations to measurements due to the small dimensions of the arteries. Thus the alternative to measure the wall shear stress is to use CFD to simulate flow in CABG geometry and thereafter compute the wall shear stress.Unfortunately, most of the modeling work in graft-flow is limited to only a part of the total bypass conduit geometry, namely the anastomosis site. As regards to the anastomosis geometry, Song MH et al developeEven three-dimensional CFD simulations have been performed on end-to-side anstomosis of a stenosed coronary bypass, by Bertolotti and Deplano . Herein,An important factor is the effect of proximal artery flow condition on the hemodynamics at the distal end-to-side anastomosis. This effect has been analysed by Kute and Vorp , using CThe combined use of imaging techniques and CFD may be necessary for meaningful clinical studies. With the availability of imaging techniques like coronary angiography, magnetic resonance angiogram (MRA), computed tomography (CT), several researchers have analysed the flow in realistic bypass graft models. There have also been some studies combining experimental measurements and CFD analyses -17.Doppler analysis is generally preferred since it is safe, accurate and rapid when compared to angiography. Even though angiography certainly gives a definite answer about graft patency, there is more risk involved and it is time consuming and requires the assistance of a cardiologist. Doppler methods have been used by Bertolotti et al and Lin It is clear from literature review that an important step towards realistic flow simulations concerns the generation of appropriate coronary vasculature geometry (involving the entire flow domain from the aorta to the perfused artery) with physiological boundary conditions. Unfortunately, a lot of work in these areas has been carried out on subsections of this bypass flow domain ,15,16,18The bypass models simulating the flow field of the anastomosis in the right and left aorto-saphenous bypass grafts are illustrated in Figures In Figures μ) of 0.00408 Pa and a density (ρ) of 1050 kg/m3. The blood vessel walls are assumed to be rigid and impermeable. For a quasi-steady, three-dimensional and laminar flow, the Navier-Stokes equations (for mass and momentum conservation) governing fluid motion is written as follows:The blood is assumed to be incompressible, with a Newtonian behaviour having dynamic viscosity , the flow field is automatically updated during each time-interval, by adopting the time-varying input data of the aorta at the inlet to the aorta, from the left ventricle (LV). The velocity magnitude is computed from the physiologically representative stroke volume over the ejection period, based on the flow wave form shown inDuring systole, the inputs to the model consist of (i) monitored time-varying flow-rate waveform at the inlet of the aorta, the calculated uniform velocity at the ascending aorta, and (ii) the flow conditions at the left and right coronaries obtained from the monitored time-varying input flow-rate waveforms Figures &3d.The fluid dynamics simulations are performed by using a control-volume-based technique, implemented in the computational fluid dynamics (CFD) code Fluent . The comThe geometry of the aorto-coronary bypass graft models is constructed in Gambit, using the dimensions provided by our cardiac-surgeon joint-author (TYS). The elements employed to mesh the computational domain consisted primarily of regular structured hexahedral elements as well as wedge elements wherever necessary. In order to carry out the mesh sensitivity analysis, numerical simulations were carried out by varying the number of mesh elements in the computational domain. Initially, the domain was discretized into 120974 hex/wedge elements. The accuracy of the simulation results was then improved by employing a finer mesh that contained 419765 elements. This number was further increased to 623138, but resulted in no significant improvement in the results. Thus to maintain a balance between the computational cost and the numerical accuracy, we concluded (based on the mesh independence test) that the appropriate number of elements for our study is 419765.In the solution algorithm used by Fluent, the governing equations are solved sequentially. Because the governing equations are non-linear (and coupled), several iterations of the solution loop need to be performed before a converging solution is obtained. Using this approach, the resultant algebraic equations for the dependent variables in each control volume are solved sequentially by a point implicit (Gauss Seidel) linear equation solver, in conjunction with an algebraic multi-grid (AMG) method. The governing equations are solved iteratively until convergence of all flow variables is achieved. The solutions of all the flow variables are deemed to have converged once their residuals computed from two successive iterations are below the set desired convergence criteria of 10-3. The study was also carried out by setting the convergence criteria as 10-4; this did not alter the results obtained earlier.For these numerical simulations we have incorporated the unsteady flow character by dividing the cardiac cycle into a number of small time interval, and analyzing for steady flow within these time intervals. To observe the velocity distribution features of the entire flow field, the computed velocity vectors are illustrated in the plane of symmetry at two different instants of the cardiac cycle.At the onset of ejection, at t = 0.13 sec, Figure The computed wall shear stress is depicted in Figure At the mid-diastolic instant (at t = 0.7 sec), when the aortic valve is closed, Figure In the anastomosis domain, a strong region of recirculation is observed near the occluded end of the artery, which forces the flow to move into the perfused right coronary artery as indicated in Figure The flow pattern variations give us insight into the wall-shear distribution. The high velocity gradients in the anastomosis give rise to large spatial variations in the resulting wall shear stress. The largest value of wall shear stress is seen in Figure The distribution of velocity-vectors in the flow field at the onset of ejection, t = 0.13 sec is shown in Figure During early ejection, as the left coronary vessel is embedded in the myocardium, the myocardium is subject to intramyocardial pressure which causes some reverse flow. The effect of LV contraction on the graft flow is incorporated by adopting the measured flow profile in the left coronary vessel at a point prior to its anastomosis with graft. In this way, we simulate the reversal of flow in the graft at the onset of ejection.A parabolic velocity profile of the back-flow in the graft is seen in Figure The velocity vectors plots at mid-diastole are displayed in Figure In the anastomosis domain, a strong recirculation region is seen in the heel region of the anastomosis at the distal end of the bypassed vessel, Figure In the anastomosis heel region there is negligible shear stress. However, elevated shear stress (of magnitude 59.43 Pa) is seen in the anastomotic toe region both the proximal and distal anastomotic regions are included, while other works ,15,16,18Our results for the aorto-right coronary bypass model and the aorto-left coronary bypass model at two different instants of the cardiac cycle, clearly reveal the following features: (i) at the onset of ejection, in the aorto-right coronary bypass model, very little flow enters the graft with a velocity magnitude around 0.0495 m/s; the maximum flow velocity inside the graft is around 0.2 m/s; (ii) at the mid-diastolic instant, the flow profile in the graft is skewed towards the outer wall, with the peak velocity increasing as it travels downstream; close to the graft exit, the maximum flow velocity attained is around 1 m/s; (iii) at the onset of ejection, in the aorto-left coronary bypass model, there is a backflow from the left coronary artery into the graft; the peak velocity of flow at the entrance to the graft is 0.0788 m/s, and very little perfusion is given to the host artery; (iv) at the mid-diastolic instant, the graft perfusion is maximum, with peak velocity magnitude of 1.1 m/s.Our study confirms that blood flow through the coronary artery bypass graft primarily occurs only during the diastolic phase of the cardiac cycle. This is in agreement with the physiological observation of coronary blood flow. There is however some difference between the flow patterns in the right and left coronary graft at the onset of ejection, with some backflow from the left coronary artery into the bypass graft which is not obtained in the case of the right coronary arterial bypass. The phenomenon can be explained by the predominant intra-cardiac course of the left coronary artery system. This aspect is also an original feature of our work.Lastly, our study has also shown (i) a low wall shear-stress region near the heel region of the anastomosis domain, and (ii) a high wall shear-stress in the toe region of the anastomosis domain, making it prone to intimal hyperplasia. This may have some clinical significance. We should take this into consideration in designing a coronary anastomotic device, so as to minimize biomechanical injuries to the coronary arterial wall. In doing so, we can alleviate or retard the development of intimal hyperplasia, which is the Achilles heel of the effectiveness of coronary artery surgery with saphenous vein.The computed results have revealed that (i) maximum perfusion of the occluded artery occurs during mid-diastole, and (ii) the maximum wall shear stress variation was observed around the toe of the anastomotic region. According to our cardiac surgeon joint author (TYS), this preliminary result can enable the clinicians to have a better understanding of vein graft disease, and hopefully we can offer a solution to alleviate or delay the occurrence of vein graft disease.MS carried out the computational fluid dynamic studies and drafted the manuscript. DNG and LPC guided the study, helped in interpretation of results and critically reviewed the manuscript. YST provided the surgical aspect of the study, and the dimensions for the model. All authors have read and approved the final manuscript.

In contrast, glioma patients treated with HSV-TK/ GCV did not show significant treatment benefit, most likely due to insufficient transgene delivery to tumor cells. Therefore, this study aimed at developing a strategy for real-time noninvasive HSV-TK gene was fused to the firefly luciferase (Luc) gene and the fusion construct HSV-TK-Luc was expressed in U87MG human malignant glioma cells. Nude mice with subcutaneous gliomas stably expressing HSV-TK-Luc were subjected to GCV treatment and tumor response to therapy was monitored in vivo by serial bioluminescence imaging. Bioluminescent signals over time were compared with tumor volumes determined by caliper.The in vivo.Transient and stable expression of the HSV-TK-Luc fusion protein in U87MG glioma cells demonstrated close correlation of both enzyme activities. Serial optical imaging of tumor bearing mice detected in all cases GCV induced death of tumor cells expressing the fusion protein and proved that bioluminescence can be reliably used for repetitive and noninvasive quantification of HSV-TK/ GCV mediated cell kill in vivo evaluation of gene therapy strategies for treatment of malignant disease.This approach may represent a valuable tool for the Presently, the standard method for assessing delivery of therapeutic transgenes to tumors relies on ex vivo analysis of explanted tumor tissue [Treatment with the suicide gene/ prodrug activating system herpes simplex virus type I thymidine kinase/ ganciclovir (HSV-TK/ GCV) is highly efficient in animal models of malignant glioma -3. In cor tissue ,9,10. Tir tissue -13.Luc) from the North American firefly Photinus pyralis as a reporter has several advantages compared to other imaging methods: (1) the technique is very sensitive using luciferase and detin vivo, ,15, (2) in vivo, , backgroin vivo monitoring of the activity of a therapeutic transgene by fusing the bioluminescent reporter gene Luc to the bioactivating "suicide" gene HSV-TK. We investigated whether this fusion construct could be used to monitor HSV-TK mediated cytotoxicity in malignant glioma by serial optical imaging in vivo. Noninvasive real time evaluation of localization, activity and persistence of a therapeutic gene in living animals may represent an important step towards optimization of gene therapy protocols.This study aimed at generating a sensitive tool for noninvasive HSV-TK cDNA from the retroviral vector G1Tk1SvNa ([Luc) gene from the pGL3 vector (Promega) were ligated into pCDNA 3.1(-) (Invitrogen). For the fusion construct, EGFP in the pEGFPLuc vector (BD Biosciences) was exchanged for HSV-TK cDNA, which had been amplified from G1Tk1SvNa by PCR. The resulting HSV-TK-Luc fusion gene contained a humanized form of the firefly Luc gene to ensure high expression in mammalian cells [HSV-TK cDNA was inserted in frame upstream of the Luc cDNA, and both genes were separated by a linker sequence of 33 nucleotides. All transgenes were expressed under the control of the CMV promoter. The correct sequence of the fusion construct HSV-TK-Luc was confirmed by DNA sequencing.The Tk1SvNa according to the manufacturer's protocol. For selection of stable clones transfected cells were replated at low density 48 h after transfection and incubated with 1 mg/ml geneticin for 4 weeks. Colonies were picked and analyzed for transgene expression.The human glioblastoma cell lines U87MG, T98G, LN18, U343, LN-Z308 and human embryonic kidney 293 cells were cultured under standard conditions. Cells were seeded in 6-well plates at a density of 3 – 5 × 103 cells/ well in a 96-well plate. GCV was added at final concentrations of 0 – 10 μg/ml and cells were incubated at 37°C/ 5% CO2 for 4 days. MTT was added at a final concentration of 0.5 mg/ml for 2 h. Absorbance was measured in a microplate reader at 590 nm (reference 660 nm). Experiments were performed in quadruplicates and repeated at least twice. Results are reported along with the standard deviation (SD).Transiently or stably transfected U87MG cells were seeded at 4 × 10Transiently transfected U87MG cells were lysed in CCLR lysis buffer (Promega) 2 days after transfection. Stably transfected cells were lysed in the same buffer when they had reached ~90% confluence. Protein content of all cell lysates was determined by the Bradford Protein assay . Equal amounts of protein were analyzed luminometrically for luciferase activity with a microplate reader (Victor2) using the Luciferase Assay System reagent (Promega). All experiments were repeated at least twice and mean values are reported along with the SD.For bioluminescence imaging of intact cells HSV-TK-Luc expressing U87 glioma cells were transferred to a black microtiter plate in order to minimize light scattering, and MTT assay was performed in quadruplicates as described above. On day 4 after addition of GCV, D-Luciferin was added to a final concentration of 500 μM to the culture medium. Cells were placed in a dark box and light emission was imaged using a cooled CCD camera . Light emitted from a region of interest (ROI) drawn over each well was quantified and mean values from quadruplicate measurements were compared with MTT results.Immunohistochemistry on paraffin sections using a rabbit polyclonal anti-Luc antibody was performed essentially as described by Lee et al . HE stai6 human U87MG glioma cells stably expressing the HSV-TK-Luc fusion protein. When xenografts had reached a size of ~5 mm in diameter, in general on days 7 to 9 post tumor implantation GCV therapy was initiated. Mice were injected twice daily i.p. with 30 mg/ kg GCV for 14 days. Control mice with xenografts (n = 3) received saline injections. Tumor size was measured every 2 to 4 days by caliper. Tumor volume was calculated according to the formula 0.52 × width2 × length.All animal protocols were approved by the Animal Care and Use Committee at Martin-Luther-University Halle-Wittenberg. Six week old male NMRI nu/nu mice (Charles River) were injected s.c. at four sites, each with 2 × 10For BLI animals were anesthetized with ketamine/xylazine and injected i.p. with 150 mg/ kg D-Luciferin. Approximately 8 minutes after D-Luciferin injection mice were placed in a dark box and a grayscale image was acquired at low light (exposure time 2 seconds). Bioluminescence was measured in the dark by a CCD camera cooled to -120°C (VisiLuxx Imager), using an acquisition time of 15 min and binning 6. Bioluminescent signals were displayed in pseudocolors and superimposed on the grayscale image using Metamorph software (Visitron). Mice receiving GCV were imaged at least on days 7, 15, 22, 29, and 56 post tumor implantation , while untreated control animals were subjected to BLI on days 7, 22, 29 and 35. In each animal a region of interest (ROI) was drawn over a single tumor or over all tumors as indicated in the text. Integrated as well as maximum light units (= counts) within this area were calculated after background subtraction. Final values are reported as the mean of the integrated or maximum counts obtained from all mice within one group. The CCD camera in use has a quantum efficiency approaching 90% at wavelengths between 550 and 770 nm, indicating that one photon is converted to ~0.9 electrons. One photoelectron corresponds to 4.52 counts. For serial quantification of light emission the conditions for image acquisition were kept constant.Statistical analysis was performed using the ANOVA and Student's t test . A p value of <0.05 was considered significant.HSV-TK cDNA was fused in frame with the Luc cDNA in 2 ways: one fusion protein contained HSV-TK N-terminally, in the other construct Luc preceded the HSV-TK moiety. Both constructs were expressed under control of the CMV promoter. Several human glioma cell lines as well as 293 cells were transiently transfected with these constructs. In general, Luc activity was found to be up to 50-fold higher in cells expressing the HSV-TK-Luc construct compared to cells expressing the Luc-HSV-TK construct (data not shown). Therefore, all further studies were performed with the HSV-TK-Luc fusion construct.To achieve a strictly equimolar coexpression of a therapeutic and a reporter gene, the HSV-TK, Luc, or HSV-TK-Luc transgenes, respectively. As all 3 vectors were of equal size, equal amounts of DNA corresponded to equimolar amounts of plasmid. Cytotoxic activity as measured by MTT assay was compared to luminometrically determined light production and found to be tightly correlated in HSV-TK-Luc transfected cells . Photon emission above background levels was not detectable in cells that had been transfected with HSV-TK only, while no cytotoxic activity was conferred to cells expressing only Luc.In order to characterize this fusion construct more thoroughly, both transient and stable transfection experiments were performed using the human U87MG glioma cell line Figures and 2. FHSV-TK or Luc alone. The overall cytotoxic activity of the fusion construct proved to be 60% of that measured in cells transfected with HSV-TK alone. Representative curves for 2 μg and 0.1 μg of transfected DNA are shown in Figure HSV-TK-Luc was 22% of that seen in cells transfected with Luc only of y Figure . A tight2 = 0.79; p < 0.001, data not shown).Having demonstrated in transient transfection experiments that Luc could be employed as a reporter for monitoring the therapeutic effect of HSV-TK, U87MG cell clones stably expressing the HSV-TK-Luc fusion protein were generated by selection of transfected cells with geneticin. Comparison of 18 of these clones for Luc and cytotoxic activity revealed a good correlation between both enzymatic activities . Photon emission from as few as 500 intact cells expressing the fusion construct was detectable by the CCD camera while the lower detection limit for the Luc expressing U87MG cell clone was 125 cells.Enzymatic activity in the U87MG clone with both the highest Luc and HSV-TK activity was compared with U87MG clones expressing unfused HSV-TK or Luc. Cells stably expressing HSV-TK did not luminesce upon addition of D-Luciferin while Luc expressing cell clones were resistant to GCV mediated cell killing (data not shown). The dual function fusion protein compared favorably to the respective clones with the highest HSV-TK or Luc activity. Light production in the HSV-TK-Luc expressing cell clone was ~41% of that seen in Luc expressing U87MG cells while cytotoxic activity of the HSV-TK-Luc labeled U87MG clone was ~84% of that seen with the most active HSV-TK expressing U87MG clone (data not shown). Photon emission determined luminometrically was found to be linearly correlated with cell number over a range of at least 5 orders of magnitude . These data demonstrate that both enzyme activities were also preserved in U87MG cells stably expressing the HSV-TK-Luc fusion construct.We further examined whether the cytotoxic activity of the HSV-TK moiety could be visualized by monitoring light emission from 3, 2 × 104 and 2 × 105 cells were injected s.c. on the back of the animals. Although not palpable, 2 × 104 cells expressing the fusion construct were detected by the CCD camera immediately after injection (= day 0), either when injected alone or mixed with 1.8 × 105 (90%) non-luminescent parental U87MG cells prior to injection, while 2 × 103 cells injected s.c. were not seen (data not shown). This high level of detectability by BLI proves the usefulness of the HSV-TK-Luc construct as a highly sensitive reporter in vivo.The above high expresser U87MG cell clone was used for xenograft experiments in nude mice. For sensitivity testing, 2 × 106 HSV-TK-Luc labeled U87MG cells at four different sites on the back and the flanks, respectively measured on day 7 were 122961 ± 22155. Serial measurements (during and after GCV therapy) of tumor volumes and integrated light intensity units within a region of interest (ROI) including all tumors were plotted against each other , thus confirming our cell culture data . In this mouse a significant correlation between light emission and tumor volume could be demonstrated, when tumor volumes were plotted against the maximum light emission within a ROI instead of integrated light units. In general, integrated light units within a ROI correlated closely with maximum light emission from this ROI: correlation coefficients (R2) for all tumors in treated mice varied between 0.97 and 0.99, all p values were <0.003.Regarding therapeutic efficacy in these mice on an individual basis, photon emission and tumor volumes showed a significant correlation, with RFive weeks after end of GCV therapy (day 56) light emission was no longer detectable in 5 of the 7 GCV-treated mice, while in 4 of them small residuums at the tumor site were still visible. Two mice still showed very weak light emission from one of their flank tumors which also disappeared in subsequent imaging studies. All GCV treated mice survived and tumor recurrence was not observed until closure of the study at day 90 post tumor implantation.2 = 0.98; p = 0.010; Figure The 3 untreated control mice were imaged on days 7, 22, 29, and 35 post tumor implantation and had to be sacrificed on weeks 5 to 6 post cell injection due to massive tumor growth. Although 2 tumors with relatively strong light emission on day 7 post tumor implantation regressed within 4 weeks in one of the control mice, overall tumor growth in all control animals plotted against light emission from these tumors still showed a tight correlation , R2 was 0.90 (p = 0.050) for this large tumor. When serially determined maximum light emission was used for quantification in this particular tumor, R2 dropped to 0.37 (p = 0.4).The control mouse shown in Figure When control mice were sacrificed, tumors were explanted, and immediately reimaged. Bioluminescence imaging confirmed reduced light emission from hemorrhagic and necrotic areas in large tumors Figure . ImmunohHSV-TK/ GCV in cell culture and in vivo. The HSV-TK-Luc fusion protein was successfully used in a brain tumor animal model for serial and sensitive real time quantification of the cytotoxic effect of HSV-TK by BLI.This study demonstrates that firefly luciferase is a valuable tool for monitoring noninvasively the efficacy of the prodrug activating system Fusion of two enzymes is the only way to guarantee stoichiometric, and thus correlated expression of both fusion partners. We chose this approach because coexpression of two separate transgenes from either one or separate promoters has been reported to result in severely impaired gene expression, e.g. due to inefficient internal ribosome entry site (IRES)-mediated translation or to prThe HSV-TK protein contains several nuclear targeting signals and is usually located predominantly in the nucleus . The enzRenilla Luc [cytotoxic activity of HSV-TK was not examined in the study. Renilla Luc activity in the above construct was found to be ~6 – 8-fold higher than seen with its unfused counterpart. As this enzyme is structurally unrelated to firefly Luc and has a lower molecular weight (36 kDa vs. 62 kDa), the study cannot be directly compared to our data. Notably, the authors mention that their attempt to fuse HSV-TK to firefly Luc resulted in a "poorly active" fusion protein [A decrease in HSV-TK activity of up to 80% compared to unfused HSV-TK has also been observed by Ray et al. when fusing the enzyme to illa Luc . N2A neu protein .Renilla Luc [in vivo with bioluminescence, fluorescence, and PET. Cytotoxicity generated by enzymatic conversion of prodrug by HSV-TK was however not measured in either of the above studies.Recently, the generation of several triple fusion proteins for imaging with different modalities was reported by two groups ,25. Thescorrelated expression of the three reporters within the triple fusion protein (HSV-TK-EGFP-Luc), nor were the activity levels of the different fusion partners compared to those of their unfused counterparts. Despite these limitations, the study confirms that Luc remains functional if fused N-terminally to other proteins, and that enzymatic activity is sufficient for in vivo BLI.Ponomarev et al. did not Renillla or firefly Luc at the NH2-terminus, followed by a fluorescent protein and a mutated HSV-TK enzyme. This orientation of the fusion partners as well as the use of mutated HSV-TK optimized for use with PET limits the direct comparison of the presented data to our results. Bioluminescent activity of the 4 fusion constructs containing firefly Luc was reduced to 22 – 63% of the activity of unfused Luc, which is similar to our findings when expressing HSV-TK-Luc in U87MG glioma cells. One of the 4 constructs (Luc-mRFP-mutant HSV-TK) fully retained HSV-TK PET reporter activity while in the others HSV-TK activity (as assessed by intracellular radiotracer accumulation) was reduced to 30 – 61% of the activity of the corresponding unfused enzyme. This is in line with our findings when expressing HSV-TK-Luc transiently in U87MG glioma cells.Ray et al. comparedRenilla Luc: (1) light emission of Renilla Luc peaks at 480 nm and thus shows only limited tissue penetration, (2) coelenterazine, the Renilla Luc substrate is prone to autoluminescence, resulting in high background if injected i.p. [Although it seems attractive to perform BLI with different luciferase enzymes, the following facts argue in favor of firefly Luc instead of ted i.p. , (3) coeted i.p. , and (4)HSV-TK and Luc genes residing on different plasmids. The time point of peak activity of both reporters differed by ~19 hours, most likely due to differences in half lives of the two enzymes. This finding supports our approach of expressing both enzymes as one molecule as this should greatly diminish differences in protein stability.Iyer et al. examinedAttenuation of both enzymatic activities is most likely a result of steric hindrance and might be substantially reduced by selecting another linker sequence. Longer intervening sequences as well as introduction of flexible polyglycine linkers may contribute to an increase in enzyme activity ,29.2 = 0.86). In contrast to our study, GCV treatment in vivo resulted in a decrease in light emission while the tumors continuously grew in size. Most likely, this reflects the relatively poor cytotoxic activity of the mutant HSV-TK used in these experiments, implying that engineered HSV-TK optimized for use as a PET reporter may not retain its full cytotoxic activity when substrates such as GCV are used. Data on the cytotoxic potential of the virus construct in cell culture were not presented.Recently, De et al. introducWe show here for the first time that a HSV-TK-Luc fusion protein in conjunction with GCV treatment can confer a curative effect on glioma bearing animals. While HSV-TK-Luc expressing glioma cells in culture were not killed completely when using GCV concentrations of up to 10 μg/ml, xenografts consisting of these cells were fully eliminated in all GCV treated mice. It has been shown by several groups that HSV-TK expressing tumor cells can elicit an antitumor immune reaction even in immunocompromised animals such as nude mice, most likely mediated by natural killer (NK) cells, activated in vivo by GCV induced cell killing ,31. We s6 cells intracerebrally in nude mice (data not shown), confirming the high sensitivity of BLI. Indeed, it has already been demonstrated in a murine orthotopic pituitary tumor model that bioluminescent light can travel through skull [Our study used a subcutaneous glioma model for "proof of concept" to allow for simultaneous bioluminescence imaging and measurement of tumour size by caliper. HSV-TK-Luc expressing U87MG glioma cells were also detected by the CCD camera after inoculation of 2 × 10gh skull .The cooled CCD camera system we used allows for quantification of emitted light. Some authors suggested that the level of transgene expression could be more reliably quantified by maximum light emission than by integrated light units within a ROI . Althougintegrated light signals emitted from tumor ROIs seem to be the measure of choice for serial imaging of transgene expression in growing tumors. The fact that tumor volume and photon emission are less tightly correlated in large tumors as compared to smaller ones implies that photon emission reflects mainly the presence of viable tumor cells within a tumor and is a more precise measure for cytotoxic efficacy than tumor size, as has also been suggested by others [If maximum and integrated light units within a ROI over single tumors were compared, we consistently found that both parameters tightly correlated with tumor size in tumors up to ~1 cm in diameter. In larger tumors (maximum diameter examined = 2 cm) the increase in size was in general less closely correlated with both integrated and maximum light units, but light emission from a ROI drawn over the entire tumor was still far more accurately mirroring tumor growth than maximum light units emitted from this region. With the increase in tumor thickness, maximum light emission from the tumor core is reduced due to necrosis, light scattering, and reduced supply of oxygen and D-Luciferin to tumor cells. On the other hand, the increase in tumor length and width is better reflected by light signals integrated over the entire tumor area, while a concomitant change in maximum light signal does not necessarily have to occur. Therefore, y others ,35.-15 – 10-17 vs. 10-11 – 10-12 mole/L of reporter probe are detectable, [Herpes simplex virus type I thymidine kinase has also been used as a reporter gene for monitoring therapeutic success with PET ,36. Howeectable, ) in combin vivo by BLI, when the bioluminescent reporter luciferase is fused in frame to the therapeutic gene HSV-TK. We used a clonal human glioma cell line stably expressing the HSV-TK-Luc fusion construct, thus guaranteeing high level transgene expression. Despite the somewhat attenuated activity of both fusion partners, a high degree of cytotoxicity by HSV-TK mediated GCV bioactivation as well as strong bioluminescent signals upon administration of D-luciferin were consistently demonstrated. In order to mirror more closely in vivo gene therapy of malignant brain tumors, experiments are underway to insert the HSV-TK-Luc fusion gene (and an improved version of it) into appropriate viral vectors and subsequently use them for treatment of orthotopically established gliomas in mice. Serial assessment of transduction levels, transgene localization and time course of fusion gene expression in living animals by BLI can aid in developing more potent gene therapy vectors for treatment of malignant glioma.We showed that therapeutic efficacy of a suicide gene/ prodrug activating system can be accurately monitored CCD, charged coupled device; CMV, cytomegalovirus; cps, counts per second; EGFP, enhanced green fluorescent protein; GCV, ganciclovir; HSV-TK, herpes simplex virus type 1 thymidine kinase; Luc, luciferase; mRFP, monomeric red fluorescent protein, PET, positron emission tomography; ROI, region of interest; SD, standard deviation, SEM, standard error of the mean.None declared.AS constructed the plasmids, performed the animal experiments together with CT and set up the in vitro assays. CT and SJ participated in the cell culture experiments and enzymatic assays. SJ and AS carried out the immunohistochemical studies. NGR and AS designed the experiments and evaluated the data. All authors have read and approved the manuscript.

CSF was measured in 9 normal subjects as part of an intensive study of physiological responses stressors in chronic pain and fatigue states. CRHCSF was first correlated with demographic, vital sign, HPA axis, validated questionnaire domains, baseline and maximal responses to pain, exercise and other stressors. Significant factors were used for linear regression modeling.Define covariates of cerebrospinal corticotropin-releasing hormone (CRH) levels in normal humans. CRHCSF with blood glucose and sodium ; (b) CRHCSF with resting respiratory and heart rates ; and (c) CRHCSF with SF-36 Vitality and Multidimensional Fatigue Inventory Physical Fatigue domains .Highly significant correlations were found despite the small number of subjects. Three models were defined: (a) CRHCSF was predicted by lower glucose, respiratory and heart rates, and higher sodium and psychometric constructs of well being. Responses at peak exercise and to other acute stressors were not correlated. CRHCSF may have reflected an overall, or chronic, set-point for physiological responses, but did not predict the reserves available to respond to immediate stressors.Low CRH CSF) and neuropeptide Y (NPY), another important neuropeptide involved in pain, autonomic and stress responses [CSF based on (a) metabolic, (b) autonomic, and (c) psychometric measures.Corticotropin – releasing hormone (CRH) plays a major role in regulating the hypothalamic – pituitary – adrenal (HPA) axis, acute responses to stressors, and other neurological functions . The ceresponses ,3, were esponses . Subjectesponses , chronicesponses , and vetesponses were incesponses were defThe mean age for the group was 35.2 yr (95% C.I.: 30.5 to 39.9) years. There were 2 females, 5 African-Americans, 3 Caucasians, and 1 Caucasian Hispanic (TABLE CSF was 121.9 (87.8 to 156.0) pg/ml. NPY did not correlate with any variable.Mean NPYCSF could be grouped into: metabolic, autonomic function, and perceptional and cognitive functions (TABLE CSF (FIGURE 2) were 0.97 and 0.66, respectively. Glucose and sodium were also negatively correlated (R2 = 0.85). Resting norepinephrine levels at 2 time points and heart rate at 4 time points were colinear and positively correlated to CRHCSF.Significant covariates of CRHns TABLE . Serum gF FIGURE . ExplainCSF was negatively correlated with the Holter monitor-derived measure of log heart rate summed for the daytime, and the threshold temperature causing an initial, mild sensation of burning pain (Stressor I). The latter indicated that subjects with higher CRHCSF perceived the burning pain of the cutaneous forearm hyperthermic stimulation at a lower threshold temperature than their peers. This may indicate an increased sensitivity to nociceptive stimuli. However, there was no correlation with deep pressure – induced pain. Negative correlations were also found with the SF-36 Vitality and SES Manage Symptoms domains. High scores were normal for these questionnaires, with lower scores indicating dysfunction.CRHCSF. Perceptions of vulnerability, physical functioning and self-efficacy were more highly related.All parts of the study were completed by at least 5 males. Analysis of the male subgroup gave some information about the role of gender. The pattern of significant covariables was different from the total group TABLE . RespiraCSF. The 3 models had high significance levels to be so significant (p < 0.02). The explained variances were very high (R2 > 0.85) suggesting that the factors may be causally connected.Sets of the significant metabolic, autonomic, and perceptual variables were grouped and analyzed by 3 linear regression models in order to detect significant relationships between independent variables and CRHls TABLE . A metabCSF did not correlate with variables associated with maximum exercise, heat – and pressure – (dolorimetry) induced pain, or other rapid onset stressors receptors and the HPA axis . The othCSF were positively correlated. The statistical model indicated that a low CRHCSF was predicted by low respiratory and heart rates. Respiratory and cardiac functions are rigorously controlled by brainstem and other nuclei that integrate incoming signals of plasma O2, CO2 and H+ concentrations, activity needs, anxiety and other stressors. Efferent cardiovascular and other autonomic reflexes are modulated by CRH in man [CSF. This is supported by studies in mice that genetically overexpress CRH. They develop chronic stress – like autonomic and physiological alterations [Resting respiratory and pre-exercise heart rates and CRHH in man . These cerations . Stressoerations that acterations . An examerations .CSF should be present in persons lacking these stressor states. This was supported by the negative correlations of CRHCSF with scores for the SF-36 Vitality and Change in Health, Self Efficacy Scale Manage Symptoms, MIQ Vulnerability, and MFI Physical Functioning domains. Each scale has an idealized "normal" end of the range of scores. Deviation towards either higher (MFI) or lower (SF-36) ends of the scales provides an estimate of dysfunction. For each of these domains, the lower CRHCSF were associated with more normal scores, while higher CRHCSF was associated with scores that were beginning to shift away from the normal pole of each scale. These trends were further supported by the statistical model where the optimum covariates of CRHCSF were SF-36 Vitality and MFI Physical Fatigue domains. The model predicted that CRHCSF would be in the low normal range when Vitality and Physical Fatigue domain scores were high . Taken together, these results confirm the consistent physical status, mental coping skills, and general health of these subjects.Extrapolation of these concepts suggests that elevated CRH may be related to anxiety, depression, or other disorders associated with chronic stress responses. If so, then lower, but normal, CRHStudies of intraventricular CRH injection in primates support our findings . The CRHCSF and metabolic, autonomic, and psychometric measures. These are novel findings in humans, but are consistent with data on CRH in acute and chronic stress models, and proposed CRH neural circuits. It will now be of great interest to contrast these statistical models identified for normal subjects with the other chronic pain and fatigue patient subsets to determine if CRHCSF correlates with different sets of variables.This small but intensively studied group of normal humans demonstrated surprisingly robust relationships between CRHNine normal, healthy subjects gave informed consent for this paid, Institutional Review Board – approved protocol. They had a comprehensive screening evaluation to exclude: severe physical impairment, morbid obesity, autoimmune/inflammatory diseases, cardiopulmonary disorders, uncontrolled endocrine or allergic disorders, malignancy, severe psychiatric illnesses , factors known to affect the HPA axis or autonomic function , or medication use. Subjects had a history and physical examination, were administered the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) (SCID II) ,20, CompSubjects were admitted to the G-CRC on the evening of Day 1. An 18-gauge catheter was inserted antecubitally and infused with normal saline at 50 ml/hr. A Holter monitor was attached to monitor autonomic regulation of cardiac rhythm -31. Upon125-CRH or -NPY was added, tubes gently vortexed, and again incubated overnight. Goat anti-rabbit antibodies were added, tubes incubated, and immune complexes precipitated by centrifugation. Radioactivity was counted for the standards, and the concentrations for each sample interpolated from the standard curves. Spiking CSF with fixed amounts of I125-peptides and performing the RIA shifted the curves to the left by the anticipated concentrations. Standard curves were reproducible to within 10%.Tubes of CSF were immediately placed on ice and then centrifuged at 4°C. The supernatants were rapidly frozen at -80°C. Tube 2 or 3 was removed from the freezer and thawed at 4°C. Peptides were extracted by precipitating high molecular weight proteins by adding an equal volume of 100% ethanol, 0.1 M acetic acid, 0.2% sodium bisulfite ,37. The Plasma catecholamine levels were measured by HPLC .All the data from the provocation studies, questionnaire domains, blood work, and neuropeptide analysis were entered into a SAS spreadsheet using sequential hand or scanner entry followed by data checking routines.Means with 95% confidence intervals were reported.CSF) and other objective and subjective patient variables were conducted as an exploratory tool. Given the small sample size (n = 9), both parametric and non-parametric correlations were used to evaluate the robustness of the correlation coefficients. Partial and intra-class correlations examined the structure of the relationships between CRHCSF and the independent variables. Original data were always checked to make sure that correlations were not spurious, due to outliers, colinearity, or sets of virtually identical scores.Simple correlations between our outcome measure , or that had < 5 recorded values were excluded from further analysis.Our data included a very wide range of covariates which spanned the physical and psychological attributes of the patients. Scatter plots were used to determine the response slopes with CRHCSF, the dependent variable. Separate models were required because of the low degrees of freedom available for the analyses, and to ensure that the explained variance (R2) was not inflated due to overloading the model with potentially collinear variables. Variance inflation factors and tolerance values were incorporated to optimize the selection of the most independent combinations of variables that also maximized the R2 and p values. The linear regression procedure took into account the multiple comparisons for each model.The remaining suitable independent variables fell into 3 categories: metabolic, autonomic and psychometric function. Three 3 separate linear regression models were con2, explained variance; SCID II, Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV); SF-36, Short Form 36; SES, Self Efficacy ScaleCESD, Center for Epidemiologic Studies Depression scale; CFS, Chronic Fatigue Syndrome; CIDI, Composite International Diagnostic Interview; CRH, corticotropin-releasing hormone; CSF, cerebrospinal fluid; MFI, Multidimensional Fatigue Inventory; MIQ, Meaning of Illness Questionnaire; NPY, neuropeptide Y; RJNB directed the RIA studies, correlation of clinical variables, and wrote the manuscript. HM confirmed the accuracy of the study results by double data entry methods, vetted the SAS database, performed the statistical analysis, and co-wrote the manuscript. GW performed the CRH and NPY assays and maintained the repository of these samples. DJC was the Principal Investigator for the overall study and helped review the draft and final manuscript.

This paper presents a simple method to increase the sensitivity of protein family comparisons by incorporating secondary structure (SS) information. We build upon the effective information theory approach towards profile-profile comparison described in [Yona & Levitt 2002]. Our method augments profile columns using PSIPRED secondary structure predictions and assesses statistical similarity using information theoretical principles.Our tests show that this tool detects more similarities between protein families of distant homology than the previous primary sequence-based method. A very significant improvement in performance is observed when the real secondary structure is used.Integration of primary and secondary structure information can substantially improve detection of relationships between remotely related protein families. Detecting an evolutionary relationship between proteins is the basis for functional inference. Existing methods most often rely on sequence information in an attempt to quantify the evolutionary divergence or similarity between the sequences compared. A significant similarity would suggest that the proteins are related. However, in many cases sequences have diverged to the extent that their similarity is undetectable by standard sequence comparison algorithms. Nevertheless, they may still have similar structures and functions ,2.It has long been postulated that evolutionary pressure acts upon the three-dimensional structure of proteins and intra-protein interactions rather than at the level of the primary sequence ,4. IndeeThere have been many attempts to build algorithms that predict protein structure from amino acid sequence. Unfortunately, this is a hard problem, and existing methods are only partially successful . On the Alternatively, one can use both representations to assess protein similarity. Incorporating secondary structure information into protein comparison is not a new idea. Several researchers have attempted to boost performance and sensitivity of various methods by adding this extra degree of information with some success. Yu et al. encoded functionally conserved sequence patterns into probabilistic structural models (that comprise a family of hidden Markov models) . The modThese and similar studies have indicated that the incorporation of secondary structure information, even if predicted, can increase sensitivity and specificity of a protein comparison model. Here we describe a method that integrates secondary structure information with primary sequence information in a single scoring scheme, using a single statistical representation. The model can be applied to any protein family and does not require the application of expensive threading algorithms. Our method extends our previous work on profile-profile comparison . SpecifiWe use a data set of domain families derived from the SCOP classification of protein structures , releasen-tuple of probability distributions of amino acids, derived from a group of related proteins, where n is the length of the multiple alignment of these proteins. It is represented in software as a two dimensional matrix of 20 rows and n columns, where each column (known as a profile column), is a probability distribution p over the 20 amino acids in one position in the multiple alignment. These profile columns form the basis of profile-profile comparisons.The PSI-BLAST profiles are the basis for our representation of a protein family. Each profile is a true secondary structure information is gleaned from the PDB files of the seed proteins using STRIDE is the Kullback-Leibler (KL) divergence γ described in section 'Integrating secondary structure with primary structure'. The significance measure reflects the probability that the source distribution, r, could have been obtained by chance. The higher r is, the more distinctive the common source distribution, and the lower the probability that it could have been obtained by chance.In this study the background distribution is composed of two components: the background distribution of amino acids (estimated from a large sequence database) and the background distribution of secondary structure elements . The components are mixed using the same mixing parameter similarity score of two probability distributions p and q as a combination of the divergence score and the significance score:We define the D → 0) whose common source is far from the background distribution (S → 1), tends to one. On the other hand, the similarity score of two dissimilar distributions (D → 1) whose most likely common source distribution resembles the background distribution (S → 0) tends to zero. This scoring scheme also distinguishes two distributions that each are similar to the background distribution (D → 0 and S → 0 giving Score - 1/2) from two dissimilar distributions, but whose common source is similar to the background distribution (D → 1 and S → 0 giving Score = 0). In a recent study [With this expression, the similarity score of two similar distributions (nt study it has bp over a set X that is obtained by "mixing" two probability distributions over two disjoint subsets: p1 over the subset X1 and p2 over the subset X2 (where X = X1 ∪ X2 and X1 ∩ X2 = θ). Let γ be the mixing parameter, i.e. the total weight of the first distribution p1 in the combined distribution p. Assume q is obtained in a similar manner from q1 and q2. Then,Note that our measures are functionals of the probability distributions, based on variations of the entropy function, and specifically the KL divergence function. One of the nice properties of this function is that it is additive in the following sense. Assume we have a probability distribution X1 (the secondary structure) and X2 (the primary structure).In other words, this measure preserves independence between the two subsets. Therefore, with our extended profile representation, the new functionals are simply a weighted sum of the individual functionals over the subsets γ and (1 - γ) respectively.Note however that this property holds for the divergence and the significance measures but not for the final similarity score that is a combination of the divergence and the significance scores. An alternative would be to compute the divergence, significance and similarity scores independently for the secondary and primary structures, and then combine the two similarity scores into one, with weights i of the seed sequence (see section 'Data sets') as the seed amino acid of the i-th profile column.It is interesting to compare the similarity scores before and after the addition of secondary structure information. To assess the impact of this information, we computed the distribution of similarity scores for five types of profile columns, depending on the type of their seed amino acid. We refer to the amino acid at position Two seed amino acids are defined as similar, neutral, or dissimilar based on their BLOSUM62 scoring matrix , with poidentical columns), (2) different columns that are associated with the same seed amino acid (strongly similar columns) (3) different columns that are associated with similar seed amino acids (similar columns), (4) different columns with mutually neutral seed amino acids , and (5) different columns with dissimilar seed amino acids (dissimilar columns). We repeated this calculation before and after the integration of true secondary structure information and the results are plotted in Figure The five types of column pairs are: 1) a column with itself .ALLR (Average Log Likelihood Ratio) scoring function that was suggested in [p and q, their ALLR score is defined asWe also tested the ested in . This scnp (nq) is the number of total counts from which p (q) is derived, and P0 is the background distribution.where We computed the correlation scores and ALLR scores for the same sets of columns defined in the previous section and compared it to the information-theoretic scores Figure . Note thprof_ss) depends on several parameters: (1) a shift parameter is introduced to convert the similarity scores to scores that are suitable for local protein comparison . The tradeoff between the primary sequence information and secondary structure probabilities was varied from zero to one. With zero dependence on secondary structure the method is equivalent to prof_sim (profile-profile comparison based on just primary structure). The results are shown in Figure γ = 0.055 (i.e. 0.055 weight on the secondary structure information and 0.945 on the sequence information) gave the best performance. (Note that if each secondary structure was given as much weight as a single amino acid γ would be or ~0.13).To estimate the best value for γ = 1), the performance was much worse than when only sequence information was used (γ = 0). These corner-case results and the fact that the best results were obtained with γ << 0.5 suggest that for protein family comparison, the coarse-grained secondary structure information is noisier and less reliable than sequence information. However, as the graphs indicate, using both sources of information clearly improves performance. Our tests were done using actual secondary structure information in the profile; however, similar results were obtained when the predicted information was used for one or both of the profiles Marquardt-Levenberg algorithm.To differentiate true similarity values from those that may be observed by chance, it is essential to establish a baseline empirical distribution for the scores. Here we used the statistical framework of the extreme value distribution (EVD). Although rigorous mathematical proof has not been found for local gapped similarity scores, empirical studies have shown that the distribution of these scores can be approximated by this distribution. An empirically fit EVD also has the benefits of being a true fit to the quirks of a particular protein family. Three such distributions were established to assess the significance of the profile-profile matches. All distributions were fit with the 'fit' function in gnuplot uniform approach (uniform parameters).The first distribution is based upon comparisons between unrelated families . This distribution is useful in that it can be used to assess the significance of a score in comparing any pair of protein families, without further need for computations. Practically, this aggregates all comparisons between non-related families into a single list. This is essentially the distribution of similarity scores of random profiles, as shown in Figure The second distribution is similar to the first, except a correction was made for the length of a profile, similar to the approach employed by FASTA . By chanThe third distribution proves to be the best approach in assessing significance of matches with a particular profile. This distribution is created on a per-family basis. The scores of each family against all (unrelated) SCOP families were fit to an EVD. Since many of the family profiles are unrelated to the query family, the corresponding scores provide a relatively reliable baseline distribution. This approach is a robust method to assessing the significance of matches for a particular profile since it allows for any unusual properties of the query profile and the parameters are adjusted accordingly that can be detected with our method. Specifically, each family in our test set is compared with all other protein families and the results are sorted based on the p-value. Given the sorted list we count the number of true family-family relationships that are detected before the orted in ).true relationship if both families belong to the same superfamily, a possible relationship if both families belong to the same SCOP fold, a weak relationship if they belong to the same class, suspicious if they belong to different classes and an error if one family is all-alpha and the second is all-beta. We repeat the procedure described above, each time using a different definition of a false positive. The results are summarized in Table Usually a false positive is defined as a relation between families that do not belong to the same superfamily. This popular criterion, however, is somewhat strict as relations between families that belong to the same fold can also be considered as positives. We use the following terminology to distinguish between the different types of "false positives". We define a relationship between two protein families to be a prof_ss improves over prof_sim although the improvement is smaller compared to the one reported in Table γ was set to 0.055), the new algorithm almost doubles the number of pairwise relationships that are detected.The second measure we use is the receiver operator characteristic (ROC) measure, a common measure in assessing sensitivity and selectivity. Given a sorted list of results, the ROC index measures the area under the curve that plots the positives versus the negatives. Maximal performance translates to a perfect separation and a maximal normalized ROC score of 1. The ROC-N measure is a variation over the ROC measure, where the plot is truncated at N negatives. In other words, the ROC-N measure is the number of true positives detected up to N false positives. Here we used the popular ROC-50 measure. To obtain the ROC-50 scores for each method we pool together all pairwise comparisons for all protein families, and sort them by their normalized e-value. The number of true positives is aggregated until 50 false positives occur. As before, we repeated this procedure with different definitions of false positives, and the results are summarized in Table In this section we give several interesting examples of alignments between remote protein families that exemplify the differences between sequence-based profile-profile alignments and the new generalized profile alignments.prof_sim is able to roughly match up the helices but not the beta strands with a rms of 11.96. The predicted secondary structure does not improve the alignment in this case, however, when the true secondary structure is used, prof_ss is able to completely align the helices as well as most of the strands with a much better rms of 4.45 , and the replication terminator protein . Although designated as all-alpha, proteins in this superfamily contain a small beta-sheet at the core. The similar substructures have three alpha helices and a couple beta strands, 5 Figure .This superfamily belongs to the concanavalin A-like lectins/glucanases fold, characterized by a sandwich structure with 12–14 strands in 2 sheets. We compared two families in this superfamily: the beta-Glucanase-like family and the vibrio cholerae sialidase, N-terminal and insertion domains .prof_ss both when using the predicted information and the true secondary structure information. On the other hand, prof_sim is unable to align the sheets at all and the bromoperoxidase A2 family . These are large and complex proteins with many helices and strands. This paper presents a simple method to improve remote homology detection between protein families. We use statistical models of protein families in the form of profiles, and by incorporating secondary structure information within that model, we can reuse existing comparison methods for comparing profiles. It is shown that this method improves over the previous method that is based only on primary sequence information.As opposed to other methods that compare single proteins, our method compares models of protein families. Instead of summing over different models, our model combines structural and primary sequence information within the profile itself. Our method allows us to explore a wide range of scenarios, between purely sequence-based representation and a purely secondary-structure based representation. The optimization of the single mixing parameter shows that the slight incorporation of predicted secondary structural information is invaluable. Since predicted structure information in PSIPRED comes from neighboring profile columns, this proves that each profile column confers extra information that is relevant to its neighbors and is useful to inferring protein relationships.Furthermore, it is shown that if true secondary structure information is used, performance improvements are very significant and the number of relationships that can be detected is almost doubled. We conclude that despite the high overall accuracy of the secondary structure prediction method, its imperfect nature can greatly affect the performance. However, our method can be generalized to any secondary structure prediction method that produces estimated probabilities for secondary structure, so should a new prediction method be found that performs better than the current methods, the model presented here is expected to reflect the improved performance and consequently improve homology detection.prof_sim program and integrated secondary structure information, optimized the model, ran experiments, and analyzed the result sets. GY conceived of the study, designed the model and analyzed the results.RC extended the

However, knockout in the mouse of cftr-/- mouse. Lungs showed decreased compliance and increased airway resistance in young animals as compared to cftr+/+ littermates. These changes were noted in animals less than 60 days old, prior to any long term inflammatory effects that might occur, and are consistent with structural differences in the cftr-/- lungs. Surprisingly, the cftr+/- animals exhibited a lung phenotype distinct from either the homozygous normal or knockout genotypes. The heterozygous mice showed increased lung compliance and decreased airway resistance when compared to either homozygous phenotype, suggesting a heterozygous advantage that might explain the high frequency of this mutation in certain populations.Using measurements of pulmonary mechanics, a definitive lung phenotype was demonstrated in the cftr results in distinct differences in pulmonary mechanics of the adult. Distinct phenotypes were demonstrated in each genotype, cftr-/-, cftr +/-, and cftr+/+. These results are consistent with a developmental role for CFTR in the lung.In the mouse the gene dosage of CF is one of the most common autosomal recessive diseases in Caucasians with a carrier rate of 3–4% [1), functional residual capacity (FRC), and other parameters of lung function prior to the onset of recurrent infection [Cystic fibrosis (CF) is a progressive disease primarily affecting the intestines, lungs, and pancreas. The gene responsible for CF was identified in 1989 [ of 3–4% , and is nfection .cftr knockout mouse does not develop overt lung disease, which has severely limited its usefulness. However, the availability of new methods for pulmonary testing in rodents [cftr knockout mouse for functional lung changes. In the present study, therefore, we examined pulmonary function in young adult cftr -/-, cftr +/-, and cftr+/+ S489x mice in an effort to establish a lung phenotype.Soon after the CF gene was discovered, a knockout mouse was developed. This mouse demonstrates subtle changes in epithelial cell phenotype, including alterations in secretory glycoconjugates and changes in secretory vesicles . Monocyt rodents ,9 now prPV) curve which is dependent upon both lung structure and interfering pathology. PV curves were measured in triplicate, starting from positive end-expiratory pressure (PEEP) values of 0, 3, and 6 cmH2O in S489X mice at 30–60 days of age following genotyping for the normal and mutant cftr alleles. The 3 cmH2O PEEP curves obtained for each genotype are presented in Figure PV curves all begin at V = 0 ml, which is the FRC defined by the 3 cmH2O PEEP. The PV curves obtained at PEEP levels of 0 and 6 cmH2O were similar.Routine evaluation of dynamic lung function employs the stepwise variation in air volume on both the inflation and deflation phases of a single breath. Measurement of airway pressures at each step results in the classic pressure-volume (Cst) of the lungs, which reflects elastic recoil at a given pressure, was calculated from the slopes of the PV curves. As shown in Figure Cst was significantly reduced in both cftr+/+ (p < 0.001) and cftr-/- (p < 0.001) as compared to age-matched cftr+/- mice. Hysteresis was altered among the 3 genotypes and cftr+/+ mice (p < 0.05) compared to cftr-/- mice. These data suggest the presence of a gene dosage effect in which an altered lung structure in the heterozygous animals leads to an elevated compliance relative to the two homozygous animals. Lung weights were measured and no statistically significant differences were observed among the three genotypes and determined values for airway resistance (Raw), tissue damping (G), and tissue elastance (H). Figure Raw, G, and H were significantly reduced in the cftr+/- mice as compared to both cftr+/+ and cftr-/- animals at all PEEP levels. Raw also decreased with PEEP in a similar fashion in all three genotypes. In contrast, Raw, H and G were significantly increased in the cftr-/- mice compared with cftr+/+ and cftr+/-, and showed a greater dependence on PEEP . The ratio G/H, termed hysteresivity (both the low and high frequency), was not significantly affected by either genotype or PEEP (Panel E). Thus, the absence of either 1 or 2 copies of the cftr gene had significantly different effects on the phenotype of the lung. Paradoxically, the absence of only one cftr copy resulted in a greater lung compliance (lower elastance) than if neither or both copies were present.We applied the forced oscillation method to the mice and determined respiratory mechanical input impedance ,11. We fcftr knockout mouse as a model of cystic fibrosis has been severely limited by its failure to demonstrate readily measurable lung disease, the primary cause of morbidity and mortality in humans [cftr in the lung of young adult animals resulted in decreased Cst and η and increased Raw, G and H as compared to normal littermate controls.The usefulness of the n humans . HoweverCst and hysteresis in cftr+/- mice was significantly higher than in cftr+/+ animals while G and H were decreased. As this was not associated with any pathology such as emphysema, we conclude that it represents a functionally different lung from that of the cftr+/+. Our data thus reveal a remarkable inverse correlation between the effect of one and two non-functional copies of the cftr gene.A particularly intriguing further observation was that η[cftr is a chloride channel and is thought to be involved in water balance, a change in surface tension in the lung, and consequently in η, might be expected. However, as shown in Figure G and H both increase, they do so in the same proportion so there is no significant change in η between the three cftr genotypes. On the other hand, lung weights were not different among the different groups of mice, so the decreased compliance and increased resistance of the cftr-/- animals was not simply due to their having smaller lungs than control animals. This suggests that the parenchymal structure in the lungs of the homozygous and heterozygous animals were organized differently, in a manner that affected G and H similarly.What do these data mean in terms of lung structure? The knockout animal has cystic fibrosis by definition, and our data now show it to also have lung disease manifest as a reduced compliance and increased resistance. These changes could reflect changes in the intrinsic mechanical properties of the parenchyma, or simply a reduction in lung volume. The former effect could include alterations in the biophysical properties of the air-liquid interface in the lungs, and would be expected to result in a change in η. Indeed,cftr levels are highest in the developing lung and decrease 75-fold at birth. In utero over-expression of cftr has also been shown to affect lung growth and development[cftr may affect the early development of the lung in a manner that is affected by the interaction of other genes.As documented in numerous publications, the mouse strain used in the present study does not develop chronic inflammatory disease up to the age (30–60 days) used in this study (for review see ). On thevelopment, and thevelopment. These dcftr animals? Interestingly, there is no decrement in lung function in human heterozygotes [cftr, something we term a "Goldilocks Effect". That is, while two defective copies of the gene are detrimental and two normal copies are satisfactory, one normal and one defective gene may results in an optimal dosage for lung development.Are there any functional consequences for increased lung compliance in the heterozygous ozygotes -21. Alsoozygotes ,23. Indeozygotes . When onozygotes is perhaozygotes . The rescftr knockout mice should reveal additional genetic loci that modulate the influence of cftr on lung growth and development. Corresponding studies in humans should be useful in evaluating the effect of therapies on reversing altered pulmonary function in the CF patient.Further studies of pulmonary mechanics in cftr knockout mouse. In addition, the cftr+/- mouse had a distinguishable pulmonary function phenotype from that observed in either the homozygous normal or mutant genotype mice. These data are consistent with CFTR-dependent, physiologic changes in the structure and function of the lung.Using sophisticated techniques to a evaluate rodent pulmonary function; a distinct, readily quantifiable lung phenotype was identified in the th generation backcross to C57Bl/6 has been maintained by random mating for the past 8 years. This colony has a 100% mortality rate among cftr knockouts by 45 days of age unless the animals are placed on an elemental liquid diet and corncob bedding upon weaning [cftr alleles. Age and litter matched cftr+/+ and cftr+/- were used for each cftr-/- mouse examined. Six animals were included in each group. All experiments were approved by the animal care and use committee.The S489X mouse 5 weaning . Mice 30flexiVent, SCIREQ Inc. Montreal, PQ, Canada) and ventilated with a tidal volume of 10 ml/kg; inspiratory:expiratory ratio of 66.67%, respiratory rate of 150 breaths/minute, and maximum pressure of 30 cmH20. PEEP was controlled by submerging the expiratory limb from the ventilator into a water trap. Each animal was paralyzed with pancuronium bromide (0.5 mg/kg) and allowed to equilibrate on the ventilator until spontaneous breathing ceased (5 minutes).The mice were anesthetized with intra-peritoneal pentobarbital (90 mg/kg) and the trachea was dissected free of surrounding tissue and cannulated with a 20-gauge cannula. The animals were then connected to a small animal ventilator , i is the imaginary unit, α links G and H, and f is frequency. We also calculated a quantity known as hysteresivity (η = G/H), which is believed to increase when regional heterogeneities develop in the lung [Raw, G and H were all normalized by multiplication by lung weight.where g airways, Iaw is the lung . Raw, G FRC defined by the PEEP, the flexiVent was programmed to deliver seven inspiratory volume steps for a total volume of 0.8 ml followed by seven expiratory steps, pausing at each step for 1 s. Plateau pressure (P) at each step was recorded and related to the total volume (V) delivered to produce a quasi-static PV curve. Cst was calculated from the slope of each curve [Starting at the ch curve , and wasZrs measurements at each PEEP level and PV curves were obtained in triplicate. Data were statistically evaluated using paired t-test with p < 0.05 being taken as significant

Caenorhabditis elegans retroviruses, are class II viral fusion proteins. Comparisons of divergent viral fusion proteins can reveal features essential for virion:cell fusion, and suggest drug and vaccine strategies.The Bunyaviridae family of enveloped RNA viruses includes five genuses, orthobunyaviruses, hantaviruses, phleboviruses, nairoviruses and tospoviruses. It has not been determined which Bunyavirus protein mediates virion:cell membrane fusion. Class II viral fusion proteins (beta-penetrenes), encoded by members of the Alphaviridae and Flaviviridae, are comprised of three antiparallel beta sheet domains with an internal fusion peptide located at the end of domain II. Proteomics computational analyses indicate that the carboxyl terminal glycoprotein (Gc) encoded by Sandfly fever virus (SAN), a phlebovirus, has a significant amino acid sequence similarity with envelope protein 1 (E1), the class II fusion protein of Sindbis virus (SIN), an Alphavirus. Similar sequences and common structural/functional motifs, including domains with a high propensity to interface with bilayer membranes, are located collinearly in SAN Gc and SIN E1. Gc encoded by members of each Bunyavirus genus share several sequence and structural motifs. These results suggest that Gc of Bunyaviridae, and similar proteins of Tenuiviruses and a group of Two classes of viral envelope proteins that mediate virion:cell fusion have been described. Class I and II fusion proteins (aka α-and β-penetrenes) are distinguished, in part, by the location of the "fusion peptide," a cluster of hydrophobic and aromatic amino acids that appears critical for fusing viral and cell membranes. The fusion peptides of class I fusion proteins are located at or near the amino terminus, whereas fusion peptides of class II fusion proteins are internal. The overall structures of these two classes of viral fusions proteins are also distinct. Class I fusion proteins have a pair of extended α helices that are separated by sequences variable in length, but usually containing one or more dicysteine linkages. Several otherwise disparate viruses, including orthomyxoviruses, paramyxoviruses, retroviruses, arenaviruses, filoviruses and coronaviruses encode class I fusion proteins -4. ClassCaenorhabditis elegans retroviruses, are class II viral fusion proteins (β-penetrenes).The Bunyaviridae family of enveloped RNA viruses includes five disparate genuses. Orthobunyaviruses, phleboviruses, nairoviruses and tospoviruses are spread by insect vectors, whereas hantaviruses are spread by rodent vectors . MembersCaenorhabditis elegans, including Cer13 . We also compared Bunyavirus M sequences to structural proteins of Flaviviruses, including members of the flavivirus genus tick-borne encephalitis virus, strain Neudoerfl ; Japanese encephalitis virus, strain JaOARS982 , yellow fever virus, strain 17D-204 , dengue virus type 2, strain PR-159/S1 , and West Nile virus, strain NY 2000-crow3356 . The prototype hepaciviruses, strain H (subtype 1a) of hepatitis C virus , and several pestiviruses, including the Alfort 187 strain of classical swine fever virus, aka hog cholera virus , bovine viral diarrhea virus genotype 1 aka pestivirus type 1, stain NADL and border disease virus, strain BD31 , were used in other comparisons.For sequence and structural comparisons of Bunyavirus M encoded proteins representatives of the five genuses were used, including pleboviruses Sandfly fever virus, Sicilian strain and Rift Valley fever virus , orthobunyavirus Bunyamwera virus , hantavirus Hantaan virus, strain 76–118 , nairovirus Crimean-Congo hemorrhagic fever virus, strain IbAr , and tospovirus tomato spotted wilt virus, ordinary strain . Additional phlebovirus M sequences compared included those of Uukuniemi virus and Punta Toro virus . Bunyavirus M sequences were compared to sequences encoded in Alphavirus subgenomic RNA, including structural proteins of Sindbis virus , Semliki Forest virus , Venezuelian equine encephalitis virus, strain TC-83 , Western equine encephalitis virus, strain McMillan , O'nyong-nyong virus, strain GULU , Mayaro virus, strain TRVL4675 , Barmah Forest virus strain BH2193 and Ross River virus, strain NB5092 . Comparisons were also made with proteins encoded by RNA2 of the HZ isolate of Rice stripe virus, a Tenuivirus , and with certain retroviruses of ) was the preferred method of secondary structure prediction [). TMpred is based on a statistical analysis of TMbase, a database of naturally occurring transmembrane glycoproteins [Xplorer version 2.2a from the Stephen White laboratory using default settings [ was used to predict mucin type GalNAc O-glycosylation sites. RasMac , developed by Roger Sayle, was used to render a 3D model of SFV E1, which was extrapolated to SIN E1 and SAN GC.Methods to derive general models of surface glycoproteins have been described previously . PRSS3, ediction . PHDsec proteins . Sequencsettings . The NetSimilar sequences or common structural/functional motifs are located collinearly in the carboxyl terminal glycoprotein of Sandfly fever virus and Sindbis virus envelope glycoprotein E1.Previously, Gallaher and coworkers modeled the structure of the retroviral transmembrane glycoprotein (TM) onto thePrior X-ray crystallographic studies have demonstrated that SFV E1 is a class II viral fusion protein (β-penetrene) . BecauseTo provide support for the proposed alignment of SAN Gc and SIN E1, another proteomics computational tool was used to compare potential membrane interactive domains in the glycoproteins. Besides fusion peptides, a motif that can be important in virus:cell fusion and is present in many class I and class II viral fusion proteins is an aromatic aa rich domain proximal to the transmembrane anchor . The preRecently, Gibbons and coworkers determined the structure of a fragment of the SFV E1 ectodomain after exposure to low pH and liposomes . Under tThere are many possible alternatives to the cysteine linkages and secondary structures of SAN Gc drawn in Figure -18). As noted previously [-8). As with phlebovirus Gc, the prototype of the hantavirus genus, Hantaan virus (HAN), showed a modest sequence alignment (p < 0.05) with SIN E1, further supporting the proposed similarities between Bunyavirus Gc and Alphavirus E1. Significant alignments were not detected between Bunyavirus Gc or Alphavirus E1 and TBEV E or other flavivirus class II viral fusion proteins. Limited local similarities were observed between some Bunyavirus Gc and pestivirus E2. It is noteworthy that the significance of the overall sequence similarities between certain phlebovirus Gc and Alphavirus E1 is higher than some similarities among Gc of some prototypic members of the Bunyaviridae . These results further validate the use of the PRSS3 algorithm to identify limited similarities amongst viral proteins. Alignment of the carboxyl terminal portion of the pvc2 protein of rice stripe virus (RiSV), a Tenuivirus, and the envelope protein encoded by Cer13 retrovirus with two phlebovirus Gc reveals a collinear arrangement of fusion peptide consensus sequences . HAN Gn also showed a significant alignment with Gn of RVF, a phlebovirus. Both HAN Gn and Gn of TSWV, a tospovirus, also showed significant alignments with envelope protein 2 (E2) of SIN. SIN E2 has been implicated as the virion protein responsible for binding to the cell surface receptor [The longest open reading frames of M segments of all members of the Bunyaviridae are antisense to the virion RNA. mRNAs transcribed from Bunyavirus M segments are translated into large polyproteins that are subsequently cleaved by into functional proteins ,43. Gc oreceptor . These rThe simplest M polyprotein, encoding only Gn and Gc, is that of hantaviruses Fig. . In addiC. elegans retroviruses previously shown to have remarkable sequence similarities to phlebovirus Gc. These results also indicate that Gallaher's "Rosetta Stone" strategy can be used to identify potential class II viral fusion proteins, as demonstrated previously for class I fusion proteins [Proteomics computational analyses suggest that Bunyavirus Gc proteins are class II viral fusion proteins (β-penetrenes), with a structure similar to the fusion proteins of Alphaviruses and Flaviviruses. Similar sequences or common structural/functional motifs are collinearly located in Bunyavirus Gc and Alphavirus E1. Features common to other class II fusion proteins, including an internal fusion peptide, a carboxyl terminal transmembrane domain and regions with a high propensity to interface with bilayer membranes, are conserved and in similar locations in Gc of viruses in each genus of the Bunyaviridae. These features are also present in glycoproteins encoded by nonenveloped Tenuiviruses of plants, and a group of proteins ,49. The Many viral fusion proteins fit neither class I or II and it is likely that other classes of viral fusion protein also exist. However, among major classes of enveloped RNA viruses, there are at least six, myxoviruses, retroviruses, paramyxoviruses, filoviruses, arenaviruses and coronaviruses, that encode class I viral fusion proteins -4. AlphaAlphaviruses appear to use separate envelope proteins for fusion (E1) and attachment (E2) . BecauseThe remarkable similarities in both the pre- and post-fusion forms of the fusion proteins of SFV E1, an Alphavirus, and DEN and TBEV, members of the flavivirus genus of the Flaviviruses, in the absence of detectable sequence similarities, suggest that Alphavirus and Flavivirus class II fusion proteins may have diverged from a common progenitor. Alternatively, there may have been convergent evolution towards the common structure. Likewise, the sequence similarities detected between phlebovirus Gc and SIN E1 are consistent with divergent evolution from a common progenitor, but are insufficient to directly establish a phylogenic relationship. The results presented here suggest that Gc of members of the Bunyaviridae may have a common ancestor. Gn and Gc are in analogous locations in the polyproteins encoded by the five genuses of the Bunyaviridae. The simplest Bunyavirus M polyprotein, that of hantavirus members, encodes only Gn and Gc, whereas M of members of other Bunyavirus genuses encode several additional proteins. Therefore, divergence of Bunyavirus M segments may have occurred either through acquisition of sequences and/or lose of sequences in a cassette manner constrained in part by the locations of the major glycoproteins.Comparisons of divergent viral fusion proteins with internal fusion peptides can reveal features essential for virion:cell fusion. Regions of high membrane interfacial propensity including the fusion peptide and the transmembrane anchor, appear in similar locations in Bunyaviruses, Alphaviruses and Flaviviruses. The presence of several additional sequences with the propensity to interact with bilayer membranes in class II viral fusion proteins has not been considered in previous virion:cell fusion models ,11,54. CCurrent fusion models do not consider that the transmembrane domain and fusion peptide, while anchored into the viral and cellular membranes, would still be free to move laterally without distorting the membranes. More importantly, the virion is quite small compared to the cell, and would be freely mobile. Rearrangement of the fusion proteins may simply draw the virus closer to the cell without distorting either the viral or cellular membranes. An alternative to the models involving apposing membrane nipple formation is suggested by the observation that sequences of class II viral fusion proteins, including the fusion peptide, the transmembrane anchor and other sequences with high WWIHS scores, potentially form a nearly continuous track of membrane interactive regions that could channel the movement of lipids during virion:cell fusion Fig. , black. In the absence of structural determinations by X-ray crystallography, models such as proposed here can provide useful hypotheses to guide experimental strategies for development of vaccines or drugs to prevent or treat infection by viruses with class II fusion proteins. Prior to the availability of X-ray structural data, several potent HIV-1 TM inhibitors were developed ,57 basedThe authors declare that they have no competing interests.CEG performed the sequence alignments and assisted in the preparation of figures. RFG supervised the work and wrote the manuscript.

Grafting is a powerful but complex means to study the spread of RNA silencing Dactylosphera vitifoliae devastated European grapewine varieties over the course of the late 1800s and early 1900s, the varieties were saved by grafting them onto resistant rootstocks from the New World. Since then, these rootstocks have been used to maintain the susceptible Old World cultivars. But grafting is also an excellent tool for scientists studying systemic signals traveling between the rootstock and distal parts of the plants, and vice versa. For example, two important studies refers to the phenomenon whereby specific gene transcript levels are reduced in the presence of a related RNA. From studies of RNA silencing in several systems, much is now known about the mechanisms involved , but theAs expected, the discovery of this process triggered a quest for the “systemic inducer” of the process: a signal that travels through the plant and is able to initiate silencing in a remote location within the plant. Grafting was an obvious tool to use in the quest for this signal, as it allowed silencing source and sink tissues to be of different origin.Nia. Some of the transgenic lines generated always showed higher levels of Nia transcripts than the wild type—as expected from the presence of an additional gene—and were termed class I lines. However, other transgenic lines had undergone silencing for both the endogenous and exogenous Nia genes and those were termed class II lines. Nia transgene, which has an endogenous counterpart in the wildtype plant, green fluorescent protein has no homolog in nontransgenic lines. Therefore, silencing spreading in the wild-type “spacer” in the sandwich grafts could not be assisted by an endogenous sequence. Rather, the systemic signal must have traveled all the way to the scion and induced gene silencing there. The establishment of systemic silencing took 4 wk in the “direct” grafts and 6 wk in the sandwich grafts. However, silencing spread to the scion only in some of the grafts: in ten out of 16 direct grafts and five out of 11 sandwich grafts.Related experiments by I found these papers were very important not only for what they proved—the existence of a systemic signal of silencing—but also because they gave an unequivocal answer to the scientific questions they posed, using relatively simple methodology. Although excited by these successful examples of the transmission of silencing, I kept coming back to two questions: (1) what prevents transmission in some of grafts, and (2) why does it take longer to transmit silencing to the scion than it takes systemic silencing to reach the most remote parts of an intact plant?When we started working on the silencing signal ourselves, we repeated some of the above experiments but found somehow lower efficiencies in the initiation of silencing in the scions. We soon realized that our results were influenced by the developmental stage of our scions. A paper from Jeff Meins's laboratory in Switzerland shed light on some aspects of the grafting puzzle. Researchers there introduced additional chitinase genes using bolistics, in sense or antisense orientation under the control of a strong promoter (35S) into chitinase transformant lines of tobacco that never exhibited spontaneous gene silencing . In lineSurprisingly, only top grafting resulted in scions that were systemically silenced by a rootstock signal. Furthermore, even transmission after top grafting was less effective than expected; in one stock/scion combination only 27 out of 71 grafts exhibited transmission of the silencing signal. The authors also found that antisense-induced silencing was never transmitted to the scion. These findings do not answer the many questions about the mechanisms underlying systemic silencing, but they point us in certain directions.The individual parts of a whole plant are, in terms of import and export, in an equilibrium that changes with development. When grafting takes place, how this equilibrium is altered depends on the individual “parts” that contribute to the “new” whole plant. In addition, there is now indirect evidence that whaFrom the grafting experiments to date, it is now evident that the transporting capacity of the vascular tissue bypass that is formed at the graft junction does not fully reach the level of the original vascular tissue. The basis for these restrictions is not known. In a way, the graft interface functions as an unintentional filter. If the specificities of this “filter” were known, it would help us comprehend some transmission inconsistencies. Keeping in mind these limitations, grafting remains an invaluable tool in the search for the systemic silencing signal.

Tamoxifen is being used successfully to treat breast cancer. However, tamoxifen also increases the risk of developing endometrial cancer in postmenopausal women. Raloxifene also decreases breast cancer in women at high risk and may have a lower risk at developing cancer of the uterus. Tamoxifen has been shown to stimulate arachidonic acid release from rat liver cells. I have postulated that arachidonic acid release from cells may be associated with cancer chemoprevention.3H] arachidonic acid. The release of the radiolabel from these cells during incubation with tamoxifen and the raloxifene analog LY117018 was measured. The prostaglandin I2 produced during incubation of the rat liver cells with μM concentrations of tamoxifen and the raloxifene analog was quantitatively estimated.Rat liver, rat glial, human colon carcinoma and human breast carcinoma cells were labelled with [2 production and that induced by lactacystin and 12-O-tetradecanoyl-phorbol-13-acetate. LY117018, however, blocks the tamoxifen stimulated prostaglandin production. The stimulated prostaglandin I2 production is rapid and not affected either by preincubation of the cells with actinomycin or by incubation with the estrogen antagonist ICI-182,780.Tamoxifen is about 5 times more effective than LY117018 at releasing arachidonic acid from all the cells tested. In rat liver cells only tamoxifen stimulates basal prostaglandin ITamoxifen and the raloxifene analog, LY117018, may prevent estrogen-independent as well as estrogen-dependent breast cancer by stimulating phospholipase activity and initiating arachidonic acid release. The release of arachidonic acid and/or molecular reactions that accompany that release may initiate pathways that prevent tumor growth. Oxygenation of the intracellularly released arachidonic acid and its metabolic products may mediate some of the pharmacological actions of tamoxifen and raloxifene. The successful treatment and prevention of estrogen-dependent breast cancer in women by tamoxifen is attributed to its estrogen receptor (ER) occupancy . In tTamoxifen and raloxifene have several properties in common; e.g. prevention of tumors in the DMBA induced rat mammary model, maintenance of bone density in the ovariectomized rat and reduction of low density lipoprotein cholesterol. The partial estrogen agonist activity of tamoxifen on uterine tissue, however, increases the risk of developing endometrial cancer. This does not appear to occur with raloxifene.2 production by the rat liver cells. Although both compounds release AA from these cells, LY117018 is less effective. Only tamoxifen stimulates both basal and PGI2 production induced by incubation of rat liver cells with lactacystin in the presence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). LY117018, however, inhibits the PGI2 production stimulated by tamoxifen.Tamoxifen stimulates arachidonic acid release from rat liver cells . In thisThe intracellular release of AA and/or the cellular reactions that accompany that release may initiate pathways that prevent tumor growth. The tissue specific effects of tamoxifen and LY117018 may be associated with the AA or with cyclooxygenase (COX) activity and/or one of the many bioactivities resulting from oxygenation and metabolism of the released AA.3H]AA (91.8 Ci/mmol) was purchased from NEN Life Science Products, Inc. ; ICI-182,780 from Tocris Cookson, Inc. ; tamoxifen and 4-OH-tamoxifen were from Sigma Chemical Co. . LY117018 was obtained from Dr. David A. Cox, Eli Lilly and Co. . Raloxifene was extracted from EVISTA® tablets with dimethylsulfoxide.The C-9 rat liver and BT-20 human breast carcinoma cells were purchased from the American Type Culture Collection and maintained in MEM supplemented with 10% fetal bovine serum. The C-6 rat glial cell line was obtained from Dr. Elaine Y. Lai in the Department of Biology, Brandeis University and maintained in medium 199. The human colon carcinoma cells (HT-29) were obtained from Dr. Basil Rigas, American Health Foundation, Valhalla, NY and maintained in McCoy's medium. AA (0.2 μCi/ml) was added and the cells incubated for 24-h. The cells were washed 4 times with MEM and incubated for various periods of time with 1.0 ml of MEM, medium 199 or McCoy's containing 1.0 mg bovine serum albumin (BSA)/ml and different concentrations of each test compound. The culture fluids were then decanted, centrifuged at 2000 × g for 10 min, and 200 μl of the supernate counted for radioactivity. Radioactivity recovered in the washes before the 6-h incubation was compared to input radioactivity to calculate the % radioactivity incorporated into the cells released from radiolabelled methylcholanthrene transformed fibroblasts (about 20% after a 3-h stimulation by serum), 92% was AA, 4% was PGE2, 0.6% and 1% were PGF2α and phospholipids respectively [3H] AA had been incorporated into phosphatidylcholine (50%), phosphatidylethanolamine (36%), phodphatidylserine (9%) and triglycerides (10%) [3H] AA label released from human colorectal cancer cell lines (HCT116 and SW180) was AA as measured by TLC AA, was added after the first 24-h incubation. The cells were incubated for another 24-h, washed three times with MEM, then incubated with lactacystin and TPA in the presence and absence of the test compounds in MEM/BSA for various periods of time. The culture fluids were decanted and analyzed for 6-keto-PGF1α, the stable hydrolytic product of PGI2, by radioimmunoassay AA is presented as a percentage of the radioactivity incorporated by the cells. Except for the time-course experiments, which used duplicate dishes would be 0.25 nM (the specific activity of the [3H] AA is 92 Ci/mmole). Since TLC or HPLC analyses were not done, the concentration of the total released AA could not be quantitatively estimated. However, even treatment of a colorectal cancer cell line with 200 μM AA for 48-h leads to apoptosis . Similar to AA release [2 production by tamoxifen is not affected by preincubation of the cells for 2-h with 1 μM actinomycin D. The induction obtained by incubation with lactacystin in the presence of TPA is inhibited of basal activity is shown in Fig. μM of ba release , stimulated Fig. . Stimula780 Fig. . This es, GW7845 . The rap2 production stimulated by tamoxifen or Aramidex ® (anastrozole) do not release AA from cells in culture (unpublished data). These two drugs inhibit estrogen synthesis by blocking aromatase enzymes and also prevent estrogen-dependent breast cancer [These studies demonstrate that, at μM concentrations, tamoxifen and the raloxifene analog, LY117,018 stimulate the release of AA from cells in culture. At nM levels, the release is not observed. The effectiveness of these two compounds at prevention of estrogen-dependent breast cancer reflects competition for the ER . In addit cancer . In view2+ ionophore A-23187 reflects phospholipase activities. It is regulated by phosphorylation of the enzyme [A mechanism that most simply explains the release of AA by tamoxifen and LY117018 is the ability of such compounds to intercalate into cell membranes and affect phospholipase activities. The release of AA from endothelial cells by the Cae enzyme . The enze enzyme and woulThe AA release by tamoxifen and other reagents studied in my laboratory occurs with μM concentrations ,19,23,24When measured after a 6-h incubation, 10 μM tamoxifen stimulates deesterification of membrane phospholipids as measured by extracellular AA release, Fig. . The 6-h2 produced by the rat liver cells can be quantitatively determined. The effects of tamoxifen and LY117018 on the COX activity of the rat liver cells may be similar to their effects on other cells that express COX. Tamoxifen and LY117018 affect COX activity differently; only tamoxifen stimulates PGI2 production. AA, which regulates the production of lipoxygenase, COX and cytochrome P-450 epoxygenase products could impact many of the pharmacological actions of these two selective estrogen receptor modulators. AA can be oxygenated by COX isoforms, lipoxygenases, and cytochrome P-450 epoxygenases and their products converted to the prostaglandins, leukotrienes, epoxyeicostetranoic acids [2 to the major physiologically active products, PGD2, PGE2, PGF2α, PGI2 and thromboxane A2 [Some tissue specific effects of tamoxifen and raloxifene, e.g. on endometrial cells of the uterus, may be related to COX activity. At the low cell densities used in the present studies, the PGIic acids and AA tic acids . In the oxane A2 . They, ioxane A2 ,31.None declared.L.L. is the sole author and all of the experiments, with the exception of the western blots referred to as Levine L and Tashjian A (unpublished data), were conducted by L.L.The pre-publication history for this paper can be accessed here:

Cervical cerclage is a surgical procedure involving suturing the cervix with a purse type stitch to keep it closed during pregnancy. This procedure has been used widely in the management of pregnancies considered at high risk of preterm delivery. Several observational studies into the efficacy of cervical cerclage have claimed high rates of successful pregnancy outcome in women with a poor obstetric history attributed to cervical incompetence. However, a recent aggregate data Cochrane review found no such conclusive evidence from seven included randomised studies. Current data suggests that cervical cerclage is likely to benefit women considered to be 'at very high risk' of a second trimester miscarriage due to a cervical factor, however identifying such women remains elusive and many women may be treated unnecessarily. Undertaking an individual patient data (IPD) meta-analysis of the studies will allow us to investigate whether treatment is more effective in particular subgroups. Such an analysis will also provide a more powerful analysis of the predictors of preterm delivery and pregnancy loss, including ultrasound measurement of cervical length, and will allow a more complete analysis of 'time to event' outcomes.The analysis will include data from randomised trials comparing the intervention of elective cerclage versus no cerclage or bedrest to prevent miscarriage or pre-term labour. A specific list of data will be requested for each trial, including demographic and obstetric history data. The primary outcomes of interest will be neonatal mortality/morbidity. Attention will also be given to secondary outcomes such as time from randomisation to delivery, preterm delivery before 32 weeks and maternal morbidity. An intention to treat analysis will be performed, with attention paid to assessing clinical and statistical heterogeneity. Multilevel models with patients and trials as the two levels will be explored to investigate treatment effect on various outcomes. Patient-level covariates will be incorporated into the models in an attempt to account for statistical heterogeneity as well as to investigate interactions with treatment effect.Predictive models generated from our analysis should lead to more effective counselling of women at risk and a more cost effective use of cerclage. Cervical cerclage is a surgical procedure carried out during pregnancy. The operation involves suturing the neck of the womb (cervix) with a purse type stitch to keep the cervix closed. This surgical procedure has been used widely in the management of pregnancies considered to be at high risk of preterm delivery.Several observational studies in the last 50 years have claimed high rates of successful pregnancy outcome in women that had a poor obstetric history attributed to cervical incompetence. However, a recent Cochrane review found no conclusive evidence from seven included randomised studies that inserting a cervical stitch in women perceived to be at risk of preterm birth or second trimester pregnancy loss attributed to cervical factors, reduces the risk of pregnancy loss, preterm delivery or morbidity associated with preterm delivery (Drakeley 2003).In the Cochrane review, the data for important clinical outcomes including preterm delivery and maternal infection showed significant heterogeneity due to inconsistency in clinical definitions used, including the cut off gestational age defining preterm delivery, and different patient populations studied.Practically, methods of undertaking a meta-analysis of several studies may involve collecting either aggregate data, or data on each patient individually. The advantages of the latter approach, described as the 'yardstick' include One of the main concerns regarding current evidence related to cervical cerclage and other interventions for preventions of preterm delivery is a possibility that the 'primary outcomes' may have been selected to give results in greatest accord with the a priori beliefs of the authors. The evidence to support this phenomenon of within-study selective reporting comes from empirical research, which demonstrates discrepancies between research protocols and subsequent publications . A regreThe aim of this project is to undertake an IPD meta-analysis of randomised trials of cervical cerclage. Specific objectives are as follows.1. To estimate the effect of cervical cerclage on gestational age at delivery.2. To investigate whether cervical cerclage is more likely to prevent extreme prematurity (<28 weeks) or delivery at later gestations.3. To investigate risk factors for preterm delivery.4. To investigate interactions between risk factors and cervical cerclage.5. To model the effect of cervical cerclage and other risk factors on neonatal and maternal morbidity.The types of studies considered for inclusion in the analysis will be all randomised trials comparing cervical cerclage with expectant management or no cerclage during pregnancy. The previous Cochrane review (Drakeley 2003) identifiThe data collected in the studies will relate to women with confirmed, or suspected of having, cervical incompetence who desire future pregnancies and women who present as an emergency and are thought to have a diagnosis of cervical incompetence. The intervention investigated in the studies will be elective cerclage by whichever method , versus no cerclage or bed rest as interventions to prevent miscarriage or pre-term labour as defined in the original Cochrane review (Drakeley 2003).The methods of trial identification described in the original Cochrane review (Drakeley 2003) were excluded in the original review.The following data for each woman/infant pair will be requested from all trials: date of randomisation and gestational age, maternal demographics and obstetric characteristics at randomisation including cervical length on ultrasound, fibronectin and bacterial vaginosis data if available, treatment allocated, complications during pregnancy including ruptured membranes, maternal pyrexia or chorioamnionitis, date of delivery, gestational age at delivery and all neonatal data including birthweight, length of stay at NICU and morbidity related to prematurity.The following methodological data will also be requested for all trials: method of generation of randomisation list, method of concealment of randomisation, stratification factors and blinding methods.Data will be accepted either in electronic (floppy disk/CD/internet) or paper form. A desired format and coding will be specified but trialists may supply data in the most convenient way open to them, providing details of coding are sent with the data.A copy of the original data sent (before checking) will be held in a separate file. The following procedures will then be performed and documented for all trial data supplied. Trial details will be crosschecked against any published report of the trial. Range and consistency checks will be applied – missing data, errors and inconsistencies will be followed up with a nominated individual. The chronological randomisation sequence will be reviewed. The balance of prognostic factors will be checked, taking into account of factors stratified for in the randomisation procedure.The primary outcome of interest will be neonatal mortality/morbidity. Choice of primary outcome is about what should determine clinical decision-making. However it is recognised that trials to date may have insufficient power and there is a need to consider secondary outcomes of time from randomisation to delivery, preterm delivery before 32 completed weeks (<32+0 weeks) and maternal morbidity as defined in the Cochrane Review (Drakeley 2003). We willReporting of these outcomes in the original trial report is not an eligibility requirement for this review.Data on all randomised patients will be requested to perform an intention-to-treat analysis as far as possible. Clinical heterogeneity will be assessed by reviewing the differences across trials in characteristics of randomised patients.Initially, an aggregate data analysis will be undertaken although treatment effect estimates will be obtained from the individual patient data. Binary outcomes will be summarised in terms of odds ratios or relative risks, depending on the degree of heterogeneity observed. Time-to-event outcomes will be summarised in terms of the log (hazard ratio). The I square statistic and chi-square test for statistical heterogeneity will be applied to these summary data.Regression models, stratified by trial, will be used to explore the effects of treatment, risk factors and treatment-covariate interactions on the various outcomes of interest. These will include Cox and accelerated life models for time-to-event outcomes treatment effect across trials. Patient-level covariates (as listed above) will then be incorporated into the model in an attempt to account for some of the remaining statistical heterogeneity. An attempt will be made to incorporate these covariates first of all by assuming their effect to be constant across all trials and subsequently by assuming some heterogeneity in the covariate effect across trials by modelling them either as fixed or random effects. Finally, treatment-covariate interactions will be investigated by including additional variables and adopting a similar approach.If IPD are not available for some trials, the potential for bias will be investigated as follows. The reasons for not being able to obtain the data will be assessed for the potential for bias. Results using aggregate data from these trials will be compared with results using aggregate data from trials where IPD have been supplied, and any difference investigated. The analysis plan will be reviewed in light of the availability of IPD but prior to any comparative analyses.Predictive models generated by our analysis should allow more effective counselling of women at risk of preterm delivery and thus more cost effective use of cerclage.ZA and PRW were authors of a paper that will be included in the IPD meta-analysis . ZA was an author of the non-IPD systematic review on this topic . The autPRW conceived the idea, CTS drafted the initial protocol, and all authors commented on and approved this final version.The pre-publication history for this paper can be accessed here:

A 38-year-old woman presented with acute hematological toxicity from her anticonvulsants, even though she had been taking them for many years Six months prior to admission, her weight was 175 lbs, and her body mass index was 36. On admission, her complete blood count was normal, but over a 2-wk period she developed acute pancytopenia . During 6/l; and WBC, 10.7 × 106/l. After starting carbamazepine, her WBC dropped to 4.5–5.5 × 106/l, and her absolute neutrophil count dropped from 9 × 106/l to about 2.5 × 106/l. When the sodium valproate was added, her MCV increased to 112 fl, her platelet count fell to 100–150 × 106/l, and her Hb dropped to 110–120 g/l.She had been taking carbamazepine for 17 y and sodium valproate for 13 y for a mixed seizure disorder. At age 22 y, before starting any anticonvulsants, her baseline hematological parameters were as follows: Hb,123 g/l; MCV, 106 fl; platelets, 296 × 10The patient's other medications on admission were carnitine and low-dose L-thyroxine for hypothyroidism; she had been taking both for several years. She had not been recently exposed to any new medications, environmental toxins, or over-the-counter dietary supplements. There was no family history of aplastic anemia. The patient lived at home with her mother, who cares for her and has legal guardianship.A bone marrow biopsy showed a6/l and her platelet count to 248 × 106/l. Serial complete blood counts showed worsening anemia over the next 15 wk, requiring two packed red blood cell transfusions at week 6 and week 14, when her Hb was 31 and 53 g/l, respectively. Throughout this period, her reticulocyte count was low, at about 0.18% , while her WBC and platelet count were normal. We made a diagnosis of pure red cell aplasia (PRCA). A second bone marrow biopsy under minimally conscious sedation was unsuccessful, and the patient declined further attempts. After the first transfusion she was started on prednisone and erythropoietin for her PRCA. Meanwhile, because of her worsening seizures, we increased her sodium valproate dose, increasing her serum valproate level from 70 mcg/ml to 110 mcg/ml. After the second transfusion, we discontinued her sodium valproate and started her on clonazepam and oxcarbazepine as alternative anticonvulsants.The patient's carbamazepine was discontinued. Within 10 d, her WBC had risen to 6.7 × 10The patient had a brisk reticulocyte response. Her reticulocyte count rose from 0.36% to 0.78% in the second week and to 6.61% in the fourth week after stopping the valproate. Six weeks after stopping the drug, her Hb was 125 g/l and her MCV was 106 fl, suggesting replacement of transfused red blood cells with newly formed red blood cells (which have a higher MCV than older transfused cells). Over the next 30 mo of follow-up, she had no relapse of her aplastic anemia.We were unable to identify a specific cause for the patient's anorexia and weight loss. We found no evidence of malignancy on admission or subsequent follow-up. Within 1 wk of stopping her sodium valproate, her appetite improved and she put on 15 lbs over the next 6 wk. By the fourth month after stopping the drug, she had gained 45 lbs.6/l; Hb, 147 g/l; MCV, 104.9 fl; and platelets, 262 × 106/l.We last saw the patient in August 2004. Her seizure control had worsened, and she had developed signs of early dementia. Her current anticonvulsants are clonazepam, oxcarbazepine, and zonisamide, and she continues taking synthroid. Her last complete blood count was stable: WBC, 6.4 × 10Sodium valproate and carbamazepine are associated with rare but potentially lethal hematological complications. There are five case reports of PRCA shortly after initiation of sodium valproate, with the longest interval between the initiation of therapy and the onset of aplasia being 2 y ,2,3,4,5.Our investigations ruled out most of the known causes of acute bone marrow suppression, making the anticonvulsants the most likely cause. Malnutrition was an unlikely cause: her WBC and platelet counts had recovered before any increase in appetite or weight gain; her body mass index was normal despite her weight loss; and her bone marrow iron stores were adequate and her serum folate and vitamin B12 levels were high, suggesting adequate nutrient supply.We considered and rejected the possibility of Down syndrome–associated aplastic anemia. There have been six case reports of this condition, and in all cases it occurred in young children, suggesting a genetic predisposition . Half ofThe brisk return of her WBC and platelet counts upon discontinuing carbamazepine, and her brisk reticulocytosis upon discontinuing sodium valproate, were both consistent with previous reports of hematological toxicity due to these drugs ,4,5,6,7.We found no cause for the patient's anorexia and weight loss, but her appetite returned and she gained weight after stopping the sodium valproate. There is a known association between this drug and anorexia .Acute hematological complications of anticonvulsant therapy can still occur after many years of therapy, so continued vigilance is warranted.Although sodium valproate is generally associated with increased appetite and weight gain, it can also be associated with anorexia, nausea, vomiting, and weight loss.

Asparagus acutifolius L. is a dioecious and native plant species, widely distributed in the Mediterranean Basin. It is known for its fine flavour and could represent an important resource for cultivation programs in desert areas. Few molecular studies have been performed on this species. In the present paper, the ISSR technique was employed to study genetic diversity in Italian A. acutifolius.ST (0.4561) and Theta B (0.4776) values indicate a wide genetic variation among the samples examined. The distance UPGMA tree grouped together the genotypes strictly according to their geographical origin, showing that each sample is genetically structured and can be considered a distinct population. AMOVA analysis further confirmed genetic structuring of the populations. Population-specific fragments were also detected.Twenty-three primers produced a total of 228 polymorphic fragments used to evaluate genetic variation. FA. acutifolius according to geographical origin, and confirm the importance of genetic studies for designing germplasm conservation strategies.The results suggest that ISSR markers are useful in distinguishing the populations of The availability of a variety of DNA markers, such as restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and intersimple sequence repeat (ISSR) has enabled researchers to investigate genetic diversity among various plant species across natural populations -5. Amongin situ and ex situ of genetic diversity in natural populations is imperative to guarantee sustainable development [Local populations of traditional cultivars provide a valuable resource for plant breeding as well as for the preservation of genetic diversity . The expelopment .Asparagus acutifolius L. (Liliaceae) is a native, perennial plant species widely distributed throughout the Mediterranean areas, whose flowers are classified as dioecious and are mainly bee-pollinated; it generally does not reproduce by self-pollination. It grows in bushy and semi-dry places, sunny or semi-shade, mainly on limestone.A. officinalis and does not require rich soils for cultivation; for these reasons, it could be an economically important resource for the recovery of arid rural areas where controlled introduced programs could be achieved.This species is known for its strong taste compared to the cultivated A. officinalis, for which many molecular markers have been characterized [A. acutifolius are drawn from RAPD analyses [Viola pubescens [Oryza [To date, there is little information available on the genetic variability of this species. At present, the most widely studied species is P, AFLP) . The fewanalyses and the analyses . The ISSanalyses . They haanalyses , Viola pubescens , potato ubescens , and Orys [Oryza .A. acutifolius collecting samples in eight different scattered rural areas: six continental and one each from the Italian islands of Sardinia and Sicily.In this study we used ISSR markers to analyse the genetic diversity of Italian A. acutifolius was collected and their characteristics.Figure A. acutifolius is summarized in Table Among the 42 primers tested, 23 proved useful to characterize the samples was exhibited in the Sassari and the lowest (35.09%) in the Recco samples.Genetic structuring was evident due to the detection of specific bands in each sample examined. Spoleto and Caserta samples showed one and three fixed specific fragments, respectively, found to be statistically significant (P < 0.0001). For the other samples, 27 ISSR specific polymorphic fragments were detected, with a varying degree of statistical significance ranging from P < 0.0400 to P < 0.0001.Genetic distances were exaSamples collected at different geographic site grouped together, as shown in the UPGMA tree Fig. , and theThe values of gene diversity are summarized in Table T) was 0.2618 ± 0.0240 and the average variation within samples (average HS) was 0.1424 ± 0.0084. The mean diversity among the samples (DST) was 0.1194. The fixation index FST = (HT-HS)/HT was 0.4561, indicating a reduction of genetic diversity of about 45%. The Theta-B value obtained by Hickory analysis is an estimate of FST under a random-effects model of population sampling. Its mean value is 0.4766 ± 0.0173; the HT and HS values are, respectively, 0.2859 ± 0.0115 and 0.1619 ± 0.0026 showing that there is a general agreement between the results obtained using the two different approaches.As summarized in Table A. acutifolius in an Italian population. ISSRs also revealed diversity within each sub-population. The results obtained are in accordance with the principle that the number of individuals used to estimate average heterozygosity can be very small if a large number of loci is studied [ISSR markers can be used in population genetic studies of plant species as they effectively detect very low levels of genetic variation . They al studied .The gene diversity values Table ranged fST and the Theta-B value (0.4766) demonstrated a very great genetic differentiation among samples, possibly caused by random genetic drift. Further statistical support to the genetic structuring of the samples examined comes from the AMOVA analysis. Despite the continuous distribution of A. acutifolius, the eight samples represent genetically differentiated populations.The fixation index is 0.4561, which indicates a substantial reduction of genetic diversity (about 45%), probably due to the high genetic isolation of samples analysed. The FA. acutifolius populations.In each population it was possible to identify ISSR specific fragments. As shown in Table A. acutifolius throughout the Italian peninsula, there is poor gene flow through the isolates. The high genetic differentiation of the A. acutifolius populations examined might be attributed to the kind of pollinators (mainly bees) that can act at short distances, preventing the gene flow, and to the effects of anthropogenic habitat fragmentation.The high degree of genetic differentiation is confirmed by the UPGMA tree topology, in which all accessions from the same population grouped together Fig. . The popA. acutifolius populations [The results obtained using ISSR markers are in agreement with the RAPD analysis that also identified population-specific fragments in different Italian ulations .A. acutifolius has well-differentiated populations, despite their morphological low variability. These results show that to maintain genetic diversity within A. acutifolius it is necessary to conserve many populations.Information about the spatial organization of genetic variability is essential for the conservation of genetic resources . Our resA. acutifolius collected in the eight different locations in Italy and 1X Taq DNA polymerase buffer, in a total volume of 25 μL. The amplification programme was 1.5 min at 94°C; 35 × 40 s at 94°C, 45 s at the primer annealing optimal temperature and negative and positive controls were loaded and run at constant voltage (150 V) for 2 hours. After running, the gels were UV visualised and recorded using a Kodak Digital Science dS1D DC40/DC120 Camera. To verify the repeatability of the results, each DNA extraction, PCR amplification, and gel running was repeated twice.Unequivocally scorable and consistently reproducible amplified DNA fragments were transformed into binary character matrix .T), heterozygosity within population (HS), diversity among populations (DST), fixation index (FST), and genetic distance [Genetic variation within and among sub-populations was analysed on the basis of the banding profile using various parameters such as percentage polymorphism (P), total heterozygosity (Hdistance ,22-24, udistance . Since Idistance based onAMOVA analysis, implemented in Arlequin , was conThe Mantel test of genetic and geographic distances was carried out to evaluate the correlation between the two data matrices.The UPGMA tree was generated using the PAUP*4.0 software , and BooMS carried out part of the sample collection, designed ISSR primers and carried out ISSR work; GG did part of DNA extraction; SM carried out part of the sample collection and DNA extraction; LG participated in the manuscript preparation and revision; SA conceived the study, carried out data analysis, co-ordination and interpretation of the results.

In a previous study, when adult subjects were exposed to a level of 400 lux light for more than 30 min or a level of 300 lux light for more than 2 hours, salivary melatonin concentration during the night dropped lower than when the subjects were exposed to dim illumination. It was suggested that such light exposure in adolescents or children during the first half of subjective night in normal life might decrease the melatonin level and prevent the falling into sleep. However, there has been no actual study on the effects of light exposure in adolescents.Effects of exposure to the bright light (2000 lux) from fluorescent light bulbs during a period of three hours from 19:30 to 22:30 in one evening were examined on evening salivary melatonin concentrations from 19:45 to 23:40. The control group was exposed to dim light (60 lux) during these three hours. Both the dim light control group [DLCG] and the bright light experimental group [BLEG] consisted of two female and three male adolescent participants aged 14–15 y.The salivary melatonin level increased rapidly from 3.00 pg/ml at 21:45 to 9.18 pg/ml at 23:40 in DLCG, whereas it remained at less than 1.3 pg/ml for the three hours in BLEG. Melatonin concentration by BLEG at 22:30 of the experimental day was lower than that at the same time on the day before the experimental day, whereas it was significantly higher in the experimental day than on the day before the experimental day in DLCG.Bright lights of 2000 lux and even moderate lights of 200–300 lux from fluorescent light bulbs can inhibit nocturnal melatonin concentration in adolescents. Ancient Japanese lighting from a traditional Japanese hearth, oil lamp or candle (20–30 lux) could be healthier for children and adolescents because rapid and clear increase in melatonin concentration in blood seems to occur at night under such dim light, thus facilitating a smooth falling into night sleep. Night sleep duration of Japanese children aged 10–18 y has become shorter by one hour during the last 30 years in Japan . Student"Convenience stores" are open for 24 hours and provide several kinds of food and other goods for general civilian life. Convenience stores are now common all over Japan even in suburban areas. Illumination inside the convenience stores is very bright (2000 lux or more at the level of the eyes). Bright lighting in retail stores seems to be a merchandising technique which has been in use worldwide for at least 60 years. Unconscious use of bright light in the evening or at night inside the convenience store may promote a circadian phase delay in students exposed to the bright light during the first half of subjective night. This hypothesis is based on a "light-pulse" experiment in the laboratory as folloMelatonin, which is synthesized in the pineal organ and secreted to the blood, is well known as a key substance which may be effective in promoting the falling into night sleep by humans . Blood mCurrently, more than 80% of junior high school students of the third grade aged 14–15 y in Kochi go to private school in the evening. If they take a short stop at the convenience store to get some fast food and enjoy talking with their colleagues in front of the store before or after going to the private school in the evening, they suffer the double exposure to bright lights at the school and at the convenience store. Such bright lights are from fluorescent light bulbs and include blue or blue-green lights with 470–500 nm wave lengths which were reported to be powerful to suppress melatonin concentration . Based oIn this study, two light conditions were investigated. One was bright and high color-temperature light of more than 2000 lux, which is used in the evening and at night inside the convenience stores, at preliminary and private school for entrance examination, and at rental video shops in Japan. The other condition was a dark and low color-temperature light with less than 60 lux which is usual in the evening for traditional Japanese settings .Experimental participants were ten Japanese junior high school adolescent students aged 14–15 y who were attending Motoyama junior high school located in the mountain area of Reihoku district (33.5°N) in Kochi Prefecture. They had enjoyed New Year holidays for 7 days before the experiment. They were instructed to keep usual diurnal rhythm (for example bed time and wake-up time) during the holidays. Before the experiment, participants were divided into the two groups of "bright light experimental group (BLEG)" and "dim light control group (DLCG)". Participants in BLEG were selected to show similar circadian typology to those in DLCG based on the scores in the morningness-eveningness (M-E) questionnaire of Torsvall and Åkerstedt under fluorescent light bulbs based on our unpublished questionnaire study on 950 families having small children aged 0–6 yrs in Kochi. More than 85% of the 950 families enjoyed evening life under fluorescent light bulbs. We measured the illumination at the level of 1 m above floor just under a usual type of round-shaped fluorescent light bulb in a typical one-room apartment for students and it was 340 lux.th January 2003, all the ten participants got together in front of Motoyama junior high school at 8:00 in the morning. A wagon car took them to the experimental place which was a Japanese style hotel located at a mountain area, Yusuhara town in Kochi Prefecture, 126 km west from Motoyama town. During the driving, illumination inside the car was 350–500 lux. The car arrived at the hotel around noon. It was snowing through the day. Behavior of all the participants was controlled during the stay in the hotel till the next morning of the experimental day. All the participants played outside exposed to the sun light with 6000–7500 lux at the eye level during 12:30–13:30 and 14:00–14:50. They were allowed to have a rest in a living room in which the floor was filled with 12 tatami mats and the illumination at the eye level was 250 lux from fluorescent light bulbs during the rest of the time till 16:30. Participants took bath one by one between 16:30–18:00 and had supper all together between 18:15 and 19:20 in the living room. At 19:25, the participants of BLEG moved to a Japanese style room with 8 tatami mats where they were exposed to the light with 2000 lux at the eye level from fluorescent light bulbs, whereas DLCG group members moved to another Japanese style room with 8 tatami mats where they were exposed to the light of 60 lux and relatively low color-temperature from a electronic light bulb. All the participants included in both groups were home-working or making a small wooden folk craft object that is typical in the Yusuhara district, under each light condition till 22:30. Room temperature was controlled at 15 ± 2°C with an oil heater in both groups. Then they came back to the former living room (12 tatami mats) and stayed there under the light of 250 lux till 23:40. Then female and male participants moved to separate rooms and went to bed just before 24:00. Salivary samples were collected in collection tubes at 21:45, 22:30, and 23:40, and these salivary samplings were preserved in a refrigerator at less than -20°C. Melatonin concentration in the samples was analyzed by a professional analyzing company (MSL Co. Ltd.) which was a specialist for several chemical and microbiological analyses. All the participants from both groups were called out to get up at 7:00 in the next morning. All the participants got up between 7:00–7:15 responding to the calling out. After taking breakfast, they left the experimental place at 9:00 back for Motoyama junior high school. Throughout the study, light exposure was measured on the eye level with a digital illumination meter.On the experimental day of the 5Detailed explanation of the objectives and methods of the experiment was provided before the experimental performance to the participants and their parents. The research project received full and complete agreement from all of them.The results are shown in Fig. In Japan, bright and high color-temperature light of more than 2000 lux is available in the evening or night inside convenience stores which are open for 24 hours and private schools for the preparation to go through the entrance examination to upper schools. Also in usual life, such exposures to bright lights in the evening private school and convenience store can suppress the night increase in blood melatonin level as a direct effect and possibly delay the circadian system that drives the melatonin secretion rhythm and sleep-wake cycle.The results of this study suggest that ancient Japanese lighting in the evening and at night, which could be supplied by a traditional Japanese hearth fire or a oil lamp or candle (20–30 lux), might be healthy for adolescents and children, because the ancient lights could allow rapid and clear increase in melatonin level leading to a smooth falling into night sleep .Bright lights of 2000 lux and even moderate lights of 200–300 lux can inhibit, as a direct effect, nocturnal melatonin concentration in children. Ancient Japanese light conditions which could be supplied by a traditional Japanese hearth fire or a small oil lamp or candle might be healthy for children, because the ancient lights could allow rapid and clear increase in melatonin level in the evening, leading to a smooth falling into night sleep.None declared.

There is a possibility of clock genes having pleiotropic effects on clock period and pre-adult development time. In order to avoid such pleiotropic effects we have used wild type flies of same genotype under environments of different periodicities, which phenotypically either speeded up or slowed down the eclosion clock of D. melanogaster.In insects, circadian clocks have been implicated in affecting life history traits such as pre-adult development time and adult lifespan. Studies on the D. melanogaster, under five different light regimes, continuous light (LL), continuous darkness (DD), and light-dark (LD) cycles of 10:10 h (T20), 12:12 h (T24), and 14:14 h (T28). Although the development time was significantly different in most light regimes, except for females under T24 &T28, pre-adult survivorship remained largely unaffected. The development time was shortest under LL, followed by T20, DD, T24 and T28 regimes, in that order. Interestingly the development time showed a positive correlation with the period of eclosion rhythm, i.e., faster oscillations were associated with faster development, and slower oscillations with slower development.We assayed pre-adult development time and pre-adult survivorship of four laboratory populations of D. melanogaster in a manner that does not involve direct pleiotropic effects of clock genes on both clock period and development time.Based on these results we conclude that periodicity of imposed LD cycles, and/or of eclosion rhythm plays a key role in regulating the duration of pre-adult development in The Kruskal-Wallis test thus confirmed the results obtained in ANOVA. Although eclosion waveforms under no two light regimes were similar, the dissimilarity was even more striking under T24 and T28. The test revealed that mean development time of males was shortest under LL, followed by T20, DD, T24, and T28, in that order. While the mean development time of females followed a similar trend, it did not differ significantly between T24 and T28.ANOVA on pre-adult development time data revealed a significant main effect of light regime showed a significant positive correlation with the mean period of eclosion rhythm under the corresponding environments , the ancestral populations of the flies used in the present study, we had reported shortest development time under LL regime, followed by LD 12:12 h, and DD [T20, DD, and T24 and T28, in that order. As opposed to LL, eclosion under DD is gated in a circadian manner, and as a result flies took longer to develop compared to that in LL. We believe that the slight discrepancy in the results of our two studies could be due to the fact that different set of flies (about 100 generations apart) with different clock periods were used in the two experiments. The periodicity of JB populations under DD was greater than 24 h, whereas those of LL populations was lesser than 24 h. Thus, consistent with our proposal, development time of JB populations was greater under DD compared to development time in LD 12:12 h regime, whereas those of LL populations was shorter in DD compared to those under LD 12:12 h.In several insect species adult eclosion is gated in a manner that it occurs only during a narrow window of time, generally around dawn when environmental humidity is the highest . In D. m neurons , and it neurons . Develop, and DD . In the T20, DD, T24, and T28) showed a significant positive correlation with the mean period of eclosion rhythm under the corresponding light regimes; i.e. shorter period of eclosion rhythm under T20 was associated with faster pre-adult development, followed closely by DD, whereas flies took longest to develop under the light regimes wherein their eclosion periodicities were 24 h and 28 h, suggesting that development in D. melanogaster is regulated by eclosion rhythm. The peak of eclosion under three periodic light regimes closely matched phase relationships of eclosion rhythm relative to LD cycles, suggesting that phases of eclosion rhythm also play a key role in the regulation of development time [D. melanogaster is not entirely regulated by the periodicity of the environment and/or the periodicity of eclosion rhythm. These results are in agreement with the findings of previous studies, where the duration of pre-adult development was positively correlated with clock period [The mean development time under four different light regimes , at constant temperature of 25°C (± 1°C), and constant humidity of 70 ± 5%, on a 21 day discrete generation cycle . These populations were maintained at moderate larval densities of ~ 60–80 larvae per 8 dram vial (9.0 mm height × 2.4 mm diameter) containing banana-jaggery food medium (henceforth banana food). The ancestry and maintenance of these populations has been described in detail in an earlier paper [3). From these petri plates, 60–80 eggs are collected into each of 40 vials in which larvae then develop into adults. Adults eclosing from these vials are transferred to plexiglass cages on 12th day after egg lay. On the 18th day after egg lay, adult flies are supplied with banana food supplemented with live yeast paste for two days, after which eggs are collected to initiate the next generation and the adults discarded. The breeding population typically consists of about 1500 flies.The four populations of er paper . In brieT20), 12:12 h (T24), and 14:14 h (T28). Thus a total of 200 vials were set up for the assay . These vials were introduced into five different light regimes at 20:00 h, when lights went off simultaneously in all LD regimes. Fluorescent white light of intensity ~ 100 lux was used during light phase, whereas in dark phase red light of λ >650 nm was used. Temperature and relative humidity in the five light environments monitored continuously using a Quartz Precision Thermo-Hygrograph, Isuzu Seisakusho Co, LTD, were found to be comparable. The vials were monitored for eclosion of adult flies after the pupae became dark. Eclosing adults were collected every 2 h, sexed, and counted until all the flies eclosed.From the running culture of each population (LL1..4), eggs laid on banana medium over a 2 h window were collected for the assay. Exactly 30 eggs were dispensed into 8 dram vials containing ~ 6 ml banana food and 50 such vials were set up from each population. Ten vials from each population were introduced into continuous light (LL), continuous dark (DD), light-dark (LD) cycles of 10:10 h , in which replicate populations were treated as random blocks, and light regime and sex were treated as fixed factors.In order to detect differences in the eclosion profiles of the flies under different light regime, development time data of individual flies from all four replicate populations were pooled and compared using Kruskal-Wallis test.Pre-adult survivorship was calculated as the fraction of eggs that successfully developed into adults in each vial. The mean pre-adult survivorship values of each population in each light regime were used as data in a mixed model ANOVA, with replicate populations as random blocks, and light regime as a fixed factor.DAP and AD were involved in collecting eggs, collecting and counting flies for pre-adult development time, estimating pre-adult survivorship, data entry and analyses. VKS conceived of the study and participated in its design and coordination. MKC and AJ gave valuable comments and suggestions throughout the study. All authors read and approved the manuscript.

Public health practitioners and researchers for many years have been attempting to understand more clearly the links between social conditions and the health of populations. Until recently, most public health professionals in English-speaking countries were unaware that their colleagues in Latin America had developed an entire field of inquiry and practice devoted to making these links more clearly understood. The Latin American Social Medicine (LASM) database finally bridges this previous gap. brings important information, originally known mostly within professional networks located in Latin American countries to public health professionals worldwide via the Internet. The LASM database uses Spanish, Portuguese, and English language trilingual, structured abstracts to summarize classic and contemporary works.This public health informatics case study describes the key features of a unique information resource intended to improve access to LASM literature and to augment understanding about the social determinants of health. This case study includes both quantitative and qualitative evaluation data. Currently the LASM database at The University of New Mexico This database provides helpful information for public health professionals on the social determinants of health and expands access to LASM. Public health practitioners have long recognized the connections between patients' socioeconomic conditions and their health -8. Yet tAmerican Journal of Public Health that focused on LASM [Until recently, however, most of the knowledge base in this discipline has remained largely unknown outside Latin America. Language barriers and disincentives to distribute this information more widely are two major reasons for this lack of awareness. Some readers might have first learned about Latin American social medicine (LASM) through recent critical reviews or throu on LASM ,11.LASM traces its historic origins to European researchers such as Rudolf Virchow and the belief systems of indigenous cultures. Both the European and indigenous sources of current social medicine practices emphasized the importance of linking social conditions to health status. Contemporary social medicine in Latin America continues to emphasize these linkages between social conditions and the health of populations. Social medicine professionals participate in a wide array of settings in Latin America and represent diverse specialties. Their integration into healthcare systems has varied by era and country .Innovative approaches to disseminating work in LASM have become increasingly available due to Internet technology. The project "Enhanced Access for Latin American Social Medicine" at The University of New Mexico with funding from the U.S. National Library of Medicine seeks to make information on the connections between social conditions and health problems available to a wide audience. The project has sought to bridge the prior information gap primarily through delivering structured abstracts of social medicine publications in Spanish, English, and Portuguese via the Internet on the LASM database beginning in 2001. Other goals of this project include: publishing full text social medicine electronic journals on behalf of medical societies in Latin America; and, maintaining a repository for key classic and contemporary social medicine publications.Structured abstracts are posted in three languages in the LASM database at The University of New Mexico for both the classic and contemporary social medicine literatures. The first phase of this project involved preparing and posting the structured abstracts of 25 landmark books, 50 book chapters, and 100 journal articles from the classic social medicine literature in Spanish, Portuguese, and English.A peer selection committee identified and agreed upon the specific selections of classic books, book chapters, and journal articles to be abstracted for this project. Table The contemporary literature summarized in the LASM database has been drawn primarily from the 12 journals currently or previously published in Latin America. Table Saúde em Debate (Brazil) and SaluCo Bulletin (Cuba), are available from the host website. The host website also posts structured abstracts of important articles from these full text journals.As a pilot, the project has made two social medicine journals available in electronic full text format. Several issues of these journals, The LASM database encompasses several broad themes within LASM. Subject areas include the history, theories, methodologies, and organizational dimensions of social medicine. Other subjects pertain to institutional analysis, social/critical epidemiology, and strategic planning. Table The LASM database is a web application developed using the Cold Fusion application server. The data are stored in a Microsoft SQLServer relational database. The data sources are the original Latin American publications, which are summarized in Spanish, Portuguese, and English languages in structured abstract format. The English-language structured abstracts are quality checked by the principal investigator, who reviews the abstracts for substantive content, and then by the librarian investigator who reviews the structured abstracts for final quality assurance purposes. Each record contains fields for author, title , place of publication, publisher and structured abstracts in each language. Records contain volume, number, and pagination when applicable. All records contain terms the from Medical Subject Headings (MeSH) system, a controlled vocabulary developed and maintained by the National Library of Medicine. Users can search the database on controlled fields of author, title, and MeSH terms in any of the three available languages. Full text abstract searching is also available. In addition to the abstract searching facilities, the LASM database can be browsed alphabetically by title. The browsing interface offers a convenient way to become familiar with the extent and diversity of the LASM literature.From January 2002 through December 2003, a total of 17, 853 visits were made to the website hosting the LASM database. The largest numbers of visits in descending rank order originated from Brazil, Mexico, Argentina, Spain, and Colombia. A preliminary, qualitative evaluation has been favorable. Following completion of this project, we will conduct and publish a comprehensive summative evaluation. A total of 250 structured abstracts in Spanish, Portuguese, and English had been posted to the LASM database as of June 30, 2004. The host website presents more detailed information about this project, as well as the structured abstracts themselves.The LASM database comprises a dynamic and searchable web application containing structured abstracts in English, Portuguese, and Spanish. It is designed using industry standard web application design principles, but its content is unique to the LASM domain.A database searching design and utility problem unique to the LASM database, and other web applications like it, relates to the problem of web-based multilingual searching. Widely available search engines cannot preprocess a multilingual language translation of search terms (e.g. retrieving results containing the Spanish word "pública" for the English search term "public"). Additionally, search engines do not recognize that the unaccented "publica" (as it might be entered by an English speaker as a search string) might be the same word as the Spanish accented "pública" and therefore will not return the user's expected search result. Key combinations and modifiers that are used to create special and accented characters in word processing programs like Microsoft Word often do not work in browser based search and form fields. Our primary instruction to users for constructing search terms containing special and accented characters suggests that they use a separate text editor that accepts keyboard modifiers to construct special and accented characters to build the search term and then copy and paste the completed search string into the field. On our search pages we also list common special and accented characters in Spanish and Portuguese that users can copy and paste into browser based search fields.Although there are no other alternatives for entering special and accented characters into browser form fields, both methodologies are somewhat cumbersome. Therefore, to improve the searching utility of the LASM database we also preprocess entered search strings to allow the search engine to perform selected character substitution on the entered search string and attempt to solve the accented character problem from the search engine side. All search strings are first passed through a regular expression routine, which substitutes single character search engine wildcards for the following characters: A, E, I, O, U, a, e, i, o, u, N, C, n, c. This effectively removes potential special and accented character misspellings. To return to the previous example, a user might enter the search string "publica" ("public" in English) in a search field as an attempt to find article titles that contain the Spanish or Portuguese word "pública". Properly spelled, "Pública" uses the accented character "ú" rather than "u" (ASCII 163 rather than ASCII 117). Unfortunately, current search engines are not intelligent enough to infer that "pública" is a match for the search string "publica". Therefore, the literal search for publica (unaccented) will not return any search results that contain "pública" although there are hundreds of instances of the word "pública" in the LASM database.In the case of "publica", the preprocessed search string that is finally submitted to the search engine is "p*bl***". The search engine will return all seven-letter word matches that contain the letters p, b, and l in the first, third, and fourth positions respectively. The net effect of this technique is to under-specify the search result. That is, the search engine may possibly return records that contain other words that happen coincidentally to match the submitted search string. On the other hand, the returned result set can be guaranteed to contain the desired search result.Problems created from under-specifying the search are limited based on experience gained from using this technique. The positional constraints of submitted characters within the search string generally are restrictive enough to prevent most problems. Specifically, within a limited domain search surface like the LASM database, the likelihood of the occurrence of most alternative word matches is very low.The character substitution methodology described here is not perfect, and many other alternative strategies for addressing the multilingual search problem have been explored by LASM technical staff. Most of the alternative strategies considered, however, involved much higher costs in terms of acquiring or developing specialized search engine capabilities or much higher abstract preparation costs. Therefore, we chose the character substitution strategy as a compromise between implementation cost and search utility.Internet technology via websites and web browsers has created numerous opportunities for public health colleagues to inform one another and to collaborate across wide geographic space. The LASM database clearly demonstrates the efficacy of the Internet for communicating its informative structured abstracts posted in the Spanish, Portuguese, and English languages. This database provides useful information that would otherwise be unavailable for public health professionals on the social determinants of health. Furthermore, it expands access to LASM through its inclusion of both the classic and contemporary literature.. As the data reported above indicate, the LASM database already has been accessed steadily since its initial small-scale publicity began in 2002. We hope that public health readers will utilize the LASM database to improve their research, teaching, and practice.Anyone with access to the World Wide Web and a web browser can access all structured abstracts in the LASM database Neither the authors nor any support personnel possess competing interests related to the LASM database or to this article's publication.JE conceived of and wrote the initial version of this article, served as an early collaborator on and helped design the project, was responsible as an investigator for acquiring journals for the Latin American social medicine collection, provided quality assurance editing on structured abstracts, played a major role in the formative evaluation of the project, and coordinated all revisions to this article. HW initiated and designed the project, served as principal investigator, obtained funding, translated and edited abstracts in the English-language, and edited this manuscript. HSB served as co-principal Investigator, helped design this project, obtained funding, and led the effort for the formative evaluation of this project. JT helped administer the project and edited this manuscript. CI coordinated the project, wrote Spanish-language structured abstracts, and provided reference materials for this article. KW and JT developed all programming aspects of the website including the search strategies, managed the web interface and underlying database and contributed the text for portions of this article.The pre-publication history for this paper can be accessed here:

Daily energy expenditure consists of three components: basal metabolic rate, diet-induced thermogenesis and the energy cost of physical activity. Here, data on diet-induced thermogenesis are reviewed in relation to measuring conditions and characteristics of the diet.Measuring conditions include nutritional status of the subject, physical activity and duration of the observation. Diet characteristics are energy content and macronutrient composition.Most studies measure diet-induced thermogenesis as the increase in energy expenditure above basal metabolic rate. Generally, the hierarchy in macronutrient oxidation in the postprandial state is reflected similarly in diet-induced thermogenesis, with the sequence alcohol, protein, carbohydrate, and fat. A mixed diet consumed at energy balance results in a diet induced energy expenditure of 5 to 15 % of daily energy expenditure. Values are higher at a relatively high protein and alcohol consumption and lower at a high fat consumption. Protein induced thermogenesis has an important effect on satiety.In conclusion, the main determinants of diet-induced thermogenesis are the energy content and the protein- and alcohol fraction of the diet. Protein plays a key role in body weight regulation through satiety related to diet-induced thermogenesis. Diet induced thermogenesis (DIT) can be defined as the increase in energy expenditure above basal fasting level divided by the energy content of the food ingested and is commonly expressed as a percentage. It is, with basal metabolic rate and activity induced thermogenesis, one of the three components of daily energy expenditure. Although DIT is the smallest component, it could play a role in the development and/or maintenance of obesity. De Jonge and Bray evaluateMethodological issues include: was the baseline appropriate, what was the energy content and nutrient composition of the test food consumed, what was the duration of the postprandial measurement period, and what was the error associated with the calculation of DIT from the measured energy expenditure. Weststrate et al investigHere, the focus is on DIT as a function of the energy content and nutrient composition of the test food consumed and the duration of the postprandial measurement period in adult subjects with a normal bodyweight. The review is based on literature published over the last 15 years.The experimental design of most studies on DIT is a measurement of resting energy expenditure before and after a test meal, with a ventilated hood system. The observation is started after an overnight fast, where subjects are refrained from eating after the last meal at 20.00 h at the latest. Thus, with observations starting between 08.00 and 09.00 h the next morning, the fasting interval is at least 12 h. Postprandial measurements are made for several hours where subjects have to remain stationery, most often in a supine position, for the duration of the measurements. In some studies, measurements are 30 min with 15 min intervals allowing i.e. for sanitary activities.The use of a respiration chamber to measure DIT has the advantage of reproducing more physiological conditions over a longer period of time while regular meals are consumed throughout the day ,6. The DStudies on DIT were selected from Medline. Studies were selected when information was presented on energy intake, diet composition with respect to carbohydrate, protein fat and alcohol of the test food, duration of the postprandial measurement, and DIT.Reported intra-individual variability in DIT, determined with ventilated-hood systems, is 6 to 30% ,8. ReporThe mean pattern of DIT throughout the day is presented in figure Fifteen studies on DIT with information on energy intake, on diet composition and on the postprandial measurement period were selected from literature Table . Five stFor a comparison of DIT between studies as a function of the nutrient composition of the test food consumed, the energy content of the test food was divided by the length of the measurement interval after food consumption and expressed in MJ/h. Only three of the 22 studies presented in table The main determinant of DIT is the energy content of the food, followed by the protein fraction of the food. The thermic effect of alcohol is similar to the thermic effect of protein.Diet induced thermogenesis is related to the stimulation of energy-requiring processes during the post-prandial period. The intestinal absorption of nutrients, the initial steps of their metabolism and the storage of the absorbed but not immediately oxidized nutrients . As suchTheoretically, based on the amount of ATP required for the initial steps of metabolism and storage, the DIT is different for each nutrient. Reported DIT values for separate nutrients are 0 to 3% for fat, 5 to 10% for carbohydrate, 20 to 30% for protein , and 10 Of the studies presented in table The higher DIT value of alcohol and protein compared with carbohydrate and fat has implications for the effect of these nutrients on energy balance. However, the main effect on energy balance does not seem to be primarily linked to the lower bioavailability of alcohol-and protein energy than that of fat and carbohydrate. Alcohol energy is largely additive to the normal diet but does not seem to affect energy balance positively . ProteinAlcohol forms a significant component of many diets and it supplements rather than displaces daily energy intake. Alcohol consumption as an aperitif has even been shown to result in a higher subsequent intake with no intake compensation afterwards . Yet, alThe main effect of protein on energy balance is thought to be DIT related satiety. Satiety scores were higher during meals with a high-protein/high-carbohydrate diet, as well as over 24 h, than with a high-fat diet . The obsIn conclusion, the main determinants of diet-induced thermogenesis are the energy content and the protein-and alcohol fraction of the diet. Protein plays a key role in body weight regulation through satiety related to diet-induced thermogenesis.

In view of clinical observations and laboratory results that support a central role of the spleen in idiopathic thrombocytopenic purpura (ITP) pathophysiology, we studied the effect of splenectomy on type-1 and type-2 cytokine gene expression in an adult ITP case, refractory to conservative treatment.6/L within 10 days after the operation. Two consecutive blood samples were obtained from the patient, 3 and 7 months after the splenectomy for the purposes of this study. A control group consisted of 11 healthy adults. Peripheral blood mononuclear cells were prepared from each blood sample and cultured in vitro for 8 h with the addition of the mitogens phorbol myristate acetate and ionomycin. Total cellular RNA extracted from 106 cells was submitted to semiquantitave reverse transcriptase-polymerase chain reaction (RT-PCR) for the amplification of IL-2, IFN-γ, IL-4, IL-5, and IL-10 metagraphs. The PCR products were run on ethidium-stained agarose gels, photographed and quantified by densitometry.The patient was subjected to splenectomy 9 months after the diagnosis with complete response, attaining platelet counts over 150 × 106/L to 265 × 106/L. The change of type-2 cytokine expression was slight and the Th2 activity (IL-4+IL-5) remained largely unchanged. The Th1/Th2 ratio, that reflects the pathogenic disease-specific T-cell immune deviation, was accordingly reduced 7 months post-splenectomy (Th1/Th2 = 1.3) compared to 3 months (Th1/Th2 = 3.5).A steep decrease of type-1 cytokine expression and their calculated sum expressing Th1 activity was observed at 7 months post-splenectomy compared to 3 months post-splenectomy, in parallel with a rise of platelet count from 190 × 10The reduction of the Th1/Th2 cytokine ratio that was observed over time after splenectomy was accompanied by full clinical remission. Nevertheless, the persistence of a type-1 polarization, even after several months following spleen removal, is suggestive of a more basic abnormality of the immune function in these patients. Adult autoimmune thrombocytopenic purpura (ITP) is a chronic acquired organ-specific autoimmune thrombocytopenic syndrome . The lowThe spleen is considered to be the primary site of the autoimmune response where initiation, maintenance and regulation of the autoimmune attack take place. The spleen is the site of autoreactive T- and B-cell interaction and activation, and autoreactive anti-platelet antibody production . Platelein vitro cultures [+CD4+, CD3+CD8+, CD3+HLADR+, and CD3+CD25+ cells increases significantly in ITP patients refractory to splenectomy [Splenectomy is followed by reduction of autoantibody peripheral blood titre ,13. Splecultures . The percultures . The numenectomy . Changesenectomy .Splenectomy is the most clinically effective therapeutic intervention in ITP patients, resulting in complete remission in two thirds of the patients with more than 60% maintaining the therapeutic effect in the long term ,18. IrreGiven the important immunoregulatory role of the spleen, we looked at the effects of splenectomy on immune activation and immune deviation indices in the peripheral blood of an ITP patient after splenectomy in association with peripheral platelet counts.6/L). Following clinical exclusion of causes of secondary thrombocytopenia [A 42 year old woman presented in October 1999 in Patras University Hospital (PUH) with lower limb purpura and low platelet count . Informed consent was obtained from the patient. PUH abides by the Helsinki declaration on ethical principles for medical research involving human subjects.in vitro for 8 h with the addition of 20 ng/ml phorbol myristate acetate (PMA) and 1 μM ionomycin .Peripheral blood mononuclear cells (PBMC) were prepared from each blood sample by centrifugation over a Ficoll-Paque gradient . The cells were cultured 6 cells was submitted to semiquantitative RT-PCR for the amplification of IL-2, IFN-γ, IL-4, IL-5, and IL-10 metagraphs [Total cellular RNA extracted from 10tagraphs . Primerstagraphs .6/L to 265 × 106/L. Regarding type-2 cytokine gene expression, IL-4 increased, IL-5 decreased, and IL-10 remained unchanged, whereas the change in Th2 activity (IL-4 units plus IL-5 units) was slight (Figure A sharp decrease in the expression of the type-1 cytokines IL-2 and IFN-γ and their calculated sum expressing Th1 activity was observed at 7 months after splenectomy compared to 3 months after splenectomy (Figure The above results show that in this patient type-1 polarization persists after removal of the spleen and attainment of clinical remission. This may mean that the spleen is not exclusively responsible for the coordination or the maintenance of the pathological immune response in this patient, provided that no accessory splenic tissue exists. Other disease centres may control the autoimmune reaction as well, such as the liver or the bone marrow. Alternatively, it is possible that ITP is the manifestation of a general immune system malfunction that pre-existed before the development of thrombocytopenia and persists after removal of what seems to be the effector of a manifestation of an autoimmune proclivity.Unfortunately, a pre-splenectomy sample was not available for analysis. As a result, no conclusions can be drawn about the effect of splenectomy on the direction of change of Th1 activity. Based on phenotypic studies showing increased presence of T lymphocytes with activated phenotype after splenectomy in ITP patients , it is pThe clinical remission may be due to the removal of a major platelet destruction site, although the underlying immune activity that drives the destruction may remain unaffected. Complete remission does not mean that increased platelet destruction has stopped after splenectomy. Platelet life span may still be shortened in this patient and/or her normal platelet count may be even higher than what was achieved after splenectomy.The pathological immune activity seems to decrease over time after splenectomy, as reflected by the lower Th1/Th2 ratio that is indicative of the degree of immune polarization. This may be explained by reduced stimulation of the immune system by activated spleen reticuloendothelial cells that present platelet antigens to T helper lymphocytes. In this way, it may be hypothesized that removal of one vital component of the self-attacking immune process can break the vicious circle that culminates in even greater immune activation, polarization, and platelet destruction. Removal of a major site of autoimmune activity may have abrogated recruitment of naïve T-cells. As a result, overall autoimmune activity wears off, as existing activated Th1 effector cells perish leaving behind a much smaller population of peripheral memory cells that retain the initial Th phenotype.Another consideration that stems from the results of this case study is that immune polarization and immune deviation of the pathological response depend more on upregulation of type-1 mediators rather than on suppression of type-2 cytokines, or that type-2 response is inadequate to control excess type-1 response in active disease.Clinical improvement after splenectomy is associated with reduced but not normalized immune activation and polarization in the patient studied. However, the spleen seems not to be absolutely necessary for the maintenance of the autoimmune reactivity.The authors declare that they have no competing interests.FPP prepared the case report, performed the experiments and drafted the manuscript. AM conceived of the study, participated in its design and coordination and co-wrote the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:

A healthcare provider's recommendation to undergo screening has been shown to be one of the strongest predictors of completing a colorectal cancer (CRC) screening test. We sought to determine the relationship between the general quality of self-rated patient-provider communication and the completion of CRC screening.A formative study using qualitative data from focus groups and quantitative data from a cross-sectional survey of church members about the quality of their communication with their healthcare provider, their CRC risk knowledge, and whether they had completed CRC screening tests. Focus group participants were a convenience sample of African American church members. Participants for the survey were recruited by telephone from membership lists of 12 African American churches located in rural counties of North Carolina to participate in the WATCH (Wellness for African Americans Through Churches) Project.Focus Groups. Six focus groups (n = 45) were conducted prior to the baseline survey. Discussions focused on CRC knowledge, and perceived barriers/motivators to CRC screening. A theme that emerged during each groups' discussion about CRC screening was the quality of the participants' communication with their health care provider. Survey. Among the 397 participants over age 50, 31% reported CRC screening within the recommended guidelines. Participants who self-rated their communication as good were more likely to have been screened (36%) within the recommended guidelines than were participants with poor communication (17%) . Participants who had adequate CRC knowledge completed CRC screening at a higher rate than those with inadequate knowledge (p = 0.011). The percentage of participants with CRC screening in the recommended guidelines, stratified by communication and knowledge group were: 42% for good communication/adequate knowledge; 27% for good communication/inadequate knowledge; 29% for poor communication/adequate knowledge; and 5% for poor communication/inadequate knowledge.Participants who rated their patient-provider communication as good were more likely to have completed CRC screening tests than those reporting poor communication. Among participants reporting good communication, knowledge about colorectal cancer was also associated with test completion. Interventions to improve patient-provider communication may be important to increase low rates of CRC screening test completion among African Americans. Colorectal cancer (CRC) screening among average risk adults 50 years and older can decrease the incidence and mortality rates for CRC -7. NatioA healthcare provider's recommendation to undergo screening has been shown to be one of the strongest predictors of completing a CRC screening test -17, and . The WATCH Project was a church-based colorectal cancer prevention study designed to increase fruit and vegetable consumption, reduce fat intake, increase moderate physical activity, and increase CRC screening among church members. In this study of patient-provider communication and CRC screening, we used data from two different sources: 1) focus groups of African American men and women, and 2) surveys of church members. The Institutional Review Board from the University of North Carolina at Chapel Hill approved this study.Data for this study were obtained as part of a larger study, the WATCH (Wellness for African Americans Through Churches) Project As part of the formative data collection in early 1998, we conducted six focus groups of African American adults. There were three focus groups of African American adult men and three of African American adult women. The focus groups comprised of a convenience sample drawn from members of African American churches located in central North Carolina. The focus groups were conducted in the churches, lasted approximately 60 minutes, were tape recorded, transcribed, and reviewed for accuracy. A $15 incentive and refreshments were provided for the participants. The focus groups were led by sex-matched African American moderators and explored issues associated with colon cancer, CRC screening (barriers and motivators), nutrition, and physical activity. The information from the focus groups was used to develop the questionnaires and to help guide the intervention strategies used in the culturally appropriate colon cancer prevention program.The second source of participants for the survey study was the baseline intervention sample for the larger WATCH project. The telephone survey collected information focused on general health status, nutrition, physical activity, patient-provider communication, CRC risk knowledge and screening behaviors. The baseline survey was completed, on average, in approximately 40 minutes and was administered by trained interviewers prior to the intervention. The baseline telephone surveys were conducted between October 1998 and October 1999.In this study, a history of CRC screening was defined as the self-report of undergoing a fecal occult blood test (FOBT), flexible sigmoidoscopy, or colonoscopy within the recommended time period. Participants were asked whether they had each of the CRC screening tests, and if they responded yes, the participants were asked when they had their last test. The items in the survey included a brief explanation of each screening test. Items were described as follows: FOBT, "which is stool slides"; sigmoidoscopy, "which is a tube inserted in the rectum to look at the colon and the bowel"; and colonoscopy, "which is a tube inserted in the rectum to look at the entire intestine, usually given in a hospital or specialist's office." Responses included, "less than 1 year"; "1–2 years"; "3–5 years"; or "more than 5 years."CRC screening was considered to be within the recommended time period based on an algorithm using ACS guidelines considering which test and how recent the test was performed (e.g. a person who reported having FOBT within the past year was considered within the recommended time period). Participants were considered to have been screened within the recommended time period if they had a FOBT within the preceding year, and sigmoidscopy within the preceding 5 years. Although current CRC screening guidelines recommend colonoscopy every 10 years for average-risk adults, we examined colonoscopy use in the past 5 years because of the limitations of the survey instrument. Self-report of CRC screening behavior has been demonstrated to be a reliable endpoint for intervention trials .Analyses of data from this study included factor analysis, analysis of variance, and logistic regression and were conducted using SPSS, version 10.1. Logistic regression analyses were performed to evaluate whether the level of perceived patient-provider communication was significantly related to CRC screening behavior in this population. Sociodemographic variables were identified as potential covariates if there was plausible theoretical or empirical evidence that the variable might be associated with the communication variable or with CRC screening. Variables that were significantly associated with communication level (p < 0.05) were retained and tested as covariates in the logistic models. Only the sex of the participant, receiving healthcare at a doctor's office versus a clinic/emergency room, and knowledge of CRC risk were significantly associated with communication and only these three covariates were entered into the initial logistic model. Variables in the model were evaluated by the Wald test and interpreted using odds ratios and confidence intervals. The overall fit of each model was evaluated using the Hosmer-Lemeshow Goodness of Fit test and by examining classification tables .Several major themes associated with CRC emerged from the personal experiences expressed about the medical community during the focus groups. One of the themes discussed by the participants in each focus group was patient-provider communication. Selected comments by focus group members about CRC and patient-provider communication are listed in Table Originally, 2480 names were obtained from the 12 church rosters located in five rural counties in North Carolina. Many members were ineligible or we were unable to contact them by telephone, and 239 members who declined to participate in the WATCH Project. There were 850 church members who participated in the WATCH Project and completed the baseline survey. The adjusted response rate was 66% using a calculation method, suggested by the Council of American Survey Organizations (CASRO) , that acThe participants in this study were the 397 church members who participated in the WATCH Project and were 50 years and older. The characteristics of the 397 participants are shown in Table Factor analysis of the five communication items was performed from the baseline survey responses and two factors were identified; one with three items and the other with two items. The second factor was dropped because it had only two items and did not add reliability to the scale. The three communication items about shared decision-making and patient satisfaction demonstrated good reliability and were summed to calculate a communication score. The communication score was used to categorize the participants into three groups: good, fair, and poor communication with providers. Participants were categorized as having "good" communication if they perceived receiving enough information from their provider, being involved in medical decisions, and thinking that their provider understood their health needs almost all the time or always. Participants who rated all three items 'sometimes', 'rarely', or 'never' scored "poor" on the communication scale, and individuals who rated the items with a mix of the above listed responses were assigned to the "fair" group.In terms of quality of communication, 75% 298/397) responded positively to all 3 questions and were considered have "good" communication; 10% (42/397) responded positively to none of the 3 questions and were considered to have "poor" communication; and 13% (50/397) had fair results. Participants in the good communication group were more likely to be female (p = 0.031), and were more likely to receive their healthcare at a doctor's office versus a clinic/emergency room/health department (p = 0.028). None of the other sociodemographic factors listed in Table 97 responOnly 45% (175/389) of the participants reported that their providers had recommended CRC screening, and just 31% (120/389) of all participants reported being screened within the recommended time interval. Of the individuals who reported being screened, 65% (78/120) stated that their doctor had recommended CRC screening, compared with 36% (97/269) of those who did not report screening.Knowledge of CRC was assessed using seven items Table with a mKnowledge about CRC was considered adequate (knowledge score > = 4) for 57% (228/397) and inadequate for 43% (197/397). Participants with adequate CRC knowledge were more likely to have completed a CRC screening test within the recommended time period compared to those with inadequate CRC knowledge (21% vs. 10%). Adequate knowledge was associated with a higher level of education (p < 0.001), a higher level of income (p < 0.001), having health insurance (p < 0.001), and having Medicare/ Medicaid as one's health insurance (p < 0.001).Results of the logistic regression analyses are shown in Table CRC screening within recommended guidelines by perceived communication and knowledge is listed in Table Our study found that participants had higher rates of CRC screening when their self-rated communication with their healthcare provider was classified as perceived as more positive. In addition, we found that participants who self-rated their communication as good and who had adequate CRC knowledge completed recent CRC screening at higher rates than those with good communication and inadequate knowledge. In addition, screening rates are higher with both good communication and adequate CRC knowledge than when only one factor is present. These findings suggest that both good patient-provider communication and CRC knowledge are important for CRC screening. Because of the cross-sectional nature of our study, we cannot determine causality, and it is possible that CRC knowledge improves by going through the screening process.Improving CRC screening rates in the African American population may require strategies that address both improving physician-patient communication skills and increasing CRC knowledge. Good patient-provider communication is fundamental to a patient's perceived quality and satisfaction with their healthcare. Better communication, including the use of shared decision making, is associated with trust between patients and providers . HoweverThe association between the lack of physician recommendations for cancer screening tests and low patient utilization of those tests has been documented in previous investigations ,19,27-30The results of our study and other previously published work ,35-39 suAlthough the results from our cross-sectional study have implications for developing colorectal cancer prevention programs, it must be recognized that prospective longitudinal studies are needed that specifically address the effect of patient-provider communication on CRC screening. Because of its cross-sectional design, we cannot infer causal relationships. In addition, since all participants were recruited from 12 churches, there may be some clustering of exposures and outcomes by physician that may affect the results; we did not measure this effect. Patient-provider communication was measured by using only three self-reported communication items, which may not fully capture the full extent of the patient-provider relationship. In addition, we do not know whether our participants had racially discordant or concordant physicians, and we did not have information regarding the physician's beliefs or practices about CRC screening or the physicians' assessment of the quality of communication with the patients. The outcome measure of having undergone CRC screening was self-reported, and these results were not validated. Finally, because of the relatively small number of participants and the use of convenience sampling, results from this study may not be generalizable.The findings from this cross-sectional study suggest that not only do patients need to be informed about their CRC risk and the importance of screening tests but that having good communication with their healthcare provider may also important. The burden of having good communication in the patient-provider relationship is the responsibility of both individuals. Both the patient and the provider should strive to improve their communication skills so that patients who want to participate in the decision about how to be screened for colorectal cancer may do so effectively.Because culturally distinct factors may contribute to poor communication and mistrust for African-Americans, specific new strategies need to be developed . BeliefsThe author(s) declare that they have no competing interests.M.L.K. participated in the conception of the study, the analyses, the interpretation of the results, writing the first draft and revising the manuscript. A.S.J. participated in the conception of the study, performed the statistical analyses, participated in the interpretation of the results, and the editing of the manuscript. M.P.P. participated in the interpretation of the results and the writing of the manuscript. M.H., E.J., and V.O. assisted with the qualitative analysis and surveys. M.K.C. participated in the conception of the study, the statistical analyses, interpretation of the results, and the writing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:

Essential hypertension is a common, polygenic, complex disorder resulting from interaction of several genes with each other and with environmental factors such as obesity, dietary salt intake, and alcohol consumption. Since the underlying genetic pathways remain elusive, currently most studies focus on the genes coding for proteins that regulate blood pressure as their physiological role makes them prime suspects.The present study examines how polymorphisms of the insertion/deletion (I/D) ACE and M235T AGT genes account for presence and severity of hypertension, and embeds the data in a meta-analysis of relevant studies.The I/D polymorphisms of the ACE and M235T polymorphisms of the AGT genes were determined by RFLP (restriction fragment length polymorphism) and restriction analysis in 638 hypertensive patients and 720 normotensive local blood donors in Weisswasser, Germany. Severity of hypertension was estimated by the number of antihypertensive drugs used.No difference was observed in the allele frequencies and genotype distributions of ACE gene polymorphisms between the two groups, whereas AGT TT homozygotes were more frequent in controls . This became significant (p = 0.035) in women only. AGT TT genotype was associated with a 48% decrease in the risk of having hypertension , and this risk decreased more significantly in women . The meta-analysis showed a pooled odds ratio for hypertension of 1.21 in Caucasians. No correlation was found between severity of hypertension and a specific genotype.The ACE I/D polymorphism does not contribute to the presence and severity of essential hypertension, while the AGT M235T TT genotype confers a significantly decreased risk for the development of hypertension in the population studied here. This contrasts to the findings of meta-analyses, whereby the T allele is associated with increased risk for hypertension. Essential hypertension is a common, polygenic, complex disorder resulting from interaction of several genes with each other and with environmental factors such as obesity, dietary salt intake, and alcohol consumption. Since the underlying genetic pathways remain elusive, currentThe Renin-Angiotensin System (RAS) has a central role in regulating blood pressure and sodium homeaostasis. Genes encoding components of RAS, including angiotensinogen (AGT), angiotensin-converting enzyme (ACE), angiotensinogen II type-1 receptor (AGTR1), and renin, have been extensively investigated as genetic determinants of essential hypertension . PolymorIn 1992, the M235T AGT TT polymorphism was first reported to be associated with hypertension . This fiThe inconsistent results might be explained in part by the genetic and environmental heterogeneity among different ethnic groups . On the The present study investigates the relationship between variants of the I/D ACE gene and M235T AGT gene, and the presence and severity of essential hypertension in a large homogeneous German population. The effect of a combination of ACE and AGT gene polymorphisms on hypertension was also examined.The design of the study followed the guidelines proposed by Cooper et al , and the2 or dialysis), 15 were due to diabetic nephropathy, 5 to chronic pyelonephritis, 1 to chronic glomerulonephritis, 1 light chain deposits, 1 polycystic kidney disease, and 14 of unknown cause (no biopsy obtained). The severity of hypertension was estimated based on the number of antihypertensive medications used, a surrogate marker for the severity of hypertension[This cross-sectional study comprised a total of 1358 individuals from Weisswasser, a county town of 25,000 in Saxony, Germany. After giving informed consent, 720 normotensive subjects were selected from local blood donors and 638 hypertensive patients from the local renal care center. All hypertensive patients included in the study had been diagnosed as suffering from primary hypertension by the attending consultants on first contact with the clinic. Hypertensives were defined as those who received at least one antihypertensive medication. At the time of blood sampling, 34.2% were diabetic , and 65.1% were suffering from kidney disease. Of the 37 patients with K/DOQI stage 5 and restriction analysis were used to determine the frequencies of the I/D polymorphisms of the ACE gene, and homo-/ heterozygoty of the M235T AGT gene,21. ACE AGT M235T polymorphism was studied by first amplifying a 104 bp long fragment of the AGT gene using the following primer sequences: sense- 5'CCGTTTGTGCAGGGCCTGGCTCTCT3', and antisense: 5'CAGGGTGCTGTCCACACTGGACCCC3'.Tth 111I.The M -> T point mutation at position 235 creates a detection site for the restriction enzyme t test for continuous data and Pearson's χ2 test for categorical data. Allele frequencies were calculated with the gene-counting method. χ2 test was used for assessment of the Hardy-Weinberg equilibrium for the distribution of genotypes. Odds ratios were calculated with a 95% confidence interval. A P < .05 was considered significant.Statistical analysis was carried out using SPSS personal computer statistical package . Demographic characteristics were compared by 2 test using P-value < 0.05. The Mantel-Haenszel odds ratios were calculated by applying both fixed effect model and random effect model in case of heterogeneity.A meta-analysis was performed using Review Manager 4.2 (The Cochrane Collaboration) to further examine the association of the AGT M235T gene polymorphisms with essential hypertension in Caucasians. A systematic literature search in PubMed Medline for articles published between April 2002 and June 2004 was carried out using the following MESH-headings: "angiotensinogen/genetics", "hypertension/genetics", "blood pressure/genetics", and "adult". The search was limited to articles published in English and studies on Caucasian human subjects. Only 2 studies were left after strict examination according to the exclusion criteria listed in . A totalDemographic data are summarized in Table AGT M/T genotyping was successful in 637 hypertensives and 720 normotensives, and ACE I/D genotype was analyzed in 636 hypertensive and 719 control subjects. The roles of age and gender in the association between hypertension and ACE and AGT gene polymorphisms were examined by comparing the effects of ACE and AGT genotypes for hypertension in men and women, young and elderly subjects respectively. "Young" was defined as "age < 50 years old", and "elderly" was characterized as "age ≥ 50 years old".No differences were observed in ACE allele and genotype frequency distribution between hypertensives and controls with respect to gender and age, and no deviations from Hardy-Weinberg equilibrium were observed in any of subgroups P > 0.1). ACE genotypes I/I, I/D and D/D were of almost identical frequency within both groups compared with those with MM genotype Figure . The stuThe effect of eight combinations on hypertension was examined. No statistically significant association was observed between any combination above and hypertension in all subjects combined. Nonetheless, in women, both genotypes of TT, DD/ID and TT, II/ID were significantly associated with lower prevalence of hypertension and a more significant association was found in women , compared to men. This is in stark contrast to findings from previous studies, including two German datasets [AGT 235 T-allele and/or TT genotype significantly increased the risk for essential hypertension in Caucasians: odds ratio T vs. M was 1.20 (95% CI: 1.11 to 1.29) [Surprisingly, the cross-sectional study presented here shows a higher prevalence of the T/T M235T AGT gene in the control group compared to the hypertensive group. AGT TT genotype was associated with a datasets ,32, and datasets ,22,47, wto 1.29) , odds rato 1.29) includedto 1.29) AGT T allele among normotensives was higher than that among hypertensives (0.36 vs. 0.33). This was inconsistent with one previous meta-analysis in Caucasians, which showed that among controls, the mean allele frequency for the AGT T allele was 0.41 (95% CI: 0.34 to 0.48), and among cases, increased to 0.45 (95% CI: 0.38 to 0.52) [AGT T allele among hypertensives (0.33) was outside the lower border of the 95% confidence interval (0.38 to 0.52) reported in [Although no significant difference was observed in AGT T allele frequency distribution between hypertensives and controls with respect to age and gender, the frequency of the to 0.52) . In the orted in . This maThe AGT genotype distribution is not in Hardy-Weinberg equilibrium (P < 0.001) while no deviations from Hardy-Weinberg equilibrium are observed on the ACE genotype distribution in the same population. This may be explained by a shift toward a higher frequency of MT individuals (62.4%) instead of TT individuals (3.7%) in this specific population.The study population presented here contains a large proportion (65%) of patients with renal disease. While selection of participants based on patient records excluded those patients that had symptoms suggesting the diagnosis of secondary hypertension at first contact, the possibility remains that at least a part of the study population suffers from renal rather than essential hypertension. It should be noted, however, that the majority of studies included in the presented meta-analysis does not give specific information regarding renal function of hypertensives, and the largest study is populAnother possible explanation is that the AGT T allele frequency may decrease with age, which was reported from the United Arab Emirates . The preA number of studies -54 examiThe effect of a combination of RAAS genes' polymorphisms on blood pressure has been investigated in the participants to the Olivetti Heart Study , in whicAlthough in most surveys the prevalence of hypertension appears to be equal in women and men , sex-speIn addition to the factor declared above, the result may be influenced by the study design and the composition of the sample population. The study design itself may influence the results. As mentioned above, this study was a cross-sectional study where subjects were assessed at a single time, and at that time most of the controls were younger than 50 years old. Animal studies have shown that hypertension genes may be activated for only certain periods during the life history of an organism . Hence, The population can be described as static population with a mixed Germanic-Slavonic background. Due to the location and political and historical setting , the population may be assumed as homogeneous. Several recent studies -61 have In the meantime, it should be noted that the present study was carried out on an unusually large population . Given Despite the limitations mentioned, this cross-sectional study does not support the notion that the ACE I/D polymorphism contributes to the prevalence and severity of essential hypertension. However, the M235T TT genotype of AGT gene was detected to confer a significantly decreased risk for the prevalence of hypertension in women from this particular population. Despite the large sample size, the present study fails to revise the odds ratio in a meta-analysis of a total of 25 studies on the association between the AGT M235T polymorphisms and hypertension in caucasians. This observation may reflect a very specific local inheritance pattern of the AGT genotypes. If this holds true, studies aiming at drug development based on genomic traits must be scrutinized rigorously as therapeutic recommendations may be valid for selected subpopulations only.The author(s) declare that they have no competing interests.M.N. designed and initiated the study, analyzed the samples and wrote part of the manuscript. A-L.Z. and M.L. did the statistical analysis. A.M. devised the concept for meta-analysis and wrote the manuscript. L.P. created the figures. M.N. and A.M. should be considered as joint co- authors.The pre-publication history for this paper can be accessed here:

Transport of fatty acids within the cytosol of adipocytes and their subsequent assimilation into lipid droplets has been thoroughly investigated; however, the mechanism by which fatty acids are transported across the plasma membrane from the extracellular environment remains unclear. Since triacylglycerol-rich lipoproteins represent an abundant source of fatty acids for adipocyte utilization, we have investigated the expression levels of cell surface lipoprotein receptors and their functional contributions toward intracellular lipid accumulation; these include very low density lipoprotein receptor (VLDL-R), low density lipoprotein receptor-related protein (LRP), and heparan sulfate proteoglycans (HSPG).We found that expression of these three lipoprotein receptors increased 5-fold, 2-fold, and 2.5-fold, respectively, during adipocyte differentiation. The major proteoglycans expressed by mature adipocytes are of high molecular weight (>500 kD) and contain both heparan and chondroitin sulfate moieties. Using ligand binding antagonists, we observed that HSPG, rather than VLDL-R or LRP, play a primary role in the uptake of DiI-lableled apoE-VLDL by mature adipocytes. In addition, inhibitors of HSPG maturation resulted in a significant reduction (>85%) in intracellular lipid accumulation.These results suggest that cell surface HSPG is required for fatty acid transport across the plasma membrane of adipocytes. The adipocyte plays a central role in overall metabolic regulation serving as a storage depot for fatty acids and as an endocrine cell to regulate energy utilization and feeding behavior ,2. The mExtracellular fatty acids that are available for adipocyte uptake are either 1) associated with circulating albumin, 2) hydrolyzed from triacylglycerol-rich lipoprotein particles by lipoprotein lipase, or 3) in the form of VLDL particles which can be directly internalized by adipocyte lipoprotein receptors. In the circulation, VLDL represents the major source of fatty acids for peripheral tissues in the form of triacylglycerols and provides a concentrated source of esterified fatty acids. It is interesting that in light of the well studied processes of cytosolic transport and assimilation of free fatty acids into triacylglycerol-rich storage droplets, the mechanism of transport of fatty acids across the adipocyte plasma membrane remains controversial. Two mechanisms, which are not mutually exclusive, have been proposed: one involves passive diffusion across the plasma membrane ,7, the oIn vivo studies corroborate these observations as uptake of long-chain fatty acids is impaired in CD36 knock-out mice [Several cell surface proteins are expressed by adipocytes which potentially contribute to receptor-mediated uptake of extracellular fatty acids; these include CD36, fatty acid transport protein-1 (FATP1), very low density lipoprotein receptor (VLDL-R), low density lipoprotein receptor-related protein (LRP), and heparan sulfate proteoglycans (HSPG). CD36 is a cell surface glycoprotein that binds long-chain fatty acids with high affinity and demonstrates a subcellular distribution that is consistent with a role in fatty acid transport across the plasma membrane -23. Follout mice or when out mice . Althougout mice . FATP1 iout mice . In adipout mice . Similarout mice ,28.Studies using a VLDL-R knock-out animal model have suggested a role for this receptor in the accumulation of fatty acids by adipose tissue; mice lacking VLDL-R demonstrate only modest weight gain and a reduction in adipose tissue mass when placed on a high-fat diet . VLDL-R To better understand the process of fatty acid transport across the plasma membrane of adipocytes, we have examined the expression levels and functional contributions of lipoprotein receptors, VLDL-R, LRP and HSPG toward intracellular lipid accumulation. Our findings suggest that cell surface HSPG play an essential role in fatty acid uptake and intracellular lipid accumulation in adipocytes.in vitro model for adipocyte differentiation [Since members of the LDL receptor family are known to play a predominant role in the uptake of lipoproteins, we chose to examine the protein expression levels of VLDL-R and LRP during adipocyte differentiation using 3T3-L1 cells , which antiation ,43. 3T3-ntiation and LRP ntiation .125I-RAP at 4°C and p-nitrophenyl-β-D-xylopyranoside (pNP-Xyl) are reagents that can serve as alternative acceptors within cells for heparan sulfate moieties and are thus able to compete for heparan sulfate chain addition to proteoglycan core proteins ,52. The To determine if HSPG are involved in intracellular lipid accumulation by mature adipocytes, we assessed cytosolic lipid droplet formation in induced 3T3-L1 cells following treatment with either 4-MUmb or pNP-Xyl. Again 3T3-L1 pre-adipocytes were incubated with 4-MUmb or pNP-Xyl concurrently with the addition of differentiation reagents and intracellular lipid accumulation was assessed by Oil Red O staining. Upon Oil Red staining, untreated mature adipocytes demonstrated large intracellular droplets; the characteristic morphologic feature of cytosolic lipid accumulation Fig. . TreatmeIntracellular lipid accumulation is a hallmark event of adipocyte development and the major factor in adipocyte hypertrophy. This prompted us to investigate the molecular mechanism by which adipocytes take up extracellular lipid components. In the present study, we provide new information on the expression levels and functional activities of certain cell surface receptors in mature adipocytes that are known to play primary roles in lipoprotein processing and clearance. These receptors have been extensively studied in liver, the major organ for dietary lipoprotein clearance , howeverThese findings offer a novel observation that cell surface HSPG appear to be essential for lipid accumulation and lipid droplet formation in adipocytes. However, this observation also raises the question as to the exact role of HSPG in the transport of fatty acids across the adipocyte plasma membrane. Based on the results presented in this study and those reported elsewhere, we have drawn a testable model to define how HSPG contribute to intracellular lipid accumulation by adipocytes Fig. . HSPG arThe identity of this HSPG is currently unknown; however, its large relative molecular mass and composition containing both heparan and chondroitin sulfates are consistent with the structural properties of the syndecan ,61 and pThe availability of circulating triacylglycerol-rich lipoproteins for adipocyte utilization and storage relies on transport of these particles across the endothelial barrier. Transcytosis of albumin across endothelium has been shown to occur by a vesicular transport pathway -70 and iHow HSPG might coordinate its activity with CD36 or FATP1 for fatty acid uptake or possibly with members of the LDL receptor family for assisted internalization is under current investigation. Presently, our results provide novel findings indicating that cell surface HSPG activity is necessary for lipid accumulation by adipocytes. It is anticipated that these observations will aid in our understanding of the complex mechanism leading to adipose hypertrophy and obesity, and provide a novel avenue to explore for targeted reduction of intracellular lipid accumulation35SO4 were purchased from MP Biomedicals . p-nitrophenyl-β-D-xylopyranoside and 4-methylumbelliferyl-β-D-xylopyranoside were from Calbiochem . Heparin, heparinase I, chondroitinase ABC, p-nitrophenylbutyrate, lipoprotein lipase, and Oil Red O were purchased from Sigma-Aldrich. Optiprep was from Greiner Bio-One . DiI was purchased from Molecular Probes . Apolipoprotein E was obtained from Calbiochem. RAP-GST fusion protein was purified as previously described [Anti-LRP polyclonal antibody was raised against an 18 amino acid peptide from the cytoplasmic tail of human LRP and antiescribed . Tissue 2 and passaged twice weekly. To differentiate 3T3-L1 cells into adipocytes, cells were incubated with 100 ng/ml dexamethasone, 100 μg/ml 3-isobutyl-1-methylxanthine, and 1 μg/ml insulin for 4 days, followed by 1 μg/ml insulin for an additional 4–8 days.3T3-L1 cells were obtained from American Type Culture Collection and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum , 1 mM sodium pyruvate, 100 μg/ml streptomycin sulfate, and 100 units/ml penicillin. Cells were cultured at 37°C with 5% COCell lysates were prepared with 20 mM Tris pH 7.4, 150 mM NaCl (TBS) containing 1% (v/v) Triton-X100. For leptin, culture supernatants were concentrated by mixing with 3 volumes ice cold acetone, incubating at -20°C for 1 h, followed by centrifugation at 10,000 × g at 4°C for 15 min. Protein pellets were resuspended with SDS-PAGE sample buffer supplemented with 2% (v/v) β-mercaptoethanol. Proteins were then separated by SDS-PAGE and transferred to Immobilon-P using a wet tank transfer system . Membranes were blocked with TBS, 0.1% (v/v) Tween-20, 5% (w/v) non-fat dry milk for 20 minutes at 23°C and incubated with the indicated antibody for 2 h at 23°C. Membranes were washed three times (10 min each) with TBS, 0.1% (v/v) Tween-20, and bound antibodies were detected with species-specific HRP-conjugated secondary antibodies followed by chemiluminescence detection according to the manufacturer's instructions . Images were captured using a Syngene GeneGnome system equipped with a Peltier-cooled 16-bit CCD camera and saturation detection. Densitometric analysis was performed using Scion Image, version 4.0.2.125I as previously described [125I-RAP-GST (500 ng/ml) diluted into 20 mM Hepes, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 1% (w/v) bovine serum albumin (buffer A) at 4°C for 3 h in the presence or absence of a 50-fold molar excess of unlabeled RAP-GST. Unbound 125I-RAP-GST was removed by rinsing cells three times with cold buffer A after which cells with bound ligand were solubilized with 0.1 N NaOH. Solubilized proteins were added to EcoLume and subjected to scintillation counting . Results were normalized to total cellular protein . Specificity was determined as the difference between total binding (without competition) and non-specific binding (non-competable) [125I-labeled ligand. All data points represent averages of duplicates or triplicates with standard errors of <5%.RAP-GST was purified and labeescribed . Differepetable) . The act2 for 3 h with 500 ng/ml 125I-RAP-GST diluted into DMEM containing 1% (w/v) bovine serum albumin in the presence or absence of a 50-fold molar excess of unlabeled RAP-GST. Media was then removed and processed for trichloroacetic acid (TCA) precipitation [Differentiated 3T3-L1 cells were incubated at 37°C/5% COpitation . TCA-sol2 35SO4 for 20 h at 37°C/5% CO2. Cells were detached by incubating with phosphate buffered saline containing 5 mM EDTA for 20 min at 23°C. Following centrifugation to pellet cells , they were lysed by incubation with solubilization buffer for 1 h at 4°C. Insoluble material was removed by centrifugation and supernatant was batch adsorbed onto 0.5 ml Macro-Prep DEAE Support (BioRad) (pre-washed with solubilization buffer) with gentle agitation for 1 h at 4°C. DEAE resin was applied to a 1.0 cm × 5.0 cm column and washed with 10 column volumes solubilization buffer followed by 10 column volumes 100 mM Tris, pH 7.5, 150 mM NaCl. Bound proteins were eluted with 100 mM Tris, pH 7.5, 1.0 M NaCl and 120 μl fractions were collected. Fifteen μl from each fraction was mixed with 3 ml EcoLume and subjected to scintillation counting. Peak fractions from DEAE enrichment of proteoglycans obtained from labeled differentiated adipocytes or chondroitinase ABC (5 units/ml) or both for 20 h at 37°C. The material was applied to a 1.0 cm × 12 cm Sephacryl S-200 HR gel filtration column and eluted with 20 mM Tris, pH 7.4, 150 mM NaCl. 350 μl fractions were collected of which 200 μl was mixed with 3 ml EcoLume and analyzed by scintillation counting.3T3-L1 pre-adipocytes and differentiated adipocytes grown in 100 mm tissue culture dishes were incubated with sulfate-free DME containing 10% (v/v) fetal calf serum and 125 μCi/ml NaNew Zealand White rabbits were placed on a high-fat chow diet (10% peanut oil/1% cholesterol) for a minimum of 4 d, blood was drawn into in 1 mM EDTA and centrifuged at 2000 × g, 15 min to remove cells. Chylomicrons were floated by centrifuging plasma at 100,000 × g for 10 min and removed by pipetting. Plasma was then mixed with OptiPrep™ and centrifuged at 350,000 × g for 3 h (SW55Ti rotor) with slow acceleration and deceleration. VLDL particles (density of 1.006 g/ml) were removed from the top of the gradient by pipetting. Purified VLDL was analyzed by SDS-PAGE and Coomassie R staining to confirm the presence of apoB100 (515 kD) and apoE (35 kD). Animal protocol (#2032) was approved by the University of New Mexico, Health Sciences Center Laboratory Animal Care and Use Committee. For DiI labeling, a working stock of 3 mg/ml was made in dimethylsulfoxide and 0.15 mg was slowly added to 1.67 mg VLDL (in 1.9 ml) with vortexing to rapidly mix. The mixture was then wrapped in foil and incubated for 8 h at 37°C. Unbound DiI was removed from DiI-labeled VLDL by OptiPrep gradient centrifugation as described above.3T3-L1 cells were plated on glass coverslips and incubated with differentiation reagents as described above. After conversion to mature adipocytes, cells were rinsed twice with DMEM and incubated with DiI-VLDL (4 μg/ml) and apolipoprotein E (3 μg/ml) diluted into DMEM in the presence or absence of either heparin (500 μg/ml) or RAP-GST (50 μg/ml). After 3 h at 37°C, cells were rinsed with phosphate buffered saline, fixed with 1.5% (w/v) paraformaldehyde for 30 min, and mounted in Gelvatol containing 1 mg/ml p-phenylenediamine. Cells were observed with a Zeiss Axioskop microscope equipped for epifluorescence. Images were capture with a Hamamatsu digital/video camera and AxioVision software.Treated and non-treated 3T3-L1 cells (as indicated in figure legends) were cultured overnight in serum-free DMEM without phenol red. Media was removed and combined with an equal volume of 100 mM sodium phosphate buffer, pH 7.2, 150 mM NaCl, 0.5% (v/v) Triton-X100. p-nitrophenyl butyrate in acetonitrile was added to a final concentration of 0.5 mM and absorbance at 400 nm was recorded every 10 s for 5 min using a Genesys UV Spectrophotometer. A standard curve for LPL activity was generated by plotting absorbance values obtained with varying concentration of purified LPL (from bovine milk). Quantitation of LPL activity in the individual media samples was determined from the standard curve.Cells were fixed with 10% (v/v) formaldehyde in phosphate buffered saline for 1 h at 23°C, rinsed twice with water, then stained for 2 h at 23°C with 0.1% (w/v) Oil Red O in 75% (v/v) isopropanol, followed by rinsing twice with water to remove unincorporated dye. Stained cells were viewed and photographed using a Zeiss AxioVert microscope with phase contrast optics and Hamamatsu digital/video camera. For quantitation, cells were dried for 2 h at 37°C, followed by incubation with 100% isopropanol for 15 m at 23°C to extract bound dye. Solubilized dye was then quantitated by spectrophotometry in a BioRad Model 680 Microplate Reader by measuring absorbance at 510 nm. Statistical significance was determined by performing a paired t-test.ApoE = apolipoprotein EHSPG = heparan sulfate proteoglycanVLDL = very low density lipoproteinVLDL-R = VLDL receptorLRP = low density lipoprotein receptor-related proteinSyn-1 = Syndecan-1FATP1 = fatty acid transport protein-1RAP = receptor associated proteinLPL = lipoprotein lipaseDiI = 1,1'-dictadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate4-MUmb = 4-methylumbelliferyl-β-D-xylopyranosidepNP-Xyl = p-nitrophenyl-β-D-xylopyranosidebFGF = basic fibroblast growth factorLCW carried out the majority of studies and drafted the manuscript. SC performed the xyloside titration study and DN performed the LPL assays. RAO provided the original conceptual framework for the study, carried out pilot experiments, participated in the experimental design and finalized the manuscript for submission. All authors read and approved the final version.

The fusion protein RUNX1-CBFA2T1 associated with t-positive acute myeloid leukaemia is a potent inhibitor of haematopoetic differentiation. The role of RUNX1-CBFA2T1 in leukaemic cell proliferation is less clear. We examined the consequences of siRNA-mediated RUNX1-CBFA2T1 depletion regarding proliferation and clonogenicity of t-positive cell lines.The t-positive cell line Kasumi-1 was electroporated with RUNX1-CBFA2T1 or control siRNAs followed by analysis of proliferation, colony formation, cell cycle distribution, apoptosis and senescence.Electroporation of Kasumi-1 cells with RUNX1-CBFA2T1 siRNAs, but not with control siRNAs, resulted in RUNX1-CBFA2T1 suppression which lasted for at least 5 days. A single electroporation with RUNX1-CBFA2T1 siRNA severely diminished the clonogenicity of Kasumi-1 cells. Prolonged RUNX1-CBFA2T1 depletion inhibited proliferation in suspension culture and G1-S transition during the cell cycle, diminished the number of apoptotic cells, but induced cellular senescence. The addition of haematopoetic growth factors could not rescue RUNX1-CBFA2T1-depleted cells from senescence, and could only partially restore their clonogenicity.RUNX1-CBFA2T1 supports the proliferation and expansion of t-positive leukaemic cells by preventing cellular senescence. These findings suggest a central role of RUNX1-CBFA2T1 in the maintenance of the leukaemia. Therefore, RUNX1-CBFA2T1 is a promising and leukaemia-specific target for molecularly defined therapeutic approaches. The chromosomal translocation t8;21) , which is associated with 10–15% of all cases of acute myeloid leukaemia, fuses the DNA binding domain of the transcription factor RUNX1 to the almost complete open reading frame of CBFA2T1 -propansulfonat (CHAPS), 1% (w/w) Dithiothreitol) and treated for 30 min on ice with 2.5 u/ml Benzonase . Total (10–20 μg) and nuclear lysates (10 μg) were analyzed as described . Band in- as suggested by the manufacturer. Real time PCRs were performed using Sybr-Green Mix , 300 nM primers and 20% v/v diluted RT reaction mix using standard conditions on a 7000 Sequence detection system . The primer sequences for STAT1 (5'-CAT CAC ATT CAC ATG GGT GGA (forward primer) and 5'-GGT TCA ACC GCA TGG AAG TC (reverse primer)), for CD34 (5'-TCC AGA AAC GGC CAT TCA G (forward primer) and 5'-CCC ACC TAG CCG AGT CAC AA (reverse primer)), for G-CSF (5'-CCC ACC TTG GAC ACA CTG C (forward primer) and 5'-AGT TCT TCC ATC TGC TGC CAG (reverse primer)) and for GAPDH (5'-GAA GGT GAA GGT CGG AGT C (forward primer) and 5'-GAA GAT GGT GAT GGG ATT TC (reverse primer)) were designed with Primer-Express software .Total RNA was isolated using the RNeasy Kit . Reverse transcription was performed with random hexamers using MMLV-RT, RNase HTo determine proliferation rates and doubling times, cells were counted using trypan blue. For single cell expansion assays, 24 h after electroporation cells were diluted to a density of 10 cells/ml and plated in 100 μl aliquots into 96 well tissue culture plates. Colony formation assays were performed 24 h after electroporation with a density of 5,000 cells/ml in semisolid medium . Colonies were counted 14 days after plating. The cytokine concentrations were 20 ng/ml for G-CSF and GM-CSF in the cell cycle and senescence assays, and 10 ng/ml for all growth factors in the colony formation assays.6 cells were suspended in 200 μl citrate buffer followed by the addition of 800 μl staining solution NonIdet P40, 500 μM EDTA) and 10 μl boiled RNase A (10 g/l). Cells were kept for 30 minutes on ice and analyzed by flow cytometry . Data analysis was performed with FCSPress 1.3 .Cell cycle analysis was performed as described . Four da for data analysis.For quantification of apoptotic cells, cells were stained with FITC-labeled annexin V as suggested by the supplier . CD34 expression was monitored by staining with anti-human CD34-FITC . Analysis was performed by flow cytometry using FCSPress 1.3 Electroporation of t-positive cells with the RUNX1-CBFA2T1 siRNA siAGF1, but not with the mismatch control siRNA siAGF6, resulted in a three- to fourfold reduction of RUNX1-CBFA2T1 fusion protein in both Kasumi-1 cells fig. . This fiTransfection with siRNAs may induce an interferon response leading to non-specific effects on cellular processes such as proliferation or differentiation . Since SIn comparison to mock-transfected cells, electroporation with RUNX1-CBFA2T1 siRNAs resulted in a ten- to twenty fold decreased clonogenicity in both single cell expansion (data not shown) and colony formation assays -negative myeloid leukaemia cell lines MV4-11, NB4, HL60, U937 and K562 was not affected upon electroporation with RUNX1-CBFA2T1 siRNA (data not shown), which argues against a general, unspecific effect of the siRNAs used in this study on leukaemic clonogenicity. Therefore, the application of RUNX1-CBFA2T1 siRNAs is sufficient to inhibit the clonogenic growth of Kasumi-1 cells, indicating that RUNX1-CBFA2T1 is essential for the clonogenicity of these leukaemic cells.To examine the effects of RUNX1-CBFA2T1 on the progenitor status of t-positive leukaemic cells, we analyzed the CD34 expression levels in siRNA-treated Kasumi-1 cells. When compared to mock-transfected cells, depletion of RUNX1-CBFA2T1 caused a twofold decrease in CD34 surface expression 7 days after electroporation . Two or more consecutive electroporations with RUNX1-CBFA2T1 siRNA caused a reduction of the amount of S phase cells by 50% and a 30% reduction in G2/M phase cells, with a corresponding increase of the G1/G0 fraction by 20% fig. . TreatmeKip1), a general inhibitor of Cyclin-CDK complexes and, thereby, of cell cycle progression. Two electroporations with RUNX1-CBFA2T1 siRNA caused a twofold increase in CDKN1B protein levels in RUNX1-CBFA2T1-depleted cells, but not in control cells (fig. The changes in the cell cycle distribution are associated with changed expression levels of CDKN1B (p27lls fig. . TherefoA possible reason for the observed inhibition of proliferation is the induction of apoptosis upon suppression of RUNX1-CBFA2T1. We addressed this possibility by examining the amount of apoptotic cells by annexin V staining and of hypodiploid (subG1) cells by flow cytometry. Electroporation of Kasumi-1 cells together with the RUNX1-CBFA2T1 siRNA siAGF1 neither increased the amount of hypodiploid cells nor the amount of annexin V positive cells when compared to control cells. Instead, after siAGF1 treatment we observed a slight but reproducible decrease in the amount of apoptotic cells in both assays. This decrease was more pronounced after two consecutive electroporations with siRNAs. Repeating the siAGF1 electroporation after 4 days resulted in a threefold decrease of the amount of annexin V stained cells fig. indicatiThe observed G1 arrest upon RUNX1-CBFA2T1 depletion could be either reversible or irreversible. An irreversible G1 arrest is a hallmark of cellular senescence. To address the question of the nature of the G1 arrest, and to analyze a possible influence of RUNX1-CBFA2T1 on cellular senescence, we stained siRNA- and mock-treated cells for senescence-associated β-galactosidase activity. After two or more electroporations with RUNX1-CBFA2T1 siRNA, but not with control siRNA, a significant fraction of the Kasumi-1 cells stain positive for β-galactosidase fig. . Cell coA possible mechanism of how RUNX1-CBFA2T1 prevents cell cycle arrest and senescence is the autocrine production of growth factors. Kasumi-1 cells express, for instance, G-CSF fig. , but notDepletion of RUNX1-CBFA2T1 for 16 days reduced the fraction of S phase cells twofold compared to control cells. This decrease is independent of G-CSF or GM-CSF fig. . When anFinally, we examined the effects of several haematopoetic growth factors on colony formation of siRNA-treated Kasumi-1 cells. Supplementing the semisolid medium with the corresponding growth factors increased colony numbers for both control cells and RUNX1-CBFA2T1-depleted cells fig. . G-CSF eTaken together, the addition of G-CSF or GM-CSF delays, but does not prevent the G1 arrest in RUNX1-CBFA2T1-depleted cell. Furthermore, senescence is not inhibited by these growth factors. In line with these findings, neither of these growth factors completely restored the impaired clonogenicity of such cells. These results indicate that neither G-CSF nor GM-CSF can rescue leukaemic cells suffering of siRNA-mediated RUNX1-CBFA2T1 depletion.3 receptor [Arf [ex vivo and in vivo [The leukaemic fusion protein RUNX1-CBFA2T1 inhibits haematopoetic differentiation by repressing gene expression via recruitment of histone deacetylases -7, and breceptor -11. In areceptor ,46, in stor [Arf . The not in vivo ,21. In aWe show that RUNX1-CBFA2T1 also supports proliferation, CD34 expression and colony formation in the t-positive leukaemic cell line Kasumi-1. Thus, RUNX1-CBFA2T1 functions are similar in HSCs and in Kasumi-1. In the latter, support of proliferation is not caused by an inhibition of cell death. Instead, as observed with many other cell types ,46, RUNXKip1 (CDKN1B) during RUNX1-CBFA2T1 depletion are in line with the recently demonstrated central role of this CDK inhibitor in the establishment of senescence [The reduced proliferation of Kasumi-1 cells upon RUNX1-CBFA2T1 depletion is paralleled by a cell cycle arrest in G1. Particularly from a therapeutic point of view, it is important to know, whether this G1 block will be relieved by recovering RUNX1-CBFA2T1 expression, or by growth factors, or whether this arrest is irreversible. We observe that RUNX1-CBFA2T1 depleted cells show enhanced levels of senescence-associated β-galactosidase activity, a hallmark of cellular senescence. Therefore, the inhibition of G1 to S transition is associated by the induction of senescence indicating that this G1 arrest is irreversible. Interestingly, the elevated levels of p27nescence ,50.Arf [Arf and of its human homologue, p14Arf [Furthermore, RUNX1 was shown to induce senescence in murine embryonic fibroblasts, probably by inducing p19Arf . Since R, p14Arf , an incr3 [An important point to consider is a possible relation between the effects of RUNX1-CBFA2T1 on clonogenicity, proliferation and senescence, and its anti-differentiation activity. For instance, increased β-galactosidase activity may also be associated with late monocytic differentiation . Moreove3 . However3 , and datin vivo might interfere with the development and maintenance of leukaemia. However, a remaining possibility might be that the stroma environment with its complex mixture of growth factors and cell-cell interactions may be able to rescue RUNX1-CBFA2T1-depleted cells. We are currently investigating this question both in cell culture and in vivo.The haematopoetic growth factors G-CSF and GM-CSF were shown to support clonogenicity and proliferation in Kasumi-1 cells . HoweverA major point of concern for siRNA applications is the specificity of the corresponding siRNA . ReasonsIn summary, we show that RUNX1-CBFA2T1 does not only inhibit the myeloid differentiation , but is HPV is an employee of Alnylam Europe AG, which also provided some of the siRNAs used in this study.NM performed the proliferation, senescence and cell cycle assays. BD analyzed the clonogenicity of growth-factor treated Kasumi-1 cells and of t negative cell lines. HR performed the immunoblot analyses and was involved in the analysis of clonogenicity and apoptosis. CC examined the expression levels of CD34 and of STAT1. HPV, AG, GH and AN participated in the coordination of the study. JK and OH conceived and designed the experiments. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:

By aligning probe sequences with expressed sequence tags (ESTs) in NHP, inter-species conserved (ISC) probesets, which have two or more probes complementary to ESTs in NHP, were identified on human GeneChip® platforms. The utility of human GeneChips® for the assessment of NHP expression patterns can be effectively evaluated by analyzing the hybridization behaviour of ISC probesets. Appropriate normalization methods were identified that further improve the reliability of human GeneChips® for interspecies (human vs NHP) comparisons.While researchers have utilized versions of the Affymetrix human GeneChip® platforms were identified for both monkey and chimpanzee. Expression data was generated from peripheral blood mononuclear cells (PBMCs) of 12 human and 8 monkey (Indian origin Rhesus macaque) samples using the Focus GeneChip®. Analysis of both qualitative detection calls and quantitative signal intensities showed that intra-species reproducibility (human vs. human or monkey vs. monkey) was much higher than interspecies reproducibility (human vs. monkey). ISC probesets exhibited higher interspecies reproducibility than the overall expressed probesets. Importantly, appropriate normalization methods could be leveraged to greatly improve interspecies correlations. The correlation coefficients between human (average of 12 samples) and monkey (average of 8 Rhesus macaque samples) are 0.725, 0.821 and 0.893 for MAS5.0 (Microarray Suite version 5.0), dChip and RMA (Robust Multi-chip Average) normalization method, respectively.ISC probesets in each of the seven Affymetrix GeneChip® platforms to assess the expression profiles of NHP for intra-species studies. Caution must be taken for interspecies studies since unsuitable probesets will result in spurious differentially regulated genes between human and NHP. RMA normalization method and ISC probesets are recommended for interspecies studies.It is feasible to use Affymetrix human GeneChip An underlying assumption in such studies is that transcripts of humans and NHP are highly conserved, and probe sequences designed to detect human genes will detect their orthologs in NHP samples. It is estimated that chimpanzees (Pan troglodytes) and humans shared 98.77 % DNA similarity [® for NHP expression analysis.Microarray studies on non human primates (NHP) have been used to address viral pathogenesis ,2, neuromilarity comparedmilarity ,12, and ® platforms for NHP expression profiling have robustly addressed the quantitative aspects of cross platform performance. Vahey and colleagues [® and demonstrated that there was no significant difference in the dynamic range of the raw fluorescence distribution for equivalent amounts of human cRNA and macaque cRNA hybridized to the chip. Chismar and colleagues [® platform and compared the expression patterns of humans with that of the rhesus macaque. They concluded that the percentage of 'present' calls observed in the transcriptome of macaque brain is lower than that of human brain, and that this is especially true for genes with lower signal intensity. Caceres and colleagues [® platforms for the study of expression profiles in NHP is to employ a sequence analysis approach.Few published studies employing human GeneChiplleagues used thelleagues used thelleagues used the® platforms to assess expression patterns in NHP samples by: a) identifying ISC probesets based on sequence analysis; b) assessing intra (within NHP species)- and interspecies (between NHP and human samples) reproducibility of GeneChip® data; and c) applying appropriate normalization methods to improve interspecies reproducibility.In this study, we address the power of human GeneChip® hybridizes with the transcriptome of a NHP, there are three possible outcomes: 1) it hybridizes with the ortholog of the NHP; 2) it cross-hybridizes with a non-ortholog transcript, or 3) it fails to hybridize due to sequence divergence. In Affymetrix GeneChip® system, a probeset is composed of 11–20 probes and each probe is a 25-mer oligo. We identified probes on the human GeneChip that are complementary to ESTs in NHP postulating that these probes would hybridize most optimally with the transcripts of NHP. We defined a probeset as an ISC probeset if it had at least two complementary probes. The rationale used to define the criterion that defines an ISC probeset is described in the methods section. The procedure used to generate ISC probesets is shown in Figure ® platforms were generated for both monkey and chimpanzee. Detailed information about each ISC probeset such as probe sequence, GenBank accession and the position and degree of matching is provided in the supplemental materials (Macaca mulatta) than for chimpanzee (Pan troglodytes). This is not because monkey EST sequences are more similar to human sequences than chimpanzee EST sequences, but because we have a much greater amount of EST sequences available for monkey. At the time of writing this manuscript, there were 33,474 monkey ESTs available, while there were only 6,943 ESTs available for chimpanzee. As the number of defined ESTs will increase in the future, additional ISC probesets could be identified for both monkey and chimpanzee using this method.When a probe sequence on the human GeneChipaterials . Table 1®s, there are three probesets that target the gene CXCR4 at different positions in its transcript. In order to address this redundancy issue, we converted the number of probesets into the number of unique UniGene clusters based on the GeneChip® annotation file provided by Affymetrix Website [® and the U133Plus2.0 GeneChip® have the lowest and highest frequency of redundant probesets for a given gene, respectively.It is not uncommon, especially in the U133Plus2.0 platform, that multiple probesets target the same gene. For example, in the U133A and the U133 Plus 2.0 GeneChip Website . While a® from Affymetrix and covers the human genome most extensively. Figure The U133Plus2.0 is the most current version of human GeneChip® data observed in intra- and interspecies samples. The intra-species reproducibility is displayed in Figure ® for NHP intra-species studies.The qualitative detection call (present / absent) output from MAS5.0 was the initial approach used to examine the reproducibility of GeneChip® platforms are used to compare the expression pattern of humans with those of NHPs, care must be taken in the interpretation of data. If we consider a probeset as being expressed when 50% or more of replicates have present calls, then 3445 (2059+1386) and 2321 (2059+262) probesets are expressed in the PBMC fraction of humans and monkeys, respectively was created. Probesets that are not expressed in human PBMCs were excluded in this analysis. Pair-wise correlation coefficients were calculated for all 20 samples . The correlation coefficients were visualized using heat spectrum graphs where colors ranging from red to white correspond to correlation coefficients of 0.5 to 1.0, respectively. In figure To assess the intra- and interspecies reproducibility of GeneChip® data variation [® data at the probe level using a non-linear algorithm while MAS5.0 normalizes data at probeset level using linear scaling. Sequence divergence usually leads to one or very few probes in a probeset being problematic while the majority of probes in that probeset may still work reasonably well. If the variation generated from these problematic probes were normalized, the interspecies reproducibility should improve. Figure Different normalization methods have been shown to significantly affect GeneChipariation -17. We cariation -16 and d® expression data as applied to the derivation of meaningful expression profile data from NHP. The utility of the human Affymetrix GeneChip® for the assessment of expression profiles in NHP depends on the experimental design and on the approach to data normalization and analysis. Our observations suggest that: 1) it is feasible to use the human GeneChip® in the evaluation of expression profiles of NHP samples for intra-species comparisons; 2) use of ISC probesets and RMA normalization are recommended for interspecies studies; and 3) with the increasing amount of ESTs of NHP, additional ISC probesets (and perfect probesets) will be identified in the near future.This paper presents a comprehensive analysis of probe sequences and GeneChipMacaca mulatta) and chimpanzee (Pan troglodytes) were downloaded from NCBI website [Affymetrix GeneChip probe sequences were downloaded from Affymetrix website . The EST website .® probe sequences and monkey /chimpanzee EST sequences. The length of a probe sequence is always 25 nucleotides while the number of probes in a probeset varies from 11 to 20 depending on GeneChip® platforms in comparison with an RT-PCR experiment, the primer length is equivalent to our probe length, and two primers (one forward and one backward) usually generate a unique sequence in a whole genome; 3) a probe sequence on the Affymetrix GeneChip® is a well designed sequence with a single probe hybridizing with a unique transcript in whole transcriptome; and 4) since the EST sequences in NHP are very limited so far, most of them do not cover whole transcript such that a false negative could be generated if we require all the probes in a probeset being complementary to known ESTs. In order to convert probeset IDs to UniGene IDs and map them onto chromosomes, probeset annotation files were downloaded from Affymetrix website [Stand alone BALST program was downloaded from NCBI website . Perl sc website . No animRhesus macaque) was collected and peripheral blood mononuclear cells (PBMCs) were separated by Histopaque-Ficoll (Sigma) gradient centrifugation. RNA preparation, Hybridization, staining and scanning of the GeneChip® was carried out as described by Vahey et al. [®. Signal values and detection calls (present or absent) for all samples were determined by using MAS5.0 . Signal values were scaled to the default target signal intensity of 500). A matrix of detection calls and a matrix of signal intensities for all samples across all probesets were constructed. A gene must exhibit 50% or more of 'present' calls in all samples to be considered 'expressed'. In this study, an expressed probeset in human is a probeset that has 6 or more present calls among 12 human samples. Similarly, an expressed probeset in monkey means there were 4 or more present calls among 8 monkey samples. The signal intensities output from MAS5.0 were log2 transformed. Model-based normalization was performed using dChip version 1.3 [rma function in the package was used at its default setting, that is, 'RMA' background correction, 'quantile normalization', 'PM only model' and 'median polish summarization'. By default, the signal intensities were already log2 transformed.Briefly, peripheral blood from healthy human and NHP and set color scales between 0.5 (red) and 1.0 (white).Intra- and interspecies correlation coefficients of signal intensities were calculated by built in function 'n 1.9.0. . VisualiNHP: non human primateEST: expressed sequence tagISC: inter-species conservedRMA: robust multi-chip averageMAS5.0: Microarray suite version 5.0ZW developed the original hypotheses, performed the bioinformatics analyses to test them and drafted the manuscript. MV provided critical input on design and execution of the laboratory experiments and with ZW interpreted the data sets and revised the manuscript. ML conducted all aspects of the animal handling including the harvest of well characterized primary samples. MN and AA are technical staff who extracted the nucleic acid and performed the laboratory portions of the microarray experiments. All authors read and approved the final manuscript.Macaca mulatta) in GeneChip® platforms Plus2.0, U133A and U133B, respectively. Note: All the four additional files are multiple-sheets MS excel files. Within each worksheet of a file, the rows are ISC probesets, the columns are probeset IDs, positions of a probe (x and y coordinates in the chip), GenBank accessions of a matching EST, probe sequence, matched EST sequence and position, BLAST e- values and matching identities, in that order.There are three worksheets in this file. Worksheet 1, 2 and 3 are ISC probesets for monkey (Click here for fileMacaca mulatta) in GeneChip® platforms Focus, FL, U95Av2 and U95B, respectively. Note: All the four additional files are multiple-sheets MS excel files. Within each worksheet of a file, the rows are ISC probesets, the columns are probeset IDs, positions of a probe (x and y coordinates in the chip), GenBank accessions of a matching EST, probe sequence, matched EST sequence and position, BLAST e- values and matching identities, in that order.There are four worksheets in this file. Worksheet 1, 2, 3 and 4 are ISC probesets for monkey (Click here for filePan troglodytes) in GeneChip® platforms Plus2.0, U133A and U133B, respectively. Note: All the four additional files are multiple-sheets MS excel files. Within each worksheet of a file, the rows are ISC probesets, the columns are probeset IDs, positions of a probe (x and y coordinates in the chip), GenBank accessions of a matching EST, probe sequence, matched EST sequence and position, BLAST e- values and matching identities, in that order.There are three worksheets in this file. Worksheet 1, 2 and 3 are ISC probesets for chimpanzee (Click here for filePan troglodytes) in GeneChip® platforms Focus, FL, U95Av2 and U95B, respectively. Note: All the four additional files are multiple-sheets MS excel files. Within each worksheet of a file, the rows are ISC probesets, the columns are probeset IDs, positions of a probe (x and y coordinates in the chip), GenBank accessions of a matching EST, probe sequence, matched EST sequence and position, BLAST e- values and matching identities, in that order.There are four worksheets in this file. Worksheet 1, 2, 3 and 4 are ISC probesets for chimpanzee (Click here for file

Patients suffering from diseased and injured organs may be treated with transplanted organs. However, there is a severe shortage of donor organs which is worsening yearly due to the aging population. Scientists in the field of tissue engineering apply the principles of cell transplantation, materials science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Both therapeutic cloning (nucleus from a donor cell is transferred into an enucleated oocyte), and parthenogenesis (oocyte is activated and stimulated to divide), permit extraction of pluripotent embryonic stem cells, and offer a potentially limitless source of cells for tissue engineering applications. The stem cell field is also advancing rapidly, opening new options for therapy. The present article reviews recent progress in tissue engineering and describes applications of these new technologies that may offer novel therapies for patients with end-stage organ failure. Cells may be heterologous (different species), allogeneic , or autologous . Autologous cells are preferred because they will not evoke an immunologic response and thus the deleterious side effects of immunosuppressive agents can be avoided.The goal of tissue engineering is to repair organ pathologies such as those acquired congenitally or by cancer, trauma, infection, or inflammation. It is based upon the foundations of cell transplantation and materials science. Tissue can be engineered 1) 2 to one that covers a surface area of >4000 m2 within 8–14 weeks. These studies indicate the possibility of collecting autologous bladder cells from human patients, expanding them in culture, and returning them to the human donor in sufficient quantities for reconstructive purposes [The ideal autologous cells can often be found within the organ itself. These cells (committed precursors) may be isolated, expanded and transplanted back into the same patient, thus representing an autologous transplantation resource. Previously, urothelial cells could be grown in the laboratory setting with only limited expansion. Several protocols were developed over the last 20 years which identified the undifferentiated cells and kept them undifferentiated during their growth phase -4. Usingpurposes -11. Majoi.e. pancreas, liver). Stem cells are envisioned as being an alternate source of cells from which the desired tissue can be derived. Human embryonic stem cells (HESC) can be derived from discarded non transferred embryos and have the advantage of being pluripotential and able to self-renew indefinitely. However, their clinical application is limited because they represent an allogeneic resource and thus their use would require high dose immunosuppressant therapy.While autologous cells are recognized as the ideal transplantation resource, many patients with end-stage organ disease are unable to yield sufficient cells for expansion and transplantation. Furthermore, some primary autologous human cells cannot be expanded from particular organs (New stem cell technologies such as somatic cell nuclear transfer (therapeutic cloning) and parthenogenesis offer an exciting alternative to create an inexhaustible supply of ESC that can differentiate into all cell types of the embryo, while not being rejected by the patient's immune system. Although many tissues have been created with ESC, they are not used clinically because of an inability to control differentiation. Hence, their ability to form multiple tissue types also becomes their limitation. New genomics and bioinformatics technologies have and will continue to offer new insights into the understanding of ESC growth and differentiation and their application to engineering tissues. In the near future, these new technologies will allow for the generation of an unlimited supply of any cell type in the body.ex vivo, ESC can potentially be the ideal resource for tissue engineering because of two fundamental properties, 1) the ability to self-renew indefinitely, and 2) the ability to differentiate into all three germ layers.The political and ethical controversy surrounding stem cells began in 1998 with the creation of HESC derived from discarded, non-transferred human embryos. The HESn = 7,334) are expected to survive the freeze/thaw process. From this, 25% are expected to develop to blastocyst stage . If one assumes a 15% efficiency rate for establishment of a HESC line from blastocysts , it Somatic cell nuclear transfer (SCNT) entails the removal of an oocyte nucleus followed by its replacement with a nucleus derived from a somatic cell obtained from that patient. Activation with chemicals or electric shock stimulates cell division up to the blastocyst stage at which time the inner cell mass is isolated and cultured, resulting in ESC. This approach is distinct from reproductive cloning because the blasotcyst is not transplanted back to the uterus. Hence, development does not proceed beyond the 100 cell stage. This process also differs from fertilization since no sperm is used in this process. The resulting ESC are perfectly matched to the patients immune system and no immunosuppressants would therefore be required to prevent rejection.adult somatic cell using nuclear transfer [While interest in the field of nuclear cloning remains high since the birth of Dolly 1997), the first successful nuclear transfer was actually reported over fifty years ago by Briggs and King . Cloned 97, the ftransfer , goats [transfer ,22, micetransfer , and pigtransfer -27.A better understanding of the differences between reproductive cloning and therapeutic cloning may help alleviate some of the controversy surrounding these technologies ,29. BannWhile promising, somatic cell nuclear transfer technology has certain limitations requiring further improvement before it can be applied widely in clinical practice. Currently, the efficiency of the overall cloning process is quite low as the majority of embryos derived from animal cloning do not survive after implantation -35. In pIt must be noted that abnormalities have been found in liveborn clones including macrosomia with an enlarged placenta ("large-offspring syndrome") , respiraBos taurus) [in vivo growth. After 12 weeks cloned renal cells were harvested, expanded in vitro, then seeded onto biodegradable scaffolds. The constructs (consisting of cells + scaffolds) were then implanted into the subcutaneous space of the same steer from which the cells were cloned to allow for tissue growth.We applied principles of both tissue engineering and therapeutic cloning in an effort to produce genetically identical renal tissue in an animal model ( taurus) . Bovine ex vivo renal replacement devices are known to be life-sustaining, there are obvious benefits for patients with end-stage kidney disease if such devices could be implanted long-term without the need for an extracorporeal perfusion circuit or immunosuppressive drugs.The kidney is a complex organ with multiple cell types and a complex functional anatomy rendering it one of the most difficult organs to reconstruct ,47. PrevCloned renal cells were seeded on scaffolds consisting of three collagen-coated cylindrical polycarbonate membranes figure . The endChemical analysis of the urine-like fluid revealed that the implanted renal cells possessed filtration, reabsorption, and secretory capabilities. Histological examination of the retrieved implants revealed extensive vascularization and self-organization of the cells into glomeruli- and tubule-like structures. A clear continuity between glomeruli, tubules, and the polycarbonate membrane was noted that allowed the passage of urine into the collecting reservoir figure . ImmunohAs previous studies have confirmed bovine clones harbor mitochondrial DNA (mtDNA) of strictly oocyte origin -57, the in vivo and cytotoxic T lymphocytes expansion in vitro [in vivo and Elispot analysis of interferon-gamma secreting T-cells in vitro. Both analyses revealed that the cloned renal cells showed no evidence of T-cell response, suggesting that rejection will not necessarily occur in the presence of oocyte-derived mtDNA is production of offspring by a female with no genetic contribution from a male and without meiotic chromosome reduction. The process is common reproductive strategy among insects such as aphids, flies, ants, and honeybees, but is also known to occur in vertebrates including lizards, snakes, fish, birds, and amphibians. The first demonstration of artificially-stimulated parthenogenesis in vitro was made by Jacques Loeb (1899), who was able to activate oocytes from sea urchins and frogs by pricking them with a needle or by changing the ambient salt concentration. Pincus (1939) demonstrated parthenogenetic activation of mammalian eggs using temperature and chemical stimuli. Thus far, parthenogenetic activation of eggs has been studied in a variety of mammals including mice, goats, cows, monkeys, and humans. Plachot et al. described parthenogenesis in humans by examining 800 human oocytes and showed that 12 activated parthenogenetically and four underwent normal cleavage[Parthenogenesis to elevate Ca2+ levels transiently, followed by application of an inhibitor of protein synthesis (cycloheximide) or protein phosphorylation (6-dimethylaminopurine). Success rates and viability appear to be organism dependent. Mouse parthenotes are capable of developing beyond the post-implantation stage in vivo [Callithrix jacchus) have only been shown to implant [2n polar body under the zona pellucida and a 2n protonucleus in the cytoplasm. After chemical activation to mimic the effects of sperm penetration on changes in cellular Ca2+ gradient, the cell fails to complete meiosis II. Instead, the second polar body is never extruded, resulting in a diploid protonucleus derived from two sets of sister chromatids. These chromatids then begin to undergo mitosis resulting in a parthenote manifesting uniparental disomy. Although the derivation of embryonic-like stem cells from oocytes is relatively inefficient (perhaps due to complexities of genomic imprinting), when they are differentiated into adult tissues, they appear fully functional.There is no confirmed example of in vivo ,67; porc in vivo ; primate implant . The rea implant ,71. Inde implant . In mostin vitro in an undifferentiated state for up to 14 months. In vitro, they have been differentiated into cardiomyocyte-like cells, smooth muscle, beating ciliated epithelia, adipocytes, several types of epithelial cells, as well as dopaminergic and serotoninergic neurons. Almost all of these neurons express TUJ1 (beta-tubulin III), and up to 25% of the TUJ1+ cells co-express tyrosine-hydroxylase. This latter enzyme marker is considered diagnostic for catecholaminergic neurons . Furthein vivo, since injection of monkey PSC into immunocompromised mice induces formation of benign teratomas containing tissue derivatives from all three germ layers including cartilage, muscle, bone, neurons, skin, hair follicles, and intestinal epithelia [These observations are recapitulated pithelia ,75. Of ppithelia . The reain vivo tumorigenic event) the de facto acceptance of experiments using teratoma tumor tissue lends some legitimacy to experimentation on parthenotes. These contradictions await reconciliation in a comprehensive ethical framework.To be sure, parthenotes are not free from ethical controversy and are viewed by some in society as artificial entities that in some sense represent 'tampering with nature.' Since a parthenote is analogous to a mature ovarian teratoma , and NP that were further differentiated for 8 days (PSC-neurons). We have identified numerous targets such as receptors and ligands present at each of these distinct time points, and are modifying our culture system in order to improve the quality and quantity of differentiation. Furthermore, we are comparing the gene expression profiles of PSC derived neurons to gene expression profiles of reference neurons. Not only will this provide new insight into the type of neurons that may be generated, but it offers clues into what our stem cell derived neurons might be lacking. We can then go back to the culture system and try to target these specific genes/signaling pathways.Further study of stem cell genomics will give additional insight into pluripotentiality. An understanding of pluripotentiality might allow for a somatic cell to be de-differentiated into an intermediate stage, which could then be expanded, differentiated and transplanted back into the patient. We are presently characterizing the genetic signature of pluripoteniality by analyzing gene expression among primate stem cells derived from a variety of methods . By identifying "stemness" genes by comparing undifferentiated stem cells to their differentiated counterpart, and comparing this to stem cells of different origins, a core set of pluripotential target genes may be mapped. Of particular interest are the 1,075 genes that are similarly down-regulated in IVF derived human ESC and monkey PSC. Furthermore, we have detected paternally imprinted genes in our HESC but not in our PSC data sets. From this we conclude that paternal imprinting might not be necessary for pluriopotentiality.Our systems biology approach incorporates the fields of genomics, cell biology, nuclear transfer, and materials science, and utilizes personnel who have mastered the techniques of bioinformatics, cell harvest, culture, expansion, transplantation, as well as polymer design essential for the successful application of these technologies. Experimental efforts are currently underway involving virtually every type of tissue and organ of the human body. Various tissues are at different stages of development with some already being used clinically, a few in pre-clinical trials, and some in the discovery stage. Recent progress suggests that engineered tissues may have an expanded clinical applicability in the future and may represent a viable therapeutic option for those who require tissue replacement or repair.

A review of "On Intelligence", a book exploring brain function by entrepreneur Jeff Hawkins and science writer Sandra Blakeslee Is Michael Moore liberal America's Rush Limbaugh? If so, is he filling a much needed, or a much lamented, gap in turning issues that are really cast in pastel shades into Day-Glo relief? In this hale monograph, Jeff Hawkins (rendered by Sandra Blakeslee) plays exactly this role for theoretical neuroscience. As a pastel practitioner myself, but furtively sharing many of Hawkins' prejudices and hunches about computational modelling in neuroscience, I am caught between commendation and consternation.Hawkins is an engineer, entrepreneur, and scientist who founded and led the companies Palm and then Handspring. He created, against what must have been considerable obstacles, the first widely successful PDA, and continued the development of this platform. He has thus amply earned a bully pulpit. The autobiographical segments of this book detail that, throughout his career, he has been interested in understanding how the brain works, using his substantial knowledge and intuition about the architecture and design of conventional computers as a counterpoint.More recently, Hawkins has generously put his money where his ideas about mentation dictate, founding the Redwood Neuroscience Institute and also funding various conferences and workshops. The institute is dedicated to ‘studying and promoting biologically accurate mathematical models of memory and cognition.’Despite its youth, the Institute already has attracted notable attention as a centre for theoretical neuroscience. Hawkins' quest, and-depending on which statements of the book you read-its endpoint (‘… a comprehensive theory of how the brain works … describ[ing] what intelligence is and how your brain creates it’) or just its tipping point , are the subject here.There are really three books jostling inside the covers. One is the (highly abbreviated) autobiography. The history of modern computing is very brief and very glorious, and this story is most entertaining. Don't miss the wonderfully faux naive letter from Hawkins to Gordon Moore asking, in 1980, to set up a research group within Intel devoted to the brain. That Hawkins prospered in clear opposition to accepted wisdom is perhaps one of the key subtexts of the book.The second, and rather less satisfying, book is about the philosophy of mind and the history of artificial intelligence and neural network approaches to understanding the brain and replicating cognition. With respect to the fields of artificial intelligence and neural nets, the text seems rather to be fighting yesterday's battles. The importance of learning, flexibility in representation and inference, and even decentralisation of control has been more than amply recognised in the inexorable rise of probabilistic approaches in both fields.With respect to the philosophy of mind, there seems to be something of an enthusiast's disdain for the niceties of philosophical pettifogging, even arguing by assertion. The discussions at the end on creativity and consciousness all seem a bit gossamer. The book is somewhat careless about functionalism, a key doctrine for computational theorists about how brains give rise to minds. According to this doctrine, at least roughly, it is the functional roles of, and functional interactions among, the physical elements of brain that matter, and not their precise physical nature. If you can capture those functional aspects correctly, for instance, in a computer program, then you can (re-)create what's important about mental states. Functionalism licenses a form of inquiry into the computational jobs played by structures in the brain. However, although formally agreeing that ‘there's nothing inherently special or magical about the brain that allows it to be intelligent,’the book slips into statements such as ‘brains and computers do fundamentally different things,’which are, at best, unfortunate shorthand.The book is a little apt to sneak plausible, but misleading, claims under the radar. Just to give one instance, it compellingly compares a six year old hopping from rock to rock in a streambed with a lumbering robot failing to do the same task. However, this is a bit unfair. One of Hawkins' self-denying ordinances is to consider the cortex pretty much by itself. As aficionados of the cerebellum would be quick to point out, the singular role for the cortex in such graceful behaviour is rather questionable.The third book is what I think is intended to be the real contribution. This contains a (not wholly convincing) attempt to conceptualise the definition of intelligence in terms of prediction rather than behaviour, and then to describe its possible instantiation in the anatomy (and mostly only the anatomy) of the cortex.To situate Hawkins' suggestions, it is instructive to consider current models of how the cerebral cortex represents, and learns to represent, information about the world without being explicitly taught. Being a popular account, the book fairly breezes by these so-called unsupervised learning models see , in whicFirst, where does the structure in the inputs come from? For the sake of concreteness, think of the input as being something like movies on a television screen. Movies don't look like white noise, or ‘snow’, because of their statistical structure. For instance, in movies, pixel activities tend to change rather slowly over time, and pixels that are close to each other on the screen tend to have relatively similar activities at any given time. Neither of these is true of white noise. More technically, movies constitute only a tiny fraction of the space of all possible activations of all the pixels on your screen. They have a particular statistical structure that the cortex is supposed to extract.What is the cortex supposed to do with this structure? The idea is that the cortex learns to model, or ‘parameterize’, it. Then, the activities of cortical cells over time for a particular input, for example, a particular face in a movie, indicate the values of the parameters associated with that face. Thereby the cortical activities represent the input. The parameters for a face might include one set for its physical structure , another set for the expression, and yet others, too.Cortical representations are thus intended to reflect directly the statistical structure in the input. Importantly, for inputs such as movies, this structure is thought to be hierarchical and, concomitantly, to provide an account of the observed hierarchical structure of sensory cortical areas. One source of hierarchical structure in movies is the simple fact that objects (such as the faces) have parts (such as eyes and cheeks) whose form and changes in form over time are interdependent. Another source of hierarchical structure is that the same face can appear in many different poses, under many different forms of illumination, and so on. Pattern theory , one of How does the cortex do all this? Of course, some fraction of this structure was built in over evolution. However, the unsupervised learning tradition concentrates on ontogenic adaptation, based on multiple presented input movies. An additional facet of the lack of supervision is that this adaptation is taken as not depending on any particular behavioural task.Finally, what does this process allow the cortex to do? The whole representational structure is intended to support inference. Crudely, this involves turning partial or noisy inputs into the completed, cleaned-up patterns they imply, using connections between areas in the cortical hierarchy. Construed this way, probabilistic inference actually instantiates a very general form of computation. Crucially, over the course of the development of unsupervised learning methods, it has been realised that the best way to approach the extraction of input structure, and inference with it, is through the language and tools of probability theory and statistics. The same realisation has driven substantial developments in artificial intelligence, machine learning, computer vision, and a host of other disciplines.We can now return to the book. Hawkins compactly sums up his thesis in the following way. ‘To make predictions of future events, your neocortex has to store sequences of patterns. To recall appropriate memories, it has to retrieve patterns by their similarity to past patterns . And finally, memories have to be stored in an invariant form so that the knowledge of past events can be applied to new situations that are similar but not identical to the past.’In fact, to take the latter points first, the sort of auto-associative storage and recall to which Hawkins refers is a theoretically and practically hobbled version of unsupervised learning's probabilistic inference. Invariance is closely related to the deformations we described above in the context of pattern theory.Unsupervised learning has certainly paid substantial attention to sequences of inputs and prediction, and to some good effect. For instance, speech recognition programs are based on a probabilistic device called a hidden Markov model, which is a key element in a wealth of unsupervised learning approaches to prediction. However, despite heroic efforts, these modelling methods are incapable of capturing the sort of complex structure seen in inputs such as natural languages. They fail on phenomena like long-distance dependencies, for example, the agreement between the cases of subjects and verbs, which are rife. This does tend to offer a vaccine against Hawkins' otherwise infectious optimism.Once place in which Hawkins goes beyond existing unsupervised learning models is in an extension to actions and control, and in an ascription of parts of the model to cortical anatomy. The hierarchical conception of cortex here goes all the way down to primary motor cortex . This allows auto-associative recall of sequences of past inputs and outputs to be used to specify actions that have formerly been successful. The discussion of this possibility is, unfortunately, rather brief. Central issues are omitted, such as the way that planning over multiple actions might happen. Also, the way that value is assigned to outcomes to determine success or failure is not discussed. The latter is widely believed to involve the neuromodulatory systems that lie below the cortex and that the book's cortical chauvinism leads it cheerfully to ignore.By contrast, the book has a rather detailed description of how the model should map onto the anatomy of the cerebral cortex. Like many unsupervised learning modellers, Hawkins is a self-confessed ‘lumper’. He ignores huge swathes of complexity and specificity in cortical structure and connections in favour of a scheme of crystalline regularity. Though this will doubtless irk many readers constitute a terrible waste of cortical representational power, or, worse, (b) interfere with, or warp, the parameterization of the aspects of the input that are important, making it harder to extract critical distinctions. The book does not address this issue, relying on there being enough predictive power to capture any and all predictions, including predictive characterisation of motor control.Second, although our subjective sense is that we build a sophisticated predictive model of the entire sensory input, experiments into such phenomena as change blindness show thiAs a final example of a spur to insomnia, unsupervised learners worry that To sum up, in terms of the adage that genius is 1% inspiration and 99% perspiration, the book's enthymematic nature suggests that not quite enough sweat has been broken. Were it 1% inspiration and 99% aspiration, though, then the appealing call to arms for a new generation of modellers should more than suffice.

The Depression Network Study (DeNt) is a multicentre study designed to identify genes and/or loci linked to and/or associated with susceptibility to unipolar depression in Caucasian families. This study presents the method and socio-demographic details of the first 470 affected sibling pairs recruited from 8 different sites in Europe and the United States of America.th edition (DSM-IV) or the International Classification of Diseases 10th edition (ICD-10) criteria for recurrent unipolar depression of moderate or severe degree and who had at least one similarly affected sibling were eligible for the study. Detailed clinical and psychological assessments were undertaken on all subjects including an interview using the Schedules for Clinical Assessment in Neuropsychiatry. Blood samples were collected from all participants to extract DNA for linkage analysis.Probands fulfilling either the Diagnostic and Statistical Manual 4The different sites used different recruitment strategies depending on local health care organisation but despite this there was remarkable similarity across sites for the subjects recruited. Although the Bonn site had significantly older subjects both for age of onset and age at interview, for the sample as a whole, subjects were interviewed in their mid-40s and had experienced the onset of their recurrent depression in their 20s. Preliminary genome screening was able to include 929 out of the 944 subjects (98.4%) typed at 932 autosomal and 544 X chromosome markersThis paper describes the methodology and the characteristics of the subjects from the 414 families included in the first wave of genotyping from the multi-site DeNT study. Ultimately the study aims to collect affected sibling pairs from approximately 1200 families. Genetic risk factors are well established for major affective disorders and a recent twin study has suggested that unipolar depression has a stronger genetic influence than was previously thought. McGuffin and colleagues have estThe majority of studies suggest a relative risk to siblings (λs) of affective disorder is in the region of 3 .The inheritance of unipolar depression is complex and involves an inter-play of genetic and environmental factors. For unipolar depression these include certain types of severe and threatening life events such as events associated with humiliation or loss ,6.Despite an excess of females to males of about 2 to 1 for unipolar depression, the heritability in a clinically ascertained sample was the same in men and women while otMost previous linkage studies have been carried out in families identified by a bipolar proband and where unipolar and bipolar relatives are frequently grouped together into a broad definition of affective disorders. Most such studies have focussed on multiple affected extended pedigrees on the assumption that there may be a sub-set segregating a gene of major effect. This approach has been successful in complex disorders such as early onset Alzheimer's disease and breast cancer. However, consistent evidence of major gene effects in bipolar disorder has not been forth-coming. In addiSibling pairs affected with recurrent unipolar depression were recruited from 8 clinical sites: Aarhus, Denmark; Bonn, Germany; Dublin, Ireland; Lausanne, Switzerland; St Louis, USA and London, Cardiff and Birmingham, UK. In addition, where available, parents of the affected sibling pairs were also included in the study.th edition operational criteria (DSMIV) [th edition operational criteria (ICD10), for unipolar depression [Subjects were identified from psychiatric clinics, hospitals, general medical practices and from volunteers responding to media advertisements. Caucasian subjects over the age of 18 were included if they had experienced 2 or more episodes of unipolar depression of at least moderate severity separated by at least 2 months of remission as defined by the Diagnostic and Statistical Manual 4 (DSMIV) or the Ipression . ProbandSubjects were also excluded if they experienced psychotic symptoms that were mood incongruent or present when there was no evidence of a mood disturbance. Other exclusion criteria were intravenous drug use with a lifetime diagnosis of dependency; depression occurring solely in relation to alcohol or substance abuse or depression only secondary to medical illness or medication, and a clear diagnosis of bipolar disorder, schizophrenia, schizo-affective disorder or acute or transient psychotic disorders in first or second-degree relatives.All subjects were interviewed using the Schedules for Clinical Assessment in Neuropsychiatry (SCAN) ,16. ItemAll interviewers from each site attended a 4-day SCAN training course in the UK. Each site also undertook further inter-rater reliability meetings regularly and annually all interviewers from all sites took part in a joint inter-rater reliability exercise.All sites obtained ethical approval for the DENT study within their own countries and institutions. All study participants gave written informed consent for participation in the study.In addition to the SCAN interview all study participants completed the Eysenck Personality Questionnaire and a deAt the time of the SCAN interview interviewers obtained 25 ml of whole blood that was collected in 37.5 ml (EDTA containing) monovettes. In addition drops of blood were placed on a Guthrie blood spot card. The blood samples were labelled with a bar code, gently mixed and stored frozen upright in a -20 degree centigrade freezer pending DNA extraction.All phenotypic information from interviews and questionnaires was coded by assigning a number to each subject, and removing any personal identifying information. The same codes were used on the blood sample tubes using a bar code system. The phenotypic information was first entered on an EXCEL spread sheet after which a data file was created using Statistical Procedures for the Social Sciences (SPSS) version 10 for Windows for the statistical analyses.All the interviewers from all sites took part in a joint inter-rater reliability exercise (in English) involving both audio-taped interviews with study subjects and videotaped interviews with actors, role-playing a depressed subject. Item by item kappa statistics for SCAN items, were calculated comparing each interviewer's ratings against AF's "master" rating. A mean item by item kappa coefficient across all the sites of 0.77 (range 0.63 – 0.89) was obtained indicating a substantial level of inter-rater agreement.For inclusion in the first part of the linkage analysis, 944 affected subjects were genotyped from the 8 study sites as follows: Aarhus 48, Birmingham 146, Bonn 110, Cardiff 126, Dublin 154, Lausanne, 56, London 111 and St Louis 193. The age at interview and age of illness onset by gender of the subjects recruited at each site are shown in Table Mean age at interview for both sexes combined for each site were as follows: Aarhus 44.13 years (standard error of the mean (SEM)1.5), Birmingham 48.44 years (SEM 1.1), Bonn 51.46 years (SEM 1.2), Cardiff 44.06 years (SEM 0.9), Dublin 43.32 years (SEM 1.0), Lausanne 47.21 years (SEM 1.3), London 45.67 years (SEM 1.0), St Louis 47.14 years (SEM 0.9). These mean age differences were statistically significant : F = 6.26 degrees of freedom (df) 7, 936, p < 0.001. Tukey Post hoc test: Dublin, Cardiff, Aarhus, London, St Louis, Lausanne, Birmingham < St Louis, Lausanne, Birmingham, Bonn).Mean age at illness onset for both sexes combined per site were as follows: Aarhus 22.67 years (SEM 1.3), Birmingham 24.30 years (SEM 0.8), Bonn 27.28 years (SEM 1.2), Cardiff 23.47 years (SEM 0.9), Dublin 22.27 years (SEM 0.8), Lausanne 24.85 years (SEM 1.4), London 22.08 years (SEM 1.1), St Louis 18.17 years (SEM 0.8). These mean age differences were statistically significant .However, there were no significant differences between sites for the numbers of men and women recruited .Of the 944 subjects, 670 (71%) were female and 274 (29%) were males and hence, the female/male ratio was 2.45:1.Mean age at interview for all female subjects was 45.40 years (SEM 0.5) and for all males subjects was 45.69 (SEM 0.8). There were no significant gender differences for age at interview The mean age of illness onset for depressed male subjects was 22.61 years (SEM 0.7) compared to 22.52 years (SEM 0.4) for depressed female subjects. There was no significant sex difference for age of onset..Fifty five percent of male subjects and 61 % female subjects were living with a partner (married or cohabiting), while 45 % male subjects and 39% female subjects were living alone . Female subjects were significantly more likely to be living with a partner compared to male subjects. (Chi squared test = 26.89 df = 1 p < 0.001).There were 295 female and 117 male probands, 335 female and 144 male siblings and 40 female and 13 male parents included in the total sample. There were no significantly differences for the gender of probands, siblings or parents .The mean age at interview for probands was 45.94 years (SEM 0.6) and for siblings was 45.80 years (SEM 0.5). There were no significant differences for age at interview between probands and their siblings Probands gave a mean age of illness onset of 20.22 years (SEM 0.6) while siblings reported a mean age of onset of 21.04 years (SEM 0.6). Again these differences were not statistically significant There were also no significant differences between probands and their siblings for marital status; 161 probands and 170 siblings were living alone while 242 probands and 290 sibings were living with a partner (chi squared test = 0.81 df = 1 p = ns).Genotyping was carried out by DeCode and the results checked for mis-specified relationships by the programs RELPAIR and Graphical Representation of Relationships (GRR) at the Institute of Psychiatry. RELPAIR compares the multipoint probability of the data conditional on the possible relationships, while GRR calculates the IBS mean and SD for each pair and plots these values, representing each type of relative pair using a different colour. Decisions about each problem pair were made on the basis of the results from both programs, although where there was discrepancy between the programs the GRR results were used.To check genotypes with Mendelian and other pedigree errors the PEDSTAT and MERLIN programs were used.These data cleaning processes resulted in 929 individuals being genotyped at 932 autosomal markers and 44 X chromosome markers. Success rates for the autosomal markers were above 61% and for 90% were above 86%. For the X chromosome the success rate was above 66%. For individuals the genotyping success rate was above 73% for autosomal markers and 61% for the X chromosome.The Depression Network study has recruited affected sibling pairs and some of their parents from 7 European and 1 North American site for a linkage analysis of recurrent unipolar depression. Because of differences in local service organisation, different recruitment strategies have been employed at the different sites. This may account for the significant differences for age at interview and age at illness onset between sites. The Bonn site recruited the oldest sibling pairs, both in terms of when subjects were interviewed and also when their illnesses had commenced. The Bonn subjects had a mean age at interview of 51.46 years compared to the Dublin subjects whose mean age at interview of 43.31 years was the youngest. Similarly the Bonn subjects mean age at illness onset was 27.28 years compared to a mean age of illness onset nearly a decade earlier for the St Louis subjects (18.17 years). It is noteworthy however that the St Louis sample included several large affected sibships. Subjects from families where there are many affected relatives may have a more genetic form of the disorder that might be contributing to an earlier age of onset.Despite these inter-site differences, the results show that there are also considerable similarities across the sites for the subjects recruited. Subjects have been mainly interviewed in their mid 40s and have experienced the onset of their recurrent depression in their early to mid 20s. Consequently subjects had on average around 20 years of history of episodes of depression when interviewed.As expected the study has shown the same preponderance of female to male subjects as many other studies with a gender ratio of around 2.45:1 [We would also not expect to find any significant differences between probands and siblings in terms of gender, age at interview, age at illness onset or marital status, which the results show is the case. Indeed for the purpose of finding genes for depression we would require siblings to have experienced similar forms of the illness.Although some subjects were excluded following genotyping due to errors that could not be reconciled, this preliminary genome linkage screen was able to include 929 subjects (98.4%) genotyped at 932 autosomal and 544 X chromosome markers. The results of the whole genome screen will be presented in due course.The Depression network study is the first co-ordinated international collaboration of its kind on the genetics of depression and one of the largest ever neuropsychiatric linkage study collection to use a uniform methodology to define and describe the phenotype. Despite taking place across eight sites and in six different countries good inter-rater agreement has been achievable as has good comparability of data.The study has been designed to overcome the difficulties that have been encountered in linkage studies of other psychiatric disorders such as schizophrenia and bipolar disorder. These started out optimistically with the assumption that genes of large effect would exist in at least some multiply affected families. However, after over a decade of contradictory findings and non replications, there is now consensus that such families are very rare or perhaps nonexistent. Rather it seems likely that common familial psychiatric disorders result from the combined effect of multiple genes none of which is either necessary or sufficient to cause the condition . ConsequThis study was funded by 3 year research grants to each participating site from Glaxo Wellcome Research and Development.AF & PMcG were overall study Principal Investigators (PIs), conceived the study and were co-ordinators of the study design, diagnostic reliability and data analysis. AF trained the interviewers from all the sites and wrote the paper. SB, LM and JP obtained the funding from Glaxo Wellcome, recruited site PIs and oversaw the quality of data collection, handling and analysis. GB analysed the genotyping data. The following authors were the individual site PIs in charge of all aspects of subject recruitment and data quality locally: OM Aarhus site, NC, LJ and IJ (joint) Birmingham site, MR and WM (joint) Bonn site, AK and MO (joint) Cardiff site, MG Dublin site, MP Lausanne site AF and PMcG (joint) London site and TR St Louis site. All authors have read and approved the contents of the final manuscript.The pre-publication history for this paper can be accessed here:

Neo-adjuvant chemotherapy is an integral part of multi-modality approach in the management of locally advanced breast cancer and it is vital to predict the response in order to tailor the regime for a patient. The common final pathway in the tumor cell death is believed to be apoptosis or programmed cell death and chemotherapeutic drugs like other DNA-damaging agents act on rapidly multiplying cells including both the tumor and the normal cells by following the same common final pathway. This could account for both the toxic effects and the response. Absence or decreased apoptosis has been found to be associated with chemo resistance. The change in expression of apoptotic markers (Bcl-2 and Bax proteins) brought about by various chemotherapeutic regimens is being used to identify drug resistance in the tumor cells. A prospective clinical study was conducted to assess whether chemotherapy induced toxic effects could serve as reliable predictors of apoptosis or response to neo-adjuvant chemotherapy in patients with locally advanced breast cancer.50 cases of locally advanced breast cancer after complete routine and metastatic work up were subjected to trucut biopsy and the tissue evaluated immunohistochemically for apoptotic markers (bcl-2/bax ratio). Three cycles of Neoadjuvant Chemotherapy using FAC regime were given at three weekly intervals and patients assessed for clinical response as well as toxicity after each cycle. Modified radical mastectomy was performed in all patients three weeks after the last cycle and the specimen were re-evaluated for any change in the bcl-2/bax ratio. The clinical response, immunohistochemical response and the drug-induced toxicity were correlated and compared.Descriptive studies were performed with SPSS version 10 and the significance of response was assessed using paired t-test. Significance of correlation between various variables was assessed using chi-square test and coefficient of correlation calculated by Pearson correlation coefficient.There was a statistically significant correlation observed between clinical, immunohistochemical response (bcl-2/bax ratio) and the drug-induced toxicity.Responders also had significant toxicity while non-responders did not show significant toxicity following neoadjuvant chemotherapy. The chemotherapy-induced toxicity was observed to be a cost effective and reliable predictor of response to neo-adjuvant chemotherapy. Neo-adjuvant chemotherapy (NACT) is an integral part of multi-modality approach in the management of locally advanced breast cancer (LABC). It is required both for the local control and distant or systemic control -5. In thApoptosis is a closely regulated form of active cell death defined by characteristic biochemical and morphological criteria. A large number of anti-cancer agents with widely differing modes of action have been demonstrated to induce apoptosis in vitro, suggesting this as a significant final common pathway for exerting their clinical effects. Mechanisms that suppress apoptosis may be important in the development of intrinsic and acquired resistance to cytotoxic drugs .It was suggested more than 20 years ago that apoptosis might account for much of the spontaneous cell loss (known from kinetic studies) to occur in many tumors and its extent often is enhanced by well-established modalities such as chemotherapy, irradiation and hormone ablation. However, during the past few years, advances in the understanding of the control of apoptosis at the molecular level has extended its potential oncologic significance far beyond the mere provision of a mechanistic explanation for tumor cell deletion. In particular, the discovery that the products of certain proto-oncogenes can regulate apoptosis has opened up exciting avenues for future research .The protean effects of various neoplastic agents on synthesis of DNA, RNA, and inhibition of synthesis which may or may not lead to cell death, requires only that some critical concentrations of active drug or metabolite be present in a cell. Proliferating normal cells may therefore be subject to the same detrimental effects of chemotherapy experienced by neoplastic tissue and successful chemotherapy dictates that recuperative abilities of normal tissues are greater than those of cancer. The two tissues generally most adversely affected by antineoplastics are the hemopoetic cells of the bone marrow and the epithelium of the aero digestive tract as a result of high growth fractions and short cycling times of the cells. The ability of the cancer patients to perform normal activities and function is also recognized both as a determinant of how well the patient may respond to chemotherapy and an index of the general toxicities of the anti cancer agents.A variety of anti-cancer drugs have been shown to induce extensive apoptosis in rapidly proliferating "normal" cell population and the tumors alike. Thus enhanced apoptosis is also likely to be responsible for many of the adverse effects observed following chemotherapy . ApoptosApoptosis is a regulated phenomenon capable of being inhibited and activated. Indeed there is evidence that stimulation of some cells by trophic cytokines or increase in their levels of expression of Bcl-2 proto-ontogeny can greatly increase their resistance to the apoptosis-inducing effects of anticancer drugs. Thus Bcl-2 proto-ontogeny expression may be implicated in the development of resistance of tumors to therapeutic agents and may contribute to tumor growth and perhaps to ontogenesis by allowing the inappropriate survival of cells with DNA abnormalities .Deregulated expression of the Bcl-2 protein represents the best-known example of a potent blocker of apoptosis. Over expression of Bcl-2 has now been shown to protect a wide variety of cell types from induction of apoptosis by many different anticancer agents. Several homologues of Bcl-2 protein have also been shown to act as inhibitors of apoptosis, including Bcl-Xl and others as apoptotic proteins such as Bax. In vitro data suggest that it is the relative ratios of anti-apoptotic and pro-apoptotic proteins that determine the likelihood of cells to undergo apoptosis in response to chemotherapeutic drugs in vivo test bed to further confirm the clinical relevance of these observations. The clinical response or the absence of response along with the toxic effects observed could well help predict the outcome with a particular chemotherapeutic regime and facilitate planning of an alternate regime for better response [The increasing use of pre-operative chemotherapy (PCT) in breast cancer offers an response -8.It is vital to assess the response to NACT in order to tailor the regime for a particular patient to predict the intrinsic or acquired chemo resistance. DNA-damaging agents such as chemotherapeutic drugs can induce apoptosis and increased resistance to chemotherapeutic agents, which has been found to be associated with decreased capacity to undergo apoptosis -9. CentrThe clinical response along with complete pathological response (CPR) is still considered a surrogate marker of response against which all other predictive markers are compared. The need to have a reliable and inexpensive predictor of response in a third world scenario can not be over emphasized since majority of patients present relatively late and the resources are limited and scarce ,3. Since1. To assess the clinical and immunohistochemical response to NACT in patients with LABC.2. To correlate immuno-histochemical (apoptotic markers i.e. Bcl-2/Bax ratio) and clinical response to the drug induced toxicity.3. To ascertain whether the drug induced toxicity could be utilized as a reliable indicator and predictor of response to neoadjuvant chemotherapy.50 FNAC proven cases of locally advanced breast carcinoma according to AJCC (American Joint Committee On Cancer) classification were included in the studyA thorough clinical and ultrasonographic examination (USG) of all the patients including the opposite breast was performed to stage the disease accurately. A core biopsy using a tru-cut needle was performed for immuno-histochemical estimation of the apoptotic markers i.e. base-line Bcl-2/Bax ratio before initiating the chemotherapy. Routine and metastatic work up was done including complete blood examination , chest radiograph, ECG (Echocardiography when ECG had a positive finding), liver function tests, Bone Scan, USG abdomen, KFT (Kidney function tests).2, adriamycin -50 mg/m2, 5-fluorouracil-600 mg/m2) at an interval of three weeks. Before each cycle the patient was clinically and sonologically examined for the breast tumor size, axillary lymph node status & appearance of systemic metastasis. All patients were given the same antitoxicity treatment according to a standardized unit protocol including adequate hydration and inject able antiemetics before initiating the chemotherapy.Patients were subjected to three cycles of FAC regime solution and incubated with blocking antibodies at 37°C. Sections were washed with TBS solution. Incubation with avidin-biotin complex (ABC) was done at 37°C for one hour and washed with TBS solution. 3,3 Diaminobenzidine tetra hydrochloride solution applied for 3–5 min. Counter-staining with haemotoxylin solution done for 3–5 min. Sections were washed with distilled water, air dried and mounted using DPX mountant.For Bax, positive controls were taken as germinal centers of the lymphoid follicles and normal breast tissue and negative control was taken as the test slide without primary antibody. For Bcl-2, positive controls were the mantle zone of lymphoid follicles and the negative controls were the test slides without primary antibody.The pattern of positive staining for bcl-2 and bax was cytoplasmic, granular.The primary antibodies for bcl-2 and bax were procured from DAKO.Bax-Rabbit Anti-Human code no. A 3533.Bcl-2 Monoclonal Mouse Anti-Human code no. M 0887.Dilution for both was 1: 40.The results were interpreted on the basis of two criteria:(1) Percentage of cells showing immune bodies; <5%: score = 0, 5–25%: score = 1, 25–75%: score = 2, >75%: score = 3(2) Intensity of staining; mild: score = 1, moderate: score = 2, intense: score = 3.Since there was a strong correlation between the intensity of staining and percentage of cells showing immune bodies, the percentage of cells showing immune antibodies alone was considered for calculating the bcl-2/bax ratio"."Descriptive studies were performed with SPSS version 10. The significance of response assessed using paired t-test. Significance of correlation between various variables assessed using chi-square test and coefficient of correlation was calculated by Pearson correlation coefficient.50 cases of locally advanced breast cancer were included in this study. Mean age of the patients was 45.5 years (range: 28 to 71 years) and 53.3% patients were pre-menopausal. Size of the tumor was measured clinically as well as by ultrasound and the patients were subdivided into four groups: <5 cm(0%), 5–8 cm(56.6%), 8–10 cm(26.6%), >10 cm(16.6%). According to the axillary lymph node status the patients were divided into three groups: N0 (0%), N1 (33%), N2 (67%).Objective clinical response was defined as more than 50% reduction in the tumor size after three cycles of neoadjuvant chemotherapy. Immuno-histochemical response was defined as decrease in the Bcl-2/Bax ratio.Clinical response including the reduction in the tumor size and axillary lymph node status was observed in 70% of patients and was found to be statistically significant p < 0.0001). There were no patients in the No group and 29.4%of the N1 patients were down staged to N0 while70.6% remained N1. In patients with N2 disease 7.7% were down staged to N0 status while 46.2% were downstaged to N1 status and 46.2% did not show any response. Immuno-histochemical response was observed in 60% and was also found to be statistically significant (p = 0.008). Correlation between immuno-histochemical and clinical response was also found to be statistically significant (p < 0.0001) [Table . There wAcute vomiting was observed in 63.3% patients. 81% clinical responders had vomiting (p = 0.002) and 78% immunohistochemical responders also had vomiting which was statistically significant (p = 0.04). Alopecia was observed in 86% clinical responders (p = 0.000) and 94% immuno-histochemical responders (p = 0.000), which was also significant. Leucopoenia was observed in only 14% and 17% of clinical and immuno-histochemical responders respectively and was found to be an insignificant predictor of response in the present study. When multiple toxicities were correlated with the clinical and immuno-histochemical response, 46.7% of patients had both acute vomiting and alopecia. 67% clinical responders (p = 0.001) had both vomiting and alopecia.72% immunohistochemical responders (p = 0.001) had both vomiting and alopecia.A significant positive correlation was observed between the presence of vomiting (r = +0.558), alopeciar = +0.802) and response to neoadjuvant chemotherapy. A significant negative correlation was observed between the absence of side effects and poor response to neoadjuvant chemotherapy in 65% of patients after two courses of FAC . NACT haesection -13.Introduced by Kerr et al (1972) to describe characteristic morphological changes seen during programmed cell death . It is d(1) Chromophin condensation(2) Membrane blebbing(3) Appearance of apoptotic bodies(4) Fragmentation of genomic DNACertain biochemical and genetic events have been identified that are associated with multiple cell types including mammary epithelium. These include the DNA fragmentation via end nuclease activation and cleavage of intracellular proteins, expression of bcl-2 family members, tumor suppressor gene p-53 directed events, proto-oncogene activation and activation of transmembrane receptor signaling pathways such as tumour necrosis factor ,17-22. AThe heterogeneous nature of breast cancer has resulted in overwhelming interest in search for prognostic markers to identify patients who might benefit most from the therapeutic modalities available.Assessment of apoptosis and individual components of apoptotic pathway are therefore relevant in determining prognosis in a particular patient . DNA damBcl-2 is anti-apoptotic in function, whereas bax is proapoptotic and it is the interaction between the two that determines the likelihood of a tumor to undergo cytotoxic drug mediated regression. Therefore any increase in bcl-2 or decrease in bax will push the balance towards chemo resistance and an increase in bax or decrease in bcl-2 will result in increased apoptosis -30.It was observed in a study conducted by Kymionis et al that incIn the present study the clinical response in terms of reduction of tumor size and immuno-histochemical response in terms of change of bcl-2/bax ratio correlated significantly with the drug induced toxicity following NACT.The time course of various toxic manipulations depends on the drug, its dose and frequency of administration, intrinsic characteristics of the tissue of interest and any local circumstances . There are few general rules however like mucosal toxicities of pain, erythema, ulceration etc. occur 3–10 days after the administration of most offending drugs. Bone marrow effects can be manifestated a few days later averaging 7–14 days. The recovery of normal functioning tissues in both cases is well under way 4–5 days after the zenith of the toxic effects.Alopecia may involve the scalp or the whole body can present within 2 weeks of the drug dose or be a progressive cumulative event. Other cumulative toxicities seen only after administration of a certain quantity of drug over some length of time include cardiomyopathy from anthracyclins.An alkylating agent belonging to the nitrogen mustard subgroup. It is inactive as such produces few acute effects and is not locally damaging. It is transformed in to active metabolites like aldophosphamide and phospharimide mustard in the liver, which then produces the wide variety of antitumour action. The prominent side effects are alopecia and cystitis, which are caused by another metabolite acrolein.It is a pyrimidine antagonist and is converted in the body to the corresponding nucleotide, 5-fluro-2-deoxy-uridine monophosphate, which inhibits thymidine synthetase and blocks the conversion of deoxyuridilic acid to deoxythymidilic acid. Selective failure of DNA synthesis occurs due to non-availability of thymidylate. Flourouracil may itself get incorporated in to nucleic acids and this may contribute to its toxicity. Even resting cells are affected, though multiplying cells are more susceptible like the cells in the gastrointestinal tract (GIT) and bone marrow.It is an anti tumor antibiotic and is capable of causing breaks in the DNA strands by activating topoisomerase II and generating quinone type free radicals. They have mutagenic potential. Maximum action is exerted at S phase, but toxicity is usually exerted in G2 phase. Cardiotoxicity is a unique side effect. Rapidly multiplying cells are more susceptible therefore it also acts on cells of GIT, bone marrow in addition to the tumor cells.Leucopenia has not been observed to be a frequently encountered chemotherapy induced toxicity using commonly used regimen in most of the studies -24. ThisThe rapidly proliferating normal and tumor cells are more susceptible to the action of chemotherapeutic agents, which could explain the significant correlation observed between the effects and the toxicity in the present study. There was a strong correlation observed between the immunohistochemical response (bcl-2/bax ratio), clinical response and drug toxicity. This indirectly indicates a correlation between chemotherapy induced apoptosis and the toxicity and therefore like apoptotic markers, chemotherapy induced toxic effects along with objective clinical response could serve as reliable and cost-effective indicators or predictors of response to NACT in patients with LABC. While many biological markers are in use and many are under trial to tailor the chemotherapy for a particular patient, most of these markers including apoptotic markers or p-glycoprotein etc. are not very frequently available and are expensive for a third world cancer set up. Thus the chemotherapy-induced toxicity along with clinical response may be utilized as a cost effective and reliable predictor of response to NACT in patients with LABC. This would also serve as an intermediate end point in determining drug sensitivity for adjuvant treatment, especially when adjuvant therapy is planned with the same regimen as induction chemotherapy. This can also help in planning an alternative regime in non-responders.None declared.CM, the principal and the corresponding author was the Supervisor and the Chief surgeon who performed and standardized surgery on the patients and designed the study.• VS participated in the designing of the study, performed the statistical analysis and was the first surgical assistant and Senior Postgraduate in charge of the cases in the study.• JP, Postgraduate surgical resident was the second assistant in charge of the cases and participated in the sequence alignment.• AL, Resident surgery participated in the data processing and statistical analysis.• SS was in charge of the molecular genetic studies at the Tumor biology lab ICMR.• AB participated in the genetic studies and data processing.• All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:

Strictly defined, evolution is a change in the gene pool, or total set of genes, of a given population over time. Genetic changes that increase the fitness of an organism—that is, increase survival or fertility—are more likely to be retained, through natural selection, and passed on to succeeding generations. In the classic case of Darwin's finches, different ecological niches exerted different selective pressures on an original population, and resulted in 14 different species, each sporting a beak uniquely adapted to harvesting particular available food sources.When it comes to microbial evolution, an ecological niche often takes the form of a host. If the microbe is a pathogen, its presence might trigger strong selective pressure from the host's immune system, precipitating an evolutionary two-step between microbe and host. Hosts with strong immune defenses can typically tolerate relatively virulent pests: conversely, ill-defended hosts die, which is bad news for the parasite. When the myxoma virus first infected a population of European rabbits in Australia in 1950, the virus was particularly lethal. Over time, less virulent strains were selected for—killing off your habitat would be an unsustainable fitness cost by most standards—and the rabbits developed resistance.In keeping with evolutionary theory, host immunity should affect the evolution of parasite virulence. Though theory predicts that immunity could potentially heighten virulence, there's no evidence that this is true. Being able to predict how natural selection will act on, and thus shape, virulence is vital for developing effective public health policies—and desperately needed vaccines—to deal with the ever growing roster of rapidly evolving pathogenic threats.Plasmodium in the mouse. Mackinnon and Read first directly injected two groups of mice with infectious parasites: “immunized” mice, which had been exposed to Plasmodium and then treated with the antimalarial drug mefloquine, and “naïve” mice, which had not. Parasites were serially transferred twenty times via a syringe from one mouse host to another. The virulence and infectiousness of the respective strains were evaluated by introducing the strains into another set of immunized and naïve mice.To investigate whether immune system defenses escalate pathogen virulence, Margaret Mackinnon and Andrew Read studied the malarial parasite As theoretically predicted, parasites evolved in the immunized mice were indeed more virulent than parasites evolved in the naïve mice. But what if the parasites were first transmitted through their natural vector, the mosquito, rather than through a syringe? Would they be as virulent? Interestingly, infection was not as severe after mosquito transmission. But parasites evolved in the immunized mice retained a higher level of virulence than those evolved in the naïve mice. This means that immunity accelerates the evolution of virulence in malaria, even after mosquito transmission, making them more dangerous to nonimmunized hosts.How does immune selection create more virulent pathogens? One possibility is that even though many parasites die in immunized hosts, those that “win the race to the syringe”—or the mosquito—are likely genetically equipped to stay ahead of advancing immune system defenses.It's not entirely clear why selection would favor more virulent parasites, but since the virulent strains showed no problems transmitting infection to new hosts, it's likely that such strains would spread throughout an immunized population. While mosquito transmission likely plays a significant role in virulence evolution—it clearly reduced virulence here—the molecular mechanics of this effect are also mostly speculative at this point. Many questions remain, but these results make a strong case that vaccine development aimed at protecting individuals against infectious pathogens would do well to consider the evolutionary implications, or increased pathogen virulence could be an unintended consequence.

Although the effects of resistant starch (RS) on postprandial glycemia and insulinemia have been extensively studied, little is known about the impact of RS on fat metabolism. This study examines the relationship between the RS content of a meal and postprandial/post-absorbative fat oxidation.14C]-triolein with breath samples collected hourly following the meal, and gluteal fat biopsies obtained at 0 and 24 h. RS, regardless of dose, had no effect on fasting or postprandial insulin, glucose, FFA or TAG concentration, nor on meal fat storage. However, data from indirect calorimetry and oxidation of [1-14C]-triolein to 14CO2 showed that addition of 5.4% RS to the diet significantly increased fat oxidation. In fact, postprandial oxidation of [1-14C]-triolein was 23% greater with the 5.4% RS meal than the 0% meal (p = 0.0062).12 subjects consumed meals containing 0%, 2.7%, 5.4%, and 10.7% RS . Blood samples were taken and analyzed for glucose, insulin, triacylglycerol (TAG) and free fatty acid (FFA) concentrations. Respiratory quotient was measured hourly. The 0%, 5.4%, and 10.7% meals contained 50 μCi [1-These data indicate that replacement of 5.4% of total dietary carbohydrate with RS significantly increased post-prandial lipid oxidation and therefore could decrease fat accumulation in the long-term. Resistant starch (RS) is any starch that is not digested in the small intestine but passes to the large bowel for fermentation . RetrogrAcute human studies describe variable postprandial glycemic and/or insulinemic responses to RS ingestion. In general, it is accepted that RS consumption lowers postprandial glucose concentrations marginally and postprandial insulin concentrations markedly. Many groups report a decrease in postprandial glycemic or insulinemic responses to RS ingestion relative to digestible starch (DS) consumption -7, whereMany studies have examined the relationship between RS ingestion and postprandial metabolite and hormone concentrations. Fewer studies have documented the effect of RS on lipid metabolism. In humans, five weeks of RS feeding lowered fasting cholesterol and triglyceride concentrations and postprandial plasma insulin concentrations relative to digestible starch (DS) feeding ,13. It hIt is possible that strong physical association between RS and dietary lipid may slow the absorption, and thereby increase the oxidation, of dietary lipid. Currently, there is no evidence pertaining to the dose-response relationship for RS ingestion and postprandial glycemia, insulinemia, fat oxidation, or meal fat storage. It is important that these parameters be defined before designing and conducting long-term, prospective RS feeding studies.No difference in fasting or postprandial insulin, glucose, FFA, or triglyceride concentration was observed between any of the RS doses examined Figure .Overall, the dose of RS in the meal had a significant influence on ΔRQ (respiratory quotient) values . Meal fat oxidation at the 5.4% RS dose was significantly higher than both the 0% (p = 0.0062) and 10.7% doses (p < 0.0001). Separate tests at 6 h or 24 h following the test meal gave comparable results suggest that the inclusion of 5.4% RS in the meal elevated postprandial fat oxidation. Unexpectedly, this effect was lost if the dose was increased to 10.7% RS.Similarly, the oxidation of -triolein suspended in olive oil and the tests were conducted as 24 h inpatient stays at the GCRC. The fat tracer was fed as a triglyceride (glycerol tri [1-14C]oleate) rather than a FFA (eg. [1-14C]oleate) in order to reflect any change in the absorption of triglyceride FFA which might be due to a strong physical association with RS thereby slowing absorption. At hourly intervals following the meal, then at 8, 10, 12, 14 and 24 hours, breath samples were collected via exhalation through a tube with a one-way valve into scintillation vials containing 2 mmol benzethonium hydroxide (to trap 2 mmol CO2), 1 ml methanol, and 1 mg phenolpthalene as a pH indicator. Gluteal fat biopsies were collected by aspiration through a 14 g stainless steel needle at baseline and 24 h after ingestion of the test meal. All breath and fat samples were assayed for the presence of 14C (as described below). For these 24 h tests, subjects received 30% of daily energy needs at each of breakfast, lunch, and dinner, with the remaining 10% of calories received in an evening snack. The timing of meals/snacks was kept constant over all tests. All food was provided by the GCRC on an inpatient basis and the macronutrient content of each meal was the same as that of the test meal. Only the test breakfast contained RS during these 24 h tests, all other meals were composed of standard, commercially available products.In three of the test meals , the bread product in the test meal was spiked with 50 μCi [1-All glucose, FFA, and TAG assays were conducted by the GCRC Core Laboratory using an automated Cobas Mira Plus . Serum insulin measurements were also performed by the GCRC Core Laboratory using a human insulin RIA kit .Fat samples, frozen in liquid nitrogen and stored at -80°C until processing, were incubated in 450 μl Solvable at 50°C for 12 h before the addition of 100 μl 30% (v/v) hydrogen peroxide (for sample bleaching). Fat samples were counted in Aquasol whereas breath samples were counted in Scintisafe 30% using a Beckman LS6500 scintillation counter . After scintillant was added, all samples were kept in the dark at room temperature for 48 h before being counted to reduce chemiluminescence.et al. .24h/g tissue weight) - (dpmbaseline/g tissue weight) × 1/specific activityμg fat stored/g fat tissue = 2)/vCO2 (min.ml) = (dpmt - dpmbackground) × 1/vCO2 =counts from sample (dpm/mol CO2/ min.mldpm.mol CO2/ min.ml × 0.446 (as 1 ml CO2 = 0.446 mol)dpm/min = dpm.mol COg fat oxidized = AUC(dpm/min) × 1/specific activity2 is the rate of CO2 production as assessed during indirect calorimetry. t is sample time (min). AUC is the incremental area under the curve.where vCOAll statistical analyses were performed using the statistical analysis software SAS, version 8.1 with a significance level of p = 0.05 and p = 0.01 for interaction terms. All results are presented as mean ± SEM, except for subject characteristics which are described as mean ± SD. To investigate each of the outcomes we used a mixed model with fixed effect terms for RS DOSE, TIME and the interaction of the two, RS DOSE*TIME. Subjects were included as random effects. The interaction term was not significant for any of the outcomes tested so an additive model was used to test the overall effect of RS DOSE and the differences between doses. To test the effects of RS DOSE at different TIMES, a model that included RS DOSE, TIME and RS DOSE*TIME was used. The repeated measures nature of the study design was taken into account by using the covariance structures available in SAS PROC MIXED. For example, measurements within a subject are assumed to be more highly correlated than between subjects, and within a particular treatment, within a subject, the measurements are assumed to be more correlated. Measurements closer in time to one another were modeled with an autoregressive, or AR(1) covariance structure.RS, resistant starch; DS, digestible starch; TAG, triacylglycerol; FFA, free fatty acid; FFM, fat free mass; SCFA, short-chain fatty acids; GCRC, General Clinical Research Center; RQ, respiratory quotientJanine Higgins and Ian Brown are listed as inventors on RS patents filed by Penford Australia Limited. Both Drs. Higgins and Brown are listed as inventors on these patents as they have intellectual property ownership of some of data used in these but receive no financial benefit.JH conceived of the study design and was responsible for overall study coordination, conducting patient visits, data analysis, and manuscript preparation. DH was responsible for patient scheduling, day-to-day study coordination, conducting patient visits, and data entry. WD contributed to the study design and manuscript preparation, and conducted patient physical examinations and fat biopsies. IB contributed to the study design, selection of RS foods, and assisted with manuscript preparation. MB conducted all statistical analysis. DB contributed to the study design and manuscript preparation, and conducted patient visits, patient physical examinations and fat biopsies. All authors read and approved the final manuscript.Individual meal (a) and total fat oxidation (b) in response to the RS content of a test breakfast. Meal fat oxidation, assessed via measurement of 14CO2 in expired air, and total fat oxidation, assessed via indirect calorimetry and calculated from non-protein RQ, and was measured in 12 healthy adults.Click here for fileIndividual area under the glucose curve vs. meal (a) and total fat oxidation (b) in response to a test breakfast. Meal fat oxidation, assessed via measurement of 14CO2 in expired air, and total fat oxidation, assessed via indirect calorimetry and calculated from non-protein RQ, and was measured in 12 healthy adults. Data from all three test meals is shown. The relationship between area under the glucose curve and fat oxidation remains the same (i.e. no relationship) when represented as individual doses or, as in this plot, for all doses .Click here for fileIndividual area under the glucose curve vs. meal fat oxidation in response to a 0% (a) or 5.4% (b) RS test breakfast. Meal fat oxidation, assessed via measurement of 14CO2 in expired air, and total fat oxidation, assessed via indirect calorimetry and calculated from non-protein RQ, and was measured in healthy adults. Data from individual test meals is shown.Click here for fileIndividual area under the insulin curve vs. meal (a) and total fat oxidation (b) in response to a test breakfast. Meal fat oxidation, assessed via measurement of 14CO2 in expired air, and total fat oxidation, assessed via indirect calorimetry and calculated from non-protein RQ, and was measured in 12 healthy adults. Data from all three test meals is shown. Click here for file

Familial hypercholesterolemia is a human monogenic disease caused by population-specific mutations in the low density lipoprotein (LDL) receptor gene. Despite thirteen different mutations of the LDL receptor gene were reported from Russia prior to 2003, the whole spectrum of disease-causing gene alterations in this country is poorly known and requires further investigation provided by the current study.Forty-five patients with clinical diagnosis of FH were tested for the apolipoprotein B (apoB) mutation R3500Q by restriction fragment length analysis. After exclusion of R3500Q mutation high-sensitive fluorescent single-strand conformation polymorphism (SSCP) analysis and automatic DNA sequencing were used to search for mutations in the LDL receptor gene.We found twenty one rare sequence variations of the LDL receptor gene. Nineteen were probably pathogenic mutations, and two were considered as neutral ones. Among the mutations likely to be pathogenic, eight were novel , and eleven have already been described in other populations. None of the patients had the R3500Q mutation in the apoB gene.Nineteen pathogenic mutations in the LDL receptor gene in 23 probands were identified. Two mutations c.925-931del7 and L380H are shared by St.-Petersburg population with neighbouring Finland and several other mutations with Norway, Sweden or Denmark, i.e. countries from the Baltic Sea region. Only four mutations were recurrent as all those were found in two unrelated families. By this study the number of known mutations in the LDL receptor gene in St.-Petersburg area was increased nearly threefold. Analysis of all 34 low density lipoprotein receptor gene mutations found in St.-Petersburg argues against strong founder effect in Russian familial hypercholesterolemia. Familial hypercholesterolemia (FH) OMIM #143890) is one of the most common monogenic human diseases. It is inherited as an autosomal dominant trait with the prevalence of the heterozygous form conventionally considered to be about 1 of 500 in most populations 43890 is . FurtherThe spectrum of LDL receptor mutations varies between different human populations and more than 900 mutations in the LDL receptor gene have been characterized worldwide -9. A recPatients with FH were recruited from two lipid clinics of St.-Petersburg, namely Institute of Human Brain and Institute of Experimental Medicine. The clinical diagnosis of familial hypercholesterolemia was based on the following criteria: highly elevated plasma total cholesterol and LDL-cholesterol, presence of tendon xanthomata, corneal arcus or both, and positive family history of myocardial infarction and hypercholesterolemia with at least one first-degree relative affected. Forty-five probands fulfilling at least two of three criteria listed above were selected for the study. Full data of patients including their lipid data are given in the Discussion section in exon 2 that allows a simple detection of this mutation. Restriction enzyme test was performed for the proband and her 4 descendants that was identified by means of SSCP and verified by sequencing in probands from two unrelated families. SSCP patterns on silver-stained gels differ strikingly in patients with and without the mutation. The presence of the mutation was confirmed in the son of the proband by SSCP.In heterozygotes a deletion of 41 nucleotides at c.1291-1331 in exon 9 (mutation FsV409:S423X) results in occurrence of specific heteroduplexes. Besides, a PCR fragment of smaller size as compared to the normal allele is revealed in silver-stained polyacrylamide gels were found to be recurrent, i.e. all of those were found in two apparently unrelated families. Together with the data obtained earlier ,38 our rThe author(s) declare that they have no competing interests.FMZ, MYM, VIG, PHN, GGN, AS performed cloning, SSCP, sequencing and familial analysis, BML, VOK, DD, ADD selected patients, VBV and OF participated in the study design and coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:

Sign-language comprehension activates the auditory cortex in deaf subjects. It is not known whether this functional plasticity in the temporal cortex is age dependent. We conducted functional magnetic-resonance imaging in six deaf signers who lost their hearing before the age of 2 years, five deaf signers who were >5 years of age at the time of hearing loss and six signers with normal hearing. The task was sentence comprehension in Japanese sign language.The sign-comprehension tasks activated the planum temporale of both early- and late-deaf subjects, but not that of hearing signers. In early-deaf subjects, the middle superior temporal sulcus was more prominently activated than in late-deaf subjects.As the middle superior temporal sulcus is known to respond selectively to human voices, our findings suggest that this subregion of the auditory-association cortex, when deprived of its proper input, might make a functional shift from human voice processing to visual processing in an age-dependent manner. Sign languages involve the use of the hands and face, and are perceived visually -3. Usinget al. observed [et al. found thPerformance on the JSL comprehension task was similar across the groups F2, 14) = 1.279, P = 0.309, one-way ANOVA). The patterns of activity evoked during the sign-comprehension task in the hearing signers and the deaf groups are shown in Figure 4 = 1.2794 = 1.279The onset of deafness is related to language acquisition. Prelingual deafness occurs before spoken language is learned. Hearing people generally learn their first language before 5 years of age; hence, prelingual deaf individuals are either deaf at birth or became deaf prior to developing the grammatical basis of their native language, which is usually before the age of 5 years. Postlingual deafness is the loss of acoustic senses, either suddenly due to an accident or as a gradual progression after native-language acquisition . Hence, The posterior STS was activated in all groups during sign comprehension, which is consistent with the proposed neural substrates that subserve human movement perception . The posBoth the early- and late-deaf groups showed activation in the planum temporale, whereas hearing signers did not. Anatomically, the anterior border of the PT is the sulcus behind Heschl's gyrus and the medial border is the point where the PT fades into the insula. The posterior border of the PT involves the ascending and descending rami of the Sylvian fissure . Functioet al. [It has been shown that non-linguistic visual stimuli (moving stimuli) activate the auditory cortex in deaf individuals, but not in hearing subjects ,16. McSwet al. showed tet al. revealedet al. ,20 that et al. indicateet al. .The middle STS, anterior to the Vpc line, was activated more prominently in the early- than the late-deaf subjects. This difference is probably not related to linguistic processes, as both early- and late-deaf subjects are equally capable of learning JSL with the same amount of training. The middle STS region is presumably the area that is selective to human voice processing . This arper se but rather to vocal features that carry nonlinguistic information [Considering that the STS voice-selective area is not sensitive to speech ormation , the funThe results of the present study suggest that in early-deaf subjects, non-auditory processing, such as that involved in the perception and comprehension of sign language, involves the under-utilized area of the cortex that is thought to be selective to the human voice (middle STS). This indicates that the sensitive period for the establishment of human voice processing in the STS might be during the first 2 years of life.The subjects comprised six early-deaf signers (mean age: 22.8 ± 3.1 years), five late-deaf signers (mean age: 34.4 ± 16.2 years) and six hearing signers and a standard birdcage head coil. Each volume consisted of 11 slices, with a slice thickness of 8 mm and a 1-mm gap, which covered the entire cerebral cortex. The time interval between two successive acquisitions of the same image was 3,000 ms, the echo time was 50 ms and the flip angle was 90 degrees. The field of view was 22 cm. The digital in-plane resolution was 64 × 64 pixels. For anatomical reference, T1-weighted images were also obtained for each subject.x, y and z axes.The first three volumes of each fMRI session were discarded because of unstable magnetization. The remaining 40 volumes per session were used for statistical parametric mapping implemented in Matlab ,27. Follt statistic (SPM{t}).Statistical analysis was conducted at two levels. First, the individual task-related activation was evaluated. Second, the summary data for each individual were incorporated into the second-level analysis using a random-effects model to make inferences at a population level. The signal was proportionally scaled by setting the whole-brain mean value to 100 arbitrary units. The signal time course for each subject was modeled using a box-car function convolved with a hemodynamic-response function and temporally high-pass filtered. Session effects were also included in the model. The explanatory variables were centered at zero. To test hypotheses about regionally-specific condition effects , estimates for each model parameter were compared using the linear contrasts. The resulting set of voxel values for each contrast constituted a statistical parametric map (SPM) of the a priori hypothesis that there would be more prominent activation in the early- than late-deaf subjects, we focused on the temporal cortex, which was anatomically defined in standard stereotaxic space [t} was set at P < .001 without a correction for multiple comparisons.The weighted sum of the parameter estimates in the individual analyses constituted 'contrast' images that were used for the group analysis. Contrast images obtained via individual analyses represent the normalized task-related increment of the MR signal of each subject. To examine group differences in activation due to the sign-comprehension task, a random-effect model was performed with the contrast images (1 per subject) for every voxel. Using the ic space . The thrNS carried out the fMRI studies, data analysis and drafted the manuscript. HY and TO conducted the MR imaging. MY, TH and KM prepared the task materials. YY and HI participated in the task design and coordination. All authors read and approved the final manuscript.

Falls are common among community-dwelling elderly people and can have a considerable impact on quality of life and healthcare costs. People who have sustained a fall are at greater risk of falling again.We replicated a British randomised controlled trial which demonstrated the effectiveness of a multidisciplinary intervention programme to prevent falls.The objective is to describe the design of a replication study evaluating a multidisciplinary intervention programme on recurrent falls and functional decline among elderly persons at risk. The study consists of an effect evaluation, an economic evaluation and a process evaluation.The programme is aimed at community-dwelling elderly people aged 65 years or over who have visited an accident and emergency department (A&E department) or a general practitioners' cooperative (GP cooperative) because of a fall.The design involves a two-group randomised controlled trial. Participants are followed for twelve months after baseline. The intervention programme consists of a detailed medical and occupational therapy assessment with referral to relevant services if indicated. People in the control group receive usual care.The main outcome measures of the effect evaluation are number of falls and daily functioning. The economic evaluation will be performed from a societal perspective. A process evaluation will be carried out to evaluate the feasibility of the intervention programme. This article describes the design of a replication of a randomised controlled trial (RCT) evaluating the effectiveness and cost-effectiveness of a multidisciplinary intervention programme to prevent further falls among elderly people at risk.Publishing the design and protocol of a study before results are available is important for several reasons. A published protocol allows easier comparison between what was originally intended and hypothesised and what was actually done , and it About one-third of people over the age of 65 fall at least once a year . People Since preventing falls has been a matter of interest for years, many programmes aimed at preventing falls have been developed and evaluated. Unfortunately, many of these have turned out to be ineffective . HoweverBecause details of the status of the participants, the context of the intervention and the content and presentation appear to be critical, it has been recommended to re-evaluate effective intervention programmes in different healthcare systems . We therThe main objective of our current study is to evaluate the effects of a multidisciplinary intervention programme on recurrent falls and functional decline among elderly persons who have visit a general practitioners' cooperative (GP cooperative) and/or an accident and emergency department (A&E department) because of a fall. This objective has resulted in the following research questions.• Is a multidisciplinary intervention programme more effective than usual care in preventing new falls and functional decline among community-dwelling elderly people who visit a GP cooperative and/or A&E department at a hospital because of a fall?• Is the multidisciplinary intervention programme cost-effective compared to usual care when assessed from a societal perspective?Besides the effect evaluation and economic evaluation, a process evaluation is being carried out to assess the feasibility and applicability of the intervention programme for those receiving and implementing the intervention.Figure We have taken various measures to ensure blinding in the data collection process. Questionnaires are collected anonymously and sorted by number. Follow-up measurements by phone are contracted out to an independent call centre, whose operators are unaware whether the subjects have been allocated to the intervention or the control group.The study design and protocols were approved by the Medical Ethics Committee of the University Hospital and University of Maastricht.Various studies have been conducted to assess the effectiveness of programmes to prevent falls. Although most studies were aimed at the general population of elderly people, details of the status of the participants appear to be critical ,6. SeverRecruitment of subjects took place at the local GP cooperative and the A&E department of the University Hospital in Maastricht. The Maastricht GP cooperative is a group of GPs from practices in the town of Maastricht and the surrounding area who have founded a non-profit organisation to provide care for their own patients after hours . The MaaThe following inclusion criteria were used: age 65 years or older, community-dwelling, having visited the A&E department or GP cooperative at the University Hospital Maastricht for the consequences of a fall, and living in Maastricht or its surrounding area. People were only allowed to enter the programme after completing and returning an informed consent form. Exclusion criteria were: not able to speak or understand Dutch, not able to complete questionnaires or interviews by telephone, cognitive impairment ,13, longSample size calculations were based on the expected effects of the intervention on the main outcome measure, the percentage of people sustaining a fall during one year of follow-up. The study by Close et al. found thTo adapt the programme developed by Close et al. to the DThe final programme includes a medical and occupational therapy assessment resulting in recommendations. The medical assessment consists of an examination performed by a geriatrician, a geriatric nurse and a rehabilitation physician to identify and address risk factors for falling. The examination includes a comprehensive general examination, but in addition focussed on a more detailed assessment of visual acuity, stereoscopic vision, mobility, balance, cognition, affect, use of medication and examination of feet and footwear. Recommendations or indications for referral resulting from this examination are sent to the patient's GP. After the medical assessments, an occupational therapist visits the patients to identify possible risk factors for falling in the home environment. The therapist makes recommendations regarding adaptations to the home environment, assistive devices, home care and behavioural change. Recommendations by the occupational therapist are sent directly to the subjects themselves and to their GPs. As stated before, the intervention period is scheduled to last for a maximum of 3.5 months after the baseline measurement.An important addition to Close et al.'s protocol is the cPeople in the control group receive usual care. At present, no guidelines exist for the systematic assessment of the underlying causes of an injurious fall presented at an A&E department or GP cooperative in the Netherlands. In usual care, medical risks and other risk factors such as environmental hazards in the home and patients' risk behaviour are not systematically registered and addressed by hospital physicians, specialists or general practitioners. Moreover, no systematic attention is currently being paid to the specific consequences of an injurious fall for the daily functioning of individual patients in their unique situation. We placed no restrictions on co-interventions.The primary outcome measures of the effect evaluation are number of falls and daily functioning. Number of falls is subdivided into three separate measures: the percentage of elderly people sustaining a fall during the one-year follow-up period, recurrent falls during follow-up , and injurious falls during follow-up . Falls are recorded continuously by means of a fall calendar during the twelve-month follow-up period. Subjects are called monthly to report their falls as recorded on the fall calendar relating to the previous month.We decided to measure daily functioning by means of the Frenchai Activity Index (FAI) , in contOur secondary outcome measures are: recuperation from the fall, health complaints, perceived health measured by means of the first two items of the RAND-36 , ADL andBesides the primary and secondary outcome measures, we assess some background variables which are considered to be predictors, confounders or effect modifiers. The following personal characteristics are assessed: age, sex, marital status, living alone and socio-economic status. In addition, we assess the circumstances and causes of the falls reported at the GP cooperative and/or A&E department, the consequences of the falls , the tyThe economic evaluation in our study is being performed from a societal perspective, which implies that all costs and outcomes are taken into account if possible. The economic evaluation will be a combination of a cost-effectiveness and a cost-utility analysis. The primary outcome measure for the cost-effectiveness analysis will be the percentage of people sustaining a fall during one year of follow-up. As mentioned above, falls are recorded by means of a calendar. Within the cost-utility analysis, the effects are measured in terms of generic health-related quality of life descriptions, measured according to the standard Dutch version of the EuroQol (EQ 5-D) in self-We will assess programme costs, healthcare costs and patient and family costs. All costs are measured by means of a cost diary , in whicThe process evaluation involves assessing the extent to which the intervention programme is performed according to protocol, the nature of the recommendations made to the participants, participants' compliance with these recommendations and the opinions of participants, physicians and therapists about the intervention programme and recommendations. Data on these topics are collected using the following methods: structured registration forms for the medical and occupational parts of the intervention programme; self-administered evaluation forms filled in by the participants after the medical intervention; interviews by telephone with the participants six weeks or longer after the recommendations are sent and interviews with all participating physicians and therapists at the end of the intervention period.Data will be primarily analysed according to the intention-to-treat principle, i.e., including all participants with valid data, regardless of whether they received or did not receive the intervention. Subsequently, the results of the intention-to-treat analysis will be compared with the results of an on-treatment analysis, to assess whether protocol deviations have caused bias. Participants with documented deviations from the study protocol will be excluded from this on-treatment analysis.Comparability between the intervention and control groups will be assessed at baseline to check for differences between the two groups. Outcome at four and twelve months will be compared between the intervention and control groups by both univariate and multivariate techniques. We will use multivariate analysis to adjust for possible differences in baseline scores and background variables between the intervention and control groups. Dropouts and losses-to-follow up will be described.The economic evaluation will involve calculating cost-effectiveness and cost-utility ratios. The additional costs and additional benefits of the intervention programme compared with usual care will be examined by calculating incremental cost-effectiveness and cost-utility ratios. These incremental ratios represent the difference in mean costs between the intervention and usual care groups in the numerator and the difference in mean effects in the denominator.Since the recruitment period is only 14 months, and the follow-up period is also relatively short (12 months), it is unlikely that there will be substantial differences between costs made by and for patients who started in the first part of the recruitment period and those who started in the last part. Therefore, discounting of costs is not required. Finally, a sensitivity analysis will be done to assess the generalisability of the assumptions made in the costing process. This sensitivity analysis, which involves calculating the upper and lower limits of the confidence interval of cost and effect variables, will allow us to explore and quantify the uncertainty not related to sampling variations.The process evaluation will mainly be analysed by means of descriptive techniques.Recruitment of eligible subjects commenced in December 2002 and ended in February 2004, resulting in a total of 333 eligible subjects being included in the trial. Randomisation resulted in the allocation of 166 participants to the intervention group and 167 to the control group. Of the 333 persons recruited, 105 (32 %) are male and 228 (68 %) are female.The follow-up period is to end in May 2005. Results on the effects of the programme will be available in 2006 and will be published in the relevant journals.Although the intervention has been subject of earlier research, this study will provide new information about the effectiveness in the Dutch situation. Furthermore, the results of the economic evaluation can provide information about the cost-effectiveness of the intervention and the effects on quality of life. In case of shown effectiveness and cost-effectiveness, we aim to implement this intervention into usual healthcare.The author(s) declare that they have no competing interests.All author's read and approved the final version of the manuscript.MH is the investigator and performed most of the writing of this manuscript.JH supervises the planning of the project and wrote the study design.JD supervises the planning and methodological aspects of the project.SA supervises the economic evaluation.HC supervises the clinical part of the project.JE is the principal investigator of the study.The pre-publication history for this paper can be accessed here:

Additional PEEP to 10 cm H2O was added after injury. Animals were randomized to one of the 3 modes of ventilation and followed for 5 hr after injury.We compared gas exchange, respiratory mechanics, and measured bronchoalveolar fluid for inflammatory cytokines, cell counts and surfactant function. Lung injury was scored by light microscopy. Pigs received mechanical ventilation (F2 and respiratory system compliance was significantly greater with biologically variable ventilation compared to the other 2 groups. Mean and mean peak airway pressures were also lower. There were no differences in cell counts in bronchoalveolar fluid by flow cytometry, or interleukin-8 and -10 levels between groups. Lung injury scoring revealed no difference between groups in the regions examined. No differences in surfactant function were seen between groups by capillary surfactometry.PaOIn this porcine model of acute lung injury, various indices to measure injury or inflammation did not differ between the 3 approaches to ventilation. However, when using a low tidal volume strategy with moderate levels of PEEP, sustained improvements in arterial oxygen tension and respiratory system compliance were only seen with BVV when compared to CMV or CMV with a recruitment manoeuvre. T) in patients with acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) is alveolar collapse [T) approach the addition of higher PEEP levels offers no further improvement in outcome [IO2, it remains unclear how best to improve and sustain oxygenation, during low VT ventilation strategies for ALI/ARDS management.A negative consequence of mechanical ventilation using lower tidal volumes , a unique computer-controlled version of control mode ventilation (CMV). With BVV, gas exchange and respiratory mechanics improved in animal models, with [T protocols using an ARDSNet algorithm [T ventilation with a recruitment manoeuvre in ALI/ARDS using a low VT approach.Buchman and othels, with and withls, with , during lgorithm and in hlgorithm . After dlgorithm . A recenlgorithm . Other ilgorithm and a malgorithm . While plgorithm , not a m2O of continuous positive airway pressure for 40 sec performed hourly for 5 hrs. This approach has been shown to improve oxygenation in patients with early ARDS who do not have any chest wall impairment [Therefore, in this study in pigs with oleic acid lung injury, we compared BVV to conventional CMV or CMV with a recruitment manoeuvre (CMV-RM) of 40 cm Hpairment . A multipairment ,20 at en2 of 35–45 mm Hg. Anaesthesia was maintained with 2% isoflurane in 100% oxygen during surgical preparation. Intravenous rocuronium bromide (1 mg/kg/hr) was administered by continuous infusion for muscle relaxation. Lactated Ringer's solution was given intravenously during the surgical preparation and for the duration of the experiment.Pigs (weighing 20–30 kg) received 0.6 mg atropine, 15 mg midazolam, and 300 mg ketamine intramuscularly for sedation. Isoflurane 5% in 100% oxygen was delivered via facemask to induce anaesthesia. When sufficient depth of anaesthesia was achieved, the pigs were intubated orotracheally with a 6.0 mm cuffed endotracheal tube. Mechanical ventilation was instituted with an Ohio 7000 ventilator with minute ventilation adjusted to maintain a PaCO® ventilator capable of delivering either CMV or BVV. The ventilator was set to deliver a square wave inspiratory flow pattern with an I:E ratio of 1:2. Isoflurane was discontinued, and a propofol/ketamine infusion at 10/2.5 mg/kg/hr substituted to maintain anaesthesia.A thermodilution pulmonary artery catheter (7.5-Fr) was inserted and advanced into the right external jugular vein until a satisfactory pulmonary capillary wedge tracing was obtained. Temperature was measured from the tip of the pulmonary artery catheter. The right femoral artery was cannulated for continuous pressure transduction and arterial blood gas (ABG) analysis. A 5-Fr single lumen femoral venous catheter was advanced into the inferior vena cava for infusion of oleic acid. A surgical tracheotomy was performed and the animal was switched to an Esprit2O recruitment manoeuvre for 30 sec, animals were ventilated with a VT of 10 ml/kg at an FIO2 of 0.3, with 5 cm H2O of PEEP. Respiratory rate was adjusted to maintain PaCO2 between 35–45 mm Hg.After a 30 cm HAfter 15 min to stabilize, baseline measurements were obtained. Haemodynamic measurements included mean arterial pressure (MAP), heart rate, central venous pressure (CVP), mean pulmonary artery pressure (MPAP), and pulmonary artery occlusion pressure (PAOP). All haemodynamic data were continuously recorded on a Gould 2600 Oscillograph . Cardiac output (CO) was measured by thermodilution, in triplicate, at stated measurement periods.T intermittently; this data was recorded using an advanced CODAS data acquisition system.A pneumotachograph with the sensor immediately proximal to the tracheotomy was used to measure airway pressures and VArterial and mixed venous gases were analyzed using a Radiometer ABL 500 . Arterial and mixed venous oxygen content, oxygen saturation, and haemoglobin concentrations were measured with a Radiometer OSM3 set for porcine blood.T used was that which the animal was receiving at that time.Static respiratory system compliance (Crs) was measured in triplicate by clamping the expiratory limb of the ventilator circuit at the end of inspiration for 1 sec to obtain a plateau pressure. The V2 <60 mm Hg for two consecutive measurements (PaO2/FIO2 <200). Dopamine was started at 5 μg/kg/min and was titrated to keep the MAP >60 mmHg. When the oxygenation target was achieved, the oleic acid infusion was stopped and the PEEP was increased to 10 cm H2O. Ten minutes after the increase in PEEP, an arterial blood gas sample was obtained to determine if PaO2 had increased. PaO2 had to be >75 mm Hg but <90 mm Hg. This was considered to represent adequate lung injury, but indicate that collapsed alveoli could be recruited with the additional PEEP. If the PaO2 did not increase, the experiment was terminated. If the PaO2 increased to >90 mm Hg, additional oleic acid was infused until the PaO2 decreased to <60 mm Hg.Oleic acid was infused via the 5-Fr femoral venous catheter at 0.2 ml/kg/hr until PaOT (7 ml/kg) protocol was initiated and the respiratory rate increased to 30 bpm. After 10–15 min arterial blood gas sampling was done to assess the stability of the PaO2. If the PaO2 remained stable, the animals were then randomized into one of three groups: conventional ventilation with a VT of 7 ml/kg (CMV); conventional ventilation with VT of 7 ml/kg with a 40 sec, 40 cm H2O recruitment manoeuvre performed hourly (CMV-RM). The recruitment manoeuvre was performed at end-expiration with the PEEP level maintained at 10 cm H2O at an FIO2 of 0.3; or biologically variable ventilation (BVV) with a mean VT of 7 ml/kg.A low VFollowing stable oleic acid lung injury, haemodynamic, gas exchange and respiratory system compliance (Crs) measurements were recorded and obtained hourly thereafter. Measurements in the CMV-RM group were obtained 5 min after each recruitment manoeuvre (RM). An additional measure of gas exchange and Crs was made in the CMV-RM group 50 min after the RM, to ascertain the duration of effect of the RM.en bloc. Following removal, the lungs were suspended upside down for 10 min to collect bronchoalveolar (BAL) fluid. Samples of BAL fluid were collected in heparinized saline and then frozen immediately at -80oC. Fluid was then sent for cytokine analysis, measures of surfactant function and flow cytometry. The lungs and previously collected BAL fluid were weighed and the lungs were suspended and aerated overnight. The following day, the lungs were placed in an oven to dry, and following a stable dry measurement, wet:dry weight ratio was calculated.At the end of the experiment, a sternotomy was performed. Animals were sacrificed with a lethal dose of thiopental. The trachea was then clamped at end inspiration and the heart and lungs were removed Surfactant function was assessed on BAL fluid samples using a capillary surfactometer in the manner of Enhorning and colleagues ,22. Such1. Raw BAL fluid surfactant function analysis – centrifuging at 200 g for 5 min to rid large debris, then the supernatant spun at 10,000 g and pellet resuspended in 100 μL saline and analyzed using the surfactometer.2. Surfactant resuspended in 100 μL of saline after chloroform/methanol extraction. Chloroform/methanol extraction permits the lipid and phospholipid fraction to dissolve in the organic nonpolar solvent (chloroform) and the solvent evaporated to dryness. Volume of the final ratio for chloroform:methanol:water was 1:1:0.9. Following extraction, the pellet was resuspended and analyzed using the surfactometer.The percentage of time that the capillary tube was open for 2 min was determined for each sample, in triplicate, then averaged. Standards were saline; 0% patent for a 2 min time period and bovine surfactant; >98% patent for a 2 min time period. Each sample was measured in a blinded fashion in both conditions.The BAL fluid sample was passed through a cell strainer and 100 μL was transferred into a 5 × 75 mm tissue culture tube. Red cells were lyses by the addition of 500 μL Optilyse C and following 15 min incubation at room temperature, the cell suspension was diluted by the addition of 500 μL Isoton II and 100 μL FlowCount Fluorospheres (Beckman Coulter). The cell suspension was immediately analyzed on a Beckman Coulter EPICS Altra cell sorter configured with a high-speed quartz flow cell tip and a water-cooled argon laser emitting 150 mW at 488 nm. Forward and side light scatter signals were employed to derive 2-parameter histograms which clearly defined the fluorescent beads and three populations of cells, subsequently defined as mononuclear cells, neutrophils and eosinophils. At least 20,000 cells were analyzed for each sample. The acquisition software provided with the instrument automatically calculated the concentrations of each cell population based on the number of events in each of the 4 analysis regions.The incubation times and washes were preformed as specified in each respective kit: . Immunoassay kits for swine IL-8 and IL-10 were used. Sample incubation times were kept as constant as possible by pre-plating on a blank 96 well plate before transferring to the coated assay plate. ELISA plates were read at 450 nm by an SLT Rainbow plate reader . Standard curves and concentration calculations were performed according to kit directions.For light microscopy, four blocks of tissue from the right lung – upper lobe, middle lobe, nondependent and dependent lower lobe, fixed in buffered formalin, processed and embedded in formalin. Sections were cut and stained with haematoxylin and eosin. Under light microscopy, a lung injury scoring system modified from Rotta et al. ,20 was uParametric data were analyzed by repeated measures ANOVA using least squares means test matrices to identify differences within and between groups from group × time or group effects. Bonferroni's correction was applied where appropriate. Non-parametric comparison of the lung injury scores was by Kruskal-Wallis test. In all circumstances a p-value ≤0.05 corrected for multiple comparisons was considered significant.Twenty-five experiments were undertaken. One animal was discarded because oleic acid injury could not be established after 1.5 hrs of infusion. In the CMV-RM group, one animal died 2 hr into the experiment but data are included in analysis in this experiment up to time of the animal's demise. Complete experiments were done in twenty-three animals; .Temperature and haemodynamic data are shown in Table 1: see 2 over time . In all groups, the PaCO2 increased following oleic acid injury and was essentially stable at these elevated levels for the remainder of the experiment. The group × time for PvO2 was significant at p = 0.002 with significantly lower PvO2 over time in the CMV-RM group. Shunt fraction was lower in the BVV group (group × time interaction p = 0.003).Table 2: see 2 tended to decrease immediately following a recruitment manoeuvre, but increased over time as measured at 50 min. PaO2 significantly decreased in RM4 and 5 (-11.1 ± 5.2 mm Hg and -5.2 ± 4.4 mm Hg respectively). Following recruitment, PaO2 increased slowly early in the experiment at RM2 and RM3 (10.1 ± 5.5 mm Hg and 11.8 ± 9.1 mm Hg respectively), but failed to do so in the later time periods. For respiratory system compliance, no statistically significant differences were seen at any time period over the course of the experiment in the CMV-RM group.PaO2O with BVV; 15.1 ± 0.2 with CMV-RM and 15.1 ± 1.0 with CMV respectively). In all 3 groups, mean Paw increased following oleic acid. A more pronounced effect was seen with peak Paw with mean peak pressures over time least with BVV . There was no group × time interaction for VT in ml/kg measured over time (p = 0.617).A group × time interaction for mean Paw was seen (p = 0.002) with Paw lowest with BVV and exponential (R2 = 0.41) curve fits.The ELISA results to assess the cytokine IL-8 concentration in BAL fluid indicated no statistically significant difference for the 3 groups: 564 ± 551 pg/mL with BVV; 653 ± 639 pg/mL with CMV and 300 ± 235 pg/mL with CMV-RM. As in the measurement of neutrophil counts, there was significant variability with IL-8 levels ranging over a 385-fold concentration difference. Such a broad range of data in both neutrophil counts and IL-8 concentration suggested the possibility of power law behaviour and correlation between these variables. A very strong power law relationship was found between IL-8 pg/mL and absolute neutrophil count/mL for pooled data; y = 77609x-6x1.38; n = 13, R2 = 0.83. In this situation the linear and exponential fits were R2 = 0.61 and R2 = 0.69 respectively. In this analysis, IL-8 concentration is presumed to be the dependent variable – being released by the monocytes (counts on the x-axis).The number of mononuclear cells from cell cytometry of BAL fluid was not different between groups: for BVV, 1346 ± 743 cells/μL; for CMV, 1767 ± 905 cells/μL; and for CMV-RM, 1068 ± 579 cells/μL. These cells presumably in large part represent alveolar macrophages. A power law relationship was seen between pooled data for a correlation between IL-8 concentration in pg/mL and monocyte count/mL – 1.0 × 100.15; n = 13, R2 = 0.07.The IL-10 results from BAL fluid, as well, indicate no statistically significant difference for the 3 groups: 119 ± 173 pg/mL for BVV; 124 ± 146 pg/mL for CMV; and 149 ± 168 pg/mL for CMV-RM. Unlike IL-8 concentrations the range of variation for IL-10 concentrations was significantly less – the maximum range differing by only 16-fold. There was no power-law relationship found between IL-10 and absolute monocyte count for pooled data: y = 7.5xT but at lower peak and mean airway pressure and greater respiratory system compliance.The main finding in this study of acute lung injury in a porcine model is that BVV significantly improved oxygenation and respiratory mechanics with no difference in indices of lung injury, inflammation or surfactant function compared to the more conventional ventilation techniques – CMV or CMV with a standard recruitment manoeuvre. The improvements seen with BVV were sustained over the course of the experiment; an effect not seen with recruitment. Such improvement was obtained at similar measured mean VT model of ALI/ARDS; not in healthy lung. As well, we have confirmed the differences between BVV and CMV seen in other work from the laboratory using the same model [These findings are similar to previously published results from our laboratory showing that BVV was superior to both CMV and CMV with sigh breaths. These sigh breaths were of no advantage in a model of healthy lung recruitment – lung reinflation after a controlled collapse for one hr with contralateral one-lung ventilation . In thatme model . No impo2. Thus, lower PvO2 in the CMV-RM group should have minimized shunt but here the shunt fraction is greatest. The lowest shunt with BVV indicates that the numerator of the shunt equation is less in this group, indicating enhanced blood flow in aerated lung units.Shunt fraction was significantly lower with BVV. Lynch et al. and SandA multimodal approach to assess lung injury, lung inflammation and surfactant function indicated no discernable differences between the 3 approaches to ventilation:1. Light microscopy studies demonstrated no significant differences between groups for lung injury in any region examined – from upper to dependent lower lobes. This finding indicates no difference between groups in lung injury as assessed by histology, with the caveat that only small areas of lung in each region were examined in a condition known to be heterogeneous.2, wet:dry weight ratios, respiratory system compliance, or peak airway pressure. Arold et al. [2. No difference in surfactant function was seen between groups as assessed by capillary surfactometry. Following oleic acid lung injury, surfactant function was markedly depressed when compared to a surfactant standard – see Figure d et al. have sho3. No difference between groups is seen for cell counts in the BAL fluid. There is considerable variation in this analysis and a full data set is not present. A significant number of studies of cell count could not be undertaken because of the nature of the BAL fluid. When thick with proteinaceous debris the samples often were unable to be prepared for meaningful cytometry analysis. Of the data represented, no difference in the proportion of neutrophils, monocytes or eosinophils is apparent for any of the 3 approaches to ventilation. Allen and Bates have sho2 = 0.007 in this experiment, n = 24). Failure to reconfirm these findings may, in part relate to the nonlinear relationships highlighted above. Small changes in initial conditions may preclude similar findings at end experiment.4. IL-8 is considered to be the major neutrophil chemoattractant cytokine in lung diseases like ARDS . IL-10 m2 and respiratory system compliance. BVV has been shown to recruit in a pure model of lung collapse – re-expansion of lung following cessation of one lung ventilation [The above observations, collectively, indicate no fundamental differences between the ventilation strategies studied in regards oleic acid or superimposed ventilator associated lung injury. No one technique seems clearly advantageous. However, BVV alone improves oxygenation significantly following acute lung injury over time, an effect that was sustained, suggesting it is the best technique of the 3 studied to recruit atelectatic lung as assessed by greater PaOtilation . With BVtilation .2O to 2 cm H2O above Pflex in an attempt to prevent derecruitment, we maintained PEEP at 10 cm H2O throughout. In a porcine model of lung lavage, lung remained recruited if PEEP was at 10 cm H2O: the value chosen in this study [2 will not increase if the sustained inflation does not restore aeration to the atelectatic regions because a significant fraction of pulmonary blood flow is shunted to nonaerated regions [2O for 60 sec – was not beneficial in another animal model and that a more aggressive recruitment manoeuvre – PEEP 40 cm H2O and pressure control ventilation of 20 cm H2O, with a respiratory rate of 10, and I:E ratio 1:1 for 2 min – was successful only after the second hour. Allen et al. [2O) did not influence the time constants for recovery in elastance both before and after lung lavage. They conclude that for a deep inflation to be beneficial in their model that it may be necessary to apply the inflation several times a minute. Thus, the appropriate PEEP level, with and following recruitment, remains controversial [It could be argued that we have not chosen the optimal recruitment strategy to compare to the two other modes of mechanical ventilation. That said, we have chosen a well recognized approach to recruitment , one assis study . This leis study . Furtheris study . Failure regions . Such ma regions have shon et al. have shooversial ,39, but The human variability file for respiratory rate used to program the ventilator is shown in Figure T strategies are used for management of ALI/ARDS. A well accepted recruitment strategy has been compared to BVV in this animal study. It is possible that other approaches for recruitment could be superior to the one chosen. However, all chosen modes of recruitment would increase airway pressures over and above those seen with BVV, potentially increasing the risk of volutrauma.This study shows that BVV with a human variability file was superior to either CMV or CMV with a recruitment manoeuvre (CMV-RM) for sustained improvement in gas exchange and respiratory mechanics in a porcine model of acute lung injury. Prior work has demonstrated an advantage of BVV in a clinical setting of atelectasis . The curD.J.F. was responsible for conduct of the experiments as a fellow in the Anesthesia Laboratory. M.R.G. supervised conduct of the experiments, helped analyze data and helped write the paper. L.G.G. helped with the experiments, data retrieval and collation and table and figure production. J.A.T. did the histological assessments and analysis. B.M.M. supervised the cytokine assays and their interpretation and helped write the paper. E. K-Y. W. conducted the cytokine assays. C.H. did the surfactant assays while a fellow in the Oral Biology Laboratory. J.E.S. supervised and interpreted the results of the surfactant assays. W.A.C.M. conceived the study, analyzed and interpreted data and helped write the paper.Table 1: Temperature and HaemodynamicsClick here for fileTable 2: Respiratory Gas and Derived DataClick here for file

Embryo implantation plays a major role in embryogenesis and the outcome of pregnancy. Plasminogen activators (PAs) have been implicated in mammalian fertilization, early stages of development and embryo implantation. The invasion of trophoblast cells into the endometrium during the implantation process can be blocked by inhibitors of serine proteases, illustrating the role of these enzymes in the invasion process. As in vitro developing embryos resulted in lower implantation rate than those developed in vivo we assume that a reduced PAs activity may lead to it. There is hardly any information regarding qualitative or quantitative differences in expression of PAs in preimplantation embryos, or comparisons between in vivo and in vitro developed embryos. The purpose of this study was to assess the expression of urokinase type (uPA) and tissue type (tPA) plasminogen activators in in vivo and in vitro preimplantation development in rat embryos using immunofluorescence confocal microscopy and computerized image analysis.Zygotes, 2-cell, 4-cell, 8-cell, morula and blastocyst stages of development were flushed from the reproductive tract (control groups) of Wistar rats. Zygotes were flushed and grown in vitro to the above mentioned developmental stages and comprised the experimental groups. Immunofluorescence microscopy and computerized image analysis were used to evaluate both qualitative and quantitative expression of plasminogen activators.uPA and tPA were found to be expressed in rat embryos throughout their preimplantation development, both in vivo and in vitro. While uPA was localized mainly in the cell cytoplasm, the tPA was detected mainly on cell surface and in the perivitelline space. In blastocysts, both in vivo and in vitro, uPA and tPA were localized in the trophectoderm cells. Total uPA content per embryo was higher in the in vivo as compared with the in vitro developed embryos at all stages measured. Blastocyst uPA content was significantly low as compared with the four-cell, eight-cell, and morula stages. Total tPA content was higher in embryos developed in vivo than those developed in vitro except for the 4-cell and 8-cell stages.In vitro embryo development leads to lower PAs expression in a stage dependent manner as compared with in vivo developing controls. The enzymes studied vary probably in the ratio of their active and inactive forms as there is no correlation between their content and the activity observed in our previous study. The localization of both PAs in the blastocysts' trophectoderm supports the assumption that PAs plays a role in the implantation process in rats. The inv failure ,13. In t failure .In the implantation process, two major factors participate: the uterus undergoes changes that prepare it for the arrival and implantation of embryos, and the embryos undergo cellular reorganization that enables them to penetrate the endometrium and to form the placenta. We assume that one of the reasons for low implantation rate of embryos developed in vitro involves reduced PAs activity.In a previous study we demonstrated differences in PAs activities between in vivo and in vitro preimplantation developed embryos. In both, uPA activity increased from the zygote towards the blastocyst stage while tPA activity remained relatively unchanged. However, tPA and uPA activities were lower in in vitro developed embryos as compared with in vivo developing ones, at all developmental stages, which may lead to a reduced implantation rate of in vitro developed embryos .There is hardly any information regarding qualitative or quantitative differences in expression of PAs in preimplantation embryos, or comparisons between in vivo and in vitro developed embryos. Therefore, the purpose of this study was to investigate the PAs expression and localization during embryo development in vivo and in vitro by immunofluorescence confocal microscopy.The following study was approved by the Institutional committee for animal care and ethics at Ben-Gurion University of the Negev, Beer-Sheva, Israel.Mature female Wistar rats 2–3 months old, weighing 180–230 g were used. The animals were kept in a temperature-controlled room maintained at 22–24°C with lighting regimen of 14 hours light 10 hours dark (light on 5:00 AM – 7:00 PM). The rats were allowed free access to rat chow and tap water.Daily vaginal smears were taken at 10:00 AM, and the stage of estrous cycle was determined. Overnight caging of a proestrous female with a male of proven fertility induced pregnancy. The next day, the presence of a vaginal plug or spermatozoa in the vaginal smear was designated as day 1 of pregnancy.Zygotes, two-cell, four-cell, eight-cell embryos and morulae were flushed with rat 1-cell embryo culture medium (R1ECM) from oviZygotes in their cumulus mass were flushed and the cumulus cells were removed by gentle aspiration through a micropipette several times in R1ECM containing 80 U/mL of hyaluronidase. Clean zygotes were washed 3 times by transfer into fresh R1ECM to remove traces of hyaluronidase. Flushed embryos were collected with a mouth-controlled micropipette .Blastocysts were flushed from the uterine horns by insertion of a 23-gauge needle attached to a syringe containing R1ECM. Flushed, free-floating blastocysts were collected into a polypropylene tube inserted through the vagina and pushed gently to surround the cervical openings. Tubes were removed, and their contents were poured into Petri dishes. Blastocysts were washed and collected as described for zygotes and embryos. These embryos developed in vivo were the control groups.2 in air. This medium was shown by Miyoshi et al. [As described for the in vivo embryos, clean zygotes were grown in vitro to the same developmental stages as controls; these were the experimental groups. Each group of embryos consisted of 25–35 embryos collected from six pregnant females. This was repeated three times for each developmental stage . Groups of 25–35 zygotes were placed into 35-mm-diameter culture dishes containing 50μL of R1ECM medium under a layer of mineral oil and cultured at 37°C under 5% COi et al. to enablThe method used was basically that of Dubey et al. with varThe distribution and concentration of PAs in the embryos were visualized by fluorescent microscopy on a Zeiss laser scanning confocal microscope equipped with an X100 objective. Z-sections and XZ-sections were obtained from 3D scanning by using LSM510 software The PAs density for each embryo was computed by image analysis based on the same principles as manual counting described elsewhere . The embData are expressed as means ± SEM. Statistical analysis was performed with two-way analysis of variance, followed by the least significant differences test for multiple comparisons using computer software . P < 0.05 was defined as statistically significant difference.Immunohistochemical staining for the location of tPA and uPA in preimplantation embryos developed in vivo and in vitro are shown in figure 3, respectively, Fig. 3, respectively).Quantitative measurement of total uPA in an embryo at each stage showed significantly lower expression (p < 0.01) in in vitro developed embryos from the 4-cell stage up to the blastocyst stage compared with the in vivo developed corresponding timepoint. The highest expression of uPA was found in in vivo developed embryos from 4-cell to the morula stage (90.58, 78.78 and 79.35 Pixels per embryo × 103, respectively, Fig. Total tPA expression in a whole embryo showed highest expression in in vivo developed embryos at the 2-cell, 8-cell, morula and blastocyst stages (61.59, 52.71, 48.03 and 52.34 Pixels per embryo × 10Our study demonstrates that uPA and tPA are expressed throughout all stages of preimplantation development of rat embryos. Zhang et al. reportedThe results show localization of immunoreactive uPA in the embryonic cell cytoplasm and plasma membrane in all developmental stages both in vivo and in vitro, while tPA is detected on the cell membrane and in the perivitelline space. In the blastocyst stage, PAs are localized mainly in the trophectoderm. In our previous study we showePro-uPA is synthesized as an inactive single chain that can be stored or secreted. The secreted pro-uPA can be cleaved to produce the two-chain active molecule, uPA, by the aid of limited proteolytic activity of plasmin . The secHigh tPA expression was detected at the zygote stage which is in accordance with high tPA activity found in this stage . This isThe embryonic extracellular matrix is in a continuous turnover during the embryonic development. The 8-cell stage is characterized by structural changes taking place in the embryo during the compaction process. It is therefore very likely that such changes at the 8-cell stage could be associated with increased tPA expression and activity which is known to participate in tissue remodeling [Lower expression of uPA was observed in in vitro developed embryos as compared with in vivo ones from the 4-cell up to the blastocyst stage while tPA expression was lower only in the morula and blastocyst stages. This could be explained by reduced metabolic activity in the in vitro developed embryos as suggested by Krisher et al. . In addiAdditional studies addressing the regulation of PA/Plasmin system by adding exogenous factors may provide insights into its role in early embryo development and implantation.The purpose of the study was to determine the relative importance of tPA and uPA in preimplantation embryo development. In vitro embryo development leads to lower PAs expression in a stage dependent manner as compared with in vivo developing ones. The localization of both PAs in the blastocysts' trophectoderm supports the assumption that PAs may play a role in the implantation process in rats.EDA participated in the planning of the project, carried out the animal experimentation, immunohistochemistry and the image analysis studies. USM participated in the planning of the project, animal experimentation and participated in preparation of the manuscript. GP participated in preparation of the manuscript. IHV participated in the planning of the project, statistical analysis and in preparation of the manuscript.