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gabaa receptors (gabaars) are essential mediators of inhibitory neurotransmission in the central nervous system and are critical for maintaining the correct balance of excitation and inhibition in the brain (smith and kittler, 2010). gabaergic synapses undergo extensive synaptic plasticity that alters the strength and efficacy of synaptic inhibition (luscher., 2011a). inhibitory synapse strength can be rapidly controlled by changing the number of gabaars in the postsynaptic domain, which is achieved by receptor insertion into and removal from the plasma membrane at extrasynaptic sites and by dynamic movements of gabaars to and from the synapse via lateral diffusion in the plasma membrane (arancibia - crcamo., 2009 ; bannai., 2009 ;. however, the molecular mechanisms and regulatory signaling pathways that locally control gabaar surface levels and synaptic stability remain unclear. the stabilization of synaptic gabaars opposite gabaergic presynaptic terminals is crucial for efficient synaptic inhibition, circuit excitability, and animal behavior (blundell., 2009 ; crestani. gabaar clustering is mediated by a complex inhibitory postsynaptic density, the major constituent of which is the hexameric scaffold, gephyrin (fritschy., 2008). however, in the absence of gephyrin, subsets of inhibitory synapses remain (essrich., 1998 ; kneussel., 1999), suggesting the existence of other inhibitory synaptic scaffold molecules. the inhibitory postsynaptic specialization also contains key adhesion molecules such as neuroligin 2 and slitrk3 (takahashi., 2012 ; varoqueaux., 2004), in addition to cytoskeletal - associated proteins, which together contribute to controlling the formation and stabilization of gabaergic synapses. interestingly, several filamentous actin (f - actin) regulatory proteins have been associated with the inhibitory postsynaptic density and gephyrin (luscher., 2011a), suggesting a potential role for the actin cytoskeleton at inhibitory synapses. however, little is known regarding the regulatory signaling scaffolds that can act locally to coordinate cytoskeletal dynamics to tune gabaar synaptic stability and synaptic inhibition. the rho family of small gtpases rho, rac, and cdc42and their regulators play essential roles in modulating actin dynamics and are increasingly implicated in synaptic pathology and neurological dysfunction. the activation state of small gtpases is determined by guanine nucleotide exchange factors (gefs) and gtpase activating proteins (gaps), which together control gtp - gdp exchange and thereby promote gtpase activation and inactivation, respectively (nobes and hall, 1999). local regulation of gtpase signaling can be further controlled by subcellular compartmentalization of gefs and gaps determined by protein scaffolds (kiraly., 2010). currently the key gtpases, regulatory gefs and signaling scaffolds acting to regulate gabaar trafficking and inhibitory transmission are poorly understood. in this study, we have identified a git1/pix / rac1/pak signaling complex that is important for maintaining gabaar surface clusters and synaptic inhibition in neurons. using a combination of imaging, biochemical, and electrophysiological approaches, we show that the signaling scaffold protein git1 (g protein - coupled receptor kinase - interacting protein 1), which interacts with the rac1 gef pix, forms complexes with gabaars and is essential for normal gabaar clustering. furthermore, we demonstrate that downstream rac1 activity is also crucial to maintain inhibitory synapse stability and works in concert with the key rac1 effector and actin - regulator, pak. we find that rac1 activity stabilizes gabaars at inhibitory synapses while disrupting git1, rac1 or pak all lead to impaired inhibitory transmission. thus git1 and pix, in complex with gabaars, play a key role in locally coordinating rac1 and downstream effector activity to regulate gabaar surface stability and inhibitory synapse strength. git1 is a signaling scaffold that can recruit pix, a gef for the small gtpase rac1, to locally control the activation of rac1 (zhang., 2005). git1 has been localized to synaptic sites in neurons but its potential association with gabaars and role in regulating signaling in the inhibitory postsynaptic domain remains unstudied. we hypothesized that git1 and pix might control rac1 signaling at inhibitory synapses and are important for gabaar clustering and inhibitory synaptic transmission. we initially determined if git1 and pix were localized at inhibitory synapses using immunocytochemistry and confocal laser scanning microscopy (clsm) of hippocampal neurons. neurons labeled with antibodies to git1, vgat (vesicular gaba transporter) to label inhibitory presynaptic terminals, and the 2 gabaar subunit to label synaptic gabaar clusters showed git1 was distributed along dendrites and exhibited colocalization with synaptic gabaar clusters (figure 1a ; figure s1a). approximately 55% of git1 localized at inhibitory synapses, whereas 78% of synaptic gabaar clusters colocalized with git1. in addition, git1 is known to be at excitatory synapses (zhang., 2003), which we confirm by showing that the excitatory synapse marker, homer, demonstrates 60% colocalization with git1 puncta (figure 1b). git1 and pix are known to form supramolecular signaling platforms and are consistently found in a tight signaling complex in many cell types (premont. we therefore imaged neurons labeled with git1, pix, and 2 gabaar antibodies, showing that 79% of inhibitory synapses colocalized with pix and thereby verifying the localization of both git1 and pix at inhibitory synapses (figure 1c). interestingly, git1 localizes with synaptic gabaars (2, 3, 2 subunits, figures s1b and s1c) but showed little overlap with extrasynaptic gabaars (subunits, figures s1d and s1e). we also demonstrated that gabaar 3 and 2 subunit antibodies readily coimmunoprecipitate git1 or pix from rat brain lysate as analyzed by western blotting (figures 1d1f), confirming that git1 and pix can form native complexes with synaptic gabaars in vivo. git1 did not interact in a complex with extrasynaptic subunits (figure s1f). we also found exogenous flag - git1 to interact with the intracellular domain of the gabaar-3 subunit, by cos7 cell pull - down assays (figure s1 g), supporting git1 s interaction with gabaars. coimmunoprecipitation of git1 with the inhibitory postsynaptic scaffold gephyrin confirmed this postsynaptic localization (figure 1 g), although our data suggest that this interaction is indirect, as demonstrated by lack of coimmunoprecipitations in transfected cos7 cells (figure s1h). as an alternative approach for further validating gabaar and pix complexes in neuronal dendrites, we performed proximity ligation assays (plas), which provide valuable information about native protein interactions in situ (ko., 2012). using plas, we demonstrate that pix complexes with gabaars in neuronal dendrites (figures 1h and 1i). furthermore, we show that git1 and gephyrin also interact in situ via plas (figures s1j and s1k), providing additional evidence to support an inhibitory postsynaptic and close association for these proteins in dendrites. to investigate the role of git1 at inhibitory synapses, we utilized rnai to knock down its protein expression in neurons. rnai caused a significant reduction of git1 expression levels in addition to causing a small reduction in dendrite length as previously shown (figures s2a s2d ; menon., 2010). to determine the consequences of git1 knockdown on inhibitory synapse and surface gabaar cluster area, we performed immunocytochemistry and clsm of neurons expressing git1 or control rnai constructs, using an extracellular 2 subunit antibody to label surface gabaars and antibodies to vgat to identify inhibitory synapses. git1 knockdown neurons exhibited a significant decrease in surface gabaar and vgat cluster area compared to control (figures 2a2c), suggesting a possible role for git1 in maintaining the integrity of inhibitory pre- and postsynaptic domains in neuronal dendrites. importantly, this effect could be rescued by coexpression of rnai - resistant human git1 (hgit1 ; figures s2e and s2f). git1 knockdown also caused a large decrease in gephyrin cluster area (figures 2d and 2e), suggesting that git1 is important for maintaining both gabaar clusters and the gephyrin scaffold in neurons. this was further confirmed by surface biotinylation assays, which revealed that surface gabaar levels were reduced in git1 knockdown neurons compared with control (figures 2f and 2 g). considering its role in other cell types, we hypothesized that git1 may be important for localizing f - actin regulatory pathways to inhibitory synapses. therefore, we sought to determine whether surface gabaars were sensitive to short - term disruption of the actin cytoskeleton by treating neurons with latrunculin - a, an inhibitor of actin polymerization (renner., 2009). we found that a 30 min application of 3 m latrunculin - a to neurons caused a significant decrease in surface gabaars (figures 2h and 2i), with no effect on extrasynaptic gabaar populations (figures s2 g and s2h), suggesting that actin polymerization does indeed play an important role in the maintenance of inhibitory synapses. we then asked whether the inhibitory synapse effects we observed upon knockdown of git1 were due to altered f - actin regulation. we therefore treated control or git1 rnai - expressing neurons with the f - actin - stabilizing drug, jasplakinolide (hering., 2003), prior to surface biotinylation and western blot analysis (figure 2j). as predicted, git1 rnai caused a significant loss of surface gabaars compared to control, which was restored by treatment with jasplakinolide (figures 2j and 2k). this suggests that the decrease in surface gabaars observed in git1-deficient neurons is caused by impaired f - actin regulation, and points toward a mechanism involving actin - regulatory proteins. git1 s primary binding partner pix is one such actin - regulatory protein and is a strong candidate to collaborate with git1 in mediating actin regulation at inhibitory synapses. to test this hypothesis, we utilized rnai to pix, which caused a significant reduction of pix expression levels (figures s3a and s3b). pix knockdown in neurons caused a similar effect to that of git1 rnai, reducing both surface gabaars and vgat cluster area (figures 3a3c). surface biotinylation assays revealed the same phenotype, with pix knockdown neurons exhibiting reduced surface gabaar levels compared with control cells (figures 3d and 3e). we found git1 or pix knockdown had no effect on ampa receptor clustering or extrasynaptic - containing gabaars (figures s3c s3f), suggesting that this protein complex is important for synaptic gabaar clustering only. we then sought to determine whether git1 and pix are important in controlling actin regulation at inhibitory synapses. we treated control neurons with phalloidin to label f - actin and found that 80% of inhibitory synapses were positive for f - actin. git1 and pix knockdown caused a significant decrease in the percentage of inhibitory synapses containing f - actin (figures 3f and 3 g), suggesting that git1 and pix have an important role in controlling f - actin at inhibitory synapses. the effect of pix knockdown on surface gabaar clusters was rescued by rnai resistant human pix (hpix) showing the specificity of the rnai. in contrast, versions of hpix that no longer have gef activity for rac1 (hpix - dn), or that contain a mutation of the pix sh3 domain (which is critical for coupling to a downstream effector, pak [hoelz., 2006 ]), were unable to rescue pix knockdown induced gabaar declustering (figures 3h and 3i). this suggests that the ability of pix to activate rac1 and interact with pak is essential for its role in maintaining inhibitory synapses and supports the idea that there exists a key actin regulatory mechanism controlling inhibitory synapse maintenance. git1 can anchor pix, which directly interacts with the small gtpase rac1 and mediates its activation (ten klooster., 2006). because we could not rescue the effects of pix knockdown with a version of pix with impaired gef activity toward rac1, we hypothesized that rac1 might be responsible for the changes in gabaar clustering in git1 or pix knockdown neurons. we verified that rac1 was localized to synaptic gabaar clusters in neurons using immunocytochemistry with antibodies to rac1, the 2 gabaar subunit and vgat, followed by clsm (figure 4a). we found that 45% of rac1 colocalized with synaptic gabaar clusters along the dendritic shaft (figure 4b), suggesting that rac1-positive signaling complexes may influence synaptic gabaar trafficking or function. to test whether rac1 activity has a role in maintaining synaptic gabaar clustering, we utilized a dominant - negative rac1 mutant (nobes and hall, 1999) (rac1-dn) to block rac1 activation in neurons. immunostaining and clsm of hippocampal neurons transfected with gfp or rac1-dn revealed a decrease in surface gabaar cluster area and vgat area in neurons expressing rac1-dn compared with control neurons (figures 4c4e). in addition, surface biotinylation assays revealed that blocking rac1 activation with the rac1-dn caused a decrease in gabaar surface levels (figures 4f and 4 g). as an alternative approach, we determined if acute short - term inhibition of rac1 would cause similar effects on gabaar surface levels. to achieve this, we incubated neurons for 1 hr with the rac1 inhibitor eht 1864 (eht ; shutes., 2007), followed by immunocytochemistry and clsm. similar to the results with rac1-dn, analysis of these neurons showed that acute inhibition of rac1 activity reduced the area of gabaar and gephyrin clusters (figures 4h4j). moreover, surface biotinylation assays with neurons treated with eht caused a decrease in surface gabaars (figures 4k and 4l), indicating that rac1 contributes to gabaergic synapse stability in neurons. we then wanted to explore the mechanism downstream of active rac1 leading to the stabilization of gabaar clusters. pak is a major effector of rac1 that modulates f - actin to stabilize essential cellular structures (kreis and barnier, 2009). we hypothesized that rac1 might mediate its action at inhibitory synapses by activating pak, which is supported by our observation that an sh3-domain mutant of pix (which can no longer couple to pak) is unable to rescue the effects of pix rnai on gabaar clusters (figures 3h and 3i). active pak is autophosphorylated ; therefore, we tested whether we could detect phospho - pak at inhibitory synaptic sites by performing immunocytochemistry and clsm of neurons labeled with antibodies to gabaar 2 subunit, the inhibitory presynaptic marker gad6 and phospho - pak (figure 5a). active pak was found clustered along dendrites and colocalized with both synaptic gabaar clusters and gad6, confirming its presence at inhibitory synapses. to determine whether pak activity is important for maintaining gabaar and gephyrin cluster stability, we incubated hippocampal neurons with ipa (deacon., 2008), a specific pak inhibitor and assessed gabaar 2, gephyrin and vgat cluster area. treatment with ipa caused a substantial decrease in gabaar and gephyrin cluster area, with little effect on vgat cluster area (figures 5b5e). surface biotinylation assays showed a decrease in surface gabaar expression in neurons treated with ipa compared with vehicle - treated neurons (figures 5f and 5 g), showing that pak activity is required for maintaining surface gabaars in neurons. to further verify a role for pak in controlling gabaar surface stability, we utilized the autoinhibitory domain (aid) of pak fused to gfp, which has widely been used to inhibit pak activity in culture by blocking its autophosphorylation (kreis and barnier, 2009). surface biotinylation of neurons transfected with gfp or gfp - pak - aid revealed that gfp - pak - aid expression caused reduced surface gabaar expression compared with control (figures 5h and 5i). to confirm that pak acts downstream of git1 and pix at inhibitory synapses, we performed rescue experiments with neurons cotransfected with git1 or pix rnai and a constitutively active (ca) mutant of pak. cotransfection with pak - ca effectively prevented the depletion of gabaar clusters observed with git1 or pix rnai alone (figures 5j and 5k), suggesting that pak acts downstream of git1 and pix to control inhibitory synapse maintenance in neurons. together, these data suggest that a git1/pix / rac1/pak signaling pathway plays an important role in stabilizing gabaar and gephyrin clusters and the maintenance of inhibitory synapses. our results suggest that components of a signaling pathway involving git1, pix, rac1, and pak are critical for stabilizing surface and synaptic gabaars and maintaining gabaergic synapse integrity in neurons. whole - cell patch - clamp recordings were performed to measure inhibitory synaptic transmission in neurons expressing the git1 rnai, pix rnai or gfp - pak aid constructs. analysis of spontaneous ipsc (sipsc) traces from these cells revealed that git1 or pix knockdown, or inhibition of pak all caused a considerable decrease in sipsc amplitude compared to control neurons (figures s4a s4e). these reduced amplitudes can be seen in representative sipsc traces (figure s4a) and the leftward shift to smaller amplitudes in cumulative probability plots (figures s4b and s4d). analysis of these data showed there was no significant change in the sipsc frequency (figures s4c and s4e). to further explore the impact of inhibiting the git1 signaling pathway, we measured the impact on miniature ipscs (mipscs). we recorded mipscs from neurons transfected with control or git1 rnai (figures 6a6e). analysis of traces from these neurons showed that knockdown of git1 caused a significant decrease in both mipsc amplitude and frequency, which could be rescued with rnai resistant hgit1, again showing the specificity of the rnai knockdown. we also recorded mipscs from neurons transfected with pix rnai and pak - aid, analysis of which demonstrated a similar reduction in mipsc amplitude and frequency as that of git1 rnai - expressing neurons (figures 6a, and 6c6e). the effects of the pix rnai were successfully rescued by coexpression with hpix, and also by pak - ca, suggesting that pak indeed mediates the effects of pix on gabaergic synaptic transmission. importantly, there was no significant difference between the decay time constants of the events recorded from neurons expressing git1 rnai, pix rnai, or pak - aid compared with control neurons, indicating that the receptor properties are unaltered (figure s4f). in addition, pix rnai expression had no significant effect on miniature excitatory postsynaptic currents (figures s4g s4i), suggesting that inhibition of this pathway under these conditions has specific effects on inhibitory synaptic transmission. to determine the impact of inhibiting this pathway on gabaergic transmission in an intact network slices were incubated with either ipa or eht to inhibit pak or rac1, respectively (figure 6f). analysis of the strength of inhibition with input / output curves of evoked ipscs (eipscs) generated by a series of stimulus intensities showed that inhibition of the rac1-pak pathway in brain slices caused a substantial depression of the input / output eipsc curves (40%50% reduction for ipa and 30%50% reduction for eht ; figures 6f and 6 g). this indicates that rac1 and pak are indeed critical to maintain gabaergic synaptic transmission in the brain. together, these electrophysiological experiments reveal that inhibiting the git1/pix / rac1/pak pathway in neurons not only reduces surface gabaar cluster and gephyrin cluster area, but also leads to reduced inhibitory synaptic transmission. clustering of gabaars at inhibitory synapses is imperative for correct synaptic inhibition in the brain and is tightly controlled by components of the inhibitory postsynaptic density. reduced gabaar synaptic clustering equates to reduced inhibition in neuronal circuits and subsequent disruption of excitatory / inhibitory (e / i) balance, producing defects in information processing at the network level. our results describe a signaling complex localized to the inhibitory postsynaptic domain that is crucial for correct inhibitory neurotransmission and the maintenance of gabaergic synaptic transmission. we show surface gabaar clusters are maintained by a git1/pix / rac1/pak signaling complex that modulates f - actin, thereby stabilizing the inhibitory postsynaptic density and synaptic gabaars (figure s4j). we demonstrate that git1 interacts with synaptic gabaar subunits and is localized at inhibitory postsynaptic sites, suggesting that it is intimately involved with the inhibitory postsynapse and its function. indeed, the interaction between git1 and gabaars places it in the exact location to scaffold pix at the inhibitory synapse where it could locally activate rac1 signaling (zhang., 2003). consistent with this, we also show that pix is located at inhibitory synapses and interacts in a complex with gabaars. gefs such as pix are essential signaling coordinators that localize and regulate small gtpase signaling at specific sites within the cell (kiraly., 2010). therefore, our data showing the presence of pix and rac1 at the inhibitory synaptic site, combined with the effects of pix knockdown and rac1 inhibition on gabaar clustering and inhibitory synaptic transmission, strongly suggest that a rac1 signaling pathway is important for maintaining synaptic inhibition. our results also point to a scaffolding role for git1 at the inhibitory synapse, potentially as an additional scaffolding protein to increase the stability of gephyrin and gabaar surface clusters. git1 and pix are also shown to be important for excitatory synapse function, acting via a rac1/pak / actin pathway (zhang., 2005), in an activity - dependent manner (saneyoshi., 2008). our findings are supportive of similar scaffolding mechanisms at inhibitory synapses, and suggest that the git1/pix signaling pathway may represent a key coordinator of actin pathways at synapses. indeed, this is in agreement with the role of this git1/pix signaling module at regions of cell - cell contact in multiple cell types (hoefen and berk, 2006). further study will now be required to define how the git1/pix complex may coordinate potential crosstalk between excitatory and inhibitory synapses. the number of gabaars at the neuronal surface and synaptic sites directly correlates with the strength of inhibitory synaptic transmission ; therefore, the modulation of gabaar synaptic accumulation is a key mechanism for plasticity of inhibitory synapses. here, we show that git1 or pix knockdown causes reduced gabaar clustering and a decrease in the number of gabaars at the neuronal surface, and this effect is mimicked by inhibition of rac1 or pak in neurons. indeed, disruption of gabaar clustering by pix knockdown can not be rescued by pix mutants lacking gef activity for rac1 or the ability to couple to pak, implicating pak as a downstream effector. importantly, the effects of rnai mediated knockdown of either git1 or pix on gabaar clustering is rescued by active pak. these biochemical and imaging data are supported by electrophysiological data, which show that knockdown of git1 or pix, or inhibition of rac1 or pak, causes reduced gabaergic currents in neurons, suggesting the reduction in gabaar clusters does indeed correlate with reduced inhibition. in addition to these postsynaptic effects, we also observe reductions in vgat clustering and mipsc frequency, suggestive of additional presynaptic effects of git1/pix knockdown. in our imaging and electrophysiological experiments, we analyze git1/pix knockdown in the postsynaptic neuron, suggesting the presynaptic effects we observe are due to destabilization of the presynaptic gabaergic synaptic bouton concurrent with the loss of postsynaptic receptors, scaffolds, and adhesion molecules. previously, disruption of gephyrin has been shown to reduce gabaar clusters postsynaptically, accompanied by a loss of presynaptic gabaergic innervation (marchionni. similarly, loss of the 2 subunit (as we demonstrate here occurs upon disruption of git1/pix) causes loss of both postsynaptic clustering and presynaptic innervation (li., it is becoming clear that crosstalk between the pre- and postsynaptic sites of inhibitory synapses is essential for their plasticity, demonstrated by the observation that inhibitory pre- and postsynaptic structures are highly mobile and can move as a single entity (dobie and craig, 2011). because our rnai experiments are targeting the postsynaptic cell, our results are consistent with alteration of the git1/pix complex disrupting inhibitory postsynaptic domains, which also causes subsequent disruption of presynaptic innervation. the git1/pix / rac1/pak pathway we have presented here documents a signaling pathway that links gabaar stability to the actin cytoskeleton via a gtpase signaling cascade. by treating neurons with latrunculin - a for 30 min, we show that an intact actin cytoskeleton is important for gabaar stability. this is in contrast with earlier findings showing no effect of latrunculin - a on gabaar clustering (allison., 2000) ; however, this study differed in both age of neurons and length of treatment (24 hr). we conclude that, although actin is not structurally required at inhibitory synapses as in dendritic spine heads, it is emerging that actin is required for the integrity of the inhibitory postsynaptic site (charrier., 2006) and for postsynaptic scaffold mobility in general (kerr and blanpied, 2012). gephyrin also interacts with collybistin (kins., 2000), a gef for the small gtpase cdc42. the role of collybistin in region - specific inhibitory synapse formation has been investigated (papadopoulos and soykan, 2011), although it is still unclear whether cdc42 activity is required for gephyrin clustering with several studies suggesting that collybistin functions independently of its gef activity (reddy - alla., 2010). thus, other rho gtpase signaling and scaffolding mechanisms are likely to be present at inhibitory synapses. in agreement with this, we show that pix and rac1 activity is required for inhibitory synapse function, by maintaining gabaar surface levels at synapses, gephyrin clustering, and gabaergic currents. git1 knockout (ko) mice have been investigated in the context of neuronal function in two independent reports. reduced dendritic spine density and dendrite length in the hippocampal ca1 region have been reported in one git1 ko model, in addition to impaired performance in learning tasks (menon., 2010). in a second study, investigators also observed memory and learning impairments, with increased hyperactivity and reduced inhibitory presynaptic input in ca1 (won., 2011). here, we demonstrate the effects of acute git1 knockdown and the short - term effects of inhibiting the git/pix / rac1/pak pathway on gabaar clustering and synaptic inhibition and find reduced clustering of essential inhibitory synapse components including gephyrin. we therefore attribute the differences in our findings and those of won., to the use of global ko strategies, which cause git1 knockdown in all cell types throughout development, which is in contrast to the short - term rnai targeting and pharmacological treatment that we employ here. reduced inhibition due to depletion of gabaars from the neuronal cell surface can alter the e / i balance of neuronal circuits, causing disrupted information processing, which may lead to altered animal behavior (blundell., 2009 ; crestani., 1999 ; papadopoulos., 2007 ; tretter., 2009 ; yizhar., deficits in gabaergic neurotransmission leading to an altered e / i balance have also been implicated in multiple neuropsychiatric disorders including depression (luscher., 2011b), bipolar disorder (craddock., 2010), schizophrenia (charych., 2009), and attention deficit hyperactivity disorder (adhd ; won. therefore, identifying the molecular mechanisms that are essential for maintaining inhibitory synaptic transmission in the brain is also critical to understanding the development of these neuropsychiatric disorders, where it may become necessary to readdress pathological alterations in e / i balance. our findings showing that a rac1 signaling pathway is important for regulating inhibitory synaptic transmission may shed light on the molecular mechanisms underlying mental illness. indeed, many of the proteins involved in the gtpase signaling pathway we describe here have been directly linked to mental disorders (allen., 2004 ; govek., 2004 ; won.. altered pak signaling due to mutations in the pak3 gene has been linked to patients with mental retardation, and pak signaling is additionally implicated in models of fragile - x syndrome and schizophrenia (chen., 2007 ; hayashi - takagi., 2014), making it an important molecule in the synaptic pathology of psychiatric disorders. in addition, git1 was recently shown to harbor a single nucleotide polymorphism causing reduced git1 expression that is strongly linked to adhd in humans (won., 2011). our results suggest how a postsynaptic git1 signaling complex plays a key role in controlling synaptic inhibition, by stabilizing gabaars at the inhibitory synaptic site, and may be an important locus for altered animal behavior and psychiatric and cognitive deficits. we have characterized a git1/pix / rac1/pak signaling pathway that controls gabaar clustering from a molecular and physiological viewpoint. we found that git1, pix, rac1, and pak are all essential to maintain surface gabaar clusters and inhibitory currents in neurons, therefore making this signaling pathway an important regulator of inhibitory signaling in the brain. details regarding antibodies, immunocytochemistry and analysis, coimmunoprecipitations, biotinylations, and cdna cloning are included in the supplemental experimental procedures. all experimental procedures were carried out in accordance with institutional animal welfare guidelines and the uk animals (scientific procedures) act 1986. cultures of cortical and hippocampal neurons were prepared from e18 sprague - dawley rat embryos as described previously (pathania., 2014 ; twelvetrees., 2010) ; see also the supplemental experimental procedures. standard voltage - clamp techniques were used for whole - cell recordings of spontaneous ipsc in cultured neurons (twelvetrees., 2010). electrodes were filled with the following internal solution (in mm) : 100 cscl, 30 n - methyl - d - glucamine, 10 hepes, 1 mgcl2, 5 egta, 2 qx314, 12 phosphocreatine, 5 mgatp, 0.5 na2gtp, 0.2 leupeptin (ph 7.27.3), and 270280 mosm. the external solution consisted of the following (in mm) : 130 nacl, 26 nahco3, 3 kcl, 5 mgcl2, 1.25 nah2po4, 1 cacl2, and 10 glucose (ph 7.4), 300 mosm. cnqx (20 m) and apv (50 m) were added to block ampa and nmda receptors. data were analyzed with kaleidagraph (albeck software) and mini analysis program (synaptosoft). to measure gabaergic transmission in cortical slices, we used standard whole - cell recording techniques (yuen., 2011). slices were incubated at room temperature (20c 22c) in artificial csf (acsf) bubbled with 95% o2, 5% co2, and then slices were treated with various agents for 1 hr before recordings. the in situ proximity ligation assay (pla) was used according to the manufacturer s instructions (olink bioscience). neurons were fixed in 4% pfa/30% sucrose, blocked (10% horse serum, 0.5% bsa, and 0.2% triton x-100, 10 min at room temperature), and incubated with primary antibodies. for control pla, a single antibody was applied. cells were washed in 1 pbs and then incubated with secondary antibodies conjugated to oligonucleotides. ligation and amplification reactions were conducted at 37c, before mounting and visualization with clsm. e.c.d. performed molecular biology and performed and analyzed some of the biochemistry and confocal imaging experiments.	summaryeffective inhibitory synaptic transmission requires efficient stabilization of gabaa receptors (gabaars) at synapses, which is essential for maintaining the correct excitatory - inhibitory balance in the brain. however, the signaling mechanisms that locally regulate synaptic gabaar membrane dynamics remain poorly understood. using a combination of molecular, imaging, and electrophysiological approaches, we delineate a git1/pix / rac1/pak signaling pathway that modulates f - actin and is important for maintaining surface gabaar levels, inhibitory synapse integrity, and synapse strength. we show that git1 and pix are required for synaptic gabaar surface stability through the activity of the gtpase rac1 and downstream effector pak. manipulating this pathway using rnai, dominant - negative and pharmacological approaches leads to a disruption of gabaar clustering and decrease in the strength of synaptic inhibition. thus, the git1/pix / rac1/pak pathway plays a crucial role in regulating gabaar synaptic stability and hence inhibitory synaptic transmission with important implications for inhibitory plasticity and information processing in the brain.
since their discovery in 1953, human adenovirus strains (hadvs) have been classified into seven species (a g). hadvs are widely distributed5and cause a broad spectrum of clinical diseases, including febrile respiratory illness (fri), pneumonia, conjunctivitis, gastroenteritis, cardiomyopathy, and urinary tract infection. severe disease and death caused by hadv infections are rare among otherwise healthy individuals.6,7 certain hadv species, including b, c, and e, are well - known causes of respiratory disease outbreaks.8,9 hadvs belonging to species c (types 1, 2, 5, and 6) are generally endemic in children and adolescents.911 species e, comprising only hadv type 4, is the most common pathogen in outbreaks of fri among military recruits.12,13 the large hadv species b includes two subspecies : b1 (types 3, 7, 16, 21, and 50) and b2 (types 11, 14, 34, and 35). hadv-3, 7, and 21 generally cause outbreaks of fri and pneumonia among adolescents and adults7, whereas hadv-34 and 35 are often associated with sporadic kidney and urinary tract infections.1 hadv-11, 14, and 16 have been infrequently associated with outbreaks of fri14,15 until the strain hadv-14p1 emerged in the united states in 2006.15 in china, hadvs have not been well studied. however, outbreaks of hadv infection (i.e., hadv-3, 7, and 11) have been previously described, particularly among primary and middle school students.1619 in april 2011, the institute for viral disease control and prevention in the chinese center for disease and prevention was notified of a cluster of hadv respiratory infections in tongwei county in the gansu province. this is the first report of an fri outbreak caused by hadv-14p1 at a primary and middle school in china, as well as the first report of hadv-14p1 infections in school - aged children in the world. staff members from the gansu center for disease control and prevention (cdc) collected a throat swab specimen from each of the 17 patients. the specimens were collected in 2-ml viral transport media, transported at 2c8c, and preserved at 80c. the first round of studies was conducted at the gansu cdc, and the second round, at the institute for viral disease control and prevention at the chinese center for disease and prevention, beijing. a commercially available seeplex rv 15 ace detection kit (seegene, inc, seoul, korea), a multiplex one - step reverse transcription polymerase chain reaction (rt - pcr) assay was used to screen for 15 different viruses as the cause of the respiratory illness. the kit included assays for respiratory syncytial virus a and b, influenza virus a and b, parainfluenza virus 14, human adenovirus, human rhinovirus, human enterovirus, human coronavirus nl63 - 229e and oc43-hku1, human metapneumovirus, and human bocavirus. specimens that were positive for adenovirus were cultured and further analyzed to type the viruses. adenovirus - positive throat swab specimens were inoculated onto a549 cells and monitored for cytopathic effect (cpe). at the end of the observation period, if no cpe was observed, the culture was further incubated for another 7 days. cultures exhibiting adenovirus - like cpe were passaged again to confirm the presence of the virus. viral dna was extracted from hadv isolates by using a qiaamp dna mini kit (qiagen, valencia, ca, usa). hadv type was identified by amplifying and sequencing the complete hadv hexon, fiber, and e1a genes. gene amplification was performed using a geneamp 9700 thermal cycler (applied biosystems, carlsbad, ca, usa). briefly, the pcr chemistry included a 25-l reaction mixture containing 2 pcr mix (promega, fitchburg, wi, usa), 1 l of each primer (listed in table 1), and 2 l of template dna. pcr conditions included an initial denaturation at 94c for 10 minutes, followed by 35 cycles of denaturation at 94c for 30 seconds, annealing at 55c for 40 seconds, extension at 72c for 70 seconds, and a final extension at 72c for 10 minutes. the amplification products were prepared through capillary gel electrophoresis using the qiaxcel dna high resolution kit (qiagen, venlo, the netherlands). the pcr products were purified (qiagen, valencia, ca, usa) and sequenced using the dye terminator method (bigdye terminator, version 3.1, cycle sequencing kit ; applied biosystems) with an abi prism 3100 genetic analyzer (applied biosystems). primers used for amplification and sequencing of the entire hexon, fiber, and e1a genes primers are designed according to the prototype strain of hadv-14 de wit (genbank accession : ay803294). the sequence data were analyzed with sequencher software (version 4.0.5 ; genecodes, ann arbor, mi, usa). the nucleotide sequence homology was inferred from the identity scores obtained using the blastn program (national center for biotechnology information, bethesda, md, usa). sequence alignments were created with bioedit sequence alignment editor software (version 5.0.9 ; tom hall, north carolina state university, raleigh, nc, usa)21, and the phylogenetic dendrogram was constructed using the neighbor - joining method with the mega software (sudhir kumar, arizona state university, phoenix, az, usa). staff members from the gansu center for disease control and prevention (cdc) collected a throat swab specimen from each of the 17 patients. the specimens were collected in 2-ml viral transport media, transported at 2c8c, and preserved at 80c. the first round of studies was conducted at the gansu cdc, and the second round, at the institute for viral disease control and prevention at the chinese center for disease and prevention, beijing. a commercially available seeplex rv 15 ace detection kit (seegene, inc, seoul, korea), a multiplex one - step reverse transcription polymerase chain reaction (rt - pcr) assay was used to screen for 15 different viruses as the cause of the respiratory illness. the kit included assays for respiratory syncytial virus a and b, influenza virus a and b, parainfluenza virus 14, human adenovirus, human rhinovirus, human enterovirus, human coronavirus nl63 - 229e and oc43-hku1, human metapneumovirus, and human bocavirus. specimens that were positive for adenovirus were cultured and further analyzed to type the viruses. adenovirus - positive throat swab specimens were inoculated onto a549 cells and monitored for cytopathic effect (cpe). at the end of the observation period, if no cpe was observed, the culture was further incubated for another 7 days. cultures exhibiting adenovirus - like cpe were passaged again to confirm the presence of the virus. viral dna was extracted from hadv isolates by using a qiaamp dna mini kit (qiagen, valencia, ca, usa). hadv type was identified by amplifying and sequencing the complete hadv hexon, fiber, and e1a genes. gene amplification was performed using a geneamp 9700 thermal cycler (applied biosystems, carlsbad, ca, usa). briefly, the pcr chemistry included a 25-l reaction mixture containing 2 pcr mix (promega, fitchburg, wi, usa), 1 l of each primer (listed in table 1), and 2 l of template dna. pcr conditions included an initial denaturation at 94c for 10 minutes, followed by 35 cycles of denaturation at 94c for 30 seconds, annealing at 55c for 40 seconds, extension at 72c for 70 seconds, and a final extension at 72c for 10 minutes. the amplification products were prepared through capillary gel electrophoresis using the qiaxcel dna high resolution kit (qiagen, venlo, the netherlands). the pcr products were purified (qiagen, valencia, ca, usa) and sequenced using the dye terminator method (bigdye terminator, version 3.1, cycle sequencing kit ; applied biosystems) with an abi prism 3100 genetic analyzer (applied biosystems). primers used for amplification and sequencing of the entire hexon, fiber, and e1a genes primers are designed according to the prototype strain of hadv-14 de wit (genbank accession : ay803294). the sequence data were analyzed with sequencher software (version 4.0.5 ; genecodes, ann arbor, mi, usa). the nucleotide sequence homology was inferred from the identity scores obtained using the blastn program (national center for biotechnology information, bethesda, md, usa). sequence alignments were created with bioedit sequence alignment editor software (version 5.0.9 ; tom hall, north carolina state university, raleigh, nc, usa)21, and the phylogenetic dendrogram was constructed using the neighbor - joining method with the mega software (sudhir kumar, arizona state university, phoenix, az, usa). the outbreak began on april 6, 2011 when a male student presented with fever (oral temperature of 38c) and a series of fri symptoms, including cough, sore throat, and headache. the student attended a school (grades 19 ; 11 classes, 28 teachers, and 487 students) located in tongwei county, gansu province, china. he continued to attend school and recovered from his symptoms after taking an unknown cold medication administered by the village health center for 3 days. through a retrospective survey, we learned that the boy did not have contacts with patients exhibiting similar symptoms during 2 weeks prior the onset of the illness. he had also never travelled outside the town. after the index case, the outbreak peaked on april 10, 2011, with 10 cases detected. the last case was reported on april 19 (figure 1a). during the outbreak, 43 students (88%, 43/487 students) from the same school developed into fri. the patients were 815 years old, and 535% (23/43) of them were 1213 years old. children from seven classes were affected in this outbreak. among them, 21 patients were from the fifth grade (figure 1b), the same grade as the index patient. all the patients presented with fever (oral 38c40c), sore throat, cough, headache, and fatigue, similar to influenza - like symptoms. some of the patients experienced pharyngeal congestion, swollen tonsils, and submandibular lymphadenopathy, and a few patients were diagnosed with mild pneumonia. given that no patient presented with severe symptoms, all the patients were treated at the village health center and continued to attend school without isolation. public health officials at the local cdc office were engaged, and they undertook measures to prevent disease spread, such as morning temperature screening of children, ensuring ventilation through open doors and windows, and disinfecting school surfaces with 02% peroxyacetic acid spray. on average, there were no severe illnesses associated with this outbreak, and all the patients fully recovered. all the patients denied any history of travel in the 2 weeks prior to the onset of illness. (a) the number of cases appearing each day from april 5 to april 20, 2011. (b) the number of cases by students (blue line) and cases by grade (columns). local cdc officials also conducted a field epidemiological investigation that included collecting pharyngeal swabs from 17 symptomatic children (table 2) within a week of the disease onset. eleven specimens had evidence of hadv infection (table 2), whereas no other pathogens were detected. characteristics of patients and their laboratory results (2011) by sequence data from the hexon gene hypervariable region (hvr1 - 6). a characteristic adenovirus - like cpe was observed in a549 cells from six of the throat swab specimens (table 2). of the 11 adenovirus - positive samples, the hypervariable region (hvr1 - 6) of the hexon gene was amplified, and blast sequence analysis revealed 100% identical with hadv-14p1 (strain isolate portland/2971/z2007, usa hadv-14p1 ; genbank accession number fj841901 ; table 2). amplifications and nucleotide identity of gansu2011 - 0111 strains were 100% identical to the hadv-14p1 reference sequence (genbank accession number : fj841901). cdc public health officials concluded that all the patients were infected with the same hadv-14p1 virus. one (gansu2011 - 01 strain) of the 6 hadv isolates was selected for further sequence studies. the complete hexon, fiber, and e1a genes were amplified, sequenced, and compared with the sequences of hadv - b2 species found in genbank (figure 2a c). the hexon gene (2838 nt) had 999% nucleotide identity with 5 hadv-14p1 strains deposited in genbank : hadv-14p1 usa 2007 strains (genbank accession numbers : fj841909 and fj822614), hadv-14p1 dublin 2009 strain (genbank accession number : hq163915), and hadv-14p1 china 2010 strains (genbank accession numbers : jf420882 and jq824845). the fiber gene (972 nt) had 100% nucleotide identity to the same 5 hadv-14p1 strains. the e1a gene (873 nt) showed 100% nucleotide identity to hadv-14p1 usa 2007 strains and one hadv-14p1 dublin 2009 strain (genbank accession numbers : fj822614, fj841915, and hq163915). the identity score was 998% compared to the hadv-14p1 china 2010 strain (genbank accession number : jf438997) and 997% compared to another hadv-14p1 china 2010 strain (genbank accession number : jq824845). nucleotide sequence data from strain gansu2011 - 01 were deposited in genbank under accession the numbers jx310315jx310317. because the hypervariable regions of hexon gene of the 11 hadv - positive specimens were identical, we chose the complete hexon gene of gansu2011 - 01 as the representative strain to be deposited in genbank. phylogenetic analysis of the complete hexon, fiber, and e1a genes of the hadv-14 gansu2011 - 01 strain by the neighbor - joining method. (a) phylogenetic tree based upon the complete hexon gene of the hadv-14 gansu2011 - 01 strain and reference strains of subspecies of hadv - b2. (b) phylogenetic tree based upon the complete fiber gene of the hadv-14 gansu2011 - 01 strain and reference strains of subspecies of hadv - b2. (c) phylogenetic tree based upon the complete e1a gene of the hadv-14 gansu2011 - 01 strain and reference strains of subspecies of hadv - b2. in recent decades, outbreaks of hadv - associated fri have been reported in various regions worldwide,8,9,23,24 including china. hadv-3 and hadv-7 have been most frequently implicated in chinese fri outbreaks.1619 here, we report the first outbreak of fri associated with hadv-14p1 at a primary and middle school in china and the first school - based hadv-14p1 outbreak in the world. hadv-14 was initially discovered in 1955 in the netherlands,25 and it was subsequently isolated in great britain in the same year,26 uzbekistan in 1962,27 and czechoslovakia in 1963.28 its detection was not reported for the next 40 years, with the only exception of the sporadic isolation in the netherlands during 1974. hadv subspecies b2 was found only in eurasia before hadv-14 was detected in north america in 2006.15 afterward, hadv-14p1 spread widely and caused severe cases and deaths in adults in the united states.29 deaths associated with hadv-14p1 infection were also recently reported in europe.30 this hadv-14p1 outbreak produced a series of mild fri symptoms among children of 815 years of age. the outbreak differed from that in the us hadv-14p1 outbreak reports in that the us adults often suffered severe pneumonia and death. the age of the patients in china likely played an important role in attenuating the disease severity.7 in our study, male patients accounted for 628% (27/43) of the cases, which was similar to other observations.3032 the significance of gender as a risk factor for hadv-14p1 infection was unknown. we hypothesized that it was due to the poor hygienic behaviors of school - aged boys. it is possible that the virus was already circulating in the school when the index case was first identified. as children with fri symptoms were not isolated, this also likely influenced the virus transmission. the subclinical rate of hadv-14p1 infection was unknown and seemed an important characteristic to study. trei.33 reported a respiratory disease outbreak associated with hadv-14 occurred at a large military basic training facility in texas during 2007. after the 6-week basic training course, the trainees were immediately assigned to advanced training sites worldwide, including south korea, which is a neighboring country of china. therefore, the possibility that the origin of hadv-14p1 in china derived from that outbreak can not be ruled out. in 2012, tang submitted to genbank (jn0321321) the genome sequence of hadv-14p1 isolated in beijing in 2010 from a 6-month - old child with an acute respiratory tract infection (arti). recently, another genome sequence of hadv-14p1 isolated in guangzhou city in 2010 from a 17-month - old child with acute suppurative tonsillitis was published.34 increasing evidence suggested hadv-14p1 was found in china as far back as 2010. the high homology between hadv-14p1 isolated from beijing, guangzhou, and gansu between 2010 and 2011 supported the hypothesis of a stable evolution and transmission of hadv-14p1 in china. in our study, amplifications of the hypervariable regions of the hexon gene from 11 hadv - positive specimens were identical to hadv-14p1 and classified as such. as reported by kajon.,35 further molecular typing and analysis aimed at the complete amplification of hexon, fiber, and e1a gene evidenced that the outbreak hadv-14p1 isolates were nearly identical to other hadv-14p1 strains isolated in the united states, europe, and china between 2006 and 2010. sequence analysis of the fiber gene from the hadv-14p1 isolated in the united states, europe,30,35 and china (gansu2011 - 01 and 2 isolates in 2010) showed a 6-nt deletion resulting in a 2-amino acid (aa) deletion (i.e., lysine and glutamine) at positions 251 and 252 in the knob region with respect to the prototype strain hadv-14p de wit.36 however, whether the deletion affects the pathogenicity and virulence of hadv-14p1 requires further studies.37,38 although our study did not provide further evidence to explain whether the 2-aa deletion might alter the virulence and increase the pathogenicity of hadv-14p1, it suggested that the strain hadv-14p1 had been stable for the last decade and was likely co - circulating in europe, the united states, and asia. 39detected and characterized hadvs infections in 10 310 adults with artis between may 2005 and july 2010 in china. hadvs were detected in 86 patients, among which the infecting virus was found to belong to species b in 779% of the cases, with only one strain found to be the same hadv-14 as that identified in 2010. this result indicated that hadv-14 was not widely circulating in beijing and was likely imported from other countries. a single viral type would dominate in a specific region, and then be quickly replaced by other types within a few years.40 moreover, the development and spread of hadv-14p1 in the united states illustrates why china needs to develop a national surveillance system for the etiology of severe pneumonia and deaths among adults and school - aged children. it will take a long time for the scenario of china 's poor sanitary conditions, crowded schools, and rural areas to improve. a simple laboratory method to distinguish hadv-14 infection from fri caused by other pathogens has not been established yet ; therefore, the improvement of laboratory detection methods for rapid identification of the hadv is crucial. moreover, there is a critical need to strengthen the surveillance system for adenovirus in china.	objectiveshuman adenovirus (hadv) type 14 had been infrequently associated with outbreaks of febrile respiratory illness (fri) until the hadv-14p1 emerged in 2006 and rapidly spread in the united states. here, we report an outbreak of fri caused by hadv-14p1 that occurred in 2011 at a primary and middle school in china.designthe basic information of the outbreak was recored ; throat swabs were collected from 17 patients, polymerase chain reaction, a549 cell culture, and sequencing were used to identify the pathogen of the outbreak.. resultstotal of 43 students were infected in this outbreak. boys were more than girls. we identified 11 hadv - positive specimens and 6 hadv isolates. genetic analysis showed that the complete hexon, fiber, and e1a sequences of isolates were nearly 100% identical with other hadv-14p1 sequences deposited in genbank.conclusionshadv-14p1 has caused outbreaks of pneumonia and mortality among adults in the united states and europe. it may cause similar conditions among chinese adults due to poor hygiene and sanitation. it seems prudent for china to develop a national surveillance system to determine the etiology of severe respiratory diseases and deaths among adults and school - aged children.
most commonly, hantaviruses are transmitted to humans via aerosols of infectious excreta (urine, feces, saliva) from chronically infected wild rodents (6). the old world hantaviruses are most often associated with hemorrhagic fever with renal syndrome (hfrs), while the new world hantaviruses cause hantavirus pulmonary syndrome (hps). this dichotomy, however, is not completely clear in all cases (7). the natural reservoirs of hantaviruses are small rodents or insectivores, and the virus is believed to cause asymptomatic life - long infection in these animals. hantaviruses are usually species - specific, although spill - overs have been described (8). puumala hantavirus (puuv) causes the majority of hantavirus - related clinical cases in europe. puuv causes a mild form of hfrs and is carried and shed by the bank vole (myodes glareolus) (9). other hfrs - causing hantaviruses circulating in europe include dobrava and saaremaa hantaviruses, which are carried by the yellow - necked mouse (apodemus flavicollis) and the striped field mouse (a. agrarius), respectively (6). seov is the only known hantavirus with a potentially world - wide distribution due to the ubiquitous occurrence of its reservoir hosts, the brown and the black rat (rattus norvegicus and r. rattus, respectively) (10). nevertheless, prior to the last decade, human and wild rat infections of seov were, for the most part, detected only in asia with human infections in europe limited to laboratory rat handling (4). during recent years, however, seov has been detected in wild rats in france, belgium, and the united kingdom (2, 3), and in pet rats in sweden, england, and wales (1, 4). human cases of severe seov - caused hfrs have been reported in france and the united kingdom (3, 5). in neither of these severe seov cases were there any connections to pet or laboratory rats, strongly indicating that the infections were contracted from nature. this study proves the occurrence of seov in the wild rat population in the netherlands. analyses were conducted at the zoonosis science center at the department of medical biochemistry and microbiology, uppsala university, the public health agency of sweden, stockholm, sweden, and at the department of microbiology, graduate school of medicine, hokkaido university, japan. rat hearts were vortexed together with 1 ml of phosphate - buffered saline (pbs) and centrifuged. the samples were initially screened by 1) enzyme - linked immunosorbent assay (elisa), 2) indirect immunofluorescence assay (ifa), and 3) immunoblotting (ib):an in - house seov elisa (will be described elsewhere, verner - carlsson, unpublished) was used for initial screening. briefly, elisa - plates were coated by the hantavirus - reactive bank vole mab 1c12 (4 g / ml), followed by post - coating (3% bovine serum albumin (bsa) in pbs), and incubation of seov (strain 8039) native antigen. rat samples were diluted 1:16 (equivalent to a final serum dilution of 1:400) and rat control sera were incubated at a dilution of 1:400, followed by alkaline phosphatase (alp)-conjugated goat anti - rat antibodies (jackson immunoresearch). all incubations lasted 1 h at 37c, and the plates were washed four times between each step. cut - off was set to od 0.100 at 405 nm.ifa using seov (strain sr-11) infected vero e6 cells was used as described earlier (11). briefly, rat blood collected on filter paper was diluted in pbs and used for ifa analyses and ib analyses. serum from a wistar rat experimentally infected with seov (strain sr-11) was used as positive control. rat samples were diluted 1:50, and control rat serum was diluted 1:100 and put onto glass slides with prefixed seov - infected vero e6 cells for 1 h at room temperature. after washing, fitc - conjugated goat anti - rat antibodies were incubated for 1 h at room temperature. fluorescence staining was visualized by an immunofluorescence microscope.the samples were further subjected to ib using seov (strain sr-11) infected vero e6 cells and uninfected vero e6 cells as antigens, as described earlier (12). briefly, after transfer onto pvdf membranes, the membranes were blocked in block ace buffer (ds pharma biomedical) for 1 h at rt. the membranes were subsequently incubated with the rat samples diluted 1:50 and positive / negative control rat serum at 1:100 dilutions, for 1 h rt, using a microwell system, followed by the addition of horseradish peroxidase - conjugated goat anti - rat igg for 1 h at rt. an in - house seov elisa (will be described elsewhere, verner - carlsson, unpublished) was used for initial screening. briefly, elisa - plates were coated by the hantavirus - reactive bank vole mab 1c12 (4 g / ml), followed by post - coating (3% bovine serum albumin (bsa) in pbs), and incubation of seov (strain 8039) native antigen. rat samples were diluted 1:16 (equivalent to a final serum dilution of 1:400) and rat control sera were incubated at a dilution of 1:400, followed by alkaline phosphatase (alp)-conjugated goat anti - rat antibodies (jackson immunoresearch). all incubations lasted 1 h at 37c, and the plates were washed four times between each step. cut - off was set to od 0.100 at 405 nm. ifa using seov (strain sr-11) infected vero e6 cells was used as described earlier (11). briefly, rat blood collected on filter paper was diluted in pbs and used for ifa analyses and ib analyses. serum from a wistar rat experimentally infected with seov (strain sr-11) was used as positive control. rat samples were diluted 1:50, and control rat serum was diluted 1:100 and put onto glass slides with prefixed seov - infected vero e6 cells for 1 h at room temperature. after washing, fitc - conjugated goat anti - rat antibodies were incubated for 1 h at room temperature. the samples were further subjected to ib using seov (strain sr-11) infected vero e6 cells and uninfected vero e6 cells as antigens, as described earlier (12). briefly, after transfer onto pvdf membranes, the membranes were blocked in block ace buffer (ds pharma biomedical) for 1 h at rt. the membranes were subsequently incubated with the rat samples diluted 1:50 and positive / negative control rat serum at 1:100 dilutions, for 1 h rt, using a microwell system, followed by the addition of horseradish peroxidase - conjugated goat anti - rat igg for 1 h at rt. two out of 16 samples, # 22 and # 33, were found clearly positive by all three screening methods ; rat # 84 was positive by elisa and ifa, but negative in ib (table 1). to finally confirm the hantavirus reactivity and a seov - specificity, we tested the three positive samples # 22, # 33, and # 84 by frnt, as previously described (13). briefly, the rat samples were serially diluted and mixed with diluted viruses containing 3070 focus forming units/100 l. confluent vero e6 cell monolayers in six - well tissue culture plates were used and incubated at 37c for 1 h after addition of the virus / antibody solution. to create a secluded environment, a mixture of tissue culture and agarose medium was subsequently added to the wells, which were then incubated for 8 days. to ensure visibility of the results, the agarose was removed from the wells, and the cells were fixed with methanol. to indicate virus - infected cells, the anti - hantavirus bank vole mab 1c12 followed by peroxidase - labeled goat antibodies to mouse igg (jackson) 3, 3, 5, 5-tetramethylbenzidine substrate (sigma) was used as substrate, and foci were enumerated. the samples # 22, # 33 and # 84 all showed high titers of neutralizing antibodies (table 1). summary of elisa, ifa, ib, frnt, and pcr results elisa : enzyme - linked immunosorbent assay ; ifa : immunofluorescence assay ; ib : immunoblotting ; frnt : focus reduction neutralization test ; pcr : polymerase chain reaction ; nt : not tested ; od : optical density at 405 nm, adjusted to the positive control=1.00. a hantavirus genus - specific nested rt - pcr was applied as described earlier (14). samples # 22, # 33 and # 84 all showed positive results, while the serologically negative sample # 31 was found negative (table 1). this is the first discovery of seov - specific antibodies in wild rats in the netherlands. in previous attempts to discover seov, during 20082011, 161 brown rats and 61 black rats were trapped at different types of environments (farmlands, urban locations, and nature reserves) in rijn and ijssel regions in the netherlands. despite initial findings of seov - reactive antibodies in several animals based on an adapted commercial ifa, and two different elisas, seov - infection could never be confirmed by neutralization test or viral sequence data (data not shown). it is therefore possible that the dispersal of seov in the area has happened relatively recently. whereas the detection of antibodies can, in case of a number of pathogens, indicate a previous infection that is no longer an infectious threat, similar conclusions can not be made concerning hantaviruses. as demonstrated in the case of puuv infections, hantaviruses can be secreted by bank voles for several months after the initial infection (15), lundkvist., unpublished observations). furthermore, even at room temperatures, the virus is stable and infectious over a few weeks outside the rodent (16) making virus transmission control a huge challenge. as seen from the example of west nile virus emergence in the united states, the existence of wildlife reservoirs makes eradication of a disease that has had time to establish itself unrealistic. thus, the recommendations for handling the situation must be made with a long - term perspective. since aerosols, contaminated by virus - infected rodent excreta, are the main source for human infection with hantaviruses, actual physical contact between rodent and man is not necessary for disease transmission (6). consequently, in many cases it will be difficult for both patients and medical specialists to suspect transmission of seov. therefore, disseminating information about the occurrence of seov in wild rat populations in the netherlands has great importance for both medical workers and the general public. in addition, tailored messages are needed for people exposed to seov due to their working environments. transmission preventive measures in the form of gloves and protective facemasks may be necessary for people working in rat - infested environments, particularly for professional rat controllers. steps have been taken by the netherlands centre for infectious disease control (rivm) to provide this type of information. further studies are planned to collect evidence on the circulation of this hantavirus among the wild rat population in other geographical areas in the netherlands, to generate and compare dutch seov sequences to seov from other countries, while the occurrence of human infections will be assessed through increased surveillance of clinical cases with hfrs - like symptoms and sero - surveillance of, for example, blood donors. the recent discoveries of seov circulating in wild rat populations in france, belgium, and the united kingdom (2, 3), and now also in the netherlands, together with the cases of severe human seov - infections so far confirmed in the united kingdom and france (1, 4), strongly highlight the importance of further studies all over europe. the authors have not received any funding or benefits from industry or elsewhere to conduct this study.	we report the first detection of seoul hantavirus (seov)-specific antibodies in the wild brown rat population in the netherlands. seov - reactive antibodies were found in three rats out of 16 in a repeated series of tests including immunofluorescence assay, immunoblot, and enzyme - linked immunosorbent assay. focus reduction neutralization test confirmed the presence of seov - specific antibodies, and reverse - transcription polymerase chain reaction (rt - pcr) confirmed the presence of hantaviral rna. this discovery follows the recent findings of seov infections in wild and pet brown rats and humans in england, wales, france, belgium, and sweden, indicating an even higher importance of this hantavirus for public health in large areas of europe.
brugada syndrome (bs) is a fatal inherited arrhythmogenic disease due to syncope, ventricular arrhythmia, and sudden death. the type 1 pattern which is considered as a bs has a coved st - segment elevation 2 mm followed by a down - sloping concave or rectilinear st - segment with a negative t wave. the type 2 pattern is characterized by a high take - off (r) that is 2 mm from the isoelectric baseline, followed by st - segment elevation that is convex to the isoelectric baseline with elevation 0.05 mv, with variable t wave in lead v1 and positive or flat t wave in lead v2. a 39-year - old male with a medical history of hypertension and diabetes mellitus presented to the emergency department with complaints of intermittent chest pain and dyspnea on exertion for 3 days. chest pain was located at the left side of the chest, six out of ten in intensity, sharp in character without radiation. the patient denied syncope, orthopnea, paroxysmal nocturnal dyspnea, palpitations, dizziness, headache, syncope, presyncope, history of heart attack, arrhythmia, seizure, blurred vision, urinary, or bowel problems. his home medications include ibuprofen as needed for chest pain and hydralazine 25 mg per os twice daily. family history revealed his mother had an unknown cardiac problem, and his sister had sudden cardiac death (scd) while she was at age 30. initial vital signs included temperature 98.7f, pulse rate 96 beats per minutes, respiratory rate 22 breaths per minutes, blood pressure 130/90 mmhg, and oxygen saturation 92% on room air. he was put on nonrebreather mask with 40% of oxygen for hypoxia and oxygen saturation was maintained with 95%. on auscultation, normal regular first and second heart sound without murmur, rub, or gallop was noted. comprehensive metabolic panel revealed potassium 2.9 mmol / l, magnesium 2.7 mg / dl, sodium 136 mmol / l, chloride 101 mmol / l, carbon dioxide 28 mmol / l, blood urea nitrogen 10 mg / dl, creatinine 1 mg / dl, glomerular filtration rate 90 ml / min, calcium 9 mg / dl, phosphorus 3.1 mg / dl, 3 sets of troponin 6 h apart were 0.01 ng / ml, and b type natriuretic peptide 5 pg / ml. coagulation profile and arterial blood gasses with 40% of oxygen with nonrebreather mask were within normal limit. computed tomography of the chest revealed moderate to marked ground glass opacities in both lung fields and normal heart size. an ekg revealed a new right bundle branch block pattern with coved st - segment elevations and inverted t wave in leads v1-v2 consistent with type 1 brugada pattern ekg [figure 1 ]. v2 hypokalemia was corrected with potassium chloride 40 meq per os once and 20 meq twice intravenously. six hours later, ekg was repeated and brugada pattern ekg was disappeared, and ekg returned to its baseline [figure 2 ]. disappearance of brugada pattern electrocardiogram after normalization of potassium the patient was treated with antibiotics and antivirals for bilateral interstitial pneumonia likely due to viral or bacterial etiology. no fever was noted throughout hospital course. chest pain on admission day was thought to be pleuritic chest pain and patient was discharged. although the patient has a strong family history of cardiac problem, patient and family members were recommended to follow - up with an electrophysiologist for drug challenge test for possible bs, but they refused. up to 5 months after discharge, the patient was contacted over the telephone and he was in good health without any syncope, chest pain, arrhythmia, or cardiac symptoms. type 1 brugada pattern ekg is a rare condition, and the prevalence of the typical ekg changes of the brugada pattern in two groups of urban populations in the united states was about 0.4 and 0.012%, respectively. bs shows autosomal dominant inheritance with variable expression and found to have mutations in the scn genes scn5a and scn10a which are encoding subunits of a cardiac sodium channel. the abnormal myocardial sodium channels decrease sodium inflow currents, thus reducing the duration of normal action potentials. a number of conditions may induce brugada pattern ekg such as fever, hyperkalemia, hypokalemia, hypercalcemia, cocaine abuse, and medications such as sodium channel blockers, heterocyclic antidepressants, and beta - adrenergic blockers. hypokalemia may cause qt prolongation and other ventricular arrhythmias due to its effect of raising resting membrane potential and increases both the duration of the action potential and the refractory period leading to reentrant arrhythmias. although there is no definite value of serum potassium level that can induce brugada pattern ekg, some case reports revealed hypokalemia may unmask brugada pattern ekg. in an animal model, it was revealed that loss of the action potential dome due to transient outward current (ito)-medicated phase 1 of potassium in the right ventricle induces a transmural voltage gradient which accentuates st - segment elevation which is similar to the mechanism of bs. therefore, hypokalemia may expand transmural dispersion of repolarization in the right ventricle leading to a brugada pattern ekg. ekg changes in hypokalemia are usually t wave inversion, appearance of u wave and st - segment depression of 0.5 mm or more in lead ii or leads v1, v2, and v3. the decision to follow - up with an electrophysiologist is determined by the patient 's age, family history, prior episodes of syncope due to cardiac disorders, and patient 's preferences. the consensus recommendations for the treatment of bs focus on implantable cardioverter - defibrillator (icd) to abort ventricular arrhythmia. icd was preferred over antiarrhythmic agents such as quinidine or amiodarone in patients with a history of scd or syncope. awareness of its appearance should be made by all physicians since patients with a family history of arrhythmia or scd are at the high risk of developing scd.	coved - type st - segment elevation in the right precordial leads are the characteristics of brugada syndrome, an inherited arrhythmogenic ion channel disease, which could lead to ventricular arrhythmia and sudden death. hypokalemia alone may induce type 1 brugada pattern electrocardiogram (ekg), and the association has rarely been reported. we describe a patient with hypokalemia 2.9 mmol / l and the appearance of new right bundle branch block pattern with coved st - segment elevations with inverted t wave in leads v1-v2. serum potassium was corrected and repeated ekg 6 h later revealed disappearance of type 1 brugada pattern. although there is no definite value of serum potassium level that can induce brugada pattern ekg, hypokalemia may unmask type 1 brugada ekg pattern. awareness of its appearance should be made by all physicians since patients with a family history of arrhythmia or sudden cardiac death (scd) are at the high risk of developing scd.
it is the 10th anniversary of the mailing of the anthrax spores, a tragic event that resulted in five deaths, seventeen other known infections, and untoward fear among the population. the cleanup is estimated to have cost $ 1 billion. what have we learned and accomplished in the interim of course, one can not consider the anthrax mailings in a vacuum, coming on the heels of the september 11 attacks on the world trade center, the pentagon, and united airlines flight 93. the reaction from congress was swift : the united states patriot act was signed into law in october 2001, and the public health security and bioterrorism preparedness response act followed in june 2002. together, these pieces of legislation put restrictions and limits on who could access and possess select agents, those pathogens deemed by the cdc and usda to be the most dangerous. the government solicited studies from the national academies on various topics relating to science and security, other federal advisory panels were formed, and the government itself has addressed bioterrorism at various levels. numerous nongovernmental organizations throughout the world, including the american association for the advancement of science (aaas), have also engaged scientists, security experts, ethicists, and others in an ongoing, fruitful discussion. while the specific charge to each of these groups differed, they all have addressed a common question : how do we ensure a vibrant research enterprise without compromising national security ? this question is of central importance given that the 21st century promises to be the biological century, which will likely spawn many new life science - based technologies and industries that will improve our lives. the answer to this question is not straightforward, mainly because of the nature of life science research. the risks that present themselves as a result of the acquisition of new knowledge are difficult to predict or quantify, while the potential benefits are more concrete. therefore, determining whether certain types of research should be prohibited or regulated is difficult. the consensus reached by both the scientific and national security communities is that research should proceed, with all involved being vigilant for the potential of misuse. the national science advisory board for biosecurity, which has been charged since 2005 with providing recommendations to the u.s. government on the oversight and conduct of dual use research, has developed a series of thoughtful reports that suggest a balanced approach to the topic (see http://www.biosecurityboard.gov). perhaps the most important contribution of this body was to acknowledge that most if not all biological research had dual use capabilities while creating a special category known as dual use research of concern (durc) and the tools to identify it. this was important because it had the effect of walling off the small part of the scientific effort that was most relevant to the threat of bioterrorism and thus leaving the vast majority of biological research undisturbed and unregulated. another valid concern is whether certain individuals should not be allowed to access select agents. while some groups have now been excluded categorically by the above - mentioned statutes, vigorous discussions concerning personnel reliability in a broader sense insider threat was amplified by the identification of bruce ivins, a scientist working in a u.s. the emerging consensus is that ongoing attention to lab workers trustworthiness, behavior, and attitude is the best means to reduce the risk that someone might deliberately cause harm. what has the overall effect of these activities been on the life science research enterprise ? the infusion of extra research dollars has been welcome, especially at a time when federal funding of biology research overall has suffered. increased attention to the possible risk of misuse however, a small but significant number of investigators have chosen to discontinue working in the field due to the added regulatory burden. it is difficult to assess how many others are not entering into this area, but anecdotal evidence suggests this is occurring more than one would like. perhaps of greater concern is that important research, such as the development of new vaccines against anthrax, has been slowed by the need to work within the new regulatory and statutory framework. compliance with select agent rules has significantly increased the cost of research on certain pathogens (1). the enactment of the select agent rules led to the destruction of several microbial collections (2) and is almost certainly interfering with the establishment of new collections or saving of new clinical isolates, given the enormous work involved in having such isolates transferred to secure facilities. furthermore, the focus on containing microbial threats by generating lists of organisms for special consideration may have created a false sense of security while greatly increasing the regulatory burden (3). we are very fortunate that 10 years have passed with no additional bioterrorism events. this is a testament to the effort of scientists behaving responsibly and working together with the national security community to minimize the risks. while ideally, one would like to reduce the risk to zero, living in the real world, we know that zero risk would mean little progress and that an impaired research enterprise will leave us more vulnerable. our nation was built by, and has thrived on the efforts of, risk takers. the current oversight system for life science research is functional and robust, ensuring that the pace to discovery is limited only by intellectual and fiscal resources.	abstractin the fall of 2001, bacillus anthracis spores were spread through letters mailed in the united states. twenty - two people are known to have been infected, and five of these individuals died. together with the september 11 attacks, this resulted in a reevaluation of the risks and benefits of life science research with the potential for misuse. in this editorial, we review some of the results of these discussions and their implications for the future.
these patients may develop dyspnea, stridor, intractable cough, hemorrhage, atelectasis, or postobstructive pneumonia, or a combination of the aforementioned manifestations because of tracheobronchial tumor extension (1). these patients are poor surgical candidates judged on the basis of either physiological or oncological criteria. the optimal management of patients with central airway obstruction includes radiotherapy, laser therapy, photodynamic therapy, cryotherapy and airway stenting, which can be done either with or without bronchoscopic guidance. airway stent can especially achieve immediate and durable airway patency resulting in an improved quality and length of life span in patients with lung cancer (2). hemoptysis particularly occurs in about 30% of all the lung cancer patients, and can be lethal to them. the treatment options of hemoptysis are pharmacology, bronchoscopic intervention, vascular embolization and surgical resection (3). in selected cases, however, these measures only offer transient control of bleeding and need prolonged mechanical ventilation (4). in the present report, we have described three cases of selective bronchial stent placement in patients with uncontrolled hemoptysis from lung cancer. between september 2009 and july 2010, 3 men (mean age 65.3 17.0 years) with uncontrolled hemoptysis from lung cancer underwent bronchial stent insertion. patients who had dyspnea and recurrent or prolonged hemoptysis despite continued conservative management were selected for the airway stenting. all bronchial stent placements were performed in the angiography room under fluoroscopic and bronchoscopic guidance. a 0.035 hydrophilic guidewire was advanced to the distal portion of stenotic and bleeding segment of bronchus under bronchoscopic guidance. a 5 fr angiographic catheter was then inserted and consecutively a small amount of non - ionic contrast material was injected into the airway to obtain bronchogram. then, a covered retrievable stent (hercules airway stent, s&g biotech, seongnam, korea) was deployed in the target area under fluoroscopic guidance. finally, follow - up bronchoscopy was performed immediately after stenting to confirm the location and patency of the inserted stent. a 78 year - old male was diagnosed with central non - small cell lung cancer 12 months earlier. the primary lung cancer was located in the proximal right upper lobar bronchus associated with postobstuctive atelectasis of the right upper lobe. however, at the time of diagnosis, the patient refused all the curative means. we performed ct imaging on outpatient clinics, which showed a completely obstructed right upper lobar bronchus by a tumor with postobstructive pneumonia of the right upper lobe. the wall of the right main bronchus and bronchus intermedius was thickened and the lumen of the right main bronchus was almost narrowed by tumor infiltrations. a bronchoscopic examination demonstrated that orifice of the right main bronchus was nearly completely obstructed by the endoluminal mass and there was diffuse bleeding from the right main bronchus. the patient received urgent external beam radiotherapy to control bleeding from the friable endobronchial tumor and bleeding gradually decreased for several days. however, eleven days after the radiation therapy, a sudden onset of dyspnea developed. chest radiography showed that the right lung had totally collapsed and bronchoscopic examination revealed complete obstruction of the right main bronchus with diffuse bleeding. bronchial artery embolization could not be considered as a treatment option for hemoptysis because bleeding conditions were not because of active arterial bleeding and there was no prominent contrast enhancement of the mass as seen on the ct scan. we decided that bronchial stent insertion in the right main bronchus was necessary to both, re - open the airway and to control the bleeding of tumor through the tamponade effect. after the bronchogram, which was performed to identify the airway on a fluoroscopic image, we decided to sacrifice superior segment of the right lower bronchus to preserve basal segments of the right lower lobe. the 12 mm - 4.3 cm self - expanding silicone - covered nitinol stent (hercules airway stent, s & g biotech, seongnam, korea) was deployed in the right lower lobar bronchus to the right main bronchus. however, proximal portion of the right main bronchus was not completely covered due to its insufficient length. for this reason, another 12 mm - 5.3 cm self - expanding silicone - covered nitinol stent was reinserted, overlapping the first stent. at the end of the procedure, an immediate improvement in the aeration of the right basal lung was observed on fluoroscopy and hemoptysis was subsided (fig. nevertheless, the patient died two days later due to lack of expectoration that resulted in whole lung aspiration pneumonitis or pneumonia. a 72 year - old man, with a history of tuberculosis ten years ago and an exposure to asbestosis, was diagnosed with squamous cell carcinoma on the left lower lobe about 20 months earlier. the patient underwent left lower lobectomy after a month of the diagnosis and had been treated with postoperative chemotherapy, but it failed to prevent recurrence and progression of the disease. the ct scan, which was performed on his visit, demonstrated an extremely stenotic left main bronchus by metastatic lymphadenopathy in mediastinum and both the hila (fig. fresh bloody sputum developed on the fourth day of admission and frequency of hemoptysis gradually increased up to three times per hour. respiration rates were raised up to a 40/min and the hemoglobin level dropped from 10.1 g / dl to 7.1 g / dl. we decided to insert the covered bronchial stent into the left main bronchus. on the fifth day after admission, 12 mm - 5.3 cm self - expanding silicone - covered nitinol stent (hercules airway stent : s & g biotech, seongnam, korea) was deployed in the left main bronchus under bronchoscopic and fluoroscopic guidance. bleeding through the endotracheal tube gradually decreased and stopped within two days after the procedure. he required continuous renal replacement therapy (crrt) due to acute renal failure by hypotension. sixteen days after the bronchial stent placement, he expired because of multi - organ failure. a 46 year - old male was diagnosed with non - small cell lung cancer 30 months earlier. the primary cancer was located in the orifice of the left upper lobe bronchus without metastasis. he was treated with left pneumonectomy after neoadjuvant chemotherapy along with postoperative radiation therapy and systemic chemotherapy. eleven months after the surgery, chest ct scans showed recurring mass at the stump of the left main bronchus, which was also confirmed by bronchoscopic biopsy. the patient underwent second and third line systemic chemotherapy, but it was unable to stop the disease from progressing. approximately four months prior to this visit, he was admitted due to presence of blood - tinged sputum. at that time, a bronchoscopic examination showed that the bleeding was from recurred mass in the left main bronchus. he visited again within several weeks due to dysphagia and recurrent blood - tinged sputum. we performed ct imaging, which depicted progression of a recurred mass at the stump of the left main bronchus. ct scan also showed narrowing of distal trachea and right proximal main bronchus (fig., it showed invasion of adjacent esophagus by the recurred mass with conglomerated lymphadenopathy. on the eighth day of admission, he coughed up approximately 500 ml of fresh blood and the patient was transferred to the intensive care unit. first, a 18 mm - 4 cm self - expanding silicone - covered nitinol stent (hercules airway stent : s & g biotech, seongnam, korea) was deployed in the distal trachea and another 12 mm - 3 cm self - expanding silicone - covered nitinol stent was deployed in the distal trachea to the right main bronchus, overlapping the former one (fig., there was a gradual decrease in the hemoptysis except for an episode of scanty bleeding that halted after conservative treatment. however, the patient needed daily bronchoscopic toileting because of lack of self - expectoration and retained mucus secretion. between september 2009 and july 2010, 3 men (mean age 65.3 17.0 years) with uncontrolled hemoptysis from lung cancer underwent bronchial stent insertion. patients who had dyspnea and recurrent or prolonged hemoptysis despite continued conservative management were selected for the airway stenting. all bronchial stent placements were performed in the angiography room under fluoroscopic and bronchoscopic guidance. a 0.035 hydrophilic guidewire was advanced to the distal portion of stenotic and bleeding segment of bronchus under bronchoscopic guidance. a 5 fr angiographic catheter was then inserted and consecutively a small amount of non - ionic contrast material was injected into the airway to obtain bronchogram. then, a covered retrievable stent (hercules airway stent, s&g biotech, seongnam, korea) was deployed in the target area under fluoroscopic guidance. finally, follow - up bronchoscopy was performed immediately after stenting to confirm the location and patency of the inserted stent. a 78 year - old male was diagnosed with central non - small cell lung cancer 12 months earlier. the primary lung cancer was located in the proximal right upper lobar bronchus associated with postobstuctive atelectasis of the right upper lobe. however, at the time of diagnosis, the patient refused all the curative means. we performed ct imaging on outpatient clinics, which showed a completely obstructed right upper lobar bronchus by a tumor with postobstructive pneumonia of the right upper lobe. the wall of the right main bronchus and bronchus intermedius was thickened and the lumen of the right main bronchus was almost narrowed by tumor infiltrations. a bronchoscopic examination demonstrated that orifice of the right main bronchus was nearly completely obstructed by the endoluminal mass and there was diffuse bleeding from the right main bronchus. the patient received urgent external beam radiotherapy to control bleeding from the friable endobronchial tumor and bleeding gradually decreased for several days. however, eleven days after the radiation therapy, a sudden onset of dyspnea developed. chest radiography showed that the right lung had totally collapsed and bronchoscopic examination revealed complete obstruction of the right main bronchus with diffuse bleeding. bronchial artery embolization could not be considered as a treatment option for hemoptysis because bleeding conditions were not because of active arterial bleeding and there was no prominent contrast enhancement of the mass as seen on the ct scan. we decided that bronchial stent insertion in the right main bronchus was necessary to both, re - open the airway and to control the bleeding of tumor through the tamponade effect. after the bronchogram, which was performed to identify the airway on a fluoroscopic image, we decided to sacrifice superior segment of the right lower bronchus to preserve basal segments of the right lower lobe. the 12 mm - 4.3 cm self - expanding silicone - covered nitinol stent (hercules airway stent, s & g biotech, seongnam, korea) was deployed in the right lower lobar bronchus to the right main bronchus. however, proximal portion of the right main bronchus was not completely covered due to its insufficient length. for this reason, another 12 mm - 5.3 cm self - expanding silicone - covered nitinol stent was reinserted, overlapping the first stent. at the end of the procedure, an immediate improvement in the aeration of the right basal lung was observed on fluoroscopy and hemoptysis was subsided (fig. nevertheless, the patient died two days later due to lack of expectoration that resulted in whole lung aspiration pneumonitis or pneumonia. a 72 year - old man, with a history of tuberculosis ten years ago and an exposure to asbestosis, was diagnosed with squamous cell carcinoma on the left lower lobe about 20 months earlier. the patient underwent left lower lobectomy after a month of the diagnosis and had been treated with postoperative chemotherapy, but it failed to prevent recurrence and progression of the disease. the ct scan, which was performed on his visit, demonstrated an extremely stenotic left main bronchus by metastatic lymphadenopathy in mediastinum and both the hila (fig. fresh bloody sputum developed on the fourth day of admission and frequency of hemoptysis gradually increased up to three times per hour. shortly afterwards, shortness of breath developed. respiration rates were raised up to a 40/min and the hemoglobin level dropped from 10.1 g / dl to 7.1 g / dl. we decided to insert the covered bronchial stent into the left main bronchus. on the fifth day after admission, 12 mm - 5.3 cm self - expanding silicone - covered nitinol stent (hercules airway stent : s & g biotech, seongnam, korea) was deployed in the left main bronchus under bronchoscopic and fluoroscopic guidance. bleeding through the endotracheal tube gradually decreased and stopped within two days after the procedure. he required continuous renal replacement therapy (crrt) due to acute renal failure by hypotension. sixteen days after the bronchial stent placement, he expired because of multi - organ failure. a 46 year - old male was diagnosed with non - small cell lung cancer 30 months earlier. the primary cancer was located in the orifice of the left upper lobe bronchus without metastasis. he was treated with left pneumonectomy after neoadjuvant chemotherapy along with postoperative radiation therapy and systemic chemotherapy. eleven months after the surgery, chest ct scans showed recurring mass at the stump of the left main bronchus, which was also confirmed by bronchoscopic biopsy. the patient underwent second and third line systemic chemotherapy, but it was unable to stop the disease from progressing. approximately four months prior to this visit, he was admitted due to presence of blood - tinged sputum. at that time, a bronchoscopic examination showed that the bleeding was from recurred mass in the left main bronchus. he visited again within several weeks due to dysphagia and recurrent blood - tinged sputum. we performed ct imaging, which depicted progression of a recurred mass at the stump of the left main bronchus. ct scan also showed narrowing of distal trachea and right proximal main bronchus (fig. in addition, it showed invasion of adjacent esophagus by the recurred mass with conglomerated lymphadenopathy. on the eighth day of admission, he coughed up approximately 500 ml of fresh blood and the patient was transferred to the intensive care unit. first, a 18 mm - 4 cm self - expanding silicone - covered nitinol stent (hercules airway stent : s & g biotech, seongnam, korea) was deployed in the distal trachea and another 12 mm - 3 cm self - expanding silicone - covered nitinol stent was deployed in the distal trachea to the right main bronchus, overlapping the former one (fig., there was a gradual decrease in the hemoptysis except for an episode of scanty bleeding that halted after conservative treatment. however, the patient needed daily bronchoscopic toileting because of lack of self - expectoration and retained mucus secretion. hemoptysis is a potentially life - threatening manifestation of underlying lung disease that requires prompt diagnosis and management. the etiology of hemoptysis varies ; bronghogenic carcinoma is one of the most common causes of hemoptysis with 19 - 32% of occurrence rate (5). the bleeding results from necrosis of a tumor, the rupture of small blood vessels in the area, or a tumor invading one of the pulmonary blood vessels. it is normally associated with mild to moderate amounts. however, massive bleeding can occur if the tumor erodes into one of the large pulmonary vessels and can signify a serious situation, where rapid treatment is crucial. management of hemoptysis aims to stop hemorrhage, replace blood loss, and treat underlying etiologies. in addition, they should be selectively intubated with a double - lumen endotracheal tube to prevent aspiration to the unaffected contralateral lung and preserve gas exchange. endobronchial tamponade using a balloon tamponade catheter is an alternate site - specific therapy through the bronchoscopy (6). however, endobronchial tamponade only offers temporary relief and prolonged mechanical ventilation is necessary (4). surgery removes the source of bleeding and is the most definitive form of therapy for patients with hemoptysis. however, a large proportion of patients are not the suitable candidates for surgery due to their advanced condition of pulmonary diseases with poor pulmonary reserves and preexisting co - morbidities. in addition, mortality rates of up to 40% have been reported following emergency surgical interventions (7). bronchial artery embolization is considered a successful procedure to control hemoptysis in various pulmonary diseases and is the most effective non - surgical treatment available for massive hemoptysis (7). however, prognosis remains poor in the patients with tumor - related hemoptysis (8). airway stents are traditionally used in the palliation of lung cancer to maintain patency of reopened airways, to secure patency of a collapsed or extrinsically compressed central airway, and to seal fistulas (1, 9). there have been two case reports that described airway stent placement to treat pulmonary hemorrhage from lung cancer (3). airway stent placement has been regarded as a successful treatment option with not only tamponade and isolation of bleeding source but also for the maintenance of airway. in the three patients mentioned in our series, the main indication for bronchial stent placement was airway obstruction with hemoptysis in advanced stages of lung cancer. immediate re - opening of airway with improvement of aeration on fluoroscopic images was also observed in all the cases. one patient died due to disease progression while surviving for three months after the procedure. the remaining two patients died within 16 days after the procedure due to non - procedural related complications like, multi - organ failure or aspiration pneumonitis. the retention of mucous secretion occurred in one stent placement and required daily bronchoscopic toileting. so, patient 's life expectancy and general condition should be considered before selecting the patients for treatment. the retention of mucous secretions is a common complication resulting from airway stent placement. covered airway stents or plastic stents prevent normal clearance of secretions and may lead to obstruction and infection. the risk with a covered stent is much higher than with a bare one (10). covered airway stents have decreased risks of tumor in - growth and granulation tissue formation. however, granulation tissue developed at the end because of irritation by the stent or the migration of a stent may block the bronchus (1, 10). other complications such as, breakage of the stent and fistula between bronchus and major vessels can also occur. in conclusion, using airway stenting for endobronchial tamponade may be considered as a treatment option for hemoptysis arising from lung cancer that can not be successfully dealt with other treatments. however, identifying the patient 's underlying condition and morbidity is essential for patient 's selection. airway stent placement also resulted in preservation of airway patency in patients with obstructive airway lesions.	malignant airway obstruction and hemoptysis are common in lung cancer patients. recently, airway stent is commonly used to preserve airway in malignant airway obstruction. hemoptysis can be managed through various methods including conservative treatment, endobronchial tamponade, bronchoscopic intervention, embolization and surgery. in our case studies, we sought to investigate the effectiveness of airway stents for re - opening the airway as well as tamponade effects in four patients with malignant airway obstruction and bleeding caused by tumors or lymph node invasions.
chronic diseases are the leading cause of mortality and morbidity and contribute significantly to the overall health expenditures from both a societal perspective as well as an individual one. common chronic diseases include heart disease, stroke, cancer, emphysema, diabetes, and osteoporosis. furthermore, cancer and cardiovascular diseases are the leading causes of mortality and morbidity worldwide, with cancer expected to result in 75,000 deaths per year in canada and 571,950 in the us. while the leading risk factor for cancer continues to be tobacco use, evidence shows that obese men and women have a greater likelihood of developing and dying from cancer than those who are not [2, 57 ]. obesity is defined as a body mass index (bmi) of 30 kg / m or greater. there are many contributing factors to obesity such as physical inactivity, unhealthy diet, genetics, and others such as metabolic, environmental, social, economic, and psychological factors. it is estimated that globally, every year, three to four million cases of cancer could be prevented by eating healthier and being more physically active. with respect to morbidity, research demonstrates that obesity increases the risk of cancers of the esophagus, breast (postmenopausal), endometrium, colon and rectum, kidney, pancreas, thyroid, gallbladder, and possibly other cancers as well. other evidence suggests that obesity leads to an increased risk for thirteen cancers including esophageal adenocarcinoma, thyroid, colon, rectal, renal, endometrial, pancreatic, gallbladder, postmenopausal breast, malignant melanoma, multiple myeloma, leukemia, and non - hodgkin lymphoma. the cost of obesity to the health care system in canada is estimated to be 4.3 billion per year. elsewhere the cost of obesity to the health care system has been found to represent 2.3% of annual hospital care costs. physical inactivity, which contributes to obesity, has been associated with significant health care expenditures for numerous chronic diseases including cancer. canadian research estimates the economic burden of obesity as $ 2.1 billion in both direct and indirect costs. data from that same study suggests that a 10% decrease in inactivity would result in health savings of $ 150 million per annum. the purpose of this meta - analysis is to synthesize the evidence evaluating the association between obesity and cancer. specifically the association between obesity and thirteen cancers shown previously to be significantly associated with obesity is the focus of this meta - analysis. in addition, among cancers for which a statistically significant positive association with obesity is observed, population - attributable risk for the canadian population is calculated and reported. several activities were included in the search strategy to identify primary studies for this paper. first, the primary studies included in renehan., for which significant associations between obesity and cancer were reported, were identified and retrieved in full document version. the search strategy employed by renehan. identified studies published between 1985 and 2007, indexed in medline and embase. three additional studies, noted by renehan., but published after their search was conducted, and therefore not included in their review, were also retrieved. the electronic searches performed by renehan. were replicated from january 2007 to may 2011 in medline and embase through ovid. all references chosen by one or both of the reviewers as being potentially relevant were selected for further review and imported into systematic review software (srs) from the centre for evidence - based medicine. finally, the reference lists of relevant studies were screened to identify additional potentially relevant studies, as well as the reference lists of published systematic reviews or meta - analyses on this topic. the following four criteria were used to assess relevance to the research question : (1) is the article a primary study ; (2) is the focus of the study to explore the relationship between obesity and cancer incidence in adults aged 18 years and older ; (3) does the study report on any one or more of the following 13 cancers : esophageal adenocarcinoma, thyroid, colon, renal, endometrial, gallbladder, rectal, malignant melanoma, postmenopausal breast, pancreatic, leukemia, multiple myeloma, and non - hodgkin lymphoma ; (4) is data on the risk ratio or odds ratio between obesity and incidence of any one or more of the 13 cancers in adults aged 18 years and over provided. both reviewers independently assessed each study for relevance and met to resolve discrepancies through discussion. all studies judged to be relevant were assessed for methodological quality by two independent reviewers using an existing quality assessment tool, based on the work of the evidence - based medicine group at mcmaster university. the assessment criteria consisted of the following components : (1) research design ; (2) identification of comparison groups, (3) comparison groups compared on important confounders at baseline, (4) outcomes and exposures measured in the same way in all groups being compared, (5) data collection tools shown to be valid, (6) data collection tools shown to be reliable, (7) follow up sufficiently long for the outcome(s) of interest ; (8) completeness of followup, (9) temporality (exposure is known to precede outcome), (10) dose - response gradient, (11) significant baseline differences controlled for in the analysis, (12) appropriate statistical tests for the research design, (13) precision of estimate of effect, and (14) sufficient detail describing study participants. studies were given an overall score out of 20 possible points and were then classified into three categories : strong, moderate, and weak. those obtaining a score of 1115 points received a rating of moderate, and those obtaining a score of 10 or less were rated as weak. reviewers independently rated each study and met to resolve discrepancies in overall ratings through discussion. studies deemed as being of weak methodological quality were excluded from further analysis as the validity of the results was questionable given the many limitations inherent in these studies. data on the population, study methods, and outcomes were extracted for each study independently by two reviewers. estimates of association were measured as a risk ratio and meta - analysis performed as a weighted average of the log risk ratios and 95% confidence intervals. in instances where odds ratios were reported for primary studies (e.g., case - control studies) these were first converted into risk ratios as suggested by renehan., and then the log risk ratios and coinciding 95% confidence intervals were calculated. tests of heterogeneity were conducted among studies using a chi square procedure, where p < 0.05 was considered an indication of heterogeneity. risk ratios were pooled using the dersimonian and laird random effects model, given that there was considerable variation in how independent and dependent variables were measured across studies, and because in most cases statistically significant heterogeneity across study results was observed. risk ratios / odds ratios and 95% confidence intervals extracted from each primary study, where those that adjusted for the greatest number of confounding variables including behavioural factors. analyses were performed and reported separately by sex. in all instances reference body mass index (bmi) was 18.524.99 which was compared to the bmi category of 30 or more. population - attributable risk (par) is the portion of the incidence of a disease in the population that is due to exposure. par% is the percent of the incidence of a disease in the population that is due to exposure. it is the percent of the incidence of the disease in the population that would be eliminated if exposure was eliminated. 1) ], where, p is the population prevalence of obesity, and rr is the pooled risk ratio. the most current obesity rates for men and women available in canada were used to represent population prevalence. in total 141 articles were identified from renehan., 1723 from the database searches and 69 from reference lists ; the total is 1933 papers. of the 141 articles included in renehan., 94 articles were relevant to the 13 cancers included in this paper. the remaining 47 articles from renehan 's review explored the association between obesity and cancers other than the thirteen of interest in this paper, and therefore were excluded. of the 1723 articles identified in the database searches, 75 were judged to be relevant. finally, of the 69 articles identified from the reference lists of relevant studies, 33 were deemed relevant. a total of 202 articles were deemed relevant for this paper. when papers were grouped according to independent studies, a total of 101 unique studies were relevant. the kappa score for agreement between reviewers on relevance assessment was 0.835, indicating high agreement. reasons studies found to be not relevant were data not reported on the 13 cancers, and/or the association between obesity and cancer in adults aged 18 years and older was not the focus of the study. it was identified during quality assessment that three studies (judged to be of moderate quality) reported data in a way that was inconsistent with the other studies, therefore could not be aggregated with other studies. one was assessed as being of strong methodological quality, 56 were rated as moderate and 41 as weak. the following criteria distinguished strong and moderate studies from weak studies : strong and moderate studies tended to use measurement tools with proven validity and reliability, demonstrated that obesity preceded cancer incidence, and established a dose - response gradient. studies of weak methodological quality rated poorly on these criteria as well as research design. a summary of the quality assessment of the 57 studies included in the meta - analysis is presented in figure 1. quality assessment of the weak studies not included in this publication can be requested from the primary author. the study designs included 43 cohort and 14 case - control studies published between 1985 and 2011. the majority of studies were conducted in the united states (19), followed by sweden (7), norway (4), and japan (5). sixteen studies were included in the meta - analysis assessing the association between obesity and colon cancer among men (figure 2(a)) and 13 studies among women (figure 2(b)). the pooled risk ratio illustrated that obese men had an increased risk of colon cancer compared to men of normal weight (rr 1.57 ; 95% confidence interval 1.48 to 1.65). the pooled risk ratio illustrated that obese women had an increased risk of colon cancer compared to women of normal weight (rr 1.19 ; 95% confidence interval 1.04 to 1.36). the pooled risk ratio from 16 studies illustrated that obese women had an increased risk of endometrial cancer compared to women of normal weight (rr 1.85 ; 95% confidence interval 1.3 to 2.65) (figure 3). there were 3 studies that assessed the association between obesity and esophageal adenocarcinoma in men (figure 4(a)) and 4 studies among women (figure 4(b)). the pooled risk ratio demonstrated that obese men did not have an increased risk for esophageal adenocarcinoma compared to men of normal weight (rr 1.23 ; 95% confidence interval 0.58 to 2.60). there was significant heterogeneity across studies (heterogeneity p = 0.02). among obese women, however, the pooled risk ratio indicated a sizable increased risk of esophageal adenocarcinoma compared to women of normal weight (rr 2.04 ; 95% confidence interval 1.18 to 3.55). there were 3 studies each assessing the association between obesity and gallbladder cancer in men (figure 5(a)) and women (figure 5(b)). the pooled risk ratio illustrated that obese men had an increased risk of gallbladder cancer compared to normal weight men (rr 1.47 ; 95% confidence interval 1.17 to 1.85). similarly, among obese women the pooled risk ratio demonstrated an increased risk of gallbladder cancer compared to women of normal weight (rr 1.82 ; 95% confidence interval 1.32 to 2.50). there were 2 studies that assessed the association between obesity and leukemia among men (figure 6(a)) and women (figure 6(b)). the pooled risk ratio illustrated that obese men had no increased risk of leukemia compared to men of normal weight (rr 1.16 ; 95% confidence interval 0.88 to 1.52). for obese women, however, the pooled risk ratio illustrated an increased risk of leukemia compared to women of normal weight (rr 1.32 ; 95% confidence interval 1.08 to 1.60). four studies assessed the association between obesity and malignant melanoma among men (figure 7(a)) and 3 among women (figure 7(b)). the pooled risk ratio illustrated that obese men had an increased risk of malignant melanoma compared to men of normal weight (rr 1.26 ; 95% confidence interval 1.07 to 1.48). the pooled risk ratio for obese women showed no increased risk of malignant melanoma compared to women of normal weight (rr 0.95 ; 95% confidence interval 0.84 to 1.07). only one study for men (figure 8(a)) and two studies for women (figure 8(b)) assessed the association between obesity and multiple myeloma. the results reported by samanic., 2006 showed obese men had significantly lower risk of multiple myeloma compared to men of normal weight (rr 0.58 ; 95% confidence interval 0.36 to 0.93). the pooled ratio for women showed an increased risk of multiple myeloma compared to women of normal weight, although the 95% confidence interval was just short of reaching statistical significance (rr 1.20 ; 95% confidence interval 0.99 to 1.45). there was no significant heterogeneity across the two studies (heterogeneity p = 0.39). four studies in men (figure 9(a)) and six in women (figure 9(b)) assessed the association between obesity and non - hodgkin lymphoma. the pooled risk ratio indicated there was no increased risk of non - hodgkin lymphoma among obese men compared to men of normal weight (rr 1.09 ; 95% confidence interval 0.98 to 1.21). there was no significant heterogeneity across studies (heterogeneity p = 0.46). among obese women, however, the pooled risk ratio showed a reduced risk of non - hodgkin lymphoma compared to women of normal weight (rr 0.91 ; 95% confidence interval 0.86 to 0.97). nine and ten studies, respectively, assessed the association between obesity and pancreatic cancer in men (figure 10(a)) and women (figure 10(b)). the pooled risk ratio illustrated that obese men had an increased risk of pancreatic cancer compared to men of normal weight (rr 1.36 ; 95% confidence interval 1.07 to 1.73). there was significant heterogeneity across studies (heterogeneity p = 0.01). among obese women the pooled risk ratio also showed an increased risk of pancreatic cancer compared to women of normal weight (rr 1.34 ; 95% confidence interval 1.22 to 1.46). eleven studies assessed the association between obesity and postmenopausal breast cancer (figure 11). the pooled risk ratio demonstrated that obese women had an increased risk of postmenopausal breast cancer compared to women of normal weight (rr, 1.25 ; 95% confidence interval 1.07 to 1.46). there were 11 studies for men (figure 12(a)) and nine for women (figure 12(b)) that assessed the association between obesity and rectal cancer. the pooled risk ratio illustrated that obese men had no increased risk of rectal cancer compared to men of normal weight (rr 1.22 ; 95% confidence interval 0.91 to 1.64). similarly among obese women the pooled risk ratio illustrated no increased risk of rectal cancer compared to women of normal weight (rr 1.03 ; 95% confidence interval 0.74 to 1.44). there were 3 studies each for men (figure 13(a)) and women (figure 13(b)) that assessed the association between obesity and renal cancer. the pooled risk ratio illustrated that obese men had an increased risk of renal cancer compared to men of normal weight (rr 1.57 ; 95% confidence interval 1.38 to 1.77). similarly, among obese women the pooled risk ratio illustrated an increased risk of renal cancer compared to women of normal weight (rr 1.72 ; 95% confidence interval 1.58 to 1.88). there were 6 studies included in the meta - analysis assessing the association between obesity and thyroid cancer among men (figure 14(a)) and 4 studies among women (figure 14(b)). the pooled risk ratio illustrated that obese men had no increased risk of thyroid cancer compared to men of normal weight (rr 1.12 ; 95% confidence interval 0.72 to 1.72). likewise, among obese women the pooled risk ratio demonstrated no increased risk of thyroid cancer compared to women of normal weight (rr 1.03 ; 95% confidence interval 0.87 to 1.23). table 1 summarizes the results of the observed associations between obesity and the 13 cancers included in this meta - analysis. among men a statistically significant increased risk of cancer was observed for the following 5 cancers : colon, gallbladder, malignant melanoma, pancreatic, and renal cancer. in addition, obese men had significantly lower risk of multiple myeloma in comparison to men of normal weight. among women a statistically significant increased risk of cancer was observed for the following 8 cancers : colon, endometrial, esophageal, gallbladder, leukemia, pancreatic, postmenopausal breast, and renal. obese women also had significantly lower risk of non - hodgkin lymphoma compared to women of normal weight. as an example to illustrate the use of meta - analysis results reported in this study, the par% for men and women in canada was calculated on cancers for which a statistically significant association between obesity and cancer incidence was observed (table 2). the population prevalence of obesity in canada in 2002 was 22.9% in men and 23.2% in women. among men par% ranged from a low of 5.6% to a high of 11.5%. the par% for obesity among men was greatest for colon and renal cancer (11.5%), gallbladder cancer (9.7%), pancreatic (7.6%), and malignant melanoma (5.6%). when the 95% confidence intervals for par% for men are considered the results are particularly noteworthy. for example for colon cancer par% is as low as 9.9% but at the upper limit of the confidence interval par% is 13.1%. likewise, for renal cancer the 95% confidence interval for par% ranged from 8.0% to 15%, while for pancreatic cancer, it ranged from 1.6% to 17.5%, and 1.6% to 9.9% for malignant melanoma. among women par% ranged from a low of 4.2% to a high of 19.4%. the par% for obesity among women was greatest for esophageal adenocarcinoma (19.4%), endometrial (16.5%), gallbladder (16%), and renal cancer (14.3). when the 95% confidence intervals for par% for women are considered the results are also noteworthy. for example, at the upper limit of the confidence interval, par% was as high as 37.2% for esophageal adenocarcinoma, 27.7% for endometrial cancer, and 25.8% for gallbladder cancer. however, at the lower limit the par% was as low as 0.9% for colon cancer, 1.6% for postmenopausal breast cancer, and 1.8% for malignant melanoma. the results of this meta - analysis demonstrate a significant association between obesity and some of the 13 cancers for men and many of the 13 cancers for women. obesity was significantly and positively associated with 5 of the possible 11 cancers relevant to men including, colon, gallbladder, malignant melanoma, pancreatic, and renal. a significant association was not observed among obese men for esophageal adenocarcinoma, leukemia, multiple myeloma, non - hodgkin lymphoma, rectal, and thyroid cancer. among women obesity was significantly and positively associated with 8 of the 13 cancers including, colon, endometrial, esophageal adenocarcinoma, gallbladder, leukemia, pancreatic, postmenopausal breast, and renal cancer. a significant association was not observed among women for malignant melanoma, multiple myeloma, non - hodgkin lymphoma, rectal cancer, and thyroid cancer. the magnitude of the associations between obesity and cancer incidence in men and women, including the 95% confidence intervals, is particularly noteworthy. for example, obese men have an increased risk of developing colon and renal (rr both 1.57), gallbladder (1.47), pancreatic cancer (1.36), and malignant melanoma (1.26) in comparison to nonobese men. for example the risk ratio is as high as 1.77 for renal cancer, 1.73 for pancreatic cancer and 1.65 for colon cancer. even at the low end of the 95% confidence interval, cancer risk is still noteworthy at 1.48 for colon cancer, and 1.38 for renal cancer. the results for obese women are equally concerning. obese women are not only at higher risk for developing cancer at more sites than men, the risk associated with some of the cancers is significantly higher. for example, obese women have an increased risk for developing esophageal adenocarcinoma (rr 2.04), endometrial (1.85), gallbladder (1.82), and renal cancer (1.72). where statistically significant associations are observed only one risk ratio is less than 20% (colon 1.19), while the remaining risk ratios range between 1.25 (postmenopausal breast), 1.32 (leukemia), and 1.34 (pancreatic). for example, at the upper limit of the confidence interval the risk ratio is as high as 3.55 for esophageal adenocarcinoma, 2.65 for endometrial, and 2.50 for gallbladder cancer. the results also clearly demonstrate that a significant proportion of cancer incidence could be avoided, in canada, by reducing the percentage of obese adults. the population - attributable risk percent for obesity in canada was highest in men for five cancers : colon, renal, gallbladder, pancreatic, and malignant melanoma. for women the greatest benefits from obesity reduction and prevention efforts would be observed for esophageal adenocarcinoma, endometrial, gallbladder, renal, pancreatic, leukemia, postmenopausal breast, and colon cancer. significant reductions in these cancers for men and women in canada would result in decreased health care expenditures and improved quality of life for many men and women in canada. when the results of this meta - analysis are compared to those of renehan., who reviewed similar but earlier literature on obesity and cancer, a number of important similarities and differences are observed. first, there were some differences in the associations found between the two meta - analyses for men. reported a significant and positive association for the following five cancers that was not reported in this meta - analysis : leukemia, multiple myeloma, non - hodgkin lymphoma, rectal, and thyroid cancer. furthermore, renehan. did not report a significant and positive association for gallbladder and pancreatic cancer whereas a significant and positive association was reported in this paper. for example, there were only two cancers, multiple myeloma and thyroid cancer where renehan. reported a positive and significant association with obesity for women, and this paper did not. there were no differences for the remaining eleven cancers on the relationship between obesity and cancer in women. the second important difference between the two meta - analyses concerns the observed magnitude of the association and the coinciding 95% confidence intervals. generally the results of our meta - analysis demonstrated higher magnitude of associations and confidence intervals. the most notable differences occurred for four cancers among men : renal, colon, pancreatic, and gallbladder cancer. for example, we reported a pooled risk ratio and 95% confidence interval of 1.57 (1.38, 1.77) for renal cancer, while renehan. reported 1.24 (1.15, 1.34). likewise, for colon cancer, we reported 1.57 (1.48, 1.65), while renehan. reported 1.24 (1.20, 1.28), and for pancreatic cancer, we reported 1.36 (1.07, 1.73) and renehan. finally, for gallbladder cancer, we reported 1.47 (1.17, 1.85) while renehan. reported 1.09 (0.99, 1.21). similar differences existed for obese women with the greatest differences in the pooled risk ratios and 95% confidence intervals being for three cancers : endometrial, renal, and gallbladder. for these cancers the magnitude of associations we reported is larger than those reported by renehan. for endometrial cancer we reported a risk ratio of 1.85 (1.30, 2.65), while renehan. for renal cancer we reported 1.72 (1.58, 1.88) while renehan reported 1.34 (1.25, 1.43), and for gallbladder cancer we reported 1.82 (1.32, 2.50) and renehan, 1.59 (1.02, 2.47). reported a larger risk ratio than we did, which was for non - hodgkin lymphoma. likely the most significant factor contributing to the differences in the results of the two meta - analyses are the different approaches to the statistical methods used to aggregate the data across studies. the approach used by renehan assessed the risk in developing cancer as bmi increased every 5 points, from the lowest bmi category up to the highest category. this strategy incorporates the risk of those who have a bmi slightly above normal, to above normal to those identified as obese. given the risk of developing cancer at lower bmi levels is generally less than that of obese persons which provides some explanation why renehan 's results generally illustrate lower overall risk and more narrow confidence intervals. in this paper we compared the risk of developing cancer between those in the lowest bmi category (normal healthy weight) and those considered obese (bmi 30 +) category. our meta - analysis therefore did not incorporate into the pooled risk ratio and 95% confidence intervals, the risk associated with developing cancer among those with slightly above normal and above normal bmi. given the primary objective of this paper was to assess the relationship between obesity and cancer incidence we believe this was the most appropriate statistical analysis to answer the research question. answered was broader in that they were interested in assessing across the spectrum of bmi from normal to obese, the relationship between bmi and cancer risk. a second major difference between the two reviews was our exclusion of studies judged to be of weak methodological quality. the exclusion of these studies likely contributed to the higher pooled risk ratios observed in this meta - analysis in comparison to renehan. the combined effect of the different statistical approaches and the exclusion of studies of weak methodological quality likely explains much of the observed differences between the reviews. meta - analysis, report higher relative risks than had been reported previously. given several studies published since 2007 were added to this paper, this may further explain the higher pooled risk ratios and 95% confidence intervals observed in this paper. it is important to note that the results from some of the studies included in this paper may not be relevant to a canadian context. for example, studies conducted in south east asian countries may have limited applicability in canada. however, a rigorous and systematic meta - analysis process should not limit study inclusion by the country in which the study was conducted. as a result a decision was made to be more rather than less inclusive. furthermore, in most instances, studies conducted in countries quite dissimilar to canada constituted relatively little overall weight in the point estimate, and therefore the impact of the results of these studies on the point estimates is relatively small. first, the evidence reported here continues to establish the evidence base suggesting a statistically significant and positive association between obesity and cancer risk. in comparison to nonobese men and women, obese men and women women are at particularly high risk for as many as eight cancers, and the magnitude of the association is very high. similarly among obese men, there is an increased risk for five cancers. given obesity generally is preventable public health efforts to implement effective population wide programs to reduce and prevent obesity are needed.	background. cancer and cardiovascular diseases are the leading causes of mortality and morbidity worldwide. the purpose of this meta - analysis is to synthesize the evidence evaluating the association between obesity and 13 cancers shown previously to be significantly associated with obesity. methods. relevant papers from a previously conducted review were included in this paper. in addition, database searches of medline and embase identified studies published from the date of the search conducted for the previous review (january, 2007) until may, 2011. the reference lists of relevant studies and systematic reviews were screened to identify additional studies. relevance assessment, quality assessment, and data extraction for each study were conducted by two reviewers independently. meta - analysis was performed for men and women separately using dersimonian and laird 's random effects model. results. a total of 98 studies conducted in 18 countries from 1985 to 2011 were included. data extraction was completed on the 57 studies judged to be of strong and moderate methodological quality. results illustrated that obese men were at higher risk for developing colon (risk ratio (rr), 1.57), renal (1.57), gallbladder (1.47), pancreatic (1.36), and malignant melanoma cancers (1.26). obese women were at higher risk for esophageal adenocarcinoma (2.04), endometrial (1.85), gallbladder (1.82), renal (1.72), pancreatic (1.34), leukemia (1.32), postmenopausal breast (1.25), and colon cancers (1.19). conclusions. the results of this meta - analysis illustrate a significant, positive, and, for some cancers, strong association between obesity and cancer incidence. given that approximately 23% of canadians are obese, a significant proportion of cancer in canada could be avoided if obesity was eliminated or significantly reduced.
ion channels are integral membrane proteins that govern the passage of ions across cell membranes. encoded by approximately 400 ion channel genes in the human genome, this superfamily of membrane proteins is involved in many important physiological functions such as the regulation of blood pressure, neurotransmission, and hormonal secretion. ion channels are implicated in a wide range of diseases including hypertension, neuromuscular disorders and parkinson s disease. consequently, they constitute the third largest class of targets in drug discovery after protein kinases and g - protein coupled receptors. despite its wide implication in disease conditions, the development of drugs targeting this membrane protein superfamily has remained underexploited, with only about 10% of drugs on the current market known to bind to ion channels. since the advent of high - throughput screening (hts) for drug discovery in the 1980s, hts has become an important tool for hit identification in pharmaceutical research. in the past decade, strategies have evolved from traditional diverse hts to the screening of focused libraries for a particular class of biological targets. for example, protein kinase - focused screening libraries have been reported by us and other research groups using well - documented structural motifs to select compounds satisfying a defined pharmacophore. however, in comparison to protein kinases, the assembly of a focused screening library targeting ion channels is more challenging owing to limited structural information about the targets, their structural diversity, and the absence of well - defined pharmacophores required for binding to ion channels.(10) the release of the chembl database(11) has facilitated open - access to a large volume of small - molecule bioactivity data for various therapeutic target families, including ion channels. here, we describe an efficient workflow for compiling a focused screening library for ion channel targets using a combination of database mining and various chemoinformatics analytical tools for structural class generation and compound selection. the established workflow can easily be adopted to assemble focused screening libraries for other therapeutic target classes with diverse recognition motifs. to enable an efficient assembly of the ion channel - focused screening library, the procedure was divided into five stages (figure 1). bioactivity data of compounds active against ion channel targets were retrieved from the chembl database.(11) this data set (chemblinitial data set) contained 25150 compounds reported to be active against 337 different molecular targets. the majority of the classification of ion channel categories followed those as defined in chembl, apart from the groups of ligand- (lgic) and voltage - gated ion channels (vgc) which were further divided to facilitate data handling. acetylcholine and serotoninergic 5ht3 receptors were grouped together to form the cationic cys - loop channels (cationiccysloop), whereas gabaa and glycine receptors were categorized as anionic cys - loop channels (anioniccysloop). for vgc, each of sodium (na), calcium (ca) percentage composition per ion channel category in the chemblinitial, chemblfiltered, and ddu_ic data sets. no desirable bioactive compounds were found for four categories of ion channels (amiloride - sensitive sodium channels (asic), cgmp - gated channels, ryanodine receptors, and ip3 receptors). various filters were applied to the chemblinitial data set for the selection of compounds to form the chemblfiltered data set (figure 3). first, filters were introduced to exclude compounds for which bioactivity data was reported only for species other than rat, mouse or human. next, the chembl confidence scores for all bioactivity data were checked to ensure there was no experimental data representing activity against nonmolecular or nonprotein targets (chembl confidence score between 1 and 3 inclusive). further, only compounds with reported bioactivity (ki, kd, ic50, or ec50) of 10 m were kept. although this cutoff value would allow compounds that violate lipinski s rule - of - five,(12) it was considered appropriate at this stage of the analysis to minimize loss of core structural information when generating structural classes (see below). finally, compounds containing unwanted groups(8) were excluded. this led to a collection of 7102 compounds representing 10 ion channel categories (figure 2). commercial availability search for compounds of the chemblfiltered data set using our in - house database(8) revealed only 329 available compounds. in light of this, bioactive templates representing the different structural classes of ion channel modulators were generated for subsequent substructure searches. bioactive templates were identified by searching for maximum common substructures (mcs) of compounds within each ion channel category in the chemblfiltered data set. during this process, singletons and under - represented classes (see experimental procedures) afterward, the structures of the bioactive templates were visually inspected, and any synthetically intractable structures were rejected to avoid the presence of synthetically challenging compounds in the final screening library that would not be proceeded as hit or lead candidates. 307 bioactive templates out of 548 generated templates were selected in the final collection and annotated against their respective categories of ion channels (table 1). after merging identical templates present in multiple categories, 297 unique bioactive templates were used as substructures to search for commercially available lead - like compounds in our in - house database.(8) this search identified 92340 compounds representing 149 bioactive templates, forming the commavail data set which contained on average 620 compounds per template. to avoid over - representation of certain templates and to keep the library at an affordable size, a maximum of 50 compounds per bioactive template was imposed.(8) in the 77 templates which were represented by more than 50 compounds (figure 4), molecular diversity of compounds was analyzed using molecular fingerprints and the 50 structurally most diverse compounds were retained in the data set. subsequently, the data set was visually inspected to remove any compounds containing synthetically intractable structures attached to the templates. finally, all bioactive templates which had five or fewer examples remaining were considered under - represented, and therefore these compounds were excluded from the data set. number of compounds per bioactive templates across different ion channel categories in the commavail data set. the 3241 compounds that passed all filter steps were purchased to form the final focused screening library (ddu_ic data set) (figure 2). covering 123 bioactive templates across nine ion channel categories, these compounds were annotated with their respective ion channel category to assist back - tracing of prospective molecular targets from phenotypic screening results. despite the number of templates progressively decreasing from 297 templates in the chemblfiltered data set to only 123 templates in the ddu_ic data set, the percentage composition of each ion channel category remained approximately constant throughout the process (figure 5). on average, out of the nine ion channel categories represented, only the anioniccysloop category showed a considerable reduction, with eight out of the 36 templates (22%) in chemblfiltered represented in ddu_ic. in contrast, 61% of the 34 templates for transient receptor potential channels were represented in the final library. percentage composition of each ion channel category by number of templates in the chemblfiltered, commavail, and ddu_ic data sets. screening of focused libraries is considered to be a cost - effective strategy for hit discovery. compiling focused libraries requires analysis of relevant chemical space in order to enrich compounds that are likely to interact with the desired target class.(5) this is commonly achieved by defining pharmacophoric or structural motifs satisfying specific binding interactions for the desired target class, which consequently requires a thorough understanding of the structural features and patterns of the protein ligand interactions. therefore, focused library construction is more challenging when the recognition motifs of the target class are less established.(10) with the first crystal structures of ion channel targets just emerging(13) and due to their heterogeneous nature involving 16 subfamilies,(14) the assembly of focused screening libraries for ion channels clearly represents a demanding task. the workflow described here (figure 1) provides an efficient protocol to analyze relevant bioactive data for ion channels and to use this information for compiling a focused library. however, since most of the compounds in chembl are not commercially available,(15) the identification of bioactive templates using mcs represents an effective method to derive substructures which can subsequently be used to retrieve commercially available compounds that are likely to modulate ion channel activity. in addition to improving compound availability, these bioactive templates, unlike many descriptors derived to predict bioactivity of chemical compounds, are easy to interpret and do not require expert chemoinformatics knowledge, hence synthetically intractable templates can be rejected at an early stage by visual inspection. besides, this mcs approach also allows the identification of promiscuous templates which appear across multiple ion channel categories. indeed, we identified eight bioactive templates which are common to multiple ion channel categories using this workflow. such observation would have been much more difficult if using more traditional similarity - based approaches such as molecular fingerprints comparison. however, promiscuous inhibitors(18) in chembl erroneously reported to be active against certain targets are difficult to be detected using this workflow. the selected compounds can be annotated with the respective ion channel category they were derived from, which facilitates target identification when using the library for phenotypic screening. this workflow is not limited to ion channels but can be adapted to any target family for which chemical information is available in chembl or other related databases. the presence of gaps for ion channel - active compounds in lead - like commercial chemical space became apparent when assembling the ion channel library. only 329 compounds (< 5%) in the chemblfiltered data set were commercially available based on our in - house database. this is perhaps not surprising, since many compounds in chembl are retrieved from medicinal chemistry literature, which often describes hit or lead optimization efforts. when searching for lead - like commercially available compounds(8) using the 297 bioactive templates derived from mcs (table 1), compound availability was vastly improved, although a significant proportion of the selected templates remained unrepresented (figure 5). these unavailable templates are present across all ion channel categories (table s1 in the supporting information), and many of them do not appear to be synthetically more challenging than the available ones (table 2). a similar observation was noted before when we assembled a focused kinase screening library.(8) the observed absence of ion channel templates in commercial chemical space is also supported by a recent publication by chuprina. who estimated a generally low occurrence of prospective ion channel modulators relative to other target classes from a collection of commercial chemical suppliers similar to those in our in - house database.(19) although the collection of suppliers in our database may not represent a comprehensive coverage of the entire commercial chemical space, our observations here, together with that of chuprina., indicate there is still scope for compound vendors to increase the diversity of lead - like compounds on offer in their libraries. we have demonstrated an efficient workflow to assemble a focused screening library for ion channel targets using bioactivity data retrieved from chembl. the workflow is based on the efficient mining of an open - access database containing bioactivity data for structurally diverse therapeutic target families and demonstrates an effective solution using bioactive templates to overcome the problem associated with limited compound availability. the final screening library contained 3241 compounds representing 123 templates across nine ion channel categories. these compounds were annotated with their respective ion channel category to enable efficient back - tracing of prospective molecular targets from phenotypic screening results. the screening library is currently being used in campaigns to identify new chemical starting points toward ion channel targets for various neglected disease programs within the drug discovery unit at dundee. these results will offer valuable data to evaluate the quality of this newly assembled screening library. bioactivity data of compounds annotated with associated ion channel targets (337 molecular targets in 14 categories) were retrieved from the chembl database (accessed feb 16mar 4, 2010). all filters applied were carried out using pipeline pilot professional client 7.5 (accelrys, inc.). unwanted groups were described as smarts strings,(8) and compounds were matched against the smarts description using substructure mapping. structural classes of the chemblfiltered data set were generated using classpharmer 4.7 (simulationsplus, inc.). the criteria of structural classes were a minimum of 2 rings (either 2 single rings linked together or 1 fused ring), 13 functional groups attached to any ring system, a maximum of 5 bonds between a ring and a functional group, and a maximum of 5 bonds between ring - connected fragments. under - represented classes were defined as those containing fewer than five compounds of which no bioactivities were better than 5 m. these templates were used as substructures to search for commercially available compounds in our in - house database containing 5.9 million unique compounds from 20 commercial chemical suppliers (as of june 2010). compounds in the commavail data set were chosen according to the lead - like criteria as previously described.(8) compound clustering and the selection of representative examples followed the same procedure as previously published.(8)	the chembl database was mined to efficiently assemble an ion channel - focused screening library. the compiled library consists of 3241 compounds representing 123 templates across nine ion channel categories. compounds in the screening library are annotated with their respective ion channel category to facilitate back - tracing of prospective molecular targets from phenotypic screening results. the established workflow is adaptable to the construction of focused screening libraries for other therapeutic target classes with diverse recognition motifs.
a 49-year - old woman diagnosed with stage iv ovarian cancer underwent extensive debulking surgery. due to the presence of right hydronephrosis, a right nephrostomy tube was inserted prior to the second surgery. during surgery, additional excision was performed because of ovarian tumor invasion of the bladder and right ureter. her right distal ureter was partly resected ; the distal margin of the remaining ureter was at the l5 level. as the remaining ureter was not long enough for anastomosis and the level of the injury site was high, ureteroureterostomy or ureterocystostomy were not possible. therefore, ureter ligation was performed by suturing the ureter three times and clipping it once. however, four days postsurgery, an antegrade ureterogram through the nephrostomy tube showed contrast extravasation at the site of ligation. we believed that the ureter was unlikely to seal up spontaneously owing to the relatively large size of the wall defect seen on the ureterogram. considering the diameter of the ureter at the transection site, the high level of the distal margin of the remaining ureter, and the patient s generally poor condition, an intervention consisting of complete ureter occlusion was planned. the occlusion stent was made of nickel / titanium alloy, and was super - elastic and self - expandable. the middle of the stent was constricted in a candy - wrapper configuration, leading to complete obstruction of the central portion (fig. when fully expanded, the stent was 10 mm in diameter and 4 cm in length. the stent was internally coated with a thin silicone membrane, leaving a bare portion at either end ; the bare portion at the distal end was intended to permit tissue growth through the stent interstices and thus prevent stent migration. the stent was constructed according to our specifications by a local manufacturer (s&g biotech, seongnam, korea). with the patient in a prone position, the right nephrostomy tube was replaced with a 9-fr vascular sheath (pinnacle tif tip, terumo, tokyo, japan). the exact leakage site at the distal margin of the ureter was confirmed with contrast - medium injection through a 5-fr kumpe catheter (cook, bloomington, in, usa). using a guidewire, an 8-fr introducer sheath was passed through the 9-fr vascular sheath, into the ureter. stent placement was technically successful and there were no immediate procedure - related complications (fig. injection of contrast medium through the ureter showed no contrast material passing beyond the occlusion stent, and no further ureteral leakage (fig. 2c). an 18-month follow - up antegrade ureterogram showed neither contrast - medium leakage nor migration of the occlusion stent (fig. although ureteral leakage is an uncommon complication following abdomino - pelvic surgeries or procedures, it can lead to serious conditions such as ureterovaginal fistula, intra - abdominal sepsis, renal failure, and loss of the ipsilateral kidney. numerous approaches have been attempted for the management of ureteral leakage, depending on the patient s general condition and the anatomic location of the leakage. in general, surgical management should be considered as the final option since repeated surgery could be harmful if the patient is unstable. in many cases, there are very limited reconstructive options available, considering the extent and location of the injury. transrenal ureter occlusion with permanent urinary diversion may be indicated as a palliative treatment for patients with a surgically or endourologically unmanageable anatomic defect of the ureter. several techniques have been suggested using tissue adhesive, balloons, coils, electrocautery, stents, and siliconeoccluding devices, but all have varying degrees of success. the reasons for incomplete occlusion of ureteral leakage have been continuous urinary flow at a blockage site, recanalization of an occlusion, or migration of the inserted materials. in the vascular system, the coagulation cascade is triggered around a foreign device, which finally leads to the desired obstruction. however, as there are no platelets or clotting factors in normal urine, some additional anchoring, such as a fixing component or a self - expandable feature, would be necessary for the device to remain in the urinary tract without migration. here, we review various techniques reported till date. isobutyl-2-cyanoacrylate has previously been used for intravascular embolization. gunther. reported its use in several patients, for achieving complete blockage of ureteral fistula via the transrenal approach. however, moldwin and smith subsequently announced that the results with isobutyl-2-cyanoacrylate were inconsistent, because the material gradually softens and may allow migration by ureteral peristalsis. two approaches of balloon occlusion have been reported using detachable balloons and nondetachable balloons. in 7 patients, gunther. successfully used detachable balloons inflated with a silicone mixture, and reported complete ureteral occlusion without complications at 16 months. additionally, papanicolaou. described the application of nondetachable balloons for temporary ureteral occlusion. although no complications have been reported following the procedures, long - term use of balloons carries potential risks for ureteral necrosis or rupture. the outcome of using coils and gelatin sponges in 22 patients, were described by farrell.. except for four patients who died owing to progression of underlying malignancy, all the other patients remained symptom - free throughout the follow - up period ranging from 2 to 29 months. coils may produce foreign body reactions in ureters, namely localized inflammation with the ingrowth of granulation tissues. ureteral occlusion with coils has been reported to have relatively low complication rates compared with other methods. recently, the latex - covered amplatzer vascular plug (avp) showed promising results in the occlusion of ureteral leakage ; in the study, 9 of 10 ureters were completely sealed off as the expandable characteristics of avps and the latex that covers avps before implantation seemed to provide an effective watertight barrier. metallic stents have generally been used for retaining the patency of vessels and nonvascular luminal structures. they have also been applied to the urinary tract, and there is a high tendency for obstruction due to reactive tissue hyperplasia. we paid attention to this occlusive mechanism and designed our occlusion stent. to our knowledge, there is no report about the specific shape and function of a ureteral metallic stent designed based on the occlusive principle. in our study, the expandable characteristics of the nitinol stent frame, as well as the silicone membrane with midline constriction, resulted in a completely watertight arrangement. in addition, the bare metallic portions at both ends might precipitate tissue ingrowth through the mesh of the stent, allowing the desirable ureteral occlusion. moreover, the combination of the expansile feature of the nitinol and the two ends with bare metallic portion in our stent would serve as an antimigration system. this would make it superior to some other devices using only the expansile characteristic to prevent migration. in addition, the distinctive candy - wrapper configuration would prevent the separation of the silicone membrane and stent meshes. the durability and long - term effectiveness of this occlusion stent were confirmed in the 18-month follow - up ureterogram. a similar kind of occlusion stent with a wine - glass shape has been reported to successfully treat bronchopleural fistulas, following pneumonectomy ; in this study, a silicone - covered occlusion stent with a self - expandable feature and antimigration barbs was used for the remaining left main bronchus. we propose the use of our specially designed, silicone - covered nitinol ureteral occlusion stent with a candy - wrapper configuration for the treatment of ureteral leakage. this stent is a less invasive, simple, and practical solution for patients with urinary leakage and in whom surgery is not indicated.	ureteral fistula is a serious complication of abdomino - pelvic surgeries, often resulting in poor outcomes owing to lack of proper treatment. we report the case of a 49-year - old woman who underwent placement of a silicone - covered ureteral occlusion stent in her right ureter for the management of ureteral leakage after pelvic surgery. a ureterogram obtained 18 months following the stent placement confirmed that there was no stent migration or additional urine leakage. we propose that the silicone - covered ureteral occlusion stent is practical, fast, and safe for the management of ureteral leakage.
much of current cancer research is focused on exploring novel targets and discovering new anticancer drugs. while the availability of new molecules certainly offers hope to patients who do not respond to existing drugs, development of each new molecule many highly active drug candidates have been shelved because of poor physicochemical or pharmacokinetic characteristics. reformulation of such molecules can overcome their unfavorable biological behavior and would offer a less expensive approach to anticancer drug development. tylocrebrine, a phenanthropiperidine alkaloid, is an example of a drug with potent anticancer activity and whose clinical trial was discontinued following the discovery of severe central nervous system (cns) toxicities. the cns toxicities of the drug were likely caused by its extensive penetration into the brain. we hypothesized that encapsulation of tylocrebrine in poly(lactide - co - glycolide) (plga)-based polymeric nanoparticles will limit the distribution of the drug to the cns, potentially decreasing its neurological toxicities. additionally, nanoparticles are known to passively accumulate in the tumor tissue through the enhanced permeability and retention (epr) effect. the combination of these properties should result in significantly improved therapeutic index. to further enhance the tumor cell uptake and retention of nanoparticles, we functionalized the nanoparticle surface with a peptide capable of targeting the epidermal growth factor receptor (egfr), which is overexpressed on the cell membrane of multiple solid tumors. using in vitro and in vivo models of egfr - overexpressing tumors our studies show that this reformulation strategy significantly improved the antitumor efficacy while reducing the brain penetration of tylocrebrine. amine - terminated poly(ethylene glycol) (molecular weight 3400 da) was purchased from laysan bio inc. plga (50:50 molar ratio of lactide glycolide, molecular weight 40 kda) was purchased from lactel (birmingham, al). poly(vinyl alcohol) (molecular weight 3070 kda) and lactic acid were obtained from sigma - aldrich co. (st. louis, mo). egfr - targeting peptide (yhwygytpqnvi) and scrambled peptide (hwpyahpthpsw) were obtained from peptide 2.0, inc. radioimmunoprecipiation (ripa) buffer and bicinchoninic acid assay kit were obtained from thermo scientific (rockford, il). all other chemicals were obtained from sigma - aldrich co. (st. louis, mo). a549 cells were cultured in dulbecco s minimum essential media (dmem), while a431 cells were grown in roswell park memorial institute media (rpmi-1640). both media were supplemented with 10% v / v fetal bovine serum and 1% v / v penicillin streptomycin. the cells were grown in a humidified environment consisting of 5% co2/95% air and were maintained at 37 c. we determined the effect of extracellular ph on the cell accumulation of tylocrebrine. for low ph conditions, serum - free rpmi was acidified with 9.1% v / v 0.1 m hcl. the ph of acidified rpmi was maintained between 6.3 and 6.7 for 6 h when placed under routine cell culture conditions. aliquots of 5 10 a431 cells were seeded in a 24-well plate and allowed to adhere overnight. tylocrebrine was first dissolved in 1 m hcl (1:1 molar ratio) and then diluted in serum - free rpmi to prepare a stock solution of 1 mg / ml. the stock solution was then diluted to 5 g / ml in neutral or acidic serum - free rpmi and added to the cells. treatments were removed 1 h later, and the cells were washed with cold 1x phosphate buffered saline (pbs). cells were digested with ripa buffer (0.1 ml) for 15 min, and the cell lysate was divided into two parts. one part (20 l) was analyzed by bicinchoninic acid assay (bca) to determine cell protein concentration (elx800 absorbance microplate reader, biotek inc., winooski, vt). the other part (80 l) was extracted overnight with methanol and tylocrebrine concentration in the methanol extract and was analyzed using high - performance liquid chromatography (hplc). hplc was performed on a beckman coulter hplc system equipped with a system gold 508 autosampler. a beckman coulter c18 column (4.6 mm 250 mm, 5 m) was used as the stationary phase. the mobile phase consisted of 80:20 mix of acetonitrile and 87 mm ammonium acetate (ph 4.2), run isocratically at a flow rate of 1 ml / min. tylocrebrine was analyzed by measuring absorbance at 265 nm using a system gold uv detector. a block copolymer of poly(lactide) (pla) and carboxyl - terminated poly(ethylene glycol) (peg - cooh) was synthesized in a two - step process. in the first step, lactic acid was reacted with amine - terminated peg to generate an amine - terminated block copolymer (pla peg - nh2). in the second step, the terminal amino group was reacted with succinic anhydride to produce pla peg - cooh. all glassware was rinsed with toluene and dried overnight at 100 c prior to both reactions. for step one, amine - terminated peg (400500 mg) was dissolved in 80 ml of dichloromethane and added to a round - bottom flask. lactic acid (2 g) was added to this mixture and stirred for 10 min. about 20 l of 1,8-diazabicycloundec-7-ene was added as a catalyst, and the reaction was allowed to proceed for 1 h. after 1 h, the solvent was reduced to 20 ml using a rotovap. the solution was added dropwise to chilled diethyl ether to precipitate the amine terminated block copolymer. the suspension was filtered, and the solid was dried at 25 c in a vacuum oven. the product was dissolved in deuterated chloroform and characterized using h nmr (varian 400 mhz). for step two, the amine terminated block copolymer (1 g) was dissolved in 50 ml of tetrahydrofuran and 5 ml of triethyl amine in a round - bottom flask. succinic anhydride (molar ratio of succinic anhydride to block copolymer was 1.1:1) was added to this solution. the reaction was allowed to proceed at 50 c for 2 h. the reaction mixture was then concentrated using a rotavap and added to cold diethyl ether to precipitate the polymer. plga nanoparticles loaded with tylocrebrine and surface functionalized with carboxyl - terminated peg were synthesized by the interfacial activity assisted surface functionalization technique developed by our lab. briefly, plga (3035 mg) and tylocrebrine (5 mg) were dissolved in 1 ml of chloroform. an aqueous solution of 2% w / v poly(vinyl alcohol) in pbs (0.15 mm, ph 7.4 ; henceforth referred to as 1x pbs) was prepared. drug mixture was added to the aqueous surfactant to form an o / w emulsion. the emulsion was probe sonicated at an output of 1821 w for 5 min over an ice bath (sonicator xl, misonix, ny) and then stirred at 650 rpm on a magnetic stir plate. peg - cooh (8 mg) dissolved in 0.2 ml of chloroform was added dropwise to the emulsion. the organic solvent was evaporated overnight under ambient conditions and then for 2 h under vacuum. the nanoparticle dispersion was washed twice with 30 ml of 1x pbs by ultracentrifugation (35 000 rpm, 35 min, 4 c) (beckman, palo alto, ca). after the second wash, nanoparticles were dispersed in 1.5 ml of 1x pbs. to this dispersion, 0.7 mg of n - hydroxysuccinimide (nhs), 1.14 mg of 1-ethyl-3-[3-(dimethylamino)propyl ] carbodiimide hydrochloride (edc), and 1.8 mg of targeting or control peptide were added, and the reaction was allowed to proceed for 3, 5, or 8 h. following the conjugation reaction, nanoparticles were dispersed in 30 ml of deionized water and centrifuged (35 000 rpm, 35 min, 4 c) to remove unconjugated peptide. the nanoparticle pellet was redispersed in 10 ml of deionized water, frozen below 50 c for 2 h, and lyophilized (labconco freezone 4.5, kansas city, mo). the lyophilized formulation was stored at 20 c. to determine particle size and zeta potential, a dispersion of nanoparticles (1 mg / ml) was analyzed by dynamic light scattering (delsa nano c, beckman coulter, fullerton, ca). to determine drug loading, nanoparticles were dispersed in a mixture of methanol and acetic acid (95:5 v / v). nanoparticles were separated from the extract by centrifugation (14 000 rpm, 15 min, 4 c). the amount of peptide on the surface of nanoparticles was determined using bca. nanoparticles were dispersed in deionized water and incubated with the bca reagent for 20 min at 37 c. the nanoparticle dispersion and standards were then centrifuged (14 000 rpm, 15 min, 25 c), and the absorbance of the supernatant was measured at 562 nm (elx800 absorbance microplate reader, biotek inc., nanoparticles were dispersed in a sufficient volume of 1x pbs (ph 7.4 or 6.5) to maintain sink conditions. the dispersions were kept on a shaker at 37 c and 100 rpm [c24 incubator shaker, new brunswick scientific (now eppendorf inc. the released drug was separated from nanoparticles using a microkros filter module (spectrum laboratories, rancho dominguez, ca). drug solution was lyophilized, dissolved in a mixture of methanol and acetic acid (95:5 v / v), and analyzed by hplc. we first determined whether cellular uptake of egfr - targeted nanoparticles was higher than nontargeted nanoparticles and if their uptake was mediated by egfr. a549 cells were seeded in a 24-well plate at a seeding density of 5 10 cells / well and allowed to adhere overnight. on the next day, nanoparticles loaded with a fluorescent dye (coumarin 6), in the presence or absence of excess free targeting peptide, were added to the cells. cells were incubated with the treatments at 4 c for 1 h, washed twice with 1x pbs, and incubated with fresh media at 37 c. after 1 h, the media was aspirated, cells were washed with 1x pbs, and lysed with ripa buffer. one part of the lysed cells was extracted with methanol, and coumarin 6 content was determined using hplc. the other part was analyzed by bca assay to determine the amount of cell protein. we also compared the cell uptake of targeted and nontargeted nanoparticles loaded with tylocrebrine. a431 cells were seeded in a 24-well plate as described previously. on the day of the experiment, tylocrebrine - loaded nanoparticles, dispersed in serum - free media, after 1 h, the media was aspirated, and the cells were washed thrice with cold 1x pbs. the cells were then lysed with 0.1 ml of ripa buffer for 15 min. one part (20 l) was analyzed by bca assay to determine the amount of cell protein. the other part (80 l) was extracted with a mixture of methanol and acetic acid (95:5 v / v). we compared the tumor cell kill efficacy of free drug, nontargeted, and targeted nanoparticles in both neutral and acidic media. a549 cells or a431 cells were seeded in a 96-well plate (8 10 cells / well) and allowed to attach overnight. nanoparticles or free drug were dispersed in serum - free rpmi media to form a stock solution. this stock solution was then diluted with either neutral or acidified serum - free media and added to the cells at various dilutions. the treatments were removed 6 h later, and the cells were washed with cold 1x pbs. the cells were then incubated with serum containing media for further 90 h. cell viability was measured using celltiter 96 aqueous one solution cell proliferation assay kit (promega, madison, wi) according to the supplier s protocol. all the animal studies described here were approved by the university of minnesota s institutional animal care and use committee. a431 cells (12 10) were dispersed in 0.05 ml of 1x pbs and injected subcutaneously in female athymic nude mice (46 weeks old, taconic biosciences, hudson, ny). mice were lightly anesthetized using isofluorane and treated with various formulations of tylocrebrine (dose, 12 mg / kg ; dosing volume, 10 ml / kg) via retroorbital injection. the free drug formulation was prepared by dissolving the drug in 1 m hcl (1:1 molar ratio) and diluting the solution with saline. excess acid was neutralized with 1 m naoh to obtain a final drug concentration of 1.2 mg / ml. to prepare nanoparticle treatments, nanoparticles were dispersed in saline and probe sonicated at an output of 1821 w thrice for 30 s each on an ice bath. the nanoparticle dispersion was centrifuged (1000 rpm, 5 min) to remove any large aggregates, and the supernatant was used. at various time points, cohorts of mice were sacrificed, and blood was collected by cardiac stick. the dry organs were then extracted overnight with a mixture of methanol and acetic acid (95:5). the dried residue was redispersed in a mixture of acetonitrile and acetic acid (95:5). the resulting suspension was centrifuged (14 000 rpm, 15 min, 4 c), and the supernatant was used for lc ms / ms was performed using an acquity ultra - performance liquid chromatography (uplc) system equipped with a waters / micromass quattro ultima mass spectrometer. lc was performed using agilent xdb - c18 column (4.6 mm 50 mm, 1.8 m) as the stationary phase. a mixture of 10 mm ammonium acetate with 0.06% v / v acetic acid and acetonitrile (55:45 v / v) was used as the mobile phase. the flow rate was 0.4 ml / min, and the run time was set to 6 min. the mass spectrometer conditions were as follows : cone voltage, 50 v ; collision voltage, 20 v ; dwell time, 0.4 s. tylocrebrine was detected by monitoring the m / z transition of 394 324.9. concentration of tylocrebrine was normalized to tissue weight and injected dose and was represented as % injected dose (id)/g of organ weight. drug exposure in each tissue was determined by calculating the area under the concentration curve (auc). the relative benefit of using the nanoparticulate formulation over the free drug was determined using the drug targeting index (dti). the dti was calculated as shown below : the therapeutic efficacy of various formulations of tylocrebrine was determined in a mouse model of epidermoid cancer. about 1 10 a431 cells, suspended in 50 l of 1x pbs, were injected subcutaneously in female nude mice. when tumor volume reached 75 mm, animals were treated with saline, free drug, nontargeted nanoparticles, or egfr - targeted nanoparticles (three doses, every 96 h, 12 mg / kg). the treatments were prepared and administered as described in the biodistribution study. at the end of the study, animals were sacrificed, and the tumors were excised. the tumors were fixed using 5% formalin solution in 1x pbs for 24 h. after the initial fixation, tumors were preserved in 70% ethanol. microtome sectioning was performed on the fixed and mounted tumor samples, and the sections were stained for cleaved caspase 3 (to determine apoptosis) and ki67 (as a marker of proliferation). staining of tumor sections was quantified using imagej 1.48v software. to determine apoptotic and proliferative indices, the fraction of the total cellular area that stained positive for the individual markers was determined and presented as apoptotic and proliferative indices. all statistical analyses were performed using one - way analysis of variance (anova) and posthoc tukey test. for the efficacy study, slopes of the tumor growth profile were analyzed at each time point for each animal. one - way anova and posthoc tukey test were performed to determine if the differences in various treatments were statistically significant. tumors are often characterized by increased production of lactic acid due to the warburg effect. impaired drainage of the acid from the tumor microenvironment leads to low extracellular ph in the tumor. ionization of the drug molecule can decrease its diffusion across the cell membrane and hence lead to decreased intracellular drug availability. we measured the effect of extracellular ph on drug accumulation in a431 cells (figure 1). decreasing the ph of the media from 7.4 (physiologic) to 6.36.7 (to mimic tumor microenvironment) resulted in reduced drug uptake (60% reduction). this is likely due to the protonation of the indolizidine nitrogen, leading to a positive charge on the molecule. tylocrebrine (as free drug) was incubated with a431 cells at an extracellular ph of 7.4 (physiological) or 6.5 (acidic). data represented as mean sd, n = 6, indicates p 10). thus, the advantage gained by nanoencapsulation strongly depends on the drug being investigated. additionally, a431 tumors were found to be fluid - filled (not shown), suggesting that the interstitial fluid pressure in these tumors is high, as previously reported. high interstitial fluid pressure (ifp) is likely to affect the intratumoral transport of nanoparticles more significantly than that of free drug molecules. the relatively large hydrodynamic particle size of the nanoparticles used in our studies may also decrease their tumor penetration. decreasing the particle size can potentially increase the delivery of nanoparticles into the tumor. alternately, tumors with a low ifp may prove to be better candidates for treatment with these formulations. the in vivo tumor growth inhibition studies show that free drug inhibited tumor growth even at the low dose used. while treatment efficacy did not improve with nontargeted nanoparticles, targeted nanoparticles resulted in a considerably greater tumor growth inhibition than other treatments. this effect may be a manifestation of both the higher tumor tissue accumulation and higher cytotoxicity of targeted nanoparticles. the dose of tylocrebrine used in our studies was relatively low (12 mg / kg). this dose was effective only if the treatment was started before the tumors reached a volume of 75 mm. larger tumors did not respond to this dose of tylocrebrine in any formulation (data not shown). the overall dose of tylocrebrine that could be administered was limited by its loading in nanoparticles. additionally, burst release of drug from nanoparticles may limit the overall efficiency of targeting and chemotherapeutic efficacy of nanoparticles. strategies that enable higher drug loading and prolonged drug release will allow for larger doses to be administered, and this could further enhance the antitumor efficacy of the drug. using polymers that have greater interaction with the drug can help achieve this goal. to this end, micelles formed from polymers consisting of aromatic rings have been shown to improve drug loading and stability as compared to micelles formed from aliphatic polymers. this has been attributed to the formation of stacks between the drug and polymer. future studies could investigate the use of such polymers for improving the therapeutic index of tylocrebrine. tylocrebrine is a potent anticancer agent, but significant penetration into the brain and low tumor cell uptake limit its use. encapsulation of the drug in plga nanoparticles significantly limited its cns penetration. moreover, surface functionalizing nanoparticles with an egfr targeting peptide led to enhanced tumor cell uptake, tumor tissue accumulation, and in vivo antitumor efficacy. we expect that the reformulation approach presented here will enable further clinical testing of a number of previously abandoned drug candidates while potentially minimizing drug development costs.	several promising anticancer drug candidates have been sidelined owing to their poor physicochemical properties or unfavorable pharmacokinetics, resulting in high overall cost of drug discovery and development. use of alternative formulation strategies that alleviate these issues can help advance new molecules to the clinic at a significantly lower cost. tylocrebrine is a natural product with potent anticancer activity. its clinical trial was discontinued following the discovery of severe central nervous system toxicities. to improve the safety and potency of tylocrebrine, we formulated the drug in polymeric nanoparticles targeted to the epidermal growth factor receptor (egfr) overexpressed on several types of tumors. through in vitro studies in different cancer cell lines, we found that egfr targeted nanoparticles were significantly more effective in killing tumor cells than the free drug. in vivo pharmacokinetic studies revealed that encapsulation in nanoparticles resulted in lower brain penetration and enhanced tumor accumulation of the drug. further, targeted nanoparticles were characterized by significantly enhanced tumor growth inhibitory activity in a mouse xenograft model of epidermoid cancer. these results suggest that the therapeutic index of drugs that were previously considered unusable could be significantly improved by reformulation. application of novel formulation strategies to previously abandoned drugs provides an opportunity to advance new molecules to the clinic at a lower cost. this can significantly increase the repertoire of treatment options available to cancer patients.
on october 8, 2015, the case - patient in conakry, guinea, became ill with fever and appetite loss. his brother - in - law, an evd survivor and nurse living in the same household, diagnosed malaria, but the case - patient did not respond to malaria treatment ; diarrhea, headache, and abdominal pain developed. on october 13, reverse transmission pcr (rt - pcr) of the case - patient s blood was positive for ebov (11). from september 2015 until confirmation of evd for this case - patient, only 7 cases of evd in guinea had been reported, all part of the same chain of transmission. of these cases, virus was not sequenced for 2 because the epidemiologic link was not clearly established. because the case - patient we report did not have any link with ongoing evd clusters investigated in guinea or sierra leone, the most likely source of infection was the survivor. because ebov is shed in survivor s semen long after recovery (7), within - marriage sexual transmission from the survivor to the woman was inferred. blood, urine, and buccal swab samples from the couple were collected, together with semen and vaginal swab samples ; all were negative for ebov by rt - pcr. blood, urine, buccal, and vaginal swab samples were processed as in (11) and seminal fluid samples as in (10). the woman s serum was positive according to igg and igm elisa testing (both titers > 1:6,400) ; the survivor s serum was positive for igg only (titer > 1:3,200) (12). serum from the only other household member, the couple s 11-year - old daughter, was negative for igg and igm. although the woman s igm titer was suggestive of a recent ebov infection, she reported having had only a mild 2-day clinical episode back in early september, characterized by myalgia, joint pain, and low - back pain (figure 1). the couple declared that they had had no sexual intercourse for 6 months after the survivor was discharged from an ebola treatment unit. timeline of the reported chain of transmission of ebola virus involving 3 persons in conkary, guinea, 20142015. etu, ebola treatment unit ; rt - pcr, reverse transcription pcr ;, negative ; +, positive. the near full - genome sequence of virus from the case - patient, obtained as previously described (10), was available on october 15, 2015. phylogenetic analysis demonstrated that it belonged to the gn1 lineage rather than the sl3 lineage that was circulating in conakry during the second half of 2015 (figure 2, panel a). thus, the cases were probably not linked to the known ongoing chain of transmission in this area because sequencing of the viruses involved in the guinea epidemic had been thoroughly performed at this time and no gn1 lineage viruses had been detected in guinea since the end of june 2015 (10). the sequence of the virus isolated from the case - patient, however, could not be closely linked to any known subclusters within gn1. the most closely related viruses were recovered from evd patients in march 2015, but phylogenetic inference suggests that the new sequence had not evolved from these patients and instead shared a common ancestor from late 2014 (figure 2, panel b). a) maximum - likelihood tree of 1,067 ebola virus sequences from the west africa epidemic, showing country of origin of each isolate. ebola virus disease cases from conakry, guinea, obtained in the second half of 2015 (from june on) are denoted by a red dot on the tree branches. b) expanded view of the gn1 lineage from panel a. blue dots indicate sequences from cases described in this article (survivor, ipd_2197 ; person who infected the survivor, ipd_2163 ; case - patient, con12930) ; red dots indicate other cases from 2015. c) root - to - tip plot for the entire gn1 lineage ; colors match those in panel b. a linear model line of best fit is shown ; gray shading represents the confidence level around the regression line as se. because various samples from the survivor (including semen) and from the woman were negative for ebov (figure 1), serum collected during the survivor s acute phase of evd (november 28, 2014) was retrieved from an archived collection and sequenced, together with a sample from the person who was thought to have transmitted the infection to him (ipd_2163) (figure 2, panel b). these 2 ebov sequences were indistinguishable and clustered with virus sequences from the case - patient, differing at 6 nt sites. this difference over an 11-month period is smaller than expected from the estimated ebov evolutionary rate that would predict 22.5 (95% ci 1333) mutations (figure 2, panel c). most likely, the first patient sexually transmitted ebov to the woman 9 months after his onset of evd in 2014, and then either the woman (with a recent history of undetected ebov infection) or a third unknown person transmitted the virus to the case - patient. after 10.5 months, virus from the case - patient differed from that of the survivor by only 6 nt substitutions. these substitutions were not present in other virus sequences from the gn1 lineage, demonstrating that the isolates are closely related to each other and more distantly related to others. the number of substitutions between the 2 genomes was 3.7 times lower than would be expected after human - to - human transmission and may indicate virus persistence in 1 person. this hypothesis is in line with reduced evolutionary rates of persistent ebov reported in liberia and sierra leone (7,8,13). however, none of her samples (i.e., blood, urine, and vaginal swab) were positive for ebov by rt - pcr ; therefore, no genomic data were available. the serial interval between the case - patient s onset of symptoms and that of the woman (30 days) is longer than the usual evd maximum incubation period (21 days). we have no molecular evidence that the woman was part of this chain of transmission ; it is plausible that a third unknown person infected the case - patient. a likely explanation of our observations, integrating genomic, serologic, and epidemiologic data, is that within - marriage sexual transmission occurred (inferred from the positive igm titers of the woman) and then the woman infected her brother through close contact. they all lived in the same small household in poor hygienic conditions ; shared toilets, meals, and at times beds ; and cared for each other. alternatively, a third unknown person may have been the link in the transmission from the survivor to the case - patient. interim advice with regard to sexual transmission of ebov has been updated recently (14). all evd survivors and their sex partners should receive condoms and counseling to ensure safer sex practices. safer sex practices should continue until the results of semen testing are negative twice or, if testing is unavailable, for 12 months. the safer sex strategy and testing could be complemented by new medical countermeasures that need to be assessed and include use of antiviral drugs, vaccination of relatives and sex partners of survivors, or both.	in october 2015, a new case of ebola virus disease in guinea was detected. case investigation, serology, and whole - genome sequencing indicated possible transmission of the virus from an ebola virus disease survivor to another person and then to the case - patient reported here. this transmission chain over 11 months suggests slow ebola virus evolution.
the terms stunned myocardium and hibernating myocardium refer to abnormality in the systolic and diastolic function of the heart following reperfusion.1 in both abnormalities, myocardial contractility and relaxation are deteriorated, while the cardiac enzymes are still viable.2 in the hibernating myocardium, however, a programmed cell death (apoptosis) pattern has been described. myocardial ischemia results in the utilization of adenosine triphosphate atp stores secondary to the paralysis of aerobic metabolism and oxidative phosphorylation.3 stunning was defined by braunwald as post - ischemic cardiac dysfunction of viable myocardium.4 clinical myocardial stunning was first reported by bolli and klonar, who separately characterized its experimental models. stunning has been documented in post - percutaneous coronary intervention and thrombolytic therapy for coronary artery stenosis5, 6 and also in the wake of cardiopulmonary bypass (cpb).6, 7 one of the technical challenges in off - pump coronary artery bypass grafting surgery (opcab) is myocardial ischemia caused by the proximal and distal snaring of the coronary artery, which gives rise to post - ischemic ventricular dysfunction.8 nonetheless, the occurrence of myocardial stunning in this setting has yet to be fully investigated. we herein report the case of a patient who developed temporary left ventricular dysfunction after an opcab procedure. a 53-year - old man presented with unstable angina due to the severe stenosis of the left anterior descending coronary artery and obtuse marginalis, although the right coronary artery was normal. laboratory findings, including a complete blood count, erythrocyte sedimentation rate, and c reactive protein, were normal. chest x - ray revealed no abnormal findings, and there was no valvular abnormality on preoperative echocardiography. the patient had no co - morbid disorders, but his left ventricular ejection fraction was reduced (4045%)., there was no respiratory distress, blood pressure was 130/80 mm hg, heart rate was 80 beats per minute, respiratory rate was 23 per minute, the neck veins were not distended, and there was no ankle edema. cardiovascular system examination showed regular first and second heart sounds with no gallop or murmur. for temporary coronary artery occlusion, 4/0 viline sutures and a bulldog clamp were used, and warm blood was employed to de air the graft. temporary coronary artery occlusion was not prolonged, and the electrocardiogram and hemodynamic variables and objective data showed no signs of ischemia or contractile dysfunction. six hours later, however, he developed low cardiac output. at exploration, cardiac tamponade there were no findings as regards pericarditis, and the patient s postoperative erythrocyte sedimentation rate, c - reactive protein, and cardiac enzymes were normal. a high dose of adrenalin and dobutamine was administered, and an intra - aortic balloon pump was used. intraoperative transesophageal echocardiography demonstrated a depression in the left ventricular function due to an akinetic lateral left ventricular wall in the region of the obtuse marginalis. after hemodynamic stabilization, the patient left the intensive care unit without an intra - aortic balloon pump and inotropic support. on the fifth postoperative day, a coronary angiogram demonstrated patent grafts and correct anastomotic sites (figure 1 & 2). on the seventh postoperative day, the akinetic lateral wall of the left ventricle changed to dyskinesia. finally, after hospital discharge on the thirtieth postoperative day, an echocardiogram showed a normal left ventricular function without regional wall motion abnormalities. cpb may contribute to the mortality and cost associated with cabg.9 recently, opcab has emerged as an alternative technique allowing coronary revascularization without the need for cpb. we have performed 300 consecutive opcab procedures in our hospital over the years. in the case reported in this paper, the anesthesia protocol was comprised of a combination, of fentanyl and pancuronium bromide, supplemented with isoflurane and nitrous oxide, to permit early extubation. an arterial and central venous line was utilized as is the standard in this modality. conduits for cabg, including the left internal mammary artery and saphenous vein, were harvested in the standard fashion. deep pericardial traction sutures were placed to facilitate elevation of the apex of the heart and exposure of the lateral wall of the myocardium. the right pleural space was opened routinely to allow displacement of the heart to facilitate the exposure of the circumflex artery. revascularization on the left anterior descending artery with the left internal mammary was typically performed first, followed by revascularization of the left circumflex artery and the right coronary artery distribution. to assist further in providing good presentation of the target arteries, especially the posterior and inferior walls, an optimal combination of pharmacological and mechanical methods was drawn upon to reduce the coronary artery movement. stabilization of the target arteries was accomplished with an octopus stabilizer (medtronic, ts 300). intravenous heparin (1 mg / kg) was given to maintain activated clotting time (act) between 200 and 300 seconds. the target coronary artery was occluded proximal and distal to the proposed arteriotomy site by widely placing double - looped 4 - 0 viline sutures. proximal anastomosis to the aorta was made on a punch aortotomy after applying a side clamp to the ascending aorta. visualization of the anastomosis was enhanced with the use of humidified carbon dioxide blower. before the application of the octopus stabilizer, amiodarone and esmolol were administered to the patient and communication with the anesthesia team was maintained to monitor changes in the patient s hemodynamic and to treat cardiac arrhythmias. after distal anastomosis, proximal anastomoses were carried out on the ascending aorta with a partially occluding clamp. serial electrocardiograms and estimation of serum creatinine phosphokinase and its mb fraction were done to detect perioperative ischemia. ventricular dysfunction developed postoperatively in 2 patients, and 2 patients developed severe left ventricular dysfunction due to the poor quality of the anastomotic site of the left internal mammary artery to the left anterior descending coronary artery graft. intraoperative flowmetry demonstrated normal graft flow and cardiac enzymes were not significantly elevated and postoperative angiography showed patent bypass graft and good quality of the anastomotic sites. (figure 1 and 2) because the bypass grafts were patent, the only ischemic event that would have caused left ventricular dysfunction was temporary occlusion of the coronary arties. we think that post - ischemic contractile dysfunction of the left ventricle has its pathophysiological background in myocardial stunning. the best approach in the postoperative period is to support the acutely failing heart by inotropic drugs and intra - aortic balloon pumps. alkholaifie reported that ultimate objective must be to prevent ventricular dysfunction by ischemic preconditioning, which could be achieved by repetitive short - time occlusion and reperfusion of the coronary vessel.9 grubitzsch did not observe segment depression or elevation after coronary artery occlusion in his patients, which usually indicates the necessity of preconditioning.8 in contrast, mulkowski, in a clinical setting of opcab, showed that transient ischemia did not limit subsequent ischemic regional dysfunction.10 this controversy in the management of post - operative left ventricular dysfunction with patent bypass graft led to this recommendation by rivetti that the use of the intra - coronary shunt must be considered if the duration of temporary coronary artery occlusion exceeds fifteen minutes. recently, opcab has emerged as an alternative technique allowing coronary revascularization without the use of cpb. because opcab is associated with temporary myocardial ischemia we think that the most important issue in performing opcab is the short ischemia time.	the term stunned myocardium refers to abnormalities in the myocardial function following reperfusion and is common in on - pump coronary artery bypass grafting (cabg) and is exceedingly rare in off- pump cabg. a 53-year - old man presented with unstable angina due to the severe stenosis of the left anterior descending coronary artery (lad) and the obtuse marginal. laboratory findings and chest x - ray revealed nothing abnormal. the intraoperative course was uneventful. the patient left the operating room without any inotropic support. six hours later, however, he developed low cardiac output. at exploration, cardiac tamponade was excluded and flowmetry showed that the graft had adequate function. cardiac enzymes were normal. high - dose adrenalin and dobutamine were administrated and an intra - aortic balloon pump was used. after hemodynamic stabilization, the patient left the intensive care unit without an intra - aortic balloon pump and inotropic support. on the fifth postoperative day, coronary angiography showed patent grafts and correct anastomotic sites. on the seventh postoperative day, the akinetic lateral wall of the left ventricle changed to dyskinesia. finally after hospital discharge on the thirtieth postoperative day, an echocardiogram showed normal left ventricular function without regional wall motion abnormalities.
a better understanding of the oral environment and cariology has resulted in extensive changes in caries management towards a more conservative approach. the development of a caries lesion is now recognized as an imbalance in the demineralization and remineralization cycle. many researchers are now investigating the use of techniques to induce remineralization of the affected tooth structure. the use of fluoride is an effective method for promoting the remineralization of an early lesion in enamel through the formation of fluorapatite. however, for every two fluoride ions, ten calcium ions and six phosphate ions are required to form one unit cell of fluorapatite (ca10(po4)6f2). hence, when topically applying fluoride, an inadequate amount of available calcium and phosphate ions can limit net enamel remineralization. another option to enhance remineralization is to supply calcium and phosphate, and to deliver fluoride more effectively. amorphous calcium phosphate (cpp acp) consists of casein phosphopeptide (cpp) aggregated with calcium phosphate to form clusters of amorphous calcium phosphate (acp). this aggregation prevents calcium phosphate precipitation, resulting in a state of supersaturation with respect to enamel, preventing demineralization and promoting remineralization. acp has been demonstrated to have anticariogenic activity in in vitro, animal and human in situ experiments, including a randomized controlled clinical trial. acp is available as a professional dental product (gc tooth mousse ; gc corporation, tokyo, japan). gc tooth mousse containing 0.09% fluoride is available as a cpp acpf paste (gc tooth mousse plus ; gc corporation, tokyo, japan). cpp acpf has been reported to have a greater potential for remineralization than cpp acp. another calcium phosphate compound is functionalized tricalcium phosphate (f tcp), which is produced by the solid - state ball milling of beta - tricalcium phosphate and sodium lauryl sulfate. f tcp can prevent calcium from prematurely interacting with ionic fluoride and forming calcium fluoride, thus delivering more fluoride and calcium ions to the enamel surface. f tcp has been shown to have remineralizing effects in both in vitro and in situ studies. f tcp has been commercially developed, and is now available as a 950 ppm fluoride toothpaste (clinpro tooth crme ; 3 m espe, saint paul, mn, usa). the aim of our study was to compare the efficacy of cpp acpf and f tcp (calcium phosphate pastes) with that of conventional 0.1% fluoride toothpaste in remineralizing enamel artificial caries lesions in a randomized, controlled, double - blind, crossover in situ trial. freshly extracted human premolar teeth were collected and sterilized with ethylene oxide gas for 18 h. the minimum number of specimens required for the study was determined based on our pilot study. we calculated that we would need at least 52 specimens per group to give a two - sided 5% significance level and a power of 80%. fifty - four teeth without caries, cracks or enamel malformation were selected and cleaned with pumice to remove debris / stains from the enamel surface. artificial caries lesions were created by immersing each tooth in demineralization solution (80 mgl carbopol, 50 mgl hydroxyapatite, 85% lactic acid, 6 moll sodium hydroxide, ph 4.8), for 21 days at room temperature to produce lesions approximately 100150 m deep. after demineralization, a low - speed cutting machine (isomet 1000 ; buehler, lake bluff, il, usa) was used to vertically section the proximal surfaces of each tooth into two enamel slabs approximately 2 mm long1 mm wide2 mm thick. the four enamel slabs from each tooth were randomly assigned to either the initial lesion group or one of the three test groups. the components of the appliances consisted of a labial arch and two adam 's clasps. two troughs (7 mm long, 5 mm wide and 3 mm deep) three enamel slabs were retained in each of the troughs by flowable composite resin to produce a 1 mm deep trough above the enamel surface to allow the establishment of plaque (figure 1). all study protocols were approved by the ethics committee of the faculty of dentistry, chulalongkorn university. nine healthy adults (eight females and one male) with an average age of 197 years participated in our study. after gaining informed written consent, the subjects were given a thorough intra - oral examination and completed a medical history questionnaire. the study inclusion criteria consisted of having at least 22 teeth without clinically active caries, periodontal disease or other oral pathology. the subjects ' mean unstimulated salivary flow rate was (0.80.3) mlmin, and their mean stimulated salivary flow rate was (3.70.8) mlmin. this double - blind, randomized, controlled in situ study comprised three distinct phases lasting 14 days each, which were preceded by an 8-day washout period to eliminate any residual effects of the previous product (figure 2). the three products used were : (i) tooth mousse plus (gc corporation, tokyo, japan) ; (ii) clinpro tooth crme (3 m espe, saint paul, mn, usa) ; and (iii) 0.1% fluoride toothpaste (colgate regular flavor ; colgate - palmolive, bangkok, thailand). the three products were given to the subjects contained in syringes and were identical in appearance. on the last day of the washout periods, the subjects wore the appliances without using any products to check for any irritation (day before the first phase only) and to allow for plaque accumulation. the assignment of the test products was based on a randomization scheme (figure 3). we used a computer - generated list of random numbers for the sequential allocation of the subjects into the groups. 79 began as group c. all subjects were asked to maintain their normal dietary habits. the participants were instructed to wear the appliances for at least 12 h a day, including during eating and drinking. the test pastes were applied twice a day, after breakfast and dinner, based on the manufacturers ' directions. for group a, the subjects brushed their teeth with 1.0 g of fluoride toothpaste for 2 min while wearing their appliances and taking care not to brush the slabs, then expectorated the slurry and rinsed their mouths with 15 ml of tap water for 10 s. after brushing, the subjects squeezed 0.25 g of tooth mousse plus onto their fingers and applied it on the enamel slabs and their upper teeth. the subjects used another 0.25 g of tooth mousse plus to apply on their lower teeth. for group b, the subjects were instructed to brush their teeth with 0.25 g of clinpro tooth crme and then expectorated the slurry and gently rinsed their mouths with tap water. for group c, the subjects were instructed to use the same brushing method as group b, using 1.0 g of fluoride toothpaste. the subjects were instructed not to eat or drink for 30 min after any of the treatments (figure 4). after the appliances had been worn for 12 h, they were removed and cleaned with a toothbrush. the subjects were instructed to avoid brushing directly on the enamel slabs. after cleaning, the appliances were stored in sealed moist plastic containers at room temperature. at the end of each 14-day period, the enamel slabs were detached and kept in deionized water at room temperature until they were embedded in resin, and the appliances were stored in tap water at room temperature until the next phase. after the washout period, new enamel slabs were used for the next experimental phase. this procedure was repeated until the subjects had rotated through all three treatment groups. the enamel slabs were embedded in resin, and 100150 m sections were prepared using a microtome (leica sp 1600 ; leica, solms, germany). the label containing the information of each slab : subject number, product used and specimen number were concealed and randomized, and the examiner was blinded to which treatment has been administered. fifty - four specimens per group were examined with a polarized - light microscope (9300 meiji ; meiji techno, chikumazawa, japan) after imbibition in deionized water. digital images were taken with a color video camera (meiji cx 3800 ; meiji techno, chikumazawa, japan) (figure 5) and were transformed into black - and - white images using microsoft office picture manager version 12.0.6412.1000. the lesion areas were measured using image - pro plus version 4.5.0.29 (media cybernetics, bethesda, usa). statistical analysis of the data was conducted using spss statistical software version 17.0 with significance () set at 0.05. the pre- and post - test parameters within each group were compared using the paired t - test at the 95% confidence level. freshly extracted human premolar teeth were collected and sterilized with ethylene oxide gas for 18 h. the minimum number of specimens required for the study was determined based on our pilot study. we calculated that we would need at least 52 specimens per group to give a two - sided 5% significance level and a power of 80%. fifty - four teeth without caries, cracks or enamel malformation were selected and cleaned with pumice to remove debris / stains from the enamel surface. artificial caries lesions were created by immersing each tooth in demineralization solution (80 mgl carbopol, 50 mgl hydroxyapatite, 85% lactic acid, 6 moll sodium hydroxide, ph 4.8), for 21 days at room temperature to produce lesions approximately 100150 m deep. after demineralization, a low - speed cutting machine (isomet 1000 ; buehler, lake bluff, il, usa) was used to vertically section the proximal surfaces of each tooth into two enamel slabs approximately 2 mm long1 mm wide2 mm thick. the four enamel slabs from each tooth were randomly assigned to either the initial lesion group or one of the three test groups. the components of the appliances consisted of a labial arch and two adam 's clasps. two troughs (7 mm long, 5 mm wide and 3 mm deep) three enamel slabs were retained in each of the troughs by flowable composite resin to produce a 1 mm deep trough above the enamel surface to allow the establishment of plaque (figure 1). all study protocols were approved by the ethics committee of the faculty of dentistry, chulalongkorn university. nine healthy adults (eight females and one male) with an average age of 197 years participated in our study. after gaining informed written consent, the subjects were given a thorough intra - oral examination and completed a medical history questionnaire. the study inclusion criteria consisted of having at least 22 teeth without clinically active caries, periodontal disease or other oral pathology. the subjects ' mean unstimulated salivary flow rate was (0.80.3) mlmin, and their mean stimulated salivary flow rate was (3.70.8) mlmin. this double - blind, randomized, controlled in situ study comprised three distinct phases lasting 14 days each, which were preceded by an 8-day washout period to eliminate any residual effects of the previous product (figure 2). the three products used were : (i) tooth mousse plus (gc corporation, tokyo, japan) ; (ii) clinpro tooth crme (3 m espe, saint paul, mn, usa) ; and (iii) 0.1% fluoride toothpaste (colgate regular flavor ; colgate - palmolive, bangkok, thailand). the three products were given to the subjects contained in syringes and were identical in appearance. on the last day of the washout periods, the subjects wore the appliances without using any products to check for any irritation (day before the first phase only) and to allow for plaque accumulation. the assignment of the test products was based on a randomization scheme (figure 3). we used a computer - generated list of random numbers for the sequential allocation of the subjects into the groups. 79 began as group c. all subjects were asked to maintain their normal dietary habits. the participants were instructed to wear the appliances for at least 12 h a day, including during eating and drinking. the use of any other oral hygiene product was prohibited. the test pastes were applied twice a day, after breakfast and dinner, based on the manufacturers ' directions. for group a, the subjects brushed their teeth with 1.0 g of fluoride toothpaste for 2 min while wearing their appliances and taking care not to brush the slabs, then expectorated the slurry and rinsed their mouths with 15 ml of tap water for 10 s. after brushing, the subjects squeezed 0.25 g of tooth mousse plus onto their fingers and applied it on the enamel slabs and their upper teeth. the subjects used another 0.25 g of tooth mousse plus to apply on their lower teeth. for group b, the subjects were instructed to brush their teeth with 0.25 g of clinpro tooth crme and then expectorated the slurry and gently rinsed their mouths with tap water. for group c, the subjects were instructed to use the same brushing method as group b, using 1.0 g of fluoride toothpaste. the subjects were instructed not to eat or drink for 30 min after any of the treatments (figure 4). after the appliances had been worn for 12 h, they were removed and cleaned with a toothbrush. the subjects were instructed to avoid brushing directly on the enamel slabs. after cleaning, the appliances were stored in sealed moist plastic containers at room temperature. at the end of each 14-day period, the enamel slabs were detached and kept in deionized water at room temperature until they were embedded in resin, and the appliances were stored in tap water at room temperature until the next phase. after the washout period, new enamel slabs were used for the next experimental phase. the enamel slabs were embedded in resin, and 100150 m sections were prepared using a microtome (leica sp 1600 ; leica, solms, germany). the label containing the information of each slab : subject number, product used and specimen number were concealed and randomized, and the examiner was blinded to which treatment has been administered. fifty - four specimens per group were examined with a polarized - light microscope (9300 meiji ; meiji techno, chikumazawa, japan) after imbibition in deionized water. digital images were taken with a color video camera (meiji cx 3800 ; meiji techno, chikumazawa, japan) (figure 5) and were transformed into black - and - white images using microsoft office picture manager version 12.0.6412.1000. the lesion areas were measured using image - pro plus version 4.5.0.29 (media cybernetics, bethesda, usa). statistical analysis of the data was conducted using spss statistical software version 17.0 with significance () set at 0.05. the pre- and post - test parameters within each group were compared using the paired t - test at the 95% confidence level. after 14 days of use in situ, each product produced a statistically significant reduction in lesion area (p0.05). the results of the present study demonstrate that both cpp acpf paste and sodium fluoride paste containing functionalized tricalcium phosphate can enhance the remineralization of artificial caries lesions in enamel when used in situ. however, when comparing the remineralization induced by these two calcium phosphate pastes with that of regular 0.1% fluoride toothpaste, no statistical differences were found. the results of our study are inconsistent with a prior study conducted by shen., where cpp acpf induced significantly more remineralization compared to 0.1% fluoride toothpaste. this may be because in their study, cpp acpf paste was applied four times a day in the form of a slurry made from 1 g of cpp acpf in 4 ml of distilled deionized water. in contrast, in the present study, the participants used tooth mousse plus following the manufacturer 's instructions that recommended using a pea - size amount (approximately 0.25 g) of tooth mousse plus on each arch after brushing with fluoride toothpaste. these differences in application methods may alter the remineralization effect of the product. in the study of shen. similarly, the increased frequency of cpp acpf use may have affected the results of their study. rinsing with cpp acpf slurry four times daily likely allowed more cpp acpf to accumulate in the subjects ' dental plaque and saliva compared with the present study. 's study also received fluoride from their normal oral hygiene procedures (brushing with 0.1% fluoride toothpaste). this may have resulted in their cpp acpf group demonstrating a greater remineralizing effect. however, in the present study, we chose application methods simulating typical product use, rather than equal amounts of each product, the results of which could be expected to reflect what would occur clinically. although studies have indicated that cpp acp paste has a remineralization effect, the results of the current study did not show any additional effect of cpp acp on the remineralization of artificial caries when used as a supplement to regular fluoride toothpaste. these findings are likely because the prior studies used protocols that were different from the manufacturer 's instruction, which the present study employed. in the prior studies, specimens were immersed in diluted cpp acp paste or were applied with cpp acp paste for 30 min, which could enhance remineralization more than using the paste following the manufacurer 's instructions. in contrast, the results of the present study were consistent with a clinical study by brchner., which compared subjects using cpp acp as a supplement to daily fluoride toothpaste with subjects who brushed with fluoride toothpaste alone. brchner. showed that the remineralization was not superior to the natural regression following daily use of fluoride toothpaste. thus, based on our results, using cpp acp could make daily oral hygiene procedures more complicated and increase costs. the results of our study showed no significant difference in remineralization between clinpro tooth crme and regular 0.1% f toothpaste. however, the participants used clinpro tooth crme in only one - fourth the amount (0.25 g) of the regular toothpaste. where 0.05% fluoride toothpaste containing f tcp tended to produce greater remineralization compared to 0.05% fluoride toothpaste when used in the same amounts. the subjects in our study followed the manufacturer 's instructions and used clinpro tooth crme in pea - size amounts (0.25 g) and received a lower amount of fluoride, but we saw the same remineralizing effect as was seen with using 1.0 g of 0.1% fluoride toothpaste or fluoride toothpaste followed by cpp acpf paste. as a result, clinpro tooth crme may be of benefit for patients who want to avoid using fluoride or for children at risk of developing fluorosis. our investigation also shows that regular 0.1% fluoride toothpaste is effective in the remineralization of artificial caries in situ. fluoride toothpaste is an effective product for the control of dental caries and can be used by the majority of the population. brushing with fluoride toothpaste is an easy procedure that is low in cost, and this product is readily available. indeed, the evidence on the effect of fluoride toothpaste on the prevention of dental caries has been extensively reviewed. therefore, dentists should advise patients to use fluoride toothpaste, especially in terms of cost - effectiveness. an in situ model is an appropriate method for the evaluation of the anticaries potential of new compounds added to dentifrices or pastes. there are many advantages to in situ studies that are performed in the human mouth, in contrast to in vitro or animal studies. in situ studies allow the control of experimental variables and a flexibility of experimental design that is impossible to achieve in clinical trials. however, this type of study depends heavily on compliance by the test subjects. to increase the subject 's compliance, we gave them an instruction checklist and directed them to check off each step as it was completed. in addition, we randomly called the subjects during the experiment to remind them to use the products. to provide a scientifically supported clinical recommendation for the use of the products examined in our investigation, further study should examine a larger population, include a group of high caries risk participants (e.g., have active caries lesions or low salivary flow rate), have a longer treatment time and include additional remineralizing products such as a 0.05% fluoride toothpaste. moreover, additional studies should include a paste without therapeutic ingredients, which would allow for a better delineation between the effect of these pastes and saliva only, and alternative methods of using these products. for example, using cpp acpf paste 12 h after brushing with fluoride toothpaste may permit the discrimination of the remineralizing effect of cpp acpf from that of fluoride toothpaste. in conclusion, all three groups remineralized the enamel slab lesions, indicating model sensitivity to fluoride. given the differences in usage amounts and treated regimens, clinpro tooth crme provided similar benefits to the 0.1% fluoride toothpaste, however, no additional benefit of tooth mousse plus was observed when used in conjunction with the 0.1% fluoride toothpaste.	to test the efficacy of two calcium phosphate pastes compared to that of fluoride toothpaste on remineralizing artificial caries in situ, this study had a double - blind crossover in situ design, involving three experimental phases of 14 days each, with an 8-day washout period between phases. nine healthy subjects participated in the study. the subjects wore removable palatal appliances mounted with six human enamel slabs with artificial caries lesions, and in each of the experimental phases, used one of the following methods two times / day : group a, brushing with 1.0 g of colgate regular flavor, followed by applying 0.25 g of tooth mousse plus ; group b, brushing with 0.25 g of clinpro tooth crme ; and group c, brushing with 1.0 g of colgate regular flavor. after 14 days, the enamel slabs (54 slabs / group) were embedded in resin, sectioned and examined with a polarized - light microscope, and the lesion areas were quantified using image - pro plus. all experimental groups showed a significant reduction in lesion area compared to the initial lesion area (paired t - test, p0.05). all three groups remineralized the enamel slab lesions, indicating model sensitivity to fluoride. given the differences in usage amounts and treated regimens, clinpro tooth crme provided similar benefits to the fluoride toothpaste ; however, no additional benefit of tooth mousse plus was observed when used in conjunction with the fluoride toothpaste.
tonsil plays an important role in immune defense mechanism especially in the production of iga and regulation of secretory immunoglobulin production against many exogenous microorganisms. it also protects from the invading pathogens as a part of waldeyer s ring, which is responsible for b- and t - cell activities in response to a variety of antigens. recurrent tonsillitis is defined as five or more episodes of true tonsillitis a year, symptoms for at least a year, and episodes that are disabling and prevent normal functioning. although the lifetime prevalence of common recurrent tonsillitis is 7%11% and has significant burden on families, most of the previous studies on tonsillitis evaluated only the role of upper respiratory tract infections and not enough attention has been given to improve children s quality of life (qol). because of the incomplete development of the immune organs in childhood, the immune activity of tonsil is considered to be more important in children than in adults. main indications for tonsillectomy are obstructive sleep apnea because of the enlarged tonsils, suspicion of malignant disease, and recurrent infections. an ideal tonsillectomy operation usually results in little morbidity and mortality, and improves patients qol. conventional dissection method is still the most common standard procedure for tonsillectomy with the advantage of being a safe procedure without any tonsillar remnants. in general, tonsillectomy also affects the patient s immune system, especially significant levels of interleukin is diminished postoperatively. bhattacharyya and kepnes showed that tonsillectomy resulted in significant improvement in the qol of patients by decreasing the burden of recurrent tonsillitis. the levels of igg, iga, and igm in the tonsillectomy patient group significantly decreased compared with those in the age - matched healthy control group. but in long - term use, it was found that treatment with penicillin resulted in hypersensitivity reactions, anaphylaxis, irritative responses, gluteal abscesses, severe local pain, and dysfunction. it is rapidly absorbed and widely distributed throughout the body, achieving higher concentrations in tissues with the therapeutic levels present in tonsil tissue during weekly medication with minimal side effects. gopal. mentioned the use of 500 mg once weekly oral azithromycin was effective in the prevention of streptococcal throat infection compared with oral penicillin therapy. pediatric qol is an important endpoint in health outcomes researches, as the life - threatening illness usually affects the progression of the child in school and increases the burden to the extended families, affects satisfaction and safety experienced by both family and patients, and results in child being usually upset, expressing anger, and appearing flustered. the aim of this study was to compare the qol outcomes after conventional dissection tonsillectomy versus azithromycin treatment controlling recurrent tonsillitis. a double - blind, randomized clinical trial was carried out in the otolaryngology department, suez canal university hospital, ismailia, egypt from march 2005 to may 2012. the study protocol was approved by the local ethics committee and written informed consent was obtained from all the patients. children attending the ent outpatient department with recurrent tonsillitis (five or more episodes of true tonsillitis a year and with symptoms for at least a year) of both sexes (age range 512 years) were included. the main inclusion criteria for this study were recurrent tonsillitis and tonsillar hypertrophy (grades 12 according to brodsky) with seronegative for anti - streptolysin o (aslo) titer, while the exclusion criteria included rheumatic heart, patients receiving long - acting penicillin, previous tonsillectomy, diabetes mellitus, grades 34 tonsillar hypertrophy according to brodsky, history of obstructive sleep apnea, or seropositive for aslo titer. complete medical history, including recurrent tonsillitis symptoms, of all the children was recorded and visual analog scale (vas) was used for the assessment of symptom severity. the assessment of qol relevant to all children was rated on a 4-point scale consisting of 23 questions in four subscales : physical (8 items), emotional (5 items), social (5 items), and school functioning (5 items). age - adjusted questions and rating scales were used for parents reporting for children or self - reporting child, and the scores of all subscales are then transformed to scale from 0 (worse score) to 100 (best score). all children underwent complete ent and oral examinations, nasal and paranasal sinus examination, and x - ray nasopharynx. complete investigations including complete blood count, aslo titer, erythrocyte sedimentation rate, and c - reactive protein were also carried out. all children were required to complete a relevant questionnaire assessing their recurrent tonsillitis symptoms using a vas to assess subjective symptoms (0 = no symptoms and 10 = severe and/or constant symptoms). blocked randomization scheme using computer - generated random numbers was performed to divide children into two groups : group a and group b. group a (n = 92) was subjected to conventional dissection tonsillectomy, whereas group b (n = 92) received single 250 mg (children 25 kg) and 500 mg (children 25 kg) of oral azithromycin once weekly. a signed consent was obtained from the parents of all the children. in the conventional blunt dissection series, boyle davis mouth gag was applied, tonsil was retracted medially, incision was made using waugh s dissection forceps and tonsillectomy was performed by blunt dissection, and tonsil was removed with control of bleeding if present using ligatures and/or electrocautery. antibiotic therapy was continued for 10 days postoperatively (90 mg / kg of amoxicillin and clavulanate). all patients received a combined analgesics non - steroidal anti - inflammatory drugs (1 mg / kg of diclofenac) with paracetamol (15 mg / kg) given every 8 h for the pain control. the objective was to clinically evaluate the recurrent throat infection and qol in both the groups. in this study, 5-year follow - up after the tonsil surgery and azithromycin treatment assessments were performed in the same manner as before and 1 year after surgery, using vas for recurrent throat infection and pediatric qol assessment scale questionnaires. parent proxy version of the pedsql 4.0 consisting of 23 questions that cover four domains (physical, emotional, social, and school functioning) was used to assess the pediatric qol. a domain - specific score is calculated from the corresponding questions, ranging from 0 (worst qol) to 100 (best qol), which can be combined for a total functioning score poor (0.2), fair (0.20.49), average (0.50.79), and good (0.8). parents of all children underwent a brief interview with the physician to complete a questionnaire and provided demographic and disease - related information. outcomes after tonsillectomy and azithromycin treatment were assessed again each year by interviews for five consecutive years. statistical analysis of the data was processed using software package for statistical analysis (spss) version 15 (spss inc., quantitative data were expressed as means sd, whereas qualitative data were expressed as numbers and percentages. the student s t - test was used to compare the significance of difference for quantitative variables that followed a normal distribution. a double - blind, randomized clinical trial was carried out in the otolaryngology department, suez canal university hospital, ismailia, egypt from march 2005 to may 2012. the study protocol was approved by the local ethics committee and written informed consent was obtained from all the patients. children attending the ent outpatient department with recurrent tonsillitis (five or more episodes of true tonsillitis a year and with symptoms for at least a year) of both sexes (age range 512 years) were included. the main inclusion criteria for this study were recurrent tonsillitis and tonsillar hypertrophy (grades 12 according to brodsky) with seronegative for anti - streptolysin o (aslo) titer, while the exclusion criteria included rheumatic heart, patients receiving long - acting penicillin, previous tonsillectomy, diabetes mellitus, grades 34 tonsillar hypertrophy according to brodsky, history of obstructive sleep apnea, or seropositive for aslo titer. complete medical history, including recurrent tonsillitis symptoms, of all the children was recorded and visual analog scale (vas) was used for the assessment of symptom severity. the assessment of qol relevant to all children was rated on a 4-point scale consisting of 23 questions in four subscales : physical (8 items), emotional (5 items), social (5 items), and school functioning (5 items). age - adjusted questions and rating scales were used for parents reporting for children or self - reporting child, and the scores of all subscales are then transformed to scale from 0 (worse score) to 100 (best score). all children underwent complete ent and oral examinations, nasal and paranasal sinus examination, and x - ray nasopharynx. complete investigations including complete blood count, aslo titer, erythrocyte sedimentation rate, and c - reactive protein were also carried out. all children were required to complete a relevant questionnaire assessing their recurrent tonsillitis symptoms using a vas to assess subjective symptoms (0 = no symptoms and 10 = severe and/or constant symptoms). blocked randomization scheme using computer - generated random numbers was performed to divide children into two groups : group a and group b. group a (n = 92) was subjected to conventional dissection tonsillectomy, whereas group b (n = 92) received single 250 mg (children 25 kg) and 500 mg (children 25 kg) of oral azithromycin once weekly. a signed consent was obtained from the parents of all the children. in the conventional blunt dissection series, boyle davis mouth gag was applied, tonsil was retracted medially, incision was made using waugh s dissection forceps and tonsillectomy was performed by blunt dissection, and tonsil was removed with control of bleeding if present using ligatures and/or electrocautery. antibiotic therapy was continued for 10 days postoperatively (90 mg / kg of amoxicillin and clavulanate). all patients received a combined analgesics non - steroidal anti - inflammatory drugs (1 mg / kg of diclofenac) with paracetamol (15 mg / kg) given every 8 h for the pain control. the objective was to clinically evaluate the recurrent throat infection and qol in both the groups. in this study, 5-year follow - up after the tonsil surgery and azithromycin treatment assessments were performed in the same manner as before and 1 year after surgery, using vas for recurrent throat infection and pediatric qol assessment scale questionnaires. parent proxy version of the pedsql 4.0 consisting of 23 questions that cover four domains (physical, emotional, social, and school functioning) was used to assess the pediatric qol. a domain - specific score is calculated from the corresponding questions, ranging from 0 (worst qol) to 100 (best qol), which can be combined for a total functioning score poor (0.2), fair (0.20.49), average (0.50.79), and good (0.8). parents of all children underwent a brief interview with the physician to complete a questionnaire and provided demographic and disease - related information. outcomes after tonsillectomy and azithromycin treatment were assessed again each year by interviews for five consecutive years. statistical analysis of the data was processed using software package for statistical analysis (spss) version 15 (spss inc., quantitative data were expressed as means sd, whereas qualitative data were expressed as numbers and percentages. the student s t - test was used to compare the significance of difference for quantitative variables that followed a normal distribution. a total of 184 children with recurrent tonsillitis (112 males and 72 females) aged between 5 and 12 years (mean age 7.4 years) were randomly divided into two groups : group a (n = 92) subjected to conventional dissection tonsillectomy, whereas group b (n = 92) received single 250 mg (children 25 kg) and 500 mg (children 25 kg) of oral azithromycin once weekly. children in group a were hospitalized for 12 days for conventional dissection technique with the operating time ranging from 20 to 45 min (average 30 min). five children in group a had complication of reactionary hemorrhage after conventional dissection, and tonsillectomy required homeostasis in operative room under general anesthesia. the reported postoperative minor complaints like halitosis and blood - stained saliva were treated conservatively with mouthwashes containing hydrogen peroxide. the return to a regular solid diet was achieved in 9.5 2.5 days. in group a, 86 children (93.4%) reported no illnesses or recurrent throat inflammation, whereas in group b, five children (5.4%) reported recurrent pharyngotonsillitis in 5-year follow - up duration without any significant difference between the two groups. the mean intensity of recurrent tonsillitis symptoms severity according to vas before treatment among the groups a and b were summarized in table i without any statistically significant difference between both groups. mean degree of different recurrent tonsillitis symptoms in both groups before treatment insignificant p > 0.05 five - year follow - up from starting the treatment (tonsillectomy vs. azithromycin), the mean intensity of pharyngitis or tonsillitis symptoms according to vas among groups a and b were compared. there were marked improvement from the pretreatment regimen, but there was no statistically significant difference between the two groups (table ii). mean degree of different recurrent tonsillitis symptoms in both groups after treatment insignificant p > 0.05 qol scale was calculated and assessment at 5-year follow - up after the treatment was done in both groups. there was a better qol in both groups compared with the pretreatment (tables iii and iv), but similar in both groups qol after treatment (fig. quality of life scale assessment in group a pre- and post - tonsillectomy highly significant at p 0.05 quality of life scale assessment in both groups after treatment there were no significant differences between the groups with regard to ent infections. but during the final year of the study period, four children in group a (4.3%) and five children in group b (5.4%) complained of ent infections. recurrent tonsillitis is considered as one of the common primary care visits to physicians, and tonsillectomy represents the most common pediatric operations ; however, its effectiveness, safety, and the net benefit of tonsillectomy is unclear. hence, research for long - term outcomes is needed. although the absolute indications for tonsillectomy is grade 4 tonsillar hypertrophy (kissing tonsil), which usually leads to obstructive sleep apnea, still more than 75% of tonsillectomies operated due to recurrent tonsillitis in the great proportion neglected the number of pharyngitis episodes and upper respiratory tract infections after tonsillectomy. no consensus has yet been reached concerning the number of annual episodes [20, 21 ]. tonsillectomy morbidity had marked impact on the qol of patients such as socioeconomic factors and increased burden to parents from the suffering of the child. some studies did not find any significant difference between patients with mild symptoms of recurrent tonsillitis and patients who have undergone tonsillectomy. on one hand, tonsillectomy should not be considered as the only solution as there is a possibility of immunological deficit that must be carefully considered when selecting the actual need for operative intervention as the function of tonsils in the immune system is not completely clear as an important constituent of the upper respiratory tract defense system. on the other hand, some studies have shown that patients who undergone tonsillectomy are at high risk of developing bronchial asthma, ulcerative colitis, goiter, and arterial hypertension at a later stage because of the loss of the immunologic barrier proving its immunological basis. azithromycin is an azalide antibiotic, which penetrates to the cell membranes and concentrates within the lysosomal compartment. consequently, it is widely distributed throughout the whole body, achieving higher concentrations in tissues, and thus, serum delivery to infected tissue is further enhanced by inflammatory processes. there were no significant differences between the groups with regard to ent infections in 5-year follow - up duration. casey and pichichero showed that azithromycin treatment for group a streptococcal tonsillopharyngitis in children and adults is more effective than other treatment regimens in eradicating and providing clinical cure of tonsillopharyngitis. odoherty also showed that azithromycin treatment is safe, well tolerated, and effective, given the longer duration of action, better side effect profile and lack of p450 interaction, greater stability in the presence of acid, better absorption, and without gastroparesis action as more than or penicillin. qol is defined as physical, social, and emotional aspects of a patient s well - being that are relevant and important to the individual. this is based primarily on the multidimensional concept of health used as assessments of the outcome of medical care, the impact of disease, and treatments. many studies have mentioned marked improvement in qol after tonsillectomy from reducing infections with increase in the body weight of children. better qol was observed in both groups when compared with the pretreatment, but similar outcomes in both groups after 5-year follow - up without statistically significant difference. our data showed the benefit from the use of azithromycin (500 mg oral once weekly) in preventing recurrent tonsillopharyngitis similarly to tonsillectomy outcomes. finally, it should be noted that qol is also affected by family situation and other physical activity outcomes. in conclusion, tonsillectomy is not the only solution to prevent recurrent throat infection as patients could be properly treated well with better qol outcomes if prophylactic azithromycin was used. azithromycin is an effective method as a prophylaxis against recurrent tonsillitis with a great benefit for better qol. both authors made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data, participated in drafting the article and gave final approval of the version to be submitted and any revised version to be published.	backgroundrecurrent tonsillitis is a common disease with marked evidence of affecting children quality of life (qol) such as their progression in school and increased burden to extended families. the aim of this study was to compare the qol outcomes after conventional dissection tonsillectomy versus azithromycin treatment in controlling recurrent tonsillitis.methodsa double - blind, randomized clinical trial was carried out in 184 children with recurrent tonsillitis randomly divided into two groups : group a was subjected to conventional dissection tonsillectomy, whereas group b received single 250 mg (children 25 kg) and 500 mg (children 25 kg) of oral azithromycin once weekly.resultsthere were no significant differences between the groups with regard to ear, nose, and throat infections during the 5-year follow - up. better qol was observed in both groups when compared with the pretreatment, but similar qol in both groups qol after treatment.conclusionazithromycin is an effective method as a prophylaxis against recurrent tonsillitis with a great benefit for better qol outcomes.
it results from the developmental failure of the posterior nasal cavity (choanae) to communicate with the nasopharynx. it is postulated to be secondary to an abnormality during the rupture of the buccopharyngeal membrane in the embryological period. obstruction of the nasal airway may also result from turbinate hypertrophy, pyriform aperture stenosis, and nasal cavity stenosis in association with craniofacial anomalies. the anatomic classification of choanal atresia has been revised. it was previously thought that choanal atresia was bony in 90% of cases and membranous in 10% of cases ; however, the development of more accurate evaluation techniques indicated that most, if not all, patients with choanal atresia have bony abnormalities. the major bony component of atresia is an abnormal widening of the vomer. the posteromedial maxilla is bowed medially and approximates, or is fused with, the lateral margin of the vomer (figure 1). the membranous components of atresia are characterized by soft tissue filling the posterior choanae associated with a narrowing of the posterior choanae just anterior to the pterygoid plates (figure 1). it is considered that combined bony and membranous malformations are found in approximately 70% of cases and purely bony atresia in approximately 30% of cases. it is a potentially life - threatening disorder because the affected infant is an obligate nose breather. symptoms range from intermittent to severe respiratory distress with cyanosis that is aggravated by feeding and alleviated by crying. approximately, 75% of children with bilateral choanal atresia have other congenital abnormalities, as exemplified by charge syndrome (coloboma of eye or microphthalmia, heart malformation, choanal atresia, retarded growth, genital hypoplasia, and ear abnormalities, typically external). surgical correction of bilateral choanal atresia is performed as soon as possible after the diagnosis has been made ; however, there is little consensus regarding the optimal surgical approach. transpalatal, transnasal, transseptal, and sublabial approaches have been utilized with varying degrees of success. recently, the transnasal endoscopic procedure has been advocated as a safe and efficacious method with the best possibility for long - term nasal patency [8, 9 ]. the surgical approach is determined by the thickness of the bony atresia plate as determined by computed tomography (ct). ct scanning is often used as a diagnostic tool for choanal atresia because it can generate information regarding the extent and type of atresia. the objective of this study was to illustrate the role of multislice ct and local contrast instillation for the diagnosis and characterization of choanal atresia as well as to highlight other associated congenital anomalies. following the approval from the hospital ethics committee, newborn patients with suspected choanal atresia due to symptoms of respiratory distress with cyanosis that is aggravated by feeding and alleviated by crying or suspected during nasal suction by attending pediatrician. 9 patients (5 males and 4 females) were included in this study between january 2007 and march 2010. all patients were examined by multislice ct using a ge high - speed 8-slice machine. the patients were positioned in the supine position with their head positioned to make their face perpendicular to the gantry (chin raised) ; the head was supported by a positioning pad. nasal suction was performed and topical nasal vasoconstriction was installed just prior to examination the images were reformatted in different planes : axial (so that the posterior choanae were at the same level as the nasopharynx), coronal, and sagittal views. the posterior choanae and their communication with the nasopharynx were clearly delineated in all patients (table 1). one patient (11.1%) had bilateral bony atresia with an ear abnormality (figure 2). one patient (11.1%) had congenital nasal pyriform aperture stenosis and a single central mega incisor (figure 3). three patients (33.3%) had unilateral mixed membranous and bony atresia and local contrast delineated that thin membrane (figure 4). three patients (33.3%) had unilateral pure bony atresia (figure 5). of the 9 patients included in our study, the patients with the single central mega incisor were diagnosed with aperture stenosis, where the width of the pyriform aperture was 5 mm at the level of the inferior turbinate. only 1 of 7 patients (14.3%) was found to have bilateral bony atresia. one of these patients had several congenital abnormalities, including cardiac and renal deformities and a hypoplastic lateral semicircular canal. of the 6 patients diagnosed to have unilateral mixed or purely bony atresia, 1 patient had esophageal atresia and a tracheoesophageal fistula. the remaining patients had no other charge syndrome lesions (charge stands for cluster of characteristics that include coloboma of the eye, heart defects, atresia of the choanae, retardation of growth or development, genital or urinary abnormalities, and ear abnormalities and deafness). choanal atresia, consisting of a unilateral or bilateral bony membranous septum between the nose and the pharynx, occurs in approximately 1 in 5000 to 7000 live births. approximately 50% of children with bilateral choanal atresia have other congenital abnormalities the most commonly described association is with charge syndrome the major diagnostic criteria occur commonly in charge syndrome, but they are relatively rare in other conditions. these are coloboma, choanal atresia, characteristic ear abnormalities, and brainstem and cranial nerve dysfunction. the minor diagnostic characteristics occur less commonly or are less specific for charge : heart defects, genital hypoplasia, orofacial clefting, tracheoesophageal fistula, short stature, and developmental delay. the nature and extent of the lesion causing the nasal obstruction (including congenital causes) is best determined by ct. when the diagnosis of choanal atresia is questionable, ct can differentiate other causes of bilateral nasal obstruction, such as pyriform aperture stenosis (figure 3) or a bilateral nasolacrimal duct cyst, from choanal atresia. unilateral causes of nasal obstruction, such as a nasal foreign body, turbinate hypertrophy (figure 6), septal deviation, antrochoanal polyp, or nasal tumor, can also be differentiated from choanal atresia by ct. the main disadvantages of using ct in pediatric populations are the high radiation exposure dose and the need for sedation to avoid movement artifacts, which render the examination of fine details less reliable. using contrast material in the nose will help to overcome that. over the last decade, ct has benefited from two major advances : the introduction of helical ct imaging in the early 1990s and multidetector ct acquisition in 1998 [13, 14 ]. this technique offers significant advantages, including improved temporal and spatial resolution, retrospective determination of slice thickness, and shorter acquisition time. the isotropic effect of multislice ct combined with the ability to take different reformatting angles is again considered an advantage of this technique. during the examination of patients with a questionable diagnosis of choanal atresia, it is necessary to have a scanning angle that enables us to delineate the communication of the nasal cavity with the nasopharynx. this can be achieved by changing the angle of the gantry so that the imaging plane becomes parallel to the hard palate. the difficulty of this technique is that the inadequate positioning of some patients in such an age group necessitates the repetition of the scan, resulting in a subsequent increase in the radiation dose. in addition, the angle of the imaging plane increases the risk to the child 's lens. multislice ct generates good isotropic reformatted images with a straight gantry and, hence, decreases the likelihood of repeating the scans and avoids the direct exposure of the lens. sagittal reformatting, although not crucial, may provide a more accurate diagnosis of choanal abnormalities (figure 7). since reformatted scan is not as accurate as actual scan and depends upon the thickness of the scan, hence the local contrast in the nose will enhance the findings without extra radiation exposure. in our institute, we apply low dose (50 ma) volumetric scans of the paranasal sinuses in thin axial cuts using the multislice technique with reconstruction in different planes. this low - dose technique does not affect the measurement accuracy of choanal parameters and can, therefore, be used with a subsequent decrease in the radiation dose. in postsurgical or recurrent choanal stenosis, the cause is usually a postoperative scar or an incompletely resected bony atretic plate. in such patients, we found that the local instillation of 13 drops of diluted nonionic contrast medium is helpful for the more accurate delineation of the communication of the posterior choanae with the nasopharynx. in some patients, even with good suction and the administration of a topical nasal vasoconstrictor, the characterization of the nature and thickness of the atretic membrane may be not accurate. the application of a local contrast agent helped to avoid such pitfalls, providing a more accurate delineation of the abnormality. this instillation did not affect the measurement of the thickness of the vomer or the width of the posterior choanae. in our study, one patient was normal with no clear evidence of choanal atresia or other causes that may lead to nasal obstruction, for example, turbinate hypertrophy ; however, the clinical diagnosis of choanal atresia was most likely due to mucosal edema. congenital nasal pyriform aperture stenosis was first described as a distinct entity in the radiology literature in 1988 and first described clinically in 1989. a pyriform aperture width of less than 11 mm in a term infant, as measured by ct, is considered to be diagnostic of this condition. as an otolaryngologist cleansing the nose and vasoconstriction will help to delineate the pathology. local contrast is an extra tool that can enhance this information especially the thickness of the atretic plate and the condition of the mucosa as well as the vomer thickness. multislice ct can clearly delineate the posterior choanae and their communication with the nasopharynx. the application of a low - dose technique using 5060 ma can avoid a high radiation dose to the child without any effect on the diagnostic accuracy. local instillation of a contrast medium helps in postsurgical cases. it could be routinely used, even in patients without previous surgery, for a more accurate delineation of the cause of the nasal obstruction.	objective. to illustrate the role of multislice computed tomography and local contrast instillation in the diagnosis and characterization of choanal atresia. to review the common associated radiological findings. methods. we analyzed 9 pediatric patients (5 males and 4 females) with suspected choanal atresia by multislice computed tomography. we recorded the type of atresia plate and other congenital malformations of the skull. results. multislice computed tomography with local contrast installed delineated the posterior choanae. three patients had unilateral mixed membranous and bony atresia. three patients had unilateral pure bony atresia. only 1 of 7 patients have bilateral bony atresia. it also showed other congenital anomalies in the head region. one patient is with an ear abnormality. one patient had congenital nasal pyriform aperture stenosis. one of these patients had several congenital abnormalities, including cardiac and renal deformities and a hypoplastic lateral semicircular canal. of the 6 patients diagnosed to have choanal atresia, 1 patient had esophageal atresia and a tracheoesophageal fistula. the remaining patients had no other charge syndrome lesions. conclusions. local contrast medium with the application of the low - dose technique helps to delineate the cause of the nasal obstruction avoiding a high radiation dose to the child.
systemic lupus erythematosus is characterized by the production of plethora of autoantibodies which potentially drive immune - complex related inflammation in various tissues and organs. breakdown of immune tolerance is believed to be one of the major mechanisms which triggers the production of autoantibodies by b cells and antibody forming cells, leading to inflammation upon binding to autoantigens and consequent tissue damage. as such recent compelling evidence has demonstrated that t cells are actually crucial in the pathogenesis of sle in that they enhance the production of autoantibodies by offering substantial help to b cells through stimulating the latter to differentiate, proliferate, and mature, in addition to their support on class - switching of autoantibodies which b cells are expressing. therefore, sle is currently believed to be a t cell - driven condition and, indeed, targeting molecules expressed on t cells and their signalling pathways can be one of the potential therapeutic strategies in sle. in comparison with healthy subjects, a number of studies have demonstrated that t cells isolated from patients with sle are abnormal, with regard to their phenotypes and functions [4, 5 ]. phenotypic and functional alterations in lupus t cells including expansion of the th17 population, perturbations of the physiology of t - cell receptors (tcrs) and postreceptor downstream signalling, oxidative stress, and epigenetic changes result in exaggeration of tcr response to stimuli and the propensity of lupus t cells to get activated. additionally, the failure of the regulatory cd4 + and cd8 + t lymphocytes in alleviating the proinflammatory milieu occurring in sle is contributory to the pathogenicity of the condition [7, 8 ]. in this brief review, a detailed account of the putative mechanisms by which the normal physiology of t cells are disturbed and why regulatory t cells fail to alleviate proinflammatory response in sle will be discussed. the current state of clinical trials evaluating therapeutic agents which target molecules expressing on and inside t cells for the treatment of sle will be updated. t cells recognize antigens presented to them by the major histocompatibility complex of antigen - presenting cells via the tcrs expressed on their surface. stimulation of tcrs upon antigen binding triggers downstream signalling pathways which enables various physiological functions of the t cells. the majority of tcrs (95%) are heterodimers which compose of an and a chain (receptors) and are anchored into the plasma membrane by a short cytoplasmic tail. a minor group (15%) of tcrs comprise a and a chain (receptors) which are expressed in certain populations of thymic t cells and peripheral t cells in the epithelia [10, 11 ]. tcrs are associated with cd3 which is a series of polypeptides with consistent amino acid sequences and is responsible for signal transduction upon antigen recognition by the tcrs [9, 12 ]. cd3 consists of four invariant polypeptides, namely,,,, and, and the cd3-tcr complex is arranged in such a way that the four tcr chains (two and two positively charged chains) are associated with two, two, one, and one chain polypeptides of the cd3 which are all negatively charged [9, 12 ]. the cd3 has extracellular, transmembrane, and cytoplasmic tails whereby the 2 chains (or its variant the chain) are the longest cytoplasmic chains amongst the rest. the cytoplasmic portions of and chains are critically involved in tcr signal transduction for they possess the immunoreceptor tyrosine - based activation motifs (itams) which are targets of phosphorylation by various specific protein kinases in the signal transduction processes. itams and the subsequent pathways activated, such as the -associated protein 70 (zap-70) pathway, are essential for t - cell activation. closely related to cd3, fcr also associates with the itams. however, instead of stimulating the zap-70 pathway, the spleen tyrosine kinase (syk) pathway is preferentially utilized [15, 16 ]. syk stimulation characteristically results in higher calcium influx into cells than that involves the zap-70 pathway, probably regulated by transcription factors c - jun and ets2. such rewiring of postreceptor downstream signalling mechanism has a strong pathological implication in lupus t lymphocytes (discussed in the next section). moreover, reduction of stability and increase in degradation of cd3 in lupus t cells are evident [1921 ]. to replace the deficient cd3 subunits, fcr receptors are reciprocally activated and expressed on lupus t cells. instead of coupling with zap-70 for signalling by the cd3 subunits, fcr associates with the syk pathway and such rewired downstream signalling confers stronger phosphorylation of signalling molecules and higher calcium influx which intensifies the tcr - derived signals in lupus t cells. increase in intracellular calcium activates calcineurin in the cytoplasm which enhances the action of the nuclear factor of activated t cells (nf - atc2) through the dephosphorylating action of calcineurin. activated nf - atc2 alters the expression of certain genes including the cd40l gene of lupus t cells by binding to the promoter of the cd40l gene. cd40l is a costimulatory molecule expressed on t cells and its cognate interaction with cd40 expressed on b cells promotes differentiation, proliferation, and antibody production of the latter, as well as class switching, in conjunction with the action of il-10 and il-21. another mechanism whereby lupus t cells exhibit a lower threshold of activation is the presence of preaggregated lipid rafts on their cell membrane. the lipid rafts are lipid - rich areas on the cell surface where tcrs and the associated signalling molecules are concentrated [25, 26 ]. during inactivated state, lipid rafts are evenly distributed throughout the cell membrane but, in lupus t cells, clustering of lipid rafts has been demonstrated even when they are minimally stimulated [24, 27 ]. clustered lipid rafts enhance lower threshold of signal transduction as molecules necessary for receptor activations are physically clustered in lupus t cells. to prove these potential mechanisms, intraperitoneal administration of pharmacologically active compounds which disrupt (mcd) and enhance (cholera toxin b) lipid raft clustering demonstrated reduction and promotion of t - cell activation, respectively, in a murine lupus - prone model. abnormalities in certain signalling pathways in lupus t cells which lead to defects in t - cell activation in patients with sle have been increasingly identified. impairment of cyclic adenosine monophosphate (camp-)dependent phosphorylation due to the reduction of protein kinase a levels has been reported [29, 30 ]. in addition, the activities of pathways involving protein kinase c and p56-lymphocyte - specific protein tyrosine kinase (p56lck) have also been found to be compromised [31, 32 ]. on the other hand, the activities of protein kinase pkr and phosphatidylinositol-3 kinase (pi3k) were found to be increased in a lupus - prone animal model [33, 34 ]. pharmacological inhibition of pi3k can ameliorate glomerulonephritis and decrease mortality in the mrl / fas murine lupus model. the activity of the mitogen - activated protein (map) kinases, which is crucial in the proliferation and apoptosis of t cells, is reduced in t cells of patients with sle. animals which are deficit in pkc (an activator of map kinase) have been shown to develop spontaneous lupus - like disease. as described above, upregulation of cd40l in lupus t cells is evident as a result of the activation of nf - atc2 secondary to high calcium flux [17, 22 ]. increase in cd40l upregulates expressions of cd80 and other costimulatory molecules on the antigen - presenting cells which further intensify the stimulatory signals to the t cells. being a sle susceptible gene, the cd40l gene is methylation sensitive. dna methylation which has been shown to be reduced in t cells, is linked to t cell auto - reactivity. hypomethylation in one of the x - chromosomes which is inactive in female lupus patients induces overexpression of cd40l mrna and hence cd40l expression on lupus t cells. altered map kinase and pkc activities are also caused by hypomethylation secondary to the deficiency of dna methyltransferase 1 in lupus t cells [23, 38 ]. il-2 is essential in reducing the polarization of nave cd4 + cells towards the th17 phenotype (see figure 1). reduced production of il-2 demonstrated in patients with sle enhances the expansion of th17 population which promotes local inflammation and recruitment of immunocytes in part due to the increased production of il-17. expression of il-2 by t cells is in fact tightly regulated by the transcription factors camp response element (cre) binding protein (creb) and the cre - modulator (crem). creb enhances the transcription of the il-2 gene while crem suppresses it by competing for the cre binding site with creb. the balance between creb and crem activity, which is important in determining whether il-2 is upregulated or downregulated, is altered in lupus t cells. there are at least 2 proposed mechanisms to explain the increased crem and reduced creb activities in lupus t cells. first, high levels of antilymphocytic antibodies in patients with sle activate calcium / calmodulin - dependent kinase iv (camkiv) which enhances crem activity through phosphorylation. second, the increased intranuclear level of protein phosphatase 2a (pp2a) in lupus t lymphocytes dephosphorylates and inactivates creb [45, 46 ]. one point of note is that elf-1, an important transcription factor of cd3, is dephosphorylated by the increased level of intranuclear pp2a in lupus t cells. dephosphorylated elf-1 fails to associate with the dna and initiate transcription of cd3 transcription, leading to the increased fcr and cd3 ratio, favouring subsequent activation of the syk instead of zap-70 pathways in lupus t cells (see figure 1). increase in oxidative stress has been demonstrated in lupus lymphocytes as evidenced by ultrastructural changes in the form of tubuloreticular structures of organelles in lymphocytes from patients with active lupus. oxidative stress induces nitric oxide activity and elevation of mitochondrial transmembrane potential which lead to activation of the protein kinase named mitochondrial transmembrane potential and mammalian target of rapamycin (mtor) in lupus t cells. increase in mtor activity causes rab4a - mediated cd3 downregulation and results in high calcium flux when lupus t cells are stimulated. increase in intracellular calcium activates camp response element modulator (crem) which inhibits il-2 and enhances il-17 expressions. mtor activation also suppresses foxp3 transcription by inhibiting dna methyltransferase 1 (dnmt1) which results in hypomethylation of the foxp3 promoter. rapamycin, an inhibitor of mtor, was demonstrated in a small clinical study of nine lupus patients to be able to normalize t - cell activation - induced calcium influx and reduce overall lupus disease activity. other potential mechanisms of mtor in immune response inhibition will be discussed in a subsequent section. cd4 + regulatory t cells (cd4 + tregs) were shown to be reduced in the secondary lymphoid organs of the nzb / w f1 lupus - prone mouse model as compared with age - matched nonautoimmune mice. deficiency of cd4 + tregs is linked to the development of lupus - like disease, while adoptive transfer of cd4 + tregs slowed the progression of renal disease and reduced mortality in nzb / w f1 mice. besides thymic cd4 + tregs, peripheral - induced cd4 + tregs (cd4 + itregs) conferred by the action of il-2 and tgf were shown to be able to reduce serum anti - dsdna levels and alleviate immune complex glomerulonephritis secondary to the reduction of t - cell help to b cells in nzb / w f1 mice. in humans, the number of cd4 + tregs was generally found to be lower in patients with active sle as compared with those with inactive disease and healthy individuals. reduced levels of forkhead box p3 (foxp3) in cd4 + tregs in patients with active lupus are generally believed to be the reason why these patients have less tregs - suppressive activity than their counterparts with inactive disease [5557 ]. interestingly, effective immunosuppressive therapies with glucocorticoids and rituximab have been shown to restore the number of functional tregs in patients with sle [5860 ]. despite the prevailing belief of the inferior quantity and functional quality of tregs in patients with sle, the lack of truly reliable markers which allow identification and isolation of the genuine treg population renders reliability and reproducibility of treg studies in sle an issue. helios, which is a transcription factor that belongs to the ikaros family, has recently been shown to be expressed by most of the foxp3 + t - cells in humans and it has been demonstrated to be able to upregulate foxp3 expression by binding to the foxp3 promoter. in contrast to the previous findings which advocated the lower quantity of tregs in lupus patients with more active disease, the population of foxp3 + helios+ tregs was indeed shown to be significantly expanded in patients with active sle when compared with those with inactive disease and healthy controls [61, 62 ]. in addition, the foxp3 + helios+ t cells isolated from 20 lupus patients were shown to have lower il-2 and ifn productions when compared with those from foxp3 + helios t cells. in both the nzb / w f1 and human monoclonal anti - dna - induced experimental mouse models, expansion of cd8 + tregs by tolerogenic peptide suppressed anti - dsdna production, cd4 + t cell proliferation, and type-2 interferon production, probably as a result of tgf and foxp3 produced by the cd8 + itregs [63, 64 ]. similar to the findings of cd4 + tregs, studies addressing the number of circulating cd8 + tregs in patients with sle have yielded inconsistent results [65, 66 ]. cd8 + tregs from patients with active sle failed to suppress effector t cells, while cd8 + tregs from patients with inactive sle demonstrated comparable suppressive ability as those from healthy individuals. of particular note, since the data of cd8 + tregs in sle are based on a small number of clinical studies, more robust studies are required to further characterize the quantity and functional aspects of cd8 + tregs in patients with sle. recently, a group of rare t cells which express high levels of cd25 and cd27 and low level of cd45ra has been found to possess regulatory and suppressive activities (cd27cd45ra treg cells), particularly the v1 subset. enumeration of the peripheral blood mononuclear cell (pbmc) populations revealed a significantly lower number of circulating cd27cd45ra treg cells in patients with sle as compared to that of healthy controls. furthermore, a significant inverse correlation was found to exist between lupus disease activity and the level of circulating cd27cd45ra treg cells. in vitro experiment confirmed the ability of lupus cd27cd45ra treg cells to express foxp3 in a cd27-dependent fashion when the cells were cultured in the presence of tgf. in addition, cd27cd45ra treg cells were demonstrated to be able to suppress the proliferation of autologous effector cd4 + cells in coculture systems. though rare in the pbmc population, further experiments are required to fully characterize the phenotype and function of these treg cells which may play an important immunopathogenic, as well as potential therapeutic, roles in suppressing the disease activity of sle. the most commonly used calcineurin inhibitors including cyclosporin and tacrolimus have been proven in randomized controlled trials to be at least as efficacious and safe as conventional treatment for proliferative and membranous lupus glomerulonephritis [6870 ]. a one - year quasirandomized trial revealed proteinuria remission rates of 83%, 60%, and 27% in patients who were in the cyclosporine, intravenous cyclophosphamide, and prednisolone groups, respectively, although the relapse rate of proteinuria was higher in patients receiving cyclosporine than those who received cyclophosphamide. as an induction therapy, the combination of prednisolone and intravenous cyclophosphamide (a total of six 4-week pulses starting at 750/m of body surface area) or tacrolimus (starting at 0.05 mg / kg / day and being titrated to a trough level of 510 ng / ml) has been shown to be equally efficacious in achieving complete renal remission. tacrolimus appeared to be safer as adverse events including leucopenia and gastrointestinal complaints were less frequent as compared to subjects in the cyclophosphamide group. as discussed previously, cd40l, which is overexpressed on lupus t cells, stimulates cd40 expressed on b cells to produce autoantibodies. two main clinical trials testing the blockade of the cd40-cd40l pathway in the treatment of sle are, however, disappointing [71, 72 ]. in addition to the failure of satisfying the predefined study end - points, the unfavourable side - effect profile of anti - cd40l unfortunately led to the premature termination of a multicentre phase ii trial of bg9588 in sle. in a double - blind, placebo - controlled trial, 85 patients with mild to moderately active sle were randomized to receive 6 infusions of anti - cd40l at doses of 2.5, 5, and 10 mg / kg and placebo at 0, 2, 4, 8, 12, and 16 weeks. after 20 weeks of treatment, lupus disease activity improved in all groups from baseline but no statistical significance was detected amongst the different groups. no difference in fatigue score and quality of life open - label trial evaluating bg9588 in the treatment of 28 patients with proliferative lupus glomerulonephritis, the occurrence of 2 myocardial infarctions in the subjects led to premature termination of the trial although significant reduction of proteinuria, haematuria, and anti - dsdna titre with increase in serum c3 levels were demonstrated. being a safe and well - tolerated drug clinically used for preventing transplant rejection, rapamycin, a macrolide antibiotic which regulates mitochondrial transmembrane potential and calcium influx, was evaluated in a small uncontrolled trial for its effectiveness in patients with sle. in 9 lupus patients who were refractory to conventional treatment, mitochondrial calcium level and t - cell activation - induced calcium fluxing were normalized in rapamycin - treated patient. in a recent prospective open - label study, rapamycin was shown to inhibit il-4 production by and necrosis of double negative (dn) t cells in patients with sle. in addition, rapamycin enhanced foxp3 expression in cd25+/cd4 + t - cells and expansion of cd25+cd19 + b cells, signifying that mtor can trigger il-4 production by and necrosis of dn t cells in active sle. recently, n - acetylcysteine (nac), the precursor of glutathione, was shown in a small clinical trial that at doses 2.4 gm and 4.8 gm daily it could reduce lupus disease activity and fatigue after 3 months of treatment as compared with placebo. nac reduced mtor activity and enhanced apoptosis of t cells, accompanied by reversed expansion of the cd4/cd8 populations. interestingly, nac was shown to induce foxp3 expression in cd4 + treg cells and reduce serum anti - dsdna levels. larger clinical trials are certainly required to validate the efficacy of this exciting therapeutic agent, especially it is anticipated that adverse effects of nac due to immunosuppression are very minimal. in both murine system and human disease of sle, t cells are found to be abnormal based on their alterations in the phenotype, receptor and signalling physiology, gene transcription, and perturbed suppressor activities of regulatory lymphocytes. the substantial involvement of t cells in the pathogenesis of sle and the apparent success in therapeutics directing at t cells in patients with sle lead to the firm belief that sle is indeed a t - cell driven autoimmune disease. while manipulating the b cells and their families with the use of b - cell depleting therapy (bdt) appears very promising in the treatment of sle and it is argued that b cells are relatively more important in the pathogenesis of sle than other immunocytes, the discrepantly prolonged beneficial effects of bdt against the much shorter half - life of rituximab invariably explain the potential importance of the participation of t cells in the pathogenic process of sle [58, 59 ].	systemic lupus erythematosus (sle) is characterized by the production of a wide array of autoantibodies. thus, the condition was traditionally classified as a b - cell disease. compelling evidence has however shown that without the assistance of the helper t lymphocytes, it is indeed difficult for the helpless b cells to become functional enough to trigger sle - related inflammation. t cells have been recognized to be crucial in the pathogenicity of sle through their capabilities to communicate with and offer enormous help to b cells for driving autoantibody production. recently, a number of phenotypic and functional alterations which increase the propensity to trigger lupus - related inflammation have been identified in lupus t cells. here, potential mechanisms involving alterations in t - cell receptor expressions, postreceptor downstream signalling, epigenetics, and oxidative stress which favour activation of lupus t cells will be discussed. additionally, how regulatory cd4 +, cd8 +, and t cells tune down lupus - related inflammation will be highlighted. lastly, while currently available outcomes of clinical trials evaluating therapeutic agents which manipulate the t cells such as calcineurin inhibitors indicate that they are at least as efficacious and safe as conventional immunosuppressants in treating lupus glomerulonephritis, larger clinical trials are undoubtedly required to validate these as - yet favourable findings.
during july 2007july 2011, six patients from the mediterranean coast city of elche, spain, who had high fever and inoculation eschars received a diagnosis of infection with r. sibirica mongolitimonae (table). for laboratory confirmation, dna was extracted from eschars, lymph nodes (fine - needle aspiration), and blood samples by using the qiaamp tissue kit (qiagen, hilden, germany), according to the manufacturer s instructions. for molecular detection, 200400 ng of dna from each sample was subjected to pcr targeting the 23s-5s rrna intergenic spacer, followed by hybridization with specific probes by reverse line blotting, as described (15). when using the probe for r. sibirica mongolitimonae, a positive hybridization signal was obtained from eschar samples from all 6 patients ; this result was confirmed by sequencing (100% similarity to a reference r. sibirica mongolitimonae strain [genbank accession no. hq710799 ] in all cases in the 357 bp sequenced). to further confirm this result, nested pcr targeting the gene for outer membrane protein a was performed as described (15) ; these sequences (514 bp) also showed 100% similarity to a reference r. sibirica mongolitimonae strain (genbank accession no. na, not available ; ast, aspartate aminotransferase ; alt, alanine aminotransferase. serologic response was analyzed by using an in - house microimmunofluorescence assay for igg and igm, performed as described (4) ; r. conorii and r. sibirica mongolitimonae were used as antigens, and cutoff values were 1:40 for igg and 1:20 for igm. acute- and convalescent - phase serum samples were obtained from 3 case - patients and single serum samples from the other 3 case - patients. results for samples from 2 case - patients were negative, but results for the remaining 4 samples showed low to medium titers. these results are consistent with previous reports (6), in which 30% of cases had a positive igm result and 50% had negative or near - cutoff igg results. all 6 case - patients lived in elche and its surroundings (230,112 inhabitants). three of the cases occurred during the spring, which is when 10/18 cases reported in the literature occurred (414). all 6 case - patients had fever (38.5c39.5c), myalgia, and headache ; in the cases from the literature, 18/18 patients had fever, 13/18 myalgia, and 11/18 headache. in our study, 1 case - patient was confused and drowsy on arrival at the emergency department. all 6 case - patients had a single inoculation eschar develop : 2 on the neck, 2 on a lower limb (figure), 1 on the scalp, and 1 on an upper limb. five (83%) case - patients had enlarged lymph nodes in the region from which the eschar drained, as reported for 10/18 (55%) cases from the literature. three case - patients (50%) had lymphangitis extending from the eschar to the draining lymph nodes (figure), compared with 6/18 (33%) in cases from the literature. inoculation eschar on popliteal area and discrete maculopapular elements in patient with lymphangitis infected with rickettsia sibirica mongolitimonae, spain, 2011. for 4/6 (67%) case - patients, a generalized maculopapular rash developed on the palms and soles but not the face ; for 2 case - patients these findings are consistent with our review of the literature, which indicated rash occurring in 13/18 (72%) cases. all 6 case - patients recovered without sequelae after antimicrobial drug treatment using doxycycline or azithromycin. fournier. (4), who reported 7 cases of r. sibirica mongolitimonae infection in 2005, proposed the name lymphangitis - associated rickettsiosis for the disease, on the basis of associated clinical features. however, for the case - patients reported here, the most common clinical signs and symptoms were fever and skin eschar, similar to those from previously reported case series ; 5 of the case - patients reported here showed regional lymph node enlargement, 4 rash, and 3 lymphangitis. because only 24 total cases have been reported and other rickettsioses produce lymphadenopathy and lymphangitis, the term lympangitis - associated rickettsiosis may be unwarranted for this disease. in our case series, 1 case - patient had mental confusion after 10 days of a febrile disease before hospitalization and was found to be hyponatremic. in this patient, the eschar was located on the scalp, and neither rash nor other clinical clues were suggestive of rickettsiosis. the patient had increased c - reactive protein plasma levels and the highest serologic antibody titers for r. sibirica mongolitimonae of the 6 case - patients (table, patient 1). since r. sibirica mongolitimonae was isolated from h. asiaticum ticks in 1991 (1), it has been recovered from h. truncatum ticks in sub - saharan africa (6) and h. anatolicum excavatum ticks in greece (7). however, in spain, r. sibirica mongolitimonae has been detected in 2/8 tick species tested (rhipicephalus pusillus and rh. bursa) at similar percentages (3.7% and 3.6%, respectively) but not from hyalomma spp. pusillus ticks from portugal was positive for r. sibirica mongolitimonae (8), while testing of other tick species, including h. lusitanicum, rh. (h. lusitanicum and h. marginatum) ticks have not been found to be infected in spain does not mean that these ticks are not vectors for r. sibirica mongolitimonae. ticks are a vector for this rickettsia on the iberian peninsula, and our findings confirm that this rickettsia species is circulating in spain, where specific vectors have yet to be described. in summary, we report 6 cases of human infection with r. sibirica mongolitimonae that occurred in the same geographic area of spain. our results indicate that pcr of eschar samples is the most useful diagnostic procedure for this pathogen ; samples from all 6 case - patients had positive results, while test results for 1 whole blood sample and 2 lymph node samples were negative. however, the limited number of samples does not make it possible to infer specific diagnostic sensitivities. the epidemiology and pathogenicity of illness caused by r. sibirica mongolitimonae infection require further investigation. an active search for the vector of r. sibirica mongolitimonae in countries of the mediterranean region is necessary to complete the epidemiology of this rickettsiosis, which is likely to be more widespread than originally assumed. in particular, clinicians caring for patients who have traveled to the mediterranean coast of spain should consider this rickettsiosis in the differential diagnosis.	human infection with rickettsia sibirica mongolitimonae was initially reported in 1996, and reports of a total of 18 cases have been published. we describe 6 additional cases that occurred in the mediterranean coast region of spain during 20072011. clinicians should consider this infection in patients who have traveled to this area.
chondral lesions in the patellofemoral joint are the most common cause for acute pain at the anterior aspect of the knee. when the fissure is located at the medial retro patellar aspect and a plica mediopatellaris exists, it is assumed that the fissure is caused by the plica as incision injury so that the removal of the plica is indicated to prevent further cartilaginous incision injuries. there are cartilage specific gradient echo sequences like the 2d multiple - echo data image combination sequence (medic) which are used in musculoskeletal imaging to delineate cervical disc herniation, tiny ligaments such as the scapholunate ligament, labrum injuries such as the bankart lesion, calcific deposits like in tendinitis calcarea or loose bodies in the joint. this sequence is used to determine the thickness of the cartilage and can be performed in 3d to quantify and map the volume of the cartilage. on the other hand, there are spin echo sequences which in contrast to the gradient echo - sequence better demarcate bone marrow edema when fat suppressed. in addition, these sequences provide a kind of indirect arthrography due to the high contrast between cartilage and surrounding fluid. thus, the short - tau inversion recovery sequence (stir) seems to be the optimal sequence to demarcate both, tiny cartilaginous fissures and bone marrow edema as their consequence. the medic has been only absolutely assessed but not relatively to other sequences schmid mr.. thus, the purpose of our study was to determine the value of the 2d multiple - echo data image combination sequence relative to the short - tau inversion recovery sequence regarding the depiction of chondral lesions in the patellofemoral joint. during a period of 6 month patients with acute pain at the anterior aspect of the knee, joint effusion and suspected chondral lesion in the patellofemoral joint underwent mri including axial medic and stir imaging. mri was performed on a 70 cm open bore and 125 cm short 1.5 t scanner (magnetom espree, siemens healthcare, erlangen, germany) using a circular polarized knee coil. only patients with chondral lesions in the patellofemoral joint on at least one mri sequence were elected for sequence comparison. the medic and stir sequence were quantitatively compared regarding the patella cartilage - to - effusion contrast - to - noise ratio (cnr). for that purpose the mean signal intensity (si) of retropatellar cartilage and effusion as well as the standard deviation (sd) of air were measured by standardized region - of - interest (roi) measurement at the level of the chondral lesion with 36 mm diameter depending on the thickness of the retropatellar cartilage. the standard deviation of air served as noise, so that cnr was calculated according to the formula : cnr : = (sicartilage sieffusion)/sdair the medic and stir sequence were qualitatively compared by two radiologists with 2 and 6 years experiences in musculoskeletal imaging who independently scored the depiction of the chondral lesion on a 3-point scale (1 = not depicted ; 2 = blurred depicted ; 3 = clearly depicted). the comparison in objective cnr and subjective rating was performed by the wilcoxon - mann - whitney - test using a p - value less than 0.05 as level of significance. inter - observer agreement was analyzed with cohen analysis using the four categories in agreement : fair (= 0.210.40), moderate (= 0.410.60), substantial (= 0.610.80), almost perfect (= 0.811.00). the medic and stir sequence were quantitatively compared regarding the patella cartilage - to - effusion contrast - to - noise ratio (cnr). for that purpose the mean signal intensity (si) of retropatellar cartilage and effusion as well as the standard deviation (sd) of air were measured by standardized region - of - interest (roi) measurement at the level of the chondral lesion with 36 mm diameter depending on the thickness of the retropatellar cartilage. the standard deviation of air served as noise, so that cnr was calculated according to the formula : cnr : = (sicartilage sieffusion)/sdair the medic and stir sequence were qualitatively compared by two radiologists with 2 and 6 years experiences in musculoskeletal imaging who independently scored the depiction of the chondral lesion on a 3-point scale (1 = not depicted ; 2 = blurred depicted ; 3 = clearly depicted). the comparison in objective cnr and subjective rating was performed by the wilcoxon - mann - whitney - test using a p - value less than 0.05 as level of significance. inter - observer agreement was analyzed with cohen analysis using the four categories in agreement : fair (= 0.210.40), moderate (= 0.410.60), substantial (= 0.610.80), almost perfect (= 0.811.00). 30 of 58 patients (male : female, 21:9 ; age : 44 12 yrs) revealed cartilage lesions (fissures, n = 5 (fig. 1, fig. 6) ; osteoarthritis, n = 3) and were included in this study. the stir - sequence was significantly (p < 0.001) superior to the medic - sequence regarding both, the patella cartilage - to - effusion cnr (mean cnr : 232 61 vs. 40 16) as well as the depiction of chondral lesion (mean score : 2.83 0.4 vs. 1.75 0.7) with substantial inter - observer agreement in the rating of both sequences (= 0.76 - 0.89). our results show that the stir - sequence is significantly superior to the medic - sequence regarding the depiction of chondral lesions in the patellofemoral joint (fig. 1, fig. 2, fig. 3, fig. thus, the stir - sequence should be preferred over the medic - sequence for the depiction of chondral lesions in the patellofemoral joint. although the medic - sequence provides a higher spatial resolution (0.5 0.4 mm vs. 0.7 0.6 mm) (tab. 3) or delamination (fig. 6). due to the high prevalence of mediopatellar plica in up to 80% of cases and its association with cartilage damage, in contrast to the medic - sequence the stir- sequence also depicts reactive edemas in the bone marrow as indirect sign of cartilage stress or damage (fig. 3). furthermore, edema depiction facilitates the assessment of severity, acuteness and consequence of cartilage stress or damage (fig. 3). also, the stir - sequence gave better depiction of the liquid film under the delamination, highlighting this pathology (fig. both, the objective analysis and subjective analysis with substantial inter - observer agreement accordingly confirm the superiority of the stir - sequence. we saw no advantage for the t2 -weighting of the medic - sequence in the depiction of chondral lesions in the patellofemoral joint. on the medic the cartilage signal was homogeneous in every case thus any inner inhomogeneity reflecting pressure the medic - sequence may be superior to the stir - sequence in the depiction of loose bodies in the joint due to its higher (not saturated) contrast between fat in tissue and bone. however, in the one case with a loose body in the patellofemoral joint there was no difference in the depiction of the loose body between the medic and stir - sequence. there was also no difference in the depiction of the plica mediopatellaris, which is anyway also and even better depicted on the sagittal fat - saturated t2 w sequence in the protocol of the entire knee. a mixed t1/t2 -weighted so - called double - echo steady state (dess) sequence has also been proposed as a cartilage specific sequence. the dess - sequence is currently being used in the osteoarthritis initiative to assess the cartilage of the knee joint. regarding the cartilage - to - effusion contrast there seems to be no significant difference between the medic- and dess - sequence. also used the model of the patellar cartilage to evaluate the diagnostic value of the dess - sequence and found that the dess - sequence was inferior to the conventional t2 w spin echo (se)-sequence. both, the medic- and dess - sequence provide less contrast than the stir - sequence for both, bone edema and cartilage injuries. thus, the stir - sequence combines the advantage of indirect arthrography with delineation of subchondral edema. however, the medic and dess 3d - sequences provide mapping of the cartilage volume with the intention to facilitate the prevention of osteoarthritis, thus providing prophylactic meaning. in contrast, the stir - sequence depicts focal damage of the cartilage and bone marrow edema as causes of pain. if other prior performed sequences reveal an effusion as indirect sign of damage we therefore prefer the stir - sequence to better depict the cause for the effusion. in the younger patient group (here with a mean age of 44 12 years (range : 2168 years)) with acute pain and effusion in the pfj in particular, the stir - sequence seems to be more helpful in directing therapy decisions as the pretest - probability cause of pain and effusion is less likely to be chronic. we did not compare the stir sequence with the fat - saturated t2w or pd sequences because the stir - fat saturation is known to optimally depict bone edema, which seems crucial to indicate lateral release or any other surgical procedure to prevent or treat osteoarthritis. in this manner, the knee protocol (table 1) also includes the maximum of bone edema - to - bone contrast. thus, for the benefit of non - invasiveness we compared the sequences regarding the cartilage - to - effusion cnr and depiction of lesion and not regarding their accuracy in the lesion detection. but the significant superiority of the stir over the medic sequence in both, the objective and subjective analysis is so clear that it seems not likely that the lack of a standard of reference biases the message of the study. the stir sequence should be preferred over the medic sequence for the depiction of chondral lesions in the patellofemoral joint. the stir sequence should be preferred over the medic sequence for the depiction of chondral lesions in the patellofemoral joint. there is no actual or potential conflict of interest in relation to this article (no disclosures).the work has not been published beforeno funding was received for this work.this paper describes the author own work.it is not under consideration elsewhere, and all authors have read and approved the manuscript. there is no actual or potential conflict of interest in relation to this article (no disclosures).the work has not been published before no funding was received for this work. it is not under consideration elsewhere, and all authors have read and approved the manuscript.	purposeto determine the value of the 2d multiple - echo data image combination (medic) sequence relative to the short - tau inversion recovery (stir) sequence regarding the depiction of chondral lesions in the patellofemoral joint.materials and methodsduring a period of 6 month patients with acute pain at the anterior aspect of the knee, joint effusion and suspected chondral lesion defect in the patellofemoral joint underwent mri including axial medic and stir imaging. patients with chondral lesions in the patellofemoral joint on at least one sequence were included. the medic and stir sequence were quantitatively compared regarding the patella cartilage - to - effusion contrast - to - noise ratio (cnr) and qualitatively regarding the depiction of chondral lesions independently scored by two radiologists on a 3-point scale (1 = not depicted ; 2 = blurred depicted ; 3 = clearly depicted) using the wilcoxon - mann - whitney - test. for the analysis of inter - observer agreement the cohen 's weighted kappa test was used.results30 of 58 patients (male : female, 21:9 ; age : 44 12 yrs) revealed cartilage lesions (fissures, n = 5 including fibrillation ; gaps, n = 15 ; delamination, n = 7 ; osteoarthritis, n = 3) and were included in this study. the stir - sequence was significantly (p < 0.001) superior to the medic - sequence regarding both, the patella cartilage - to - effusion cnr (mean cnr : 232 61 vs. 40 16) as well as the depiction of chondral lesion (mean score : 2.83 0.4 vs. 1.75 0.7) with substantial inter - observer agreement in the rating of both sequences (= 0.760.89).conclusionfor the depiction of chondral lesions in the patellofemoral joint, the axial stir - sequence should be chosen in preference to the axial medic - sequence.
the human vitreous humour (vh) is a transparent, highly - hydrated gel, which occupies the posterior segment of the eye between the lens and the retina. it is comprised almost entirely of water (99%) with the remainder consisting of a mixture of collagen fibres, hyaluronic acid, hyalocytes, inorganic salts, and lipids. the average protein concentration of the healthy vh is 0.5 mg / ml, consisting largely of albumin (6070%). further components are globulins, coagulation proteins, complement factors, and low - molecular - weight proteins. the ciliary body provides a constant fluid exchange by diffusion, ultrafiltration, and active transport of aqueous fluid into the posterior segment. proteins may accumulate in the vitreous by local secretion (e.g., glycoprotein), filtration from blood (e.g., albumin), or diffusion from the surrounding tissues. because of the close contact between the vitreous and the inner retina, physiological and pathological conditions of the retina affect both the proteome and the biochemical properties of the vh. various vitreoretinal diseases induce changes in specific vitreous proteins, especially when the blood - retinal barrier is disrupted. because vh can be totally or partially removed without marked detriment to the eye, surgical vitrectomy and vitreous biopsies are performed as part of routine clinical practice, providing abundance of human vh samples for analysis. many earlier studies investigated levels of individual proteins in vh from healthy and diseased eyes, using biochemical or immunological techniques, in particular enzyme - linked immunoabsorbent assay (elisa) [710 ]. this approach, however, is not suitable for the discovery of networks of functionally related proteins ; hence it can further our understanding of the pathophysiology of a disease only to a limited degree. proteomics is the large - scale study of the entire complement of proteins, the so - called proteome, present in a cell, tissue, biofluid, or organism in any given state. a novel hypothesis can be generated from global protein expression analysis of disease tissue, which can then be addressed with cellular and in vivo functional studies. proteomic analyses of healthy and diseased vh have been performed [5, 6, 10, 1224 ] to scrutinize the protein profile of vitreoretinal diseases, with the ultimate aim of identifying disease markers that could become the diagnostic and pharmaceutical targets of the future. the search so far has not been conclusive, but as proteomics is still an evolving field, better technologies and deeper understanding of the peculiar nature of the vh bear promising potential. this paper aims to guide biological scientists through the different proteomic techniques that have been used to analyse the vh. a second objective is to present future perspectives for the study of intravitreal inflammation using proteomics. proteomics experiments are categorised according to their objective : assay or discovery. assay or targeted studies typically seek to quantify a predefined set of proteins or peptides, whereas discovery experiments aim to analyse larger, unbiased sets of proteins. all proteomic analyses conducted on vh have used mass spectrometric discovery techniques to facilitate the identification and quantification of the many proteins occurring in the vh, expanding the spectrum of suitable candidates for targeted analyses. of the discovery methods that have been developed, all involve a multistep process, which includes sample acquisition, digestion of the protein sample into peptides, fractionation of the peptide mixture (or prefractionation of the proteins, depending on the technique chosen), protein identification by mass spectrometry, and data analysis. the various methods differ in their requirements for sample preparation, the extent and the level of sample fractionation (proteins or peptides), the type of ms, and the data processing tool used. each step will be described, reporting the different experimental strategies used for analysis of the vh and discussing their advantages and limitations. anatomically, the vitreous body can be subdivided into three main regions : the vitreous core, the vitreous base, and the vitreous cortex. the vitreous core (or central vitreous) comprises the main bulk of the vh and is a highly hydrated extracellular matrix, which is normally a cellular. the vitreous base and cortex both contain a low concentration of cells, named hyalocytes, and dense bundles of collagen fibrils. skeie and mahajan recently demonstrated by one - dimensional (1d) sodium dodecyl sulphate - polyacrylamide gel electrophoresis (sds - page) that the different substructures of the human vitreous, when individually isolated from post - mortem eyes, are characterised by an unique protein profile. hence, the dissection technique and the size of the sample are likely to influence the proteome composition. for ethical reasons, vitreous surgery necessitates a pathological state, even in retinal conditions such as macular pucker or macular hole. for this reason, some authors argue that examining vh from a carefully selected biobank eye is more representative of the normal vitreous proteome. whilst such opinion is debatable because of the postmortem changes that can occur, being able to harvest the entire vitreous body offers a definite advantage over the small sample produced by a core vitreous biopsy. vh can also be extracted from eyes enucleated because of a trauma or an ocular malignancy. in such cases, it is important to preserve the integrity of the globe for pathological examination. in the authors ' experience, the majority of the vh can be harvested anyway using a 23 g needle on a 10 ml syringe, which is inserted transclerally in the posterior segment of the intact globe. this yields at least 3 ml (out of the 4 ml total volume of the vitreous body). it is not advisable to harvest the vh following sectioning of an enucleated eye, as on opening the globe the more liquid part of the vh tends to spill, leaving the scientist with a highly viscous residue, which is nonrepresentative. in the vast majority of studies undiluted core vitreous biopsies are taken at the time of surgical vitrectomy for an underlying vitreoretinal disease, most often proliferative diabetic retinopathy. approximately 1 ml of undiluted vh can be obtained at the onset of pars plana vitrectomy, with closed infusion line, by manual aspiration with cutting on through the vitrectomy probe into a 2.5 ml syringe connected along the aspiration line. core vitreous biopsies from patients undergoing vitrectomy for macular hole (normal controls, as mh is an idiopathic condition that develops as the result of vitreofoveal traction and is therefore unlikely to affect the protein composition of the vh. most proteomic studies have been conducted on vitreous fluid obtained from diabetic patients undergoing surgery for proliferative diabetic retinopathy (pdr), which is a major cause of vitreous haemorrhage. this is an important element to consider when collecting vitreous fluid for proteomic analyses, as the haemorrhage can cause a massive influx of serum proteins into the vh, confounding results. for this reason, sim and colleagues have measured vitreous haemoglobin levels with a spectrophotometer and excluded all samples containing more than 5 mg / ml of haemoglobin [14, 19 ]. the preservation of biological state and sample quality prior to proteomic processing and analysis are extremely important. the proteins should be protected against loss or change as a consequence of proteolytic degradation. ideally, vh should be snap - frozen in liquid nitrogen immediately and stored at 80c until used. the ability to extract proteins is the key limiting factor in all subsequent proteomic identification and profoundly influences differential protein identification associated with diseased states. the main problem when handling vh specimens is the viscous nature of such samples. the collagen fibrillar network and associated surface macromolecules with age, the vitreous undergoes progressive liquefaction, starting in the vitreous core as pockets of fluid that then coalesce. neal. have measured the viscosity coefficient of different regions of the human vh in phakic and pseudophakic donor eyes. in phakic eyes, viscosity is higher near the lens than near the retina, whilst this trend is reversed in pseudophakic ones. hence, the macromolecular composition and the viscosity of vh samples differ according to the anatomical region where the sample is taken, the age of the patient, the state of the lens, and the presence of any vitreous pathology. viscosity prevents accurate pipetting, posing a problem when small accurate aliquots are needed for antibody - based assays or for assessing the protein content of a large specimen (e.g., bradford assay) prior to proteomic analyses. various preanalytical treatments have been proposed to reduce viscosity, including boiling, high - speed centrifugation, microfiltration, dilution, and hyaluronidase treatment [31, 32 ]. the effect of these treatments on the vh has been investigated in forensic science for the postmortem analysis of chemical analytes such as glucose, urea, and creatinine, but there is no comparative study on the effect of such pre - treatments on proteins. high - speed centrifugation (12000 rpm for 15 minutes) is the most common technique that is used to separate the liquid component of the vh from its structural one. centrifugal filters, such as the 0.22 m gv durapore filter (millipore, carrigtwohill, cork, ireland) have also been used to clarify vitreous samples. because proteomes are very complex mixtures, a number of techniques have been employed to extract them prior to analysis. protein fractionation is an important first step in facilitating access to the low abundant proteins of interest for clinical research. the most common techniques for this purpose are affinity chromatography for protein depletion and gel electrophoresis for protein separation. peptide fractionation is used in shotgun proteomics where the entire proteome is digested into peptides, which are then fractionated and identified by ms. this approach is thought to introduce less bias into a biological sample ; hence it is most frequently used in quantitative protein expression profiling. albumin and immunoglobulin account for over 80% of the whole - vitreous protein content, possibly preventing the detection of less abundant proteins. this is particularly relevant in 2d - page experiments, when large spots of albumin and immunoglobulin can overlap small spots, thereby obscuring less abundant proteins. affinity chromatography is frequently used in proteomic studies of body fluids to deplete highly abundant proteins and enhance the detection of low abundance ones. in vh, igg removal prior to electrophoresis has been achieved using protein a sepharose 4 fast flow (amersham pharmacia biotech) or with the proteoextract albumin / igg removal kit (calbiochem, san diego, ca, usa). immunoaffinity subtraction (is) is an alternative approach that allows bounding and retrieval of the 12 most abundant plasma proteins (hsa, igg, fibrinogen, transferrin, iga, igm, apolipoprotein a - i, apolipoprotein a - ii, haptoglobin, 1-antitrypsin, 1-acid glycoprotein, and 2-macroglobulin) from biological fluids using a commercially available system (beckman coulter proteomelab igy-12 column, beckman coulter, fullerton, ca, usa). kim. treated vh samples from eyes with pdr using igy-12 columns and subsequently compared the low and high abundance protein fractions obtained by 2-de. forty - seven spots were excised from the low abundance protein gel and 5 proteins were identified, while 116 spots were excised from the high abundance protein gel and 25 proteins were identified. the identification rate was low in the low abundance protein gel, hence the authors abandoned this prefractionation technique suggesting that high abundance proteins account for the most protein in vh and that low abundance proteins of interest may have also been removed by the is column, as verified in other studies. the sample is first denatured with a buffer containing sds, which charges each protein with a negative charge, identical per unit mass, so that the electrophoretic run leads to fractionation based solely on size. depending on gel size and resolution, sds - page enables separation of proteins into about 1050 fractions, which are recovered by excision and digested into peptides for sequencing by ms. for separation of complex protein mixtures with a higher resolution, sds - page has been combined with isoelectric focusing (ief), which separates proteins based on isoelectric points. this is called two - dimensional (2d) gel electrophoresis and has been used for several decades in proteomics. the use of immobilised ph gradient strips for ief is an improved technique that allows resolution of hundreds of denatured proteins in a single 2-de gel. after electrophoresis, the proteins in the gel are stained for visualisation, quantification, and comparison. the various detection methods (radioactivity, dyes, fluorescence, and silver) as well as the data analysis issues that must be taken into account when quantitative comparative analysis of 2d gels is performed have been critically reviewed in a recent work. 2-de has been the prefractionation technique of choice in the majority of proteomic studies on vh conducted to date [5, 6, 15, 17, 18, 21, 23, 24 ]. the stain and detection software used evolved over time, moving from coomassie brilliant blue (cbb) for global protein detection to fluorescent dyes with higher sensitivity and dynamic range such as sypro ruby protein stain. relative quantification of protein expression levels between samples was estimated based on the assumption that the optical density of the spots (od%) had to be proportional to the protein concentration. differences in apparent protein expression levels between the vh samples were considered potentially significant when matched spots exhibited at least a twofold difference in their averaged od%. using this technique, ouchi. performed the first quantitative comparison of 2d gel protein expression in vitreous from patients with and without diabetic macular oedema (dmo), detecting 72 spots from dmo vh and 64 spots from non - dmo vh. the intensity of 8 spot was significantly different, leading to the identification of six proteins (pedf, apolipoprotein a4, apolipoprotein 1, thyroid hormone receptor interacting protein-11, plasma retinol - binding protein, and vitamin d - binding protein) with higher expression in the dmo group. a more reliable and reproducible method of relative protein quantitation from two or more samples is 2d fluorescence difference gel electrophoresis (dige), a version of 2d - page where the proteins of each sample are labelled with a different fluorophore prior to electrophoresis. gels are scanned at wavelengths unique to each fluorescent label and the images are analysed for differences in protein patterns such as spot density or mass shift. using dige, hernndez. compared vh from eight diabetic patients with dme and eight nondiabetic controls and detected 1300 protein spots. the analysis of spots of differing intensity leads to the identification of 25 proteins, four of which were specifically associated with dmo. garca - ramrez. had been the first to apply dige for analysis of the vh. using this technique, they identified 11 proteins as differentially produced in the vh of pdr patients in comparison with vh from non - diabetic subjects ; 8 were overproduced (zag, apolipoprotein a1, apolipoprotein h, fibrinogen a, c4b, factor b, c3, and c9) and 3 were significantly under produced (pedf, irbp, and itih2). the higher expression of apoliprotein a1 and h in pdr patients has been confirmed in a later study by the same group by dige and western blot of vh samples, as well as mrna expression in the retina. mass spectrometry (ms) is the key analytical technique in proteomics for the identification and, increasingly, for the quantification of proteins. the principle of ms is to measure the mass (m) to charge (z) ratio of ions in the gas phase, hence the peptides need to be first transferred into the gas phase and ionised. the two relevant techniques for ionization of peptides, proteins, and protein - like molecules (e.g., glycoproteins) are matrix - assisted laser desorption / ionization (maldi) and electrospray ionization (esi). for maldi, the analyte is dissolved and cocrystallised with a matrix on a probe surface, which is then irradiated by a uv laser pulses. the ionised analyte is then separated by the time - of - flight (tof) analyser, most commonly employed in maldi - ms. the m / z value of peptides is measured by recording the time ions require to travel over a fixed distance inside the mass analyser. in esi, highly charged analyte droplets from a fine spray outlet are ionised at atmospheric pressure in the presence of a strong electric field, to generate a series of charged gas - phase ions. the charged ions are then emitted and focused into the high - vacuum region of the mass analyser, which records the various charge states of the molecule separated according to their m / z ratios. there are a number of mass analysers in addition to the above - described tof : quadrupole, ion trap, orbitrap, and fourier transform cyclotron ion resonance (ft - icr). each one works differently, having their own strengths and weaknesses and can be used alone or in combination. the mass spectra can be directly compared with protein databases for matching the molecular weights using appropriated scoring algorithm (peptide mass fingerprinting). this technique, however, is limited by the database (as it should contain prior information on the protein for matching) and by the complexity of the protein mixture (as it becomes difficult to select the right peptide mass from a lot of peaks). tandem mass spectrometry (ms / ms) involves two consecutive steps : peptide mass determination and generation of partial amino acid sequence information for a particular peptide based on further fragmentation. the m / z values of the fragments are then recorded in the tandem mass spectrum. tandem ms can be done by two separate analysers (e.g., tof - tof) or inside the same mass analyser (e.g., ion trap). to enhance detection of proteins from very complex mixtures, frequently used platforms are the lc - ms / ms instruments, where ion - pair reversed chromatography or nanohigh performance liquid chromatography (hplc) is used prior to tandem ms. advances in lc - ms / ms have greatly improved the dynamic range and sensitivity for analysis of complex protein mixtures. large - scale proteome profiling has been verified for different organisms, as well as mammalian tissues and cell lines by using multi - dimensional lc - ms / ms. by adopting this technique, yu. have scrutinised the protein profiles of vh from 24 patients undergoing vitrectomy for proliferative vitreous retinopathy (pvr) and 8 biobank eyes, identifying 363 proteins. an even better example of how proteomics is strictly dependent on the technology employed has been provided by kim., who could identify 49 proteins using 2-de and 531 proteins using lc - ms / ms on the same set of vh from pdr eyes. algorithms have been developed for amino acid sequence and protein identification by matching the information contained in mass spectra against a database of theoretical or previously identified spectra. algorithms can generate both false - positive and false - negative assignments, which are influenced by the stringency of spectra to sequence criteria. discerning a true match from a false match is critical in proteomic data analysis. the most common tools for ms / ms - based peptide identification and data analysis have been comprehensively reviewed elsewhere. because of the complexity of the proteomic workflow and data analysis, it is essential to validate the identified candidate proteins using independent techniques, such as western blot. moreover, the experimental design needs to take into consideration the influence of technical and biological variabilities, which are particularly relevant in biological samples like the vh. fifteen studies conducted over the last decade have used a range of proteomic methodologies including 2de, dige, esi - ms, maldi - ms, and lc - ms / ms to compare the vitreous proteome of patients with various stages of diabetic retinopathy (dr) and pvr to that from non - diabetic patients and those with mh [5, 6, 10, 1216, 1824, 37 ]. one other study investigated the proteome of vh from human phakic and pseudophakic donor eyes. in general, the total protein content reported for the vitreous of patients with dr is higher than that measured in the non - diabetic and control samples. as already discussed above, this may be due, however, to an influx of serum due to vitreous haemorrhage and/or disruption of the blood - retinal barrier, leading to elevated levels of proteins not associated with intravitreal protein production. indeed, in the study of sim., a comparison of proliferative vitreoretinopathy and normal vitreous demonstrated upregulated levels of intraocularly produced lipoproteins in the former. overall, studies analysing the vitreous proteome in patients with dr have varied greatly both in terms of the total number of proteins identified and the number of proteins differentially expressed between the test group(s) and controls, as well as the specific proteins then proposed to play a role in the pathogenesis of vitreoretinal disease states. although a detailed discussion of the specific proteins identified by these studies is beyond the scope of this chapter, it is clear that as proteomic technologies have evolved over this period, so the number of identified proteins has increased. whether any of these proteins and the pathways that they regulate is of importance in the pathogenesis of dr remains a very interesting translational question, which is being investigated by more quantitative targeted approaches. intraocular inflammation accounts for 1015% of bilateral and 22% of unilateral blindness in the united states, and 10% of visual impaired registration in the uk. many efforts are being made to deepen our understanding of the different aspects of the inflammatory process, evaluate new therapeutic strategies, and ultimately be able to deliver personalised care for patients with intraocular inflammatory diseases. proteomics analyses of intravitreal inflammation have not yet been performed on human samples, whilst they have been successfully performed on vh from animal models. endotoxin - induced uveitis (eiu) is an animal model of acute ocular inflammation. to characterize the mechanism of eiu, bahk. analysed the infiltration of proteins in the vitreous bodies of rats with eiu and normal rats using 2-de - maldi - tof / ms and micro lc / lc - ms / ms, identifying specific modifications in the crystallin family proteins. spontaneous equine recurrent uveitis (eru) is a recurrent uveitis that develops in the horse and results in blindness. the vitreous is the body fluid closest to the disease - affected tissue and possibly also an effector of pathological processes relevant for eru. surgical removal of the vh can lead to a considerable decrease in the frequency and severity of relapses, therefore vitreous composites are likely to contribute to disease progression. deeg and coworkers have been systematically comparing vh from healthy and disease - affected equine eyes by proteomic profiling [53, 54 ]. in an earlier study, they applied 2-de - maldi - tof / ms, identifying a total of 42 proteins, 9 of which differentially expressed in eru. these are functionally related to immune response, inflammation, and maintenance of the blood - retinal barrier. more recently, they identified eru - related functional protein networks and affiliated molecular signalling pathways using lc - ms / ms - based label - free quantification followed by pathway enrichment analyses. the increased sensitivity gained by omitting gel - based prefractionation resulted in overall detection of 119 different proteins. a large fraction of these proteins were differentially expressed in eru samples as opposed to controls (26 upregulated, 44 downregulated). pathway enrichment analyses were performed using the consensuspathdb program, suggesting the participation of the wnt pathway in the pathogenesis of the uveitis. this shows how the development of ms - based methods significantly improved quantitative proteomic analyses of the vh, enabling comprehensive identification of differentially regulated proteins and detection of novel molecular pathways that could become the therapeutic targets of the future.	the human vitreous humour (vh) is a transparent, highly hydrated gel, which occupies the posterior segment of the eye between the lens and the retina. physiological and pathological conditions of the retina are reflected in the protein composition of the vh, which can be sampled as part of routine surgical procedures. historically, many studies have investigated levels of individual proteins in vh from healthy and diseased eyes. in the last decade, proteomics analyses have been performed to characterise the proteome of the human vh and explore networks of functionally related proteins, providing insight into the aetiology of diabetic retinopathy and proliferative vitreoretinopathy. recent proteomic studies on the vh from animal models of autoimmune uveitis have identified new signalling pathways associated to autoimmune triggers and intravitreal inflammation. this paper aims to guide biological scientists through the different proteomic techniques that have been used to analyse the vh and present future perspectives for the study of intravitreal inflammation using proteomic analyses.
the basic mechanism of bonding to enamel and dentin is essentially an exchange process involving replacement of minerals from the hard dental tissues with resin monomers, which, upon setting, become micro - mechanically interlocked in the created porosities.1 contemporary adhesives can be classified on the basis of underlying adhesion strategy into etch - and - rinse, self - etch, and resin - modified glass - ionomer adhesives.2 the success of the etch - and - rinse adhesives for bonding resin - based restorative materials to enamel and dentin is well supported by numerous studies and many years of clinical experience.3 the concept of self - etching adhesives is based on the use of polymerizable acidic monomers that simultaneously condition and prime both dentin and enamel.4 these adhesives are subdivided into three categories based upon their ph value : strong systems have a ph of 1 or below, intermediary strong systems have a ph of approximately 1.5, and mild systems have a ph of 2 or more.5 the bond strength of self - etching adhesive systems to enamel is controversially discussed in the literature ; some studies have reported comparable data to that observed with etch - and - rinse systems,68 while other studies considered them less reliable when bonding to enamel.912 there is still some concern that manufacturers are sacrificing enamel bond strength in their struggle for simplified and strengthened bonding to the more complex substrate, dentin, despite the fact that enamel is the front - gate determinant of a restoration s longevity and durability. many conditioning agents have been used for surface pretreatment of enamel and dentin, including phosphoric, maleic, nitric, citric, and ethylenediaminetetraacetic (edta) acids. these acids are used to remove the smear layer and to demineralize the underlying enamel and dentin.13 one might consider pre - etching the enamel with phosphoric acid prior to application of a self - etching adhesive system. the effect of such additional etching on enamel bond strength is also controversially discussed in the literature. its use might be beneficial with some self - etching adhesives, but this depends largely on the properties of the adhesive itself.14 it has been previously reported that etching of enamel surfaces with edta is not recommended because of its negligible non - uniform effect.15,16 however, the effect of edta pretreatment on bond strength of enamel in conjunction with self - etching adhesives has not, to our knowledge, yet been addressed in literature. the interaction of such a mild conditioning agent as a pretreatment agent with different ph categories of self - etching adhesive systems is a matter of speculation. thus, this study was designed to determine the effect of two surface pretreatment agents on the enamel bond strength of self - etching adhesive systems with different ph values. the null hypotheses were tested first : pretreatment of the ground enamel surfaces with phosphoric acid or edta had no effect on the shear bond strength of self - etch adhesives to enamel. second, there was no difference in bond strengths between self - etching adhesive systems with different ph values when bonding to ground enamel. a total of 90 sound extracted maxillary human premolars were used for shear bond strength testing. the teeth, excluding the buccal surface, were embedded in self - curing acrylic resin (rapid repair, degudent gmbh, hanau, germany) by using a specially fabricated cuboidal teflon mould (321.3 cm). the buccal enamel surface of the embedded premolars was ground on a water - cooled mechanical grinder (tf250, jeanwirtz, dsseldorf, germany) by using 180-grit abrasive paper to obtain flat enamel surfaces with a clinically relevant smear layer. the acrylic resin blocks were placed in the mould. to standardize the bonding area, a piece of vinyl tape with a 3-mm diameter punctured hole the teeth were assigned into three groups according to bonding procedure. in the first subgroup (control), no pretreatment agent was applied. in the second subgroup, etching was performed using 37% phosphoric acid (pa) for 15 seconds (total etch ; ivoclar vivadent ag, schaan, liechtenstein). in the third subgroup, surfaces were pretreated with 18.85% edta for 60 seconds (edta odahcam ; dentsply, latin america, rio de janeiro, brazil). adper prompt l - pop (aplp) (3 m espe ag dental products, seefeld, germany), adhese (se) (ivoclar vivadent ag, schaan, liechtenstein), or frog (fg) (sdi limited, bayswater, victoria, australia) self - etch adhesive systems (n=10) were then applied to the demarcated bonding area following manufacturers instructions (table 1). all adhesives were cured using a bluephase c5 (ivoclar vivadent ag, schaan, liechtenstein) light emitting diode curing unit for 10 seconds at a light intensity of 500 mw / cm. the light intensity was periodically checked with the light meter integrated in the handpiece holder of the curing unit. the 2-mm - thick roof composed of two equal halves with a circular 3-mm diameter hole was used for the packing of the tetric evoceram (shade a3) (ivoclar vivadent ag, schaan, liechtenstein) resin composite. the composite was then light cured for 20 seconds using the same light curing unit according to manufacturer instructions. after 24-hour storage in distilled water, the samples were subjected to compression testing using a mono - bevelled, chisel - shaped metallic rod in a computerized universal testing machine (model lrx - plus ; lloyd instruments ltd., the specimens were stressed in shear at a cross - head speed of 0.5 mm / min. the shear force at failure was recorded and converted to shear stress in mpa units using computer software (nexygen-4.1 ; lloyd instruments ltd., the fracture sites of the debonded surfaces were examined using a binocular stereomicroscope (smz-10, nikon, melville, ny, usa) at 15x magnification. representative samples were chosen for examination under scanning electron microscopy (sem) (xl 30, philips, eindhoven, netherlands). samples were mounted on sem stubs and sputter - coated (ladd sputter coater, ladd research industries, williston, vermont, usa) with a thin layer of gold under vacuum. examination was done at 30 kv of accelerating voltage at different magnifications and characteristic photomicrographs were obtained at 1000x magnification. two - way analysis of variance (anova) was used in testing significance for the effect of both variables studied (adhesive system versus surface pretreatment agent) on the mean shear bond strength. post - hoc tukey s test was used for pair - wise comparison between the means when the anova test was significant. representative samples (n=2) were prepared and treated with the corresponding surface pretreatment agents and adhesive systems for each subgroup as described earlier. however, the steps of bonding component application and light curing were skipped in the case of the two - step self - etching adhesive systems used while the light curing step was skipped in the case of the single - step self - etching adhesive system. all specimens were then treated with a 60-second acetone rinse under ultrasonic movement (ultrasonic steri - cleaner uc-150, sturdy industrial co., taipei, taiwan) for the removal of any crystals and other residues from primers and left to air dry. additional representative samples (n=2) were prepared using the same surface pretreatment agents, adhesive systems, and restorative resin composite used for shear bond strength testing. after 24-hour storage in distilled water, the bonded specimens were cut perpendicular to the resin / enamel interface using a slow - speed diamond disc (k6974, komet, lemgo, germany) under water lubrication. the cross - sectioned specimens were finished and polished under running water with optidisc (kerr corporation, orange, california, usa) resin composite finishing and polishing discs from coarse / medium to fine to extra - fine grits. polished interfaces were demineralized with 50% pa for 15 seconds, rinsed thoroughly with distilled water, and air dried. all specimens were gold sputtered under vacuum and examined using sem at 30 kv accelerating voltage. images of the enamel surfaces topography were viewed at 2000x magnification, while those for resin - enamel interface analysis were examined at 3500x magnification. a total of 90 sound extracted maxillary human premolars were used for shear bond strength testing. the teeth, excluding the buccal surface, were embedded in self - curing acrylic resin (rapid repair, degudent gmbh, hanau, germany) by using a specially fabricated cuboidal teflon mould (321.3 cm). the buccal enamel surface of the embedded premolars was ground on a water - cooled mechanical grinder (tf250, jeanwirtz, dsseldorf, germany) by using 180-grit abrasive paper to obtain flat enamel surfaces with a clinically relevant smear layer. the acrylic resin blocks were placed in the mould. to standardize the bonding area, a piece of vinyl tape with a 3-mm diameter punctured hole was placed over the mid - coronal ground enamel surface. the teeth were assigned into three groups according to bonding procedure. in the first subgroup (control), no pretreatment agent was applied. in the second subgroup, etching was performed using 37% phosphoric acid (pa) for 15 seconds (total etch ; ivoclar vivadent ag, schaan, liechtenstein). in the third subgroup, surfaces were pretreated with 18.85% edta for 60 seconds (edta odahcam ; dentsply, latin america, rio de janeiro, brazil). adper prompt l - pop (aplp) (3 m espe ag dental products, seefeld, germany), adhese (se) (ivoclar vivadent ag, schaan, liechtenstein), or frog (fg) (sdi limited, bayswater, victoria, australia) self - etch adhesive systems (n=10) were then applied to the demarcated bonding area following manufacturers instructions (table 1). all adhesives were cured using a bluephase c5 (ivoclar vivadent ag, schaan, liechtenstein) light emitting diode curing unit for 10 seconds at a light intensity of 500 mw / cm. the light intensity was periodically checked with the light meter integrated in the handpiece holder of the curing unit. the 2-mm - thick roof composed of two equal halves with a circular 3-mm diameter hole was used for the packing of the tetric evoceram (shade a3) (ivoclar vivadent ag, schaan, liechtenstein) resin composite. the composite was then light cured for 20 seconds using the same light curing unit according to manufacturer instructions. after 24-hour storage in distilled water, the samples were subjected to compression testing using a mono - bevelled, chisel - shaped metallic rod in a computerized universal testing machine (model lrx - plus ; lloyd instruments ltd., the specimens were stressed in shear at a cross - head speed of 0.5 mm / min. the shear force at failure was recorded and converted to shear stress in mpa units using computer software (nexygen-4.1 ; lloyd instruments ltd., fareham, uk). the fracture sites of the debonded surfaces were examined using a binocular stereomicroscope (smz-10, nikon, melville, ny, usa) at 15x magnification. representative samples were chosen for examination under scanning electron microscopy (sem) (xl 30, philips, eindhoven, netherlands). samples were mounted on sem stubs and sputter - coated (ladd sputter coater, ladd research industries, williston, vermont, usa) with a thin layer of gold under vacuum. examination was done at 30 kv of accelerating voltage at different magnifications and characteristic photomicrographs were obtained at 1000x magnification. two - way analysis of variance (anova) was used in testing significance for the effect of both variables studied (adhesive system versus surface pretreatment agent) on the mean shear bond strength. post - hoc tukey s test was used for pair - wise comparison between the means when the anova test was significant. representative samples (n=2) were prepared and treated with the corresponding surface pretreatment agents and adhesive systems for each subgroup as described earlier. however, the steps of bonding component application and light curing were skipped in the case of the two - step self - etching adhesive systems used while the light curing step was skipped in the case of the single - step self - etching adhesive system. all specimens were then treated with a 60-second acetone rinse under ultrasonic movement (ultrasonic steri - cleaner uc-150, sturdy industrial co., taipei, taiwan) for the removal of any crystals and other residues from primers and left to air dry. additional representative samples (n=2) were prepared using the same surface pretreatment agents, adhesive systems, and restorative resin composite used for shear bond strength testing. after 24-hour storage in distilled water, the bonded specimens were cut perpendicular to the resin / enamel interface using a slow - speed diamond disc (k6974, komet, lemgo, germany) under water lubrication. the cross - sectioned specimens were finished and polished under running water with optidisc (kerr corporation, orange, california, usa) resin composite finishing and polishing discs from coarse / medium to fine to extra - fine grits. polished interfaces were demineralized with 50% pa for 15 seconds, rinsed thoroughly with distilled water, and air dried. all specimens were gold sputtered under vacuum and examined using sem at 30 kv accelerating voltage. images of the enamel surfaces topography were viewed at 2000x magnification, while those for resin - enamel interface analysis were examined at 3500x magnification. representative samples (n=2) were prepared and treated with the corresponding surface pretreatment agents and adhesive systems for each subgroup as described earlier. however, the steps of bonding component application and light curing were skipped in the case of the two - step self - etching adhesive systems used while the light curing step was skipped in the case of the single - step self - etching adhesive system. all specimens were then treated with a 60-second acetone rinse under ultrasonic movement (ultrasonic steri - cleaner uc-150, sturdy industrial co., taipei, taiwan) for the removal of any crystals and other residues from primers and left to air dry. additional representative samples (n=2) were prepared using the same surface pretreatment agents, adhesive systems, and restorative resin composite used for shear bond strength testing. after 24-hour storage in distilled water, the bonded specimens were cut perpendicular to the resin / enamel interface using a slow - speed diamond disc (k6974, komet, lemgo, germany) under water lubrication. the cross - sectioned specimens were finished and polished under running water with optidisc (kerr corporation, orange, california, usa) resin composite finishing and polishing discs from coarse / medium to fine to extra - fine grits. polished interfaces were demineralized with 50% pa for 15 seconds, rinsed thoroughly with distilled water, and air dried. all specimens were gold sputtered under vacuum and examined using sem at 30 kv accelerating voltage. images of the enamel surfaces topography were viewed at 2000x magnification, while those for resin - enamel interface analysis were examined at 3500x magnification. table 2 shows the results of statistical analysis using two - way anova test to describe the effect of both studied variables (adhesive system and surface pretreatment agent). both adhesive systems and surface pretreatment agents had statistically significant effects on mean shear bond strength (p<.001 and p=0.041, respectively). the interaction between adhesive systems and surface pretreatment agents had a statistically significant effect on mean shear bond strength (p=0.049). the results of tukey s test for the comparison between different interactions of adhesive systems with surface pretreatments are shown in table 3. comparing the 3 adhesive systems when applied according to manufacturer instructions, the intermediary strong self - etch adhesive system (se) showed statistically highest shear bond strength values followed by the strong self - etching adhesive system (aplp) while the mild self - etch adhesive system (fg) showed the statistically lowest shear bond strength values. with regard to the effect of the different surface pretreatments, it was revealed that different surface pretreatments did not statistically affect the mean shear bond strength values of the intermediary strong self - etching adhesive system (se). pa pretreatment did not affect its bond strength values of the aplp system ; on the other hand, edta significantly reduced its bond strength values. however, pa pretreatment significantly increased the mean shear bond strength values of the mild self - etching adhesive system, which was not affected by edta pretreatment. each fractured surface was allocated to one of five types : type 1, adhesive failure between the bonding resin and enamel ; type 2 : partial adhesive failure between the bonding resin and enamel and partial cohesive failure of the bonding resin ; type 3 : partial adhesive failure between the bonding resin and enamel and partial cohesive failure of the enamel ; type 4 : 100% cohesive failure of the bonding resin ; or type 5 : 100% cohesive failure of the enamel. figure 1 shows a bar chart of the percentage distribution of failure modes, while figure 2 represents sem photomicrographs for the different types of failure modes. fractographic analysis of the fractured sites revealed that adhesive failure (type 1) was the predominating failure type. without additional surface pretreatment, only the intermediary strong self - etching adhesive system showed cohesive failure of the enamel (type 5). the highest percentages of failure (type 1) were found in the strong and mild self - etching adhesive system groups with 100% in no pretreatment and edta pretreatment and 90% in the pa pretreatment subgroups. in the intermediary strong self - etching adhesive system, type 1 failure was seen 60% of the time with no pretreatment and pa pretreatment and 50% with edta pretreatment. type 2 failure (20% and 30%) was seen only in the intermediary strong self - etching adhesive system with pa and edta pretreatment subgroups, respectively. type 3 failure was seen only in the same two subgroups (10% in both). type 5 failure was seen in the intermediary strong self - etching adhesive system group with different surface pretreatments and in the strong self - etching adhesive system and the mild self - etching adhesive system groups with pa pretreatment. according to cehreli and altay,15 each of these types was used in the interpretation of the enamel surface topography photomicrographs : type i, preferential dissolution of the prism cores resulting in a honeycomb - like appearance ; type ii, preferential dissolution of the prism peripheries creating a cobblestone - like appearance ; type iii, a mixture of type i and type ii patterns ; type iv, pitted enamel surfaces as well as structures that look like unfinished puzzles, maps, or networks ; and type v, flat, smooth surfaces. sem photomicrographs of the surface topography and resin / enamel interface of the strong self - etching adhesive system are shown in figure 3. with no surface pretreatment, topographical ultra - morphological characterization (figure 3a) showed predominant homogeneous deep preferential dissolution of the enamel prism peripheries with areas of prism core dissolution (a type iii etching pattern). meanwhile, interfacial ultra - morphological characterization (figure 3b) depicted resin infiltration in the form of a very thin hybrid - like layer with sparse, broad, and shallow tag - like structures. a thin continuous adhesive layer was evident between the tags and the over - laying resin composite. with pa pretreatment, enamel surface topography (figure 3c) showed progressively homogeneous and deeper type iii etching patterns. the interface (figure 3d) revealed resin infiltration in the form of a broader hybrid - like layer with numerous tag - like structures penetrating deeper into the etched enamel. with edta pretreatment, topography (figure 3e) revealed a milder, homogeneous type i etching pattern. the enamel prisms were hollowed out to deep pits or craters placed side by side separated by thick interprismatic enamel persisting in the form of rings. the interface (figure 3f) closely resembled that of no surface pretreatment (figure 3b). sem photomicrographs of the surface topography and resin / enamel interface of the intermediary strong self - etching adhesive system are shown in figure 4. with no surface pretreatment, the interface (figure 4b) depicted resin infiltration in the form of a very thin hybrid - like layer with thick and shallow penetrating tag - like structures., the topography (figure 4c) had a deeper mixed etching pattern (type iii) with areas that were less intensely etched. the interface (figure 4d) revealed resin infiltration in the form of a broader hybrid - like layer with numerous deeply penetrating tag - like structures. cracks may be attributed to the high vacuum employed for sem examination. with edta pretreatment, topography (figure 4e) certain areas were markedly etched while others were very mildly involved with merely delineation of the prismatic morphology. the interface (figure 4f) showed resin infiltration in the form of a very thin hybrid - like layer with very shallow sparse tag - like structures. sem photomicrographs of the surface topography and resin / enamel interface of the mild self - etching adhesive system are shown in figure 5. with no surface pretreatment, the topography (figure 5a) had a very mild irregular etch pattern not related to prism morphology (type iv etch pattern) in the form of shallow craters with areas remaining unetched. the interface (figure 5b) had resin infiltration in the form of a lamina - like, thin, and hybrid - like layer with no resin tag formation. a thin continuous adhesive layer was evident. with pa pretreatment, (figure 5c) a homogeneous and regular type ii etch pattern with deep interprismatic dissolution the interface (figure 5d) revealed resin infiltration in the form of a very thin hybrid - like layer. with edta pretreatment, topography (figure 5e) revealed preferential dissolution of interprismatic enamel (type ii) with areas remaining unetched. the interface (figure 5f) showed a close resemblance to that of no surface pretreatment (figure 5b). table 2 shows the results of statistical analysis using two - way anova test to describe the effect of both studied variables (adhesive system and surface pretreatment agent). both adhesive systems and surface pretreatment agents had statistically significant effects on mean shear bond strength (p<.001 and p=0.041, respectively). the interaction between adhesive systems and surface pretreatment agents had a statistically significant effect on mean shear bond strength (p=0.049). the results of tukey s test for the comparison between different interactions of adhesive systems with surface pretreatments are shown in table 3. comparing the 3 adhesive systems when applied according to manufacturer instructions, the intermediary strong self - etch adhesive system (se) showed statistically highest shear bond strength values followed by the strong self - etching adhesive system (aplp) while the mild self - etch adhesive system (fg) showed the statistically lowest shear bond strength values. with regard to the effect of the different surface pretreatments, it was revealed that different surface pretreatments did not statistically affect the mean shear bond strength values of the intermediary strong self - etching adhesive system (se). pa pretreatment did not affect its bond strength values of the aplp system ; on the other hand, edta significantly reduced its bond strength values. however, pa pretreatment significantly increased the mean shear bond strength values of the mild self - etching adhesive system, which was not affected by edta pretreatment. each fractured surface was allocated to one of five types : type 1, adhesive failure between the bonding resin and enamel ; type 2 : partial adhesive failure between the bonding resin and enamel and partial cohesive failure of the bonding resin ; type 3 : partial adhesive failure between the bonding resin and enamel and partial cohesive failure of the enamel ; type 4 : 100% cohesive failure of the bonding resin ; or type 5 : 100% cohesive failure of the enamel. figure 1 shows a bar chart of the percentage distribution of failure modes, while figure 2 represents sem photomicrographs for the different types of failure modes. fractographic analysis of the fractured sites revealed that adhesive failure (type 1) was the predominating failure type. without additional surface pretreatment, only the intermediary strong self - etching adhesive system showed cohesive failure of the enamel (type 5). the highest percentages of failure (type 1) were found in the strong and mild self - etching adhesive system groups with 100% in no pretreatment and edta pretreatment and 90% in the pa pretreatment subgroups. in the intermediary strong self - etching adhesive system, type 1 failure was seen 60% of the time with no pretreatment and pa pretreatment and 50% with edta pretreatment. type 2 failure (20% and 30%) was seen only in the intermediary strong self - etching adhesive system with pa and edta pretreatment subgroups, respectively. type 3 failure was seen only in the same two subgroups (10% in both). type 5 failure was seen in the intermediary strong self - etching adhesive system group with different surface pretreatments and in the strong self - etching adhesive system and the mild self - etching adhesive system groups with pa pretreatment. each of these types was used in the interpretation of the enamel surface topography photomicrographs : type i, preferential dissolution of the prism cores resulting in a honeycomb - like appearance ; type ii, preferential dissolution of the prism peripheries creating a cobblestone - like appearance ; type iii, a mixture of type i and type ii patterns ; type iv, pitted enamel surfaces as well as structures that look like unfinished puzzles, maps, or networks ; and type v, flat, smooth surfaces. sem photomicrographs of the surface topography and resin / enamel interface of the strong self - etching adhesive system are shown in figure 3. with no surface pretreatment, topographical ultra - morphological characterization (figure 3a) showed predominant homogeneous deep preferential dissolution of the enamel prism peripheries with areas of prism core dissolution (a type iii etching pattern). meanwhile, interfacial ultra - morphological characterization (figure 3b) depicted resin infiltration in the form of a very thin hybrid - like layer with sparse, broad, and shallow tag - like structures. a thin continuous adhesive layer was evident between the tags and the over - laying resin composite. with pa pretreatment, enamel surface topography (figure 3c) showed progressively homogeneous and deeper type iii etching patterns. the interface (figure 3d) revealed resin infiltration in the form of a broader hybrid - like layer with numerous tag - like structures penetrating deeper into the etched enamel. with edta pretreatment, topography (figure 3e) revealed a milder, homogeneous type i etching pattern. the enamel prisms were hollowed out to deep pits or craters placed side by side separated by thick interprismatic enamel persisting in the form of rings. the interface (figure 3f) closely resembled that of no surface pretreatment (figure 3b). sem photomicrographs of the surface topography and resin / enamel interface of the intermediary strong self - etching adhesive system are shown in figure 4. with no surface pretreatment, the interface (figure 4b) depicted resin infiltration in the form of a very thin hybrid - like layer with thick and shallow penetrating tag - like structures. with pa pretreatment, the topography (figure 4c) had a deeper mixed etching pattern (type iii) with areas that were less intensely etched. the interface (figure 4d) revealed resin infiltration in the form of a broader hybrid - like layer with numerous deeply penetrating tag - like structures. cracks may be attributed to the high vacuum employed for sem examination. with edta pretreatment, topography (figure 4e) certain areas were markedly etched while others were very mildly involved with merely delineation of the prismatic morphology. the interface (figure 4f) showed resin infiltration in the form of a very thin hybrid - like layer with very shallow sparse tag - like structures. sem photomicrographs of the surface topography and resin / enamel interface of the mild self - etching adhesive system are shown in figure 5. with no surface pretreatment, the topography (figure 5a) had a very mild irregular etch pattern not related to prism morphology (type iv etch pattern) in the form of shallow craters with areas remaining unetched. the interface (figure 5b) had resin infiltration in the form of a lamina - like, thin, and hybrid - like layer with no resin tag formation. a thin continuous adhesive layer was evident. with pa pretreatment, (figure 5c) a homogeneous and regular type ii etch pattern with deep interprismatic dissolution the interface (figure 5d) revealed resin infiltration in the form of a very thin hybrid - like layer. with edta pretreatment, topography (figure 5e) revealed preferential dissolution of interprismatic enamel (type ii) with areas remaining unetched. the interface (figure 5f) showed a close resemblance to that of no surface pretreatment (figure 5b). this study evaluated the effect of surface pretreatments (pa or edta) on the bond strength of three self - etching adhesive systems to ground enamel surfaces. the self - etching adhesive systems were selected based on their ph values ; one was chosen to represent each ph category. all of the selected adhesives had the same solvent (water - based) and contained 2-hydroxyethylmethacrylate. adhesives were also devoid of functional monomers that are claimed to chemically interact with tooth substrates. the buccal surface was ground parallel to the tooth long axis to flatten the enamel surface for shear testing and to standardize the orientation of enamel prisms.9 this process removes the outer hypermineralized and acid - resistant enamel and it is also consistent with clinical practice when the outer 0.5 mm of labial enamel is removed during bevelling or for veneering.17 results of the present study revealed that pa pretreatment of the enamel surface led to a significant increase in bond strength values with the mild self - etching adhesive only, while edta pretreatment did not enhance the bond strength values of any of the tested self - etching adhesive systems. both the strong and intermediary strong self - etching adhesive systems revealed definite etching patterns as depicted in figures 3a and 4a. pretreating enamel surfaces with pa led to further deepening of the same etching pattern created by both adhesive systems (figures 3c and 4c). this deepening was consistent with the increase in length of the tag - like structures at the interface (figures 3d and 4d). this observation is in agreement with that reported in shinchi,18 who showed that both the depth of etching and the length of the resin tags contribute little to bond strength in pa - etched enamel. in addition, brackett found that the depth of etching and the subsequent depth of resin permeation induced by self - etching adhesive systems do not correlate with the attained bond strength. this may be due to the fact that increasing the depth of the resin tag does not contribute substantially to the increase in cumulative surface area created by acid etching of cut enamel.20 a marked increase in surface area is achieved via the creation of regular microporosities among the apatite crystallites ; resins can infiltrate these microporosities and result in the formation of an enamel resin composite consisting of inter- and intra - crystallite resin encapsulation as well as resin infiltration into the interprismatic boundaries.21 moreover, it was recently reported that resin - to - enamel bonding with self - etching systems is based on a similar mechanism of inter- and intra - crystallite hybridization of the enamel surface rather than resin tag formation.22 this is in partial agreement with perdigao,23 who found that pa pretreatment did not enhance the sealing ability of the strong self - etching adhesive system, aplp, in non - thermocycled specimens. however, this result is in disagreement with lhrs,5 who reported a significant increase in enamel shear bond strength of the strong single - step system xeno iii and the intermediary strong two- and one - step systems se and futurabond nr after additional pa etching. in addition, erhardt reported significant increases in enamel shear bond strength of the single - step intermediary strong self - etching adhesive one up bond f after pa pretreatment. this supports the fact that the bonding performance of adhesives is still material - dependant. on the other hand, pa pretreatment enhanced the bond strength of the mild self - etching adhesive system in the current study, which is in agreement with other studies.13,2429 the data from these studies collectively suggest that the mild self - etching adhesive systems used were unable to provide an adequate level of demineralization to achieve optimum bonding to enamel. pretreatment with pa created adequate microporosities, which enhanced resin permeation. in the topographical sem photomicrographs (figures 5a and 5c), pa pretreatment converted the etching pattern of the mild self - etching adhesive from an indefinite form (type iv) to the more definite retentive form (type ii). this observation was also contiguous with the appearance of resin tags at the resin / enamel interface of pa - treated enamel (figures 5b and 5d). meanwhile, erhardt explained this by the fact that pa might remove the smear layer, lowering its buffering capacity and leaving the enamel surface more receptive to self - etching primer diffusion. this result is, however, in contrast with weerasinghe,17 who reported no statistically significant difference in enamel bond strength with pa pretreatment in conjunction with clearfil se bond. however, clearfil se bond contains the functional monomer 10-methacryloxydecyl dihydrogen phosphate (10-mdp), which is thought to chemically interact with tooth tissues. the effect of edta pretreatment on enamel bond strength in conjunction with self - etching adhesive systems has not yet been addressed in the literature. however, studies that tested enamel etching with edta did not recommend its use because of the negligible non - uniform effect falling into the type iv etching pattern category on ground enamel16 or the type v etching pattern category on unground enamel.15 these phenomena may be due to the neutral ph of edta (6.47.4). in addition, the concentration and application time might not have been sufficient to obtain a desirable effect on enamel.16 this result conforms to sem findings of topography and interface (figures 3e, 3f, 4e, 4f, 5e, and 5f), as it was evident that edta pretreatment had a negligible and unpronounced effect when compared to the action of each of the three adhesive systems that were applied according to manufacturers instructions (figures 3a, 3b, 4a, 4b, 5a, and 5b) without further pretreatment, especially with the mild self - etching adhesive system (figures 5e and 5f). when the three self - etch adhesives were applied according to manufacturer instructions, the intermediary strong system (se) showed the best performance followed by the strong self - etching adhesive system (aplp) this result is in agreement with de munck,3 who showed that the strong self - etching aplp adhesive system scored the lowest in microtensile bond strength of all of the experimental and control adhesives including the intermediary strong systems se and optibond solo plus se. the authors speculated that etching aggressiveness does not entirely correlate with bonding effectiveness as the individual features of the adhesive resin itself plays a role. variation in adhesive viscosity, surface tension, chemical interaction of acidic monomers with enamel, water concentration, and cohesive strength of the adhesive are all examples of such features.30 although water is a major component of all self - etching adhesives that allows the ionization of the acidic monomers to perform a demineralizing reaction, strong self - etching adhesives have high solvent contents to promote the complete ionization of the acidic monomer.31 the high water content in aplp (80%) could be difficult to remove by air blowing.30,32 this in turn could decrease the polymerization efficacy and degree of conversion, thus altering the mechanical properties of the adhesive.33 in addition, excess water may also dilute the primer and reduce its effectiveness.34 it has also been speculated that the high acidity of unpolymerized monomers remaining at the oxygen inhibited layer after light curing may attack the polymerization initiation system of the resin composite, resulting in lower bond strength.32 aplp and fg contain pa ester as the acidic polymerizable monomer unlike se, which contains phosphonic acid acrylates. the latter is reported to have improved hydrolytic stability and reactivity in free radical polymerization. moreover, se also contains the hydrolytically stable cross - linking monomer bis - acrylamide.4,10 the strong system aplp is a single - step system (all - in - one) while the intermediary strong system se is a two - step system. van landuyt reported that the amounts of ingredients applied on the tooth surface differ considerably between one- and two - step adhesives. two - step adhesives consist of pure priming solution containing only functional etching monomers dissolved in organic solvent and water, and a solvent - free bonding containing hydrophobic cross - linking monomers that allow for a thicker and more hydrolytically stable adhesive layer. this layer can probably act as a shock absorber between tooth tissues and composites. on the other hand, one - step adhesives are complex mixtures of both hydrophobic and hydrophilic ingredients.9 on the other hand, this result is in contrast with findings of goracci,10 atash and van den abbeele,32 and perdigo.35 the first two studies reported no significant difference in bond strength between aplp compared to se. this contradiction may be attributed to differences in testing methodologies and the substrate examined. in the current study, the mild self - etching adhesive system (fg) showed the lowest bond strength compared to the strong and intermediary strong adhesive systems. this could be partially attributed to the relatively higher numbers of retentive etching patterns created by the strong and intermediary strong adhesive systems (figures 3a and 4a) compared to the indefinite non - retentive pattern created by the mild self - etching adhesive system (figure 5a). the ph of the mild self - etching adhesives might be optimal for dentin but may not be sufficiently aggressive for enamel.19 the intermediary strong, self - etching adhesive system (adhese) might have higher potential for bonding to enamel than the strong and mild, self - etching adhesive systems (adper prompt l - pop and fg). phosphoric acid pre - treatment could be beneficial for bonding to enamel using mild self - etching adhesive systems. edta pre - treatment is not a viable alternative for enamel bonding to self - etching adhesive systems. the uniformity rather than the depth of the etching pattern affected the bonding of self - etching adhesives to enamel.	objectives : this in vitro study determined the effect of enamel pretreatment with phosphoric acid and ethylenediaminetetraacetic acid (edta) on the bond strength of strong, intermediary strong, and mild self - etching adhesive systems.methods:ninety sound human premolars were used. resin composite cylinders were bonded to flat ground enamel surfaces using three self - etching adhesive systems : strong adper prompt l - pop (ph=0.91.0), intermediary strong adhese (ph=1.61.7), and mild frog (ph=2). adhesive systems were applied either according to manufacturer instructions (control) or after pretreatment with either phosphoric acid or edta (n=10). after 24 hours, shear bond strength was tested using a universal testing machine at a cross - head speed of 0.5 mm / minute. ultra - morphological characterization of the surface topography and resin / enamel interfaces as well as representative fractured enamel specimens were examined using scanning electron microscopy (sem).results : neither surface pretreatment statistically increased the mean shear bond strength values of either the strong or the intermediary strong self - etching adhesive systems. however, phosphoric acid pretreatment significantly increased the mean shear bond strength values of the mild self - etching adhesive system. sem examination of enamel surface topography showed that phosphoric acid pretreatment deepened the same etching pattern of the strong and intermediary strong adhesive systems but converted the irregular etching pattern of the mild self - etching adhesive system to a regular etching pattern. sem examination of the resin / enamel interface revealed that deepening of the etching pattern was consistent with increase in the length of resin tags. edta pretreatment had a negligible effect on ultra - morphological features.conclusions:use of phosphoric acid pretreatment can be beneficial with mild self - etching adhesive systems for bonding to enamel.
for many years, surgery was the only conventional treatment for colorectal obstruction, with a price of high rates of mortality and morbidity. surgical treatment options included decompressing colostomy or large bowel resection with or without a stoma. however, since the introduction of colorectal stenting in 1990 by dohmoto, large bowel obstruction relief may be achieved with better outcomes, especially in palliative patients. by alleviating the need for emergency surgery, stenting also enables preoperative comorbidity evaluation, proximal large bowel assessment, and staging of cancer, if needed. after the emergency treatment of obstruction and in cases when surgery is indicated, stenting enables laparoscopic colorectal resection with better short - term outcomes. the aim of this study was to assess morbidity and mortality in elderly patients undergoing stenting for large bowel obstruction at darent valley hospital from 1997 to 2010. a retrospective case review was performed of all patients registered in a prospective database who underwent self - expanding metal stent (sems) insertion for colorectal obstruction within the colorectal unit at darent valley hospital (dartford, uk) between january 1997 and december 2010. data including demographic details, stricture location, indication for the procedure, success, failure, and complications of the procedure were collected. data related to perforation, stent migration, reobstruction, fracture of the stent, and other major complications were included. stenting - related mortality was defined as death caused by direct stenting - related complications (mainly perforation). calculation of mortality during the same admission and poststenting 30-day mortality rates were recoded on the basis of the national health service 's computerized system. patients were divided into 3 groups : those 80 years of age (group iii). colorectal stenting was carried out in the interventional radiology room under light sedation and appropriate monitoring, with the patient placed in the left lateral decubitus position. in most cases, sems were inserted as a joint procedure between a colorectal surgeon and a radiologist using the over - the - wire method. the colonoscope was introduced to the lesion site, and a flexible guidewire was inserted to bypass the stricture. the scope was then retracted to enable introduction of the stent, which later was positioned and deployed. method, predominantly for proximal tumors, whereby the stent was introduced through the colonoscope. fluoroscopy was required to ensure successful wire placement and to delineate the size of the tumor for the assessment of stent length. final assessment of stent positioning was made using water - soluble contrast material, ensuring colon decompression. we used 2 types of stent : the metallic wallflex colonic stent (boston scientific corporation, natick, ma) and the memotherm colonic stent (bard medical, covington, ga). in all cases of the through - the - scope method, undisclosed data were collected on a personal computer, using microsoft excel software (microsoft corporation, redmond, wa). continuous variables such as age were categorized into subgroups and compared using student t tests or mann - whitney u tests, depending on their distribution. we constructed probability curves using the kaplan - meier method and compared them using the log - rank test. statistical analysis was performed using statsdirect version 2.7.8 (statsdirect ltd, cheshire, uk). colorectal stenting was carried out in the interventional radiology room under light sedation and appropriate monitoring, with the patient placed in the left lateral decubitus position. in most cases, sems were inserted as a joint procedure between a colorectal surgeon and a radiologist using the over - the - wire method. the colonoscope was introduced to the lesion site, and a flexible guidewire was inserted to bypass the stricture. the scope was then retracted to enable introduction of the stent, which later was positioned and deployed. method, predominantly for proximal tumors, whereby the stent was introduced through the colonoscope. fluoroscopy was required to ensure successful wire placement and to delineate the size of the tumor for the assessment of stent length. final assessment of stent positioning was made using water - soluble contrast material, ensuring colon decompression. we used 2 types of stent : the metallic wallflex colonic stent (boston scientific corporation, natick, ma) and the memotherm colonic stent (bard medical, covington, ga). in all cases of the through - the - scope method, undisclosed data were collected on a personal computer, using microsoft excel software (microsoft corporation, redmond, wa). continuous variables such as age were categorized into subgroups and compared using student t tests or mann - whitney u tests, depending on their distribution. we constructed probability curves using the kaplan - meier method and compared them using the log - rank test. statistical analysis was performed using statsdirect version 2.7.8 (statsdirect ltd, cheshire, uk). during the study period, 132 sems were deployed in a similar number of patients for treatment of colorectal pathology, with an average age of 72.1 years. of these, 53 were included in group i (average age, 59.7 years ; range, 2869 years), 42 were in group ii (average age, 75.2 years ; range, 7079 years), and 37 were in group iii (average age, 86.3 years ; range, 8095 years). similar diversity of gender, indication for the procedure, and colonic stricture location was found (table 1). there were no significant differences in the technical success rates of the procedure (88.7%, 76.2%, and 78.4%, p =.23) and clinical success rates (88.7%, 73.8%, and 78.4%, p =.16). data concerning complications were available for 124 of 132 patients and were comparable for the different groups (25.5%, 30.0%, and 12.1%, p =.17). the mortality rate during the same admission was significantly higher in the older groups (4.0%, 15.0%, and 22.2%, p =.027), but stent - related mortality and 30-day mortality were similar. kaplan - meier 30-day survival curves showed a similar tendency of probability of survival for the 2 older groups (7080 and 80 years) compared with the young group (1870 years), with a log - rank p value of.19 (figure 1). colonic stenting : data per age group complications : data per age group percentages were calculated from known data. kaplan - meier 30-day survival curve per age group (log - rank p =.1904). of all stented patients, 48 (36.4%) underwent surgery. of these, 27 (20.4% of all patients) were operated on urgently because of failure of stent insertion, failure to achieve bowel decompression, or stent complications. twenty - one patients (15.9%) underwent planned surgery, of whom 11 (8.3%) underwent laparoscopic surgery. when comparing age groups, it is notable that the younger groups had more surgery (50.9%, 38.1%, and 13.5%, respectively, p =.0013 ; figure 2), though there was no statistical difference between the indication for surgery (planned vs urgent) or the surgical approach (laparoscopic vs open). ratio (%) of surgery versus no surgery per age group (p =.0013). stoma prevention was achieved in 105 of 132 patients (79.4%), and only 27 patients (20.6%) had stomas. a significant difference in stoma rate was discovered when comparing age groups, with a higher rate of stoma among the young group (17 of 53 [32.1% ], 7 of 42 [16.7% ], and 3 of 37 [8.1% ], p =.0147). the risk for a patient 80 years of age. colorectal cancer accounts for about 1 million new cases per year worldwide, with 15% of all cases presenting as acute colonic obstructions, making it the leading cause of large bowel obstruction. colonic obstruction necessitates urgent decompression that traditionally required surgery, which carries high risk for mortality and morbidity, as well as high stoma rates. however, in recent years, the use of colorectal stenting proved to be a successful method for relief of symptoms of large bowel obstruction. it also enables management and correction of fluid and electrolyte imbalance, cardiac arrhythmias, and respiratory complications. sems insertion can also be used in benign strictures such as diverticular disease, postradiation, and anastomotic stricture. the array of colonic stenting technical success rates in the literature is wide, with a mean of 92% (range, 66.6%100%). clinical success rates are slightly lower, reaching a mean of 88% (range, 46%100%). technical failure is due mainly to the inability to place a guidewire through the stricture. a recent systematic review and meta - analysis of randomized clinical trials of sems insertion versus emergency surgery by tan found lower technical and clinical success rates of 70.7% and 69.0%, respectively, which were linked partially to complete large bowel obstruction. tight strictures, such as in complete obstruction, can cause marked abdominal distension and difficulties in guidewire placement, leading to lower rates of stent deployment. it demonstrates a technical stent placement success rate of 81.8% and a clinical success rate of 81.1%, both within reported ranges. with the increase in life expectancy and the link between colorectal cancer and age, there is a higher likelihood that an elderly population will exhibit acute mechanical colonic obstruction due to advanced cancerous disease. previous strategies for the treatment of colonic obstruction were associated with high mortality rates of 15% to 34%. in an association of coloproctology of great britain and ireland study of large bowel obstruction that included data on nearly 8000 patients, the average mortality rate was 16.5%, but when adding risk factors such as age > 85 years and american society of anesthesiologists score of iii, the mortality rate has reached 50%. the results of our study outline a tendency toward a higher proportion of 30-day mortality rates among the different age groups (13.2%, 23.8%, and 27.0%, p =.153 ; 12.7% overall). although this difference might be linked to a small sample size, it also indicates better outcomes for sems insertion compared with surgery. higher rates of mortality during the same admission for the older groups, ii and iii, were found (4.0%, 15.0%, and 22.2%, p =.027 ; 20.4% overall), even though similar bowel decompression rates were achieved, with comparable complication rates. an additional effect was the different curvature tendency shown in the kaplan - meier diagram regarding the 30-day probability of survival, which was similar for groups ii and iii compared with group i (figure 1). we believe that the disturbance of homeostasis, which is caused by colonic obstruction including dehydration due to shift of fluids, electrolyte imbalance, reduced intestinal blood flow, and mucosal edema with bacterial translocation, causes a physiologic insult that is more challenging for the aged population, thus influencing survival rates. alleviating obstruction with stent insertion buchanan published their experience in stenting elderly patients (median age, 82 years ; range, 6996 years), with successful sems placement in 9 of 11 patients and a median survival time of 5 months. when analyzing outcomes in a population > 70 years of age with colorectal obstructions, guo compared stent insertion and primary surgery, with better results for the stenting group in terms of primary anastomosis and 30-day mortality rate. although without discussing the issue of age, other studies of large bowel obstruction have published better results for sems insertion compared with urgent surgery in terms of primary anastomosis and stoma rates, with comparable complications and mortality results. on the other hand, elderly patients undergoing urgent colorectal surgery have worse 1-year survival rates. in a study by mamidanna, patients were classified into 3 age groups : 70 to 75, 76 to 80, and > 80 years. thirty - day mortality rates were 17.0%, 23.3%, and 31.0%, respectively (p <.001). advanced age was an independent risk factor for mortality in risk - adjusted regression analyses. surgery for octogenarians with colorectal cancers was also linked to significant morbidity and mortality rates in a study by tan. an association between advanced age, emergency surgery, and comorbidity was found. in our opinion, the data in the literature suggest that elderly patients would probably have difficulties in regaining homeostasis after an incident of large bowel obstruction, but they would probably do better with stenting than with emergency surgery. another remarkable issue is the significant difference between the proportions of patients undergoing surgery among the different age groups, with higher rates in younger patients causing higher rates of stoma (figure 2). we can not fully explain this difference, because of the retrospective nature of our study. nevertheless, we suspect that this difference might be linked to patients ' comorbidity issues (discussed earlier in this article) as well to patients ' and surgeons ' preferences relating to end - of - life decision making. we believe that surgeons have a greater reluctance to pursue aggressive management in the poststenting period, although in some cases, patients were probably too sick to undergo surgery. we also think that older patients, as well as surgeons treating the elderly, tend to prefer less invasive techniques such as sems insertion and avoid the choice of surgery. failure to rescue in those cases is partially a consequence of attitude rather than a result of disease and aggressive management. similar tendencies are found in other areas of colorectal cancer treatment. a study by serra - rexach examined differences in therapeutic approach in 503 patients with colorectal malignancies who were divided into a young group (age < 75 years) and an older group (age 75 years). although no differences were observed between the groups in terms of tumor differentiation, extension, tumor stage, or comorbidities, young patients were more likely to receive surgery, radiotherapy, and chemotherapy and were less likely to receive palliative care. similar attitudes toward the elderly are found when observing research in the field of chemotherapy, in which the elderly are underrepresented in clinical trials. our study demonstrates similar indications and stricture locations when comparing sems placement in young and elderly patients. comparable success, failure, and complication rates were found, with a higher same - admission mortality rate among the older patients. we believe that this dissimilarity represents the significant divergence in the disease burden due to large bowel obstruction, especially in elderly patients. we also observed a relationship between age and the tendency to operate, with aging patients less likely to undergo surgery for large bowel obstruction. this, we believe, reflects on the different attitudes of both patients and surgeons in terms of decision making. prospective randomized trials are needed to better determine the benefits of sems compared with emergency surgery.	background and objectives : emergency surgery for large bowel obstruction is associated with high morbidity and mortality rates, especially in elderly patients. colonic self - expanding metal stents may provide temporary relief of obstructions and enable preoperative evaluation. the aim of this retrospective study was to assess the clinical outcomes of emergency stenting in elderly patients with large bowel obstructions.methods:between 1997 and 2010, patients presenting with large bowel obstructions were treated predominantly with self - expanding metal stent insertion. clinical data, including age, site of obstruction, success rate, and surgery and mortality rates, were collected. patients were divided into 3 groups (i, ii, and iii) according to age : 80 years.results:one hundred thirty - two consecutive patients underwent stent implantation, with a mean age of 72.1 years (range, 2895 years). similar diversity of sex, indication, and stricture location was found. there were no significant differences in clinical success (88.7%, 73.8%, and 78.4%, p =.16) and stent - related mortality (2.1%, 3.3%, and 3.6%, p = 1.00). similar rates of stoma creation were also found (59.3%, 46.7%, and 60.0%, p =.76). in contrast, rates of surgery were lower in older patients (50.9%, 38.1%, and 13.5%, p =.0013), and mortality during the same admission was significantly higher in patients > 70 years of age (4.0%, 15.0%, and 22.2%, p =.027). kaplan - meier 30-day survival curves for the 3 groups showed a trend toward earlier death among patients > 70 years of age.conclusions:this study demonstrates that stenting provides similar success rates in all age groups but is associated with higher mortality rates in older patients.
prostate cancer (pca) is one of the most common cancers and the second most common cause of cancer - related death in men in the united states. however, the age - adjusted incidence rate of pca in korea increased from 10.1 per 100,000 people in 2002 to 27.0 per 100,000 people in 2012. according to the annual report of the national cancer center in 2012, pca was the fifth most common cancer and the seventh most common cause of cancer - related death in korean men. to detect pca, the results of prostate biopsy may include pca, benign diagnosis (e.g., benign prostate hyperplasia [bph ], inflammation, and prostatitis), high - grade prostatic intraepithelial neoplasia (hgpin), and atypical small acinar proliferation (asap). in patients with a benign diagnosis hgpin is defined as proliferation of the acini and ducts in epithelial cells similar to that of pca. it has been reported that the cancer detection rate on second biopsy in patients with hgpin ranges from 25% to 79%. asap is a term used by pathologists when the obtained tissue is not sufficient to be diagnosed with pca but exhibits abnormal morphology. it has been reported that the cancer detection rate on second biopsy in patients with asap ranges from 21% to 60%. hgpin and asap have been recognized as premalignant lesions and are potential risk factors for pca. however, there is no consensus regarding the management of patients with hgpin and asap. therefore, we compared the cancer detection rate, gleason scores on second biopsy, and unfavorable disease rate after radical prostatectomy (rp) in order to investigate the differences in patients with benign diagnosis, hgpin, and asap on first biopsy. these results will provide important information for establishing the appropriate clinical management of korean patients with hgpin and asap. this study was approved by the institutional review board of samsung medical center in seoul, korea (irb no. we retrospectively reviewed data from 7,477 patients who underwent prostate biopsy between march 1995 and november 2012. initial prostate biopsy was performed when the serum prostate - specific antigen (psa) level was more than 2.5 ng / ml and/or there were abnormal findings on the digital rectal examination. patients who were diagnosed with pca on a first biopsy and those who did not undergo a second biopsy were excluded. the second biopsy was done when the result of the first biopsy included hgpin and asap, and the psa level was continuously elevated. finally, a total of 1,323 patients who underwent a second biopsy were enrolled in this study. the patients were divided into three groups according to the results of the first biopsy : benign diagnosis, hgpin, and asap. additionally, patients with hgpin or asap were subdivided into two groups according to the number of positive cores : single core of hgpin (hgpin1), multiple cores of hgpin (hgpin2), single core of asap (asap1), and multiple cores of asap (asap2). clinicopathologic parameters such as age, prostate volume, psa level on first and second biopsy, and the time between the first and second biopsy were compared among the three groups. psa density (psad) was calculated by using the formula of the psa level divided by the prostate volume on first biopsy. psa velocity was also calculated by dividing the time between the first and second biopsy into the change in the psa level. the cancer detection rate was compared and univariate and multivariate analyses were performed among the three groups to predict the diagnostic factors of pca on a second biopsy, including clinical factors, hgpin, and asap. gleason scores and the rate of unfavorable disease were compared in patients who underwent rp. unfavorable disease was defined as a gleason score8, pathologic staget3b (involving the seminal vesicle), and/or spread to the regional lymph nodes after rp. we used an automatic biopsy gun with an 18-gauge needle to harvest the prostate tissue. to minimize hemorrhagic complications, patients were advised not to take any oral anticoagulants including aspirin for 7 days before the procedure. laboratory studies including prothrombin time and activated partial thromboplastin time were checked routinely to confirm normal coagulation profile. all patients received an intramuscular dose of aminoglycoside just before the procedure, and oral ciprofloxacin and/or cephalosporin for 7 days after the procedure. sextant biopsy was the standard method before 2009, which was later replaced by 12-core biopsies. all procedures were performed on a hospital - affiliated outpatient clinic basis, and all patients provided informed consent prior to the procedure. continuous and categorical variables were described as meanstandard deviation and absolute values (percentage), respectively. the distributions of clinicopathologic variables among the bph, hgpin, and asap groups were compared by using analysis of variance. comparisons of gleason scores and subgroups of hgpin (hgpin1 vs. hgpin2) or asap (asap1 vs. asap2) were performed by using pearson chi - square test. comparisons of the cancer detection rate, unfavorable disease rate, and predictors for pca were performed by using logistic regression.. 20.0 (ibm co., armonk, ny, usa), and a two - tailed p - value 0.05). overall, 214 patients (16.2%) were diagnosed with pca on the second biopsy (table 2). pca was detected in 164 patients (14.6%) with a benign diagnosis, in 33 patients (22.1%) with hgpin, and in 17 patients (32.1%) with asap. the cancer detection rate was significantly higher in patients with hgpin and asap than those with a benign diagnosis (hgpin vs. benign diagnosis, p=0.027, and asap vs. benign diagnosis, p=0.004, respectively). however, there was no significant difference between the asap and hgpin groups (p=0.150). when patients were divided into subgroups according to the number of positive cores, pca was detected in 15 patients (16.7%) with hgpin1 and in 18 patients (30.5%) with hgpin2 (table 3). patients with hgpin1 and those with a benign diagnosis had similar cancer detection rate (16.7% and 14.6%, p=0.567, respectively). on the other hand, pca was detected in 13 patients (31.0%) with asap1 and in 4 patients (36.4%) with asap2. the cancer detection rate was not significantly different between patients with asap1 and asap2 (p=0.732). however, patients with asap1 showed a much higher rate of cancer detection than in patients with a benign diagnosis (31.0% and 14.4%, respectively, p=0.003). the cancer detection rate was significantly higher in patients with hgpin2 and asap on first biopsy. in patients who were diagnosed with pca on second biopsy, the hgpin and asap groups had greater proportions of patients with a gleason score6 than did the benign diagnosis group (57.6%, 70.6%, and 51.8%, respectively) and lower proportions of patients with a gleason score8 than did the benign diagnosis group (6.1%, 5.9%, and 16.5%, respectively). however, there were no statistically significant differences in gleason scores among the three groups (p=0.324). in patients diagnosed with pca unfavorable disease was detected in 8 patients (8.3%) with benign diagnosis, 2 patients (8.0%) with hgpin, and 1 patient (10.0%) with asap. however, there were no meaningful differences in the unfavorable disease rate (benign diagnosis vs hgpin, p=0.857, and benign diagnosis vs asap, p=0.957 respectively) (table 5). table 6 shows the logistic regression analysis of predictors for pca detection among the three groups. in the univariate analysis, age (p=0.003), psa on first (p=0.001) and second biopsy (p<0.001), and psad (p=0.001) were significantly different in patients who were diagnosed with pca. in the age - adjusted analysis, of these factors, psa on first (p=0.005) and second biopsy (p<0.001) and psad (p=0.001) were significantly different. in the multivariate analysis, age (p=0.038), psa on second biopsy (p<0.001), and psad (p<0.001) in the present study, we investigated the differences in the cancer detection rate, gleason scores on second biopsy, and unfavorable disease rate after rp among patients with benign diagnosis, hgpin, and asap on first prostate biopsy. a total of 214 patients (16.2%) were diagnosed with pca on a second biopsy. patients with older age, a higher serum psa level on second biopsy, and a high psad had an increased risk of pca detection in age - adjusted and multivariate analysis. the cancer detection rate was significantly higher in patients with hgpin2 and asap on first biopsy. therefore, when we interpret the results of the first biopsy, we should consider the potential risk of pca according to the number of positive cores in patients with hgpin and asap. to the best of our knowledge, this is one of the largest studies in korea that has examined the significance of hgpin and asap cores on first biopsy. several studies have reported that the cancer detection rate on second biopsy ranges from 25% to 79% and from 21% to 60% in patients with hgpin and asap, respectively. in this study, 33 patients (22.1%) and 17 patients (32.1%) were diagnosed with pca in the hgpin and asap groups, respectively. reported that the cancer detection rate was higher in patients with multiple cores of hgpin (35.9%) than in those with single - core hgpin (22.0%). in our study, the cancer detection rate was significantly higher in patients with hgpin2 than in those with hgpin1 (30.5% vs. 16.7%, p=0.047). analyzed a total of 295 patients with asap and reported that pca was detected in 125 patients (42.4%) on second biopsy. in our study, patients with asap also had a significantly higher cancer detection rate than did those with a benign diagnosis (32.1% vs. 14.6%, p<0.001). reported that, when patients were diagnosed with pca on second biopsy, the proportion of gleason scores were as follows : gleason6 was 83%, gleason 7 was 15%, and gleason8 was 4%. in our study, gleason6 was 53.8%, gleason 7 was 25.0%, and gleason8 was 7.1%. there was a relatively higher proportion of patients with a gleason score8 and a lower proportion of patients with a gleason score6 in our study. some studies have reported that patients diagnosed with pca in korea have higher gleason scores than do western patients, and that tendency may be reflected in our study. reported that the percentages of patients with gleason6 and gleason8 were 48.1% and 13.6%, respectively. however, there were no statistically significant differences in gleason scores among three groups (p=0.324) in this study. of the 214 patients diagnosed with pca on a second biopsy, 131 patients (61.2%) patients overall underwent rp. unfavorable disease after rp was detected in 8 patients (8.3%) with benign diagnosis, 2 patients (8.0%) with hgpin, and 1 patient (10.0%) with asap. there were no meaningful differences in the rate of unfavorable disease among the three groups (benign diagnosis vs. hgpin, p=0.857, and benign diagnosis vs. asap, p=0.957, respectively). taken together, the results of hgpin and asap on first biopsy were not correlated with gleason scores on second biopsy or the unfavorable disease rate after rp in this study. the mean time between the first and second biopsy was 32.8 months in the benign diagnosis group, 30.1 months in the asap group, and 7.5 months in the hgpin group. if the result of the first biopsy was a benign diagnosis, regular follow - up was performed in 6 months, and each time the patients came to our hospital, their serum psa levels were checked. if the result of the first biopsy is hgpin or asap, the follow - up interval should be shorter than that for bph. usually, follow - up was performed in 3 months, and at that time we considered whether a repeat biopsy should be performed. if the psa level was high, we recommended a repeat biopsy more strongly. in this study, there was a significant difference in the intervals between biopsies among the three groups (p<0.001). short - term repeat biopsy was done in the hgpin group, whereas the benign diagnosis and asap groups did not differ significantly (p=0.605). although the higher rate of cancer detection in patients with asap on repeat biopsy has been reported previously, the timing of the repeat biopsy was delayed more than in those with hgpin. one possible explanation of this finding is that we may underestimate the risk of asap and perform a delayed biopsy in some of these patients. recently, on the basis of the results of this study, we have changed the institutional policy for performing a repeat biopsy. short - term repeat biopsy in 6 months is recommended more strictly in patients with asap. the differences in the number of biopsy cores could result in an underestimation of positive cores in the hgpin and asap groups. we can not exclude the possibility of actual hgpin2 patients being misclassified as hgpin1 in the sextant biopsy group. third, we did not analyze the number of the cohort receiving a 5- reductase inhibitor between the first and second biopsy, which could affect the serum psa level. also, the time between the first and second biopsy differed among the three groups, which also affected the bias with 5- reductase inhibitor. the effect of a 5- reductase inhibitor may be more significant in some groups and not significant in other groups. thus, we can not rule out that the use of 5- reductase inhibitors may have influenced the results of the present analysis. in summary, we compared the cancer detection rate, gleason scores, and the rate of unfavorable disease to investigate differences in patients with benign diagnosis, hgpin, and asap on first biopsy. the cancer detection rate was much higher in patients with multiple cores of hgpin and at least one core of asap. old age, a high psa level on second biopsy, and high psad were associated with an increased risk of pca detection.	purposeto investigate the differences in the cancer detection rate and pathological findings on a second prostate biopsy according to benign diagnosis, high - grade prostatic intraepithelial neoplasia (hgpin), and atypical small acinar proliferation (asap) on first biopsy.materials and methodswe retrospectively reviewed the records of 1,323 patients who underwent a second prostate biopsy between march 1995 and november 2012. we divided the patients into three groups according to the pathologic findings on the first biopsy (benign diagnosis, hgpin, and asap). we compared the cancer detection rate and gleason scores on second biopsy and the unfavorable disease rate after radical prostatectomy among the three groups.resultsa total of 214 patients (16.2%) were diagnosed with prostate cancer on a second biopsy. the rate of cancer detection was 14.6% in the benign diagnosis group, 22.1% in the hgpin group, and 32.1% in the asap group, respectively (p<0.001). when patients were divided into subgroups according to the number of positive cores, the rate of cancer detection was 16.7%, 30.5%, 31.0%, and 36.4% in patients with a single core of hgpin, more than one core of hgpin, a single core of asap, and more than one core of asap, respectively. there were no significant differences in gleason scores on second biopsy (p=0.324) or in the unfavorable disease rate after radical prostatectomy among the three groups (benign diagnosis vs. hgpin, p=0.857, and benign diagnosis vs. asap, p=0.957, respectively).conclusionspatients with multiple cores of hgpin or any core number of asap on a first biopsy had a significantly higher cancer detection rate on a second biopsy. repeat biopsy should be considered and not be delayed in those patients.
building and renovation measures can not only cause significant contamination of dust but can also liberate great amounts of fungal spores and therefore can pose the risk of invasive aspergillosis in severely immunocompromised patients with e.g. bone marrow or organ transplants and acute leukaemias,. in acute care hospitals ward closure or the transferring of patients is often impossible in cases of sudden events. the aim of this study was to monitor the load of particulate matter and fungal spores after two incidents of considerable damage caused by water which occurred in university hospital of essen and which had to be managed during ward activity. in january 2009 water damage occurred in the bathroom of a surgical ward of university hospital of essen. the wall of the bathroom and the floors of two adjacent patient rooms and the adjacent ward 's corridor were wet and had to be scraped. dust protecting walls were installed insulating the ward from the affected rooms and the affected part of the corridor (figure 1 (fig. 1) and figure 2 (fig. exhaust air was conducted outside through a sealed hole in the window (figure 3 (fig. between january and april 2009 and between september and october 2009 respectively we measured concentrations of particles 0.3, 0.5, 1 and 5 m and fungi in the air of affected wards. measuring devices were placed 1 m to 1.50 m above ground level and 1 m distant from the doors of dust protecting walls. on the surgical ward measurements were performed on both sides of the corridor, the left side from the renovation area directed towards the intensive care unit, the right side directed towards the stairway. control measurements were performed on two surgical wards and on an intermediate care ward respectively. air supply of the cardiac surgery intensive care unit and intermediate care unit was hepa filtered. 2x 50 litres of air were collected by mas-100 (merck) on sabouraud agar, which were incubated aerobically at 25c for 5 days. we analysed data of the hospital documentation system for diagnosed or suspected aspergillus infections of patients from both intensive care wards. statistical analysis was performed on a personal computer using the statistical package for social science (spss). results of the measurements during the renovation work on the surgical ward are shown in table 1 (tab. 1) and table 2 (tab. 2) and results of measurements during the renovation on the cardiac surgical intensive care unit are shown in table 3 (tab. 4). during the renovation work on the surgical ward concentrations of moulds and particles 5 m were significantly higher on the left side of the renovation area than on the right side (p=0.036 and p 0.3 m, > 0.5 m and > 1 m were significantly higher on the control ward on the other floor than on the ward with renovation work going on. during the renovation work on cardiac surgical intensive care unit concentrations of particles of all sizes there was no difference in fungal concentration between the cardiac surgical intensive care ward and intermediate care ward. aspergillus fumigatus could neither be cultured of the air of the cardiac surgical intensive care unit nor of the intermediate care unit. during renovation concentrations of moulds and particles 5 m at two different measuring points which were only few meters apart from each other on the affected surgical ward were significantly different. where the concentrations of particles were elevated doors were kept closed whereas rubbish was carried outside on the right side. similar to cooper. who could not find any statistically significant difference in the levels of viable fungi during construction work performed with protective measures we could not find any increase of concentrations of particles and moulds on the surgical ward compared to both control wards. on the contrary particle and fungal concentrations on the control ward on another floor of the building were significantly increased. it is unlikely that this increase was caused by dust blown through the opened windows because exhausted air of the renovated rooms passed through a hepa filter. indoor concentration of particles and moulds is not only supposed to be influenced by outdoor air but also by indoor activity. on the control ward there were a lot of movements of patients and members of staff which might have influenced particle and fungal concentrations in the air. the mean concentrations of moulds on the surgical ward as well as on both control wards were higher than those measured by ortiz. in the air of hospital rooms in a hospital in spain but were less than those found by kolk. in outdoor air in germany. peak concentrations of moulds during well partitioned construction and renovation works were found to be in the same range as concentrations of outdoor air in germany,. the cardiac surgery intensive care unit and intermediate care unit are provided with hepa filters. although particle concentrations were increased on surgery intensive care unit during renovation work there was no rise of fungal concentration and no aspergillus fumigatus was cultured from the air. reports about the effect of hepa - filtration on aerial fungal concentration during renovation or construction works are different.. found a significant reduction of aspergillus spores during renovation works by use of mobile air filtration system. reported an increase of aerial fungal concentration on a ward adjacent to hospital renovation despite being protected with hepa filtration. an explanation could be renovation and construction works producing different amounts of dust and thus liberating different numbers of fungal spores. but goodly. concluded from measurements performed during one year that spore concentrations could not be predicted from the nature of building work. the volume of air sampled by mahieu. as well as by us was 100 liters whereas that of cornet. they found that the detection rates of aspergillus spores were dependent of sampled volume. provided that the renovation area is tightly insulated from the areas of patient care on a ward, closure does not seem to be necessary during renovation works because variation of airborne fungi is similar to that of outdoor and control air. similar conclusions were drawn by rautiala. who measured aerial fungi adjacent to construction zones which were placed under negative pressure and which were isolated with a plastic barrier. this team should perform risk assessment and determine necessary protective measures before starting any construction, renovation or maintenance work in health care settings. patients with increased risk of invasive aspergillosis should be identified and should be moved to other wards far away from construction and renovation areas and use of mobile hepa filters should be considered. nosocomial outbreaks of fungal infections have been caused by aerial concentrations as low as 1.1 cfu / m aspergillus fumigatus and no minimal infective dosis of aerial viable fungal spores can be given. there are controversial indications as to whether fungal spore concentration should be measured during construction or renovation works in healthcare settings, but air pressure gradients are advised to be regularly verified in artificially ventilated areas. provided that the renovation area is tightly insulated from the areas of patient care on a ward, closure does not seem to be necessary during urgent renovation works because variation of airborne fungi is similar to that of outdoor air or air in control wards. this team should perform risk assessment and determine necessary protective measures before starting any construction, renovation or maintenance work in health care settings.	background : construction and renovation work in hospitals pose risks of fungal airborne infections for immunosuppressed patients. if possible, reconstruction work will be postponed to periods without patient treatment. however, in many situations urgent damage demands immediate refurbishment works before the transferring of patients to other wards or closure of wards is possible. reported here are infection control related measures and implemented procedures after two incidents of water damage which occurred on a surgical ward and an intensive care unit at the university hospital of essen.methods : between january and april 2009 and between september and october 2009, respectively, concentration of air - borne particles and number of viable fungi were measured at two surgical wards and one icu. preventive infection control measures included erection of protective walls and hepa filtration of air from the renovation area.results : during the renovation work on the surgical ward concentrations of moulds and particles 5 m were significantly higher on the left side of the renovation area than on the right side (p=0.036 and p<0.001). concentrations of particles 1 m and particles 5 m on both sides of the renovation area were significantly increased when compared with the control ward on the same floor but not when compared with the control ward on the other floor. particles of all size were significantly elevated on the icu during the renovation work. aspergillus fumigatus could neither be cultured of the air of cardiac surgery intensive care unit nor of the intermediate care unit (control ward). during renovation works there was no nosocomial mould infection of patients treated on the two wards.conclusion : provided that the renovation area is tightly insulated from the areas of patient care on a ward, closure does not seem to be necessary during renovation works because variation of airborne fungi is similar to that of outdoor or control air. however a multidisciplinary team should be established. this team should perform risk assessment and determine necessary protective measures before starting any construction, renovation or maintenance work in health care settings.
currently this genus includes only one validly published species named dermabacter hominis, which was formerly known as the coryneform bacteria of the centers for disease control groups 3 and 5,. members of this genus are gram - positive, non spore forming, non acid fast, nonmotile, short rods, facultatively anaerobic, catalase positive and oxidase negative. d. hominis has also been detected in clinical samples such as wound swabs, bronchial washings, abscesses and ear smears,,,. recently, high - throughput genome sequencing and matrix - assisted laser desorption / ionization time - of - flight mass spectrometry (maldi - tof) analyses of bacteria have given unprecedented access to an abundance of genetic and proteomic information,. thus, a polyphasic approach is currently proposed in our laboratory to describe new bacterial taxa, including their genome sequence, maldi - tof spectrum, and major phenotypic characteristics such as gram staining, culture conditions, metabolic characteristics, habitat and, if applicable, pathogenicity. here in may 2014, while working at dakar, the index finger of a researcher was bitten by a fish. strain ff11 (table 1) was isolated from this wound by culture on 5% sheep 's blood enriched columbia agar (biomrieux, marcy letoile, france). in order to identify the strain ff11, maldi - tof protein analysis was performed using a microflex lt (bruker daltonics, leipzig, germany), as previously reported,. the scores previously established by bruker to identify or validate species compared to the instrument 's database were applied. in short, a score of 2.000 with a species with a validly published name allows identification at the species level ; scores of 1.700 and < 2.000 allow identification at the genus level ; and a score of < 1.700 does not allow any identification to be made. they were then imported into maldi biotyper software (version 2.0, bruker) and analysed by standard pattern matching (with default parameter settings) against the main spectra. scores ranging from 1.315 to 1.511 were obtained for ff11, suggesting that this strain was not a member of any known species. the reference mass spectrum from strain ff11 was incremented in our database (fig. 1). moreover, strain ff11 exhibited 97.9% 16s rrna sequence similarity with dermabacter hominis (genbank accession no. x91034), the phylogenetically closest bacterial species with standing in the nomenclature (fig. 2). this value was lower than the 98.7% 16s rrna sequence identity threshold recommended by meier - kolthoff. in 2013 to delineate a new species within the firmicutes phylum without carrying out dna - dna hybridization. different growth temperatures (25, 28, 37, 45 and 56c) were tested. growth of the strain was also tested under anaerobic and microaerophilic conditions using genbag anaer and genbag microaer systems (biomrieux), respectively, and under aerobic conditions, with or without 5% co2. optimal growth was observed under aerobic and microaerophilic conditions, but weak growth was observed under anaerobic conditions at 37c. strain ff11 shows white convex colonies measuring approximately 1 mm in diameter on 5% sheep 's blood enriched columbia agar (biomrieux). cells are gram - positive, nonmotile, non spore forming short rods (fig. 3). the negative staining of the cells and observation under transmission electron microscopy (fei company, hillsboro, oregon, usa) displays cells lacking flagella (fig. 4). dermabacter indicis is catalase positive and oxidase negative. using an api 50ch strip (biomrieux), fermentation was observed for d - galactose, d - glucose, n - acetyl - d - glucosamine, d - lactose, d - saccharose, d - trehalose, d - melezitose, d - raffinose, starch and d - turanose. using the api coryne strip (biomrieux), positive reactions were also observed for pyrazinecarboxamide, pyroglutamic acid--naphthylamide, esculin ferric citrate, urea and d - maltose. negative reactions were noted for potassium nitrate (reduction of nitrates), -glucuronidase, gelatin, d - ribose, d - xylose, d - mannitol and glycogen. using the api zym strip (biomrieux), enzymatic reactions were observed for esterase, esterase lipase, lipase, acid phosphatase, alkaline phosphatase, naphthol - as - bi - phosphohydrolase, cystine arylamidase, trypsin, -glucosidase, -galactosidase, -mannosidase, -fucosidase and n - acetyl--glucosaminidase. negative reactions were observed for leucine arylamidase, valine arylamidase, -glucosidase, -galactosidase and -glucuronidase. strain ff11 is susceptible to ciprofloxacin, amoxicillin / clavulanic acid, ticarcillin, ceftriaxone, imipenem, doxycycline, gentamicin and cefalotin, but it is resistant to colistin, trimethoprim / sulfamethoxazole, erythromycin and nitrofurantoin. a comparison of phenotypic characteristics with dermabacter hominis, brachybacterium faecium, brachybacterium muris, and helcobacillus massiliensis is summarized in table 2. the organism was selected for sequencing on the basis of its phylogenetic position, 16s rrna similarity and phenotypic differences with other members of the dermabacteraceae family. here table 3 shows the project information and its association with migs (minimum information about a genome sequence) version 2.0 compliance. dermabacter indicis strain ff11 (= csur p1488 = dsm 100283) was grown on 5% sheep 's blood bacteria grown on four petri dishes were resuspended in 5 100 l of tris - edta buffer and 150 l of this suspension was diluted in : 350 l tris - edta buffer 10, 25 l proteinase k and 50 l sodium dodecyl sulfate for lysis treatment. extracted dna was then purified using three successive phenol chloroform extractions and ethanol precipitations at 20c overnight. the genomic dna concentration was measured at 69.3 ng/l using the qubit assay with the high sensitivity kit (life technologies, carlsbad, ca, usa). genomic dna (gdna) of dermabacter indicis ff11 was sequenced on the miseq technology (illumina, san diego, ca, usa) with the mate pair strategy. the gdna was barcoded in order to be mixed with 11 other projects with the nextera mate pair sample prep kit (illumina). the mate pair library was prepared with 1.5 g of gdna using the nextera mate pair illumina guide. the pattern of the fragmentation was validated on an agilent 2100 bioanalyzer (agilent technologies, santa clara, ca, usa) with a dna 7500 labchip. the dna fragments are ranged in size from 1.5 to 11 kb with an optimal size at 6.730 kb. no size selection was performed, and 636 ng of tagmented fragments were circularized. the circularized dna was mechanically sheared into small fragments with an optimum at 653 bp on the covaris device s2 in t6 tubes (covaris, woburn, ma, usa). the library profile was visualized on a high sensitivity bioanalyzer labchip (agilent), and the final concentration library was measured at 59.1 nmol / l. after a denaturation step and dilution at 15 pm, the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. automated cluster generation and sequencing runs were performed in a single 39-hour run at a 2 251 bp read length. total information of 5.9 gb was obtained from a 624k / mm cluster density with cluster passing quality control filters of 96.33% (12 040 000 clusters). within this run, the index representation for dermabacter indicis ff11 the 1 918 640 paired reads were filtered according to the read qualities. open reading frames (orfs) were predicted using prodigal with default parameters, but the predicted orfs were excluded if they spanned a sequencing gap region. the predicted bacterial protein sequences were searched against the genbank database and the clusters of orthologous groups (cogs) database using blastp. the trnascan - se tool was used to find trna genes, while ribosomal rnas were found using rnammer and blastn against the genbank database. orfans were identified if their blastp e value was lower than 1e-03 for an alignment length greater than 80 aa. if alignment lengths were smaller than 80 aa, we used an e value of 1e-05. artemis was used for data management and dna plotter for the visualization of genomic features. the mauve alignment tool (version 2.3.1) was used for multiple genomic sequence alignment. briefly, this software combines the proteinortho software for detecting orthologous proteins in pairwise genomic comparisons, then retrieves the corresponding genes and determines the mean percentage of nucleotide sequence identity among orthologous orfs using the needleman - wunsch global alignment algorithm. annotation and comparison processes were performed in the multi - agent software system dagobah, including figenix libraries that provide pipeline analyses. genome - to - genome distance calculator (ggdc) analysis was also performed using the ggdc web server as previously reported,. here, the genome of dermabacter indicis strain ff11 (embl / ebi accession no. cyug00000000) is compared to those of dermabacter hominis strain 1368 (jdrs00000000), brachybacterium faecium strain dsm 4810 (cp001643), brachybacterium paraconglomeratum strain lc44 (agso00000000), brachybacterium squillarum strain m-6 - 3 (agbx00000000) and brachybacterium muris strain ucd - ay4 (aorc00000000). the embl / ebi bioproject number is prjeb10922 and consists of 248 large contigs. finally, the draft genome of d. indicis ff11 generated a 2 222 902 bp long genome with a 63.2% g+c content (fig. 5). of the 2124 predicted genes, 2074 were protein - coding genes and 50 were rnas (three 5s rrna genes, one 16s rrna gene, one 23s rrna gene and 45 trna genes). the remaining genes were annotated as hypothetical proteins. the properties and statistics of the genome the draft genome of d. indicis is smaller than d. hominis, b. faecium, b. paraconglomeratum, b. squillarum and b. muris (2.22, 2.51, 3.61, 3.78, 3.19 and 3.26 mb respectively). the g+c content of d. indicis is higher than that of d. hominis (63.2 and 62.7%, respectively) but lower than that of b. faecium, b. paraconglomeratum, b. squillarum and b. muris (72.0, 72.4, 72.8 and 70.0% respectively). the gene content of d. indicis is smaller than that of d. hominis, b. faecium, b. paraconglomeratum, b. squillarum and b. muris (2124, 2302, 3191, 3432, 2869 and 2914 respectively). the distribution of genes into cogs categories was similar in all the genomes compared (fig. 6). in addition, d. indicis shared 2074, 2226, 3068, 3341, 2765 and 2806 orthologous genes with d. hominis, b. faecium, b. paraconglomeratum, b. squillarum and b. muris (fig. 6). the genomic similarity between strain ff11 and the closely related brachybacterium species the results of phenotypic, phylogenetic and genomic analyses allow us to propose the creation of dermabacter indicis sp. nov., which contains strain ff11. n. indicis, pertaining to the latin name of the index finger, from which the type strain was isolated). strain ff11 is a gram - positive bacterium, facultatively anaerobic, with small (1 mm) and white colonies on 5% sheep 's blood enriched columbia agar. strain ff11 is nonmotile, non spore forming, oxidase negative and catalase positive. strain ff11 presents positive reactions for d - galactose, d - glucose, d - trehalose, d - melezitose, d - raffinose, starch, d - turanose, esterase, esterase - lipase, pyrazinecarboxamide, pyroglutamic acid--naphthylamide, esculin ferric citrate, urea, d - lactose, d - maltose, d - saccharose, acid phosphatase, naphthol - as - bi - phosphohydrolase, cystine arylamidase, trypsin, -glucosidase, -galactosidase, -mannosidase, -fucosidase and n - acetyl--glucosaminidase. dermabacter indicis strain ff11 is susceptible to ciprofloxacin, amoxicillin / clavulanic acid, ticarcillin, ceftriaxone, imipenem, doxycycline, gentamicin and cefalotin but resistant to colistin, trimethoprim / sulfamethoxazole, erythromycin and nitrofurantoin. the 16s rrna and genome sequences are deposited in genbank under accession numbers ln810502 and cyug00000000, respectively. the type strain ff11 (= csur p1488 = dsm 100283) was isolated from a human finger wound in dakar, senegal.	strain ff11 t was isolated from the wound on a researcher 's finger who had been bitten by a fish (protopterus annectens) in senegal. analysis by matrix - assisted laser desorption / ionization time - of - flight mass spectrometry did not provide any identification, but the 16s rrna sequence exhibited 97.9% identity with dermabacter hominis. phenotypic and genomic analyses demonstrated that strain ff11 t is gram - positive, facultatively anaerobic, nonmotile and non spore forming ; it exhibited a genome of 2 222 902 bp encoding 2074 protein - coding and 50 rna genes, with a 63.2% g+c content. we consequently proposed the creation of dermabacter indicis strain ff11 t.
rigid internal fixation with metallic materials is a standard technique, in use for the last 35 years and performed to align bone segments during healing periods. rigid fixation methods have till now, relied almost exclusively upon metallic components because of their high strength and contourability. however, there are inherent drawbacks associated with metallic devices, as susceptibity to corrosion, need to remove these plates / screws due to factors like infection, stress shielding, plate palpability, temperature sensitivity ; interference with advanced imaging technique, possible growth restrictions and implant migration in pediatric population and problem in patients who have to be irradiated as they may cause backscatter. in an effort to overcome the disadvantages of metallic osteofixation devices, the advantages of biodegradable osteosynthesis devices include the gradual transference of physiological forces to the healing bone, the reduced need for a second operation to remove the material, its potential to serve as a vehicle to deliver bone - healing proteins to fracture / osteotomy sites and no growth restrictions or possible migration in growing patients. the implant degrades naturally and totally after fracture has healed thus preventing the stress shielding effect while fibrous tissues or bone fills the space previously occupied by the implant. ideally, these materials are sufficiently rigid, biocompatible, provide stability without affecting bone healing and bone strength adversely, with no interference with post - operative imaging techniques or radiotherapy. today most commercially available materials which have optimal characteristics for use as a fixation modality in maxillofacial trauma are co - polymers consisting of polylactide acid (80 - 82%) and polyglycolic acid (18 - 20%). however, these have not been recommended for use in loaded and functional bone such as adult mandible because of the lesser strength and stiffness of the material. the present study was undertaken to evaluate the efficacy of biodegradable plates and screws as a method for internal fixation in patients of midface fracture and children with fractures of the mandible and to study the morbidity associated with the use of these devices. all patients with maxillofacial trauma reporting to the outpatient clinic of the department of oral and maxillofacial surgery of our institute were screened for inclusion in the present study. patients with major systemic diseases, presence of infection at fracture site, pathologic fractures, comminuted fractures, fractures with extensive bone loss and with history of hypersensitivity reaction to bioresorbable material were excluded. finally, eight adult patients with midface fractures requiring open reduction and internal fixation and two pediatric patients with mandibular fractures (excluding condylar fractures) were included in the study. the patients participating in this study were thoroughly informed of the advantages and disadvantages of this treatment, and their consent was documented. the patients were evaluated preoperatively and post operatively for the following information : location and type of fracture (displaced vs. nondisplaced), derangement of occlusion, paraesthesia along the distribution of infra - orbital nerve (in case of midface fractures) or mental nerve (in case of mandible fractures), presence or absence of diplopia, enopthalmos / exopthalmos in case of mid face fractures. on radiological assessment the site, nature and displacement of fractures were assessed using sub - mento - vertex view and occipitomental view for mid - face and orthopantomograph for mandible [figure 1 ]. dental impressions were taken in all patients and arch bars were placed in patients with deranged occlusion. orthopantomogram showing right angle fracture biodegradable inion cps (2.0 mm) plating system (inion, tempere, finland) approved by the food and drug administration, was used as a method of internal fixation [figure 2 ]. the material is copolymer of l - lactide acid (lpla), poly d - lactide acid (dlpla) and trimethyl carbonate (tmc) and polygycolic acid (pga). these plates have been reported to resorb slowly, maintaining 70% of their initial strength at nine to 14 weeks, with 42% bulk resorption by 40 weeks, and are completely resorbed by two to four years (in vitro data performed by inion for the us food and drug administration). plates are malleable after activation in the inion thermo water bath (55c). after water bath treatment, plates are most malleable for 10 - 15 seconds for easy adaptation. they can also be re - heated for further contouring or bent once cooled. surgical access was gained either through an existing skin laceration or a standardized surgical approach, reduction of fracture segments was achieved and segments were stabilized for plate fixation under general anesthesia. the patients with zygomatic complex fractures were stabilized with one or two point fixation at the maxillary buttress and frontozygomaic suture, using 2 mm four hole and two hole plates respectively. pediatric patients with developing or mixed dentition, presenting with fractures of mandible were stabilized with 2 mm plates and screws placed at the inferior border, below the developing tooth buds, for angle and parasymphysis fracture. the plate was placed into a water bath at 55c for one to two minutes, allowing the plate to become malleable. once the plate was adapted across the fracture line, a screw hole was created by using 1.75 mm drill bit at a speed of 2000 rpm with constant irrigation. the screw was selected depending on the site of application (2 mm diameter and 5 - 7 mm length). the screw with the screw driver was inserted and tightened. in the event of screw head fracture during placement of the screw, the head was removed and the previously drilled osteotomy was re - drilled through the remnant of the previous screw shank. the site was re - tapped in standard fashion with placement of a new screw of equal size. intra - operative details like time taken for plate fixation, plate fracture, and screw breakage was noted. intermaxillary fixation was done for a variable period of time ranging from two - four weeks. all patients received amoxicillin with clavulanate and metrogyl in the perioperative period, continuing for seven days postoperatively [figures 3 and 4 ]. fracture site exposed and fracture segments reduced a 4-hole bioresorbable plate fixed to the fractured segments all patients with maxillofacial trauma reporting to the outpatient clinic of the department of oral and maxillofacial surgery of our institute were screened for inclusion in the present study. patients with major systemic diseases, presence of infection at fracture site, pathologic fractures, comminuted fractures, fractures with extensive bone loss and with history of hypersensitivity reaction to bioresorbable material were excluded. finally, eight adult patients with midface fractures requiring open reduction and internal fixation and two pediatric patients with mandibular fractures (excluding condylar fractures) were included in the study. the patients participating in this study were thoroughly informed of the advantages and disadvantages of this treatment, and their consent was documented. the patients were evaluated preoperatively and post operatively for the following information : location and type of fracture (displaced vs. nondisplaced), derangement of occlusion, paraesthesia along the distribution of infra - orbital nerve (in case of midface fractures) or mental nerve (in case of mandible fractures), presence or absence of diplopia, enopthalmos / exopthalmos in case of mid face fractures. on radiological assessment the site, nature and displacement of fractures were assessed using sub - mento - vertex view and occipitomental view for mid - face and orthopantomograph for mandible [figure 1 ]. dental impressions were taken in all patients and arch bars were placed in patients with deranged occlusion. biodegradable inion cps (2.0 mm) plating system (inion, tempere, finland) approved by the food and drug administration, was used as a method of internal fixation [figure 2 ]. the material is copolymer of l - lactide acid (lpla), poly d - lactide acid (dlpla) and trimethyl carbonate (tmc) and polygycolic acid (pga). these plates have been reported to resorb slowly, maintaining 70% of their initial strength at nine to 14 weeks, with 42% bulk resorption by 40 weeks, and are completely resorbed by two to four years (in vitro data performed by inion for the us food and drug administration). plates are malleable after activation in the inion thermo water bath (55c). after water bath treatment, plates are most malleable for 10 - 15 seconds for easy adaptation. surgical access was gained either through an existing skin laceration or a standardized surgical approach, reduction of fracture segments was achieved and segments were stabilized for plate fixation under general anesthesia. the patients with zygomatic complex fractures were stabilized with one or two point fixation at the maxillary buttress and frontozygomaic suture, using 2 mm four hole and two hole plates respectively. pediatric patients with developing or mixed dentition, presenting with fractures of mandible were stabilized with 2 mm plates and screws placed at the inferior border, below the developing tooth buds, for angle and parasymphysis fracture. the plate was placed into a water bath at 55c for one to two minutes, allowing the plate to become malleable. once the plate was adapted across the fracture line, a screw hole was created by using 1.75 mm drill bit at a speed of 2000 rpm with constant irrigation. the screw was selected depending on the site of application (2 mm diameter and 5 - 7 mm length). the screw with the screw driver was inserted and tightened. in the event of screw head fracture during placement of the screw, the head was removed and the previously drilled osteotomy was re - drilled through the remnant of the previous screw shank. the site was re - tapped in standard fashion with placement of a new screw of equal size. intra - operative details like time taken for plate fixation, plate fracture, and screw breakage was noted. intermaxillary fixation was done for a variable period of time ranging from two - four weeks. all patients received amoxicillin with clavulanate and metrogyl in the perioperative period, continuing for seven days postoperatively [figures 3 and 4 ]. fracture site exposed and fracture segments reduced a 4-hole bioresorbable plate fixed to the fractured segments ten patients of maxillofacial fractures were treated by this method including nine males (90%) and one female (10%). they ranged in age from six to 45 years and the mean age was 32.8 years. the most common etiological factor was road traffic accident (60%) followed by interpersonal violence (30%) and fall (10%). two pediatric patients with angle (10%) and parasymphysis (10%) fracture and eight patients with midface fractures, out of which 5 (50%) were zygomatico - maxillary complex fractures, two (20%) were le fort ii and one (10%) patient was of le fort iii fracture, were included in this study [graph 1 ]. distribution of patients according to fracture site in preoperative evaluation, displacement of fracture segments was noticed in five (50%) patients and five (50%) patients had deranged occlusion. two patients (20%) (1 with le fort ii and 1 with le fort iii) reported paraesthesia along the distribution of infra - orbital nerve. diplopia was present in two patients (25%) and one patient presented with enophthalmos (12.5%) who had le fort ii fracture. time taken to fix one plate with the required number of screws varied from 20 - 40 minutes. a total of 20 plates were used and there was no incidence of intra - operative plate fracture. out of the total number of 68 screws used, only three screws broke during fixation (4.14%). post - operatively, all the patients were reviewed clinically and radiographically after 24 hrs, one week, four weeks, 12 and 24 weeks for adequate reduction, stability of fracture segments, centricity of occlusion and post operative complications [figure 5 ]. reduction of the fracture segments was satisfactory and occlusion relationships were judged as normal in all the cases at every follow up. adequate stability was maintained throughout the post - operative period in all the patients (100%). there was no incidence of (0%) post - operative paraesthesia, diplopia, enopthalmos / exopthalmos, wound dehiscence and plate exposure. in one patient (10%), signs of post - operative infection were noticed including swelling and pain, which was managed conservatively with administration of oral antibiotics and analgesics [graph 2 ]. in modern maxillofacial surgery, rigid osteosynthesis has become one of the major breakthroughs. for 25 years now, titanium has shown its excellent qualities and is currently regarded as the golden standard. when using metallic materials ; sometimes it is necessary to perform a second operation for the removal of these screws. all the disadvantages inherent to metallic materials such as palpation, sensitivity, migration, obstruction in the x - ray, possible bone resorption, allergies, and growth delays in children have led to the development of resorbable materials. since the introduction of biodegradable devices in 1966 bioresorbable materials mostly used in maxillofacial surgery are a mixture of rigid and elastic polymers selected for their strength, malleability, and degradation properties. the degradation profiles have been tailored to provide initial stability and then progressively load bone to stimulate regeneration. the results of various studies suggested that the use of biodegradable fixation in selected patients with facial fractures is acceptable. in the present study, ten cases of maxillofacial fractures were treated with internal fixation using inion cps biodegradable plates and screws. eight patients with mid face fractures and two pediatric patients with developing or mixed dentition presented with fractures of mandible. in two of the adult patients where midface fractures were associated with mandible fractures, the mandibular fractures were managed with titanium plates and screws because the strength of the bioresorbable system is not sufficient to provide stability for fracture mandible in adults as it is a load bearing bone. this was in accordance with observations of bryan bell r, cavusoglu t and wood gd. although the tensile strength of a biodegradable plate is less as compared to the titanium, it provides enough stability to the fracture segments in the non - load bearing areas so that healing can take place. no intra - operative late fracture occurred during this study which in accordance with study by enislidis g. out of the total number of 68 screws used for plate fixation, three screws broke, intra - operatively (4.41%) during screw tightening. this is more than the 1.87% incidence of screw fracture noted in the study conducted by enislidis g. this was probably due to the inadequate pre - tapping of screw holes and consequently excessive friction between the screw and the bone. bell and kindsfater recommended that these screws need only be"finger tight, " and care must be taken when placing them, especially in thin bones. if excessive torque is applied, the screw head will break. the angulation and the pressure at the time of drilling and tapping are important factors in this system. post - operative reduction and stability of the fracture segments was maintained in all the patients throughout the follow - up period which reflects upon the strength of the bioresorbable plates and screws which maintains reduction and provides enough stability for healing to take place. in literature, in two series, authors could achieve and maintain reduction ranging from acceptable to excellent in 100% of patients. centricity of occlusion was observed from immediate post - operative till 24 weeks and was maintained in all the patients which further indicated the stability provided by this system. pre - operative paraesthesia along the distribution of infra - orbital nerve, present in two patients of mid face fractures (le fort ii and le fort iii), was found to recover completely at four weeks post operative period which is in accordance to study by benoliel. at 12 weeks follow up, one patient of zygomatic fracture presented with pain and swelling at operative site. in this patient, plate was placed at zygomatic buttress approached through an intra - oral buccal vestibule incision. the infection at the intra - oral surgical site could be attributed to the wound contamination in the oral cavity. this finding goes in agreement with incidence of infection reported in literature, where one in ten patients had symptoms of mild infection according to a study conducted by wittwer. in the present study, none of the patient had wound dehiscence and plate exposure in the post - operative period. this is somewhat contradictory to incidence of wound dehiscence reported in literature which varies from 2% to as high as 6%. these results can be attributed to the tight closure of the incision obtained with meticulous suturing, adequate soft tissue cover over the implant site and the newer thinner implants. no local tissue reaction and foreign body type of reaction was noticed in the evaluation of surgical site during the follow up period which is contrary to study by wittwer. this difference is probably related to small sample size, compared to other series, which had larger number of patients. although, implant material could not be seen on radiographs, yet these were obtained in all patients, post - operatively, at predecided follow - up intervals. satisfactory reduction was seen in all patients, as judged with the position of fracture segments in 24 hrs post operative radiographs and compared with all subsequently obtained images. the undisturbed reduction was considered as a marker of the stable fixation provided by the bioresorbable system. the screw holes visible as radiolucencies on the radiograph, were also seen to maintain their position throughout the observed period of follow up. this again was considered as a marker for maintenance of reduction which is in accordance with ferretti and bell. rozema., studied the effect of these plates and screws when radiation is applied. they concluded that these can be regarded as tissue equivalents that do not interfere with the dose distribution of radiotherapy and can safely be used for fracture fixation of bone segments when post operative radiotherapy is needed. still, there are apprehensions to the use of these materials and they have not been able to find place in the armamentarium of the common practicing surgeon. their exorbitant cost and questionable ability to be used in load bearing areas remains a major hurdle. another significant factor of the limited use is the resistance by surgeons to modify their conventional, well experienced, treatment techniques. however, if these problems could be completely overcome and these implants could offer the same benefits as metallic implants, they would be able to find a greater place in craniofacial fracture fixation. since it was a short term study and sample size was very small, the possibility of late reaction to biodegradable implants needs to be evaluated and there is always a scope for long term follow up in such cases. biodegradable plates and screw system provided satisfactory stabilization when used as internal fixation for fractures of midface fractures in patients of all ages and mandibular fractures in early childhood.the biodegradable implant material did not evoke any significant immune response and is not toxic.the fixation system did not alter the healing process.the fixation technique was not difficult to master.a second surgical procedure was not carried out in any of the patients to remove the implant as the bioresorbable implant resorbed over a period of time. this is more beneficial in case of children where removal of a metallic implant is recommended even in asymptomatic cases because of growth restrictions.meticulous procedures along with special instruments provided by the manufacturer were mandatory to achieve the best results. the study had few shortcomings in terms of its small sample size and short term follow up. the encouraging results as obtained in this study seem to reinforce the use of biodegradable plates and screws in fixation of maxillofacial fractures. biodegradable plates and screw system provided satisfactory stabilization when used as internal fixation for fractures of midface fractures in patients of all ages and mandibular fractures in early childhood. the biodegradable implant material did not evoke any significant immune response and is not toxic. a second surgical procedure was not carried out in any of the patients to remove the implant as the bioresorbable implant resorbed over a period of time. this is more beneficial in case of children where removal of a metallic implant is recommended even in asymptomatic cases because of growth restrictions. meticulous procedures along with special instruments provided by the manufacturer were mandatory to achieve the best results. the study had few shortcomings in terms of its small sample size and short term follow up. the encouraging results as obtained in this study seem to reinforce the use of biodegradable plates and screws in fixation of maxillofacial fractures.	aims : the present study was undertaken to evaluate the efficacy of biodegradable plating system for fixation of maxillofacial fractures and to study the morbidity associated with the use of biodegradable plates and screws.materials and methods : this prospective study consisted of 10 patients with maxillofacial fractures requiring open reduction and internal fixation. fractures with infection, comminuted and pathological fractures were excluded. all were plated with biodegradable system (inion cps) using standard plating principles and observed for a total period of 24 weeks. characteristics of the fractures, ease of use of bioresorbable plate / screw system and post operative complications were assessed.results:of total 10 patients, eight patients were of midface fracture and two pediatric patients with mandibular fracture, with nine male and one female. the mean age was 32.8 years. out of 20 plates and 68 screws applied to the 10 fractures sites ; there were three incidences of screw breakage with no other intraoperative difficulties. paresthesia of the infraorbital nerve was present in two patients, and recovered completely in four weeks after surgery. fracture reduction was considered to be satisfactory in all cases. one patient developed postsurgical infection and was managed with oral antibiotics and analgesics.conclusions:favorable healing can be observed through the use of biodegradable plates and screws to stabilize selected midface fractures in patients of all ages, as well as mandible fractures in early childhood, however further studies with more sample size are required.
brown adipose tissue (bat) is a kind of fat tissue different from white adipose tissue (wat). in mammals, bat has its function mainly in nonshivering thermogenesis, responsible for control of body temperature and regulation of energy balance. bat is commonly found in human newborns and small mammals, and it is previously thought that there is only a vestigial amount of bat in adulthood. recently, f - fdg pet / ct demonstrated a relatively broad distribution of functional bat in adult human, mainly along the way of sympathetic nerve control, such as paravertebral and periadrenal regions [3, 4 ]. these functional bats have drawn our attention as a potential therapeutic target for inducing weight loss through its energy expenditure pathway [2, 5 ], because excision or denervation of interscapular bat can produce abnormal increase in the amounts of wat in those animals, meaning that they are becoming fatter. some other studies, though showing limited success, have already tried to activate bat by stimulating sns to control body weight, using, for instance, 3-adrenoceptor agonists cl-316243 or l-796568 [7, 8 ]. but cold stimulus has been proved to increase f - fdg uptake, an indicator of bat metabolism, in either animal or human studies [9, 10 ]. cold can activate sympathetic nerve system (sns) to excrete norepinephrine, and then the bat, which is rich in sympathetic nerve terminals, will produce more heat. genomic studies have indicated that the heat production is related to uncoupling protein 1 (ucp1), which is highly expressed in the mitochondria of bat cells and can convert glucose and free fatty acid into heat. for further evaluation of how the influence factors related to sns changed the bat metabolism, this study investigated several interventions to stimulate or inhibit sns and bat metabolism of mice, and under the application of micropet, bat metabolic statuses were clearly seen in vivo, showing different degree of fdg uptake under different interventions which could be semiquantitatively analyzed in real time. micropet served as a useful tool in monitoring bat function under different interventions and showed the potential to guide further study of the relationship between bat metabolism and obesity and diabetes. five - to - ten - wk female kunming mice (provided by beijing medical college animal breeding center) were enrolled in this study (mean body weight = 25 g). all animals were kept in the animal breading center of pumch with constant room temperature of 21c. control mice (n = 12) were examined under room temperature of 20 - 21c. three intervention groups were involved in this study (table 1). in physical intervention group, mice were exposed to cold (n = 6, 6c-7c, t = 1 h), cold and then warm (n = 6, 6c-7c, t = 1 h, and then 35c-36c, t = 1.5 h), or only warm (n = 6 and t = 1.5 h). for pharmacological stimulation group, under 21c mice received intraperitoneal injection of norepinephrine (ne, 0.4 mg / kg, n = 5), epinephrine (0.02 mg / kg, n = 5), isoprenaline (0.016 mg / kg, n = 6), respectively, or both cold and ne (ne : 0.04 mg / kg, n = 7). for pharmacological inhibition group, cold pre - exposure mice received intragastric administration of propranolol (13.2 mg / kg, n = 3) or saline (n = 3). control mice were kept in the examination room with constant temperature of 20c-21c without physical intervention or pharmacological stimulation. for cold intervention, we put the mice in a plastic box and kept them in a temperature - regulative refrigerator (bc-185fa, aucma, china) with a constant temperature of 6c-7c (measured by a thermometer in the refrigerator) for 1 h. for warm stimuli, we placed the mice in a plastic box laid above a power - regulative heating pad (ml-1.5 - 4, tianjin tester company, china) with the temperature of 35c-36c (measured by a thermometer in the box) for 1 h and kept warming the mouse for 0.5 h after fdg injection. norepinephrine (tianjin jinhui amino acids co.), epinephrine (beijing yongkang phar co.), and isoprenaline (shanghai harvest phar co.) were diluted with saline and injected into peritoneal cavity of mouse in the volume of 100 l (0.4 mg / kg), 100 l (0.02 mg / kg), and 30 l (0.016 mg / kg), respectively, 1 h before fdg injection. propranolol tablets (tianjin lisheng phar co.) were dissolved in saline to 1 mg / ml and administered intragastrically through a small animal lavage needle in the volume of 300 l (13.2 mg / kg) per mouse 1 h before fdg injection. to monitor the combination effect of cold stimuli and ne, we performed the cold stimuli protocol as stated and gave ne injection with 0.04 mg / kg 1 h before fdg injection. all of these pharmacological interventions were performed under room temperature of 21c, and after interventions, water and food were provided. after physical or pharmacological interventions, 3.7 mbq f - fdg was injected into peritoneal cavity for each mouse. anesthesia using summit as-1 - 000 - 7 animal anesthesia system (usa) was performed with 1.5% isoflurane (with o2 combination of 2 liter per minute) 40 min later. pet data acquisition procedure was performed under siemens inveon system 10 min after anesthesia when mice were totally unconscious. anesthesia was continued during the scanning process with the same flow rate through a facemask designed for small animal. all micropet images were studied by two researchers (wu and cheng) on a high - resolution computer screen applying asipro vm software., the two researchers compared the interscapular bat f - fdg uptake color intensity of mouse from different intervention groups. for semiquantitative analysis, 3d round regions of interests (roi) were placed carefully over the interscapular bat and brain on micropet images for each mouse, respectively. f - fdg uptake value was acquired in the form of nanocurie per cubic centimeter (nci / cc) automatically after placing rois. uptake ratio (r) of maximum interscapular bat uptake and mean brain uptake was calculated for each mouse and compared between different interventions. data were reported as means sd. in visual analysis, different interscapular bat f - fdg uptake intensity was found under different interventions as demonstrated by the micropet images (figure 1). cold and ne exposure caused the highest bat uptake, followed by cold exposure and the control, while propranolol lavage and warming up showed decreased bat uptake in cold pre - exposed mice. in semiquantitative analysis, bat f - fdg uptake was significantly higher under cold exposure compared to control mice under room temperature (r : 10.22 4.13 versus 4.08 1.32, p = 1.96 10) and highest with both cold exposure and ne stimulations (r : 15.64 5.58 versus 4.08 1.32, p = 2.19 10). a stimulation with only ne (r : 10.55 5.85), epinephrine (r : 4.57 0.86), or isoprenaline (r : 4.64 2.67) at room temperature all increased bat uptake compared with the controls (r : 4.08 1.32), but only ne showed significant difference compared with the controls (p =.002) (figure 2). for bat inhibition, warming could significantly reduce bat uptake in mice with cold pre - exposure (r : 2.13 0.43 versus 10.22 4.13, p =.001) and without cold pre - exposure (r : 2.48 0.88 versus 4.08 1.32, p =.017). in addition, propranolol could significantly reduce bat uptake in cold pre - exposed mice (r : 1.30 0.16 versus 3.09 0.90, p =.027) (figure 3). brown adipose tissue (bat) has been shedding new light on weight control of human recently, but few researches focused on monitoring bat metabolic function using molecular imaging. it is well known that bat has more sympathetic innervations than wat, which means that bat may be activated by stimulating sns. for future investigation of new methods to control obesity through activation of bat function, the present study aimed mainly at establishing a technology platform using micropet to evaluate the bat function through interventions with factors related to sns activation or inhibition. considering that the sns might probably be modulated by short - term interventions, the temperature control or drug administration was generally set as 1 h before fdg injection in this study. moreover, bat can bring in false positive results in pet images of human by showing increased radioactive uptake especially in the supraclavicular area [14, 15 ] ; many studies, therefore, try to find methods to inhibit bat uptake in humans, for instance, by keeping the patients warm or giving orally propranolol administration [1618 ]. our study provided new data concerning these two methods in inhibiting bat uptake that only 1.5 hour of warm intervention (1 h before and 0.5 hour after fdg injection) could quickly and markedly inhibit bat metabolism (r : 2.48 0.88 versus 4.08 1.32, p =.017), and compared with propranolol intervention, warming seemed to be more effective in reducing bat activity (p =.001). based on this result, in clinics, to prevent bat uptake in pet images, providing a warm environment for patients right before pet scan would be a more effective, convenient, and safer way than propranolol intake. cold stimulus was proved useful to increase bat metabolism in either animal or human studies [9, 10 ] because it can activated sympathetic nerve terminals surrounding the bat to excrete ne for more heat production. in our study, cold exposure also significantly increased bat activity (r : 10.22 4.13 versus 4.08 1.32, p = 1.96 10), and this effect was more obvious when combined with ne injection (p = 2.19 10), well confirming that bat metabolism was correlated with catecholamine system. moreover, cold effect could be quickly suppressed by warming (r : 2.13 0.43 versus 10.22 4.13, p =.001) or propranolol administration afterwards (r : 1.30 0.16 versus 3.09 0.90, p =.027), both of which were antagonistic to sympathetic - adrenal system, thus prohibiting bat activity. in our present study the -adrenoceptor agonists (ne, epinephrine and isoprenaline) all increased interscapular bat activity in mouse, but only ne showed a significant difference to controls (p =.002,.459, and.293, resp.). we think the dosage insufficiency of epinephrine and isoprenaline we gave mice may answer this question. with limited researches and few examples to follow, we gave a tentative dosage of epinephrine (0.02 mg / kg) and isoprenaline (0.016 mg / kg) to mice, which is about the clinical dosage for children (about half of adult dose), much smaller than the advised dose for mouse (about 9-fold clinical dosage for adults) based on surface area per kilogram. however, the dosage of ne (0.4 mg / kg) we gave mice was 11 times the dosage for human adult, so only ne was effective in activating bat metabolism. baba. used epinephrine in 5 mg / kg, and micropet scan showed significant bat uptake compared to controls ; therefore, we think increasing dosage in future study may overcome this problem. there are some limitations in this study. for instance, only female mice were enrolled in this study, and the mouse number in each intervention group was not big enough (n = 3 ~ 7). furthermore, some uncontrollable factors such as environmental noise and stimuli caused by experimental handling may potentially influence the nerve system of mouse and thus the activity of bat. in future studies in general, this preliminary study gives new prospective in studying the stimulation and inhibition of bat of mouse by a quantitative analyzing tool of micropet. by applying micropet, environment temperature control can significantly stimulate or alleviate bat uptake of f - fdg within 1 h in mice. stimulating sympathetic nerve system by norepinephrine can significantly increase the metabolism of bat within 1 h, while inhibiting sns by warming or propranolol can alleviate bat function. this preliminary study with f - fdg micropet warrants further investigations of the mechanism of bat and various methods of intervention according to clinical purposes. however, better optimization of different intervention conditions as well as some other methods for activating bat should be further explored and studied in the hope of future human application.	brown adipose tissue (bat) is emerging as a potential target for treating human obesity. it has been indicated that bat is rich in innervations of sympathetic nerve control. using 18f - fdg micropet imaging, this study aims at evaluating how factors related to sympathetic activation / inhibition changed bat metabolism of mice. bat 18f - fdg uptake were semiquantitatively evaluated in different groups of mice under temperature (cold or warm stimulus) or pharmacological interventions (norepinephrine, epinephrine, isoprenaline, or propranolol) and were compared with the corresponding controls. it was found that bat activation can be stimulated by cold exposure (p = 1.96 104), norepinephrine (p =.002), or both (p = 2.19 106) within an hour before 18f - fdg injection and can also be alleviated by warming up (p =.001) or propranolol lavage (p =.027). this preliminary study indicated that bat function could be evaluated by 18f - fdg pet imaging through short - term interventions, which paved the way for further investigation of the relationship between human obesity and bat dysfunction.
although intravenous thrombolysis has become a standard treatment for acute ischemic stroke in eligible patients, the recommendations for treating patients aged over 80 years are still a subject of debate. many studies have revealed a higher mortality rate and lower favorable outcome rate in patients aged over 80 years. in some studies, the rate of symptomatic intracerebral hemorrhage (ich) was higher in patients aged over 80 years while in others it was similar to that observed in younger patients. however, a large study and a meta - analysis have both recently shown that older patients benefit from treatment at least as much as younger patients. menon. assessed risk score for intracranial hemorrhage after intravenous thrombolytic treatment in 10,242 patients with acute ischemic stroke and found that asian race was one of the independent risk predictors. thrombolytic studies from some asian countries, such as taiwan and vietnam, have not included patients aged over 80 years. previous studies in thai patients showed that older age (70 years old) was inversely associated with early improvement. significantly higher rates of mortality and symptomatic ich were also found in patients aged 70 years or over as compared with younger patients after treatment with recombinant tissue - plasminogen activator (rtpa). however, whether treatment with thrombolysis will have a greater beneficial effect on outcomes than treatment without rtpa in thai elderly patients is unknown. the purpose of this study was to compare stroke outcomes in patients aged over 70 years treated with and without intravenous thrombolysis. the number of patients on each arm was calculated by the following formula (equal to 82) : p1=0.40, p0=0.20, power of the test 80%, confidence level 95%. one - hundred and five patients aged over 70 years with acute ischemic stroke treated with intravenous rtpa at thammasat university hospital, thailand, between june 2007 and january 2011 were identified from the thammasat stroke registry and were included in the study. one - hundred and five patients aged over 70 years who were seen from may 2010 to january 2011, and who were not treated with intravenous thrombolysis, were included as control. intravenous rtpa was prescribed for acute ischemic stroke patients within 3 h of onset if there were no contraindications. however, after the publication of the european cooperative acute stroke study iii (ecass iii), and the recommendations with regard to widening the time window for the treatment of acute ischemic stroke with intravenous rtpa issued by the american heart association / american stroke association, we extended the time window for treatment with rtpa to 4.5 h. most contraindications were the same as in the guidelines for treatment of acute ischemic stroke from the american heart association / american stroke association. however, older age (> 80 years old) was not an exclusion criterion. patients with high blood pressure (systolic blood pressure > 185 mmhg or diastolic blood pressure > 110 mmhg) were not excluded as long as blood pressure could be controlled by intravenous nicardipine (as defined by achieving target systolic blood pressure 8 points) 24 h after admission. the base - line characteristics of patients, including age, gender, cardiovascular risk factors, blood glucose at presentation, platelet count, prothrombin time, severity of stroke, and stroke subtypes were studied. the measured outcome variables of this study were early improvement at 24 h, symptomatic intracerebral hemorrhage, favorable outcome (mrs 0.1) and death at three months. patients demographics, vascular risk factors and measured outcome variables were compared between those who were treated with and those without intravenous thrombolysis ; an independent - samples t - test for continuous variables and the test for dichotomous variables were used. the data are presented as means for continuous variables and percentage (number) for dichotomous variables. this study was approved by the human ethics committee of the faculty of medicine, thammasat university, thailand. one - hundred and five patients aged over 70 years were not treated with intravenous rtpa during the study period ; of these, 49 patients presented within 4.5 h after stroke onset. the reasons for not prescribing rtpa were : delays in the intervention process, i.e. late presentation, delay in laboratory or ct process (n=14, 28.6%) ; excessively mild stroke severity, i.e. nihss under 4 (n=12, 24.5%) ; rapid improvement (n=5, 10.2%) ; abnormal ct findings i.e. hypodense lesion over one - third of the mca distribution (n=16, 32.7%) ; recent major surgery (n=1, 2%) ; and history of intracerebral hemorrhage, ich (n=1, 2%). follow - up ct or magnetic resonance imaging (mri) was performed in 48 patients who did not receive rtpa. the base - line characteristics of the patients with and those without rtpa treatment are presented in table 1. there were significant differences in base - line characteristics associated with older age (78 vs 76 ; p=0.03), milder stroke (10 vs 13 ; p=0.001), lower blood sugar at presentation (116 vs 135 ; p=0.026) and reduced frequency of cardioembolic stroke (25% vs 42% ; p=0.009) in patients without the rtpa treatment as compared with those with the rtpa treatment. however, there was no significant difference in the rates of favorable outcomes (41% vs 37% ; p=0.56) or mortality (25% vs 22% ; p=0.69) in association with a marginally higher rate of symptomatic intracerebral hemorrhage (4% vs 14% ; p=0.09) in those receiving rtpa treatment. a significantly higher rate of early improvement at 24 h was found in patients treated with rtpa (4% vs 23% ; p=0.001). univariate analysis and multiple logistical regression analysis were carried out to look for the factors associated with early improvement. only rtpa treatment was related to early improvement (or 14.1, 95% ci : 1.6 - 124.7) after adjustment for gender, stroke subtypes, hyperlipidemia, coronary artery disease, atrial fibrillation, antithrombotic treatment and symptomatic intracerebral hemorrhage. stroke outcomes at three months as measured by the modified rankin scale (mrs) are presented in figure 1. there were significant differences in some base - line characteristics in the subgroup of patients aged over 80 years, with a greater history of hypertension and higher mean age, milder stroke, and a reduced risk of cardioembolic stroke in patients not receiving rtpa treatment (table 1). patients (> 80 years old) treated with intravenous rtpa had a higher rate of favorable outcomes (40% vs 16% ; p=0.137) and a lower rate of mortality (22% vs 44% ; p=0.128) than patients not receiving rtpa treatment. the number of elderly patients is increasing in most countries and consequently strokes in the very old (> 80 years) subgroup occur more often ; approximately one - third of all stroke patients are in this subgroup. mortality rates and non - dependency were higher in patients aged over 80 years as compared with the younger patients. higher 1-month mortality rates were 31 - 34.6% in the very old patients (> 80 years) as compared with the younger patients (13.4 - 16.7%) ; one study reported a 1-year mortality rate of 51.6%. disability after stroke (mrs 3) was reported in 78% of the very old patients who survived. our study showed that the 3-month mortality rate was 44% in patients aged over 80 years who did not receive rtpa treatment. causes of high mortality may result from the reduced utilization of health care resources or the comorbidity and high proportion of atrial fibrillation reported in association with stroke. comorbidity, cognitive impairment, marital status, absence of caregivers and a lack of motivation that reduced the effectiveness of rehabilitation programs were potential contributors to the high level of disability observed in stroke survivors. thrombolytic treatment in very old patients with acute ischemic stroke is still a subject of debate. the national institute of neurological disorders and stroke (ninds) included all age ranges of the patients whereas the european cooperative acute stroke study (ecass) excluded patients aged over 80 years. thus, the european medicine evaluation agency has not approved thrombolysis with alteplase for use in patients aged over 80 years. a systemic review and meta - analysis included 13 studies comparing outcomes after intravenous thrombolysis among 764 elderly patients (age 80 years) and 2792 patients under 80 years of age. elderly patients achieved less favorable outcomes (or 0.49, 95% ci : 0.40 - 0.61) and showed higher mortality rates (or 2.77, 95% ci : 2.25 - 3.40) but not significantly higher symptomatic ich rates (or 1.31, 95% ci : 0.93 - 1.84) as compared with patients under 80 years old. ford. studied outcomes and symptomatic ich rates in 19,411 patients aged 80 years or under and 1831 patients aged over 80 years in the safe implementation of treatment in stroke - international stroke thrombolysis register (sits - istr). here patients over 80 years of age had a higher mortality rate (30% vs 12%, or 1.53, 95% ci : 1.43 - 1.65) and were less independent (35% vs 57%, or 0.73, 95% ci : 0.68 - 0.78). there was a nonsignificant increase in the symptomatic ich rate according to ninds criteria in the older subgroup (9.5% vs 7.8%, or 0.96, 95% ci : 0.87 - 1.06). mishra. assessed the effect of age in response to alteplase by comparing 23,334 patients who received thrombolysis from the sits - istr (during 2002 - 2009) with a control group made up of 6166 patients who participated in vista neuroprotection trials (during 1998 - 2007) and were not treated with rtpa. among the subgroup of patients aged over 80 years, 2235 and 1237 patients were treated with and without rtpa, respectively. as compared with a control subgroup, very old patients (> 80 years) treated with rtpa had a higher rate of favorable outcomes (mrs 0 - 2) (or 2.1, 95% ci : 1.7 - 2.5) and a marginally lower mortality rate (or 0.89, 95% ci : 0.76 - 1.0). the third international stroke trial (ist-3) compared the efficacy and safety of rtpa vs placebo, with a sample size of 3035 patients and including 1617 patients aged over 80 years. in a subgroup of patients aged over 80 years, the rate of favorable outcome was higher in those receiving rtpa treatment (27.3% vs 23.5%, or 1.35, 95% ci : 0.97 - 1.88) and this did not differ significantly from the younger patients. our study showed non - significant differences in the rates of favorable outcomes, symptomatic intracerebral hemorrhages and mortality in patients aged over 70 years with or without thrombolytic treatment. however, there were significant differences in several base - line characteristics between the groups. older age, severe stroke, high blood glucose, cardioembolic stroke had been reported to be the independent factors of poor outcomes and death. thus, it is not possible to conclude that thrombolytic treatment is ineffective in aging patients. differences in favorable outcomes and death between those with and without rtpa treatment were obvious in subgroups of patients aged over 80 years. patients with the rtpa treatment had a higher rate of favorable outcomes (40% vs 16% ; p=0.137) and a lower rate of mortality (22% vs 44% ; p=0.128) compared with those without the rtpa treatment. however, the difference did not reach statistical significance, which may largely be due to the small number of patients in this subgroup. a transcranial doppler (tcd) study in patients treated with rtpa found that the timing of arterial recanalization, as determined by tcd, correlated with early clinical recovery from stroke. several studies reported a similar proportion of successful recanalization in patients aged over 80 years to that achieved in younger patients. our study showed that early improvement within 24 h after stroke onset was found more frequently in patients who received the rtpa treatment, which may represent a higher rate of recanalization. firstly, this was a retrospective review of sequential cases and was not a randomized controlled study. non - thrombolytic patients (> 70 years of age) were classified by the duration of time required to reach similar numbers to those achieved in patients treated with rtpa. the differences in stroke severity between the case and control groups may represent more rapid stroke awareness in patients with more severe stroke, as this subgroup of patients was more likely to receive thrombolytic treatment. however, for the subgroup of the patients aged over 80 years, characteristics were fairly similar. second, a follow - up brain ct or mri was not performed in some of the patients who did not receive rtpa treatment ; therefore, the rate of asymptomatic intracerebral hemorrhage may be underestimated. this is the first study in thailand comparing stroke outcomes in aging patients with and without rtpa treatment. the study showed that the very old patients receiving rtpa did not have poorer outcomes. further randomized controlled studies are still needed to confirm the suggested benefit of thrombolysis in aging asian patients. in conclusion, in subgroups of patients aged over 80 years, this retrospective review revealed that there was a trend of higher rate of favorable outcome and lower mortality in patients receiving rtpa treatment.	higher mortality was found in very old patients with acute ischemic stroke treated with intravenous recombinant tissue - plasminogen activator (rtpa) as compared to younger patients. the benefit of thrombolytic treatment in this particular subgroup is still a subject of debate. the purpose of this study was to compare stroke outcomes in thai patients aged over 70 years treated with and without intravenous rtpa. this was a retrospective review of sequential cases and was not a randomized controlled study. one - hundred and five patients with acute ischemic stroke aged over 70 years who were treated with intravenous rtpa and 105 patients without rtpa treatment (control group) were included in the study. patients base - line characteristics and study outcomes of interest were compared. there were significant differences in the base - line characteristics of the two groups. however, for the subgroup of patients aged over 80 years, these characteristics were similar. those who were treated with intravenous rtpa had a higher rate of favorable outcomes (40% vs 16% ; p=0.137) and a lower rate of mortality (22% vs 44% ; p=0.128) than patients who did not receive rtpa treatment. in well - matched subgroups of patients aged over 80 years, our retrospective review revealed there was a trend of a higher rate of favorable outcome and lower mortality in patients receiving rtpa treatment. more study is needed to further confirm the suggested benefit of thrombolysis in asian octogenarian acute stroke patients.
serum samples were obtained during a nationwide influenza vaccine serologic study in taiwan that started in 2006. children (75 years of age) were recruited. serum samples were obtained immediately before and 3 weeks after intramuscular injection with 1 dose of nonadjuvanted, trivalent, inactivated influenza vaccine formulated for the 200809 northern hemisphere winter season (samples were obtained from some participants > 75 years of age before and after receiving 1 dose of the vaccine formulated for the 200708 winter season). microneutralization (mn) and hemagglutination inhibition (hi) assays were performed according to the world health organization manual on animal influenza diagnosis and surveillance (4). using these assays with 0.75% guinea pig erythrocytes, we assayed samples for antibodies against a / california/07/2009 (h1n1) virus. the seroprotection rate was defined as the percentage of serum titers > 40 by hi or titers > 160 by mn. the seroconversion rate was defined as the percentage of vaccine recipients whose serum hi titers or mn titers increased by at least 4-fold after vaccination. stata software version 8.2 (statacorp lp, college station, tx, usa) was used for analysis. a total of 176 participants (40 children, 36 adults, 50 older adults, and 50 elderly adults) were enrolled (table). few or no preexisting cross - reactive antibodies against pandemic (h1n1) 2009 virus were detected by hi assay in samples from children (prevaccination seroprotection rate 0%). after vaccination, seroprotection rates and geometric mean titers measured by hi assay were essentially unchanged but increased significantly in the 3 adult groups when measured by mn assay (p 80. however, itoh. (6) reported that blood donors from japan who were born after 1920 had almost no appreciable neutralizing antibodies against this virus. because the hemagglutinin gene of pandemic (h1n1) 2009 virus is similar to that of viruses that circulated in humans during 19181957 (7), itoh. suggested that pandemic (h1n1) 2009 virus is antigenically divergent from human influenza viruses (h1n1) that circulated during the 1920s1950s. (5), who suggested that human influenza virus (h1n1) circulating in taiwan after 1920 resembled the 1918 pandemic virus (h1n1) and pandemic (h1n1) 2009 virus and could lead to cross - protection against the current virus. furthermore, unlike the situation in the united states, there was no program for vaccination against the 1976 swine influenza virus (a / nj/76) in taiwan. the results of our study also explain why only 7% of patients hospitalized for pandemic (h1n1) 2009 virus in taiwan were > 65 years of age (8). similar epidemiologic observations have been reported in the united states (3) and new zealand (9). whether elderly persons still have cross - reactivity several decades after exposure to 1918 (h1n1) virus is unknown. original antigenic sin has been described in relation to influenza virus, dengue virus, hiv, and several other viruses (1012). for persons > 65 years of age, the 1918 (h1n1) virus is likely the first influenza virus to which they were exposed, and their antibody response should have increased in subsequent years. regression analysis showed that older persons with high hi titers were more likely than younger adults to have higher mn titers. because the hi assay detects only antibodies against hemagglutinin, the mn assay provides more information about the level of protective antibodies against influenza viruses. as a person ages, production of antibodies against hemagglutinin or other components of influenza virus should protect against potential infections. although a titer of 40 by hi is accepted as a cutoff value for seroprotection, a consensus for protective titers by mn is lacking (5,13). we suggest that mn is probably more sensitive than hi for evaluating neutralizing antibodies against pandemic (h1n1) 2009 virus.	we studied preexisting immunity to pandemic (h1n1) 2009 virus in persons in taiwan. a total of 18 (36%) of 50 elderly adults in taiwan born before 1935 had protective antibodies against currently circulating pandemic (h1n1) 2009 virus. seasonal influenza vaccines induced antibodies that did not protect against pandemic (h1n1) 2009 virus.
nephrotic syndrome (ns) is a minimal - change disease (mcd) in about 90% of children younger than 10 years and in about 5070% of older children. occasionally, however, ns is induced by self - limited renal diseases, such as infection - related glomerulonephritis (irgn). irgn is usually characterized by hematuria, proteinuria, edema, and often by hypertension and a mild degree of acute renal injury. when these characteristic findings are not present, misdiagnosis may be made without histological study. here, we report a rare case of ns due to irgn without hematuria and hypertension. a 14-year - old boy was referred to our hospital because of a 5-day history of low - grade fever, nausea, weight gain and recent leg edema. he had been in his usual health until approximately 1 week before admission and had no symptoms suggestive of upper respiratory or skin infection. six months before admission, his urinalysis on school examination was normal. on admission, his blood pressure was 118/66 mm hg, his pulse 66/min, and his temperature 36.7c. he weighed 63 kg and had experienced 7 kg of weight gain in the preceding week. the hematocrit was 41.6%, hemoglobin concentration 15.1 g / dl, platelet count 248,000/mm, and leukocyte count 8,860/mm. the serum urea nitrogen level was 10.1 mg / dl, the creatinine level 1.03 mg / dl, uric acid 4.5 mg / dl, total cholesterol 199 mg / dl, total protein 5.2 g / dl, and albumin 2.4 g / dl. the c - reactive protein level was 0.22 mg / dl, igg 985 mg / dl (normal range : 8701,700), iga 134 mg / dl (80140), and igm 116 mg / dl (34220). the total complement level was 27.0 iu / l (3045), c3 91 mg / dl (80140), c4 9.5 mg / dl (1130), and c1q < 1.5 g / ml. antistreptolysin o was 164 iu / l (< 166). hepatitis b virus surface antigen, hepatitis c virus antibody, human immunodeficiency virus antibody, antinuclear antibody, anti - neutrophil cytoplasmic antibodies, and cryoglobulin were all negative. renal ultrasound and computed tomography showed normal kidneys. on the 2nd hospital day, kidney biopsy was performed to investigate the cause of ns. the patient 's renal biopsy showed mild endocapillary hypercellularity, mainly of mononuclear cells, with several neutrophilic leukocytes (fig. mitotic figures and several apoptotic changes of the tubular epithelial cells were observed (fig. mild cellular infiltrations around blood vessels, which were not clear enough to be conclusively diagnosed as vasculitis, were observed, but arteriosclerosis was not found. by immunofluorescent study, prominent fine granular igg (2 +) and weakly positive c3 (1 +) deposits were found along the capillary walls and in the mesangium (fig. 2), but iga, igm, c4, and c1q deposits were very modest in the same pattern. electron - microscopically, many relatively small subepithelial hump - like electron - dense deposits were observed. a few subendothelial, intramembranous and mesangial deposits were also observed (fig. electron - microscopic findings were roughly compatible with the early phase of post - streptococcal acute glomerulonephritis (psagn). from the clinical and histopathological findings, the diagnosis of ns due to infection - related endocapillary proliferative glomerulonephritis (engn) was made, and we observed the patient with just rest and dietary cure. on the 11th hospital day, serum complement levels and renal function returned to normal : the total complement level was 43 iu / ml, c3 134 mg / dl, c4 20.9 mg / dl, and the serum creatinine level was 0.71 mg / dl. on the 14th hospital day, his edema subsided and his proteinuria decreased to 1.0 g / day. on the 20th hospital day, he was discharged without any subjective symptoms. to find the causative organism of his renal lesion the levels of antibodies to the following pathogens were measured : parvovirus b19 (b19), herpes simplex virus, varicella zoster virus, parainfluenza virus, coxsackievirus a and b, cytomegalovirus, measles virus, rubella virus, mumps virus, and epstein - barr virus, as well as fecal adenovirus antigen, which has been reported as the other responsible organism of post - infectious glomerulonephritis (pign). nasal swabs for influenza type a and b, and serum antibody to mycoplasma and legionella were negative. we investigated his serum using human parechovirus and enterovirus polymerase chain reaction primer sets, which could detect human parechovirus and enterovirus (including poliovirus, coxsackievirus a and b, echovirus, and rhinovirus). immunofluorescence staining of the renal biopsy tissues with anti - b19, anti - antigen nephritis - associated plasmin receptor antibody and anti - streptococcal pyrogenic exotoxin b were all negative. from the above findings on the 2nd hospital day, kidney biopsy was performed to investigate the cause of ns. the patient 's renal biopsy showed mild endocapillary hypercellularity, mainly of mononuclear cells, with several neutrophilic leukocytes (fig. mitotic figures and several apoptotic changes of the tubular epithelial cells were observed (fig. mild cellular infiltrations around blood vessels, which were not clear enough to be conclusively diagnosed as vasculitis, were observed, but arteriosclerosis was not found. by immunofluorescent study, prominent fine granular igg (2 +) and weakly positive c3 (1 +) deposits were found along the capillary walls and in the mesangium (fig. 2), but iga, igm, c4, and c1q deposits were very modest in the same pattern. electron - microscopically, many relatively small subepithelial hump - like electron - dense deposits were observed. electron - microscopic findings were roughly compatible with the early phase of post - streptococcal acute glomerulonephritis (psagn). from the clinical and histopathological findings, the diagnosis of ns due to infection - related endocapillary proliferative glomerulonephritis (engn) was made, and we observed the patient with just rest and dietary cure. on the 11th hospital day, serum complement levels and renal function returned to normal : the total complement level was 43 iu / ml, c3 134 mg / dl, c4 20.9 mg / dl, and the serum creatinine level was 0.71 mg / dl. on the 14th hospital day, his edema subsided and his proteinuria decreased to 1.0 g / day. on the 20th hospital day, he was discharged without any subjective symptoms. to find the causative organism of his renal lesion the levels of antibodies to the following pathogens were measured : parvovirus b19 (b19), herpes simplex virus, varicella zoster virus, parainfluenza virus, coxsackievirus a and b, cytomegalovirus, measles virus, rubella virus, mumps virus, and epstein - barr virus, as well as fecal adenovirus antigen, which has been reported as the other responsible organism of post - infectious glomerulonephritis (pign). nasal swabs for influenza type a and b, and serum antibody to mycoplasma and legionella were negative. we investigated his serum using human parechovirus and enterovirus polymerase chain reaction primer sets, which could detect human parechovirus and enterovirus (including poliovirus, coxsackievirus a and b, echovirus, and rhinovirus). immunofluorescence staining of the renal biopsy tissues with anti - b19, anti - antigen nephritis - associated plasmin receptor antibody and anti - streptococcal pyrogenic exotoxin b were all negative. from the above findings based on the abrupt onset of nephrotic - range proteinuria, mild hypocomplementemia, its benign clinical course, and renal pathological findings, our patient was diagnosed as having ns due to irgn. the diagnostic criteria for irgn are : (1) clinical or laboratory evidence of infection preceding or at the onset of glomerulonephritis ; (2) depressed serum complement ; (3) endocapillary proliferation and exudative glomerulonephritis ; (4) c3-dominant or co - dominant glomerular immunofluorescence staining, and (5) hump - shaped subepithelial deposits on electron microscopy. at least three of the above criteria were required for the diagnosis of irgn, and our patient almost fulfilled the criteria. although histological findings of the present case showed diffuse engn, the clinical characteristic findings of irgn, such as hematuria, obvious low serum complement level and hypertension, were not recognized. we could not detect the causative organism of his irgn from the laboratory and renal histopathological findings. the nomenclature for glomerulonephritis associated with infections has become confusing. in a recent study, glassock. proposed a nomenclature for glomerulonephritis associated with infections, which were classified into two, pign and irgn. our patient exhibited some clinical findings, such as fever, weight gain and leg edema, simultaneously, and there was no latent period. such a clinical course suggested that our patient had irgn, which was directly related to the infection itself, but without any evidence of an ongoing infection. another possibility must be considered, i.e., that the causative agent could directly damage the kidney tissue, followed by immunocomplex - type glomerulonephritis, as suggested in parvovirus b19 irgn., engn is reported to be recognized in only 0.5% of primary glomerular disease except iga nephropathy. in addition, ns due to psagn is uncommon, occurring in approximately 0.4% of patients. psagn is the major disease among glomerulonephritis associated with infections, and the most typical histopathological finding is engn. in the case of psagn, sudden onset of edema together with gross hematuria and hypertension is a common finding and correlates with volume expansion due to sodium and fluid retention. alternative complementary pathways are activated in psagn ; more than 90% of patients with psagn have low levels of total complement and c3, and normal or mildly depressed c4 levels. our patient exhibited abrupt onset of edema, normal anti - antistreptolysin o titer, hypoalbuminemia and high - selectivity proteinuria, but no hematuria or hypertension. our patient was a 14-year - old nephrotic boy with mild renal dysfunction and mild hypocomplementemia. these abnormalities were minor ; however, renal biopsy was performed to determine the accurate treatment plan. from the renal histopathological findings however, without renal histopathological examination, our patient could have been diagnosed with corticosteroid - sensitive ns. glomerulonephritis associated with infection is commonly triggered by streptococci, although many other organisms, including bacterial, viral, parasitic, rickettsial and fungal infections, can also cause the condition [8, 9 ]. in spite of the detailed examinations acute glomerulonephritis associated with several viral infections, such as b19, hepatitis a virus, measles, yellow fever and epstein - barr virus, was reported [8, 9 ]. in such cases, extrarenal specific symptoms associated with viruses (such as rash, liver injuries, lymph adenopathy, and arthralgia) were present. however, our patient showed no extrarenal findings. recently, various renal findings associated with b19 infection have been reported. acute nephritic syndrome with hypocomplementemia often following a prodrome of fever, rash, and arthritis is common, and nephrotic - range proteinuria is seen as well. using renal histology, immune complex - mediated acute engn hypercellularity composed of mainly mononuclear cells and a few neutrophils, mesangiolytic changes, and mitosis of endothelial cell were observed. such findings resemble those of irgn following b19 infection, although from the laboratory and renal immunohistopathological findings, b19 infections were not probable. using frozen serum, we examined human parechovirus, which could cause injury of endothelial cells as well as b19 ; however, its preceding infection was not proven. recently, several cases of iga - dominant acute pign were reported. in our patient, the immunofluorescence pattern was obviously igg dominant, and it was different from iga - dominant pign. we speculate that another unrecognized pathogen, perhaps a virus, could cause ns due to irgn. therefore, it may not be completely denied that cases who have self - limited renal diseases received corticosteroid therapy. corticosteroids have well - recognized and potentially serious adverse effects such as poor growth, obesity, hypertension, diabetes mellitus, osteoporosis and behavioral disturbances, especially in childhood. as shown here, in childhood, ns could be induced by irgn, and clinical and laboratory findings may resemble those of mcd. when an acute nephrotic child shows atypical clinical or laboratory findings, such as very transient hypocomplementemia, acute renal injury, and so on, irgn should be considered as the differential diagnosis.	nephrotic syndrome without hematuria due to infection - related glomerulonephritis is uncommon. the present report describes a case of nephrotic syndrome due to infection - related glomerulonephritis without hematuria and hypertension in an older child. a 14-year - old boy was referred to our hospital because of a 5-day history of fever, nausea, weight gain and recent leg edema without hypertension. laboratory data showed nephrotic - range proteinuria, hypoalbuminemia, mild hypocomplementemia and acute renal injury without hematuria. although, due to the clinical presentation, minimal - change nephrotic syndrome was mostly suspected, a renal biopsy showed endocapillary hypercellularity mainly of mononuclear cells with segmental mesangiolytic changes. fine granular igg and c3 deposits were noted by an immunofluorescent study ; many relatively small electron - dense deposits were observed electron - microscopically. these findings led to the diagnosis of nephrotic syndrome due to infection - related endocapillary proliferative glomerulonephritis, although the causative organism of his nephritis was not detected. he recovered with rest and dietary cure. when we examine an acute nephrotic child, infection - related glomerulonephritis should be considered as the differential diagnosis to avoid unnecessary use of corticosteroids.
a 42-year - old man (patient a) was admitted on july 31, 2000, for a fever of 2 weeks duration. he had had diabetes mellitus for 20 years and alcoholism for > 10 years. physical examination and abdominal echography showed hepatomegaly and a huge abscess (12 cm x 10 cm x 8 cm) over the right lobe of the liver. abscess aspirate culture yielded k. pneumoniae with two colonial morphotypes (isolates on trypticase soy agar plates supplemented with 5% sheep blood [bbl microbiology systems, cockeysville, md ] after 24 hours of incubation in ambient air) (figure 1) and two resistotypes (by the routine disk diffusion method). one (isolate a1) had mucoid, opaque colonies and was resistant to ampicillin but susceptible to cefazolin and cefoxitin, and the other (isolate a2) had nonmucoid, white colonies and was resistant to ampicillin, cefazolin, and cefoxitin. both isolates were susceptible to amoxicillin - clavulanate and cefotaxime. the antibiotic was changed to imipenem (500 mg every 6 hours) on the 11th hospital day. imipenem was continued for a total of 24 days, followed by ciprofloxacin (750 mg every 12 hours) for 3 weeks. a follow - up echography 4 months after antibiotic treatment ended showed that the abscess had disappeared. colonial morphology of klebsiella pneumoniae grown on a primary isolation plate (trypticase soy agar supplemented with 5% sheep blood) from the abscess aspirate of patient a after 24 hours of incubation. a 66-year - old man (patient b) with diabetes mellitus was admitted on august 4, 2000, with fever and hiccups of 4 days duration. laboratory tests showed elevated levels of alkaline phosphatase (585 u / l ; reference range 66 - 220 u / l) and gamma - glutamyltranspeptidase (301 u / l ; reference range < 52 u / l) but normal levels of aspartate and alanine aminotransferases. abdominal echography and computed tomography revealed an abscess (6 cm x 4 cm x 4 cm) over the right lobe of the liver and a gallbladder stone. two isolates (isolates b1 and b2) of k. pneumoniae with different colonial morphotypes and resistotypes (as in isolates a1 and a2, respectively, from patient a) were found in one aspirate culture, and two isolates (isolates b3 and b4) of the same species with different phenotypes (as in isolates b1 and b2, respectively) were recovered from one set of blood cultures, performed at admission. cefoxitin was discontinued, and cefotaxime (2 g every 6 hours) was administered. the patient received cefotaxime for 15 days, followed by oral cefixime (200 mg every 12 hours) for 7 weeks. follow - up echography showed the abscess had disappeared. in vitro susceptibilities of 14 antimicrobial agents for these isolates biotyping of thee isolates was performed with the api id32 gn system (biomerieux, marcy i'etoile, france). random amplified polymorphic dna (rapd) patterns of the six isolates were determined by means of arbitrarily primed polymerase chain reaction, as described in our previous report (8). a total of four primers were used : m13 (5'-ttatgtaaaacgacggccagt-3 '), oph2, opa3, and opa9 (operon technologies, inc., alameda, ca). pulsotypes of these isolates were determined by pulsed - field gel electrophoresis ; plasmid analysis and conjugative experiment were performed as described (3). for comparisons, molecular typing of an additional two k. pneumoniae the cefoxitin- and cefuroxime - resistant isolates (isolates a2, b2, and b4) showed two- to four - fold higher mics of cefotaxime and ceftriaxone (mics 0.5 - 1 g / ml), ceftazidime (mics 1 g / ml), flomoxef (mics 0.5 - 2 g / ml), ticarcillin - clavulanate (mics 8 g / ml), piperacillin - tazobactam (mics 4 - 8 g / ml), cefepime (mics 0.5 - 1 g / ml), cefpirome (mics 0.12 - 1 g / ml), imipenem (mics 2 g / ml), and meropenem (mics 0.12 g / ml) than those of the cefoxitin - susceptible isolates (isolates a1, b1, and b3). all isolates were susceptible to the above antibiotics and aminoglycosides (gentamicin and amikacin). as shown in the table, the identity of biotypes, rapd patterns with four primers, and pulsotypes (figure 2) was clearly demonstrated for the isolates with two different phenotypes from each patient, suggesting that they belonged to a single clone in each patient (clone 1, isolates a1 and a2 ; clone 2, isolates b1 to b4). different clones isolated from two patients seen within 1 week indicate that no outbreak occurred. isolates a1 and a2 both had two plasmids (50 kb and 5 kb). only one plasmid (30 kb) was found in each of the four isolates (b1 to b4) of patient 2. amp, ampicillin ; cz, cefazolin ; fox, cefoxitin, cxm, cefuroxime, ctx, cefotaxime, cro, ceftriaxone. pulsed - field gel electrophoresis profiles of xbai - digested genomic dnas from eight klebsiella pneumoniae isolates. lanes 1 and 2, profiles for isolates a1 and a2 (from patient a) ; lines 3 to 6, profiles for isolates b1 to b4 (from patient b), respectively ; and lanes 7 and 8, isolates of k. pneumoniae from two other patients used as control strains. although infection caused by a single clone of one bacterial species that simultaneously possessed two obviously different colonial morphotypes has been noted previously (8), infection caused by a single clone of k pneumoniae exhibiting two discrete colonial morphotypes and resistotypes has never been reported. primary liver abscess due to k. pneumoniae having resistance to second - generation cephalosporins (mic up to 128 g / ml for isolate b2 and 256 g / ml for isolate b4 in patient b) and higher mics of third - generation cephalosporins (mics up to 1 g / ml for isolate a2 in patient a) has also not been previously reported. we believe that this decreased in vitro susceptibility contributed to the unsatisfactory response to treatment with these agents. the genetic basis for the resistance to cephalosporins and for the mucoid material synthesis by some gram - negative bacteria may be chromosomally determined or dependent on the acquisition of a specific plasmid or phage (5,9,10). the resistant and mucoid material synthesis may also be regulated by the environment in which the bacteria grow (5,9,10). the association of a specific plasmid with the presence of mucoid phenotype was not found in our isolates because both mucoid and nonmucoid strains of the two clones of k. pneumoniae had identical plasmid profiles. the mechanisms of the coexistence of two phenotypically distinct isolates within a single clone k. pneumoniae in abscess fluid, blood, or both should be investigated. the mechanism of cefoxitin resistance in our isolates was unclear because of the failure of transferability of the plasmids existing in our isolates to the susceptible recipient. whether this resistance was chromosome mediated or caused by other mechanisms these two cases suggest that primary liver abscess caused by multiresistant strains of k. pneumoniae in diabetic patients may be an emerging problem in taiwan. the belief that only strains of k. pneumoniae that are susceptible to cephalosporins cause primary liver abscess has now been disproved.	two diabetic patients with primary liver abscess, who initially responded unsatisfactorily to intravenous ceftriaxone or cefoxitin treatment and had abscess drainage, were found to be infected with a single clone of klebsiella pneumoniae with two different colonial morphotypes and resistotypes. primary liver abscess caused by second - generation cephalosporin - resistant k. pneumoniae strains may be an emerging problem in taiwan.
the progression of hiv disease involves various stages characterized by changes in lymphocyte subsets and viral load. after initial infection, an acute viremic stage occurs, which is accompanied by a rapid decline in the number of cd4 + t lymphocytes and a slow increase in cd8 counts. during the clinical latency phase, cd4 counts gradually decline, and hiv load is lower than during the acute stage, although virus is still present. finally, the late stage of hiv disease is reached, where cd4 + t - cells fall severely, and the number of viral particles surge. in the case of the hiv infected patient, emphasis is placed on cd4 counts because when the number of cd4 + t - cells falls below a critical level, prophylaxis against opportunistic infection is initiated. flow cytometry is not useful for samples that have been frozen or stored, therefore retrospective studies involving hiv - infected patients ' samples are unable to view the data in the light of information regarding the immune status at the time the samples were frozen. we also perceived that studies of hiv-1 status, particularly in infants, could be facilitated by a technique using smaller amounts of blood, such as that from a heelstick or finger - prick. quantitative methods for reverse transcriptase polymerase chain reaction (rt - pcr) are rapidly surpassing all other methods of quantifying mrna levels. we therefore set out to develop an rt - pcr based method to quantitate cd4 and cd8 mrna in the hopes that this could be used to predict cell counts. although it has long been known that hiv-1 down - regulates cd4 cell surface expression, the loss of cd4 is not complete and the hiv - infection does not reduce cd4 mrna levels. thus, a method based on quantitating cd4 and cd8 mrna could provide a means of assessing immune status without requiring repetitive and large blood draws, and could be exquisitely sensitive. this method may also be useful in assessing the immune status of patients retrospectively from archived or frozen samples. qc - rt - pcr, and related methodologies, however, did not prove useful as substitutes for assessment of cd4 and cd8 cell numbers in hiv - infected persons. samples were acquired from special hematology, louisiana state university medical center, after exempt status was obtained from tulane institutional review board. the relationship between cd4 and cd8 cell number by flow cytometry, and the amount of mrna as quantitated by qc - rt - pcr, was determined. flow cytometry was performed as previously described along with white blood cell and differential counts, including percent lymphocytes, in order to determine numerical values. intact mrna was successfully extracted from as little as 400 l of whole blood, comparable amounts to that obtained from infants via heelstick. this mrna was used to quantify the amount of cd4, cd8, as well as gapdh, mrna that is being transcribed in t lymphocytes within the blood. the corrected quantity of cd4 and cd8 mrna as determined by qc - rt - pcr and the corresponding cd4 and cd8 counts of that patient as determined by flow cytometry are depicted in table 1. the patients with unknown status all have inverted cd4:cd8 cell ratios and are therefore likely to be hiv infected (the majority of the samples sent for flow cytometry are hiv - positive). the relationship between the mrna and cell number for cd4 and cd8 are depicted (fig. the relationship between cell counts and the quantity of mrna as determined by qc - rt - pcr. (a) cd4, and (b) cd8. the hiv status of each sample is depicted. represents hiv positive status, represents hiv negative status, and reepresents unknown status. our results indicate that there is no correlation between cd4 and cd8 mrna levels and numbers of cells expressing these proteins on their surface. the formation of cd4-gp160 complexes in intracellular compartments may contribute to cell surface cd4 down - modulation during hiv-1 structural protein expression. these and other mechanisms may explain the lack of correlation between message and surface expression in hiv - positive individuals. hiv infection may also interfere with the translation or surface expression of cd8, although it is not used as a viral receptor. whatever the mechanism, no correlation between cd4 and cd8 cell counts and mrna levels of cd4 and cd8 could be determined by qc - rt - pcr. there was also no correlation between cd4 and cd8 mrna levels and the number of cells expressing these proteins on their surface. given the magnitude of the variations observed by qc - rt - pcr it is unlikely that other methods to quantitate these mrnas, e.g. northern blotting or real - time pcr, would reveal a correlation either. qc - rt - pcr and related techniques such as real - time pcr are not useful as substitutes for assessment of cd4 and cd8 cell status in hiv - infection by flow cytometry. the strategy for development of competitor templates for quantitative - competitive rt - pcr (qc - rt - pcr) was similar to that of lipman and coworkers. oligonucleotide primers (clontech, palo alto, ca) were chosen that span introns of cd4 and cd8 genes in order to avoid amplification of cellular dna contaminating rna preparations. products are generated from cdna and deletional mutants are constructed by restriction endonuclease digestion and religation. the deletional mutants were then subcloned using the ta cloning vector pcr2.1 (invitrogen, carlsbad, ca), and sequenced for confirmation. the method used to generate a competitor for cd8 was slightly different than previously reported methods. the wild type pcr product was inserted into the ta cloning vector before digesting with bbsi, an enzyme with no recognition sites inside the vector and only one site in the insert. the linearized plasmid was gel purified, the free ends of the plasmid were then shortened using bal31 nuclease. the ends were filled using the klenow fragment of dna polymerase, and then religated. the competitor template for glyceraldehyde phosphate dehydrogenase (gapdh), used to control the amount of rna in each extraction, have already been constructed by lipman., (1994), and were kindly provided by dr. known concentrations of the competitor cdna was amplified in the same reaction tube as unknown amounts of sample derived rt - cdna. total rna is extracted from 400 l to 1 ml of whole blood using a similar method to zhang and yunis. first strand cdna is synthesized using oligo dt primers and maloney murine leukemia virus reverse transcriptase (promega, madison, wi), diluted 1 to 3, then 3 l is used as template in a 25 l pcr. the remainder of the reactions were made up by 5 l pcr buffer (gibco brl, grand island, ny), 0.25 g of each primer, 6 mm mgc12 in each cd4 reaction and 4 mm mgc12 for cd8 reactions, 2 mm total dntp 's, and 0.5 u of taq polymerase. for cd4 and cd8 reaction conditions were : 94 for 3 mins, then 33 cycles of 94 for 45 secs, 55 for 45 secs, and 72 for 2 mins on a perkin elmer thermocycler. 33 cycles was chosen as this was the largest number still within the linear range of the pcr (data not shown). in each assay we performed at least six different reactions for each sample using increasing amounts of competitor dna. the amounts of sample were quantitated by extrapolating across at least three reactions in which approximately equal amounts of sample and competitor products were produced.. quantity of sample derived mrna can be determined when the intensity of its pcr band is equal to that of the competitor 's upon co - amplification. staining with ethidium bromide the standard curve plots the log cdna / competitor band intensity (as determined by analysis of pict files with nih image) on the x - axis versus the log of incremental dilutions of competitor dna on the y - axis. solving for the antilog of x when y = 0 will give the concentration of unknown amount of dna that of cd4, cd8 or gapdh. the success of the rna extraction and reverse transcription is determined by quantification of gapdh mrna by qc - rt - pcr. each value for quantity of cd4 or cd8 the strategy for development of competitor templates for quantitative - competitive rt - pcr (qc - rt - pcr) was similar to that of lipman and coworkers. oligonucleotide primers (clontech, palo alto, ca) were chosen that span introns of cd4 and cd8 genes in order to avoid amplification of cellular dna contaminating rna preparations. products are generated from cdna and deletional mutants are constructed by restriction endonuclease digestion and religation. the deletional mutants were then subcloned using the ta cloning vector pcr2.1 (invitrogen, carlsbad, ca), and sequenced for confirmation. the method used to generate a competitor for cd8 was slightly different than previously reported methods. the wild type pcr product was inserted into the ta cloning vector before digesting with bbsi, an enzyme with no recognition sites inside the vector and only one site in the insert. the linearized plasmid was gel purified, the free ends of the plasmid were then shortened using bal31 nuclease. the ends were filled using the klenow fragment of dna polymerase, and then religated. the competitor template for glyceraldehyde phosphate dehydrogenase (gapdh), used to control the amount of rna in each extraction, have already been constructed by lipman., (1994), and were kindly provided by dr. known concentrations of the competitor cdna was amplified in the same reaction tube as unknown amounts of sample derived rt - cdna. total rna is extracted from 400 l to 1 ml of whole blood using a similar method to zhang and yunis. first strand cdna is synthesized using oligo dt primers and maloney murine leukemia virus reverse transcriptase (promega, madison, wi), diluted 1 to 3, then 3 l is used as template in a 25 l pcr. the remainder of the reactions were made up by 5 l pcr buffer (gibco brl, grand island, ny), 0.25 g of each primer, 6 mm mgc12 in each cd4 reaction and 4 mm mgc12 for cd8 reactions, 2 mm total dntp 's, and 0.5 u of taq polymerase. for cd4 and cd8 reaction conditions were : 94 for 3 mins, then 33 cycles of 94 for 45 secs, 55 for 45 secs, and 72 for 2 mins on a perkin elmer thermocycler. 33 cycles was chosen as this was the largest number still within the linear range of the pcr (data not shown). in each assay we performed at least six different reactions for each sample using increasing amounts of competitor dna. the amounts of sample were quantitated by extrapolating across at least three reactions in which approximately equal amounts of sample and competitor products were produced.. quantity of sample derived mrna can be determined when the intensity of its pcr band is equal to that of the competitor 's upon co - amplification. staining with ethidium bromide the standard curve plots the log cdna / competitor band intensity (as determined by analysis of pict files with nih image) on the x - axis versus the log of incremental dilutions of competitor dna on the y - axis. solving for the antilog of x when y = 0 will give the concentration of unknown amount of dna that of cd4, cd8 or gapdh. the success of the rna extraction and reverse transcription is determined by quantification of gapdh mrna by qc - rt - pcr. each value for quantity of cd4 or cd8 heather jaspan conceived of the study, performed all the molecular analyses, and wrote the manuscript.	backgrounda polymerase chain reaction (pcr)-based method for quantitating cd4 and cd8 mrna could provide a means of assessing immune status of aids patients and other immunologically compromised persons without requiring large blood draws, and could be exquisitely sensitive. such a method would also be useful in assessing the immune status of patients retrospectively.resultsquantitative competitive reverse transcription pcr (qc - rt - pcr) assays were developed for measurement of cd4 and cd8 mrna. samples were obtained from hiv - positive and negative patients whose cd4 and cd8 counts had been determined via flow cytometry. the quantity of cd4 (n = 13) and cd8 (n = 28) mrna standardized according to gapdh mrna quantities, all determined by qc - rt - pcr, were compared to cell number as determined by flow cytometry. there was no correlation between cd4 and cd8 cell counts and mrna levels of cd4 and cd8 as determined by qc - rt - pcr. there is no correlation between cd4 and cd8 mrna levels and the number of cells expressing these proteins on their surface.conclusionqc-rt-pcr, and related methodologies are not useful substitutes for assessment of cd4 and cd8 cell numbers in hiv - infected persons.
private hospitals are unwilling to take emergency cases. government run primary and secondary care hospitals in india are still focused on vaccine preventable diseases, maternal child health, and at best provide the most basic first aid to trauma cases. resuscitation, specialist care, and operative facilities are lacking in these hospitals. yet, these facilities are the only option available for providing initial care to a trauma victim. conventionally, trauma mortality is predicted using scores based on anatomical, physiological, or a combination of both types of criteria. mechanism of injury is also included as a critical variable in the revised trauma score and trauma injury severity score (triss). these include age, sex, pre - existing pathologiesi, hypotension, number of units of blood transfused, serum lactate, base deficit, massive blood transfusion, early coagulopathy, early hyperglycemia defined as levels more than 200 mg / dl, iatrogenic mistakes, prehospital care, trauma center volume, and designation. trauma victims cared for at the primary and secondary hospitals in uttar pradesh are subsequently referred to the king george medical university (kgmu) trauma center. the trauma center of kgmu is the only trauma center in the state of uttar pradesh and caters to the need of about 200 million. such transfers of trauma patients delay critical time for appropriate definitive care and may be associated with adverse outcomes. the objectives of the study were to identify predictors of 1 year mortalityin trauma patients presenting at kgmu a resource constrained setting which does not allow for full international standard of trauma careii. the secondary objective was to describe interhospital referral and determine its effect on trauma mortality. adult injured patients without burns and more than 18 years of age presenting to the trauma center were included in the study, subject to written informed consent. two hundred and sixty patients were required for the necessary power using the thumb rule of 10 patients per variable studied ; but to increase the representativeness of the sample, data was collected for 1 year. in order to check the representativeness of our sample over different days of a week, patients admitted on mondays over a 1 year period and eight randomly selected wednesdays and saturdays were consecutively recruited to the study. patients admitted on wednesdays and saturdays were to be included in final analysis to predict mortality if they were found to be clinically and demographically similar to patients admitted on mondays. consecutive recruitment for 24 hours beginning 8:00 am was employed on the days mentioned above. all 572 patients were followed - up for mortality for a period of 1 year by means of post discharge hospital visits, or phone calls or home visits every month. characteristic factors knowniii to be associated with mortality were recorded in patients enrolled in the study, subject to written informed consent from the patient or his kin. due to lack of an ongoing audit and record keeping at peripheral hospitals, there was no method to ascertain patients for iatrogenic mistakes other than missed injuries. base deficit, serum lactate, and obesity assessment are not performed routinely during the course of treatment of every trauma victim and thus not included. information regarding the patient 's socioeconomic status identified as below poverty line (bpl) was also collected.iv information about referral and interhospital transfer included details of the transfer process, training of the transfer personnel, and adequacy of the transfer. data communicated to the trauma center from the referring hospitals was collected from the transfer records. data was collected using a standardized questionnaire in which item analysis had been done for inter- and intraobserver agreement. time trend analysis of mortalitywas done using actuarial survival analysis and cox proportionate hazard model. during the study period, 592 eligible patients were admitted to the kgmu trauma center. of these, 572 consented to the study. fifty - seven percent (327/572) of patients were referred admitted and 43% (245/572) were directly admitted patients. patients were predominantly male (83.5%) with median age 38 years, median injury severity score (iss) 9, and mean glasgow coma scale (gcs) 12.20 4.1 (median 15). the demographic and clinical characteristics as well as mortality of the patients admitted on different days of the same week were found to be similar [table 1 ]. during follow - up, 143 patients died (24.96% ; three due to unrelated cause and hence excluded from analysis). clinical and demographic characteristics of patients admitted on different days documentation about date and time of injury (0%), referral time (13.71%), pulse rate (34.38%), and blood pressure (bp ; 34.25%) was generally suboptimal in the facility notes accompanying the referred patient. transit notes were not available in any of the patients. on arrival to kgmu, 71 patients had a gcs score of less than 8. no patient had a systolic bp 15 ; odds ratio (or) = 18.94 ; 95% confidence interval (ci) 11, 33) and severe head injury (gcs 26 or 10) and presence of cervical spine injury were found to be significant predictors on logistic regression, but not significant on cox proportionate hazard analysis. bootstrapping of the logistic regression model and cox proportionate hazard model identified age, iss, aptt, and gcs score at admission to be significant predictors of 1-year mortality. however, systolic bp and cervical spine injury were found to be insignificant [table 2 ]. summarizing the results for analysis to identify predictors of 1 year mortality kaplan meier survival curve showed two distinct phases of mortality, namely within 6 days of sustaining injury and another after more than 6 days of sustaining injury [figure 1 ]. of the 140 deaths, 86 occurred within 6 days of sustaining injury, while 54 occurred after 6 day. kaplan meier survival curve of the injured admitted patients on logistic regression, mortality within 6 days of injury was found to be predicted by age, iss, aptt, and gcs score at admission. mortality after 6 day was found to be predicted exclusively by gcs score at the time of admission. various best possible cutoffs for the logistic regression model to predict 1 year mortality were diagnostically evaluated to estimate diagnostic values for 1 year mortality. this series of trauma patients validates previously reported predictors of mortality such as age, iss, abnormal respiratory rate (> 26 or 10) at admission, and increased aptt and gcs score at admission. however, our results do not validate systolic bp at admission and presence of cervical spine injury as valid predictors of mortality as these factors were found to be insignificant in the analysis. we report higher age to a be significant predictor of mortality on logistic regression as well as cox proportionate hazard model, which indicates that the effect of age on mortality lasts for 1 full year. we also found abnormal respiratory rate at admission to be positively associated with mortality on logistic regression. however, the same were not found to be positively associated with mortality on cox proportionate hazard model. this is because the effect does not last beyond the first few days of injury, a finding which has been reported in literature. systolic bp at the time of admission was found to be significant on logistic regression as well as cox proportionate hazard model. however, it was found to be insignificant on bootstrapping, and thus it should not be treated as universally valid. injury - induced hemorrhage accounts for the largest proportion of mortality within the 1 hour of trauma center care, causes 50% of injury - associated death within the first 24 hours of trauma care, and claims more lives than any other injury - induced pathology within the first 48 hours of care. a limitation of the study was that time to death since injury was not recorded in hours, and hence we are unable to comment on the value of hypotension in predicting mortality within 1hour injury. another notable limitation is that a significant number of injured died post discharge at their homes or at another hospital where the exact time of death was not available. due to the software excluding patients with incomplete data sets, 26 patients of cervical spine injury with neurological deficit were excluded from multivariate analysis. a negative beta coefficient for cervical spine injury as reported by us is due to inclusion of cervical spine injury patients without neurological injury and hence a complete data set in the logistic regression model. due to this selective exclusion of cervical spine injury with neurological deficit we are unable to comment on the role of cervical spine injury with neurological defect in predicting mortality. a significant finding of the study is that after 6 day of injury gcs score at admission is the only significant predictor of 1 year mortality. gcs score within 4 hours of injury has been reported to predict 2 week mortality. in our study, 110 patients (78.5%) died within 2 weeks of injury and another 30 died after 2 weeks up to 16 week (87.14% mortality occurred within 3 weeks and 97.85% mortality occurred within 8 weeks). this suggests that the predictive value of gcs score at the time of admission extends beyond 2 weeks. it has been reported that mortality prediction using gcs may not be very accurate due to presence of paralysis, use of sedating medications and coexisting injuries. in an organized system of trauma care with prehospital care incorporating advanced life support system protocols, use of paralytics in the field and during transport this trend in pre - hospital management may account for the results seen by stocchetti., who described a subset of patients wronglyclassified as severe. the same trend may explain significant correlation between gcs and 6-month glasgow outcome scale demonstrated between 1992 and 1996 and subsequent loss of correlation in the period 19972001. very good predictive power as demonstrated in our study may be due to lack of prehospital care and lack of intubation facilities in peripheral hospitals and during transport. we report lack of an association between sex and mortality in our study. this could be because we did not segregate female patients by age groups ; and hence potential hormonal status, thereby resulting in the mixing of premenopausal, perimenopausal, and postmenopausal patients. several studies that stratify patients by age and injury severity did find a gender - based survival advantage for premenopausal women with iss > 15 and others documented a reduced incidence of sepsis and multiple organ dysfunction syndrome (mods). random blood sugar done at the time of admission and early blood transfusion were significant on bivariate analysis, but insignificant on multivariate analysis. this could be due to a very small number of patients with hyperglycemia at admission. in contrast to other studies, blood transfusion was not found to be significantly associated with mortality. one important findings of our study is the positive association between referral and bpl status. possible explanations include bpl patients tending to primarily present at nearer hospitals (with subsequent referral) due to the patient 's lack of funds to hire transportation to another hospital, lack of knowledge of other health facilities, or selective referral of poorer patients by the peripheral hospital due to lack of adequate financial reimbursement for the services provided. none of the 71 patients with gcs scores < 8 had airway protected by endotracheal intubation, contrary to current recommendations that seek to prevent hypoxia and hypercarbia, and thereby poorer neurological outcome from head trauma. this leads to a strong emphasis on ensuring airway protection during retrieval / transfer in the indian settings. the lack of cervical spine immobilization and intubation may reflect the lack of suitably trained emergency department staff and/or lack of equipment at the referral hospitals. the choice of inadequate resuscitative intravenous fluids (other than ringer lactate of normal saline which are recommended by atls course) in 22 (5.94%) patients and the prevalence of untreated hypotension at arrival found in 106/317 (32.41%) referred patients without neurogenic shock may signify delayed recognition of shock states and under resuscitation in patients. however, this is a contentious issue as there is decent literature to suggest that relative hypotension may be actually helpful. the paucity of documentation of the patient 's clinical status, the time of injury, time of referral, rampant use of non - ambulance vehicles for transferring referred patients, and lack of transfer request was alarming. our finding that admission and subsequent transfer from a peripheral hospital is associated with significant delay in admission to trauma center confirms the finding of other studies. a surprising finding of our study was lack of significantly higher mortality in the transferred group, despite the fact that it was more severely injured and presented significantly later to the trauma center when compared with the directly admitted group. the reason for this could be a very high number of injuries that are neither time critical nor affected by the place of treatment and very low numbers of patients in subgroups (major trauma, severe head injury, subdural hematoma (sdh), and extradural hematoma (edh)) where early treatment at trauma center is known to be beneficial. an important finding in our study was a significantly higher percentage of multisystem major trauma patients and severe head injury patients in the referred group. an important trend, though statistically insignificant seen in our study was lower mortality in severe head injury patients, subdural hematoma patients, and major trauma patients in the directly admitted group. this study showed that age, iss, aptt, and gcs may be considered as externally valid predictors of mortality in the trauma patients. however, since bootstrapping only provides limited estimates of external validity, there is a need to test these factors against the well accepted requirements of external validity namely population, ecological, and temporal validity. studies focusing on gcs as predictor of mortality should consider following patients upto at least 3 weeks and preferably up to 8 weeks. compared to the directly admitted patients, referred admitted patients at the kgmu trauma center present significantly late to the trauma center, are more critically ill, have higher percentages of multisystem major trauma and severe head injury patients. however, mortality is similar in both the groups. positive association of bpl status with referral as reported by us needs to be further researched in order to find the exact cause. there is a need for a rational referral policy contributed to and agreed by all service providers which must be strongly enforced without delay. on the basis of evidence generated by this paper and similar papers published elsewhere our recommendations are that : all hospitals and interfacility transfer ambulances share a unified electronic medical recordevidence - based standard international guidelines should be adapted locally and disseminated to all healthcare providers who care for the traumatically injuredall transfer of substantially injured patients should be accompanied by trained healthcare providersall vehicles involved with the transfer of injured patients should contain agreed upon basic equipment.standardized communication with the receiving trauma facility should be mandated before patient transfer. all hospitals and interfacility transfer ambulances share a unified electronic medical record evidence - based standard international guidelines should be adapted locally and disseminated to all healthcare providers who care for the traumatically injured all transfer of substantially injured patients should be accompanied by trained healthcare providers all vehicles involved with the transfer of injured patients should contain agreed upon basic equipment.	background : traditional approach to predicting trauma - related mortality utilizes scores based on anatomical, physiological, or a combination of both types of criteria. however, several factors are reported in literature to predict mortality independent of severity scores. the objectives of the study were to identify predictors of 1 year mortality and determine their magnitude and significance of association in a resource constrained scenario.materials and methods : prospective observational study enrolled 572 patients. information regarding factors known to affect mortality was recorded. other factors which may be important in resource constrained settings were also included. this included referral from a peripheral hospital, number of surgeries performed on the patient, and his socioeconomic status (below poverty line (bpl) card). patients were followed till death or upto a period of 1year. logistic regression, actuarial survival analysis, and cox proportionate hazard model were used to identify predictors of 1year mortality. limited estimate of external validity of the study was obtained using bootstrapping.results:age of patient, injury severity score (iss), abnormal activated partial thromboplastin time (aptt), glasgow coma scale (gcs) score at admission, and systolic blood pressure (bp) at admission were found to significantly predict mortality on logistic regression and cox proportionate hazard models. abnormal respiratory rate at admission was found to significantly predict mortality in the logistic regression model, but no such association was seen in cox proportionate hazard model. bootstrapping of the logistic regression model and cox proportionate hazard model provide us with a set of factors common to both the models. these were age, iss, aptt, and gcs score at admission.conclusion:multivariate analysis (logistic and cox proportionate hazard analysis) and subsequent bootstrapping provide us with a set of factors which may be considered as valid predictors universally. however, since bootstrapping only provides limited estimates of external validity, there is a need to test these factors against the well accepted requirements of external validity namely population, ecological, and temporal validity.
there are more than 20 regulator of g - protein signaling (rgs) proteins, and rgs5 is a member of the r4/b subfamily (1 - 4). rgs proteins are components of g - protein (guanine nucleotide binding protein) coupled receptor (gpcr) complexes, and they act to shorten the duration of signaling resulting from ligand binding to gpcrs by acting as activators of the gtpase activity of the subunit of g proteins (5). gtpase activation of rgs5 is apparently specific for the gi and gq g protein subunits (6, 7). gi and gq, respectively, can inhibit adenylyl cyclase and activate phospholipase c (8, 9). in addition to g proteins, there is emerging evidence that rgs proteins also interact with other proteins downstream of gpcrs (10). there is abundant evidence for roles of rgs5 in vascular and cardiac remodeling and blood pressure homeostasis (11 - 13). it has been suggested that it is a stimulator of apoptosis of endothelial cells (14). a role for rgs5 in various cancers has also been reported (15 - 19). with respect to ocular functions, it was first found that rgs5 mrna is up - regulated in hypertensive monkey eyes (20). subsequently, high expression of rgs5 mrna was reported in human ciliary body and trabecular meshwork (tm) tissues and a novel alternative spliced variant of rgs5 mrna was identified in human ocular tissues (21). it is a major component of the conventional outflow pathway of aqueous humor and significantly modulates outflow of this fluid from the anterior chamber to venous blood via schlemm s canal (22). increased tm resistance causes decreased outflow and increased intraocular pressure, the latter being a major risk factor for glaucoma (23). our long standing interest in glaucoma prompted a study aimed at identification of non - housekeeping genes expressed in human tm cells (24). a meta - analysis on tm gene expression data derived from four microarray, two cdna library, and one sage (serial analysis of gene expression) experiment identified only ten non - housekeeping genes that were reported to be expressed in the human tm by all studies (24 - 30). their identification in all studies was taken to mean that these are highly and consistently expressed non - housekeeping genes in the human tm (24). micrornas (mirnas), which are small (~22 nucleotides) single stranded non - coding rnas, are now considered major components of the cellular machinery for gene expression regulation in multicellular eukaryotes (31, 32). they have important roles in various biological processes, including development, cell growth, cell signaling, and apoptosis (33). mirnas have been implicated in the etiology of various diseases (34 - 36). they act at the post - transcriptional level, by promoting mrna degradation or by having inhibitory effects on translation, and they usually exert fine tuning regulatory effects ; on average, they affect a two - fold decrease in production of target proteins (37 - 39). each mirna can have multiple mrna targets, and the expression of up to 60% of human genes is thought to be affected by mirnas (40, 41). the most critical factor in mirna targeting is complementary pairing of sequences within the 3 untranslated (3-utr) regions of mrnas with a seven nucleotide seed region within the mirnas (41, 42). various bioinformatics tools use this feature and more subtle criteria to predict mirnas that may target specific genes and to predict genes that an mirna of interest may target (43, 44). mirna expression and the genes and pathways they affect in ocular tissues have now been investigated in several studies (45 - 49). we recently reported the effect of mir-204 on the expression of foxc1 and other genes in human tm cells (50). in a preliminary effort to gain a more complete understanding of aspects of rgs5 expression in ocular tissues, we here aimed to identify mirnas which may target rgs5 mrnas candidate mirnas to be tested for targeting rgs5 mrna were selected in three steps. initially, mirwalk was used to identify mirnas with predicted binding sites within the mrna s 3-utr segment ((51) ; http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/). this in silico bioinformatics tool relies on information accrued from predictions made by nine (rna22, miranda, mirdb, targetscan, rnahybrid, pita, pictar4, pictar5, and diana - microt) established programs for binding of mirnas to 3-utrs of all known human genes. mirwalk ultimately reports predictions made by ten algorithms, including its own algorithm and those of the nine programs specified above. thus identified, those whose expression in the eye had been experimentally validated were chosen at the second level of selection. expression in the eye was assessed on the basis of information available at mirwalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/) and review of the literature (47). finally, seven mirnas with high prediction scores were chosen to be tested for experimental verification of targeting the 3-utr segment of rgs5 mrna by the dual luciferase assay. an eighth mirna that is a family member of the seven selected mirnas was also tested. mirnas were purchased either as cloned sequences within pcmv - mir vectors (origene, rockville, md, usa) or as un - cloned mirs (ambion, austin, tx, usa). empty pcmv vector and a scrambled mimic mirna were used as negative controls for assessing the effects, respectively, of the cloned and un - cloned mirnas that were tested. scrambled mimic mirna does not produce identifiable effects on known human mrna functions (reference for snord ; http://www.invitrogen.com/1/1/11216-mir-vana-mirna-mimic-negative-control-1.html). the 3-utr segment of rgs5 mrna consists of 5038 nucleotides (nm_003617). to facilitate cloning, genomic dna encoding the 3-utr segment was amplified by pcr in three shorter amplicons, rgs5 - 1 (nucleotides 687 - 2162), rgs5 - 2 (2899 - 3992), and rgs5 - 3 (3995 - 4967). forward and reverse primers for all amplicons were designed to include, respectively, xho1 and not1 restriction enzyme recognition sites. the three fragments collectively contained almost all recognition sites predicted by the mirwalk analysis (figure 1a). each fragment was cloned downstream of the renilla luciferase reporter gene, directly 3 to its amino acid coding region, in psicheck2 to create vectors psicheck2- rgs5 - 1, psicheck2-rgs5 - 2, and psicheck2-rgs5 - 3 (promega corporation, madison, wi, usa). the psicheck2 vector, in addition to the renilla gene which is expressed under the t7 promoter, contains the firefly luciferase gene adjacent to the hsv - tk promoter. for verification the sequences of primers used for the cloning reactions are presented in table 1. dual luciferase assay. nucleotide positions are with reference to the translation termination codon, and the position of the first nucleotide after that codon is considered + 1. the thick lines represent rgs5 - 1, rgs5 - 2, and rgs5 - 3 that were cloned ; the nucleotide positions at the termini of the fragments are shown. indicates p<0.05, indicates p<0.01, and indicates p<0.001 for comparison of renilla / firefly luciferase ratios between control transfections and transfections with mirnas. rlu, relative light units sequences of primers used for pcr - amplification of rgs5 - 1, rgs5 - 2, and rgs5 - 3 xhoi recognition sites on forward primers and noti recognition sites on reverse primers are shaded the effects of the mirnas were assessed in human embryonic kidney (hek)-293 cells (national institute of genetic engineering, tehran, iran). empty or recombinant psicheck2 vectors (250 ng) were co - transfected in 24-well plates (2 10 cells / well) with cloned mirnas (250 ng pcmv - mir dna (origene ; mir-7 : cat # sc400648, mir-9 : z cat # sc400676, mir-23a : cat # sc400294, or mir-23b : cat # sc400295)) or with un - cloned mirnas (final concentration of 25 nm (ambion ; mir-96 : pm10422, mir-182 : pm12369, mir-204 : pm11116, or mir-211 : pm10168)) using lipofectamine ltx reagent (invitrogen, carlsbad, ca, usa). forty eight hours after transfections, renilla and firefly luciferase activities were measured using dual luciferase assays (promega corporation) according to the manufacturer s instructions. expression of cloned mirnas was verified by real time pcr using snord-47 mirna as control. candidate mirnas to be tested for targeting rgs5 mrna were selected in three steps. initially, mirwalk was used to identify mirnas with predicted binding sites within the mrna s 3-utr segment ((51) ; http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/). this in silico bioinformatics tool relies on information accrued from predictions made by nine (rna22, miranda, mirdb, targetscan, rnahybrid, pita, pictar4, pictar5, and diana - microt) established programs for binding of mirnas to 3-utrs of all known human genes. mirwalk ultimately reports predictions made by ten algorithms, including its own algorithm and those of the nine programs specified above. thus identified, those whose expression in the eye had been experimentally validated were chosen at the second level of selection. expression in the eye was assessed on the basis of information available at mirwalk (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/) and review of the literature (47). finally, seven mirnas with high prediction scores were chosen to be tested for experimental verification of targeting the 3-utr segment of rgs5 mrna by the dual luciferase assay. an eighth mirna that is a family member of the seven selected mirnas was also tested. mirnas were purchased either as cloned sequences within pcmv - mir vectors (origene, rockville, md, usa) or as un - cloned mirs (ambion, austin, tx, usa). empty pcmv vector and a scrambled mimic mirna were used as negative controls for assessing the effects, respectively, of the cloned and un - cloned mirnas that were tested. scrambled mimic mirna does not produce identifiable effects on known human mrna functions (reference for snord ; http://www.invitrogen.com/1/1/11216-mir-vana-mirna-mimic-negative-control-1.html). the 3-utr segment of rgs5 mrna consists of 5038 nucleotides (nm_003617). to facilitate cloning, genomic dna encoding the 3-utr segment was amplified by pcr in three shorter amplicons, rgs5 - 1 (nucleotides 687 - 2162), rgs5 - 2 (2899 - 3992), and rgs5 - 3 (3995 - 4967). forward and reverse primers for all amplicons were designed to include, respectively, xho1 and not1 restriction enzyme recognition sites. the three fragments collectively contained almost all recognition sites predicted by the mirwalk analysis (figure 1a). each fragment was cloned downstream of the renilla luciferase reporter gene, directly 3 to its amino acid coding region, in psicheck2 to create vectors psicheck2- rgs5 - 1, psicheck2-rgs5 - 2, and psicheck2-rgs5 - 3 (promega corporation, madison, wi, usa). the psicheck2 vector, in addition to the renilla gene which is expressed under the t7 promoter, contains the firefly luciferase gene adjacent to the hsv - tk promoter. for verification nucleotide positions are with reference to the translation termination codon, and the position of the first nucleotide after that codon is considered + 1. the thick lines represent rgs5 - 1, rgs5 - 2, and rgs5 - 3 that were cloned ; the nucleotide positions at the termini of the fragments are shown. indicates p<0.05, indicates p<0.01, and indicates p<0.001 for comparison of renilla / firefly luciferase ratios between control transfections and transfections with mirnas. rlu, relative light units sequences of primers used for pcr - amplification of rgs5 - 1, rgs5 - 2, and rgs5 - 3 xhoi recognition sites on forward primers and noti recognition sites on reverse primers are shaded the effects of the mirnas were assessed in human embryonic kidney (hek)-293 cells (national institute of genetic engineering, tehran, iran). empty or recombinant psicheck2 vectors (250 ng) were co - transfected in 24-well plates (2 10 cells / well) with cloned mirnas (250 ng pcmv - mir dna (origene ; mir-7 : cat # sc400648, mir-9 : z cat # sc400676, mir-23a : cat # sc400294, or mir-23b : cat # sc400295)) or with un - cloned mirnas (final concentration of 25 nm (ambion ; mir-96 : pm10422, mir-182 : pm12369, mir-204 : pm11116, or mir-211 : pm10168)) using lipofectamine ltx reagent (invitrogen, carlsbad, ca, usa). forty eight hours after transfections, renilla and firefly luciferase activities were measured using dual luciferase assays (promega corporation) according to the manufacturer s instructions. expression of cloned mirnas was verified by real time pcr using snord-47 mirna as control. based on mirwalk and review of the literature, targeting of the 3-utr of rgs5 mrna has not yet been experimentally validated for any mirna. seven hundred seventy six mirnas were computationally predicted to target this 3-utr by the algorithm used by mirwalk or at least one of the mirna prediction programs included therein. four hundred thirty five of these met our threshold selection criterion of being predicted by at least four programs. of these, the expression of at least 55 has been experiment- tally evidenced in the eye (http://www.umm. as em - pirical evaluation of all these was beyond the scope of the present study, seven of the mirnas were chosen on the basis of predictive scores to be tested by the dual luciferase assay. the seven mirnas were mir-7, mir-9, mir-23a, mir-23b, mir-96, mir-182, and mir-204. an eighth mirna (mir-211) was also selected because it belongs to the same family as mir-204 which is known to be an important mirna with respect to various ocular functions, and because mir-211 has been shown to be relevant to glaucoma related functions of the tm (45, 46, 49, 52). the distribution of the predicted recognition sites of these mirnas on the 3-utr of rgs5 mrna and on these cloned fragments is shown in figure 1a. the sequences of the 3-utr fragments cloned in psicheck2- rgs5 - 1, psicheck2-rgs5 - 2, and psicheck2-rgs5 - 3 were shown to be correct. the number of predicted target sites for each mirna in each 3-utr fragment tested was either one or two. expression of the cloned mirnas after transfection into hek293 cells was confirmed by real time pcr (not shown). the results of effects of the mirnas as assessed by dual luciferase assays are presented in figure 1b and table 2. effects on fragment rgs5 - 2 were comprehensively tested, but effects of mir-182 on rgs5 - 1 and mir-23a, mir-23b, and mir-182 for rgs5 - 3 were not assessed. results of replicate assays were impressively similar as evidenced by small standard deviations in the renilla / firefly luciferase ratios (figure 1b). renilla / firefly luciferase ratios were not significantly different in cells transfected with only psicheck2 backbone, scrambled mirna, or any of the mirnas being tested (not shown). this means that the differences observed in the presence of psicheck2 vectors containing 3-utr sequences are due to mirna effects that were elicited only in the presence of those sequences. among the thirteen assays performed with psicheck2 vectors containing 3-utrs, the tested mirnas decreased target gene expression at a statistically significant level in seven of the reactions this was evidenced by decreased renilla / firefly luciferase ratio in presence of the tested mirna as compared to control reactions (figure 1b). unexpectedly, mir-182 caused increased renilla / firefly luciferase ratio in the presence of psicheck2-rgs5 - 2. the effect of mir-204 in presence of psicheck2-rgs5 - 2 (1.4-fold) was notable because it reached statistical significance at the level of p<0.0002. mir-23a and mir-23b, that share common seed sequences, are each predicted to have four recognition sites in the 3-utr of rgs5. effects of these mirnas on the single site present in rgs5 - 3 were not tested ; however they caused decreased renilla luciferase expression in the presence of rgs5 - 1 and rgs5 - 2 that contain the remaining recognition sites (figure 1b, table 2). effects of mirn-23a and mir-23b on rgs5 - 2, respectively, were 2.0- and 1.8-fold. having two as compared to one recognition site did not necessarily result in increased mirna effect (table 2). among the five assays in which statistically significant effects of mirnas were not observed, four had two recognition sites for the tested mirna ; among the seven reactions in which the mirnas reduced renilla luciferase expression, four had one recognition site and three had two. we have shown by the luciferase assay that all mirnas tested except mir-182 had a highly reproducible effect on down regulation of the renilla luciferase gene when the gene was associated with a partial 3-utr segment of the rgs5 gene. the up - regulatory effect of mir-182 in the presence of rgs5 - 2 may have been indirect and caused by effects of this mirna on other regulatory components active in hek-293 cells. the observed effects of mir-7, mir-9, mir-23a, mir-23b, mir-96, mir-204, and mir-211 on rgs5 - 1, rgs5 - 2, and/or rgs5 - 3 were modest. although we did not observe that having two rather than one binding site was correlated with increased down regulation ; it is generally believed that the presence of multiple target sites does increase effects of mirnas. in this light, it is possible that the presence of the entire 3-utr segment (5038 200 nucleotides) downstream of the renilla luciferase gene would have a more pronounced effect. mirnas mir-7, mir-9, mir-23a, and mir-23b are each predicted to have four or five target sites within the entire 3-utr segment, whereas there was a maximum of two target sites in the segments tested (figure 1a). known ocular related functions of the mirnas here tested, were reviewed with the consideration that the functions may shed light on their putative effects on rgs5 expression. mir-7 has been reported to be among the mirnas whose levels where changed in tm cells upon treatment with mechanical stress which is a factor that potentially contributes to the pathogenesis of glaucoma (53). mir-9 is necessary for proper eye development, and has a role in inflammatory responses of retinal microglia cells (54). mir-23a and/or mir-23b are thought to be essential for proper eye development, to protect retinal epithelial cells from oxidative damage, and to be involved in choroidal neovascularization (54). both are present in human aqueous humor and may have roles in the etiology of cataract (47, 48). among the mirnas tested, mir-204 is likely to be the most important with respect to ocular development and ocular functions. some of its functions are mediated through the important eye transcription factors meis2 and foxc1 (46, 50). mir-204 is known to be expressed in murine eye tissues from early developmental stages through adulthood, and has been shown to regulate multiple aspects of eye development in the medaka fish (45, 46, 55) ; it is expressed in human tm cells (49, 52). the mirna may have roles in the etiology of cataract and is involved in the regulation of apoptosis, endoplasmic reticulum stress response, and inflammation in human tm cells (48, 49). the above findings do not provide an exact signal for the biological significance of the effects of the mirnas on rgs5 expression. nevertheless, given that rgs5 is definitively expressed in the human tm (23, 25 - 27), and that it is up - regulated in hypertensive eyes (20), the results presented here justify further investigations on the regulatory functions of the mirnas on rgs5 expression and on genes downstream of g - protein coupled receptors that associate with gi and gq. this having been said, we emphasize that our results are preliminary, and that the effects of the mirnas need to be studied at the protein level, and most importantly, in ocular relevant cells such as tm cells.	objective(s):an earlier meta - analysis on gene expression data derived from four microarray, two cdna library, and one sage experiment had identified rgs5 as one of only ten non - housekeeping genes that were reported to be expressed in human trabecular meshwork (tm) cells by all studies. rgs5 encodes regulator of g - protein signaling-5. the tm tissue is the route of aqueous fluid outflow, and is relevant to the pathology of glaucoma. micrornas constitute the most recently identified components of the cellular machinery for gene regulation in eukaryotic cells. given our long standing interest in glaucoma, we aimed to identify mirnas that may target rgs5.materials and methods : eight mirnas were selected for study using bioinformatics tools and available data on mirnas expressed in the eye. their effects were assessed using the dual luciferase assay. 3-utr segments of rgs5 mrna were cloned downstream of a luciferase coding gene in psicheck2 vectors, and these were co - transfected with each of the mirnas into hek293 cells.results:the outcomes evidenced that one or more of the segments are in fact targeted by mir-7, mir-9, mir-96, mir-23a, mir-23b, mir-204, and mir-211. down regulations by the mirnas were statistically significant. the effect of mir-204 is considered particularly important as this mirna is well known to regulate eye development and to affect multiple ocular functions.conclusion:our results justify further studies on regulation of rgs5 expression and rgs5 downstream functions by these mirnas.
a healthy macula offers the most acute vision and is crucial to tasks with high visual demands. any disorder a macular hole or macular edema, for instance reduces visual acuity. the success of the treatment for these conditions is often determined by the presenting thickness and depends on posttreatment thickness. the conventional practice of assessing the macula for its thickness, involving slit - lamp biomicroscopy, fundus photography, and fluorescein angiography, is less sensitive to subtle changes and is only qualitative,1 and is not a common practice even in developing countries such as nepal where a more accurate and sophisticated instrument, optical coherence tomography (oct), is used. oct, an optical analog of ultrasound,2 is unique on account of its combined features : an objective method of quantitatively determining the macular characteristics,3 ability to produce high resolution and cross - sectional images accurately and precisely,4,5 and yet purely noninvasive ; all of which enable diagnosis, management, and monitoring of patients with retinal diseases. decision making while comparing the normative population database information stored in the oct software with results obtained from the population under examination in nepal has the probability to be flawed, as we might have different values for the macular variables compared with the normative database in the software, and hence puts the reliability of the test in question. as a unique study, this aims to determine the normal macular thickness and volume measurements in normal nepalese eyes. this cross - sectional, quantitative, and hospital - based study comprised 126 eyes of 28 male and 35 female subjects (mean age, 21.176.72 years ; age range, 1037 years). they were recruited between february and december 2013 from the department of ophthalmology, institute of medicine in nepal. ethical clearance approval was obtained from the institutional review board at the institute of medicine. the study fully adhered to the tenets of the declaration of helsinki. before their inclusion, informed verbal consent was sought from the subjects who were 18 years or over and from the parents or attendants when the subjects were under 18 years. sixty - three subjects (126 eyes) diagnosed as having healthy normal eyes following a complete anterior and posterior segment evaluation and refraction, and not having diseases and conditions (diabetes mellitus ; hypertension ; transplant ; autoimmune disease ; high intraocular pressure, ie, greater than 21 mmhg ; and refractive error, ie, greater than 0.25 d spherical equivalent) underwent a fast mode macular scanning with the commercially available spectral domain (sd) oct (spectra lis hra + oct ; heidelberg engineering, inc., heidelberg, germany) immediately after retinoscopy was carried out by an optometrist when the pupils were still dilated (> 5 mm diameter). the basic working principles have already been explained in great detail.6,7 the scan was performed over a 66 mm area in the posterior pole to achieve a high quality image. the center point of each scan direction represented minimum foveal thickness (central minimum thickness, or foveola).8 a traditional early treatment diabetic retinopathy study (etdrs) grid which contains three concentric rings of diameters 1, 3, and 6 mm, and two reticules to divide the macula into nine sections was employed. axial length measurements were taken using an ultrasound a - scan biometer (axis - ii pr ; quantel medical, inc. central subfield thickness (cst), also known as foveal thickness, was defined as the average thickness of the macula in the central 1 mm etdrs grid.9,10 average macular thickness was defined as the mean of thicknesses in nine sections.11 macular volume was defined as the sum of all volumes of all nine sections. all data were entered into epidata v3.1 (the epidata association, odense, denmark) and, then for analysis, were exported to spss version 20 (ibm corporation, armonk, ny, usa). the shapiro wilk test (p>0.05) was used to test the normality of the distribution.12,13 a student s t - test and one - way analysis of variance (for approximately normally distributed data) and mann whitney u - test and kruskal wallis test (for skewed data) were used to generate p - values between groups. a regression model was used to assess any correlation between 1) age and axial length, 2) age and macular thickness, 3) age and macular volume, 4) axial length and macular thickness, 5) axial length and macular volume, and 6) macular thickness and macular volume. this cross - sectional, quantitative, and hospital - based study comprised 126 eyes of 28 male and 35 female subjects (mean age, 21.176.72 years ; age range, 1037 years). they were recruited between february and december 2013 from the department of ophthalmology, institute of medicine in nepal. ethical clearance approval was obtained from the institutional review board at the institute of medicine. the study fully adhered to the tenets of the declaration of helsinki. before their inclusion, informed verbal consent was sought from the subjects who were 18 years or over and from the parents or attendants when the subjects were under 18 years. sixty - three subjects (126 eyes) diagnosed as having healthy normal eyes following a complete anterior and posterior segment evaluation and refraction, and not having diseases and conditions (diabetes mellitus ; hypertension ; transplant ; autoimmune disease ; high intraocular pressure, ie, greater than 21 mmhg ; and refractive error, ie, greater than 0.25 d spherical equivalent) underwent a fast mode macular scanning with the commercially available spectral domain (sd) oct (spectra lis hra + oct ; heidelberg engineering, inc., heidelberg, germany) immediately after retinoscopy was carried out by an optometrist when the pupils were still dilated (> 5 mm diameter). the basic working principles have already been explained in great detail.6,7 the scan was performed over a 66 mm area in the posterior pole to achieve a high quality image. the center point of each scan direction represented minimum foveal thickness (central minimum thickness, or foveola).8 a traditional early treatment diabetic retinopathy study (etdrs) grid which contains three concentric rings of diameters 1, 3, and 6 mm, and two reticules to divide the macula into nine sections was employed. axial length measurements were taken using an ultrasound a - scan biometer (axis - ii pr ; quantel medical, inc. central subfield thickness (cst), also known as foveal thickness, was defined as the average thickness of the macula in the central 1 mm etdrs grid.9,10 average macular thickness was defined as the mean of thicknesses in nine sections.11 macular volume was defined as the sum of all volumes of all nine sections. all data were entered into epidata v3.1 (the epidata association, odense, denmark) and, then for analysis, were exported to spss version 20 (ibm corporation, armonk, ny, usa). the shapiro wilk test (p>0.05) was used to test the normality of the distribution.12,13 a student s t - test and one - way analysis of variance (for approximately normally distributed data) and mann whitney u - test and kruskal wallis test (for skewed data) were used to generate p - values between groups. a regression model was used to assess any correlation between 1) age and axial length, 2) age and macular thickness, 3) age and macular volume, 4) axial length and macular thickness, 5) axial length and macular volume, and 6) macular thickness and macular volume. the age distribution of participants as shown in table 1 was based on sturges formula and followed an approximately normal distribution curve (p=0.194 and 0.333 for males and females, respectively). the measures of central tendency involved the mean and/or the median, depending on the distribution of data. the statistics of the macular thickness section - wise are shown in tables 2 and 3. the average macular thickness was higher in males than in females (u=334, p=0.031, right eyes ; t (61) = 2.094, p=0.04, left eyes) (tables 4 and 5). using the wilcoxon signed - ranks test, we observed that the right and the left eyes differed significantly over inner temporal thickness (p=0.007), inner superior thickness (p=0.019), and outer nasal thickness (p=0.001). only total macular volume (p=0.007), inner superior volume (p=0.000), and outer nasal volume (p=0.007) were different between the right and the left eyes. also, intersex variability in the left eyes was observed to be the same as that of the right eyes for macular volume measurements (tables 6 and 7). unless otherwise stated, the values will pertain to right eyes only. upon comparing three axial length groups (22.0522.70 mm, 22.7023.19 mm, and 23.1924.44 mm), the average macular thickness (kruskal wallis test, p=0.202) (figure 2) and macular volume measurements (kruskal wallis test, p=0.543) were not statistically different. average macular thickness and macular volume correlated with each other (r=0.944, p=0.000). average macular thickness (r=0.30, p=0.017) and macular volume (r=0.335, p=0.007) negatively correlated with age (figures 3 and 4). macular thickness and macular volume decreased by 0.556 m and 0.0156 mm, respectively, for each year of increasing age. however, cst and foveal volume (r=0.216, p=0.09) did not significantly correlate with age (r=0.243, p=0.055). macular thickness and volume increased with axial length ; however, not all sections had a significant correlation. nevertheless, the correlation study showed that the average macular thickness (r=0.254, p=0.044), cst (r=0.363, p=0.003), and foveal volume (r=0.387, p=0.002) increased with axial length. the upper limits of the average macular thickness and cst never overshot 330 and 287 m, respectively. the thinnest region was within the central section and measured only 180 m, while the thickest region was the inner superior section, which measured as high as 372 m. the outer sections were significantly relatively thin compared with inner sections. for both the right and left eyes, the central maximum thickness carried the smallest p - value to show the difference in thickness between males and females. except for the inner nasal volume in the inner macula, and the outer nasal volume and outer inferior volume in the outer macula, used as a diagnostic and monitoring tool for vitreomacular disorders, sd - oct on account of increased scan resolution and reliability, is becoming increasingly useful. unlike time domain - oct (td - oct) with an axial resolution of ~10 m, sd has an increased axial resolution of ~5 m. more information on axial resolution and image acquisition protocols of various commercially available oct instruments can be found elsewhere.14 the increase in resolution, which is a cutting - edge technology, makes possible the visualization of even imperceptible pathologic changes and helps with much better clinical use. similar previous study results10,11,1420 involved either sd - oct or td - oct or both in healthy eyes with refractive error. this study is unique because 1) it involved only healthy emmetropic eyes undergoing sd - oct (spectralis hra + oct, heidelberg engineering inc.) and 2) it is of a maiden kind. we can not therefore claim to accurately compare the findings from similar studies with those from the current study. td - oct marks the inner outer segment interface as the posterior retinal boundary, while sd - oct generally marks the retinal pigment epithelium as the posterior surface. in light of this, td - oct underestimates the thickness and volume measurements by 5060 m.21 in the same vein, the type of sd - oct used in this study uses the bruch s membrane as the posterior boundary, and hence there is an additional increased 20 m thickness compared to sd - oct in general.15,16 other sources of discrepancy include, but are not limited to, ethnicity variation and scan (radial versus linear). this, therefore, calls for exercising caution while comparing the data obtained from different studies. we tabulated data of right and left eyes because we can not deny that anatomical differences may exist between two eyes, and we believe normative data of right and left eyes separately help in comparing corresponding eyes. in addition, this study serves as a pool of data for the nepalese population, which will be used for comparison with findings from future studies to be conducted in nepal and abroad. this study is in line with an earlier study17 that reported 1) a significant correlation between age and minimum foveal thickness and 2) no significant correlation between age and cst. cst bears no correlation with age.18 on the contrary, studies22,23 reported a relationship of cst with age. the finding from a study22 that foveola does not thin with age is countered by our study while the same study had a finding of parafoveal attenuation consistent with ours. similar to previous studies,11,19,2427 the average macular thickness was significantly greater in males compared to females. in contrast, studies9,15 reported no difference in retinal thickness between males and females. the fact that thickness and volume measurements were almost always greater for males than for females could be explained by the smaller, thinner physique of females. further studies are required of the nepalese eyes to demonstrate differences, if any, in ocular biometry between male and female eyes. average macular thickness observed in this study was different from other studies, with measurements ranging from 258 to 300 m reported by studies.10,18,28 based on studies,15,19,20,26,27,2931 our understanding is that ethnicity has an effect on macular thickness, for we have observed higher values for almost every macular region. we side with chauhan and marshall32 on the effect of much darker pigmentation of the retinal pigment epithelium on the light signal, which is rendered attenuated, leading to reduced retinal thickness in african americans. in line with one study,11 where the male foveae measured significantly thicker than the female foveae, our study findings are consistent with previous findings that inner regions are thicker than outer ones, which are thicker than cst. nevertheless, there is a debate over which of the inner four sections measures thickest. studies33,34 reported maximal thickness in the superior and inferior regions, which could be attributed to the papillomacular bundle course along these regions. some reports10,11,16,17 mentioned the inner nasal section as the thickest region, which is in keeping with the dense ganglion nerve fiber layer in the nasal section. quite the opposite, reports20,3537 and our study showed that the inner superior region was the thickest of all. just as a study17 had reported, the intersex difference in thickness in the outer regions and inner nasal region was not observed, while a significant difference was observed in relation to the rest of the regions. this could indicate that the sex - wise variation occurs predominantly more toward the center of the macula. this variation of thickness between males and females may account for a female preponderance in macular hole.38,39 the study40 done in nepal also reported a female preponderance. cst obtained in our study differs by ~3 to 31 m from that obtained in the literature11,16,37 which used sd - oct, whereas cst was thicker by ~35 to 60 m upon comparing this study and the literature,10,17,20,37 which used td - oct. our study therefore reports larger values for almost all nine sections compared to other studies with the exception of one study,11 which reports a value less by 3.68 m. minimum foveolar thickness was not statistically different between males and females, and the range of mean minimum foveolar thicknesses spanned from 149 to 182 m.10,17,20,37 however, our study reports a larger value and an intersex difference. the studies11,16 that reported smaller macular thickness in all nine regions in comparison with our study had mean total macular volumes of 9.950.49 mm and 10.010.6 mm, respectively. in addition, one study24 that reported all nine thinner regions except the fovea which was thicker had a mean total macular volume of 9.740.71 mm. interestingly enough, the macular volume in our study was smaller. nevertheless, this statistics was larger than other studies.17,19 reports11,17,24 that males had greater macular volume compared to females are consistent with our study. all inner sections, except for the inner superior section, differed between males and females.17 the same observation was observed in this study. our study and a previous study17 agreed that outer nasal volume is greatest and foveal volume is lowest. the increase in axial length was associated with decreased retinal thickness and volume.36 in contrast, we observed an increase in thickness and volume. such an observation could be limited to emmetropes only and once ametropia occurs, our understanding of the relationship of axial length with thickness and volume may not apply. we therefore hypothesize that some other retinal changes might follow, which can bring about anatomical changes. we recruited participants from an age group of 1037 years because in children under ten, biometry and oct measurements were difficult to obtain as the children remained uncooperative, while in participants over 40, cataract (however mild it may be), undiagnosed diabetes mellitus, and systemic hypertension could be present. a small sample size should not deter us from interpreting and generalizing our findings because oct is a highly reliable and reproducible sophisticated technology. nonetheless, a small sample size, the oct model, and acquisition protocol may have given rise to discrepancy. this study does not take into account healthy eyes with refractive error. by providing a pool of normative data of the macular measurements, this study will help differentiate a healthy macula from a diseased one, and assist with diagnosis, monitoring, and management of macular diseases, and aid similar studies in the future.	purposeto record the normative values for macular thickness and macular volume in normal nepalese eyes.methodsin all, 126 eyes of 63 emmetropic subjects (mean age : 21.176.76 years ; range : 1037 years) were assessed for macular thickness and macular volume, using spectral domain - optical coherence tomography over 66 mm2 in the posterior pole. a fast macular thickness protocol was employed. statistics such as the mean, median, standard deviation, percentiles, and range were used, while a p - value was set at 0.05 to test significance.resultsaverage macular thickness and total macular volume were larger in males compared to females. with each year of increasing age, these variables decreased by 0.556 m and 0.0156 mm3 for average macular thickness and total macular volume, respectively. the macular thickness was greatest in the inner superior section and lowest at the center of the fovea. the volume was greatest in the outer nasal section and thinnest in the fovea. the central subfield thickness (r=0.243, p=0.055) and foveal volume (r=0.216, p=0.09) did not correlate with age.conclusionmales and females differ significantly with regard to macular thickness and macular volume measurements. reports by other studies that the increase in axial length reduced thickness and volume, were negated by this study which found a positive correlation among axial length, thickness, and volume.
peripheral ossifying fibroma (pof) is a reactive soft tissue growth that is usually seen on the interdental papilla. it may be pedunculated or broad based, usually smooth surfaced and varies from pale pink to cherry red in color. it is believed to comprise about 9% of all gingival growths and to arise from the gingival corium, periosteum, and the periodontal membrane. it has also been reported that it represents a maturation of a pre - existing pyogenic granuloma or a peripheral giant cell granuloma. a 12-year - old girl reported with the chief complaint of soft tissue growth in the palate. intraoral examination revealed a painless pedunculated, cauliflower - like rubbery mass on the palatal aspect of the maxillary left permanent molar extending towards the occlusal surface [figure 1 ]. the lesion was abnormally large about 2.5 cm mesiodistally and 1.5 cm buccopalatally and the side of the lesion facing the occlusal surface was focally ulcerated. the maxillary first permanent molar was buccally displaced. history revealed that the lesion started growing on its own since she first noticed it about a month back when it was a small nodule. the lesion was painless and occasionally bled on its own or when traumatized with toothbrush and in its present state was interfering with occlusion. radiograph revealed only soft tissue shadow and space between maxillary second premolar and first molar [figure 2 ]. soft tissue growth extending toward the occlusal surface iopa showing the soft tissue shadow and space between premolar and molar after routine blood examinations, excisional biopsy of the growth was done [figure 3 ] under antibiotic coverage and thorough curettage of the adjacent periodontal ligament, and periosteum was carried out to prevent recurrence. histomorphological examination revealed evidence of calcifications in the hypercellular fibroblastic stroma [figure 4 ] confirming the lesion as pof. the follow - up of the case showed normal healing of the area [figure 5 ]. pof has also been described by various synonyms such as peripheral cemento ossifying fibroma, peripheral odontogenic fibroma (podf) with cementogenesis, peripheral fibroma with osteogenesis, peripheral fibroma with calcification, fibrous epulis, calcifying fibroblastic granuloma, etc. almost 60% of the lesions occur in the maxilla and mostly occur anterior to molars. dental calculus, plaque, microorganisms, dental appliances, and restorations are considered to be the irritants triggering the lesion. the lesion though usually smaller than 1.5 cm in diameter can reach a much larger size and can cause separation of the adjacent teeth, resorption of the alveolar crest, destruction of the bony structure and cosmetic deformity. the term pof should not be confused with podf, which is the rare peripheral counterpart of central odontogenic fibroma. in north america, it is still synonymously used by many for pof as the lesion is thought by them to be derived from the periodontal ligament and hence to be odontogenic. the evidence for its odontogenic origin is circumstantial, being based partly on the demonstration of oxytalan fibers within its calcified structures and its exclusive occurrence on gingiva. however, oxytalan fibers have also been reported in the sites other than the periodontal ligament. a pof is more common in females and in the anterior maxilla, but podf has a predilection for males and the posterior mandible. the pof and ossifying fibroma (of) are the lesions that exhibit similar histomorphologic features and both originate from periodontal ligament cells. but a pof is a reactive lesion where as an of is a benign neoplastic lesion included in the group of benign fibro - osseous lesions of the jaws and both pof and of show different proliferative activities. the ulcerated lesions are more likely to be painful but in this case it was not painful. gingival lesions that imitate pof are peripheral giant cell granuloma, pyogenic granuloma, fibroma, calcifying epithelial odontogenic cyst, calcifying odontogenic cyst, etc. radiographically radiopaque foci within the soft tissue tumor mass are observed if the calcified element is significant, but in this case no radiopaque foci were seen but only shadow of the lesion was seen probably because the lesion was of short duration of time. histologically, pof can exhibit either ulcerated or intact stratified squamous epithelium. in a typical ulcerated lesion, three zones could be identified : zone i : the superficial ulcerated zone covered with the fibrinous exudate and enmeshed with polymorphonuclear neutrophils and debris. zone ii : the zone beneath the surface epithelium composed almost exclusively of proliferating fibroblasts with diffuse infiltration of chronic inflammatory cells mostly lymphocytes and plasma cells. zone iii : more collagenized connective tissue with less vascularity and high cellularity ; osteogenesis consisting of osteoid and bone formation is a prominent feature, which can even reach the ulcerated surface in some cases. the calcified material can generally take one or more of the following four forms : (a) mature lamellated trabecular bone ; (b) immature, highly cellular bone ; (c) circumscribed amorphous, almost acellular, eosinophilic, or basophilic bodies, and (4) minute microscopic granular foci of calcification. the nonulcerated lesions are typically identical to the ulcerated type except for the presence of surface epithelium. cementum - like material is found in less than one - fifth of the lesions and dystrophic calcifications are more prevalent in ulcerated lesions. treatment requires proper surgical intervention that ensures deep excision of the lesion including periosteum and affected periodontal ligament. thorough root scaling of adjacent teeth and/or removal of other sources of irritants should be accomplished. in children, reactive gingival lesions can exhibit an exuberant growth rate and reach significant size in a relatively short period of time. in addition, the pof can cause erosion of bone, can displace teeth, and can interfere or delay eruption of teeth. early recognition and definitive surgical intervention result in less risk of tooth and bone loss. it is suggested that there is no absolute histological distinction between bone and cementum, and as the so - called cementum - like globules of calcification are seen in fibro - osseous lesions in all membrane bones, it is unrealistic to separate the ossifying and cementifying lesions and it is speculated that the fibro - osseous lesions might represent stages in the evolution of a single disease process passing through the stages of fibrous dysplasia to ossifying fibroma to cementoid lesions. in conclusion, clinically it is difficult to differentiate between most of the reactive gingival lesions particularly in the initial stages. regardless of the surgical technique employed, it is important to eliminate the etiological factors and the tissue has to be histologically examined for confirmation.	the gingiva is often the site of localized growths that are considered to be reactive rather than neoplastic in nature. many of these lesions are difficult to be identified clinically and can be identified as specific entity only on the basis of typical and consistent histomorphology. peripheral ossifying fibroma is one such reactive lesion. it has been described with various synonyms and is believed to arise from the periodontal ligament comprising about 9% of all gingival growths. the size of the lesion is usually small, located mainly in the anterior maxilla with a higher predilection for females, and it is more common in the second decade of life. a clinical report of a 12-year - old girl with a large peripheral ossifying fibroma in the posterior maxilla showing significant growth and interference with occlusion is presented.
impaired sitting balance is a common problem for stroke patients1, yet a sitting position is essential for safe execution of a variety of movements2. several interventions have been devised to improve balance, including task - oriented training4 and treadmill training5. dean and shepherd demonstrated that task - oriented training was effective at improving reach while sitting6. recently, whole - body vibration (wbv) exercise has been developed as a new modality for physical therapy7. previous studies have suggested that wbv exercise increases muscle strength and improves muscular performance and balance8, 9, and the positive effects of wbv on muscle performance should help to improve balance10, 11. wbv acts through repetitive sensorimotor stimulation and therapies with wbv have been conducted for elderly patients as well as patients with cerebral palsy, multiple sclerosis, and stroke12,13,14,15. some authors have reported wbv training combined with other physical therapies improves balance16,17,18, and wbv was shown to positively influence the postural control and mobility of chronic hemiparetic stroke patients11. recently, studies have emphasized the importance of sitting balance for stroke patients and have recommended various balance training methods for its improvement. however, physical therapies for improved sitting balance are underpinned by little empirical evidence19. some studies have focused on the use of wbv, but no study has been conducted to verify the effects of wbv for stroke patients in the sitting position. therefore, we investigated the effects of task - oriented training with wbv on the sitting balance of stroke patients. we hypothesized that a group of stroke patients performing task - oriented training with wbv would show greater improvement of sitting balance than a control group that performing task - oriented training alone. the inclusion criteria were history and clinical presentation (hemiparesis) of stroke (> 6 month post - event) ; ability to sit independently for at least 10 minutes ; no participation in any balance training program during the previous six months ; no orthopedic problems, such as a fracture, deformity, or severe osteoarthritis ; and sufficient cognitive ability to participate in the training : korean version of mini - mental state examination (mmse - k) scores of 21 or higher. the exclusion criteria were comorbidity or disability other than stroke, and an uncontrolled health condition for which vibration is contraindicated. participation in the study was voluntary and the subjects fully understood the contents of this study. after providing an explanation of the study purpose, as well as the experimental method and processes, written informed consent to participation in the study was obtained from all the subjects. subjects sat, with their feet unsupported, on a wii balance board (nintendo, kyoto, japan) and were asked to keep their arms folded across their chests. their thighs were kept parallel, with 75% of their length supported on the wii balance board. data were acquired at 100 hz, and the mean value of three measurements collected over 30 seconds was used. cop total path length and average velocity were the outcome measures used in this study. this was performed using a level yardstick mounted on a wall at the height of each subject s acromion of the nonparetic side, while sitting on a chair with no back or arm rests. the subjects were seated with their hips, knees, and ankles positioned at 90 of flexion, with their feet positioned flat on the floor. three measurements were made under each of the following conditions : (1) sitting with the nonparetic side near the wall and leaning forward, (2) sitting with the back to the wall and leaning toward the nonparetic side, and (3) sitting with the back to the wall and leaning toward the paretic side. the subjects were instructed to lean as far as possible in each direction without rotating or touching the wall. if the patient could not raise the paretic arm, the distance covered by the acromion during leaning was used. thirty people fulfilled the inclusion criteria and voluntarily agreed to participate in this study. the participants were randomly assigned to the experimental group (n1 = 15) or the control group (n2 = 15). both groups performed one session (15 minutes) a day (5 days / week) of task - oriented training in the sitting position. the experimental group received wbv during task - oriented training (galileo pro ; novotec medical gmbh, germany). the control group performed only task - oriented training. the wbv frequency (1522 hz) and amplitude (05.8 mm) were adjusted relative to subjects physical abilities10. the four exercise tasks were (1) sitting alone at a table and correcting body alignment ; (2) reaching in different directions for objects located beyond arm s length using the nonparetic side ; (3) reaching in different directions for objects located beyond arm s length using the paretic side ; and (4) a bilateral reaching task, such as throwing a ball, lifting a box, and inserting a ring. each exercise session was 15 minutes in duration, and subjects practiced each of the four tasks for 3 minutes with a 1-minute rest in between. investigators supervised each session and were responsible for ensuring that the amount and intensity of the exercise at each exercise station was graded to each subject s level of functioning. the independent t - test was used to compare differences between group means and changes in values, and the paired t - test was used to test differences in continuous variables within groups. all patients completed the intervention and assessments. there were no significant differences in gender, paretic side, age, weight, height or duration of onset between the groups (table 1table 1.general characteristics of subjectsexperimental group(n1=15)control group(n2=15)gendermale / female9/67/8paretic sideright / left6/95/10age (year)62.89.0 65.115.7weight (kg)63.36.258.09.0height (cm)162.76.2161.16.5duration (month)13.05.412.65.7meansd). differences in static sitting balance are presented in table 2table 2.comparison of cop results between pre- and post - testexperimental group(n1=15)control group(n2=15)va (cm / s)pre3.00.2 3.00.3post2.90.23.00.3change0.60.10.10.1tl (cm)pre89.56.989.49.3post87.86.991.18.7change1.73.21.64.1va : velocity average of cop, tl : total path length of cop. there were no significant differences in the average velocity and total path length of cop sway between the groups. va : velocity average of cop, tl : total path length of cop. p<0.05 differences in dynamic sitting balance are presented in table 3table 3.comparison of mfrt results between pre- and post - testexperimental group(n1=15)control group(n2=15)mfrt - a (cm)pre26.09.9 21.011.4post32.18.023.510.7change6.15.92.44.2mfrt - n (cm)pre10.95.413.64.9post16.65.715.56.2change5.74.11.94.8mfrt - p (cm)pre9.94.410.75.6post13.23.912.15.6change3.43.41.34.2mfrt - a : modified functional reach test - anterior reach. non - paretic and paretic reach were significantly higher in the experimental group (p<0.05). after the intervention, anterior reach was significantly higher (p<0.05) in the control group. there were no significant differences in non - paretic and paretic reach in the control group. differences in total length of cop between pre- and post - intervention differed significantly between the two groups (p<0.05). in this study we investigated the effect of task - oriented training with wbv on the sitting balance of stroke patients. average velocity and total path length of cop sway were used to evaluate the static balance of the sitting position. force platforms have previously been used to investigate the balance control of unsupported sitting of post - stroke individuals through analysis of the cop sway21, 22. previous research reported that a leg exercise with wbv group and a leg exercise group without wbv showed similar improvements in the average velocity of cop in a standing position23 with no significant difference between them. these results were similar to our present results, as we found no significant differences in average velocity or total path length of cop sway between the groups. it is possible that the training was not sufficiently intense or long enough to elicit improvements in the subjects ; it is also possible that the sensitivity of the assessment method was insufficient. in the current study, the mfrt was used to evaluate improvements in dynamic balance in the sitting position. the average anterior and lateral reach distances of adults between 60 and 70 years old are 346 mm and 206 mm, respectively24. in our study, the anterior, nonparetic, and paretic reach of both groups were below the normal averages at pretest, indicating that both groups had impaired sitting balance. however, at posttest, the experimental group showed significant improvements (p<0.05) in the anterior, nonparetic, and paretic reach, demonstrating that task - oriented training with wbv in a sitting position is a useful intervention for improving the dynamic sitting balance of stroke patients. vibration is a useful method for stimulating proprioception and is capable of long - lasting postural improvement25. this demonstrates that task - oriented training is a useful intervention for improving anterior reach while sitting. in the experimental group, our study suggests that task - oriented training with wbv in the sitting position has beneficial effects on some aspects of sitting balance of chronic stroke patients. we anticipate that this training method will be used in physical therapy at stroke patient care centers as it is an effective form of training for balance functions. further research is needed with larger numbers of subjects to confirm and generalize our present findings.	[purpose ] the purpose of this study was to determine the effects of task - oriented training with whole body vibration (wbv) on the sitting balance of stroke patients. [subjects ] the subjects were 30 stroke patients who were randomly divided into experimental (n1=15) and control (n2=15) groups. [methods ] subjects in both groups received general training five times per week. subjects in the experimental group practiced an additional task - oriented training program with wbv, which was performed for 15 minutes, five times per week, for four weeks. the center of pressure (cop) path length and average velocity were used to assess subjects static sitting balance, and the modified functional reach test (mfrt) was used to assess their dynamic sitting balance. the paired t - test was performed to test the significance of differences between before and after the intervention. the independent t - test was conducted to test the significance of differences between the groups. [results ] following the intervention, the experimental group showed a significant change in mfrt. [conclusion ] the results of this study suggest that task - oriented training with wbv is feasible and efficacious for stroke patients.
lo sr decade in y emettendo particelle beta, con un tempo di dimezzamento pari a 28.5 anni ed una energia massima di emissione pari a 546 kev. tale decadimento caratterizzato da un tempo di dimezzamento pari a 64.1 ore e da una energia massima di emissione pari a 2284 kev (stamoulis., 2007). caratteristica molto importante dal punto di vista analitico la capacit di sr e y di raggiungere il cosiddetto equilibrio secolare ovvero lo stato in cui le concentrazioni di attivit dei due isotopi sono equivalenti. essendo lenergia massima di emissione delly circa 5 volte maggiore rispetto a quella dello sr, risulta favorevole la sua determinazione, in luogo di quella del radioisotopo progenitore, con evidente guadagno in sensibilit analitica (delloro., 2013). losr considerato un pericoloso contaminante delle filiere agroalimentari in quanto, possedendo una spiccata affinit chimica con il calcio, pu essere rapidamente assorbito e si deposita nelle ossa nelle quali viene trattenuto (torres., 2002). le problematiche di carattere sanitario connesse allaccumulo di sr nel tessuto osseo umano ed animale consistono nella possibile comparsa di osteosarcoma ed altre neoplasie. inoltre, questo radionuclide pu influenzare negativamente alcuni processi enzimatici e di trasporto nei quali coinvolto il calcio (bronner., 1963). relativamente alla possibilit di accumulo di sr nelle materie prime per mangimi e quindi nei prodotti finiti, tale rischio risulta particolarmente rilevante in relazione alla capacit di trasferimento del radionuclide fino ai prodotti alimentari di origine animale. nelle zone particolarmente contaminate, il passaggio di radionuclidi (cs e sr) dallambiente ai foraggi e da questi ai prodotti di origine animale (latte e carni bovine e suine) stato pi volte messo in evidenza da diversi studi effettuati dal disastro di chernobyl ad oggi (annenkov e averin, 2011). nelle stesse condizioni di contaminazione radioattiva laccumulo di sr risulta molto maggiore rispetto a quello relativo al cs. in uno studio, pubblicato nel 1998 (fesenko., 1998), sono stati definiti inoltre dei coefficienti di passaggio piante - foraggio, sia per il cs che per lo sr, espressi come bq / kg di piante / kbq / m. per lo sr tali coefficienti variano da 4.52 per il frumento invernale a 8.50 per il mais, fino a 11.63 per le erbe graminacee - leguminose annue da foraggio. in condizioni di contaminazione media corrispondente a circa 1 kbq / m questi coefficienti di accumulo portano ad una concentrazione di attivit di sr, per singola unit foraggera, variabile da 1.3 bq per lorzo ed il grano, a 8.45 bq per linsilato di mais, fino a 33.7 bq per le erbe graminacee leguminose pluriennali. dal punto di vista della contaminazione della filiera agroalimentare, la materia prima che risente maggiormente della contaminazione da sr senza dubbio il latte. questo dovuto, come gi accennato in precedenza, alla spiccata similarit chimica di questo radioisotopo con il calcio, elemento molto abbondante nel latte. diversi studi hanno dimostrato che il passaggio di sr nel latte indipendente dal tipo di mangime utilizzato (fieno, leguminose, cereali) e che il contenuto finale pu variare dallo 0.2 al 6.2% (annenkov, 1964). per questo motivo, il regolamento (euratom) no 2218/89 della commissione europea prevede controlli ufficiali sulla radiocontaminazione del latte, in caso di incidenti nucleari, e stabilisce i massimi limiti consentiti per la contaminazione da sr. tali limiti corrispondono a 125 bq l (o bg kg) per il latte ed i prodotti lattiero caseari ed a 75 bq l per gli alimenti per linfanzia (compreso il latte) (commissione europea, 1989). laccumulo di radiostronzio nei muscoli e negli organi risulta invece molto meno rilevante, dal momento che dopo la penetrazione nel sangue la maggior parte dei radionuclidi viene rimossa mediante gli organi escretivi o accumulata nello scheletro (iammarino., le concentrazioni residue nel muscolo risulteranno dunque esigue e variabili tra 0.011 bq kg per i bovini a 0.03 bq kg per i suini. per quanto riguarda le percentuali di accumulo nelle uova, essendo queste un prodotto di organi di escrezione, tali valori risultano non trascurabili. la percentuale di accumulo di sr nelle uova pu raggiungere l1.4% del contenuto totale della razione giornaliera. in questo caso il potenziale di accumulo dello sr risulta inferiore (circa la met) rispetto a quello del attualmente, il regolamento com (2010)184 definitivo 2010/0098 della commissione europea non fissa dei limiti massimi consentiti per la contaminazione da sr nei mangimi, definendo solo quelli relativi al cs. tali limiti sono stati invece definiti in altre regolamentazioni internazionali, come il regolamento rdu-99 che in bielorussia fissa alcuni limiti, come ad esempio 260 bq kg nel fieno, 185 bq kg nella paglia, 100 bq kg nellerba e nel grano da foraggio, 50 bq kg nellinsilato, 137 bq kg nei tuberi e 37 bq kg nellerba verde (repubblica della bielorussia, 2001). risulta evidente, per quanto detto sinora, che il monitoraggio della contaminazione da sr dei mangimi deve essere considerato un importante compito per gli organi preposti ai controlli sulla salute animale e sulla sicurezza alimentare. da un punto di vista metodologico, la determinazione di sr nelle matrici solide ha sempre presentato numerose problematiche analitiche, inerenti soprattutto la separazione radiochimica, dovute alla presenza di diversi cationi dalle caratteristiche chimicofisiche simili allo sr (ca, pb, bi, etc.) inoltre, non sono mai state definite e sviluppate le necessarie procedure di validazione del metodo (maxwell iii e culligan, 2009 ; brun., 2003 ; heilgeist, 2000 ; kabai., 2011 ; kim., 2009 ;, 2007 ; wilken e joshi, 1991 ; vajda e kim, 2010). nel presente lavoro viene, pertanto, presentata lattivit di sviluppo e validazione di un metodo radiochimico per la determinazione di sr in varie tipologie di mangime per lapplicazione di questa tecnica nelle attivit routinarie di controllo. la metodica analitica impiegata stata la scintillazione liquida ad ultra basso fondo (lsc), tecnica che consente di effettuare la determinazione di concentrazioni di attivit di sr al di sotto di 0.01 bq kg, dopo opportuno trattamento del campione (estrazione / purificazione / separazione del radioisotopo) e raggiungimento dellequilibrio secolare sr / y. laffidabilit di questa procedura analitica stata verificata mediante procedure di validazione implementate seguendo un protocollo sviluppato in - house, che consente di determinare le pi importanti performances analitiche in accordo con gli attuali riferimenti legislativi (commissione europea, 2002, 2004). del campione ne vengono inceneriti 250 g in muffola secondo la rampa di temperatura mostrata in figura 1. le ceneri vengono disciolte in una miscela hno3 8m / hf al 50% e viene aggiunto 1 ml di carrier (sr stabile 10,000 mg l). viene effettuata una lisciviazione della miscela a 300c, quindi una filtrazione e laggiunta di acido ossalico anidro e sodio acetato. laggiunta di acido ossalico consente di precipitare lo stronzio presente nel campione come ossalato e di separarlo da altri metalli affini, una volta corretto il ph della soluzione a 4.5 mediante aggiunta di nh4oh al 30%. il precipitato viene quindi disciolto con una miscela h2o2/hno3 8 m e la soluzione viene portata a secco. il residuo viene disciolto in hcl 0.5 m e si aggiungono altri 4 volumi di h2o. successivamente viene effettuata la rimozione di altri due importanti interferenti radiometrici, il pb ed il bi mediante precipitazione, sotto forma di solfuro, aggiungendo 1 ml dei rispettivi standard di carrier stabili (10,000 mg l) e circa 50 mg di sodio solfuro. ii precipitato formatosi viene rimosso mediante filtrazione con carta bibula, il ph del filtrato viene corretto ad 1.0 per rimuovere ly eventualmente presente, mediante complessazione con acido etil - esil ortofosforico (hdehp), effettuando una doppia aggiunta di una soluzione di hdehp al 20% in toluene, agitazione vigorosa ed eliminazione della fase organica. la fase acida viene portata a volume (200 ml) con hcl 0.1 m e posta a temperatura ambiente fino al raggiungimento dellequilibrio secolare sr / y (min. 15 viene aggiunto 1 ml di carrier yttrio stabile 10000 mg l, il ph della soluzione viene corretto a 1.0 e si procede con la separazione delly eventualmente presente mediante complessazione con hdehp, ovvero aggiungendo 200 ml di una soluzione di hdehp al 5% in toluene, agitando vigorosamente e recuperando la fase organica che viene quindi lavata due volte con hcl 0.1 m. lestrazione finale delly dalla soluzione si ottiene mediante separazione liquidoliquido utilizzando due aliquote da 150 ml di hno3 3 m. ly, estratto nella fase acida, viene quindi precipitato come ossalato aggiungendo acido ossalico all8%, correggendo il ph a 2.5 mediante aggiunta di nh4oh al 15% e riscaldando la soluzione fino ad ottenere abbondante precipitato bianco. il precipitato viene filtrato, disciolto in una miscela hno3/h2o2, portato a secco, quindi ridisciolto con hcl ed essiccato nuovamente. il residuo finale viene ripreso con 8 ml di acido cloridrico 0.1 n, trasferito in apposita vial dove viene aggiunto il cocktail di scintillazione (ultima gold ab ; perkin elmer, waltham, ma, usa). la lettura lsc stata effettuata mediante scintillatore liquido ad ultra basso fondo (wallac 1220 quantuls ; perkin elmer) impostato con un tempo di conteggio di 1000 minuti. il metodo stato sottoposto ad una procedura di validazione su matrici alimentari solide (carni fresche e prodotti della pesca), implementata seguendo un approccio validativo in - house per verificare laffidabilit del metodo allo scopo preposto. sono stati verificati i seguenti parametri : selettivit, linearit, precisione, recupero, efficienza di conteggio, robustezza ed incertezza di misura. il decision threshold e il detection limit sono stati calcolati in accordo con la norma iso 11929:2010 (iso, 2010) ; la selettivit stata valutata mediante prove ripetute di fortificazione e verificando la forma dello spettro ed i conteggi totali ; la linearit stata verificata mediante conteggio di tre sorgenti di attivit pari a 1.5, 95.48 e 494,40 bq ; laccuratezza stata verificata effettuando prove di fortificazione ripetute (n=6) in condizioni di ripetibilit intermedia, a due livelli di concentrazione di attivit pari a 0,5 e 1.0 bq kg ; infine, lefficienza di conteggio e la minima concentrazione di attivit rivelabile sono state valutate trattando tre sorgenti di sr, di attivit pari a 1.0 bq, con la stessa procedura di separazione dellyttrio utilizzata per i campioni (iammarino., 2012). al fine di estendere il campo di applicazione ai mangimi, e considerando che tale tipologia di campione caratterizzata da concentrazioni di attivit di sr non trascurabili, 6 tipologie diverse di mangime e materie prime per mangimi sono stati analizzate ; quindi, sugli stessi campioni, sono state effettuate prove di fortificazione a 1.0 bq kg. il metodo cos ottimizzato e validato stato impiegato per condurre un monitoraggio della contaminazione da sr su 32 campioni di foraggi e mangimi (18 insilati, 7 fieni e 7 mangimi). il metodo radiochimico validato in questo studio ha mostrato diversi aspetti che lo rendono preferibile rispetto ad altri attualmente disponibili (maxwell iii e culligan, 2009 ; brun., 2003 ; heilgeist, 2000 ; kabai., 2011 ; kim., 2009 ; stamoulis., 2007 ; wilken e joshi, 1991 ; vajda e kim, 2010). la specifica serie di precipitazioni e purificazioni garantisce la separazione radiochimica dello sr da altri isotopi ed interferenti ; inoltre, la tecnica lsc consente di registrare spettri sia di particelle alfa che beta con una efficace discriminazione delle sorgenti di emissione. la tecnica lsc fornisce, inoltre, valori di efficienza di conteggio (0.87), di decision threshold (0.003 bq kg) e di detection limit (0.008 bq kg) pi efficienti rispetto alle tecniche tradizionali. in figura 2 mostrato il caratteristico spettro di emissione beta dellyttrio-90. altri importanti parametri di validazione (tabella 1) che caratterizzano il metodo sono la linearit (r=0.9987), il recupero medio (92 e 89%), il coefficiente di variazione percentuale (14 e 15%) per livelli di additivazione pari rispettivamente a 0.5 bq kg e 1.0 bq kg, lincertezza di misura, pari al 16.0% e la robustezza, valutata in condizioni di major changes, ovvero variando la matrice da analizzare, caratterizzata da un campo di applicazione composto da carni fresche (bovine, suine ed ovi - caprine), prodotti carnei (insaccati e prosciutti), prodotti ittici (molluschi bivalvi, cefalopodi e teleostei), prodotti lattiero caseari (mozzarella, ricotta e formaggi a breve e lunga maturazione), grano e prodotti derivati del grano (pane e pasta). importante ricordare che, al fine di verificare ulteriormente laccuratezza del metodo stato analizzato un materiale di riferimento (iaea 152 : coriandolo). tale materiale di riferimento, caratterizzato da un valore certificato di concentrazione di attivit dello sr pari a 7,7 bq kg alla data del 31 agosto 1987, ha fatto registrare un valore medio di concentrazione di attivit di sr pari a 7.161.67 bq kg (p=95%, k=2). tenuto in considerazione lintervallo di accettabilit, dichiarato sullo stesso materiale di riferimento certificato (7.08.3 bq kg ad un livello di confidenza del 95%), si potuta confermare lattendibilit dellintera procedura analitica. la sessione supplementare di validazione, relativa allestensione del campo di applicazione ai mangimi, ha portato ai risultati riassunti nella tabella 2. le contaminazioni registrate nelle varie tipologie di mangime sono risultate molto variabili (da 0.23 bq kg relativa al mangime per conigli, a 1.61 bq kg relativa al mangime complementare minerale), tuttavia non possibile trarre conclusioni relativamente alla relazione tra entit della contaminazione e tipo di mangime a causa del basso numero di campioni analizzati non statisticamente significativo. futuri studi, gi in fase di progettazione, andranno ad approfondire e definire leventuale specificit nella contaminazione da sr nelle varie tipologie di mangime. per quanto riguarda le prove di fortificazione, i risultati hanno confermato lestrema versatilit e robustezza del metodo validato. la percentuale media di recupero risultata infatti pari al 90% (recupero medio di validazione al livello 1.0 bq kg=89%) con un coefficiente di variazione percentuale pari a 14.3% (cv% di validazione al livello 1.0 bq kg=15%). in figura 3 sono riportati i confronti tra gli spettri relativi alle varie tipologie di mangime testate, analizzate prima e dopo la fortificazione con sr. il monitoraggio su campioni di foraggi e mangimi ha confermato la necessit di un controllo radiochimico, soprattutto per quanto concerne le materie prime, in quanto su tutti i campioni stata verificata una contaminazione da sr ben superiore al detection limit (0.008 bq kg) del metodo (tabella 3). in particolare, i campioni di fieno hanno fatto registrare la concentrazione di attivit media di sr pi elevata (2.93 bq kg) ; questo in accordo con quanto definito dal regolamento bielorusso rdu-99 che fissa il limite massimo di contaminazione a 260 bq kg nel fieno ed a 50 bq kg nellinsilato. tale netta differenziazione nella stima del limite massimo di contaminazione, probabilmente, dovuta alla percentuale di sostanza secca che caratterizza queste due tipologie di matrice, ovvero 85% circa nel fieno e 40% circa nellinsilato. la concentrazione di attivit media registrata nei mangimi risultata la pi bassa (0.77 bq kg) ed anche questo dato sostanzialmente in accordo con quanto ad oggi disponibile in letteratura. infatti, attualmente, non sono stati definiti limiti massimi ammissibili per la contaminazione da sr nei mangimi e ci essenzialmente dovuto al fatto che, secondo i dati presenti in letteratura scientifica, questa matrice infine, grazie allanalisi statistica dei dati ottenuti (f - test a due campioni per varianze), stato possibile verificare una omogeneit dei dati tra i campioni di fieno e quelli di insilato, mentre i campioni di mangimi sono risultati contaminati a livelli statisticamente inferiori. il metodo radiochimico descritto in questo studio, validato per la determinazione di radionuclidi beta emittenti (sr) nei mangimi presso il centro di referenza nazionale per la ricerca della radioattivit nel settore zootecnico - veterinario (foggia), ha mostrato diversi aspetti (selettivit, sensibilit e robustezza) che lo rendono preferibile rispetto ad altri attualmente disponibili. inoltre, le prove di validazione hanno permesso di verificare lidoneit allo scopo di tale metodica grazie alla valutazione delle pi importanti performance analitiche : selettivit, sensibilit, linearit, accuratezza e robustezza. per quanto concerne il monitoraggio su campioni di foraggi e mangimi, tale lavoro ha permesso di confermare la necessit di controlli di tipo radiochimico, relativi alla contaminazione da sr, soprattutto per quanto concerne le materie prime. infatti su tutti i campioni stata verificata una contaminazione da sr ben superiore al detection limit (0.008 bq kg) del metodo. in particolare, i campioni di fieno hanno fatto registrare la concentrazione di attivit media di sr pi elevata (2.93 bq kg), seguita da quella relativa ai campioni di insilato (2.07 bq kg) e da quella relativa ai campioni di mangime (0.77 bq kg).	90sr is considered as a dangerous contaminant of agri - food supply chains due to its chemical affinity with calcium, which makes its absorption in bones easy. 90sr accumulation in raw materials and then in final products is particularly significant in relationship to its ability to transfer into animal source products. the radionuclides transfer (137cs and 90sr) from environment to forages and then to products of animal origin (milk, cow and pork meats) was studied and evaluated in different studies, which were carried out in contaminated areas, from chernobyl disaster until today. in the present work, the development and validation of a radiochemical method for the detection of 90sr in different types of animal feed, and the application of this technique for routinely control activities, are presented. liquid scintillation counting was the employed analytical technique, since it is able to determine very low activity concentrations of 90sr (< 0.01 bq kg1). all samples analysed showed a 90sr contamination much higher than method detection limit (0.008 bq kg1). in particular, the highest mean activity concentration was registered in hay samples (2.93 bq kg1), followed by silage samples (2.07 bq kg1) and animal feeds (0.77 bq kg1). in fact, all samples were characterized by 90sr activity concentrations much lower than reference limits. this notwithstanding, the necessity to monitor these levels was confirmed, especially considering that 90sr is a possible carcinogen for human.
tetanus is a disease characterized by hypertonia and muscular spasms due to the toxin tetanospasmin formed by clostridium tetani. tetanus continues to be widespread, especially in the developing world though incidences have come down due to immunization. we present a case of tetanus in a young girl which initially mimicked a striatal toe though the potential cause of inoculation was trauma to nose. a 16-year - old girl presented with a history of sudden onset of pain in left leg with difficulty in walking for 2 days. an upturned big toe without fanning of other toes was seen on the left [figure 1 ]. a clinical diagnosis of a striatal toe was made and the patient was admitted for further evaluation [figures 1 and 2 ]. contrast - enhanced magnetic resonance imaging of the brain revealed no abnormality of the basal ganglia. liver function test was normal and slit lamp examination did not show a kf ring. corrected serum calcium was 9.8 mg / dl. on the 3 day after admission, she developed stiffness of bilateral lower limbs followed by episodic spasms of the back and neck. there were no signs of autonomic hyperactivity and she did not have any seizures. on further probing, patient 's mother revealed that she had suffered a fall a day before onset of her symptoms and hurt her nose. her mother said that she had received her primary series of tt in infancy and received her last booster 6 years back at around 10 years of age. she was given tetanus immunoglobulin (tig) 5000 units intramuscularly stat and also started on intravenous metronidazole and diazepam infusion. on the 5 day after admission, she developed mild trismus and opisthotonus completing the clinical picture of generalized tetanus. she started showing signs of recovery by day 10 and made a complete recovery in 3 weeks from onset of symptoms. tetanus is caused by the toxin tetanospasmin released by the germinating spores of the anaerobic gram - positive bacilli c. tetani. as spores of the bacilli occur in the soil, tetanus frequently occurs following a contaminated wound, but cases have also been reported after dental procedures, surgeries, burns, intravenous drug abuse, and intramuscular injections. our patient presented with a localized form of tetanus affecting initially the left leg that mimicked a striatal toe even though there was no local injury in the leg. it is possible that our patient had another insignificant trauma to her leg that had gone unnoticed. a striatal toe is a finding in clinical neurology that reliably localized the lesion to the caudate nucleus and putamen. the absence of fanning of the rest of the toes differentiates the upturned big toe from the babinski extensor plantar response. tetanus presenting initially with the classic clinical picture of striatal toe has not been reported before. thus, it is important to keep localized tetanus in the differential diagnosis of extrapyramidal disorders presenting with such spasticity, especially in the absence of definite history of trauma. dystonias can be ruled out in such cases by checking for resolution after a dose of benztropine. severity of tetanus can be graded according to the ablett classification into four grades. according to the ablett classification, the world health organization recommends a 5 dose schedule for tetanus vaccination while in india additional doses are recommended in the universal immunization program (uip). our patient had received three doses as dtp in infancy and another dose between 12 and 24 months of age. she also had received two booster doses of dtp and td at 5 years and 10 years according to her mother as per the uip. the advisory committee on immunization practices of the centers for disease control does not recommend either td vaccination or tig for patients who have received more than three doses of tt with the last dose within 5 years. td is recommended in clean wounds with last booster > 10 years back and other wounds with last booster > 5 years back. tig should be given in unclean wounds when vaccination status is unknown or patient has received 6 h old, > 1 cm deep, stellate, ischemic, denervated, contaminated or infected. our patient had received tt immunization at the time of primary wound care as per the recommendations. she did not receive tig as she had received more than 3 doses of immunization in the past. identification of vulnerable patients by testing for protective tetanus antibodies is not viable in our setting. this may be due to a defect in the patients humoral immunity due to which she could nt mount an antibody response. rapid bedside tests to screen for tetanus immunization status have been found to be more effective than medical interview and wound assessment in some studies, but cost effectiveness remains an issue. wound care, antimicrobial therapy with metronidazole and human tig are cornerstones of therapy of established tetanus. a full course of active immunization with tt should be started early as neither clinical infection nor tig provide long - term immunity. cases of tetanus continue to come to medical attention in spite of widespread immunization in this age and time. we must be alert to the possibility and institute prompt treatment after a clinical diagnosis as early passive immunization reduces morbidity and mortality.	we report the case of a 15-year - old girl who was initially diagnosed to have a striatal toe. her condition progressed and she later developed clinical features consistent with tetanus. history of blunt trauma to nose was elicited retrospectively. antimicrobial therapy with metronidazole and both active and passive immunization was started immediately. the patient went on to make a complete recovery.
so - called scent glands (syn. defensive glands) are not only considered an important synapomorphic character of all harvestmen (opiliones), but also represent by far the largest exocrine system in this arachnid order. generally, these glands constitute paired sacs in the opilionid prosoma, opening onto the body surface via one large orifice (ozopore) on each of the lateral margins of the carapax dorsal to the coxae of legs i or ii. their main biological role is thought to be predator defense (e.g., martens, 1978), but scent glands also are sites of production of antimicrobial agents (e.g., fieser and ardao, 1956) or, in certain species, may emit pheromones (holmberg, 1986 ; machado., 2002). moreover, opilionid scent glands are the sources of a diversity of rare natural products, and the chemistry of their secretions is characteristic for specific opilionid groups. for example, the secretions of cyphophthalmi are complex and contain specific blends of naphthoquinones and methyl ketones (raspotnig. hitherto known secretions of the palpatores include acyclic ketones, alcohols, and aldehydes, present in some sclerosomatid eupnoi (blum and edgar, 1971 ; meinwald., 1971 ; jones., 1976, 1977 ; ekpa., 1985), and naphthoquinones reported from one species of phalangiid eupnoi (wiemer., 1978 only one study is available on scent gland secretions of the dyspnoi, demonstrating the presence of naphthoquinones, methoxy - naphthoquinones, and anthraquinones in the scent glands of a nemastomatid species (raspotnig., 2010). however, with respect to the number of species investigated, secretions of the mostly tropical laniatores appear to be best studied. laniatorean secretions are chemically distinct from the secretions of cyphophthalmids and palpatoreans, and specific patterns of phenols and benzoquinones have been demonstrated from a growing number of species since the 1950s (e.g., estable., 1955 ; eisner., 1971, 1977, 2004 ; roach., 1980 ; hara., 2005 ; review by gnaspini and hara, 2007 ; shear., 2010a, b ; pomini., 2010 ; fttinger., essentially, nearly all published studies deal with representatives of groups of more - derivative laniatores. thus, the current knowledge on laniatorean secretions is strongly biased in favor of the grassatores. a second, more basal laniatorean group is almost unknown, but is crucial for a consistent picture of opilionid scent gland chemistry. only one example of insidiatorean chemistry has been published so far (ekpa., 1984), revealing a series of completely aberrant components from an american travunioid species, sclerobunus robustus. these compounds include terpenes, such as bornyl esters, camphene, and limonene, and two alkaloids, namely n, n - dimethyl - - phenylethylamine and the tobacco alkaloid nicotine. one important and well recognized taxon within the insidiatores is the cladonychiidae (superfamily travunioidea), including about 15 species in 5 extant genera (a 6th genus was described from baltic amber by ubick and dunlop in 2005). while genera cryptomaster, erebomaster, speleomaster, and theromaster are restricted to the northern united states, an exclusively european clade is represented by eight species of holoscotolemon, occurring from western europe to romania and southwards to serbia and montenegro. as a first contribution to scent gland research in european insidiatores, we here report on the scent gland chemistry of four species of holoscotolemon, covering a representative part of european cladonychiidae. collection of species specimens of holoscotolemon jaqueti (corti, 1905), h. lessiniense martens, 1978, h. oreophilum martens, 1978, and h. unicolor roewer, 1915, were collected by hand under stones and litter, and from samples of leaf litter by means of a soil sifter. while h. jaqueti is a species with a disjunct south - east european and carpathian distribution (hungary, slovakia, romania, ukraine, bosnia and hercegovina, serbia), h. unicolor is restricted to the eastern alps (austria, slovenia, north eastern italy), and both h. lessiniense and h. oreophilum are local endemics of the southwestern alps (italy). holoscotolemon oreophilum can be found in a small area in the sea alps / alpes maritimes and ligurian alps in italy, and h. lessiniense inhabits the monti lessini close to the lake garda in the southern alps. the investigated material was collected in serbia (h. jaqueti), italy (h. oreophilum, h. lessiniense), and austria (h. unicolor) (table 1). for determination of the holoscotolemon species, we used descriptions of martens (1978). komposch (graz), g. raspotnig (graz), and i. karaman (novi sad). table 1collection of holoscotolemon - species and preparation of extractsspecieslocation (co - ordinates, altitude)dateextracts no.(individuals)h. jaquetiserbia : ovar - kablar gorge, above ovar banja (4353n, 2011e, 375 m)20 mai 20091754 (1 juv), 1755 (1), m430 (1 juv)h. oreophilumitaly : alpi marittime, coli de tende s limonette (4409 n, 732 e, 1,475 m)29 july 20102447 (1), 2448 (1)h. lessini, monte pasubio (4546 n, 1107 e, 760 m)17 july 2009m353 (1), m354 (1), m355 (1), m356 (1)italy : mt. lessini, nne verona, s velo veronese (4535n, 1104 e, 920 m)31 july 20102449 (1)h. unicoloraustria : carinthia, villach, eichholzgraben (4638 n, 1350 e, 590 m)5 april 20101935 (1), 1936 (1)austria : styria, graz, maria trost (47 06 n, 15 29 e, 445 m)20 april 20101973 (1 juv), 1983 (3 juv)25 april 20101995 (3 juv)10 may 20102000 (2 juv), 2001 (2 juv)austria : styria, aibl (46 41 n, 15 13 e, 400 m)27 june 20102127 (1 juv)austria : carinthia, soboth, road between krumbach and rothwein (4681 n, 1507 e, 1,000 m)23 june 20102243 (1), 2244 (1), 2245 (1)refers to intern labelling of extracts collection of holoscotolemon - species and preparation of extracts refers to intern labelling of extracts extraction and analysis of secretions scent gland secretion was obtained by whole body extraction of individuals in 150 l of hexane or ethyl acetate for about 30 min as previously described and standardized for other opilionids (raspotnig. 21 extracts were prepared, mostly extracts of single individuals (adults), but in some cases also pooled extracts (juveniles), as summarized in table 1. aliquots of extracts (2 l) were subject to gas chromatographic - mass spectrometric (gc - ms) analysis, using a trace gc2000 coupled to a voyager ms, both from thermo (vienna, austria). the gc was equipped with a zb-5ms fused silica capillary column (30 m 0.25 mm i.d. injection was splitless with helium (at 1.5 ml / min) as carrier gas. the column temperature was programmed from 50c (held for 1 min) to 200c at 10c / min, and then to 300c at 15c / min. the ion source of the ms and the transfer line were kept at 170c and 310c, respectively. electron impact (ei) reference compounds authentic nicotine (mixture of (r)- and (s)-nicotine) for comparison of gc - ms data was purchased from sigma (vienna, austria). as a reference for authentic anabaseine, we used anabasine (from sigma, vienna, austria), which also contained a small amount of anabaseine (about 1%). quantification of secretion components determination of absolute amounts of nicotine in secretions are based on the integration of peak areas in the chromatograms, and comparisons to a calibration curve established for authentic nicotine. scanning electron microscopy for scanning electron microscopy (sem), specimens were fixed in bouin, washed, dehydrated, air - dried, and mounted on aluminum stubs prior to sputter coating with gold (agar sputter coater, grpl, tulln, austria). micrographs (sem) were taken with a philips xl30 esem (philips / fei, vienna, austria) at high vacuum mode and 20 kv accelerating voltage. chemical identification of extract components gc - ms analyses of extracts of adult holoscotolemon jaqueti, h. oreophilum, and h. lessiniense revealed two major (components a and e), and three minor compounds (components b, c, and d) (fig. 1). all components were identified as pyridine alkaloids as outlined in the following and as summarized in table 2. compound a [nicotine ], compound b [3-(1-methyl-2-piperidinyl)-pyridine ], compound c [anabasine ], compound d [2,3-bipyridyl ], compound e [anabaseine ]. compounds at rt = 13.56 min and rt = 14.55 min were found inconsistently in extracts of holoscotolemon lessiniense, and possibly represent isomeric, not fully characterized pyridines with a molecular weight of m = 174table 2gas chromatographic and mass spectral data to components from extracts of holoscotolemon lessiniense, h. oreophilum, and h. jaquetipeakretention time (min)ei - fragmentation (m / z)identified asa10.54162 (m, 49), 161 (46), 133 (79), 119 (24), 118 (16), 92 (23), 84 (100), 65 (21), 51 (22), 42 (61)nicotine = (s)-3-(1-methyl-2-pyrrolidinyl)-pyridineb11.85176 (m, 12), 175 (7), 147 (7), 133 (9), 119 (24), 98 (100), 42 (28)3-(1-methyl-2-piperidinyl)-pyridinec12.29162 (m, 26), 161 (23), 133 (45), 119 (45), 106 (49), 105 (60), 92 (19), 84 (100), 80 (22), 78 (20), 57 (29), 51 (28), 41 (38)anabasine = 3-(2-piperidinyl)-pyridined12.89157 (12), 156 (m, 100), 155 (71), 130 (20), 128 (12), 104 (7), 79 (13), 78 (22), 51 (13)2,3-bipyridinyl = 2-(3-pyridinyl)-pyridinee13.35161 (12), 160 (m, 97), 159 (100), 145 (46), 132 (14), 131 (79), 118 (8), 105 (44), 104 (76), 78 (27), 77 (29), 51 (36), 41 (29)anabaseine = 3,4,4,6-tetrahydro-2,3-bipyridinetentatively identified on the basis of mass spectral data chemical profiles of scent gland secretions in three species of holoscotolemon. compound a [nicotine ], compound b [3-(1-methyl-2-piperidinyl)-pyridine ], compound c [anabasine ], compound d [2,3-bipyridyl ], compound e [anabaseine ]. compounds at rt = 13.56 min and rt = 14.55 min were found inconsistently in extracts of holoscotolemon lessiniense, and possibly represent isomeric, not fully characterized pyridines with a molecular weight of m = 174 gas chromatographic and mass spectral data to components from extracts of holoscotolemon lessiniense, h. oreophilum, and h. jaqueti tentatively identified on the basis of mass spectral data major components component a showed an ei - mass spectrum consistent with that of nicotine [3-(1-methyl-2-pyrrolidinyl)-pyridine ], exhibiting a molecular ion (m) at a mass charge / ratio (m / z) of 162, loss of a hydrogen atom (m / z 161), and a base ion at m / z 84. the base ion corresponded to loss of the pyridyl moiety (m 78). further fragments were observed at m / z 133 (m 29) and m / z 119 (m 43), both of which are reported as characteristic of nicotine, and correspond to losses of c2h5- and c3h7-radicals from the molecular ion by different processes of rearrangement (duffield., 1965). as a reference for comparison of gc retention, we used a racemic mixture of nicotine (nicotine has an asymmetric carbon atom in position 2 of the pyrrolidine - ring). the specific enantiomers, however, were not separable due to the chromatographic conditions used, and both enantiomers eluted as a single peak at rt = 10.54 min, exactly matching the retention time of component a. thus, we did not determine the chirality of nicotine from holoscotolemon spp. so far, only the (s)-enantiomer is known to occur in nature.the ei - mass spectrum of the second major component (e) had a molecular ion at m / z 160, again exhibiting loss of hydrogen (base ion at m / z 159), and losses of 15 (leading to the ion at m / z 145), 29 (m / z 131), and 56 (m / z 104) mass units. the spectrum appeared to be similar, but not fully consistent, with reported spectra of anabaseine (a nicotine - related tobacco nicotinoid) from the literature (e.g., wheeler., 1981). however, a comparison of mass spectral fragmentation and retention times to an authentic sample of anabaseine [3,4,5,6-tetrahydro-2,3-bipyridine ] showed full correspondence. minor components on the basis of mass spectral fragmentation patterns of components b and c, two further nicotinic alkaloids were indicated, both showing mass spectra similar to that of nicotine : component b appeared to be a higher homolog to nicotine with m at m / z 176 and a base ion at m / z 98, suggesting a piperidine - structure instead of the pyrrolidine - ring. the compound was tentatively identified as 3-(1-methyl-2-piperidinyl)-pyridine by mass spectral comparisons to literature, and to spectra from the nist - library. the mass spectral fragmentation of component c differed from that of nicotine mainly in the presence of prominent twin ions at m / z 105 and m / z 106, being characteristic of anabasine [= 3-(2-piperidinyl)-pyridine ]. the compound was identified by comparisons of spectra and retention times with an authentic sample of anabasine. only the mass spectrum of component d was clearly different from all other spectra, although again showing an intense molecular ion at m / z 156 (base ion) along with loss of hydrogen (m / z 155). the isotopic m + 1-ion (about 12% relative intensity) still indicated the presence of 10 carbon and 2 nitrogen atoms, suggesting a molecular formula of c10h8n2, and thus corresponding to the fully aromatic homolog of anabasine and anabaseine, 2,3-bipyridyl [2-(3-pyridinyl) pyridine ]. subsequent mass spectral comparisons confirmed a mass spectral fragmentation pattern indistinguishable from spectra of authentic 2,3-bipyridyl from literature and from the nist - library. extract profiles two different extract patterns were clearly distinguished : (1) nicotine - rich and (2) anabaseine - rich extracts. the extracts of holoscotolemon oreophilum and h. jaqueti were dominated by compound a (nicotine : > 97% of the secretion, based on calculation of peak areas, see also table 2 and fig. 1). components b (3-(1-methyl-2-piperidinyl)-pyridine) and c (anabasine) were found constantly as accompanying minor or trace components (each about 1% of the secretion). in h. lessiniense, anabaseine (compound e) was the major compound in the extracts (> 95%, see table 2 and fig. 1), accompanied by trace amounts of anabasine and 2,3-bipyridyl (each about 13% of the secretion). in two out of five extracts, two trace components were also found at rt = 13.56 min and 14.55 min (each about 1% of the secretion), both showing a pyridine - like pattern and a molecular weight of 174, but both remain unidentified.it is noteworthy that some individuals did not show any components. in particular, extracts of three specimens (all collected at one locality in serbia, see table 1) of h. jaqueti were investigated, and two of these (one female, one juvenile) consistently showed the nicotine - rich composition mentioned above. in the extract of the remaining specimen (a juvenile), no components could be detected. for h. lessiniense, we analyzed extracts of five individuals (all female), which were from two different collections (see table 1) : three extracts (two of collection no. 1, one of collection no. 1). for the largest species of the genus, h. oreophilum, only two individuals (one male, one female) were available to us, both of which discharged large amounts of nicotine. absolute amounts of secretions were determined for h. oreophilum, and were calculated to be about 15 g nicotine per individual (11.8 g measured for one specimen, 16 g for the other specimen).thus, only for h. oreophilum (nicotine - rich), could male and female extracts be compared, showing no differences. for h. jaqueti (nicotine - rich) and h. lessiniense (anabaseine - rich), only females and juveniles (subadult females) were available ; between subadults and adults, holoscotolemon unicolor contrasting the results obtained from h. jaqueti, h. oreophilum, and h. lessiniense, no components were detected in any of the nine extracts from adults of h. unicolor. this was surprising since these specimens were from several different collection sites in austria and were collected at different times of the year (see table 1). scent glands in h. unicolor, as well as openings (ozopores) appeared to be as well developed as in the other holoscotolemon species (fig. 2). however, in one extract of an early - instar juvenile, traces of three nitrogen - containing compounds were detected (not shown), whereas extracts of 11 other juveniles did not show any compounds. the trace components in the single juvenile extract were tentatively identified as dimethyl - isoalkylpyrazines by mass spectral comparisons to literature, and to spectra from the nist - library (compound 1 : a dimethyl - isobutylpyrazine, m at m / z 164 ; compounds 2 and 3 : isomeric dimethyl - isopentylpyrazines, m at m / z 178). choloscotolemon oreophilum ; dholoscotolemon lessiniense ; e and f details of left ozopore in holoscotolemon jaqueti position and morphology of ozopores in species of genus holoscotolemon. choloscotolemon oreophilum ; dholoscotolemon lessiniense ; e and f details of left ozopore in holoscotolemon jaqueti origin of extract components we assume that the compounds found in the whole body extracts of holoscotolemon herein investigated are from the scent glands of these species. this view is based on several facts, and on corroborating evidence from previous studies. first, scent glands are the only glandular structures capable of producing such amounts of secretions ; they are large and well developed in all species of the genus holoscotolemon. corroborative evidence for the scent gland origin of extract components comes from extraction - attempts of individuals with obviously depleted gland reservoirs. we assume that we may have examined many empty specimens, as indicated by the lack of any compound in the gas chromatograms of extracts. from previous investigations, we know that depletion of gland reservoirs can be a rapid process, mainly depending on external irritation stimuli. refill of glandular reservoirs is slow ; according to holmberg (1970), this process takes a few days in phalangiids. in our study, the relation of specimens with well - filled gland reservoirs to specimens with depleted reservoirs was about 1:1 for h. jaqueti and h. lessiniense. glandular depletion may be explained by irritating stimuli in the course of collection and transport. absolute amounts of secretion, as shown for nicotine in extracts of h. oreophilum, were calculated to be about 15 g per individual. such amounts correspond to a volume of 15 nl of nicotine (which is a liquid with a density of 1.01 g / ml at room temperature), and hence to two hypothetical spherical droplets of about 130 m in diameter. moreover, droplets of that size would be consistent with the expected dimension and filling capacity of glandular reservoirs in holoscotolemon, at least if glandular dimensions in other opilionids of comparable size are considered (e.g., juberthie, 1976 ; schaider and raspotnig, 2009).in general, the whole body extraction - method is widely used and commonly accepted for the assessment of exocrine products in small arthropods that are too tiny to sample or dab their secretions directly from the gland orifices. the method has been standardized for investigations of exocrine glandular products in oribatid and astigmatid mites over many years (sakata and norton, 2001 ; raspotnig., 2001, 2008), and also has been used to assess scent gland exudates of opilionids of different groups (raspotnig., 2005, 2010 2010a, b). for instance, in sironids (cyphophthalmi) direct sampling of secretions from ozopores leads to the same chemical results as working with hexane whole body extracts (raspotnig., nicotine and anabaseine in general, tobacco alkaloids are unusual components in arthropod exocrine secretions. nicotine is known only from sclerobunus robustus (a travunioid laniatorean) (www.pherobase.com). anabaseine, on the other hand, is new for scent gland secretions of opiliones. nicotine is a defensive neurotoxin of some plants, and anabaseine is a neurotoxin from nemertine worms that use it for paralyzing prey and possibly for deterring predators (kem, 1971 ; kem., 1971, 1976). one example, however, is the poison glands of certain myrmicine ants (wheeler., 1981 ; leclercq., 2001 ; co., 2003), where anabaseine also may act as a part of the trail pheromone (attygalle., 1998). in certain hymenopterans, anabaseine is found as a by - product in anabasine - rich secretions (co., 2003 ; cruz - lopez., 2006). anabaseine is remarkable for exhibiting striking neurotoxic effects by interacting with nicotinic receptors, and a synthetic anabaseine - derivative has proved to be a promising test agent to study and cure cognitive function loss associated with several human diseases (kem., 1997, 2006). to our knowledge, h. lessiniense is the only arthropod species producing an exocrine secretion almost exclusively based on anabaseine. it is further interesting to note that h. lessiniense (anabaseine - rich) and h. oreophilum (nicotine - rich), both occurring in the italian southern alps and separated by an air - line distance of just 300 km, use differently composed secretions ; this might indicate more specific communicative functions of scent gland secretions in cladonychiid harvestmen, in addition to assumingly defensive roles. pyridines and alkyl - pyrazines three minor components in the holoscotolemon - secretions were classified as pyridines, chemically close to nicotine and anabaseine. these compounds may represent by - products of the biosynthetic pathways to nicotine and anabaseine, but the possibility of their artificial formation from major compounds in the hot injector of the gas chromatograph can not be excluded. in fact, small amounts of 2,3-bipyridyl also could be detected when analyzing an authentic sample of anabasine, calling the scent gland origin of 2,3-bipyridyl into question. on the other hand, 2,3-bipyridyl is known to represent a second neurotoxic component in nemertine worms, in addition to anabaseine (kem., 1976), and also occurs in exocrine glands of certain aphaenogaster and messor ants (attygalle., 1998 ; co., 2003). the alkyl - pyrazines detected in the extract of one single juvenile of h. unicolor also are widespread exocrine compounds in several species of ants and wasps (cavill and houghton, 1974 ; borg - karlson and teng, 1980 ; wheeler., 1982 ; tecle., 1987 ; brophy, 1989 ; jones. in some cases, alkyl pyrazines seem to accompany alkaloids of the anabasine - type as minor components in exocrine secretions of some hymenopterans (e.g., cruz - lopez., 2006). however, our preliminary chemical data for h. unicolor, deviating from the chemistry of the other three holoscotolemon species, need further clarification, and we hope to unravel this question in the near future. chemosystematics in genus holoscotolemon our study provides an analysis of the scent - gland chemistry of all four alpine and carpathian holoscotolemon species. in this context as well as from a zoogeographical and evolutionary point of view, questions concerning the presently inhabited areas of these species are of interest, particularly with respect to the hypothetical regions where they outlasted the last ice - age (wrm). holoscotolemon unicolor is endemic to the eastern alps and, following holdhaus (1954), thaler (1966, 1976), and komposch (2009), is a re - wanderer of long distance, which probably survived the last ice age in holoscotolemon oreophilum and h. lessiniense are local endemics of the southern alps, and should have survived the wrm ice - age in massifs de refuge in the areas of their present distribution in the sea alps (h. oreophilum) and monti lessini (h. lessiniense), respectively. martens (1978) found no close relationship between h. lessiniense and h. unicolor, and believed the former species is closer to h. oreophilum. therefore, it is surprising that the available chemical information points toward a high similarity between the most western (h. oreophilum) and the most eastern species (h. jaqueti). thus, tentatively, nicotine may represent a plesiomorphic compound in holoscotolemon (and possibly also in travunioidea, see below) while h. lessiniense, occurring in an area in between, shows a clearly distinct although related chemistry (i.e., anabaseine - rich), possibly representing the derivative status. scent gland chemistry in insidiatores the major part of laniatorean scent gland research has been performed on grassatorean laniatores, and this strong bias may have contributed to the picture of a typical, rather homogenous, phenolic and benzoquinone - rich phenol- and benzoquinone - rich secretions have been identified from representatives of all grassatorean families hitherto investigated, such as many gonyleptidae (see gnaspini and hara, 2007 and references therein ; hara., 2005 ; machado and pomini, 2008 ; fttinger., 2010), roach., 1980), stygnommatidae (duffield., 1981), stygnopsidae (pomini., 2010 ; shear., 2010b), and phalangodidae (shear., 2010a). even though components other than phenols and benzoquinones (i.e., some accompanying acyclic compounds and alkyl - dihydro - pyrans) have been found in certain gonyleptidae (hara., 2005 ; rocha., 2011), nitrogen - containing compounds or even tobacco alkaloids do not seem to occur in the scent glands of the grassatores. on the other hand, there is growing evidence that insidiatorean secretions or at least those of superfamily travunioidea are based on a completely different chemistry that relies on nitrogen - containing compounds as major secretory constituents. (1984) who found two nitrogen - containing compounds (- dimethyl - phenylethylamine and nicotine) in the scent gland secretion of sclerobunus robustus, a travunioid species from both another continent (america) and a second travunioid family (travuniidae ; kury 2000 onwards). for grouping of northern hemisphere triaenonychoidea such as sclerobunus with travunioidea see giribet and kury (2007). preliminary chemical results from representatives of travuniids in europe (unpublished) also indicate consistently that tobacco alkaloids constitute major secretory components in species of travunia and abasola (for possible synonymies see kury and mendes 2007) as well as in the so - far systematically unplaced but travunioid trojanella (karaman, 2005). in this respect, nitrogen - containing compounds : (1) may be widespread or even common in the travunioidea ; and, (2) according to the basal phylogenetic position of travunioidea within laniatores, these compounds also may represent the ancestral scent gland equipment of laniatoreans.opilionid scent gland secretions generally seem to be a promising pool of group- or even species - specific chemical characters, representing an independent set of data of phylogenetic value, in addition to characters from traditional morphology and molecular genetics. the importance of scent gland chemistry for studies of opilionid phylogeny has been emphasized previously (roach., 1980 ; duffield., 1981), and more recent papers have added to this topic (raspotnig., 2005, 2010 ; jones., so far, an evolutionary line may be traced from the naphthoquinone- and acyclic ketone - rich secretions of the cyphophthalmi to the similarly structured secretions in eupnoid and dyspnoid palpatores (see introduction), but any evolutionary link to the chemically distinct secretions of laniatores is still missing. additionally, within the laniatores, the chemical gap between the alkaloid - rich secretions of the travunioidea on the one hand and the generally phenol- and benzoquinone - rich chemistry in the grassatores on the other hand is not yet well explained. in this regard, information on the unknown scent gland chemistry of the true triaenonychoidea (sensu kury, 2000 onwards) would be important, as this mainly southern hemisphere - distributed group of insidiatores (see pinto - da - rocha and giribet, 2007) may represent the missing link in the chemosystematic puzzle of harvestmen.	the exocrine secretions from prominently developed prosomal scent glands in four species of the european laniatorean harvestman genus holoscotolemon (laniatores, travunioidea, cladonychiidae) were analyzed by gas chromatography mass spectrometry. two major alkaloidal compounds were detected : nicotine accounted for more than 97% of the secretion in holoscotolemon jaqueti and h. oreophilum, whereas the chemically related nicotinoid alkaloid anabaseine was the major compound in h. lessiniense. in addition, a series of minor nitrogen - containing components was found, namely 3-(1-methyl-2-piperidinyl)-pyridine and anabasine, in h. jaqueti and h. oreophilum, and anabasine together with 2,3-bipyridyl in h. lessiniense. by contrast, extracts of adult h. unicolor did not show any components. in one juvenile specimen of h. unicolor, however, low amounts of alkyl pyrazines (dimethyl - isobutyl- and dimethyl - isopentylpyrazines) were detected. nitrogen - containing components previously were found in sclerobunus robustus (an american travunioid harvestman), so scent gland - derived alkaloids may be widespread or even common in the travunioidea. alkaloids have not been reported for other opilionid scent gland secretions outside the travunioidea, and we hypothesize that they may be the phylogenetically ancestral allomones in the laniatores, having been reduced and replaced by a phenol- and benzoquinone - rich chemistry in the more derived grassatorean taxa.
gene expression dynamics holds vital information about how a cell responds to environmental changes. in the case of an infecting microbe, gene expression data could provide clinically relevant insights on how a pathogen adapts to the infection environment and causes damage to the host. in the past two decades, gene expression studies both in vitro and in vivo have generated large amount of data and laid a foundation for understanding infection biology. however, current profiling technologies including microarray and rnaseq, are not optimal for profiling of pathogen gene expression during infection. host rna constitutes an overwhelming portion (usually > 99%) of the total rna isolated from infected tissue samples. the host rna contributes to high background on microarrays, and dominates sequence reads from rnaseq. pathogen cell isolation from infected tissue, in theory, can help to enrich for pathogen rna, but it poses additional problems : it requires large quantities of infected tissue ; the procedure can be tedious ; and most importantly, it is difficult to conserve the native state or integrity of rna during the lengthy process. in order to generate more comprehensive and accurate data on pathogen gene expression during real infections, we set out to develop a protocol that allows reliable and cost - effective quantification of minority rna species in a mixed rna population. nanostring ncounter is a recently developed platform that makes direct multiplexed measurement of gene expression using digital bar - coded probe pairs. this platform has a sensitivity level comparable to that of qrt - pcr but does not require enzymatic amplification. the technology is not genome - wide, it allows detection of up to 800 different genes in each assay. here we use gene expression profiling study of a major human pathogen, candida albicans, during an invasive infection as an example, to demonstrate : 1) technical details of the experimental procedures (protocol), 2) the sensitivity, reproducibility and dynamic range of this method (representative results), and 3) important considerations for designing and performing experiments based on this protocol (discussion). this protocol can be easily adapted for various pathogens and has been successfully applied in a number of infection models. all animal procedures were approved by the institutional animal care and use committee at the los angeles biomedical research institute (protocol 011000) and carried out according to the national institutes of health (nih) guidelines for the ethical treatment of animals. the mice were caged in an aaalac - accredited facility located on the campus of harbor - ucla research and education institute. caging and husbandry was provided according to the guidelines in the us public health service publication " guide for the care and use of laboratory animals ". obviously sick, lethargic mice were segregated from the group and euthanized to minimize suffering. the mice were euthanized by pentobarbital overdose (210 mg / kg), as recommended by the panel on euthanasia of the american veterinary medical association. inoculate three mice per experimental group intravenously with 1 x 10 yeast - phase c. albicans cells. place each kidney into a screw cap tube, snap freeze in liquid nitrogen, and store at -80 c for later rna extraction. note : the animal experiments were carried out as described in detail in xu.. prepare the following reagents and instruments before removing kidneys from the -80 c freezer : open a total rna extraction kit. add 2-mercaptoethanol (1% v / v) to buffer rlt and mix well (prepare 2 ml for each sample).prepare phenol : chloroform : isoamyl alcohol 25:24:1 (need 600 l for each sample).label m - type homogenization tubes (m - tube) and chill on ice.label 2 ml screw cap tubes, and add approximately 300 l zirconia beads to each tube.check instruments are available : tissue dissociator, beadbeater, tabletop centrifuge with adaptors for 50 ml tubes and 96 well plates, photometer. add 2-mercaptoethanol (1% v / v) to buffer rlt and mix well (prepare 2 ml for each sample). prepare phenol : chloroform : isoamyl alcohol 25:24:1 (need 600 l for each sample). label m - type homogenization tubes (m - tube) and chill on ice. label 2 ml screw cap tubes, and add approximately 300 l zirconia beads to each tube. check instruments are available : tissue dissociator, beadbeater, tabletop centrifuge with adaptors for 50 ml tubes and 96 well plates, photometer. add 600 l phenol : chloroform : isoamyl alcohol 25:24:1 to the tubes from the previous step. close the lids tightly and vortex on beadbeater for 3 min in a 4 c cold room. centrifuge the tubes at 15,000 x g for 5 min in a 4 c cold room. carefully transfer the aqueous phase to a new 1.5 ml microfuge tube, mix well with equal volume of 70% ethanol, then load onto the spin column (load twice). wash the spin column once with 700 l buffer rw, followed by twice with 500 l buffer rpe. centrifuge one extra minute in a dry collection tube to remove remaining liquid in the spin column. thaw reporter codeset (green cap tube) and capture codeset (gray cap tube) on ice. add 130 l hybridization buffer to the reporter codeset, invert to mix and spin down. add 10 g of total tissue rna (in a volume of 5 l) to each tube. incubate the reactions at 65 c in a thermal cycler with heated lid o / n (~18 hr). remove one sample cartridge from -20 c and let it warm to rt. remove two reagent plates from 4 c and centrifuge at 670 x g for 2 min in a tabletop centrifuge. set up the prep station following step - by - step instructions on the screen, select the high sensitivity option. remove the reactions from the thermal cycler and immediately load on the prep station. select to run the high sensitivity program (3 hr). when the prep station program is complete, apply mineral oil to the bottom of the cartridge (for generation one ncounter only, later generations do not require this step), then load the cartridge onto the digital analyzer. set up the digital analyzer following step - by - step instructions on the screen, select the high resolution scan option. select the high resolution (600 fields) option, run the scanning program (~4.5 hr). when the scanning program is complete, download the results (or choose to receive the results by email), and import raw data into manufacturer 's software. the software will automatically check data quality and raise flags if the quality of the data falls out of the normal range. perform technical adjustment using the built - in function (optional, follow instructions in the software), then export the data as an excel file. normalize the data using one of the following methods : total counts from all genes in the codeset ; one or a few internal control genes ; geometric mean of highly expressed genes (see discussion). calculate the mean expression values for each gene (if the experiment was done in replicates or triplicates), then calculate the ratio of expression levels among different experimental groups. (optional) analyze and visualize the profiling results by built - in functions in the nsolver software or a clustering program such as multiexperiment viewer. use male balb / c mice weighting 20 - 22 g for all studies. inoculate three mice per experimental group intravenously with 1 x 10 yeast - phase c. albicans cells. place each kidney into a screw cap tube, snap freeze in liquid nitrogen, and store at -80 c for later rna extraction. note : the animal experiments were carried out as described in detail in xu.. prepare the following reagents and instruments before removing kidneys from the -80 c freezer : open a total rna extraction kit. add 2-mercaptoethanol (1% v / v) to buffer rlt and mix well (prepare 2 ml for each sample).prepare phenol : chloroform : isoamyl alcohol 25:24:1 (need 600 l for each sample).label m - type homogenization tubes (m - tube) and chill on ice.label 2 ml screw cap tubes, and add approximately 300 l zirconia beads to each tube.check instruments are available : tissue dissociator, beadbeater, tabletop centrifuge with adaptors for 50 ml tubes and 96 well plates, photometer. open a total rna extraction kit. add 2-mercaptoethanol (1% v / v) to buffer rlt and mix well (prepare 2 ml for each sample). prepare phenol : chloroform : isoamyl alcohol 25:24:1 (need 600 l for each sample). label m - type homogenization tubes (m - tube) and chill on ice. label 2 ml screw cap tubes, and add approximately 300 l zirconia beads to each tube. check instruments are available : tissue dissociator, beadbeater, tabletop centrifuge with adaptors for 50 ml tubes and 96 well plates, photometer. remove tubes containing kidneys from -80 c freezer and put on ice. add 1.2 ml buffer rlt with 2-mercaptoethanol to each kidney. add 600 l phenol : chloroform : isoamyl alcohol 25:24:1 to the tubes from the previous step. close the lids tightly and vortex on beadbeater for 3 min in a 4 c cold room. centrifuge the tubes at 15,000 x g for 5 min in a 4 c cold room. carefully transfer the aqueous phase to a new 1.5 ml microfuge tube, mix well with equal volume of 70% ethanol, then load onto the spin column (load twice). wash the spin column once with 700 l buffer rw, followed by twice with 500 l buffer rpe. centrifuge one extra minute in a dry collection tube to remove remaining liquid in the spin column. thaw reporter codeset (green cap tube) and capture codeset (gray cap tube) on ice. add 130 l hybridization buffer to the reporter codeset, invert to mix and spin down. add 20 l of the mix to each of the 12 reaction tubes. add 10 g of total tissue rna (in a volume of 5 l) to each tube. incubate the reactions at 65 c in a thermal cycler with heated lid o / n (~18 hr). remove one sample cartridge from -20 c and let it warm to rt. remove two reagent plates from 4 c and centrifuge at 670 x g for 2 min in a tabletop centrifuge. set up the prep station following step - by - step instructions on the screen, select the high sensitivity option. remove the reactions from the thermal cycler and immediately load on the prep station. select to run the high sensitivity program (3 hr). when the prep station program is complete, remove the cartridge and seal the lanes with a clear tape. apply mineral oil to the bottom of the cartridge (for generation one ncounter only, later generations do not require this step), then load the cartridge onto the digital analyzer. set up the digital analyzer following step - by - step instructions on the screen, select the high resolution scan option. select the high resolution (600 fields) option, run the scanning program (~4.5 hr). when the scanning program is complete, download the results (or choose to receive the results by email), and import raw data into manufacturer 's software. the software will automatically check data quality and raise flags if the quality of the data falls out of the normal range. perform technical adjustment using the built - in function (optional, follow instructions in the software), then export the data as an excel file. normalize the data using one of the following methods : total counts from all genes in the codeset ; one or a few internal control genes ; geometric mean of highly expressed genes (see discussion). calculate the mean expression values for each gene (if the experiment was done in replicates or triplicates), then calculate the ratio of expression levels among different experimental groups. (optional) analyze and visualize the profiling results by built - in functions in the nsolver software or a clustering program such as multiexperiment viewer. one main challenge for pathogen gene expression profiling in vivo is to get enough reads from pathogen rna. given the low percentage of pathogen transcripts in total rna, large amount of total rna (> 10 g) has to be used for each reaction. the platform has a unique advantage in this perspective : it uses both a capture probe and a report probe to increase the specificity, so that the overwhelming amount of host rna does not cause a significant level of noise. as shown in figure 1 (adapted from ref. 6), background raw counts from an uninfected tissue sample (red dots) were all below 10, while raw counts from two infected tissue samples (blue dots) were all above 10. 6), raw counts from two biological replicates (blue dots, 48 hr post - infection kidney samples) had very good correlation, with r - square value equals 0.945. the platform also provides sufficient dynamic range to encompass natural biological expression levels (figure 1, raw counts ranged between 10 and 10). using a probe set specifying 248 environmental response genes, we were able to discern two phases of pathogen gene expression (figure 2, adapted from ref. 6) : an early gene expression response comprises genes with rna levels significantly different between the inoculum and 12 hr post - infection samples (p 2-fold change in expression), and a late gene expression response comprises genes with rna levels that are significantly different between the 12 hr time point at 48 hr post - infection samples (p 2-fold change in expression). these results indicate that c. albicans gene expression is dynamically regulated during invasive infection of a mammalian host. probe counts for two infected (48 hr post - infection, blue data points) and one uninfected (red data points) kidney samples are presented as a scatter plot. expression of c. albicans environmentally responsive genes during invasive infection (adapted from ref. 6). changes in expression levels during mouse kidney invasion for 248 c. albicans genes are presented in a heat map format. mean values of biological triplicates are shown for up - regulation (yellow) and down - regulation (blue) of genes at 12, 24, and 48 hr post - infection relative to mean inoculum levels (0 hr). color saturation represents the extent of the expression change, with full saturation at 10 fold up- or down - regulation. portions of the heat map are expanded to illustrate representative early up - regulated genes (top), late genes (middle), and early down - regulated genes (bottom). in these portions, we define early expression changes as significant differences between the inoculum and 12 hr samples. we define late expression changes as significant differences between the 12 and 48 hr samples. significance refers to changes of > 2 fold and a p - value 100 genes) containing randomly selected probes, using total counts for normalization can be a good choice, because the total counts of a large number of unrelated genes may faithfully reflect the amount of rna input. for a small codeset (< 100 genes) containing probes focused on a specific process (such as hyphal growth, given that hyphal growth genes tend to be co - regulated), choosing one or a few housekeeping genes as control for normalization is essential. tdh3, a robustly expressed metabolic gene, has served well as control for many experiments. the third method is a hybrid of method 1 and 2, with an emphasis for equal contribution from highly expressed genes. though the ncounter platform is not genome - wide, it allows quantification of expression level for up to 800 genes in a single assay. therefore, choosing the most informative genes to analyze is critical for the success of the profiling. information from published expression and functional data is compiled to screen for genes that are of potential interest to the current study, such as the environmental response genes. this approach allows easy comparison of in vivo profiling data to many existing datasets, hence providing context to data interpretation. a category of genes in a microbe genome, such as all genes specifying transcription factors, kinases or cell wall proteins are chosen for probes. this approach allows identification of novel virulence factors and discovery of novel regulatory relationships based on the in vivo profiling data.	for most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene transcripts from infected tissues. pathogen rna constitutes a tiny portion of the total rna isolated from infected tissue samples. both microarray and rnaseq technologies have difficulties in generating reliable reads for weakly expressed pathogen genes. mutant pathogen strains with reduced in vivo proliferation pose an even bigger challenge. here we describe an in vivo gene expression profiling protocol that is very fast, extremely sensitive and highly reproducible. we developed this protocol during our investigation of the fungal pathogen candida albicans in a murine model of hematogenously disseminated candidiasis. using this protocol, we have documented time courses of dynamically regulated c. albicans gene expression during kidney infection, and discovered unexpected features of gene expression responses to antifungal drug treatment in vivo.
although it is textbook knowledge that the functions of biomacromolecules are strongly coupled to their conformational motions and fluctuations, computer simulation of such motions has been a challenge for decades. typically, distinct algorithms are employed to estimate equilibrium quantities (e.g., refs (3) and (4)) and dynamical properties (e.g., refs (510)). in principle, a single long dynamics trajectory would be sufficient to determine both equilibrium and dynamical properties, but such simulations remain impractical for most systems of interest. aside from straightforward simulations, more technical approaches that can yield both equilibrium and dynamical simulation, sometimes under minor assumptions, a number of approaches employ markov state models (msms) as part their overall computational strategy. on the basis of replica exchange molecular dynamics (remd), it is possible to extract kinetic information from continuous trajectory segments between exchanges and thereby construct an msm. the adaptive seeding method (asm) similarly builds an msm based on trajectories seeded from states discovered via remd or another of the so - called generalized ensemble (ge) algorithms. msms have also been used in combination with short, off - equilibrium simulations to construct the equilibrium ensemble of folding pathways of a protein. another general strategy is to employ a series of nonintersecting interfaces that interpolate between states of interest selected in advance. milestoning generates and analyzes transitions between interfaces assuming prior history does not affect the distribution of trajectories. transition interface sampling (tis) and its variants also analyze such transitions and can yield free energy barriers in addition to rates while accounting for some history information. forward flux sampling (ffs) again samples interface transitions : it accounts for history information and can yield rates and equilibrium information. the weighted ensemble (we) simulation strategy (see figure 1), which has a rigorous basis as a path - sampling method, has also been suggested as an approach for computation of both equilibrium and nonequilibrium properties. although we was originally developed as a tool for characterizing nonequilibrium dynamical pathways and rates (e.g., refs (5), (2528)), the strategy was extended to steady - state conditions including equilibrium. the simultaneous computation of equilibrium and kinetic properties using we was demonstrated with configuration space separated into two states by a dividing surface and later for arbitrary states defined in advance of a simulation. in contrast to many other advanced sampling strategies, we generates an ensemble of continuous trajectories, all at the physical condition (e.g., temperature) of interest. (a) ensemble of trajectories with arrow tips indicating the instantaneous configuration and tails showing recent history in the space of two schematic coordinates q1 and q2. states a and b, shown in gray, are two arbitrary regions of phase space. (b) dissection into two subsets based on whether a trajectory was most recently in state a (black solid arrows, the steady state) or state b (red dashed, the steady state). (c) statistically equivalent ensemble of weighted trajectories, with arrow thickness suggesting weight. configuration space has been divided into cells (bins) which each containing an equal number of trajectories. here, we further develop the capability of we simulation to calculate equilibrium and nonequilibrium quantities simultaneously in several ways that may be important for future studies of increasingly complex systems. (i) the approach described below permits the calculation of rates between arbitrary states, which can be defined after a simulation has been completed. in a complex system, the most important physical states, including intermediates, generally will not be obvious prior to simulation. further, the present approach opens up the possibility to use rate calculations to aid in the state - definition process. (ii) the non - markovian analysis described here enables unbiased rate calculations in the typical case where bins used by we simulation do not exhibit markovian behavior. the analysis is general and can be applied outside the we context, including the analysis of ordinary long trajectories. (iii) the non - markovian analysis can improve the efficiency of we simulations by yielding accurate estimates of observables from shorter simulations. the analysis is based on a previously suggested decomposition of the equilibrium ensemble into two nonequilibrium steady states. we is easily parallelizable because it employs multiple trajectories and was recently used with 3500 cores. because there is no need to catch trajectories at precise transition interfaces, we algorithms lend themselves to a scripting - like implementation which has been employed to study a wide range of stochastic systems via regular molecular dynamics, monte carlo, the string strategy, and gillespie - algorithm dynamics of chemical kinetic networks. we simulation uses multiple simultaneous trajectories, with weights that sum to one, that are occasionally coupled by replication or combination events every units of time. the coupling events typically are governed by a static partition of configuration space into bins (figure 1c), although dynamical / adaptive bins may be used. in the case of static bins, when one or more trajectories enters an unoccupied bin, those trajectories are replicated so that their count conforms to a (typically) preset value, m. replicated daughter if more than m trajectories are found to occupy a bin, trajectories are combined statistically in a pairwise fashion until m remain, with weight from pruned trajectories assigned to others in the same bin. these procedures are carried out in such a way that dynamics remain statistically unbiased. this study does not adjust weights according to previously developed reweighting procedures during the simulation. rather, the we simulations described here are long enough to permit relaxation to the equilibrium state. once the equilibrium state is reached in a we simulation, meaning that there is a detailed balance of probability flow between any two states, equilibrium observables such as state populations or a potential of mean force can be calculated simply by summing trajectory weights in the corresponding regions of phase space. estimation of observables. to calculate rates, the equilibrium set of trajectories (figure 1a) is decomposed into two steady states as shown in figure 1b : the steady state consisting of trajectories more recently in a than b, and the steady state with those most recently in b ; these were denoted ab and ba steady states, respectively, in ref (31). trajectories are labeled according to the last state visited, i.e., classified as or, during a we simulation or in a postsimulation analysis (post - analysis). the direct rate kab estimate is computed from the probability arriving at the final state via1where mfpt is the mean - first - passage time, flux(a b\|) is the probability per unit time arriving at state b in the steady state, and p() is the total probability in the steady state. by construction p() + p() = 1. normalizing by p() effectively excludes the reverse steady state, and the rate calculation only sees the unidirectional steady state as in ref (23). an expression analogous to eq 1 applies for kba. also note that the effective first order rate constant, defined by flux(a b\|)/paeq, can be determined from equilibrium we simulation because paeq can be directly computed by summing weights in a. we note that analogous direct calculation of observables can be performed from an equilibrium ensemble of unweighted (i.e., brute force) trajectories by assigning equal weights to each. beyond the direct estimates of observables based on trajectory weights, we also generalize previous matrix formulations for nonequilibrium steady states into an equilibrium formulation that explicitly accounts for the embedded steady states (as in figure 1b, c). these non - markovian matrix estimates are tested below and may prove important for future we studies using shorter simulations, as described in the discussion. our matrix approach explicitly uses the decomposition of the equilibrium population into and components for each bin i:2which implies p() = ipi and p() = ipi. we called this a labeled analysis. thus, with n bins, a set of 2n probabilities is required rather than n. similarly, a 2n 2n rate matrix is required : kijv, where and can be either the or subsets of trajectories each of the previously considered kij rate elements is thus decomposed into four history - dependent elements which account for whether the particular trajectory was last in state a or b and whether the trajectory transitions between the and subsets. the analysis assumes states consist strictly of one or more bins, but this is always possible in a post - analysis without a loss of generality. in other words, given the flexibility we have when we define the bins, it is not a real limitation that the states have to be strictly constituted by bins. constructing a labeled rate matrix for unbiased calculations. for purposes of illustration, here state a consists solely of bin 1 and state b solely of bin 3. left : a traditional rate matrix with history - blind elements. the rate kij gives the conditional probability for transitioning from bin i to bin j in a fixed time increment, regardless of previous history. the element kijv is the conditional probability for the i to j transition for trajectories initially in the subensemble which transition to the subensemble, where and are either or. the labeled rate matrix correctly assigns the and subpopulations of each bin, whereas the traditional matrix may not. we wish to emphasize that this analysis is non - markovian because we are explicitly including history information (i.e., and labels) in the new 2n 2n rate matrix. once the matrix is built, the steady state observables are obtained using the same mathematical formalism that would be used in a regular markov model. however, the matrix should be seen as a tool of linear algebra and not as embodying any physical assumptions. for example, consider a bin in the intermediate region (neither a nor b), such as bin 2 in figure 2. in this region, an trajectory can not change into a trajectory, nor vice versa ; hence rates for these processes are zero. similarly, an trajectory in the intermediate region which enters a bin in b must turn into a trajectory, so the rate will always be zero to the components of bins in b. the non - markovian results below stem from the division into and steady states, but several steps are required. first, rates among bins are estimated in a post - analysis as3where ij is the probability flux, for a given iteration, from bin i to j of trajectories only with initial and final labels and, respectively, while i is the population labeled as which is initially in i. the subscript 2 in the numerator indicates that the rate kijv is estimated to be nonzero only when more than one transition is observed ; after the second event, all events are included, from the first one, to avoid bias. the requirement for two transitions was found to greatly enhance numerical stability in estimating fluxes and rates between macroscopic states : rates estimated from single events exhibit large fluctuations. notice that eq 3 is a ratio of averages and differs from the average ratio ij/i, which might seem equally or more natural. however, our data show that eq 3 yields unbiased estimates, while the average ratio may not (data not shown). the difference between the two estimators indicates that transitions are correlated with trajectory weights. perhaps more importantly, the average ratio places less importance on high weight transitions due to the instantaneous normalization and that is, low - weight transitions count as heavily as high - weight events, which evidently biases the rate estimate. in the ratio of averages, high - weight events appropriately count more. to obtain macroscopic rates between states consisting of arbitrary sets of bins (noting that arbitrary bins can be employed in a post - analysis), we calculate labeled fluxes for use in eq 1 via4the labeled bin populations pi and pi are obtained from the steady - state solution of the labeled rate matrix k = { kij}. a summary of the labeled or non - markovian matrix procedure for estimating rates between arbitrary states is as follows. first, we obtain the labeled rate matrix k = { kijv } using eq 3 to average interbin transitions. second, we solve the matrix problem kpss = pss, yielding the steady state solution pss. then, the steady state solution pss along with the labeled rate matrix elements are used to calculate the flux entering state b and the flux entering a (eq 4). finally, the mfpt values are obtained from eq 1. in the graphs below, each non - markovian estimate shown is from the matrix solution using the kij rates calculated based on all data obtained until the given iteration of the simulation. the non - markovian matrix formulation exhibits a number of desirable properties : (i) unlike with unlabeled (i.e., implicitly markovian) analysis, kinetic properties will be unbiased as shown below. (ii) solution of both the and steady states is performed simultaneously via a standard markov - state - like analysis of the kij rate matrix. by contrast, if the and steady states are independently solved within a markov formalism, there can be substantial ambiguity in how to assign feedback from the target to initial state when the initial state consists of more than one bin. (iii) the labeled formulation guarantees, by construction, the flux balance intrinsic to equilibrium, namely, flux(a (iv) the analysis can be performed using arbitrary bins (and states defined as sets of these bins). it is not necessary to employ the bins originally used to run the we simulation because a post - analysis can calculate rates among any regions of configuration space. (v) the analysis is equally applicable to ordinary brute - force simulations. for reference, we also perform a traditional markov analysis of the trajectories, which will prove to yield biased rate estimates because most divisions of configuration space (e.g., we bins) are not true markovian states. the markov analysis proceeds without labeling the trajectories. elements of the rate matrix are estimated as5where the subscript 2 again means that we only estimate a rate as nonzero once at least two transitions from i to j have occurred. bin populations are then computed by solving for the steady - state solution of the markov matrix with elements kij. the computation of an mfpt requires the use of source (a) and sink (b) states. hence, we determine markovian macroscopic rates by substituting the markovian kij for all nonzero elements of the kij. we emphasize that this is merely an accounting trick to establish sources and sinks and simultaneously measure both a - to - b and b - to - a fluxes / rates. we perform a smoothing operation on the macroscopic markovian rates because otherwise the data are fairly noisy. the mfpt results shown for the markovian matrix analysis are running averages based on the last 50% of the estimates (where each estimate is from the matrix solution using kij estimates from all data obtained until the particular iteration). we confirmed numerically that such smoothing did not contribute bias to any of the mfpt estimates. weighted ensemble simulations were performed on two systems : the alanine tetrapeptide (ala4) solvated implicitly and a pair of explicitly solvated methane molecules. all simulations were performed at 300 k with a stochastic thermostat (langevin thermostat). friction constants of 5.0 and 1.0 ps were used for ala4 and methane systems, respectively. the molecular dynamics time step used for all systems was t = 2 fs. an iteration is defined to be the simultaneous propagation of all trajectories in the ensemble for some amount of time,. in these studies, a value of = 2500t is used for ala4 and = 250t for the methane methane system. for ala4, the all - atom amber ff99sb force field with implicit gb / sa solvent and no cutoff for the evaluation of nonbonded interactions was simulated using the amber 11 software package. the hawkins, cramer, and truhlar pairwise generalized born model is used, with parameters described by tsui and case (option igb=1 in amber 11 input file). the progress coordinates were selected and binned using a 10 10 partition of a 2d space. a dihedral distance d = ((1/n)idi) with respect to a reference set of torsions is used in the first dimension, where n is the number of torsional angles considered and di is the circular distance between the current value of the ith angle and our reference, i.e., the smaller of the two arclengths along the circumference. this dimension was divided every 14 from 0 to 126 and then a final partition covering the space (126,180 ]). in the second dimension, a regular rmsd, using only heavy atoms, is measured with respect to an -helical structure. in this case, the space was divided every 0.4 from 0 to 3.6 and then a final partition covering the space [3.6,). values and coordinates for the references used to compute the order parameters are given in the supporting information (si). the methane molecules were simulated using the gromacs 4.5 software package with the united - atom gromos 45a3 force field and dodecahedral periodic box of tip3p water molecules (about 900 water molecules in a 34 34 24 box). van der waals interactions were switched off smoothly between 8 and 9 ; real - space electrostatic interactions were truncated at 10. long range electrostatic interactions were calculated using particle mesh ewald (pme) summation. the single progress coordinate was the distance r between the two methane molecules, following ref (28). the coordinate r [0,) was partitioned with a bin spacing of 1 from 0 to 16 and a last bin covering the space r [16,). for the post analysis of methane, the coordinate r [0,) was partitioned so that the first bin is the space r [0,5), then a bin spacing of 2 was used from 5 to 17, while the last bin covers the space r [17,). the results shown below include all data generated in all trajectories : no transient or relaxation period has been omitted. for ala4, populations and mfpts are estimated using we and compared to independent measurements based on ordinary brute force (bf) simulation. rates are estimated in both directions between the two sets of states a1,b1 and a2,b2 shown in figure 3 (see si to visualize representative structures). the second set is less populated and consequently expected to be more difficult to sample. figure 3 also shows the bin definitions used in the post - analysis, which were the same as those used during the we simulation. however, as we shall see in our second system, we can use any partition of the space for the post analysis. the ala4 free energy surface. the surface is projected onto two coordinates : d = ((1/n)idi) from one reference structure (see si) and the rmsd with respect to an ideal -helix. the surface was computed using 3.0 s of ordinary brute force simulation. the set of states a1,b1 is highlighted in green, while the second set a2,b2 is highlighted in red. the grid shows bins that were used both for we simulation and for the post - analysis calculation of observables via the non - markovian matrix formulation. the data shown below are based on the same total simulation times in bf and we. the bf estimates and confidence intervals are based on a single long trajectory of 3.0 s where thousands of transitions between states were observed. five independent we simulations were run, each employing a total of 3.0 s accounting for all the trajectories. the use of independent we runs permits straightforward error analysis for comparison with bf. as described above, direct we measurements sum trajectory weights for population and flux calculations. figures 4 and 5 show direct estimates for both equilibrium and kinetic quantities for both sets of states. we estimates as a function of simulation time are compared to 95% confidence intervals for bf simulation. direct we estimates for populations and mean first passage times (mfpts) for ala4 states a1,b1 from figure 3. five independent we runs are shown, each based on 3.0 s of total simulation time. dashed lines indicate roughly a 95% confidence interval based on 3.0 s of brute force simulation. each nanosecond of molecular (single - trajectory) time corresponds to approximately 200 ns of we simulation including all trajectories in a single run. direct we estimates for populations and mean first passage times for ala4 states a2,b2 from figure 3. five independent we runs are shown, each based on 3.0 s of total simulation time. dashed lines indicate roughly a 95% confidence interval based on 3.0 s of brute force simulation. each nanosecond of molecular time corresponds to approximately 200 ns of we simulation accounting for all trajectories in a single run. as with all observables, data from five is the estimate using all data from the run and thus is based on a total simulation time equal to that of bf (3 s). the spread of the rightmost we data points therefore can be compared with the bf confidence interval to gauge statistical quality. the mean values of the direct estimates are in agreement with bf confidence intervals in all cases. in some cases, the spread of we estimates is significantly less than that for bf prior to the full extent of we simulation. each nanosecond of molecular time in figures 4 and 5 (i.e., single - trajectory time) corresponds to approximately 200 ns of total simulation in a single we run accounting for all trajectories. hence, in some cases, considerably less we simulation is required for an estimate of the same statistical quality as resulted from the full bf simulation of 3.0 s. we also show results of the non - markovian matrix analysis for select observables. figure 6 shows that the non - markovian analysis yields unbiased estimates of the same equilibrium and nonequilibrium properties calculated with direct estimates. (results for other observables, like the population of a1 and the a1b1 mfpt, not shown, exhibit qualitatively similar agreement.) the agreement contrasts with a purely markovian matrix formulation, which does not account for the labeling described above, which can yield statistically biased estimates for kinetic quantities (see methane results, below). unbiased matrix - based estimates are important when reweighting is used in we as noted in the discussion. reweighting was not used in the present study, however. population of a2 and mean first passage time for ala4 from a2 to b2, estimated by the non - markovian matrix analysis of we data. dashed lines indicate roughly a 95% confidence interval from brute force simulation, as in figures 4 and 5. the states are defined in figure 3. in the methane system, we simulation is used to measure first - passage times based on a range of state definitions. for a complex system, analyzing the sensitivity of the mfpt to state definitions could aid in the definition of states. the mfpt was estimated directly, as well as by both non - markovian and markovian matrix analysis. to assess statistical uncertainty, the bins used for post - analysis differ from those used in the original we simulation, as a matter of convenience underscoring the flexibility of the approach. figure 7 shows passage times measured as a function of the boundary position for the unbound state. the boundary of the bound state a was held fixed at a separation of 5 while the definition of the unbound state was varied from 5 to 17. the passage times were measured in increments of 2 and compared with bf results as shown in figure 7. the bf confidence intervals are based on a single long trajectory of 0.4 s, the same total simulation time used in each we simulation. the mean first passage time for methane association (b to a) and dissociation (a to b) measured directly and from the non - markovian matrix analysis from we simulation as a function of the boundary of state a. the inset displays the pmf along with the definitions of the unbound and bound states, indicated by b and a, respectively. dashed lines indicate roughly a 95% confidence interval based on 0.4 s of brute force simulation. figure 7 shows that both direct and non - markovian matrix estimates are in agreement with bf confidence intervals. for fixed state definitions, figure 8 shows the evolution of state populations mfpts, as was done for ala4. we fix the movable boundary position in figure 7 (inset), defining state b as all configurations with r > 11. direct and non - markovian we estimates for populations and mean first passage times (mfpts) are plotted vs molecular time. five independent we runs are shown, each based on 0.4 s of total simulation time. dashed lines indicate roughly a 95% confidence interval based on 0.4 s of brute force simulation. each nanosecond of molecular time corresponds to approximately 80 ns of we simulation accounting for all trajectories in a single run. the bound state (a) is defined by distances less than 5, and b is defined by distances greater than 11. the performance of the non - markovian matrix estimates are particularly noteworthy in figure 8. the matrix estimates converge faster than direct estimates to the exact results for the state populations. presumably, this is because the direct approach requires relaxation of the full probability distribution to equilibrium, whereas the matrix approach requires only relaxation of the distribution with each bin (in order to obtain accurate interbin rates kij). in contrast to the unbiased mfpt estimates obtained by both direct and non - markovian analysis, the markov analysis can be significantly biased for the mfpt. figure 9 shows that applying the markovian analysis (section 2.3) leads to mfpt estimates clearly outside the bf confidence interval. data in the si show that the use of a more sophisticated model such as a maximum - likelihood estimator for reversible markov models yields similar results and does not correct the bias. populations of a (r 11) and mfpts for the methane system, estimated by the non - markovian matrix analysis and the markovian analysis without history information. dashed lines indicate roughly a 95% confidence interval from brute force simulation based on 0.4 s of total simulation time. equilibrium properties, however, can be estimated without bias in a markovian analysis because history dependence is immaterial. figure 9 also illustrates correct (equilibrium) population estimates based on the markovian analysis. to our knowledge, this is the first weighted ensemble (we) study using the original huber and kim algorithm to simultaneously calculate both equilibrium and nonequilibrium quantities. the present study estimates observables (populations and mfpts) based on arbitrary states defined in a postsimulation analysis, permitting the examination of different state definitions and their effects on observables. two qualitatively different estimation schemes were examined, including a non - markovian rate - matrix formulation which shows promise for reducing transient initial - state bias (a bias which is intrinsic to direct estimation of observables based on weights). both schemes showed substantial efficiency gains for some observables even in the test systems which appear to lack significant energy barriers in their configurational landscapes. nevertheless, as described below, the present data do point to further challenges likely to be exhibited by larger, more complex systems. one key feature of the we implementation studied here is the ability to investigate a range of state choices. as computer simulations tackle systems of growing complexity, it seems increasingly unlikely that states chosen prior to a study will prove physically or biochemically relevant. indeed, it is already the case that specialized algorithms are invoked to identify physical states, separated by the slowest time scales, from existing trajectories. with we simulation, as suggested by our methane data, one can adjust state boundaries to minimize the sensitivity of rates to those boundaries. a possible concern with postsimulation state construction is the need to store a potentially large set of coordinates to ensure sufficient flexibility in post analysis. as an illustration, storage of { x, y, z } coordinates for 1000 heavy atoms in a we run of 1000 iterations using 1000 trajectories would require 10 gb. the estimation of both equilibrium and kinetic properties from relatively short simulations is an important goal of current methods development, including for we. here, we have demonstrated as a proof of principle that we simulation can do this efficiently (compared to brute force simulation), without bias, in parallel, and with flexibility in defining states. given the relatively fast time scales (nanosecond scale) characterizing the present systems, it is somewhat surprising that we is better than brute - force simulation for some of the observables and never worse. previous studies suggest that we has the potential for greater efficiency in more complex systems. once a configuration space is discretized (e.g., bins in we simulation), one expects in general that transitions among such discrete regions will not be markovian. to take the simplest example, in a 1d system, whether a trajectory enters a finite - width bin from the left or right will affect the probability to make a transition in a given direction. so generally, discretized systems are non - markovian, even when the underlying continuous dynamics are markovian. this study compared estimation of equilibrium and nonequilibrium observables using the original we algorithm and via post - analysis. as mentioned in the introduction, the occasional rescaling of weights to match an equilibrium or nonequilibrium steady - state condition was not used to avoid any potential complications. our data clearly show that a standard markovian analysis of we simulation is inadequate (figure 9), since we bins typically are not markovian. additional information history dependence, as embodied in the / labeling scheme is needed to obtain unbiased results. inclusion of history information in the matrix analysis means it is intrinsically non - markovian regardless of the linear algebra employed. future work will incorporate the rate estimation and non - markovian matrix schemes developed here, as well as possibly the simpler markovian scheme shown in section 2.3. our data (figure 8) suggest that these could be very successful in bringing a we simulation closer to a specified steady state. but it is an open question whether reweighting simulations will prove superior to the type of post - analysis suggested here. importantly, data presented here indicate that some rate estimators could lead to biased estimates for populations, which, in turn, would bias a reweighted simulation. one practical future approach, suggested by the work of darve and co - workers, could be to define preliminary states in advance to aid sampling transitions in both directions and then to subject the data to the same post analysis performed here to examine additional state definitions besides the initial choices. the present study has not addressed some of the intrinsic limitations of the we approach, which are the related issues of correlations among trajectories (due to the replication and merging events) and sampling orthogonal coordinates not divided up by we bins. in the systems examined here, there was sufficient sampling in orthogonal dimensions to obtain excellent agreement with brute force results in all cases. however, significant future effort will be required to address correlations and orthogonal sampling, the latter being a problem common to methods which preselect coordinates such as multiple - window umbrella sampling and metadynamics. in this proof - of - principle study, the parallel weighted ensemble (we) approach has been applied to measure equilibrium and kinetic properties from a single simulation in small but nontrivial molecular systems. importantly, populations and rates could be measured for arbitrary states chosen after the simulation. for all tested observables, unbiased estimates were obtained, as validated by independent brute - force simulations. in a number of instances, we was significantly more efficient yielding estimates of a given statistical quality in less overall computing time compared to simple simulation, including all trajectories. in this sense, not only is we a parallel method but it can exhibit super - linear scaling ; e.g., 100 cores can yield desired information more than 100 times faster than single - core simulation. we also developed a non - markovian matrix approach for analyzing we or brute - force trajectories, capable of yielding unbiased results, sometimes faster than direct estimates of observables from we. the non - markovian formulation also yields simultaneous estimates of equilibrium and nonequilibrium observables based on an arbitrary division of phase space, which is not possible in a standard markovian analysis. the approaches tested here will need to be further developed and tested in more complex systems.	equilibrium formally can be represented as an ensemble of uncoupled systems undergoing unbiased dynamics in which detailed balance is maintained. many nonequilibrium processes can be described by suitable subsets of the equilibrium ensemble. here, we employ the weighted ensemble (we) simulation protocol [huber and kim, biophys. j.1996, 70, 97110 ] to generate equilibrium trajectory ensembles and extract nonequilibrium subsets for computing kinetic quantities. states do not need to be chosen in advance. the procedure formally allows estimation of kinetic rates between arbitrary states chosen after the simulation, along with their equilibrium populations. we also describe a related history - dependent matrix procedure for estimating equilibrium and nonequilibrium observables when phase space has been divided into arbitrary non - markovian regions, whether in we or ordinary simulation. in this proof - of - principle study, these methods are successfully applied and validated on two molecular systems : explicitly solvated methane association and the implicitly solvated ala4 peptide. we comment on challenges remaining in we calculations.
idiopathic hypereosinophilic syndrome (he s) is a condition of unknown origin characterized by clinical manifestations attributable to eosinophilia and eosinophilic infiltration of tissues. cardiac involvement is rare and threatening accounting for 33% to 43% of death in he s. management of pregnant patients with he s is challenging and rarely been reported, particularly in the setting of heart failure. a 29-year - old woman returning from saint martin island presented in april 2013 with dyspnea and chest pain, which were then diagnosed as clinically suspected eosinophils - associated myocarditis because of pulmonary edema, elevated troponin, and high eosinophils blood count. she received 3 pulses of steroids and was also treated with 2 doses of ivermectine as strongyloides serology was positive. in july 2013, troponin dosage, eosinophils blood count, echocardiography, and cardiac magnetic resonance imaging (mri) were normal. in december 2013, when she was pregnant with 18 weeks of amenorrhea (wa), she was admitted to the intensive cardiology unit department for respiratory distress. blood tests showed elevated ultrasensitive troponin : 139 ng / ml (n < 0.14), nt - probnp : 3350 ng / l, and markedly increased circulating eosinophils at 4.75 10/l. renal and liver tests were normal. a transthoracic echocardiogram revealed hyperkinetic left ventricular with subtotal thrombotic occlusion of left ventricle (lv) (figure 1c and e). cardiac mri revealed diffuse lv endomyocardial fibrosis with circumferential intraventricular adherent lv thrombus (figure 1a). no extra - cardiac involvement (i.e., neurological, pulmonary, cutaneous, or gastrointestinal) was detected. screening for all etiology of secondary or clonal eosinophilia was negative including extended lymphoid phenotyping, janus kinase 2 mutation (jak2), and factor interacting with papola and cpsf1 (fip1l1)-type platelet - derived growth factor receptor (pdgfra) transcript fusion testing. (a) four - chamber view during cine acquisition after gadolinium injection shows subendocardial enhancement of the left ventricle (lv) with a large adherent thrombus inside the ventricular cavity. (d and f) echocardiogram findings 6 month after delivery showing complete disappearance of lv thrombus and normal lv ejection fraction. symptomatic treatment included diuretics, low - dose -blockers, and curative anticoagulation with enoxaparin. three intravenous pulses of 1 g methylprednisolone were administrated followed by 1 mg / kg / d of oral prednisone. she quickly improved, keeping a class iii new york heart association (nyha) dyspnea. despite persistent severe postcapillary pulmonary hypertension one month later, echocardiography was stable and eosinophils were still at 1.0 g / azathioprine was introduced (2 mg / kg / d) and steroids were progressively tapered down. the newborn 's weight was 1900 g (between 10th and 25th percentile), and birth blood ph and lactate were 7.26 and 3 under azathioprine, 6 months after delivery, she was asymptomatic, and eosinophils blood count as well as echocardiography and cardiac mri had normalized (figure 1). here we described a case of severe eosinophilic endomyocardial fibrosis occurring during pregnancy. under treatment with steroids and azathioprine, despite severe pulmonary hypertension, pregnancy had a favorable outcome. acquired eosinophilia is classified as secondary (parasitic, drug - induced, vasculitis,), clonal (myeloid or lymphoid malignancies,), or idiopathic. after exclusion of secondary causes of eosinophilia, a stepwise diagnostic algorithm should include peripheral blood screening for fip1l1pdgfra transcript fusion, bone marrow biopsy, jak2 mutation testing, peripheral blood lymphocytes phenotyping, and t - cell receptor gene rearrangement studies in order to discard clonal causes of eosinophilia. the diagnostic of he s can be advocated in case of negativity of all the test cited above. cardiac involvement of he s is rare and life - threatening, accounting for 33% to 43% of deaths, with a frequency reported from 5% to 72%. treatment of chronic acquired eosinophilia strongly depends on whatever the cause of eosinophilia. in clonal he s, corticosteroids remain the first - line treatment and corticosteroid - sparing agents nowadays mainly rely on hydroxyurea, interferon-, or cyclosporine. despite a promising preliminary study, mepolizumab and other anti - interleukin-5 antibodies no specific drugs have been advocated in cardiac involvement of he s except for heart failure treatment and anticoagulation therapy because of the high frequency of intracardiac thrombus. the use of azathioprine as a corticosteroid - sparing agent for he s has been only anecdotally reported without clear information about its efficacy. only 3 cases of pregnancy during he s have been reported, all with favorable outcomes. two patients were treated with steroids (maintenance doses between 20 and 25 mg / d) and the last one was left untreated. one of those patients had a cardiac involvement consisting in right and lv thrombus but without cardiac failure. the persistence of pulmonary hypertension and elevated circulating eosinophils counts led us to introduce a corticosteroid - sparing agent, namely, azathioprine, because of the safety profile during pregnancy. six months after delivery, echocardiography and cardiac mri confirmed full resolution of interventricular thrombus with regression of endomyocardial fibrosis. in conclusion, even in a severe cardiac involvement of he s, pregnancy can successfully be conducted using steroids and azathioprine. this study has been approved by the ethics committee : comit de protection des personnes (cpp) ile - de - france vi, the authors would like to thank professor david grant for his help in editing the manuscript.	abstractidiopathic hypereosinophilic syndrome (he s) is a condition of unknown origin characterized by clinical manifestations attributable to eosinophilia and eosinophilic infiltration of tissues. cardiac involvement is rare and threatening accounting for 33% to 43% of death in he s. management of pregnant patients with he s is challenging and have rarely been reported, particularly in the setting of heart failure.we here report on the case of a 29-year - old woman with he s who developed severe endomyocardial fibrosis with heart failure during pregnancy. outcome was favorable under treatment with prednisone and azathioprine.this case illustrates a favorable outcome of endomyocardial fibrosis during pregnancy.
chronic illness and chronic pain can have profound negative effects on relationship and sexual satisfaction of both patients and partners. the effects of chronic illness on sexuality are multifactorial and have an impact on all phases of sexual response. sexual desire, arousal and activity may be altered by illness through interference with the hormonal, vascular and neural integrity of the mind and the genitalia, as well as the effects and side effects of medications. anxiety, fear, loss of self - esteem, grief and depression are strongly associated with chronic illness and may also impair sexual function. studies examining the prevalence of sexual dysfunction, and the role of psychological variables, including quality of life, on sexual activity in patients with non - cancer pain found that sexual dysfunction was common, and more frequently reported by those with greater disability and depression, shorter pain duration, and infrequent use of coping self - statements. although sexual dysfunction was commonly reported, subjects perceived it had less importance in quality of life than did other factors possibly because of adaptation to the impact of chronic illness on sexual function. migraine is an extremely prevalent disease : the lifetime prevalence of migraine in women is 33% and the 1-year prevalence of migraine in women is 25%. in men, it is a chronic episodic disease, most common in men and women between the ages of 1555 years that can impair many aspects of life. the aim of the present study was to compare the prevalence of sexual dysfunctions between the migraine sufferers and non - sufferers participants. in the present cross - sectional study, we evaluated female students from the ben - gurion university of the negev who agreed to participate in the study. all the participants were included after having received a detailed explanation on the purpose and design of the study. previously published data or the researchers assumptions regarding pain and sexual function were not presented to the participants. female students were recruited from classrooms, in which a brief presentation of the study was given. subjects who were pregnant, were not sexually active during the year prior to questionnaire administration, and subjects who suffered from any neurological disorder (other than headache) or sustained a head trauma during the last year, were excluded from the study. the participants were requested to fill a questionnaire. questionnaires were filled without the presence of an interviewer, and did not include any recognizable data to assure full anonymity of the collected data. all subjects completed the following questionnaires : demographics and background : participants were asked to answer questions regarding their personal background (e.g., age, marital status, religion, extent of religious observance, height, weight etc.).headache history : the participants were asked to report whether they suffered from troubling headache and if so, to report headache characteristics and accompanying features, using a list of items (frequency, number of headache days / month, severity, aura symptoms, localization, quality, exacerbating factors, nausea, vomiting, sensitivity to light and sound, triggers etc.). migraine diagnosis was based on the 2004 ihs criteria for migraine with and without aura.migraine disability assessment scale (midas) : the migraine disability assessment (midas) scale was used to quantify headache - related disability. midas is a brief and reliable headache - specific tool, which captures headache - related disability. it consists of five questions relating to the influence of headache on everyday activities over the preceding 3 months.the israeli sexual behavior inventory (isbi) : the isbi is a 33 items questionnaire which was primarily designed to assess the impact of sexual problems, chronic illness and disability on sexual functioning and experience.the questionnaire was designed to evaluate several dimensions (domains) of female sexuality sexual desire, orgasm, sexual avoidance, interpersonal sexual relationship, health influence, satisfaction and pain, during the previous year. the participants rated their answers in a scale of 15 according to the rate of agreement with certain phrases or according to the incidence of experiencing phenomenon asked in the questionnaire. demographics and background : participants were asked to answer questions regarding their personal background (e.g., age, marital status, religion, extent of religious observance, height, weight etc.). headache history : the participants were asked to report whether they suffered from troubling headache and if so, to report headache characteristics and accompanying features, using a list of items (frequency, number of headache days / month, severity, aura symptoms, localization, quality, exacerbating factors, nausea, vomiting, sensitivity to light and sound, triggers etc.). migraine diagnosis was based on the 2004 ihs criteria for migraine with and without aura. migraine disability assessment scale (midas) : the migraine disability assessment (midas) scale was used to quantify headache - related disability. midas is a brief and reliable headache - specific tool, which captures headache - related disability. it consists of five questions relating to the influence of headache on everyday activities over the preceding 3 months. the israeli sexual behavior inventory (isbi) : the isbi is a 33 items questionnaire which was primarily designed to assess the impact of sexual problems, chronic illness and disability on sexual functioning and experience. this instrument was proved to be a reliable and valid self - report measure of sexual behavior. the socio demographic and health related characteristics were presented as proportion for categorical variables and mean with standard deviation for continuous variables. the isbi questions were mapped on the scale from 1 to 5 as the higher grade signed the lower dysfunction. the isbi questions were combined into domains and the score for each domain was calculated as the sum of related questions grades. the socio demographic and health related characteristics were compared between the migraine sufferers and non - sufferers participants using chi square test, fisher s exact test and student s t test. whitney non - parametric test due to the small sample size in subgroups and discrete nature of the isbi scores. the multivariable analysis for sexual behavior based on linear regression. a p values of 20), % 15.2 headache characteristics among migraine suffering subjects levels of sexual activity, sexual desire, ability to experience orgasm and general satisfaction from sexual life did not differ significantly between migraine and non - migraine subjects. nevertheless, migraine patients reported a greater degree of pain during intercourse and a higher frequency of fear of sexual intercourse and penetration and lower levels of satisfaction (fig. 1). the total isbi score among migraine sufferers was significantly lower than in non - sufferers (mean rank 49.5 vs. 68.9 ; p = 0.01). multivariable analysis showed that migraine, absence of regular sexual partner, and self - reported religiousness (traditional or orthodox) was independently associated with lower isbi scores. among these predictors, migraine had the strongest relationship with the dependent variable (table 3). sexual desire, p = 0.138 ; satisfaction, p = 0.032 ; orgasm, p = 0.250 ; pain, p = 0.022 ; interpersonal sexual relationships, p = 0.661 ; health influence, p < 0.001table 3predictors of isbi total score errorbetapmigraine (yes vs. no)2.270.570.34<0.001regular partner (no vs. yes)1.260.590.180.034religiousness (traditional or orthodox vs. secular)1.980.890.190.028 comparison of isbi domain scores (mean ranks) between migraine sufferers and non - sufferers. sexual desire, p = 0.138 ; satisfaction, p = 0.032 ; orgasm, p = 0.250 ; pain, p = 0.022 ; interpersonal sexual relationships, p = 0.661 ; health influence, p < 0.001 predictors of isbi total score multivariable analysis the severity of migraine, as reflected by the midas score, was found to be correlated to lower scores in the health domain (rs = 0.404 ; p = 0.024). it was not associated with other domains of the score or with the total isbi score. female sexual dysfunction is a general term used for various disorders of the sexual process in women affecting the various segments of the sexual response cycle : desire, arousal, orgasm and resolution. inability or difficulty to enjoy any of these stages can interfere with sexual satisfaction and cause some degree of dysfunction. female sexual dysfunctions can be accordingly subdivided in the dsm iv into desire, arousal, orgasmic and sexual pain disorders. in the presented study, we attempted to evaluate sexual function and dysfunction among young women suffering from migraine compared to non - migraine sufferers, using the isbi. sexual desire, orgasm and sexual interpersonal relations were similar among migraineurs compared to non - migraineurs, and where comparable to previous reports regarding israeli women in this age group. unlike a recent study, we did not find migraine patients to have higher degrees of sexual desire. three domains were significantly impaired among migraine sufferers : health influence, sexual pain and satisfaction. positive answers regarding fear of sex and avoidance of sexual intercourse were more prevalent among migraine patients. as in other chronic pain conditions, indeed its influence, reflected by higher scores in this domain, was found to be correlated with higher midas scores. it seems reasonable, that avoidance, fear of penetration and consequently lower satisfaction are associated with higher levels of sexually associated pain. sexual pain disorders [8, 10 ] fall into one of two categories : dyspareunia and vaginismus. vaginismus is the recurrent or persistent involuntary or subconscious voluntary spasm (or contraction) of the genital perineal muscles that surround the outer third of the vagina when the insertion of any object is attempted. dyspareunia is estimated to affect 515% of women annually, according to the national health and social life survey, and vaginismus affects 1517% of women presenting to a sex therapy clinic [8, 10, 11 ]. our study questionnaire was not designed to evaluate the various aspects of sexual pain disorder, and this subject should be addressed in future studies. a possible explanation for the high prevalence of sexual pain disorder among migraine females is sensitization of pain pathways [10, 12 ] often found in migraine patients (either as a cause or consequence). clinical and experimental observations on migraine suggest that a general hyper excitability could develop along nociceptive trigeminal neurons allowing the activation of descending pathways that facilitate pain processing or the suppression of pathways that slow down pain transmission. slowly progressive dysfunction of central pain systems secondary to the repetition of attacks of the originating headache has been implicated in transforming the process from an episodic to a chronic condition. this concept of an irritable focus in the central nervous system is currently known as central sensitization and is a characteristic feature of visceral pain. dyspareunia was found to be a co - morbid phenomenon with other pain syndromes [10, 11 ] (e.g. fibromyalgia), and to involve increased sensitivity to pain and touch. it is also possible that migraineurs tend to be more attentive to somatic and pain symptoms. recurrent pain is often characterized by elevated pain - related fear and avoidance of behaviors which the headache sufferer believe will initiate or perpetuate the headache pain, a maladaptive process which serves to promote pain related disability. this process might be the underlying reason for the increased fear of sexual intercourse and penetration among migraine patients in this study. the present study was designed as a pilot study, exploring different aspects of female sexuality in migraine sufferers and non - sufferers. to avoid bias, the evaluated population is not representative of the general population, but rather a homogenous population of young, educated, secular women belonging to a relatively high socioeconomic status. one has to take into account that only in such a population asking intimate sexual questions, and getting honest answers ; is feasible. compared to israeli women in similar age group [6, 7 ], our study population reported higher sexual desire, tended more to initiate sexual intercourse with their partner, and lower fear of intercourse and penetration. the interpretation of the findings should therefore be limited to this group of women only. as the respondents were asked to report about only troubling headaches, only a small number of subjects reported headaches corresponding to ihs criteria of tension type headache. no information regarding tension headache can be concluded from this study and further investigation is required. therefore, no conclusions can be drawn from the current study regarding patients with this type of headache. despite these limitations, our findings suggest that migraine affects the sexual life of women suffering from it. this effect is both severity dependent (as represented by the health influence domain) and independent (intercourse associated pain). future studies should be directed in exploring the incidence of sexual dysfunction in general and specifically sexual pain disorders in other migraine suffering populations, and in evaluating the exact nature of the sexual pain disorders.	chronic illness and chronic pain can have profound negative effects on relationship and sexual satisfaction, yet the influence of migraine on sexuality has not been previously evaluated. to assess sexual functions in subjects with migraine compared to those with no migraine. we evaluated female university students using the israeli sexual behavior inventory (isbi). migraine was diagnosed according to self - reported symptoms according to the ihs criteria. several dimensions of female sexuality desire, orgasm, sexual avoidance, interpersonal sexual relationship, health influence, satisfaction and pain were evaluated using a structured questionnaire. thirty - three (23.9%) of the participants met the ihs criteria for episodic migraine with and without aura. sexual activity, desire, orgasm and satisfaction from sexual life did not differ significantly between migraine sufferers and non - sufferers. migraine patients reported lower isbi scores, higher health influence on sexual life, higher levels of sexual pain and lower sexual satisfaction. migraine negatively affected the sexual life of sufferers. sexual pain disorder is more common among migraine sufferers compared to non - migraineurs.
the institutional review board approved this study and did not require the approval of patients nor their informed consent for review of their images and records. from january 2002 to july 2004, 303 women underwent breast mri examinations were initially considered for this study. the primary inclusion criterion of this retrospective analysis was a preoperative mri in patients with histologically confirmed breast cancer. among the 303 women, 213 patients had confirmed breast cancer. of these patients, 61 who underwent neoadjuvant chemotherapy were excluded because of a possible chemotherapeutic effect causing different kinetics and morphology of suspicious lesions (5). the average age of the patients in the present study was 50 years (range 23 to 79 years). indications for an mr examination included preoperative staging before planned breast conserving therapy to exclude multicentricity in patients who had one mammographic or sonographic suspicious lesion (n = 123), a search for an occult tumor with axillary metastases of suspected breast origin (n = 5) and a postoperative examination to rule out residual disease (n = 21). a review of the mr images and final reports by an experienced radiologist specialized in breast imaging was conducted. we searched the cases with additional suspicious breast lesions that were initially detected by mri. by the definition of liberman (6), mr lesions were considered to be additional sites if they were located in a different breast quadrant than the index cancer, if they were in the same quadrant but were separated from the index cancer by at least 1.0 cm of intervening normal - appearing tissue on mri, or if they were in the same quadrant and contiguous with the index cancer but extended at least 4.0 cm beyond the site of the index cancer. typically benign appearing enhancing lesions, i.e. those with a circumscribed margin or delayed enhancement were excluded. if mri revealed additional suspicious breast lesions other than the index cancer, a targeted us performed by the radiologist who interpreted the mr images was recommended to look for a us correlates amenable to further biopsy or localization. although there were no standardized protocols to follow, a targeted us examination by the radiologists who had knowledge of the mri findings was performed for the clinical and mammographical occult lesions. when targeted us was performed, the additional suspicious breast lesions were classified into two groups based on whether or not they had a us correlates. we compared the rate of identifying additional cancers between the lesions with and without a us correlate using the fisher 's exact test. the 31 patients that did not receive targeted us consisted of two groups ; one of the groups in whom the breast us was performed before the breast mr imaging and did not correlate with any additional detectable lesion by mri and the other group with mammographic microcalcifications, which were not detected initially and did correlate with the mri lesions. these lesions were confirmed by surgical excision or were followed with a subsequent examination. breast mri was performed with the patient prone in a 1.5-t commercially available imager (signa ; ge medical system, milwaukee, wi) with the use of a dedicated surface breast coil. the mri sequence used in this study included a fat suppressed axial fast spin echo t2-weighted sequence (4000/120, repetition time msec / echo time msec) and fat - suppressed unilateral sagittal dynamic imaging. dynamic imaging was performed with a t1-weighted three - dimensional, fat - suppressed fast spoiled gradient - echo sequence (17.3/1.3 ms ; flip angle, 30) three times (one before and two after a rapid bolus injection of 0.1 mmol / kg gadopentetate dimeglumine [magnevist ; berlex, wayne, nj ]) (7). section thickness was held between 1.0 - 2.0 mm without an intersection gap with a 256 192 matrix, an 18 - 24 cm field of view, and a scan time of 3 - 4 minutes. two sequential post - contrast scans were obtained without a break, beginning immediately after the saline flush. standard subtraction images were obtained by subtracting the precontrast images from the early peak (or serial) postcontrast image on a pixel - by - pixel basis. reverse subtraction images were obtained by subtracting the last postcontrast image from the early peak postcontrast image. if the lesion demonstrated a kinetic pattern showing an early rise and an early washout, the lesion would be observed with the remaining high signal intensity on reverse subtraction images. if any kinetic curves were equivocal, the morphologic features were considered together. if the lesion has a spiculate or an irregular margin, we regarded it as a morphological suspicious lesion. if a lesion had morphologic features that indicated it was probably benign but reverse subtraction images demonstrated a high signal intensity lesion, this was also considered to be suspicious (8). criteria for the features of suspicious non - mass - like enhancements included linear, ductal, and segmental patterns. breast us examinations were performed by one of four breast imaging radiologists using a 7 - 10 mhz linear transducer (logiq 700 ; general electric, milwaukee, wi, or hdi 5000 ; philips medical systems, bothell, wa). at the time of breast us thirty - eight lesions in 31 patients were performed with targeted us. of 21 patients with correlated suspicious enhancing lesions that were evident on us, eight underwent us - guided core biopsy for the suspicious lesion and 10 underwent us - guided localization, one with us - guided hook - wire localization and the remaining nine with us - guided carbon marking (9). in the residual three patients, two patients had a scheduled mastectomy due to the extensive extent of an index cancer and one had breast conserving surgery because of the lesion localized 1 cm just inferior to the index cancer within the same quadrant. in the 10 patients without a us of the four that underwent a us - guided localization, us - guided needle localization was used in one and carbon marking in three. although lesions are not defined on us with certainty, localizations were performed in five lesions of four patients for subtle sonographic lesions likely consistent with the additional suspicious mr lesions. as the lesions without a us correlate could not be localized, careful histological examinations were requested at the area of any additional mr lesions. the institutional review board approved this study and did not require the approval of patients nor their informed consent for review of their images and records. from january 2002 to july 2004, 303 women underwent breast mri examinations were initially considered for this study. the primary inclusion criterion of this retrospective analysis was a preoperative mri in patients with histologically confirmed breast cancer. among the 303 women, 213 patients had confirmed breast cancer. of these patients, 61 who underwent neoadjuvant chemotherapy were excluded because of a possible chemotherapeutic effect causing different kinetics and morphology of suspicious lesions (5). the average age of the patients in the present study was 50 years (range 23 to 79 years). indications for an mr examination included preoperative staging before planned breast conserving therapy to exclude multicentricity in patients who had one mammographic or sonographic suspicious lesion (n = 123), a search for an occult tumor with axillary metastases of suspected breast origin (n = 5) and a postoperative examination to rule out residual disease (n = 21). a review of the mr images and final reports by an experienced radiologist specialized in breast imaging was conducted. we searched the cases with additional suspicious breast lesions that were initially detected by mri. by the definition of liberman (6), mr lesions were considered to be additional sites if they were located in a different breast quadrant than the index cancer, if they were in the same quadrant but were separated from the index cancer by at least 1.0 cm of intervening normal - appearing tissue on mri, or if they were in the same quadrant and contiguous with the index cancer but extended at least 4.0 cm beyond the site of the index cancer. typically benign appearing enhancing lesions, i.e. those with a circumscribed margin or delayed enhancement were excluded. if mri revealed additional suspicious breast lesions other than the index cancer, a targeted us performed by the radiologist who interpreted the mr images was recommended to look for a us correlates amenable to further biopsy or localization. although there were no standardized protocols to follow, a targeted us examination by the radiologists who had knowledge of the mri findings was performed for the clinical and mammographical occult lesions. when targeted us was performed, the additional suspicious breast lesions were classified into two groups based on whether or not they had a us correlates. we compared the rate of identifying additional cancers between the lesions with and without a us correlate using the fisher 's exact test. the 31 patients that did not receive targeted us consisted of two groups ; one of the groups in whom the breast us was performed before the breast mr imaging and did not correlate with any additional detectable lesion by mri and the other group with mammographic microcalcifications, which were not detected initially and did correlate with the mri lesions. these lesions were confirmed by surgical excision or were followed with a subsequent examination. breast mri was performed with the patient prone in a 1.5-t commercially available imager (signa ; ge medical system, milwaukee, wi) with the use of a dedicated surface breast coil. the mri sequence used in this study included a fat suppressed axial fast spin echo t2-weighted sequence (4000/120, repetition time msec / echo time msec) and fat - suppressed unilateral sagittal dynamic imaging. dynamic imaging was performed with a t1-weighted three - dimensional, fat - suppressed fast spoiled gradient - echo sequence (17.3/1.3 ms ; flip angle, 30) three times (one before and two after a rapid bolus injection of 0.1 mmol / kg gadopentetate dimeglumine [magnevist ; berlex, wayne, nj ]) (7). section thickness was held between 1.0 - 2.0 mm without an intersection gap with a 256 192 matrix, an 18 - 24 cm field of view, and a scan time of 3 - 4 minutes. two sequential post - contrast scans were obtained without a break, beginning immediately after the saline flush. standard subtraction images were obtained by subtracting the precontrast images from the early peak (or serial) postcontrast image on a pixel - by - pixel basis. reverse subtraction images were obtained by subtracting the last postcontrast image from the early peak postcontrast image. if the lesion demonstrated a kinetic pattern showing an early rise and an early washout, the lesion would be observed with the remaining high signal intensity on reverse subtraction images. if any kinetic curves were equivocal, the morphologic features were considered together. if the lesion has a spiculate or an irregular margin, we regarded it as a morphological suspicious lesion. if a lesion had morphologic features that indicated it was probably benign but reverse subtraction images demonstrated a high signal intensity lesion, this was also considered to be suspicious (8). criteria for the features of suspicious non - mass - like enhancements included linear, ductal, and segmental patterns. breast us examinations were performed by one of four breast imaging radiologists using a 7 - 10 mhz linear transducer (logiq 700 ; general electric, milwaukee, wi, or hdi 5000 ; philips medical systems, bothell, wa). at the time of breast us examination, prior mr images were made available for direct correlation. of 21 patients with correlated suspicious enhancing lesions that were evident on us, eight underwent us - guided core biopsy for the suspicious lesion and 10 underwent us - guided localization, one with us - guided hook - wire localization and the remaining nine with us - guided carbon marking (9). in the residual three patients, two patients had a scheduled mastectomy due to the extensive extent of an index cancer and one had breast conserving surgery because of the lesion localized 1 cm just inferior to the index cancer within the same quadrant. in the 10 patients without a us correlate, the lesions in six patients could not be localized. of the four that underwent a us - guided localization, us - guided needle localization was used in one and carbon marking in three. although lesions are not defined on us with certainty, localizations were performed in five lesions of four patients for subtle sonographic lesions likely consistent with the additional suspicious mr lesions. as the lesions without a us correlate could not be localized, careful histological examinations were requested at the area of any additional mr lesions. of the 149 total patients for whom mri was used for preoperative evaluation of breast cancer, 69 additional suspicious breast lesions were detected by mri in 62 patients (42%). of those 69 lesions, 26 (38%) were confirmed as cancers by histology. by mri, 48 of 69 lesions (70%) revealed a mass, and 21 (30%) demonstrated nonmass - like enhancements and the average diameter of the 69 additional enhancing lesions in these 62 patients was 1.1 cm (range, 0.4 - 5.0 cm). in addition, of these 62 patients with 69 lesions, 57 (92%) had a single additional lesion, four (6%) had two lesions, and one (2%) had four lesions. among 69 lesions, contralateral enhancing lesions were detected in ten patients (16%), of which confirmed cancers in three patients (5%). of the 62 patients with additional lesions, 31 patients with 38 lesions underwent targeted us. histological findings of these 38 lesions in 31 patients performed with targeted us revealed cancers in 18 (47%) lesions, high - risk lesions in two (5%), benign lesions in 15 (39%), and unidentified lesions in three (8%) (table 1). the latter three lesions that were not confirmed by histological examination were removed by a mastectomy in one patient and breast conservative surgery with a wide extent in two patients. of the 38 lesions examined with targeted us, 27 (71%) had a us correlate, which included 15 (56%, 15/27) cancers (fig. 1), two (7%, 2/27) high - risk lesions (lobular carcinoma in situ and atypical ductal hyperplasia in each one) and ten (37%, 10/27) benign lesions (fig. 2). of the 11 lesions without a us correlate, three (27%, 3/11) proved to be cancerous (fig. 3) and five (45%, 5/11) were benign as determined by mastectomy or breast conserving surgery with additional excision (fig. 4), however, three (27%, 3/11) were not confirmed by histology. the cancer rate was statistically higher in lesions with a us correlate than in ones without a us correlate (p = 0.028) (table 2). of 15 cancers with a us correlate us, ten (67%) were confirmed as invasive ductal carcinoma, and five (33%) as ductal carcinoma in situ. of the three cancers without a us correlate, two (67%) were ductal carcinoma in situ, and one (33%) as an invasive ductal carcinoma. the difference between invasive and in situ carcinoma was not statistically significant (p = 0.528). of us correlate lesions identified by histology, the high - risk lesions included lobular carcinoma in situ and atypical ductal hyperplasia for each one. the benign lesions were diagnosed as papilloma in three, fibroadenoma, stromal fibrosis, fibrocystic disease in two each, and columnar cell change in one. imaging findings and pathological results of us correlate lesions are summarized in table 3. of 31 patients who underwent targeted us, the surgical plan was altered in 27 patients (87%) and not in four patients. targeted us justified a change in treatment for 22 patients (81%) and misled five patients (19%) into an unnecessary surgical excision. of 21 patients with us correlates, the surgical treatment plan for 19 patients was altered preoperatively with the consent of the patient. these 19 patients included 14 with additional cancers, two with high - risk lesions and three with benign lesions (one fibroadenoma and two intraductal papillomas). even though the us - guided core biopsy for three patients confirmed benign lesions, these patients underwent conserving surgery and a wider excision, because the surgeon did not want to defer the operation because of a personal concern of the patient or delayed pathological results with the consensus of the patient. of the ten patients without us correlates, surgical treatment was changed in eight patients. however, three patients for whom cancers were confirmed subsequently underwent mastectomy at the discretion of surgeon (two), and conservative surgery with a wide excision during the operation (one). five patients underwent unnecessary wide excision due to the difficulty in localizing the additional lesions. in 13 (76%) of 17 patients (14 with us correlates and 3 without us correlates) in whom mri detected additional cancers, the surgery was converted to mastectomy in ten or additional contralateral conserving surgery in three. of the 149 total patients for whom mri was used for preoperative evaluation of breast cancer, 69 additional suspicious breast lesions were detected by mri in 62 patients (42%). of those 69 lesions, 26 (38%) were confirmed as cancers by histology. by mri, 48 of 69 lesions (70%) revealed a mass, and 21 (30%) demonstrated nonmass - like enhancements and the average diameter of the 69 additional enhancing lesions in these 62 patients was 1.1 cm (range, 0.4 - 5.0 cm). in addition, of these 62 patients with 69 lesions, 57 (92%) had a single additional lesion, four (6%) had two lesions, and one (2%) had four lesions. among 69 lesions, contralateral enhancing lesions were detected in ten patients (16%), of which confirmed cancers in three patients (5%). of the 62 patients with additional lesions, 31 patients with 38 lesions underwent targeted us. histological findings of these 38 lesions in 31 patients performed with targeted us revealed cancers in 18 (47%) lesions, high - risk lesions in two (5%), benign lesions in 15 (39%), and unidentified lesions in three (8%) (table 1). the latter three lesions that were not confirmed by histological examination were removed by a mastectomy in one patient and breast conservative surgery with a wide extent in two patients. of the 38 lesions examined with targeted us, 27 (71%) had a us correlate, which included 15 (56%, 15/27) cancers (fig. 1), two (7%, 2/27) high - risk lesions (lobular carcinoma in situ and atypical ductal hyperplasia in each one) and ten (37%, 10/27) benign lesions (fig. 2). of the 11 lesions without a us correlate, three (27%, 3/11) proved to be cancerous (fig. 3) and five (45%, 5/11) were benign as determined by mastectomy or breast conserving surgery with additional excision (fig. 4), however, three (27%, 3/11) were not confirmed by histology. the cancer rate was statistically higher in lesions with a us correlate than in ones without a us correlate (p = 0.028) (table 2). of 15 cancers with a us correlate us, ten (67%) were confirmed as invasive ductal carcinoma, and five (33%) as ductal carcinoma in situ. of the three cancers without a us correlate, two (67%) were ductal carcinoma in situ, and one (33%) as an invasive ductal carcinoma. the difference between invasive and in situ carcinoma was not statistically significant (p = 0.528). of us correlate lesions identified by histology, the high - risk lesions included lobular carcinoma in situ and atypical ductal hyperplasia for each one. the benign lesions were diagnosed as papilloma in three, fibroadenoma, stromal fibrosis, fibrocystic disease in two each, and columnar cell change in one. of 31 patients who underwent targeted us, the surgical plan was altered in 27 patients (87%) and not in four patients. targeted us justified a change in treatment for 22 patients (81%) and misled five patients (19%) into an unnecessary surgical excision. of 21 patients with us correlates, the surgical treatment plan for 19 patients was altered preoperatively with the consent of the patient. these 19 patients included 14 with additional cancers, two with high - risk lesions and three with benign lesions (one fibroadenoma and two intraductal papillomas). even though the us - guided core biopsy for three patients confirmed benign lesions, these patients underwent conserving surgery and a wider excision, because the surgeon did not want to defer the operation because of a personal concern of the patient or delayed pathological results with the consensus of the patient. of the ten patients without us correlates, surgical treatment was changed in eight patients. however, three patients for whom cancers were confirmed subsequently underwent mastectomy at the discretion of surgeon (two), and conservative surgery with a wide excision during the operation (one). five patients underwent unnecessary wide excision due to the difficulty in localizing the additional lesions. in 13 (76%) of 17 patients (14 with us correlates and 3 without us correlates) in whom mri detected additional cancers, the surgery was converted to mastectomy in ten or additional contralateral conserving surgery in three. breast mri is generally accepted as the preferred diagnostic method for patients with breast cancer because of its high sensitivity (10 - 12). in contrast, the specificity of breast mr images remains highly variable (37 - 100%) (10, 11). in situations where there is a high probability of cancer such as preoperative staging, use of mri the detection of additional lesions by mri in breast cancer patients was frequent (42%). among the 69 additional lesions detected by mri, 26 (38%) were shown to be cancers following a histological examination. because of the need to biopsy and localize these lesions initially seen only on mri, and until recently, the lack of biopsy systems that were mr compatible and commercially available, we designed an alternative method. if a lesion that is detected only on mri can be found on us through careful re - examination, the visualization of the lesion on us will enable a us - guided intervention by a skilled radiologist and allow the patients to possibly avoid a multi - step surgery. some investigators have previously reported on the reliability of targeted ultrasound for suspicious mri - detected lesions (13 - 15). because targeted us might be complementary to a subjective us by referencing mr imaging, this technical procedure is more helpful in reducing the number of missed cancers than using us alone or using us before mri. in our study, targeted us found 71% of the additional lesions seen only on mr in breast cancer patients. in comparison to a previous report where 23% of the cases were detected, we suggest the basis for these differing results include the selection of patients that had a high probability of cancer, the more frequent use of bilateral whole breast ultrasound due to unfamiliarity with a skillful mr - guided biopsy technique, and an advantage in lesion detection due to the relatively smaller - sized breasts of asian women included in the study that allowed routine use of a smaller film size (eg. biopsy or localization was successfully completed in all patients with a us correlate, and 15 (71%) of these patients had additional sites of cancer subsequently identified by mri. of these 31 patients who underwent targeted us, carcinoma was found in 56% of patients that had a us correlate ; in comparison, cancer was identified in 27% of patients lacking a us correlate. similar to a previous study, a us correlate was more often observed for cancers in the present study (3). twenty lesions (74%) out of 27 with a us correlate were detected as a mass (or small nodule) on mri and were observed more often for invasive forms (67%) than for in situ forms (33%) (p = 0.528). additional lesions that are verified by mri may lead to either a justified surgical excision or over - treatment. in a previous study on preoperative mri, planned surgical management was altered in 26% of the cases (16). in our study, we demonstrated that the targeted us results altered subsequent surgical management from what had been planned initially for 27 (87%) of all 31 patients who underwent targeted us. targeted us in addition to mri seems to contribute to an alteration in the surgical plan. targeted us justified the change in treatment for 22 patients (81%) and misled five patients (19%) to unnecessary surgical excision. the five over - treated patients who underwent additional treatment had lesions without a us correlate. a retrospective study such as this has deficiencies. first, histological confirmation was absent or unavailable in some instances, because of the inability to conduct mri - guided percutaneous biopsy. thus, excision of lesions without a us correlate sometimes resulted in sacrificing large amounts of normal tissue to examine an mr - suspicious area. therefore, we speculate that an mr - guided biopsy provides the unique ability to identify these lesions that are not visualized by us (17, 18). second, although us in this study was performed by four skilled breast imaging radiologists and carefully correlated with additional imaging studies, us interpretation is highly dependent upon both the operator and the equipment. as for the us findings in our study, radiologists tend to search for a sonographic hypoechoic lesion in order for it to be considered as suspicious. there may be a chance of missing the additional lesion that would be seen as a hyperechoic or isoechoic lesion by targeted us. it has been our experience that if a suspicious lesion in patients with breast cancer is additionally depicted only on mri, re - examination with another modality, specifically ultrasound, can improve the accuracy of identification and characterization of the lesion, thereby increasing confidence in the application of an intervention. however, our results also highlighted the need of mr - guided biopsies in some cases. in conclusion, targeted us can play a useful role in the evaluation of additional suspicious mr lesions in breast cancer patients, but is limited in lesions without a us correlate.	objectiveto investigate the usefulness of targeted ultrasound (us) in the identification of additional suspicious lesions found by magnetic resonance (mr) imaging in breast cancer patients and the changes in treatment based on the identification of the lesions by the use of targeted us.materials and methodsone - hundred forty nine patients who underwent breast mr imaging for a preoperative evaluation of breast cancer between january 2002 and july 2004 were included in the study. we searched all cases for any additional lesions that were found initially by mr imaging and investigated the performance of targeted us in identifying the lesions. we also investigated their pathological outcomes and changes in treatment as a result of lesion identification.resultsof the 149 patients with breast cancer, additional suspicious lesions were detected with mr imaging in 62 patients (42%). of the 69 additional lesions found in those 62 patients, 26 (38%) were confirmed as cancers by histology. thirty - eight lesions in 31 patients were examined with targeted us and were histologically revealed as cancers in 18 (47%), high risk lesions in two (5%), benign lesions in 15 (39%), and unidentified lesions in three (8%). the cancer rate was statistically higher in lesions with a us correlate than in lesions without a us correlate (p = 0.028). of 31 patients, the surgical plan was altered in 27 (87%). the use of targeted us justified a change in treatment for 22 patients (81%) and misled five patients (19%) into having an unnecessary surgical excision.conclusiontargeted us can play a useful role in the evaluation of additional suspicious lesions detected by mr imaging in breast cancer patients, but is limited in lesions without a us correlate.
so the cells that are metabolically or physiologically active but can not be cultured on specific media are the viable but nonculturable cells (vbnc). in fact, less than 1% of the microorganisms in natural water and soil samples are cultured in viable count procedures. in 1982, xu and coworkers introduced the term viable but nonculturable bacterial cells (vbnc) to distinguish particular cells that could not form colonies on solid media but retained metabolic activity and the ability to elongate after the administration of nutrients. according to oliver, vbnc can be defined as a metabolically active bacterial cell that crossed a threshold in this way, for known or unknown reasons, and became unable to multiply in or on a medium normally supporting its growth. most of the bacteria that enter vbnc state are gram - negative species belonging to the gamma subclass of the proteobacteria branch, except for rhizobium, agrobacterium, and helicobacter - campylobacter species. bashford and colleagues announced that they had recovered vibrio cholerae from streams and drainage ditches, including sites with negligible chance of sewage contamination. around the same time, colwell. also found vibrio cholerae in maryland, usa. she and her coworkers showed that, in artificial sea water, vibrio cholera and e. coli remained viable though they lost the capacity to form colonies on culture media. soon salmonella enteritidis, shigella sonnei, and legionella pneumophila joined the list of organisms known to be capable of entering a state in which they failed to show up on nutrient agar yet took up substrates and signaled in other ways that they were certainly not dead. the use of laboratory media to recover and enumerate bacteria and to link them with or absolve them from pathological and other activities became obsolete by the new discoveries, and the term vbnc (viable but nonculturable) was introduced. microorganisms that do not grow in culture media, but are still metabolically active and capable of causing infections in animals and plants, are said to be in a vbnc state. traditional laboratory culture conditions and methods can not meet the requirements of vbnc organisms to resume growth. vbnc cells exhibit active metabolism in the form of respiration or fermentation, incorporate radioactive substrates, and have active protein synthesis but can not be cultured or grown on conventional laboratory media. they have been detected by observing discrepancies between plate count enumeration of bacterial population and direct staining and microscopic counts. for example, viable but nonculturable cells of vibrio cholerae, enteropathogenic e. coli, legionella pneumophila, and various other bacteria have been shown to regain culturability after they have entered the intestinal tracts of animals. the vbnc state is defined as a state of dormancy triggered by harsh environmental conditions, such as nutrient starvation, extreme temperatures, and sharp changes in ph or salinity ; osmotic stress, oxygen availability [20, 21 ], and damage to or lack of an essential cellular component including dna ; exposure to food preservatives and heavy metals [23, 24 ] ; exposure to white light ; activation of lysogenic phages or suicide genes such as sok / hak or autolysins ; and decontaminating processes such as pasteurization of milk and chlorination of wastewater. the characteristics of bacteria in the vbnc state can be summarized as follows : maintaining apparent cell integrity ; possessing some form of measurable cellular activity ; possessing apparent capacity to regain culturability in vivo ; responding to external stimulus as shown by specific gene expression ; having low metabolic activity ; exhibiting dwarfing ; having reduced nutrient transport ; containing a high atp level and exhibit high membrane potential ; having extensive modifications to the fatty acid composition in cytoplasmic membranes ; within the cell wall peptidoglycan, increased crosslinking, increased muropeptides bearing covalently bound lipoprotein, and shortening of the average length of glycan strands ; higher autolytic capability than exponentially growing cells ; retained plasmids ; increased antibiotic resistance due to lower metabolic activity ; changes in outer - membrane protein profile ; continuous gene expression.the vbnc state continues to be disputed due to the difficulties of differentiating vbnc cells and dormant cells through resuscitation and phenotypic studies. however, recent molecular studies have generated data to support the existence of the vbnc state. maintaining apparent cell integrity ; possessing some form of measurable cellular activity ; possessing apparent capacity to regain culturability in vivo ; responding to external stimulus as shown by specific gene expression ; having low metabolic activity ; exhibiting dwarfing ; having reduced nutrient transport ; containing a high atp level and exhibit high membrane potential ; having extensive modifications to the fatty acid composition in cytoplasmic membranes ; within the cell wall peptidoglycan, increased crosslinking, increased muropeptides bearing covalently bound lipoprotein, and shortening of the average length of glycan strands ; higher autolytic capability than exponentially growing cells ; increased antibiotic resistance due to lower metabolic activity ; changes in outer - membrane protein profile ; continuous gene expression. the vbnc is also considered an important reservoir of many human pathogens in the environment. the following list includes but is not limited to the pathogenic bacteria that can enter the vbnc state : aeromonas hydrophila, agrobacterium tumefaciens, burkholderia cepacia, campylobacter jejuni, enterobacter aerogenes, enterobacter cloacae, enterococcus faecalis, escherichia coli (including ehec), helicobacter pylori, klebsiella pneumoniae, legionella pneumophila, listeria monocytogenes, mycobacterium tuberculosis, pseudomonas aeruginosa, salmonella typhi, salmonella typhimurium, shigella dysenteriae, shigella flexneri, shigella sonnei, streptococcus faecalis, vibrio alginolyticus, vibrio cholerae, vibrio harveyi, vibrio parahaemolyticus, and vibrio vulnificus (types 1 and 2). many believe that pathogens in the vbnc state are unable to induce infection / disease despite retaining their virulent properties. however, when vbnc pathogens pass through a host animal, resuscitation and resumption of metabolic activity have led to infections and diseases [40, 41 ]. the first evidence of pathogenicity of nonculturable cells was the demonstration of fluid accumulation in the rabbit ileal loop assay (rica) by vbnc vibrio cholerae o1, followed by human volunteer experiments. vbnc e. coli nonculturable cells were also reisolated after passaging through rabbit ileal loops 4 days after inoculation, and chick embryos died when injected with nonculturable cells of legionella pneumophila, leading to the conclusion that vbnc pathogens remain potentially pathogenic. many indicator bacteria and pathogenic bacteria which exist in aquatic habitats have been shown to have a vbnc state. water is routinely tested for such indicators and pathogens, and if they are not detected or enumerated at a concentration below guidelines, waters are deemed to be safe for public consumption. therefore, where circumstances indicate possible presence of vbnc pathogens, additional molecular methodology needs to be used to reduce the risk of infective disease outbreaks. thus, food and environmental and clinical samples no longer can be considered free from pathogens if culturing yields negative results. for the general public, the presence of vbnc in water and food may be related to low - grade infections or the so - called aseptic infections. in many cases, the infections are incorrectly attributed to viruses since no bacteria were detected. for example, vibrio cholerae o1 in the surface water have been shown to remain in nonculturable state. these water sources are used for domestic purpose regularly and pose a risk of infection. when conditions are not favorable for growth, then it transforms to the nonculturable state in association with crustacean copepods. persistence of vibrio cholerae in water in the vbnc state is an important public health factor, since detection will not be successful if only conventional cultural methods are used. similarly, shigella can undergo vbnc state in water but become a threat when they enter the human body. additional studies have indicated that a good number of pathogenic bacteria can survive food and water treatment processes and persist as well as retain virulence in processed food, pasteurized milk, potable water, and the environment. one study demonstrated that recurrent urinary tract infections in many individuals were caused by uropathogenic e. coli cells which remained in vbnc state. other studies showed that uropathogenic vbnc e. coli retain enteropathogenicity as shown by continual production of enterotoxin. nilsson. showed that vbnc helicobacter pylori cells can express virulence factors such as caga, vaca, and vrea. food and its surrounding environment are a complex system, in which physiochemical characteristics (ph, aw, and chemical composition) and environmental factors (storage temperature and time, decontamination treatments, and packaging under modified atmosphere) act simultaneously on contaminating bacteria leading to the vnbc state. for example, it has been demonstrated that refrigerated pasteurized grapefruit juice induced the vbnc state in e. coli o157 : h7 and s. typhimurium within 24 hours of incubation. reported that in pasteurized milk which has undergone thermal treatment, contaminating bacteria such as e. coli and pseudomonas putida enter into the vbnc state but retain transcription and translation functions. several foodborne outbreaks have been reported in japan, where pathogens such as salmonella enterica subsp. enterica and e. coli o157 in the vbnc state were responsible for outbreaks. the vnbc is also critical in determining shelf life and microbial stability of food and beverages. for example, acetic acid and lactic acid bacteria entered the vbnc state in wine as a consequence of lack of oxygen and presence of sulphites. nalidixic acid (2040 mg / l) is used to stop cell division. after exposure to nalidixic acid, viable cells continue to grow and will appear elongated, whereas the nonviable metabolically inactive cells will retain their original shape and size. viable cells will be seen as elongated, whereas vbnc / dormant cells will be seen as oval and large. frequently used stains are acridine orange, 4,6-diamino-2-phenyl indole (dapi), fluorescein isothiocyanate (fitc), indophenyl - nitrophenyl - phenyltetrazolium chloride (int), and 5-cyano-2,3-ditolyl tetrazolium chloride (ctc). the mode of action of these dyes and the reactions observed are summarised in table 1. in recent years, a new differential staining assay, the baclight live / dead assay, has been developed. the assay allows simultaneous counting total and viable (metabolically active) cells, by using two nucleic acid stains, that is, green - fluorescent syto 9 stain and red - fluorescent propidium iodide stain. syto 9 stains both live and dead bacteria, whereas propidium iodide penetrates only bacteria having damaged membranes. when used together, propidium iodide reduces syto 9 fluorescence in dead bacteria with damaged membranes resulting in red fluorescent cells, whereas the live bacteria will fluoresce green. are nucleic acids (dna / rna) which have been chemically or radioactively labeled and are used to detect complementary target dna / rna. specific amplification of dna targets in bulk dna extracts from environmental and clinical samples permits detection of specific organisms or groups of related organisms without the need to cultivate them, provided the appropriate unique primers are used. these procedures do not discriminate between culturable and nonculturable forms of the target organisms. due to the failure of distinguishing between dead or live cells by dna - based methods, rna - based methods are a more valuable estimate of gene expression and/or cell viability under different conditions. this technique is more able to discriminate between culturable and nonculturable forms of an organism. furthermore, reverse transcriptase pcr (rt - pcr) can distinguish between live and dead cells. this is possible because it is an mrna - based method and mrna is short - lived (half - life less than 1 minute). messenger rna is only present in metabolically active cells and not found in nature after cell death. rt pcr can detect nonculturable but active or live cells. even though traditional culture methods fail to detect the presence of specific vbnc in a sample, the presence of these microbes can be demonstrated using some of the molecular techniques described. more specifically, oligonucleotide probes of l820 nucleotides are proving most useful because they hybridize rapidly to specific dna sequences of target organisms. the detection of vbnc cells directly from the environmental samples can also be achieved using different types of blotting such as colony blot, slot blot, dot blot, and southern blot. the principle of blotting is the use of radio- or nonradioactive or fluorescence labeled probe. fluorescent in situ hybridization (fish) is an alternative format for hybridization probes in which fluorescence labeled dna or rna probes are hybridized with target nucleic acids in whole, permeabilized cells. the application of this method to the detection of single microbial cells by using this is done through selective targeting of regions of rrna, which consist of conserved and variable nucleotide regions. by choosing the appropriate rrna probe sequence, fish can be used to detect all bacterial cells (a universal probe) or a single population of cells (a strain - specific probe) of vbnc. from the above discussions, it is evident that a number of nonspore - forming human pathogenic bacteria can enter the vbnc state with maintained cellular structure and biology and persistent gene expression but remain nonculturable by traditional cultural techniques. they can survive and revert to culturable conditions when provided with appropriate conditions, hence, being a significant threat to public health and food safety. further research is needed to elucidate mechanisms leading to the vbnc and the development of methodologies to confirm their existence.	the viable but nonculturable (vbnc) state is a unique survival strategy of many bacteria in the environment in response to adverse environmental conditions. vbnc bacteria can not be cultured on routine microbiological media, but they remain viable and retain virulence. the vbnc bacteria can be resuscitated when provided with appropriate conditions. a good number of bacteria including many human pathogens have been reported to enter the vbnc state. though there have been disputes on the existence of vbnc in the past, extensive molecular studies have resolved most of them, and vbnc has been accepted as a distinct survival state. vbnc pathogenic bacteria are considered a threat to public health and food safety due to their nondetectability through conventional food and water testing methods. a number of disease outbreaks have been reported where vbnc bacteria have been implicated as the causative agent. further molecular and combinatorial research is needed to tackle the threat posed by vbnc bacteria with regard to public health and food safety.
endodontically treated teeth restored with post and core face failure due to loosening of the post, fracture of the post or the root itself.1 the common post and core complications documented were post loosening (5%), root fracture (3%), caries (2%), and periodontal disease (2%).2 fracture of the coronal tooth structure can occur on anterior teeth as they are subjected to shear stresses. very often the post and core fails, leaving behind an intact crown. root may have adequate bone support and which could be favorably used if the post is retrieved. this clinical report describes the technique of refabricating post and core to match the features of the existing crown. a 34-year - old female patient reported with fractured maxillary right canine tooth (figure 1). on examination, it was found that the tooth was the root canal treated and restored with a cast post and core and a metal ceramic crown over it (figure 2). the core was fractured leaving the cast post embedded in the root.the post was loosened using ultrasonic vibrations and retrieved using an artery forceps. the residual cement in the post space was removed and a fiber post of diameter 1.5 mm (flexi - post, coltenewhaledent) was bonded using dual - cured composite resin (paracore, coltenewhaledent) (figure 3).the crown was intact, and it was decided to use the same for restoring the fractured tooth. a 50 m polytetrafluoroethylene (ptfe) (teflon) tape was adapted on the tissue surface of the crown and a dual - cured core build - up composite resin (paracore, coltenewhaledent) was injected into the crown and positioned over the bonded post (figures 4 - 6).. it was then light polymerized for 40 s.the crown was then removed leaving the core bonded to the post and the tooth structure. the core was light polymerized again for 40 s. the teflon tape was removed from the crown, and the crown was cemented to the core using glass ionomer cement (gic gold label glass ionomer luting and lining cement) (figure 7).. the patient was followed up for 1 year, and it was uneventful. a 34-year - old female patient reported with fractured maxillary right canine tooth (figure 1). on examination, it was found that the tooth was the root canal treated and restored with a cast post and core and a metal ceramic crown over it (figure 2). the residual cement in the post space was removed and a fiber post of diameter 1.5 mm (flexi - post, coltenewhaledent) was bonded using dual - cured composite resin (paracore, coltenewhaledent) (figure 3). the crown was intact, and it was decided to use the same for restoring the fractured tooth. a 50 m polytetrafluoroethylene (ptfe) (teflon) tape was adapted on the tissue surface of the crown and a dual - cured core build - up composite resin (paracore, coltenewhaledent) was injected into the crown and positioned over the bonded post (figures 4 - 6).. it was then light polymerized for 40 s. the crown was then removed leaving the core bonded to the post and the tooth structure. the core was light polymerized again for 40 s. the teflon tape was removed from the crown, and the crown was cemented to the core using glass ionomer cement (gic gold label glass ionomer luting and lining cement) (figure 7).. the patient was followed up for 1 year, and it was uneventful. the need for retreatment of a tooth and/or prosthetic restoration may arise due to secondary caries, pulpal involvement, trauma to the restoration, and/or foundation, and subjective desires for a more aesthetic or durable restoration. crowned teeth requiring retreatment due to fracture of the underlying tooth structure and/or foundation restoration often possess an undamaged extracoronal restoration. in such cases, it may be desirable to reuse the restoration for the sake of cost and time.3 a badly distorted endodontically treated tooth is always a challenge for any dentist. presented a comprehensive literature review over this topic and described his own way to handle these emergencies. he inserted a prefabricated plastic post into the palatal canal of a maxillary molar. later on following an impression of the remaining tooth, already prepared for a crown was inserted into a fresh fabricated crown, followed by crown cementation.4 an indirect fabrication of two separate castings, first for the post and then fabricating the core for mandibular molars was suggested by sadan. this technique was similar to chiche s technique but worked on indirect fabrication method.5 cast post core systems accumulated stress within cast post cores and apical one - third region of the tooth. using fiber posts, the stresses were distributed to the cervical one - third region of the tooth and the supporting bone. specimens restored with fiber - reinforced post systems offered more homogenous stress distribution than cast posts as fiber posts possess a similar modulus of elasticity to that of dentin.6,7 hence, it was decided to use a glass fiber post for this case. the use of ptfe tape provides a space of approximately 50 m, which was used to accommodate the cement.8 in this paper, the most common difficulty in the clinical practice has been discussed. restoration for the endodontically treated teeth, where there is hardly no proper support for the post makes the work for the clinicians more complicated and challenging. this paper describes a novel technique for refabricating a post and core restoration for an existing crown using polyfluoroethylene tape.	a fractured coronal tooth structure beneath an intact crown is a common clinical occurrence. if the underlying root is healthy, the tooth is restored with a post and core followed by refabrication of the crown. this paper describes a technique of using the existing intact crown for the above - mentioned situation. a 34-year - old female was referred with a fractured right canine with an intact crown. a post was found fractured in the canal which was subsequently retrieved. a new fiber post was cemented in the post space followed by adaptation of 50 m polytetrafluoroethylene (teflon) tape on the tissue surface of the crown. dual - cured core build - up composite resin was injected into the crown and adapted to the fractured tooth. on curing and removal of the crown, a new composite resin core was found bonded to the tooth structure. the teflon tape was removed from the crown, and the crown cemented to the core using glass ionomer cement. this technique of building up the core of the tooth using teflon tape adapted to the tissue surface of the crown was found to be successful even after 1 year of follow - up.
	addition of the carbon fluorine bond of a series of perfluorinated and polyfluorinated arenes across the mg mg bond of a simple coordination complex proceeds rapidly in solution. the reaction results in the formation of a new carbon magnesium bond and a new fluorine magnesium bond and is analogous to grignard formation in homogeneous solution.
this study was a controlled clinical trial aiming to compare the outcomes of open reduction and rigid internal fixation of displaced calcaneal fractures with that of non operative treatment. the research proposal of this study was approved by research department of isfahan university of medical sciences. from december 1998 until january 2009, 72 patients with displaced intra - articular calcaneal fractures admitted in kashani university hospital and were randomly allocated to surgical and non surgical groups. first group underwent open reduction and internal fixation with reconstruction plate and screws fixation and the other group were treated with closed reduction and cast immobilization.13 data were collected by clinical observation and a questionnaire six months following surgery. some reasons that patients were not selected for surgery were as follows : patient disagreement with surgery, open fractures (not suitable for open reduction internal fixation), combined injuries (head trauma, cardiovascular disorders, severe osteoporosis...) also were not suitable for surgery179 and severe comminution. post - operative exam after two weeks, one month, three months, six months and one year was recorded. roentgenography was obtained and physical examination including pain scoring, swelling, limitation of activities, shoe wearing difficulties, range of motion and osteoarthritis in ankle and subtalar joints as well as patient satisfaction were also scored and recorded. functional scoring based on kerr 's meta - analysis and pain scoring was used.1121 data were analyzed by spss 18 software. seventy two patients with mean age 49 years (21 - 84 years old) were included. eleven patients were excluded from the study (unable to follow up after hospital discharge). eventually, 61 cases of calcaneal fractures enrolled and were also followed - up for averagely 3 years. patients in operation group sub - classified in two categories : first group comprised of 17 cases with essex - lopresti technique (under fluoroscopy - x - ray control, closed reduction, internally fixed with pin fixation, and cast immobilization).22 second group consisted of 14 cases with open reduction with lateral calcaneal approach and fixation with reconstruction plate (figure 4). ambulation with crutch walking and non - weight bearing continued for 6 - 10 weeks after surgery. lateral calcaneal approach with exostosectomy thirty cases had non - surgical treatment, with splinting, ice pack, elevation the limb, then short leg cast and ambulation with crutch walking and non weight bearing. in non - surgery group ambulation with crutches started 3 days after casting and cast was removed after 6 weeks and physical therapy, home exercises and partial weight bearing was recommended. the findings regarding the comparison between two groups for different variables are shown in table 1. fracture classification (sander s) in post operation examinations, range of motion of ankle in both groups were good (more than 50% of r.o.m) but in subtalar joints decreased range of motion in non - operative group compared to operative group was obtained (odds ratio : 6.75, p = 0.002). last x - ray examinations showed that traumatic osteoarthritis (degenerative joint disease) in non - operative group was a major problem (26% compared to 9% in operative group). shoe fitting difficulties was also a major problem (50% in non - operative compared with 12% in operative group).18 pain in walking was 63% in conservative group compared to 29% in operative group (odds ratio : 6.72, p = 0.001).2122 swelling in ankle and foot, reflex sympathetic dystrophy including focal osteoporosis and or spotty osteoporosis in non operative group was twice more than operative group (6 cases versus 3 cases ; odds ratio : 6.80, p = 0.001) (table 2). our findings showed that open reduction and internal fixation of displaced calcaneal fractures in absence of open fracture, severe osteoporosis, or comminution and poor general condition may be the preferred method of treatment. although surgical treatment of calcaneal fractures has recently received attention owing to good results and less morbidities,13911 most of orthopaedic surgeons prefer to treat them conservatively, probably because of many complications including post operative infections, wound dehiscence, malreductions and long term osteoarthritis. most of calcaneal fractures are intra - articular and displaced. on the other hand, many of these fractures have ecchymosis, swelling, and blister which increase risk of open reduction and internal fixations (fracture- blister).14 thornes.15 showed that surgically treated calcaneal fractures have better prognosis compared with those conservatively treated. burdeaux 's work also showed that shoe fitting and weight bearing difficulties were less in those underwent operation.16 pain in daily living activities were dramatically less in patients with open reduction and internal fixation, and range of motion in ankle and subtalar joints were desirable.1720 open reduction and internal fixation of displaced calcaneal fractures in absence of open fracture, severe osteoporosis, or comminution, poor general condition may be the preferred method of treatment. young and middle aged patients with calcaneal fracture who are in sander 's type ii and iii with mild comminution due to minor trauma, without soft tissue injuries may be the best candidates for open reduction and internal fixation. mhn and fmm participated in the design study, conduct the study and prepared the manuscript.	background : the aim of this study was to compare outcomes of open reduction and rigid internal fixation of displaced calcaneal fractures with that of non operative treatment.methods:seventy two consecutive patients with displaced intra - articular calcaneal fractures were selected regarding inclusive and exclusive criteria and then were randomly allocated to surgical and non surgical groups. first group underwent open reduction and internal fixation with reconstruction plate and screws fixation and the other group were treated with closed reduction and cast immobilization. data were collected by clinical observation and a check list. data was analyzed by chi - square and student 's t-test.results:the results showed significant difference between outcomes of surgical treatment and nonsurgical method (p = 0.001). there were some differences between two methods in terms of decreasing pain [odd ratio (or) : 6.72, p = 0.001 ], swelling (or : 6.80, p = 0.001), increased range of motion of the joints (p = 0.001), decreased late osteoarthritis (or : 2.33, p = 0.22) in favor of surgical group.conclusions:open reduction and internal fixation of displaced calcaneal fractures in absence of open fracture, severe osteoporosis, or comminution, poor general condition may be the preferred method of treatment.
the presence of oral synechia along with cleft palate is a rare syndrome. we encountered a case of cleft palate accompanied by congenital lateral oral synechia the infant was delivered normally at full term with a low birth weight of 1,750 g. there were no particularly notable points in the family history, medical history, or reproductive history. on examination, the patient had a restricted mouth opening resulting from congenital oral synechia due to membranous adhesion between the free margin of the cleft palate and the floor of the mouth, lateral to the tongue on the left side. the congenital oral synechia appeared as a thin membrane with a broad attachment at the floor of the mouth measuring approximately 2 cm in anteroposterior width. at the palatal margin the attachment narrowed to about 0.5 cm [figure 1 ]. congenital lateral synechia the infant faced feeding problems due to the restricted mouth opening, and therefore an immediate surgical excision of the synechia was decided on. feeding was done using an infant feeding tube till anesthetic clearance was obtained for the procedure. at 1 week after birth, the congenital synechia was excised under sedation uneventfully restoring adequate mouth opening and allowing normal feeding [figure 2 ]. the first report of oral synechia was by illera in 1875, but the first documented case of a lateral synechia between the floor of the mouth and free margin of cleft palate was by hayward and every in 1957. over the years, almost 60 cases of oral synechia have been reported out of which 52 are lateral synechia and 8 were of the median variety. these can be classified into five types : synechia by cord - like adhesion of the alveolar mucosa on one or both sides of the upper and lower jaw (alveolar synechia) ; synechia by a membranous adhesion on the hard palate and floor of the mouth, excluding the rear of the tongue (lateral synechia) ; synechia in which the hard palate and tongue are partially involved ; synechia in which the soft palate and tongue are widely involved, such that continuity is interrupted between the oral cavity and the pharynx ; and synechia by a membranous adhesion between the hard palate and lower lip., reported that five family members had cleft palates and synechia, one having a cleft palate without synechia, and one transmitted the gene but did not express it. the etiology of intra - oral bands or synechia of epithelial tissue has been debated, but many theories have been proposed. during the 7 to 8 week of embryological development, the alveolar ridges, tongue, and palatal shelves are in contact with each other. the ensuing palatal closure depends on downward contraction of the tongue. when the tongue protrudes from the mouth as a result of medial movements of the oral cavity walls, it prevents the alveolar ridges from fusing. genetic, teratogenic, or mechanical insults during this critical stage may lead to periods of close, quiescent contact between oral structures, and this predisposes to abnormal fusion. longacre asserts that oral synechia is due to the persistence of the buccopharyngeal membrane, and is for that reason associated with micrognathia and cleft palate. kruger speculates that the mechanical effect of the tongue may contribute to cases in which the periphery of the cleft palate adheres to mucous membranes on the floor of the mouth, and that adhesion in cases of cleft palate may occur as a result of obstruction by the tongue. according to mathis, when adhesion of the palatal shelf occurs during developmental stages, adhering epithelial rudiments, for some reason, lead to synechia. the general consensus on the treatment of cleft palate lateral synechia syndrome is excision of the synechia and palatal closure. dalal., have reported a case of intra alveolar synechia in two siblings, one of whom had a spontaneous resolution of the adhesion. have documented that the synechia provided additional tissue for surgical closure with less tension on the palatal flaps. we believe that such a use of the synechia has been possible, because the membranous adhesion in this case had fibromuscular bands in it unlike our situation where the synechia consisted of thin membranous tissue of unequal width. the conventional oro - tracheal intubation or the use of a laryngeal mask may not be possible due to the presence of the synechia. fiberoptic nasotracheal intubation may not be feasible due to technical difficulties of finding a bronchoscope small enough for a week old infant. performing a procedure on the highly vascular floor of the mouth under local anesthesia in an infant where immediate excision of the synechia is deemed necessary either due to breathing or feeding problems, sedation can be used under constant anesthetic monitoring for the surgical procedure as was done in our case.	cleft lip and palate are the most common congenital craniofacial anomaly in humans. the presence of oral synechia along with cleft palate is a rare syndrome. we encountered one case that had a cleft palate accompanied by congenital oral synechia due to a membranous adhesion between the floor of the mouth and the free margin of the cleft palate.
educational reform leader michael fullan5 observed that : when adults do think of students, they think of them as the potential beneficiaries of change they rarely think of students as participants in a process of school change and organizational life. fullan5 says that engaging the hearts and minds of students is the key to success in school but many schools see students only as sources of interesting and usable data. students soon tire of invitations that address matters they do not think are important, use language they find restrictive, alienating, or patronizing and that rarely result in action or dialog that affects their lives. despite demonstrated valuable and realistic ideas, student voice is not widespread and students are vastly underutilized resources. student engagement strategies must reach all students, those doing okay but bored by the irrelevance of school, and those who are disadvantaged and find schools increasingly alienating as they move through the grades.5 the research on empowering students validates the wisdom of engaging youth in education, health, and social issues. efficacy studies on youth peer mediation programs in which students are empowered to share responsibility for creating a safe and secure school environment demonstrated the value of turning to students as partners6,7 students learned peer mediation skills, reduced suspensions and discipline referrals in schools, and improved the school climate.7 research has also demonstrated that student peer educators achieve similar or better results than adult educators.8 a review article on the effects of giving students voice in the school decision - making process found evidence of moderate positive effects of student participation on life skills, democratic skills and citizenship, student - adult relationships, and school ethos while finding low evidence of negative effects.9 wallerstein,10 who has supported the use of youth empowerment strategies in all aspects of health promotion, noted that student participation enhances self - awareness and social achievement, improves mental health and academic performance, and reduces rates of dropping out of school, delinquency, and substance abuse. a 2014 review of 26 articles11 to identify the effects of student participation in designing, planning, implementing, and/or evaluating school health promotion measures found conclusive evidence showing (1) enhanced personal effects on students (enhanced motivation, improved attitudes, skills, competencies, and knowledge) ; (2) improved school climate ; and (3) improved interactions and social relationships in schools both among peers and between students and adults. both educational12,13 and health experts,10 as well as youth development experts,14 have advocated engaging students as partners to improve the health of peers, family, and community as well as improve the very process of school reform.5,12,13 pittman noted that change happens fastest when youth and community development are seen as two sides of the same coin and young people are afforded the tools, training and trust to apply their creativity and energy to affect meaningful change in their own lives and in the future of their neighborhoods and communities. toshalis and nakkula13 support the effectiveness of students as partners in promoting change within the school. they noted that student voice most often is only at the far left of the spectrum, using students as data sources (figure 1). however, in those schools that have tried involving students as leaders of change, remarkable success has occurred. various forms of youth engagement such as peer education, peer mentoring, youth action, student voice, community service, service - learning, youth organizing, civic engagement, and youth - adult partnerships provide students with a sense of safety, belonging, and efficacy ; gains in their sociopolitical awareness and civic competence ; strengthened community connections;16 and improved achievement.14 spectrum of student voice in schools and community given the opportunity to discover their true passion, students will accept the challenge and deliver. high school student zak malamed17 and a few friends decided it was time for students to speak up. they held their first twitter chat for students who were feeling frustrated about how little say they had in the school reform discussions going on around them. the question to students became what can we do to improve this school ? from the impetus of several frustrated students to the organization known as student voice (http://www.studentvoice.org), the conversation has grown to a movement dedicated to revolutionizing education through the voices and actions of students. supporters promise to advocate for students to be authentic partners in education and ensure that they have a genuine influence on decisions that affect their lives. the student voice collaboration18 was started by the new york city department of education to help students improve themselves and their schools. participating students learned how the educational system works, interviewed school leaders about decision - making, and created a 1-page map showing how decisions were made in their school. students conducted research on a challenge in their schools and then developed a student - led campaign to address the challenge. finally, they set a city - wide agenda identifying something that would benefit all new york city students. as a result of this work, one student group developed 6 recommendations that were shared with the new york city chancellor of education. the program 's goal was to show students that they can bring about change by working within the system. the student - centered schools : closing the opportunity gap evaluation study, conducted by the stanford center for opportunity policy in education,19 described how 4 student - centered high schools in california supported student success. student - centered practices focused on the needs of students through a rigorous, rich, and relevant curriculum that connected to the world beyond school. personalization was critical and students were provided with instructional supports that enabled success. each of the 4 schools supported students ' leadership capacities and autonomy through inquiry - based, student - directed, and collaborative learning within the classroom and in the community. advisory programs, a culture of celebration, student voice, leadership opportunities, and connections to parents and community were embedded in each school. the study revealed that creating high schools designed around student rather than adult needs requires a shift in beliefs that must be translated into action. the stanford study19 also showed that teachers and administrators need to be prepared to address students ' academic, social, and emotional needs in ways that empower students to take control of their own learning. this has significant implications for teacher and administrator preparation programs, teacher induction, and professional development. a culture of collaboration and partnership must go beyond traditional educator networks to include students as partners and consumers of educational programs and services. meaningful student involvement requires educators to provide learning experiences that enhance students ' skill development. effective teachers guide students to discovery, help students make meaning of what they learn, and include students as essential and legitimate contributors to achieving their own health and success. schools must continuously acknowledge the diversity of students by validating and authorizing them to represent their own ideas, opinions, knowledge, and experiences and truly become partners in every facet of school change but certainly in those programs and services that directly impact their health and well - being. student empowerment can and should begin in the elementary grades. serriere described a youth - adult partnership in one elementary school in which mixed grade level k-5 students participated in small school gatherings described as social, civic, and academic networks designed to create a sense of community and encourage student voice. group projects focused on making a difference, defined by the students as helping the poor, writing letters to military personnel, or aiding a local animal shelter. these authentic activities incorporated critical thinking, decision - making, collaboration, and planning skills and demonstrated that given the right circumstances, even the youngest students can have a voice in their work. similarly, the national health education standards21 emphasize effective communication, goal setting, decision - making, and advocacy all skills that enable students to take a more active role in their school and community. ultimately, the goal is to prepare all students for college, career, and life, educating and empowering them to become informed, responsible, and active citizens. the wscc model provides a vehicle for students to create meaningful learning experiences in education and health that help create a safe and supportive school. students can best articulate their own needs, thus maximizing the provision of health and counseling services. however, schools can not simply ask a select few for their opinions or blessings ; rather, schools must make concerted and genuine efforts to move from the contrived student voice of a few students to the meaningful student involvement of all students. at the heart of meaningful student involvement are students whose voices have long been silenced.12 every student deserves to be healthy, safe, engaged, supported, and challenged but evidence suggests that most students do not receive the supports they need to achieve these outcomes. while the five promises articulated by the america 's promise alliance22 do not use the same terms as the wscc model, they are quite similar in describing the fundamental needs of students : healthy start, safe places, caring adults (supported), opportunities to help others (engaged), and, effective education (challenged). a survey completed in 2006 by america 's promise revealed that 7 in 10 young people ages 12 to 17 (69%) received only 3 or less of the 5 fundamental resources needed to flourish. only 31% (or 153 million students out of 494 million students) in grades 6 - 12 received 4 to 5 of these fundamental resources. the 2014 quaglia institute for student aspirations ' my voice survey23 also confirmed the need for more resources. the survey, completed by a racially and socioeconomically diverse sample of 66,314 students in grades 6 - 12 representing 234 schools across the nation, was designed to measure variables affecting student academic motivation and concentrated on the following student constructs : self - worth, engagement, purpose / motivation along with peer support, and teacher support. the authors of the survey noted that the results of the 2014 survey demonstrated little change with the annual results since 2009. students with a sense of self - worth were 5 times more likely to be academically motivated, yet 45% of students did not have a sense of self - worth. those who described themselves as engaged were 16 times more likely to be academically motivated but 40% reported that they were not engaged. students with a sense of purpose were 18 times more likely to be academically motivated but 15% reported no purpose. teacher support increased academic motivation 8 times over while peer support increased academic motivation 4 times over. however, 39% of the students reported no teacher support and 56% reported no peer support.23 clearly, there is a need for a more coordinated and collaborative approach to meeting students ' basic needs the wscc model could be one mechanism schools and communities use to improve students ' feelings and experiences of self - worth, engagement, purpose, peer support, and teacher support as students become partners in the dissemination of the model. meaningful youth involvement in promoting the wscc model needs to be promoted. learning opportunities to empower youth can be divided into individual empowerment, organizational empowerment, and community empowerment.24 individual empowerment occurs when youth develop the self - management skills, improve competence and exert control over their life, while organizational empowerment refers to schools and community organizations that provide opportunities for engaging in student empowerment as well as benefit from student empowerment. community empowerment refers to the provision of opportunities for citizen participation at the local, state, and national level and the ensuing efforts to improve lives, organizations, and the community.25 successful youth - adult partnerships happen when the relationships between youth and adults are characterized by mutuality in teaching, learning, and action. while these relationships usually occur within youth organizations or in democratic schools, they could become one mechanism for disseminating the wscc model. fletcher asks adults to : imagine a school where democracy is more than a buzzword, and involvement is more than attendance. it is a place where all adults and students interact as co - learners and leaders, and where students are encouraged to speak out about their schools. picture all adults actively valuing student engagement and empowerment, and all students actively striving to become more engaged and empowered. envision school classrooms where teachers place the experiences of students at the center of learning, and education boardrooms where everyone can learn from students as partners in school change [to improve not only education outcomes but also health outcomes ]. what can schools do to empower students and support student voice ? the authors suggest adapting 4 goals identified by fletcher,25 to include a health focus as well as a school improvement focus : engage all students at all grade levels and in all subjects as contributing stakeholders in teaching, learning, and leading in school [to ensure that student needs are being met ]. expand the common expectation of every student to become an active and equal partner in school change [that includes health - promoting student support programs and services as cornerstones of school improvement ]. provide students and educators with sustainable, responsive, and systemic approaches to engaging all students [in school improvement and health promotion ] and validate the experience, perspectives, and knowledge of all students through sustainable, powerful, and purposeful school - oriented and school - community roles.25 table 1, an adaptation of a chart by fletcher,12 provides examples of empowerment roles students can assume as partners in promoting achievement and health through the implementation of the wscc model. while the table identifies opportunities at 3 grade clusters (e = elementary / k-5, m = middle grades/6 - 8, hs = high school/9 - 12), these suggestions can be easily adapted for any grade level. potential roles for students in the implementation of the wscc e, elementary school ; m, middle school ; hs, high school ; wscc, whole school, whole community, whole child. adapted from fletcher.12 a number of researchers1416 have provided guidelines and recommendations for schools and communities on how to begin this process by : assessing the needs of youth on a regular basis;14,15 developing a local database of resources for youth development;14 asking community - based organizations (cbos) to document and share with schools what they specifically accomplish related to learning outcomes14 to coordinate a scope and sequence of learning;14 developing curricula that integrates community resources for learning and teaching;14 providing youth with a supportive home base in which youth can work with dedicated and nurturing adults;15 creating youth - adult teams that are intentional about the long - term social changes to be achieved;15 balancing the need for short - term individual supports for youth with long - term goal of community change;15 recognizing and rewarding youth for their participation in youth organizations;14 providing professional development for educators to learn about the power of youth organizations to assist in providing youth with skills;14 advocating for a line item in the community budget to support youth as partners in improving the community and schools;14 providing multiple options for youth participation ensuring that youth receive the support to progressively take on more responsibility as they gain experience and skills;16 providing coaching and ongoing feedback to both youth and adults;14,15 establishing strategies to recruit and retain a diverse core of youth;15,16 providing organizational resources such as budget, staff training, and physical space aligned to support quality youth - adult partnership;16 and providing adults and youth with opportunities to reflect and learn with same - age peers.16 in addition, kania and kramer26 identified 5 conditions needed for achieving collective impact on any issue (but particularly educational reform) that could be instructive for school - community partnerships that support youth engagement and empowerment. these include a common agenda, shared measurement systems, mutually reinforcing activities, continuous communication, and backbone support functions (such as convening partners, conducting needs assessments, developing a shared strategic plan for aligning efforts, selecting success metrics, and designing an evaluation). hung in a review of the factors that facilitated the implementation of health - promoting schools which also engaged community agencies as partners in the process, identified the following effective and somewhat similar strategies : following a framework / guideline ; obtaining committed support from the school staff, school board, management, health agencies, and other stakeholders ; adopting a multidisciplinary, collaborative approach ; establishing professional networks and relationships ; and continuing training and education. hung also noted that coordination was the key to promoting school - community partnerships and encouraged a 2-pronged approach : a top - down approach, a more effective initiating force to introduce and support the coordination role ; and a bottom - up commitment, including the participation of parents and students, critical for sustaining an initiative. ten years of research,14 assessing the contributions of 120 community youth - based organizations in 34 cities across the nation, revealed that students working with cbos, when compared to youth in general, were 26% more likely to report having received recognition for good grades. almost 20% were more likely to rate their chances of graduating from high school as very high, 20% were more likely to rate the prospect of their going to college as very high, and more than 2 - 1/2 times were more likely to think it is very important to do community service or to volunteer and give back to their community.14 students can also play an important role within their own school or district. the centers for disease control and prevention28 recommends that schools and communities establish school health coordinating councils at the community level to facilitate the communication and common goals recommended by kania and kramer as well as the professional networks and relationships identified by hung as drivers of quality school health programs. members of both the district level and school councils / teams include representatives from education, public health, health and social service agencies, community leadership, and families. some schools and districts have found that including students as critical contributors to the work of the councils / teams is vital. the center consolidated school district (colorado),29 recognizing the importance of a collaborative approach to student health and learning, has a health advisory committee that represents the wscc components and includes community professionals, school staff, parents, and students. the district believes that, with support, students can achieve academically and be successful in life. health and wellness efforts are integrated into the work of the center school district as reflected by a health and wellness goal for the district 's unified improvement plan. the district sees wellness as the foundation for learning sustained by creating and maintaining environments, comprehensive health, policies, practices, access to services and resources, and attitudes that develop and support the inter - related dimensions of physical, mental, emotional, and social health.29 persons at the district 's skoglund middle school believe that educating students about leading a healthy lifestyle is important and because students educate others about a healthy lifestyle and its impact, sustainability is enhanced. as administrators, staff, parents and students understand the importance of coordinated school health efforts, they become the school 's strongest advocates.30 skoglund has discovered that when parents and students demand something, it continues.30 clearly, student voice and involvement is valued in the district which uses the wscc model to guide its work. table 2 provides examples of resources to assist school and community agency staff members to empower students as partners, enable student voice, and develop students as partners for change. every student deserves to be healthy, safe, engaged, supported, and challenged but evidence suggests that most students do not receive the supports they need to achieve these outcomes. while the five promises articulated by the america 's promise alliance22 do not use the same terms as the wscc model, they are quite similar in describing the fundamental needs of students : healthy start, safe places, caring adults (supported), opportunities to help others (engaged), and, effective education (challenged). a survey completed in 2006 by america 's promise revealed that 7 in 10 young people ages 12 to 17 (69%) received only 3 or less of the 5 fundamental resources needed to flourish. only 31% (or 153 million students out of 494 million students) in grades 6 - 12 received 4 to 5 of these fundamental resources. the 2014 quaglia institute for student aspirations ' my voice survey23 also confirmed the need for more resources. the survey, completed by a racially and socioeconomically diverse sample of 66,314 students in grades 6 - 12 representing 234 schools across the nation, was designed to measure variables affecting student academic motivation and concentrated on the following student constructs : self - worth, engagement, purpose / motivation along with peer support, and teacher support. the authors of the survey noted that the results of the 2014 survey demonstrated little change with the annual results since 2009. students with a sense of self - worth were 5 times more likely to be academically motivated, yet 45% of students did not have a sense of self - worth. those who described themselves as engaged were 16 times more likely to be academically motivated but 40% reported that they were not engaged. students with a sense of purpose were 18 times more likely to be academically motivated but 15% reported no purpose. teacher support increased academic motivation 8 times over while peer support increased academic motivation 4 times over. however, 39% of the students reported no teacher support and 56% reported no peer support.23 clearly, there is a need for a more coordinated and collaborative approach to meeting students ' basic needs one involving families, schools, communities, and peers. the wscc model could be one mechanism schools and communities use to improve students ' feelings and experiences of self - worth, engagement, purpose, peer support, and teacher support as students become partners in the dissemination of the model. meaningful youth involvement in promoting the wscc model needs to be promoted. learning opportunities to empower youth can be divided into individual empowerment, organizational empowerment, and community empowerment.24 individual empowerment occurs when youth develop the self - management skills, improve competence and exert control over their life, while organizational empowerment refers to schools and community organizations that provide opportunities for engaging in student empowerment as well as benefit from student empowerment. community empowerment refers to the provision of opportunities for citizen participation at the local, state, and national level and the ensuing efforts to improve lives, organizations, and the community.25 successful youth - adult partnerships happen when the relationships between youth and adults are characterized by mutuality in teaching, learning, and action. while these relationships usually occur within youth organizations or in democratic schools, they could become one mechanism for disseminating the wscc model. fletcher asks adults to : imagine a school where democracy is more than a buzzword, and involvement is more than attendance. it is a place where all adults and students interact as co - learners and leaders, and where students are encouraged to speak out about their schools. picture all adults actively valuing student engagement and empowerment, and all students actively striving to become more engaged and empowered. envision school classrooms where teachers place the experiences of students at the center of learning, and education boardrooms where everyone can learn from students as partners in school change [to improve not only education outcomes but also health outcomes ]. what can schools do to empower students and support student voice ? the authors suggest adapting 4 goals identified by fletcher,25 to include a health focus as well as a school improvement focus : engage all students at all grade levels and in all subjects as contributing stakeholders in teaching, learning, and leading in school [to ensure that student needs are being met ]. expand the common expectation of every student to become an active and equal partner in school change [that includes health - promoting student support programs and services as cornerstones of school improvement ]. provide students and educators with sustainable, responsive, and systemic approaches to engaging all students [in school improvement and health promotion ] and validate the experience, perspectives, and knowledge of all students through sustainable, powerful, and purposeful school - oriented and school - community roles.25 table 1, an adaptation of a chart by fletcher,12 provides examples of empowerment roles students can assume as partners in promoting achievement and health through the implementation of the wscc model. while the table identifies opportunities at 3 grade clusters (e = elementary / k-5, m = middle grades/6 - 8, hs = high school/9 - 12), these suggestions can be easily adapted for any grade level. potential roles for students in the implementation of the wscc e, elementary school ; m, middle school ; hs, high school ; wscc, whole school, whole community, whole child. adapted from fletcher.12 a number of researchers1416 have provided guidelines and recommendations for schools and communities on how to begin this process by : assessing the needs of youth on a regular basis;14,15 developing a local database of resources for youth development;14 asking community - based organizations (cbos) to document and share with schools what they specifically accomplish related to learning outcomes14 to coordinate a scope and sequence of learning;14 developing curricula that integrates community resources for learning and teaching;14 providing youth with a supportive home base in which youth can work with dedicated and nurturing adults;15 creating youth - adult teams that are intentional about the long - term social changes to be achieved;15 balancing the need for short - term individual supports for youth with long - term goal of community change;15 recognizing and rewarding youth for their participation in youth organizations;14 providing professional development for educators to learn about the power of youth organizations to assist in providing youth with skills;14 advocating for a line item in the community budget to support youth as partners in improving the community and schools;14 providing multiple options for youth participation ensuring that youth receive the support to progressively take on more responsibility as they gain experience and skills;16 providing coaching and ongoing feedback to both youth and adults;14,15 establishing strategies to recruit and retain a diverse core of youth;15,16 providing organizational resources such as budget, staff training, and physical space aligned to support quality youth - adult partnership;16 and providing adults and youth with opportunities to reflect and learn with same - age peers.16 in addition, kania and kramer26 identified 5 conditions needed for achieving collective impact on any issue (but particularly educational reform) that could be instructive for school - community partnerships that support youth engagement and empowerment. these include a common agenda, shared measurement systems, mutually reinforcing activities, continuous communication, and backbone support functions (such as convening partners, conducting needs assessments, developing a shared strategic plan for aligning efforts, selecting success metrics, and designing an evaluation). hung in a review of the factors that facilitated the implementation of health - promoting schools which also engaged community agencies as partners in the process, identified the following effective and somewhat similar strategies : following a framework / guideline ; obtaining committed support from the school staff, school board, management, health agencies, and other stakeholders ; adopting a multidisciplinary, collaborative approach ; establishing professional networks and relationships ; and continuing training and education. hung also noted that coordination was the key to promoting school - community partnerships and encouraged a 2-pronged approach : a top - down approach, a more effective initiating force to introduce and support the coordination role ; and a bottom - up commitment, including the participation of parents and students, critical for sustaining an initiative. ten years of research,14 assessing the contributions of 120 community youth - based organizations in 34 cities across the nation, revealed that students working with cbos, when compared to youth in general, were 26% more likely to report having received recognition for good grades. almost 20% were more likely to rate their chances of graduating from high school as very high, 20% were more likely to rate the prospect of their going to college as very high, and more than 2 - 1/2 times were more likely to think it is very important to do community service or to volunteer and give back to their community.14 students can also play an important role within their own school or district. the centers for disease control and prevention28 recommends that schools and communities establish school health coordinating councils at the community level to facilitate the communication and common goals recommended by kania and kramer as well as the professional networks and relationships identified by hung as drivers of quality school health programs. members of both the district level and school councils / teams include representatives from education, public health, health and social service agencies, community leadership, and families. some schools and districts have found that including students as critical contributors to the work of the councils / teams is vital. the center consolidated school district (colorado),29 recognizing the importance of a collaborative approach to student health and learning, has a health advisory committee that represents the wscc components and includes community professionals, school staff, parents, and students. the district believes that, with support, students can achieve academically and be successful in life. health and wellness efforts are integrated into the work of the center school district as reflected by a health and wellness goal for the district 's unified improvement plan. the district sees wellness as the foundation for learning sustained by creating and maintaining environments, comprehensive health, policies, practices, access to services and resources, and attitudes that develop and support the inter - related dimensions of physical, mental, emotional, and social health.29 persons at the district 's skoglund middle school believe that educating students about leading a healthy lifestyle is important and because students educate others about a healthy lifestyle and its impact, sustainability is enhanced. as administrators, staff, parents and students understand the importance of coordinated school health efforts, they become the school 's strongest advocates.30 skoglund has discovered that when parents and students demand something, it continues.30 clearly, student voice and involvement is valued in the district which uses the wscc model to guide its work. table 2 provides examples of resources to assist school and community agency staff members to empower students as partners, enable student voice, and develop students as partners for change. the wscc model places students in the center for a reason : students are the consumers of the programs and services we, the adults, provide. a student - centered school considers the thoughts and opinions of the students it serves. that means schools must seek out the opinions and ideas of every student, not just those elected to student government or acknowledged as school leaders. this dialogue must begin in elementary grades as students learn how to develop and present a convincing argument and advocate for their own health, safety, engagement, support for learning and academic challenges as well as these supports for their peers. these skills can be developed, refined, and supported by the implementation of a comprehensive, sequential prek-12 health education program aligned with the national health education standards. school administrators must regularly engage all students through social media, surveys, town hall meetings, and focus groups. creating a continuous feedback loop, where comments are welcomed and expected, is critical to supporting student voice. whereas having student representatives on the school health committee / team is important, asking all students to participate in developing and implementing school health policies is necessary in order to create a safe environment for discussion. it is critical that students become involved in the conversation at the outset and not after decisions have already been made. building trust in the system is crucial to the success of the wscc model. as schools implement the wscc approach, they must create an ongoing dialog about school health policies, programs and services and ensure that the student body is well - represented in those conversations. three simple questions are critical to that process : what do students think about the planned policy, program or service ? how will the policies, programs, and services impact all students in the school ? what would the students do differently if given the opportunity to do so ? ensuring that all students have the skills needed to become effective communicators is but a first step toward creating an environment where students feel safe and supported. empowering students as partners in the dissemination of the wscc model will help generate trust and acceptance and ensure that students ' needs are being met. ascd 's the learning compact redefined : a call to action set the stage for the development of the wscc model with this statement : we are calling for a simple change that will have radical implications : put the child at the center of decision - making and allocate resources time, space, and human to ensure each child 's success.3 creating meaningful roles for students as allies, decision makers, planners, and foremost, as consumers, ensures that our focus is truly student - centered. placing students in the center of the wscc model makes visible the commitment of education and health to collaboratively prepare today 's students for the challenges of today and the possibilities of tomorrow. we can accomplish this by engaging and empowering students and acknowledging them as capable and valuable partners in the process (figure 2). source : ascd1 the preparation of this paper involved no original research with human subjects.	backgroundstudents are the heart of the whole school, whole community, whole child (wscc) model. students are the recipients of programs and services to ensure that they are healthy, safe, engaged, supported, and challenged and also serve as partners in the implementation and dissemination of the wscc model.methodsa review of the number of students nationwide enjoying the 5 whole child tenets reveals severe deficiencies while a review of student - centered approaches, including student engagement and student voice, appears to be one way to remedy these deficiencies.resultsresearch in both education and health reveals that giving students a voice and engaging students as partners benefits them by fostering development of skills, improvement in competence, and exertion of control over their lives while simultaneously improving outcomes for their peers and the entire school / organization.conclusionscreating meaningful roles for students as allies, decision makers, planners, and consumers shows a commitment to prepare them for the challenges of today and the possibilities of tomorrow.
indomethacin 99% (auzohu - konch pharmaceutica, mexico) with an average particle size of 50 m, lactose usp (lactochem, mexico), a millipore membrane 0.45 m, a vibrating ball mill (pen walt s.s. white), a ceramic ball mill (erweka), carver press, a microbalance autobalance ad-4 (perkin - elmer), a dsc-7 (perkin - elmer), and uv-201 spectrophotometers (perkin - elmer) and ftir spectrophotometers, tensor 27 (bruker) were used. physical mixtures of il were prepared by mixing equal weights of the drug and carrier and tumbling in a plastic receptacle for 10 min to assure a homogeneous mixture. samples of indomethacin alone and the physical mixtures (il) were ground in a porcelain ball mill (60 rpm) for about 4 h in order to compare with those which were ground in a vibrating ball mill at different times at 1700 rpm for 20 and 30 min (pure indomethacin, i20 and i30, and the physical mixtures il20 and il30, respectively). the properties assessed were particle size distribution, degree of crystallinity loss, changes in the infrared spectra and apparent solubility. the morphology and the size distribution of the particles were obtained through images of secondary electrons using the jeol jsm-5600 scanning electron microscopy (sem). the samples were placed on the surface of a bronze sample holder using double sided adhesive tape. the images were digitized and the size distribution was calculated from these images using the imago web for windows and digital micrograph processor as well as the statistical package for the social sciences (spss) software, version 12.0. the degree of crystallinity loss was determined through a dsc process, where sample weights were kept within a range of 5 - 10 mg and the temperature from 50 to 250 at a constant rate of 10/min in the presence of a nitrogen draft. this was further confirmed by the use of a high resolution transmission electron microscopy (tem) using hf-2000feg at 200 kev (hitachi, tokyo, japan). the dry samples were collected on a standard lacey formvar coated electron microscope copper grid and images and diffracting patterns digitized. fourier transform - infrared spectra (ftir) were obtained on a system using the kbr disk method. solubility was evaluated from the dissolution of 10 mg of each sample in 100 ml of water at 370.5, stirring at 60 rpm for a 2 h period, then filtering and measuring absorbance at a wavelength of 254 nm. measurements were carried out in triplicate. by monitoring the dissolution for a period of 6 h, it was noticed that after the first 2 h, the value of the absorbance became constant, therefore, all other tests were carried out in 2 h periods. physical mixtures of il were prepared by mixing equal weights of the drug and carrier and tumbling in a plastic receptacle for 10 min to assure a homogeneous mixture. samples of indomethacin alone and the physical mixtures (il) were ground in a porcelain ball mill (60 rpm) for about 4 h in order to compare with those which were ground in a vibrating ball mill at different times at 1700 rpm for 20 and 30 min (pure indomethacin, i20 and i30, and the physical mixtures il20 and il30, respectively). the properties assessed were particle size distribution, degree of crystallinity loss, changes in the infrared spectra and apparent solubility. the morphology and the size distribution of the particles were obtained through images of secondary electrons using the jeol jsm-5600 scanning electron microscopy (sem). the samples were placed on the surface of a bronze sample holder using double sided adhesive tape. the images were digitized and the size distribution was calculated from these images using the imago web for windows and digital micrograph processor as well as the statistical package for the social sciences (spss) software, version 12.0. the degree of crystallinity loss was determined through a dsc process, where sample weights were kept within a range of 5 - 10 mg and the temperature from 50 to 250 at a constant rate of 10/min in the presence of a nitrogen draft. this was further confirmed by the use of a high resolution transmission electron microscopy (tem) using hf-2000feg at 200 kev (hitachi, tokyo, japan). the dry samples were collected on a standard lacey formvar coated electron microscope copper grid and images and diffracting patterns digitized. fourier transform - infrared spectra (ftir) were obtained on a system using the kbr disk method. solubility was evaluated from the dissolution of 10 mg of each sample in 100 ml of water at 370.5, stirring at 60 rpm for a 2 h period, then filtering and measuring absorbance at a wavelength of 254 nm. measurements were carried out in triplicate. by monitoring the dissolution for a period of 6 h, it was noticed that after the first 2 h, the value of the absorbance became constant, therefore, all other tests were carried out in 2 h periods. 1 shows the microphotographs of i30 and il30, where the size of the aggregates are around 5 and 2 m, respectively and morphology of both is different. in the first sample, 2 shows the thermograms of i and l, whose melting points were 162 and 219, respectively. another signal for the form of lactose monohydrate appears at 151.3, and it is associated to the loss of water. microphotographs of samples i30 (a) 1 m and il30 (b) 0.5 m dsc thermograms of indomethacin and lactose. differential scanning calorimetry thermograms of pure indomethacin (i) and lactose (l) the experiments were carried out using a common porcelain ball mill and there were no changes to the endotherm at 162 after 4 h, which are results obtained by etman and nada. the loss of crystallinity for samples i20 and i30 was observed through the decrease in the endotherms around 162 by 30.6% and about 56%, respectively, compared to the endotherm of unground indomethacin. taylor and zografi worked only with indomethacin with similar equipment and reported a 36.5% loss of crystallinity at 30 min whereas with our equipment, the efficiency was 1.5 times higher in the same amount of time. 3 shows the thermograms for the physical mixture (il), as well as the samples ground for 20 and 30 min each (il20 and il30). the thermogram of sample il20 shows a reduction of the peak around 162 resulting in a 43% loss of crystallinity for indomethacin while the endotherm at 151.3 belonging to lactose almost disappeared. differential scanning calorimetry thermograms of the physical mixture of indomethacin and lactose (il), and physical mixtures ground for 20 min (il20) and 30 min (il30) with a vibrating ball mill. sample il30 presents a vitreous transition at 136, two lactose endotherms at 151.7 and 209, and a significant decrease of the area for the melting peak of indomethacin at 162. this resulted in a loss of crystallinity of almost 81% compared to etman and nada who reported a crystallinity loss of 63% using a ball mill for about 90 min. both techniques demonstrate a high degree of crystallinity loss as proposed from the dsc analysis. 4a shows a bright field high resolution image of a grain of the il30 sample. the 2 m particles are made up of smaller nanoparticles of average 200 nm in average size, although there are smaller particles. diffraction patterns of such particles were obtained, which clearly show the amorphous nature because broad rings were observed (fig. high resolution transmission electron microphotographs and diffraction of il30 the confirmation that we were dealing with il30 was demonstrated by obtaining energy dispersive x - ray spectra, which clearly shows the presence of the cl due to the indomethacin (fig. characteristic x - ray energy dispersive spectrum of il30 particles showing the cl peaks typical of the i - phase and c peaks. cu and si come from the sustaining grid from the analysis of indomethacin ftir spectrum (fig. 6, i), it can be observed that there are hydroxyl bands at 3371 cm (free oh) and 2927_3350 cm (oh - associated), two carbonyl bands at 1716 cm (acid group) and 1692 cm (amide group). comparing the infrared spectrum of the physical mixture (fig. 6, il) with that of lactose (fig. 6, l) it is clear that the bands of indomethacin are overlapped by those of lactose. in addition therefore, there is no sign of an interaction in the physical mixture. on the contrary, with the ground indomethacin, 6, i30), and practically disappears in the case of the mixture of indomethacin - lactose that was ground for 30 min (il30). fourier transform - infrared spectra of indomethacin (i), ground indomethacin (i30), ground mixture of indomethacin and lactose (il30), physical mixture of indomethacin and lactose (il) and lactose (l). the data contained in table 1 show that the apparent solubility of indomethacin improves in the physical mixture with lactose, almost doubling when compared to that of the pure drug sample indomethacin. table 1 also shows a 2.22 fold increase when the indomethacin is ground for a 30 min period. finally, a 2.76 fold increase is reached for the mixture of il30 which was also ground for an equivalent period of 30 min. analyses of the unground drug and lactose showed an average particle size of around 50 and 30 m, respectively. after high speed grinding, indomethacin (i30) and the mixture il30 had a reduced average particle size of around 5 and 2 m, respectively, however, the latter showed cylindrical structures, which could increase the surface area and therefore, improve its wettability. from the analysis of the thermograms, there is a gradual reduction of area of the endotherms at 162 (i20 and i30) observed in fig. this could be interpreted as a change in the crystalline state of the particle because even though it is known that the grinding process reduces the particle size, this would increase the rate of dissolution, but not the solubility of a drug. as a matter of fact, the apparent solubility listed in table 1, where solubility itself has increased, allowed us to corroborate that this is due to the destruction of the crystalline net of indomethacin instead of the presence of other polymorphic forms. this can be verified since those forms would appear at 134 and 154 and there are no signals in those regions. in the case of the physical mixture, it is reasonable to expect that the improvement in the apparent solubility could be due only to the hydrophilic properties of lactose, because no change was found in the thermogram nor in the infrared spectrum of that sample (il). it can be seen from the thermogram of the mixture ground for 30 min (fig. 3) that the crystallinity loss was stronger than i30 (without lactose) and the vitreous transition as well as the endotherm at 209 could be an indication of interactions between indomethacin and lactose. furthermore, our solid dispersions present a yellow colour which coincides with the observations of cavallari., who prepared tablets containing indomethacin, lactose and polyethylene glycol with an ultrasound assisted compaction machine and the yellow colour of the tablets was due to the destruction of the crystal lattice. related to the interaction between the drug and the excipient which was strongly evidenced by the thermal analysis, it was important to determine its nature. in this case, the disappearance of the band in the infrared at 1716 cm observed in fig. 6 (il30) which was identified as an acid carbonyl stretch, supported the idea that hydrogen bonds formed between the acidic function of indomethacin and the oh groups of lactose. this idea is in agreement with other studies, such as those conducted by valizadeh., who have observed similar changes in the ftir spectra when they worked with indomethacin and dextrin. in addition, taylor and zografi reported the hydrogen bond formation between pvp amide carbonyl and indomethacin carboxylic acid hydroxyl in the amorphous solid dispersion prepared by coprecipitation. furthermore, watanabe proposed the interaction between indomethacin and the silanol groups in the ground mixture of the drug with sio2. it has been proven that the apparent solubility of the ground solid dispersion of indomethacin and lactose (il30) increased almost 3 fold when compared to that of the pure indomethacin. this could be explain with the connection of two factors, first the crystallinity loss of indomethacin in presence of lactose, corroborated by the dsc thermograms and tem. thermal analysis also enabled the exclusion of polymorphism phenomena as a consequence of the mechanical treatment. secondly, the number of interactions between the carboxyl group of indomethacin and the hydroxyl groups of lactose for the ground sample increased through the mechanoactivation using the vibrating ball mill.	mechanochemical activation is a practical cogrinding operation used to obtain a solid dispersion of a poorly water soluble drug through changes in the solid state molecular aggregation of drug - carrier mixtures and the formation of noncovalent interactions (hydrogen bonds) between two crystalline solids such as a soluble carrier, lactose, and a poorly soluble drug, indomethacin, in order to improve its solubility and dissolution rate. samples of indomethacin and a physical mixture with a weight ratio of 1:1 of indomethacin and lactose were ground using a high speed vibrating ball mill. particle size was determined by electron microscopy, the reduction of crystallinity was determined by calorimetry and transmission electron microscopy, infrared spectroscopy was used to find evidence of any interactions between the drug and the carrier and the determination of apparent solubility allowed for the corroboration of changes in solubility. before grinding, scanning electron microscopy showed the drug and lactose to have an average particle size of around 50 and 30 m, respectively. after high speed grinding, indomethacin and the mixture had a reduced average particle size of around 5 and 2 m, respectively, showing a morphological change. the ground mixture produced a solid dispersion that had a loss of crystallinity that reached 81% after 30 min of grinding while the drug solubility of indomethacin within the solid dispersion increased by 2.76 fold as compared to the pure drug. drug activation due to hydrogen bonds between the carboxylic group of the drug and the hydroxyl group of lactose as well as the decrease in crystallinity of the solid dispersion and the reduction of the particle size led to a better water solubility of indomethacin.
in all experiments, we exposed bacterial strains to the phage t4rii, which targets lipopolysaccharides embedded in the bacterial outer membrane. as our altruistic suicidal strain, we used e. coli carrying the prophage (henceforth e. coli), which encodes the rex abortive infection system that is triggered by several phages including t4rii. as the phage - sensitive strain, we used e. coli carrying the abi - free control prophage hk97 (henceforth e. coli hk97). finally, we used an e. coli hk97 strain that has evolved resistance against t4rii (henceforth e. coli t4). while we compared the performance of phage - sensitive e. coli hk97 vs. suicidal e. coli in our previous study, we here compare the competitive abilities of phage - sensitive e. coli hk97 vs. phage - resistant e. coli t4, and relate it to our previous findings. we first examined the performance of e. coli hk97 and e. coli t4 in an unstructured environment. we did this by growing the two strains together in shaken liquid medium at various frequencies (e. coli t4 varied from 0.1% to 99%) in the presence of t4rii (moi = 0.001) and followed population growth and the change in relative strain frequency over time. we found that t4rii eradicated monocultures of e. coli hk97, whereas e. coli t4 could thrive even when initially rare (fig. 1) moreover, e. coli t4 significantly outcompeted e. coli hk97 under all conditions (fig. 2, one - sample t - tests for fitness > 0 : t5 > 6.9, p 0, p 0 : t29 = 11.17, p < 0.0001) and e. coli t4 (t11 = 9.52, p < 0.0001). this comparison shows that both altruistic host - defense and individual - based resistance mechanisms are efficient means to defy bacteriophages in a structured environment. however, e. coli t4 seemed to be slightly but significantly more efficient than e. coli (fig. 3 ; two sample t - test : t40 = 4.20, p = 0.0001). a plausible explanation is that suicide always takes its toll, especially at high moi, which reduces the efficiency of suicidal host defense compared with an individual - based resistance mechanism. additionally, we also found that the relative fitness of e. coli t4 was greatly reduced in the structured compared with the unstructured environment, an observation that is compatible with the view that medium viscosity also reduces phage dispersal, such that some fraction of e. coli hk97 colonies remain unharmed. figure 3. comparing the performance of suicidal e. coli and resistant e. coli t4 in competition against e. coli hk97 in the presence of phage t4rii in a structured environment (1.5% agar plate). both strains significantly outcompeted e. coli hk97, indicating that suicidal host defense as well as individual - based resistance were efficient in withstanding phages. a direct comparison between the two strategies reveals that individual - based resistance is slightly but significantly better under these conditions. but note that although the experiments were performed following the exact same protocol, they were performed at different dates, which might explain some of the differences. to be able to put our findings in an ecological context, we need to become clear about the level of structuring that occurs in natural bacterial populations. while an unifying answer is not possible owing to the fact that bacteria populate an enormous range of different habitats, it seems justified to conclude that some degree of population structure always exists. consequently, we can deduce that altruistic host defense likely represents an efficient strategy to withstand bacteriophages in natural habitats. while our comparison revealed that both host - defense strategies can be efficient in laboratory settings, it remains unclear, which of the two strategies is more prevalent in nature. individual - based resistance might be more common because it is easier to evolve, since a single point mutation can lead to resistance. in contrast, suicidal defense systems are often based on complex machineries that eventually have first evolved for other purposes, and have only later been coopted into suicidal systems. although more complex, we propose that suicidal host - defense systems might be cheaper to maintain in the long run because they are often decoupled from other functions, and might therefore entail little pleiotropic cost. individual - based resistance, on the other hand, is typically achieved by altering a molecule on the bacterial cell surface that is recognized by a phage and to which the phage adsorbs. such an adaptation may impair the original function of the molecule and consequently reduce bacterial fitness (as observed in t4 phages), even more so if further modifications are necessary to resist different phages, or to respond to phage counter - resistance. these potential cost differences might also affect the co - evolutionary dynamics between phages and bacteria. moreover, it would be interesting to see whether both resistance mechanisms can occur in the same individual, or whether they are mutually exclusive, with one resistance mechanisms eventually replacing the other one over evolutionary time scales. while speculative at this stage, these considerations will hopefully stimulate future work. finally, another unresolved issue about abi systems is that they often involve the action of prophages. a plausible explanation for this is that prophages already possess mechanisms that can kill bacteria during induction (i.e., when a prophage enters the lytic phase), which can eventually be coopted into suicide machineries. more sinister is the possibility that bacteria are not always free to choose their doom. for instance, is has been observed that competition between two prophages in the same bacterial cell leads some prophages to induce premature cell death, possibly to prevent the other prophage from replicating. to obtain e. coli t4, we diluted overnight cultures of e. coli hk97 100-fold, and incubated 100 l together with 10 pfu of phage t4rii overnight in a plate reader. cultures whose growth pattern indicated the evolution of resistance were selected and serially passaged (three times) in 3 ml medium. before every passage, we diluted cultures 1,000-fold and added 10 pfu of phage t4. this was done to select for full resistance, and to reduce possible resistance costs through compensatory mutations. we then streaked out cultures, and picked a single colony to establish a stock culture, stored at 80c. we verified resistance by cross - streaking against lysates of both phages t4 and t4rii. we chose this procedure because we were interested in obtaining a strain that exhibits a different defense mechanism against phage t4rii, but otherwise grows comparably to e. coli and e. coli hk97 (which do not differ in their growth rate in the absence of phages). indeed, when grown in mixed cultures, we found that neither e. coli t4 nor e. coli hk97 experienced a selective advantage in the absence of phages (t4 = 0.48, p = 0.66, difference in malthusian fitness = 0.02). the downside of this procedure is, however, that we can not infer the initial cost of phage resistance. we performed all experiments following the same protocol as described previously. in short, experiments shown in figures 1 and 2 were performed in 96-well microtiter plates in 100 l of medium. initial concentration of both competing strains together was 5 10 cfu / ml, initial concentration of phage t4rii was 10 pfu / ml. experiments for figure 3 were performed in 24-well plates with wells filled with 1.5 ml lb 1.5% agar each. between 10 and 10 cfu of e. coli and 10 cfu of e. coli t4 were added together with 10 pfu of phage t4rii. to be able to distinguish bacterial strains in mixed competition, we marked all strains with plasmids pgfp and pdsred carrying genes for fluorescent proteins under an inducible promoter as described previously. competition assays in unstructured and structured environments followed protocols previously described, except that experiments in structured environments were performed on agar instead of agarose.	adaptations by hosts in response to parasitism are generally believed to reduce the susceptibility of the adapted individual. however, recent work on escherichia coli showed that bacteria can fight deadly phage attacks by committing altruistic suicide upon infection, in order to prevent parasite transmission to nearby relatives. here, we compare the efficiency of suicidal host defense with individual - based resistance. we show that in unstructured environments suicidal host defense is futile since suicide can not preferentially protect relatives, whereas individual - based resistance is highly efficient in defying phages. in contrast, we found that in structured environments suicidal host defense and individual - based resistance were both efficient in withstanding phages, with the latter type performing slightly better. we propose that the putative lower efficiency of suicidal host defense might be compensated by the fact that suicidal systems usually do not bear pleiotropic costs of resistance, as it is usually the case for individual - based resistance mechanisms.
	nuclear factor kappa b (nf - kappa b) is a transcription factor that regulates expression of several genes coding for inflammatory and immunoregulatory proteins including the neutrophil chemotactic cytokine mip-2. in previous studies we found that crocidolite asbestos activates the nuclear translocation of nf - kappa b as well as mip-2 gene expression in rat alveolar type ii cells. here we report that both crocidolite - induced nf - kappa b activation of mip-2 gene expression can be attenuated by the antioxidant tetramethylthiourea, suggesting the dependence of these responses on oxidative stress. crocidolite exposure of rle - tn cells also increased production of h2o2, a response that was inhibited by the mitochondrial respiratory chain inhibitor thenoyltrifluoroacetone (ttfa). ttfa treatment of rle-6tn cells also inhibited crocidolite - induced nuclear translocation of nf - kappa b and mip-2 gene expression. these results indicate crocidolite exposure of rat alveolar type ii cells results in increased production of mitochondrial - derived hydrogen peroxide and that mitochondrial - derived oxidants contribute to crocidolite activation of nf - kappa b and increases in mip-2 gene expression.imagesfigure 1figure 2figure 3figure 4
the national health and nutrition examination survey (nhanes), previously described (14), is a stratified multistage probability cluster survey conducted in the noninstitutionalized population (14). the survey includes 1) an in - home interview through which demographic and basic health information are obtained and 2) a physical examination and laboratory measures taken at a mobile examination center (mec). participants were adults aged 20 years who completed the 20052008 interview and mec visit and who answered yes when asked whether a physician or other health care professional ever told them that they had diabetes (n = 1,251). since there are no specific clinical guidelines for people with type 1 diabetes, participants likely to have type 1 diabetes were excluded (n = 18), based on age of diagnosis 7% (n = 26, 3%), and 19% stated that their provider did not specify an a1c goal (data not shown). for participants who had heard of the a1c measure, similar differences were found by race / ethnicity, education, income, and examinations for eye and foot complications. overall, there was no significant difference in any knowledge measure by sex, years since diagnosis, glycemic medication use, or self - monitoring of blood glucose, regardless of insulin use. population aged 20 years with diabetes (nhanes 20052008) by demographic characteristics, diabetes - related factors, and medical care (n = 1,233) sixty - three percent of participants stated their last blood pressure level ; 87% had heard of the measure ldl cholesterol, but only 22% stated their last ldl cholesterol level (table 2). reported blood pressure and ldl cholesterol levels could not be verified. similar to results for a1c, stating last blood pressure and ldl cholesterol level was greatest in non - hispanic whites compared with mexican americans and higher with more education and income. participants who reported taking antihypertensive medications or reported having several a1c tests, a dilated eye exam, or several foot exams in the past year had more knowledge of their last blood pressure level. similar to a1c, relationships by demographics, medication use, and a1c testing frequency and having examinations for eye complications persisted for those who reported their physician - specified blood pressure and ldl cholesterol goals in accordance with ada recommendations. few participants reported their physician recommending a blood pressure goal > 130/80 mmhg (n = 85, 7%) or ldl cholesterol goal > 100 mg / dl (n = 65, 8%) (data not shown). forty - seven percent and 41% of participants reported that their provider did not specify a goal for blood pressure or ldl cholesterol, respectively. similar to a1c, having heard of the ldl cholesterol measure was greatest in non - hispanic whites and higher in people with more income and education and in those with more a1c tests in the past year. population aged 20 years (nhanes 20052008) by demographic characteristics, diabetes - related factors, and medical care (n = 1,233) participant knowledge of their last abc level was not associated with meeting abc goals, with the exception of people who knew their last ldl cholesterol level (63% vs. no, 53% for a1c 7% (n = 26, 3%), and 19% stated that their provider did not specify an a1c goal (data not shown). for participants who had heard of the a1c measure, similar differences were found by race / ethnicity, education, income, and examinations for eye and foot complications. overall, there was no significant difference in any knowledge measure by sex, years since diagnosis, glycemic medication use, or self - monitoring of blood glucose, regardless of insulin use. population aged 20 years with diabetes (nhanes 20052008) by demographic characteristics, diabetes - related factors, and medical care (n = 1,233) sixty - three percent of participants stated their last blood pressure level ; 87% had heard of the measure ldl cholesterol, but only 22% stated their last ldl cholesterol level (table 2). reported blood pressure and ldl cholesterol levels could not be verified. similar to results for a1c, stating last blood pressure and ldl cholesterol level was greatest in non - hispanic whites compared with mexican americans and higher with more education and income. participants who reported taking antihypertensive medications or reported having several a1c tests, a dilated eye exam, or several foot exams in the past year had more knowledge of their last blood pressure level. similar to a1c, relationships by demographics, medication use, and a1c testing frequency and having examinations for eye complications persisted for those who reported their physician - specified blood pressure and ldl cholesterol goals in accordance with ada recommendations. few participants reported their physician recommending a blood pressure goal > 130/80 mmhg (n = 85, 7%) or ldl cholesterol goal > 100 mg / dl (n = 65, 8%) (data not shown). forty - seven percent and 41% of participants reported that their provider did not specify a goal for blood pressure or ldl cholesterol, respectively. similar to a1c, having heard of the ldl cholesterol measure was greatest in non - hispanic whites and higher in people with more income and education and in those with more a1c tests in the past year. population aged 20 years (nhanes 20052008) by demographic characteristics, diabetes - related factors, and medical care (n = 1,233) participant knowledge of their last abc level was not associated with meeting abc goals, with the exception of people who knew their last ldl cholesterol level (63% vs. no, 53% for a1c 30% ; nevertheless, we found significant values by medication use and retinopathy and found similar trends by demographics. self - reported abc levels could not be verified, but in a small subanalysis, correlation analysis suggested that among those with good control, participants were generally correct in assessing their control. in addition, reported versus actual a1c values that were documented up to a year prior were found to be very similar in the study by harwell. finally, variation in laboratory calibration methods and the time period between self- reported and nhanes tests for a1c may increase error in correlation analysis. given the enormous public health burden of diabetes, future work should delineate factors associated with meeting abc recommendations using a multifaceted approach. while a patient s abc knowledge may be important, approach must be developed to translate research on the benefits of risk factor control into better outcomes for patients with diabetes. understanding the interaction between demographics, knowledge, and other diabetes - related factors may provide insight for improving diabetes control and reducing diabetes complications. in addition, the communication that physicians, health care professionals, and diabetes educators have with patients is important for improved diabetes outcomes.	objectivewe examined the prevalence of knowledge of a1c, blood pressure, and ldl cholesterol (abc) levels and goals among people with diabetes, its variation by patient characteristics, and whether knowledge was associated with achieving levels of abc control recommended for the general diabetic population.research design and methodsdata came from 1,233 adults who self - reported diabetes in the 20052008 national health and nutrition examination survey. participants reported their last abc level and goals specified by their physician (not validated by medical record data). analysis included descriptive statistics and logistic regression.resultsamong participants tested in the past year, 48% stated their last a1c level. overall, 63% stated their last blood pressure level and 22% stated their last ldl cholesterol level. knowledge of abc levels was greatest in non - hispanic whites, lowest in mexican americans, and higher with more education and income (all p 0.02). demographic associations were similar for those reporting physician - specified abc goals at the american diabetes association recommended levels (a1c < 7%, blood pressure < 130/80 mmhg, and ldl cholesterol < 100 mg / dl). nineteen percent of participants stated that their provider did not specify an a1c goal compared with 47% and 41% for blood pressure and ldl cholesterol goals, respectively. for people who self - reported a1c < 7.0%, 83% had an actual a1c < 7.0%. otherwise, participant knowledge was not significantly associated with risk factor control, except for in those who knew their last ldl cholesterol level (p = 0.046 for a1c < 7.0%). results from logistic regression corroborated these findings.conclusionsample opportunity exists to improve abc knowledge. diabetes education should include behavior change components in addition to information on abc clinical measures.
clustering genes by their expression profiles among samples and/or across time points is a useful approach to reducing data dimension and identifying co - expressed genes that may share common biological functions or regulatory networks.1 there are three typical experimental designs, under which gene expression data are collected : (1) multiple samples at one time point, (2) one sample at multiple time points, and (3) multiple samples each at multiple time points. data collected under each experimental design possess specific distributional characteristics to that design, which should be properly accounted for in data analysis. for the analysis of gene clustering, we have previously developed a model - based clustering method, called the clustering of regression models (corm) method, to accommodate data collected from various experimental designs while accounting for data distributional characteristics specific to each design.2 corm uses regression to model the expression of each gene and clusters genes that share similar regression coefficients to sample covariates. it applies an em algorithm to iteratively assign genes to clusters and estimate the regression coefficients for each cluster. we have implemented corm for the clustering of linear models (clm) method and the clustering of linear mixed model (clmm) method, with the former applied to cluster genes using cross - sectional data2,3 and the latter time course data with or without replicates.2,4 in this paper, we report an r package that we recently developed implementing the clm method and the clmm method, and for each method, we provide two distinct data examples illustrating their uses. the corm package is available at r cran and it can be imported once installed with the r code below : the clm method is implemented by the function fit.clm. the first example is to cluster genes using microarray data derived from a set of breast cancer samples, including 38 invasive ductal carcinoma (idc) and 21 invasive lobular carcinoma (ilc) samples.5 two indicator variables, one for idc and another for ilc, were used as the covariates in the regression model for clm. a set of 474 markers, which were selected based on their differential expression between the two breast cancer subtypes idc versus ilc, are grouped into nine clusters with genes in each cluster sharing similar expression levels in both subtypes.2 the second example is to cluster candidate target genes for a microrna named hsa - let-7f using their expression data derived from a set of normal and tumor tissue samples.6 three variables were used in the linear regression model for clm : (1) let-7f expression, (2) indicator of disease status, and (3) interaction between let-7f and disease status. a set of 178 markers, which were selected based on their significant association with let-7f expression, are clustered to look for genes that share similar association with let-7f and hence may be similarly regulated by let-7f.3 the clmm method is implemented by two functions : fit.clmm and fit.clmm.2. the former can be used to cluster single - time course data or replicated time course data with replicate samples sharing the same time points. the latter can be used to cluster replicated time course with the samples belonging to two groups each having a different set of time points. a single - time course study examining yeast cell cycle is used to illustrate the use of fit.clmm.7 the fixed effects and random effects in the linear mixed effects model are both set to be the spline basis of time. a set of 256 cell cycle - dependent genes identified by zhao.8 were clustered into six groups. the expression profiles over time for genes in each group showed significant periodicity with similar peak time within each group and difference between groups.2 a replicated time course study assessing yeast cell cycle in two yeast samples of different genotypes is used to illustrate the use of fit.clmm.2.4 the two genotypes are wild - type yeast and single - mutant yeast with yox1 gene knocked out. the two samples were measured for gene expression at the same time points in the experiment ; however, each sample incurred bad time points at different times due to technical issues, which were removed from the clustering analysis. in order to accommodate the different time points for the two samples, separate arguments for the fixed and random effects are allowed for the two samples in fit.clmm.2. the same list of 256 cell cycle - dependent genes is partitioned into eight groups using the wild - type and mutant yeast data to look for genes whose expressions are similarly regulated by the mutation.4 the first example is to cluster genes using microarray data derived from a set of breast cancer samples, including 38 invasive ductal carcinoma (idc) and 21 invasive lobular carcinoma (ilc) samples.5 two indicator variables, one for idc and another for ilc, were used as the covariates in the regression model for clm. a set of 474 markers, which were selected based on their differential expression between the two breast cancer subtypes idc versus ilc, are grouped into nine clusters with genes in each cluster sharing similar expression levels in both subtypes.2 the second example is to cluster candidate target genes for a microrna named hsa - let-7f using their expression data derived from a set of normal and tumor tissue samples.6 three variables were used in the linear regression model for clm : (1) let-7f expression, (2) indicator of disease status, and (3) interaction between let-7f and disease status. a set of 178 markers, which were selected based on their significant association with let-7f expression, are clustered to look for genes that share similar association with let-7f and hence may be similarly regulated by let-7f.3 the former can be used to cluster single - time course data or replicated time course data with replicate samples sharing the same time points. the latter can be used to cluster replicated time course with the samples belonging to two groups each having a different set of time points. a single - time course study examining yeast cell cycle is used to illustrate the use of fit.clmm.7 the fixed effects and random effects in the linear mixed effects model are both set to be the spline basis of time. a set of 256 cell cycle - dependent genes identified by zhao.8 were clustered into six groups. the expression profiles over time for genes in each group showed significant periodicity with similar peak time within each group and difference between groups.2 a replicated time course study assessing yeast cell cycle in two yeast samples of different genotypes is used to illustrate the use of fit.clmm.2.4 the two genotypes are wild - type yeast and single - mutant yeast with yox1 gene knocked out. the two samples were measured for gene expression at the same time points in the experiment ; however, each sample incurred bad time points at different times due to technical issues, which were removed from the clustering analysis. in order to accommodate the different time points for the two samples, separate arguments for the fixed and random effects are allowed for the two samples in fit.clmm.2. the same list of 256 cell cycle - dependent genes is partitioned into eight groups using the wild - type and mutant yeast data to look for genes whose expressions are similarly regulated by the mutation.4 it can be applied to cluster gene expression data collected under various types of experimental designs and forms an integrative analysis framework together with regression models typically used to detect differentially expressed or time - dependent genes. table 1 lists the computing time spent for the aforementioned data examples as tested on a linux server (intel xeon). in our experience, applying the corm method, the em algorithm typically converges in just a few iterations and is quite robust to the starting values when there are relatively well - separated clusters in the data. we would recommend applying corm to about 100500 genes to partition them into reasonably sized clusters.	we report a new r package implementing the clustering of regression models (corm) method for clustering genes using gene expression data and provide data examples illustrating each clustering function in the package. the corm package is freely available at cran from http://cran.r-project.org.
human life is greatly affected by three factors : deficiency of food, health problems, and environmental issues. food and health are basic human requirements beside a clean and safe environment. with increasing world 's population at a greater rate, human requirements for food are rapidly increasing. approximately 36 million people die each year from noncommunicable and communicable diseases, such as cardiovascular diseases, cancer, diabetes, aids / hiv, tuberculosis, malaria, and several others according to http://globalissues.org/. despite extensive efforts being made, the current world food production is much lower than human requirements, and health facilities are even below standard in the third - world countries. rapid increase in industrialization has soared up the environmental pollution and industrial wastes are directly allowed to mix with water, which has affected aquatic marines and, indirectly, human - beings. unlike tradition approaches to overcome agriculture, health, and environmental issues through breeding, traditional medicines, and pollutants degradation through conventional techniques respectively, the genetic engineering utilizes modern tools and approaches, such as molecular cloning and transformation, which are less time consuming and yield more reliable products. for example, compared to conventional breeding that transfers a large number of both specific and nonspecific genes to the recipient, genetic engineering only transfers a small block of desired genes to the target through various approaches, such as biolistic and agrobacterium - mediated transformation. the alteration into plant genomes is brought either by homologous recombination dependent gene targeting or by nuclease - mediated site - specific genome modification. recombinase mediated site - specific genome integration and oligonucleotide directed mutagenesis can also be used. recombinant dna technology is playing a vital role in improving health conditions by developing new vaccines and pharmaceuticals. the treatment strategies are also improved by developing diagnostic kits, monitoring devices, and new therapeutic approaches. synthesis of synthetic human insulin and erythropoietin by genetically modified bacteria and production of new types of experimental mutant mice for research purposes are one of the leading examples of genetic engineering in health. likewise, genetic engineering strategies have been employed to tackle the environmental issues such as converting wastes into biofuels and bioethanol [47 ], cleaning the oil spills, carbon, and other toxic wastes, and detecting arsenic and other contaminants in drinking water. the advent of recombinant dna technology revolutionized the development in biology and led to a series of dramatic changes. it offered new opportunities for innovations to produce a wide range of therapeutic products with immediate effect in the medical genetics and biomedicine by modifying microorganisms, animals, and plants to yield medically useful substances [8, 9 ]. most biotechnology pharmaceuticals are recombinant in nature which plays a key role against human lethal diseases. the pharmaceutical products synthesized through recombinant dna technology, completely changed the human life in such a way that the u.s. food and drug administration (fda) approved more recombinant drugs in 1997 than in the previous several years combined, which includes anemia, aids, cancers (kaposi 's sarcoma, leukemia, and colorectal, kidney, and ovarian cancers), hereditary disorders (cystic fibrosis, familial hypercholesterolemia, gaucher 's disease, hemophilia a, severe combined immunodeficiency disease, and turnor 's syndrome), diabetic foot ulcers, diphtheria, genital warts, hepatitis b, hepatitis c, human growth hormone deficiency, and multiple sclerosis. considering the plants develop multigene transfer, site - specific integration and specifically regulated gene expression are crucial advanced approaches. transcriptional regulation of endogenous genes, their effectiveness in the new locations, and the precise control of transgene expression are major challenges in plant biotechnology which need further developments for them to be used successfully. human life is greatly threatened by various factors, like food limitations leading to malnutrition, different kinds of lethal diseases, environmental problems caused by the dramatic industrialization and urbanization and many others. genetic engineering has replaced the conventional strategies and has the greater potential to overcome such challenges. the current review summarized the major challenges encountered by humans and addresses the role of recombinant dna technology to overcome aforementioned issues. in line with this, we have detailed the limitations of genetic engineering and possible future directions for researchers to surmount such limitations through modification in the current genetic engineering strategies. recombinant dna technology comprises altering genetic material outside an organism to obtain enhanced and desired characteristics in living organisms or as their products. this technology involves the insertion of dna fragments from a variety of sources, having a desirable gene sequence via appropriate vector. manipulation in organism 's genome is carried out either through the introduction of one or several new genes and regulatory elements or by decreasing or blocking the expression of endogenous genes through recombining genes and elements. enzymatic cleavage is applied to obtain different dna fragments using restriction endo - nucleases for specific target sequence dna sites followed by dna ligase activity to join the fragments to fix the desired gene in vector. the vector is then introduced into a host organism, which is grown to produce multiple copies of the incorporated dna fragment in culture, and finally clones containing a relevant dna fragment are selected and harvested. the first recombinant dna (rdna) molecules were generated in 1973 by paul berg, herbert boyer, annie chang, and stanley cohen of stanford university and university of california san francisco. in 1975, during the asilomar conference regulation and safe use of rdna technology was discussed. paradoxically to the view of scientists at the time of asilomar, the recombinant dna methods to foster agriculture and drug developments took longer than anticipated because of unexpected difficulties and barriers to achieve the satisfactory results. however, since the mid-1980s, the number of products like hormones, vaccines, therapeutic agents, and diagnostic tools has been developed continually to improve health. a quick approach is offered by recombinant dna technology to scrutinize the genetic expression of the mutations that were introduced into eukaryote genes through cloned insulin genes insertion inside a simian virus fragment. in a similar way, tumor growth was inhibited by adenoviral vector that encodes endostain human secretory form through antiangiogenic effects. targeted gene disruption has been used to produce antitumor derivatives in other hosts which were structurally similar for the production pathways. besides, longer acting therapeutic proteins have been developed through recombinant dna technologies ; for example, sequences containing additional glycosylation site are one of the most followed approaches. a new chimeric gene has been developed through this technique which contains the fsh -subunit coding sequences and the c - terminal peptide of the hcg -subunit coding sequences. researchers have also developed vectors and combined vectors for gene therapy and genetic modification approaches. presently, viral vectors have received immense consideration in clinical settings, some of which have also been commercialized. in principle, viruses are modified to be safe for clinical purposes. they have several applications including treatment of severe diseases including cancer either through in vivo or gene therapy (ex vivo), vaccination, and protein transduction approaches. the production of clinical grade viral vectors improvement has become possible due to advance manufacturing technologies. at present, due to the severe adverse effects, retroviral vectors are losing their importance although the viral entities transfer genes quickly and correctly into a number of species. the simplest nonviral gene delivery system uses naked dna, when injected directly into certain tissues, particularly muscles, produces significant levels of gene expression with least side effects. more recently, a p1 vector has been designed to introduce the recombinant dna into e. coli through electroporation procedures. this new cloning system is used for establishing 15,000 clone library initially averagely 130150 kb pairs insert size. the construction of low copy number vectors, for example, pwsk29, pwks30, pwsk129, and pwks130, was carried out using pcr and recombinant dna technology. these vectors can also be used for generating unidirectional deletions with exonuclease, complementation analysis, dna sequencing, and run - off transcription. a broad range of applications of recombinant dna technology has been summarized in figure 1. recombinant dna technology is a fast growing field and researchers around the globe are developing new approaches, devices, and engineered products for application in different sectors including agriculture, health, and environment. for example, lispro (humalog), in comparison with regular human insulin, is a well effective and fast acting recombinant insulin. similarly, epoetin alfa is a novel and well - recognized recombinant protein that can be effectively used in curing of anemia. recombinant hgh was found with a great improvement in treating children lacking the ability to produce hgh in a required quantity. clinical testing approval by the fda in december 1997 for a recombinant version of the cytokine myeloid progenitor inhibitory factor-1 (mpif-1) was an achievement to give recognition to this technology. with its help anticancer drug 's side effects can be mitigated whereas it has the ability to mimic the division of immunologically important cells [23, 24 ]. clustered regularly interspaced short palindromic repeats (crispr), a more recent development of recombinant dna technology, has brought out solutions to several problems in different species. activation, suppression, addition, and deletion of genes in human 's cells, mice, rats, zebrafish, bacteria, fruit flies, yeast, nematodes, and crops proved the technique a promising one. mouse models can be managed for studying human diseases with crispr, where individual genes study becomes much faster and the genes interactions studies become easy by changing multiple genes in cells. the crispr of h. hispanica genome is capable of getting adapted to the nonlytic viruses very efficiently. the engineering of a strain is required with priming crispr for priming crrnas production and new spacers acceptance. crispr - cas system has to integrate new spacers into its locus for adaptive immunity generation. recognition of foreign dna / rna and its cleavage is a controlled process in sequence - specific manner. information related to the intruder 's genetic material is stored by the host system with the help of photo - spacer incorporation into the crispr system. cas9 t (gene editing tool) represents dna endonucleases which use rna molecules to recognize specific target. class 2 crispr - cas system with single protein effectors can be employed for genome editing processes. dead cas9 is important for histone modifying enzyme 's recruitment, transcriptional repression, localization of fluorescent protein labels, and transcriptional activation. targeting of genes involved in homozygous gene knockouts isolation process is carried out by crispr - induced mutations. in this way, essential genes can be analyzed which in turn can be used for potential antifungal targets exploration. natural crispr - cas immunity exploitation has been used for generation of strains which are resistant to different types of disruptive viruses. crispr - cas, the only adaptive immune system in prokaryotes, contains genomic locus known as crispr having short repetitive elements and spacers (unique sequences). crispr array is preceded by at - rich leader sequence and flanked by cas genes which encode cas proteins [32, 33 ]. in escherichia photo - spacer adjacent motif (pam) is required for interference and acquisition because the target sequence selection is not random. the memorization of the invader 's sequence starts after crispr array transcription into long precursor crrna. during the final stages of immunity process specific recognition prevents the system from self - targeting [32, 34 ]. in different species of sulfolobus, the crispr loci contain multiple spacers whose sequence matches conjugative plasmids significantly while in some cases the conjugative plasmids also contain small crispr loci. spacer acquisition is affected by active viral dna replication in sulfolobus species whereas the dna breaks formation at replication forks causes the process to be stimulated. according to the above information, crispr - cas system has obtained a unique position in advanced biological systems because of its tremendous role in the stability and enhancement of immunity. zinc - finger nucleases (zfns) and transcription activator - like effector nucleases (talens) are chimeric nucleases composed of programmable, sequence - specific dna - binding modules linked to a nonspecific dna cleavage domain. therapeutic potential of zfns and talens is more specified and targeted [25, 36, 37 ]. similarly, recombinant protein fibroblast growth factor (fgf-1) has been developed which functions in inducing the formation of new blood vessels in myocardium. its injection (biologic bypass) into a human myocardium cause an increased blood supply to the heart. apligraf, an fda approved product, which serves as a recombinant skin replacer, specified for the leg ulcer 's treatment and dermagraft, is effective in the treatment of diabetic ulcers [3840 ]. after successful production of insulin from e. coli through recombinant dna technology, currently several animals, notably cattle and pigs, have been selected as insulin producing source, which however, triggered immune responses. the recombinant human insulin is identical to human porcine insulin and comparatively infrequently elicits immunogenic responses. human growth hormone was the first protein expressed in tobacco plants [41, 42 ]. besides insulin, several new drugs related to recombinant dna technology have undergone developmental improvements and a number of protein production systems have been developed. several engineered microbial strains have been developed to carry out the formulation of drugs [41, 43, 44 ]. molecular medicine formation that is specifically based on proteins faces serious issues including methods and biology of the cells which function to produce medically important compounds through recombinant dna techniques. to overcome these obstacles, there is intense need to improve quality and quantity of medicines based on a molecular phenomenon. cell factories are considered important in recombinant dna technologies, but these needed to be explored with more details and in depth as the conventional factories are not fulfilling the needs. similarly, the endothelial growth factor and notch signaling were used to engineer oncolytic adenovirus which acts as a breast cancer selective agent for the antagonist 's expression. this decreases the total blood vessels numbers and causes a dramatic change along with the perfused vessels which indicates the improved efficacy against the tumor and vascular effects. efforts have been made to modify the influenza virus genome using recombinant dna technology for development of vaccines. the modifications are based on engineering of vectors to expression of foreign genes. in practical, the ns gene of the influenza virus was replaced with foreign gene, commonly chloramphenicol acetyltransferase gene. thereafter, the rna previously recombined is expressed and packaged into virus particles after transfection with purified influenza a virus in the presence of helper virus. it has been clarified that 5 terminal and the 3 terminal bases are sufficient from influenza a virus rna to produce signals for rna replication, rna transcription, and rna packaging into influenza virus. the abovementioned new production systems enhance pipelines for development of various vaccines and drugs and so forth. production of high quality proteins depends on physiology of a cell and the conditions provided to it. the expression of proteins becomes retarded if a cell goes under stressful conditions, which may also favor the production in some cases. thus, further improvements are required for the better and safe production at genetic and metabolic levels. these cells allow the incorporation of foreign genes with less resistant barriers and expression is easily controlled. compared to plant and mammalian cells to be taken as hosts, microbial systems provide less complicated machinery which ultimately enhances the performance and quality of proteins production. the use of common microbial species, including bacteria and yeasts, is promising but the less common strains have also been observed promising as being cellular factories to produce recombinant molecular drugs. the increasing demands of drugs and the needs of quality can be fulfilled with better results if these cellular factories of microorganisms get incorporated into productive processes of pharmaceuticals (table 1) [41, 45, 46 ]. recombinant dna technology has major uses which made the manufacturing of novel enzymes possible which are suitable in conditions for specified food - processing. several important enzymes including lipases and amylases are available for the specific productions because of their particular roles and applications in food industries. microbial strains production is another huge achievement that became possible with the help of recombinant dna technology. a number of microbial strains have been developed which produce enzyme through specific engineering for production of proteases. certain strains of fungi have been modified so that their ability of producing toxic materials could be reduced. it is suitable agent for food items including fruits, vegetables, cheese, and meat to be stored as it increases their shelf life. the inhibition of food spoiling microorganisms can be carried out through immobilized lysozyme in polyvinyl alcohol films and cellulose. lysozyme impregnation of fish skin gelatin gels increase the shelf life of food products and inhibit different food spoiling bacterial growth [4850 ]. exopolysaccharides of staphylococcus and e. coli can be hydrolyzed with the use of dspb which is engineered from t7. biofilms related to food industries can be removed by the combining activity of serine proteases and amylases. s. aureus, salmonella infantis, clostridium perfringens, b. cereus, campylobacter jejuni, l. monocytogenes, yersinia enterocolitica, and some other food spoiling microorganisms can be inhibited by glucose oxidase. it is also considered one of the most important enzymes in food industry to kill wide range of foodborne pathogens. derivation of recombinant proteins being used as pharmaceuticals came into practice from first plant recently and many others are through to be used for more production of similar medically important proteins. wide range of recombinant proteins have been expressed in different plant species to be used as enzymes in industries, some majorly used proteins in research are proteins present in milk which play a role in nutrition, and new polymeric proteins are being used in industries and medical field. with the invention of hbv vaccine production in plants, plants have been used to produce several therapeutic protein products, such as casein and lysozyme for improving health of child and polymers of protein for tissue replacement and surgery. high yielding molecular proteins is one of the major tasks under consideration in field of recombinant dna technology. traditional breeding and quantitative trade locus (qtl) analysis assisted in the identification of a rice variety with protein kinase known as pstol1 (phosphorus starvation tolerance1) help in enhancing root growth in early stages and tolerates phosphorus deficiency. overexpression of this enzyme enables root to uptake nutrients in sufficient amount in phosphorus deficient soil which ultimately enhances the grain yield. this gene contains a peptide which plays role in delivery of protein from cytosol to chloroplast. a number of important genes deleted from chloroplast have been observed to be transferred into nucleus, except ycf1 and ycf2, in order to avoid disruptions in photosynthesis and other necessary processes. trans - genesis into chloroplast is considered stable as the nuclear transgenic plants face the problems of lower expression and transgene escape via pollen. almost ten thousand copies of transgenes have been incorporated into the genome of chloroplast [5557 ]. t7gene10 engineering against salt stress has been found successful but with lower expression rate into nongreen tissues. -tmt gene insertion into chloroplast genome results in multiple layer formation of the inner chloroplast envelope. lycopene -cyclase genes introduction into the plastid genome of tomato enhances the lycopene conversion into provitamin a [57, 58 ]. cdnas with full lengths are the main resources for expression profiling of genes. 44 k agilent oligonucleotide microarray is used for field grown rice transcriptome analysis. gene expression fluctuation and transcriptome dynamics these processes and predictions are helpful to improve crop production and resistance to either environmental or microbial stresses. resistance to fungal and bacterial infections can be enhanced by wrky45 gene in rice which is induced by plant activator benzothiadiazole that activates innate immune system of plant. kala4 gene is responsible for the black color of rice which makes the rice resistant to attacking pathogens [59, 60 ]. genetic modification is needed in facilitating gene by gene introduction of well - known characters. potato, beans, eggplant, sugar beet, squash, and many other plants are being developed with desirable characters, for example, tolerance of the herbicide glyphosate, resistance to insects, drought resistance, disease and salt tolerance. recombinant dna technology has wide spectrum of applications in treating diseases and improving health conditions. the following sections describe the important breakthroughs of recombinant dna technology for the improvement of human health : gene therapy is an advanced technique with therapeutic potential in health services. the first successful report in field of gene therapy to treat a genetic disease provided a more secure direction toward curing the deadliest genetic diseases [62, 63 ]. this strategy shows good response in providing treatment for adenosine deaminase - deficiency (ada - scid), which is a primary immunodeficiency. at the beginning of this technology, several challenges including maintenance of patients on pegylated ada (peg - ada) during gene therapy and the targeting of gene transfer to t - lymphocytes were the reasons for unsuccessful results [64, 65 ]. however, later on successful results were obtained by targeting haematopoietic stem cells (hscs) by using an improved gene transfer protocol and a myeloablative conditioning regime. adrenoleukodystrophy (x - ald) and x - linked disorder are is possible through the expression of specific genes transferred by lentiviral vector, based on hiv-1. x - ald protein expression indicates that gene - correction of true hscs was achieved successfully. the use of lentiviral vector was made successful for the first time to treat genetic human disease. this success in the field of health sciences opened up new doors to extend the research to treat serious death causing diseases through immunotherapy. highly sustained levels of cells that were engineered for tumor recognition in blood using a retrovirus encoding a t - cell receptor in two patients up to 1 year after infusion resulted in regression of metastatic melanoma lesions. autologous t - cells were genetically modified to express a chimeric antigen receptors (car) with specificity for the b - cell antigen cd19 for the treatment of chronic lymphocytic leukemia. genetically modified cells undergo selective expansion for diseases such as scid - x1 and ada - scid as a consequence of in vivo selection conferred by the disease pathophysiology despite the correction of only a modest number of progenitors. combination of gene and drug therapy 's potential has recently been highlighted in a trial seeking to confer chemoprotection on human hscs during chemotherapy with alkylating agents for glioblastoma. gene transfer to a small number of cells at anatomically discrete sites is a targeted strategy that has the potential to confer therapeutic benefit. it showed impressive results for incurable autosomal recessive dystrophies such as congenital blindness and leber congenital amaurosis (lca). german phase i / ii gene therapy clinical trial aimed to treat chronic granulomatous disease in april 2006 that came up with success. mobilized cd34 + cells isolated from peripheral blood were retrovirally transduced and infused into the patient where two - thirds of the patients showed clear benefit from this treatment. after the treatment silencing of the transgene as a result of methylation of the viral promoter caused the severity of infection that leaded to the death of patient. many different cancers including lung, gynecological, skin, urological, neurological, and gastrointestinal tumors, as well as hematological malignancies and pediatric tumors, have been targeted through gene therapy. inserting tumor suppressor genes to immunotherapy, oncolytic virotherapy and gene directed enzyme prodrug therapy are different strategies that have been used to treat different types of cancers. the p53, a commonly transferred tumor suppressor gene, is a key player in cancer treating efforts. in some of the strategies, the most important strategies that have been employed until now are vaccination with tumor cells engineered to express immunostimulatory molecules, vaccination with recombinant viral vectors encoding tumor antigens and vaccination with host cells engineered to express tumor antigens. new fiber chimeric oncolytic adenoviruses vectors (ad5/35-egfp) offer an affective new anticancer agent for the better cure of hepatocellular carcinoma. a demonstration of these vectors through proper assaying was significant for transduction improvement and more progeny of the virus were produced in hcc. a higher level of transgenic expression was mediated and an enhanced antitumor effect was observed on in vitro hcc cells while keeping the normal cells protected against cytotoxicity. cancer gene therapy has become more advanced and its efficacy has been improved in recent years. treatment of cardiovascular diseases by gene therapy is an important strategy in health care science. in cardiovascular field, gene therapy will provide a new avenue for therapeutic angiogenesis, myocardial protection, regeneration and repair, prevention of restenosis following angioplasty, prevention of bypass graft failure, and risk - factor management. mutation in gene encoding wasp, a protein regulating the cytoskeleton, causes wiskott - aldrich syndrome (inherited immunodeficiency). its treatment requires stem cells transplantation ; in case matched donors are unavailable the treatment is carried out through infusion of autologous hspcs modified ex vivo by gene therapy. metastatic cancer can be regressed through immunotherapy based on the adoptive transfer of gene - engineered t - cells. accurate targeting of antigens expressed by tumors and the associated vasculature and the successful use of gene engineering to retarget t - cells before their transfer into the patient are mainly focused on in this therapy. cancer cells often make themselves almost invisible to the immune system and its microenvironment suppresses t - cells survival and migration but genetic engineering of t - cells is the solution to these challenges. t - cells in cancer patients can be modified by recombining the genes responsible for cancer - specific antigens recognition, resistance to immunosuppression, and extending survival and facilitating migration to tumors. fusion between the genes echinoderm microtubule - associated protein like 4 (eml4) and anaplastic lymphoma kinase (alk) is generated by an inversion on the short arm of chromosome confers sensitivity to alk inhibitors. vial - mediated delivery of the crispr / cas9 system to somatic cells of adult animals induces specific chromosomal rearrangements. targeting the wnt pathway in cancer is an attractive therapeutic approach, where lgk974 potently inhibits wnt signaling, has strong efficacy in rodent tumor models, and is well - tolerated. head and neck cancer cell lines with loss - of - function mutations in the notch signaling pathway have a high response rate to lgk974. codon - optimized gene, on the basis of coding sequence of the influenza virus hemagglutinin gene, was synthesized and cloned into a recombinant modified vaccinia virus ankara (mva). immunization with mva - h7-sh2 viral vector in ferrets proved to be immunogenic as unprotected animals that were mock vaccinated developed interstitial pneumonia and loss of appetite and weight but vaccination with mva - h7-sh2 protected the animals from severe disease. viral gene therapy is one of the leading and important therapies for head and neck cancer. tumor - associated genes are targeted by viruses, and p53 gene function was targeted through such therapy at first. cancer cells can be destroyed by oncolytic viruses through viral replication and by arming with therapeutic transgenes. high density lipoprotein gene abca1 mutation in cells can make the cells be differentiated into macrophages. gene knockouts in embryonic stem cells enhance the capability of cells to be differentiated into macrophages and specifically target the desired pathogens. the allele replacements in this case will assist in studying protein coding changes and regulatory variants involved in alteration of mrna transcription and stability in macrophages. plant systems have been recently used for the expression and development of different antibodies and their derivatives. most importantly, out of many antibodies and antibody derivatives, seven have reached to the satisfactory stages of requirements. transgenic tobacco plants can be used for the production of chimeric secretory iga / g known as carorx, carorx. oral pathogen responsible for decay of a tooth known as streptococcus mutants, can be recognized by this antibody. a monoclonal antibody called t84.66 can affectively function to recognize antigen carcinoembryonic, which is still considered an affectively characterized marker in cancers of epithelia [84, 85 ]. a full - length humanized igg1 known as anti - hsv and anti - rsv, which can function as the recognizing agent for herpes simplex virus (hsv)-2-glycoprotein b, has been expressed in transgenic soybean and chinese hamster ovary (cho) cells. antibodies from both sources have been shown to prevent vaginal hsv-2 transmission in mice after applying topically ; if worked similarly in humans it would be considered as inexpensive and affective prevention against diseases transmitted through sexual interactions [8688 ]. 38c13 is scfv antibody based on the idiotype of malignant b lymphocytes in the well - characterized mouse lymphoma cell line 38c13. administration of the antibody to mice resulted in the production of anti - idiotype antibodies that are able to recognize 38c13 cells, which help to protect the mice against with injected lymphoma cells, is a lethal challenge [89, 90 ]. unique markers recognizing enzymes could be produced through this system, most affectively the surface markers of a malignant b - cells to work as an effective therapy for non - hodgkin lymphoma like diseases in human. a monoclonal antibody known as the production of full - length monoclonal antibody and scfv and diabody derivatives was made possible in plants through transgenesis and agroinfiltration in tobacco transformed transiently. testosterone production by stimulated hcg can be inhibited by each of these antibodies in cells cultured by leydig and uterine weight gain could be delayed in mice, through which hcg activity is checked. diagnosis and therapy of tumors complex system of drug metabolizing enzymes involved in the drug metabolism is crucial to be investigated for the proper efficacy and effects of drugs. recombinant dna approaches have recently contributed its role through heterologous expression, where the enzyme 's genetic information is expressed in vitro or in vivo, through the transfer of gene [92, 93 ]. a fear free and painless technique to transfer adenovirus vectors encoding pathogen antigens is through nasal transfer which is also a rapid and protection sustaining method against mucosal pathogens. this acts as a drug vaccine where an anti - influenza state can be induced through a transgene expression in the airway. in vitro production of human follicle - stimulating hormone (fsh) fsh is considerably a complex heterodimeric protein and specified cell line from eukaryotes has been selected for its expression. most interestingly r - fsh and luteinizing hormone (lh) recombination was made successful to enhance the ovulation and pregnancy [94, 95 ]. as an important component of alternative medicine, traditional chines medicines play a crucial role in diagnostics and therapeutics. these medicines associated with theories which are congruent with gene therapy principle up to some extent. these drugs might be the sources of a carriage of therapeutic genes and as coadministrated drugs. transgenic root system has valuable potential for additional genes introduction along with the ri plasmid. it is mostly carried with modified genes in a. rhizogenes vector systems to enhance characteristics for specific use. the cultures became a valuable tool to study the biochemical properties and the gene expression profile of metabolic pathways. the intermediates and key enzymes involved in the biosynthesis of secondary metabolites can be elucidated by the turned cultures [96, 97 ]. some nutrition related genes for different components in strawberry including proanthocyanidin, l - ascorbate, flavonoid, polyphenols, and flavonoid are important for improving the component of interest through genetic transformation. in case of raspberry, bhlh and fruite4 genes control the anthocyanin components whereas erublrsq072h02 is related to flavonol. by specific transformation the release of genetically engineered microbes, for example, pseudomonas fluorescens strain designated hk44, for bioremediation purposes in the field was first practiced by university of tennessee and oak ridge national laboratory by working in collaboration [99, 100 ]. the engineered strain contained naphthalene catabolic plasmid putk21 and a transposon - based bioluminescence - producing lux gene fused within a promoter that resulted in improved naphthalene degradation and a coincident bioluminescent response. hk44 serves as a reporter for naphthalene bioavailability and biodegradation whereas its bioluminescence signaling ability makes it able to be used as an online tool for in situ monitoring of bioremediation processes. genetic engineering has been widely used for the detection and absorption of contaminants in drinking water and other samples. for example, atphr1 gene introduction into garden plants torenia, petunia, and verbena changed their ability for pi absorption. the atphr1 transgenic plants with enhanced pi absorption ability can possibly facilitate effective phytoremediation in polluted aquatic environments. a fragment of the atphr1 gene was inserted into binary vector pbinplus, which contains an enhanced cauliflower mosaic virus 35s promoter. this plasmid was named pspb1898 and was used for transformation in petunia and verbena using agrobacterium tumefaciens. atphr1 is effective in other plant species, such as torenia, petunia, and verbena but posttranscriptional modification of the endogenous atphr1 counterpart might be inhibited by overexpression of atphr1. tnt is only partially digested in which the nitrogen further reacts with oxygen to form toxic superoxide. to overcome this issue, the gene responsible for monodehydroascorbate reductase is knocked out which increases the plant tolerance against tnt. fine - tuning enzymatic activity and knockout engineering together enhance the plant responses to toxic metals. phytochelatin synthase, a heavy metal binding peptides synthesizing enzyme, revealed a way to enhance tolerance against heavy metals through enzymatic activity attenuation. recombinant dna technology has proven to be effective in getting rid of arsenic particles that are considered as serious contaminants in soil. pvacr3, a key arsenite [as(iii) ] antiporter was expressed in arabidopsis which showed enhanced tolerance to arsenic. seeds of plants genetically engineered with pvacr3 can germinate and grow in the presence of higher than normal quantity of arsenate [as(v) ] which are generally lethal to wild - type seeds. osnramp5 and oshma3 represent the transporters to uptake cadmium (cd) and its retention. in plants, brassino - steroid (br) recent biotechnological approaches for bioremediation include biosorption, phytostabilization, hyperaccumulation, dendroremediation, biostimulation, mycoremediation, cyanoremediation, and genoremediation, which majorly depend on enhancing or preventing specified genes activities. several microorganisms, specifically cyanobacteria, mediate hydrogen production, which is environmental friendly energy source. the specific production is maintained by utilizing the required enzymes properly as these enzymes play a key role in the product formation. but advanced approaches like genetic engineering, alteration in nutrient and growth conditions, combined culture, metabolic engineering, and cell - free technology [110112 ] have shown positive results to increase the hydrogen production in cyanobacteria and other biofuels [3, 4 ]. the commercialization of this energy source will keep the environment clean which is not possible by using conventional energy sources releasing co2 and other hazardous chemicals. also cyanobacteria can be engineered to make them able to convert of co2 into reduced fuel compounds. this approach has been successful for vast range of commodity chemicals, mostly energy carriers, such as short chain and medium chain alcohols. the conductive biofilms of geobacter sulfurreducens are potential sources in the field in renewable energy, bioremediation, and bioelectronics. deletion of pilz genes encoding proteins in g. sulfurreducens genome made the biofilm more active as compared to wild - type. the electron acceptor cl-1 produced biofilms that were 6-fold more conductive than wild - type biofilms when they were grown with electrode. this high fold conductivity lowered the potential losses in microbial fuel cells, decreasing the charge transfer resistance at the biofilm - anode surface and lowering the formal potential. the fact that microbial cells are mostly used in the production of recombinant pharmaceutical indicates that several obstacles come into their way restricting them from producing functional proteins efficiently but these are handled with alterations in the cellular systems. common obstacles which must be dealt with are posttranslational modifications, cell stress responses activation, and instability of proteolytic activities, low solubility, and resistance in expressing new genes. mutations occurring in humans at genetic levels cause deficiencies in proteins production, which can be altered / treated by incorporation of external genes to fill the gaps and reach the normal levels. the use of escherichia coli in recombinant dna technology acts as a biological framework that allows the producers to work in controlled ways to technically produce the required molecules through affordable processes [41, 116 ]. recombinant dna research shows great promise in further understanding of yeast biology by making possible the analysis and manipulation of yeast genes, not only in the test tube but also in yeast cells. most importantly, it is now possible to return to yeast by transformation with dna and cloning the genes using a variety of selectable marker systems developed for this purpose. these technological advancements have combined to make feasible truly molecular as well as classical genetic manipulation and analysis in yeast. the biological problems that have been most effectively addressed by recombinant dna technology are ones that have the structure and organization of individual genes as their central issue [117, 118 ]. recombinant dna technology is recently passing thorough development which has brought tremendous changes in the research lines and opened directions for advanced and interesting ways of research for biosynthetic pathways though genetic manipulation. actinomycetes are being used for pharmaceutical productions, for example, some useful compounds in health sciences and the manipulation of biosynthetic pathways for a novel drugs generation. these contribute to the production of a major part of biosynthetic compounds and thus have received immense considerations in recombinant drugs designing. their compounds in clinical trials are more applicable as they have shown high level activity against various types of bacteria and other pathogenic microorganisms. recombinant dna tech as a tool of gene therapy is a source of prevention and cure against acquired genetic disorders collectively. dna vaccines development is a new approach to provide immunity against several diseases. in this process transfection for cancer gene therapy with minimal toxicity, such as in case of brain cancer, breast cancer, lung cancer, and prostate cancer, is still under investigation. also renal transplantation, gaucher disease, hemophilia, alport syndrome, renal fibrosis, and some other diseases are under consideration for gene therapy. recombinant dna technology is an important development in science that has made the human life much easier. in recent years, it has advanced strategies for biomedical applications such as cancer treatment, genetic diseases, diabetes, and several plants disorders especially viral and fungal resistance. the role of recombinant dna technology in making environment clean (phytoremediation and microbial remediation) and enhanced resistace of plants to different adverse acting factors (drought, pests, and salt) has been recognized widely. the improvements it brought not only in humans but also in plants and microorganisms are very significant. the challenges in improving the products at gene level sometimes face serious difficulties which are needed to be dealt for the betterment of the recombinant dna technology future. in pharmaceuticals, especially, there are serious issues to produce good quality products as the change brought into a gene is not accepted by the body. moreover, in case of increasing product it is not always positive because different factors may interfere to prevent it from being successful. considering health issues, the recombinant technology is helping in treating several diseases which can not be treated in normal conditions, although the immune responses hinder achieving good results. several difficulties are encountered by the genetic engineering strategies which needed to be overcome by more specific gene enhancement according to the organism 's genome. the integration of incoming single - stranded dna into the bacterial chromosome would be carried out by a reca - dependent process. the introduction of genetic material from one source into the other is a disaster for safety and biodiversity. for example, it is obvious that genetically engineered plants can cross - breed with wild plants, thus spreading their engineered genes into the environment, contaminating our biodiversity. further, concerns exist that genetic engineering has dangerous health implications. thus, further extensive research is required in this field to overcome such issues and resolve the concerns of common people.	in the past century, the recombinant dna technology was just an imagination that desirable characteristics can be improved in the living bodies by controlling the expressions of target genes. however, in recent era, this field has demonstrated unique impacts in bringing advancement in human life. by virtue of this technology, crucial proteins required for health problems and dietary purposes can be produced safely, affordably, and sufficiently. this technology has multidisciplinary applications and potential to deal with important aspects of life, for instance, improving health, enhancing food resources, and resistance to divergent adverse environmental effects. particularly in agriculture, the genetically modified plants have augmented resistance to harmful agents, enhanced product yield, and shown increased adaptability for better survival. moreover, recombinant pharmaceuticals are now being used confidently and rapidly attaining commercial approvals. techniques of recombinant dna technology, gene therapy, and genetic modifications are also widely used for the purpose of bioremediation and treating serious diseases. due to tremendous advancement and broad range of application in the field of recombinant dna technology, this review article mainly focuses on its importance and the possible applications in daily life.
according to estimates by the who, there are approximately 9.4 million patients a year diagnosed with tuberculosis (tb), with an annual mortality of 1.7 million. historically, the majority of tb cases are pulmonary ; however, cases of extrapulmonary tb, including abdominal tb, are increasingly being reported in the uk. traditionally thought to be a disease of developing asian and african countries, it is now increasing in prevalence in the west. contributing factors to the rising incidence of abdominal tb in the uk are the increasing prevalence of hiv, immigration from developing countries and an ageing population [3, 4 ]. abdominal tb often involves the gastrointestinal (gi) tract, peritoneum, mesentery, abdominal lymph nodes and solid organs, and usually causes non - specific symptoms like in our case, making the diagnosis difficult and a high index of suspicion necessary. we, here, present the case of an immunocompetent patient, from a non - tb endemic country, who developed intestinal tb involving the caecum. a 43-year - old afro - caribbean gentleman was admitted to the ed suffering from haemoptysis. he had previously been noted to have an enlarged lymph node in the right posterior triangle of neck which had been biopsied. at the time of presentation, the results of the histopathology were pending. he had also been suffering from weight loss of 18 kg over the previous 3 months along with night sweats and a reduced appetite. on examination, since his history and clinical findings were very suspicious of tb, he was reviewed by the respiratory team. empirical anti - tubercular therapy was suggested after sputum cultures were sent for acid fast bacilli (afb) and a ct thorax showed a right upper lobe collapse and a 3-cm apicoposterior cavity. there was also consolidation seen in the right middle and lower zone as well as the left lung (fig. 1) figure 1:ct thorax right - sided upper and mid zone collapse + consolidation. seventy - two hours after starting his anti - tubercular therapy, he took a turn for the worse and was transferred to the icu for ventilatory and inotropic support. he had 1 l of bleeding per rectum and dropped his haemoglobin (hb) from 140 to 65 a ct abdomen + pelvis with contrast was performed, which showed a large (20 cm) inflammatory caecal mass with a moderate amount of intra - abdominal free fluid (fig. 2). selective angiography of the superior mesenteric artery demonstrated the bleeding point in the caecum and this was embolized superselectively with coils (fig. 3). the posterior wall of the caecum was perforated and visibly the nidus of the bleeding (fig. figure 4:grossly enlarged and perforated caecum with a suction device demonstrating the depth of the cavity. grossly enlarged and perforated caecum with a suction device demonstrating the depth of the cavity. postoperatively, the patient made a good recovery and had no further episodes of bleeding or drop in hb. the histology from both the initial lymph node biopsy as well as the caecal mass came back positive for tb, as did his sputum cultures. he was discharged on post - op day 10 with a plan to complete his full course of anti - tubercular therapy and undergo both surgical and respiratory outpatient follow up. gi involvement of tb can result in stricture formation, intestinal obstruction, perforation, fistulas and small bowel volvulus. intestinal perforation is uncommon but when does occur is a serious and life - threatening complication mortality rates secondary to perforation are estimated to be > 30%. early recognition of abdominal symptoms and intervention may improve the prognosis of tuberculous intestinal perforations. recommended treatment is the resection of the affected bowel followed by end - to - end anastomosis. however, for an inflammatory caecal mass of uncertain aetiology as in our case, right hemicolectomy has been recommended because of the concern of possible malignancy. some perforations occur before or shortly after antituberculous therapy begins, suggesting a natural progression of the condition, thought to be caused by a reactive thickening of the peritoneum and subsequent adhesion formations, leading to proximal dilatation and perforation. other cases had evidence of improvement during the course of treatment, before a later perforation. there have also been multiple cases of intestinal perforations occurring after completion of antituberculous therapy. it has been hypothesized that a paradoxical response to treatment may be responsible for these cases. this is similar to the disease progression in our case the previously asymptomatic patient from the gi point of view acutely decompensated and developed caecal distention and perforation within 72 h stating anti - tubercular therapy. isolated caecal perforation that too tubercular in origin is extremely rare and only one other case has been reported. if perforation does occur, it is in accordance with laplace 's law which states that the intraluminal pressure needed to stretch the wall of a hollow tube is inversely proportional to its radius. the caecum has the largest diameter of the colon and therefore requires the least amount of pressure to distend. perforation of the caecum has been found to occur when the diameter reaches 16 cm in our case, it was 20 cm and therefore perforation was not unexpected. the extent of caecal distension seen in our case is unheard of in the literature and underlines the insidious nature of intestinal tb : our patient never complained of any gi signs or symptoms to suggest intestinal tb until he decompensated and bleed massively per rectum. in conclusion, diagnosis of intestinal tb can be challenging hence requiring a high index of suspicion and caecal distension, and perforation can be paradoxically triggered by anti - tubercular therapy. furthermore, the massive distension of the caecum reported in our case can be explained by laplace 's law.	a 43-year - old man presented to the hospital with haemoptysis. when worked up, his history and examination were highly suggestive of pulmonary tuberculosis (tb). he subsequently developed a massive upper gastrointestinal bleed and underwent an emergency laparotomy, which revealed a massively dilated caecum measuring 20 cm in diameter. the caecum had perforated due to acute decompensation of intestinal tb. though common in developing countries, tb is rare in the uk, especially the intestinal kind. the most striking feature of this case is, however, the size of the caecal distension caused by the tubercular inflammation and subsequent perforation something unheard of in the literature. this massive caecal distention would be explained by the law of laplace. in conclusion, massive distension and caecal perforation are possible consequences of intestinal tb, especially in the 4872 h immediately after starting anti - tubercular therapy.
an efficient procedure was devised for the functionalization of fullerene [13 ], which involved observing the c602 ion in a reaction which occurs with the addition of benzylideneacetal. another reaction between azides and c60 was also explored because of the electrochemically attractive properties exhibited by its products. the formation of methanofullerenes or methanoannulene isomers is predicted when the c60 core is joined to the fragment unit (cr2), as well as when the ch2 is specifically linked to the c60 bond. this kind of functionalization occurs when fullerene c60 reacts with diazomethane to form an unstable molecule which converts to h2c61. spectroscopy is also used for the structural characterization, in order to identify the different electronic properties in a particular case study, where the addition of ch2 to the molecular structure of fullerene produces conformational isomers of methanofullerene. a number of studies exist which have attempted [9, 10 ] to show that this kind of addition manifests a certain energetic preference. these have focused on the nature of the c - c bond of a particular aromatic ring. two different bonds are possible contenders : the cc(6,6) and cc(6,5) bonds, both within the structural conformation of fullerene. besides this, theoretical and experimental features of magnetic susceptibility in the cycloaddition reaction between c60 and diazo compounds provide evidence of paramagnetic and diamagnetic currents. all of these studies aid in the description of the chemical characteristics of these new compounds. therefore, this study will explain the reactivity between c60 and ch2, where an interaction promoted by radicals is expected to constitute an exothermic addition reaction and where carbon atoms with pyramidalization in their bond sp are obtained as a result. in this process, the fullerene s carbon atoms change their hybridization from a trigonal sp to a tetrahedral sp. theoretically, four possible isomers can exist : cc(6,5) open, cc(6,5) closed, cc(6,6) open, cc(6,6) closed, depending on the addition of the fragment to either the (6,5) or (6,6) bond, and furthermore, taking into account whether the distance between the carbon atoms is sufficient to generate a bond or not. the isomers : cc(6,5) closed, and cc(6,6) open have not yet been observed in experiments. theoretical results have indicated that these two isomers are not propitious because they have two (6,5 closed) and three (6,5 open) double bonds in endocyclic position on the pentagonal ring. thus a controversy exists concerning the addition of ch2 as a basic molecule to c60 in these isomers. moreover, they are included in the present study in order to validate this assumption. the aim of the present work is to establish the most favorable interaction between fullerene and ch2, the simplest species that can generate stable derivatives with fullerene, considering different cycle addition channel reactions in order to achieve an energetically stable c60-ch2 compound. the different channel reactions are provoked by changing the initial conditions in terms of their dynamics ; namely the departure speeds of ch2, but with an initial preferential orientation imposed on this molecule in relation to the cc - bonds of c60. the results indicate not only a possible reaction mechanism, but also the nature of the final product. the electronic structure of c60 is commonly studied by first principle methods that include electronic correlation and spin - orbital corrections. density functional theory (dft), included in the nwchem software has been used in this instance. this is a pure dft method ; containing becke s gradient correction for exchange and perdew - wang s for correlation. b88-lyp methods were also employed for the geometry optimization. in the case of the b88-lyp functional, the non - local correlation was provided by the lyp expression and the correction was carried out by means of the vwn functional. this level of computation has provided good results in terms of the molecular geometry optimization and the energies of the bond orders for fullerene. the optimized geometry of fullerene was taken as the starting point in the molecular dynamics study, bearing in mind that the geometry was obtained with a minimum of energy in order to identify the different paths for the reaction. the algorithm of quasi - newton algorithm which seeks hessian approximations is implemented as a part of the nwchem package. the molecular dynamics simulation technique is important, because it reveals the dynamic nature of the nuclear particles. it helps to provide a deeper understanding of the reaction mechanism which takes place on the addition of ch2 to fullerene. the electronic wave function is revealed, taking into account that electrons are immersed in a field of instantaneously fixed nuclei, whereas the nuclear particle is immersed in an average electronic field. the heavy masses and very short spatial extension of the nuclear particles compared to those of electrons permit us to simplify the problem by considering the nuclei as classical particles. in order that this should occur, each nucleus follows a newtonian trajectory due to quantum forces derived from the electron potential plus the electrostatic force exerted by the other nuclear particles. this computational level has provided good results for molecular dynamic calculations [2225 ]. in all molecular dynamic calculations, the representation is described in terms of a displacement with multiplicity of triplet without charge (double free radical) or a charge of (2), as the starting point. this has a tendency to change its configuration due to the pyramidalization phenomenon, and it is also expected that the reaction will follow a mechanism where one of the double bonds on the fullerene surface [cc(6,5) or cc(6,6) ] provides the electrons for the new bond, which may also cause strain on the bucky ball. this feature is fundamental to the suggestion that the reaction with ch2 takes a single preferential direction which could involve either the cc(6,6) or the cc(6,5) bonds. correspondingly, the chosen model consists of a buckyball c60 identified by geometrical optimization which is composed of aromatic rings in a regular pattern. the buckyball c60 has 20 six - member rings and 12 five - member rings. the c60 molecule belongs to the ih point group of symmetry ; therefore it is possible to separate a symmetrical fragment for study purposes, while conserving the symmetry and permitting the selection of portions from the fullerene molecule. the buckyball c60 was identified by geometry optimization calculations, where the atoms that compose the portions selected from the fullerene molecule were unfrozen in the movement, permitting the analysis of the structural and electronic changes that take place during this interaction. the atoms of the first and second neighbours with respect to cc(6,5) and cc(6,6) bonds were also unfrozen, whereas in the case of the rest of the fullerene atoms, they were frozen when the dynamic calculation was made. the first and second portion atoms of fullerene were considered as active for the interaction between ch2 and cc(6,5), as shown in fig. 1. in order to obtain the structural and electronic properties in this interaction, the ch2 manifest a displacement with an initial speed throughout the preferential direction, sufficient to impact over aromatic rings of fullerene. a number of calculations were carried out at the level of first principles for the interaction between fullerene and the ch2 ion. 1atom assets comprising the first and second neighbours of the molecular structure of fullerene, used for calculating the energy values in the molecular dynamic in the case of an interaction between the ch2 fragment and ; a) cc(6,6), b) cc(6,5) fullerene bonds atom assets comprising the first and second neighbours of the molecular structure of fullerene, used for calculating the energy values in the molecular dynamic in the case of an interaction between the ch2 fragment and ; a) cc(6,6), b) cc(6,5) fullerene bonds although, potential repulsive interaction is present at the surface of the fullerene molecule, the most important factor is the participation of ch2 which is an electron donor in this case, and one of the hydrogen atoms joined to the ch2 fragment, because these particles present a different conformation when this fragment is considered as a receptor of an ion or a double free radical. the determination of the electronic participation of ch2 initiates in all cases with the cc(6,5) and cc(6,6) closed conformations and the dynamic process is monitored searching for the possibility to open the bond (yielding the open variations) or the remains of the single bond (in all these cases it is considered that fullerene is a weak electron acceptor and ch2 with a double negative charge donates the electrons for the whole process). total energy and charge analysis are the main sources of information relating to the reaction mechanism. the study involve two cases : total charge : 0 for the interaction c60 (singlet, charge 0) + ch2 (triplet, charge 0) which implies a c60 - --ch2 triplet system under study. in this case ch2 presents two lone electrons (bi - radical)total charge : -2 for the interaction c60 (singlet, charge 0) + ch2 (singlet, charge -2) which implies a c60 - --ch2 singlet system under study. in this case total charge : 0 for the interaction c60 (singlet, charge 0) + ch2 (triplet, charge 0) which implies a c60 - --ch2 triplet system under study. in this case ch2 presents two lone electrons (bi - radical) total charge : -2 for the interaction c60 (singlet, charge 0) + ch2 (singlet, charge -2) which implies a c60 - --ch2 singlet system under study. in this case the interaction between two molecules provides sufficient information relating to changes in electronic properties (in particular the charge transference and structural atomic bond modifications). the values are obtained from dynamic simulation which shows the entire range of changes associated with the different stages in the reactions. therefore, in the addition reaction, the ch2 fragment was positioned at a constant distance of 10 from the c60, then an impetus was applied to the fragment in order that it should collide with the c60 molecule, a process which varies in terms of the time elapsed, ranging between 120 to 500 femtoseconds, depending on the speed of the fragment. the dynamic born - openheimer calculations were applied in order to reveal the total energy values for the interaction between c60 and ch2 molecules. the different energy values correspond to different simulations associated with different charges or multiplicity, as well as depending on the initial speed acquired by the ch2 fragment. this specific speed permits the production of kinetic energy sufficient for surmounting the potential barrier of the transition state. at this point, the first one consists of when ch2 interacts with the cc(6,5) bond on fullerene, whereas the second one concerns the interaction with the cc(6,6) bond. there are different structural changes and charge distributions in each instance and the result even varies when different initial speeds are tested for each simulation, for the purpose of defining the optimal speed for collision and observing the reaction mechanism. a systematic method is to draw a graph of the electronic energy data as a function of time, taking into account a specific speed for ch2 during its interaction with the fullerene c60 cc(6,5) bond, as shown in fig. 2. the energy calculations indicated the electronic activation energy for three different speeds and it also became apparent that the addition process occurred when the speed reached 15 angstroms / femtosecond (/ fs), it is important to note that the first part of the figure shows an almost null interaction and a potential barrier due to repulsive effects, besides the first maximum is about 40 fs whereas the second one is given between 80 and 90 fs and corresponds to the activated complex. conditions were similar for all these speeds and the behaviour manifested in the first collisions for the other two speeds indicated the repulsion effect during the interaction. the energy values plotted on the graph are associated with the interaction which takes place when the ch2 fragment moves at velocities of 9, 12 and 15 / fs. 2interaction between ch2 and cc(6,5) bond fullerene molecule c60 at 15 units of speed. the phenomenon shows different behaviour in this instance ; the potential barrier is surmounted the second after first minimum when velocity is 15 interaction between ch2 and cc(6,5) bond fullerene molecule c60 at 15 units of speed. the phenomenon shows different behaviour in this instance ; the potential barrier is surmounted the second after first minimum when velocity is 15 the results for energy values of the interaction when the speed reached 9 / fs indicated that the ch2 fragment interacted slowly with the fullerene surface. the energy values presented an increase when the ch2 covered a short distance to the fullerene and showed a maximum value that corresponded to the potential barrier of repulsion. under the same conditions, the energy values that correspond to the speed of 12 / fs when compared to the last case indicated that on this occasion no addition took place between ch2 and the fullerene surface. these behaviours indicated a number of small bonding changes due to the effects of repulsion, but there was no addition of the ch2 fragment, indicating that under these conditions the potential barrier is greater than the kinetic energy associated with the movement of ch2. this effect of repulsion on the part of ch2 is represented by an almost symmetrical curve in the right zone of the plot. the effect of repulsion at speeds of 9 and 12 / fs was an indicator that partial charge is not an essential factor for the reaction mechanism and indicated that the speed needed to be increased. applying the same reaction condition, speed was increased to 15 / fs. the results of the interaction for both species were obtained by applying molecular dynamics. in this instance, there are changes in energy associated with bond length changes ; a situation which sketches a typical addition reaction mechanism, resulting in structural changes and charge transference (see below). in the three cases with three different speeds, the ch2 fragment takes a preferential direction to collide with the cc(6,5) bond, forming a straight line between the carbon atom of the ch2 fragment and the bond between both rings in the fullerene molecule. in the case of 15 / fs, it was revealed that the main part of the reaction refers to the addition of the ch2 fragment to the surface of the fullerene. this stage should be considered as an important stage in the reaction mechanism and here the electronic donation of the double bond to the ch2 fragment on the surface of fullerene is apparent, as shown in fig. 3the interaction between fullerene and the ch2 fragment produced a reaction mechanism of addition, pyramidation in the ch2 fragment was observed, shown as a cc(6,5) bond open addition the interaction between fullerene and the ch2 fragment produced a reaction mechanism of addition, pyramidation in the ch2 fragment was observed, shown as a cc(6,5) bond open addition energy values are also associated with significant changes in the atomic bond lengths where both fragments are united at the maximal energy point. this characteristic indicates similar behaviour to that occurring in the case of charge transfer. under these conditions, the first one is associated with an almost null interaction and a potential barrier due to the effect of repulsion, in the second section the reaction occurs showing an elongation around the cc(6,5) ring bond ; at this point the curved section corresponds to a maximum peak at 350 kcal mol and a minimum in the range of 2060 fs and a third section which corresponds to the relaxed bond, taking into account the activated complex at 330 kcal mol, which represents the addition of ch2 to the aromatic ring. the mulliken population analysis was applied to the interaction between ch2 and cc(6,5) of the fullerene c60 because this is the best option for attack. the dynamic charge transference is presented in fig. 4, plotted as the calculated mulliken charge versus various time intervals. the system was divided into fragments ; one fragment corresponded to the fullerene and the other to the ch2, both before and after the reaction. during the first stage (before the ch2 had made impact on the fullerene), the system exhibited a total charge of -2, made up of the ch2 with a charge of -0.25 and the fullerene with a net charge of - 1.75. the graph plot showed little variation because the fragments retained their charge, even when they were far apart. however, there was a short time period when greater variation was apparent, this corresponded to the modification in bond lengths and an increase in the energy correlated with fig. it is interesting to note that the major change occurred in the period between 40 and 60 fs where the whole charge of 2 (or bigger) is totally concentrated in the fullerene fragment, whereas the methylene fragment reduced its charge to zero (or possibly a small positive charge) allowing the system to remain as it was at the beginning, but with the new bond having been formed. the likely interpretation is that the double bond on the cc(6,5) was retained on the fullerene until the bond was formed, suggesting that the reaction speed depends mainly on the concentration of c60. 4charge transfer for the interaction between ch2 and the cc(6,5) bond fullerene molecule c60 at the moment when ch2 was added charge transfer for the interaction between ch2 and the cc(6,5) bond fullerene molecule c60 at the moment when ch2 was added on the other hand, we carried out calculations at different speeds for the interaction between ch2 and the cc(6,6) bond, with results indicating that no reaction takes place if the same conditions are applied. only an effect of repulsion was evident in the system comprising ch2 and cc(6,6). this result confirms the proposition derived from experimental evidence that the preferred mechanism was that which comprised the ch2 fragment and the cc(6,5) bond. during the process of the revision of this work, the authors found out about a similar study in which other kind of methods carried out on similar systems leads to the same conclusions that in the present case, this is an excellent situation that validates both studies. the energy analysis carried out by applying molecular dynamics brings us to the conclusion that the main condition necessary for ensuring that the addition of ch2 to c60 depends on certain energy being present, exceeding that of the potential barrier presented by the ch2 ion fragment with a charge of -2 or a biradical with multiplicity of 3. this pathway is more likely to occur with the bond cc(6,5), taking specific speed into consideration. these results agree with experiments which suggest that there is a certain conformation which favours this reaction, as shown in the case of the electronic configuration of the bonding interaction.	the theoretical study of the interaction between ch2 and fullerene (c60) suggests the existence of an addition reaction mechanism ; this feature is studied by applying an analysis of electronic properties. several different effects are evident in this interaction as a consequence of the particular electronic transfer which occurs during the procedure. the addition or insertion of the methylene group results in a process, where the inclusion of ch2 into a fullerene bond produces the formation of several geometric deformations. a simulation of these procedures was carried out, taking advantage of the dynamic semi - classical born - oppenheimer approximation. dynamic aspects were analyzed at different speeds, for the interaction between the ch2 group and the two bonds : cc (6, 6) and cc (6, 5) respectively on the fullerene (c60) rings. all calculations which involved electrons employed dft as well as exchange and functional correlation. the results indicate a tendency for the ch2 fragment to attack the cc (6, 5) bond.
overactive bladder (oab) is characterized by symptoms of urinary urgency with or without incontinence, often accompanied by daytime and nighttime frequency.1 it is caused by an overactive detrusor muscle, in many cases with no apparent cause, although a significant proportion of cases may be accompanied by a neurogenic dysfunction.1 the prevalence of oab in the general adult population ranges from 14% to 16%,2 usually associated with other health problems, such as sleep disorders, anxiety and/or depression, urinary tract and skin infections, etc, that not only lead to a considerable use of health care resources, but also affect the general well - being, activities of daily living, and health - related quality of life.37 antimuscarinics are the primary drugs used for the treatment of oab,3 but response and tolerability vary among patients. flexible doses have become increasingly important in society, especially among elderly patients and children.8 the use of flexible doses allows treatment to be better adapted to the patient s needs, by determining the most favorable balance for patients.8 approximately 60% of patients with oab choose to have the dose of their antimuscarinic increased, resulting in a favorable response in terms of disease management without increasing adverse effects.8 flexible doses are defined as the capacity of a treatment to shift within a certain range, in which the drug is shown to be safe, while still exerting the desired therapeutic effect.9,10 many conditions and needs of the patient may affect the treatment and benefit from the use of flexible doses, such as tolerability, safety, severity, and other clinical and demographic characteristics that the patients may present. the possibilities offered by the use of flexible doses directly affect how doctors and patients adjust treatment to maximize therapeutic response while controlling the occurrence of adverse effects.11 fesoterodine is an antimuscarinic indicated for the symptomatic treatment of oab and can be given in flexible doses.1114 in a study based on the analysis of data from two flexible - dose clinical trials of fesoterodine, cardozo observed that the highest dose of the drug was more effective in patients with more severe oab symptoms, that is, those who had at least two episodes a day of urinary incontinence with urgency. however, dose escalation can lead to increased use of health care resources (medical visits, use of pads, or concomitant drugs) because of a delay in achieving greater effectiveness in symptom control.11 therefore, in routine clinical practice, some clinicians prescribe high doses of fesoterodine, from the time of first treatment in newly diagnosed patients (0) for each additional unit of health benefit, expressed in qalys (qalys gained). the incremental costs and the qalys were calculated as the difference between the values for the fesoterodine fast scaling to high - dose group minus the values for the dose - scaling traditional group. in spain, the cost - effectiveness threshold at which a health intervention may be financed by the nhs is usually around an icer 75 years), by number of comorbidities, with or without urine leakage, and patients with oab for > 12 months. last, a third uncertainty analysis of the model included a probabilistic sensitivity analysis (psa) with nonparametric repeated sampling (bootstrapping). the psa was performed by using nonparametric resampling methods (bootstrapping), consisting of empirically studying the behavior of the icer through a large number of subsamples (simulations) obtained by randomly selecting samples of the same size as the study samples.26,27 in this study, 5,000 simulations were performed. the cost comparison was done by following thompson and barber s recommendations,28 using a general linear model (analysis of covariance), and adjusted for the following covariates : sex, history of oab, total days of treatment, and the following comorbidities : perineal surgery, perineal aortic surgery, stroke, cardiovascular disease, angina and disc diseases, spinal stenosis, and spine surgery. in addition, a multiple linear regression analysis was carried out to observe the associated variables (adjusted for covariates). a descriptive and univariate statistical analysis was performed with the mean and median values, the standard deviation and 95% confidence intervals in parametric variables and median and interquartile ranges in nonparametric variables, after verifying the normality of the distribution with the kolmogorov the following tests were used : analysis of variance, chi - squared, pearson correlation coefficient, and paired sample t - test, according to the data distribution. the confidence interval of the mean differences was calculated by resampling (bootstrapping) with 1,000 iterations. spss for windows, version 19 (ibm corporation, armonk, ny, usa), was used to establish the statistical significance for p - values at 12 months of history (table 5). the psa with 5,000 simulations showed that, in 5.5% of the iterations, the icer of the fast escalating high - dose group was in the upper right quadrant of the cost - effectiveness graph (figure 3a), indicating greater effectiveness, albeit with a higher cost ; whereas for 4.0% of the time, the fast scaling high - dose group was less effective and with higher costs than the dose - scaling group (dominated option). overall, 47.2% in the fast scaling high - dose group was dominant (more effective with lower costs) and for 43.4% of the time, it was less effective but with lower costs. the psa showed that when the cost - effectiveness threshold was set at a maximum of 30,000 per qaly gained, the fast scaling high - dose group proved to be cost - effective vs the dose - scaling group in 67.6% of simulations performed with the icer (figure 3b). this study comprised an economic evaluation of the use of an antimuscarinic drug (fesoterodine), given in flexible doses, in patients newly diagnosed with oab, in daily clinical practice in spain, using flexible doses to prescribe two different dosages. in this way numerous clinical studies have shown the effectiveness of using flexible doses of antimuscarinic drugs to treat symptomatic oab.812,14,15 data from observational studies, such as the study presented here,16 which give a more realistic view of daily practice, may also help to develop better therapeutic strategies, which complement those obtained in traditional clinical trials, and therefore it is also interesting to estimate effectiveness of drug use in these conditions.29 in different economic evaluations with fesoterodine published in spain, where a cea has been performed, the superiority of fesoterodine has been shown, compared with other drugs for the symptomatic treatment of oab.1719 these economic evaluations have shown that, compared with solifenacin, tolterodine or mirabegron, fesoterodine has an icer under the cost - effectiveness threshold normally accepted in spain for inclusion in the pharmaceutical services of nhs.25 the economic superiority of fesoterodine compared with other antimuscarinic drugs available in our health care setting has also been shown in conditions of routine or real - life practice, indicating that fesoterodine can lead to health care savings in the treatment of oab, which is nhs - funded, and offsetting its higher pharmacological cost with less use of health care resources, such as medical visits, absorbents, or concomitant medication related to oab (antidepressants, anxiolytics, etc), in patients of any age and in vulnerable subjects.3032 however, none of the above economic evaluations has addressed the cost - effectiveness of fesoterodine in flexible doses, which allows for two different dosages : a rapid scaling to high doses or the standard scaling according with the recommendation in the fesoterodine summary of product characteristics. in our study, when flexible doses of fesoterodine were used in newly diagnosed patients, fast scaling to high doses proved to be a cost - effective treatment option in most of the situations analyzed according to the current recommendations in our health care setting.25,33 this economic evaluation has confirmed the findings of other economic evaluations with fesoterodine, which show that its biggest pharmacological cost (in this case due to the use of fast scaling to high doses versus conventional titration) comes with savings in other components of nhs - funded health care costs, namely, cost for absorbents, concomitant medication related to oab, and health care costs for medical visits to primary care and to specialists, which fully offset additional pharmacological costs. these results have been able to be replicated in various alternative scenarios and subgroups of patients, which fully justify this economic evaluation, although it may be considered unusual to compare two doses of the same drug, because it is rare to find similar economic evaluations in the scientific literature.34 one of the most sensitive factors in this economic evaluation was time since diagnosis of oab. thus, the results show that high doses of fesoterodine turn out to be cost - effective when the diagnosis of oab is recent, that is, 12 months, from the current perspective in spain for the ability to pay per qaly gained, according to current recommendations.25,33 this suggests that clinicians should consider escalating fast to high doses of fesoterodine only if patients have had oab for < 12 months, because only in these circumstances is the greater pharmaceutical cost offset by savings in other components of health care costs, while there are more qalys gained. the findings of this economic evaluation could help health authorities and decision makers in health care in the process of making decisions that entail improvements in daily clinical practice (real world), although this study is not without its limitations. first, due to the study design, no data are available on the severity of the oab at the start of treatment or on dose escalation, because doing this evaluation required bladder diaries that the patients had to complete over several days, tasks that are often used in clinical trials but not in routine practice. this has prevented us from knowing whether the groups were completely homogeneous at the start of treatment with fesoterodine. however, the advantage of pragmatic studies such as this one, which represent routine or real - life medical practice, may lie in their utility in health care decision making. another possible limitation revolves around the costs used, since in this study no information on hospitalizations, cost per protocol (cost of side effects or switching), or additional testing (urodynamics, cultures, etc) was collected. still, it should be noted that these patients usually do not require hospitalization, and this cost component might not be expected to change the results of the economic study. further, the impact of possible fractures produced, particularly in vulnerable patients, on the use of health care resources, was not collected. however, this might not be a significant problem from an economic point of view, as has been observed in other economic evaluations carried out in our health care setting with antimuscarinic drugs.1719 finally, this evaluation was not performed from the society s perspective, because this study did not take into account variables such as payment for absorbents by the patient, travel costs, losses in productivity, or their impact on indirect costs. with increasing pressure on payers and prescribers to make economic decisions regarding patients, data on cost - effectiveness this is the first study to carry out an analysis of fesoterodine with different treatment regimens and show that treatment with fesoterodine scaling fast to high doses involves savings and more clinical benefits (in terms of qalys gained) versus the standard titration of this drug. according to the results from this study, treatment with fesoterodine fast scaling to 8 mg may be considered a cost - effective alternative for the spanish nhs, compared with the traditional titration of this drug, from 4 to 8 mg, in the treatment of oab in females who have had oab for < 12 months, in routine clinical practice.	objectiveto carry out cost - effectiveness analysis from the spanish national health system perspective, of treating overactive bladder (oab), in newly diagnosed patients with two flexible doses of fesoterodine in routine clinical practice.patients and methodseconomic evaluation of flexible - dose fesoterodine in newly diagnosed patients, including two treatment groups : standard escalating from 4 to 8 mg or fast escalating to 8 mg. costs were estimated from health care resources utilization related to oab, and were expressed in 2015 euros. quality - adjusted life - years (qalys) were obtained from overactive bladder questionnaire - short form. univariate and probabilistic sensitivity analyses were carried out.resultsthree hundred and ninety symptomatic oab patients treated with fesoterodine and newly diagnosed (141 in fast escalating group and 249 in standard escalating) were analyzed. adjusted health care total costs were not statistically different ; difference 4.1 (confidence interval : 153.3 ; 25.1) p=0.842. qalys were higher in fast escalating to high dose vs standard escalating group, resulting in a cost of 16,020/qaly gained for fast escalating vs standard escalating group.conclusionwhen the cost - effectiveness threshold is set at a maximum value of 30,000/qaly gained, fesoterodine fast escalating group was cost - effective vs standard escalating group 67.6% of the time. the treatment with fesoterodine, in female patients newly diagnosed, fast escalating to 8 mg was a cost - effective option relative to escalating traditionally from 4 to 8 mg, in the management of oab in routine clinical practice, from the spanish national health system perspective.
copolymerizing different monomers offers vast possibilities to tune the properties of covalent copolymers. in fact, the ability to copolymerize monomers in an alternating, gradient, random, or block manner offers superb precision in the control over the copolymer s molecular structure (composition, sequence, and length). as a result generally, the molecular structure of the copolymer can be predicted and tuned by a delicate interplay of reactivity and feed ratios of the two monomers via the classical copolymer equation, in combination with advanced controlled polymerization techniques. in the field of supramolecular copolymers, in contrast, much less attention has been devoted to developing predictive rules that allow to forecast the sequence, composition, and length of multicomponent mixtures. the dynamic nature of the non - covalent interactions makes multifunctional supramolecular copolymers versatile systems, which gives them a high potential for use as adaptive materials, in organic electronics, and as biomimetic systems. however, constructing hierarchical multicomponent co - assemblies is particularly challenging as it requires a rational design that encompasses a subtle balance between the complementary recognizing motifs for connecting different monomers into an ordered array and the compatibility between the diverse peripheral functional groups. moreover, important differences are encountered when copolymers made under thermodynamic equilibrium are compared with those prepared under kinetic control. when mixing different types of monomers, two extreme assembly cases may occur ; total self - sorting or complete co - assembly of the different monomers. many elegant examples have been presented in which multicomponent co - assembly has been achieved by aromatic donor acceptor interactions, hydrogen bonding, -stacking interactions, and others. in addition, multicomponent systems are also found to form self - sorted assemblies. a comprehensive molecular understanding recently, significant progress has been made in the elucidation of pathway complexity, mechanistic detail, feed - dependent behavior, thermodynamic stability, composition, and stack length of supramolecular (co)polymerizations. exciting examples are found in kinetically controlled living copolymerizations as developed for covalent block copolymers and small molecules, which show that kinetically trapped block structures can be formed in supramolecular copolymers. for systems under thermodynamic equilibrium, a combined experimental and theoretical approach proved to be a robust method to unravel several issues, although these endeavors were typically limited to one - component or simple two - component systems. however, in many examples reported on multicomponent systems, these characteristics remain largely elusive, particularly when the components aggregate via different supramolecular polymerization mechanisms. in the past, our group has extensively studied the helical, one - dimensional self - assembly of chiral and achiral alkyl - substituted btas (alkyl - btas), which follows a highly cooperative mechanism in dilute aliphatic solvents. the handedness of the helical superstructures, p or m, is biased by the presence of a non - racemic stereocenter in the aliphatic side chains. in contrast, the self - assembly of btas functionalized with short oligodimethylsiloxane (odmsi) chains (odmsi - bta) revealed a decrease in propensity for assembly compared to alkyl - substituted btas, which was attributed to the presence of sterically crowded siloxanes. the presence of odmsi side chains in bta - derivatives increased the critical aggregation concentration 10100 times over that of alkyl - btas, but the mechanism of self - assembly remained unclear. in addition, we also observed that unsymmetrical btas comprising both odmsi and alkyl side chains form a superlattice in the bulk due to strong phase separation of the two chemically incompatible side chains. an intriguing question is how btas with either odmsi or alkyl side chains would interact in solution. in a mixture of alkyl - bta and odmsi - bta, three outcomes are possible : (1) alternate copolymerization to minimize steric demand of odmsi - btas ; (2) complete self - sorting due to peripheral chain incompatibility ; or (3) everything in between. to elucidate these possibilities, we herein perform sergeant - and - soldiers experiments between opposite pairs of chiral and achiral alkyl - bta and odmsi - bta derivatives (scheme 1). a combination of chiral (optically active) and achiral btas permits the observation of copolymerization behavior by circular dichroism (cd) spectroscopy. an unexpected chiral induction was observed from the mixture of two independently cd - silent solutions of achiral alkyl - bta (a - bta) and chiral odmsi - bta (r - si - bta). a detailed analysis was conducted to understand how the differences in molecular structure and polymerization pathway of individual monomers affect the chiral amplification and the overall copolymerization process by combining spectroscopic studies with in - depth theoretical modeling. in the present work, we propose a general experimental / modeling approach to understand various aspects of supramolecular copolymerization under thermodynamic control. this collective approach permits us to successfully elucidate the monomer feed - dependent copolymerization behavior and thereby the composition of the supramolecular copolymers obtained. in the present report, before studying the copolymerization between alkyl - bta and odmsi - bta, we first revisit the mechanism of homopolymerization of the individual components. the copolymerization is then analyzed by the mixing of the two monomers in different ratios. finally, we put forward a new theoretical model that permits to fully explain the copolymerization process. before studying the copolymerization behavior, we first analyzed the mechanism of homopolymerization of the individual monomers. the self - assembly of chiral r - si - bta in methylcyclohexane (mch) was probed using temperature- and concentration - dependent cd measurements and compared to that of achiral a - bta under identical conditions. the experimental cooling curves were fitted with a one - component mass - balance model (figures s1 and s2). this model is based on the equilibrium between monomers, oligomers, and polymers, and the aggregation process is divided into a nucleation regime and an elongation regime. the equilibrium constant kn describes the formation of a nucleus, whereas ke describes the equilibrium constant in the elongation phase. the nucleation step is highly unfavorable in a cooperative process so that kn 25 mol%), the cd spectrum changes to the spectrum of pure sergeant, with a single maximum at 225 nm. such differences in the cd spectra of the mixtures can be attributed to a different packing of the hydrogen bonds within the helical stacks below 10 mol% and above 25 mol% r - si - bta. the difference in molecular packing was also evident from the ft - ir data of 1 mm mch solutions of 10 mol% and 90 mol% of r - si - bta that showed resemblance with pure a - bta and r - si - bta, respectively (figure s5). to further elucidate this change in the shape of the cd signal as a function of increasing fraction of r - si - bta, variable - temperature uv and cd experiments were carried out at various compositions of r - si - bta / a - bta in mch at a total bta concentration of 50 m (figures 2 and 3). interestingly, two different trends in the uv and cd cooling curves are observed. up to 5 mol% of r - si - bta sergeant added, all the uv cooling curves are superimposable to the curve of pure a - bta, indicating a similar cooperative mechanism of copolymerization as observed for a - bta homopolymer (figure 2a). however, above the threshold of 5 mol% of r - si - bta, the melting curves gradually become more sigmoidal (figures 2b and 3b), indicating a change toward an isodesmic growth mechanism. temperature - dependent uv spectra of solutions containing different ratios of r - si - bta / a - bta probed at = 220 nm with ctot = 50 m in mch (cooling rate = 2 kmin). (a) going from violet curve (0 mol% r - si - bta) to red curve (5 mol% r - si - bta), upon addition of 1 mol%, 2 mol%, 3 mol%, and 4 mol% of r - si - bta to a - bta. (b) going from violet curve (10 mol% r - si - bta) to red curve (100 mol% r - si - bta), upon addition of 25 mol%, 50 mol%, and 75 mol% of r - si - bta to a - bta. experimental and computed (vide infra) cd cooling curves probed at = 220 nm with ctot = 50 m in mch containing different ratios of r - si - bta / a - bta (cooling rate = 2 kmin). (a) going from violet curve (0 mol% r - si - bta) to red curve (5 mol% r - si - bta), upon addition of 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, and 4 mol% of r - si - bta to a - bta. (b) going from violet curve (10 mol% r - si - bta) to red curve (100 mol% r - si - bta), upon addition of 25 mol%, 50 mol%, and 75 mol% of r - si - bta to a - bta. experimental data are represented by dashed lines, while computed values are represented by solid lines. to rationalize these observations, we hypothesize that the two monomers can copolymerize into aggregates in which the monomer present in majority dictates the molecular packing / aggregate properties. at lower fractions of r - si - bta, a - bta dominated cooperative helical aggregates are formed contributing to the huge cd signal whereas at higher fractions of r - si - bta, r - si - bta dominated isodesmic aggregates (x - type) are assembled but participate with low cd intensity. as the current experimental techniques available do not allow defining the precise nature of the assemblies at such low concentration, we resort to theoretical models to understand the copolymerization in more detail. a model describing two - component copolymerization was put forward to explain the origin of the remarkable chiral amplification followed by steep fall in the cd signal observed in the sergeant - and - soldiers experiments performed between independently cd - silent solutions of a - bta (named a in the model) and r - si - bta (named si in the model). this new model is an extension of the one - component model, but now with two monomers and three types of aggregates, namely p, m, and x. the current model takes into account not only distinct dimers and elongation steps but also the types of bonds formed between different monomers in the stacks. the reaction scheme is described as a sequence of stepwise monomer associations and dissociations, where the equilibrium constants are assumed to depend not only on the aggregate type but also on the types of the two monomers involved in the bond formed / broken, and whether it concerns the nucleation of a new aggregate or the elongation of an existing one (see supporting information section b for details). the equilibrium concentrations of all species are obtained by solving the mass - balance equations for the two monomers, and these are subsequently used to calculate theoretical cd and/or uv absorption cooling curves. this methodology has been applied previously to describe both the majority - rules and the sergeant - and - soldiers principles in cooperative two - component bta supramolecular polymerization. herein, we assume that r - si - bta forms enantiomeric isodesmic x - type aggregates (with thermodynamic parameters as reported in table 1) while the a - bta forms racemic cooperative aggregates with two opposite helicities, i.e., p- and m - type aggregates, with equal likelihood (again with thermodynamic parameters as reported in table 1, ga - pm). moreover, we assume that a - bta can mix into x - type aggregates (ga - x) and, vice versa, that r - si - bta can mix into both p- (gsi p) and m - type (gsi - m) aggregates, by forming a - bta / r - si - bta bonds. additionally, we consider that the formation of r - si - bta / r - si - bta bonds within cooperative p- and m - type aggregates and a - bta / a - bta bonds in isodesmic x - type aggregates are energetically unfavorable, therefore they are not considered in the model. an optimal fit of the theoretical cd curves (figure 3, solid lines) with the experimental cd curves (figure 3, dashed lines) is obtained with the thermodynamic parameters shown in table 2 (see supporting information section b for details). figure 4 shows that the uv melting curves calculated by this newly developed two - component model also demonstrate nice agreement with the experimental curves (figure 2). note that these uv curves are results of the model, with parameters that were fitted only with cd curves, which further illustrates the potential of the model to describe the experimental data. calculated uv curves obtained from the model showing the theoretical normalized absorbance as a function of temperature. (a) going from violet curve (0.5 mol% r - si - bta) to red curve (5 mol%), upon addition of 1 mol%, 2 mol%, 3 mol%, and 4 mol% of r - si - bta to a - bta. (b) going from violet curve (10 mol% r - si - bta) to red curve (100 mol% r - si - bta), upon addition of 25 mol%, 50 mol%, and 75 mol% of r - si - bta to a - bta. these model results show a similar spectral trend as observed in the experimental uv cooling curves in figure 2. as is evident from the thermodynamic parameters of the copolymerization as a function of the temperature in figure 5, the mixing of r - si - bta into m- or p - type aggregates is less favorable than the addition of a - bta (ga - pm gsi p). in addition, the incorporation of a - bta and r - si - bta into x - type aggregates is less energetically favorable than their incorporation into cooperative p- or m - aggregates (ga - pm 0.5 mol%), the p - type aggregates rapidly prevail over the m - type aggregates. the lower the temperature, the smaller the fraction of sergeant needed to solely have p - type aggregates. the largest quantity of p - type aggregates is obtained with a fraction of r - si - bta of around 10 mol%, which is in agreement with the experimental observations. moreover, for higher fractions of sergeants (> 25 mol%), the isodesmic x - type aggregates start to compete with the p - type aggregates (figure 6d, e). when the quantity of r - si - bta is preponderant (> 75 mol%), the isodesmic x - type aggregates prevail over the cooperative p - type aggregates (figure 6f), and a dominant isodesmic mechanism is observed in the mixture resulting in a fall in the cd intensity. figure 7a shows the calculated amounts of different species formed as a function of the addition of sergeants at 293 k and predicts the coexistence of p- and m - type aggregates above 10 mol% of sergeant added. for p - type aggregates, the number of monomers per aggregate decreases when the fraction of sergeants increases (figure 7b). calculated concentration of btas in the various species types (p, m, and x) and concentration of free monomers (a and si) as a function of the temperature for several fractions of sergeants r - si - bta : 0.5 mol% (a), 4 mol% (b), 10 mol% (c), 25 mol% (d), 50 mol% (e), and 75 mol% (f). (a) calculated concentration and (b) average stack length (number of monomers per stack) of the various species types over the fraction of sergeants r - si - bta at 293 k, ctot = 50 m in mch. to gain insight into the composition of the distinct aggregate types, the percentages (figure s7) and concentrations (figure s8) of sergeants r - si - bta constituting the p- and x - type aggregates over sergeants added were plotted, clearly indicating that the compositions of the two aggregate types are not constant. with increasing fraction of sergeants in the mixture, up to approximately 85 mol%, where the fraction of p - type aggregates becomes negligible, the fraction of r - si - bta in the p - type aggregates increases sub - stoichiometrically, up to a limit of approximately 30 mol%. this analysis suggests that above the fraction of 10 mol% of r - si - bta (where the cd signal is maximum), its propensity to intercalate into p - type aggregates becomes lower, which initiates the formation of segregated r - si - bta - dominated x - type aggregates in the mixture. as per the model prediction, we conclude that the drop in the cd intensity with increasing fraction of the sergeants is the outcome of a reduction in both the amount and the average stack length (number of monomers per stack) of cooperative p - type aggregates. since in the sergeant - and - soldiers experiment the total monomer concentration remains constant, the amount of a - bta dominated p - type aggregates continuously decreases with increasing r - si - bta / a - bta feed ratio thereby causing depletion in the cd signal. by working instead in a solution with increasing fraction of r - si - bta for a fixed concentration of the achiral a - bta, the model with the same thermodynamic parameters predicts a steady rise in the cd signal rather than the drop observed in the sergeant - and - soldiers experiment (figure 8a), due to continuous formation of x - type aggregates in the presence of an invariant amount of p - type aggregates. (a) experimental and calculated cd signal at 220 nm and (b) calculated concentrations of the various species types over increasing fraction of sergeants r - si - bta at a fixed concentration of a - bta 25 m at 293 k. to experimentally validate this prediction, we performed a new set of experiments. cd spectra were measured from a set of solutions prepared at fixed ca - bta = 25 m and increasing concentration of r - si - bta (figure 8a, figure s9). indeed an overall rise in the cd signal is observed, with two different slopes in the two regimes that can be rationalized by the model. the first regime, up to 2.5 m of r - si - bta (i.e., 9 mol% of r - si - bta), exhibits a chiral amplification corresponding to the intercalation of r - si - bta into p - type aggregates. in the second part of the titration curve (cr - si - bta > 2.5 m), the continuous addition of chiral r - si - bta displays a small enhancement in the cd signal due to limited further intercalation of r - si - bta in p - type aggregates as well as the formation of x - type aggregates (figure 8b). since the contribution of the helical columnar p - type aggregates to the cd signal is significantly larger than the contribution of the short x - type aggregates, only a small rise is observed in the cd intensity in the second regime compared to the sharp jump in the first regime. the continuous increase in the cd intensity in this experiment rules out the possibility of copolymerization of all the monomers into a single stack type (x - type). the formation of only x - type aggregates would result in a significant drop in the overall cd signal due to the dominance of r - si - bta, analogous to the drop observed in the sergeant - and - soldiers experiment described in figure 1c. in fact, the presence of two regimes perfectly matches with the coexistence of p - type and x - type aggregates, as predicted by the model. based on the experimental data and the mathematical model, the entire copolymerization process as studied by the sergeant - and - soldiers up to nearly 10 mol% of r - si - bta, favorable incorporation of r - si - bta into p - type aggregates takes place in a cooperative mechanism, such that the concentration of p - type aggregates rapidly increases at the expense of m - type aggregates, leading to chiral amplification. below 10 mol% of r - si - bta in the mixture, the steric hindrance of bulky r - si - bta does not adversely affect its behavior as a sergeant for a - bta, rather r - si - bta prefers to randomly intercalate within predominantly a - bta - based aggregates to minimize its own steric repulsion. however, at 25 mol% of r - si - bta added, the shape of the cd spectra starts to change indicating a change in the packing within the aggregate. most likely, the incompatibility between the alkyl and the odmsi side chains in combination with the isodesmic assembly of the increasing amounts of r - si - bta overrules the favorable steric parameter for copolymerization. this plausibly results in the formation of segregated r - si - bta dominated short x - type aggregates, in the presence of p - type aggregates in the mixture. with increasing r - si - bta / a - bta feed ratios, the amount of a - bta dominated p - type aggregates continuously drops in the mixture leading to a consistent fall in the cd signal observed in the second regime. additionally, due to the coexistence of a significant population of the free r - si - bta monomer in the mixtures (figure 7a), the compositions of the p- and x - type aggregates change continuously with added r - si - bta. illustrated are various species types present below 10 mol% and above 25 mol% of r - si - bta added. for clarity, the presence of free a - bta monomers in the system is omitted, as its concentration is very low. in conclusion, we reported supramolecular copolymerization under thermodynamic equilibrium conditions between two bta monomers that bear structurally different alkyl and odmsi side chains and are distinguished by distinct self - assembly mechanisms. their co - assembly behavior was analyzed by sergeant - and - soldiers cd experiments and in - depth theoretical modeling. a remarkable chiral induction was observed by mixing of two individually cd silent diluted solutions resulting from supramolecular copolymerization between two monomers stacking via cooperative and isodesmic mechanisms. the two regimes observed in the variation of the cd signal of the mixtures were related to the combined effect of sterically demanding self - assembly of r - si - bta, the odmsi side chain s incompatibility with a - bta, and the difference in mechanism of the homopolymerization of the individual monomers. below 10 mol% of r - si - bta, the huge chiral amplification originates from the favorable cooperative copolymerization of a - bta soldiers with chiral r - si - bta sergeant into p - type aggregates, in which the r - si - bta sergeants completely copolymerize with a - bta to minimize their own steric hindrance. beyond 25 mol%, the effect gradually diminishes due to both side - chain incompatibility and isodesmic assembly of the dominant bulky r - si - bta monomer, resulting in the segregated formation of r - si - bta dominated x - type aggregates along with p - type aggregates. as the amount of a - bta monomers and thereby p - type aggregates continuously decreases with increasing r - si - bta / a - bta feed ratios, we observe a continuous drop in the cd signal that predominantly arises from the helical columnar p - type aggregate, explaining the biphasic behavior. an excellent agreement between the experimental results and the model allowed us to quantify the thermodynamic parameters, the concentrations, and the compositions of different aggregates present in the mixtures as a function of various monomer compositions and temperatures during the copolymerization. this example is an illustration of how subtle balances between several parameters such as different monomer compatibilities, relative monomer reactivities, and individual mechanism of homopolymerization are more critical in the construction of supramolecular copolymers than established for covalent copolymerization. the present work is another step toward developing the understanding on the largely unknown area of multicomponent supramolecular copolymerizations in the context of the mechanistic elucidation, thermodynamic stability, and monomer feed - dependent copolymerization behavior. we propose that such a generalized understanding combining spectroscopic studies with explicit theoretical modeling on the complexity of supramolecular copolymerization will pave the way for constructing more complex functional supramolecular materials for future applications.	supramolecular copolymers, non - covalent analogues of synthetic copolymers, constitute a new and promising class of polymers. in contrast to their covalent counterparts, the details of their mechanism of formation, as well as the factors determining their composition and length, are still poorly understood. here, the supramolecular copolymerization between two slightly structurally different benzene-1,3,5-tricarboxamide (bta) monomers functionalized with either oligodimethylsiloxane (odmsi) or alkyl side chains is unraveled by combining experimental and theoretical approaches. by applying the sergeant - and - soldiers approach using circular dichroism (cd) experiments, we are able to obtain detailed insights into the structure and composition of these supramolecular copolymers. moreover, we observe an unexpected chiral induction upon mixing two independently cd - silent solutions of the achiral (soldier) and chiral (sergeant) monomers. we find that the subtle differences in the chemical structure of the two monomers impact their homopolymerization mechanism : whereas alkyl - btas cooperatively self - assemble, odmsi - btas self - assemble in an isodesmic manner. the effect of these mechanistic differences in the supramolecular copolymerization process is investigated as a function of the composition of the two monomers and explicitly rationalized by mathematical modeling. the results show that, at low fractions of odmsi - bta sergeants (25 mol%), the isodesmic assembly of the increasing amounts of sergeant becomes more dominant, and different species start to coexist in the copolymerization process. the analysis of the experimental data with a newly developed theoretical model allows us to quantify the thermodynamic parameters, the distribution of different species, and the compositions and stack lengths of the formed supramolecular copolymers existing at various feed ratios of the two monomers.
	colorectal cancer (crc) is the third most common cause of cancer - related deaths with treatment of advanced and metastatic crc (mcrc) remaining palliative at best.1 the epidermal growth factor receptor (egfr) has been identified as a therapeutic target for a multitude of malignancies, including mcrc. ligand - binding to egfr results in the subsequent activation of multiple signal transduction pathways including the pi3k / akt and ras / raf / mapk pathways, which are vital for cell growth and survival.2 constitutive activation of these signaling pathways leads to deregulated cellular proliferation, malignant progression, and invasion.3
the case control study was conducted in metropolitan atlanta, georgia, usa, during march 2003february 2004, among children with asthma who were > 2 years of age (10). case - patients were defined as patients with asthma exacerbation ; controls were defined as patients with stable asthma. the definitions, epidemiologic and laboratory methods, and clinical description of patients are available from table 1 and the previously published report (10). pcr (rt - pcr) targeting the 5-noncoding region (ncr) (10). for further genetic characterization, hrv - positive samples were extracted from a previously unopened aliquot and amplified by using a nested rt - pcr that targeted the virus capsid protein 1 (vp1) gene at positions 24322781, based on hrv 1b (genbank accession no. d00239) for species a and positions 25312799, based on hrv 14 (genbank accession no. nc_001490) for species b. we used sequencher 3.1.1 software (gene codes, ann arbor, mi, usa) for sequence assembly and editing. nucleotide and predicted amino acid sequences were aligned with previously published hrv vp1 sequences (genbank accession nos. ay355180ay3552831, ef186077, ef077279, ef077280, ef582385ef582387) by using clustalw as implemented in bioedit (version 7.0.5) (www.mbio.ncsu.edu/bioedit/bioedit.html). phylogenetic trees were constructed by using the neighbor - joining algorithm implemented in paup version 4.0.d10 (11). partial vp1 sequences for the novel hrv strains were submitted to genbank (accession nos. as reported previously (10), hrvs were detected by a 5-noncoding region seminested rt - pcr in 53 (37%) of 142 children with asthma, including 39 (60%) of 65 case - patients and 14 (18%) of 77 controls. of these, the hrvs from 29 (55%) (24 [62% ] of the 39 hrv - positive case - patients and 5 [36% ] of the 14 hrv - positive controls) were subsequently genotyped. vp1 sequences from the remaining 24 hrv - positive specimens could not be obtained because of low amplicon yield (table 2). specimens from patients with symptoms of acute viral respiratory infection (table 1) were more likely than those from patients without viral symptoms to yield sufficient vp1 amplicon for genotyping (percent genotyped 85% and 36%, respectively ; odds ratio [or ] 9.1 ; 95% confidence interval [ci ] 2.150.0 ; p 90% vp1 amino acid sequence identity with respective prototype strains. genogroup c hrvs form a clade phylogenetically distict from species a and b hrvs. of the 29 hrvs successfully genotyped, species a accounted for 18 (62%) strains, species b accounted for 3 (10%), whereas 8 (28%) strains formed a phylogenetically distinct clade, which we provisionally named genogroup c (table 2, figure). of the 18 hrv - a strains, 17 showed close genetic relatedness (80.7%93.8% nucleotide and 89.6%98.8% predicted amino acid sequence identity) to hrv prototype strains. one hrv - a strain (ga23584) was highly divergent from the closest prototype, hrv80 (73.2% nucleotide and 73.0% amino acid sequence identity), which suggests that it could represent a distinct previously undescribed hrv. the 3 hrv - b strains were closely related to prototype strains (84.0%88.6% nucleotide and 89.7%93.4% predicted amino acid sequence identity). phylogenetic tree of partial virus capsid protein 1 (vp1) amino acid sequences of human rhinoviruses (hrvs) identified in 29 hrv - positive pediatric asthma patients, march 2003february 2004, atlanta, georgia, usa (designated), previously published sequences of strains qpm (genbank accession no. the partial vp1 sequences of genogroup c strains were phylogenetically distinct from hrv species a and b and showed a substantial intragroup diversity (figure). vp1 sequence identity of these viruses with the closest match within the same genogroup ranged from 68.4% to 74.6% for nucleotide and from 68.5% to 85.5% for amino acid sequences. these novel viruses were related to other recently described hrvs : hrv qpm detected in specimens from australia (4), c024c026 detected in specimens from hong kong (6), and nat001 and nat045 detected in specimens from california (8) (figure). their identity scores compared with hrv qpm were 66.0%82.7% for nucleotide and 65.2%86.9% for amino acid sequences. one of the strains (ga23592) was almost identical in partial vp1 sequence to c026 (figure). the degree of genetic diversity among the genogroup c viruses far exceeded that between hrvs defined as distinct serotypes by classical serologic methods, which suggests that at least 7 of 8 of these viruses are antigenically distinct from each other rather than minor variants of the same serotype. the genogroup c hrv identity scores were substantially lower when compared with their closest matches from species a and b : 48.2%51.1% for nucleotide and 38.5%49.8% for amino acid sequences, and 35.9%42.8% for nucleotide and 29.3%35.8% for amino acid sequences, respectively. in our study, the association of asthma exacerbations with hrv infection appeared to be largely driven by the novel genogroup c, which was found exclusively in case - patients, and species a. the association was statistically significant for species a (detected in 15 [23% ] of 65 case - patients vs. 3 [4% ] of 77 controls ; or 7.4 ; 95% ci 1.943.1 ; p0.05) or for hrvs that could not be genotyped (15 [23% ] cases vs. 9 [12% ] controls ; p>0.05). the distribution of hrvs between case - patients and controls still differed when the analysis was limited to the hrv - positive group (p = 0.05) or to genotyped hrvs only (p<0.05). the results of the only other study that reported novel hrvs in asthma patients (2 of which, nat001 and nat045, were related to genogroup c viruses in our study) are difficult to interpret because that study of adults with cold symptoms showed an unexpected lack of association of hrvs with asthma exacerbation (8). patients infected with genogroup c hrvs had lower forced expiratory volumes during the first second (fev1) than did those infected with other hrvs (median 58.5% vs. 93% ; p = 0.01), but the distribution of demographic and other clinical variables did not differ significantly between the 2 groups. lower fev1 with genogroup c infection than with other hrvs suggests a potentially greater severity of asthma exacerbation in patients infected with these hrvs. when one considers the great variation among hrv serotypes in levels of sensitivity to candidate antiviral compounds (12,13), genogroup - related differences in associated disease patterns have implications for clinical management of hrv infections in asthma patients and for development of antiviral drugs against hrvs. preliminary data suggest that hrv - qpm and related hrv - c strains from hong kong share certain vp1 sequence characteristics with hrvs that are resistant to a candidate antipicornavirus drug, pleconaril (4,6,13). these data raise the possibility that these novel hrvs might also be resistant to this compound. the hrv - positive specimens from which vp1 gene sequences could not be obtained derived predominantly from patients without symptoms of acute respiratory viral illness. the absence of symptoms in hrv - infected persons likely reflects subclinical, asymptomatic infection, which is common for hrvs (14), or hrv persistence after a recently resolved infection (15), both of which are likely associated with lower viral loads (as opposed to acute symptomatic infections), thus leading to lower detection rates in a vp1 assay that uses highly degenerate primers. in conclusion, we found a striking genetic diversity of hrvs among children with asthma and confirmed the existence and wide geographic distribution (usa, australia, hong kong) of hrvs distinct from both previously recognized hrv species, a and b. our finding supports the role of the novel hrvs as human pathogens. additional studies are needed to further explore clinical and public health implications of these findings.	to determine links between human rhinoviruses (hrv) and asthma, we used data from a case control study, march 2003february 2004, among children with asthma. molecular characterization identified several likely new hrvs and showed that association with asthma exacerbations was largely driven by hrv - a and a phylogenetically distinct clade of 8 strains, genogroup c.
parkinson 's disease (pd) is one of the most common neurodegenerative diseases with a prevalence of 100200/100000 people worldwide [13 ]. the clinical manifestation is characterised by specific motor deficits : bradykinesia, tremor, rigidity, and postural instability. while the uk brain bank criteria require the presence of bradykinesia combined with at least one of the other symptoms [4, 5 ] for diagnosis of parkinson 's disease, others do not consider that bradykinesia has to be always present and make the diagnosis of parkinson 's disease when two of these symptoms are present [6, 7 ]. the pathological definition of pd includes the loss of dopaminergic cells in the substantia nigra pars compacta with the subsequent lack of the neurotransmitter dopamine [812 ] and the presence of lewy bodies. medical and surgical treatments provide symptomatic relief, but even with optimal therapy there is no cure for the disease and symptoms progress further. pd patients tend to adapt a sedentary lifestyle very early in the course of the disease [15, 16 ] and have lower levels of strength [17, 18 ] and functional ability. this is caused by a combination of physical impairments, such as walking and balance problems, cognitive dysfunction with focus on executive dysfunction, depression, fatigue, and pain. a review on the effectiveness of exercise in pd has shown that exercise is effective at improving physical functioning, health - related quality of life (hrql), leg strength, walking and balance [19, 20 ]. however, there have not been sufficient data regarding dose, type and components of exercise programmes, and stage specific effects. despite the interest in this area, only a few randomized clinical trials comparing different approaches of sports therapy are published so far. we selected from our data base 500 pd patients who participated in sports. to choose appropriate sports activities for the study, we asked this group to complete a questionnaire. according to the patients replies, they liked the social aspects of sports activities and wanted to improve their health, fitness and mobility. nordic walking is a physical activity consisting of walking with poles similar to ski poles. the poles are equipped with rubber or spike tips and the walking itself resembles nordic style skiing. patients are asked to walk in an upright position, not leaning forward or backward. the head should be up and looking forward and the poles are held close to the body. when the leading foot is moving forward, the opposite arm swings forward to waist height. the poles remain pointing diagonally backward and the pole is pushed as far back as possible. the arm straightens and the hand is opening off the grip by the end of the arm swing. compared to walking, there is a stronger involvement of the upper body in nordic walking which might burn more calories [21, 22 ]. furthermore, by using the poles, the muscles in the upper body can be activated, and the length of each step taken is supposedly increased, resulting in a faster gait [23, 24 ]. the aim of the present study was to evaluate the effects of two aerobic training programmes, nordic walking and walking and the effects of a combined flexibility and relaxation programme without aerobic components and the adherence to the allocated sports programmes after completion of the study period. the first hypothesis of the study was that all exercise programmes, irrespective of the mode of the training programme, improve (a) walking speed, (b) gait pattern, (c) balance, (d) symptoms of parkinson 's disease, and (e) cardiovascular parameters at maximal and submaximal intensity. the second hypothesis was that the effects of nordic walking are superior to those of the walking and the flexibility and relaxation programme. 90 pd patients, 45 men and 45 women (uk brain bank criteria), and a hoehn & yahr stage ii and iii were included in the study. exclusion criteria were severe concomitant diseases, which limit physical performances, and a second neurological disease. demographic data included age, body length, body weight (patients were weighed at the beginning and end of the training programme), body mass index (bmi), duration of disease, weekly sports activity, smoking habits, and concomitant diseases (hypertension, chronic obstructive pulmonary disease, thyroid disease, diabetes mellitus, hypercholesterinaemia, osteoarthritis) prior to the training patients underwent a medical examination including a cardiovascular exercise test. a medical history was taken, and patients were screened for coronary heart disease, hypertension, pulmonary function, cardiopulmonary diseases, diabetes and osteoarthritis. patients kept an activity log one week prior to the training programme and during the last week of the training period. patients did not participate in other sports programmes during the study but were allowed to continue physiotherapy and participated in family leisure activities. one training group performed nordic walking (nw) training, 3 times per week for 6 months. each session lasted 70 minutes and consisted of a warming up including some flexibility and strength exercises with and without the poles. one session per week was dedicated to practising nw technique, the other sessions focused on endurance training. patients were encouraged to increase the intensity of the training by walking faster or uphill and to increase the distance walked. training sessions took place in a park and a forest near to the university hospital. in addition, nw instructors used a checklist for assessment of the technical skills (i.e., diagonal sequence, opening of the hands, and planting of the poles). the training session also lasted 70 min and consisted of a warming up, technique training, endurance training and a cooling down. the flexibility and relaxation programme did not include aerobic exercises, while aerobic fitness and endurance were additionally addressed by walking and nw training. three physiotherapists who were trained as nw instructors conducted the training sessions of the three groups. the borg scale was applied as a measure for exertion to estimate the intensity of the training. since training should also help patients to adapt a more active life style, partners were offered six training sessions of the training programme, which was allocated to the patient. primary outcome measures were maximal walking speed on a treadmill ergometer, a 12-m and 24-m walking test, stride length and gait variability, assessed at different walking speeds, specific pd disability on the updrs (unified parkinson 's disease rating scale) and hrql (parkinson 's disease questionnaire 39 ; pdq39). outcome was assessed in all patients at baseline and after completion of the training period. secondary outcome measures were physical activity in everyday life and adverse effects of the training. adverse effects included cardiovascular side effects such as exercise - induced hypotension and injuries, caused by fall or overuse. outcome was assessed by two movement disorder specialists (ir, sm), who were blinded to the treatment arm. the unified parkinson 's disease rating scale (updrs) is the most frequently used outcome measure in clinical trials in parkinson 's disease. the updr scale has four subscales : part 1, which has 4 questions on mentation, behaviour, and mood (range 016 points), part 2, which has 13 questions on activities of daily living (adl) (range 052 points), part 3, which has 14 questions on motor functions (range 0108 points), and part 4, which has 11 questions on motor and other complications of advanced disease (023 points). the updrs - sum score ranges from 0 to 199 points, with a higher score indicating greater problems. posture, postural stability, alternating movements and leg agility were assessed by the single items of the updrs motor scale (score of each item ranging between 0 and 4 points). for assessment of health - related quality of life, patients filled in the parkinson 's disease - specific questionnaire (pdq 39), which consists of 8 subscales : subscale 1 mobility (max 40 points) ; subscale 2 activities of daily living (max 24 points), subscale 3 emotional well - being (max 24 points), subscale 4 stigma (max 16 points), subscale 5 social support (max 12 points), subscale 6 cognition (max 16 points), subscale 7 communication (max 12 points), and subscale 8 bodily discomfort (max 12 points). the sum score of raw data ranges from 0 to 156 points, with high scores indicating lower health - related quality of life. for better comparison of the results, raw data were transformed and expressed in percentages of maximal possible sum score. for exclusion of significant depression and anxiety the hospital anxiety and depression scale was applied. the scale consists of two subscales, an anxiety scale and a depression scale ranging from 0 to 21 points, respectively. questions related to anxiety are marked with a and to depression with d. depression and anxiety are scored separately. on each scale 0 to 7 points indicate a normal, 8 to 10 points a borderline abnormal and 11 or more points an abnormal result. the test assesses orientation, registration, attention, calculation, recall, language, writing, and copying. pain was rated by using a visual analogue scale for several regions of the body (neck, arms, hands, back, iliosacral joint, hip, knees, feet, and toes). pain was recorded to evaluate potential adverse events of the training, health risks, injuries and overuse injuries during the training period. the 14 items have a choice of 5 answers, numerically scored from 0 to 4. maximal sum score adds up to 56 points with a higher score indicating good balance. patients were asked to walk as fast as possible a distance of 12 m, and after a 5-minute break a distance of 24 m with a turn after 12 m. the time was taken for the 12 m distance and the 24 m distance. stride time (time from initial contact of one foot to subsequent contact of the same foot), percentage of double stance time ([time of bilateral foot contact / stride time ] 100), stride length (distance from initial contact of one foot to subsequent contact of the same foot) and the coefficient of stride length ([standard variation / mean ] 100) [3638 ] were assessed at different walking speeds on a motorized medical treadmill ergometer. furthermore, increased stride - to - stride variability might reflect a failure of automatic stepping mechanisms. increased gait variability can be seen throughout the course of parkinson 's disease and has been found to be one aspect of walking, closely associated with risk of falls in the elderly [4042 ]. for better comparison of the training effects patients walked at 6 different walking speeds (1.5 km / h, 1.8 km / h, 2.1 km / h, 2.4 km / h, 2.7 km / h, and 3 the treadmill was equipped with force platforms, that allowed an accurate determination of foot - ground contact. patients performed a maximal exercise test on a medical motorized treadmill ergometer (woodway pps med) and were instructed to walk on the treadmill. switching to jogging was prohibited, since walking and jogging put different demands on the cardiovascular and muscular system. every 2-minute the speed was increased by 0.5 km / h. at the end of each 2-minute stage blood pressure (bp) and heart rate (hr) were taken. however, fatigue, pain, and muscle weakness might hinder patients from successfully completing the test. therefore, we assessed as well performance at submaximal exercise intensity. blood pressure and heart rate changes between resting conditions and maximal walking speed were recorded at baseline and final assessment. maximal exertion was defined as reaching a heart rate of 220age or a perceived exertion of 17 on the borg scale ; this means walking with very hard effort. for assessment of exercise capacity at submaximal level after completion of the training programme, two approaches were chosen : the bp and hr responses to increasing work load between a walking speed of 3 km / h and 5 km / h were assessed, bp and hr changes between resting conditions and the walking speed which corresponded to the maximal walking speed at the first assessment were recorded and compared to the bp and hr change between resting conditions and maximal walking speed at the first assessment. patients were secured with a safety harness, suspended from the ceiling, without weight support. before testing patients were given 5 min to familiarise themselves with the treadmill locomotion. to assess the attractiveness of the exercise programme for the patients, all patients were contacted by phone six months after completion of the training period and a short telephone interview was conducted. do you continue the instructed training regime practiced during the course of the study ? if not, did you stop exercising or did you switch to another sports activity ? statistical analysis was conducted using ibm spss statistics 18.0 (ibm, somers, usa) statistical software. the power analysis regarding the updrs was based on an improvement of the updrs motor scale by 5 points, which is clinically relevant according to the findings of schrag.. demographic data on ordinal level were analysed by using a nonparametric test (kruskall - wallis). the kruskall - wallis test was also applied for the analysis of improvement in posture, postural stability, alternating movements, freezing, gait pattern, and leg agility. results of the updrs, gait parameters and cardiovascular data were assessed by using the repeated measure analysis. the repeated measure analysis provides information about between and within subjects effects. within subject effects linear trends were extracted by orthogonal polynomials and analysed for days and for trials (gait parameter). the interaction between groups and the linear trend of days yielded information about difference in the rate of improvement between groups. the study was approved by the ethical committee of the justus - liebig university giessen, and all patients gave written informed consent. there were 4 smokers in the flexibility and relaxation group and 5 smokers in the walking and nordic walking group, respectively. baseline assessments did not reveal any statistical differences in demographic data, health condition, and fitness between the groups. there were no significant differences in severity of the disease 14 patients of the nw group, 15 of the walking group, and 16 of the flexibility and relaxation group were in hoehn & yahr stage ii. medication had to be adapted in 2 patients of the walking and the flexibility and relaxation group respectively and in 1 patient of the nordic walking group. three patients reported wearingoff, 1 patient dyskinesias and 1 patient early morning dystonia. since change of medication had been minor (increase of levodopa dosage by 50 mg in 3 patients, reduction of levodopa dose by 100 mg in one subject, and addition of entacapone in one case), the patients continued the study. three patients scored in a range of mild depression at baseline on the had, two patients at the final assessment. figure 2 shows the overall rating of perceived exertion during the training sessions ; participants of the flexibility and relaxation group rated the intensity of the training significantly lower (f = 22.88 ; p 25. despite the mild increase of daily activity, six months after completion of the study, patients were asked whether they were still exercising. 60% of the participants of the walking group continued walking 2 - 3 times per week, 10% gave up exercising and 30% started practising nw. it was difficult to find a gym, and patients were more dependent on a physiotherapist to run the sessions. in summary, the main results of the study were that all training programmes provided some benefit for the patients. pain of the neck, hips, and iliosacral joint, health - related quality of life, and balance improved in all groups. in contrast maximal walking speed, stride length, double stance, exercise capacity at submaximal level, subscale 6 (cognition) of the pdq 39 and the updrs sum score and updrs - motor score improved only in the walking and nw group significantly. patients, who completed a walking or nordic walking training, had a better posture and postural stability, showed less freezing, and were faster in alternating movements. nw was superior to walking and the flexibility and relaxation programme in improving postural instability, gait pattern, stride length and stride length variability. the exercise programmes were not associated with an increased risk of falls or injuries. based on the results of the study, the first hypothesis that all training programmes improve walking speed, gait parameters, balance, cardiovascular parameters, parkinson specific symptoms, pain, and hrql can be partly corroborated only. the flexibility and relaxation programme did not improve significantly maximal walking speed on the treadmill, cardiovascular capacity, stride length and parkinson 's specific disability. neurological symptoms served rarely as outcome measures for exercise programmes, and only a few studies found significant benefit in direction of exercise intervention. in the present study, we found a clinical relevant improvement on the updrs score. analysis of the results shows that the improvement can be referred to the improvement of the motor scale, especially freezing, posture, alternating movements, walking pattern, and postural instability. have shown that a reduction of the updrs motor scale by 5 points is clinically relevant. the nordic walking training resulted in a mean improvement of 6.4 4.1 points of the updrs motor scale, while the walking group failed to reach a 5-point improvement despite the significant reduction of motor disability compared to baseline. 83.3% of the nw group, 63.3% of the walking group, and 33% of patients of the flexibility and relaxation group improved by more than 5 points on the updrs motor score as stated above. neurological signs such as rigidity and tremor were not improved by exercise treatment, but it is to note that walking and nordic walking had positive effects on some key symptoms of pd such as posture, alternating movements, freezing, and postural stability. however, the updrs might not be sensitive enough to detect the differences generated by the exercise training. schenkman and butler proposed that a restricted range of motion of axial structures might contribute to loss of postural control, gait impairment and decline in overall function. exercises, designed to improve axial range of motion, were shown to improve functional reach distance and timed gait tasks. this might explain that improvement of balance was observed in all groups even in the flexibility and relaxation group. posture and postural instability were most improved by nw. supported by the poles patients walked cross - country during the training, and it has been shown that a training, which challenges the balance control, can improve the postural stability and gait control. the cross country training has possibly improved balance so far that subjects of the nw group are also more stable without poles. furthermore, the poles represented external triggers, and patients remembered to use the more affected arm to handle the pole. the increased involvement of the more affected hand in motion sequences might have led to an improvement of the agility of the more affected hand. in contrast to the findings of schenkman., the flexibility and relaxation programme did not improve the walking speed in the webster walking test in the present study. therefore, walking and nw training resulted in more specific adaptations with regard to the outcome measures. the importance of the specificity of training is shown in the results of the 12-m and 24-m walking tests and the exercise test on the treadmill. walking and nordic walking improved walking speed and gait parameters most. thus, the specific walking training was superior to the flexibility and relaxation programme regarding walking speed, stride length and gait variability. in agreement with other studies [5, 49, 50 ], the majority of patients reported a better health - related quality of life at the end of the study independent of the training programme. since patients enjoyed the social aspects of the training, emotional aspects might contribute to this improvement. interestingly, the patient group with the greatest perceived exertion improved most on the subscale 6 (cognition) of the pdq39 and reported better concentration and memory. there is a large body of studies reporting beneficial effects of endurance exercises on cognitive functions especially executive functions. however, we did not perform a formal cognitive test battery after completion of the 6-month training period. at baseline assessment, all patients scored in a normal range on the mmse although this test does not assess executive functions in particular. the second hypothesis that nordic walking is generally superior to the flexibility and relaxation programme and walking could not be confirmed. in most of the tests, nordic walking was only superior to the flexibility and relaxation programme but not superior to the walking group, especially in the exercise test. earlier studies purported an enhancement of cardiovascular metabolism and an increase in walking speed by nw. however, recent studies have shown that the physiological effects of nw depend largely on the technical skills of the individuals. without being competent in using the participants of the nw group had difficulties to employ the correct technique in the beginning of the training period, and it took a long time until they involved the upper body. although, at the end of the training period nearly all patients showed at least good technical skills, at the assessment after 3-month training, only 50% of the patients showed good technical skills. probably due to these technical difficulties, most of the patients of the nw group did not walk significantly faster with poles for most of the training period. therefore, the metabolic and cardiovascular demands on the body might not have been much higher than in the walking group. consequently, the effects of walking and nw walking might have been quite similar. there is growing evidence that the capacity to learn remains in early stages of pd [52, 53 ]. in a previous study (unpublished data so far), we had shown that pd patients learnt a throwing task comparably to healthy age - matched subjects, but their performance level dropped significantly more than that of healthy subjects between the training sessions. a similar learning pattern was observed in the present study, patients who managed the push - off phase at the end of a training session had again difficulties in the next training session. therefore, the 6-month study period might have been still too short to detect different effects of walking and nw training. cardio - respiratory capacity increases by adaptations of muscle fibres, better muscle recruitment, and improvement of cardio - respiratory parameters. pd patients have deficits in muscle recruitment and development of dynamic muscle strength. during the first weeks of training, the body learns to recruit the correct muscles in a proper sequence while inhibiting unnecessary muscle recruitment. since walking was not practised in the flexibility and relaxation group, gait parameters and walking speed did not improve significantly. in addition, fitness training involves systematic progressions that apply the overload principle to purposeful short- and long - term goals. the overload principle states that, for adaptations to occur, a threshold level must be exceeded. the demands of the walking and nw training on the body were greater than the demands of the flexibility and relaxation programme. accordingly patients of the walking and nw group perceived a higher degree of exertion during the training than the flexibility and relaxation group. the ability of the patients of the walking and nw group to realise a higher walking speed with lower blood pressure levels and a lower heart rate showed the successful adaptation to exercise. although studies on cardiovascular parameters are inconsistent, there is some evidence that pd patients can improve oxygen consumption by aerobic conditioning programmes [46, 54 ]. since oxygen consumption was not measured in the present study, we can not claim that cardiovascular fitness of the patients had improved. at least lower increases of blood pressure and heart rate during the exercise test in patients having completed a walking or nordic walking training programme can likely be attributed to a better efficiency of the cardiovascular system. however, the capacity of the patients to respond to demands on the cardiovascular system with a lower blood pressure increase after completion of the training has to be regarded as a positive effect of the training. it is always desired to have a carry - over effect of the exercise training into daily life. however, the carry - over effect in pd is often small. as long as the patients are cognitively intact and do not have marked postural instability, they are immediately able to walk faster and with long steps. during the training sessions, patients are focused on walking and their attention is directed to the desired movement patterns. patients might compensate for the neurotransmitter imbalance through bypassing the defective basal ganglia and instead using the frontal cortex to regulate movement size and timing [55, 56 ]. therefore, patients are often not able to keep the gait pattern, when they are distracted. we did not assess whether there was an improvement of gait pattern in every day life. however, the walking and nordic walking training had an impact on the activities of daily life. patients became more active and spent less time sitting during the day and instead more time doing heavy work during the week. pain is one of the most common nonmotor symptoms in pd and leads to reduction of physical activity, since patients fear an increase of pain by movements. pain in pd is thought to have several origins ; either neuropathic pain that is central in nature, increasing during off - phases, or musculoskeletal pain. this was confirmed in the present study. about 36% of the subjects participating in the study suffered from osteoarthritis. osteoarthritis is often associated with pain and leads to a decreased range of joint motion and to a reduction of physical activity which is especially detrimental to pd patients. the neurodegenerative disease already induces rigidity, a reduction of joint movement leads to slow movements. pain can start a vicious circle and leads to a significant loss of muscle strength and mobility. there is now sufficient evidence that muscle weakness, immobility, and neurodegenerative diseases are risk factors for osteoarthritis [57, 58 ]. furthermore, exercises, especially weight - bearing exercises, decrease pain scores, increase muscle strength, and might improve position sense. therefore, the impact of walking and nw on back and leg pain might have been stronger. there was also a tendency that hand pain was most improved by nw, which might be due to the required rhythmic hand opening and closing. nordic walking was only in some aspects superior to walking but improved postural stability, stride length, stride length variability, and percentage of double stance phase more than the other training programmes. these gait parameters seem to be important for a steady gait, and have major effects on mobility. reduced stride length variability and a shorter double stance phase are associated with a lower risk of falling [3941 ], while a reduction of stride length is associated with fear of falling. although we did not assess the most comfortable walking speed of the patients, the patients with lower gait variability felt more comfortable during walking compared to those with higher gait variability. nw was most attractive for the patients, and all patients continued the training, which might be a prerequisite to maintain an active life style. supportive was that 70% of the spouses took up nw and accompanied the patients after completion of the study. since social aspects were very important for the pd patients, the participation of the spouses in nw might support further physical activity. physiotherapists should be encouraged to teach pd patients nw and to provide practise sessions with high intensity. presumably the combination between motor learning, some flexibility exercises, and endurance training produces the best training effects. three training lessons per week are necessary to learn the right technique and to improve cardiovascular fitness. a lower frequency of trainings sessions and lower intensity of the training in a previous study had been less effective. exercise training should already be offered to patients in early stages of the disease to prevent a loss of muscle strength and a rapid decline of mobility. nordic walking might also be suitable in advanced stages of pd, since the support of the poles reduces the risk of falls. however, in advanced stages, fluctuations of motor symptoms and autonomic disturbances, such as exercise - induced hypotension, have to be considered. in addition, at least some of the training sessions should be supervised, since patients do not notice their technical deficits. the risk of injuries due to accidents during the training was low, but attention has to be drawn to a small risk of overuse injuries, especially of the shoulder, in nw. therefore, an accompanying physiotherapy treatment is recommended, training should be supervised, and training frequency and intensity has to be gradually increased. we included patients in mild - to - moderate stages of the disease and without a history of falls in the past. in another study (personal communication), we assessed the risk of falls in nw training in pd patients with frequent falls. patients had been able to perform the training and did not sustain severe injuries. patients participating in the current study did not suffer from severe concomitant diseases, which might have resulted in a positive selection of patients. many patients of this age have significant concomitant diseases and might be less suitable for an aerobic exercise programme. thus, the perception of the nw group, that they can better memorize and recall information, might be confounded by improved emotional well - being. due to the lack of technical facilities, the exercise test did not include the measurement of v02 consumption. better muscle coordination and recruitment, improvement of strength flexibility and increase of range of movements and balance reflect the peripheral mechanisms of adaptation to exercise. cardiovascular effects and reduction of risk factors might contribute as well. in recent years, there is growing evidence from animal and human studies, that physical exercise enhances brain plasticity [6265 ]. physical activity modifies the release of dopamine and dopamine turnover and enhances the release of bdnf (brain - derived neurotrophic factor). these mechanisms might play an important role in central effects of physical activity [66, 67 ]. thus, nw, when performed with higher intensity, might provoke a greater release of bdnf., the study has shown that all forms of exercises had some positive effects on symptoms and deficits caused by pd, but walking and nordic walking, which involve aerobic endurance training, were superior to the flexibility and relaxation programme. walking and nordic walking reduced parkinson - specific disability and pain, improved health - related quality of life, gait and led to a more active life style. however, pd patients needed more time to practise nw in order to obtain the necessary technical skills. therefore, it might take longer for training effects to appear, since improvement of motor function is a prerequisite to participate in training sessions with higher intensity. participation in sports activities including aerobic training sessions has positive effects on daily activity, gait and pd - specific disability. prospective randomised long - term studies are needed to decide whether exercise therapy might also slow down the progression of parkinson 's disease.	symptoms of parkinson 's disease (pd) progress despite optimized medical treatment. the present study investigated the effects of a flexibility and relaxation programme, walking, and nordic walking (nw) on walking speed, stride length, stride length variability, parkinson - specific disability (updrs), and health - related quality of life (pdq 39). 90 pd patients were randomly allocated to the 3 treatment groups. patients participated in a 6-month study with 3 exercise sessions per week, each lasting 70 min. assessment after completion of the training showed that pain was reduced in all groups, and balance and health - related quality of life were improved. furthermore, walking, and nordic walking improved stride length, gait variability, maximal walking speed, exercise capacity at submaximal level, and pd disease - specific disability on the updrs in addition. nordic walking was superior to the flexibility and relaxation programme and walking in improving postural stability, stride length, gait pattern and gait variability. no significant injuries occurred during the training. all patients of the nordic walking group continued nordic walking after completing the study.
type 2 diabetes and prediabetes have emerged as significant health issues in overweight / obese african - american and latino - american pediatric populations in the united states (us). data from the search for diabetes in youth study indicate that incidence rates for type 2 diabetes were three times higher in african - americans and latino - americans aged 1519 years compared to non - latino whites. the studies to treat or prevent pediatric type 2 diabetes also reported a larger proportion of african - american and latino - american children and adolescents with high fasting insulin levels compared to their non - latino white counterparts (29.3%, 44.3%, and 20.5%, resp.). this ethnic disparity in diabetes and prediabetes has been linked to more severe insulin resistance and pancreatic beta - cell dysfunction in these ethnic minority children and adolescents. while several behavioral mechanisms have been proposed to explain the increased diabetes risk in african - american and latino - american children and adolescents [4, 5 ], research investigating the role of sociocultural factors (i.e., cultural attitudes, beliefs, and behaviors) is limited. with the rapid increase in cultural diversity of the us, african - american and latino - american cultures are quickly becoming a part of mainstream american culture, evolving within the us, while simultaneously integrating aspects of different african and latin american cultures. these alternative cultures have aspects that are uniquely shaped by historical, social, political, and economic forces present in the us. consequently, african - american and latino - american children and adolescents who come of age in the us, a multicultural society, interact with people from different cultural backgrounds which can lead to an interchange of cultural attitudes, beliefs, and behaviors. unger. have argued that ethnic minority children and adolescents who interact with non - latino whites may chose to adopt one of four sociocultural orientation patterns : (a) integration combining aspects of their family 's culture with aspects of mainstream american culture ; (b) assimilation replacing their family 's culture with mainstream american culture ; (c) separation retaining their family 's culture while rejecting mainstream american culture ; or (d) marginalization becoming alienated from both cultures. this approach to conceptualizing sociocultural orientation is unique in that it emphasizes the psychological aspects of culture rather than assessing proxy indicators such as language use, nativity, and time in the us. the limited number of studies assessing the influence of sociocultural orientation on type 2 diabetes risk suggests that, for african - americans, integrating into mainstream american culture while retaining aspects of their own family 's culture is inversely associated with diabetes risk through health - related behaviors. with one notable exception, much research suggests integrating and/or assimilating into the mainstream american culture is positively associated with obesity [1214 ] and suboptimal dietary choices [5, 1517 ], whereas separation from mainstream american culture is positively associated with increased insulin resistance. although the influence of sociocultural factors on subsequent diabetes risk in african - americans and latino - americans is striking, these findings may be confounded by socioeconomic position. sociocultural attitudes, beliefs, and behaviors are heavily influenced by an individual 's socioeconomic environment. ethnic minority children and adolescents who reside in low socioeconomic households may be more likely to be segregated from non - latino whites both at school and in their neighborhood providing less exposure to a multicultural environment and limited sociocultural options. in contrast, ethnic minority children and adolescents living in middle - to - high socioeconomic households are more likely to live in racially mixed neighborhoods and/or attend predominantly white schools thereby increasing interaction with individuals from different cultural backgrounds and expanding sociocultural orientation options. despite the well - characterized relationships between sociocultural orientation, socioeconomic position, and type 2 diabetes risk, few researchers have attempted to disentangle these associations to better understand the increased diabetes risk reported in minority children and adolescents. therefore, the primary objective of this study was to examine the independent relationships between sociocultural orientation, household social position, and type 2 diabetes risk in overweight / obese african - american and latino - american children and adolescents. it was hypothesized that, for both minority groups, low household social position would be associated with increased diabetes risk defined as decreased insulin sensitivity (si), decreased acute insulin response (airg), and decreased disposition index (di) derived from a frequently sampled intravenous glucose tolerance test. independent of household social position, integration (combining aspects of one 's family 's culture with aspects of mainstream american culture) would be associated with lower diabetes risk in overweight / obese african - american children and adolescents, whereas this same sociocultural adaptive style would be associated with increased diabetes risk in latino - americans. all participants met the following inclusion criteria : age- and gender - specific bmi 85th percentile, african - american or latino - american ethnicity (self - report and based on all four grandparents being of the same ethnic group as the child in the study), and between the ages of 818 years. prior to any testing, informed written consent and assent were obtained from the participants and parents. participants arrived at the general clinical research center (gcrc) where a licensed pediatric health care provider conducted a medical / family history and physical examination which included an assessment of tanner stage [22, 23 ]. body composition was measured by air displacement plethysmography (bodpod ; life measurement instruments, concord, ca). total dietary intake was assessed with three - day dietary records given to participants to complete at home, which were later returned to research staff for nutritional analysis. three - day physical activity recall was used to assess self - reported physical activity. a frequently sampled insulin - modified intravenous glucose tolerance test (fsigt) was used to assess type 2 diabetes risk. fasting plasma insulin and glucose concentrations were used to estimate insulin resistance using homeostasis model assessment (homa - ir), which was calculated as homa - ir = [(fpi fpg)/22.5 ]. plasma collected during the fsigt was analyzed for glucose and insulin, and values were entered into the minmod millennium 2003 computer program (version 5.16, bergman, usc) to calculate si, airg, and di. si was defined as the net capacity for insulin to promote the disposal of glucose and to inhibit the endogenous production of glucose. airg was defined as the area under the plasma insulin curve between 0 and 10 minutes. di, an index of beta - cell function, was calculated as the product of airg and si. detailed descriptions of the fsigt methods and protocols used in this study have been previously published [2628 ]. during the gcrc visit, participants also completed a questionnaire regarding sociocultural orientation and parents of participants answered questions regarding parental educational attainment and occupational rank. sociocultural orientation was assessed using the acculturation, habits, and interests multicultural scale for adolescents (ahimsa) questionnaire. the eight items on the ahimsa as well as the four response items are listed in table 1. the ahimsa responses were divided into four sociocultural orientation subscales : the us was categorized as assimilation (cronbach alpha : 0.79) ; the country my family is from was categorized as separation (cronbach alpha : 0.68) ; both was categorized as integration (cronbach alpha : 0.79) ; and neither was categorized as marginalization (cronbach alpha : 0.50). for the african - american participants, the ahimsa responses for assimilation and separation were modified to read white - american culture and my family 's culture, respectively (cronbach alphas : 0.680.79). scores on each orientation scale ranged from 0 to 8 and are presented as percentages of a total possible out of 8. for example, 0 on the assimilation scale indicated that the respondent did not answer the us or white - american culture to any of the items which is the equivalent of 0%. an 8 indicated that the respondent answered the us or white - american culture to all eight items, which represents a score of 100%. household social position was measured using the hollingshead 's two - factor index of social position. the hollingshead scale was used because it is one of the most commonly used socioeconomic measures. the hollingshead score is a composite measure of educational attainment and occupational rank and was computed in the following manner. an education score (1 through 7, with 1 equal to less than a seventh - grade education and 7 equal to graduate training) and an occupation score (1 through 7, with 1 equal to unskilled employee and 7 equal to higher executives, proprietors of large businesses, and major professionals) were assigned for each parent / guardian based on information provided by them. education and occupation scores were then weighted to obtain a single score for each parent / guardian (range of 8 to 49) that reflects one of five social strata (1 through 5, with 1 being a reference to unskilled laborers or low social position and 5 a reference to major professionals or high social position). for families with multiple caretakers, scores for each individuals whose primary activities were homemaking, school, or who received state assistance did not have categorizable occupations according to the hollingshead method and were not included in the household social position score (n = 42). the hollingshead method has relatively good interrater agreement (6796%) and correlates well with other measures of social position (r = 0.730.86) [30, 31 ]. data analysis included data summarization, spearman correlations, and multivariate regression modeling. data were evaluated for normality before analysis and natural log transformations were made when necessary. of the 156 participants, 20 participants were missing data for dietary intake, moderate - to - vigorous physical activity, and/or sedentary time and were not included in the multivariate regression analyses. mean variable differences by ethnicity were analyzed by independent t - tests, chi - square, and ancova. spearman correlations were used to explore the associations between sociocultural and socioeconomic variables (i.e., ahimsa subscales, household social position) and fsigt parameters (i.e., si, airg, and di). multivariate regression models were used to further explore the relationships between the independent variables (i.e., ahimsa subscales, household social position) and dependent variables (i.e., si, airg, and di). specifically, partial correlations and parameter estimates were used to describe the relationship between sociocultural and socioeconomic variables and fsigt variables after controlling for a priori covariates. a priori covariates included sex, tanner stage, fat mass, fat - free mass, energy intake, moderate - to - vigorous physical activity, sedentary time, household social position (for sociocultural variables only), and si (for airg only). statistical analyses that addressed the objectives of this study were stratified by ethnicity for the reason that both qualitative and quantitative variables were examined in this study with african - americans using a modified version of the ahimsa questionnaire (i.e., response items were modified for assimilation and separation) and latino - americans using the standard version. a priori significance level was set at p < 0.05. all assumptions for multiple linear regression were satisfied and fsigt variables were log transformed in order to meet these assumptions. table 2 displays the participant characteristics for the 43 african - american and 113 latino - american boys and girls included in this study. for household social position, 60.4% of african - american households classified themselves in either the middle or upper - middle categories, compared to 17.7% for latino - american households. for behavioral factors, african - american children and adolescents participated in significantly fewer minutes of moderate - to - vigorous physical activity per week, compared to latino - americans (p < 0.05) ; however, dietary patterns were similar across ethnic groups. for biological factors, african - american adolescents were significantly taller and heavier than their latino - american counterparts, with higher bmis, volumes of fat mass and fat - free mass (all p 's < 0.05). after controlling for sex, tanner stage, fat mass, fat - free mass, and si (for airg only), fasting glucose and si were significantly lower in african - american children and adolescents compared to latino - americans (both p 's < 0.01). in addition, homa, airg, and di were significantly higher in african - americans compared to latino - americans (all p 's < 0.05). ethnic differences were not observed in age, tanner stage, bmi percentile, total dietary intake, and sedentary time. spearman correlations revealed that, for african - american children and adolescents, assimilation was positively associated with log si (= 0.33, p < 0.05) and marginalization was positively associated with log airg (= 0.39, p < 0.05). however, these relationships were no longer significant in our multivariate regression analyses suggesting confounding of biological and behavioral covariates included in our model. for latino - american children and adolescents, spearman correlations revealed a negative association between integration and di (= 0.20, p < 0.05) ; again this relationship was no longer significant after controlling for biological and behavioral covariates in our regression analyses. household social position was negatively associated with di (= 0.37, p < 0.05) in latino - americans ; this relationship remained significant in our regression analyses. table 3 displays the results of the multiple regression analysis for african - american children and adolescents. we observed a positive parameter estimate and partial correlation between the ahimsa subscale integration, log airg and log di. also, log airg was positively associated with integration (= 0.27 0.09, r = 0.48, p < 0.01) after controlling for sex, tanner stage, fat / fat - free mass, energy intake, moderate - to - vigorous physical activity, sedentary time, and si. additionally, log di was also positively associated with integration (= 0.28 0.09, r = 0.55, p < 0.01), after controlling for covariates. from these results and with all confounding effects of covariates being equal, predicted mean airg was 92% higher for african - american children and adolescents at the 75th compared to 25th percentile of the ahimsa integration subscale. predicted mean di was 93% higher for african - american children and adolescents at the 75th compared to 25th percentile of the ahimsa integration subscale. although the bivariate analysis did not reveal a significant correlation between integration, log airg, and log di, once other variables were accounted for, these relationships became significant, suggesting negative confounding by covariates in our model. there were no significant relationships between household social position, other ahimsa subscales (i.e., separation, assimilation, and marginalization), and fsigt parameters. table 4 displays the results of the multiple regression analysis for latino - american children and adolescents. we observed a negative parameter estimate and partial correlation between household social position, log airg, and di. moreover, log airg was inversely associated with household social position (= 0.010 0.004, r = 0.19, p = 0.02), after controlling for sex, tanner stage, fat / fat - free mass, energy intake, moderate - to - vigorous physical activity, sedentary time, and si. in addition, di was inversely associated with household social position (= 20.44 7.50, r = 0.27, p < 0.01), after controlling for biological and behavioral covariates. to better understand which of the two socioeconomic indicators measured was driving the inverse relationship between household social position and diabetes risk, we also calculated parameter estimates and partial correlations for educational attainment, occupational rank, log airg, and di. these analyses revealed that log airg and di were significantly associated with parental educational attainment (airg : = 0.09 0.36, r = 0.19, p = 0.01 ; di : = 189.56 61.72, r = 0.29, p < 0.01), whereas the associations between parental occupational rank, log airg, and di were nonsignificant (data not shown). from these results and with all covariates being equal, predicted mean airg was 129% lower for latino - american children and adolescents at the 75th compared to 25th percentile of parental education. the model for di contained an intercept of 3600 10 min and a parameter estimate of 189.6 10 min for every one - unit increase in parental education. from these results and with all covariates being equal, di was 31% lower for latino - american children and adolescents at the 75th compared to 25th percentile of parental education. pancreatic beta - cells have the ability to increase insulin secretion (via airg) in response to insulin resistance. this nonlinear hyperbolic relationship between sensitivity and secretion is best described as di [25, 32 ]. hence, higher airg and di typically represent an ability to compensate for insulin resistance in order to maintain normal glucose tolerance (i.e., lower diabetes risk). in contrast, lower airg and di represent an inability of the pancreas to secrete enough insulin at a given level of insulin resistance where impaired glucose tolerance may arise (i.e., higher diabetes risk). indeed, our laboratory has shown both increased airg and di as potential compensatory mechanisms for decreased si in minority children and adolescents [3, 27 ]. the underlying determinants that contribute to increased insulin resistance and pancreatic beta - cell dysfunction in overweight / obese african - american and latino - american children and adolescents are unknown ; however sociocultural and socioeconomic factors each play a unique role in shaping diabetes risk in ethnic minorities. in the present analysis, the sociocultural adaptive style of combining aspects of both mainstream white - american culture while retaining aspects of their own family 's culture was negatively associated with type 2 diabetes risk in overweight / obese african - american children and adolescents (as reflected by higher airg and di). these relationships remained significant after adjusting for household social position and other behavioral and biological covariates. in contrast, household social position was positively associated with type 2 diabetes risk in latino - american children and adolescents (via decreased airg and di). taken together, these findings suggest that sociocultural factors may be important predictors of type 2 diabetes risk in overweight / obese african - american children and adolescents whereas socioeconomic factors, rather than culture, may be more important for latino - americans. african - americans are a heterogeneous ethnic group who vary in the extent to which they both retain their black - american culture and also adopt aspects of white - american culture. previous research on adults has documented the relevance of these adaptive cultural styles to health and health - related behaviors in african - american adults [10, 34 ]. dressler. reported african - americans living in accordance with culturally constructed local community norms or cultural consonance in lifestyle were a stronger independent predictor of smoking and hypertension than were indicators of socioeconomic position (i.e., occupation, income and education). reported that positive identification with african - american culture and a self - perception of being successful in both the black and white ways of life were associated with healthy behaviors, including reduced fat consumption, more participation in leisure - time physical activity, reduced smoking, and, in women only, reduced alcohol consumption. our results are generally consistent with these findings and suggest that the protective health effects of integrating two cultures also extend to overweight / obese african - american children and adolescents at increased risk for type 2 diabetes. in essence, integrating aspects of both black - american and white - american cultures was associated with lower diabetes risk (via increased airg and di), independent of household social position, physical activity, sedentary time, dietary intake, sex, tanner stage, and fat / fat - free mass. an association between culture and type 2 diabetes risk, independent of physical activity and diet, is plausible, given what is known about the physiological mechanisms linking psychosocial stress to insulin resistance and subsequent type 2 diabetes risk via hypothalamic - pituitary - adrenal axis activation [35, 36 ]. in general, integration of two or more cultures is viewed as a less stressful, more adaptive process, because this orientation allows ethnic minorities to function effectively in a multicultural society while still maintaining supportive connections to their own family 's culture. hence, integration may be associated with lower psychological stress in african - americans, thereby influencing type 2 diabetes risk independent of physical activity and diet. additional research is needed to better understand the associations between integration, psychological stress, and diabetes risk in this ethnic minority group. many more researchers have investigated the influence of sociocultural factors on diabetes risk in latino - americans. the influence of culture on behavior and subsequent diabetes risk is inconsistent and may be confounded by socioeconomic position. in the present study, household social position, not sociocultural orientation, was positively associated with type 2 diabetes risk in latino - american children and adolescents. moreover, post hoc analyses revealed that, of the two socioeconomic indicators measured (educational attainment and occupational rank), parental education was driving the relationship between household social position and diabetes risk. a protective effect of socioeconomic position and educational attainment in particular on type 2 diabetes risk has been well established among adults and non - latino whites ; however, in the present study, this relationship was not present in either ethnic group. the rationale for the absent relationship in african - americans and paradoxical relationship in latino - americans is unclear. nevertheless similar findings have been previously reported between socioeconomic position and other metabolic outcomes in minority children and adolescents. using data from the national health and nutrition examination survey and national health interview survey, sobal and stunkard reported that ethnic minority children from higher socioeconomic households were just as likely to be overweight and obese as compared to children residing in lower socioeconomic households. these findings taken together with those in the present study suggest that residing in higher socioeconomic households may not be protective against obesity and subsequent type 2 diabetes risk in ethnic minority children and adolescents as has been previously reported in non - latino whites. moreover, parental education may be a stronger independent predictor of type 2 diabetes risk than culture in latino - americans ; additional research is warranted. first, data limitations precluded analysis of other factors known to influence diabetes risk in this analysis including genetic admixture, smoking status and alcohol consumption, social desirability, and self - reported psychological stress. similarly, proxy indicators of acculturation such as language use, nativity, and time in the us were not available for our participants. second, although prior research suggests that household social position and sociocultural orientation are predictors rather than consequences of diabetes risk [10, 21, 34 ], the cross - sectional nature of this study impeded our ability to make causal inferences. third, these findings in a small sample of overweight / obese african - american and latino - american children and adolescents living in the greater los angeles area can not necessarily be generalized to all adolescents living in the us. finally, post hoc power calculations revealed that some of our analyses were underpowered given the large variability in fsigt - derived insulin and glucose indices. despite being underpowered, we were able to detect significant associations between the ahimsa subscale integration, airg, and di in african - americans as well as significant associations between household social position, airg, and di in latino - americans. thus, our findings may be an underestimation of the true effect of sociocultural orientation and household social position on type 2 diabetes risk in overweight / obese african - american and latino - american children and adolescents. nevertheless, additional research examining these relationships in a larger, more homogenous sample may better elucidate the role of sociocultural and socioeconomic factors in shaping type 2 diabetes risk in overweight / obese ethnic minority pediatric populations. in summary, sociocultural orientation and household social position appear to play distinct and opposing roles in type 2 diabetes risk in overweight / obese african - american and latino - american children and adolescents. for african - americans, maintaining a sense of their own family 's culture while integrating into mainstream white - american society was independently associated with decreased diabetes risk (as represented by increased airg and di). for latino - americans, increased diabetes risk was independently associated with increased household social position, higher parental education in particular, via decreased airg and di. future research should continue to examine these factors over time to better understand the relationships between the sociocultural orientation, household social position, and type 2 diabetes risk in overweight / obese african - american and latino - american children and adolescents. moreover, behavioral interventions and public policies are needed to better address sociocultural and socioeconomic factors associated with type 2 diabetes risk in ethnic minority pediatric populations.	purpose. it is unclear whether sociocultural and socioeconomic factors are directly linked to type 2 diabetes risk in overweight / obese ethnic minority children and adolescents. this study examines the relationships between sociocultural orientation, household social position, and type 2 diabetes risk in overweight / obese african - american (n = 43) and latino - american (n = 113) children and adolescents. methods. sociocultural orientation was assessed using the acculturation, habits, and interests multicultural scale for adolescents (ahimsa) questionnaire. household social position was calculated using the hollingshead two - factor index of social position. insulin sensitivity (si), acute insulin response (airg) and disposition index (di) were derived from a frequently sampled intravenous glucose tolerance test (fsigt). the relationships between ahimsa subscales (i.e., integration, assimilation, separation, and marginalization), household social position and fsigt parameters were assessed using multiple linear regression. results. for african - americans, integration (integrating their family 's culture with those of mainstream white - american culture) was positively associated with airg (= 0.27 0.09, r = 0.48, p < 0.01) and di (= 0.28 0.09, r = 0.55, p < 0.01). for latino - americans, household social position was inversely associated with airg (= 0.010 0.004, r = 0.19, p = 0.02) and di (= 20.44 7.50, r = 0.27, p < 0.01). conclusions. sociocultural orientation and household social position play distinct and opposing roles in shaping type 2 diabetes risk in african - american and latino - american children and adolescents.
the facilitation or inhibition of gastrointestinal motility due to stress has been previously reported (1, 2). as gastrointestinal motility is partially reflected by egg activity (3, 4), the effects of stress on the epigastric, supraumbilical and infraumbilical egg data can be correlated with anxiety scores. our previous study demonstrated that the local differences in the power content during 16-location egg were more clearly shown at rest, during the postprandial state and during the mirror drawing test (mdt) (5). furthermore, we demonstrated the epigastric egg inhibition (seen in 3 cpm activity) and infraumbilical egg facilitation (seen in 6 cpm activity) during mdt stress in a numerical comparison of the power content ratio of the mdtr and topographic egg mapping of mdtr (power content during mdt / power content at rest) (6). therefore, in the present study, we compared the effects of mdt - related stress on each of the 16 locations of egg using the power content ratio of the mdtr to clarify the correlation between stress and egg, which partially reflects gastrointestinal motility (3, 4, 6). this project was conducted under the approval of the ethics committee of niigata university, faculty of medicine (project no. 179). informed consent was obtained from all of the subjects after explanation about the informed consent immediately prior to the egg recording. there were 58 subjects, (52 males and six females), with 23 of the subjects ranging in age from 2038 years (23.1 1.0, n=23), while the age of other 35 subjects was not known. the methods used for recording and analyzing eggs were the same as those used in the previous studies (7, 8). briefly, unipolar eggs were recorded from 16 locations (channels, ch) from the thoraco - abdominal skin surface (fig. location of the electrodes. superimposed images of the body and the location of the 16 electrodes on the thoraco - abdominal body surface based on the xiphoid process, costal arch, and iliac line. the numbers (1 - 16) by the filled circles indicate the roughly averaged location of the electrodes. the length of x0~xmax was assumed to be 32 cm, and that of y0~ymax 36 cm based on the superimposed body lines (5). 2012 ; 48(23) : 4757) (6).), using a reference electrode on the right leg. the amplifier was a modified electroencephalographic (eeg) amplifier, with time constant set at 5 sec, with a high cut at 0.5 hz, a low cut of 6 dboct and a high cut of 12 dboct, (biotop 6r 124, nec - sanei, japan). after cleaning the skin with ethanol, electrode cream was applied to the disc electrode for the eeg (diameter=11 mm). resting eggs were recorded for about 20 min, in subjects who had fasted for at least eight hours, and were sampled every 128 sec (1 file). after recording the resting control data, subjects were exposed to the stress of the mdt. the mdt involves tracing the cue figure of a metal star, reflected onto a mirror with an electric pen, which gives a click alarm when the tracing runs off the edge of the star (error). the mdt stress was applied for about 5 min to obtain 23 egg files. compiled running spectra were obtained after the files were analyzed using the maximal entropy method (mem). the spectral frequency readings were classified into five groups : the 1-cpm group (02.4 cpm), 3-cpm group (2.54.9 cpm), 6-cpm group (5.07.4 cpm), 8-cpm group (7.59.9 cpm) and 10-cpm group (10.012.9 cpm). ensemble means were obtained during rest, during the stress of the mdt, and after the mdt. with regard to the egg parameters, this study focused on the power content ratio of mdtr for each channel. the egg power content ratio of electrode locations1 cpm (02.4)3 cpm (2.54.9)6 cpm (5.07.4)8 cpm (7.59.9)10 cpm (10.012.9)ch.22.17.580.95.590.52.077130.86.0640.86.12ch.51.95.2610.90.05440.89.060140.96.0890.84.10ch.81.71.161.01.066121.08.10150.84.0730.83.096ch.101.76.181.11.07151.04.089160.85.0780.76.088ch.111.59.141.36.1461.03.091170.84.0910.64.079ch.121.64.171.22.09070.96.074180.82.0880.67.096ch.131.63.191.21.07481.03.084190.74.0760.66.081ch.141.48.131.30.1090.99.081200.84.121.28.57ch.151.32.1121.17.076101.04.073100.87.100.79.11ch.161.34.1131.19.092111.04.077220.81.0730.69.070the effect of the mdt on the power content ratio (mdtr, n=5258, means s.e.m.). 46, 47, 48, 49, 410, and 1314, p<0.01. 1315, 1316, 1317, 1318, 1319, 1320, 1321, and 1322, p<0.001. lists the figures for the epigastric 2, 5 and 8 channels and the infraumbilical 1216 channels found in our previous studies for simplicity (5, 6). the anxiety scores were estimated using the hads (hospital anxiety and depression scales) (9, 10). the electrode positions were represented by two - dimensional standard coordinates, xi and yi, and a spectral peak at a certain electrode position was expressed as zi = (xi, yi) (7, 8, 11, 12). superimposed images of the body and the location of the 16 electrodes on the thoraco - abdominal body surface based on the xiphoid process, costal arch, and iliac line. the numbers (1 - 16) by the filled circles indicate the roughly averaged location of the electrodes. the length of x0~xmax was assumed to be 32 cm, and that of y0~ymax 36 cm based on the superimposed body lines (5). 2012 ; 48(23) : 4757) (6). the effect of the mdt on the power content ratio (mdtr, n=5258, means s.e.m.). 46, 47, 48, 49, 410, and 1314, p<0.01. 1315, 1316, 1317, 1318, 1319, 1320, 1321, and 1322, p<0.001. the mean and standard errors (sem) were calculated, and the student 's t test was used to determine the level of statistical significance. the epigastric (ch.2, 5 and 8) and infraumbilical (ch.1216) power content ratio of the mdtr of five spectral frequencies in addition to umbilical channels 10 and 11 are shown in table 1. the power content ratio of mdtr of 3- and 6 cpm in the epigastric channels was generally, significantly lower than that of the infraumbilical channels. the significant linear correlations (p<0.05) between the anxiety scores and power content ratio of mdtr are shown in table 2table 2. the linear correlation parametersprch.2 (3 cpm)1.610.0460.071ch.10 (6 cpm)1.310.0130.11ch.11 (6 cpm)1.110.0330.080ch.13 (6 cpm)1.180.0370.077ch.15 (6 cpm)1.710.0080.123ch.16 (6 cpm)1.280.0380.076tabulation of the the significant linear correlation parameters (p<0.05) between the anxiety scores and the power content ratio of the mdt to that at rest (mdtr). and fig. 2. a : the correlation between the anxiety scores and the egg power content ratio of 3 cpm. the linear correlation between the anxiety scores (y axis) and the power content ratio (mdtr) of 3 cpm of epigastric channel 2 (x - axis). slope () = 1.61, p = 0.046, and r = 0.071. b : the correlation between anxiety scores and the egg power content ratio of 6 cpm. the linear correlation between anxiety scores (y - axis) and the power content ratio (mdtr) of 6 cpm of infraumbilical channel 15 (x - axis). slope = 1.71, p = 0.008, and r = 0.123.. the slope of the ch.2 correlation (3 cpm) was negative (fig. 2a) and the slopes of ch.10, 11, 13, 15, and 16 (6 cpm) were positive (fig. tabulation of the the significant linear correlation parameters (p<0.05) between the anxiety scores and the power content ratio of the mdt to that at rest (mdtr). a : the correlation between the anxiety scores and the egg power content ratio of 3 cpm. the linear correlation between the anxiety scores (y axis) and the power content ratio (mdtr) of 3 cpm of epigastric channel 2 (x - axis). slope () = 1.61, p = 0.046, and r = 0.071. b : the correlation between anxiety scores and the egg power content ratio of 6 cpm. the linear correlation between anxiety scores (y - axis) and the power content ratio (mdtr) of 6 cpm of infraumbilical channel 15 (x - axis). it is well known that egg records gastrointestinal electrical activity or myoelectric activity, which reflects some of the motility as power content (3, 4). the power content ratio or the normalization of egg change, mdtr (power content during mdt / power content at rest) reflects the real change of the local egg for each electrode as demonstrated in topographic egg maps (6). it is also well known that stress influences the gastric and colonic activity measured with egg. in fact, stress induces dual excitatory and inhibitory effects that can be observed with egg. cold pressor test stress, interviews and performing arithmetic calculations increased the colonic egg (13). electric shock significantly decreased the percentage of the 3 cpm frequency and tachyarrhythmia (%) component of the egg, but forehead cooling increased the percentage of the 3 cpm frequency (14). the induction of similar gastric inhibition or facilitation by stress has been reported using manometry (14, 15, 16, 17). we have previously reported the effects of the acute stress of mdt on the gastric and colonic facilitation or inhibition with egg. however, mdt stress did not appear to exert effects on the intestinal egg activity (6, 8, 12). it is generally accepted that the normal gastric spectral activity of egg is 3 cpm (3, 18, 19). however, the gastric and colonic egg activity includes both 3- and 6- cpm egg activity according to gastrectomy and colectomy studies (7, 20, 21, 22, 23, 24). therefore, the infraumbilical 6 cpm egg activity in this study is considered to reflect the colonic myoelectric activity. both colonic facilitation and inhibition however, the finding of a significantly higher power ratio of the mdtr of the infraumbilical 3 cpm than that in the epigastric recording during mdt suggested that the mdt stress inhibited gastric egg and facilitated colonic egg. in addition, topographic egg maps drawn according to the power content ratio of the mdtr and the absolute power ratio of mdtr supported this idea (6). similar findings of colonic facilitation have been reported with manometry (13, 25, 26, 27, 28, 29). mdt - related stress significantly increased bowel evacuation frequency, while it is known that depressed patients tend to be constipated (30)., a linear correlation with a negative slope was found between the anxiety scores and the mdtr of 3 cpm egg of channel 2 in this study (table 2, fig., the correlation between the anxiety scores and the 3 cpm mdtr was not calculated in channel 2 (ch2) alone, and the mean of mdtr for channels 3, 4, 5, and 6 channels was defined as the epigastric ch1 (8, 12). a significant linear correlation was not found between the anxiety scores and the 3 cpm of ch1 mdtr (12). a significant linear correlation with a positive slope was also found in the mdtr of 6 cpm of umbilical channels 10 and 11 (table 2). similarly, the infraumbilical ch3 was calculated using the mean of channels 12, 13, and 14 (12). however, a linear correlation with a positive slope was found between the anxiety scores and infraumbilical 6 cpm of ch3 mdtr 6 cpm (12) and channel 13 (one of the ch3 substituents) in this study in addition to channel 15 (fig. 2b) and channel 16 (table 2). a significant linear correlation with a positive slope was also found in the mdtr of 6 cpm of umbilical channels 10 and 11 (table 2). the locations of channels 10 and 11 may correspond to the right and left flexure of the colon. it is has been suggested that various stressors depress stomach contractility and emptying, and facilitate colonic motility, transit and defecation through the limbic, hypothalamic and autonomic nervous system via rf - r2 (corticotrophin - releasing factor receptor and crf - r1, respectively (32, 33, 34, 35). our egg findings in human subjects provide further supports for these studies (6). finally, the present results further support the idea that mdt stress inhibits stomach motility and facilitates colonic motility. the author does not have any financial relationship with the organization that sponsored the research.	electrogastrograms (eggs) were recorded at 16 locations on the thoraco - abdominal surface at rest and then both during and after the acute stress of performing the mirror drawing test (mdt). a significant linear correlation with a negative slope was found between the anxiety scores and the ratio of the power content during mdt to the power content at rest (r) (mdtr1) of the 3 cpm component from the epigastric channel 2 recording. in contrast, significant linear correlations with positive slopes were found between the anxiety scores and mdtr1 of the 6 cpm component of the recordings from the infraumbilical channels (channels 13, 15, and 16). the epigastric 3-cpm egg activity reflects gastric myoelectric activity, while the infraumbilical 3- and 6-cpm activity reflects that of the colon. therefore, these results seem to further support the previous report of the inhibition of gastric egg by stress and the stress - mediated facilitation of colonic egg (homma s, j smooth muscle res. 2012 ; 48(23) : 4757).
systemic lupus erythematosus (sle) is an autoimmune disease with wide - ranging clinical manifestations and the potential to affect multiple organ systems in the body. healthy immune responses help prevent or overcome harmful antigens and infections but immune dysfunction with loss of self - tolerance can lead to autoimmunity with self - antigens becoming the target of immune attack. patients with sle develop t and b cell - mediated autoimmune responses manifested by the production of autoantibodies. the actual mechanism of autoimmunity, and factors that contribute the progression to clinical autoimmune disease, vitamin d deficiency has been implicated as one of the environmental factors contributing to the prevalence of several autoimmune diseases, including sle. vitamin d is an essential steroid hormone with well - established effects on mineral metabolism, skeletal health, and more recently described effects on cardiovascular and immune health. it has become recently apparent that vitamin d deficiency contributes to the morbidity and mortality of multiple chronic diseases. lifestyle factors have led to an increased prevalence of vitamin d deficiency in the general population, while improved availability and reliability of the serum 25-hydroxyvitamin d [25(oh)d ] test have led to better awareness of the widespread deficiency. because patients with sle are advised to avoid direct sunlight, a common trigger of disease flares but also the primary source of vitamin d3, the risk of vitamin d deficiency is even higher among sle patients than in the general population. without oral supplementation, the primary source of vitamin d3 (cholecalciferol) is the skin upon exposure to ultraviolet - b radiation (uvb). vitamin d2 (ergocalciferol) from dietary sources is typically a minor contributor to overall vitamin d status. interestingly, solar radiation, particularly uvb (280315 nm), is a risk factor for sle and sle - related mortality. one study found over 90% of patients with sle exposed to uv radiation had an abnormal reaction. the importance of vitamin d in immune regulation has gained increased interest over the past decade with the discovery of the vitamin d receptor (vdr) being expressed by cells of the immune system and manipulation of 1,25-dihydroxyvitamin d [1,25(oh)2d ] having downstream immune effects. the effects of 1,25(oh)2d are mediated by complex nuclear receptor and the binding of the activated complex to regulatory dna sequences of target genes. the vdr gene is a direct target of its own receptor, thus facilitating an upregulation of the vdr protein in certain target tissues. several hundred vitamin d - regulated genes have been identified, including multiple genes involved with the innate and adaptive immune system. the overall immunologic effects of 1,25(oh)2d include downregulating th1 immune responses, modulating the differentiation of dendritic cells (dcs), and lowering proliferation of activated b cells, while upregulating regulatory t cells and preserving innate immune responses. each of the immune pathways influenced by 1,25(oh)2d has profound potential implications for patients with sle. in general, 1,25(oh)2d promotes the development of dcs with tolerogenic properties. 1,25(oh)2d inhibits dc il12 production and secondarily limits the development of th1 helper cells shifting to a th2 phenotype. interferon (ifn), primarily produced by activated plasmacytoid dcs, plays a key role in sle, with activation of the ifn pathway being associated with increased anti - dsdna antibodies and increased disease activity among patients with sle. in vitro and ex vivo studies have shown the ability of 1,25(oh)2d to inhibit dc maturation and the ifn signature typical of active sle. vitamin d deficiency also contributes to b - cell hyper - activation and autoantibody production in genetically susceptible individuals. linker - israeli. showed that 1,25(oh)2d and its analogs inhibit polyclonal and anti - dsdna igg production by stimulated peripheral blood mononuclear cells from patients with sle. compared 32 female patients with sle to 32 healthy matched controls and found 25(oh)d deficiency to be associated with antinuclear antibody (ana) positivity among controls and with increased b cell activation among patients. additional immune modulating actions of 1,25(oh)2d of importance in suppressing autoimmunity include increasing treg cells, decreasing autoantibody production, suppressing the release of inflammatory mediators and the potential reestablishment of immune tolerance. vitamin d deficiency is a global health problem being detected at all ages, particularly among populations with darker skin pigmentation living further from the equator. african americans have a 3-fold increased prevalence of sle, develop sle at an earlier age, and have increased sle - related morbidity and mortality compared with caucasians. it is notable that the same ethnic disparities seen in the prevalence of vitamin d deficiency are seen in the prevalence of sle. multiple studies have shown that the majority of patients with sle have insufficient levels of 25(oh)d, especially among african american and hispanic patients with sle. to examine whether vitamin d deficiency is a potential environmental trigger of sle, nurses health study ii prospective analysis of over 180,000 women followed for up to 22 y found no significant evidence of association between dietary vitamin d intake and subsequent development of sle. there were limitations which include use of self - reported exposure data without serum 25(oh)d measurements, observational study design and applicability of results to caucasian female populations only. there was also limited power to detect a small effect of vitamin d intake on sle development and the confidence intervals were relatively wide (0.51.4). in the population - based carolina lupus inception - cohort study it was noted that lower 25(oh)d levels were found in 123 cases with newly diagnosed sle compared with 240 controls, which was statistically significant in caucasians (p = 0.04), controlling for age, sex, season and smoking. overall, 67% of the subjects were vitamin d deficient, with mean levels significantly lower among african americans (15.9 ng / ml) compared with caucasians (31.3 ng / ml). critically low vitamin d levels (10 ng / ml) were found in 22 of the sle cases. these baseline results within an inception - cohort of sle suggest vitamin d deficiency as a possible risk factor for the development of sle. many, but not all, studies of 25(oh)d among patients with established sle have shown an association of 25(oh)d deficiency with increased sle disease activity. a summary of the studies which have examined the relationship between sle and vitamin d status is presented in table 1. patients with sle have a higher risk for osteoporosis and fragility fractures, compared with age - matched controls. the increased osteoporosis and fracture risk among young patients with sle is attributed to systemic inflammation, frequent corticosteroids use, and more recently the high prevalence of vitamin d deficiency. low levels of 25(oh)d results in the calcium reserves in bone being depleted in an attempt to correct for the reduced calcium that will be absorbed from the gut. as the reduction in intestinal calcium absorption associated with low levels of 25(oh)d triggers the release of parathyroid hormone (pth), which stimulates the absorption of calcium through increased production of 1,25(oh)2d. pth also mediates the mobilization of calcium from bone by stimulating bone resorption, which results in a reduction in bone mineral density. vitamin d deficiency is one of several osteoporosis risk factors common among patients with sle, including high disease activity, renal disease, corticosteroid use, and premature ovarian failure from cytotoxic medications such as cyclophosphamide. since most patients with sle have multiple disease - associated and traditional osteoporosis risk factors, bone mineral density loss tends to run a rapid course, making vitamin d status even more important. sle is associated with higher incidence of cardiovascular problems at a younger age. a leading cause of morbidity and mortality in women with sle, including those who are premenopausal, is cvd. patients with sle have an increased incidence of myocardial infarction up to 5 times that of the general population, with an age - specific incidence in young women up to 50 times higher. there is evidence to show that, like diabetes, sle itself is an independent risk factor for the development of atherosclerosis. the excess cardiovascular risk, which may be up to 52 times in sle, is not explained by traditional cardiovascular risk factors. excess mortality in sle follows a bimodal pattern, with the early peak predominantly a consequence of active sle or its complications, and the later peak largely attributable to atherosclerosis. patients with sle are also at increased risk of nonfatal ischemic heart disease. as overall survival for patients with sle improves with better, more targeted, immune suppression therapies, the prevention of morbidity and mortality from atherosclerosis becomes an increasingly critical need. vascular stiffness can be partly driven by inflammation, and better disease control in patients with inflammatory arthritis results in a reduction in pulse wave velocity (pwv) noted that inflammatory biomarkers in sle were particularly associated with aortic pwv. in study by reynolds., the association between 25(oh)d and stiffness was at least in part accounted for by disease activity since in a regression model that includes sledai score the association was no longer significant. the results suggest that the association between 25(oh)d and disease activity is strongest in those patients with the most active disease / lowest vitamin d. vitamin d deficiency may therefore augment the inflammatory response in sle, underpinning both increased disease activity and vascular stiffness. a recent study by zhao., has shown that concentrations of 25(oh)d were inversely associated with all - cause and cvd mortality among adults with hypertension in the united states. due to the high prevalence of vitamin d deficiency seen worldwide and the conditions associated with deficiency, we recommend all patients at risk for sle or with established disease be screened for vitamin d deficiency. the only lab test usually required to ascertain the patient s status is the 25(oh)d level. ideal level of 25(oh)d for the general population and in subpopulations with certain health conditions such as sle. as our knowledge expands, we may find out that higher thresholds are needed for optimal health, however at this time the minimally adequate level of 25(oh)d is 30 ng / ml. achieving sufficient levels of vitamin d is a particularly complex issue for patients with sle because they are told to avoid the sun, the primary source of vitamin d. there is good evidence that even sensible sun exposure of 1 minimal erythema dose daily could trigger a disease flare. for that reason, there will be an even greater dependence on adequate dietary vitamin d intake. unlike other vitamins, currently very little of our daily vitamin d comes from food. many experts are recommending increased vitamin d fortification of common foods to help counteract widespread deficiency. but for now, oral vitamin d supplementation is needed for most, if not all, patients with sle. oral vitamin d supplementation is recommended in the form of vitamin d3 (cholecalciferol) rather than d2 (ergocalciferol). vitamin d3 is preferred over vitamin d3 due to vitamin d2 being less efficacious in raising serum 25(oh)d, having diminished metabolite binding to vitamin d binding protein (dbp) and a shorter shelf life. the dose of oral vitamin d3 required to achieve adequate 25(oh)d levels can be difficult to predict and will depend on the patient s baseline serum level and the presence of other risk factors for deficiency, such as obesity and corticosteroid use. the american college of rheumatology published guidelines in 2001 recommending calcium and vitamin d supplementation for all patients starting corticosteroids. therefore patients on corticosteroids often require higher daily doses of vitamin d to maintain adequate levels. a phase i study of daily oral vitamin d3 showed that doses up to 4,000 iu / day for 3 mo was safe and well - tolerated among african american patients with sle, and other trials among non - sle patients have shown similar safety of vitamin d3 4,000 iu / day. until further prospective trial results in patients with sle are available, we recommend correcting vitamin d deficiency with 50,000 iu capsule of vitamin d3 weekly for 8 weeks, followed by 2,0004,000 iu of vitamin d3 daily. the dose required to achieve and maintain adequate levels of 25(oh)d depends on the starting level, with roughly 100 iu of additional daily oral vitamin d3 required to raise the serum 25(oh)d level by 1 ng / ml. it takes approximately 3 mo to achieve steady - state once supplementation is started, but higher doses achieve steady - state sooner. individual responses may vary and known risk factors for deficiency should be taken into account. the importance of vitamin d in immune regulation has gained increased interest over the past decade with the discovery of the vitamin d receptor (vdr) being expressed by cells of the immune system and manipulation of 1,25-dihydroxyvitamin d [1,25(oh)2d ] having downstream immune effects. the effects of 1,25(oh)2d are mediated by complex nuclear receptor and the binding of the activated complex to regulatory dna sequences of target genes. the vdr gene is a direct target of its own receptor, thus facilitating an upregulation of the vdr protein in certain target tissues. several hundred vitamin d - regulated genes have been identified, including multiple genes involved with the innate and adaptive immune system. the overall immunologic effects of 1,25(oh)2d include downregulating th1 immune responses, modulating the differentiation of dendritic cells (dcs), and lowering proliferation of activated b cells, while upregulating regulatory t cells and preserving innate immune responses. each of the immune pathways influenced by 1,25(oh)2d has profound potential implications for patients with sle. in general, 1,25(oh)2d promotes the development of dcs with tolerogenic properties. 1,25(oh)2d inhibits dc il12 production and secondarily limits the development of th1 helper cells shifting to a th2 phenotype. interferon (ifn), primarily produced by activated plasmacytoid dcs, plays a key role in sle, with activation of the ifn pathway being associated with increased anti - dsdna antibodies and increased disease activity among patients with sle. in vitro and ex vivo studies have shown the ability of 1,25(oh)2d to inhibit dc maturation and the ifn signature typical of active sle. vitamin d deficiency also contributes to b - cell hyper - activation and autoantibody production in genetically susceptible individuals. linker - israeli. showed that 1,25(oh)2d and its analogs inhibit polyclonal and anti - dsdna igg production by stimulated peripheral blood mononuclear cells from patients with sle. compared 32 female patients with sle to 32 healthy matched controls and found 25(oh)d deficiency to be associated with antinuclear antibody (ana) positivity among controls and with increased b cell activation among patients. additional immune modulating actions of 1,25(oh)2d of importance in suppressing autoimmunity include increasing treg cells, decreasing autoantibody production, suppressing the release of inflammatory mediators and the potential reestablishment of immune tolerance. vitamin d deficiency is a global health problem being detected at all ages, particularly among populations with darker skin pigmentation living further from the equator. african americans have a 3-fold increased prevalence of sle, develop sle at an earlier age, and have increased sle - related morbidity and mortality compared with caucasians. it is notable that the same ethnic disparities seen in the prevalence of vitamin d deficiency are seen in the prevalence of sle. multiple studies have shown that the majority of patients with sle have insufficient levels of 25(oh)d, especially among african american and hispanic patients with sle. to examine whether vitamin d deficiency is a potential environmental trigger of sle, prospective studies and inception cohorts are needed. in the nurses health study and nurses health study ii prospective analysis of over 180,000 women followed for up to 22 y found no significant evidence of association between dietary vitamin d intake and subsequent development of sle. there were limitations which include use of self - reported exposure data without serum 25(oh)d measurements, observational study design and applicability of results to caucasian female populations only. there was also limited power to detect a small effect of vitamin d intake on sle development and the confidence intervals were relatively wide (0.51.4). in the population - based carolina lupus inception - cohort study it was noted that lower 25(oh)d levels were found in 123 cases with newly diagnosed sle compared with 240 controls, which was statistically significant in caucasians (p = 0.04), controlling for age, sex, season and smoking. overall, 67% of the subjects were vitamin d deficient, with mean levels significantly lower among african americans (15.9 ng / ml) compared with caucasians (31.3 ng / ml). critically low vitamin d levels (10 ng / ml) were found in 22 of the sle cases. these baseline results within an inception - cohort of sle suggest vitamin d deficiency as a possible risk factor for the development of sle. many, but not all, studies of 25(oh)d among patients with established sle have shown an association of 25(oh)d deficiency with increased sle disease activity. a summary of the studies which have examined the relationship between sle and vitamin d status is presented in table 1. patients with sle have a higher risk for osteoporosis and fragility fractures, compared with age - matched controls. the increased osteoporosis and fracture risk among young patients with sle is attributed to systemic inflammation, frequent corticosteroids use, and more recently the high prevalence of vitamin d deficiency. low levels of 25(oh)d results in the calcium reserves in bone being depleted in an attempt to correct for the reduced calcium that will be absorbed from the gut. as the reduction in intestinal calcium absorption associated with low levels of 25(oh)d triggers the release of parathyroid hormone (pth), which stimulates the absorption of calcium through increased production of 1,25(oh)2d. pth also mediates the mobilization of calcium from bone by stimulating bone resorption, which results in a reduction in bone mineral density. vitamin d deficiency is one of several osteoporosis risk factors common among patients with sle, including high disease activity, renal disease, corticosteroid use, and premature ovarian failure from cytotoxic medications such as cyclophosphamide. since most patients with sle have multiple disease - associated and traditional osteoporosis risk factors, bone mineral density loss tends to run a rapid course, making vitamin d status even more important. a leading cause of morbidity and mortality in women with sle, including those who are premenopausal, is cvd. patients with sle have an increased incidence of myocardial infarction up to 5 times that of the general population, with an age - specific incidence in young women up to 50 times higher. there is evidence to show that, like diabetes, sle itself is an independent risk factor for the development of atherosclerosis. the excess cardiovascular risk, which may be up to 52 times in sle, is not explained by traditional cardiovascular risk factors. excess mortality in sle follows a bimodal pattern, with the early peak predominantly a consequence of active sle or its complications, and the later peak largely attributable to atherosclerosis. patients with sle are also at increased risk of nonfatal ischemic heart disease. as overall survival for patients with sle improves with better, more targeted, immune suppression therapies, the prevention of morbidity and mortality from atherosclerosis becomes an increasingly critical need. vascular stiffness can be partly driven by inflammation, and better disease control in patients with inflammatory arthritis results in a reduction in pulse wave velocity (pwv) noted that inflammatory biomarkers in sle were particularly associated with aortic pwv. in study by reynolds., the association between 25(oh)d and stiffness was at least in part accounted for by disease activity since in a regression model that includes sledai score the association was no longer significant. the results suggest that the association between 25(oh)d and disease activity is strongest in those patients with the most active disease / lowest vitamin d. vitamin d deficiency may therefore augment the inflammatory response in sle, underpinning both increased disease activity and vascular stiffness. a recent study by zhao., has shown that concentrations of 25(oh)d were inversely associated with all - cause and cvd mortality among adults with hypertension in the united states. due to the high prevalence of vitamin d deficiency seen worldwide and the conditions associated with deficiency, we recommend all patients at risk for sle or with established disease be screened for vitamin d deficiency. the only lab test usually required to ascertain the patient s status is the 25(oh)d level. ideal level of 25(oh)d for the general population and in subpopulations with certain health conditions such as sle. as our knowledge expands, we may find out that higher thresholds are needed for optimal health, however at this time the minimally adequate level of 25(oh)d is 30 ng / ml. achieving sufficient levels of vitamin d is a particularly complex issue for patients with sle because they are told to avoid the sun, the primary source of vitamin d. there is good evidence that even sensible sun exposure of 1 minimal erythema dose daily could trigger a disease flare. for that reason, there will be an even greater dependence on adequate dietary vitamin d intake. unlike other vitamins, currently very little of our daily vitamin d comes from food. many experts are recommending increased vitamin d fortification of common foods to help counteract widespread deficiency. but for now, oral vitamin d supplementation is needed for most, if not all, patients with sle. oral vitamin d supplementation is recommended in the form of vitamin d3 (cholecalciferol) rather than d2 (ergocalciferol). vitamin d3 is preferred over vitamin d3 due to vitamin d2 being less efficacious in raising serum 25(oh)d, having diminished metabolite binding to vitamin d binding protein (dbp) and a shorter shelf life. the dose of oral vitamin d3 required to achieve adequate 25(oh)d levels can be difficult to predict and will depend on the patient s baseline serum level and the presence of other risk factors for deficiency, such as obesity and corticosteroid use. the american college of rheumatology published guidelines in 2001 recommending calcium and vitamin d supplementation for all patients starting corticosteroids. therefore patients on corticosteroids often require higher daily doses of vitamin d to maintain adequate levels. a phase i study of daily oral vitamin d3 showed that doses up to 4,000 iu / day for 3 mo was safe and well - tolerated among african american patients with sle, and other trials among non - sle patients have shown similar safety of vitamin d3 4,000 iu / day. until further prospective trial results in patients with sle are available, we recommend correcting vitamin d deficiency with 50,000 iu capsule of vitamin d3 weekly for 8 weeks, followed by 2,0004,000 iu of vitamin d3 daily. the dose required to achieve and maintain adequate levels of 25(oh)d depends on the starting level, with roughly 100 iu of additional daily oral vitamin d3 required to raise the serum 25(oh)d level by 1 ng / ml. it takes approximately 3 mo to achieve steady - state once supplementation is started, but higher doses achieve steady - state sooner. individual responses may vary and known risk factors for deficiency should be taken into account. it is well established that many people worldwide have inadequate levels of 25(oh)d, particularly patients with sle who often have additional risk factors for deficiency inherent to their disease. there is mounting evidence that vitamin d plays a key role in the pathogenesis and progression of autoimmunity. the hope is that something as nontoxic, inexpensive and widely available as vitamin d turns out to be effective as a disease suppressing intervention for patients with sle. in addition to the potential benefit of vitamin d replacement on sle activity, patients will also avoid the excess morbidity and mortality associated with long - term deficiency of vitamin d. continued research will help us better understand the immunomodulatory role of vitamin d and determine the ideal range of serum 25(oh)d for immune health.	systemic lupus erythematosus (sle) is a complex multi - system autoimmune disease. vitamin d deficiency has been proposed as an environmental trigger of disease onset and as a contributor to increased sle activity. sle patients are prone to develop vitamin d deficiency because of photosensitivity leading to sun avoidance and other sun protective measures. the impact of vitamin d on immune function previously seen in vitro and in cross - sectional studies has now been shown in prospective human studies, strengthening the evidence that there is a connection between sle and vitamin d status. this review describes the role of vitamin d on immune function, prevalence of vitamin d deficiency in patients with sle, identify risk factors for deficiency, describe the consequences of deficiency in sle patients, and review current vitamin d recommendations for patients with sle.
breastfeeding, especially exclusive breast - feeding (ebf), is the ideal method for feeding and growth in the first 6 months of birth. breast milk not only reduces the risk of incidence of many acute and chronic diseases such as diarrhea and respiratory infections in breast - fed infants, but also it has numerous benefits to mothers (for example, the reduced risk of breast cancer, uterus cancer, ovarian cancer, and osteoporosis). among all the possible problems arising during the breastfeeding period, breast conditions and those occurring in the early days of breastfeeding are most important. nipple problems include the development of macroscopic lesions that occurs on the nipples and areola. these lesions may appear as eroded skin, wounds, and fissure as well as clinical signs of erythema, edema, white or yellow blisters, dark spots, and ecchymosis. the most common consequences of traumatic nipples include infants deprivation of breast milk benefits and it may lead to stress and dissatisfaction in mothers. studies indicate that problems occurring in the first days following birth may lead to the cessation of breastfeeding. moreover, according to the statement issued by the world health organization (who), less than 40% of infants below 6 months of age are exclusively fed by breast milk. the incident of traumatic nipples varies between 29% and 76%, while nipple pain caused from breastfeeding varies between 34% and 96%. if these pains remain untreated, 26% of nipple pains will develop into nipple fissure and severe pain. unfortunately, treatment of traumatic nipple is complicated because of the repeated sucking by the infant, infections caused by the entrance of microorganisms through the nipple fissure, and constant exposure of nipple skin with the infant s oral flora. treatment of traumatic nipple dates back to the seventeenth century, but there is no explicit statement on the most suitable topical treatment for traumatic nipples. in this regard, complementary medicine, especially herbal medicine, plays a major role in improving the quality of postpartum care. currently, 40% of common medicines are derived from plants and natural resources. among the available treatments, this composition is capable of absorbing up to 30% of water. in 2008, dennis indicated that the application of lanolin is effective for the treatment of sore nipples. he stated that lanolin along with teaching the correct breastfeeding technique is the best solution to the treatment of nipple fissure. in contrast, gartner showed that recovery of nipple fissure in the lanolin group took longer. in another study, dodd & chalmens carried out a comparison between the effects of hydrogen dressing and lanolin on the treatment of traumatic nipples and showed that the hydrogen dressing group experienced a significant decrease in pain as compared to the lanolin group. however, no significant difference was observed in wound healing between two groups. dexpanthenol is one of the skin healers which belongs to the family of b complex vitamins. this medicine functions as a moisturizer by reducing water loss through the epidermis and maintaining the softness and elasticity of the skin. peppermint herb which is a native plant to iran, is widely used for the treatment of skin numbness, burns, scars, itching and inflammation. in 2003, ahluwalia made a comparison between the contributions of peppermint, lanolin, and teabag to the treatment of traumatic nipples and reported a significant decrease in pain and a significant increase in the nipple trauma healing of the peppermint group.they concluded that peppermint increases tissue flexibility and prevents fissure. in another study, sayyah melli showed that the administration of peppermint juice was three times more effective than breast milk in the prevention of nipple fissure., akbari indicated that peppermint essence can improve nipple fissure in primiparous women. after a vast review of the existing literature, no study was found to have confirmed the effects of dexpanthenol and peppermint on the treatment of traumatic nipple in iran, as all of the studies in this country were conducted on the prevention of trauma. considering the high prevalence of traumatic nipple and its complications, the lack of any agreed treatment for traumatic nipples, and the necessity of early treatment of traumatic nipples with the lowest side effects, the present study was carried out to compare the effects of lanolin, peppermint, and dexpanthenol creams on the treatment of traumatic nipples this randomized controlled trial conducted to compare the effects of lanolin, peppermint, and dexpanthenol on the treatment of traumatic nipples in breastfeeding mothers. the target population included the breastfeeding mothers who visited at the abbas abad and farabi health centers and sanandaj children s hospital in 2014. these centers were selected for this study because the number of breastfeeding mothers visiting these centers was high., the sample size was calculated to be 35 for each group by confidence interval of 95% and statistical power of 80% and using the three - group comparison formula. finally, by considering a sample loss of 20%, 42 women were selected in each group. the research samples were all of the breastfeeding mothers, who visited at the children s hospital and health centers and who had no drug prohibitions. after obtaining the permission of the ethics committee and the research deputy of tabriz medical sciences university, the samples were selected from breastfeeding mothers, who met the inclusion criteria and visited at the abbas abad and farabi health centers and sanandaj children s hospital. after explaining the research and its method and objectives to the mothers afterwards, the personal and social information forms were completed and midwifery histories of the subjects were recorded. the forms and records were completed based on the observation of the breastfeeding methods of the mothers using the store and champion scales. the samples were finally selected based on the following inclusion criteria : 1) primiparous mothers ; 2) exclusive feeding with breast milk ; 3) term infant ; 4) infant with below two months of age ; 5) obtaining the minimum score of 1 from the store and champion scales ; 6) mothers free of any medical disorders ; 7) lack of sensitivity of mothers to lanolin, peppermint, and dexpanthenol ; 8) infant weight of 2500 to 4000 grams ; and 11) lack of any nipple disorders (flat and depressed nipples). the exclusion criteria included the followings : 1) infants suffering from tongue and tooth disorders ; 2) using pacifiers, bottles, and plastic nipples for infants ; 3) history of maternal psychological disorders reported by mothers ; and 4) mother or infant suffering from any nipple - irrelevant condition that lead to cessation of breastfeeding. the allocation sequence was determined by a computer - generated randomization scheme with the block sizes of 3 and 6 and the allocation ratio 1:1:1. then, the tubes were filled with 50 ml of lanolin, lanolin, or dexpanthenol by the laboratory of pharmaceutics of tabriz school of pharmacy. the peppermint tube contained carbopol, methylparaben, triethanolamine, and glycerin along with 0.2% of peppermint oil, and the dexpanthenol tube contained 5% b complex analogue. every woman entering the study received sealed, opaque pockets (of apparently similar sizes). numbers ranging from 1 to 126 were printed on the pockets with identical uniform tubes. the pockets included three types of cream : lanolin cream, peppermint cream, and dexpanthenol cream. the subjects were randomly assigned into three groups receiving lanolin, peppermint, and dexpanthenol, and neither the researcher nor the subjects were aware of the assignment sequence and the position of each subject. that is to say, this study was a double blind trial. the breastfeeding mothers were not allowed to use any other treatment method during the study period and they were asked to report any case of sedatives consumption during the study. the participants were asked to apply a thin layer of the cream to the traumatic nipple and its areola three times a day immediately after breastfeeding for two weeks. the participants had recontamination of hand washing with warm water and soap before applying the cream. the information was collected through four steps (prior to intervention and on the third day, seventh day, and fourteenth day). the participants, who were trained on completion of the store and champion scales in advance, completed the scales on the third, seventh, and fourteenth days and submitted them to the researcher on the fourteenth day (the appointment day). in addition, a follow - up appointment was set for all of the participants one week after entering the research to record the intake of any sedatives or development of any complication caused by the treatment. the patients were given a cell phone number to be able to call and ask any questions related to the disease during the daytime until 12 am. the first part questioned the demographic and social information and the maternal history of subjects. this questionnaire covered information such as the patient age, marriage age, marital status, education, profession, residence location, midwifery information (including the number of pregnancies, number of delivery, number of living children), history of medical surgeries and history of breastfeeding (number of infants, duration of breastfeeding period, sedatives received for breastfeeding pains, treatment effect, breastfeeding status, and breast care during pregnancy, following birth and during breastfeeding). the positive response (yes) was assigned the 1 score while the negative response (no) was assigned the 0 score. score 0 was assigned to no pain and score 5 (the maximum score) was assigned to the highest level of pain. it is also used to assess the recovery of wound depth and the extent of damage caused to the tissue. the breastfeeding mothers used this scale and its numerical scale to assess the extent of nipple trauma and its recovery. lack of trauma was assigned the 0 score and larger numbers were recorded for deeper traumas. the scale ended with the maximum score (being 5). this tool has been used in various studies inside and outside the country, and its validity have been confirmed by champion. this tool has been used in various studies inside and outside the country, and its validity have been confirmed by storr. the reliability of the store and champion scales was measured using the test - retest method (r>0.7). the data was analyzed using spss software ver.13 and the following test methods : test - retest method, kruskal wallis method, chi - square test, anova, and repeated measures anova. in this clinical trial, 150 breastfeeding mothers were assessed were assessed for eligibility and finally 126 mothers were included in the research (figure 1). consort flow diagram results of this study revealed that the three study groups were similar in demographic, social, and fertility properties. the mean (sd) age of study samples in the lanolin, peppermint, and dexpanthenol groups was 27.7 years (8.6), 27 years (6.1), and 27.2 years (5.9), respectively. the mean (sd) marriage age was 19.8 years (4.4), 20.4 years (3.5), and 21.6 years (4.2) for the lanolin, peppermint, and dexpanthenol groups, respectively. more than half of mothers in the three groups had delivered once, and only in the dexpanthenol group 3 mothers (7.8%) had a history of four deliveries or more. most mothers in the three groups had just delivered their first infants and most of the mothers had an experience of natural childbirth. only 8 (20%), 4 (9.5%), and 7 (16.7%) patients in the lanolin, peppermint, the mean (sd) score of nipple pain in the lanolin, peppermint, and dexpanthenol groups prior to intervention was 3.12 (0.93), 3.07 (1.02), and 3.07 (1.02), respectively. the mean (sd) score of nipple pain on the third day of intervention was 1.29 (0.60), 1.26 (0.54), and 1.33 (0.65) for the lanolin, peppermint, and dexpanthenol groups, respectively. the mean (sd) nipple pain on the seventh day of intervention was 0.24 (0.49), 0.21 (0.47), and 0.14 (0.42) for the lanolin, peppermint, and dexpanthenol and dexpanthenol groups had a history of cesarean section. most mothers in the three groups had no history of previous surgeries and only one participant in the dexpanthenol group (2.6%) had surgery history (table 1). all of the figures, except for the specified figures, are expressed as frequency (%).chi - square test ; one - way analysis of variance (anova), two - tailed chi square test groups, respectively. more than 80% of mothers entered the painless phase on the seventh day of intervention. the number of mothers who entered the aforementioned stage was 32 (78%), 33 (80.5%), and 37 (88.1%) in the lanolin, peppermint, and dexpanthenol groups, respectively. on the fourteenth day of intervention 100% of mothers in three groups entered the painless phase. the result showed that the mean score of nipple pain at the prior to intervention stage, third, seventh, and fourteenth days of intervention was not significantly different between three group (p>0.05). but, repeated measures anova showed a significant difference in comparison of the three time periods of intervention in each group (p0.05) (table 3). anova with repeated measure, anova however, the results of repeated measures anova indicated a significant decrease in each group the comparison of the three phases (p<0.001) (table 3). in the research procedure all of the three groups were asked to first apply the cream to their forearms and administer the drug no infant reaction was also observed in the three groups to nipples coated with the aforementioned creams. that is to say, the infants easily started and continued drinking breast milk. therefore, no difference was made to the breastfeeding procedure and no complication was observed. there was also no statistically significant difference between the groups in terms of side effects when taking creams. research results revealed the equivalent effects of the 0.2% peppermint, lanolin, and dexpanthenol creams on the treatment of nipple pain and trauma. a thin layer of these creams was administered every 8 hours for two weeks. in the present study, according to the store scale, the pain of participants in the lanolin group declined by 0%, 78%, and 100% on the third, seventh, and fourteenth days of intervention, respectively. moreover, 2.4%, 80.5%, and 100% of reduction were observed in the peppermint group in the aforementioned days and 2.4%, 88.1%, and 100% of decline were observed in the dexpanthenol group, respectively., were in line with the results of our study regarding the effectiveness of lanolin. in the study by abou - dakn., 80% decline was observed on the seventh day and full recovery was obtained on the fourteenth day. these findings are also consistent with our findings, but in their research the effect of peppermint water on the treatment of nipple pain was reported to be more than lanolin cream. therefore, their study does not comply with our study in general and the difference in the results was resulted from the fully uniform intervention in our study. moreover, in their research the groups were not equally exposed to intervention, because 100 patients in the lanolin group, 50 patients in the teabag group, and 50 patients in the peppermint group were undergone the intervention. in the present study, according to the champion scale, similar levels of improvement in term of nipple trauma resulted from administration of the peppermint, lanolin and dexpanthenol creams. in a study conducted by kuscu., the results of using dexpanthenol for the treatment and recovery of trauma were similar to our results. that is to say, dexpanthenol contributes to the healing of skin wounds through epithelialization and granulation. it also leads to wound improvement by storing water through the epidermis. in the research by dennis., the patients were mainly complaining about pain and 78% and 71% of pain declines were observed in the multipurpose ointment and lanolin groups on the seventh day of treatment, respectively. the results of their study are consistent with our study regarding the decreased pain symptoms. vieira., conducted a systematic review and concluded that administration of lanolin with or without breast milk was the most effective intervention for the treatment of traumatic nipples as compared with other the treatments such as breast milk, hydrogel, adhesive polyethylene film dressings, a spray containing chlorhexidine with alcohol, and distilled water. in the other study by mohammadzadeh., the duration of trauma recovery in the lanolin group was higher than the breast milk and control groups. the reason for the difference between our results and the above research was the limitations that were found on this research. for example, no scale was used to determine the scores for nipple trauma andthe ranking was carried out under the supervision of a researcher, who was aware of the study groups. one of the advantages of this study was the innovation in using 0.2% peppermint essence in the form of cream for the treatment of traumatic nipples. moreover, the recovery of patients on the 3, 7, and 14 days of treatment was also followed. since no pre - term and abnormal infants were included in the study, the research findings can not be generalized to these infants. moreover, this research was not carried out in medical centers of villages, and other cities. therefore, the trainings, the program resulted from the native specifications of sanandaj city, and its results may not be applicable to other areas and cities of iran. few studies have been conducted in iran and the world on traumatic nipple treatment, it is recommended to carry out further studies on this topic. it is recommended to conduct studies on the possible maternal and neonatal side effects of substances used for traumatic nipple treatment. the contribution of peppermint to the treatment of traumatic nipples is similar to that of lanolin and dexpanthenol. therefore, peppermint could be considered as an effective alternative to the treatment of traumatic nipples in patients willing to use herbal medicines. hereby we express our gratitude to the personnel of bessat hospital, abbas abad and farabi clinics in sanandaj, and the laboratory of pharmaceutics of tabriz school of pharmacy who assisted us in preparing the medicines. we also would like to thank the authorities of the school of nursing and midwifery and research deputy of tabriz university of medical sciences for their financial support and also other colleagues who aided us in the course of this research.	introduction : traumatic nipple is among the most common problems of the breastfeeding period which leads to early cessation of breastfeeding. the study aimed to compare the effects of the lanolin, peppermint, and dexpanthenol creams on the treatment of traumatic nipples. methods : this double - blind randomized controlled trial was carried out on 126 breastfeeding mothers. the mothers had visited at the health centers and children s hospitals in sanandaj city. the selected participants were randomly divided into the following three groups of lanolin, peppermint, and dexpanthenol cream groups. nipple pain was measured using the store scale while trauma was measured with the champion scale. analyses were carried out through the kruskal wallis test, chi - square, anova, and repeated measures anova by using spss software ver. 13. results : the result showed that the mean score of nipple pain and nipple trauma at the prior to intervention stage, third, seventh, and fourteenth days of intervention was not significantly different between three groups. but, repeated measures anova showed a significant difference in comparison of the four time periods of intervention in each group. conclusion : results of this study revealed that the lanolin, peppermint, and dexpanthenol medicines had similar therapeutic effects on traumatic nipple.
the prevalence of heart failure (hf) continues to increase and is now the most common cause of hospitalization in the united states.1,2 routine management of patients with hf requires careful attentiveness to fluid status and diuretic treatment efficacy. although diuretics have been in clinical use for decades, patient response and clinical effectiveness are uncertain.3 although the physical examination is a critical component for decision - making, it is limited by body habitus or subclinical hemodynamic fluid changes. in addition to rales, edema, and elevated jugular venous pulse, clinicians measured weight, urine output, and serum creatinine on a daily basis in order to determine when euvolemia is achieved. heterogeneity of hf and disease management present significant challenge, and often other comorbidities and conditions complicate presentation of acute decompensated heart failure (adhf ; ie, cardiorenal syndrome).4 the two basic hemodynamic explanations for the development of cardiorenal syndrome are (1) inadequate forward perfusion typically after one or two parenteral doses of loop diuretic and (2) passive venous congestion that temporarily worsens with plasma refill from the extravascular tissues in the setting of diuresis for adhf. daily point - of - care ultrasound is ideal to better explore these hemodynamic changes in this patient population. assessment of cardiac structure and function is an important component in the clinical management of adhf and to understand the pathophysiology of the individual patient. transthoracic echocardiography is the primary method of imaging individuals with hf ; however, echocardiography is used infrequently and typically not serially secondary to cost and resource utilization.5 current appropriateness guidelines do not address the use of point - of - care echocardiography in clinical scenarios of adhf to guide acute response to therapy.6 the recent availability of miniaturized ultrasound technology enables physicians to perform real - time, point - of - care ue assessment of patients.7 when applied to patients with dynamic cardiovascular disorders such as adhf, this technology allows for daily objective assessment of intravascular fluid status. although the benefit of these devices seems obvious, little data exist on the type of information or additive benefit these devices provide in the routine care of hf patients particularly with respect to inferences concerning passive venous congestion of the kidneys. this prospective, observational trial examined the use of handheld, ultraportable echocardiography (ue) by physicians in patients admitted with adhf. the purpose of this study was to measure daily changes in ue in patients with adhf and compare those who respond to diuretic therapy to those who did not. this prospective comparative trial examines physiologic measures of intravascular fluid volume derived from ue and explores predictors of diuretic response in adhf. this study was conducted from december 2011 to january 2012 at genesys regional medical center (grmc), a 410-bed community - based teaching hospital. symptomatic patients admitted to grmc with the primary diagnosis of adhf who were able to provide informed consent were considered for selection during initial cardiac consultation. subjects with adequate visualization with ue (as determined by preliminary imaging by the echocardiologist study investigator [tev ]) were invited to be a part of the study. patients were excluded if they had poor, nondiagnostic imaging on ue, unable to provide informed consent, younger than 18 years, or if a prisoner or other institutionalized individual. ultimately, 77 subjects were enrolled and were typically scanned with the ultrasound device on a daily occurrence during routine physical examination. during data analysis, patients were divided into two groups based on whether or not they achieved a negative fluid output of 3 l within 48 hours. achievement of 3 l of fluid removal represented the median urine output of all study participants and was therefore selected as the criterion for patient inclusion in the diuretic responder cohort. the ultraportable two - dimensional ultrasound device used in this study measures 3 wide by 5.3 tall and weighs 3,000 ml of fluid output at 48 hours), independent of other variables. goodness - of - fit calculation revealed that the model appropriately fit the data, and the area under the receiver operating characteristics curve was 0.85 as described in table 3 and figure 2. there were a total of 77 patients who fulfilled study criteria and were included in this analysis. the overall mean age was 67.0 (sd 13.1) years, 58% of patients were male, and the average body mass index (bmi) was 29.5 kg / m. the minimum, average, and maximum inpatient length of stay was 3.0, 6.7, and 14 days, respectively. of the total 77 patients, 25 patients (32.4%) presented with new - onset adhf and the remainder presented with acute exacerbation of chronic hf. baseline demographic information of the patients are presented in table 1. in total, with all patient days combined, the study population consisted of 544 inpatient days, in which 431 days (79.2%) were spent for ue. in total, 73 of 77 patients survived to hospital discharge and one patient expired during the study. patients with new - onset hf comprised 32.4% of the study, while 67.5% were found to have an acute exacerbation of chronic hf. the most frequent comorbid conditions were hypertension (53.3%), hyperlipidemia (44.2%), coronary artery disease (41.6%), anemia (42.9%), and tobacco use (41.6%). atrial fibrillation was present in 32.5%, chronic obstructive pulmonary disease was present in 11.7%, and diabetes was present in 19.5%. overall formal echocardiography showed a decreased mean left ventricular ejection fraction (lvef) of 39.6% (sd 16.7%). the majority of patients (79.2%) also had diastolic dysfunction and mitral valve regurgitation was another common finding. the mean left ventricular internal diastolic dimension was 6.22 cm (sd 1.12 cm), mean left ventricular internal systolic dimension was 4.89 cm (sd 1.52 cm), interventricular septum diastolic thickness was 1.20 cm (sd 0.12 cm), left ventricular posterior wall diastolic thickness was 1.14 cm (sd 0.11 cm), left ventricular relative wall thickness was 0.38 cm (sd 0.062 cm), and mean right ventricular systolic pressure was 45.5 mmhg. of the 77 subjects, 12 patients were observed to have noticeable improvement in cardiac function during the course of the hospital stay by ue. of the 12 patients with improved lv function, the majority of these patients had new - onset hf and most were presumed to be nonischemic cardiomyopathy (only one patient had coronary angiography performed). this patient presented with acute on chronic decompensated hf secondary to acute on chronic myocardial ischemia / infarction. this patient had an initial lvef of approximately 45% visualized by ue, and over the next three days, the lvef deteriorated to approximately 15%. unfortunately, the patient was deemed nonrevascularizable, and despite maximal therapy, the patient developed cardiogenic shock and expired from a malignant arrhythmia on hospital day 4. the diameter of the ivc was measured on each participant on day 1 of hospitalization and on subsequent hospital days. the change in ivc diameter at 48 hours the mean ivc diameter for all subjects upon initial hospitalization was 2.04 cm (0.37 cm), the mean ivc diameter for all subjects after 48 hours was 2.03 cm (0.49 cm), and the mean 48-hour change in ivc diameter was 0.013 cm (0.270 cm). figure 1 demonstrates a patient who was treated over four days and the change in ivc diameter and other relevant clinical variables. on average, the ivc dimension decreased by 0.23 cm (0.37 cm) over the study course. in all but three patients, the ivc dimension decreased over the total examination period. in the three patients in whom the ivc dimension increased, the absolute measurement on the final day study remained within the normal range (3,000 ml of fluid output at 48 hours), independent of other variables. goodness - of - fit calculation revealed that the model appropriately fit the data, and the area under the receiver operating characteristics curve was 0.85 as described in table 3 and figure 2. attributable to its novelty, no previous studies have described the use of the latest generation of ue in routine clinical management of adhf. the data from this small observational study demonstrate the ability of ue to predict patient response to diuretics when treated for adhf. at the present time, ue is approved by regulatory bodies for clinical use in medicine and is commercially available7 ; however, little data exist on the type of information or additive benefit these devices provide in the routine care of hf patients. when used as an extension of the daily physical examination, fluid overload can be monitored noninvasively with ultrasound measurements of global left ventricular function and change in ivc diameter.8 until recently, significant barriers to ultrasound device technology prevented their implementation for routine use. reduction in device size, weight, and cost, with improvements in battery life, and ease of use are making inroads for this bedside tool. factors such as time constraints, noisy intensive care units, and patient body habitus have threatened the time - honored physical examination. this movement away from relying solely on older practices may be justifiable, as evidence - based scrutiny is lacking for many of these subjective examination techniques when applied in contemporary settings.9,10 this new era is moving ultrasound technology from the realm of formalized diagnostic imaging into the realm of a tool used to supplement the physical examination much the same as the stethoscope (table 4).11 ue ultimately fulfills its promise by allowing us to finally visualize pathology directly than traditional methods and adds to other established examination methods (ie, percussion, palpation, and auscultation). hf is a very heterogenous clinical disorder and requires physicians to utilize multiple data points to make the diagnosis including history, physical examination, and testing. previous literature suggests that ultrasound assessment of structures like the ivc can provide valuable supplemental data to aid in diagnosis and management.1214 as described, the significant change observed in these patients was a decrease in mean ivc size over the study period. novel to our report, we demonstrate the ability of the ue to provide these data. ivc diameter is an estimation of right atrial pressure and is analogous to the changes that occur with jugular venous distention in patients treated for fluid overload. the value of sequential assessment of the jugular venous distention is that it helps monitor patient clinical status and individually tailor diuretic therapy.15 it can be very difficult to assess response to diuretic therapy within the first few days of initiation. it can also be very difficult to predict which patients will respond favorably to diuretic therapy as evident by the lack of difference in clinical variables between the responders and nonresponders observed in this study. as expected, impaired renal function and lower bnp levels in patients predicted less responsiveness to diuretic therapy based on the multivariate regression. the change in the ivc diameter, however, was very robust in prediction of diuretic response. importantly, if the ivc diameter remained relatively unchanged or increased with diuresis, it predicted a poor response in urine output over the next several days, suggesting increased plasma refill and temporary increases in passive renal congestion. this is consistent with a previous report that bnp, ivc size, and collapsibility were able to predict 30-day readmission in adhf patients.16 our study has all the limitations of small prospective cohort studies and unblinded assessments using novel technology. we present data from a small number of patients where clinical utility was observed ; therefore, a considerably larger study without selection bias is still needed. this study did not capture data on the exact dose of diuretic administered, daily medication changes, and many other clinical variables that would be expected to be present in patients admitted with adhf. patients with poor acoustic visualization were excluded, so future studies should compare conventional 3d echocardiography to its pocket counterpart to further describe limitations. showing raw data obtained from routine clinical care does not yield consistent information among all patients enrolled, particularly with variations in daily laboratories and the number of days patients scans. it is thought that primary care providers and residents would benefit from ue,17 so further investigation is needed to support what level of basic training is necessary for reproducible results in the hf population. while ultrasound is noninvasive and cost - effective, use of handheld echocardiography requires initial investment by the provider. finally, we did not have advanced biomarkers of acute kidney injury (aki) or imaging measures of intrarenal venous congestion or intracapsular pressure, and thus, our inference on passive renal congestion and aki is indirect. with daily ultrasound measurement, this study demonstrates that a negative change in ivc diameter, the absence of diagnosed ckd, and bnp value independently predicted higher likelihood of responding to diuretic therapy within the first 48 hours.	backgroundroutine management of patients with acute decompensated heart failure (adhf) requires careful attentiveness to fluid status and diuretic treatment efficacy. new advances in ultrasound have made ultraportable echocardiography (ue) available to physicians for point - of - care use. the purpose of this study is to explore physiologic measures of intravascular fluid volume derived from ue and explore predictors of diuretic response in adhf.methodsvarious echocardiography imaging measurements, particularly diameter and collapse of inferior vena cava (ivc), were collected from 77 patients admitted with a primary diagnosis of adhf. patients were divided into two groups based on whether or not they achieved a net negative fluid output of 3 l within 48 hours. the demographic information, serum laboratory markers, and physical characteristics of the subjects were obtained to correlate with daily ultrasound measurements. univariate and multivariate analyses were used to compare diuretic responders to nonresponders.resultsa negative change in the ivc diameter at 48 hours was robust in prediction of diuretic response. for every 1 mm decrease in the ivc diameter at 48 hours, there was an odds ratio of 1.62 (95% ci : 1.202.19) for responding to diuretic therapy independent of other variables including baseline renal filtration function and blood b - type natriuretic peptide.conclusionassessment of central venous pressure as a proxy for passive renal congestion independently predicts initial diuretic response in adhf. future research is needed to further understand the individual variation in response to loop diuresis and to identify optimal treatment approaches that utilize anatomic and physiologic measures such as venous ultrasound.
traumatic tooth intrusion is a type of dental injury that involves displacement of the tooth into the alveolar socket. because of their locked position in the socket, the percussion test often elicits a high - pitched metallic sound, similar to an ankylosed tooth. radiographically, the tooth is shifted in an apical direction with partial or complete disappearance of the periodontal ligament (pdl) space. traumatic tooth intrusion usually involves the maxillary anterior teeth and is more common in the primary than the permanent dentition. intrusion represents a very complex wound, involving disruption of the marginal gingival seal, alveolar bone, pdl fibers, cementum and the neuro - vascular supply to the pulp. andreasen found that only 3% of permanent teeth injuries were intrusive luxation and all intruded teeth with closed root apices lost their vitality regardless of the degree of intrusion so endodontic intervention should be done as soon as possible. traumatic tooth intrusion might result in unexpectedly severe complications, such as pulp necrosis, inflammatory root resorption, ankylosis, replacement resorption, and loss of marginal bone support. pulp necrosis is the most common complication after luxation injuries, and has been reported to occur in almost 100% of fully formed, intruded permanent teeth so, endodontic intervention is required as soon as tooth exposed to oral environment after intrusion to prevent complications. the management of intruded permanent tooth may consist of (i) allowing spontaneous re - eruption, (ii) surgical repositioning and fixation, (iii) orthodontic repositioning, and (iv) a combination of surgical and orthodontic therapy. immediate orthodontic force on traumatically intruded teeth would facilitate the dental extrusion, allow an early endodontic access and it can be considered a way to prevent the appearance of ankylosis. this method, although, can increase the risk of external root resorption and marginal bone loss. this case report describes conservative management of extensive cervical and apical inflammatory root resorption of traumatically intruded tooth developed as a result delayed referral for endodontic management. a healthy 10.5-year - old female patient had history of dental trauma which resulted in complete intrusion of tooth [figure 1a and b ]. patient initially seen by an orthodontist for forced orthodontic extrusion of intruded tooth. after 6 months of orthodontic treatment intruded tooth repositioned back to its original position [figure 1c ] ; and direct composite filling was done to build the broken incisal edge [figure 1d ]. after removal of orthodontic appliance grade 2 mobility was noticed in tooth 11 and post - treatment radiograph revealed sever external apical and cervical root resorption with large periapical radiolucency around tooth 11 [figure 2a ] ; so patient was referred for management of external root resorption. (a) complete traumatic intrusion of tooth 11 at the time of trauma. (b) (d) after completion of fixed orthodontic extrusion and final composite build - up of 11 (a) radiograph after orthodontic treatment showing external root resorption and periapical radiolucency around tooth 11. (b) x - ray findings at 3 months follow - up with calcium hydroxide in tooth 11. (c) x - ray findings at 6 months follow - up with calcium hydroxide in tooth 11. (d) final obturation radiograph with gutta percha in tooth 11 at 2 year follow - up her clinical examination showed mixed dentition with satisfactory oral hygiene. tooth 11 had grade 2 - 3 mobility, was in good occlusion and not tender to palpation or percussion. tooth 11 responded negatively to pulp sensibility testing (thermal and electrical), while teeth 12, 21, and 22 responded positively. although, clinical and radiographic findings were alarming for the treatment success and long - term prognosis of tooth 11 but still after discussing plan with parents decided to preserve the tooth 11. a pulpless and an infected root canal system with chronic apical periodontitis, apical inflammatory root resorption, and lateral inflammatory root resorption (at the cervical level, on both mesial and distal aspects). a pulpless and an infected root canal system with chronic apical periodontitis, apical inflammatory root resorption, and lateral inflammatory root resorption (at the cervical level, on both mesial and distal aspects). immediate treatment plan was to arrest inflammatory root resorption process by removing necrotic pulp from canal space and placement of calcium hydroxide based intra canal medication. regular, long - term clinical and radiographic assessment of traumatized and adjacent teeth on follow - up visit for vitality. on initial visit root canal treatment started in tooth 11. after pulpectomy working length was determined roughly on periapical radiograph, although the root apex anatomy was distorted because of external apical root resorption. after chemo - mechanical cleaning and shaping with standardized step back filing technique (using master apical file) calcium hydroxide based intracanal medication was placed and coronal sealing of access opening was done with glass ionomer cements. on follow - up visit after 3 month mobility in tooth 11 was reduced to grade 1 and periapical radiograph revealed arrested root resorption process and decrease in size of periapical radiolucency [figure 2b ]. subsequently at 6 months follow - up, tooth mobility reduced to its physiological limit and periapical radiograph revealed arrested root resorption process, disappearance of periapical radiolucency and continued bony defect filling around tooth 11 [figure 2c ] ; so obturation completed by using master apical gutta percha points along with accessories (cold lateral condensation technique). clinical and radiographic assessment of tooth 11 showed normal healing process with no sign of replacement root resorption or ankylosis [figure 2d ]. regular, long - term clinical and radiographic assessment of traumatized and adjacent teeth on follow - up visit for vitality. on initial visit root canal treatment started in tooth 11. after pulpectomy working length was determined roughly on periapical radiograph, although the root apex anatomy was distorted because of external apical root resorption. after chemo - mechanical cleaning and shaping with standardized step back filing technique (using master apical file) calcium hydroxide based intracanal medication was placed and coronal sealing of access opening was done with glass ionomer cements. on follow - up visit after 3 month mobility in tooth 11 was reduced to grade 1 and periapical radiograph revealed arrested root resorption process and decrease in size of periapical radiolucency [figure 2b ]. subsequently at 6 months follow - up, tooth mobility reduced to its physiological limit and periapical radiograph revealed arrested root resorption process, disappearance of periapical radiolucency and continued bony defect filling around tooth 11 [figure 2c ] ; so obturation completed by using master apical gutta percha points along with accessories (cold lateral condensation technique). clinical and radiographic assessment of tooth 11 showed normal healing process with no sign of replacement root resorption or ankylosis [figure 2d ]. management of preadolescent with such an injury is determined by two main variables : the stage of tooth development and the amount of intrusion. studies have shown that, whereas intrusions of less than 3 mm have an excellent prognosis, those with an intrusion of more than 6 mm will ultimately be lost owing to progressive inflammatory resorption following overwhelming injury of pdl cells. it has been reported that intrusion and avulsion injuries carry a high - risk of inflammatory root resorption, replacement root resorption and dentoalveolar ankylosis due to irreversible damage to the pdl. the follow - up management of dental trauma in which tooth intrusion occurs requires special attention for all structures involved : the pulp, the pdl, and the alveolar bone. the early complications are mainly loss of pulp vitality and external root resorption so pulp extirpation should be initiated as soon as possible after intrusion with complete root development to prevent inflammatory root resorption. the main late complications that arise when endodontic treatment is not initially performed include loss of vitality and concomitant inflammatory root resorption in teeth with closed apices and obliteration of the pulp tissue in the teeth that remain vital. in this case, sever inflammatory external root resorption of central incisor could be a result of delay in referral for extirpation of necrotic pulp and exaggerated with forced orthodontic extrusion. it has been reported that almost 100% of fully formed, intruded permanent teeth develop pulp necrosis, so pulp extirpation should be initiated as soon as possible after intrusion with complete root development to prevent inflammatory root resorption. elimination of infected materials from the root canal has been shown to arrest inflammatory resorption with a high degree of success, and the use of calcium hydroxide dressing has been reported to result in a high frequency of periapical healing. although, 97% of all inflammatory resorptions are arrested after long - term calcium hydroxide treatment. this has been explained by its antibacterial property and denaturing effect on tissues in the resorption lesion, including resorbing cells, after diffusion through the dentinal tubules. in this case, we found excellent response to arrest the inflammatory process and periapical healing after removal of infected necrotic pulp and use of calcium hydroxide dressing on follow - up visits. it seems that local routes of antibiotic administration are a more effective mode than systemic applications. they are typically used in conjunction with corticosteroids and these combinations have anti - inflammatory, anti - bacterial and anti - resorptive properties, all of which help to reduce the periapical inflammatory reaction including clastic - cell mediated resorption. there is no current consensus on optimal treatment for intruded permanent teeth. in 1996, oulis. recommended that each case should be considered individually and the treatment should take into account the severity of the intrusion and tooth mobility. forced orthodontic eruption has been suggested as a possible treatment option, which might allow for remodeling of bone and the periodontal apparatus. the intruded tooth may be sufficiently repositioned for endodontic treatment within 2 - 3 weeks, so that inflammatory resorption can be prevented or treated if present. in this case, completely intruded tooth was successfully repositioned with forced orthodontic extrusion but delayed referral for endodonic management developed a severe complications. traumatic injury to teeth presents a considerable challenge for the practitioner. to cover all these various demands and necessities in a proper and adequate manner, a sound knowledge and experience of many different dental fields is needed and combined surgical, restorative, endodontic, and orthodontic approach are required to achieve an adequate rehabilitation of the patient. it is important that treatment provision is evidence based and holistic to ensure that the best possible dental and personal outcomes are achieved.	intrusive luxation is one of the most severe types of dental trauma. the risk of development of inflammatory or replacement root resorption is high if not timely managed. endodontic intervention is required soon after the occurrence of trauma, in an attempt to prevent or delay inflammatory root resorption. this case report emphasized timely referral for endodontic management of intruded tooth to prevent inflammatory root resorption.
severe ocular injuries involving partial or total loss of visual acuity, particularly those associated with maxillofacial fractures, are accordingly rare. while ophthalmologists treat most ocular injuries, maxillofacial surgeons also frequently face severe cases associated with facial fractures in emergency department patients. this is especially true at hospitals lacking ophthalmology divisions ; maxillofacial surgeons are often responsible for first ophthalmic assessments at these institutions. this study presents recommendations based on our experiences, as maxillofacial surgeons, in the management of patients with facial fractures and associated severe ocular injuries. between january and december 2009, 1779 patients with maxillofacial fractures were admitted to our division of maxillofacial surgery. inclusion criteria were : (1) preoperative coronal and axial computed tomography (ct) scans ; and (2) partial or total loss of vision at the time of emergency consultation, determined by a rapid ophthalmological assessment (roa), according to the guidelines of hammer and soparkar and patrinely. all ocular lesions responsible for the visual damage were classified as type 3 (severe), based on the severity scale of rao. patients with ocular motility deficits and type 1 and type 2 ocular injuries, those with incomplete clinical and radiological records, and those who had not completed a maxillofacial and ophthalmologic 6-month follow - up examination were excluded. data collected from the patient medical records included age, gender, mechanism of injury, location and type of facial fractures, type of ocular injury and cause of vision loss, method of treatment, and duration of hospitalization (days). among the 1779 admitted patients, 40 (2.2% ; 32 male, 8 female) presented with severely reduced vision or blindness caused by type 3 ocular injuries. severe ocular injuries are associated only with fractures that affect the middle third of the face and involve one or more orbital walls ; the 40 patients included in this study constituted 4.1% of the 969 admitted patients with such fractures. principal causes of the severe ocular injuries in this sample were workplace accidents (n = 11) and motor vehicle accidents (mva ; n = 11), followed by falls (n = 9), assaults (n=4), and sports injuries (n = 4) [table 1 ]. all of the ocular lesions that were secondary to sports related trauma were caused by horse kicks incurred during horseback riding. etiology of the severe ocular injuries association between causes and type of ocular injuries in total, 23 patients exhibited pure orbital fractures (12 floors, 4 medial walls, 3 roofs, 1 lateral wall, and 3 floors plus medial walls), 12 presented with orbito - maxillo - zygomatic (omz) complex fractures, and 5 patients suffered other complex facial fracture types, such as naso - orbito - ethmoidal or le fort ii / iii fractures. in total, 23 orbital floors, 16 lateral walls, 11 medial walls, and 3 roofs were involved [table 3 ]. summary of patients parameters the severe reduction or loss of vision following these fractures was the result of one or more of the following mechanisms, consistent with perry and moutray : direct lesion of the globe (10 penetrating lesions with successive rupture, 8 lesions of the anterior compartment);indirect traumatic optic neuropathy (deceleration / shearing forces ; 11);direct traumatic optic neuropathy (two (nos. 12 and 13) due to intracanalicular bony impingement of the optic nerve, one (no. 14) due to transaction of the optic nerve with traumatic avulsion of the eye);critical reduction of endorbital tissue perfusion (eight due to orbital compartment syndrome caused by a retrobulbar hematoma). direct lesion of the globe (10 penetrating lesions with successive rupture, 8 lesions of the anterior compartment) ; indirect traumatic optic neuropathy (deceleration / shearing forces ; 11) ; direct traumatic optic neuropathy (two (nos. 12 and 13) due to intracanalicular bony impingement of the optic nerve, one (no. 14) due to transaction of the optic nerve with traumatic avulsion of the eye) ; critical reduction of endorbital tissue perfusion (eight due to orbital compartment syndrome caused by a retrobulbar hematoma). of the 10 patients with global rupture, six received retinal assessment, suturing of the globe, and (during the same intervention) treatment of facial fractures within 24 h of arrival in the emergency department. this treatment was conducted in collaboration with consulting ophthalmologists from the ophthalmology institute (oi). the severity of the remaining four global ruptures required enucleation, with epithesis placement at least 6 months later. two (nos. 33 and 39) of the eight patients with direct lesions of the anterior compartment were transferred to the oi within 48 h of arrival at the hospital [figure 1 ]. 3438, 40) underwent surgical intervention for orbital and maxillofacial fractures within 48 h, and were subsequently transferred to the oi for management of the ocular injuries. (a) clinical image showing post - traumatic cataract of the left eye and (b) coronal ct scan showing a blow - out fracture of the left orbital floor steroids were administered to the 11 indirect neuropathy patients, in accordance with the protocol developed by the second national acute spinal cord injury study (nascis ii ; methylprednisolone 30 mg / kg as bolus + 5.4 mg / kg per h/24 h). the two patients suffering a direct neuropathy due to intracanalicular bony impingement of the optic nerve received immediate steroid treatment and subsequent surgical decompression of the optic nerve. the surgeries were performed in another hospital 7 and 10 days after the traumatic event. (a) coronal and (b) axial ct scans show lateral wall fracture involving the optic canal seven of the eight patients with retrobulbar hematomas underwent orbital decompression within 24 h of the traumatic event [figure 3 ]. the decompressions were performed according to the surgical protocols of our division, and resulted in partial or complete recovery of vision. 15) with hematoma - associated blindness was referred from another hospital 48 h after the traumatic event. (a) coronal and (b) axial ct scans show retrobulbar hematoma of the superior orbital compartment the mean length of hospital stay was 9.3 days, and the mean follow - up period was 56 months (range, 12 - 97). no patient in this study experienced a new - onset of blindness after facial fractures were repaired. this is unfortunately not always possible, particularly in hospitals like ours that lack ophthalmology divisions. the literature displays a consensus that an accurate ocular assessment must precede the surgical treatment of midfacial ocular fractures.[618 ] identification of the ocular lesion is important because its management may take precedence over the treatment of midfacial and orbital fractures. for example, the repair of an orbital fracture may exacerbate a misdiagnosed ocular rupture or laceration, resulting in permanent damage to the vision. jamal. reported that preoperative diagnosis of an ocular lesion is also a crucial medicolegal issue, so that responsibility for a permanent loss of vision is not ascribed to the treatment of maxillofacial fractures. maxillofacial surgeons are important components of emergency department teams, because they can manage the airways, oral and maxillofacial bleeding, and craniofacial fractures, as well as perform ocular assessments. the early identification of ocular lesions causing partial or complete loss of vision allows the maxillofacial surgeon to request an early ophthalmological consultation or referral (if the patient 's condition permits), to plan the eventual treatment of facial fractures. we use the roa to investigate ocular injuries in conscious and aware patients, especially when midfacial fractures are present. the roa consists of inspection (e.g., for redness or laceration), and testing of visual acuity, red and brightness saturation, pupillary functions, and double vision. when an accurate anamnesis can not be obtained (e.g., patient is intoxicated, unconscious, sedated, or intubated), pupillary dimensions and light reactivity must be clinically assessed. it is also essential to know the timing of vision loss ; several authors have demonstrated that better prognoses for vision recovery when the loss was slow and progressive after the traumatic event. we found a 2.2% incidence of severe ocular injuries among the 1779 emergency department patients with maxillofacial fractures who were admitted to the division of maxillofacial surgery. this incidence increased to 4.1% when only patients with middle third facial fractures were considered. these results fall within reported ranges among midfacial fracture patients, which vary from 0.32% described by zachariades. and 0.8% by mackinnon. pelletier. and brown. have suggested that such wide variation in occurrence may reflect ophthalmologists participation in the assessment of facial trauma patients ; such participation increases the incidence of ocular injuries identified. the literature reflects widespread agreement about the causes and sites of the facial fractures most frequently associated with severe ocular injuries.[61011131618 ] as we found in this study, mvas and workplace accidents are the principal causes of ocular lesions. the fracture sites most frequently associated with severe ocular injuries are orbital (mainly involving the orbital floor, followed by the lateral wall), as demonstrated in this study and in previously published research. although magnetic resonance imaging (mri) can provide detailed information about the orbital soft tissues, the additional resolution is not a significant factor in surgical planning decisions. ct scans are routinely used to diagnose facial fractures (including those involving the optic canal), optic nerve lesions, and intraorbital hematomas ; they are also used to exclude the presence of intracranial lesions that might impact visual acuity. patients presenting at the emergency department of our hospital with severe loss of vision caused by severe ocular injuries associated with maxillofacial fractures were managed by a maxillofacial surgeon alone or, more rarely, with an oi ophthalmologist providing emergency consultation. such consultations were performed for 18 patients with facial fractures associated with direct global lesions, the most frequent cause of partial or total vision loss in our sample. an ophthalmologist successfully repaired six of the 10 global ruptures in this sample, but none of these patients recovered vision. following ophthalmological intervention, an ophthalmologist diagnosed anterior compartment lesions of the eye in the eight patients with partial global lesions. he performed immediate surgical treatment in only two of these cases ; six patients first underwent maxillofacial fracture repair. a maxillofacial surgeon performed emergency decompression on the eight patients with orbital compartment syndrome due to increased endorbital pressure caused by a retrobulbar hematoma. this procedure resulted in the partial or complete recovery of vision in all but one case (no. this patient was referred from another hospital 48 h after the traumatic event, causing delays in diagnosis and treatment that led to the failure of vision recovery. ophthalmologic assessment was requested within 12 h after surgical decompression, if the patient 's condition allowed. megadoses of steroids were preoperatively administered to seven hematoma patients, according to the nascis ii protocol. the use of megadose steroid therapy in patients with severe visual deficits following traumatic optic neuropathy has been extensively discussed.[2024 ] in 1999, the international optic nerve trauma study found sufficient evidence to conclude that neither corticosteroids nor optic canal surgery should be considered the standard of care for patients with traumatic optic neuropathy. given their beneficial effects on spinal cord injuries, however, physicians are reluctant to not use megadose steroid therapy to treat traumatic optic neuropathies. the orbital contents are enclosed in a fascial sheath and surrounded by rigid bony walls, except for the semi - flexible anterior border created by the orbital septum and eyelids. increased volume from even minimal bleeding in this space may increase the orbital pressure. perfusion pressure to the retina is thereby reduced, resulting in compression of the long and short ciliary vessels and successive ischemic damage to the retina. therefore, stated that the most critical aspect of orbital hematoma treatment is the rapidity of the diagnosis and the decision to perform surgical drainage. drainage was performed in the patients in our study with the same surgical approach used to treat orbital wall fractures. the role of surgery in the management of ocular injuries is controversial ; some centers routinely explore all causes of traumatic vision loss, while others perform only selective exploration. postsurgical improvements have been reported among many ocular injury patients, including those with no initial light perception and those operated upon several months after injury.[2630 ] spontaneous improvement without medical or surgical intervention has also been reported. the 11 patients in our study with indirect traumatic optic neuropathy received megadoses of steroids within 8 h after the traumatic event. steroid therapy was associated with the surgical treatment of facial fractures in seven of these patients, and partial or complete recovery of vision was achieved in nine patients. in contrast, megadose steroid therapy had no apparent effect in two patients with direct traumatic optic neuropathy. the maxillofacial surgeon may encounter severe ocular injuries during the emergency assessment of patients with facial fractures. the maxillofacial surgeon must, therefore, be able to perform an roa and to evaluate the severity of ocular lesions. the ophthalmic assessment of an unconscious patient should minimally include observation of the pupils, ocular structures, and changes in global pressure, even in the presence of significant post - traumatic eyelid swelling. a conscious patient is able to provide an accurate anamnesis, alerting the physician to previous ocular pathologies or ophthalmologic surgical interventions that may contribute to the diagnosis. the roa should be performed as soon as the patient 's condition allows, quickly assessing pupillary reactions and the patient 's ability to count fingers. in cases of severe vision loss, knowledge of the timing can allow the surgeon to distinguish among retinal damage, optic nerve damage, and other causes. the vision should be monitored for several hours, because further deterioration may suggest an indirect traumatic optic neuropathy or a compartment syndrome. the cause and site of the lesion are important factors ; previously reported data and our results show a higher frequency of severe ocular injuries associated with fractures of one or more orbital walls, following a workplace accident or mva. ct scans should be used to assess the risks presented by the ocular lesion by confirming the orbital fracture, identifying potential fracture extensions to the bony canal of the optic nerve, and excluding the presence of endorbital hematomas. in consultation with an ophthalmologist, the maxillofacial surgeon will be able to plan the treatment of skeletal and ocular lesions. some ocular injuries may require management before repair of the bony lesions, whereas other lesions of the ocular adnexa may be treated simultaneously or following the repair of maxillofacial fractures.	aim : this study was designed to evaluate the incidence of severe ocular injuries associated to maxillofacial fractures and report their management in the emergency department.patients and methods : among the 1779 patients admitted for maxillofacial fractures, those with partial or total loss of vision at the time of emergency consultation were included in the study. data collected from the patients medical records included age, gender, mechanism of injury, location and type of facial fractures, type of ocular injuries and cause of blindness, methods of treatment, and days of hospitalization.results:forty patients (2.2%), 32 men and 8 women, ranging from 17 to 85 years of age, presented with severely reduced vision or blindness associated to fractures of the facial middle third with involvement of one or more orbital walls, mainly caused by motor vehicle and work accidents. in 18 patients, severe ocular injuries were determined by direct lesion of the globe, in 14 by direct or indirect traumatic optic neuropathy and in 8 by a retrobulbar hematoma. direct lesion of the eyeball was treated by prompt repair or enucleation of the globe, though no or little recovery of vision was obtained. ophthalmologic and/or maxillofacial treatment of the anterior compartment lesions of the eye allowed a partial or total recovery of the vision. a partial or total recovery of the vision was observed in almost all the patients with indirect traumatic optic neuropathy after administration of steroids according to nascis ii protocol. likewise, an evident improvement of the vision was obtained by immediate drainage of retrobulbar hematoma.conclusions:early diagnosis of the nature of the ophthalmic injury and treatment are important, and involvement of the ophthalmologist is mandatory.
regeneration techniques require an effective antibacterial regimen to disinfect the necrotic root canal space of immature teeth. triple antibiotic paste (tap), a combination of metronidazole, ciprofloxacin, and minocycline, has been the most commonly used medicament to disinfect the root canal during endodontic regeneration. a recent review reported that tap was used in more than half of all published endodontic regeneration cases. therefore, studies have suggested eliminating the minocycline in a double antibiotic paste (dap), keeping only metronidazole and ciprofloxacin, or substituting the minocycline with another antibiotic, such as clindamyxin, cefaclor, or amoxicillin. the use of ethylenediaminetetraacetic acid (edta) has been suggested as a final irrigation step in endodontic regeneration after the removal of the intracanal medicament. irrigation with edta has been proposed to wash out the remaining intracanal medicament and expose the collagen and other organic proteins that may improve the biological environment for endodontic regeneration. recently published in vitro studies reported a negative effect of intracanal antibiotic medicaments on hardness, resistance to fracture, and indentation properties of radicular dentin. however, no previous studies have explored the effect of antibiotic medicaments and edta on mechanical properties of radicular dentin. the predentin is a less mineralized collagen - rich dentin layer adjacent to the pulp of immature teeth with a thickness ranging from 10 to 47 m. this layer may play an important role in endodontic regeneration since minimal to no instrumentation is recommended. exploring the mechanical properties of the root canal surface that includes predentin and its underlying newly formed dentin in immature teeth is challenging due to the required metallographic preparation of dentin to perform any valid traditional mechanical test such as flexural strength, microhardness, or nanohardness. recently, a new biodent indenter was suggested to measure the indentation properties of bone, as well as root canal dentin surfaces without the need for any metallographic preparation using a technique known as reference point indentation (rpi). the rpi approach relies on a reference probe, which stays on the hard tissue surface, and a test probe, which slides relative to the reference probe while indenting the measured hard tissue. some parameters obtained from biodent indenter in bone studies were suggested to correlate with and predict energy to fracture obtained through traditional mechanical testing. the purpose of this study was to investigate the effect of intracanal antibiotic medicaments used in endodontic regeneration and edta on the microindentation properties and hardness of root canal dentin surfaces and polished radicular dentin using the rpi technique and a traditional microhardness technique, respectively. ten immature human mandibular premolars previously stored in 0.1% thymol at 4c were used within 6 months of extraction after obtaining indiana university institutional review board approval to use human teeth (irb number ; 1305011353). furthermore, each immature tooth had to have at least a 1 mm - diameter opening at the apical foramen with two - thirds of the root formed. each tooth was decoronated, and two 4 mm root dentin cylinders were obtained from the coronal and middle thirds of each root utilizing a water - cooled low - speed diamond saw (buehler, lake bluff, il, usa). the pulp tissue was extirpated with a barbed broach without touching the root canal surface. four specimens were obtained from each root by sectioning each cylinder longitudinally across the maximum diameter of the root canal into two specimens without touching the root canal dentin surface. a tap was prepared by mixing usp - grade antibiotic powders compounded of equal portions of metronidazole, ciprofloxacin, and minocycline (champs pharmacy, san antonio, tx, usa) with sterile water (1 g / ml). a dap was prepared by mixing usp - grade antibiotic powders compounded of equal portions of metronidazole and ciprofloxacin (champs) with sterile water (1 g / ml). the four specimens obtained from each root were randomly assigned to three treatment groups (tap + edta, dap + edta, and only edta) and one hank 's balanced salt solution (hbss) control group. then, specimens were placed in a 2 ml conical sample cup (fisher scientific, florence, ky, usa) containing 0.15 ml of one of the treatment pastes or hbss for the edta and control groups. thus, the root canal dentin surface of each specimen in the antibiotic groups was covered with 0.3 mm layer of the treatment paste. after that, the specimens were taken out and rinsed thoroughly with sterile water for 30s. each specimen in the three treatment groups was immersed for 5 min in a magnetic stirrer bath that contained 5 ml of 17% edta (henry schein, melville, ny, usa) followed by immersion in sterile water for 5 min before being subjected to rpi testing. rpi was performed using a biodent hfc [figure 1 ] (active life scientific, santa barbara, ca, usa) with bone probe 3 (active life scientific) as described in a previous study. in summary, each root dentin specimen was immobilized on an acrylic block, the bone probe assembly was passively placed on the root canal surface and three indentations 1 mm apart were measured and averaged from each dentin specimen. ten indentation cycles of a 5 n indentation force were applied at a frequency of 2 hz. the most common outcome variables reported in the literature were included in this study. those are the first cycle indentation distance (i d) which is the distance indented on the first of the 10 cycles, the i d increase (idi) which is the increase in an i d from the first to the tenth cycle, and the total i d (total i d) which is the maximum indentation after the end of the tenth cycle. furthermore, the hardness values were estimated using first cycle i d according to the following equation of cone geometry : illustration of the major components of the biodent reference point indenter. a force generator is used to move a test probe relative to a reference probe and the force in monitored by a force transducer. a distance transducer is used to monitor the movement of the test probe relative to the reference probe. the reference probe, a modified hypodermic needle, is connected to the instrument body with a luer lock fitting. the probe assembly (test probe and reference probe) is disposable and replaced after every 150 indentations where p is the constant load applied = 5 newton ; r and h are the first cycle i d values obtained from biodent. forty 4 4 radicular dentin specimens were obtained from the coronal and middle thirds of immature roots. each specimen was embedded in acrylic resin (varidur, buehler, lake bluff, il, usa) with the pulpal surface facing outside. the resulting blocks (10 10 8 mm) were ground flat and polished according to a standardized protocol described in a previous study. as a final cleaning step, the polished specimens were sonicated in neutral detergent solution (2% micro 90 liquid soap, international product corporation, burlington, nj, usa) and rinsed with de - ionized water for 3 min. the specimens were randomly assigned to the three treatment groups and a control group as described in the biodent experiment. for antibiotic - treated groups, approximately 0.15 ml of tap or dap was applied on each embedded specimen to cover each specimen with a 0.3 mm layer of antibiotic paste. a close fit custom made ethylene vinyl acetate dome shaped caps (soft - try, ultradent products, south jordan, ut, usa) were used to cover each treated embedded specimen. then, each specimen from the three treatment groups was immersed in 17% edta followed by sterile water as described in biodent experiment before being subjected to vickers hardness testing. microhardness measurements were performed using a vickers microhardness tester (leco, lm247, st. three indentations, spaced 200 m apart, were made on each specimen using a 50-g load and a 10-s dwell time. the representative hardness value for each specimen was obtained as the mean of the results for the three indentations. one root specimen from each group of the biodent experiment was randomly selected for scanning electron microscopy (sem) analysis to confirm the presence of indentations on the intact root canal surfaces after various treatments. each selected specimen was irrigated with 5 ml of distilled water, sonicated in de - ionized water for 10 min, and desiccated for 48 h. then, specimens were sputter coated for 2 min with gold / palladium and images were taken with a jeol 6390 lv scanning electron microscope (peabody, ma, usa). the effect of treatment type on biodent parameters and vickers microhardness values was examined using one - way anova. pair - wise comparisons were performed using fisher 's protected least significant differences to control the overall significance level at 5%. to satisfy the anova assumptions, ten immature human mandibular premolars previously stored in 0.1% thymol at 4c were used within 6 months of extraction after obtaining indiana university institutional review board approval to use human teeth (irb number ; 1305011353). furthermore, each immature tooth had to have at least a 1 mm - diameter opening at the apical foramen with two - thirds of the root formed. each tooth was decoronated, and two 4 mm root dentin cylinders were obtained from the coronal and middle thirds of each root utilizing a water - cooled low - speed diamond saw (buehler, lake bluff, il, usa). the pulp tissue was extirpated with a barbed broach without touching the root canal surface. four specimens were obtained from each root by sectioning each cylinder longitudinally across the maximum diameter of the root canal into two specimens without touching the root canal dentin surface. a tap was prepared by mixing usp - grade antibiotic powders compounded of equal portions of metronidazole, ciprofloxacin, and minocycline (champs pharmacy, san antonio, tx, usa) with sterile water (1 g / ml). a dap was prepared by mixing usp - grade antibiotic powders compounded of equal portions of metronidazole and ciprofloxacin (champs) with sterile water (1 g / ml). the four specimens obtained from each root were randomly assigned to three treatment groups (tap + edta, dap + edta, and only edta) and one hank 's balanced salt solution (hbss) control group. then, specimens were placed in a 2 ml conical sample cup (fisher scientific, florence, ky, usa) containing 0.15 ml of one of the treatment pastes or hbss for the edta and control groups. thus, the root canal dentin surface of each specimen in the antibiotic groups was covered with 0.3 mm layer of the treatment paste. after that, the specimens were taken out and rinsed thoroughly with sterile water for 30s. each specimen in the three treatment groups was immersed for 5 min in a magnetic stirrer bath that contained 5 ml of 17% edta (henry schein, melville, ny, usa) followed by immersion in sterile water for 5 min before being subjected to rpi testing. rpi was performed using a biodent hfc [figure 1 ] (active life scientific, santa barbara, ca, usa) with bone probe 3 (active life scientific) as described in a previous study. in summary, each root dentin specimen was immobilized on an acrylic block, the bone probe assembly was passively placed on the root canal surface and three indentations 1 mm apart were measured and averaged from each dentin specimen. ten indentation cycles of a 5 n indentation force were applied at a frequency of 2 hz. the most common outcome variables reported in the literature were included in this study. those are the first cycle indentation distance (i d) which is the distance indented on the first of the 10 cycles, the i d increase (idi) which is the increase in an i d from the first to the tenth cycle, and the total i d (total i d) which is the maximum indentation after the end of the tenth cycle. furthermore, the hardness values were estimated using first cycle i d according to the following equation of cone geometry : illustration of the major components of the biodent reference point indenter. a force generator is used to move a test probe relative to a reference probe and the force in monitored by a force transducer. a distance transducer is used to monitor the movement of the test probe relative to the reference probe. the reference probe, a modified hypodermic needle, is connected to the instrument body with a luer lock fitting. the probe assembly (test probe and reference probe) is disposable and replaced after every 150 indentations where p is the constant load applied = 5 newton ; r and h are the first cycle i d values obtained from biodent. ten immature human mandibular premolars previously stored in 0.1% thymol at 4c were used within 6 months of extraction after obtaining indiana university institutional review board approval to use human teeth (irb number ; 1305011353). furthermore, each immature tooth had to have at least a 1 mm - diameter opening at the apical foramen with two - thirds of the root formed. each tooth was decoronated, and two 4 mm root dentin cylinders were obtained from the coronal and middle thirds of each root utilizing a water - cooled low - speed diamond saw (buehler, lake bluff, il, usa). the pulp tissue was extirpated with a barbed broach without touching the root canal surface. four specimens were obtained from each root by sectioning each cylinder longitudinally across the maximum diameter of the root canal into two specimens without touching the root canal dentin surface. a tap was prepared by mixing usp - grade antibiotic powders compounded of equal portions of metronidazole, ciprofloxacin, and minocycline (champs pharmacy, san antonio, tx, usa) with sterile water (1 g / ml). a dap was prepared by mixing usp - grade antibiotic powders compounded of equal portions of metronidazole and ciprofloxacin (champs) with sterile water (1 g / ml). the four specimens obtained from each root were randomly assigned to three treatment groups (tap + edta, dap + edta, and only edta) and one hank 's balanced salt solution (hbss) control group. then, specimens were placed in a 2 ml conical sample cup (fisher scientific, florence, ky, usa) containing 0.15 ml of one of the treatment pastes or hbss for the edta and control groups. thus, the root canal dentin surface of each specimen in the antibiotic groups was covered with 0.3 mm layer of the treatment paste. after that, the specimens were taken out and rinsed thoroughly with sterile water for 30s. each specimen in the three treatment groups was immersed for 5 min in a magnetic stirrer bath that contained 5 ml of 17% edta (henry schein, melville, ny, usa) followed by immersion in sterile water for 5 min before being subjected to rpi testing. rpi was performed using a biodent hfc [figure 1 ] (active life scientific, santa barbara, ca, usa) with bone probe 3 (active life scientific) as described in a previous study. in summary, each root dentin specimen was immobilized on an acrylic block, the bone probe assembly was passively placed on the root canal surface and three indentations 1 mm apart were measured and averaged from each dentin specimen. ten indentation cycles of a 5 n indentation force were applied at a frequency of 2 hz. the most common outcome variables reported in the literature were included in this study. those are the first cycle indentation distance (i d) which is the distance indented on the first of the 10 cycles, the i d increase (idi) which is the increase in an i d from the first to the tenth cycle, and the total i d (total i d) which is the maximum indentation after the end of the tenth cycle. furthermore, the hardness values were estimated using first cycle i d according to the following equation of cone geometry : illustration of the major components of the biodent reference point indenter. a force generator is used to move a test probe relative to a reference probe and the force in monitored by a force transducer. a distance transducer is used to monitor the movement of the test probe relative to the reference probe. the reference probe, a modified hypodermic needle, is connected to the instrument body with a luer lock fitting. the probe assembly (test probe and reference probe) is disposable and replaced after every 150 indentations where p is the constant load applied = 5 newton ; r and h are the first cycle i d values obtained from biodent. forty 4 4 radicular dentin specimens were obtained from the coronal and middle thirds of immature roots. each specimen was embedded in acrylic resin (varidur, buehler, lake bluff, il, usa) with the pulpal surface facing outside. the resulting blocks (10 10 8 mm) were ground flat and polished according to a standardized protocol described in a previous study. as a final cleaning step, the polished specimens were sonicated in neutral detergent solution (2% micro 90 liquid soap, international product corporation, burlington, nj, usa) and rinsed with de - ionized water for 3 min. the specimens were randomly assigned to the three treatment groups and a control group as described in the biodent experiment. for antibiotic - treated groups, approximately 0.15 ml of tap or dap was applied on each embedded specimen to cover each specimen with a 0.3 mm layer of antibiotic paste. a close fit custom made ethylene vinyl acetate dome shaped caps (soft - try, ultradent products, south jordan, ut, usa) were used to cover each treated embedded specimen. then, each specimen from the three treatment groups was immersed in 17% edta followed by sterile water as described in biodent experiment before being subjected to vickers hardness testing. microhardness measurements were performed using a vickers microhardness tester (leco, lm247, st. joseph, mi, usa) on the polished pulpal side of the specimen. three indentations, spaced 200 m apart, were made on each specimen using a 50-g load and a 10-s dwell time. the representative hardness value for each specimen was obtained as the mean of the results for the three indentations. one root specimen from each group of the biodent experiment was randomly selected for scanning electron microscopy (sem) analysis to confirm the presence of indentations on the intact root canal surfaces after various treatments. each selected specimen was irrigated with 5 ml of distilled water, sonicated in de - ionized water for 10 min, and desiccated for 48 h. then, specimens were sputter coated for 2 min with gold / palladium and images were taken with a jeol 6390 lv scanning electron microscope (peabody, ma, usa). forty 4 4 radicular dentin specimens were obtained from the coronal and middle thirds of immature roots. each specimen was embedded in acrylic resin (varidur, buehler, lake bluff, il, usa) with the pulpal surface facing outside. the resulting blocks (10 10 8 mm) were ground flat and polished according to a standardized protocol described in a previous study. as a final cleaning step, the polished specimens were sonicated in neutral detergent solution (2% micro 90 liquid soap, international product corporation, burlington, nj, usa) and rinsed with de - ionized water for 3 min. the specimens were randomly assigned to the three treatment groups and a control group as described in the biodent experiment. for antibiotic - treated groups, approximately 0.15 ml of tap or dap was applied on each embedded specimen to cover each specimen with a 0.3 mm layer of antibiotic paste. a close fit custom made ethylene vinyl acetate dome shaped caps (soft - try, ultradent products, south jordan, ut, usa) were used to cover each treated embedded specimen. then, each specimen from the three treatment groups was immersed in 17% edta followed by sterile water as described in biodent experiment before being subjected to vickers hardness testing. microhardness measurements were performed using a vickers microhardness tester (leco, lm247, st. joseph, mi, usa) on the polished pulpal side of the specimen. three indentations, spaced 200 m apart, were made on each specimen using a 50-g load and a 10-s dwell time. the representative hardness value for each specimen was obtained as the mean of the results for the three indentations. one root specimen from each group of the biodent experiment was randomly selected for scanning electron microscopy (sem) analysis to confirm the presence of indentations on the intact root canal surfaces after various treatments. each selected specimen was irrigated with 5 ml of distilled water, sonicated in de - ionized water for 10 min, and desiccated for 48 h. then, specimens were sputter coated for 2 min with gold / palladium and images were taken with a jeol 6390 lv scanning electron microscope (peabody, ma, usa). the effect of treatment type on biodent parameters and vickers microhardness values was examined using one - way anova. pair - wise comparisons were performed using fisher 's protected least significant differences to control the overall significance level at 5%. to satisfy the anova assumptions, the effect of treatment type on root canal dentin surfaces as well as on polished radicular dentin was significant for indentation properties and microhardness measurements. figure 2a shows that first - cycle i d of tap + edta and dap + edta - treated dentin were significantly greater than edta - treated and control dentin (p < 0.0001). furthermore, first - cycle i d of the edta group was significantly greater than the control dentin (p = 0.0126). however, first - cycle ids of tap + edta and dap + edta - treated dentin were not significantly different from each other (p = 0.26). (a) mean and standard error (se) of the first - cycle indentation distance (i d) of root canal dentin exposed to various treatments and a no - treatment control group. (b) mean and se of estimated hardness of intact root canal dentin exposed to various treatments and a no - treatment control group. (c) mean and se of i d increase of intact root canal dentin exposed to various treatments and a no - treatment control group. (d) mean and se of total i d of intact root canal dentin exposed to various treatments and a no - treatment control group. (e) mean and se of vickers microhardness of polished radicular dentin exposed to various treatments and a no - treatment control group. different upper - case letters represent a significant difference the estimated hardness was significantly higher for the control group compared to edta (p = 0.0021), dap + edta (p < 0.0001), and tap + edta (p < 0.0001) treated dentin [figure 2b ]. in addition, the estimated hardness was significantly higher for edta treated dentin than for dap + edta (p < 0.0001) and tap + edta (p < 0.0001) treated dentin. however, no significant difference in estimated hardness was observed between dap + edta and tap + edta treated dentin (p = 0.27). figure 2c shows that the idi of tap + edta and dap + edta - treated groups were significantly greater than edta - treated and control dentin (p < 0.0001). however, the idi of tap + edta and dap + edta - treated dentin was not significantly different from each other (p = 0.17), nor were edta - treated and control groups (p = 0.63). figure 2d shows that the total i d of tap + edta and dap + edta - treated dentin was significantly greater than edta - treated and control dentin (p < 0.0001). however, the total i d of tap + edta and dap + edta - treated dentin was not significantly different from each other (p = 0.26). edta - treated dentin had significantly greater total i d than the control group (p = 0.020). vickers microhardness was significantly higher for the control dentin than for edta (p = 0.0039), dap + edta (p < 0.0001), and tap + edta (p < 0.0001) treated dentin [figure 2e ]. in addition, microhardness was higher for edta - treated dentin than for dap + edta (p < 0.0001) and tap + edta (furthermore, microhardness was higher for dap + edta than for tap + edta (p the order of means of the four study groups was the same for both estimated hardness using rpi and vickers microhardness. scanning electron microscopy images taken at 750 magnification showed the presence of microindentations as well as microcracks created during the repetitive loading. qualitatively, the micro - indentations were larger and deeper in tap + edta [figure 3a ] and dap + edta - treated dentin [figure 3b ] compared to edta - treated dentin [figure 3c ] and untreated control [figure 3d ]. (a) biodent indentation in triple antibiotic paste + ethylenediaminetetraacetic acid (tap + edta) treated root canal dentin surface. the use of various indentation techniques to determine the hardness of radicular dentin after exposure to various root canal irrigants and medicaments is a common approach in endodontic research. previous studies have used segments of polished radicular dentin due to the difficulties in performing a standardized indentation test on the actual root canal surface. however, polishing root canal dentin would remove the superficial predentin layer as well as the underlying newly formed inner radicular dentin with higher tubular density. therefore, trying to study the indentation properties of the intact root canal surface without metallographic manipulation would be more representative of the actual clinical situation. this study proposes a novel direct mechanical test to characterize the intact root canal dentin surface after exposure to antibiotic medicaments and edta solution. a traditional microhardness mechanical test was also performed in this study on polished radicular dentin to compare the magnitude of hardness reduction in both approaches. however, no statistical correlation was performed between the two types of hardness due to the different nature of the examined dentin (unpolished intact dentin versus polished dentin). dentine hardness property has been related to other mechanical properties such as modulus of elasticity, compressive strength and tensile strength. indeed, a recent investigation observed that a significant reduction in root fracture resistance after 3 months intracanal treatment with dap and tap was proceeded by a significant reduction in dentin microhardness after 1-month treatment with the same medicaments. in the current study, the percentage reduction in estimated dentin hardness from the rpi experiment were significantly higher in dap + edta (53%) and tap + edta (58%) treated dentin compared to edta - treated dentin (26%). furthermore, the percentage reduction in hardness of dap + edta and tap + edta - treated dentin reported in this study was higher than 4-week dap, and tap treated dentin reported in a recent study using the same rpi technique, which was 40% and 50%, respectively. this indicates that the use of antibiotic intracanal medicaments followed by edta irrigation have an additive effect on the reduction of dentin hardness. this could be explained by the ability of edta to chelate calcium and cause superficial dentin demineralization as well as the acidic nature of antibiotic medicaments and their ability to demineralize dentin. in this study, the percentage reduction in hardness reported using rpi of both edta - treated dentin (26%) and dap + edta (53%) treated dentin were higher than the percentage reduction in vickers hardness, which was 14% for edta - treated dentin and 39% for dap + edta treated dentin. this could be explained by the ability of the rpi technique to test the root canal surface dentin which is expected to be weaker due to the high density of dentin tubules in this area and the presence of predentin. on the other hand, the percentage reduction in hardness reported using rpi of tap + edta (58%) treated dentin were lower than the percentage reduction in vickers microhardness using a traditional experiment, which was 70% for tap + edta - treated dentin. the inability of the rpi technique to detect more reduction in microhardness for tap + edta treated dentin compared to the vickers microhardness technique could be explained by the severe dentin erosive effect caused by the highly acidic nature of tap. a recent study has shown that 4-week dentin treatment with tap followed by edta caused an average surface loss of 67 m. the estimated hardness values obtained from first - cycle i d depends mainly on the depth of penetration of the test probe into dentin in relative to the reference probe. the presence of surface loss on the measured surface after treatment may greatly decrease the first cycle i d and thus affect the magnitude of the estimated hardness. this might be considered as one of the limitations of the rpi technique when used to measure the effect of solutions / medicaments with high erosive potential on a hard tissue. the above reason could also explain the inability of the rpi technique to significantly differentiate between dap + edta and tap + edta treated dentin. a significant difference was detected between dap + edta and tap + edta treated dentin when vickers microhardness was used. the significantly lower vickers microhardness of tap compared to dap in the current study could be explained by the relatively low ph of tap compared to dap as well as the presence of minocyclin in tap, which was suggested to chelate calcium and demineralize dental hard tissues. therefore, 10 indentation cycles were used in the current study as an attempt to obtain more quantitative variables regarding the mechanical behavior of the treated dentin. the total i d variable reported in this study showed the same significant trends to first cycle i d and the estimated hardness, which might be considered as another reliable variable for differentiating between various dentin treatments. the idi of tap + edta and dap + edta - treated dentin was significantly greater than edta - treated and control dentin. recent studies on bone have shown a significant correlation between idi and modulus of toughness estimated by three - point bending mechanical tests as well as crack growth toughness. however, idi of tap + edta and dap + edta treated dentin were not significantly different from each other, nor were edta - treated and control dentin. this indicates that the idi variable was less sensitive in detecting significant differences after various dentin treatments compared to first cycle i d and total i d. in the current study, 1 g / ml of tap and dap were used since these are the concentration required to create a pasty consistency that can be applied into the infected immature root canal. some concerns have been raised regarding the use of these relatively high concentrations during endodontic regeneration. these concerns include the negative effects of tap and dap on the mechanical properties of radicular dentin and direct or indirect cytotoxic effects of tap and dap on human stem cells of the apical papilla and human dental pulp cells. the american association of endodontists has recommended the use of low concentrations of the antibiotic medicaments. however, nondiluted tap is still the most commonly used medicaments in the majority of the recently published clinical studies and there is no clinical evidence supporting the efficiency of low concentrations of antibiotic medicaments in endodontic regeneration. the use of lower concentrations of these antibiotic medicaments might be a good approach to minimize their negative effect on the mechanical properties of radicular dentin. both polished and intact root canal dentin treated with antibiotic pastes followed by edta had significant increases in indentation properties and significant reduction in microhardness compared to untreated control dentin and edta - treated dentin. however, the rpi technique was not able to significantly differentiate between dap + edta- and tap + edta - treated dentin. the currently used clinical endodontic regeneration protocols that include intracanal application of tap followed by edta irrigation may cause a significant reduction in hardness of radicular dentin ranging from 58% to 70%.	objective : the objective was to investigate the effect of intracanal antibiotic medicaments followed by ethylenediaminetetraacetic acid (edta) on the indentation properties and hardness of radicular dentin using a biodent reference point indenter and a traditional microhardness technique, respectively.materials and methods : specimens with intact root canal dentin surfaces and polished radicular dentin specimens were obtained from immature human premolars. each type of specimen was randomly assigned (n = 10 per group) and treated with either double antibiotic paste (dap) for 4-week followed by edta for 5 min, triple antibiotic paste (tap) for 4-week followed by edta for 5 min, edta for 5 min or hank 's balanced salt solution (control). the biodent reference point indentor and vickers microhardness tester were used to measure the indentation properties of root canal surfaces and the hardness of polished dentin specimens, respectively. one - way anova followed by fisher 's protected least significant differences were used for statistical analyses.results:both types of radicular dentin treated with antibiotic pastes and/or edta had a significant increase in the majority of indentation properties and a significant reduction in hardness compared to the untreated dentin. furthermore, treatment of dentin with antibiotic pastes and edta caused significant increases in indentation properties and a significant reduction in hardness compared to edta - treated dentin. however, the rpi technique was not able to significantly differentiate between dap + edta and tap + edta - treated dentin.conclusion:dentin treated with antibiotic medicaments followed by edta had a significant increase the indentation properties and significantly reduction in hardness of radicular dentin.
contemporary restorations of dental defects with implants are widely applicable, clinically prospective and comfortable treatment method. although it is important to realize that this method, as all other restorative procedures, is aimed not to change the tooth but exactly restore what has been lost in biological and mechanical aspects. an implant success rate is a numerical quantitative expression, which values the success of the implant as a matter of persistence until its fatal loss. this rate reaches 95% and is based on the osseointegration of the implant. if the osseointegration does not occur, the implant will be rejected and will not have a successful outcome because of early complications. there are two types of failures : early complication, which occur before the prosthetics and late complications after osseointegration and restoration [2,4 - 8 ]. it means that all 95% of successful implants still have risks of late failures, which are related with longevity and quality of treatment. under functional loading condition physiologically we can expect 1 - 1.5 mm bone loss throughout the first year and < 0.2 mm every following year. this process could be accelerated by mechanical, chemical and biological factors. with lack of attention and control, this physiological process may turn into a pathological inflammation of peri - implant soft tissues as peri - implant mucositis or its later form : peri - implantitis. despite high implant survival rate, epidemiological studies and clinicians insufficiently paying attention to the quality factor and sustainability of our final restoration. however, there should be understood that implants can not sufficiently replace natural teeth despite their survival rates. some of the main reasons for late implant failures can be bacterial factors, host health conditions (diabetes mellitus and bisphosphonates), smoking, overloading and iatrogenic factors [9 - 11 ]. major debates and the absence of consensus prevail especially due to overloading effect on implant complications. the intact root of the tooth is covered by periodontium and has 25 to 100 m micro - movements on axial direction. in comparison, osseointegrated dental implants can move just 3 to 5 m at the same axis. tooth mobility is determined by deformation of periodontal ligaments while implant mobility is determined by limited deformation of the bone. in a micro - movement like this, the periodontal ligament works as a damper and can reduce the effect of the stress on surrounding structures. moreover, periodontium could adjust the load because of proprioceptive sensors. according to mechanostat theory by frost, however, there are no clear guidelines : how much functional and para - functional loading can be injurious for dental implant, what mechanical factors can decrease load effect for such kind of restorations and what should be avoided. the aim of this review is to find out risks of mechanical impacts of peri - implant bone loss and prosthetic influence on bone stability. protocol and registration the review was registered on the international prospective register of systematic reviews prospero. this protocol could be found in prospero register : http://www.crd.york.ac.uk/prospero/display_record.asp?id=crd42016037224 this review was performed following prisma statements (preferred reporting item for systematic review and meta - analyses). types of publications the review included publications in english, which were published from january 2011 till april 2016. the selection of studies consists of randomized control trials, cohort studies, case - control studies, case reports, animal and in vitro studies. information sources and search a literature search was performed of two databases - medline (pubmed) and embase websites. the specific keywords were selected on purpose to establish a maximal informative and an accurate search. keywords were as follows : absorbing, bone, damping, dental implant, dis - integration, occlusal, overloading, peri - implant, shock, strain, stress. according to these, the search was performed on pubmed and embase search systems : dental and implant and (overloading or stress or shock or strain) and ((bone and loss) or disintegration or damping or absorbing). two reviewers independently screened the title and abstract of articles derived from this broad search. after primary screening decisions of both reviewers were compared and discussed. the third experienced reviewer accomplished a secondary screening for those cases where the disagreement was. the inclusion of articles was done according to : the description of the usage of dental implants, the estimation of implant loading, the load transmission and distribution over peri - implant bone or bone level changes. fixed prosthetic treatment and functional loading or over - loading should be applied in studies. in order to review the latest data, the studies over 5 years old also, studies that analyse the effect of masticatory forces in restoration level only (crown, abutment, screw) without changes in the bone level were excluded. the selected studies were divided into groups according to the type of the study : experimental in vitro, experimental in vivo and clinical. data of experimental finite element analysis (fea) studies were systematized in assessing load vector and value, stress and strain in peri - implant bone, implant length, diameter, bone layers, type of bone, bone level, osseointegration level, implant design, type of implant - abutment connection, restoration (table 1). c = cortical bone ; t = trabecular bone ; d1, d2, d3, d4 = bone density rate ; mpa = megapascals ; n = newtons ; na = not appropriate ; nd = no data ; ps = platform switch ; nps = non - platform switch ; strain = a common engineering unit measuring strain. data were collected from the included fea studies and arranged in the following fields : bone layers - shows types of applied bone layers ; type of bone - describes the density of bone ; osseointegration level characterizes the conditions of implant osseointegration ; bone level - describes the depth of implant position ; design of implant - describes the shape of implant model ; diameter of implant - describes diameter dimension ; length of implant - describes length dimension ; implant - abutment connection - shows type of connection (platform switch or non - platform switch) ; type of supra - construction - description of prosthesis ; axial load - strength value in an axial direction ; oblique load - strength value in an oblique direction ; lateral load - strength value in a lateral direction ; stress / strain units - measuring units. assessment of methodological quality the intrinsic methodological and lacks of design were independently screened by two reviewers. the qualitative assessment was accomplished for each of elected study and their risk of bias was evaluated according to the cochrane library. they were grouped according to the type of the study : 20 - in vitro fea ; 8 - in vitro (tests) ; 5 - in vivo ; 3 - prospective, 6 - retrospective. the distribution of publications shows the prevalence of virtual fea mechanistic studies. with the intention of achieving methodical homogeneity, all fea studies (n = 20) were involved in data analysis (figure 1). study characteristics the analysis of articles, which describes the fea study, illustrates that both conditions and the obtained data are heterogeneous (table 1). according to methods, studies differ on the different simulated conditions : a different bone base. mainly, double layered (cortical + trabecular) bone model was applied and only a few models were single layered. the authors programmed various bone types (d1 - d4) and some were evaluating stress distribution on bone following density. for example, it could be programmed as a peri - implant cylinder, as a section of the jaw or a full dental arch. some authors provided a real scanned situation. a different osseointegration. in reality, implant is not fused with bone absolutely. some referred to it as an ideal for simplicity (100% integration) and others as a partial osseointegration for more realistic model. there were different selection of implant length, diameter, thread and other properties in the studies. some of the investigations were carried out without a reference of the implant - abutment connection or completely without it. also, there were studies in which the investigation was performed only at implant - abutment model. the angle of oblique load was a variable too. some studies analysed stress distribution depending on load direction but mostly dominated free of choice. the loading conditions are simulated not only at different vectors but also at different loads, which were used from 50 to 500 n. different outcome units. the dominant measuring of stress was expressed in von mises mpa, but studies with tangential stress were measured in mpa or strain in strain. the impact of loading resulted in stress or strain at the implant - bone interface was described in all 20 publications. mainly there were stress dependence on the force acting axis [20 - 28 ] and all authors assured that non - axial loading increased peri - implant stress. also, implant design characteristics were described as factors causing the stress. the alteration of stress was related with implant length, width [21,25,26,28 - 31 ], macro - relief as thread, and micro - relief as porosity. some of the articles emphasized that stress changes were caused by platform switching connection [20 - 22,33 ]. the influence of abutment - implant connection was also described by another two articles, who revealed impact of inaccuracy at this interface. prosthetic related factors as crown / implant (c / i) ratio and restorative materials were also analysed. all the selected studies were assessed for their risk of bias according to the cochrane library. the included studies did not report it. the sequence generation could be debatable for in vitro or perhaps impossible, especially for fea. evaluation of allocation concealment and blinding of participants and outcomes of in vitro studies was confirmed severe and not appropriate. the incomplete outcome data was not explained in most cases and assessment unclear means that no missing data was reported (table 2). na = not appropriate. due to the study design and conditions, heterogeneity of outcomes may not achieve a comparison of results and statistical analysis. this review was performed with an aim to systematically review risks of mechanical impact on peri - implant strain and prosthetic influence for stability across fea studies. despite the heterogeneity between the fea studies, the established trends correlate with other in vitro, in vivo, prospective and retrospective studies from the same search. according to newton s third law it is undisputed that chewing forces are transmitted to restoration and these forces do not decrease but transform into energy, which is distributed in certain amounts through the restoration - implant complex. energy could be distributed into restorative materials, cement layer, abutment, screw, implants and peri - implant bone. according to reviewed studies, an overloading of peri - implant bone fea studies have shown dependence between stress distribution and loading direction. assessing the effect of the axial force, was detected the distribution of uniform stress in peri - implant bone without concentrations in a specific surface area of the implant. the non - axial loading of the implant increased the stress concentrations in peri - implant bone because of bending. it is important to understand, that the axis of rotation locates at the top of the bone. in fea models under load of 100 n without any changes of other conditions except of loading direction (axial vs. oblique), the stress increased and its trend of concentration was revealed under oblique loading. angled abutments created the same effect because they shifted the load from the implant axis. without changes of load direction following the increased abutment angulation from 0 to 15, stress concentrations increased in cortical peri - implant bone. consequently, the oblique load creates a bending effect, which affects the bone, especially around the rotation area but the different mechanical properties of bone layers determine different displacement of the implant body. other parameters of restorations such as cantilevers and c / i ratio could increase bending of the implant and stress or strain in the bone. the behaviour of different sized restorations was compared : single - unit, fixed partial, fixed full. another retrospective study did not find significant bone loss around implant when distal cantilevers were constructed with full arch prosthesis. so the number of implants could create a counterpoise and reduce the bending effect of cantilevers. the similar bending effect could be caused by the extended width of the implant crown. however, the study did not find an influence between the width of the crown and peri - implant bone loss. performed an fea study, in which the influence of different c / i ratio to stress distribution was investigated. the dependence between peri - implant bone loss and c / i ratio was confirmed in prospective clinical studies. otherwise, c / i ratio depends not only on crown length, but also on implant length. this supports the fea study with the conclusion that short implants create higher stress at peri - implant cortical bone. the shape of the implant could affect the stress distribution around the implant surface. according to fea results, as the diameter of the implant decreased, all studies comparing the effectiveness of conventional (non - platform switch) and platform switch abutments revealed that platform switch type abutments decreased stress concentrations in peri - implant bone [20 - 22,33 ] and its positive effect was higher for cortical bone than trabecular. on the other hand, platform switch reduced stress from the bone, but accelerated on the interface of connection surfaces and that could provoke strain for mechanical parts such as implant, screw and abutment. the concept of passive fit implies that there was no gap or strain induced by misfit of the framework prior to functional loading. this fea investigated peri - implant stress when theoretical misfit was recorded on implant - abutment level and on abutment - crown level. approved similar findings in their study, where misfit of abutment was generated with changing of conical angle of abutment. the passive fit of implant and related prosthetic components were considered to be very important and its absence was thought to cause complications in biological tissues and mechanical failures. consequently, imperfections at the connection regions could generate overloading of peri - implant bone as well as in prosthetic parts. it may be worth considering, that analog abutments or bases could cause the similar stress because of unmanaged imperfections. study detected that shock - absorbing capacity of implant restorations depends on damping behaviour of different dental materials under the cyclic loading conditions. the usage of composite materials damping behaviour was growing due to visco - elasticity of such material. changes could exist in restorative layers but that could not be detected by static linear fea. if the damping effect of platform switch was detected, elastic prosthetic parts could accelerate the same phenomenon. described damping behaviour of periodontal ligament and its shock - absorbance. in clinical cases, when an implant restoration had a natural opposite tooth, the periodontal ligament of antagonistic could absorb significant part of the loading energy. authors established 0.2 mm peri - implant bone loss in cases with natural antagonist and 0.6 mm bone loss in cases with opposite implant restoration at the same time, and concluded, that higher bone level is in cases with opposite natural tooth. implant loss because of direct overloading reasons was described in in vivo study with rats and in retrospective study. in vivo based older studies detected that in case of bacterial peri - implant inflammation and overloading could accelerate bone loss. however, we have to agree with pellegrini. review, which stated that the detrimental effect of occlusal overload on bone - implant interface is still a controversial issue. peri - implant strain could be generated by non - axial loading, cantilever prosthetic elements, crown / implant ratio, type of implant - abutment connection, misfits, properties of restoration materials and antagonistic tooth. finite element analysis studies are not erroneous methodically and the results correlate with other experimental and clinical findings. due to the heterogeneity of finite element analysis studies and expression of their results, it is impossible to perform meta - analysis or systematic reviews.	abstractobjectivesto systematically review risks of mechanical impact on peri - implant strain and prosthetic influence on stability across finite element studies.material and methodsan online literature search was performed on medline and embase databases published between 2011 and 2016. following keywords tiered screening and selection of the title, abstract and full - text were performed. studies of finite element analysis (fea) were considered for inclusion that were written in english and revealed stress concentrations or strain at peri - implant bone level.resultsthere were included 20 fea studies in total. data were organized according to the following topics : bone layers, type of bone, osseointegration level, bone level, design of implant, diameter and length of implant, implant - abutment connection, type of supra - construction, loading axis, measurement units. the stress or strain at implant - bone contact was measured over all studies and numerical values estimated. risks of overloading were accented as non - axial loading, misfits, cantilevers and the stability of peri - implant bone was related with the usage of platform switch connection of abutment.conclusionsperi-implant area could be affected by non - axial loading, cantilever prosthetic elements, crown / implant ratio, type of implant - abutment connection, misfits, properties of restoration materials and antagonistic tooth. the heterogeneity of finite element analysis studies limits systematization of data. results of these studies are comparable with other findings of in vitro, in vivo, prospective and retrospective studies.
natural killer (nk)/t - cell lymphoma is the most common subtype of primary nasal lymphoma1). nasal nk / t cell lymphoma is a very aggressive form of lymphoma, with over 50% of the tumors showing involvement of the adjacent alveolar bones, hard palate, orbits, nasopharynx and an association with an extensive soft - tissue mass2). relapse patterns of nasal nk / t cell lymphoma after treatment have not been systematically evaluated, and mediastinal nodal relapse at a controlled primary site has not been previously reported. here we report a case of mediastinal single nodal relapse of a nk / t cell lymphoma, detected by positron emission tomography (pet) and confirmed pathologically, which was removed completely with surgery using video - assisted thoracoscopy (vats). a previously healthy 40-year - old korean male was admitted to asan medical center, seoul, korea, in december 2003 with symptoms of nasal obstruction and fever. a radiological examination of the paranasal sinus disclosed a soft tissue mass in the left middle meatus and associated obstructive sinusitis in the left maxillary sinus (figure 1). a rhinoscopic examination revealed an irregular, and partly ulcerated protruding mass occupying the nasal cavity. a punch biopsy showed the presence of extensively necrotic tissue with a few viable foci of sheets of atypical medium- to large - sized lymphocytes with irregularly shaped nuclei. the tumor cells were positive by immunostaining for cd3, cd 56, cytotoxic granule marker tia-1, and uchl-1 (cd45ro), but negative for cd20, cd79a, cd4, and cd8. in situ hybridization showed the presence of epstein - barr virus in the majority of the tumor cell nuclei. the patient was diagnosed with an extranodal cd 56 + nasal nk / t cell lymphoma. to evaluate the disease status, computed tomography (ct), bone marrow aspiration and biopsy, and pet fluorine-18 fluorodeoxyglucose (fdg) pet showed a focal hypermetabolic lesion with a 4.8 maximal standardized uptake value (suv) in the left nasal cavity, but no abnormally hypermetabolic lesions in the chest or abdomen (figure 2, figure 3a). all of these findings were consistent with a stage ieb nasal nk / t cell lymphoma. hematological levels were hemoglobin 12.8 g / dl, hematocrit 36.9%, platelet count 17210/mm, and white blood cell count 4,200/mm (66% neutrophils, 29% lymphocytes, 5% monocytes, and 0.5% eosinophils). all blood chemistry findings were normal, except for a slightly elevated lactate dehydrogenase level of 379 iu / l (normal range 120 - 250 iu / l). a cytological examination of the cerebrospinal fluid showed no abnormalities. the patient was treated with three cycles of chemotherapy, consisting of cyclophosphamide, doxorubicin, vincristine, and prednisolone (chop), followed by 28 fractions of local radiation therapy to the primary site, with a total dose of 5,040 cgy. in april 2004, about one month after completion of the radiation therapy, fdg pet - ct revealed a newly developed focal hypermetabolic lesion with a maximal suv of 3.6 in the aorto - pulmonic nodal station. there were no abnormal findings at other sites, except for post - radiation sinusitis and thyroiditis (figure 3b). microscopically, the node showed numerous histiocytes and epithelioid granulomas and scattered medium - sized cd56-positive lymphocytes in the paracortex ; the nuclei of these cells were positive for epstein - barr virus. the tissue specimen of the mediastinal lymph node showed histology similar to that of the nasal mass (figure 4a - d). thirty - five months after the initial diagnosis and thirty months after the initial relapse, the patient is still alive, without any evidence of further relapse. an extranodal nk / t cell lymphoma is characterized by extensive mucosal ulceration and angiocentric or angiodestructive lymphomatous infiltration. these tumors have an immunophenotype of nk / t cell neoplasms, including expression of cytoplasmic cd 3 epsilon and cd56, and many are positive for ebv3, 4). these nk cell lymphomas show a geographic predilection, in that they are more common in asian regions such as hong kong, japan, and korea, and in latin american countries including mexico, peru, and guatemala5 - 7). " nasal " nk / t cell lymphomas are classified as lesions confined within the nasal cavity and nasopharynx, whereas " nasal - type " nk / t cell lymphomas are lesions involving sites outside the nasal cavity / nasopharynx, such as the oral cavity, palate, larynx, tonsil, skin, soft tissues, and the visceral organs4, 8, 9). the prognosis for patients with primary non - hodgkin 's lymphoma of the nasal cavity is poor, and the rates of distant metastasis and local relapse are high. the 5-year overall survival and disease - free survival rates for 102 patients with stage ie nasal nk / t cell lymphoma have been recently reported to be 71.7% and 60.9%, respectively10). outcomes of 262 extranodal nk / t cell lymphomas were recently used to develop a prognostic model of these tumors, with new prognostic factors including b symptoms, stage, lactate dehydrogenase (ldh) level, and regional lymph node status11). according to this model, our patient, who had b symptoms and an elevated ldh level, is in group 3 (two risk factors). because of the low incidence and geographic occurrence of these tumors, a systematic evaluation of the treatment for nasal nk / t cell lymphomas has not been fully performed. therefore, the optimal therapy for nasal nk / t - cell lymphoma has not yet been established. although a prospective randomized trial about optimal treatment has not been performed, several retrospective studies have shown radiotherapy to be superior to chemotherapy alone for stage i / ii disease12, 13). some studies report that the addition of chemotherapy to radiotherapy does not appear to confer any survival benefit in early stage patients13, 14). consequently radiotherapy, either as the initial treatment or as part of the chemotherapy regimen, is presently the mainstay of a treatment program for early stage nk / t cell lymphoma. the patient in this case was treated with three cycles of chemotherapy, followed by in field radiation therapy as and initial treatment program. secondary lymph node involvement is rarely encountered until late in the course of disease15). in the patient described here, we diagnosed mediastinal relapse promptly using pet, a highly sensitive diagnostic tool for various cancers that can detect increased glucolytic activity of neoplasms16). pet is usually performed to diagnose and stage tumors and to monitor response to therapy17). although ct is frequently performed for patients with lymphoma, it is less sensitive for small tumor foci. following treatment of a lymphoma, ct can reveal residual masses or enlarged lymph nodes, which may or may not contain a viable tumor18, 19). lymphoma evaluation using ct is primarily dependent on the size and morphologic criteria used to differentiate a malignant from a reactive lymph node. the patient described here had a single mediastinal lymph node of 1 cm in size. evaluation by ct alone, without pet, would have made the diagnosis difficult and would have affected the decision in deciding whether the patient should have been further evaluated by the use of vats. various techniques can be used to perform diagnostic mediastinal biopsies, the most widely used being the use of percutaneous fine - needle aspiration, cervical mediastinoscopy, parasternal mediastinotomy, open biopsy through a thoracotomy, and more recently vats20). vats is currently indicated not only for diagnostic procedures, but also for its versatility, permitting other surgical treatments at the same time. mediastinoscopy is still the most widely used procedure for mediastinal lymph node biopsy, but access to the aorticopulmonary window may be difficult and is limited by the aorta and left main bronchus. however, vats makes it possible to reach all lymph node stations, including the posterior subcarinal, paraesophageal, and prevertebral stations20). the potentially aggressive behavior of relapsed nk / t - cell lymphomas makes early and correct pathological diagnosis and prompt treatment important. the patient described here developed a single mediastinal nodal relapse one month after initial treatment of the nasal nk / t cell lymphoma, and was diagnosed and treated using pet and vats. this patient is still alive 35 months after the initial diagnosis and 30 months after the initial relapse without any evidence of further relapse.	a nasal nk / t cell lymphoma is a very aggressive form of lymphoma. patterns of relapse after treatment have not been systematically evaluated, and mediastinal nodal relapse at a primary site has never been documented. we describe here a 40-year old man who presented with a nasal obstruction caused by a protruding mass that was identified as a nasal nk / t cell lymphoma. the initial work - up, including chest and abdominopelvic computed tomography (ct) and positron emission tomography (pet), showed no regional or distant metastasis. a ct scan performed following three cycles of chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisolone (chop) showed that the mass had nearly disappeared. radiation therapy undertaken following chemotherapy was given to the primary site. however, pet performed following radiotherapy revealed a single mediastinal lymphadenopathy, with no evidence of residual tumor in the nasal cavity. a biopsy using video - assisted thoracoscopy (vats) showed the presence of a recurrent nk / t cell lymphoma with an immunophenotype identical to that of the primary nasal lymphoma. an additional three cycles of chop chemotherapy were administered, and the patient remains alive, with no evidence of disease 30 months after the initial relapse. these findings indicate that early detection with pet and prompt surgical excision with the use of vats can lead to successful treatment of a relapsed nasal nk / t cell lymphoma.
in target - based approaches to drug discovery, linking the observed phenotypic response to a ligand of interest with on - target modulation is a critical step. to this end, both on- and off - targets of a drug candidate need to be identified and characterized prior to clinical development. among many target identification methods, photoaffinity - labeling is particularly attractive, as the transient association of the molecular targets with a drug candidate becomes permanent after photo - cross - linking in the native cellular environment. in addition, for targets that are part of a fragile multiprotein complex, the in situ covalent capture prevents potential loss of the targets after cell lysis. because photoaffinity probes are generally used in excess relative to their targets in order to drive the formation of the target drug complexes, nonspecific targets can also be captured during photo - cross - linking. to overcome this problem, two strategies have been employed : (i) use ligands with higher affinity so that the photoaffinity probes can be used at lower concentrations ; and (ii) use photoaffinity labels that generate reactive intermediates with high, yet selective reactivities toward the ligand - bound targets. to this end, only a few photoaffinity labels have been reported in the past 40 years (chart 1), including phenyl azide, diazirine (da), and benzophenone (bp). while these photoaffinity labels have shown tremendous versatility in biomedical research, nevertheless they have two major shortcomings : (i) the photogenerated nitrene, carbene, and diradical intermediates exhibit extremely short half - lives, leading to very low target capturing yields, and (ii) the nitrone, carbene, and diradical intermediates are prone to react nonselectively with any proximal c h / x h bonds (x = n, o, s), resulting in high background. to balance reactivity with specificity, we envisioned that alternative photogenerated intermediates may exhibit longer half - lives and greater functional group selectivity. indeed, hamachi and co - workers have reported elegant ligand - directed chemistries based on the electrophilic tosyl and acyl imidazolyl groups and demonstrated their exquisite specificity in selective protein target labeling in situ. inspired by this work, we hypothesized that an appropriately functionalized photoreactive tetrazole could serve as a highly selective, electrophilic photoaffinity label for in situ target capture. here we report the development of 2-aryl-5-carboxytetrazole (act, chart 1) as a robust photoaffinity label for identification of the targets and off - targets of dasatinib and jq-1two drugs profiled extensively in the literature. compared with da and bp, act gave higher yields in the ligand - directed photo - cross - linking reactions with the recombinant target proteins. in addition, the act - based probes facilitated the in situ target identification in a manner similar to the da - based ones. whereas the tosyl and acyl imidazolyl groups were successfully employed in labeling endogenous targets in living cells, they are not ideal affinity labels for general target identification because of concerns about their stability in cellular milieu. we hypothesized that act should serve as an ideal photoaffinity label based on the following considerations : (i) 5-carboxy - substituted 2-aryltetrazoles are photoactive ; (ii) placement of a carboxyl group at the c - position of 2h - tetrazole increases the electrophilicity of the photogenerated carboxy - nitrile imine intermediate ; (iii) nucleophilic thiol - addition of 2-mercaptobenzoic acid and 3-mercaptopropionic acid to the base - generated carboxy - nitrile imine was reported in the literature ; and (iv) the photogenerated carboxy - nitrile imine intermediate should undergo rapid medium quenching when a proximal nucleophile is not available (figure s1), minimizing the undesired reactions with nonspecific targets. thus, two series of photoaffinity probes were prepared (see supporting information for synthetic schemes) : one series is based on dasatinib, a potent inhibitor of bcr - abl kinase, the src family kinases, as well as btk (bruton s tyrosine kinase) ; and the other is based on jq-1, a potent inhibitor of the bet family of bromodomain proteins (figure 1a). specifically, three photoaffinity labels, act, da, and bp, were attached to dasatinib or jq-1 via the previously reported linkage sites. the linker length was varied to adjust the distance of the reactive intermediate from the target binding site (figure 1a). an alkyne tag was placed on the photoaffinity labels to enable the click chemistry - mediated detection and enrichment of the targets from cell lysates. to determine how the attachment of photoaffinity label affects the inhibitory activity and specificity, kinome profiling was carried out for dasatinib - derived probes (figure 1b and table s1), while the in vitro binding assay was performed for jq-1-derived probes (figure 1c and table s2). we found that da probes 2a and 2b retained most of their inhibitory activities while act probes 1a and 1b showed modest reduction (220-fold), particularly for 1a with the shorter linker. in comparison, the bp probes exhibited the largest reduction in inhibitory activity (25400-fold), presumably due to the positioning of a large, flat aromatic structure in the solvent - exposed hinge region. for the jq-1 series, almost all the photoaffinity probes demonstrated greater inhibitory activities than jq-1, indicating that the hydrophobic photoaffinity labels form additional interactions with brd2 - 4 outside the shallow and flat canonical binding pocket. in the cell proliferation assays, the photoaffinity label - linked jq-1 probes showed potencies similar to the parent jq-1 against leukemic cell line skm-1, but reduced activities against breast carcinoma mx-1 as well as nonsmall cell lung carcinoma nci - h1299 (table s2), likely due to the permeability difference of the photoaffinity probes and/or the disparate dependency of these cell lines on brd proteins for proliferation. dasatinib and jq-1-derived photoaffinity probes containing 2-aryl-5-carboxy - tetrazole (act), diazirine (da), or benzophenone (bp) photoaffinity label (pal) and their biological activities. (a) structures of the photoaffinity probes. a panel of 82 protein kinases were surveyed in this assay, and inhibition constants, ki, are given in micromolar. (c) plots of the inhibition of brd-2, -3, and -4 by jq-1-derived photoaffinity probes. see table s2 in the supporting information for ki values. to compare the efficiency of these three photoaffinity labels in covalently labeling their targets, we treated recombinant btk and brd4 proteins with appropriate probes, and we detected the photo - cross - linked adducts using in - gel fluorescence analysis after the copper - catalyzed click chemistry with rhodamine - azide. for btk, all probes showed irradiation and ligand - dependent labeling, with 1a and 3a giving the strongest fluorescence (figure 2a and figure s2), suggesting other factors, e.g., click chemistry yield, may also affect the overall labeling efficiency. these probes also selectively labeled btk in the k562 cell lysate spiked with recombinant btk protein (figure s3). for brd4 protein, both act- and da - based probes showed uv light- and ligand - dependent labeling, while bp - based probes 6a/6b exhibited strong background labeling, evidenced by lack of signal attenuation in the presence of jq-1 (figure 2b) as well as labeling of bsa which was added to the reaction mixture to prevent nonspecific binding of brd4 to the plastic surface (figure s4). in the k562 cell lysate spiked with recombinant brd4, act - based probe 4a showed stronger labeling of brd4 than da - based probe 5a, while bp - based probe 6a showed no labeling (figure s5), presumably due to its nonspecific associations with many cellular proteins. evaluating the efficiency and selectivity of photoaffinity - labeling of recombinant proteins by the small - molecule probes. (a) evaluating the btk labeling efficiency using in - gel fluorescence (top panels). for reaction setup, 0.5 g of btk (final concentration 0.1 m), 0.2 m small - molecule probe, 10 m dasatinib (for competition only) in 50 l of pbs were used. for photoirradiation, a hand - held uv lamp with a wavelength of 302 nm for act (5 min) and bp (20 min) or 365 nm for da (10 min) was used. (b) evaluating the brd4 labeling efficiency using in - gel fluorescence (top panels). for reaction setup, 0.4 g of brd4 (final concentration 0.4 m), 0.2 m small - molecule probe, 5 g of bsa, and 10 m jq-1 (for competition only) in 50 l of pbs were used. the equal loading of proteins was verified by sypro ruby staining of the same gels (bottom panels). see supporting information for procedures of click chemistry with tamra - azide and polyacrylamide gel electrophoresis. because the photoaffinity probes with the short linker in general exhibited higher labeling efficiency (figure 2), we decided to focus on these probes in the following comparison studies. to quantify the photo - cross - linking yield, we incubated recombinant protein targets with appropriate dasatinib or jq-1 probes, subjected the mixture to a brief uv irradiation, and analyzed the mixtures by lc - ms. gratifyingly, act - based probes 1a and 4a showed robust photo - cross - linking with their targets, reaching 60% cross - linking yield for 1a (figure 3a) and 95% cross - linking yield for 4a (figure 3d). in contrast, da - based probes 2a and 5a gave the desired photo - cross - linked products in much lower yields (figure 3b, 3e). the control experiment showed that the photoactivation efficiencies are similar between the act and da probes (figure s6). surprisingly, bp - based probes 3a and 6a did not yield any detectable photo - cross - linked adducts ; instead, the recombinant target proteins showed significant broadening of their mass peaks, suggesting the initial photoadducts, if they are formed, may have undergone fragmentation to generate less than expected lower - molecular weight adducts (figure 3c, 3f). an alternative explanation is that the benzophenone serves as a photosensitizer to cause nonspecific oxidative damage to the proteins. importantly, the act - mediated photo - cross - linking with the target protein is ligand - dependent, as addition of dasatinib or jq-1 into the reaction mixture abolished the photoadducts (figure s7). in addition, the photo - cross - linking yield showed probe - concentration dependency as increasing amount of act - probe 4a used in the reaction led to a higher photo - cross - linking yield (figure s8). quantifying the cross - linking efficiency of the photoaffinity labels with recombinant target proteins by lc - ms. (a c) deconvoluted masses of the product mixture after incubating 2.5 m human btk387659 with 25 m dasatinib probe 1a, 2a, or 3a in 100 l of pbs for 15 min followed by photoirradiation with a hand - held uv lamp for 5 min (302 nm for act and bp, 365 nm for da) on ice. (d f) deconvoluted masses of the product mixture after incubating 2.5 m brd444168 with 5 m jq-1 probe 4a, 5a, or 6a in 100 l of pbs followed by photoirradiation with a hand - held uv lamp for 5 min (302 nm for act and bp, 365 nm for da) on ice. the cross - linking yield was calculated using the following equation : yield% = iphotoadduct/(itarget protein + iphotoadduct), where itarget protein and iphotoadduct represent the ion counts of the target protein and the photoadduct, respectively, and marked at the upper - right of the spectra. to identify cross - linking sites on the target protein, we digested the probe 1a - treated recombinant btk protein with trypsin, and analyzed the product mixture by lc - ms / ms. a tripeptide fragment corresponding to btk488490 with the carboxy - nitrile imine linked with the glu-488 side chain was identified (figure 4a). it is noted that recombinant btk387659 protein contains 25 glu and 14 asp residues and only glu-488 was detected as labeled, indicating that the photo - cross - linking is ligand - dependent. this ligand - directed proximity - driven reactivity is consistent with the probe docking model (figure 4b) in which the binding of probe 1a to the kinase active site brings the c of the act ca. 6.9 away from the carboxylate of glu-488 ; indeed, it is the only nucleophilic side chain within 9.0 from the electrophilic site. certainly, because the act is completely solvent exposed and highly mobile, these distances may vary as the act orients itself dynamically relative to the btk protein. we propose that the photoadduct is formed via nucleophilic addition of the glu-488 carboxylate to the carboxy - nitrile imine intermediate followed by a 1,4-acyl shift (figure 4c). this mechanism is consistent with a literature report in which quenching of the in situ generated diaryl nitrile imine by an excess carboxylic acid produced the n-acyl - n-aryl - benzohydrazide product in good yield. it is conceivable that other nucleophiles such as cys (figure s9), if they are in close proximity, may also participate in the cross - linking reactions with act for other targets. since a recent report suggested that the photoreactivity of diaryltetrazole can be harnessed for photo - cross - linking with target proteins through their acidic side chains, we compared the intrinsic reactivity of the carboxy - nitrile imine to that of the diaryl - nitrile imine toward glutamic acid (10 mm) in mixed pbs / acetonitrile (1:1) solution. in the model study, the glutamate - quenching product was clearly detected for the diphenyl - nitrile imine (figure s10). in contrast, the carboxy - nitrile imine underwent predominant chloride quenching when a weak nucleophile such as glutamic acid is present in solution (figure s9), suggesting that the observed photo - cross - linking of 1a with glu-488 of the btk enzyme is not merely the result of elevated local concentration of the glutamate near the in situ generated carboxy - nitrile imine. indeed, because of the rapid chloride quenching of the reactive carboxy - nitrile imine, act should be more suitable as a photoaffinity label than the diaryltetrazoles, as the background cross - linking reactions with the nucleophilic side chains present on protein surfaces would be minimal. determination of the cross - linking site on btk protein and the proposed ligand - directed cross - linking mechanism. the ms / ms spectrum for probe 1a - modified tripeptide fragment, emr, is shown with the fragment ions annotated on the structure. (b) a docking model of probe 1a bound to btk (pdb code : 3k54) showing a proximal glu-488 residue located on a loop 6.9 away from the c of the tetrazole ring. (c) proposed mechanism of the ligand - dependent nucleophilic addition to the carboxy - nitrile imine followed by the o n acyl shift to generate the specific photoadduct. encouraged by high in vitro photo - cross - linking efficiency, we sought to assess the efficiency and selectivity of act as a new photoaffinity label for in situ target identification. for comparison, we included the da - based probes 2a and 5a, as they exhibited excellent biological activities (figure 1) and moderate photo - cross - linking reactivity (figures 2 and 3). in brief, suspended k562 cells were treated with 1 m probe 1a, 2a, 4a, or 5a for 5 h before uv irradiation (5 min for act probe - treated cells at 302 nm ; 10 min for da probe - treated cells at 365 nm). the cells were lysed, and the lysates were reacted with biotin azide prior to pulldown with the streptavidin agarose beads. western blot analyses revealed that the dasatinib targets, btk, src, and csk, and the jq-1 target, brd4, were successfully captured by their respective photoaffinity probes, and pretreating the cells with 50 m parent drug, dasatinib or jq-1, abolished the capture (figure s11). in - gel digestion of the streptavidin captured proteins on sds - page gel followed by lc - ms / ms analyses produced lists of potential targets. to ensure that the captured proteins are derived from the ligand - dependent photo - cross - linking, high - confidence targets were compiled based on the following two criteria : (1) at least two unique peptides were identified in the ms, and (2) the area under the curve (auc)a measurement of ms signal intensity and reliabilityfor the parent drug - pretreated sample is not detectable. using these criteria, six kinases were identified by probe 1a, five of which also appeared in probe 2a - treated cells, indicating act works similarly to da (figure 5a, table s3). however, probe 1a failed to identify abl protein, presumably due to a lack of proximal nucleophilic side chains near the kinase active site. for jq-1 targets, probes 4a and 5a successfully captured the bromodomain proteins brd-2, -3, and -4 with minimum off - targets (figure 5b, table s4), suggesting both act and da are efficient in the in situ target identification. comparison of our data with other literature - reported ms - based target identification studies revealed that these act- and da - based photoaffinity probes performed exceptionally well (tables s5s6). venn diagrams of the identified protein targets by (a) dasatinib - derived photoaffinity probes 1a and 2a ; and (b) jq-1-derived photoaffinity probes 4a and 5a. taken together, we show that act can serve as an effective photoaffinity label for target identification both in vitro and in live cells. compared to the existing photoaffinity labels such as bp and da, the main advantage of act lies in its unique photo - cross - linking mechanism, which in principle should lead to reduced background reactions with nonspecific targets as well as a facile mapping of the ligand - binding site. structurally, act is comparable in size to bp and the electronically stabilized da derivatives such as trifluoromethylaryl diazirine, and it features a modular design with the carboxy group at the c - position of 2h - tetrazole, providing the conjugation handle for a drug molecule and the aryl group responsible for the photoreactivity. compared to da and bp, act showed higher cross - linking yields with the desired targets in vitro (figure 3), but it produced similar efficiency in target capture in situ in a two - step cross - linking / capture procedure (figure s11), suggesting additional optimization of the capture step may be necessary in order to achieve higher overall target capture yield. at present, an alkyne tag was appended onto the aryl ring to enable the click chemistry - mediated target capture. however, alternative chemical moieties that are captured covalently by engineered enzymes, e.g., the haloalkane moiety for halotag and the benzoguanine moiety for snap tag, will be explored in the future for more efficient target capture. because of its unique cross - linking mechanism, a potential drawback of act is that a suitable nucleophile needs to be present near the ligand - binding site for a target to be captured and identified, which may result in false negative ; for example, abl kinase was not identified by 1a in this study. in principle, this limitation can be potentially overcome by increasing the linker length between act and the ligand to allow the survey of a larger area surrounding the ligand - binding pocket. in summary, we have developed a new photoaffinity label, 2-aryl-5-carboxytetrazole (act), for efficient in situ target capture and subsequent identification. the attachment of act to two drug molecules was generally well tolerated without significantly altering the binding affinity and specificity. compared with da and bp, act provides a unique mechanism of target capture through which the photogenerated carboxy - nitrile imine intermediate reacts with a proximal nucleophile near the target active site. as a result, act displayed the cleanest and most efficient cross - linking with the recombinant target proteins in vitro among the three photoaffinity labels tested. in the in situ target identification studies with two previously profiled drugs, dasatinib and jq-1, act successfully captured the desired targets in both cases with an efficiency comparable to da. while aniline was used as the aryl group in the present study, a wide range of heterocycles will be explored in the future with a goal to identify acts with enhanced solubility and photo - cross - linking reactivity. one microliter of 0.5 mm dasatinib in dmso (for competition experiments) or dmso (without dasatinib competition) was added to 0.5 g of btk in 50 l of pbs. after incubation at r.t. for 15 min, 1 l of 10 m photoaffinity probe in dmso was added. after additional incubation at r.t. for 30 min, the mixture was irradiated with a hand - held 302 nm uv lamp, ca. a premixed click reaction cocktail (6 l, 1:3:1:1 of 50 mm cuso4 in water/1.7 mm tbta in 1:4 dmso - buoh/50 mm tcep in water/1.25 mm tamra - azide in dmso) was added, and the reaction mixture was incubated at r.t. for 1 h. after 1 h, 500 l of cold acetone was added, and the mixture was left at 20 c overnight. the mixture was then centrifuged at 17,200 g at 4 c for 20 min and the pellet was collected. to the pellet was added 30 l of 1 sds sample buffer, and the mixture was boiled at 95 c for 10 min before sds - page with 420% bis - tris gel using mops as running buffer. one microliter of 0.5 mm dasatinib in dmso (for competition experiments) or dmso (without dasatinib competition) was added to 0.5 g of btk in 50 l of 2 mg / ml k562 cell lysate in pbs. after incubation at r.t. for 15 min, 1 l of 10 m photoaffinity probe in dmso was added. the mixture was irradiated with a hand - held 302 nm uv lamp, ca. 23 cm from the top of the sample. a premixed click reaction cocktail (6 l, 1:3:1:1 of 50 mm cuso4 in water/1.7 mm tbta in 1:4 dmso - buoh/50 mm tcep in water/1.25 mm tamra - azide in dmso) was added, and the reaction mixture was incubated at r.t. for 1 h. after 1 h, 500 l of cold acetone was added and the mixture was left at 20 c overnight. the mixture was then centrifuged at 17,200 g at 4 c for 20 min, and the pellet was collected. to the pellet was added 30 l of 1 sds sample buffer, and the mixture was boiled at 95 c for 10 min before sds - page with 420% bis - tris gel using mops as running buffer. one microliter of 0.5 mm (+) -jq-1 in dmso (for competition experiments) or dmso (without competition) was added to 0.4 g of brd4 and 5 g of bsa (added to reduce nonspecific binding to the vial surface) in 50 l of pbs. after incubation at r.t. for 15 min, 1 l of 10 m photoaffinity probe in dmso was added. after additional incubation at r.t. for 30 min, the mixture was irradiated with a hand - held 302 nm uv lamp, ca. a premixed click reaction cocktail (6 l, 1:3:1:1 of 50 mm cuso4 in water/1.7 mm tbta in 1:4 dmso - buoh/50 mm tcep in water/1.25 mm tamra - azide in dmso) was added, and the reaction mixture was incubated at r.t. for 1 h. after 1 h, 500 l of cold acetone was added and the mixture was left at 20 c overnight. the mixture was then centrifuged at 17,200 g at 4 c for 20 min and the pellet was collected. to the pellet was added 30 l of 1 sds sample buffer, and the mixture was boiled at 95 c for 10 min before sds - page with 420% bis - tris gel using mops as running buffer. one microliter of 0.5 mm (+) -jq-1 in dmso (for competition experiments) or dmso (without competition) was added to 0.1 g of brd4 in 50 l of 2 mg / ml k562 lysate in pbs. after incubation at r.t. for 15 min, 1 l of 10 m photoaffinity probe in dmso was added. after additional incubation at r.t. for 30 min, the mixture was irradiated with a hand - held 302 nm uv lamp, ca. 23 cm from the top of the sample. a premixed click reaction cocktail (6 l, 1:3:1:1 of 50 mm cuso4 in water/1.7 mm tbta in 1:4 dmso - buoh/50 mm tcep in water/1.25 mm tamra - azide in dmso) was added, and the reaction mixture was incubated at r.t. for 1 h. after 1 h, 500 l of cold acetone was added and the mixture was left at 20 c overnight. the mixture was then centrifuged at 17,200 g at 4 c for 20 min and the pellet was collected. to the pellet was added 30 l of 1 sds sample buffer, and the mixture was boiled at 95 c for 10 min before sds - page with 420% bis - tris gel using mops as running buffer. plates were stamped with 5 l of kinase buffer (life technologies # pr4940d) containing recombinant kinase (2.510 nm final concentration), eu or tb labeled antibodies (his or gst ; 0.52 nm final concentration), and fluorescently tagged probe (3200 nm final concentration). appropriate probes were diluted in kinase buffer, and 120 l of compound was added to the plate using biomex fx. example for btk kinase : btk (invitrogen, pv3363) was added to 5 l of kinase buffer (# pr4940d) to a final concentration of 10 nm, supplemented with 2 nm tb labeled anti - his antibody and 200 nm oregon green labeled probe. afterward, 120 l of diluted compound in kinase buffer was added and the plate was incubated at r.t. for 2 h. the plate was read on an envision plate reader, and the ki values were calculated using the assay explorer software. one hundred million k562 cells were plated in 20 ml of dmem media (5 million cells / ml) without fbs and antibiotics. twenty l of 50 mm unmodified ligand (dasatinib or (+) -jq-1) in dmso (competition experiments) or dmso (without competition, control) was added to the cells, and the mixture was incubated for 30 min (37 c, 5% co2, gentle shaking). afterward, 20 l of 1 mm probe in dmso (all experiments except the control) or dmso (control) was added (final competitor concentration = 50 m, final probe concentration = 1 m), and the sample was kept in the incubator for 5 h (37 c, 5% co2, gentle shaking). after 5 h of incubation, cells were washed twice with 2 ml of pbs and then resuspended in 2 ml of pbs in 35 mm petri dishes. the mixture was irradiated with a hand - held 302 nm uv lamp, ca. pbs was changed to 2 ml of 0.02% tween-20 in pbs, and a protease inhibitor cocktail was added (amresco, # m250). the suspended cells were lysed with sonication (10 10 s with 10 s breaks, 40% power) on ice. the lysate was centrifuged (20 min, 17,200 g, 4 c) and filtered through a 0.2 m membrane. the protein concentration was measured to be 812 mg / ml using the bca assay. click reaction was performed by following a published procedure. in brief, 10 mg of cell lysate was diluted with pbs to 5 ml to obtain a final concentration of 2 mg / ml. to the above solution, 113 l of 5 mm azide - peg3-biotin (aldrich, # 762024) in dmso, 113 l of 50 mm tcep in pbs, 340 l of 1.7 mm tbta in 1:4 dmso - buoh, and 113 l of 50 mm cuso4 were added. the mixture was gently mixed at r.t. for 1 h before 45 ml of acetone after centrifugation, the protein pellet was collected, washed with 2 10 ml of cold methanol, and redissolved in 14 ml of 0.1% sds in pbs. prewashed streptavidin agarose beads (60 l, thermo scientific, # 20347) were added, and the mixture was rocked at 4 c overnight. the beads were washed with 3 1 ml of 0.1% sds in pbs followed by 5 1 ml of pbs. then, 60 l of 2 sds sample buffer was added and the mixture was boiled at 95 c for 12 min before samples were loaded onto sds - page gel. rplc - ms was performed using an agilent 1100 hplc coupled to an agilent lc / msd tof running masshunter workstation acquisition b.04.00. data was deconvoluted in masshunter qualitative analysis b.07.00 using the maximum entropy algorithm with a 0.5 da mass step, proton mass adduct, and baseline subtract factor 7.0. the site of modification of btk by probe 1a was determined by in - gel trypsin digestion of the band corresponding to the protein after labeling with 1a as described in the following reference : shevchenko, a. evaluation of the efficiency of in - gel digestion of proteins by peptide isotopic labeling and maldi mass spectrometry. lc - ms / ms analysis was performed using a waters nanoacquity hplc system coupled to a thermo fisher scientific fusion mass spectrometer. separation of the peptides was achieved using a thermo easyspray pepmap column (es802 ; c18, 2 m, 100, 75 m 25 cm) at a flow of 0.25 l / min, with a gradient starting at 5% b (b = 0.1% formic acid in acetonitrile, a = 0.1% formic acid in water), ramping to 15% b at 2 min, 1535% b over 20 min, followed by a 5 min ramp to 80% b, washing for 6 min at elevated flow (0.4 l / min), before returning to the starting conditions. the fusion source was operated at 1.9 kv in positive ion mode with ms detection in the orbitrap using 120 k resolution. the modified tripeptide was identified by its fragmentation spectrum that resulted from quadrupole isolation of the triply charged ion using an isolation window of 1.8 m / z, and fragmentation via hcd at 26% collision energy, with fragment ion detection in the orbitrap at 15k resolution. protein from in situ enriched samples was eluted from beads with 100 l of 2 lds - page sample buffer (invitrogen ; 141 mm tris base, 106 mm trishcl, 2% lds, 10% glycerol, 0.51 mm edta, 0.22 mm serva blue g, 0.175 mm phenol red, ph 8.5), and the mixture was heated to 80 c for 10 min. a 20-l sample was applied to sds - page running with 412% bis - tris gel and mops running buffer to remove the detergent. in - gel digestion five microliters, representing 10% of each sample, was loaded via waters nanoacquity autosampler onto an acclaim pep map precolumn (p / n 164535) with online trapping and salt removal (trapping flow rate at 5 l / min for 3.5 min). analytical separation was performed over a 90 min run using an easy spray column (es802) heated to 45 c. reverse phase gradient was delivered at a flow rate of 0.225 l / min by waters nanoacquity hplc as follows : 0 min 10% b, 55 min 25% b, 60 min 40% b, 60.1 min 98.0% b, 65.1 min 10% b, 89.0% b, where b is 0.1% formic acid in acetonitrile. spectra were collected on a thermo fisher scientific fusion mass spectrometer using the following parameters : 2.1 kv spray voltage, 275 c transfer tube temperature, 3501500 m / z scan range with a quadrupole isolation window of 1.6 m / z, ms1 in the orbitrap at 120 k resolution, ms2 by cid in the ion trap with rapid speed, ms2 scans collected with top speed 3 s cycle, dynamic exclusion with repeat count 1 if occurs within 30 s and exclude for 60 s. mips on with charge states 27 allowed with 4e5 agc orbitrap and 2e3 ion trap settings. raw files were processed by proteome discoverer (v 2.1.081) and searched by mascot (v 2.4.0) using the uniprot human database (downloaded 08 - 10 - 2015). ms1 tolerance was set to 20 ppm, and ms2 tolerances were set to 0.8 da. label - free quantitation was performed with the precursor ions area detector function. areas under the curve (aucs) less than 2.0e5 were determined below loq based on previous studies on the performance of the instrument using proteomic reagent standards. thresholds per experiment were set for significant differences dependent upon the determination of potential sample loading bias by comparing total ion chromatogram (tic) intensity between paired injections (competition) and average signal from nonspecifically binding protein background.	photoaffinity labels are powerful tools for dissecting ligand protein interactions, and they have a broad utility in medicinal chemistry and drug discovery. traditional photoaffinity labels work through nonspecific c h / x h bond insertion reactions with the protein of interest by the highly reactive photogenerated intermediate. herein, we report a new photoaffinity label, 2-aryl-5-carboxytetrazole (act), that interacts with the target protein via a unique mechanism in which the photogenerated carboxynitrile imine reacts with a proximal nucleophile near the target active site. in two distinct case studies, we demonstrate that the attachment of act to a ligand does not significantly alter the binding affinity and specificity of the parent drug. compared with diazirine and benzophenone, two commonly used photoaffinity labels, in two case studies act showed higher photo - cross - linking yields toward their protein targets in vitro based on mass spectrometry analysis. in the in situ target identification studies, act successfully captured the desired targets with an efficiency comparable to the diazirine. we expect that further development of this class of photoaffinity labels will lead to a broad range of applications across target identification, and validation and elucidation of the binding site in drug discovery.
bacterial cells from the species escherichia coli specifically interact with the lower gastrointestinal tracts (including the microbiotas) of animals and humans, a relationship resulting from a long - term coevolutionary process that has shaped the well - defined e. coli core genome. we fully agree with the proposal that e. coli should be treated as a single microorganism, in spite of the fact that strains are often classified according to their intestinal pathotypes, including as enteropathogenic, enterotoxigenic, enterohemorrhagic, enteroaggregative, enteroinvasive, adherent - invasive (1), and, in the case of o104:h4, enteroaggregative hemorrhagic e. coli (eahec). all of these e. coli pathotypes have essentially the same core genome (comprising about 2,000 genes) maintained by vertical descent. during the course of e. coli s evolution, a basic type of the e. coli genome seems to have been complemented by the acquisition by horizontal transfer of different adaptive (including pathogenic) genes, genes that are propagated by introgressive descent (2). the outcome is the emergence of a variety of strains with different colonization or pathogenic abilities. from this perspective, it may be inappropriate to link the term pathogenic to particular serotypes or e. coli multilocus sequence types (mlsts). classic pathogenic types might correspond to those types where the acquisition of pathogenicity traits (pts) has been documented with particular frequency. however, not all e. coli serotypes or sequence types (sts) are equally distributed in all habitats, indicating that a number of noncore genes have evolved to provide different degrees of ecological specificity, eventually leading to some kind of ecological barrier for intraspecies genetic exchanges among e. coli genomes from different ecological environments (3). certainly not all pathogenicity traits are equally distributed among e. coli serotypes or sts. that probably means that a highly pathogenic clone emerges when an e. coli type able to sustain particular environmental interactions accumulates pathogenic genes. the strains belonging to the o104:h4 serotype (st678, phylogroup b1), responsible for the severe german outbreak of bloody diarrhea and hemolytic - uremic syndrome in 2011, illustrate this concept. in their paper in mbio, comparative genomics of recent shiga toxin - producing escherichia coli o104:h4 : short - term evolution of an emerging pathogen, grad. (5) indicate that a rapid introgressive evolution has occurred in these strains by sequential acquisition of foreign genetic material, including such pathogenicity traits as shiga toxin and aggregative - adherence fimbriae. the result is a highly pathogenic behavior for e. coli in humans, but what are the evolutionary benefits for the bacterial organism ? it seems likely that e. coli o104:h4 has undergone selection in some way or another in the recent past and has enlarged its population size and/or improved its adaptation to multiple habitats. the short - term evolution indicated by grad. (5) and the cumulative acquisition of pathogenic traits require frequent and extensive ecological and genetic interactions with other bacterial (donor) cells, probably requiring a large number of cells and/or very effective dispersal, according to the genetic - capitalism principle (6). studies based on whole - genome sequencing of several e. coli o104:h4 (st678) strains isolated along the last few years revealed strong genetic differences in chromosomal and plasmid content (7). an unexpectedly high number of recombinant genes (125 genes) was found, and interestingly, the possible donors of these genes were not clustered in a single e. coli phylogenetic group. in fact, in half of cases, the recombinant genes contained sequences from donors in six phylogenetic groups. even though the possibility of extensive recombination with a highly mosaic donor strain of another phylogroup can not be totally excluded, differences among e. coli o104:h4 strains suggest separate sites and events in their recent evolutionary history. this implies a dense network of interactions with other bacteria. what could have been the necessary context for these interactions ? the simple answer is that organisms evolve from their natural reservoirs, where a sufficient population size can be reached. this natural reservoir, that is, the optimal environment for the reproduction, maintenance, and evolution of e. coli o104:h4, remains uncertain (8). e. coli is a normal component of the microbiotas of humans and animals, the natural place where e. coli strains might genetically interact with other e. coli strains and with many other proteobacteria. strains of serotype o104:h4 have been found mostly in association with sporadic cases of persistent or severe diarrhea since the 1990s, but little is known about their prevalence in healthy humans. classic and modern studies from the 1960s indicate that o104 strains were scarcely found among normal human hosts at that time. it is worth noting that investigations during the outbreak in germany also discarded the possibility of a cattle reservoir, unlike with other shiga toxin - producing e. coli strains. we agree with grad. that the information about o104 epidemiology depends on the resolution of the technology used and the number of samples studied (9). at this time, the enemy - within - us hypothesis (10), that is, that humans are the only natural reservoir for e. coli o104:h4, should be seriously considered. it seems possible that humans can be colonized with such low numbers of the strain that they would be effectively invisible to classical culturing methods. this possibility is compatible with the observation of poor interhuman transmission among individuals in highly affected areas who share a household (11). these data seem to indicate that e. coli o104:h4 has gained little reproductive advantage from its amazing ability to collect pathogenic determinants, either in human hosts or in cattle. work (5) require an understanding of the circumstances that allow fertile interactions between e. coli o104:h4 and other bacteria that may donate pathogenicity genes. of course, big population sizes would facilitate these contacts, but it seems that o104:h4 does not form large populations. the evolutionary benefits of acquiring these donated traits are not expressed as high reproduction rates. however e. coli o104:h4 might have evolved not as an r - strategist (oriented to maximal reproduction) but as a k - strategist (with lower numbers of individuals but higher efficiency in niche exploitation). well established in its intestinal niche, and with a population density below the threshold for producing illness, e. coli o104:h4 might have had the opportunity of interacting with many other transient and resident e. coli strains and eventually with donors of pathogenicity traits. donors of pathogenicity genes are probably available in human feces with a higher frequency than is usually assumed. genes encoding one of the key pathogenicity traits of e. coli o104:h4, shiga toxin (stx genes), were detected in norway in 13% of healthy volunteers and in 36% of samples from patients (12). each gain of new pathogenicity genes by e. coli o104:h4 contributes to a deeper exploitation of the intestinal niche. note that along this process, a physical and functional convergence with other e. coli strains which have expanded their populations as a result of harboring colonization and/or antibiotic resistance traits is expected to occur. increased connectivity facilitates further community genetic exchanges building, as in the case of shigella (an e. coli derivative), a quasi - species group (fig. 1). schematic space - time diagram illustrating the possible dynamics of the combinatorial evolution of pathogenicity traits (pts) in e. coli. at the bottom of the figure, in light red, yellow, light blue, and violet, are the isolated strains containing single pts. such traits might increase the local spread of these strains ; genetic interactions between pairs of them (white squares) produce novel strains with 2 pts (the central box in the white square arises from the interaction of the flanking strains). following new local spreads, strains with 2 pts (like those in green), interact with other pt - containing strains, giving rise to strains with increasing numbers of pts, resulting in multi - pt strains (shown in dark red, dark blue, and gray at the top of the figure) of different evolutionary trajectories. other possibilities to explain the promiscuous life of e. coli o104:h4 in spite of its seemingly low population density remain to be explored. this strain was consistently found in a common food (salad) vehicle involved in the germany and france outbreaks, fenugreek sprouts (trigonella foenum - graecum, family fabaceae), presumably of african origin. might this legume, used on this continent both for animal pasture and for human food, be something more than a vehicle ? could o104:h4 be ecologically associated (and genetically interact) with the root nodule bacteria of this plant ? the possibility of an endemic status of e. coli o104:h4 in humans in central africa has recently been suggested (8). was this plant exposed during agricultural farming to animal and human sewage ? we should clarify the field (network) of interaction giving rise to the outbreak strain. as in a crowdsourcing software project, the evolution of o104:h4 starts with an initial plastic bacterium project onto which new elements, including pathogenicity genes, are anonymously contributed by similar bacteria. at the end, we have different combinations of elements that develop and grow continuously within the crowdsourcing community (fig. 1). surveilling a reduced number of conserved genes can produce deceiving results, because pathogenicity genes are frequently allocated to mobile modules with little sequence conservation. the availability of next - generation sequencing (ngs) technologies allows us to reveal the complete gene landscape of a bacterium and, moreover, of a community of bacteria. this new perspective on outbreaks circumvents the old bias of looking only at specific conserved genes belonging to the causative agents. ngs enables scientists to trace the importance of gene units that move freely between strains and to analyze the flux of these genes between bacterial communities. in the postgenomics era, ngs technologies are providing public health efforts with advanced and quick tools that allow researchers not only to retrospectively analyze the epidemiological evolution of an outbreak but also to predict its future evolution.	abstracta recent study published in mbio [y. h. grad., mbio 4(1):e00452 - 12, 2013 ] indicates that a rapid introgressive evolution has occurred in escherichia coli o104:h4 by sequential acquisition of foreign genetic material involving pathogenicity traits. o104 genetic promiscuity can not be readily explained by high population sizes. however, extensive interactions leading to cumulative assemblies of pathogenicity genes might be assured by small k - strategist populations exploiting particular intestinal niches. next - generation sequencing technologies will be critical to detect particular gene cocktails as potentially pathogenic ensembles and to predict the risk of future outbreaks.
crustaceans like other invertebrates only have innate immunity, including many immune molecules to eliminate exogenous pathogens. to date, many studies have verified that hemocyanin is an important nonspecific innate immune defense molecule and can provide an effective immune defense in arthropods [28 ]. its primary function is to transport and store oxygen and also to participate in osmoregulation, molt cycle, exoskeleton formation, and melanin synthesis [1012 ]. so far, studies on the immunologic function in crustacean are mainly focused on the hemocyanin itself or its degraded peptides, which have hemolytic activity, agglutination property, and antiviral function [2, 4, 5, 7, 13 ]. hemocyanin genes have been cloned and characterized in certain crustacean species such as litopenaeus vannamei, eriocheir sinensis, cherax quadricarinatus, homarus americanus, and caridina multidentata and atyopsis moluccensis. copper is a central component of hemocyanin and a cofactor of many other enzymes, like superoxide dismutase, cytochrome oxidase, and lysyl oxidase [19, 20 ]. the dietary copper level can affect crustacean immune responses in penaeus monodon and e. sinensis. it was found that the expression of hemocyanin mrna in e. sinensis was affected by dietary copper level. investigation of the functional relationship between dietary copper and hemocyanin mrna expressions can provide better understanding on crustacean innate immunity and offer insight into disease control through dietary management in shrimp farming. we hypothesize that copper as a major component of hemocyanin can impact prawn innate immunity through dietary copper manipulation. oriental river prawn (macrobrachium nipponense) is an important aquaculture species in china and other southeast asian countries. various diseases have been found in the m. nipponense farming population due to intensive culture and environmental pollution. for example, the bacterial disease induced by aeromonas hydrophila is one of the major diseases which can cause 30% death of prawn, sometimes as high as 70%. as a result, the investigation on the m. nipponense innate immune mechanism against a. hydrophila has become a key issue of health management in crustacean farming. the hemocyanin subunit is a functional group for crustacean immunity, and a hemocyanin subunit has been cloned and characterized in the freshwater prawn m. nipponense. in this study, we discovered another hemocyanin subunit in m. nipponense (mnhc-1). to understand the role of mnhc-1 in m. nipponense immunization, (1) the full - length cdnas of mnhc-1 was cloned, (2) the distribution of mnhc-1 in different tissues was examined, (3) the mrna expression of mnhc-1 in immune defense after prawn challenge with a. hydrophila was examined, and (4) the relative expression level of mnhc-1 in the hepatopancreas of m. nipponense fed different levels of copper was quantified. all adult oriental river prawns (m. nipponense) were obtained from a local farm in shanghai. the hepatopancreas, muscle, gill, ovary, intestine, heart, and stomach were collected from healthy prawn, flash - frozen in liquid nitrogen, and stored at 80c until rna extraction. a total of 1 ml hemolymph was collected from the ventral sinus using a sterile syringe and diluted using half volume of anticoagulant solution, then centrifuged at 8000 g for 10 min at 4c to collect the hemocyte, and stored at 80c immediately until rna extraction. prior to the challenge experiment, the adult prawns were acclimatized in the laboratory for 2 weeks. a total of 300 healthy prawns were randomly divided into two groups with five replicates. according to the preliminary experiment, the prawn in the bacterial challenge trial was injected with 100 l a. hydrophila in saline suspension (1 10 cfu / ml) obtained from shanghai ocean university, while each prawn in the control group received the same volume of saline injection. after injection, prawns were put back to the rearing tanks, and hemocyte samples were collected at 0, 3, 6, 12, 24, and 48 h after injection, centrifuged, and stored at 80c for rna extraction. the juvenile m. nipponense were obtained from the same farm as the adults and acclimated for two weeks in the laboratory conditions prior to the feeding trial. the basal diet was supplemented with copper sulphate (analytical reagent, shanghai chemical co., shanghai, china) at 0, 10, 20, 30, 40, 80, and 160 mg cu kg diet, respectively. the actual copper concentrations in the feeds were analyzed to be 2.8, 12.2, 20.9, 29.8, 43.1, 78.9, and 157.1 mg kg, respectively, by the flame atomic absorption photometry. prawn juveniles (0.101 0.001 g) were randomly placed in 21 of 300-l tanks with 30 prawns per tank in triplicate. prawns were fed to apparent satiation twice daily (8:00 and 17:00 h) for 56 days. to maintain water quality, one - third of the tank water was exchanged daily. during the feeding period, l, and total ammonia nitrogen 0.05). those subunits differ considerably in their primary structures and are encoded by distinct genes. in this study, we cloned and characterized the expression pattern of one hemocyanin subunit from m. nipponense (mnhc-1). mnhc-1 was a polypeptide of 675 amino acids with a 21-amino acid putative signal peptide. the signal peptide ends ala - x - ala motif, which is a frequent accordance prior to the cleavage site of signal peptides, suggesting that a cleavage site is located at the 21 - 22 amino acids [32, 35 ]. structurally, mnhc-1 has conservative copper - binding domains including six histidine residues (h212, h216, h242, h362, h366, and h402) of the copper - binding sites ; this domain agrees with other crustaceans [4, 14, 15 ]. based on immunological methods, the crustacean hemocyanin subunits are classified into three distinct subunit types : alpha, beta, and gamma. the phylogenetic analysis showed that mnhc-1 and mnhc-2 belong to separate clade ; mnhc-1 belongs to the gamma subunit which has evolved at a later time compared to alpha and beta subunits in freshwater shrimps. the present study showed that the highest level of mnhc-1 mrna expression occurred in the hepatopancreas. the result is the same as the findings in other crustaceans, such as h. americanus, fenneropenaeus chinensis, and e. sinensis, and consistent with the report that hemocyanin synthesis occurs mainly in the hepatopancreas. mnhc-1 expression was detected in all the examined tissues except for muscle, which is different from another subunit of hemocyanin in m. nipponense (mnhc-2) expressed in the muscle. we also found that mnhc-1 was expressed in ovary, whereas mnhc-2 was hardly expressed in ovary. the discrepant expression patterns of these two subunits of hemocyanin may be owing to their functional specialization in different tissues. hemocyanin is an important multifunctional protein in mollusks and arthropods. besides its role as an oxygen carrier, its immune functions including antibacterial activities, agglutination property, and po activity have become hot topics of immunological research [2, 4, 5, 7 ]. zhang. found that the main protein directly bound to the vibrio alginolyticus, vibrio harveyi, a. hydrophila, and staphylococcus aureus in l. vannamei serum was hemocyanin, suggesting that hemocyanin possesses antibacterial functions. the c - terminus of l. vannamei hemocyanin is possibly related to the immunity in shrimp to different pathogens. gram - negative bacteria a. hydrophila are a common species of the aeromonas genus in water and water habitants. the infection of a. hydrophila in fish and prawn including m. nipponense has been one of the major diseases under farming conditions [25, 41 ]. sun. showed that the hemocyanin gene expression of e. sinensis was significantly upregulated by a. hydrophila infection. in our study, temporal and spatial expressions of mnhc-1 in the hemocytes of prawn infected with a. hydrophila showed a clear time - dependent pattern. the level of mnhc-1 mrna expression significantly decreased at 3 h after injection, then started to significantly increase after 6 h and 12 h, and then reached the peak at 12 h, implying that the hemocyanin is involved in the antibacterial defense of prawn. in another study, the level of mnhc-2 mrna expression in prawn significantly increased over time and peaked at 3 h after the a. hydrophila challenge. lei. found a similar result in p. japonicus that pjhcl transcriptional upregulation occurred after 4 h of injection of the active wssv. it is possible that pjhcl may be triggered by the fast expressed proteins in virus. it is also likely that mnhc-1 may be induced by fast expressed protein in bacteria, but the detailed defensing mechanism of hemocyanin against bacterial infection needs further study. the expression of hemocyanin subunits varies with environmental or nutritional changes [42, 43 ]. our present study showed that the level of dietary copper affected the hemocyanin gene expression in prawn. the level of mnhc-1 mrna in hepatopancreas of the prawn fed 43.1157.1 mg cu kg diet was significantly higher compared to that fed 2.829.8 mg cu kg. the increase of dietary cu concentration also increased the cu content in hepatopancreas, especially in the 43.1157.1 mg cu kg groups. therefore, we suggest that the high level of copper content in the hepatopancreas can trigger mnhc-1 mrna expression, whereas the optimal level of dietary copper (2040 mg kg diet) can increase hemocyanin mrna expression in the hepatopancreas and hemocytes of crab. these studies suggest that the effect of dietary copper level on hemocyanin mrna expression is species - specific. it seems that the two subunits of hemocyanin may have structural functions in their own hemocyanin and show different response to the level of dietary copper. dietary copper may be first used for hemocyanin synthesis as low dietary copper did not reduce the level of mnhc-1 mrna. in conclusion, we cloned the hemocyanin subunit gene (mnhc-1) from the hepatopancreas of m. nipponense. our results suggest that mnhc-1 may play a critical role in antibacterial defense in prawn. these results provide the foundation for further studies in biological function and regulation of hemocyanin in crustacean.	hemocyanin is a copper - containing protein with immune function against disease. in this study, a hemocyanin subunit named mnhc-1 was cloned from macrobrachium nipponense. the full - length cdna of mnhc-1 was 2,163 bp with a 2,028-bp open reading frame (orf) encoding a polypeptide of 675 amino acids. the mnhc-1 mrna was expressed in the hepatopancreas, gill, hemocytes, intestine, ovary, and stomach, with the highest level in the hepatopancreas. in the infection trial, the mnhc-1 mrna transcripts in the hemocytes were significantly downregulated at 3 h after injection of aeromonas hydrophila and then upregulated at 6 h and 12 h, followed by a gradual recovery from 24 to 48 h. the mnhc-1 transcriptional expression in the hepatopancreas was measured after m. nipponense were fed seven diets with 2.8, 12.2, 20.9, 29.8, 43.1, 78.9, and 157.1 mg cu kg1 for 8 weeks, respectively. the level of mnhc-1 mrna was significantly higher in the prawns fed 43.1157.1 mg cu kg1 diet than in that fed 2.829.8 mg cu kg1 diet. this study indicated that the mnhc-1 expression can be affected by dietary copper and the hemocyanin may potentially participate in the antibacterial defense of m. nipponense.
gastric neuroendocrine tumours (nets) account for more than 20% of all diagnosed gastric tumours. in recent years, the incidence of these tumours has increased substantially, probably due to an increase in the number and quality of diagnostic tests. these tumours can be classified into three main types according to their clinical and histological characteristics. the most common are type 1 tumours (accounting for 7080% of all gastric nets), which present with achlorhydria - induced hypergastrinemia secondary to chronic atrophic gastritis and are commonly associated with b12 malabsorption and pernicious anemia. most type 1 nets are grade 1 (g1), with metastasis present in only 25% of patients at diagnosis. type 2 tumours are the least common (56% of gastric nets) and are sporadic tumours associated with zollinger - ellison 's syndrome or, more commonly, related to type 1 multiple endocrine neoplasms (men-1 syndrome). type 3 gastric nets are sporadic, often solitary, and usually higher - grade lesions (g3 being the most common). type 3 nets, unlike types 1 and 2, are not associated with elevated gastrin levels. they are typically larger lesions, may be ulcerated, and are capable of inducing carcinoid syndrome (producing 5-hydroxytryptophan). treatment of metastatic gastric nets is highly complex, and a multidisciplinary approach is considered essential to assure that all therapeutic options are considered. in oligometastatic cases, nccn and somatostatin analogues have been used to decrease gastrin levels, and there is evidence to suggest that use of these drugs can also reduce recurrences [5, 6, 7, 8 ]. here we present a highly unusual case involving a patient diagnosed with a well - differentiated g2, type 3 gastric net with exclusive metastatic bilateral ovarian involvement. to our knowledge, this is the first such case reported in the literature, as the cause of ovarian involvement is usually due to local invasion in the context of patients with major abdominal involvement rather than metastasis. a 35-year - old hpv-18-postive female, without any previous personal or family medical history of interest, underwent a routine follow - up examination for cervicitis in december 2013. a cone biopsy was performed, and a diagnosis of early infiltrating cervical adenocarcinoma was made. the maximum tumour thickness was 0.7 mm extending 2 mm horizontally and adjacent to an in situ adenocarcinoma (5 mm in the largest diameter) located 2 mm from the upper border of the endocervix. the clinical stage was pt1a2. given these findings, together with the fact that the patient was nulligravida but with reproductive desires, a radical trachelectomy plus sentinel node biopsy was planned. in accordance with established protocol, pelvic magnetic resonance imaging was performed (january 2014) prior to surgery, revealing a solid, slightly heterogeneous lesion in the right ovary measuring 27 24 mm and an intracapsular lesion (10 mm 8 mm) in the left ovary. no pathological findings were observed at the cervical level, and no evidence of lymphadenopathy was observed. given these highly unusual radiological findings bilateral involvement of the ovaries due to leiomyomas the multidisciplinary tumour board recommended a biopsy of both ovaries. a bilateral ovarian laparoscopy (january 29, 2014) revealed a solid 25-mm lesion in the right ovary and a 15-mm lesion in the left ovary. based on the pathological examination of the intraoperative biopsy, the preliminary finding was a right ovarian cyst with areas of carcinoma. consequently, trachelectomy was ruled out and instead a right adnexectomy was performed. the definitive pathological findings were an infiltrating low - grade net tumour (ki-67 = 5%) of likely gastrointestinal origin. the gastroscopy revealed a tumour measuring approximately 4 cm in diameter with central ulceration located in the greater curvature of the stomach. the biopsy revealed a well - differentiated, non - gastrin - producing g2 net (ki-67 = 7%). in addition to the endoscopic tests, somatostatin receptor scintigraphy (octreoscan) and fdg - positron emission tomography (fdg - pet / ct) were performed to assess disease dissemination : both tests revealed pathological uptake in the body - fundus junction, in the gastrohepatic lymph nodes, and in the left ovary. chromogranin levels were 105 ng / ml, while urinary 5-hydroxyindoleacetic acid (5-hiaa) levels were 21 mg / day. 2 show the results of the fdg - pet / ct scans. based on these findings, the case was presented to the multidisciplinary net board, summarized as follows : 35-year - old female with a history of endocervical microinfiltrating adenocarcinoma leading to an incidental diagnosis of a well - differentiated g2 gastric net with metastatic nodal involvement in a single locoregional area with bilateral involvement of the ovaries, treated previously with right adnexectomy. given the patient 's young age and lack of comorbidities, the tumour board following clinical guidelines recommended surgical treatment of the affected structures (left ovary, stomach, and gastrohepatic lymph nodes). in late march 2014, a complete laparoscopic gastrectomy was performed, with esophagojejunal anastomosis and d1 lymphadenectomy expanded to include nodal stations 79. the postoperative pathological report showed the following : complete gastrectomy with a well - differentiated g2 net invading the entire gastric wall into the serosa, with metastasis in 1 out of 17 dissected nodes. on immunohistochemistry, the tumour was positive for ae1ae3, chromogranin, synaptophysin, cdx-2, and negative for gastrin. given these findings, consequently, a laparoscopic hysterectomy with left adnexectomy was performed in may 2014 without complications. in the postoperative period, the patient progressed favourably with acceptable gastrointestinal tolerance except for clinical signs of occasional dumping. pathological findings from the surgical specimens indicated metastasis in the left ovary from a well - differentiated g2 tumour, no evidence of disease in the uterus, and foci of mild dysplasia in the endocervix. after both surgeries, a thoracoabdominal - pelvic ct and an octreoscan (fig. the images showed that the patient was free of macroscopic disease, and laboratory results for chromogranin a and 5-hiaa acid levels were normal. in july 2014, the patient had recovered from both surgeries and was in good general condition with tolerable symptoms associated with early onset menopause and total gastrectomy (occasional episodes of dumping in addition to hot flushes). currently, the patient is free of disease, with excellent performance status (100%). the case reported here involves a young woman with an incidental diagnosis of a type 3, g2 gastric net with bilateral ovarian involvement reminiscent of krukenberg 's tumour, which is typical in gastric adenocarcinomas. at present, the route of spread from the stomach to the ovaries in such cases is not well understood, although diverse mechanisms have been postulated, including lymphatic dissemination (case reports have described gastric adenocarcinomas confined to the mucosae with lymphatic infiltration and ovarian metastases), and haematogenic or peritoneal dissemination [10, 11 ]. gastroscopy is the main diagnostic and follow - up technique in patients with a gastric net. ultrasound - guided endoscopy is indicated to assess the extent of gastric wall invasion. for full staging, the present case was managed in accordance with published guidelines, which recommend surgical treatment of oligometastatic disease whenever possible [3, 4 ]. after surgery, the patient was free of disease, and we elected to perform active surveillance with regular ct and octreoscans. we did not administer any adjuvant treatment in this case because the current evidence does not support the use of adjuvant therapy in patients without postsurgical evidence of disease. gastric nets should be managed in a center with an experienced multidisciplinary team that can offer the patient a full range of therapeutic alternatives. although progress has been made in the systemic treatment of advanced nets in recent years, systemic treatment options remain limited [5, 6 ]. other reported cases are mostly in the context of very advanced disease in which curative - intent surgery is not feasible. in this case, however, in patients with a positive octreoscan following surgery, adjuvant treatment with somatostatin analogues may be beneficial in low - grade tumours such as the one reported here written informed consent was obtained from the patient for publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor of this journal. the translation and editing of the manuscript was funded by ipsen pharma s.a., who also paid for the article processing charges related to publication.	treatment of metastatic gastric neuroendocrine tumours (nets) is challenging. in oligometastatic cases, surgical resection is recommended whenever possible. somatostatin analogues have been used to decrease gastrin levels, and available evidence suggests that these drugs can also reduce recurrences. here we present a highly unusual case involving a patient with a well - differentiated grade 2, type 3 gastric net with exclusive metastatic bilateral ovarian involvement. to our knowledge, this is the first such case reported in the literature, as the cause of ovarian involvement is usually due to local invasion rather than metastasis. we believe this case is of interest not only due to the unusual presentation, but also because it makes us consider adjuvant treatment with somatostatin analogues in patients with low - grade tumours and a positive postoperative octreoscan.
moxifloxacin is a fourth - generation fluoroquinolone antibiotic that exerts its effects by trapping a dna drug enzyme complex and specifically inhibiting atp - dependent enzymes topoisomerase ii (dna gyrase) and topoisomerase iv. currently, moxifloxacin is being extensively used in the treatment of respiratory system diseases such as community - acquired pneumonia (cap), chronic bronchitis (cb) and chronic obstructive pulmonary disease (copd) for the broad spectrum of antimicrobial activity against respiratory tract pathogens, including gram - positive and gram negative organisms, anaerobic bacteria, and atypical respiratory tract pathogens [14 ]. the favorable pharmacokinetics of moxifloxacin, including a high mean apparent volume of distribution and a long terminal half life, supports a once - daily dosing regimen in the treatment of infectious disease. it is revealed that moxifloxacin is primarily eliminated in the liver. in recent years, a variety of methods on high - performance liquid chromatography (hplc) for measuring moxifloxacin concentration in plasma have been reported. fluorescence detector was applied in several methods for its advantage of sensitivity [712 ]. however, some complex techniques such as gradient elution and on - column focusing, precolumn derivatisation, or special column, were employed. in addition, these expensive specific instruments would increase the experiment cost and not brief enough for clinical application. although a few methods applied hplc with uv detector to determine moxifloxacin in plasma [1316 ], automated extraction methods with a polymeric cartridge, poor extraction recovery, or complicated flow phase were involved. lc / esi - ms / ms methods have also been reported [17, 18 ], but these advanced techniques are not suitable for clinical routine. in this paper, we describe a rapid and simple hplc method for determination of moxifloxacin in human plasma using ultraviolet (uv) detection that allows rapid processing of large number of plasma samples. the method employed a simple mobile phase of weak acidity, permitting the stability of moxifloxacin, and the application of kromasil c18 column ensures the efficient separation of the drug. ciprofloxacin was used as is in the method, which exhibit goods separation of the two kinds of fluoroquinolones. hplc grade acetonitrile, methanol (tianjin xiehe haopeng chromatography technology co., ltd., china), triethylamine (tianjin bodi chemical industry co., ltd., china), and phosphoric acid (shijiazhuang chemical factory, china) were used for the hplc analysis. the hplc system (shimadzu chromatography division, japan) consisted of a pump (lc-10at), a uv detector (shimadzu, spd-10avp plus), and autosampler with built - in system controller. chromatographic separation of moxifloxacin was achieved using a kromasil c18 (250 mm 4.6 mm, 5 m) reverse phase analytical column. the mobile phase consists of a mixture of 0.1% triethylamine (adjusted ph to 4.8 with phosphoric acid)/acetonitrile (80/20, v / v). the mobile phase was pumped at an isocratic flow rate of 1 ml / min at room temperature. the wavelength of uv detection was set at 296 nm for moxifloxacin. a stock solution (5 g / ml) of moxifloxacin was prepared by dissolving 500 g of drugs in 100 ml of mobile phase. working solutions were prepared from the stock solution by sequential dilution with mobile phase just before use. all solutions were protected from light by covering them with aluminium foil and stored in brown volumetric flask at 4c. plasma samples 200 l were transferred to a 1.5 ml centrifuge tube, and then 40 l of is stock solution (5 g / ml) was spiked., 1 ml acetonitrile was added. after being vortexed for 3 min, the organic layer was transferred to another centrifuge tube and evaporated at 40c under a stream of air. then, the dried extract was reconstituted with 200 l of mobile phase, and a 60 l aliquot was injected into chromatographic system. selectivity of the assay method was assessed by evaluating potential interference from endogenous compounds in the blank plasma as well as the separation of moxifloxacin and is. the separation efficiency of moxifloxacin and is and probable endogenous compounds from plasma was checked by comparing the chromatograms of blank plasma, blank plasma with is, and blank plasma with is plus moxifloxacin. the peak and the retention time for each drug under the chromatographic conditions of the moxifloxacin assay were recorded. in addition, chromatogram of pure standard analyte and is in mobile phase was evaluated as a control. to 200 l of blank human plasma 1, 2, 4, 20, 40, 100, and 200 l of moxifloxacin working solution were added, yielding final concentrations of 0.025, 0.05, 0.1, 0.5, 1.0, 2.5, and 5.0 g / ml moxifloxacin. to this mixture, 40 l of is working solution was added to yield is concentration of 1.0 g / ml. each concentration point in the calibration curve was analyzed three times under the same hplc conditions as described above. the peak area ratios of moxifloxacin to the is for each of the standard solutions were calculated and plotted as a function of drug concentrations in human plasma. the calibration curves were acceptable only if they had correlation coefficients (r) of 0.99 or greater. repeatability was evaluated at 0.5 g / ml of moxifloxacin with 1.0 g / ml of is in 10 ml flow phase. this was measured for six times per day and the relative standard deviation (rsd) was calculated. to examine the precision of the method, plasma spiked with three concentrations consisting of low, middle, and high concentrations (0.05, 0.5, and 2.5 g / ml) of the analyte was prepared. intra - day precision was evaluated by analyzing the spiked controls five times a day. this was repeated on five consecutive days to permit an assessment of inter - day precision. recovery of moxifloxacin was determined by comparing the peak area of the analyte extracted from the plasma with peak area obtained by the direct injection of pure standard analyte in mobile phase at three different concentrations containing low, middle and high concentrations (0.05, 0.5, and 2.5 g / ml). moxifloxacin stoking solution 2, 20, and 100 l was added to 200 l blank plasma, respectively ; then 40 ml is working solution was added. following the extraction procedure, the sample was prepared and then determined for five times for each concentration. the concentration of moxifloxacin was obtained by putting the peak area ratio into the calibration curve. the validated hplc method was applied in a population pharmacokinetics research of moxifloxacin in 37 chinese adult patients with infectious disease including cap, cb, urinary tract infection (uti), and bacterial diarrhea. moxifloxacin was administered as a single intravenous dose of 400 mg once daily for 514 days. two venous blood samples (2 ml) were collected into heparinised tube right after the first administration and before the fourth dose. the blood samples were immediately centrifuged (3500 rpm, 10 min) to separate and stored at 80c until assayed. the index of age, gender, height, weight, blood creatinine (cr), urea nitrogen (bun), glutamic - pyruvic transaminase (alt), and glutamic - oxalacetic transaminase (ast) concentration was recorded. a correlation between moxifloxacin blood concentration and the chromatogram showed no interfering peaks near the same retention times as moxifloxacin or internal standard derivatives (figures 1(b) and 1(c)). was the chromatogram of pure standard analyte and is in mobile phase, which demonstrated the peaks in figures 1(b) and 1(c), was derived from moxifloxacin and is. the linearity of the calibration curve for moxifloxacin in spiked drug - free plasma over the concentration range of 255000 ng / ml was evaluated. the peak area ratio of each concentration in determining the calibration curve was exhibited in table 1. calibration curve obtained by plotting peak area ratio (moxifloxacin / internal standard) versus concentration was linear over the range of 255000 ng / ml. the calibration curve of moxifloxacin in plasma was linear and was represented by the regression equations y = 3.0505x 0.0234 (figure 2), where y presents the peak area ratio (moxifloxacin / is) and x represents the plasma concentration of moxifloxacin (g / ml). the mean correlation coefficient (r) for the moxifloxacin calibration curves was 0.9998. the results of repeatability tests were displayed in table 2. the intra - assay rsds at the three concentrations were 4.12, 3.12, and 1.85, while the inter - assay rsds were 4.70, 1.16, and 3.38. the mean percentage extraction recoveries of moxifloxacin at the three concentrations were 69.88 3.19, 78.86 4.12, and 78.51 2.44, respectively (table 5). the use of is, namely, ciprofloxacin, was crucial to decrease the error brought by the extraction procedure. in this study, ciprofloxacin was used as is because the structure and properties of ciprofloxacin were similar to moxifloxacin. mean value of the method recovery was 98.85%, which ranged from 96.6 to 101.8 (table 6). application of the method to a population pharmacokinetics study in patients with infectious diseases. to demonstrate its utility in a pharmacokinetics study, the method was applied to measure the concentrations of moxifloxacin in plasma samples obtained from patients with infectious disease who were given intravenous moxifloxacin 400 mg once daily. peak moxifloxacin concentrations in plasma ranging from 1.652 to 6.743 g / ml with an average of 3.364 1.686 g / ml were achieved right after intravenous administration of moxifloxacin. trough concentrations ranged from 0.359 to 2.676 g / ml with an average of 0.914 0.481 g / ml. the present study to determine the blood concentration in plasma principally focused on hplc with uv detection, on account of its low sample amounts, easy operation, and high sensitivity. however, tetrabutylammonium hydrogen sulfate, which was expensive and not easy to get, has to be used as ion - pair reagent. moxifloxacin, which had strong ultraviolet absorption, could be absorbed in 296 nm. therefore, hplc - uv method was applied in this experiment, which was more economical. internal standard method was adopted in order to reduce the system error caused by the instruments and operations. this method was easy to operate, simple in sample preparation, and highly sensitive. in the pretest, the solutions of acetonitrile0.01 mol / l potassium dihydrogen phosphate and 0.01 mol / l potassium dihydrogen phosphate - methanol - acetonitrile peaks more than expected were observed leading to uncertain drug peak due to unknown reason. the higher ph value was, the longer the retention time of moxifloxacin would be. moxifloxacin did not separate completely with endogenous substances in plasma when ph value of flow phase was 4.5. the problem was solved after ph value was increased to 4.8. according to the results above, 0.1% triethylamine (adjusted ph to 4.8 with phosphoric acid)/acetonitrile (80/20, v / v) were confirmed as flow phase which exhibited good separation among compounds in plasma, is, and moxifloxacin with a relatively short retention time. internal standard method was adopted in order to reduce the system error caused by the instruments. in this study, we were aiming to find an internal standard that exhibited good separation with moxifloxacin and could be obtained as easy as possible. some previous methods used internal standards which were difficult to obtain commercially [8, 15 ]. in this study, ciprofloxacin was used as is and the retention time was about 5 min, which allowed moxifloxacin to separate completely. therefore, a more suitable method having a wider range of capability is required for evaluating the precise pharmacokinetics of these drugs. moxifloxacin concentrations of 37 patients with a continuous intravenous dose of 400 mg once daily were determined in the tests. peak concentration in the first dose and trough concentration in the fourth day were studied, respectively. according to the results, liver function would be responsible for the peak concentration while age and renal function may influence the trough concentration of the medicine, although the number of patients in the study was limited. found the pharmacokinetic feature varied little between normal and liver dysfunction patients, so that it was not necessary to adjust treatment dosage in patients with liver damage. in this study, the peak concentration of no1 patient (weight as 45 kg, alt, ast as 53.8 and 39.8, resp.), 4.116 g / ml, may lead to the conclusion that liver dysfunction may increase the peak concentration. moxifloxacin would be metabolized to m1 and m2 after the second phase biotransformation and be proved not to increase as the age of the patients is growing [1, 6 ]. skalioti. demonstrated the plasma concentrations of moxifloxacin in patients undergoing continuous ambulatory peritoneal dialysis were 5.86 0.60 mg / l, 1 hour after an oral dose of 400 mg, which was higher than the concentrations in healthy patients, 2.53.4 mg / l, leading to a conclusion that renal dysfunction may influence the metabolism of moxifloxacin and increase the concentration. the method validation indicates that this method is a simple, rapid, precise, and accurate assay for the determination of moxifloxacin concentrations in human plasma. moreover, the method requires only a small volume (200 l) of plasma, which makes it suitable for studying the pharmacokinetics in patients, especially for the elderly. the sensitivity and simplicity of the method makes it suitable for routine therapeutic drug monitoring or clinical pharmacokinetic studies of moxifloxacin. in conclusion, this optimized method is sensitive and reproducible enough to be used in pharmacokinetic studies.	objective. to develop a simple and rapid high - performance liquid chromatography (hplc) method for measuring moxifloxacin concentration in human plasma. methods. following a single step liquid - liquid extraction, analytes along with an internal standard (is) were separated using an isocratic mobile phase of 0.1% triethylamine (adjusted ph to 4.8 with phosphoric acid)/acetonitrile (80/20, v / v) at flow rate of 1 ml / min on reverse phase kromasil c18 column (250 mm 4.6 mm, 5 m) at room temperature. results. total analytical run time for selecting moxifloxacin was 15 min. the assays exhibited good linearity (r2 = 0.9998) over the studied range of 25 to 5000 ng / ml. the absolute recovery rate of low, medium, and high concentrations were 69.88%, 78.86%, and 78.51%, respectively. the relative recovery rates were 98.50%, 96.61%, and 101.79%, respectively. coefficient of variation and error at both of the intraday and interday assessments were less than 4.7%. conclusions. the results indicated that this method is a simple, rapid, precise and accurate assay for the determination of moxifloxacin concentrations in human plasma. this validated method is sensitive and reproducible enough to be used in pharmacokinetic studies.
the plan will contain three components : (1) the nei framework for vision research, a draft of which is available containing six goals, (2) vision research in 2011, which is expected to be released in early 2012, and (3) ongoing planning workshops to supplement emerging needs and opportunities being identified by planning panels. recent planning workshops have included ocular pain and sensitivity, ophthalmic genetics, advances in optical imaging and biomedical science, and ocular epidemiology. nei solicited opinions from members of the scientific, medical, and patient communities to help shape its research agenda through a respondents were asked to provide suggestions on the most significant scientific discoveries in vision research since 2004 and the most significant scientific research needs, gaps, and opportunities that can be addressed by nei. similarly, the european commission is currently undertaking an open consultation process for unmet research / clinical needs in vision science and ophthalmology in europe. the consultation process was open from november 15, 2011 to december 31, 2011 and will culminate in a white book entitled a vision for horizon 2020 a european strategic roadmap for vision research and ophthalmology, stakeholders in the vision community, including scientists, clinicians, patient organizations, academic and private institutions, and companies were asked to suggest innovative research goals that they would like to get funded. the strategic plan for 20092013 of the international agency for the prevention of blindness (iapb) includes six strategic objectives, with approaches and measures of success provided for each of the objectives. the plan includes advocacy to develop sustainable income resources and promote vision 2020 to eliminate the main causes of avoidable blindness by the year 2020. the international council of ophthalmology 's research committee met in 2002 to develop a research agenda for the prevention of global blindness. they divided research priorities into three domains research : operations research, epidemiologic risk profile, and basic biologic research. they considered research opportunities for cataract, trachoma, onchocerciasis, xerophthalmia, glaucomas, diabetic retinopathy and age - related macular degeneration, and refractive error. action items to move this agenda forward included developing regional consortia to pool resources to estimate burden of disease ; monitoring outcomes of interventions and sharing lessons learned ; getting a consortium of scientists from wealthy and poor countries to systematically monitor new drugs and discoveries testing them for application in the developing world. a novel opportunity for collaboration the approach taken by these organizations to develop research agendas that allow for input from all relevant parties in the vision community has proven to be successful. inherent in the development of research agendas is the need to document what is known about the biology of a given condition as well as the prevalence and incidence of disease and success of interventions. figs 1a d demonstrate the state of funding for vision research by the nei in the us. the actual dollars spent on vision research from fy2007 to fy2012 remained relatively constant, along with the relatively constant inflation rate reflected by the consumer price index. over that same time frame, the percentage of the national institutes of health (nih) to eye diseases and disorders of vision fell [fig. 1b ] as did the success rate for new competitive awards from nei [fig. the number of applications for new competitive awards rose between fy2002 and fy2012 [fig. 1d ]. in the uk, the success rate for research grants fell from 17% in 20092010 to 15% in 20102011. success rates for project grants from the national health and medical research council (nhmrc) in australia from 2005 to 2010, showed an increase for 2 years and then fell again recently (21.1%, 21.3%, 27.6%, 26.5%, 22.8%, 23.3%). these data highlight the need for researchers to consider all potential sources of funding for their research. (a) national institutes of health funding for eye diseases and disorders of vision relative to 228 other research / disease areas, fy 2007 to fy 2011. (b) percent of nih funding allocated to eye disease and disorders of vision, fy 2007 to fy 2012. (c) success rates for all new awards by the national eye institute, 2002 to 2011. (d) number of applications for new awards to the national eye institute, 2002 to 2011 the universal consideration for funding is that the reason for funding should align with the mission of the funding organization. a statement on the preliminary grant application web site for the macular degeneration foundation sums it up wellthe foundation carefully considers all eligible applications to determine whether a project fits its current program interests and funding principles. three current areas of focused funding are then outlined, followed by funding principles that include scientific merit, achievability and real world applicability. researchers need to ensure that a potential funding agency would be interested in funding their area of research. the first considerations when looking for research funding are likely to be the amount of funding available and whether institutional indirect charges or administrative overheads are allowable grant expenses. this is because some institutions do not allow scientists to apply for grants where these costs are not allowed.turn-around time from application to funding availability may also be an important consideration when considering possible funding sources. these issues are summarized for various funding sources in table 1 and are discussed in more detail below. the majority of research and development is funded by private industry but federal grants and contracts remain the primary source of research funding for many academic researchers. as shown earlier, the success rates for research funding from the nei in the us, the medical research council in the uk, and the nhrmc in australia have fallen in recent years.researchers should check the web sites of these funding organizations for grant submission deadlines, funding priorities and any restrictions, such as country of residence. the nei will fund research that takes places outside the us professional associations, such as the association for research in vision and ophthalmology, often offer grant writing sessions at their annual meetings. nongovernmental organizations (ngos) are very heterogeneous groups, but share a vision and passion for improving their communities or the world in relation to a specific concern, such as glaucoma or blindness. they tend to rely heavily on volunteers and fund - raising to support their missions. depending on the type of ngo, some of these funds are disbursed as grants to researchers. a summary of some of the ngos that fund vision research is contained in table 2. the list includes the research on disorders that they fund, and ngos that fund projects outside the us. as highlighted in table 1, ngos generally do not cover institutional indirect costs but can have a much shorter turn - around time from grant application to receipt of funding. for - profit organizations such as device manufacturers and pharmaceutical companies have research and development budgets. they seek research collaborators to evaluate their products and they occasionally fund external investigator - initiated projects. for instance, pfizer inc. has a global investigator - initiated research program. types of research that are eligible for support include clinical studies involving pfizer drugs, observational studies such as epidemiology or outcomes studies.other types of research on diseases states that include novel diagnostic tools where pfizer has no direct commercial interest and in - vitro or animal studies are also eligible. information is available on the pfizer web site and interested researchers are advised to contact pfizer representatives. pfizer also has a competitive grant programs with specific research criteria and timelines ; again, information is available on their website. the alcon research institute (ari), established in 1981, is a virtual institute that honors outstanding ophthalmic researchers through a symposium and unrestricted research grants. most nonprofit research institutions receive some funding from donors for their nonprofit mission. larger organizations may have development departments. researchers can work with their development officers to help prepare the case to receive the support of specific research programs or projects. again, it is important to align the interest of the donors with the needs of the researchers. service organizations, such as lions clubs international and rotary international, are committed to improving their communities. they provide volunteer time and small grants to research projects with a generally very fast turn - around time. the lions eye health program, in conjunction with the nei, is a community - based education program to promote healthy vision. while websites contain some information about the priorities of service organizations, a call or visit to a local chapter is the best way to determine if a specific research project is a good match to the service organization. local chapters regularly look for speakers for their meetings ; this is a good opportunity to raise the level of interest in vision research in the community. well established processes exist to develop and update national and international priorities for vision research. with the number of grant applications increasing and the pay lines generally decreasing, applicants need to be sure that their funding requests are well matched to the vision and mission of the funding organization. collaboration is encouraged between senior and junior investigators and between researchers in developed and developing countries. as can be inferred from reading the reference list in this manuscript, there is a lot of information available on web sites about potential funding sources, grant deadlines, and other necessary information for grant seekers. researchers are advised to check web sites regularly for updated information and to sign up for email updates and newsletters from funding organizations, where available.	a number of organizations have employed a consultative process with the vision community to engage relevant parties in identifying needs and opportunities for vision research. the national eye institute in the us and the european commission are currently undergoing consultation to develop priorities for vision research. once these priorities have been established, the challenge will be to identify the resources to advance these research agendas. success rates for federal funding for research have decreased recently in the usa, uk, and australia. researchers should consider various potential funding sources for their research. the universal consideration for funding is that the reason for funding should align with the mission of the funding organization. in addition to federal research organizations that fund investigator - initiated research, other potential funding sources include nongovernmental organizations, for - profit companies, individual philanthropy, and service organizations. in addition to aligning with organizational funding priorities, researchers need to consider turn - around time and total funds available including whether an organization will cover institutional indirect costs. websites are useful tools to find information about organizations that fund research, including grant deadlines. collaboration is encouraged.
giant cell lesions of the maxillofacial area can vary from asymptomatic radiolucency of slowly growing lesion to aggressive tumours showing high recurrence rate as well as rapid expansive progression characterized by root resorption and pain. gcgs of the jaws arise either peripherally in periodontal ligament, mucoperiosteum, or centrally in the bone. the world health organization has defined the central giant cell granuloma as an intraosseous lesion consisting of cellular fibrous tissue that contains multiple foci of hemorrhage, aggregations of multinucleated giant cells and occasionally trabeculae of woven bone. histologically, both peripheral and central variants of giant cell granuloma are characterized by the presence of numerous multinucleated giant cells (mgcs) in a prominent fibrous stroma. foci of hemorrhage with liberation of hemosiderin pigment and newly formed osteoid or bone are often seen. the mgcs are concentrated in the areas of hemorrhage and are adjacent to blood vessels. jaffe separated cgcg from gct of the bone on clinical and histologic grounds and suggested that mgcs in cgcg represent a phagocytic response to hemorrhage. however, it affects females more often than males, in a 2:1 ratio and is seen most frequently under the age of 30 years. one study of 38 patients shows 74% to be less than 30 years of age and 61% to be less than 20 years of age. the lesion commonly presents as a solitary radiolucency with a multilocular appearance or less commonly, a unilocular appearance. it is more prevalent in the anterior than the posterior jaws, often crossing the midline, and the mandible is more commonly affected than the maxilla. this lesion has also been reported in the small bones of the hands and feet. the behavior of cgcg is variable, most commonly producing an asymptomatic expansion of the jaws. however, it can be clinically aggressive, associated with pain, osseous destruction, cortical perforation, root resorption, and recurrence. cases of cgcg occurring with neurofibromatosis (type 1), noonan - like syndrome, or both have been reported. these lesions may possibly lead to a confusion in their correct diagnosis as many pathologists report them taking into consideration one of the prominent histopathologic feature. such misinterpretation may be because of the small number of cases reported in the literature with uncertain clinical, radiographic and histopathologic features of these lesions. so even surgeons may end up treating these lesions inadequately or patients may need to undergo multiple surgeries. the present case report highlights a case of recurrent and aggressive form of cgcg in the mandible. a 22-year - old man presented with a swelling in the left ramus of the jaw 2 years ago. examination revealed a unilocular radiolucent lesion, with a scalloped inferior border [figure 1 ]. the ct scan revealed a well defined hyperdense soft tissue seen in the region of and below the left coronoid process of mandible, with suspicion of sclerosis. a partial mandibulectomy was performed and a reconstruction plate with a mini plate at the anterior region along with a fibular graft in the jaw was inserted to repair the defect [figure 2 ]. the inferior border of the lesion is scalloped orthopantomograph showing the reconstruction plate repairing the defect in the left jaw after one year, the patient, now 23 years old, complained of a recurrent swelling in the same region. intraorally, the patient presented with a growth in the left buccal mucosa at the level of the occlusal plane, which was excised and microscopically reviewed. the first molar along with the premolars were removed, the region was curetted and a new reconstruction plate was given. photomicrograph confirming the presence of granulomatous lesion (h and e, 40) a year later, the patient now 24 years old, was referred to the department of oral surgery with the complaint of pain and recurrent swelling of the left jaw [figure 4 ]. extra oral swelling of the left lower jaw clinically the lesion extended from the corner of the mouth to the anterior part of tragus on the left side, which was 4 4 cm in size, irregular in shape with a rough texture. the swelling was hard in consistency, showed no secondary changes and was non tender on palpation. intraoral examination revealed an exophytic growth present posteriorly near the junction of the buccal mucosa and pterygomandibular fossa region, at the level of the occlusal plane, sized 1 1.5 cm and soft in consistency. presently the ct scan revealed an evidence of an expansile destructive mass (4.3 3.8 4.3 cm in the maximum anteroposterior, transverse and superoinferior dimensions) in the expected location of the left coronoid process, with thin residual septae like areas of osseous density seen in a large soft tissue mass. this soft tissue mass showed near isodensity compared to the adjacent muscles of the left masseteric space. the lesion expanded the insertion of the left temporalis muscle and bulged anteriorly into the left buccal space and posteriorly into the left condylar head and neck and left parotid gland. medially, the lesion led to mild pressure erosion with thinning of the buccal cortex of the left maxillary tuberosity and bulged against the left medial pterygoid muscle [figure 6 ]. intra oral photograph depicting exophytic growth in the junction of the left pterygomandibular fossa region and buccal mucosa ct scan showing the extent of the lesion during the second recurrence routine hemogram and urine examination were normal. on the basis of clinical and radiological examination a provisional diagnosis of cgcg was made. the serum chemistry of calcium, phosphorous, parathyroid hormone was normal, there by excluding the possibility of hyperparathyroidism. surgery was performed by a submandibular incision at the site of the previous scar, with the removal of the reconstruction plate, mini plate and graft, along with the condyloid process. histopathological examination of excised specimen revealed evenly dispersed (2 - 3/hpf) giant cells each having 2 - 8 nuclei in them, in close approximation with proliferating blood vessels admixed with areas of haemorrhage. even the bone graft attached to the condyle showed the presence of tumor giant cells [figure 8 ]. no recurrence was noticed in post operative follow - up phase of 3 years [figures 9 and 10 ] and further reconstruction of mandible using iliac crest graft is intended. photomicrograph showing giant cells in a vesiculated fibroblast connective tissue stroma (h and e, 40) photomicrograph showing presence of giant cells near the condylar region (h and e, 40) post - operative follow up opg verifying the absence of lesion post - operative intraoral view the etiopathogenesis of the cgcg of jawbones has not been clearly established but it has been suggested that it is the result of an exacerbated reparative process related to previous trauma and intraosseous hemorrhage that triggers the reactive granulomatous process. donoff and rosenberg discussed a case record of an uncomplicated extraction because of pericoronitis in the area of the lesion and claimed the local changes in the blood flow throughout the bone and local bone dysplasia could be probable etiologic factors. unal., presented a 12-year - old girl cgcg in the mandible caused by a molar tooth extraction and explained the pathogenesis by a traumatic aetiology. association of t (x ; 4)(q22;q31.3) in the etiology of gcg has been reported. although, cgcgs are benign osseous lesions, some authors separate cgcg into two types, referring to its clinical and radiographic features : (a) nonaggressive lesion is usually slow growing and asymptomatic, does not show cortical resorption by the lesion or root perforation in teeth affected, and it is significantly less likely to recur than the aggressive type ; and (b) aggressive lesions, is usually found in younger patients and is painful, grows rapidly, is larger, often causes cortical perforation and root resorption and has a tendency to recur. predicting the behavior of cgcgs that will exhibit a higher risk of recurrence after treatment has been problematic. waldron reported a mean interval between diagnosis and initial treatment and treatment of a recurrence was 21 months, and stated that very few recurrences were manifested after 2 years of initial treatment. the most reliable factors related to an increased risk of recurrence include clinical activity of lesions (72% of recurrence in the aggressive forms, 3% of recurrence in the nonaggressive forms), younger patients, demonstrated perforation of cortical bone and tumor size. there has been studies suggesting that the greater functional surface area occupied by giant cells and larger relative size of giant cells may identify tumors with aggressive behavior. recently, kruse - loser., also proved that the aggressive variant of cgcg presented a high number of giant cells, an increased mitotic activity, and a high fractional surface area. however, other studies have not been able to predict the clinical course of cgcgs from known histological or immunohistochemical features. we reviewed the archival cases of 10 cgcgs from our department which were nonaggressive and non recurrent, the demographical information, location, radiographic features and histopathological features of which are shown in table 1. the demographic information, location, radiographic features and histopathological features of 10 nonaggressive cgcgs are as follows the present case showed 2 - 3 giant cells per high power field, which was less compared to that seen in our archival cases. the connective tissue was minimal, but with a high cellularity and a vesiculated fibroblast population. the nonaggressive cases of cgcg showed a minimal - moderate cellularity and a non vesiculated fibroblast population. the vascularity in the present case was minimal, which was not a differentiating factor, as cases in the archives showed a varied vascularity from minimal to marked.. it may be well - defined or ill - defined and shows variable expansion and destruction of the cortical plate. the radiological appearance of the lesion is not pathognomonic and may be confused with that of many other lesions of jaws. the final diagnosis eventually rests on histopathology because the clinical and radiological features are not specific. cgcg of the jaw usually presents as a painless solitary radiolucent expansion in most of the cases. rankl (receptor activator of nuclear factor kb ligand) present on stromal cells influences the differentiation of giant cells from rank expressing mononuclear cells. amongst all, gct is most difficult to differentiate from cgcg without clinical and histological aids. histologically, cgcg has a hemorrhagic background with presence of plump bland fibroblast, hemosiderin and fewer giant cells with smaller number of nuclei, which are less uniformly distributed. while in case of gct, giant cells are uniformly scattered with larger number of nuclei and absence of fibroblasts and hemorrhage. diffuse sheets of large giant cells and polygonal mononuclear cells seen in gct are lacking in cgcg. cystic areas (the aneurysmal bone cyst component) are lesser as compared to gct. differential diagnosis from brown tumor is based mainly on clinical and laboratory data, as well as age of onset and multiplicity of lesions. immunohistochemical studies on cgcg have helped to establish the lineage of the cells, but not to predict the aggressiveness of the lesion. supporting the theory that the multinucleated giant cells are derived from macrophages is the immunoreactive response to muramidase, -1antichymotrypsin, and -1antitrypsin. aggressive and nonaggressive cgcgs stained for antibodies to cd34, cd68, factor xllla, and smooth muscle actin, prolyl 4-hydroxylase, ki-67, p53 protein, rank, and glucocorticoid receptor alpha have revealed no phenotypic differences between the types. calcitonin receptor expression, however, has been found to exhibit a statistically significant difference with more expression in the aggressive type. generally, curettage of well - defined localized lesions is associated with a low rate of recurrence. in extensive lesions with radiographic evidence of perforation of cortex, the medical management of cgcg as an adjunct to surgery includes treatment with steroids or calcitonin which inhibits osteoclastic activity. interferon - alpha appears useful in the management of aggressive cgcg, presumably due to its anti - angiogenic effects. although extensive literature has been made available to the readers who envisage a keen interest in cgcg of the jaw, clarity to this entity with respect to terminology, behavior and its adjunctive nature to the gct occurring in long bones has rarely been lucid in its understanding. the concomitant presence or initiation of this entity with various other diseases like aneurysmal bone cyst and also its histopathological similarities to diseases associated with hormonal imbalances like the present case highlights the perplexity in diagnosing cgcgs, which are aggressive in nature due to its close proximity with respect to pathology, behavior and prognosis from gct. the recurrent nature of the present case and the extensive destruction caused in the hard and soft tissues convinces us the need of exploring the possibilities of the so called true	central giant cell granuloma is a fairly common lesion in the jaws aetiology of which is still completely unknown but thought to be of a reactive process to some unknown stimuli. it usually arises either peripherally in periodontal ligament, mucoperiosteum, or centrally in the bone. the histological hallmark for both peripheral and central giant cell granuloma (cgcg) is the presence of distinctive multinucleated giant cells (mgcs) in a prominent fibrous stroma. central giant cell granuloma is an uncommon benign proliferative lesion that almost exclusively occurs within the jaw. eventually, it may become aggressive leading to the expansion and perforation of cortex resulting into mobility and displacement of teeth with root resorption. the present case focuses on the dilemma and perplexity in diagnosing aggressive cgcgs, due to its close proximity with respect to pathology, behavior and prognosis from giant cell tumors (gct). central giant cell granuloma persuaded extensive destruction to the hard and soft tissues with high rate of recurrence encourage us the need of exploring the possibilities of giant cell tumors having a definitive presence in the jaws.
squamous cell carcinoma (scc) is the second most frequent form of skin cancer superseded in occurrence only by basal cell carcinoma and it is commonly seen in elderly white men. like basal cell carcinoma, scc is predisposed for by excessive ultraviolet light exposure, hence, its association with advancing age and cumulative sun exposure, exposed anatomic sites and the highest incidence in sunny geographic localities. several histological subtypes of scc have been described including, keratoacanthoma, acantholytic, spindle cell, verrucous, clear cell, papillary, signet ring, pigmented, and desmoplastic scc. clear cell scc is a rare entity and only a total of six cases were previously reported as at 2006. we present a case of clear cell scc of skin with an exophytic mass on the face in order to add to the scarce literature of this rare variant of scc. a case of a 62-year - old male, a concrete block maker / bricklayer who presented at the dental centre, university college hospital, ibadan on account of a non - healing ulcer on the left side of his face of 6 months duration. the lesion was said to have started as a small firm painless swelling in the left infra - orbital region, which gradually increased in size until 3 months later when it became ulcerated with associated pain and purulent discharge. physical examination revealed a cachectic and pale elderly man with an obvious facial asymmetry due to a fleshy exophytic mass on the left side of the face. the patient was not a known diabetic or hypertensive and had no other known systemic disease. there was no history of tobacco or alcohol use and he was not being treated for any chronic condition. the mass measured about 16 cm in its widest diameter and extends from the left supra - orbital region to the left maxillary area and also to the left temporal area about 1 cm anterior to the left auricle. the mass caused a deviation of the lateral wall of the left nostril to the right, though clinically the nasal wall did not appear infiltrated by the tumor. it also extended superiorly such that the left eye globe could not be visualized [figure 1 ]. clinical picture showing an exophytic growth the exophytic mass had a necrotic central area which was covered with slough tissue. there was impaired mouth opening ; the left buccal sulcus was fully obliterated by the tumor mass but the palate appeared clinically normal. fine needle aspiration cytology suggested a malignant epithelial neoplasm possibly a salivary gland malignancy or a skin adnexia malignant tumor. following incisional biopsy, sections showed a malignant epithelial neoplasm composed of islands of large oval to polyhedral malignant squamous cells with eosinophilic to amphophilic cytoplasm and vesicular nuclei with some of the nuclei peripherally placed. there were areas showing clear cell differentiation of the malignant squamous cells with some cells arranged in a pseudo - glandular pattern and isolated areas showing keratin pearl formation [figure 2 ]. figure 3 shows lesion to be negative for periodic acid schiff (pas), mucicarmine and alcian blue stains but was strongly positive for ae1/ae3 (immunostain). h and e, (100/400) showing malignant epithelial cells with some areas of clear differentiation sections (400) of mucicarmine, periodic acid schiff and alcian blue are negative while ae1/ae3 is strongly positive clear cell carcinoma also referred to as hydropic scc was first described by kuo in 1980 as a variant of scc with extensive hydropic changes. the hydropic degeneration of neoplastic cells and the accumulation of intracellular fluid and not the accumulation of glycogen, lipid, or mucin, results in its clear cell appearance. all cases reported so far have been in the head and neck region with the mandible being the most common site and all including the present case occurred in men who work out doors. the clear cell variant of scc is histologically similar to sebaceous neoplasms but distinguishing features include evidence of squamous differentiation in clear cell scc which was seen in this case. other differential diagnosis include clear cell acanthoma, clear cell hidradenoma, clear cell hidradenocarcinoma, tricholemmoma, pilar tumor, balloon cell nevus, balloon cell melanoma, and metastatic renal cell carcinoma. the clear cells of clear cell acanthoma, clear cell hidradenoma, clear cell hidradenocarcinoma, tricholemmoma, pilar tumor all have a high content of cytoplasmic glycogen, which was absent in this case as demonstrated by negative staining with pas, mucicarmine, and alcian blue. clear cell hidradenoma is an adnexal tumor which in addition to clear cells also has granular cells that surround sweat duct - like structures and a hyalinized stroma. tricholemmoma is a superficial, lobular tumor growing around hair follicles with central clear cells and palisading peripheral cells. pilar tumors may show focal or extensive clear - cell change and usually show glassy keratinization. balloon cell nevus and balloon cell melanoma show nests of melanocytes at the dermo - epidermal junction. metastatic renal cell carcinoma has rich vasculature and will show clinical evidence of a renal tumor. although, the etiology of clear cell scc is not completely understood, immune - suppression, arsenic exposure, radiation, chronic ulceration, have been suggested as possible etiologic factors. all the previously reported cases occurred in white elderly men that work out doors suggesting a role for ultra violet radiation. this is supported by this case which also occurred in elderly man who works out doors (bricklayer), though as far as we know this is the first reported case in a dark - skinned adult. cohen., had previously reported two cases of clear cell carcinoma in - situ in a married couple in which human papilloma virus (type 5 in the husband and type 21 in the wife) was positive in both spouses which suggested that contact transmission may be responsible, however, the validity of this factor in the present case can not be commented on. the clinical presentation of this case appears to be quite aggressive as the lesion which started as an ulcer became a large exophytic mass after 6 months. more report of cases is required to characterize the clinical behavior and prognosis of this rare variant of scc.	clear cell squamous cell carcinoma (scc) is a rare variant of scc of skin in which ultraviolet radiation has been suggested as possible etiology. this case is that of a 62-year - old male concrete block maker / bricklayer who presented with a 6 months history of a non - healing ulcer on the left side of his face. histology showed features of malignant epithelial neoplasm composed of islands of large oval to polyhedral malignant squamous cells with eosinophilic to amphophilic cytoplasm and vesicular nuclei and there were areas showing clear cell differentiation and isolated areas of keratin pearl formation. the lesion was also negative for periodic acid schiff, mucicarmine, and alcian blue stains but was strongly positive for ae1/ae3 (immuno - stain). this case showed an aggressive and bizarre clinical presentation but more report of cases are needed to have a better characterization of the clinical presentation and prognosis of this variant of scc.
revision of medical training programs have become a necessity to keep pace with the population growth, changing disease patterns and health priorities of the society. this is why the iranian document for medical education revolution obliges universities to train human resources so that they are able to respond to the health needs of individuals and society by performing their professional duties. the revision of educational programs has led to the introduction of a new concept called socially accountable universities of medical sciences. in line with the global shift in the attitude towards social accountability, this concept has also been emphasized in iran as a philosophical approach toward higher education. it focuses on the responsibility of universities for training students who are responsive to the real needs of the iranian society and capable of fostering the achievement of goals for the country as stipulated in the fifth development plan and national 2025 vision plan. in addition to addressing the values, the world health organization has defined social accountability of medical universities as the obligation to direct their education, research and service activities towards addressing the priority health concerns of the community, region, and/or nation they have a mandate to serve. in order to fulfill social accountability, universities of medical sciences should be committed to the continuous development of new and effective approaches / strategies for the development of health, and demonstration of positive and tangible effects of university products on community health. therefore, it is crucial to examine to what extent universities of medical sciences have achieved social accountability. it is obvious that in order to achieve social accountability, social responsibility should also be considered ; because accountability should improve from the lowest level, namely responsibility, to the highest level, namely accountability. in fact, responsibility refers to the commitment and awareness of the authorities to train competent individuals in order to meet the needs of the society that mostly has a theoretical feature while accountability tends to guide and provide the education, research and services to meet the needs of the society and evaluate the process. in this regard, a model was presented to assess social accountability in medical schools, which was revised in 2012. conceptualization - production - usability model (cpu model) offers a list of parameters for evaluation and improvement of medical schools quality. similarly, the training for health equity network (thenet) has developed a framework by limiting the cpu model that evaluates social accountability with a heightened focus on the educational activities of medical schools (9). however, the cpu model and thenet framework only present general determinants to examine the social accountability of medical schools. therefore, they can not be used as clear, assessable and generalizable determinants for studying accountability in all departments associated with health sciences. moreover, there are plenty of global debates concerning social accountability standards and improvement of evaluation quality. in the context of iran, some projects have been performed to measure the effect of social accountability on community health this perhaps clarifies the reason for the existing emphasis on the development of certain methods and tools for social accountability assessment in the literature. due to differences in education, research and service processes across various schools of medical sciences, lack of determinants for examining social accountability of nursing and midwifery schools on one hand, and the role played by these schools in improving healthcare services offered to society through training healthcare workers, on the other hand, evaluation of their position on the way towards social accountability is crucial. hence, the current study aimed to develop determinants of social accountability for the schools of nursing and midwifery in line with the native and indigenous values of iran. this study was carried out using a classical delphi technique. given that delphi technique is used to receive comments from a group of experts on an issue or a question and reach consensus by using a series of survey questionnaire rounds and effective feedback to members, the research team employed the delphi technique to explain social accountability determinants in this study. 931101 from the ethics committee of mashhad university of medical sciences, the study was conducted in mashhad nursing and midwifery school over 5 months since february 2015. purposive sampling was used to select experts with rich information, knowledge and experience from faculty members, master and phd students as well as graduates of the nursing and midwifery school. experts needed to have at least three - year clinical and educational experience and enough time. experts who did not fill in the questionnaires and had no tendency to continue were excluded from the study. considering entry requirements for experts, nursing and midwifery school and higher education and clinical departments of imam reza and ghaem hospital in mashhad were visited and full details on social accountability subject and purpose and procedure of the study were provided for the experts and informed consent was completed by them. if the expert refused to participate in the study, another person was selected by purposive sampling method. the first and the third rounds used a non - face - to - face questionnaires whereas an in - person questionnaire was used in the second round. some studies have suggested a sample size ranging from 10 to 30. in this study, 30 and 15 experts respectively entered the first, second and third rounds with respect to probability of loss. each of the follow up rounds (i.e. two and three) was attended by 15 experts. in each round, the necessity for the presence of a determinant in the final instrument was determined by the scores assigned to each determinant of social accountability by the experts. experts needed to assign each item to three activity areas of education, research and services. in the first round, a structured questionnaire for the primary determinants of social accountability was prepared based on extensive search in the related literature across national and international databases. additionally, the researchers used documents such as fifth development plan, the national 2025 vision plan (clearing the way for development and construction of iran in various fields associated with culture, science and technology, society, administration, economics, regional development, security, defense, politics, law, and budget), and general policies for health issues by iranian authorities. the most important references used to draft the items were the general policies of health (5 items), comprehensive scientific health map (10 items), socially accountable medical education strategic plan (15 items), cpu model (7 items), thenet s social accountability framework (12 items), as well some papers presented on social accountability in related high rank journals (30 items). some questions extracted from these sources are as follows : are public health priorities identified ?, are the needs of population at risk considered in the areas of education, research and service ?, is the students competence in response to the priorities of the society measured ?, are education, research and service providing activities revised to adapt with priorities and needs of the society ?, what is the role of school in increasing the partnership of the individuals, families and society to improve public health ?, are faculty members competent enough in the field of social accountability ?, are research and research funds pushed toward health priorities of the society ?, and how are the capabilities of the institutions and organizations used to improve health care sector ? the questionnaire included 128 determinants and was scored using a 5-point likert scale (ranging from 1=strongly disagree to 5=strongly agree). participants were asked to freely write down their ideas, viewpoints, and proposed determinants (if any). the second round included the proposed new criteria and determinants which had not obtained the consensus of experts to be included in or removed from the final instrument. according to the experts opinion, a 7-point likert scale questionnaire with 83 items (from 1=strongly disagree to 7=strongly 15 experts did not tend to participate in the study after the first round, in the second round, 15 experts were asked to examine and score the questionnaire items before the start of the session. during the face to face session, each expert presented his / her viewpoints separately on determinants. the third round was held within a week from the second round and involved sending determinants which had not achieved consensus to the same experts who attended the second round of delphi process. panelists were required to score 17 determinants for inclusion in the ultimate tool by choosing yes / no. data analysis was carried out by statistical package for the social sciences (spss version 11.5). to describe the research units, descriptive statistics, frequency distribution, mean and standard deviation there are more than 15 methods to evaluate the expert s consensus between delphi rounds among which interquartile deviation (iqd) and percentage of agreement were employed in this study. moreover, interquartile deviation (iqd) and the percentage of agreement were employed to evaluate the consensus among experts. in the first round of the study which used a 5-point likert scale, determinants with of percentage of agreement of 0.9 and iqd1 were moved to the final determinant set while items with percentage of agreement of 1 were removed from the set. additionally, determinants that scored between these cut - off values were transferred to the second round of delphi. in the second round, with a seven - point likert scale, determinants that obtained an agreement percentage of 0.7 and iqd1 were transferred to the final set whereas those with the percentage of agreement of 1 were omitted. besides, other items were transferred to the third round for the final consensus. in the third round, using a yes / no scoring approach, the determinant was regarded to meet consensus if 67% of the experts confirmed a determinant by choosing yes ; otherwise, it was deleted from the final tool. in the first round, a structured questionnaire for the primary determinants of social accountability was prepared based on extensive search in the related literature across national and international databases. additionally, the researchers used documents such as fifth development plan, the national 2025 vision plan (clearing the way for development and construction of iran in various fields associated with culture, science and technology, society, administration, economics, regional development, security, defense, politics, law, and budget), and general policies for health issues by iranian authorities. the most important references used to draft the items were the general policies of health (5 items), comprehensive scientific health map (10 items), socially accountable medical education strategic plan (15 items), cpu model (7 items), thenet s social accountability framework (12 items), as well some papers presented on social accountability in related high rank journals (30 items). some questions extracted from these sources are as follows : are public health priorities identified ?, are the needs of population at risk considered in the areas of education, research and service ?, is the students competence in response to the priorities of the society measured ?, are education, research and service providing activities revised to adapt with priorities and needs of the society ?, what is the role of school in increasing the partnership of the individuals, families and society to improve public health ?, are faculty members competent enough in the field of social accountability ?, are research and research funds pushed toward health priorities of the society ?, and how are the capabilities of the institutions and organizations used to improve health care sector ? the questionnaire included 128 determinants and was scored using a 5-point likert scale (ranging from 1=strongly disagree to 5=strongly agree). participants were asked to freely write down their ideas, viewpoints, and proposed determinants (if any). the second round included the proposed new criteria and determinants which had not obtained the consensus of experts to be included in or removed from the final instrument. according to the experts opinion, a 7-point likert scale questionnaire with 83 items (from 1=strongly disagree to 7=strongly 15 experts did not tend to participate in the study after the first round, in the second round, 15 experts were asked to examine and score the questionnaire items before the start of the session. during the face to face session, each expert presented his / her viewpoints separately on determinants. the third round was held within a week from the second round and involved sending determinants which had not achieved consensus to the same experts who attended the second round of delphi process. panelists were required to score 17 determinants for inclusion in the ultimate tool by choosing yes / no. data analysis was carried out by statistical package for the social sciences (spss version 11.5). to describe the research units, descriptive statistics, frequency distribution, mean and standard deviation there are more than 15 methods to evaluate the expert s consensus between delphi rounds among which interquartile deviation (iqd) and percentage of agreement were employed in this study. moreover, interquartile deviation (iqd) and the percentage of agreement were employed to evaluate the consensus among experts. in the first round of the study which used a 5-point likert scale, determinants with of percentage of agreement of 0.9 and iqd1 were moved to the final determinant set while items with percentage of agreement of 1 were removed from the set. additionally, determinants that scored between these cut - off values were transferred to the second round of delphi. in the second round, with a seven - point likert scale, determinants that obtained an agreement percentage of 0.7 and iqd1 were transferred to the final set whereas those with the percentage of agreement of 1 were omitted. besides, other items were transferred to the third round for the final consensus. in the third round, using a yes / no scoring approach, the determinant was regarded to meet consensus if 67% of the experts confirmed a determinant by choosing yes ; otherwise, it was deleted from the final tool. the thirty experts for the first round of delphi technique included 15 faculty members, 7 postgraduate students, and 8 graduates, out of which 15 were selected for the second and third rounds. demographic features for the panel and respondents for each survey round the questionnaire was administered to all experts. 23 experts filled in the questionnaire, out of which 8 had no tendency to participate in the second round. thus, 15 experts were studied in the second round, attending the face to face session and completing rounds 2 and 3 questionnaire. the classic delphi method shown in figure 1 (indicating the number of items) depicts the process and results through each round of the questionnaire distribution. determinants examined in different delphi rounds in the first round of delphi, 15 determinants obtained consensus, but 4 were removed. based on the comments by experts on each determinant, a total of 83 determinants (6 proposed new determinants and 77 items which did not obtain the consensus of experts for presence in or removal from the final tool) reached the second round. from the 83 determinants, 39 determinants gained the experts consensus and one additional item was added based on experts suggestion in the second round of delphi. in this round, furthermore, twenty eight overlapping determinants were also merged following comments by experts. at the end only two determinants were removed in the third round and the remaining items obtained the consensus of experts. also, each item was assigned to three activity areas of education, research and services based on the panel of experts. overall, out of the 128 primary social accountability determinants for the school of nursing and midwifery, 69 reached consensus. fifteen of them reached consensus in the first round (table 2), 39 determinants in the second round (table 3), and 15 determinants in the third round (table 4). determinants reaching consensus in the first round main activity : three main activities of nursing and midwifery school. 1 : education ; 2 : research ; 3 : service determinants reaching consensus in the second round determinants reaching consensus in the third round the present study developed objective determinants to determine the social accountability of nursing and midwifery schools through delphi method. it is believed that schools related to health sciences should play their role in teaching, research and providing services to resolve the problems of society. therefore, social accountability determinants obtained from this study can be used as a yardstick to keep track of these activities for nursing and midwifery schools. it is necessary for schools to adjust and revise their educational activities and programs to ensure training students who are capable of meeting community needs. to this end, it has been suggested that educational system should pay special attention to the needs, social issues and health problems of their communities. in the event that the programs of an educational institution are presented in this way, they can be considered a measure to meet community needs and strengthen the health system, in line with social accountability. the current study addressed this issue by including determinants such as identifying health priorities of the community or regulating the mission of schools according to the health priorities of the society. providing evidence - based educational programs in another point it focuses on education according to the needs of society, students and valid scientific findings. therefore, when educational activities of the nursing and midwifery schools are based on evidence, the needs and challenges of the society will be considered, and this issue is expressed as the emphasis of educational programs on evidence - based care. in obtaining the determinants, the moral issues of the society have were emphasized, as an important aspect of professional competence. in some studies, ethical dimension of the social responsibility the review of literature shows that ethics is an important aspect of social responsibility, and consequently organizations are expected to consider the values and beliefs of people in their activities. therefore, earlier studies suggest that codes of ethics should be taught to students in order to institutionalize commitment to these codes. besides, providing educational programs to improve the care provided in the field of spiritual health is another issue of particular importance. studies suggest that today spirituality is the fourth dimension of health and plays an important role in improving the quality of people s lives. some of the earlier studies in iran show that more than 80 percent of nurses have received no training on the concept of spiritual care. as a result, it is essential for educational programs to take into account the spiritual issues related to healthcare. in the present study, another point agreed upon by experts was related to educational programs which should also cover the staff and faculty members of the universities, in the form of research opportunities and continuing education programs to improve their knowledge, attitudes and skills. this finding is consistent with those of the previous study which considered monitoring the staff and faculty members as one of the main sub - themes of social accountability in the programs of educational departments. another important dimension in nursing and midwifery schools is research. according to earlier studies, when a research activity enjoys social accountability and applies scientific methods to solve health - related problems of the society, its results can improve public health. as such, research frameworks necessary for conducting research activities related to the needs of community should be provided to students. in this regard, it has been stated that the reason why suez canal university school of medicine enjoys a high level of social accountability is the research activities of the university which are tailored to the needs of the community health, determined by the ministry of health, and supported by appropriate financial and technical resources for researchers. it is, therefore, important that universities take the right steps in the implementation and evaluation of research activities to achieve predetermined priorities. however, according to some studies, execution and evaluation plans are not based on research priorities in many countries including america. the determinants proposed in this study reflect this issue through several items, including to conduct the researches according to the now and future health needs and challenges of the community health or to support profitable and problem - oriented dissertations and studies related to the health needs of the country. apart from education and research, another point of interest is applying research results in healthcare. applying the findings of a study it enhances the efficiency and quality of healthcare and can improve social accountability situation in nursing and midwifery schools. however, in iran, due to some problems such as lack of sufficient knowledge and skills, research findings are not used properly by healthcare workers. providing high quality healthcare services are the center of gravity in the performance of quality assurance systems. according to the world health organization, quality guarantee of the provided services depends on the competency evaluation and improvement. therefore, evaluating professional competencies of the graduates and students plays an important role in their competence to meet the health needs of the society. to this end, it has been suggested that one of the main approaches to institutionalize social accountability is to train qualified students and graduates with qualifications, by educational institutions. however, according to several studies, professional competence of the students in iran is inadequate and this can seriously affect the quality of healthcare services the current study presented numerous determinants to evaluate professional competence among the students and graduates. a further problem in this area is that the faculty should guide the clients towards a correct lifestyle because a healthy lifestyle is valuable in reducing breakout and health problems, promoting health, coping with stress factors, and improving the quality of life. earlier studies show that lifestyle is related to 53 percent of mortality among people. therefore, in healthcare services a process should be created so that people take responsibility through adopting a healthier lifestyle. however, according to previous studies in the context of iran, adequate training is not provided so that people can lead a healthy lifestyle by themselves. while achieving this level of services is emphasized by the iran s supreme leader and is embedded in the health policy, increasing awareness, responsibility, empowerment and participation of the individuals, families and community depends on providing, maintaining and improving health. therefore, given that one of the goals set by the world health organization by 2020 is improving life- style of people, some strategies should be arranged in the agenda of the faculties related to the health science. these points have also been agreed upon by the study experts and they had consensus about them. finally, it should be noted that the current study had some limitations due to not having its proposed determinants evaluated for psychometric properties, validity and reliability. however, by using a comprehensive literature review and capitalizing on the high level of experience of the study experts for the delphi techniques over three rounds, the finally accepted determinants of social accountability can be regarded as suitable determinants for nursing and midwifery schools in iran. however, due to administrative constraints and because it was not possible that the experts from other universities take part in in - person rounds of the delphi, all experts were selected from mashhad university of medical sciences. social accountability determinants were explained by 69 items in iranian nursing and midwifery schools. results of this study provide the managers and authorities of nursing and midwifery school, policy makers, and associated institutions with valuable contributions. the proposed determinants in the current study can be used as a means toward realizing the goals of social accountability for medical school. it is suggestions that in future research, the psychometric properties of the proposed determinants should be verified. it is also necessary to explain and evaluate social accountability determinants in other schools affiliated to health sciences.	abstractbackground : revising the medical education programs to meet the needs of society has become both a necessity and an important priority due to the considerable increase of population, changing patterns of diseases, and new health priorities. while this necessity has been highlighted in iran s fifth development plan as well as its national 2025 vision plan, the determinants of social accountability have not been explained yet. this study aimed to develop determinants of social accountability in the iranian nursing and midwifery schools. methods : this classic delphi study included thirty experts in nursing and midwifery education, research and services selected based on purposive sampling and three rounds of delphi technique and conducted in nursing and midwifery school of mashhad university of medical sciences. the primary data were collected using an initial structured questionnaire prepared through extensive review of literature. spss 11.5 software was used to analyze the data. the interquartile deviation and percentage of agreement were also used to study the consensus of opinion by experts. results : finding obtained from the rounds of delphi resulted in selecting 69 determinants out of the initial pool of 128 primary determinants of social accountability. the items were selected based on experts consensus and categorized under three main activities of nursing and midwifery school, namely education, research, and service. conclusion : social accountability determinants were explained by 69 items for schools of nursing and midwifery in iran. the proposed determinants can be used by managers and authorities of nursing and midwifery school, policy makers, and evaluating institutions associated with them to ensure realizing social accountability goals.
the mayer - rokitansky - kster - hauser (mrkh) syndrome or mullerian duct agenesis is characterized by congenital aplasia of the uterus and the upper part (2/3) of the vagina in women showing normal development of secondary sexual characteristics and a normal 46, xx karyotype. till date the presence of alopecia with mrkh syndrome is a mere coincidence or an associated finding is a subject of research and may require further evidence. a 17-year - old girl presented with hair loss from the scalp for 1 year. the loss of hair begins at the occipital area of the scalp, and it progressed along the lateral margins of the scalp in a band like fashion clinically suggestive of ophiasis type of alopecia areata. detailed gynecological examination revealed normal secondary sexual characters with well - developed external genitalia and hypoplastic vagina [figure 1b ]. the histopathology of the scalp revealed lymphocytic infiltration around the lower third of the hair follicle, a finding suggestive of alopecia areata. to rule out the cause of amenorrhea, ultrasonograhy of abdomen and pelvis was done which revealed the absence of uterus and right kidney with normal ovaries [figure 2a and b ]. hormone levels (follicle - stimulating hormone, luteinizing hormone, estradiol, testosterone, and thyroid function test) were within normal limits. clinical findings shows (a) ophiasis type of alopecia areata, (b) well - developed external genitalia and hypoplastic vagina ultrasonography of abdomen and pelvis shows (a) absence of uterus, (b) absent right kidney the detailed review of four case reports of mrkh syndrome with alopecia in world 's literature has been given in table 1. first, three reports were from the same geographical region of middle east (jordan, lebanon, and turkey, respectively). hypothesized that the founder mutation in middle east population might be responsible for the condition to be restricted to that geographical region. however, the fourth case report from south asia (pakistan) questioned the hypothesis of founder mutation leading to mrkh syndrome restricted to that geographical region. we are reporting the fifth case report which is the second case report from south asia and first among indian population. our case report is another evidence to suggest that mrkh syndrome with alopecia is not restricted to middle east population. review of literature of mrkh syndrome with alopecia in all previously reported four case reports, there was a history of parental consanguinity and siblings were also affected with mrkh syndrome and alopecia. in our case, there was no history of parental consanguinity and siblings were normal. there are many case reports and syndromes of hypogonadism with alopecia. in all the previous case reports of mrkh syndrome with alopecia, hypergonadotropic hypogonadism was noted. this case is the first report of mrkh syndrome with alopecia with normal gonadal function. mrkh syndrome may have associated abnormalities such as renal agenesis, skeletal abnormalities, hearing loss, or cardiac defects. in our case, the right renal agenesis was the associated finding of mrkh syndrome with alopecia which is not observed in all the previous four case reports under review. in a female patient of alopecia areata, history of primary this syndrome is caused by embryologic growth failure of the mullerian duct with resultant agenesis or underdevelopment of the vagina, uterus or the both. all the reported cases of mrkh with alopecia including our case had alopecia areata which is an autoimmune disease. there seems to be no direct correlation between these entities as one is due to a genetic defect, and other is an autoimmune disease. hence, the presence of alopecia in the case of mrkh syndrome is a mere coincidence, or an associated finding of the syndrome is a matter of further study. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed. the authors certify that they have obtained all appropriate patient consent forms. in the form the patient(s) has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal. the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity can not be guaranteed.	a 17-year - old girl presented with alopecia involving lateral margins of the scalp with primary amenorrhea. there was no history of parental consanguinity, and no other siblings were having similar complaints. her secondary sexual characters were well developed with hypoplastic vagina. histopathological findings from scalp biopsy showed features of alopecia areata. ultrasonography of abdomen and pelvis revealed the absence of uterus and the right kidney. follicle - stimulating hormone, luteinizing hormone, estradiol, testosterone, and thyroid function test was within normal limits. the patient had normal 46, xx karyotype. till date, only four case reports of mayer - rokitansky - kster - hauser (mrkh) syndrome with alopecia has been reported. we are reporting the first case of mrkh syndrome with alopecia with normal gonadal function in world 's literature.
cd8 cytotoxic t lymphocytes (ctls) play an essential role in combating viral infections (zhang and bevan, 2011). during activation by antigen - presenting cells, ctls integrate t cell receptor (tcr), co - stimulatory, and cytokine receptor signaling to fine - tune proliferation and differentiation and establish various effector cell subtypes characterized by the expression of different surface markers and cytokine production abilities (marchingo., 2014). together, these mechanisms allow the generation of ctl responses that can defend the host organism during primary infection and provide protective immunity against reinfection. primed ctls are able to detect viral peptides restricted to major histocompatibility complex class i (mhc - i) and establish a tcr - triggered immunological synapse with their targets to secrete the content of their cytotoxic granules toward the infected cell (dustin, 2008). the targeted secretion of several effector proteins, such as granzymes and perforin, triggers the cell - death machinery in the infected cell while leaving antigen - negative bystander cells intact (lopez., 2012). furthermore, ctls secrete various cytokines that contribute to antiviral immunity. however, it remains unclear how important the contact - dependent killing of target cells is in relation to these indirect mechanisms of control. the efficiency of ctl - mediated contact - dependent killing of different cell types has been studied extensively in vitro. such studies have suggested that ctls can rapidly, serially, and even simultaneously kill multiple target cells within minutes (wiedemann., 2006). however, assays of in vitro killing have a limited capacity to reflect the situation of how ctls sense, reach, and interact with infected cells in a three - dimensional tissue in vivo. whereas many assays of in vitro killing involve ctls and targets co - cultured in suspension or as cell pellets, access to infected cells is likely to be limited in the intact tissue in which only selected cells are infected with viruses. also, the extracellular matrix and bystander cells might exert multiple, often suppressive effects on ctl function (zhang and bevan, 2011). in addition, in co - culture killing assays, ctls are brought passively together with target cells, whereas in vivo killing requires active ctl sensing and migration (germain., 2012). thus, it remains unclear how fast and how robustly virus - infected cells are killed by single ctls in different virus - infected tissues (elemans., 2014, elemans., 2012, hickman., 2015, hogan., 2014). in the current study, we quantified ctl killing kinetics by two - photon microscopy in mice infected with murine cytomegalovirus (mcmv) or modified vaccinia virus ankara (mva). to this end, we used ex vivo two - photon imaging of explanted lymph nodes and in vivo imaging of intact skin together with transgenic and natural ctls and virus - expressed functional reporter systems. importantly, we found that not every contact between ctls and target cells led to a perforin - dependent ca flux and target - cell death. using datasets on single - cell tracking, we estimated the average per capita killing rates (pckrs : the number of targets killed per ctl per day) of transgenic and endogenous ctls that kill different types of cells infected with several strains and species of viruses. in contrast to the conventional theory of highly efficient killing, our results consistently showed that pckrs in vivo were overall limited to a value of about 216 infected cells killed per ctl per day. furthermore, we observed that viral mhc - i immune evasion strongly reduced ctl - mediated antigen - specific contact - dependent killing in vivo. finally, we showed that by increasing the probability of target - cell death after multiple encounters, ctls could cooperate during killing of virus - infected cells. to determine killing kinetics of ctls in vivo, we infected mice with mcmv reporter strains (marquardt., 2011). mcmv-2d expresses the red fluorescent protein (fp) mcherry and a secretable gaussia luciferase, whereas mcmv-3d additionally expresses the ovalbumin (ova)-derived siinfekl peptide epitope. mcmv downmodulates surface mhc - i expression in infected cells, possibly leaving ctls incapable of recognizing their targets (gold., 2004, hansen., 2010, krmpotic., 1999). we therefore also used mcmv-3d-vrap, which lacks m06 and m152, two viral regulators of antigen presentation (vrap) genes interfering with mhc - i recognition (ziegler., 1997), to study the effect of mhc - i downregulation on ctl effector function. to determine mcmv tropism after subcutaneous (s.c.) footpad infection of c57bl/6 (b6) mice with mcmv-3d, we used fluorescence microscopy to detect bright - mcherry - expressing cells in the draining popliteal lymph node at days 1 and 2 after infection. all mcmv - infected cells lacked expression of cd45 and cd169 and were located below the subcapsular sinus floor on the afferent side of the lymph node, approximately 2030 m beneath the capsule (figures 1a1e). some mcmv - infected cells expressed the fibroblast markers podoplanin (gp38) and er - tr7-antigen, but not the cell - adhesion molecule madcam (data not shown). thus, the mcmv strains used in this study initially infect fibroblast- or pericyte - like stromal cells, but not macrophages or dendritic cells. the number of mcherry virus - infected cells as determined by microscopy strongly correlated with the mcmv - encoded gaussia luciferase activity (figure 1f and data not shown). therefore, we used the number of intact virus - infected cells to determine infected cell densities throughout this study. taken together, different microscopy techniques allowed for a clear and unbiased quantification of the density of intact virus - infected cells in defined micro - anatomical regions. we tested the ability of ctls to directly detect and kill cells infected by different variants of mcmv after adoptive transfer of tcr - transgenic, fp - expressing, siinfekl - k - specific ot - i cells into b6 recipients. these mice were immunized with siinfekl peptide and poly(i : c) or with ova protein together with mva. both procedures induced expansion and maturation of ot - i ctls within 46 days (figures 2a and 2b). the percentage of expanded cd44 ot - i ctls present in blood reliably predicted the number of ot - i ctls in lymph nodes 2 days later (figure 2c), allowing the analysis of mcmv infection in mice harboring defined numbers of virus - specific ctls. one day after mcmv-2d infection, ctls expectedly did not express the activation marker cd69, and ex vivo two - photon microscopy showed that ctls failed to attach to or kill infected cells (figures 2d and 2e ; movie s1). upon infection with mcmv-3d, ot - i ctls strongly upregulated the activation marker cd69 but remained highly motile and established only a few contacts with infected cells. consequently, most mcmv-3d - infected cells remained intact, even in the presence of high numbers of activated ctls (figures 2d and 2f ; movie s1). in contrast, in lymph nodes with high ctl densities, most mcmv-3d-vrap - infected cells were killed within 24 hr after infection (figure 2 g ; movie s1). together, these findings demonstrate that viral mhc - i immune evasion protects mcmv-3d - infected cells from ctl - mediated killing and that in the absence of viral immune evasion, high ctl densities correlate with local eradication of infected cells. next, we characterized migration parameters of ctls within the first 1420 hr after infection. compared to ctls in mock, mcmv-2d, or mcmv-3d infection, ctls that attacked mcmv-3d-vrap - infected cells showed significantly reduced track speeds (figure 3a). furthermore, ctls showed very low motility coefficients, a measure for ctl displacement over time, according to the random - walk - hypothesis framework (beltman., 2009). also, ctl turning angles were increased during local confinement to regions with infected cells (figures 3b3d). these findings demonstrate that ctls migrate at low velocities and stay confined in small tissue volumes while recognizing virus - infected cells and that viral immune evasion significantly alters ctl migration behavior and killing of virus - infected cells. mcmv-3d-vrap - infected targets usually remained intact after contact by a single ot - i ctl (figures s1a and s1b ; movie s1). in contrast, when numerous ctls interacted simultaneously or serially with mcmv-3d-vrap - infected cells, we frequently observed target cell disruption (figure 4a ; movie s2). the killing process of the virus - infected cells took some time, and morphological changes were observed over 1040 min, long before the target cells finally disappeared. typically, we observed initial morphological disturbances followed by the formation of blebs and later by the shedding of larger portions, resembling apoptotic bodies, of the mcherry - labeled cell body (figure 4a [lower panel ], figure 4b). the cognate interaction between ctls and their targets rarely led to stable synapses with complete arrest of ctls. instead, while contacting their targets, ctls remained motile with an instantaneous velocity of 24 m / min for periods of approximately 1015 min (figure 4c) before regaining velocity. kinapses observed between t cells and dendritic cells presenting antigen with intermediate or low affinity during t cell priming (dustin, 2008). throughout the manuscript, we will use the term kinapse to describe this type of migratory interaction between ctls and virus - infected cells during target - cell killing. direct observation of ctl - target - cell interaction behavior and quantification of target - cell fate revealed that killed virus - infected cells experienced a median of 3.5 distinct ctl contacts, whereas surviving cells were rarely targeted (0 median contacts ; figure 4d). killed targets encountered a cumulative median contact time of 50 min (figure 4e). individual contacts between ctls and surviving targets lasted 8.5 min (figure 4f), and individual contacts between ctls and killed targets lasted for approximately 9.0 min (median ; figure 4f). these observations indicate that successful killing of infected cells is not simply determined by the duration of individual ctl contacts. when comparing the different mcmv strains, we observed that 1% and 8% of ctls established contacts with mcmv-2d- and mcmv-3d - infected cells, respectively, whereas 38% of the ctls contacted mcmv-3d-vrap infected cells (figures 4 g and 4h). furthermore, we found that virus - infected cells were usually disrupted within 2060 min (but rarely within 10 min) after the first observed ctl contact (figure 4i). together, these findings indicate that induction and execution of pro - apoptotic signaling cascades in virus - infected cells does not ensue instantaneously after a single ctl contact but that the accumulation of multiple ctl contacts determines the fate of virus - infected cells. to study ctl - mediated killing in another infection model, we generated two reporter strains of the poxvirus mva (kremer., 2012). injection of mva - mcherry into the footpad led to infection of cd169 subcapsular sinus macrophages in the draining lymph node (data not shown). ot - i ctls killed mva - ova - mcherry- but not mva - mcherry - infected cells (figure 5a ; movie s3). as observed during mcmv-3d-vrap infection, ctls within the mva - ova - mcherry - infected lymph nodes showed reduced track speed (figure 5b), antigen - triggered dynamic contacts (figure 5c), and disruption of targets that depended on ctl density at the site of infection (figure 5d). to quantify ctl - mediated killing of the different virus - infected cells, we analyzed the expression kinetics of the mcherry reporter to test whether detection of all infected cells can be expected during the two - photon imaging time windows. in vitro infection revealed stronger and faster mcherry expression by mva at 48 hr after infection (figures s2a s2d), whereas after 12 hr, both mva and mcmv were robustly detectable both in vitro and in the infected lymph node (figures s3a s3d ; movie s4). furthermore, virus - specific ctls could already recognize their specific virus - encoded peptide at 8 hr after infection (figures s3d and s3e). thus, direct observation of single ctls and single virus - infected cells was feasible from 1224 hr after infection. next, we used two - photon - microscopy - derived datasets from the different virus - infection models to calculate pckrs of ot - i ctls, i.e., the theoretical average number of infected cells killed per ctl per day (elemans., 2012). pckr values reported in the literature are highly variable and were calculated on the basis of indirect assays or on assays relying on adoptively transferred artificial targets (elemans., 2012, regoes., 2007). on the basis of our direct observation of endogenous virus - infected cells killed, here we calculated pckrs of 4.8 (median, mcmv-3d-vrap) and 4.2 (median, mva - ova - mcherry ; figure 5e). notably, viral mhc - i immune evasion of mcmv-3d reduced the pckr to 0.0 (median), thus resembling infection with mcmv-2d (figure 5e). together, direct visualization and quantification revealed that the ot - i ctl population showed a limited killing efficiency in different infection models and that viral mhc - i immune evasion dramatically reduced ctl killing efficiency in vivo. to determine whether ctls generated from the endogenous pool of cd8 t cells show killing efficiencies similar to those of tcr - transgenic cd8 t cells, we generated ctls in wild - type or perforin - deficient b6 mice (prf) by intraperitoneal infection with mcmv-3d. after 68 days, mcmv - specific ctls were isolated by tetramer staining (altman., 1996). employing our previously developed technique of intralymphatic injection (braun., 2011), we delivered tetramer - enriched (60% purity) or tetrameter - sorted (90% purity) ctls or negatively enriched ctls (depletion of cd62l and non - cd8 t cells) into the afferent lymphatic vessel draining toward the popliteal lymph node of mcmv-3d-vrap - infected mice. one day after intralymphatic transfer, we found that the reduction of virus - infected cell numbers, i.e., killing of targets, was dependent on the local number of b6 ctls (figures 6a and 6b). similar to b6 ctls, tetramer - sorted prf ctls showed the typical migration behavior during target cell attack but were unable to disrupt virus - infected cells (figure 6c ; movie s4). to quantify ctl killing efficiencies after intralymphatic injection, we used a mathematical model to describe the killing of virus - infected cells at different ctl densities 024 hr after infection (supplemental experimental procedures ; figure 6d). this model makes no assumptions on ctl killing mechanisms and has been developed in accordance with published modeling approaches (reviewed in elemans., 2012, and regoes., 2007). furthermore, this model calculates average pckr values for the entire ctl population, and the results obtained can be directly compared to the counting approach described in figure 5. applying this simple mathematical model, we calculated pckrs from 2.0 to 10.4 for tetramer - enriched, tetramer - sorted, or negatively selected polyclonal ctls (figure 6e). in contrast thus, pckrs obtained by intralymphatic transfer of in - vivo - primed ctls and mathematical modeling were in agreement with the pckrs obtained by real - time two - photon imaging, together arguing for a limited average pckr for ctls attacking virus - infected cells. because ctl killing efficiency might be different in a non - lymphoid organ, we next studied ctl killing in the dermis of t - cell - deficient mice (cd3e ; rag2). these animals were reconstituted with fp - ot - i or fp - cd8 t cells. intraperitoneal virus infection was then used to prime and expand t cells (figure s4a). in vivo two - photon microscopy showed that 1 day after secondary s.c. ear infection with mcmv-3d-vrap, but not after infection with mcmv-3d, ot - i ctls migrating around infected dermal fibroblasts were found to exhibit the typical dynamic scanning behavior characterized by low track speed and low motility coefficients (figures s4b s4d). likewise, mice reconstituted with polyclonal cd8 t cells and primed intraperitoneally with mcmv-3d displayed ctls characteristically scanning infected cells in the dermis, as well as local eradication of mcmv-3d-vrap - infected targets (figures s4e s4 g ; movie s5). in the skin, we observed median pckrs of 16.0 and 12.5 for ot - i and polyclonal ctls, respectively (figure s4h). thus, two - photon in vivo imaging of the infected skin supports the conclusion that ctls show limited killing efficiency when attacking virus - infected stromal cells. it is currently unclear whether all ctls specific to the same epitope show the same killing rate or whether some ctls kill more efficiently than others. thus, we first tested whether all ctls that are in contact with infected cells show signs of activation. using ot - i ctls expressing retrovirally transduced nuclear factor of activated t cells (nfat)-gfp (aramburu., 1998) and h2b - morange reporter constructs, and assuming that cognate recognition results in nuclear nfat translocation within 13 min (marangoni., 2013), we addressed whether ctls in contact with infected cells can readily recognize the target. after mcmv-2d or -3d infection, the nfat - gfp fluorescent signals remained in the ctl cytoplasm, which is in agreement with the absence of killing of these virus variants. in contrast, after mcmv-3d-vrap infection, 80% of ctls in contact with infected cells but also some ctls not in contact with targets showed nuclear nfat - gfp (figures s5a s5d ; movie s6 ; data not shown). thus, ctls were usually activated during target - cell contact. however, because ctls frequently retained the nfat signal in the nucleus after target disengagement (marangoni., 2013), perforin pores in the plasma membrane have been suggested to trigger a transient calcium (ca) flux in the target cell (keefe., 2005). to gain further insights into the functional heterogeneity of ctls, we therefore qualitatively and quantitatively analyzed ctl - induced ca influx in infected targets. to study ca fluxes in virus - infected cells, we replaced the luciferase - encoding sequence of mcmv-3d and mcmv-3d-vrap with a sequence encoding the ultra - sensitive ca sensor gcamp6s (chen., 2013), resulting in the reporter viruses mcmv-3d - ca and mcmv-3d-vrap - ca, respectively (figures s6a s6c). after footpad infection of non - immunized b6 mice with mcmv-3d - ca or mcmv-3d-vrap - ca, most infected cells showed spontaneous short ca fluxes that lasted on average 6 s (median ; figures 7a7d ; movie s7). after infection with mcmv-3d-vrap - ca, ot - i ctls dynamically interacted with and triggered long - lasting ca fluxes in infected targets (figures 7e7 g ; movie s7). ctl - induced ca fluxes lasted for 80 s (median ; interquartile range [iqr ] = 40240 s ; figures 7e7 g) and started 480 s (median) after ctl encounter (iqr = 60820 s ; figure 7h). although ctls intensively contacted infected cells, 40% failed to induce a long - lasting ca flux (figure 7i), and only 10% of ctls induced three or four death - associated ca fluxes (figure 7j). perforin - deficient ctls failed to elicit long - lasting ca fluxes and cell death (figure 7k ; figure s6d). together, these findings reveal that individual ctls and ctl contacts targeting virus - infected cells show strong functional heterogeneity. because single ctl contacts frequently failed to trigger long - lasting ca influx or death of target cells, we next addressed whether ctls cooperate while killing virus - infected cells. in all of the above - described imaging models, virus - infected cells were disrupted more frequently when they were contacted by several rather than single ctls. to test for ctl cooperativity, we choose a null hypothesis that assumed that every ctl contact is an independent event and that previous contacts do not influence the target cell s death probability. this bernoulli - series null hypothesis states that the probability p(n) that a target cell will die after n interactions is given by the term p(n) = 1 (1 data from 660 individual mcmv-3d-vrap - infected target cells were grouped according to the total number of ctl contacts observed per target cell. targets without any observed ctl interactions died with a probability of p(0) = 0.01, whereas targets with a single or two ctl contacts died with a probability of p(1) = 0.15 or p(2) = 0.28, respectively. in the case of three or five ctl contacts, the probability that an infected cell would die was significantly higher than predicted from the null hypothesis of independent interaction outcomes (figures 7l and 7 m). thus, ctls were able to cooperate to kill virus - infected cells by increasing the probability of target cell death after multiple ctl encounters. in the present study, we visualized how ctls killed virus - infected cells in vivo. injection, infected draining lymph node stromal cells and subcapsular sinus macrophages, respectively. because all reporter viruses encoded mcherry, we could clearly identify single infected cells and use time - lapse ex vivo and in vivo two - photon microscopy to determine their fates after they were contacted by effector cd8 t cells. two - photon imaging revealed that in most situations, migrating ctls do not come to a complete arrest to establish a static synapse with their target. stable and static synapses have been described in lymph nodes (1) in vivo between antigen - presenting cells and t cells during defined stages of t cell priming and (2) in vitro on coated surfaces between ctls and targets (mempel., 2004, ritter., 2015). as described for static synapses, the formation of dynamic kinapses observed in the present study also relied on the cognate interaction between tcr and mhc - i - presented antigen, because kinapses do not form between ot - i ctls and cells infected with mcmv-2d (no cognate antigen) or mcmv-3d (low surface mhc - i expression). among other factors, the formation of static immunological synapses has been shown to depend on integrin activation, which allows firm adhesion and complete arrest of migrating cells (liu. interestingly, treatment of mice with neutralizing antibodies against 1- and 2-integrin or injection of ctls lacking the tcr adaptor protein adap, involved in tcr signaling leading to integrin activation, did not show differences in the slow but dynamic ctl migration behavior during the attack on infected cells or during the faster migration when ctls were not contacting infected target cells (figure s7). therefore, integrin - independent formation of dynamic kinapses might represent a mechanism that not only allows ctls to deliver the content of their cytotoxic granules but also keeps them in a motile state to successfully search for further targets. by recruiting effector cells to the site of infection, chemokine receptors such as cxcr3 and ccr5 have been shown to contribute to ctl function (hickman., 2015, kastenmller., 2013). in the present study, we observed no significant difference between wild - type and cxcr3- or ccr5-deficient ctls regarding killing efficiencies in the intralymphatic transfer model (data not shown). these findings indicate that the mode of ctl delivery, the strain of virus investigated, and the cell type infected might affect the differential requirement for chemokine receptors for ctl recruitment. in our study, ctl attack on virus - infected cells resembled the behavior of nk cells attacking tumor cells (deguine and bousso, 2013). in addition to assessing cell disruption on the basis of changes in cell morphology, we used the genetic ca sensor gcamp6s expressed in the target cell to better understand ctl - mediated killing. earlier in vitro studies have suggested that perforin pores in the plasma membrane of target cells cause a transient ca influx that is sensed by the cells as a sign to repair plasma - membrane damage (keefe. in virus - infected cells, expression of gcamp6s proved a sensitive and reliable indicator of ca fluctuations. in the absence of natural killer cells and ctls, virus - infected cells showed spontaneous short ca fluxes (data not shown), whereas ctl - induced fluxes could last for several minutes and reliably indicated subsequent cell death. our study revealed that, on average, 8 min elapsed between ctl contact and ca influx, whereas subsequent cell disruption occurred in most situations between 20 and 120 min after the first ctl contact. in one study, in vivo imaging revealed killing of lymphoid cells within 1020 min of ctl contact (mempel., 2006), whereas other studies failed to observe rapid killing of virus - infected keratinocytes, malaria - infected hepatocytes, or tumor cells (breart., 2008, cockburn., 2013, hickman., 2013). notably, when ctls attack peptide - pulsed b cells, additional b cell surface molecules (such as slamf7) could strengthen the ctl attachment and affect ctl behavior during killing. together, the specific tropism of the pathogen and the targeted cell type essentially contribute to the outcome of ctl - mediated immune reactions. the control of virus infection is critically determined by the quality of the ctls generated (plotkin, 2013, varadarajan., 2011). ctls can be heterogeneous with regard to the expression of effector molecules as well as the expression of co - stimulatory or inhibitory surface molecules (jenkins. the data shown here also reveal that ctls are heterogeneous with regard to nuclear translocation of the transcription factor nfat after cognate antigen recognition. furthermore, ctls are highly diverse with regard to their ability to induce death - associated ca influx in target cells ; for example, in movies with an average duration of 66 min, 40% of the ctls were unable to trigger ca influx, whereas 10% induced three or four ca fluxes. the local presence of antigen - specific regulatory t cells can affect ctl killing efficiency (mempel. the presence of immune - suppressive cells might also explain why intralymphatically delivered tetramer - enriched cells kill slightly less efficiently than very pure tetramer - sorted ctls. two - photon microscopy allowed us to analyze the fate of single infected cells with regard to their ctl - contact history. this approach revealed that ctls exhibited cooperativity in killing targets when more than two ctls contacted the infected cell. the underlying mechanisms for this observation are unclear but could be explained by the differential expression levels of granzymes and perforin (and potentially other molecules) in individual ctls (jenkins., 2008). two ctl contacts did not result in detectable signs of cooperativity, maybe because the effect of two ctl contacts is too small to be revealed in the assay setup used. in general, if we assume the existence of ctls with a high or low killing capacity, it is plausible that multiple contacts increase the probability that a fully armed ctl actually disintegrates the infected cell. alternatively or additionally, it seems possible that the accumulation of sub - lethal pro - death signals delivered by different single ctls might lead to faster apoptosis of the target. the average killing efficacy of individual ctls crucially affects the outcome of infectious diseases relying on ctl - mediated killing as a main control mechanism. there is currently no consensus on even the order of magnitude of in vivo ctl killing rates in either experimental models or human diseases (elemans., 2014, elemans., 2012, hickman., 2013). estimated killing rates of mouse and human ctls vary considerably, and it is unknown how much the different viruses studied, the vaccination protocols applied to ctl generation, or the killing assays used affect the killing estimates (garcia., 2015). further complicating matters, the definition of killing rates often relies on different rate constants, not taking into account the number of ctls that might be responsible for the killing (ganusov and de boer, 2008). we therefore chose to calculate the average number of virus - infected cells killed per ctl per 24 hr as a simple measure of the ctl - mediated killing to allow direct comparison between different models. we determined killing rates either by directly observing disintegration of infected cells over time by a defined ctl population size or by determining the ratio of ctls and infected cells at given time points after infection. the pckrs calculated by these approaches give an average value that describes the killing efficiency of the whole ctl population (and not of individual ctls) without assumptions about ctl cooperation or the percentage of non - active ctls. strikingly, our data show a consistent range of 216 infected cells killed per ctl per day, irrespective of the investigated epitopes recognized by the ctl, the method of calculation (counting or mathematical modeling), the type of cell infected (macrophages or stromal cells), or the virus used (mva or mcmv-3d-vrap). because two - photon imaging can not detect killing events that might occur before imaging is started, the pckr values from direct imaging - based counting might slightly underestimate killing efficiencies. however, killing of non - visible cells does not affect our mathematical model, because the time point of killing is not important in this experimental setup. both direct counting and mathematical modeling might have limitations, but all pckr estimates obtained here (from different viruses infecting different cells, different organs, and by different methods) are consistent in that they range from 2 to 16 target cells killed per ctl per day. it remains unknown for how long a single ctl can maintain a certain killing rate, and it will be interesting to measure whether single ctl killing rates increase or decrease after sequential target - cell encounter. all together, our data indicate that the average in vivo ctl killing capacity is rather limited and not in agreement with the assumption that all ctls act as rapid and serial killers that destroy hundreds or thousands of targets per day (elemans., this can possibly explain the failure of some ctl - based vaccines (plotkin, 2013, yewdell, 2010) or the development of chronic viral infections (west., 2011), in which functional ctls are initially present but fail to eradicate all virus - infected cells. all mcmv strains were derived from the psm3fr smith strain (cloned as a bacterial artificial chromosome) and were produced and titrated in vitro according to standard techniques (marquardt., 2011). gcamp6s - expressing mcmv strains were designed with plasmid pgp - cmv - gcamp6s (chen., 2013) obtained from addgene (plasmid 40753). mva - mcherry and mva - ova - mcherry were generated by homologous recombination using the plasmid vectors piiidhr - p7.5 and plw-73 according to standard procedures. c57bl/6 (b6) mice were bred at the central animal facility at hannover medical school under specific - pathogen - free conditions or purchased from charles river or the jackson laboratory (jax). the following mutant b6 mouse strains were used : c57bl/6-tg(cag - egfp) (jax 003291) mice expressing gfp under an artificial cmv - actin - globin promoter (here named gfp), c57bl/6-tg(cag - ecfp) mice expressing cfp under the beta - actin promoter (jax 004218), tcr - transgenic ot - i cd45.1 mice (derived from c57bl/6-tg(tcratcrb)1100mjb / j ; jax 003831), f1-cross between gfp and ot - i cd45.1 (named fp - ot - i), adap mice (c57bl/6j fyb b6.pl-thy1a/cyj b6-tg(tcratcrb)1100mjb), cd3e mice (c57bl/6-cd3etm1mal / orl), prf1 mice (jax 002407), and rag2 mice (jax 008449). all experiments were conducted in accordance with the local animal welfare regulations reviewed by the institutional review board and the lower saxony state office for consumer protection and food safety (laves). cd8 t cells from ot - i mice, containing 10 cd8 tcr v5 ot - i cells, were transferred into recipient mice. two to six hours after mcmv-3d-vrap infection, 2.5 10 to 1 10 ctls diluted in 5 l pbs were injected in 90 s into the afferent lymph vessel draining toward the popliteal lymph node, as described before (braun., 2011). ex vivo two - photon imaging was performed with custom - build chambers for imaging explanted lymph nodes, as described in halle. the skin at the site of infection in the ear of anesthetized mice was imaged directly in vivo. we calculated the number of infected cells killed per t cell per day on the basis of the observed time course of the number of killed virus - infected cells and the average number of ctls in two - photon microscopy movies. data from independent experiments were pooled, and average killing rates were reported. alternatively, we used the single - time - point imaging data from the intralymphatic injection together with the mathematical model to calculate average ctl killing rates and 95% confidence intervals. statistical analysis was performed with graphpad prism 4. when comparing two groups, we calculated p values with the nonparametric mann - whitney test. to compare multiple groups, we used the kruskal - wallis test with dunn s test. a. braun., k. werth, and r.a. performed the experiments ; k.a.k., h.k., and m.m.- h. designed and applied the mathematical models ; and s.h. and r.f.	summaryaccording to in vitro assays, t cells are thought to kill rapidly and efficiently, but the efficacy and dynamics of cytotoxic t lymphocyte (ctl)-mediated killing of virus - infected cells in vivo remains elusive. we used two - photon microscopy to quantify ctl - mediated killing in mice infected with herpesviruses or poxviruses. on average, one ctl killed 216 virus - infected cells per day as determined by real - time imaging and by mathematical modeling. in contrast, upon virus - induced mhc class i downmodulation, ctls failed to destroy their targets. during killing, ctls remained migratory and formed motile kinapses rather than static synapses with targets. viruses encoding the calcium sensor gcamp6s revealed strong heterogeneity in individual ctl functional capacity. furthermore, the probability of death of infected cells increased for those contacted by more than two ctls, indicative of ctl cooperation. thus, direct visualization of ctls during killing of virus - infected cells reveals crucial parameters of cd8 + t cell immunity.
liposarcomas are histologically divided into five subtypes : myxoid, pleomorphic, dedifferentiated, round cell, and well - differentiated (wd) liposarcoma (atypical lipomatous tumor). approximately 75% develop in the deep soft tissue of the limbs, followed by 20% in the retroperitoneum and a much smaller percentage in the inguinal region. other sites are uncommon ; in particular, primary wd liposarcoma in the mesentery is rare.. here, a case of wd liposarcoma in the duodenal mesentery is presented, and the literature is reviewed. the patient was a 73-year - old japanese man who received follow - up care for viral hepatitis, liver cirrhosis, and hepatocellular carcinoma by a gastroenterologist at the takii hospital of the kansai medical university. he had noticed the presence of an upper abdominal mass for approximately 1 year, but did not seek treatment due to the lack of symptoms. finally, he consulted a gastrointestinal surgeon because of the rapid growth of the abdominal mass and upper abdominal insufficiency. physical examination revealed a large and movable mass palpated in the left upper abdomen, and computed tomography showed a well - circumscribed tumor mass with no metastases. tumor markers, including carcinoembryonic antigen, ca19 - 9, and -fetoprotein, were within normal limits. the upper abdominal tumor was located in the duodenal mesentery close to the ligament of treitz with no adhesion to the surrounding organs, such as the transverse colon and liver. the patient remained healthy without any evidence of recurrence for 6 months after the surgery. the excised tumor was a well - circumscribed mass that was 12.4 9.6 cm in size and weighed 548 g. cut sections of the tumor showed a lobulated yellow and/or grayish - colored appearance with no necrotic areas (fig. different histologic areas were separated by broad fibrous septa containing cells with enlarged hyperchromatic and atypical nuclei (fig. some areas contained collagen fibrils of varying density embedded with spindle cells, including multinucleated giant cells and some cells exhibiting a floret - like pattern (fig. 2b). other areas consisted predominantly of mature fat cells that were variable in size ; the nuclei of these cells were slightly pleomorphic and hyperchromatic (fig. 2c). additionally, there were areas of dense lymphocytic infiltration and/or myxoid change. within the tumor, the labeled streptavidin biotin (lsab) technique was performed with an lsab staining kit (dako, carpinteria, denmark) and the antibodies listed in table 1. 3a), and a few cells were positive for -smooth muscle actin but negative for desmin. fat cells were s-100 positive, whereas spindle cells were s-100 negative. in tumor cells (including both spindle cells and fat cells), the average ki-67 proliferation index was 10% (fig. the tumor was determined to be a wd liposarcoma (atypical lipomatous tumor) that primary originated from the duodenal mesentery. we report a case of primary wd liposarcoma originating in the mesentery of a 73-year - old man. the most common differential diagnostic problem of wd liposarcoma is its distinction from spindle cell / pleomorphic lipoma. lipoma - like wd liposarcomas can be identified by the population of various - sized fat cells, including atypical nuclei and immature fat cells. a characteristic feature of spindle cell / pleomorphic lipomas is the presence of floret - type giant cells. however, floret - type giant cells are rarely seen in wd liposarcomas, and then only in small numbers. in the present case, floret - like multinucleated cells were observed in sclerotic regions. similar to myxoid liposarcomas, wd liposarcomas occasionally may have a predominantly myxoid appearance. however, it is not definite that widespread cd34 immunoreactivity can exclude other lipomatous neoplasms ; diffuse cd34 staining in a lipomatous tumor may simply reflect the presence of spindle cells [6, 7 ]. in the present case, cd34 showed diffuse and strong positivity in spindle - shaped and multinucleated giant cells embedded in the sclerotic stroma. immunohistochemical detection of cdk4 and mdm2 as well as gene amplification of cdk4 and mdm2 status has been shown to be a sensitive and specific means of identifying wd liposarcoma from other benign lipomatous tumors. in our study, some tumor cell nuclei were cdk4 positive, and a smaller number of cell nuclei were mdm2 positive. moreover, in agreement with a previous report, the ki-67 index was highly suggestive of wd liposarcoma in the present case. the cytogenetic pattern of wd liposarcoma is characterized by the consistent presence of circular (ring) and giant rod chromosomes [1, 5 ]. however, in the present case, fresh material was not available for a cytogenetic study. primary wd mesenteric liposarcomas are rare with only 12 well - documented cases (including the present case) in the english literature (table 2) [10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ]. wd mesenteric liposarcoma tends to occur in the fourth to seventh decades of life (mean age 57.9 years) with no difference in frequency based on the patients sex (men / women : 7/5). although immunoreactivity for androgen receptor has not been systematically studied in wd liposarcomas, the expression of androgen receptor was seen in the tumor cells in the present case. endogenous and/or exogenous steroids via the androgen receptor may be involved in the growth of this tumor. wd liposarcomas are relatively large in size, and most tumors are over 10 cm in diameter at the time of surgery. wd liposarcomas can be subdivided into the following groups : lipoma - like, sclerosing, inflammatory, and spindle cell. the presence of more than one histologic variant is common in single tumors, and subclassification does not indicate any prognostic significance. in our survey, we found that mixed variants are frequently described in wd liposarcomas. among the 12 cases, local recurrence occurred in 2 cases. surgical margin may be the most important prognostic factor in that incomplete resection may lead to recurrence, and the risk of dedifferentiation may also be considered. in conclusion, complete resection and long - term follow - up are necessary for wd mesenteric liposarcoma due to the risk of local recurrence of the tumor.	mesenteric liposarcoma is a rare neoplasm. here, we report the case of a 73-year - old japanese man with a well - differentiated (wd) liposarcoma of the mesentery. due to rapid growth of the abdominal mass and abdominal insufficiency, a tumorectomy was performed. the excised tumor was 12.4 9.6 cm in size and weighed 548 g. cut sections showed a lobulated yellow and/or grayish - colored appearance. the histological features were predominantly those of the sclerotic and lipoma - like variants of wd liposarcoma. the cytoplasm of most spindle cells was diffusely immunoreactive for cd34, while fat cells were positive for s-100 protein. some spindle cell nuclei were positive for cdk4, and a few were positive for mdm2. the average ki-67 proliferation index in tumor cells was 10%, and androgen receptor expression was detected in tumor cell nuclei. the present case and 11 cases identified from a literature search were reviewed. the wd mesenteric liposarcomas developed in patients in the fourth to seventh decades of life (mean age 57.9 years). the patients consisted of 7 men and 5 women. all tumors were larger than 10 cm in diameter at the time of surgery. complete resection might be the only curative therapy for wd liposarcomas of the mesentery, but long - term follow - up is needed because of the possibility of a local recurrence of the tumor.
esophageal cancer (ec) is the eight most common cancer worldwide, accounting for 3.2% of total new cases in 2012.(1) about 80% cases of ec worldwide occur in less developed regions.(2) according to globocan 2012 estimate, in india, there were 42,000 new cases of ec, the incidence being 4.13% of all new cancer cases, and 39,000 deaths were caused due to ec.(1) acharya tulsi regional cancer treatment & research institute, bikaner, rajasthan is a regional cancer center witnessing a large number of cancer patients from northwest india. despite various advances in the treatment of ec, it is one of the least responsive tumors to cancer therapy, and the overall prognosis remains poor. the ratio between mortality to incidence is about 0.88, more than 80% of the cases die within 2 years of diagnosis even in developed nations.(3) it is the nation 's most common malignancy involving the gastrointestinal tract.(4) in north america and western europe, alcohol and tobacco use are the major risk factors for squamous cell carcinoma (scc), accounting for 8090% of cases.(5) diets of scant amounts of fruits, vegetables, and animal products are associated with increases in scc.(6) other risk factors associated with ec include plummer - vinson syndrome (a condition characterized by iron - deficiency anemia and low riboflavin levels), achalasia, and tylosis.(7) in india, ec is being reported in increasing numbers from assam, kashmir, tamil nadu, karnataka, and kerala.(8) no literature is present from northwest india about the etiopathology of ec. therefore, it is important to understand various aetiopathological factors associated with ec to find out various schemes for primary prevention of the disease. in this clause, we have reviewed the sociodemographic parameters of the patients with ec who visited the oncology department of our center in the last 10 years. this is a retrospective analysis of medical records of the cancer patients registered in the regional cancer center of northwest india from january 2003 to december 2012. being a retrospective study, no ethical approval was required for the study as all the patients were treated with the standard departmental protocol. for extracting the data, the computerized data, hard copies of the files, and also the radiotherapy files of the patients were reviewed. a total of 55,242 patients with a variety of malignancies were registered in the 10-year duration. out of this, the sociodemographic parameters including age, gender, locality, socioeconomic status, religion, education, occupation, and the addiction patterns of the patients were studied. nonsmokers were defined as having smoked fewer than 100 cigarettes in their lifetime or less than one cigarette per day for 6 months or more. all others were counted as smokers for the purpose of recording in the history sheet. to assess alcohol consumption, subjects were asked about their usual intake of beer, wine, and liquor from the age at which they started drinking at least one alcoholic beverage per month. modified bg prasad classification(9) was used to assess the socioeconomic status of the patients as per the institutional policy. to calculate odds ratio (or) for smoking, the data of global adult tobacco survey (india) was used to define the control population.(10) the data from global survey on alcohol and health data by world health organization (who) region was utilized to calculate or for alcohol abuse.(11) all the recorded data was entered in windows excel sheet and imported to the statistical software to perform the calculations. statistical calculations were performed used statistical package for social sciences (spss) for windows version 20.0 (ibm corp., out of 55,742 patients registered in our hospital, 3,667 were diagnosed to have ec. 53.4% patients were males with male : female ratio of 1.15:1, indicating almost similar incidence of ec in males and females of northwest india. the mean age was 54.6 11.7 years ; 66.2% of the patients were illiterate and 48.6% belonged to the low socioeconomic status ; 41% cases were of > 60 years age, while 3.4% of the patients were of 30 years ; and 76% of the patients came from the rural areas. the primary location of the disease was 19.4, 43.9, and 28.9% in upper, middle, and lower third of the esophagus, respectively. squamous cell histology was identified in 75.6% of the patients, while 18% patients had adenocarcinoma. smoking was identified as a risk factor in 48% of the patients with bidi as the most commonly (26%) smoked form of the tobacco, 25% of the patients were addicted to alcohol, while 11% had the habit of chewing tobacco. strong associations was observed between smoking and ec (or = 5.7, 95% confidence interval (ci) = 5.36.1). alcohol abuse was also associated with increased risk of ec (or = 2.7, 95% ci = 2.13.3). ec is a disease with poor prognosis with approximately half of the patients presenting with unresectable or metastatic disease, thus cure rate of more than 15% are seldom achieved. most of the western literature has reported male to female ratio of 4:1.(1213) however, in our study, this ratio is 1.15:1, thus indicating a higher incidence of ec in females of northwest india. using the interactive cancer atlas which is a project of the national cancer registry programme, it was found that highest age adjusted incidence rate of ec (international classification of diseases (icd)-10 : c15) in females of india is in east khasi hills district of meghalaya (10.8 per 100,000 population) ; while for males, it is 9.9 per 100,000 at the same place.(14) however, for males, this figure is highest in aizawl, mizoram (26.7 per 100,000). studies from south india have reported a ratio of 2:1.(1516) another study from western india observed a ratio of 1.4:1.(17) kamangar., reviewed the epidemiological pattern of ec in northeastern india and reported 1:1 ratio in golestan province.(3) the epidemiologic feature of equally high incidence of ec among females is a rare one ; such ratios have also been reported in linxian, china.(18) one common feature of these areas of the world with 1:1 male to female ratio is high registry reported rate of 100 per 100,000 population.(1920) very high risks and this unusual incidence pattern may indicate the presence of a strong risk factor that is shared by both the genders.(3) thus, it can be inferred that the areas with high prevalence of ec have nearly equal ratio of females and males affected by the disease. in our study, 3.46% patients were of 30 years. it has been found that ec is rare in individuals younger than age 30 in both low and high incidence areas ; ec cases in those 30 years of age in northern china, northeastern iran, and the surveillance, epidemiology, and end results (seer) registries in the us account for 0.7, 1, and 0.5% of cases, respectively.(212223) another indian study by chitra., also reported similar age patterns with 5% cases below the age of 40 years with majority 62% cases of age group 4160 year.(4) in the us, the mean age of ec patients at diagnosis is 68 years, while it was 54.6 years in our study.(18) in our study, about two - third of the ec patients were illiterate and half belonged to low socioeconomic status as per the modified bg prasad scale. sehgal., also reported that the majority (63%) cases were illiterate and 59.5% from lower socioeconomic status.(16) thus, our data are in agreement with already published literature. this may stem from the fact that most of the patients visiting our cancer center belong to the rural area with farming being their main occupation. another plausible explanation of this observation may be the use of organophosphorus pesticides in farming practices in patients coming from sriganganagar district of rajasthan and patients of punjab and haryana. however, exact comment on this etiology is not possible in the absence of specifically designed studies investigating the level of organophosphates in the serum of these patients. such studies will further provide insight into the carnal knowledge of pesticides with cancer. in our study, 19.4% patients had the malignancy of upper third of the esophagus ; 44% patients had middle and 29% had carcinoma of the lower esophagus. in 7.6% of the patients, insufficient data were available to locate the exact site of the lesion. in another study by giri., the percentage of patients with upper, middle, and lower third cancer was 9.66, 40.57, and 49.76%, respectively.(17) in our study, 18% patients had adenocarcinoma, while 75.6% had scc. in 6.4% cases, western literature has reported alcohol and tobacco use to the major risk factor for scc, accounting for 8090% of the cases.(5) in the present study also, the addiction of smoking was present in the majority (48%) of the patients. western reports have also described the relative risk of ec in relation to the amount of alcohol and tobacco consumed, including a relative risk of 155:1 when consuming 30 g / day of tobacco with 121 g / day of alcohol.(24) esophageal scc risk is 4.2 times higher in current smokers in europe compared with never - smokers, a meta - analysis showed.(25) another meta - analysis showed that esophageal ac risk is 2.3 times higher in people with 40 + years of cigarette smoking, compared with never - smokers.(26) in our study, the or for smoking and risk of ec was estimated to be 5.7. since 75.6% patients in our study had scc, our data is in agreement with the results of these meta - analyses. esophageal scc risk is 3038% higher in people who consume around 11.5 units of alcohol per day, 2.6 times higher in those who consume around 1.56 units of alcohol per day, and 5.5 times higher in those who consume 6 + units of alcohol per day, compared with never - drinkers.(2728) esophageal ac risk is not associated with alcohol drinking overall (versus not drinking).(29) esophageal carcinoma is one of the important cancers prevalent in northwest india with a relatively higher incidence in females as compared to the findings of other studies. the etiology in majority of patients is linked to tobacco and alcohol, thus, modification of lifestyle with limiting the use of addictions may be an effective strategy in the prevention of this dreaded and mostly incurable disease. a nationwide campaign is required to generate public awareness about this dreaded disease along with identifying the high risk population. besides, further research is needed to identify the causes of geographical variations in the incidence of ec and its relatively higher incidence in females of northwest india.	background : despite various advances in the treatment of esophageal cancer (ec), being one of the least responsive tumors to cancer therapy, the overall prognosis remains poor. therefore, it is significant to understand various sociodemographic factors associated with ec to find out various schemes for primary prevention of the disease.materials and methods : this is a retrospective analysis of medical records of the ec patients registered in the regional cancer center of northwest india from january 2003 to december 2012. the site of the disease and the histology were also recorded in addition to the various sociodemographic parameters.results:out of 55,742 patients registered in our hospital ; 3,667 were diagnosed to have ec. male : female ratio was 1.15:1. the mean age was 54.6 11.74 years ; 66.15% of the patients were illiterate and 48.6% belonged to the low socioeconomic status. smoking and alcohol consumption were identified as risk factors in 48 and 25.6% of the patients, respectively.conclusions:the etiology in majority of the patients is linked to tobacco and alcohol, thus, modification of life style with limiting the use of addictions may be an effective strategy in the prevention of this dreaded and mostly incurable disease.
despite advances in breast cancer treatment, bone remains the most common site of metastasis, occurring in around 70% of patients with metastatic disease. bone metastases are incurable and associated with significant morbidity in terms of fractures, pain, and reduced quality of life. bone destruction occurs from disruption of the finely controlled balance between bone resorption (by osteoclasts) and formation (by osteoblasts), resulting in net bone breakdown. increased understanding of the pathogenesis of bone disease has resulted in the development of a number of bone - targeted agents (btas), the most widely used clinically being inhibitors of osteoclastogenesis and osteoclast activation [i.e. bisphosphonates, or receptor activator of nuclear factor kappa - b ligand (rankl) inhibitors (e.g. denosumab) ]. however, despite the use of these increasingly potent btas, progress in terms of absolute reductions in the occurrence of skeletal related events (sres) is modest, and further basic, translational, and clinical research is clearly needed,,,,,,. the bone and the oncologist new updates (bonus) meeting is an annual canadian multidisciplinary conference on the interaction of bone and cancer biology. each year, clinical oncologists, basic scientists, and other health researchers gather to discuss the discoveries in bone research and their implications for cancer patients. the most recent conference, bonus 8, was held in ottawa in april 2013 with featured speakers from across canada and the united states. topics of discussion were diverse, and ranged from prevention of skeletal metastases to the exploration of biomarkers as tools to guiding treatment. as part of this meetings mandate is to ensure publication of findings to as broad an audience as possible this commentary was written to assimilate key presentations from a number of experts in the area and focus on where the field is moving, or needs to move, if we are to make further progress. quite simply the objectives of any therapy should be the prolongation of life and/or improvement of quality of life. we all recognize that bone metastases are common in breast cancer and cause significant morbidity. they may lead to sres, including pathologic fractures, need for surgery or radiation to bone, spinal cord compression, and hypercalcemia. these complications result in loss of autonomy, pain, and consumption of significant healthcare resources. bone - targeted therapy with bisphosphonates and more recently, denosumab, has been seen as an important part of anti - cancer therapy. in the bone microenvironment, bisphosphonates may exert anti - tumor effects via attenuation of tumor adhesion and invasion, and induction of tumor cell apoptosis. this class of drugs may also indirectly suppress tumor proliferation by virtue of their anti - angiogenic effects. denosumab is an inhibitor of the rank ligand, a factor that promotes osteoclast differentiation and activation, and interruption of this interaction may lead to decreased bone resorption and destruction. both bisphosphonates and denosumab have been shown to be effective in reducing sres,,. however, while bisphosphonates and denosumab are effective in preventing and delaying sres, none have yet shown a beneficial effect on overall or progression free survival,,. moreover, the benefit in terms quality of life for patients with metastatic cancer from the use of these bone - targeted agents is also unclear. while long term follow up of two large trials comparing pamidronate to placebo showed that patients in the pamidronate arm experienced less pain, the overall quality of life was not different between the arms similarly, another trial comparing zoledronic acid to placebo showed that while zoledronic acid did improve pain control slightly compared to placebo, it did not improve the overall quality of life for patients on this drug. lastly, in a trial comparing denosumab to zoledronic acid in metastatic breast cancer, statistical improvements in quality of life was observed with denosumab only at a minority of time points during follow up and the analysis was compromised by the effects of multiplicity. in summary, current evidence suggests that bone - targeted agents reduce morbidity from metastatic disease to bone, and potentially improve quality of life in patients with metastatic breast cancer. as there are costs and potential adverse effects associated with these agents, care should be taken in their use. in addition, further work is needed in the creation of meaningful measures of quality of life in these patients. metastatic tumor growth in the bone is affected by a complex network of cellular interactions and effector molecules. the interactions between osteoclasts, osteoblasts, and tumor cells have been shown to drive tumor growth through a vicious cycle. in this framework, tumor cells secrete factors such as parathyroid hormone - related protein (pthrp) that induce osteoblast and osteoclast activity, leading to bone resorption and destruction, release of growth factors such as transforming growth factor (tgf)-, and subsequent tumor growth and perpetuation of the destructive cycle. what has become clear over the years is that while the vicious cycle was a relatively simple concept to explain some interactions occurring in metastatic bone disease and rationale for the development of bisphosphonates and denosumab the real picture is a lot more complex. increasingly, the importance of the tumor microenvironment itself in this destructive cycle has come under scrutiny. cells that are not directly involved in bone remodeling, including lymphocytes, macrophages, and stromal cells may affect tumor growth through their interactions with tumor cells. as part of the bone marrow stromal environment, adipocytes, fibroblasts, and chrondrocytes have been implicated in the differentiation and proliferation of both hematopoietic and cancer cells, in part through secretion of pro - resorption cytokines by tumor cells following their engagement with stromal cells via vcam-1 mediated interactions. myeloid - derived suppressor cells (mdsc) represent a diverse population of myeloid - lineage cells that includes macrophages, dendritic cells, and granulocytes. they are known to proliferate in the setting of cancer, can down - regulate the immune response, and have been linked to multiple aspects of cancer progression, including tumor growth, angiogenesis, and metastasis,. in a xenograft mouse model, these cells have been shown to differentiate into osteoclasts and accelerate tumor growth and bone destruction. the extracellular matrix (ecm) of the bone the ecm contains a constellation of proteins and other biomolecules that serve both as a scaffold for mineral deposition, and as signaling molecules that help direct bone formation and resorption. bone sialoprotein (bsp) is a major non - collagenous protein in the ecm, and can induce cell adhesion and promote osteoclastogenesis. high bsp levels are seen in multiple cancers, and have been associated with excessive bone resorption in animal models. indeed, preliminary experiments in a mouse model using sirna - mediated knockdown of the bsp gene revealed reductions in both osteolysis and tumor incidence. it is clear that the process of bone metastasis involves much more than the model of the vicious cycle itself, and that many types of cells, effector molecules, and the extracellular matrix participate in the development of bone metastases. better understanding of their interactions with bone and tumor cells may lead to discovery of novel protective therapies. bone resorption due to osteoclast activity can liberate growth factors from the bone matrix that can drive further tumor growth. this process is associated with significant morbidity including bone loss, pathologic fractures, and cancer - induced bone pain. aberrant signaling in these pathways not only drive tumor growth but can lead to significant symptoms such as pain. this concept of abnormal cues on the physiology of the body in the context of cancer may be termed oncodynamics. investigation of these abnormal cues may lead to novel therapies for cancer - associated symptoms. for example, glutamate is increasingly recognized as one of the important dysregulated signaling molecules in cancer biology, especially in the context of bone metastasis. commonly recognized as an excitatory neurotransmitter necessary for normal brain function, glutamate is also intimately involved in bone metabolism in both health and diseased bone. multiple cancer cell lines are known to secrete glutamate into the extracellular environment. in vitro models have shown that its secretion by cancer cell lines stimulated osteoblast differentiation, while inhibition of glutamate release led to reduction of the osteoclast population. specific glutamate receptors present on osteoclasts have been identified, and modulation of these receptors can inhibit glutamate release and bone resorption. multiple studies involving subcutaneous administration of glutamate to healthy volunteers revealed glutamate to be a potent dose - dependent inducer of the pain response,. given its secretion by cancer cells, tumor - derived glutamate may be involved in the generation or maintenance of cancer - induced bone pain, through direct stimulation of perception of pain or its disruptive effect upon bone homeostasis in the presence of bone metastases. major and minor depression can be seen in over one third of cancer patients, a rate that is far higher than in the general population. abnormalities in glutamate levels have been observed in multiple areas of the brain in patients with depression. compellingly, the glutamate receptor antagonist ketamine has been shown in multiple small studies to produce anti - depressive effects in patients. whether direct links exist between tumor - secreted glutamate and depression the bonus conference continues to be a forum that attracts oncologists, basic scientists, and other professionals interested in bone health in cancer. this years meeting reviewed our current understanding of bone biology and metastasis, as well as ongoing research in the field. what is evident is that we have likely maximized the benefits that patients will receive from current bone - targeted therapies and that the gains from increasingly potent agents while statistically significant are clinically modest,. if progress in this area is going to be made we need to use the new knowledge we are generating around the complex interactions that occur in bone to develop new treatment strategies. ultimately we all hope that this will also enhance our efforts at trying to stop breast cancer from spreading to the bones in the first case.	the annual bone and the oncologist new updates (bonus 8) conference focuses on the current understanding and dilemmas in the treatment and prevention of bone metastasis in cancer, as well as novel research on bone homeostasis and cancer - induced bone loss. we present commentaries from experts for their own take on where they feel the field of bone - targeted therapies for metastatic breast cancer is moving, or needs to move, if we are to make further progress.
hemophagocytic lymphohistiocytosis (hlh) is a hyperinflammatory disorder with clinical manifestations that include fever, organomegaly, and cytopenia1). hlh occurs either as a primary form (genetic or familial) or as a secondary form (acquired)2). the familial form of hlh (fhl) is a potentially fatal autosomal recessive disorder due to constitutionally defective cell - mediated cytotoxicity3). in particular, the onset of fhl is reported to be within one year after birth in 70% to 80% of the cases. until now, five genetic loci for fhl (fhl1-fhl5) have been reported, involving the following four causative genes : prf1 (fhl2), unc13d (fhl3), stx11 (fhl4), and stxbp2 (fhl5)3). in this report, we describe a fatal case of fhl2 due to a novel prf1 gene mutation in a newborn girl with severe clinical manifestations that mimicked severe sepsis. the proband was a 29-day - old female infant who was admitted to our institution because of four days of mild fever and rapid breathing, moaning, and mottling. she was the second child of a nonconsanguineous couple, born at 38 weeks of gestation with a birth weight of 3,500 g. family history revealed that her sister died at 28 days of age from sepsis with rapidly progressive clinical manifestations of acute hepatitis, gastrointestinal bleeding, and pulmonary hemorrhage. the initial neonatal course of the proband was not remarkable until she developed respiratory distress and uncontrolled fever on postnatal day 29. her condition deteriorated, and she developed lactic acidosis, 22.38 mmol / l (normal range, 0.5 - 2.2 mmol / l), which required mechanical ventilation and continuous veno - venous hemodiafiltration (fig. the 7th day of the admission, laboratory finding revealed leukocytes level at 4,01010/l, thrombocytes 2810/l, hemoglobin 9.7 g / dl, absolute neutropenic counts 96010/l, triglyceride 774 mg / dl, ferritin 165,000 ng / ml, fibrinogen 93 mg / dl. laboratory workup for infection revealed no diagnostic findings ; however, on day 31, broad - spectrum antibiotics and intravenous immunoglobulin and corticosteroid were administered empirically with a presumptive diagnosis of neonatal sepsis. since the patient 's manifestations met the criteria for hlh 2004, a bone marrow study was performed on day 35, which revealed frequent active hemophagocytic histiocytes. as the first step of a diagnostic workup for fhl, we performed flow cytometric analysis for the intracytoplasmic perforin expression of cytotoxic cells using the proband 's peripheral blood sample, as previously described with some modifications4). we observed a complete absence of perforin expression in the proband 's natural killer (nk) cells and cd8 + t cells. the experiments using her parents ' blood samples demonstrated partial deficiency of cytoplasmic perforin in nk cells and complete deficiency in cd8 + t cells (fig. 2a). based on the results of flow cytometric analysis, we obtained written informed consent from the parents and proceeded with a molecular genetic test for fhl2 by direct sequencing analysis of the prf1 gene, as previously described5). as a result, the proband was shown to have two deleterious point mutations of prf1, c.65delc [p.pro22argfs2 ] and c.1090_1091delct [p.leu364glufs93 ] (fig. her parents were heterozygous carriers of the respective mutant alleles (compound heterozygous for the c.65delc mutation (of maternal origin ; novel) and the c.1090_1091delct mutation (of paternal origin ; known), confirming the compound heterozygous status of the two mutations in the proband. based on the diagnosis of fhl2, the patient received hlh-2004 chemotherapy with cyclosporine, etoposide, and dexamethasone. she experienced hypotensive episodes that were initially responsive to fluid resuscitation and medications but that eventually progressed to refractory status, leading to renal failure. in this report, we described a fatal neonatal case of fhl2 due to mutations of prf1, including a novel frameshift mutation. the patient 's complicated multiorgan manifestations and fulminant clinical course mimicking severe sepsis delayed the diagnosis of fhl. the family history included a deceased sister with similar clinical manifestations, which led us to search for a genetic etiology. since the onset of fhl is typically young, frequently < 1 year6), clinical suspicion for fhl is critical for timely diagnosis and proper management to improve the prognosis of the disease in neonatal intensive care. flow cytometric analysis of the intracytoplasmic perforin expression of cytotoxic cells and, more recently, intraplatelet expression of munc 13 - 4 protein have been introduced to screen for fhl2 and fhl3, respectively4,7). our patient showed a complete deficiency of perforin in both nk cells and cd8 + t cells, while her carrier parents had a partial deficiency in nk cells and a complete deficiency in cd8 + t cells, respectively. this observation is in line with the findings described in a previous report4). at the molecular level, the patient was compound heterozygous for two point mutations, c.65delc (p.pro22argfs2) and c.1090_1091delct (p.leu364glufs93). both mutations are deleterious in nature by way of introduction of a premature stop codon due to a frameshift. the complete deficiency of perforin expression revealed by flow cytometric analysis could be interpreted in the context of a genotype - phenotype correlation : two deleterious mutant alleles resulting in severe protein deficiency and the early age of onset followed by a fulminant clinical course. based on a review of the literature and mutation database (human gene mutation database professional ; http://www.hgmd.org/), c.65delc is a novel mutation. the c.1090_1091delct mutation had been reported in a korean patient (a two - month - old girl) with homozygous status in our previous study5). interestingly, c.1090_1091delct was reported to be the most frequent fhl2 mutation in japan8). the small number of fhl2 in korea is due to the unusual predominance of fhl3 (~90%)5). collectively, c.1090_1091delct is restricted to fhl2 of korean and japanese descendents with high frequencies, suggesting a distinct geographical distribution and founder effect, as exemplified by prf1:c.50delt mutation in fhl2 in people of african ancestry9). clinically, our patient had a course that rapidly deteriorated into multiorgan failure requiring continuous veno - venous hemodiafiltration. severe lactic acidosis and a family history of a sibling who had died from similar clinical manifestations that mimicked sepsis led us to suspect bacterial sepsis with a genetic etiology of metabolic disease. the suspicion of fhl was delayed until we obtained negative results for bacteriologic and metabolic workup. however, persistent fever, sepsis - like features, and a progressively deteriorating clinical course may suggest the diagnosis of fhl in neonates10). in previous data, three cases of fhl in identical male twins and their younger brother who presented with fever and severe one of them showed reversed cd4/cd8 ratio (0.52) in flowcytometric lymphocyte subset analysis and aspirate of bone marrow revealed typical features consistent with fhl in two of them. but none of them performed direct sequencing analysis11). as exemplified by the present case, fhl needs to be considered as one of the differential diagnoses when a newborn presents with severe sepsis - like features with progressive multiorgan failure. in summary, we described a fatal neonatal case of fhl2 that mimicked severe sepsis. clinical assessment and laboratory workup for fhl is needed in critically ill neonates with severe sepsis and multiorgan failure, particularly when there is no evidence of a microbiological or metabolic cause.	hemophagocytic lymphohistiocytosis (hlh) occurs in the primary form (genetic or familial) or secondary form (acquired). the familial form of hlh (fhl) is a potentially fatal autosomal recessive disorder that occurs because of constitutional defects in cell - mediated cytotoxicity. here, we report a fatal neonatal case of type 2 fhl (fhl2) that involved a novel frameshift mutation. clinically, the newborn presented with severe sepsis - like features and required mechanical ventilation and continuous venovenous hemodiafiltration. flow cytometry analysis showed marked hlh and complete absence of intracytoplasmic perforin expression in cytotoxic cells ; therefore, we performed molecular genetic analyses for prf1 mutations, which showed that the patient had a compound heterozygous mutation in prf1, that is, c.65delc (p.pro22argfs2) and c.1090_1091delct (p.leu364glufs93). clinical and genetic assessments for fhl are required for neonates with refractory fever and progressive multiple organ failure, particularly when there is no evidence of microbiological or metabolic cause.
this study provides class iv evidence that immunoadsorption combined with immunosuppression therapy is effective in patients with autoimmune encephalitis with surface antibodies. the purpose of our study was to investigate whether addition of antibody - removing therapy to immunosuppression (ia - is therapy) accelerates recovery of patients with proven autoimmune encephalitis and surface antibodies or antibodies to intracellular antigens. from june 2011 to may 2015, 30 patients were treated with ia because of definite or suspected autoimmune encephalitis. eleven patients were excluded due to incomplete data or doubts about the validity of the diagnosis. the remaining 19 patients were either immunotherapy - naive (n = 5) or had received immunotherapeutic interventions before without effect (n = 14). fifteen patients were treated in the epilepsy centre bielefeld - bethel, germany, a tertiary referral center, and 4 in the department of neurology, university of mnster, germany. data were recorded for 3 time points : baseline (before ia treatment), early follow - up (directly after ia), and late follow - up several months after ia. patients had limbic encephalitis (with lgi1 or caspr2 antibodies, n = 7), anti - nmdar encephalitis (n = 7), or immune - mediated temporal lobe epilepsy with gad antibodies (n = 5). one patient had a neoplasm (nmdar - f, small cell lung cancer). individual patients ' characteristics at baseline modified rankin scale (mrs) scores were determined retrospectively and independently by 2 investigators per patient ; one knew the patient, the other one rated from the records. in cases of divergent ratings (maximum difference was 1 mrs point), the mean was noted. values 2 indicate independent living of the patient, values > 2 increasing degrees of dependency. seizure frequencies are expressed as per week and relate to following time periods : baseline, preceding 4 months or (if this was shorter) time from symptom onset ; at early follow - up, the previous week ; at late follow - up, time since last ia session. for memory assessment, the verbal learning and memory test, the german adaptation of the rey auditory verbal learning test, and the diagnostikum fr cerebralschdigung, revised version (dcs - r) for figural memory were used. in mnster patients, eight values out of the potential 57 (19 patients 3 time points [14% ]) are missing. five patients received 2 ia sequences because of an assumedly incomplete effect of the first one. this study provides class iv evidence because of a lacking control group and masked outcome assessment. all patients underwent ia based on an individual informed written consent of the patient or his or her representatives (compassionate use). antibody specificities and titers for all patients were determined in the bethel antibody laboratory using cell - based assays in the form of commercially available biochips (euroimmun, lbeck, germany). forty - eight hours after transfection, cells are fixed with paraformaldehyde (lgi1, caspr2, and other antigens, not relevant for this study) or acetone (nmdar with nr1 subunits only and gad65). coated cover glasses are cut into 1 1 mm fragments and assembled to mosaics in defined orders. for titration purposes, slides containing 5 double fields (1 with hek cells transfected with the antigen of interest and 1 with nontransfected control cells) we followed the manufacturer 's instructions with modifications. for detection of antibodies, serum at 1:15 and undiluted csf the secondary system consists of a goat - anti - human immunoglobulin g (igg) (heavy and light chain) antibody conjugated with alexa fluor 594 produced by jackson immunoresearch (west grove, pa, code 109 - 585 - 088) at a dilution of 1:100. both incubate at room temperature (anti - igg : 30 minutes, hoechst 33342 : 10 minutes). finally, cover glasses are put on antifading mounting medium 11,4-diazabicyclo (2.2.2) octane (1%). stained biochips are examined using a fluorescence microscope (leica dm 2000 ; wetzlar, germany) with excitation at 592 nm and emission filter at 616 nm for bound antibody and 350/462 nm for nuclear counterstain. the decision if an antibody is present in the tested material is done by 1 of 3 investigators (c.g.b. two persons rate titration stainings independently. in cases of divergent ratings, the mean is recorded. in 16 out of a potential 114 samples, no material for titration was available (14%). in bielefeld - bethel, sera were stored and titrated, having been obtained directly prior to an ia session and directly afterwards. orienting suggestions for applications, dosages, treatment durations, and follow - up schedules were agreed upon among the physicians in the participating neurologic and nephrologic departments (figure 1a). the scheme was modified individually according to patients ' properties, preferences, or organizational circumstances. for immunoadsorption, vascular access was obtained by an indwelling shaldon catheter inserted into the internal jugular vein. plasma filtrate passes through either a regenerative double column system (immunosorba fresenius medical care, bad homburg, germany) with 1.52 plasma volumes (mnster, n = 4) or 2 to 2.2 plasma volumes (bielefeld - bethel, n = 4) processed or a disposable tryptophan column (immunosorba tr-350, octonova technology, diamed medizintechnik gmbh, cologne, germany ; 2 l plasma per session, n = 11, all done in bielefeld - bethel). at onset, 2 to 3 sessions were done on consecutive days. one ia sequence consisted of 10 sessions in bielefeld - bethel (except for patient nmdar - e with 9 sessions and lgi1-c with 8 sessions) and 5 in mnster (nmdar - d received 8 sessions). all patients except one (nmdar - d) received prednisolone in parallel (median dose 4.9 g, range 1.416 g). before ia, 5 patients had been treated with other immunotherapies (plasma exchange : n = 2, nmdar - e and lgi1-c ; ivig : n = 2, nmdar - a and lgi1-c ; azathioprine : n = 1, gad - c ; and cyclophosphamide : n = 1, caspr2-a ; for further details, see table 1). (b d) changes of antibody titers in serum (b) and csf (c) at early and late follow - up in relation to the antibody targets (means with standard errors of the means). (d) antibody titers of all patients (pooled) from bielefeld - bethel (n = 15, means and standard errors of the means) on the immunoadsorption days directly prior to (dark purple bars) and after the immunoadsorptions (light purple bars). this shows the known sawtooth configuration that reflects the antibody redistribution between the vascular and extravascular compartments. even after the fifth session, caspr2 = contactin - associated protein-2 ; gad = glutamic acid decarboxylase ; ia = immunoadsorption ; lgi1 = leucine - rich glioma inactivated protein 1 ; nmdar = nmda receptor. all patients underwent ia based on an individual informed written consent of the patient or his or her representatives (compassionate use). antibody specificities and titers for all patients were determined in the bethel antibody laboratory using cell - based assays in the form of commercially available biochips (euroimmun, lbeck, germany). forty - eight hours after transfection, cells are fixed with paraformaldehyde (lgi1, caspr2, and other antigens, not relevant for this study) or acetone (nmdar with nr1 subunits only and gad65). coated cover glasses are cut into 1 1 mm fragments and assembled to mosaics in defined orders. for titration purposes, slides containing 5 double fields (1 with hek cells transfected with the antigen of interest and 1 with nontransfected control cells) we followed the manufacturer 's instructions with modifications. for detection of antibodies, serum at 1:15 and undiluted csf are incubated with biochips for 30 minutes at room temperature. the secondary system consists of a goat - anti - human immunoglobulin g (igg) (heavy and light chain) antibody conjugated with alexa fluor 594 produced by jackson immunoresearch (west grove, pa, code 109 - 585 - 088) at a dilution of 1:100. both incubate at room temperature (anti - igg : 30 minutes, hoechst 33342 : 10 minutes). finally, cover glasses are put on antifading mounting medium 11,4-diazabicyclo (2.2.2) octane (1%). stained biochips are examined using a fluorescence microscope (leica dm 2000 ; wetzlar, germany) with excitation at 592 nm and emission filter at 616 nm for bound antibody and 350/462 nm for nuclear counterstain. the decision if an antibody is present in the tested material is done by 1 of 3 investigators (c.g.b. two persons rate titration stainings independently. in cases of divergent ratings, the mean is recorded. in 16 out of a potential 114 samples, no material for titration was available (14%). in bielefeld - bethel, sera were stored and titrated, having been obtained directly prior to an ia session and directly afterwards. orienting suggestions for applications, dosages, treatment durations, and follow - up schedules were agreed upon among the physicians in the participating neurologic and nephrologic departments (figure 1a). the scheme was modified individually according to patients ' properties, preferences, or organizational circumstances. for immunoadsorption, vascular access was obtained by an indwelling shaldon catheter inserted into the internal jugular vein. plasma filtrate passes through either a regenerative double column system (immunosorba fresenius medical care, bad homburg, germany) with 1.52 plasma volumes (mnster, n = 4) or 2 to 2.2 plasma volumes (bielefeld - bethel, n = 4) processed or a disposable tryptophan column (immunosorba tr-350, octonova technology, diamed medizintechnik gmbh, cologne, germany ; 2 l plasma per session, n = 11, all done in bielefeld - bethel). at onset, 2 to 3 sessions were done on consecutive days. one ia sequence consisted of 10 sessions in bielefeld - bethel (except for patient nmdar - e with 9 sessions and lgi1-c with 8 sessions) and 5 in mnster (nmdar - d received 8 sessions). all patients except one (nmdar - d) received prednisolone in parallel (median dose 4.9 g, range 1.416 g). before ia, 5 patients had been treated with other immunotherapies (plasma exchange : n = 2, nmdar - e and lgi1-c ; ivig : n = 2, nmdar - a and lgi1-c ; azathioprine : n = 1, gad - c ; and cyclophosphamide : n = 1, caspr2-a ; for further details, see table 1). (b d) changes of antibody titers in serum (b) and csf (c) at early and late follow - up in relation to the antibody targets (means with standard errors of the means). (d) antibody titers of all patients (pooled) from bielefeld - bethel (n = 15, means and standard errors of the means) on the immunoadsorption days directly prior to (dark purple bars) and after the immunoadsorptions (light purple bars). this shows the known sawtooth configuration that reflects the antibody redistribution between the vascular and extravascular compartments. even after the fifth session caspr2 = contactin - associated protein-2 ; gad = glutamic acid decarboxylase ; ia = immunoadsorption ; lgi1 = leucine - rich glioma inactivated protein 1 ; nmdar = nmda receptor. patients had antibodies against lgi1 (n = 3 ; disease duration 13 [range 9.541 ] months), caspr2 (n = 4 ; 4.8 [range 0.916 ] months), nmdar (n = 7, all had csf antibodies ; disease duration 1.2 [range 0.336 ] months), or gad (n = 5, all with high serum titers 1:1,500 and csf antibodies ; 8 years [range 4 months28 years ]). individual data on all patients at baseline and follow - up time points are given in tables 1 and 2. at early follow - up (median 5 days after the last ia, range 043 days), serum and csf titers decreased by a median of 97% and 64%, respectively, in the whole group. at late follow - up (median lag : 3.9 months, range 2.48.7 months), median decrease rates were 97% (serum) and 88% (csf). individual patient titer changes are given in figure 1, b and c. course of serum titers during the sequence of ia is depicted in figure 1d. individual patients ' characteristics at early and late follow - up at early follow - up, 9 of 14 patients with antibodies against surface antigens had improved by 1 mrs point : 2 of 3 with lgi1 antibodies, 3 of 4 with caspr2 antibodies, and 4 of 7 with nmdar antibodies. whereas at baseline all patients with antibodies to surface antigens were dependent (mrs > 2), 6 of 14 patients were independent (mrs 2, 43%) at early follow - up (figure 2). rapid improvement is particularly evident by looking at seizure frequencies in patients with lgi1 and caspr2 antibodies : 5 became immediately seizure - free. the lgi1 patients and caspr2-b received no antiepileptic medication at all until early follow - up. caspr2-c was on the same dose until late follow - up, and caspr2-a had even been reduced (figure 3, a and b). this is similar with memory performance of patients with nmdar antibodies : 2 of 7 improved rapidly by more than 1 sd (figure 3c). remarkably, 4 of 5 gad - antibody - positive patients had disease durations of > 3 years prior to ia. modified rankin scale (mrs) values of the present series that lie in between whole numbers (because of the averaging procedure in case of divergent ratings) are rounded up to be as conservative as possible. the line in between mrs 2 and 3 separates patients who can look after themselves (mrs 2) from those who can not live independently (mrs > 2). (e h) individual mrs changes are given (reduction indicates clinical improvement). responder patients are those with mrs change of at least 1 (red lines). the dark green parts of the columns are changes from baseline to early follow - up ; the light green parts are those that evolved in addition to that until late follow - up. above each bar, patient nmdar - f improved between early and late follow - up while she underwent radio - chemotherapy of her small cell lung cancer. caspr2 = contactin - associated protein-2 ; gad = glutamic acid decarboxylase ; lgi1 = leucine - rich glioma inactivated protein 1. seizures in patients with (a) leucine - rich glioma inactivated protein 1 (lgi1) antibodies and (b) contactin - associated protein-2 (caspr2) antibodies. the antiepileptic drug defined daily dose (aed ddd) figures show that this was not due to anticonvulsive therapy. nmda receptor (nmdar) patients with 2 patients improving by > 1 sd already at early follow - up (red arrows). note the preservation of hippocampal volume in patient lgi1-b in contrast to the hippocampal sclerosis that was present from the beginning of ia in patient lgi1-c. aed ddd according to who (http://www.whocc.no/atc_ddd_index/ ; accessed september 2, 2015). at late follow - up, 12 of 14 patients (86%) with surface antibodies were responders on the mrs compared to baseline. all but 1 patient with nmdar and 2 with caspr2 antibodies had improved even beyond the performance at early follow - up, at least by 0.5 mrs scores (figure 2, e g). only 1 man with limbic encephalitis and lgi1 antibodies (lgi1-c) but already fixed hippocampal sclerosis and 1 woman with nmdar antibodies (nmdar - g) did not improve by 1 mrs point (figure 2, e and g). however, despite a good early overall recovery, there was no recovery of memory performance in patients with lgi1 or casrp2 antibodies within the observational period. no patient with gad antibodies, 4 of 5 with long time lags until ia, improved at any point in time. the only patient with a small - cell lung cancer (nmdar - f) showed no improvement at early follow - up but only after radio - chemotherapy between early and late follow - up. a median of 4.5 (3.610.0) months after the last ia of the first series, 6 patients started a second ia series due to assumed incomplete effect of the first series : nmdar - g, lgi1-c (with hippocampal sclerosis), caspr2-d, caspr2-b, gad - a, and gad - d. five had a follow - up (median 4.6 months, range 2.96.0 months). none of these patients improved by 1 mrs point compared to first late follow - up. adverse effects of ia were associated with the venous catheter : colonization of catheter tip with coagulase - negative staphylococcus requiring antibiotic therapy (lgi-2) and venous air embolism (gad65 - 1). glucocorticoid therapy led to elevated blood glucose levels (caspr2-d), acne (nmdar - e), upper respiratory infection (lgi1-a), weight gain, and skin dryness and fungal infections (gad65 - 3). tachycardia and lip herpes were related to rituximab and cyclophosphamide in 1 patient (nmdar - c) and depressive mood disturbance to mycophenolate mofetil (gad65 - 1). a 19-year - old woman (nmdar - b) had encephalopathy with predominant psychiatric / cognitive impairment. six days after symptom onset, she was treated at another hospital with 4 1,000 mg iv methylprednisolone without any effect. one day after last ia, mrs was found to have gone down from 4.5 (baseline) to 1.0. her memory returned almost into the normal range (from z score 1.3 to 1.0). three months later, she was back to school without memory impairment (mrs 0, memory z score : + 0.4) (figure 3c). a 65-year - old woman (lgi1-b) started having multiple dyscognitive seizures per day. diagnosis of limbic encephalitis with lgi1 antibodies and left hippocampal hyperintense lesion without atrophy was made 13 months after disease onset when she was not on antiepileptic drugs and had 5 seizures per day. she became seizure - free within 35 days after start of ia and 80 mg oral prednisolone (figure 3a). an mri 3 months after ia showed normalization of the hippocampus (figure 3a). two years after disease onset, immunosuppressive and antiepileptic therapy had been terminated, with total remission of symptoms. one year later, she had a relapse with 3 seizures per day. after starting prednisolone 80 mg / d (but neither ia nor antiepileptic drugs), seizures stopped within 10 days. a 60-year - old man (lgi1-c) developed le due to lgi1 antibodies with frequent pilomotor seizures, hyperhidrosis, and memory and mood problems. at disease onset, iv methylprednisolone, oral prednisolone, and immunoglobulins had been administered with transient improvement of memory deficits and hyperhidrosis. upon presentation to our center (41 months after symptom onset) and prior to ia - is, he had mri evidence of left - sided hippocampal sclerosis and right - sided amygdalar hyperintense signal (figure 3a). ia was followed by seizure reduction at early follow - up (figure 3a). a second ia course was performed about 6 months later because depression and seizures continued and lgi1 antibodies increased. a 30-year - old man (gad65-b) had pharmacoresistant temporal lobe seizures but normal memory performance and normal mri. levetiracetam was increased from 2 g to 3 g per day without effect on mrs or seizures. a median of 4.5 (3.610.0) months after the last ia of the first series, 6 patients started a second ia series due to assumed incomplete effect of the first series : nmdar - g, lgi1-c (with hippocampal sclerosis), caspr2-d, caspr2-b, gad - a, and gad - d. five had a follow - up (median 4.6 months, range 2.96.0 months). none of these patients improved by 1 mrs point compared to first late follow - up. adverse effects of ia were associated with the venous catheter : colonization of catheter tip with coagulase - negative staphylococcus requiring antibiotic therapy (lgi-2) and venous air embolism (gad65 - 1). glucocorticoid therapy led to elevated blood glucose levels (caspr2-d), acne (nmdar - e), upper respiratory infection (lgi1-a), weight gain, and skin dryness and fungal infections (gad65 - 3). tachycardia and lip herpes were related to rituximab and cyclophosphamide in 1 patient (nmdar - c) and depressive mood disturbance to mycophenolate mofetil (gad65 - 1). a 19-year - old woman (nmdar - b) had encephalopathy with predominant psychiatric / cognitive impairment. six days after symptom onset, she was treated at another hospital with 4 1,000 mg iv methylprednisolone without any effect. one day after last ia, mrs was found to have gone down from 4.5 (baseline) to 1.0. her memory returned almost into the normal range (from z score 1.3 to 1.0). three months later, she was back to school without memory impairment (mrs 0, memory z score : + 0.4) (figure 3c). a 65-year - old woman (lgi1-b) started having multiple dyscognitive seizures per day. diagnosis of limbic encephalitis with lgi1 antibodies and left hippocampal hyperintense lesion without atrophy was made 13 months after disease onset when she was not on antiepileptic drugs and had 5 seizures per day. she became seizure - free within 35 days after start of ia and 80 mg oral prednisolone (figure 3a). an mri 3 months after ia showed normalization of the hippocampus (figure 3a). two years after disease onset, immunosuppressive and antiepileptic therapy had been terminated, with total remission of symptoms. one year later, she had a relapse with 3 seizures per day. after starting prednisolone 80 mg / d (but neither ia nor antiepileptic drugs), seizures stopped within 10 days. a 60-year - old man (lgi1-c) developed le due to lgi1 antibodies with frequent pilomotor seizures, hyperhidrosis, and memory and mood problems. at disease onset, iv methylprednisolone, oral prednisolone, and immunoglobulins had been administered with transient improvement of memory deficits and hyperhidrosis. upon presentation to our center (41 months after symptom onset) and prior to ia - is, he had mri evidence of left - sided hippocampal sclerosis and right - sided amygdalar hyperintense signal (figure 3a). ia was followed by seizure reduction at early follow - up (figure 3a). a second ia course was performed about 6 months later because depression and seizures continued and lgi1 antibodies increased. we traced back this incomplete response to the fixed hippocampal sclerosis. a 30-year - old man (gad65-b) had pharmacoresistant temporal lobe seizures but normal memory performance and normal mri. levetiracetam was increased from 2 g to 3 g per day without effect on mrs or seizures. two - thirds of patients with igg antibodies to the surface antigens lgi1, caspr2, and nmdar responded within days to ia - is therapy. the rapid achievement of seizure freedom in 5 of 7 lgi1/caspr2-antibody - positive patients and the fast memory improvement in 2 of 7 patients with anti - nmdar encephalitis are particularly impressive. in this retrospective, uncontrolled design, it is challenging to disentangle the effect of ia from (1) the natural course of autoimmune encephalitis, (2) the effect of previous immunologic interventions, and (3) the concomitant prednisolone therapy. option 1 probably did not contribute to the rapid improvements during ia as documented at early follow - up. the relapse in patient lgi1-b (see case description) was successfully treated with steroids alone, but this was a different disease episode. on the other hand, 2 patients had been treated with corticosteroids alone for 1 month (nmdar - b) or with a combination of corticosteroids and cyclophosphamide for 6 months (caspr2-a). only after ia - is therapy did the patients improve. this means that in these 2 patients a specific effect of ia can be uncoupled from a steroid effect in the same disease episode. other reports have also described patients with surface antibodies benefiting from plasmapheresis in combination with immunosuppression. incomplete improvement in patients lgi1-c and caspr2-d is best explained by either preexisting or developing hippocampal atrophy that may result from complement activation with subsequent irreversible neuronal death. our data do not argue in favor of ia in patients with temporal lobe epilepsy and gad antibodies. poor responses of patients with gad antibodies have also been reported in other cohorts including recent onset patients. this suggests that gad antibodies per se predict limited therapeutic effects of ia - is. other authors, however, describe a more positive influence of apheresis therapy in patients with antibodies against intracellular antigens. however, improvement was not stringently defined, short - lived, or absent. poor response to antibody removal in these patients accounts for a primarily t - cell - based cytotoxicity and supports the idea of lack of antibody pathogenicity. in line with previous studies, our study data show that ia brought down antibody concentration in the periphery. interestingly, csf titers were also strongly reduced (by 66% at early follow - up). in a previous plasma exchange study, however, only a mean drop of 23% of csf antibody titers was observed. this is probably due to weaker reduction of peripheral igg levels (mean 75%) with reduced igg redistribution from csf across the blood this shift from csf to the bloodstream may contribute to the sawtooth - like kinetics of antibody concentrations in serum (figure 1d).long - term reduction of antibodies is probably due to the ongoing suppression of antibody secretion by corticosteroids. apart from the in part rapid effect of ia, evaluation of clinical usefulness of ia needs to take into account tolerability and costs. addition of ia to corticosteroids at first glance increases costs and risks. in 2 of 19 patients, there were side effects that would have been serious adverse events in prospective trials, all related to the venous catheter. however, accelerated clinical recovery of patients treated with ia with reduced neurologic impairment, reduced or absent seizures, and possibly shorter periods of time spent in the hospital may outweigh risks and costs introduced by ia treatment.. long disease duration until ia - is in some patients may have contributed to an unfavorable outcome. late diagnosis or failures of first therapeutic interventions were the reasons for the long time lag until ia - is therapy. we did not study control patients, including patients with steroid therapy alone. on the basis of our data, the effect of ia can not be clearly separated from that of concomitantly given steroids, ivig, or other immunosuppressive therapies in some patients. the only patient with a tumor (nmdar - f) did not improve during ia - is therapy. rapid improvement of patients with surface antibodies upon ia further supports the idea of a pathogenic effect of these antibodies. one might tentatively suggest that addition of ia to corticosteroids can speed up recovery by quick reduction of antibody titers. this might be an advantage in severely affected patients. in the absence of a control group and a masked outcome assessment, prospective controlled studies are needed to gain further data for the specific benefit of ia.., and c.g.b. antibody determination was done by c.g.b., c.b., c.g.b. wrote the manuscript draft, which was reviewed and refined by all authors for important intellectual content. diamed medizintechnik gmbh, cologne, germany, and fresenius medical care, bad homburg, germany, supported this evaluation. m. dogan onugoren received travel funding and/or speaker honoraria from fresenius medical care and eisai. d. munstermann runs a commercial laboratory, which performed the detection of vgkc antibodies described in the study. h. wiendl serves on the scientific advisory boards for bayer healthcare, biogen idec, sanofi - genzyme, merck serono, novartis, roche, and teva ; received travel funding and/or speaker honoraria from bayer vital gmbh, bayer schering ag, biogen, csl behring, emd serono, fresenius medical care, sanofi - genzyme, merck serono, omniamed, novartis, and teva ; is on the editorial board for journal of clinical practice, journal of neuroinflammation, and plos one ; has consulted for biogen idec, merck serono, novartis, omniamed, roche, and sanofi - genzyme ; and received research support from bayer healthcare, bayer vital, biogen idec, merck serono, novartis, sanofi - genzyme, teva pharma, german ministry for education and research, deutsche forschungsgesellchaft, european union, else kroner fresenius foundation, fresenius foundation, hertie foundation, nrw ministry of education and research, interdisciplinary center for clinical studies muenster, re children 's foundation, sanofi - aventis, and novonordisk. n. melzer received travel funding and/or speaker honoraria from biogen idec, glaxosmithkline, teva, and fresenius medical care ; performs immunoabsorption ; and received research support from fresenius medical care. bien serves on the scientific advisory board for eisai and ucb ; received travel funding and/or speaker honoraria from grifols, ucb, eisai, glaxosmithkline, destin honoraria, fresenius, diamed, and rasmussen encephalitis children project ; received research support from fresenius medical care, diamed, university of bonn, hagedorn foundation bielefeld, and the krankenhaus mara ; and receives payment for antibody tests (antineural antibodies) performed in the antibody laboratory (as those described in this study).	objective : it was hypothesized that in encephalitides with autoantibodies directed to cns surface antigens an antibody - removing intervention might speed up recovery.methods:the outcome of autoimmune encephalitis in 19 patients with antibodies against surface antigens (leucine - rich, glioma inactivated 1 [lgi1 ], n = 3 ; contactin - associated protein-2 [caspr2 ], n = 4 ; nmda receptor [nmdar ], n = 7) and intracellular antigens (glutamic acid decarboxylase [gad ], n = 5) after immunoadsorption in addition to corticosteroid therapy was evaluated retrospectively. modified rankin scale (mrs) scores and data on seizures, memory, and antibody titers directly after immunoadsorption (early follow - up) and after a median of 4 months (late follow - up) were compiled.results:immediately after immunoadsorption, 9 of 14 patients with antibodies against lgi1, caspr2, or nmdar (64%), but none with gad antibodies, had improved by at least one mrs point. five of the 7 patients with lgi1 or casrp2 antibodies had become seizure - free, and 2 patients with nmdar antibodies had a memory improvement of more than 1 sd of a normal control population. at late follow - up, 12 of 14 patients with surface antibodies had improved (86%), and none of the patients with gad antibodies.conclusions:it is suggested that addition of immunoadsorption to immunosuppression therapy in patients with surface antibodies may accelerate recovery. this supports the pathogenic role of surface antibodies.classification of evidence : this study provides class iv evidence that immunoadsorption combined with immunosuppression therapy is effective in patients with autoimmune encephalitis with surface antibodies.
the difficulties diagnosing the existence of autistic spectrum disorders (asds) is increasingly recognized.[24 ] the absence of a precise understanding of the condition and of reliable diagnostic tests, prevents a proper understanding of the condition. current dyslexia research fails to consider the influence of pathology, yet every organ is solely biochemical. there is no mechanism which distinguishes between the ability of teachers to teach and children to learn. the prevalence of dyslexia has increased significantly since the mid-1970s. arguably, the cognitive abilities of the developed world are in decline. are diagnosed with learning dysfunction and circa 2 m children are being prescribed methyl phenidate to aid their concentration ; 25% of grade 12 adolescents can not read at a proficient level and in the uk circa 20% of the adult population has significant problems with literacy and numeracy. the occurrences of asds are no longer exceptional. any explanation for developmental dyslexia (dd) should seek to explain, for example : dd develops in the pre - pubescent child. it becomes increasingly difficult to treat or manage dyslexia when the child has progressed beyond puberty ; therefore, its occurrence is associated with pre - pubescent physiology and is fixed by post - pubescent physiologies. the pathologies which develop during early childhood influence the development and/or function of the neural structures including the transmission of data through the magnocellular and parvocellular pathways. dd can not be due to any single factor.sense perception and coordination can be altered by pathologies, by drugs and experiences such as low gravity during space flight. asds which influence learning abilities are influenced by the brain 's biology, neural development and associated processes which regulate its function, rather than any significant failure of its structures.many dyslexic children may have excellent speech, hearing, visual perception, iq, memory and/or balance. dd affects children differently, and exists in different levels of severity and influences different aspects of cognitive functions and is not necessarily an impediment to success. some very successful people have dyslexia. they may be good with numbers, be creative, motivated, have an active life, be in good health and/or they might simply have a high iq. this indicates that, for many, the condition is more associated with the brain 's coordinated function rather than its structures.in some autistics the symptoms regress whilst the child has a fever. this indicates that asds are influenced by the processes which regulate the body 's function.the existence of prosopagnosia and/or synaesthesia illustrate, how the sense perception and sensory coordination are disrupted by genetic factors which influence protein expression.genetic changes have been linked to abnormalities in the brain development and of the associated cognitive deficits.the body 's physiology and regulation of its function is linked to different encephalograph (eeg) frequencies and how this influences the system function and/or the regulation or release of biochemicals in the ans.neurons interact in functionally coherent groups or patterns which are involved in the fixation and recall of memories. it becomes increasingly difficult to treat or manage dyslexia when the child has progressed beyond puberty ; therefore, its occurrence is associated with pre - pubescent physiology and is fixed by post - pubescent physiologies. the pathologies which develop during early childhood influence the development and/or function of the neural structures including the transmission of data through the magnocellular and parvocellular pathways. sense perception and coordination can be altered by pathologies, by drugs and experiences such as low gravity during space flight. asds which influence learning abilities are influenced by the brain 's biology, neural development and associated processes which regulate its function, rather than any significant failure of its structures. many dyslexic children may have excellent speech, hearing, visual perception, iq, memory and/or balance. dd affects children differently, and exists in different levels of severity and influences different aspects of cognitive functions and is not necessarily an impediment to success.. they may be good with numbers, be creative, motivated, have an active life, be in good health and/or they might simply have a high iq. this indicates that, for many, the condition is more associated with the brain 's coordinated function rather than its structures. in some autistics this indicates that asds are influenced by the processes which regulate the body 's function. the existence of prosopagnosia and/or synaesthesia illustrate, how the sense perception and sensory coordination are disrupted by genetic factors which influence protein expression. genetic changes have been linked to abnormalities in the brain development and of the associated cognitive deficits. the body 's physiology and regulation of its function is linked to different encephalograph (eeg) frequencies and how this influences the system function and/or the regulation or release of biochemicals in the ans. neurons interact in functionally coherent groups or patterns which are involved in the fixation and recall of memories. in general, developmental dyslexia is not an inheritable genetic condition, though some dyslexic children may inherit the condition because their parents also have a similar condition. this may not be genetic, for example, if the parents lead a sedentary lifestyle then the child is also likely to have the same lifestyle. it will influence their genetic and epigenetic profile (in ways which differ from child to child) but, in many, it may be reversible if addressed in the pre - pubescent years. the gene is a dynamic entity which influences its environment through its expression of proteins. it also responds to environmental influences, for example, someone with type 2 diabetes will have an altered gene expression profile. however, if the same person was to undertake an exercise program their gene expression profile will return to its pre - morbid state. nutrition and exercise alter the gene expression profile ; stress alters the gene expression profile and suppresses immune function ; and viruses and vaccines alter the gene expression profile, suppress immune function and introduce foreign proteins. any alteration to gene expression (genotype) and the ability of such proteins to react (phenotype) an encephalograph monitors the electrical activity of the brain. although the eeg is a widely used tool in medical research, its link to underlying neural activity during the processing of sensory data from the external and internal environments, remains poorly understood. it measures the electrical fluctuations of ionic current which flows within the neuronal structures, which are influenced by the prevailing biochemistries (proteins, ph and levels of minerals, etc.), and which also influence the functions of neurons and neuronal networks. the main clinical applications are the diagnosis of epilepsy, coma, encephalopathies, and confirmation of brain death. whilst there is a perceived link between eeg frequencies and the neuronal networks, it is also recognized that there is a poor level of understanding of this relationship. some networks appear to be better understood than others, for example, the resonant frequency involving the thalamus and cerebral cortex which is associated with sleep spindles. there is, therefore, an established link between eeg frequencies and neural networks which plays a role in the consolidation of memories and the learning process(es). the body 's biochemistry and the prevailing eeg frequencies exist in a dynamic equilibrium. exposure to flashing lights can cause photosensitive migraine whilst selected flashing lights are used to treat migraine. biochemical extremes influence the prevailing eeg frequency(s), and hence, the ability to fix and recall memories, concentrate and ultimately influence the ability of the body to function. fatty acid supplements lessen the symptoms of dyslexia and improve the behavior of children whilst stimulants raise eeg frequencies, energy levels and lower concentration. by contrast, elevated levels of ethyl alcohol or blood glucose are associated with lowered eeg frequencies, concentration and increased predisposition to sleep. the body 's multi - level function is linked to the prevailing eeg frequencies. in the case of dd the following associations with eeg frequencies have been recorded : delta activity, delta and theta, alpha, beta and gamma eeg activity. the brain records and reacts to the physiological significance of an event by using frequency i.e. in the case of most severe trauma and to recuperate (delta) ; when experiencing pain or stress (theta) ; violence, stress and/or significant achievements (alpha). that the delta and theta frequencies act at the physiological level is proven by a number of precedents e.g. for example : delta and theta are active throughout the 24 h period whereas gamma, beta and alpha are mainly active when awake;growth and repair are significantly enhanced during the periods of sleep (predominantly delta);lack of sleep (predominantly delta)increases morbidity and mortality;sleep is a physiological system. delta and theta are active throughout the 24 h period whereas gamma, beta and alpha are mainly active when awake ; growth and repair are significantly enhanced during the periods of sleep (predominantly delta) ; lack of sleep (predominantly delta)increases morbidity and mortality ; sleep is a physiological system. the development of pathologies, the introduction of foreign proteins and the consequence of exposure to viruses and/or through vaccination, alters the delicate equilibrium which exists between biochemistry and eeg frequency(s) and also alters the function and coordination of the senses, for example, influencing visual perception. in the most extreme case(s) this will manifest as a pathological functional system where the brain seeks out the best - fit solution in order to most effectively maintain the body 's physiological stability. the eeg reflects the speed of neuronal processing i.e. the regulation of a physiological significant system is structured to make slow adjustments, for example, at the delta (1 - 4 hz) or theta frequencies (4 - 8 hz), by comparison with the rapid adjustments which are required to handle cognitive input (30 - 60 hz) and subsequent behavioral responses. the effects of nicotine in smokers, decreases delta and theta frequencies and increases high alpha and beta frequencies. both hearing and visual systems handle sensory input through magnocellular and parvocellular pathways which influence color perception, visual contrast, sound frequency and loudness.there is a relationship between color perception and the autonomic nervous system.magnocellular pathways process information more quickly than the parvocellular pathways.magnocellular processing deficits have been found throughout the neuro - visual system.magnocellular and parvocellular processing is linked to color perception and visual contrast but must also be linked to the function of the brain.alterations of blood glucose levels before and after meals are associated with increased predisposition to sleep and lowered levels of concentration. both hearing and visual systems handle sensory input through magnocellular and parvocellular pathways which influence color perception, visual contrast, sound frequency and loudness. magnocellular and parvocellular processing is linked to color perception and visual contrast but must also be linked to the function of the brain. alterations of blood glucose levels before and after meals are associated with increased predisposition to sleep and lowered levels of concentration. this indicates that visual perception, in particular those deficits associated with dd, is associated with the speed of neural processing, the ans and prevailing pathologies. that speech is rhythmic is significant because the ears do not work in a continuous mode. rhythm helps the brain to identify the nature of the word (e.g., whether a word comprises of two, three, four or more sounds). this is the way that a child forms language i.e. of ever increasing complexity but from the simplest origins. a young child will often shorten a word, for example, cin - der - ella may be shortened to cin - ella. moreover, sounds can be in and out of phase. if such phasing, or speed of processing is distorted, a dyslexic child may miss part of a word or have difficulty discerning the spoken word from background noise. instability in the dynamic relationship involving frequency and biochemistry manifests in different ways, for example, the difficulty keeping the beat in music, altered vocal spectrum, poor sleep, poor fixation and recall of memories, etc. there are various light and sound - based therapies which are used to treat asds. in the treatment of dd flashing light biofeedback therapies the idea that the learning process requires synchronized neural activity is not new, however, this has excluded consideration of a link between the brain, sensory organs and visceral matrix. it illustrates how flashing lights can induce photosensitivity and also that the frequency and colors used in flashing light therapies can be selected with therapeutic effect, for example, in the treatment of hyperactivity, dyslexia, asds,[4244 ] tinnitus, hypertension, asthma, diabetes mellitus, migraine and sleep apnoea, etc. there is a principle, which contemporary medical research and/or educational psychology has yet to explain. however, the nature and significance of their function, coordinating interactions between the prefrontal cortex and hippocampus, has only recently been recognized. this is significant because the pre - frontal cortex regulates or controls the body 's executive functions. the coordination and synthesis of emotions, thinking, memory and body or physical movement. in addition the brain uses frequency to organize the activity of these functionally coherent groups of neurons. there is a significant connection between neuronal function and the ans. what we consider as executive functions it is the neural regulation of the physiological systems and ans which influence emotions, thought processes, the establishment and recall of memories and the function of the body and/or physical movement. the nature of this relationship i.e. between cognition, the neural networks, physiological systems, eeg frequencies and ultimately with cellular and molecular biochemistry ; has been incorporated into a mathematical model and technology. regulating physiological systems is necessary to ensure the supply of oxygenated blood to the body and brain, to regulate the immune system and to maintain the health and function of the inner and middle ear. antibiotics and other medications reduce or eliminate ear infections such as otitis media, although, the condition often appears to re - occur. cognitive dysfunction may be the inevitable consequence of extremes of multi - sensory input which manifests as pathologies. a lack of exercise or poor diet degrades the condition of the muscle(s) in the heart and other organs and influences the levels of minerals which are necessary for proper metabolic function and the metabolism of blood glucose. this leads to problems with neural blood flow in later life, often manifested as pathologies, for example, diabetes, cardiovascular disease, hypertension, atherosclerosis, etc. it improves neural blood flow and stimulates the biochemistries which support neuron and synapse formation. it influences the condition of the neural structures, and the ability to memorize and concentrate. for example : acidic (and/or alcoholic) drinks alter the natural metabolic processes. the metabolites alter lipid metabolism and increase weight.the prevailing ph influences the levels and bioavailability of minerals for example, the levels of sodium, potassium, magnesium and calcium, which are necessary to facilitate the flow of ions across neuronal membranes. this influences the action potentials, rates of neuronal firing, and ultimately the fixation of memories.low levels of iron influences the delivery of oxygen to the neural tissues and affects attention and concentration.low levels of magnesium influences the metabolism of blood glucose. this alters eeg frequencies, predisposition to sleep and concentration.sensorineural hearing loss has been associated with hypothyroidism. this is significant because the endocrine glands are integral aspects of the body 's function.omega-3-fatty acids are implicated in a range of neurodevelopmental disorders including add, developmental dyslexia and asds. such substrates are sources of unsaturated fatty acids and are essential for neural development and function. the use of omega-3 fatty acid supplements is associated with health improvements : the use of methyl phenidate to improve concentration indicates that developmental dyslexia has biochemical origins.higher levels of blood glucose increases blood viscosity and lowers immune function, and hence influences the function of the sensory organs.dietary protein influences the fixation of memories.cognitive dysfunction is a commonly observed feature of (i) drugs,(ii) diseases and (iii) viral infections, for example, in herpes simplex, hepatitis c, enteroviruses and epstein - barr virus, etc. visual and phonological deficits are a common feature of diabetes mellitus, heart disease(s), migraine, alzheimer 's disease, multiple sclerosis, dd, regressive autism, tetanus, add and viral infections, for example, mumps, measles, rubella, pneumococcal meningitis. the prevailing ph influences the levels and bioavailability of minerals for example, the levels of sodium, potassium, magnesium and calcium, which are necessary to facilitate the flow of ions across neuronal membranes. this influences the action potentials, rates of neuronal firing, and ultimately the fixation of memories. low levels of iron influences the delivery of oxygen to the neural tissues and affects attention and concentration. this is significant because the endocrine glands are integral aspects of the body 's function. omega-3-fatty acids are implicated in a range of neurodevelopmental disorders including add, developmental dyslexia and asds. such substrates are sources of unsaturated fatty acids and are essential for neural development and function. the use of omega-3 fatty acid supplements is associated with health improvements : the use of methyl phenidate to improve concentration indicates that developmental dyslexia has biochemical origins. higher levels of blood glucose increases blood viscosity and lowers immune function, and hence influences the function of the sensory organs. cognitive dysfunction is a commonly observed feature of (i) drugs,(ii) diseases and (iii) viral infections, for example, in herpes simplex, hepatitis c, enteroviruses and epstein - barr virus, etc. visual and phonological deficits are a common feature of diabetes mellitus, heart disease(s), migraine, alzheimer 's disease, multiple sclerosis, dd, regressive autism, tetanus, add and viral infections, for example, mumps, measles, rubella, pneumococcal meningitis. such observations indicate that the understanding of dd must include the structure of the brain and its biological function, because they are mutually dependent. poor diet, lack of exercise and/or the influence of stress (ors), particularly in early childhood, influences the development and function of the neural structures which process sensory input, the fixation and recovery of memories and concentration etc. however, they need to be activated in order to react with their substrates. if the temperature, ph or their associated biochemistries are not sustained at an optimum level this will influence protein conformation. biophotons of light deliver the energy which activates proteins and specific biochemistries ; regulates the function of the physiological systems ; and synchronizes the activity of groups of neurons and their electrical impulses. any epigenetic factors which, directly or indirectly, influence the structure of chromosome and genes will influence protein expression. accordingly the shifts in color perception which are often associated with dd are associated with the onset of pathologies. it is also plausible that sound can be used to deliver a therapeutic effect although this may be limited by the phasic way in which the ears function. the delivery of sound and music, at appropriate frequencies, have a coherent effect upon the brain waves and our sensory perception. indeed, the steady deep rhythms now employed in contemporary music such as shamanic music or aboriginal chanting and dancing, have an unexplained physiological effect. by contrast, the experiential selection of frequencies can be expected to have a hit - and - miss, and/or negative effect. problems with the inner ear inevitably influence balance and the auditory spectrum i.e. the loudness level and also the ability to discern the spoken word. if the child can not discern the spoken word, or can not relate the spoken word to the written word, it will be difficult for them to engage in spoken or written dialogue with their peers. similar outcomes will be observed in the cases of children who have the symptoms of moving words when reading (a symptom occasionally noted in patients with trigeminal neuralgia) and/or who have untypically low levels of concentration. problems with balance are associated with the function of the cerebellum in which the purkinje cells receive information from all parts of the body. the cerebellum is a significant organ, assimilating multi - sensory information from the senses and visceral organs. accordingly, balance will be affected without the coherent feed - forward of sensory and visceral data to the cerebrum by the cerebellum i.e. to transfer sensory coordinates into motor coordinates. the body uses the significance and importance of memories as a template against which it can gauge its current or future predisposition and abilities. it is the prevailing physiology which determines the continuous reconstruction of the neural structures and fixation of memories from the simplest memories borne before, during and after birth ; and of ever more complex interneuronal structures which build up throughout life. if the brain recorded only the memory of every event, the physiological correlate would not have any meaning. it is only by correlating the new memory to past memory(s) that it becomes significant. the body 's function is ultimately attributable to the rate at which proteins are expressed by genes, the rate at which proteins react with their substrates (and their outcomes), and the factors which influence the rates of their reaction. there is no part of the body 's structure or function which does not have a biochemical nature. memories are laid down from birth as a biochemical data matrix of ever increasing levels of complexity, linked to the different neural centres which store and process memories, until the body 's metabolism is no longer able to sustain the creation and function of neurons, axons and synapses and of associated memories ; and/or the body 's metabolism is no longer able to sustain the function of existing neural structures. consequently, the stimulation of past memories, for example, of musical memories, may have a therapeutic effect by its association with the memory of the body 's internal health. the body 's function and the regulation of the physiological systems is significant and is frequency dependent. it is only by understanding the multi - level significance of the eeg frequencies that dyslexia and asds can be managed. current dyslexia research is focussed upon the qualitative and quantitative determination of the extent of cognitive dysfunction in those with impaired learning and dd. however, there is little research which examines the pathological origins. in order to diagnose a condition i.e. of cognitive or medical dysfunction, it is necessary to understand the fundamental nature of the condition. without such understanding, moreover pathologies encountered in the pre - pubescent stage are likely to alter physiological development. this article illustrates that pathologies are associated with visual and auditory perception and coordination, and the ability to memorize and concentrate. it supports the earlier article(s) by ewing, ewing and parvez which indicates that the brain 's coordinated function, in particular the activity of neurons or the synchronized activity of groups of neurons, are required to facilitate and coordinate the sensory processing and memorizing of information, which is required as part of the learning process. it indicates that this process requires synchronized activity between the brain, sensory organs, ans (and visceral organs) and limbs. pathologies influence the nature and speed of transmission of data from the senses to the brain. it illustrates how, in the pre - pubescent child, the coordinated effect of the factors which influence genotype and phenotype (such as stress, lack of outdoor exercise, poor diet, exposure to viruses, and the insidious effect of vaccines i.e. factors which contribute to lowered immune function) alter the body 's physiological stability and contribute to varying degrees of sensory dysfunction, in particular, a lack of coordination between the senses arising from the influence of pathologies upon the speed of processing, involving the magnocellular and parvocellular pathways. the brain looks to establish physiological stability. if this is not achievable it looks for a best - fit and selects the most appropriate set of circumstances, involving frequency, which enables it to stabilize the body 's function. this explains why dyslexia becomes a chronic condition - although relatively benign from the physiological perspective it indicates that the range of genetic and phenotypic influences which manifest as dd are too complex to be attributed to any single genetic cause i.e. that epistasis influences the complexity of our gene expression profile. multiple gene - gene interactions influence the expression of proteins and ultimately their manifestation as pathologies. this paper is based upon a developed technology which is based upon a mathematical model of the relationship between visual perception, and the ans. this model incorporates a revised understanding of the nature and significance of the physiological systems. the argument presented as a unified explanation for developmental dyslexia appears consistent with the theme of various reference papers - published by galaburda, rosen, stein, baron - cohen, fields rd, richardson, habib, and others many of which appear in the references section of this article.	this case is presented to explain that developmental dyslexia and related autistic spectrum disorders have solely pathological origins. there is a general consensus of opinion which supports the phonological theory. however, this largely ignores the biological basis for all aspects of the brain 's development and function, and hence, for its dysfunction. a unified explanation must take into account all salient features including cognitive dysfunction, encephalograph (eeg) frequencies, neural networks, physiological systems, autonomic nervous system and the function of the cerebellum. it must explain the significance of the brain waves and neurons and their normally synchronized or coherent function. this article builds upon an earlier article by the authors, which incorporates a review and discussion of the prevailing theories or models for developmental dyslexia. it looks at the issues from a top - down systems biology perspective. it concludes that it may be only the body 's biochemistry and, in particular, the onset of pathologies that explain the phenomena which we recognize as developmental dyslexia. pathologies experienced in the early prepubescent years influence neural development. they influence the speed and coherent transmission of data between the senses and neural centers. it is proposed that this explains the nature and occurrence of what we recognize as developmental dyslexia.
coronary artery fistula (caf) is an unusual coronary artery abnormality, where connection exists between coronary artery and cardiac chamber or another blood vessel. caf originating from the left main trunk and left circumflex artery is very unusual and rarely reported in the literature. we describe a case of giant left main trunk and left circumflex artery fistula to the right ventricle. a 24-year - old male patient presented with complaints of dyspnea and palpitation during exercise. on physical examination, his blood pressure was 120/80 mmhg and pulse rate was 75 bpm. the heart was rhythmic and chest auscultation revealed a continuous murmur of grade 3/6 at the upper left sternal border. transthoracic echocardiography (tte) showed enlargement of the left main trunk and left circumflex coronary artery, with a 25-mm inner diameter, diffusely tortuous dilated and drained into the posterior wall of the right ventricle [figure 1 ]. left anterior descending artery, right coronary artery, and major side branches were normal. the left ventricular systolic function was normal with an ejection fraction of 65% and there were no abnormalities in the regional wall motion and cardiac valves. laboratory investigations were normal. in order to further evaluate the fistula in more detail, a 320-slice dynamic volume ct (aquilion one, software release v. 4.3, toshiba medical systems, japan) angiogram was performed, using 3-dimensional volume - rendered (vr) and curved - plain reconstruction (cpr). it demonstrated a large fistula arising from the left main trunk and left circumflex artery emptying to the right ventricle [figure 2 ]. color - doppler echocardiography revealed enlarged and tortuous of lm and lcx (white arrows) origin from ao, abnormal flow from ao passing lm and lcx to rv through a fistula (red arrow). (ao : aorta, lm : left main trunk, lcx : left circumflex artery, lv : left ventricle, rv : right ventricle, ra : righe atrium, la : left atrium) contrast - enhanced ct coronary angiogram. 3-dimensional volume - rendered (vr) and curved - plain reconstruction (cpr) shows origin and course of the fistula. the vessel runs from the left main trunk and left circumflex coronary artery and drained into the posterior wall of the right ventricle through a fistula (red arrow). (ao : aorta, lm : left main trunk, lcx : left circumflex artery, lv : left ventricle, rv : right ventricle) congenital caf is caused by a cardiovascular abnormality in embryo period, which accounts for 0.27 - 0.40% of all congenital cardiac defects. acquired caf has been described after surgical procedures, endomyocardial biopsy, trauma, inflammation, atherosclerosis, and collagen vascular disease. caf usually drains into the right chamber, pulmonary artery, pulmonary veins, superior vena cava, inferior vena cava, and coronary sinus. most caf patients are usually asymptomatic and are found incidentally during angiographic evaluation for other cardiac diseases. clinical symptoms of patients with caf are dependent on the size of fistula and the pressure of its terminal chamber. in our patient, the giant caf increases the volume load of the right heart chambers and the pulmonary artery. if the shunt of caf is significant, steal blood phenomenon may occur and cause myocardial ischemia or infarction, which increases risk of infective endocarditis, accelerates atherosclerosis, and even heart failure. generally, tte is an important primary non - invasive tool for diagnosis of coronary artery abnormality and confirmed by coronary angiography. with the cardiac ct technological improvement in recent years, particularly in temporal / spatial resolution and reduced radiation dose, coronary computed tomography angiography (ccta) has been a relatively new imaging modality and non - invasive method for assessment of coronary artery disease. ccta not only precisely demonstrate the origin, course, and drainage site of the fistula, but also provides surgical plan for treatment. it is suggested that caf should be treated as soon as possible to avoid the potential risk of complications, no matter whether clinical symptom exists or not. in our case, he experienced no serious cardiac events over half a year of follow - up. in conclusion, the giant left main trunk and left circumflex artery fistula to the right ventricle are rarely seen.	coronary artery fistula including the left trunk and left circumflex is uncommon. we present a 24-year - old male patient with a giant left main trunk and left circumflex artery to right ventricle fistula, which is diagnosed by transthoracic echocardiography and coronary computed tomography angiography. in this paper, the case report is to provide a better understanding of clinical characteristics for this disease.
embolic stroke of undetermined source is a common stroke subtype that accounts for 23% to 40% of all strokes.1, 2, 3 appropriate investigation of the underlying causes is important for reducing the proportion of patients diagnosed with embolic stroke of undetermined source, thereby facilitating therapy implementation to target the underlying cause of the index stroke. anticoagulation therapy, for example, is appropriate for patients with paroxysmal atrial fibrillation (paf) because recurrent strokes are often of the same subtype as the preceding index stroke. a gold standard for individualized workup after a stroke is lacking, although some procedures include brain imaging to differentiate ischemic from hemorrhagic stroke, vascular study, blood tests (eg, glucose and lipid profiles), and ecg. workup costs may differ greatly, even among regions with the same socioeconomic status.4 as the number of tests has increased with advances in diagnostic techniques, workup costs have also continuously risen. costs could be reduced by avoiding expensive but ineffective tests.4 targeted selection and judicious use of the appropriate tests for embolic stroke of undetermined source in stroke workups are crucial. although extensive pathogenic workups generally decrease the number of undetermined cases, they may also paradoxically increase the prevalence of embolic stroke of undetermined source. although transesophageal echocardiography (tee), for example, may reveal highrisk sources with indication for oral anticoagulation,5 imprudent use may inadvertently lead to a rise in cases with 2 determined causes. patent foramen ovale (pfo) is prevalent in both the general population and among stroke patients, whereas aortic arch atheroma (aaa) is commonly observed in elderly patients with multiple vascular risk factors. the probability of having pfo or aaa as a cause of, or coincident with, stroke should be weighted in patients with embolic stroke of undetermined source along with pfo or aaa.6 diagnostic investigations of suspected cases of embolic stroke of undetermined source, particularly with advanced diagnostic techniques, should be guided and chosen in accordance with patient characteristics at the time of clinical presentation. the costeffectiveness of advanced diagnostic technologies will greatly depend on the appropriate selection of patients for the various diagnostic tests. kent and colleagues recently reported on the clinical and imaging characteristics of pfo that are likely to be stroke related or incidental, using the risk of paradoxical embolism (rope) score7 ; however, no studies have evaluated the probability of paf, pfo, and aaa in patients with embolic stroke of undetermined source. in the current study, we investigated the clinical and radiological characteristics of these 3 common causes of embolic stroke of undetermined source to develop a prediction model to help with decision making during etiologic workups. two separate data sets from different hospitals were used to develop and validate a prediction model. for model development, we prospectively recruited patients with acute ischemic stroke who were admitted to the samsung medical center (a tertiary university hospital in seoul, republic of korea) from september 2008 through september 2014. we used a prospective cohort from the ajou university hospital (a tertiary university hospital in suwon, republic of korea) during the same period to test the model 's performance. from patients who experienced focal or lateralizing neurological symptoms within 7 days from onset and had relevant lesions on diffusionweighted imaging (dwi), we enrolled patients with stroke due to an undetermined cause at the time of admission. all patients underwent ecg and brain magnetic resonance imaging and magnetic resonance angiography in the emergency room. we excluded patients if they had a determined cause of stroke before admission, based on the stop stroke study trial of org 10172 in acute stroke treatment (ssstoast ; eg, significant stenosis of relevant arteries on magnetic resonance angiography or atrial fibrillation [usually permanent ] on the first ecg). the following data were systematically collected : demographic information ; medical history of vascular risk factors such as hypertension, diabetes mellitus, dyslipidemia, and smoking history ; and initial national institutes of health stroke scale (nihss) scores. in addition, we performed extensive workups, including repetitive ecgs, transthoracic echocardiography or tee, echo bubble tests or transcranial righttoleft shunt tests, multidetector row computed tomography (mdct), and cardiac telemetry (72 hours). the transcranial righttoleft shunt test is based on the intracranial detection of intravenously injected microemboli. the size and functional relevance of a righttoleft shunt can be assessed more easily using transcranial doppler ultrasound, with sensitivity and specificity similar to tee.8 based on the results of the extensive cardiology workups, patients were divided into 3 groups : aaa (n=40), pfo (n=153), and paf (n=128) (figure 1). aaa was considered as a cause of stroke if vulnerable aaa was observed on the tee or mdct. vulnerable aaa was defined as aortic plaques in the ascending aorta or proximal arch that met 1 of the following criteria : (1) 4 mm of intima media thickness on tee or 6 mm of thickness adjacent to the aortic wall on mdct or (2) ulcerated plaque or (3) mobile plaque on tee or soft plaque on mdct.9, 10 we performed the tee or transcranial doppler ultrasound agitated saline test or mdct to evaluate pfo. pfo was deemed present when 1 of following criteria was observed : (1) the passage of > 3 microbubbles to the left atrium within 3 cardiac cycles after complete opacification of the right atrium on the tee, (2) microembolic signals within 40 seconds after injection of agitated saline with microbubbles on the transcranial doppler ultrasound, or (3) a distinct flap in the left atrium at the expected location of the septum primum or a continuous column of contrast material connecting both atria or jet of contrast material into the right atrium on the mdct.11, 12, 13 patients in the paf group were those who had no history or ecg findings of atrial fibrillation at admission, but paf was diagnosed using repetitive ecgs or 72hour cardiac telemetry. if patients had paf plus pfo or aaa, patients were classified as belonging to the paf group because the current evidencebased classification system classifies paf as a highrisk embolic source and pfo and aaa as low or uncertain sources of embolism.14 few patients had both aaa and pfo, and they were excluded from this analysis. aaa indicates aortic arch atheroma ; mra, magnetic resonance angiography ; mri, magnetic resonance imaging ; paf, paroxysmal atrial fibrillation ; pfo, patent foramen ovale ; tia, transient ischemic attack. all participants underwent 3t magnetic resonance imaging including dwi (achieva ; phillips medical system). in the development set, the dwi parameters were as follows : repetition time 3000 ms, echo time 80 ms, matrix number 128128, 2 b values of 0 and 1000 s / mm, slice thickness 5 mm, interslice gap 2 mm, 22 axial slices, and field of view 240 mm. in the validation set (intera, achieva ; philips healthcare), the parameters were as follows : repetition time 3300, echo time 77 ms, matrix number 128128, 2 b values of 0 and 1000 s / mm, slice thickness 5 mm, 28 axial slices, and field of view 220 mm. we analyzed lesions noted on dwi in terms of their size, number, and distribution. the largest diameter of each lesion was measured, and each lesion was divided into small or large lesions based on a threshold of 20 mm. we evaluated dwi lesion distribution based on the involved structure (eg, cortex, subcortex, or both) or the involved vascular territory (eg, anterior or posterior circulation). the involvement of multiple vascular territories was noted when multiple lesions on dwi were located (1) in unilateral anterior and posterior circulation, (2) in bilateral anterior circulation, or (3) in bilateral anterior and posterior circulation. descriptive demographic, clinical, and radiological data are shown as meansd or numbers and frequencies, as appropriate. we analyzed the differences among the groups using a chisquare or mann whitney test for discrete variables and 1way anova or kruskal wallis tests for continuous variables. we used a multinomial regression analysis to detect the clinical and dwi factors associated with the 3 groups : aaa, pfo, and paf. to develop the prediction model, we used a generalized logit model for the nominal response data after selecting the variables with p 10. the prediction model was validated externally in a different cohort (n=117). in all analyses, all statistical analyses were performed using commercially available spss version 20 (ibm corp), sas version 9.3 (sas institute), and r version 3.1.1 (r foundation for statistical computing). two separate data sets from different hospitals were used to develop and validate a prediction model. for model development, we prospectively recruited patients with acute ischemic stroke who were admitted to the samsung medical center (a tertiary university hospital in seoul, republic of korea) from september 2008 through september 2014. we used a prospective cohort from the ajou university hospital (a tertiary university hospital in suwon, republic of korea) during the same period to test the model 's performance. from patients who experienced focal or lateralizing neurological symptoms within 7 days from onset and had relevant lesions on diffusionweighted imaging (dwi), we enrolled patients with stroke due to an undetermined cause at the time of admission. all patients underwent ecg and brain magnetic resonance imaging and magnetic resonance angiography in the emergency room. we excluded patients if they had a determined cause of stroke before admission, based on the stop stroke study trial of org 10172 in acute stroke treatment (ssstoast ; eg, significant stenosis of relevant arteries on magnetic resonance angiography or atrial fibrillation [usually permanent ] on the first ecg). the following data were systematically collected : demographic information ; medical history of vascular risk factors such as hypertension, diabetes mellitus, dyslipidemia, and smoking history ; and initial national institutes of health stroke scale (nihss) scores. in addition, we performed extensive workups, including repetitive ecgs, transthoracic echocardiography or tee, echo bubble tests or transcranial righttoleft shunt tests, multidetector row computed tomography (mdct), and cardiac telemetry (72 hours). the transcranial righttoleft shunt test is based on the intracranial detection of intravenously injected microemboli. the size and functional relevance of a righttoleft shunt can be assessed more easily using transcranial doppler ultrasound, with sensitivity and specificity similar to tee.8 based on the results of the extensive cardiology workups, patients were divided into 3 groups : aaa (n=40), pfo (n=153), and paf (n=128) (figure 1). aaa was considered as a cause of stroke if vulnerable aaa was observed on the tee or mdct. vulnerable aaa was defined as aortic plaques in the ascending aorta or proximal arch that met 1 of the following criteria : (1) 4 mm of intima media thickness on tee or 6 mm of thickness adjacent to the aortic wall on mdct or (2) ulcerated plaque or (3) mobile plaque on tee or soft plaque on mdct.9, 10 we performed the tee or transcranial doppler ultrasound agitated saline test or mdct to evaluate pfo. pfo was deemed present when 1 of following criteria was observed : (1) the passage of > 3 microbubbles to the left atrium within 3 cardiac cycles after complete opacification of the right atrium on the tee, (2) microembolic signals within 40 seconds after injection of agitated saline with microbubbles on the transcranial doppler ultrasound, or (3) a distinct flap in the left atrium at the expected location of the septum primum or a continuous column of contrast material connecting both atria or jet of contrast material into the right atrium on the mdct.11, 12, 13 patients in the paf group were those who had no history or ecg findings of atrial fibrillation at admission, but paf was diagnosed using repetitive ecgs or 72hour cardiac telemetry. if patients had paf plus pfo or aaa, patients were classified as belonging to the paf group because the current evidencebased classification system classifies paf as a highrisk embolic source and pfo and aaa as low or uncertain sources of embolism.14 few patients had both aaa and pfo, and they were excluded from this analysis. aaa indicates aortic arch atheroma ; mra, magnetic resonance angiography ; mri, magnetic resonance imaging ; paf, paroxysmal atrial fibrillation ; pfo, patent foramen ovale ; tia, transient ischemic attack. all participants underwent 3t magnetic resonance imaging including dwi (achieva ; phillips medical system). in the development set, the dwi parameters were as follows : repetition time 3000 ms, echo time 80 ms, matrix number 128128, 2 b values of 0 and 1000 s / mm, slice thickness 5 mm, interslice gap 2 mm, 22 axial slices, and field of view 240 mm. in the validation set (intera, achieva ; philips healthcare), the parameters were as follows : repetition time 3300, echo time 77 ms, matrix number 128128, 2 b values of 0 and 1000 s / mm, slice thickness 5 mm, 28 axial slices, and field of view 220 mm. we analyzed lesions noted on dwi in terms of their size, number, and distribution. the largest diameter of each lesion was measured, and each lesion was divided into small or large lesions based on a threshold of 20 mm. we evaluated dwi lesion distribution based on the involved structure (eg, cortex, subcortex, or both) or the involved vascular territory (eg, anterior or posterior circulation). the involvement of multiple vascular territories was noted when multiple lesions on dwi were located (1) in unilateral anterior and posterior circulation, (2) in bilateral anterior circulation, or (3) in bilateral anterior and posterior circulation. descriptive demographic, clinical, and radiological data are shown as meansd or numbers and frequencies, as appropriate. we analyzed the differences among the groups using a chisquare or mann whitney test for discrete variables and 1way anova or kruskal wallis tests for continuous variables. we used a multinomial regression analysis to detect the clinical and dwi factors associated with the 3 groups : aaa, pfo, and paf. to develop the prediction model, we used a generalized logit model for the nominal response data after selecting the variables with p 10. the prediction model was validated externally in a different cohort (n=117). in all analyses, all statistical analyses were performed using commercially available spss version 20 (ibm corp), sas version 9.3 (sas institute), and r version 3.1.1 (r foundation for statistical computing). of the patients with embolic stroke of undetermined source at the time of admission, we included 321 patients with 1 of 3 etiologies : aaa in 40 (12.5%), pfo in 153 (47.7%), and paf in 128 (39.9%) patients. patients with pfo were younger than patients in the other 2 groups (pfo : aged 55.5 years ; aaa : aged 72.9 years ; paf : aged 71.6 years ; p 60 minutes) transient ischemic attacks compared with arteroembolic strokes from the carotid artery or aortic arch (cholesterolcontaining). clot components reportedly determine infarct patterns ; an autopsy study revealed that emboli containing fibrin often cause large cortical infarcts, whereas emboli containing cholesterol crystals frequently result in small border zone infarcts.17 when investigating the underlying causes of embolic stroke of undetermined source, physicians need to decide on the type and extent of the ancillary procedures they will use to document a precise embolic source for proper secondary preventive management. in clinical practice, routinely ordering diagnostic tests, including tee, and workups for paradoxical embolism and deep vein thrombosis, aortogenic embolism, and prolonged cardiac telemonitoring is neither indicated nor possible. tee is considered the gold standard for the evaluation of embolic stroke of undetermined source ; however, routine application of tee is often limited in patients with acute stroke because of acute illness, mental change, coagulopathy or bleeding tendency, and lack of patient cooperation. the use of more noninvasive diagnostic techniques, such as the mdct to detect aortogenic embolism18, 19 or transcranial righttoleft shunt test to detect paradoxical embolism,8 would be practical alternatives. in contrast, current guidelines recommend performing cardiac monitoring for at least 24 hours to detect paf20 ; however, an atrial fibrillation detection rate of 24hour holter monitoring does not seem to be sufficient, so longterm cardiac rhythm monitoring should be considered in patients with a high probability of having paf. moreover, the yield of diagnostic tests may differ among patients, depending on the probability of having pfo, aaa, and paf as a cause of stroke. the yield (positive predictive value and probability of stroke cause) of cardiac telemonitoring, for example, and the workup for paradoxical embolism and deep vein thrombosis are very different. based on a report by the us centers for medicare and medicaid services, aggregate cost for intensive embolic source evaluation in a single ischemic stroke patient can be > $ 2000 (ie, ecg, $ 17 ; complete transthoracic echocardiography, $ 229 ; transcranial doppler ultrasound, $ 290 ; diagnostic tee, $ 308 ; mdct, $ 420 ; and cardiac telemetry, $ 680). considering the increasing number of stroke patients, inadvertent etiologic evaluations could be a great burden to the economy. consequently, selecting appropriate cardiologic workups for individual patients based on the probability equation would improve the costeffectiveness of advanced diagnostic technologies and may help with timely etiologic investigation and accurate diagnosis. the current study has the advantage of incorporating derivation and external validation data, but it also has several limitations. although all patients underwent dwi and magnetic resonance angiography, the cardiac workups, which included tee, mdct, and cardiac telemonitoring, varied among patients, depending on the physician 's preference in realworld practice. patients who were likely to have aortogenic or paradoxical embolism, for example, received additional tee more often, whereas those who were suspected of having paf received cardiac telemonitoring more often. nevertheless, the external validation analysis using data from a different medical center suggested that the physician 's preference did not significantly influence our conclusions. in addition, we hypothesized that it is important to differentiate embolism cause in patients with embolic stroke of undetermined source because different treatment strategies are required (eg, plaque stabilizer [ie, statin ] for aaa, closure [in some cases ] for pfo, and anticoagulation for paf) ; however, this is beyond the scope of the current analysis. last, these data are from 2 centers in south korea ; therefore, the generalizability of our results may be limited the current study has the advantage of incorporating derivation and external validation data, but it also has several limitations. although all patients underwent dwi and magnetic resonance angiography, the cardiac workups, which included tee, mdct, and cardiac telemonitoring, varied among patients, depending on the physician 's preference in realworld practice. patients who were likely to have aortogenic or paradoxical embolism, for example, received additional tee more often, whereas those who were suspected of having paf received cardiac telemonitoring more often. nevertheless, the external validation analysis using data from a different medical center suggested that the physician 's preference did not significantly influence our conclusions. in addition, we hypothesized that it is important to differentiate embolism cause in patients with embolic stroke of undetermined source because different treatment strategies are required (eg, plaque stabilizer [ie, statin ] for aaa, closure [in some cases ] for pfo, and anticoagulation for paf) ; however, this is beyond the scope of the current analysis. last, these data are from 2 centers in south korea ; therefore, the generalizability of our results may be limited our data indicate that patients with embolic stroke of undetermined source showed distinct clinical and radiological features depending on the underlying stroke cause. our probability equations can aid decision making by identifying patients who are likely to have paf during hospitalization in the first days of stroke onset or, conversely, those likely to have aortogenic pathology or paradoxical embolic sources. continuous efforts are needed to refine the approach to working up cases of suspected embolic stroke of undetermined source, incorporating other biomarkers, such as btype natriuretic peptide21 or genetic risk factors.22 this study was supported by the korean health technology r&d project, ministry of health and welfare, republic of korea (hi14c16240000). the funder had no influence on design and conduct of the study ; collection, management, analysis, and interpretation of the data ; and preparation, review, or approval of the manuscript. data s1. a microsoft excel calculator for etiology prediction. click here for additional data file.	backgroundfrom a therapeutic viewpoint, it is important to differentiate the underlying causes of embolism in patients with cryptogenic stroke, such as aortic arch atheroma, patent foramen ovale, and paroxysmal atrial fibrillation. we investigated the clinical and radiological characteristics of these 3 common causes of cryptogenic embolism to develop models for decision making in etiologic workups.methods and resultsa total of 321 consecutive patients with acute infarcts from cryptogenic embolism were included. patients were divided into 3 groups aortic arch atheroma (n=40), patent foramen ovale (n=153), and paroxysmal atrial fibrillation (n=128)based on extensive cardiologic workups. we used a multinomial logistic regression analysis to detect the clinical and diffusionweighted imaging factors associated with the probability of aortic arch atheroma, patent foramen ovale, and paroxysmal atrial fibrillation. clinical and radiological features differed among the groups. the patent foramen ovale group had a healthy vascular risk factor profile and showed posterior circulation involvement compared with other groups (p<0.01). in contrast, paroxysmal atrial fibrillation related strokes had higher initial national institutes of health stroke scale (nihss) scores and larger lesions than the other groups (p<0.001). the aortic arch atheroma group had clinical features similar to those of the paroxysmal atrial fibrillation group but showed small lesions scattered in multiple vascular territories (p<0.001). multivariate regression analysis revealed that age, initial nihss score, lesion size (20 mm), multiple (3) lesions, and involvement of posterior circulation or multiple vascular territories differentiated the 3 groups (pseudo, r 2=0.656). the prediction ability of this model was validated in the external validation cohort (n=117, area under the curve 0.78).conclusionsour data indicate that patients with cryptogenic embolic stroke show distinct clinical and radiological features depending on the underlying causes.
genetic regulation of flowering is a key element of adapting plants to cultivation in different latitudes, daylength responses and/or purposes (such as seed / grain or biomass production). in the semi - arid tropical habitats to which many of the world s most important seed / grain crops are native, flowering during short days of 700 genotypes have been converted over 50 years of effort, in most cases involving four generations of backcrossing each followed by two generations of selfing to identify recessive day - neutral flowering segregants. previously, we studied f2 progeny of a cross between s. bicolor btx623, a day - neutral breeding line, and s. propinquum, a tropical and short - day flowering wild relative. most of the f2 population, with at least one copy of the s. propinquum short day flowering allele, flowered in mid - september or later in the latitude where the experiment was done (30.6n ; college station, tx), consistent with the short - day flowering of the tropical s. propinquum parent. we showed a single quantitative trait locus (qtl), flravgd1, to account for 85.7% of this phenotypic variation in flowering time (lin. 1995), and to correspond approximately to the locus of the triticeae photoperiodic genetic loci, ppd1 - 3 and ppd - h1 (paterson, lin,. we show by integration of positional and association genetic evidence that the short - day flowering qtl flravgd1 locates on chromosome 6 (formerly linkage group d) in a 10-kb interval within the physically largest centimorgan in the sorghum genome. this interval contains a single annotated gene that we show to partly complement the day - neutral flowering phenotype. the gene accounting for flravgd1 is sb06g012260, a member of the flowering locus t (ft) family of transcription factors that is highly divergent from most known floral regulators in this family. sb06g012260 is unique to panicoids, evolving as a single gene duplication shortly after the oryzoid (rice) panicoid (sorghum, maize) divergence. sb06g012260 suppresses flowering, although it is quite distant evolutionarily from other ft family members that are floral suppressors (pin. we discuss its relationship to named sorghum flowering genes and implications of these findings for candidacy of other genes, as well as its evolution in an invasive sorghum species that has adapted to temperate latitudes. to conduct interval mapping of flowering time in sorghum, we previously analyzed an f2 population of s. bicolor cultivar btx623s. propinquum using 78 rflp loci spanning 935 cm with an average distance of 14 cm between markers (lin. a qtl explaining 85.7% of phenotypic variation in flowering time, flravgd1, was placed in the 21-cm interval between dna markers psb095 and psb428a. to more finely map flravgd1, 34 f2 plants were selected that were putatively recombinant in the 21-cm interval between dna markers psb095 and psb428a on chromosome 6. an additional 27 dna markers were applied to pooled dna from 50 to 150 selfed f3 progenies that were also grown in the field near college station, texas. about 4 of the 34 f3 families, # 10, 187, 191, and 211, were excluded because the dna marker genotypes of f2 and pooled f3 tissue were not consistent (# 211), or because the flravgd1 locus genotype of their f2 parents predicted from phenotypic segregation in f3 progenies was contradicted by both flanking markers and by virtually all other markers on the chromosome (# 10, 187, 191). each such inconsistency would have required a double recombination event to explain, and 3 such events among 34 progeny in a 21-cm interval are highly improbable. a few such incongruous plants were also observed in the f2 generation, and are an important example of the need for progeny testing to reliably fine - map flowering, which can be influenced by other genetic effects, temperature, and other factors such as some diseases (quinby 1974). based on progeny testing, flravgd1 was placed between dna markers psb1113 and cdsr084, estimated to range from 0.3 to 1.1 cm apart in two different mapping populations studied (the larger distance is indicated in fig. bac clones were identified containing each of these dna markers, but lengthy efforts to chromosome walk in this region failed due to repeatedly encountering repetitive dna near bac ends. the sorghum genome sequence revealed this 1.1 cm interval to include 34 mbp, thus having 60-fold less recombination than the genome - wide average (paterson. (a) linkage mapping of flravgd1 to sorghum chromosome 6 (lg d ; lin. 1995) ; (b) progeny testing in 30 f3 families that were recombinant in the interval containing flravgd1 delineated the locus to a 1.1-cm region including 400 genes ; (c) analysis of 90 diverse exotic sorghums and their corresponding converted derivatives delimited flravgd1 to a 4.1-mb region including 63 genes. (d) association genetics implicated sb06g012260 in short - day flowering conferred by flravgd1. (e) the day - neutral haplotype of sb06g012260 contains three deletions in the 5 region, one removing a caat box essential to many eukaryotic promoters. (a) linkage mapping of flravgd1 to sorghum chromosome 6 (lg d ; lin. 1995) ; (b) progeny testing in 30 f3 families that were recombinant in the interval containing flravgd1 delineated the locus to a 1.1-cm region including 400 genes ; (c) analysis of 90 diverse exotic sorghums and their corresponding converted derivatives delimited flravgd1 to a 4.1-mb region including 63 genes. (d) association genetics implicated sb06g012260 in short - day flowering conferred by flravgd1. (e) the day - neutral haplotype of sb06g012260 contains three deletions in the 5 region, one removing a caat box essential to many eukaryotic promoters. to better circumscribe the location of flravgd1 in the 34-mbp target region, and to confirm that the s. propinquum - derived locus also accounts for intraspecific variation within s. bicolor (the cultigen), we utilized recombination accumulated during conversion of diverse short - day tropical sorghums to day - neutral flowering (stephens. 1967). sorghum conversion involves four backcrosses each followed by two generations of selfing (stephens. 1967), a breeding scheme that in regions held heterozygous is expected to impart 3.5 more recombinational information to converted genotypes than f2 or recombinant inbred genotypes (allard 1956). ninety diverse exotic sorghums and their corresponding converted derivatives (supplementary table s1, supplementary material online) were genotyped at nine simple sequence repeat (ssr) loci distributed approximately evenly through the 34 mbp target interval inferred for the qtl based on our s. bicolors. 1995), and closely coincides with flravgd1, strongly supporting the hypothesis that the s. propinquum - derived qtl locus also accounts for intraspecific variation in short - day vs. day - neutral photoperiod response in s. bicolor. in 384 diverse sorghums (hash. 2008) phenotyped for photoperiod response index (pri : see materials and methods), we identified and genotyped dna polymorphisms in annotated genes near the conversion peak. tassel was used to perform tests of association, employing population structure covariates and a kinship matrix for the sorghum germplasm panel based on published ssrs (hash. the most significant association (p 12 h), long days accelerated flowering by 30 days for many genotypes from south africa, the most temperate part of the natural range. east asian (asiae) sorghums, the largest temperate - adapted group from the northern hemisphere, also had accelerated long day flowering. flowering of 384 diverse sorghums were compared in the 20072008 post - rainy (short day), and 2008 rainy (long day) seasons in peninsular india to determine photoperiod response index (as described in methods). hierarchical clustering of sorghum accessions was based on distances between ssr genotypes (number of bands not shared). while short - day sorghums experience delayed flowering under long days (> 12 h), long days accelerated flowering by 30 days for many genotypes from south africa, the most temperate part of the natural range. east asian (asiae) sorghums, the largest temperate - adapted group from the northern hemisphere, also had accelerated long day flowering. s1, supplementary material online) containing short - day s. propinquum sb06g012260 alleles subcloned from a bac and transformed into the day - neutral accession tx430 using published methods (howe. 2006) each delayed flowering of transgenic f2 progeny in long days (table 1). widely used for sorghum transformation because of its high efficiency (howe. 2006), tx430 has day - neutral flowering but has an unusual haplotype, with deletion of seven amino acids in the 4th exon of sb06g012260. independent t0 transformants were selfed to produce t1 segregating progenies, then 1524 plants from each t1 family were evaluated in the greenhouse under ambient long day conditions (at 33.95 n latitude), planting on 17 may (13 h, 58 min photoperiod) and recording the number of days to flower emergence. plants were genotyped by pcr to determine allele state for the transgene. among 13 transformation events carrying a 5 kb construct limited to sb06g012260 and its immediate upstream elements, two conferred statistically significant delays averaging 13.1 (p = 0.03) and 24.8 days (p = 0.09), and one line showed unexpected accelerated flowering (14.1 days, p = 0.05). among 10 independent events harboring a 10 kb construct spanning the entire haplotype (from sb06g012260 through the 4,186 nt element), transgenic f2 progeny of only three showed significantly altered flowering, with delays of 4.1 (p = 0.002), 4.2 (p = 0.07) and 5.2 (p = 0.008) days. table 1regression analysis of relationship of flowering time to genotype for transgenic constructs (transgene events with p plantgdb, last accessed june 22, 2016. a phylogenetic tree of inferred homologous protein sequences was made at http://www.phylogeny.fr/, last accessed june 22, 2016, using muscle for alignment and maximum - likelihood (phyml) to determine the tree. four distinct sub - families including ftl - like, ft / ftl - like, tfl1-like and mft - like proteins, are highlighted on the tree. the impact on flowering (promoting or suppressing) are shown for several well studied pebp genes (reviewed in klintens. the panel on the right along the tree shows multiple alignments based on the arabidopsis ft protein at amino acid position 85 at exon 2, positions 128141 (p - loop domain), positions 150152 and position 164 that were shown to have critical regulatory roles. for full alignments across the entire length of these proteins, six homologs were found in arabidopsis (prefixed at, including ft ; kardailsky. 1999), 19 in rice (os, including hd3a ; kojima. 2002), 19 in sorghum (sb), 26 in maize (grm or ac) and 8 in sugarcane (put-157a - saccharum_officinarum), identified by blast from http://www.plantgdb.org/prj/estcluster/progress.php>plantgdb, last accessed june 22, 2016. a phylogenetic tree of inferred homologous protein sequences was made at http://www.phylogeny.fr/, last accessed june 22, 2016, using muscle for alignment and maximum - likelihood (phyml) to determine the tree. four distinct sub - families including ftl - like, ft / ftl - like, tfl1-like and mft - like proteins, are highlighted on the tree. the impact on flowering (promoting or suppressing) are shown for several well studied pebp genes (reviewed in klintens. the panel on the right along the tree shows multiple alignments based on the arabidopsis ft protein at amino acid position 85 at exon 2, positions 128141 (p - loop domain), positions 150152 and position 164 that were shown to have critical regulatory roles. for full alignments across the entire length of these proteins, an sb06g012260 allele found at high frequency in weedy and invasive s. halepense may have contributed to its spread. sorghum halepense (johnson grass), a tetraploid derived naturally from wild s. bicolor and s. propinquum progenitors (paterson, schertz,. 1995), has spread across much of asia, africa, europe, north and south america, and australia. its establishment in usa is post - columbian, by introduction as a prospective forage and/or contaminant of sorghum seedlots (mcwhorter 1971). with inbreeding progenitors of tropical origin and therefore being putatively homozygous for short - day flowering, the original tetraploid s. halepense is expected to have had four copies of the short - day haplotype. among the few old world s. halepense accessions in the us national plant germplasm collection, we confirmed by pcr that pi209217 from south africa and pi271616 from india are homozygous for the short - day haplotype, and confirmed by growouts that these two genotypes do not flower in long days. with maximum seed production under 10.512 h daylengths consistent with short - day flowering, us (temperate) s. halepense nonetheless flowers and produces ample seed in photoperiods of up to 16 h (warwick and black 1983). we genotyped the four loci diagnostic of the short - day haplotype (423 nt, 4,186 nt, 3 nt, and intron indels) in 480 s. halepense plants sampled equally from populations in ga, tx (2), ne, and nj described previously (morrell. at the two terminal loci of the haplotype, 81.6% (423 nt indel) and 88.2% (intron indel) were homozygous for the short - day alleles, consistent with the expected genotype of their tropical progenitors. however, at the two internal loci of the haplotype (4,186 and 3 nt indels), only 1.1% and 8.0% of plants were homozygous for the short - day - associated alleles (fig. 4 and supplementary table s2, supplementary material online). only 39-bp upstream from the locus of the caat box deletion in day - neutral s. bicolor, 85.9% of s. halepense plants have at least one copy of a 4-nt insertion that disrupts a tc - rich repeat, a cis - acting motif enriched in promoters of photoperiod - responsive genes (mongkolsiriwatana. we have not found this mutation in any other members of the genus (unpublished data), suggesting that it is rare or novel. further, at the 4,186-nt indel, 98.9% of s. halepense also have at least one allele from the day - neutral haplotype (fig. thus, it appears that an sb06g012260 mutation in naturalized us s. halepense independent of those in s. bicolor has resulted in 4genotype distributions of five us johnson grass populations near the sb06g012260 gene. among 480 plants sampled equally from ga, tx (2), ne, and nj populations (morrell. 2005), 81.6% and 88.2% were homozygous for the short - day haplotype (blue bars) at two terminal loci (423 nt, 27 bp intron indels), but only 1.1% and 8.0% at two internal loci (4,186 and 3 nt indels). homozygosity for day - neutral alleles (green) is nearly absent from the ga sample (from the region where johnson grass is thought to have been introduced to usa), but exceeds 10% in the two northerly populations (ne, nj) where day - neutral flowering would be most advantageous, and is intermediate in the two tx populations. genotype distributions of five us johnson grass populations near the sb06g012260 gene. among 480 plants sampled equally from ga, tx (2), ne, and nj populations (morrell. 2005), 81.6% and 88.2% were homozygous for the short - day haplotype (blue bars) at two terminal loci (423 nt, 27 bp intron indels), but only 1.1% and 8.0% at two internal loci (4,186 and 3 nt indels). homozygosity for day - neutral alleles (green) is nearly absent from the ga sample (from the region where johnson grass is thought to have been introduced to usa), but exceeds 10% in the two northerly populations (ne, nj) where day - neutral flowering would be most advantageous, and is intermediate in the two tx populations. based on several lines of evidence, sb06g012260 appears to have evolved as a single - gene duplication (fig. 5) shortly after the oryzoid (rice) panicoid (sorghum, maize) divergence. first, its divergence from its nearest sorghum homolog, sb04g008320 is estimated at 40 ma (ks = 0.43, based on a widely - used evolutionary rate for cereal genes ; gaut. 1996), suggesting that it evolved after oryzoid (rice) panicoid (sorghum, maize) divergence (consistent with gene tree topography, fig. 3). sb04g008320 has a colinear rice ortholog (os02g13830.1) but sb06g012260 lacks colinear orthologs in both rice and brachypodium distachyon, although possessing them in setaria and maize (fig. 5). this pattern of orthology is indicative of an origin of sb06g012260 in panicoids, shortly after divergence from a common ancestor shared with oryzoids (rice) and pooids (brachypodium). rectangles represent predicted gene models with colors showing relative orientations (blue : same strand, green : opposite strand). matching gene pairs are displayed as connecting shades. microsynteny pattern around sb06g012260 across five grasses. rectangles represent predicted gene models with colors showing relative orientations (blue : same strand, green : opposite strand). matching gene pairs several independent lines of evidence including fine mapping, association genetics, mutant complementation, and evolutionary analysis all implicate a single gene, sb06g012260, as the cause of the flravgd1 qtl that accounted for 85.7% of variation in flowering time of a temperatetropical sorghum population (lin. identification of this gene was complicated by its location in the physically largest single cm in the sorghum genome, containing 5% of sorghum genomic dna and 1.3% (400) of genes (paterson. 2009), with enrichment of retroelements and other non - genic dna. while both its panicoid - specific origin (fig. 5) and 1) falsify the inference that ma1 corresponds to the locus of the triticeae photoperiodic genetic loci, ppd1 - 3 and ppd - h1 (paterson, lin,. 10 paralog should exist at 105 mb, near the flowering qtl (koester. 1993). however, the nearest ft homolog to this location (ac217051.3_fg006 : chr. 10, 114 mb) has a sequence so divergent from sb06g012260 that it appears non - orthologous. the wild - type sb06g012260 is the first floral suppressor discovered in the ftl clade of the ft gene family although another clade member is a floral promoter, zcn8 (meng. 3). despite being a floral suppressor, sb06g012260 resembles all four functionally characterized members of the tfl1 clade of the family (hanzawa. 2016). among the 23 transgene events that we evaluated, 5 showed statistically significant (0.1 or less, with 3 at 0.05 or less) delays in flowering, whereas only one showed the acceleration that might be expected from general overexpression of a florigen. the candidate mutation that removed a caat box upstream of the day - neutral sb06g012260 allele would be expected to reduce gene expression, also consistent with the hypothesis that wild - type sb06g012260 is a floral suppressor. there is growing appreciation that floral regulation and other aspects of growth and development are regulated by antagonistic actions of multiple members of ft and other gene families (lifschitz. exchange of a single amino acid between arabidopsis florigen ft and floral repressor tfl1 is sufficient to exchange the functions of these opposing genes (hanzawa. 2005). the finding that mutant complementation studies of sb06g012260 only partially recapitulated the short - day phenotype, underline the need for more work to understand the function and regulation of sb06g012260. it is noteworthy that the overwhelming majority of transgenic lines with significant deviations from tx430 had delayed flowering, not the general promotion of flowering that might be explained simply by overexpression of many ft - like genes (mcgarry and ayre 2012). however, it is somewhat perplexing that only one of the 23 events reached the average 24.6 (3.5) day delay between the reference genetic stock 100 m (murphy. 2011) that contains ma-1 (a dominant allele conferring short - day flowering in tropical sorghums ; quinby and karper 1945) and tx430 under our conditions. shorter flowering delays than the ma1 reference genotype 100 m relative to putatively near - isogenic sm100 (murphy. 2011) may indicate that some distant regulatory elements were missing from the transgene constructs and/or that its native heterochromatic chromatin environment is important to its natural function. a key element of the additional information needed is the exact timing and location of sb06g012260 gene expression. in more than 1.7 trillion reads from (unpublished) sorghum transcriptomic data pooled across leaves, stems, and flowers of 40 diverse genotypes grown by several different investigators in different environments, we found only four reads from sb06g012260 two each in two different genotypes. a detailed study of 19 sorghum pebp genes in roots, leaves (four stages), stems, shoot apices and floral heads by semiquantitative rt - pcr also detected no expression of sb06g012260 in any tissue (wolabu. further, two of the three other sorghum members of the subclade including sb06g012260, sb04g008320 (sbft7) and sb03g002500 (sbft13), also show no expression in any tissue, although the third member, sb10g021790 (sbft9) shows moderate expression in leaves (wolabu. finally, the nearest maize homolog of sb06g012260, zcn21, shows no discernible expression in a broad sampling of tissues (danilevskaya. in contrast, sb02g029725 (sbft6), which we implicate above in the flravgb1 sorghum flowering qtl, showed strong expression in leaves across all developmental stages and was also detected in the floral head and florets, but does not induce flowering in arabidopsis (wolabu. other members of its subclade (sbft3, 4, 5, and 11) all show expression but in different tissues and developmental states, and like sb06g012260 do not show expression patterns or genic interactions consistent with being florigens (wolabu. where does sb06g012260 fit among the named sorghum flowering loci ? based on the high frequency at which this region has long been known (lin. 1995) to be selected for during sorghum conversion, we inferred that the qtl in this region was ma1, the sorghum maturity (flowering) locus long known to have the largest effect on phenotype (quinby and karper 1945). moreover, based on relatively coarse resolution qtl data, we previously suggested correspondence of flravgd1 to a homoeologous series of triticeae photoperiodic (ppd1 - 3) loci (paterson, lin,. others have suggested prr37 to be ma1 based on positional proximity to a flowering trait mapped in genetic populations different from ours, together with its expression pattern in response to photoperiodic cues (murphy., the transition from the heterochromatic surroundings of sb06g012260 to euchromatin, and an associated increase in recombination, coincides with a precipitous drop in levels of conversion at 37 mb (fig. prr37 is in a euchromatic genomic location (40.3 mb) experiencing only 15% conversion (fig. curiously, study of 390 exotic - converted pairs genotyped at 46,062 markers (albeit with 66% missing data before imputation) suggested an introgression peak at 42 mb, different from both our peak and prr37 (thurber. the fact that this region has 60-fold less recombination than the genome - wide average (fig. 1), suggests that limited recombination rather than multiple loci under selection (thurber. 2013) accounts for the high frequency of introgression in this region, and indeed across much of the chromosome. evidence in support of prr37 as ma1 is further confounded by factors that were not known to its proponents (murphy. 2011) or their reviewers. prr37 was not tested by mutant complementation, but was a positional candidate that showed striking expression differences based on comparison of near - isogenic lines called 100 m and sm100 that differ in prr37 alleles (murphy. s2, supplementary material online) that the short - day flowering 100 m line is introgressed with not only a putatively short - day prr37 allele but also with the short - day sb06g012260 haplotype, based on genotyping at both the 423 and 4,186 nt indels that are on the distal side of the gene relative to prr37. this indicates that the chromosome segment by which 100 m and sm100 differ contains both prr37 and sb06g012260, as well as at least 82 intervening protein - coding genes and 6.75 mb dna (prr37 [sb06g014570 ] has an address of chr6 : 40,280,41440,290,602. phenotypic differences between these lines could therefore be explained by either of these two genes, interactions between the two, or other factors. further, in the genotype that was the source of the short - day flowering allele that we mapped (s. propinquum), prr37 is non - functional with a 2-nt insertion near the 5 end causing 19 nonsense mutations (supplementary fig. s3, supplementary material online), effectively ruling out that it could confer a dominant phenotype in crosses with s. bicolor. finally, the fine - scale mapping that we describe herein (fig. in addition to ma1, a more recently discovered flowering locus, ma6, also maps to chromosome 6 very near the location of prr37 (brady 2006) indeed, one report stated that prr37 corresponded to ma6 (mullet. however, members of the same group who implicated prr37 as ma1 have more recently implicated a new positional candidate as ma6 based on apparent functional polymorphism and expression differences between long - day and short - day conditions (murphy. 2014) whereas this new candidate corresponds to orthologs in several other cereals such as the rice ghd7 gene, once again evidence from mutant complementation is lacking. the time and cost associated with sorghum transformation is clearly a hindrance to validation of candidate genes in this promising botanical model. in partial summary, we consider the most probable scenario to be that sb06g012260 is the cause of the ma1 short - day flowering trait in sorghum, as supported by independent lines of evidence including fine mapping, association genetics, mutant complementation, and evolutionary analysis. evidence in support of another nearby gene (prr37) as ma1 is now known to be confounded with previously unknown factors, specifically the presence of sb06g012260 and at least 82 additional annotated genes on the crucial introgressed chromosome segment. the non - functional prr37 allele in our population rules out the possibility that the two genes are functioning in concert to confer the trait. while this new evidence does not contradict the hypotheses that each gene independently contributes to the flowering phenotype, or that prr37 may be ma6 (mullet. 2012), recent data implicating the sorghum ortholog of rice grain number, plant height and heading date-7 (ghd7) as ma6 is more compelling (murphy. 2014), albeit needing support from mutant complementation the evolution of sb06g012260 from a single gene duplicate, rather than a dosage - balanced gene from whole - genome duplication, follows a trend exemplified by key members of the c4 photosynthesis pathway (wang. wgd - duplicates are a rich potential source of genetic novelty, with longer half - lives than single gene duplicates (lynch. however, single - gene duplicates, often lacking their ancestral regulatory elements and in different chromatin environments from their ancestral gene(s), may have greater per - gene potential for the evolution of novelty as reflected by greater divergence of expression patterns than wgd - duplicates (wang. an interesting hypothesis for further investigation is whether the survival of single - gene duplicates such as sb06g012260 may be favored by location in recombinationally - recalcitrant heterochromatin, where neutral or slightly - deleterious mutations tend to survive longer than in euchromatin (bowers. 2009) and offer more opportunity for a mutation that confers new function to coincide with an environment that drives it to high frequency. recalcitrance of the ma1 region of chromosome 6 to recombination may have contributed to the evolution of a coadapted gene complex (mayr 1954). beyond flowering, qtls have been associated with this region affecting kernel weight (paterson, lin,. 1995), tillering, rhizomatousness and regrowth (paterson, schertz,. in particular, the ma1 region also holds dw2, the gene of largest effect on sorghum stature (height) (lin. 1995) but which has long been asserted to be separable from ma1 by recombination. quinby suggested that ma1 and dw2 were different closely - linked genes, with ca. 8% crossing over (quinby 1974), but only evaluating 47 families based on phenotype. based on our observation that late flowering can occasionally be a result of factors other than allelic status at the ma1 locus and that progeny testing is necessary to validate it, such a small study must be considered tenuous. among the 30 validated f3 families in our study, three showed different segregation patterns for flowering time and plant height. since these 30 individuals comprised all confirmed recombinants in the region from a population of 370 individuals, this suggests a 0.5-cm linkage distance between ma1 and dw2 (lin 1998). while the strongest association of allelic variation with plant height in the diversity panel was at sb06g012260 itself (p = 0.007), we also found a marginally significant association at sb06g007330 (p = 0.023), a putative cation efflux family protein. further study is needed to determine if sb06g007330 is dw2 however an intriguing hypothesis for further testing is that increased height confers a competitive advantage in light interception, and alleles conferring this trait might have become abundant more quickly if co - transmitted with alleles for optimal flowering time in the native tropical environment of sorghum. moreover, further dissection of the ma1 region may reveal whether qtls for other traits that have been mapped to the region (lin. 1995 ; paterson, lin,. 1995 ; paterson, schertz,., 1995) are pleiotropic consequences of ma1 or dw2, or represent additional members of a coadapted gene complex. engineering of genotypes that silence the short - day flowering trait may render obsolete the need to laboriously convert tropical grasses to day - neutral flowering by twelve (!) generations of breeding (stephens. 1967), potentially dramatically accelerating cross - utilization of temperate and tropical germplasm for sorghum, sugarcane, and many other crops. targeted selection or engineering of strong floral repressor alleles in biomass crops may confer consistent high yields (julien and soopramanien 1976 ; long 1976 ; julien. rflp and ssr mapping used published methods, with markers drawn from published maps (bowers. phenotyping of f3 families was based on 50150 selfed progenies per family, grown in the field near college station, texas, at ambient daylengths described in detail and with flowering dates recorded as described (lin. association genetics used a 384-member worldwide sorghum diversity panel from icrisat, previously characterized with 41 ssr markers (hash. 2008), evaluated in 2007 under short - day conditions (11.812.15 h light) and high humidity, under which short - day sorghums are expected to initiate flowering promptly. a 2008 planting was characterized by a transition from long to short - day (13.111.0 h) photoperiod and dry conditions, and short - day sorghums would be expected to delay flowering. flowering time was the number of days required for 50% of the plants in a single row to flower (dfl50%). photoperiod response index (pri) was defined as the mean difference in dfl50% between the two planting seasons (i.e., pri = dfl50%2008dfl50%2007). resequencing used bigdye terminator chemistry, and sequences were manually checked and aligned for single nucleotide polymorphism (snp) identification with sequencher 4.1. the quantity and frequency of haplotypes, and linkage disequilibrium were determined by haplotyper 1.0, and tassel 2.1, respectively. tassel was used to perform tests of association, employing population structure covariates and a kinship matrix for the gcp / icrisat germplasm panel based on published ssrs (hash. the bac was sequenced to confirm the integrity of sb06g012260 and identify restriction sites, then completely digested with pvuii to recover a 10.5-kb fragment that extends 5,220-bp upstream and 234-bp downstream from sb06g012260. likewise, the bac clone was completely digested with restriction enzymes stui and bstz17i to recover a 5.2-kb fragment that extends 434-bp upstream and 234-bp downstream from sb06g012260. both fragments were cloned into transformation vector pzp211, and the clones (supplementary fig. s1, supplementary material online) re - sequenced to confirm the integrity of the constructs. independent t0 transformants were selfed to produce t1 segregating progenies, then 1524 plants from each t1 family were evaluated in the greenhouse under ambient long day conditions (at 33.95 n latitude), recording the number of days from planting on 17 may to flower emergence. genotyping of the four loci diagnostic of the ma1 haplotype (423 nt, 4186 nt, 3 nt, and intron indels) in s. halepense used 480 plants sampled equally from ga, tx (2), ne, and nj populations described previously (morrell. supplementary figs. s1s4 and tables s1 and s2 are available at molecular biology and evolution online (http://www.mbe.oxfordjournals.org/).	of central importance in adapting plants of tropical origin to temperate cultivation has been selection of daylength - neutral genotypes that flower early in the temperate summer and take full advantage of its long days. a cross between tropical and temperate sorghums [sorghum propinquum (kunth) hitchc.s. bicolor (l.) moench ], revealed a quantitative trait locus (qtl), flravgd1, accounting for 85.7% of variation in flowering time under long days. fine - scale genetic mapping placed flravgd1 on chromosome 6 within the physically largest centimorgan in the genome. forward genetic data from converted sorghums validated the qtl. association genetic evidence from a diversity panel delineated the qtl to a 10-kb interval containing only one annotated gene, sb06g012260, that was shown by reverse genetics to complement a recessive allele. sb06g012260 (sbft12) contains a phosphatidylethanolamine - binding (pebp) protein domain characteristic of members of the ft family of flowering genes acting as a floral suppressor. sb06g012260 appears to have evolved 40 ma in a panicoid ancestor after divergence from oryzoid and pooid lineages. a species - specific sb06g012260 mutation may have contributed to spread to temperate regions by s. halepense (johnsongrass), one of the world s most widespread invasives. alternative alleles for another family member, sb02g029725 (sbft6), mapping near another flowering qtl, also showed highly significant association with photoperiod response index (p = 1.5310 6). the evolution of sb06g012260 adds to evidence that single gene duplicates play large roles in important environmental adaptations. increased knowledge of sb06g012260 opens new doors to improvement of sorghum and other grain and cellulosic biomass crops.
it was first reported in 1923 in an autopsy case by mandelstamm, and since then, fewer than 250 cases have been described. the prognosis of patients with pulmonary artery sarcoma is usually poor. in reported cases, the mean survival time was usually less than 2 months for those without surgical treatment and approximately 10 months for those treated surgically. surgical resection, if possible, is known to be the best therapeutic option to prolong survival. thyroid and adrenal glands are representative endocrine organs that have high vascularity, and we often encounter thyroid or adrenal metastases in autopsy cases with malignant diseases ; however, malignant metastasis to both endocrine organs during follow - up is not common in the clinical setting. as imaging techniques and fine needle aspiration skills resection of metastatic disease has been shown to prolong survival of patients in a variety of cancers. metastasectomy can be justified in liver metastases from colorectal cancer or neuroendocrine tumors and in lung metastases from colorectal cancer or from osteogenic sarcoma. there has been a limited number of reports evaluating the role of thyroid and/or adrenal metastasectomy [6 - 10 ]. here we report a case of a patient with pulmonary artery intimal sarcoma who showed long - term survival after sequential metastasectomies of the thyroid and adrenal glands. a 62-year - old woman presented with a 4-week history of dyspnea on exertion and facial edema in november 1999. electrocardiography showed t wave inversion at leads v1 - 5, iii, and avf, right axis deviation, and right ventricular hypertrophy. initial echocardiography revealed an echogenic mass in the main pulmonary artery with significant flow obstruction and right ventricular dysfunction suggesting pulmonary embolism. chest computed tomography (ct) revealed an embolism - like mass in the pulmonary trunk and in both sides of the main pulmonary arteries (fig. the surgical specimen showed an ill - defined, protruding polypoid mass that was 4.8 cm in the greatest dimension. the pathology revealed pulmonary artery intimal sarcoma, which was supported by positive smooth muscle actin and negative desmin in immunohistochemical staining (fig. she received radiation therapy (4,500 cgy fractionated) and chemotherapy (cyclophosphamide, doxorubicin, and dacarbazine) as adjuvant treatments. during follow - up, she was regularly checked by echocardiography and chest ct. initial follow - up chest ct (november 2001, 2 years after initial surgery), mild diffuse enlargement of the thyroid gland with multiple small low density lesions was discovered. it is hard to know if these findings existed before the initial operation, because the preoperative chest ct did not include the neck. this lesion was just followed up with chest ct yearly and showed no change for 3 years. in august 2004 (4.7 years after initial surgery), she was referred to an endocrinologist for abnormal thyroid function tests. miu / l (normal, 0.4 to 5.0), and total t4 was 166 nmol / l (normal, 76 to 178). ct of the neck showed two low density nodules of 2.02.5 cm and 0.90.9 cm in both lobes of the thyroid glands, which was no different from previous studies (fig. fine needle aspiration cytology was done and revealed atypical spindle cells, suggestive of metastatic intimal sarcoma (fig. she underwent total thyroidectomy, and histology confirmed metastatic intimal sarcoma with clear resection margins. she was followed by neck and chest ct (covering the adrenal gland), and there was no evidence of local recurrence or distant metastasis until august 2005. a 10.08.5 cm sized round, heterogeneous mass replacing the whole adrenal gland superior to the right kidney was newly detected by chest ct in march 2006 (6.3 years after initial surgery), suggesting a metastatic lesion in the right adrenal gland (fig. 3a). an f - fluorodeoxyglucose positron emission tomography (f - fdg pet)/ct scan showed a large hypermetabolic mass (max standardized uptake value, 6.8) with internal necrosis (fig. the tumor cells were positive for smooth muscle actin and were negative for inhibin, cytokeratin, chromogranin, and desmin, supporting the above diagnosis. during follow - up after right adrenalectomy, abdomen - pelvic ct and f - fdg pet / ct showed no evidence of recurrences or metastases, including the anastomosis site of the main pulmonary trunk, thyroid bed, and adrenalectomy site. she has been alive and well without any evidence of disease as of may 2012 (12.5 years after initial surgery). intimal sarcoma is a rare tumor that arises from large vessels, including the aorta and pulmonary artery. it is characterized by insidious growth, but it usually presents with extensive local invasion and hematogenous metastases at the time of diagnosis. metastases are most commonly found in the lungs, although deposits in the pancreas, kidney, brain, lymph nodes, and skin have been reported. although endocrine organs have high vascularity, metastasis to endocrine organs in the clinical setting is not as common ; however, recent autopsy studies have shown that the incidence of thyroid metastases ranges between 1% and 24% in patients with a known history of neoplasm. the prevalence of adrenal metastases in patients with extra - adrenal cancers ranges from 32% to 73% in different series. breast cancer and lung cancer are the most frequent malignancies showing metastases to the thyroid. in the case of adrenal metastasis, lung, kidney, breast, and gastrointestinal carcinomas are the most frequent primary malignancies. as ultrasonography - guided aspiration and various imaging modalities such as f - fdg pet / ct have been more frequently used in clinical settings, more cases with metastases to the thyroid or adrenal gland are encountered. moreover, asymptomatic solitary metastatic lesions are more likely to be found before they become widespread. thus, a thyroid or adrenal mass in a patient with a history of a previous malignancy should be regarded as potentially metastatic, even if the primary tumor was adequately treated many years before. no standard therapeutic guidelines have been established for solitary metastatic disease to the thyroid or adrenal gland. the efficacy of surgical treatment for a solitary metastasis as seen in our case is unknown. numerous case reports have suggested that metastases to the thyroid or adrenal gland are associated with a poor prognosis, because most thyroid or adrenal metastases occur late in the course of disseminated cancers. surgical treatment for metastases has not been widely adopted because of the concern about probable associated advanced diffuse metastatic diseases ; however, radical treatment for an isolated metastasis to the thyroid or adrenal gland can be curative or achieve long - term survival, and an aggressive surgical approach has been recommended by some authors [6 - 10,18 ]. surgery has been advocated for metastatic disease, provided the disease is limited and/or amenable to complete treatment. preoperative investigations should be as extensive as possible in order to detect any inaccessible metastatic sites or invasion hampering complete surgery. the prognosis of patients who have a solitary thyroid or adrenal metastasis depends on multiple factors such as the site of origin, the histological cell type of primary tumors, antecedent history of distant metastasis or discovery of other synchronous distant metastasis, and extent of local disease. there should be no extra thyroid or adrenal disease, and preoperative imaging studies should demonstrate disease that is likely to be completely resectable. furthermore, as in our case, an additional metastatic site does not have to be an exclusion criterion if it seems to be curable. we treated a rare case with intimal sarcoma of the pulmonary artery that metastasized to the thyroid and adrenal glands sequentially. the patient developed a thyroid metastasis 4.7 years after initial surgery and an adrenal metastasis 6.3 years after initial surgery. there should be more investigations regarding which cancers should be considered for metastasectomy of the endocrine organs with curative intent.	cancer metastases to the thyroid or adrenal gland are uncommon. furthermore, cases showing long - term survival after surgical resection of those metastatic tumors are rare. we report a case of pulmonary artery intimal sarcoma with metastases to the thyroid and adrenal glands sequentially that was successfully treated with sequential metastasectomies. a 62-year - old woman presented with a 4-week history of dyspnea on exertion and facial edema in november 1999. echocardiography and chest computed tomography (ct) revealed an embolism - like mass in the pulmonary trunk. pulmonary artery endarterectomy with pulmonary valve replacement was performed, and histopathology revealed pulmonary artery intimal sarcoma. a thyroid nodule was found by chest ct in november 2001 (2 years after initial surgery). during follow - up, this lesion showed no change, but we decided to obtain fine needle aspiration cytology (fnac) in august 2004 (4.7 years after initial surgery). fnac revealed atypical spindle cells suggestive of metastatic intimal sarcoma. she underwent total thyroidectomy. during follow - up, a right adrenal gland mass was detected by chest ct in march 2006 (6.3 years after initial surgery), and adrenalectomy was done, which also revealed metastatic sarcoma. she has been followed up without any evidence of recurrent disease until may 2012 (12.5 years after initial surgery).
jawbone cysts are pathological cavities containing liquid, semi - liquid or gas, which is partially or totally covered by an epithelial tissue. the odontogenic cysts covered by epithelium are divided into two groups of developmental and inflammatory based on their origin. they are resulted from odontogenic tissue (epithelial rests of malassez, dental lamina and rests of enamel organ) (1). radicular cyst (rc) is the most common odontogenic cyst originated from epithelial rests of periodontal ligaments resulting from the inflammation of the necrosis pulp. for the cyst to develop dentigerous cyst (dc) is the most common odontogenic cyst next to the radicular cyst involved in about 24% of all real cysts in the jaw (4). the dc is developed from expansion of the dental follicles resulting from the fluid accumulation between the crown of a tooth and its surrounding epithelium (5). the odontogenic keratinized cyst (okc), first introduced by philipsen, since 2005, the world health organization (who) categorized this cyst as odontogenic tumors due to its neoplastic properties and replaced the term odontogenic keratocyst with keratocystic odontogenic tumor (7). epidermal growth factor receptors (egfr) known as her1 and erbb1 are in turn recognized as the first group of erbb receptors family as a member of a bigger family called tyrosine kinase receptors (8, 9). afterwards, other members of this family were introduced including her2 (erbb2), her3 (erbb3) and her4 (erbb4). the activity of such receptors is necessary for normal growth and cell differentiation by providing atp needed by the cell (10). as an example it has been demonstrated that her3 plays an important role in some human cancers such as laryngeal, breast, lung, gastric, ovarian, thyroid carcinoma, melanoma etc. ; the her3 expression is associated with worse survival in solid tumors (12 - 19). increased her3 expression dysplastic epithelium and oral squamous cell carcinomas (sccs) showed lesser expression of her3 in developing and mature epithelium than dysplastic and malignant oral epithelium (10). (22) revealed more her3 overexpression in metastatic head and neck squamous cell carcinoma (hnscc) compared to primary hnscc. they also found a correlation between her3 overexpression and worse overall survival. according to their results, her3 overexpression, as an independent factor, related to poor prognosis and represented as a potential molecule for targeted therapy. considering limited studies performed to examine the significance of her3 factor in identifying malignant potential of odontogenic cysts, this investigation evaluated the her3 marker expression in okc, dc and rc cysts, which may potentially change to scc and other malignancies of jaws. this descriptive - analytical study was conducted on all 57 samples of odontogenic cyst (fulfilling the inclusion criteria) prepared from pathological archive of dentistry college in zahedan, iran as a referral and governmental center of oral pathology in april 2013. this project was approved as a dentistry dissertation in the ethics committee of zahedan university of medical sciences, code 90 - 1000 in july 2011. the odontogenic cysts slides were reviewed and the diagnosis was confirmed by two oral and maxillofacial pathologists. samples with incomplete information required in their files, inadequate tissue for examination or if contained necrosis and inflammation or not fixed correctly were excluded from the study. after selecting the samples, their paraffin blocks were cut by 3-micron incisions and mounted on charged glass slides. then, the incisions were paraffinized in xylene and rehydrated in alcohol. to detect antigens, the samples were placed in a citrate buffer with ph = 6 and incubated in a microwave oven. afterwards, the samples were stained immunohistochemically based on the manufacturer s instruction with the antibody erbb3 (mouse monoclonal 1 ml, novocastra, newcastle, uk ; lot no : 6003990). after staining immunohistochemically, staining intensity was examined in four grades as 0 (as non - stained), 1 (weakly stained), 2 (moderately stained), 3 (highly stained) and the percentage of stained cells was investigated by counting 1000 cells in five grades as 0 (0%), 1 (1% - 25%), 2 (26% - 50%), 3 (51% - 75%) and 4 (76% - 100%). the total score ranged from 0 to 12, where the scores equal to or higher than 4 were considered as positive and below 4 as negative. the comments of the two pathologists were finally compared ; those with any difference were rechecked by a third pathologist. all pathologists were blinded to study samples. to measure the inter - observer agreement, chicago, il). the kolmogorov - smirnov test was used to assess the normality of data. the quantitative variables with normal distribution were examined by anova and the chi - square test was used to compare qualitative data. data related to their gender and age is shown in table 1. as seen, 60% of the samples in all the three types of dc, rc and okc were male. okc was developed in the fourth decade of life and the two other cysts in the third decade of life. the data related to average percentage of stained cells is shown in table 2 as divided by the type of cysts. as seen in table 3, the staining percentage of cells was higher than 25% in more than 50% of all groups. data regarding staining intensity in the three types of cysts is shown in table 4. as seen, staining intensity in dc and okc was often weak, while it was strong in rc. (%). a, staining pattern with high intensity of epithelial covered cells of okc. b, staining pattern with moderate intensity of epithelial covered cells of okc in basal and supra - basal layers. d, non - stained epithelial cover cells of radicular cyst (magnification 400). the total score was obtained from multiplying intensity grade by percentage grade of stained cells. in 50% (8 cases) of dc samples and 52.4% of okc samples (11 cases), the staining result was positive and just for 20% (4 cases) of rc samples, positive results were achieved, while chi - square showed no significant difference (p = 0.07). however, the difference between okc and rc was significant (p = 0.03), while the difference between dc and rc was close to significant level using chi - square test (p = 0.06). the present study was performed to evaluate her3 expression in epithelial lining of rcs, dcs and okcs. the results obtained based on the total score indicated the positive her3 expression in 52.4% of okc, 50% of dc and 20% of rc samples. the staining procedure of cells was the same in cytoplasm and membrane in basal and supra - basal layers. her3 is one of the four members of epidermal growth factor receptors as erbb, which is activated by connecting to neuregulin-1 and neuregulin-2 ligands. since her3 lacks intrinsic kinase activity, induction of signal occurs through formation of heterodimers with egfr, her2 and her4 (24). most studies examining the her3 expression in oral lesions were about dysplastic and cancerous lesions of oral mucosa (10, 20 - 23), in which some significant results were obtained with respect to increased expression and lymph node metastases, prognosis and invasion (2, 20, 22). it was recently found that her3 plays an important role in response to radiotherapy of head and neck carcinomas and blocking its activity along with radiotherapy may be beneficial for the treatment of human tumors (25). (26) studied her3 expression in adenoid cystic carcinoma of salivary glands ; her3 expression was found in all samples and staining intensity of tumor cells for this marker in invasive areas was more intense. the results of another study revealed that systematic conditions such as diabetes would increase her3 expression in certain stages of oral oncogenesis, which is likely resulted by increased cellular proliferation and apoptosis inhibition (27). no similar study was found examining the effect of her3 marker on these three types of cysts. accordingly, the results of studies examining other members of erbb family in odontogenic cysts and tumors are suggested. in an investigation by shrestha. (28) egfr expression was positive in 60% of okc, 47.4% of dc and 35% of rc samples. on the other hand, in the study performed by li. (29) the higher expression of egfr was reported as the first member of tyrosine kinase receptors family in developmental cysts such as okc and dc compared to rc. inflammation was identified as a factor for decreased expression of this receptor in rc. in the present study, rc showed the lowest rate of her3 expression based on the total score. in an investigation by de - vicente, egfr expression reported as 100% of cases of ameloblastoma, 73% of okcs, 40% of dc and 30% of rcs, where egfr expression was found significantly more in ameloblastoma (30). moreover, it is suggested in some studies to consider anti - egfr factors to reduce the size and treat inoperable tumors as close to vital structures (31, 32). in the investigation conducted by de oliveira. (33) cytoplasm and membrane expression of egfr marker in basal and supra - basal layers of dc and okc were examined ; cytoplasmic expression of egfr was significantly higher in okc basal, supra - basal layers and cytoplasm and membrane expression of egfr in supra - basal layer of dc. furthermore, they stated that egfr might indicate high capability of cells in response to stimulation, which may be considered as a cause of odontogenic lesions. in addition, goncallves reported egfr expression in 100% of rc and dc cases as well as 94% of okc cases, while he considered membrane and cytoplasm placement of egfr in cells as an important factor in response to stimulating proliferation (34) ; this may be generalized to cytoplasm and membrane expression of her3 as well. kolar. (35) compared sporadic and syndromic okc and reported a high expression of bcl2, p27kip1 and c - erbb2 in syndromic keratocysts, and lower proliferative activity in basal cells of sporadic okc. furthermore, they suggested the difference between dc and rc compared to sporadic and syndromic okc in higher proliferation in basal cells layer, and by contrast, lower proliferation in supra - basal cells layer. heikinheimo examined her3 immunohistochemical expression in 12 sporadic okc samples ; their results showed that her3 has a high incidence in epithelium supra - basal layers and the epidermal growth factor receptor pathway was known as a part of okc pathogenesis (7). this result differs slightly from the present study, so that her3 expression was observed as monotonous and even a case was found as non - stained (4.8%). in some studies like shintani. (10) ones, researchers discussed merely on the percentage of stained cells while in other studies like ours, both percentage and intensity were used to evaluate her3 expression (18, 21, 22). assessment of staining only based on intensity is not an appropriate criterion, because staining intensity may be differently reported by different pathologists, especially in weak and moderate levels of intensity. evaluation of her3 expression in microscopic examination of staining slides immunohistochemicalized based on staining intensity and percentage stated as numbers would provide researchers with a full and more comprehensive result with lower error compared to her3 expression as separately in accordance with intensity or percentage of stained cells (22). in general, the results of this study showed that the average percentage of stained cells was high in dc, okc and rc, respectively. for total score of staining, okc, however, the results were quite different based on moderate and high staining intensity, and it was high in rc, okc and dc, respectively. it may be concluded that her3 has a higher expression in developmental cysts including okc and dc compared to inflammatory cysts of rc. meanwhile, the higher rate of her3 marker expression in okc may justify inherent growth potential, stimulation - independent proliferation capability, invasive growth and high recurrence rate of the cyst accepted today as a tumor. lack of research in the field of her3 expression in odontogenic cysts necessitates further investigations in this respect. finally, it is suggested to conduct further studies about her3 expression in relation to the behavior of odontogenic cysts (e.g. recurrence after treatment, transformation to malignancy) and/or in comparison with tumors such as ameloblastoma and scc.	background : it has been demonstrated that her3 plays an important role in some human cancers and the her3 expression is associated with worse survival in solid tumors.objectives:this study was conducted to compare her3 expression in epithelial lining of radicular cysts (rcs), dentigerous cysts (dcs) and odontogenic keratocysts (okcs).materials and methods : this was a descriptive - analytical study, which assessed all 57 paraffin blocks of rcs, dcs and okcs (21 rcs, 16 dcs, 20 okc) from pathological archive of dentistry college of zahedan, iran. the her3 expression in cytoplasm and membrane was examined by immunohistochemical method. the data collected was analyzed using spss16 by anova and chi - square. p < 0.05 was considered as statistically significant.results:the her3 expression had positive results in 52.4% of okc, 50% of dc and only 20% of rc samples. there was a significant difference between her3 expression in okcs and rcs.conclusions:the her3 expression in developmental odontogenic cysts was higher than that in inflammatory odontogenic cysts. the higher rate of her3 expression in okc may justify inherent growth potential, stimulation - independent proliferation capability, invasive growth and high recurrence rate of the cyst accepted today as a tumor.
appreciation of the existence of a plasma membrane receptor for thyroid hormone analogues on the extracellular domains of a structural plasma membrane protein, integrin v3 [13 ], has permitted recognition of new control mechanisms for the release of cytokines, including chemotactic cytokines or chemokines. for example, transcription of the fractalkine ligand (cx3cl1) and receptor (cx3cr1) genes is downregulated in tumor cells by tetraiodothyroacetic acid (tetrac), a deaminated, naturally occurring analogue [5, 6 ] of l - thyroxine (t4), and this action is initiated at integrin v3 [2, 4 ]. these fractalkine effects were demonstrated in human breast cancer cells, and it is tumor cells and endothelial cells that generously express v3. within the central nervous system (cns), fractalkine has been observed to have both neuroprotective and neurotoxic actions (see below) and we may ask if thyroid hormone in the cns is one determinant of the type of action that cx3cl1 will manifest. synthesis and release of a panel of cytokines the integrin is generously expressed by nervous system tumor cells, for example, glioma and glioblastoma cells, and proliferation of these cells is stimulated via v3 by l - thyroxine, the principal secretory product of the thyroid gland, and, to a lesser extent, by 3,5,3-triiodo - l - thyronine (t3), which is derived from t4 by deiodination and is the form of thyroid hormone that is active intracellularly. formulations of tetrac inhibit actions of t4 and t3 on nervous system cancer cells. it is important to note that healthy neurons also express v3 and that thyroid hormone has been shown to act on such cells to control functions such as sodium current (ina) [11, 12 ]. thus, binding to the plasma membrane of tumor cells, endothelial cells, and certain normal nervous system cells, thyroid hormone influences downstream the transcription of a number of genes relevant to inflammatory and immune responses [4, 8 ]. thyroid hormone is widely acknowledged to have critical intracellular actions on the nervous system that are initiated by t3 at thyroid hormone receptors (trs) in the cell nucleus. these hormonal effects that require primary interactions of t3 with trs are described as genomic. in contrast, nongenomic hormonal effects are those initiated at the plasma membrane or in cytoplasm [3, 13 ] ; these may involve t3, t4, or hormone analogues such as tetrac. t3-dependent genomic effects are critical to central nervous development [1416 ], neurophysiology, and neuroprotection [1721 ]. it must also be pointed out that the hormone has actions on brain development that are nongenomic and are chiefly upon the cytoskeleton and state of actin these effects are critical to early structural development of brain, particularly the cerebellum, and are dependent upon t4, rather than t3. in this position paper, we briefly review the actions of thyroid hormone analogues on chemokines that are relevant to the nervous system. much of the information we have about these factors and their nongenomic regulation by thyroid hormone has come from studies of cells or tissues that are not of nervous system origin. however, studies of apparently neuroprotective properties of thyroid hormone have of course been conducted in nervous system tissues and are of particular interest because the hormone can be antiapoptotic and proangiogenic, qualities desirable in cells of the cns in the setting of ischemia. but circumstances exist in which the hormone may also be proinflammatory and this quality may explain variability of reports in the thyroid hormone - neuroprotection literature. the current review is intended to encourage the examination in nerve cells and glia of the likely possibility that thyroid hormone analogues are modulators of chemokine actions in the nervous system. human chemokines are 48 small (up to 14 kda) chemotactic cytokines of four classes. the classes are cc, cxc, c, and cx3c, oriented about the conserved n - terminal cysteine, where x is any amino acid [25, 26 ]. appending an l indicates the protein is a ligand and an r indicates function of the molecule as a receptor. chemokines may be constitutively expressed in tissues (homeostatic chemokines), for example, where they are involved in generation and maintenance of vasculature, or they may be periodically generated in response to local inflammatory responses. both of these actions are observed in the nervous system. thyroid hormone is an important proangiogenic factor by a variety of mechanisms [27, 28 ], contributing via this and other pathways to the inflammatory response, as pointed out above. it was thus not surprising to find that the thyroid hormone analogue, tetrac, reformulated as a nanoparticle (nanotetrac or nano - diamino - tetrac), affected transcription of the genes of as many as seven chemokines with either of or both proangiogenic and proinflammatory properties (see below). the nanotetrac formulation involves the covalent binding via a linker of tetrac to a large poly[lactic - co - glycolic acid ] nanoparticle to maximize duration of exposure of tetrac to the cell exterior and integrin v3. these initial studies of thyroid hormone action on chemokine gene expression were conducted in tumor cells, but the nongenomic mechanism by which tetrac acts on chemokines involves plasma membrane integrin v3, a protein that mediates critical actions of thyroid hormone and hormone analogues on neurons [11, 12 ], granulocytes, and endothelial cells. these cells are essential components of the homeostatic and inflammatory effects of chemokines in the nervous system and other tissues. the integrin also transduces the thyroid hormone (t4) proliferative signal on glioma and glioblastoma cells. among the homeostatic chemokines in the cns that contribute to maintenance of the vasculature of the blood - brain barrier is ccl20. ccl20 is designated a homeostatic chemokine, as defined by its constitutive expression in lymphatic and thymic tissue. it is also constitutively transcribed at low levels at the choroid plexus and other epithelial barriers outside the nervous system, but its expression at these sites will respond to proinflammatory cytokines, for example, interleukin-6 (il-6). ccl20 may play a role in the importation of t cells into the cns [30, 31 ]. ccl20 gene transcription is downregulated by tetrac formulations (figure 1) via an integrin v3-dependent process. because t4 and t3 bind to the thyroid hormone - tetrac receptor on the extracellular domain of this integrin and this binding is inhibited by tetrac formulations, we propose that t4 and t3 have the capacity to stimulate ccl20 gene transcription. this possibility has not yet been investigated. ccl26 is another member of the cc family of chemokines whose gene transcription is regulated from the cell surface (integrin v3) by tetrac formulations. rather, ccl26 is involved in hepatoma cell biology and in pathogenesis of certain skin diseases. a product of microglia, chemokine cxcl2 has important chemotactic activity on granulocytes, inducing neutrophil infiltration of tissues with consequent inflammation and damage to tissues, as do other cxc products. release of cxcl2 from microglia is at least in part a response to increased tissue atp levels that are a consequence of tissue damage. atp activates signal transducing mitogen - activated protein kinase (mapk) in these cells that downstream results in cxcl2 gene expression. traumatic brain injury - related inflammation involves choroid plexus production of cxcl2 and other cxcs that stimulate neutrophil infiltration. cxcl2 is also implicated in the neutrophil infiltration of the brainstem in atypical experimental autoimmune encephalomyelitis (eae). decreased transcription of the cxcl2 gene is a tissue response to nanotetrac (figure 1), suggesting that this agent could have damage limitation activity in a variety of cns tissue damage scenarios. the integrin - mediated effect of nanotetrac also implies that agonist thyroid hormone (t4 or, possibly, t3) may act at v3 to enhance the inflammatory response to cns tissue trauma or the process of eae. like cxcl2, cxcl3 is also generated in the choroid plexus as a component of the inflammatory response to traumatic brain injury. these chemokines are secreted by choroidal epithelium both basolaterally and apically, that is, bidirectionally, in order to assure granulocyte passage across the blood - brain barrier by the paracellular pathway. in addition to its proinflammatory properties, cxcl3 has been shown to regulate the migration of cerebellar granule neuron precursor cells (gcps). a significant minority of childhood medulloblastomas originate from gcps, apparently reflecting defective migration of these cells and prolongation of their residence in the external granular layer (egl) of the cerebellum. such residence is associated with gcp proliferation and failure to differentiate. in this example of cxcl3 action, if, as we propose, t4 via its receptor on v3 enhances cxcl3 gene expression, then the presence of the hormone in the course of brain development would support the normal outmigration of gcps from the egl and contribute to minimization of risk of medulloblastoma. in contrast, the demonstrated action of tetrac in its nanoparticulate formulation to reduce cxcl3 gene expression might be a factor in decreased migration of gcps ; avoidance of exposure of the developing brain to tetrac is desirable. the pathogenesis of certain autoimmune diseases of the cns, such as multiple sclerosis (ms), remains incompletely understood. cxcl10 is a small proinflammatory, proangiogenic, interferon - (ifn--) inducible chemokine that has been implicated in the development of ms. cxcl10 is produced by white blood cells (granulocytes, monocytes), endothelial cells, and astrocytes, among others. cxcl10 binds to the cxcr3 receptor that is expressed by t lymphocytes, natural killer (nk) cells, and certain kinds of epithelial cells. vazirinejad and coworkers and others [3942 ] have found elevated circulating (serum) or csf content of cxcl10 and have proposed that cxcl10 contributes importantly to inflammatory demyelination that is an essential component of ms. but csf levels of cxcl10 are occasionally elevated in subjects with evidence of cns inflammation [38, 43 ]. because a tetrac formulation that acts exclusively at integrin v3 stimulates transcription of the cxcl10 gene (figure 1), the thyroid hormone - relevant issue that is raised here is whether thyroid hormone (t4 or t3)through cxcl10may be a factor that reduces the aggressiveness of pathogenesis of ms. thus, it is important to examine the possible protective actions of t4/t3 in models of ms and a possibly deleterious effect of tetrac formulations in such models. that tetrac can downregulate expression of specific genes and upregulate other genes which is not surprising, given that thyroid hormone analogues via v3 can, via signal transduction pathways, differentially control proactivator and corepressor nucleoproteins. we have reported elsewhere that tetrac upregulates expression of thrombospondin 1 (tsp1) gene and microrna-15a (mir-15a) but downregulates mir-21, egfr, and vegfa. against this background, it is important to note that thyroid hormone has been shown by others to induce remyelination via action(s) on oligodendrocyte precursor cells in the model of cuprizone - induced demyelination. dell'acqua and coworkers also have shown that the hormone also supports remyelination and is neuroprotective in eae. thus, the thyroid hormone analogue, tetrac (formulated as nano - diamino - tetrac), has the potential to modulate cns inflammation bidirectionally by action on expression of the genes for cxcl2 and cxcl3supporting inflammation and on expression of the cxcl10 gene, possibly reducing inflammation. testing in models of cns inflammation will determine whether there is a dominant thyroid hormone / tetrac effect on one or more specific cxc chemokines. the c chemokines are xcl1 (lymphotactin-) and xcl2 (lymphotactin-). the single receptor to which these chemokines bind is xcr1. the xcl1, xcl2, and xcr1 genes are apparently not subject to modulation by thyroid hormone or the hormone analogue, tetrac. the resident macrophages of the cns, microglia, have both proinflammatory (m1) and anti - inflammatory (m2) phenotypes. release of cx3cl1 (fractalkine) by damaged neurons supports an m1 response by binding to its receptor (cx3cr1) on microglia and fostering recruitment of circulating white blood cells. the proinflammatory state ensues with release of factors such as reactive oxygen species, nitric oxide, and inflammatory cytokines. the m2 state is the product of microglial production of anti - inflammatory cytokines and growth factors in response to cx3cl1 at the microglial cx3cr1 [5, 45 ]. thus, the m1 versus m2 phenotypic state of microglia [46, 47 ] appears to determine neuroprotective versus neurotoxic activities of cx3cl1 and the evolution / progression of brain diseases such as alzheimer 's, ischemia, and traumatic brain injury [7, 4648 ]. fractalkine and its receptor, cxcr1, may form a ternary complex with integrin v3 that results in activation of the integrin ; activation is a change in configuration of the protein to support cell migration and cell - cell interactions that are critical functions of integrins. microglia express v3. inhibiting thyroid hormone actions at integrin v3, nanotetrac decreases transcription of the cx3cl1 gene and thus we speculate that the m2 and m1 responses of microglia are both supported by nongenomic action of t4 at v3. this possibility has not been examined experimentally, nor has the possibility been considered that the transition from m2 to m1 state in microglia is a process that is subject to influence by thyroid hormone. thyroid hormone in the form of t3 is known to have genomic effects on microglia [52, 53 ]. the nongenomic downregulation of cx3cl1 gene expression by nanotetrac in damaged neurons is possibly a desirable intervention to investigate in the early phases of models of alzheimer 's. in contrast, such an intervention is to be avoided in settings in which the m2 response prevails, for example, in recruitment of microglia to developing synapses in developing brain and regulation of microglia - neuron interactions in brain development, adulthood, and aging. because thyroid hormone can provoke inflammatory cytokine production, one can ask whether this hormone permissively contributes to induction of the m1 response, in which hormonal action on fractalkine production could be either protective or neurotoxic. like lauro and collaborators, we endorse additional studies of the determinants of the neuroprotective versus neurotoxic cx3cl1 responses ; we also urge the definition of the possibly distinctive roles of thyroid hormone isoforms in the m1 and m2 responses. the actions of fractalkine on the developing brain have been recently reviewed by arnoux and audinat. the functions in developing brain of cx3cl1 on microglia through cx3cr1 on glial cells include support of neuron survival and axon outgrowth and refining synaptic circuits through microglial phagocytic activity. there is also some neuronal death that occurs normally in brain development and microglia are involved in such events. because expression of the cx3cl1 gene is downregulated by the thyroid hormone analogue, tetrac, at integrin v3, we propose that the critical relevance of t3 and t4 to normal brain development is in part dependent upon actions of the hormone on the fractalkine gene. tetrac has anticancer properties outside the cns and any clinical use of the agent or its formulations that may emerge must be avoided in the setting of pregnancy. finally, it has recently been appreciated that fractalkine can bind directly to integrin independently of cx3cr1 and thereby activate the integrin. the binding site where this occurs is near the rgd (arg - gly - asp) recognition site in the head of the integrin and thus proximal to the thyroid hormone - tetrac receptor on v3. thus, cx3cl1 may have integrin - dependent functions in cells that do not express cx3cri. the existence of this chemokine binding site on the integrin also raises the possibility of interactions / crosstalk between the hormone - binding and fractalkine - binding sites on the integrin that could be influenced by tetrac and t4. cx3cl1 is the only chemokine known to undergo constitutive cell internalization and thyroid hormone is known to drive internalization of v3. the possibility thus exists that cell uptake of fractalkine is modulated by thyroid hormone binding to the integrin. the chemokine receptor genes whose transcription is subject to modulation by thyroid hormone analogue tetrac include (1) cxcr4, the principal ligand of which is cxcl12 and the transcription of the gene which is increased by nanotetrac ; (2) ccr1, the ligands which include ccl3, ccl4, ccl6, ccl9/ccl10, ccl14, ccl15, and ccl23, and the transcription of the gene which is decreased by nanotetrac ; and (3) cx3cr4, the ligand of which is cx3cl1. transcription of this receptor gene is frankly decreased by nanotetrac. only in the case of cx3cr4/cx3cl1 are both ligand and receptor genes affected similarly from the thyroid hormone - tetrac receptor on v3. we propose that agonist thyroid hormone, for example, t4, acts contrarily to nanotetrac at the integrin this would involve a decrease in cxcr4 gene expression and increases in ccr1 and cx3cr4 gene transcription but this has not been experimentally approached. among the principal issues of this review is the relevance of integrin v3 to regulation of chemokine gene expression. fibronectin, vitronectin, and osteopontin, as examples that are critical to tissue integrity, and only recently has it been recognized that small molecule ligands of the integrin of the thyroid hormone family specifically affect transcription of at least 6 chemokines. of these agents, 5 are important to functions of the cns, particularly, maintenance of the integrity of the choroid plexus and blood - brain barrier, and contributions to inflammatory processes in the nervous system. the fact that thyroid hormone analogues can affect chemokine ligand and receptor gene transcription is not surprising, given the actions of analogues of the hormone on several aspects of the inflammatory response and on the immune response. we have previously pointed out that expression of the genes for cx3cl1 (fractalkine) and the fractalkine receptor is subject to regulation from the thyroid hormone - tetrac receptor on integrin v3. we emphasize here that observations of effects of nanoparticulate tetrac (nanotetrac) on expression of chemokine genes do not provide assurance that principal thyroid hormone isoforms t4 and t3affect expression of all of these genes and do so in directions opposite to those of nanotetrac. that this may be the case, however, is suggested in the case of regulation of demyelination / remyelination in several models. that is, thyroid hormone has remyelination activity, whereas the antithyroid hormone factor, tetrac, has been shown by us to stimulate cxcl10 expression, supporting demyelination. it is thus important to address this issue directly and compare integrin - mediated studies of t4/t3 versus tetrac on transcription of cxcl10 and other chemokine genes, particularly in cells of importance to vascular and inflammatory responses of the cns. in contrast to its upregulatory action on cxcl10 gene expression, tetrac in the case of other chemokines reviewed here serves as a factor downregulating gene expression. we know that t4 supports the inflammatory response in a variety of tissue settings, and therefore the suppressive effects of tetrac on expression of genes for cxcl2, cxcl3, and ccl20 would be anti - inflammatory. t4 and unmodified or reformulated tetrac could affect the state of inflammatory cells outside the cns, following which these cells could gain access to the nervous system. however, t4 and tetrac are avidly bound at a receptor site on plasma protein transthyretin (ttr), and ttr serves at the choroid plexus to support trafficking across the blood - brain barrier of thyroid hormone analogues. thus, two thyroid hormone receptors are of particular interest to the actions of thyroid hormone and hormone analogues on specific chemokine gene expression in the cns : ttr and integrin v3. these receptors are structurally unrelated, but essential to provision of access of hormone analogues to various cells in the brain, for example, microglia, astrocytes, neurons, and endothelial cells, that are targets of specific chemokines. in summary, thyroid hormone is proposed to support proinflammatory processes in the cns that are due to a limited number of chemokines. thyroid hormone, especially t4, is proangiogenic by a number of mechanisms in a variety of tissues and thus it is not surprising that thyroid hormone analogues affect angiogenesis in brain, at least in part via chemokines. thyroid hormone is a proliferative factor for gliomas and glioblastoma, as well as for a variety of other nonneurological cancers [1, 3, 62 ]. it is possible that the biology of another cns tumor, medulloblastoma, may be affected by thyroid hormone and hormone analogues, but in a wholly different manner and working via a chemokine, cxcl3. that is, via stimulation of cxcl3 gene transcription, thyroid hormone might increase outmigration of cerebellar granule neuron precursor cells from the external granular layer of the cerebellum and reduce the risk of medulloblastoma in pediatric patients. consistent interference with transcription of the chemokine genes that we observed in the two examples of human cancer (figure 1) may constitute an important and yet underappreciated molecular component of the mechanisms of anticancer activity of therapeutic tetrac formulations.	the extracellular domain of plasma membrane integrin v3 contains a receptor for thyroid hormone (l - thyroxine, t4 ; 3,5,3-triiodo - l - thyronine, t3) ; this receptor also binds tetraiodothyroacetic acid (tetrac), a derivative of t4. tetrac inhibits the binding of t4 and t3 to the integrin. fractalkine (cx3cl1) is a chemokine relevant to inflammatory processes in the cns that are microglia - dependent but also important to normal brain development. expression of the cx3cl1 gene is downregulated by tetrac, suggesting that t4 and t3 may stimulate fractalkine expression. independently of its specific receptor (cx3cr1), fractalkine binds to v3 at a site proximal to the thyroid hormone - tetrac receptor and changes the physical state of the integrin. tetrac also affects expression of the genes for other cns - relevant chemokines, including ccl20, ccl26, cxcl2, cxcl3, and cxcl10. the chemokine products of these genes are important to vascularity of the brain, particularly of the choroid plexus, to inflammatory processes in the cns and, in certain cases, to neuroprotection. thyroid hormones are known to contribute to regulation of each of these cns functions. we propose that actions of thyroid hormone and hormone analogues on chemokine gene expression contribute to regulation of inflammatory processes in brain and of brain blood vessel formation and maintenance.
animals : a total of 60 cattle with or without ocular abnormalities were used in this study (tables 1 table 1. summary of the japanese black cattle casescase no.agesexmajor clinical diagnosis (symptoms)ocular abnormalitiesoptic pathway lesionsno. 4120 yfno significant lesions+absent, ; present, +. d, day (s) ; m, month (s) ; y, year (s). m, male ; c - m, castrated male ; f, female. l, left side ; r, right side. and 2 table 2.summary of the holstein and f1 cattle casesholstein cattleagesexmajor clinical diagnosis (symptoms)ocular abnormalitiesoptic pathway lesionsno. 69 mmbloatabsent, ; present, +. d, day (s) ; m, month (s) ; y, year (s). l, left side ; r, right side ; b, both sides.). the examined animals included 41 jb (20 males, 20 females and 1 castrated male), 13 holstein (hol ; 2 males and 11 females) and 6 crossbreed cattle (japanese black x holstein : f1 ; 3 males and 3 females). the sample collection methods and necropsy procedure were approved by the animal care and use committee of obihiro university of agriculture and veterinary medicine. d, day (s) ; m, month (s) ; y, year (s). m, male ; c - m, castrated male ; f, female. d, day (s) ; m, month (s) ; y, year (s). m, male ; f, female. pathological examination : in addition to the major organs including the liver, spleen, kidneys, heart and lungs, we examined the bilateral retina, optic nerve, optic chiasm, optic tract, lateral geniculate body, optic radiation and cortex of the occipital lobe as much as possible. for the histopathological examinations, brain tissue and other organs, such as the eye and optic nerves, were fixed in 15% neutral buffered formalin and processed by routine procedures for paraffin embedding. five - micrometer - thick paraffin sections were stained with hematoxylin and eosin (he). in addition, sections of the optic nerve, optic chiasm, optic tract, lateral geniculate body, optic radiation and cortex of the occipital lobe were also stained with luxol fast blue - hematoxylin eosin (lfb - he). sections of the abovementioned structures were also subjected to immunostaining using a streptavidin - biotin peroxidase complex method (histofine sab - po kit ; nichirei, tokyo, japan). the following primary antibodies were used : mouse monoclonal anti - neurofilament antibodies (dianova - immunotech, hamburg, germany) and rabbit polyclonal anti - glial fibrillary acidic protein (gfap) antibodies (dako cytomation, kyoto, japan). the reactions were visualized using 33-diaminobenzidine (nichirei), and the sections were counterstained with mayer s hematoxylin. assessment of astrogliosis in the optic pathway using gfap immunostaining : for evaluation of the optic pathway (optic nerve, optic chiasm, optic tract, lateral geniculate body, optic radiation and cortex of the occipital lobe) for astrogliosis, we counted the number of gfap - positive cells in 3 randomly selected areas (using a x40 objective lens) and scored the samples according to the mean number of gfap - positive cells detected : 0 to 0.2 =, 0.3 to 1.0 =, 1.1 to 10.0 = +, 10.1 to 20.0 = + +, 20.1 +++. 15, 16, 25, 39 and 40) that could be examined, we evaluated the intensity of gfap immunoreactivity at the nerve fiber layer, astrocytes and mller cells. for comparison, 2 unaffected jb cattle (nos. measurement of vitamin a and vitamin b12 concentrations : the serum vitamin a (iu / dl) and vitamin b12 (pg / ml) concentrations of the affected and unaffected jb cattle were examined by daiichi kishimoto at their clinical examination center (obihiro, japan). 15, 16, 25, 28, 34, 39, 40 and 41) and 11 unaffected jb cattle (nos. 2, 3, 7, 17, 18, 20, 3133, 36 and 37) were measured, as were the serum vitamin b12 concentrations of 7 affected jb cattle (nos. 15, 16, 25, 28, 39, 40 and 41) and 11 unaffected jb cattle (nos. 2, 3, 7, 17, 18, 20, 3133, 36 and 37). the significance of any difference was determined using the two - sided student s t test. detection of the bvdv gene : we attempted to detect the bvdv gene in order to determine whether bvdv infection is involved in optic pathway degeneration. to detect bvdv, sample rna was extracted from the serum samples collected from the affected jb cattle with optic pathway degeneration (nos. 15, 16, 25, 28, 39 and 40) using a qiaamp viral rna mini kit (qiagen, hilden, germany). each sample was processed using the single - tube rt - pcr method to detect the bvdv gene using a geneamp ez rtth rna pcr kit (applied biosystems, foster city, ca, u.s.a.), according to the method described in a previous report. as a positive control, a 2 years old holstein cow with mucosal disease cases with optic pathway degeneration : among the 60 cattle examined, bilateral degenerative changes in the optic pathway were only observed in 8 jb cattle (nos. 15, 16, 25, 28, 34, 39, 40 and 41). the affected animals ranged in age from 1 month old to 20 years old and included 3 males and 5 females. each of the affected animals was from a different farm. of the 8 jb cattle with optic pathway degeneration, 4 jb cattle (2 cases with keratitis, nos. 15 and 39 ; 1 case with intraocular abscess, no. moreover, 33 of the 41 jb cattle without ocular lesions did not exhibit optic pathway degeneration. of the 13 hol cattle, 4 hol cattle (nos. 2, 5, 9 and 13) had ocular lesions, and 9 hol cattle did not. in addition, optic pathway degeneration was not observed in 6 f1 cattle without ocular lesions. the details of the optic pathway degeneration observed in the 8 jb cattle are as follows. lesions similar to those observed in the optic pathway were not observed in other regions of the central nervous system. however, slight to mild perivascular inflammatory cell cuffing was observed in regions of the central nervous system outside of the optic pathway in 4 of the affected jb cattle (nos. optic nerve, optic chiasm and optic tract : the degenerative changes involved diffuse axonal degeneration and astrogliosis affecting the bilateral optic nerve, optic chiasm and optic tract (fig. 1.(a, b, c, d) histological and immunohistological findings of the optic tract in a japanese black cow (no. 23) without optic pathway degeneration (control). (e, f, g, h) histological and immunohistological findings of the optic tract in a japanese black bull (no. (f) decrease in myelin sheath density, formation of myelin ovoid and macrophage infiltration are observed. (h) anti - gfap immunohistochemistry detected an increased number of gfap - positive astrocytes. bar=25 m.). the extent of astrogliosis was determined as moderate to severe based on immunohistochemical staining of gfap (fig. 1h and table 3table 3.astrogliosis in the optic pathway as evaluated by gfap immunohistochemistrycase no.optic nerveoptic chiasmoptic tractretinalrlrlrno. +, strong gfap immunoreactivity was detected in the stratum opticum and ganglionic cell layer., +, strong gfap immunoreactivity was detected from the stratum opticum to the outer granular layer. to assess the presence or absence of astrogliosis in the optic nerve, optic chiasm and optic tract, we counted the numbers of gfap - positive cells (x40 objective lens) and scored each sample according to the mean number of gfap - positive cells observed : 0 to 0.2 = -, 0.3 to 1.0 =, 1.1 to 10.0 = +, 10.1 to 20.0 = + +, 20.1 + + +. l, left side ; r, right side ; na, not available.). (a, b, c, d) histological and immunohistological findings of the optic tract in a japanese black cow (no. (e, f, g, h) histological and immunohistological findings of the optic tract in a japanese black bull (no. (f) decrease in myelin sheath density, formation of myelin ovoid and macrophage infiltration are observed. bar=25 m. (g) anti - neurofilament immunohistochemistry demonstrates a reduction in axon density. bar=25 m. (h) anti - gfap immunohistochemistry detected an increased number of gfap - positive astrocytes. bar=25 +, strong gfap immunoreactivity was detected in the stratum opticum and ganglionic cell layer., +, strong gfap immunoreactivity was detected from the stratum opticum to the outer granular layer. to assess the presence or absence of astrogliosis in the optic nerve, optic chiasm and optic tract, we counted the numbers of gfap - positive cells (x40 objective lens) and scored each sample according to the mean number of gfap - positive cells observed : 0 to 0.2 = -, 0.3 to 1.0 =, 1.1 to 10.0 = +, 10.1 to 20.0 = + +, 20.1 + + +. - he - stained sections, loss of myelin and myelin ovoid formation were noted in the same areas (fig. in addition, the axon density, which was evaluated using anti - neurofilament immunohistochemistry, was also decreased in these regions (fig. none of the affected animals had histological lesions of fibrosis at the optic nerve, optic chiasm and optic tract. lateral geniculate body, optic radiation and cortex of the occipital lobe : among the 8 jb cattle that exhibited degenerative changes in the optic nerve, optic chiasm and optic tract, slight histological changes were detected in the optic radiation. although gemistocytic astrocytes were not detected in the bilateral optic radiation in the jb cattle with optic pathway degeneration, a mild increase in the number of gfap - positive cells was observed. however, we did not detect any significant changes in the lateral geniculate body and cortex of the occipital lobe in the jb cattle with optic pathway degeneration in the applied methodology. retina : we could examine the retina in 5 of the 8 cases (nos. no significant changes in retinal structure were detected in 3 of the 5 cases in which he staining - based histological evaluations could be carried out (nos. 15, 16 and 25). however, in one of these cases (case no. 16), the retinal structures of the animal s right side had disappeared due to intraocular abscess formation. bilateral plasma cell infiltration around the retinal capillaries was noted in the remaining 2 cases (nos. none of the affected animals exhibited histopathological changes, such as loss of the photoreceptor cell, degeneration of the photoreceptor cell and retinal pigment epithelial cells, retinal atrophy, and retinal dysplasia. furthermore, none of the affected animals had histopathological changes of the optic disk cupping. in addition, immunohistological evaluations detected strong immunoreactivity of gfap in the stratum opticum and ganglionic cell layer in all of the abovementioned 5 jb cattle (fig. 2.(a, c) histological findings of the retina in a japanese black cow without optic pathway degeneration (no. 23) (a) and a japanese black bull with optic pathway degeneration (no. bar=50 m. (b, d) immunohistological evaluation of the retina in a japanese black cow (no. 23) (b) and a japanese black bull (no. 16) (d). strong gfap immunoreactivity is detected in the stratum opticum and ganglionic cell layer in no. moreover, the strong immunoreactivity of gfap was also detected in the inner and outer granular layers in 2 cases (nos. 39 and 40) in which plasma cell infiltration was observed around the retinal capillaries. (a, c) histological findings of the retina in a japanese black cow without optic pathway degeneration (no. 23) (a) and a japanese black bull with optic pathway degeneration (no. bar=50 m. (b, d) immunohistological evaluation of the retina in a japanese black cow (no. strong gfap immunoreactivity is detected in the stratum opticum and ganglionic cell layer in no. bar=50 m. vitamin a and vitamin b12 concentrations of japanese black cattle with optic pathway degeneration : there were no significant differences in the concentrations of vitamin a or b12 between the jb cattle with and without optic pathway degeneration (table 4table 4.vitamin a and vitamin b12 concentrations of japanese black cattlejapanese black cattle with optic pathway lesionsjapanese black cattle without optic pathway lesionsp - value(n=8)(n=11)vitamin a (iu / dl)51.8 59.045.9 44.3nsvitamin b12 (pg / ml)279.2 114.9231.1 188.0nsjapanese black cattle with optic pathway lesions (nos. 15, 16, 25, 28, 34, 39, 40 and 41). japanese black cattle without optic pathway lesions (nos. 2, 3, 7, 17, 18, 20, 31 - 33, 36 and 37). n=7 (nos. 15, 16, 25, 28, 39, 40 and 41). 15, 16, 25, 28, 34, 39, 40 and 41). japanese black cattle without optic pathway lesions (nos. 2, 3, 7, 17, 18, 20, 31 - 33, 36 and 37). detection of the bvdv gene in japanese black cattle with optic pathway degeneration : we attempted to detect the bvdv gene in 6 of the 8 jb cattle (nos. 15, 16, 25, 28, 39 and 40) with optic pathway degeneration. however, the bvdv gene was not detected in the sera of any of these animals. in the present study, we detected bilateral optic pathway degeneration in 8 of 41 japanese black cattle. however, optic pathway degeneration was not observed in any of the examined hol or f1 cattle. it is known that severe intraocular lesions can cause degenerative changes in the optic pathway [11, 12, 17, 25, 27, 28 ]. among the cattle examined in this study, 4 of the 8 jb cattle that exhibited optic pathway degeneration also had ocular lesions, including 2 with keratitis, 1 with intraocular abscess and 1 with keratoconjunctivitis. thus, we considered that the optic pathway degeneration observed in these 4 jb cattle did not represent secondary changes due to such ocular lesions. in the present study, the remaining 4 cattle did not have any ocular problems, and the optic pathway degeneration observed in these cases (2 of 2 could be examined) was also bilateral. these facts may indicate the presence of optic pathway degeneration in jb cattle with little clinical significance. as a central nervous system lesion outside the optic pathway, however, in this case, optic pathway degeneration characterized by severe gliosis was observed bilaterally in the same degree. therefore, we considered that the optic pathway degeneration was not related to the brain abscess. however, the degenerative change characterized by severe gliosis was observed as localized lesions in the optic pathway. thus, we considered that the meningoencephalitis was not related to the optic pathway degeneration. optic pathway degeneration has been caused by numerous causes [1, 11, 22, 28, 37 ]. in various animal species and humans, inflammation of the optic pathway (optic neuritis) ; glaucoma, in which retinal ganglion cells are affected by increased intraocular pressure ; optic nerve trauma ; and compression of the optic nerve have been listed as major causes of optic pathway degeneration [1, 11, 12, 17, 22,23,24, 28, 37 ]. among the jb cattle examined in the present study, none of the animals exhibited histopathological changes that were suggestive of glaucoma, such as cupping of the optic disk. similarly, the 8 jb cattle with optic pathway degeneration either did not display inflammation localized only in the optic pathway or traumatic or compressive lesions. therefore, we considered that the optic pathway degeneration observed in the present study was probably not caused by such conditions. in beef cattle, it has been known that the feeding method of limiting the amount of vitamin a supply improves the meat quality. as a result of excessive limitation, vitamin a deficiency may be observed in herds [2, 33, 34, 37 ]. in vitamin a - deficient cattle, narrowing of the optic canal occurred due to bone hypoplasia and eventually caused compressive optic neuropathy [5, 11, 14, 34, 37 ]. however, this pathological condition was limited to cattle that were 2 years old or less. vitamin a deficiency also causes retinal degeneration in adult animals, and squamous metaplasia of the urinary, respiratory and/or gastrointestinal epithelia has been known to develop in such animals [2, 10, 14, 26, 33 ]. in the present jb cattle affected with optic pathway degeneration, these pathological findings including optic canal stenosis, retinal degeneration and retinal atrophy were not detected. in calves associated with maternal vitamin a deficiency, macroscopical examination of affected eyes revealed congenital ocular abnormalities, such as microphthalmos, ocular dermoids covering the external surfaces of the eyes and aphakia [4, 23 ]. histopathological examination of these affected eyes revealed severe retinal dysplasia [4, 23 ]. these pathological changes were not observed in the present jb cattle affected with optic pathway degeneration. furthermore, the mean serum vitamin a concentration of these animals was not significantly reduced. these results strongly suggest that vitamin a deficiency which has been reported was not involved in the optic pathway degeneration seen in the jb cattle in the present study. in humans and nonhuman primates, it has been reported that vitamin b12 deficiency can cause bilateral optic neuropathy [3, 15, 16, 18, 29, 30 ]. vitamin b12 deficiency also causes lesions including demyelination and spongiosis to develop in the central nervous system white matter of such species [3, 15, 16, 18, 29, 30 ]. however, the jb cattle that were affected by optic pathway degeneration in the present study did not exhibit such changes in their central nervous system nor was their mean serum vitamin b12 concentration significantly reduced. these facts clearly indicate that vitamin b12 deficiency was not related to the optic pathway degeneration observed in the jb cattle. in cattle, it is known that congenital bvdv infection can cause ocular lesions, including retinal dysplasia and hypoplasia of the optic nerve [8, 37 ]. thus, we also examined whether persistent infection of bvdv was involved in the optic pathway degeneration observed in the present study. however, the bvdv gene was not detected in any of the jb cattle with the optic pathway degeneration. moreover, these pathological findings, including retinal dysplasia and hypoplasia of the optic nerve, were not detected in the present jb cattle affected with the optic pathway degeneration. therefore, we considered that there is no close relationship between optic pathway degeneration and persistent infection of bvdv in jb cattle. in sheep and cattle, helichrysum argyrosphaerum can cause visual defects like myelin vacuolation of the optic nerve fiber and retinal degeneration [4, 35 ]. in these animals, spongiosis of the white matter of the brain is also observed [4, 35 ]. the pathological findings observed in the present jb cattle affected with the optic pathway degeneration differed from those of helichrysum argyrosphaerum poisoning. furthermore, all the present jb cattle with optic pathway degeneration were being kept at different farms. also, animals which have kept in the same farms did not show symptoms suggestive of the helichrysum argyrosphaerum poisoning. therefore, we considered that poisoning caused by this plant was not related to the optic pathway degeneration observed in the jb cattle. among the jb cattle affected with the optic pathway degeneration in which it was possible to perform histological evaluations of the retina, strong gfap immunoreactivity was detected in the stratum opticum and ganglion cell layer. moreover, strong gfap immunoreactivity was also seen in the inner and outer granular layers in the 2 jb cattle that exhibited retinal perivascular inflammatory cell cuffing. it has been reported that glial cells distributed in the stratum opticum and ganglion cell layer also stained positive with gfap immunohistochemistry in the normal retina [9, 20 ]. also, it is known that the gfap immunoreactivity is increased in retinal and optic nerve injury [9, 20, 21, 38 ]. in the present study, strong gfap immunoreactivity was observed at the retina in the jb cattle with optic pathway degeneration. therefore, the strong gfap immunoreactivity in the retina may indicate the presence of retinal damage. however, the retinal areas covered by the present examination were restricted to small areas (one to two sections for each grove). therefore, we could not verify the precise time - dependent changes and significance of the retinal damage in the present optic pathway degeneration. in the present study, we could not identify the cause of the optic pathway degeneration in each case. also, the ages of the affected animals varied from 1 month old to 20 years old. however, optic pathway degeneration characterized by severe gliosis has never been reported in cattle without bilateral compressive lesions in the optic pathway or bilateral severe retinal atrophy. therefore, the present study indicates the presence of sporadically detected optic pathway degeneration in jb cattle. for an exact understanding of the clinical significance and the disease pathogenesis,	degeneration of the optic pathway has been reported in various animal species including cattle. we experienced a case of bilateral optic tract degeneration characterized by severe gliosis in a japanese black cattle without any obvious visual defects. to evaluate the significance, pathological nature and pathogenesis of the lesions, we examined the optic pathway in 60 cattle (41 japanese black, 13 holstein and 6 crossbreed) with or without ocular abnormalities. none of these animals had optic canal stenosis. degenerative changes with severe gliosis in the optic pathway, which includes the optic nerve, optic chiasm and optic tract, were only observed in 8 japanese black cattle with or without ocular abnormalities. furthermore, strong immunoreactivity of glial fibrillary acidic protein was observed in the retinal stratum opticum and ganglion cell layer in all 5 cattle in which the optic pathway lesions could be examined. as etiological research, we also examined whether the concentrations of vitamin a and vitamin b12 or bovine viral diarrhea virus (bvdv) infection was associated with optic pathway degeneration. however, our results suggested that the observed optic pathway degeneration was probably not caused by these factors. these facts indicate the presence of optic pathway degeneration characterized by severe gliosis that has never been reported in cattle without bilateral compressive lesions in the optic pathway or bilateral severe retinal atrophy.
hirayama disease, juvenile muscular atrophy of the distal upper limb, is an uncommon cervical myelopathy associated with neck flexion movements24513). this disorder usually develops in the late teens and early twenties with a male preponderance. although the underlying causative mechanism remains unclear, findings in recent studies reveal that the pathogenetic mechanism of this disease is anterior shifting of posterior dura of the lower cervical dural canal during neck flexion, often asymmetric, flattening of the lower cervical cord24578101314). this disorder, mostly nonfamilial, is typically exhibits an insidious onset, slow progression, and often a self - limiting course3451013). we report 2 cases of hirayama disease and describe the pathognomonic findings at flexion magnetic resonance imaging (mri). an 18-year - old man visited gyeongsang national university hospital with progressive weakness and atrophy of right upper arm since 3 months ago. initially, several months ago, his symptom appeared as twinge sensation on posterior thoracic to sacrum when he flexed his neck. more recently, he noted slowly progressive weakness and atrophy of upper arm, especially right shoulder. in visits, we found grossly atrophic changes in right shoulder triceps and biceps muscles. neurologic examination revealed that the deep tendon reflexes were symmetrically normal and sensation to sharp pain, vibration and light touch was intact. no pathologic signs, such as horner sign, hoffman sign, and babinski sign were detected. none of his family had the same symptoms. for further evaluation, we checked image work up. his dynamic views of cervical spine showed segmental instability in c4 - 5, c5 - 6 levels (fig. 1), and atrophic spinal cord and increased signal intensity, c5 - 6 level in mri (fig. no significant pathologic lesions at neutral position mri, but we found prominent posterior epidural space with engorged epidural venous plexus at flexion view (fig. the patient was applied with a neck collar to prevent neck flexion, and after a year of follow - up, presents no signs of disease progression in physical examination. a 15-year - old man with no prior medical history presented to our institution with a 2-year history of progressive weakness and atrophy of right hand. he had severe disability of the right hand (gross motor grade iii, especially 4th-5th finger grade ii). full abduction, adduction of the digits, opposition of the thumbs and palmar grasps were impaired. an electromyography / nerve conduction velocity study revealed active denervation change in the atrophied muscles as monomelic amyotrophy. we found straightening of the cervical cord, localized atrophy at c5-c7 level, t2-weighted hyper intensity due to myelomalacia (fig. 3). a philadelphia neck brace was placed, and the patient was doing well, with no further progression of symptoms, at follow - up study. an 18-year - old man visited gyeongsang national university hospital with progressive weakness and atrophy of right upper arm since 3 months ago. initially, several months ago, his symptom appeared as twinge sensation on posterior thoracic to sacrum when he flexed his neck. more recently, he noted slowly progressive weakness and atrophy of upper arm, especially right shoulder. in visits, we found grossly atrophic changes in right shoulder triceps and biceps muscles. neurologic examination revealed that the deep tendon reflexes were symmetrically normal and sensation to sharp pain, vibration and light touch was intact. no pathologic signs, such as horner sign, hoffman sign, and babinski sign were detected. none of his family had the same symptoms. for further evaluation, we checked image work up. his dynamic views of cervical spine showed segmental instability in c4 - 5, c5 - 6 levels (fig. 1), and atrophic spinal cord and increased signal intensity, c5 - 6 level in mri (fig. no significant pathologic lesions at neutral position mri, but we found prominent posterior epidural space with engorged epidural venous plexus at flexion view (fig. the patient was applied with a neck collar to prevent neck flexion, and after a year of follow - up, presents no signs of disease progression in physical examination. a 15-year - old man with no prior medical history presented to our institution with a 2-year history of progressive weakness and atrophy of right hand. he had severe disability of the right hand (gross motor grade iii, especially 4th-5th finger grade ii). full abduction, adduction of the digits, opposition of the thumbs and palmar grasps were impaired. an electromyography / nerve conduction velocity study revealed active denervation change in the atrophied muscles as monomelic amyotrophy. we found straightening of the cervical cord, localized atrophy at c5-c7 level, t2-weighted hyper intensity due to myelomalacia (fig. a philadelphia neck brace was placed, and the patient was doing well, with no further progression of symptoms, at follow - up study. this disease is different from the known types of motor neuron diseases because of its nonprogressive behavior and pathologic findings of focal ischemic changes in the anterior horn of the lower cervical cord5). so, the disease has also been described in the literature as juvenile muscular atrophy of the distal upper extremity, juvenile muscular atrophy of a unilateral upper extremity, juvenile asymmetric segmental spinal muscular atrophy, benign focal amyotrophy, or monomelic amyotrophy10). however, the pathologic finding of focal ischemia prompted neurologic imaging investigations, which have revealed dynamic changes in the cervical dural sac induced neck flexion23121314). the relatively short and tight dura mater seen in patients with hirayama disease is unable to compensate for the increased length of the vertebral canal during neck flexion, as a result of which the dural canal tightens up during neck flexion, resulting in an anterior shift of the posterior dural wall leading to spinal cord compression23478121314). repeated neck flexion result in multiple episodes of ischemia and chronic trauma to the spinal cord, which eventually leads to myelopathy, as evident by asymmetric lower cervical cord thining235781014). there is another hypothesis that hirayama disease is a form of intrinsic motor neuron disease rather than flexion - induced amyotrophic cervical myelopathy9). although hirayama disease is self - limiting and application of cervical collar for 3 to 4 years has been generally advocated for the treatment because progression of signs and symptoms is usually expected to cease within several years1), early diagnosis is still necessary. because avoidance of neck flexion by using neck collars remains the first - line treatment which is also effective in stopping the progression of disease11). but surgical intervention was considered in patients with particularly severe forms of disease, severe spinal cord atrophy, amyotrophy extending to unusual segment (t1), and clinical signs of pyramidal deficit suggestive of severe myelopathy.. the anterior decompressive approach may be better for patients with anterior effacement and severe cervical kyphosis on mri during neck flexion. also, cervical duroplasty with tenting sutures via laminoplasty led to spinal cord decompression with the preservation of cervical alignment and local physiological motion in young patients with hirayama disease without major complications6). in the first case, he was recommended surgical intervention. because there are a short duration of disease progression and segmental instability at image work - up, the symptoms are likely to worsen during the conservative treatment. but, he refused surgical treatment and was applied a conservative treatment, a philadelphia neck brace. although he presented no signs of disease progression in last follow - up, careful observation and more follow - up periods should be needed considering natural history of disease. in the second case, the patient was prescribed a philadelphia neck brace to prevent neck flexion other than therapeutic intervention, as disease progression was already in the late stage. to maintain the present functions of the patient, a home exercise program that included joint range of motion exercises all patients were doing well, with no further progression of symptoms, follow - up studies. hirayama disease has a tendency to be overlooked or misdiagnosed, since hirayama disease in uncommon and symptoms are similar to other type of motor neuron diseases. therefore, an early detection and a correct diagnosis may lead to therapeutic opportunity to arrest progression or to improve hand disability of young patients. a high awareness of its natural history, careful physical examination and detailed neurophysiological studies all have a role to play in narrowing down the list of differential diagnoses. dynamic flexion mri studies of the cervical spine are key for its accurate diagnosis, as the forward migration of the posterior surface of the dura mater can be demonstrated using this modality.	hirayama disease, juvenile muscular atrophy of the distal upper limb, is a rare disease predominantly affecting the anterior horn cells of the cervical spinal cord in young men. this cervical myelopathy is associated with neck flexion. it should be suspected in young male patients with a chronic history of weakness and atrophy involving the upper extremities followed by clinical stability in few years. herein, we report 2 cases of hirayama disease on emphasis of diagnostic approach and describe the pathognomonic findings at flexion magnetic resonance imaging.
brucellosis is one of the important multi - organ zoonotic diseases with annually more than 500,000 new cases worldwide (1). the epidemiological zone of this infection includes the arabian peninsula, mediterranean basin, indochina, some parts of central asia and south america (2). in the endemic regions, consumption of non - pasteurized dairy such as soft cheese, butter, ice cream is the most usual transmission manner of this infection (3). the main complaints of the infected patients are fever, chills, night sweats, myalgia, anorexia, headache, joint pain and heart attacks (4, 5). the main symptom of brucellosis is fever with unknown origin ; therefore, it can be misdiagnosed with similar pathologies such as all of fevers of unknown origin that may be caused by infectious diseases, malignancies, collagen vascular diseases including tuberculosis, malaria, rheumatic fever and leishmaniasis (6). the forms of the clinical course of brucellosis in humans are acute, sub - acute and chronic (7). to detect patients with the disease, medical history should be taken, and biochemical, hematological and serological test should be performed. isolation, and molecular tests should also be done. among serologic tests, standard agglutination test (sat), the main important purpose of brucellosis treatment was to decline the involvement and signs of the disease. irrespective of the suitable treatment, some patients have relapse of the disease symptoms since these bacteria are intracellular and can survive within macrophages. these confidants may cause chronic and relapse infection (8). the present study aimed to retrospectively analyze the clinical characteristics and complications in the clinical forms of human brucellosis. the current study included patients diagnosed with brucellosis at the infectious diseases clinic affiliated to babol university of medical sciences, babol, iran, within the past two decades. all demographic characteristics such as age, gender, residency, risk factors and clinical manifestations of all admitted patients with brucellosis were exploited from their files and recorded in the questionnaires. the diagnosis of brucellosis was confirmed by representing standard agglutination test (sat) 1.320 and 2-mercaptoethanol (2-me) 1.80 for the patients with clinical signs and symptoms compatible with those of brucellosis. patients were divided into three groups according to their history, symptoms and clinical presentation time : acute (0 - 2 months), sub - acute (3 - 12 months) and chronic (> 1 year) (9). diagnosis of musculoskeletal system complications was determined by the finding of swelling, effusion and limitation of motion in an involved joint and was confirmed by x - ray in the prone position. moreover, spondylitis was diagnosed using magnetic resonance imaging (mri). genitourinary system involvement was diagnosed by finding swelling and tenderness of scrotal skin, testis and epididymis, with confirmation by sonography. endocarditis was diagnosed by elevation of anemia, cardiac murmur, and was confirmed by the detection of vegetations using echocardiography. central nervous system (cns) involvement was made by considering the presence of sat positivity and abnormal findings obtained from cerebrospinal fluid (csf) analyses (> 10 leukocyte / mm, protein > 45 mg / dl, glucose < 2:3 of the blood glucose level). overall, 957 patients with brucellosis were included in the study, 706 (73.8%) cases were acute, 216 (22.6%) sub - acute, and 35 (3.7%) were chronic. among the patients 535 (55.9%) were male and 422 (44.1%) were female with the mean age of 34 16.9 years (rang : 1 - 90). in terms of gender distribution, involvement with acute form of brucellosis regarding other clinical forms, occurs significantly in males 418 (59.2%) more than females 288 (40.8%) (p = 0.003). most of the patients, 421 (44.1%), were 21 - 40 years. while 238 (24.9%) case were < 20 years, 221 (23.1%) were 41 - 60 years and 77 (7.9%) patients were older than 60 years ; therefore, age differences between the groups and all stages of disease were significant (p = 0.001). more patients were from rural, 705 (73.8%), than the urban, 250 (26.2%), areas. significant relationships were found between clinical forms of the disease and frequency of occupational exposure or risk factors in brucellosis (p = 0.01). the most common clinical manifestations were arthralgia, sweating, fever and backache in 679 (71%), 638 (66.7%), 547 (57.2%) and 376 (39.3%) cases, respectively. between clinical features, the most frequent involvement was arthritis with 126 cases (13.2%), gastrointestinal complications 57 (6%), splenomegaly 49 (5.1%), sacroiliitis 46 (4.8%) and spondylitis with 44 (4.6%) cases. complications were observed at all stages of infection but arthritis ; spondylitis were significantly observed in patients with acute disease (p = 0.03, p = 0.001) respectively. focal organs involvement such as epididymo - orchitis and respiratory system were presented in 40 (4.2%) and 22 (2.3%) of the cases respectively. surprisingly, osteomyelitis, bursitis, cellulitis, cns and skin involvements were not observed in chronic form of the disease. brucellosis is a public health problem that can cause severe significant disability and complications (10). according to the annual report of world health organization (who) brucellosis can occur in both genders and any age group (12). in the current study, 535 (55.9%) of the patients were male and 422 (44.1%) were female with a mean age of 34 16.9 years which was similar to some other studies (13). in some studies, the female ratio was reported more than that of the male (14). overall, the prevalence of brucellosis can occur at any age but is common in adults and young people (15). in the current study the most common age group was 21 - 40 years, 421 patients (44.1%), which was similar to other studies (16). of this age group 312 (44.2%) were acute, 95 (44.2%) sub - acute and 14 (40%) were chronic. these results show how age range reflects socio- economic and cultural status of this infection in an endemic area. some studies reported older mean age groups usually working on farms with dairy production ; therefore, brucellosis is more frequently occurs in this age group (17). the current study results were similar to some studies presenting that brucellosis was still endemic in rural areas, but rare in urban areas (18). in the north of iran, the rate of population involved in this infection in rural areas is more than that of the urban areas. in recent years human brucellosis cases a recent study found an overall 247 (25.8%) of patients in high risk occupational groups. many previous studies overlooked some of these important at risk occupational groups (20). the primary transmission route of brucellosis is by the ingestion of unpasteurized dairy products in the endemic countries ; whereas in the developed countries infection occurs mostly due to occupational exposure (21). similar to other studies, the majority of patients, 439 (45.9%) cases, had risk factors such as history of consuming unpasteurized milk or milk products (22). in contrast to the current study results about residency and risk factors, some experts showed that chronic brucellosis were from rural areas because of consumption of unpasteurized dairy products or contact with animals (9). brucellosis seems to affect human immune system and can cause acute, sub - acute and chronic clinical features. in the present study, 73.8%, 22.6% and 3.7% of the patients had the acute, sub - acute and chronic forms of the disease, respectively. but the study by eini. presented that 24% of the patients had acute brucellosis, 70.8% were in sub - acute stages and 5.2% had chronic brucellosis (23). acute form in different genders was compared (male : 59.2% vs. female : 40.8%). in another study by keramat. most patients had brucellosis with the main clinical symptoms of being arthralgia, sweating, fever and backache ; findings were similar to the results obtained by the others working in endemic regions (24). moreover, typically cases with acute brucellosis present signs such as fever, fatigue, chills and sweating (25). in this study, 71% of the patients had arthralgia and in terms of clinical type, 70.7% had the acute form. the results of the current study were similar to those of the studies in other countries ; and arthritis was the most common complications of brucellosis (22). on physical examination, sweating (66.7%) and fever (57.2%) were mostly observed in the acute form the disease. another study identified a significant correlation between fever and the acute form of brucellosis (26). in which the most frequent symptoms were fever (63.2%), sweating (62.7%), arthralgia (59.1%) and back pain (58.5%) (27). brucellosis complications are a major medical problem in countries where the infection is still endemic. the prevalence of focal involvement has been reported to range from 20% to 40% in many studies (28). arthritis involvement was observed in 14.7%, 9.7% and 2.9% of the patients with acute, sub - acute and chronic brucellosis, respectively. showed that sacroiliitis and polyarthritis were more frequent complications in acute cases (29). in a recent study, spondylitis occurred in 2.8%, 10.2% and 5.7% of the patients with acute, sub - acute and chronic disease, respectively. according to bodur. spondylitis was observed in 12 (46%) and 13 (50%) patients with acute and sub - acute brucellosis (30)., people should be taught to avoid consumption unpasteurized dairy products and contact with infected animals. therefore, due to the various clinical presentations and undiagnosed complications, diagnosis of brucellosis is difficult.	backgroundbrucellosis is one of the important multi - organ zoonotic infectious diseases. the forms of the clinical course of brucellosis in humans are acute, sub - acute and chronic.objectivesthe present study aimed to retrospectively analyze the clinical characteristics and complications in the clinical forms of human brucellosis in iran.patients and methodsthe population included 957 patients admitted in the infectious diseases clinic affiliated to babol university of medical sciences, babol, iran, within the past two decades. data for the patients were obtained and documented in questionnaires. patients were divided into three groups according to their history, symptoms and clinical presentation time : acute (0 - 2 months), sub - acute (3 - 12 months), and chronic (> 1 year).resultsmost of the patients (73.8%) were in the acute stages of brucellosis, 22.6% had sub - acute brucellosis and 3.7% had chronic brucellosis. the most frequently observed symptoms were arthralgia (71%), sweating (66.7%), fever (57.2%) and backache (39.3%). the most common complication was arthritis (13.2%) in this study.conclusionsthis infection was observed with a diversity of clinical manifestations. therefore, diagnostic difficulty because of the various clinical presentations and the way to find undiagnosed complications should be investigated in the differential diagnosis of other diseases.
empty sella is characterized by the herniation of the subarachnoid space within the sella, which is often associated with some degree of flattening of the pituitary gland. in primary empty sella (pes), several etiopathogeneic hypotheses have been proposed, including a congenital incomplete formation of the sellar diaphragm and suprasellar factors such as a stable or intermittent increase in intracranial pressure as well as volumetric changes in the pituitary (as observed in pregnancy).[13 ] on the other hand, secondary empty sella (ses) may be caused by pituitary adenomas undergoing spontaneous necrosis (ischemia or hemorrhage). other causes known to cause ses are infective, autoimmune, traumatic, radiotherapy, drugs, and surgery. numerous studies have shown that empty sella syndrome may be associated with pituitary dysfunction contrary to the notion of it being an incidental finding. hence this study was undertaken to evaluate the clinical and hormonal profile in patients with empty sella. patients undergoing a ct / mri at our center, for various reasons but with the finding of empty sella were included in this study. patients having psychiatric disturbances, critical illness, pregnancy, lactation, or on drugs known to affect pituitary function were excluded from the study. apart from routine tests, hormonal evaluation included serum thyroid stimulating hormone, t4, cortisol (8 am), prolactin (prl), total testosterone, follicle stimulating hormone, leuteinizing hormone and fasting igf 1, all done by chemiluminescence. patients with history of pituitary apoplexy, head injury or surgery for pituitary adenoma were considered as ses and patients in whom an etiology was not discernible were considered as pes syndrome. patients undergoing a ct / mri at our center, for various reasons but with the finding of empty sella were included in this study. patients having psychiatric disturbances, critical illness, pregnancy, lactation, or on drugs known to affect pituitary function were excluded from the study. apart from routine tests, hormonal evaluation included serum thyroid stimulating hormone, t4, cortisol (8 am), prolactin (prl), total testosterone, follicle stimulating hormone, leuteinizing hormone and fasting igf 1, all done by chemiluminescence. patients with history of pituitary apoplexy, head injury or surgery for pituitary adenoma were considered as ses and patients in whom an etiology was not discernible were considered as pes syndrome. a total of 34 patients, diagnosed radiologically to have empty sella, were evaluated and of them 24 had pes and 10 had ses. the mean age at diagnosis in subjects with pes was 40.6 9.4 years and that in ses was 37 9.6 years. in both pes and ses female predominance was observed, with the sex ratio being 3:1 and 2.3:1 respectively. etiology in the cases of ses was sheehans syndrome in five, head trauma in three and post pituitary surgery in two. in the female patients with pes 83.3% (15/18) were multiparous and only 11% (2/18) were nulliparous, mean numbers of pregnancies in the multiparous women being 3.26 0.79. the mean bmi in patients with pes was 26.4 4.2 kg / m and that in ses was 24.3 2.1 kg / m [table 1 ]. table showing patients with pes in different bmi groups most common presenting feature in pes was headache, present in 12 patients (50%). the headache was variable in localization, severity and duration, the most common pattern being diffuse with moderate intensity and a duration dating to few years. three patients associated vomiting and evidence of papilledema suggestive of benign intracranial hypertension. however, cerebrospinal fluid (csf) pressures were not measured in these cases. all the patients of ses had panhypopituitarism, the most common presenting symptom being secondary amenorrhea in females and features of hypocortisolism in males. in subjects with pes, 12 out of 24 (50 %) had endocrine dysfunction, though such dysfunction was clinically suspected in only eight of them. the most common endocrine dysfunction noted was hyperprolactinemia, which was seen in 5 (20.8%) patients. four of these patients were females and none of the patients had serum prl levels more than 100 g / l (mean 61.96 13.5 three of these five patients had isolated hyperprolactinemia while the other two had associated anterior pituitary hormonal deficiencies. in the pes subjects, 37.5% had at least one hormonal deficiency, the most common being isolated igf 1 deficiency seen in four patients (12.5%). other hormonal deficiencies that were seen in this group were isolated hypogonadotropic hypogonadism in two patients (8.3%), isolated central hypothyroidism in one patient (4.1%), isolated hypocortisolism in one patient (4.1%), and multiple pituitary hormone deficiency (hypocortisolism and hypogonadism) in one patient (4.1%). all the patients of ses had panhypopituitarism, the most common presenting symptom being secondary amenorrhea in females and features of hypocortisolism in males. in subjects with pes, 12 out of 24 (50 %) had endocrine dysfunction, though such dysfunction was clinically suspected in only eight of them. the most common endocrine dysfunction noted was hyperprolactinemia, which was seen in 5 (20.8%) patients. four of these patients were females and none of the patients had serum prl levels more than 100 g / l (mean 61.96 13.5 three of these five patients had isolated hyperprolactinemia while the other two had associated anterior pituitary hormonal deficiencies. in the pes subjects, 37.5% had at least one hormonal deficiency, the most common being isolated igf 1 deficiency other hormonal deficiencies that were seen in this group were isolated hypogonadotropic hypogonadism in two patients (8.3%), isolated central hypothyroidism in one patient (4.1%), isolated hypocortisolism in one patient (4.1%), and multiple pituitary hormone deficiency (hypocortisolism and hypogonadism) in one patient (4.1%). the reported prevalence of pes in general population is 8 - 35%. in our study pes accounted for most cases of empty sella and it was more commonly noted in females with higher parity, as has been seen in earlier studies. enlargement of the pituitary during pregnancy may lead to weaken of the sellar diaphragm, thus predisposing to herniation of cerebrospinal fluid into the sella. obesity causes obstructive sleep apnea leading to hypercapnia and increased csf pressure predisposing to empty sella. this is thought to be due to pituitary stalk compression as a consequence of the remodeling of the hypothalamo - pituitary region and altered csf dynamics. the most common hormonal deficiency was isolated growth hormone deficiency followed by isolated gonadotropin deficiency, which is compatible with previous studies. somatotrophs are probably a more vulnerable component of the pituitary in pes, hence systematic gh testing and gh substitution considered in pes. the high incidence of endocrine abnormalities in patients with pes, even when asymptomatic, mandates that these patients should routinely be subjected to endocrine evaluation to detect these deficiencies early, and appropriate replacement instituted where necessary, thus ensuring them of a better quality of life.	introduction : empty sella is characterized by the herniation of the subarachnoid space within the sella, which is often associated with some degree of flattening of the pituitary gland. this study was undertaken to evaluate the clinical and hormonal profile in patients with empty sella.aims and objectives : to evaluate the clinical and hormonal profile of the patients with an empty sella.materials and methods : patients undergoing a ct / mri at our center, for various reasons but with the finding of the empty sella were included in this study. a detailed history and clinical examination was done. apart from routine tests, hormonal evaluation included serum thyroid stimulating hormone, t4, cortisol (8 am), prolactin, total testosterone, follicle stimulating hormone, leutinizing hormone, and fasting insulin like growth factor 1 (igf 1) were done.results:a total of 34 patients, diagnosed radiologically to have empty sella, were evaluated and of them 24 had primary empty sella (pes) and 10 had secondary empty sella (ses). in subjects with pes, 12 out of 24 (50%) had endocrine dysfunction. the most common endocrine dysfunction noted was hyperprolactinemia, which was seen in 5 (20.8%) patients and the most common hormonal deficiency was isolated gh deficiency seen in four patients (12.5%).conclusion : the high incidence of endocrine abnormalities in patients with pes mandates that these patients should routinely be subjected to endocrine evaluation to detect these deficiencies early, and appropriate replacement instituted where necessary, thus ensuring them of a better quality of life.

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