In the present study we have investigated the influence of NMDA and GABA receptor activation on the modulation of electrically stimulated dopamine release by adenosine. Application of the adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA), concentration-dependently inhibited dopamine release to a maxiumum of 50%. Perfusion of the glutamate receptor agonist, NMDA, in low magnesium, caused a rapid and concentration-dependent inhibition of dopamine release. Prior perfusion with the adenosine A1 receptor antagonist, DPCPX, significantly reduced the effect of 5 μM and 10 μM NMDA on dopamine release. The GABAA receptor agonist, isoguvacine, had a significant concentration-dependent inhibitory effect on dopamine release which was reversed by prior application of the GABAA receptor antagonist, picrotoxin, but not DPCPX. Finally inhibition of dopamine release by CPA (1μM) was significantly enhanced by prior perfusion with picrotoxin. These data demonstrate an important role for GABA, NMDA and adenosine in the modulation of dopamine release.In the striatum many neurotransmitters including GABA, glutamate, acetylcholine, dopamine, nitric oxide and adenosine interact to regulate synaptic transmission. Dopamine release in the striatum is regulated by a number of pre- and post-synaptic receptors including adenosine. We have recently shown using isolated rat striatal slices, and the technique of fast cyclic voltammetry, that adenosine A We a in vivo -12 and iin vitro -17 inhiberminals  an indir2-like receptors are involved in the presynaptic inhibition of glutamatergic transmission [2-like [1-like receptor family. The presence and the function of D1-like receptors on corticostriatal terminals is still a matter of some debate. A1 receptors located on corticostriatal terminals inhibit transmitter release through the blockade of Ca2+ currents [1 receptors are located on glutamatergic terminals, it has been suggested that the ability of A1 receptors to modulate dopamine release is secondary to their ability to decrease glutamate release, resulting in a decrease in the activation of ionotropic glutamate receptors localized in dopaminergic terminals [1 receptors in this modulation.Dopamine-glutamate reciprocal modulations play a major integrative role in the striatum. Glutamate acts on two types of glutamatergic receptors, ionotropic glutamatergic receptors  and metabotropic glutamatergic receptors that are G-protein coupled. Ionotropic NMDA receptors are found postsynaptically on GABA neurons . These rsmission . The gen [2-like , but notcurrents . As A1 rerminals . In the 1-like receptors are present on striato-nigral medium spiny output GABAergic neurons. Conversely striato-pallidal GABAergic output neurons express D2-like receptors [2 and D5 receptors has also been demonstrated on GABAergic interneurons [1-like receptors have been implicated in this effect [A receptors with picrotoxin caused a decrease in evoked dopamine release [3H-dopamine release from striatal slices but did not alter spontaneous release [1 receptors have been reported to be present on GABAergic terminals, where A1 receptor activation has been shown to be inhibitory [GABA plays a central role in the processing of information in the striatum. There are two neuronal sources of GABA in the striatum, spiny projection neurons and intrinsic GABAergic interneurons. The spiny projection neurons are the prinicipal efferent cells of the striatum. These neurons receive excitatory input from motor cortices and thalamus and dopamine input from midbrain dopamine cells. Dopaminergic input is critical for the control of movement by the basal ganglia; its loss leads to the motor deficits observed in Parkinson's disease. Dopamine Deceptors , 29. Locrneurons  and D1-ls effect -33. Evid release . Other s release . However release -38. In v release . Adenosihibitory . With th2.2.1-1 at 30°C for 1 h before electrical stimulation.Male Wistar rats (50-75g) were killed by decapitation. The brain was quickly removed into ice-cold artifical cerebrospinal fluid (aCSF). Blocks of tissue containing the caudate putamen and nucleus accumbens were prepared. 350 μm thick slices were sectioned using a Campden vibrotome. Brain slices were then transferred to a holding chamber containing aCSF (see below) at room temperature (20-21°C) to equilibrate for 1 h. A single slice was then transferred to a recording chamber and perfused with oxygenated aCSF at 5 ml.min2.2Millar voltammeter [Neurolog modules) was evoked using a square-wave pulse of 10 V amplitude and 0.1 ms duration delivered once every 5 min. A sample and hold output before during and after a stimulus is shown in 2+-dependent [MacLab.Following 1 h equilibration, a bipolar tungsten-stimulating electrode with a tip separation of 200 μm  was placed in the dorsolateral CPu see . A carbotammeter  was appltammeter . Stimulaependent , 45. Wor2.32=0.9952, linear correlation analysis).A typical sample and hold trace (+610mV) obtained during the calibration of a carbon fibre electrode is shown in 2.44, 2; CaCl2, 2; NaH2PO4, 1.25 and D-glucose, 10 mM in H2O. aCSF was bubled with 95% O2/5% CO2. N6-cyclopentyladenosine (CPA), isoguvacine hydrochloride, and metoclopramide monohydrochloride, were obtained from Sigma, UK and dissolved in de-ionized water. 8-cyclopentyl-1,3-dipropylxanthine  and picrotoxin  were dissolved in dimethylsulphoxide (DMSO). Stock solutions of DMSO were made to obtain concentrations of DMSO lower than 0.005% in the superfusing aCSF. Control experiments for solvent were carried out in parallel. This final concentration of DMSO (0.005%) in the perfusing aCSF did not affect single pulse dopamine release, rise time or decay time  was prepared every day according to the following composition in mM: NaCl, 120; KCl, 2.5; MgSOcay time . NMDA an2.5Single pulse evoked dopamine over flow was measured as the peak release in response to electrical stimulation . Rise ti3.3.12+ ions. To unmask the NMDA effect on dopamine transmission, experiments were carried out using low Mg2+ aCSF (0.3 mM Mg2+). The rise time of single pulse evoked dopamine release was not significantly affected by perfusion with low Mg2+ aCSF  and GBR3.22 caused a concentration dependent reduction of single pulse evoked dopamine release  in low Mg3.3A receptors do not tonically modulate dopamine transmission, at least in the in vitro preparation. Addition of the adenosine A1 receptor agonist, CPA at 1 μM (this concentration has previously been shown to cause moderate inhibitory effects on dopamine release [Perfusion with picrotoxin (100 μM) for 60 min did not alter single pulse evoked dopamine release . This may imply that GABA release ) caused  release ) for 60  release ).3.4A receptor agonist, inhibited dopamine release in a concentration dependent manner. Results are expressed as dopamine release versus the log concentration of isoguvacine in Perfusion of cumulative concentrations of isoguvacine , the selective GABA1 receptors in the inhibitory effect of isoguvacine on dopamine release was investigated. Slices were pre-perfused with the A1 receptor antagonist, DPCPX (1 μM) for 30 min before addition of isoguvacine. In the presence of DPCPX, the inhibitory effect of isoguvacine on dopamine release was significantly enhanced . Using one way analysis of variance the inhibitory effect of isoguvacine in the presence of DPCPX on dopamine release was found to be significantly greater than treatment with isoguvacine alone  receptor block and that the inhibitory effect of adenosine (A1) receptor activation on electrically stimulated dopamine release can be enhanced by GABA receptor block. In addition we also demonstrate a role for A1 receptors in the inhibitory effects of isoguvacine (GABA agonist) on electrically stimulated dopamine release.In the present studies we have shown that the inhibitory effects of NMDA on electrically stimulated dopamine release can be modulated by adenosine , proposed that, activation of NMDA receptors causes a large release of dopamine, leading to a depletion of dopamine, and subsequent inhibition of electrically stimulated dopamine release. In our experiments using 10 and 20 μM NMDA, a transient increase in the charging current, followed by a decrease in the charging current was observed, as has been reported by [Electrical stimulation of the dorsal striatum releases many neurotransmitters including dopamine, glutamate, acetylcholine, adenosine and GABA. NMDA receptor activation has been shown to increase adenosine release in striatal slices , which i studies , 24 we horted by , suggest1 receptor antagonist, for 30 min had a small but significant attenuating effect on the effects of 5 μM NMDA on dopamine release. 1 μM DPCPX has been previously used in studies in the nucleus accumbens [Pre-perfusion with 1 μM DPCPX, the adenosine Accumbens . Lower cccumbens . Pre-perccumbens  and the 4.2A receptors, indicating a lack of GABAergic tone in the slice preparation [A receptors located on dopamine terminals one would expect an increase in dopamine release. In vivo microdialysis studies have shown that bicuculline, a GABAA receptor antagonist, increased striatal acetylcholine output, whereas muscimol, a GABAA receptor agonist, decreased striatal acetylcholine output [A receptors located on acetylcholine interneurons [We next investigated a role for GABARs in the adenosine-induced inhibition of dopamine release. Perfusion with 100 μM picrotoxin, at a concentration that has been used previously in brain striatal slices , 34, didparation . In our e output . It may rneurons , enhanciA receptor agonist inhibited dopamine release in a concentration-dependent manner, which was reversed by pre-perfusing the slice with picrotoxin, suggesting that isoguvacine is acting directly via GABAA receptors to inhibit evoked dopamine release. A high concentration of isoguvacine was required to significantly inhibit dopamine release. However in vitro patch clamp studies carried out in brain slices containing the ventral tegemental area used concentrations that ranged from 10-300 μM isoguvacine and they obtained an EC50 value of 62±8 mM isoguvacine [A receptors on dopamine terminals. Also GABA activity has been shown to modulate dopamine transmission, possibly by acting through GABAA receptors on nigrostriatal dopaminergic terminals [1-like receptors found on the GABAergic neurons of the direct pathway and also fast-spiking GABAergic interneurons, depolarize the cell membrane and enhances electrically evoked GABA release [1 receptor antagonist, DPCPX, enhanced the isoguvacine effect on dopamine, an effect which was significant when compared to isoguvacine alone. DPCPX may be acting on A1 receptors found on GABAergic terminals and may produce an overall increase of GABA in the synaptic cleft, thus enhancing the isoguvacine effect. D2-like receptors are located on GABAergic interneurons and striatopalidal spiny projection neurons, were they inhibit GABA release [2 receptor activation in the effects of CPA on dopamine release was ruled out, as metoclopramide has previously been shown not alter the CPA effect; see reference [Perfusion with isoguvacine, the GABAguvacine . Using tguvacine  were unaerminals , 37, 56. release . The att release , 58. A reference .In the in vitro slice preparation, connectivity between different neurons is likely to be at least partially maintained. It is possible that ligands are acting on receptors other than those found on nigrostriatal terminals to modulate neurotransmitter release. These neurotransmitter systems interact with each other and with voltage-dependent conductances to regulate dopaminergic transmission. The dopamine response in our experiements is elicited by a short, discrete stimulus, so although other neurotransmitters are being released, it is unlikely that changes in neurotransmitter levels due to electrical stimultion can act on the evoked dopamine. The dopaminergic terminals contain fast acting ionotropic receptors and it is likely that the concentration of neurotransmitters in the synaptic cleft and extracellular space plays a fundamental role in determining the excitability of the dopaminergic terminal, thus determining the amount of dopamine released due to a single pulse stimulus.In conclusion in our experiments using the technique of fast cyclic voltammetry, we have demonstrated a novel role for adenosine, glutamate and GABA in the modulation of dopamine release in the dorsolateral striatum.


How could phasic variations in dopamine level affect the learning outcome of a spiking neural network? How may neuromodulation affect the network's instantaneous response to simultaneously arriving glutamatergic inputs? How may this depend on the brain regions involved?In our spiking phenomenological model for signal transmission across the synapse and along the dendritic tree, we propose a new approach for the influence of dopamine-like neuromodulators on the ascribed aspects, which unifies diverging views on its role in (reinforcement) learning and  contrast.We call into question the common practice of simulating dopaminergic influence on an STDP rule as a third factor, and instead show how an instantaneous effect of a dopamine-like neuromodulator on postsynaptic activity can also lead to reinforced learning outcomes.As the phasic change of neuromodulator needs to be present during glutamatergic transmission in our model, we do not account for delayed reward as stated in the distal reward problem. Instead, we assume an involvement of hippocampus and cortical working memory for long delays of reward. However, as our transmission-based model does not interfere with the standard two-factor STDP rule, it may be freely combined with existing extensions to STDP if needed.


Most of the available literature on cardiac arrests is dealing with treatment. In this cohort study, we have sought to describe the events surrounding cardiac arrests such as time, cause, initial rhythm, and the final outcome.Retrospective analysis of all consecutive cardiac arrests at Sydvestjysk Sygehus Esbjerg in the years 2002 and 2007. The events were initially identified using a registry in the Department of Anaesthesia on all patient contacts. Using a unique personal identification number on all patients, we retrieved the charts and nurses notes and extracted the relevant data.We included 267 cardiac arrests, 175 out-of-hospital and 92 in-hospital arrests. The cardiac arrests were distributed with 21 % in daytime (08-16), 27 % in the evening (16-00) and 24 % at night (00-08). The causes of cardiac arrests were acute coronary syndrome in 24 %, respiratory insufficiency in 13 %, hypotension in 5 %, arrhythmias in 3 %, other causes in 15 % and unknown in 40 %. The initial rhythm was asystole in 58 %, pulseless electrical activity in 18 %, pulseless ventricular tachycardia/ventricular fibrillation in 15 %, rhythm with perfusion in 2% and unknown in 7 %. In three patients, the treatment was stopped because of a Do Not Attempt Resuscitation order in the chart. In 64 patients (20 %) the treatment was stopped due to return of spontaneous circulation and 30 (47 %) patients survived to discharge.Cardiac arrests at Sydvestjysk Sygehus Esbjerg occur around the clock with an even distribution during the day, evening and night. Most cardiac arrests are caused by acute coronary syndrome and respiratory failure. We most often witnessed asystole as the presenting rhythm. In-hospital arrests had a better chance of having return of spontaneous circulation. Even in patients with return of spontaneous circulation the final outcome is poor. There was no difference in the final outcome in 2002 and 2007.


The IDAT file format is used to store BeadArray data from the myriad of genomewide profiling platforms on offer from Illumina Inc. This proprietary format is output directly from the scanner and stores summary intensities for each probe-type on an array in a compact manner. A lack of open source tools to process IDAT files has hampered their uptake by the research community beyond the standard step of using the vendor’s software to extract the data they contain in a human readable text format. To fill this void, we have developed the illuminaio package that parses IDAT files from any BeadArray platform, including the decryption of files from Illumina’s gene expression arrays. illuminaio provides the first open-source package for this task, and will promote wider uptake of the IDAT format as a standard for sharing Illumina BeadArray data in public databases, in the same way that the CEL file serves as the standard for the Affymetrix platform. The format itself is well documented by the manufacturer, who also provides an open-source software development kit (SDK). As a result, in addition to Affymetrix’s own software suite, a large number of CEL parsing tools exist, including a parser implemented based on the file format documentation:affyio1 and a parser based on the SDK:affxparser2.The DNA microarray field is dominated by the three manufacturers: Affymetrix, Illumina and Agilent. While the basic premise behind their competing products is the same (i.e. the measurement of hybridisation between sample and immobilised probes on arrays via fluorescence), the formats in which these data are presented to end users are quite different, with each manufacturer electing to use their own proprietary format. The most ubiquitous of these is the CEL file, which has been accepted as a standard format for publishing the raw data generated on the Affymetrix platform. A search of the Gene Expression Omnibus , so-called out-of-band probes (methylation 450k)7 and meta information including software versions and scan date .The same is not true of the primary IDAT format from Illumina, with only 1.5% (49 out of 3208) of the submissions in GEO that use Illumina BeadArrays including IDAT files as supplementary material. Given that IDATs are the standard file type generated during BeadArray processing, it seems reasonable to assume that the relative dearth of IDAT files in the public domain is due to the lack of widespread support for the format. The development of alternative parsing tools has proven more challenging for IDATs for a number of reasons. The foremost amongst these is a lack of public documentation, leaving tool developers to determine the file structure themselves. A further hurdle has been the encryption of IDAT files generated from expression chips. These barriers initially left researchers reliant on the output from Illumina’se place . TheGen8 packageilluminaio that can handle IDAT files from any Illumina BeadArray platform, providing a simple unified interface to various low-level data extraction routines.Here we introduce the BioconductorThe IDAT file format varies depending upon the array platform . IDATs gOn the other hand, gene expression IDAT files are produced as encrypted XML files. Once decrypted the majority of the data are found as ten Base64 encoded strings. These ten fields include the ID, mean and standard deviation values as found in genotyping IDATs, as well as median and trimmed-mean intensity values, the mean and standard deviation of local background intensities, and the number of beads both before and after outliers have been excluded.Each array type is also associated with a manifest file (with file extension BPM or BGX) that provides details of probe sequences, intended genomic targets and whether it is a control probe or not, information that is necessary to correctly interpret the data.illuminaio is an R package9. The reading of IDAT files is achieved using thereadIDAT function. This routine is able to determine the type of IDAT file that has been passed and calls the appropriate code to read the file and return the data as a R list object .Decryption of expression IDATs is performed using the open-source DES decryption routine available inilluminaio package also supports the parsing of non-encrypted IDAT files compressed by gzip and the reading of manifest files describing the array design (readBGX andreadBPM).Theilluminaio are nearly identical to those reported usingGenomeStudio. Small discrepancies related to rounding performed byGenomeStudio are observed. The package vignette contains a detailed comparison. The time taken to read an IDAT depends on the platform, with encrypted expression arrays taking around 1 second per file , and methylation and SNP platforms between 1 to 6 seconds depending on the chip density .The summarised intensity values obtained byilluminaio that can read files from any of Illumina’s BeadArray technologies will promote greater use of the IDAT file as a primary data format in the analysis and sharing of results from BeadArray based profiling studies. Theilluminaio package is intended for use by developers to efficiently extract the content of both IDAT and bead-manifest files, thereby expanding the possibilities for conducting reproducible research with these data.The availability of an open-source IDAT reader throughhttp://cancergenome.nih.gov/). IDAT files from Illumina methylation and genotyping arrays are available in large numbers as Tier 1 data from the TCGA website (https://tcga-data.nci.nih.gov/tcga/). Of particular interest is the Illumina 450k methylation array, for which Tricheet al.7 has shown improvements in background correction by using out-of-band probes, information that is only available through IDAT files and not the GenomeStudio output. For this work Tricheet al. usedilluminaio to access the out-of-band probes, which shows the advantage of having access to low-level data.One exception to the dearth of IDAT files noted in the introduction is the The Cancer Genome Atlas  and from10.5281/zenodo.7588. et al. presents an R package to process IDAT files, the file format of Illumina BeadArrays. The packageilluminaio fills an important gap in data processing by enabling the analysis of Illumina data without Illumina software. The gap is illustrated when the authors cite the disparity between the availability of CEL files (Affymetrix file format) and IDAT files in public data repositories. In addition to eliminating the need to use Illumina software,illuminaio extracts additional information from the IDAT files allowing improved quality control analyses and additional data correction algorithms. Figure 1 of the manuscript illustrates the simplified analysis pipeline enabled byilluminaio.This manuscript written by Smith illuminaio. Figure 2 of the manuscript summarises the details of the file format for each of the BeadArray platforms. As such this is not just the development of a new tool, as the authors have provided thorough documentation for a widely used platform. In addition, the vignette accompanyingilluminaio clearly explains the packages methods and how to use them.The authors briefly describe the IDAT file format that was not publically documented before illuminaio, has already led to the development of many new analysis packages that utilise IDAT files and the additional information they provide. In turn these packages are leading to the increased exchange and availability of the raw IDAT files in collaborations and data repositories.This package,I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Dr. Morris,Thank you for your positive review. We are happy that you find our tool useful.Best,Kasper D. Hansen The article describes illuminaio, an R package to process the raw data files produced by the Illumina scanning software. This tool is valuable, because it enables researchers to use a completely open analysis workflow, without having to use a closed source, blackbox, analysis step.I have tried the package on IDAT files from several chiptypes, and only files from the beginning of 2005 scanned with beadscan 2.2 were rejected. Files scanned with beadscan 2.3 (Nov 2005) and onwards were processed properly.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Dr. Oosting,Thank you for your review.  I am especially happy that you have taken the effort to apply illuminaio on output from older versions of beadscan.We have contacted Dr. Oosting offline and have received a number of files from old scans. We have started implementing support for these older files, and initial results are promising. We expect to release a new version of illuminaio shortly, which will read these files.Best,Kasper D. Hansen


EHP received nearly 1,500 papers during 2011, and nearly 600 of those papers were sent out for peer review. Each paper is evaluated by at least 2 reviewers for scientific quality and content. Reviewing manuscripts, a difficult task performed by dedicated volunteers, is vital to the scientific process. A list of EHP reviewers is available on our website (http://ehponline.org/article/info:doi/10.1289/ehp.120-a53). EHP would also like to recognize the top 12 Reviewers of the Year. These individuals reviewed at least 5 papers during the year and received high ratings from the Associate Editors for their timeliness and the quality of their reviews. The EHP 2011 Reviewers of the Year are Adrian Barnett, Dana Barr, David Bellinger, Joe Braun, Ralph Delfino, Howard Frumkin, Kazuhiko Ito, Frederick Lipfert (not pictured), Jie Liu, Matthew Longnecker, C. Arden Pope, and Allan Smith. We at EHP are grateful to these and all other reviewers who assisted us during 2011.(127 KB) PDFEHP published 280 papers in 12 issues during 2011, and the journal is very grateful for the time and effort of the more than 1,000 reviewers who assisted us last year.Click here for additional data file.


Antibody-mediated rejection (AMR)  of cardiac allografts remains difficult to diagnose and treat. Interest in AMR of cardiac allografts has increased over the last decade as it has become apparent that untreated humoral rejection threatens graft and patient survival. An international and multidisciplinary consensus group has formulated guidelines for the diagnosis and treatment of AMR and established that identification of circulating or donor-specific antibodies is not required and that asymptomatic AMR, that is, biopsy-proven AMR without cardiac dysfunction is a real entity with worsened prognosis. Strict criteria for the diagnosis of cardiac AMR have not been firmly established, although the diagnosis relies heavily on tissue pathological findings. Therapy remains largely empirical. We review an unfortunate experience with one of our patients and summarize recommended criteria for the diagnosis of AMR and potential treatment schemes with a focus on current limitations and the need for future research and innovation. Humoral rejection is now clearly established to be a major threat to graft and patient survival after cardiac transplantation. Unfortunately, our diagnosis and treatment of cardiac allograft dysfunction has revolved mainly around understanding the cellular response and our insight into the recognition of antibody-mediated rejection (AMR) and consequently our ability to treat AMR has lagged. Humoral rejection of cardiac allografts differs from cellular rejection by targeting endothelial cells leading to the production of a capillary vasculopathy and by the infiltration of neutrophils and macrophages rather than T cells. Pathological diagnosis involves providing evidence of endothelial injury and antibody and complement deposition . Thus, tA 24-year-old woman with a complex medical history that included idiopathic thrombocytopenic purpura requiring splenectomy and recent postpartum acute respiratory distress syndrome requiring five days of ventilatory support was transferred to our facility from a local hospital at three months postpartum in severe cardiogenic shock. She was found to have nonischemic cardiomyopathy which required emergent biventricular paracorporeal ventricular assist devices . After stabilization and recovery, she was listed as a UNOS status 1A  for heart transplantation without detectable panel reactive antibodies (PRAs). Approximately two months subsequent to VAD placement, she underwent orthotopic heart transplantation with an HLA-compatible cadaveric heart . She had a persistent postoperative coagulopathy requiring transfusion of multiple packed red blood cells, fresh frozen plasma, and platelets and eventual return to the operating room for washout and re-exploration within 24 hours of transplantation. Her retrospective cross-match was negative, and subsequent tests for donor-specific antibodies and anti-HLA circulating antibodies were negative using the Luminex solid-phase assays. She was tested for circulating antibodies multiple times throughout her care and during her final admission.C. difficile diarrhea requiring oral vancomycin. Her medical complications included renal insufficiency, severe bifrontal chronic headaches requiring change in immunosuppression from tacrolimus to cyclosporine, recurrent supraventricular tachyarrhythmias with ablation therapy, antiphospholipid antibody syndrome requiring chronic warfarin anticoagulation and a thrombotic cerebrovascular accident without residual neurologic sequelae.Her transplant course was complicated by multiple complications but most notably for recurrent severe infections mandating reduction in her immunosuppressive regimen. Her infections included severe genital wart outbreaks of the perineum requiring nine separate intraoperative fulgurations, as well as debridement of recurring perivulvar and peri-anal abscesses and fistula, CMV viremia and recurrent The patient's immunosuppressive regimen included tacrolimus, mycophenolate mofetil (MMF), and corticosteroids during the initial transplant period. Her steroids were weaned during the first three months and discontinued completely in the maintenance period because of the recurrent infections described above and her preoperative history of splenectomy. By fourteen months postoperatively, she was converted to cyclosporine. Her headaches resolved after the change in calcineurin inhibitor. For the last twelve months of her life, she was treated with two drug therapy, cyclosporin and MMF. Her cyclosporin dose was intermittently decreased when she presented with clinical infections. Her MMF dose was decreased during infections or episodes of neutropenia from 1000 mg twice a day to 500 mg twice a day but maintained at 1000 mg twice a day during her final 12 months. In her final week, her cyclosporine was reduced from 150 mg twice a day to 75 mg twice a day. Her trough cyclosporine levels were maintained at around 200 ng/mL in her final months but during periods of infection were allowed to drop below 100 ng/mL.Despite the required changes in her immunosuppressive regimen, the patient had only one documented episode of mild cellular rejection diagnosed at biopsy in the first perioperative month. She gradually developed evidence for mild diastolic dysfunction over the first two years following transplantation. Based on left and right heart catheterization results and echocardiographic and biopsy results, there was concern that she was developing mild constrictive or restrictive physiology. Her ejection fraction gradually decreased from 70% in the first few months after transplantation to 50% at 29 months after-transplant. She showed no evidence of significant mitral or tricuspid regurgitation. Heart biopsies taken throughout her course and during the months that she developed cardiac dysfunction showed no evidence for cellular or humoral rejection. The evaluations included staining for C4d (complement product) and CD68 (a macrophage specific marker). She underwent two heart biopsies in her last nine months with the last being 4 months prior to her death. Again, both of these biopsies were negative for cellular and humoral rejection in the setting of worsening ventricular function. Left heart catheterization showed no evidence of coronary vasculopathy.At approximately 29 months after transplantation, the patient was admitted for diarrhea, dehydration, emesis, and acute renal insufficiency. She responded well to rehydration; however, she experienced an episode of hypoxemia leading to complete heart block from which she was easily resuscitated but required intubation. A urine screen was positive for both temazepam and alprazolam, which the patient had been prescribed as an outpatient and the cause of her arrest was attributed to hypoxemia secondary to hypoventilation and sedation. She was subsequently noted to have right upper and left lower extremity swelling, and ultrasound revealed superficial venous thrombosis, despite continued Coumadin anticoagulation. A chest scan to rule out pulmonary embolism was performed and was negative. She became febrile and was placed on broad-spectrum antibiotics. On the day of her death, she developed recurrent third-degree heart block and ventricular tachycardia. Despite aggressive resuscitation efforts, she died that evening. An autopsy was performed, and although the initial gross evaluation failed to show the cause of her graft failure, histologic and pathologic myocardial sections eventually revealed severe but patchy cellular and antibody-mediated rejection.  in two major clinical studies. The first study, entitled CARGO (Cardiac Allograft Rejection and Gene Observation), was published in 2006 and led to the development of a proprietary gene expression test called the Allomap . The hypAllomap is now available on the market but has been received with significant skepticism and the lack of enough clinical validation has made it difficult to get insurance companies to pay for it. To address this, a multidisciplinary and multiinstitutional study group was formed that included 13 US cardiac transplant centers . The stuAntibody-mediated rejection still poses unanswered and complex questions. The role of antibody-mediated rejection in acute and chronic cardiac allograft dysfunction is now firmly established –9. HowevWe may have been aided by the use of two potential approaches. One approach was published in a recent study by Kobashigawa et al.  which shAnother approach that may aid us in the future is the possibility for the potential biopsy diagnosis of antibody-mediated rejection involving the analysis of endothelial cell gene expression profiling , 16, 17.Therapy in AMR should aim toward improving graft dysfunction, prevention of long-term complications such as coronary allograft vasculopathy (CAV), and improving graft survival. As mentioned above, the International Society of Heart and Lung Transplantation 2005 guidelines included allograft dysfunction as required criteria in the definition of AMR . The recPlasmapheresis is very effective at rapid removal of circulating antibodies. Two most frequently used techniques are plasma exchange method and double-filtration plasmapheresis . Both foThe efficacy of antithymocyte globulin (ATG) in treating AMR in heart transplantation is unclear, even though it has been used to treat AMR in renal transplantation . CalcineRituximab, which is known to inhibit activation and maturation of B cells, has been used to treat AMR in heart transplant patients. Rituximab, given as a weekly dose for up to four doses and administered in combination with plasmapheresis, IVIG, and steroids, has been used successfully to treat AMR and to reduce PRA in pre-heart transplant –27. The Complement activation is an important factor by which circulating alloantibodies induce graft injury. Eculizamab is a humanized monoclonal antibody which prevents activation of complement component by binding to C5 and thus preventing generation of C5a and the membrane attack complex. Eculizamab has been approved for treatment of paroxysmal nocturnal hemoglobinuria in the USA. Early experience has shown success in treating AMR in renal transplant patients , 31.Total lymphoid irradiation has been used to treat rejection in heart transplant patients but appears to increase the risk of hematologic malignancies . PhotophNo treatment strategy in cardiac transplantation has consistently proven to be successful in treating AMR. Tacrolimus-based therapy, in combination with MMF and steroids, appears to be effective in preventing AMR. Future trials may provide more insight into the utility of newer therapies with rituximab, bortezomib, and anticomplement antibodies.In its simplest formulation, the clinical efforts of post-heart transplant management involve three basic goals: prevent rejection, minimize infection, and minimize the adverse side effects of the immunosuppressive therapy. Currently, diagnosis is highly dependent upon pathological tissue evidence. While patients can be treated empirically, the hope is that advances in molecular biology including transcriptome and proteomic analyses will help improve our ability to manage patients following heart transplantation. New diagnostic modalities will include refinement of molecular testing to provide diagnostic and prognostic markers for rejection and infection. Newer treatment modalities for antibody-mediated rejection will target antibody production and complement activation. It is likely that a percentage of patients with unclear etiology of graft dysfunction may be suffering from undiagnosed cellular and/or humoral rejection because of the heterogeneous nature of the rejection process.


Kaposi's sarcoma (KS) in HIV negative patients is rare and has to be distinguished from AIDS associated KS. Two groups are at risk to develop non-AIDS related KS: elderly men mainly of Mediterranean origin and persons with iatrogenic immunosuppression.In order to define risk-groups and major clinical features we retrospectively evaluated clinical data of all patients with non-AIDS associated KS presenting to the Department of Dermatology, University Hospital Tuebingen between 1987 and 2009. Data were extracted from the tumor registry of the Comprehensive Cancer Center Tuebingen and from patient records.20 patients with non-AIDS KS have been identified. The average age at KS onset was 66.6 years; the male-to-female-ratio was 3∶1. Most of the patients were immigrants from Mediterranean or Eastern European countries (60%). 15 cases of classic KS versus 5 cases of iatrogenic KS were observed. In 95% of the cases, KS was limited to the skin, without mucosal, lymph node or visceral manifestation. KS lesions were in all cases multiple and mostly bilateral, the most common localization was the skin of the lower extremities. Tumor control was achieved in nearly all cases by the use of local or systemic therapy. No patient died from KS.Unlike KS in AIDS patients, non-AIDS associated KS is a rather localized process which rarely involves lymph nodes or organs. It is mostly seen in elderly males from Mediterranean or Eastern European countries and in most cases responsive on local or systemic therapeutic strategies. Kaposi's sarcoma (KS) was first described in 1872 by the Hungarian dermatologist Moritz Kaposi Four groups are at risk to develop KS: elderly males of Mediterranean and Eastern European lineage; children and adults from central Africa; persons who are iatrogenically immunocomromised; and homosexual men infected with human immunodeficiency virus (HIV) Chang et al. discovered human herpes virus 8, (HHV-8), also known as Kaposi's sarcoma associated herpes virus, which is strongly implicated in the pathogenesis of all types of KS To date it remains unclear whether KS itself is a true malignancy or rather just a reactive proliferation This study focuses on non-AIDS related KS of the skin i.e. cutaneous manifestations of KS in HIV-negative patients presented to the Department of Dermatology, University of Tuebingen Medical Center between 1987 and 2009.This is a retrospective descriptive study of a series of 20 patients admitted consecutively for KS, from 1987 to 2009 in the Department of Dermatology, University of Tuebingen Medical Center. Informed consent was not obtained for all patients as the entire data was analyzed anonymously. This applies according to the German Medical Association's professional code of conduct and has been approved by the Ethics Committee, University of Tuebingen .In order to define risk-groups and major clinical features we retrospectively identified cases registered by the tumor registry of the Comprehensive Cancer Center Tuebingen and evaluated clinical data documented in the hospital records.All cases presented histopathologically approved KS lesions of the skin and negative HIV-1/2 screening by macro enzyme immunoassay. Histological diagnosis was in most cases completed by immunohistochemical tests such as HHV-8 staining, or immunostaining with antibodies against endothelial markers D2-40, CD31 and CD34. Most patients underwent tumor staging by lymph node and abdominal ultrasound as well as chest x-ray.SEM = 7.86 months). In 7 cases time of observation was determined by intercurrent death. Causes of death were other than KS in all cases.Demographic features such as origin, age at onset, gender of the patient, as well as clinical features such as clinical subtype of KS and localization of lesions were evaluated. Furthermore treatment modalities, results and tumor recurrence in the time of observation were recorded. Treatment outcome was classified according to the Response Evaluation Criteria in Solid Tumors (RECIST Guidelines) Statistical package of social sciences 16.0 software  was used to calculate means and standard deviations.SD = 15.36). The youngest patient was 36-year-old and iatrogenically immunosuppressed; the oldest developed his classic KS by the age of 90.20 cases of non-AIDS KS were identified in this study. Mean age at diagnosis of the group was 66.6 year-old (SD = 12.09).Mean age at onset of patients with classic KS was 69.6 years (n = 15) versus 25% female patients (n = 5) was shown with a male/female ratio of 3∶1.A male predominance in KS – 75% male patients (n = 12) of the patients observed in Tuebingen were immigrants from Mediterranean and Eastern European countries  versus eight German patients.60%  of the cases, KS was limited to the skin, without mucosal, lymph node or visceral manifestation.In 95% , no patient presented just one singular lesion. 70% of the patients (n = 14) were affected on both hemispheres of the body and 30% (n = 6) presented unilateral lesions.KS lesions were multiple in all patients (n = 8), six patients were affected on their feet and five patients on all four extremities. All in all 95% of the patients were affected on the skin of the lower extremities.The most frequent manifestation was the skin of the lower leg . The duration of administration of interferon ranged from five weeks to six years. In one case with post-transplant KS immunosuppressive medication was switched from ciclosporin A to sirolimus resulting in a partial response. One patient had a stable disease course and therefore did not need treatment and one patient refused therapy.Treatment outcome was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST-Guidelines). The term “complete response” means clearance of KS lesions on later visits when compared with the first lesions on admission. “Partial response” equals at least 30 percent decrease in the sum of the longest diameter of target lesions whereas “stable disease” involves less than 30 percent decrease. The terms complete and partial response as well as stable disease implicate the absence of new lesions or of progressive lesions. This was achieved in all cases. No patient presented a progressive disease course initially nor died of KS. Four patients developed multiple local recurrences. Two of them had primarily received a local therapy and one interferon-α-2a.Clinical features and treatment outcome of non-AIDS KS are presented in Non-AIDS KS is considered a rare disease, but incidence varies according to individual factors such as origin, sex, age and immune status of the patient.Between 1987 and 2009, only 20 cases of non-AIDS related KS were observed at the Department of Dermatology in Tuebingen, Germany. This is in line with the low incidence of cases appearing in North-Western Europe.No data regarding the incidence of KS in Germany currently exists. Iscovich et al. analyzed reports of classic KS from different cancer registries throughout the world between 1988 and 1992. Low incidence rates were found in Denmark (0.18 per million men aged over 65 per year), intermediate rates in France, and especially high rates in Sardinia and Sicily (13.2 per million men aged over 65 per year) It is worth mentioning that the majority of the non-AIDS KS in Tuebingen (12 out of 20) have been found in first generation immigrants from Mediterranean countries with presumably higher incidence rates e.g. in Turkey and Italy Click here for additional data file.


Finally, we demonstrate acute measurements of dopamine release in the caudate of a human patient during DBS electrode implantation surgery. The data generated are highly amenable for future work investigating the relationship between dopamine levels and important decision variables in human decision-making tasks.Fast-scan cyclic voltammetry at carbon fiber microelectrodes allows rapid (sub-second) measurements of dopamine release in behaving animals. Herein, we report the modification of existing technology and demonstrate the feasibility of making sub-second measurements of dopamine release in the caudate nucleus of a human subject during brain surgery. First, we describe the modification of our electrodes that allow for measurements to be made in a human brain. Next, we demonstrate  The experimental methodology must be safe to the patient, compatible with existing neurosurgical apparatus and the operating-room environment, and capable of sub-second detection of physiological dopamine. To achieve these goals, we fabricated an electrode assembly for use with clinical stereotactic apparatus, based upon an existing biocompatible microsensor that has been validated for in vivo detection of dopamine transmission was highly conserved relative to the conventional glass-insulated carbon-fiber recording electrode referenced to Ag/AgCl  brain tissue for 30 minutes, a period approximating the anticipated implantation time in human brain. The electrochemical response to dopamine was linear with concentration and the sensitivity was not altered by brain implantation . Furthermore, when these variables were normalized to their own standard deviations (z-scored), the linear regression slope was 0.91 indicating a near-unity relationship in these variables. Surprisingly, the slope of the dopamine signal over a period five seconds prior to a market price update correlated with subsequent market returns  demonstrating that it is a significant predictor of future market activity. To test the capacity of this prediction, we constructed a trading model based on the fluctuations in the dopamine signal leading up to the market price updates, which invested 100%  when the dopamine slope was positive and 0%  when the slope was negative. Over the 5 markets played, this trader model earned 202 points (a gain of 175%), more than two times the amount earned by the subject's expressed behavior. These data demonstrate that the information encoded in the dopamine signal of this patient is potentially useful for economic decision making.Next, we tested our electrode in a single human subject using procedures approved by Baylor College of Medicine's Institutional Review Board. This proof-of-principle demonstration was conducted in a consenting patient (MH), who suffered from late-stage Parkinson's disease and was undergoing elective surgery for DBS-electrode implantation into the subthalamic nucleus. On the day of surgery, MH was not medicated with levodopa. Following routine intraoperative electrophysiology recording for functional mapping of the DBS target site, the carbon-fiber microelectrode assembly was placed in MH's right caudate nucleus at x = 19.18 mm, y = 9.13 mm, and z = 18.28 mm from the mid-commissural point. This target was afforded due to the proximity of the target location to the trajectory of the planned DBS electrode insertion path. During insertion, the carbon fiber electrode was retracted in the protective tube and advanced into the brain for final placement. The subject then engaged in a behavioral task We have demonstrated the use of existing electrochemical methods in situ measurements of dopamine release in a human brain; and have generated preliminary evidence that dopamine release can be tracked and investigated in the context of human decision-making behavior. This methodological demonstration opens the door to future investigations utilizing sub-second chemical measurements in the human brain, which should yield important insights into the role of dopamine signaling in human decision-making.In summary, we have demonstrated the first http://www.plosone.org.All of the work presented here was performed under the approval of the University of Washington's Institutional Animal Care and Use Committee (rodent work: 4073-1) and Baylor College of Medicine's Institutional Review Board (human work: H-24018). All procedures conform to the ethical principles outlined in the Declaration of Helsinki. Baylor College of Medicine's Institution Review Board approved this study in humans and written informed consent was obtained from the patient. Detailed methods of all procedures are available in the supplementary online material at Figure S1Dopamine release in the caudate tracks the market price during the sequential investment task. (a) Sequential investment task. For each decision in the game the subject is presented three pieces of information: (1) market trace (red), (2) portfolio value  and (3) the most recent fractional change in portfolio value . The vertical grey bar (middle) is toggled by the subject to determine how much of one's own portfolio to invest in 10% increments . (b) (left) Representative market  and corresponding dopamine measurement . Scale bar: normalized units (σ = 1 standard deviation) along the vertical axis and time (seconds) along the horizontal axis. Linear regression of the dopamine response on to the market price shows a significant correlation: p<0.000001; regression slope = 0.91; and r2 = 0.549, N = 100 decisions. (middle) Scatter plot of the dopamine slope (5 seconds prior to market update) and market returns along with the fitted regression line. . (right) Bar plot comparing the performance of two agents playing the investment task: two agents are compared: patient “MH” and an agent modeled after the dopamine signal (DA). (c) Cyclic voltammograms are measured once every 100 ms in the human striatum. At each data point, a principal components regression based model  is used to derive the dopaminergic contribution to the measured signal. Here Q-values (Qt) provide a measure of the unaccounted residual variance once the PCR-model is applied. Instances where Qt surpasses an experimentally determined Q-threshold (Qσ) are plotted here  below the predicted dopamine (black) and known market value (blue) traces. By this analysis any dopamine data point accompanied by a magenta point is predicted from a cyclic voltammogram that has a significant amount of unaccounted for residual variance given the PCR model used.(TIFF)Click here for additional data file.Methods S1(DOC)Click here for additional data file.


Blood leukocytes have a remarkable capacity to bind to and stop on specific blood vessel areas. Many studies have disclosed a key role of integrin structural changes following the interaction of rolling leukocytes with surface-bound chemoattractants. However, the functional significance of structural data and mechanisms of cell arrest are incompletely understood. Recent experiments revealed the unexpected complexity of several key steps of cell-surface interaction: (i) ligand-receptor binding requires a minimum amount of time to proceed and this is influenced by forces. (ii) Also, molecular interactions at interfaces are not fully accounted for by the interaction properties of soluble molecules. (iii) Cell arrest depends on nanoscale topography and mechanical properties of the cell membrane, and these properties are highly dynamic. Here, we summarize these results and we discuss their relevance to recent functional studies of integrin-receptor association in cells from a patient with type III leukocyte adhesion deficiency. It is concluded that an accurate understanding of all physical events listed in this review is needed to unravel the precise role of the multiple molecules and biochemical pathway involved in arrest triggering. This is due to the rapid formation and dissociation of bonds such as are formed between endothelial E- and P-selectins and lymphocyte-associated ligands comprising P-selectin glycoprotein ligand 1 (PSGL-1), E-selectin ligand 1 (ESL-1), and the hyaluronan receptor CD44  enabling it to interact with intercellular cell adhesion molecule 1  expressed by endothelial cells  surrounded by a membrane under tension (∼3.5 × 10−5 N m−1). This is a minimal model  is called the wall shear rate. Typical wall shear rates of a few hundreds of s−1 are found in postcapillary venules  is different from the dimension of conventional (3D) association rates as defined in Eq. koff(F) is the rupture frequency (in s−1) of a single-bond subjected to a distractive force F. A simple interpretation of this formula can be obtained by viewing bond rupture as the passage of a molecular complex AB from a bound state at zero separation to a free state that is reached by crossing an energy barrier of height W at separation distance d , where kB is Boltzmann’s constant and T is the absolute temperature. Applying a force F will decrease W by the product Fd . Bell estimated at 0.5 nm the order of magnitude of parameter d for an antigen-antibody interaction corresponding to the depth of an antibody binding site, leading to an estimate of ∼8 pN for parameter F°= kBT/d. More detailed discussion may be found in a number of papers following Eyring’s  had to be replaced with a function kon(d) representing the frequency (in s−1) of bond formation between a ligand and a receptor molecules maintained at distance d  could in principle be derived experimentally by simultaneous determination of the binding frequency of receptor-bearing microspheres and ligand-coated surfaces and microsphere-to-surface distance , where F° is the aforementioned force constant. After the rupture of a first bond, the whole force will be applied on the remaining one, thus inducing rapid failure. The dissociation rate of the divalent attachment will thus vary as exp(F/2F°). In absence of force-sharing, the force is expected to divide attachment lifetime by exp(F/F°). As a numerical example, a disrupting force of 40 pN is expected to reduce the lifetime of a single-bond attachment mediated by an integrin of force constant F°∼ 7 pN by 300, while the lifetime of a force-sharing divalent attachment will be reduced by only 17.First, let us consider the effect of an external force F: if the force is equally shared between both bonds, the lifetime of each bond will be divided by exp, nanotopographical membrane rearrangement to allow contact with ligand-bearing surfaces (within tens of seconds), and lateral diffusion of integrin molecules to align along ligands, form clusters, or both. However, other experiments suggest that initial arrest is followed within minutes by an attachment strengthening phase including several actions, as described below.Arrest stabilization may include at least three concomitant processes Figure C That weMonitoring cell-surface detachment under flow revealed significant cell deformation during detachment  type I was identified three decades ago as a syndrome caused by a partial or complete defect of integrin β2 chain expression. Symptoms included sensitivity to bacterial infection, leukocytosis, and absence of pus formation. In 1997, a functional β2-integrin deficiency associated with a bleeding tendency and abnormal platelet spreading was reported in a patient suffering symptoms resembling but somewhat milder than LAD-I . Under these conditions a single molecular bond could induce a detectable arrest  The frequency of durable arrests (2 min or more) should account for a combination of integrin clustering and high affinity state acquisition. (iii) Finally, the molecular contact area between leukocytes and ICAM-1-coated surfaces after 15 min interaction, was used as a reporter of membrane-surface alignment and spreading. Cells were stimulated with Mn++, which stabilizes active conformations without involving intracellular cascades, or chemotactic peptide fMLF, phorbol myristate acetate (PMA), and calcium ionophore ionomycin that are know to activate cells by triggering signaling cascades. The following conclusions were obtained:(i)A clear hierarchy of measured parameters was obtained: spreading could not be normal if durable arrest frequency was normal, and durable arrest could no be stimulated in patients’ cells if total arrests were lacking.(ii)++-induced arrests were normal in patients cells, validating the possibility of detecting individual interactions provided integrin unbending was correctly induced.As expected, Mn(iii)Total arrest frequency was normal, but durable arrest frequency was decreased in fMLF-stimulated neutrophils, confirming the importance of active cell functions to stabilize arrests in contrast with short-term molecular interactions Phorbol myristate acetate (PMA) was reported to induce both mobility changes Surprisingly, T lymphocytes from LAD-III patients displayed abnormal spreading on anti-CD3-coated surfaces. Interestingly, LFA-1 was recently found to be involved in T lymphocyte activation by anti-CD3-coated surfaces, even in absence of ICAM-1 (Li et al., Analyzing the function of kindlin-3-defective cells might give valuable information on both the role of kindlin-3 in integrin function and the interrelation of the physical events described in this review. We used the availability of a LAD-III patient to investigate neutrophils and T lymphocytes: we quantified three steps of the arrest sequence triggered by several integrin activators (Robert et al., The recent expansion of molecular biology techniques allowing high throughput analysis of gene sequence and expression in individuals makes it a prominent goal to define with maximum accuracy the function of newly characterized genes and proteins. This is a formidable task due to the complexity of molecular networks driving cell functions. The current challenge is to find tractable ways of analyzing these networks. A possible way of approaching this goal may consist of determining the functional consequences of a well defined network perturbation (Ku et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HIV-associated KS is the most common reported malignancy in sub-Saharan Africa, and appropriate therapy of KS requires accurate diagnosis. In much of the region, however, KS diagnosis is limited to clinical suspicion without pathologic confirmation. Where pathology is available, specific anti-KSHV stains are rarely available and overall pathologic accuracy for KS has not been evaluated.We introduced skin punch biopsy for KS at HIV/AIDS care clinics in Uganda and Kenya within the East Africa IeDEA Consortium. Clinicians suspecting KS could obtain a biopsy same day without charge. After interpretation by local African pathologists who only had access to routine H&E staining, biopsies were read by dermatopathologists at UCSF who could, at their discretion, recut and restain specimens or stain against latency-associated nuclear antigen (LANA) of KSHV. The interpretation by the U.S. dermatopathologists, who serve a large base of HIV-infected patients, was considered the gold standard.Clinicians at 26 HIV/AIDS clinics in Uganda and Kenya referred 739 patients with clinically suspected KS for skin biopsy. Overall, 77% (95% CI: 74%--80%) of these clinically suspected cases were determined pathologically to be KS after U.S. review; 19% had another diagnosis and 4% were indeterminate. There was no significant difference in the percentage found to be KS between countries (p=0.20) or over time (p=0.11). When KS was not found, a wide variety of other diagnoses, both clinically significant and insignificant, were made by the U.S. dermatopathologists (Table Amongst clinicians at HIV/AIDS clinics in East Africa, clinical suspicion of KS alone is not optimally specific for KS diagnosis. Clinical suspicion alone often either misdiagnoses conditions which are less concerning than KS or misses other serious conditions that require different therapy than KS. Assuming the U.S. interpretation is the gold standard, pathologic determination of KS in East Africa is specific but not optimally sensitive. The findings urge for increased availability of skin punch biopsies for KS diagnosis in Africa and augmentation of pathology services.


The purpose of this study was to validate the Physical Activity Enjoyment Scale (PACES) in a sample of older adults. Participants within two different exercise groups were assessed at two time points, 6 months apart. Group and longitudinal invariance was established for a novel, 8-item version of the PACES. The shortened, psychometrically sound measure provides researchers and practitioners an expedited and reliable instrument for assessing the enjoyment of physical activity. Enjoyment is both a predictor and outcome of physical activity participation -3. ExpecLittle is known about the development of physical activity enjoyment among older adults. Within the interactionist framework of social cognitive theory (SCT) ,9, self-Physical Activity Enjoyment Scale (PACES) , CFI = .848, TLI = .828). Due to the negatively-worded items (11 of 18 items), it is quite possible that method effects may account for significant variance in the model , CFI = .911, TLI = .880), a positive latent method factor (LMF) (χ2 = 243.887(128), p < .001, RMSEA = .077 , CFI = .909, TLI = .891), CU among negatively-worded items (χ2 = 124.368(80), p = .001, RMSEA = .061 , CFI = .965, TLI = .933), a negative LMF (χ2 = 254.763(124), p < .001, RMSEA = .084 , CFI = .897, TLI = .873), and both a positive LMF and a negative LMF (χ2 = 195.493(117), p < .001, RMSEA = .067 , CFI = .938, TLI = .919). No further modifications were deemed appropriate and the full18-item model was dropped from further analyses.Kendzierski and DeCarlo's  originalhe model ,47. Follhe model  procedur2 = 78.686(20), p < .001, RMSEA = .139 , CFI = .875, TLI = .825). Several parameterizations accounting for method effects improved fit indices. Specifically, adding CU among only positively-worded items fit the data well (χ2 = 22.301(14), p = .073, RMSEA = .063 , CFI = .982, TLI = .965) as did the positive LMF approach (χ2 = 26.678(16), p = .045, RMSEA = .066 , CFI = .977, TLI = .960). The addition of CU among only negatively-worded items (χ2 = 33.860(14), p = .002, RMSEA = .097 , CFI = .958, TLI = .916) and the negative LMF approach (χ2 = 42.949(16), p < .001, RMSEA = .106, CFI = .943, TLI = .900) provided a poor fit; however, the model accounting for both a positive LMF and a negative LMF fit the data very well (χ2 = 14.461(11), p = .209, RMSEA = .046 , CFI = .993, TLI = .981). In sum, this 8-item measure appears to have an inherent response bias due to the fact that half of the items are worded in reverse. Given that out-of-range parameter estimates and other inadmissible solutions were obtained with the best-fitting models (a common problem with LMF models , CFI = .988, TLI = .983). Note that the modified scale still contains two negatively-worded items, but alternative models did not significantly improve the model fit. Reliability was calculated with standardized estimates using McDonald's omega , CFI = 1.000, TLI = 1.027). The metric invariance model, with the addition of identical factor loadings across groups, also provided an excellent fit to the data (χ2 = 40.197(47), p = .748, RMSEA = .000 , CFI = 1.000, TLI = 1.021), and the adjusted Satorra-Bentler (S-B) χ2 Δ test , CFI = 1.000, TLI = 1.026), and the S-B χ2 Δ test was not significant. Then, residual variances were constrained across groups and this residual invariance model provided a good model-data fit (χ2 = 63.170(62), p = .435, RMSEA = .016 , CFI = .997, TLI = .997), and the S-B χ2 Δ test was not significant (note that the RMSEA change did exceed the recommended cutoff by .001). We also wanted to test whether any latent mean differences existed in between the two groups. Therefore, we constrained latent factor means to be equal and this model fit the data well (χ2 = 64.390(63), p = .428, RMSEA = .017 , CFI = .996, TLI = .997), and the S-B χ2 Δ test was not significant. Finally, we constrained the latent variances and this model did not significantly change in fit (χ2 = 64.776(64), p = .449, RMSEA = .013 , CFI = .998, TLI = .998). Together, these findings suggest that latent mean scores may be compared across groups and that there were no differences in enjoyment. Factor loadings and residuals for the model are reported in Table To determine if our novel, best-fitting, 8-item model could be generalized across older adults engaging in different modes of exercise, we next tested group invariance across both exercise conditions. The configural model, with each item regressed on a single latent enjoyment factor, fit the data extremely well (χ2 Δ test  was not 2 = 123.512 (95), p = .026, RMSEA = .045 , CFI = .972, TLI = .965). The metric invariance model showed little change in overall fit (χ2 = 134.905(102), p = .016, RMSEA = .046 , CFI = .968, TLI = .962), and the S-B χ2 Δ test was not significant. Scalar invariance also provided similar fit indices (χ2 = 140.879(109), p = .022, RMSEA = .044 , CFI = .969, TLI = .966), and S-B χ2 Δ was not significant. In addition, the residual invariance model also provided an excellent fit to the data (χ2 = 148.924(117), p = .025, RMSEA = .043 , CFI = .969, TLI = .968), one which was not significantly different from the less restrictive, scalar model. Finally, constraining latent means (χ2 = 149.288(118), p = .027, RMSEA = .042 , CFI = .969, TLI = .969) and variances (χ2 = 150.742(119), p = .026, RMSEA = .042 , CFI = .969, TLI = .969) did not change the fit indices . The PACES-8 correlated positively with experienced physical change , psychological/emotional change , and functional change  at times 1 and 2, respectively. The correlation between PASE and PACES-8 was marginal  to small . PACES-8 strongly correlated with the original 18-item version at both time points .Bivariate associations .With our new, internally consistent, group and longitudinally invariant measure of PACES, we examined whether any substantive differences were evident in older adults engaging in two different exercise modalities. Interestingly, the two exercise conditions did not differ in enjoyment. This is not altogether surprising, as aerobic and resistance training have each been associated with favorable self-reported changes in vitality, and less favorable outcomes including reduced pleasure and increased fatigue ,55-57. OAs expected, the revised PACES-8 was positively correlated with five of six SPS subscales at both time points. The "opportunity for nurturance" scale did not correlate with enjoyment. Being responsible for others at home may be a value held by older adults, but caring for others in an exercise group may be less relevant. In fact, research by Carstensen and her colleagues ,60 suggeThere are some limitations of this study worth noting, including the study's demographic characteristics which consisted primarily of White (91%) females (65%). Older adults from more varied backgrounds may have a different concept of enjoyment. For example, the meaning of enjoyment may be different for older adults who may have had to use physical activity as a means of transportation to work every day. Unfortunately, invariance testing across subgroups was not possible due to limitations in sample size. The study is also limited by the fact that change in enjoyment could not be tested between baseline (inactivity) and subsequent measurements, when we might expect change. This is also a general limitation of the PACES, as it was intended for people already involved in physical activity, and it limits the scale's utility. Finally, we cannot say for sure that age differences are contributing to the problems with the original scale's factor structure, and direct comparisons across age are needed. Future studies should examine the psychometric properties of the scale in other populations across other time-frames  and exercise modalities.This study has important practical implications for any conclusions based on the original 18-item PACES. In fact, our findings suggest that studies reporting changes based on the 18-item version may reflect a change in measurement characteristics rather than a change in the construct itself. On a positive note, the revised scale is shorter and may expedite clinical assessment and reduce participant burden. Most importantly, this study has validated a measure of enjoyment in older adult populations that is invariant across exercise modality and time, and corresponds with an established measure of perceived social support.Enjoyment is an important construct in physical activity participation and maintenance. Accurate and valid measurement is critical for research comparison and integrity. Our results show that an 8-item revised scale is invariant across groups and time in a sample of community-dwelling older adults. These results are consistent with previous work and provide practical applications for future clinical and research use.The authors declare that they have no competing interests.All authors read and approved the final manuscript. SM: manuscript writing, study design, data analysis; EO: manuscript revisions, data analysis; SW: manuscript revisions, data analysis and collection; AS: manuscript revisions, data analysis and collection; TW: manuscript revisions, data analysis and collection; EM: manuscript revisions, data analysis and collection; NG: manuscript revisions, data analysis; JF: manuscript revisions, data analysis; AK: design of the parent study; manuscript revisions; EM: study design, manuscript revisions, supervision of research group


STM2209 and STM2208 are contiguous loci annotated as putative protein-coding genes in the chromosome of Salmonella enterica. Lack of homologs in related Enterobacteria and low G+C content suggest that S. enterica may have acquired STM2209-STM2208 by horizontal transfer. STM2209 and STM2208 are co-transcribed from a promoter upstream STM2209, and their products are inner (cytoplasmic) membrane proteins. Analysis with the bacterial adenylate cyclase two-hybrid system suggests that STM2209 and STM2208 may interact. Expression of STM2209-STM2208 is subjected to phase variation in wild type Salmonella enterica serovar Typhimurium. Switching frequencies in LB medium are 6.1×10−5 (OFF→ON) and 3.7×10−2 (ON→OFF) per cell and generation. Lack of DNA adenine methylation locks STM2209-STM2208 in the ON state, and lack of the LysR-type factor OxyR locks STM2209-STM2208 in the OFF state. OxyR-dependent activation of STM2209-STM2208 expression is independent of the oxidation state of OxyR. Salmonella cultures locked in the ON state show alteration of O-antigen length in the lipopolysaccharide, reduced absorption of bacteriophage P22, impaired resistance to serum, and reduced proliferation in macrophages. Phenotypic heterogeneity generated by STM2209-STM2208 phase variation may thus provide defense against phages. In turn, formation of a subpopulation unable to proliferate in macrophages may restrain Salmonella spread in animal organs, potentially contributing to successful infection. The locus, annotated as STM2209-STM2208 in the Salmonella genome database Salmonella enterica but not in Salmonella bongori nor in other enteric bacteria. Aside from the annotation of STM2209-STM2208 as putative protein-coding genes, the literature contains little information on STM2209-STM2208. An exception is a transcriptome analysis in Dam+ and Dam− strains of S. enterica which revealed that STM2209-STM2208 transcripts are more abundant in a Dam− background STM2209-STM2208 as a locus repressed by Dam methylation STM2209-STM2208 is actually a phase variation locus whose expression is locked in the ON state in Dam− mutants. We also show that lack of the LysR-like factor OxyR locks STM2209-STM2208 expression in the OFF state. STM2209 and STM2208 are part of a single transcriptional unit, and encode inner membrane proteins. Constitutive expression of STM2209-STM2208 alters lipopolysaccharide O chain length, reduces phage P22 adsorption, decreases resistance to serum, and impairs proliferation in macrophages. Altogether, our observations suggest that phase variation of STM2209-STM2208 may contribute to phenotypic heterogeneity in Salmonella populations, providing defense against phages and restraining Salmonella spread in animal organs.This study describes a new phase variation locus in Salmonella enterica used in this study  hsdR2 mcrA1 mcrB1) was used for bacterial two-hybrid assays. E. coli CC118 lambda pir [phoA20 thi-1 rspE rpoB argE(Am) recA1 (lambda pir)] and E. coli S17-1 lambda pir [recA pro hsdR RP4-2-Tc::Mu-Km::Tn7 (lambda pir)] were used for directed construction of point mutations. Plasmids constructed for this study are listed in All the strains of is study  belong t− strains on LB agar, 75 µl of a 10 mg/ml catalase solution (Sigma-Aldrich) was spread on the surface of the plates.Luria–Bertani (LB) broth was used as liquid medium. Solid LB broth contained agar at 1.5% final concentration. Green plates lac fusions in the Salmonella chromosome, FRT sites generated by excision of Kmr cassettes were used to integrate either plasmid pCE37 or pCE40 STM2209), and F2208-5 and F2208-3 (for STM2208). Transductional crosses using phage P22 HT 105/1 int201 (The oligonucleotides used in this study are listed in S. enterica stationary phase cultures (OD600 ∼3), using the SV total RNA isolation system (Promega) as described at http://www.ifr.ac.uk/safety/microarrays/protocols.html. The quantity and quality of the extracted RNA were determined using an ND-1000 spectrophotometer (NanoDrop Technologies). To diminish genomic DNA contamination, the preparation was treated with DNase I .RNA was extracted from STM2209, RT2208-5 and RT2208-3 for STM2208) at a final concentration of 0.2 µM each. Real-time cycling conditions were as follows: (i) 95°C for 10 min and (ii) 40 cycles at 95°C for 15 sec, and 60°C for 1 min. A no-template control was included for each primer set. Melting curve analysis verified that each reaction contained a single PCR product. Gene expression levels were normalized to transcripts of ompA, a housekeeping gene that served as an internal control. The Student's t test was used to determine if the differences in retrotranscribed mRNA content observed in different backgrounds were statistically significant.An aliquot of 0.6 µg of DNase I-treated RNA was used for cDNA synthesis using the High-Capacity cDNA Archive kit (Applied Biosystems). Quantitative reverse transcriptase (RT)-PCR reactions were performed in an Applied Biosystems 7500 Fast Real-Time PCR System. Each reaction was carried out in a total volume of 25 µl on a 96-well optical reaction plate (Applied Biosystems) containing 12.5 µl Power SYBR Green PCR Master Mix (Applied Biosystems), 11.5 µl cDNA (1/10 dilution), and two gene-specific primers . Bacterial cells contained in 0.25 ml of culture were collected by centrifugation and suspended in 50 µl of Laemmli sample buffer . Proteins were resolved by Tris-Glycine-PAGE using 12% gels (for STM2208) or Tris-Tricine-PAGE 15% gels (for STM2209). Conditions for protein transfer have been described elsewhere Total protein extracts were prepared from bacterial cultures grown at 37°C in LB medium until stationary phase  and pIZ1759 (constructed with PE5 and PE2208). The end-labeled primer was extended with avian myeloblastosis virus reverse transcriptase (Boehringer Mannheim) under conditions described previously The oligonucleotides PE2209 and PE2208, complementary to internal regions of the genes STM2209-STM2208 was achieved using the QuikChange® Site-Directed Mutagenesis Kit (Stratagene). Briefly, a ∼1.3 Kb fragment of the STM2209-STM2208 region containing the 4 GATC sites was cloned into the pGEMT plasmid using the oligonucleotides Clo2208-5 and Clo-2208-3. Mutations in every GATC were then introduced using oligonucleotides harboring CATC changes (labeled as DIRnuevo and INVnuevo). The resulting plasmid containing the fragment with 4 CATC sites was then digested with XbaI and SacI, cloned onto the suicide plasmid pDMS197 E. coli CC118 lambda pir. Plasmids derived from pMDS197 were transformed into E. coli S17-1 lambda pir. The resulting strains were used as donors in matings with S. enterica 14028 harboring a Cmr cassette in place of the 4 GATC sites (constructed using oligonucleotides delGATC-PS1 and delGATC-PS2) as recipients. Tcr transconjugants were selected on E plates supplemented with tetracycline. Several Tcr transconjugants were grown in nutrient broth (without NaCl) containing 5% sucrose. Individual tetracycline-sensitive segregants were then screened for cloramphenicol sensitivity and examined for the incorporation of the mutant allelle by Sau3AI digestion and DNA sequencing using external oligonucleotides. Construction of the oxyRC199S mutation was achieved in the same way, using the oligonucleotides ClooxyR-5 and ClooxyR-3 for cloning onto pGEMT, and the oligonucleotides oxyRC199SDIR and oxyRC199SINV for site-directed mutagenesis. A strain with a Cmr cassette in place of the oxyR gene (constructed using oligonucleotides deloxyR199-PS1 and deloxyR199-PS2) was used as a recipient in this case.Mutation of the 4 GATC sites contained in the promoter region of et al. S. enterica cells from an LB liquid overnight culture at a multiplicity of infection of 0.1, and the mixture was incubated at 37°C. Samples were taken every 2 min, centrifuged for 1 min at 13,000 rpm in a microcentrifuge, and the supernatant was titrated on the S. enterica wild-type strain ATCC 14028. The sample obtained at time zero (a sample taken immediately after addition of bacteriophages to the cell suspension) was considered to correspond to 100% unadsorbed phages, and the remaining numbers were calculated relative to this number. The Student's t test was used to determine if the differences in adsorption were statistically significant.The efficiency of phage adsorption was calculated as described by Gabig 600 of the washed bacterial suspension was measured to calculate cell concentration. A bacterial mass containing about 3.14×108 cells was pelleted by centrifugation. Treatments applied to the bacterial pellet, electrophoresis of crude bacterial extracts, and silver staining procedures were performed as described by Buendia-Claveria et al.To investigate lipopolysaccharide (LPS) profiles, bacterial cultures were grown overnight in LB. Bacterial cells were harvested and washed three times with 0.9% NaCl. The O.D.STM2208::lac fusion was plated on LB + X-gal and colonies displaying an ON or OFF phenotype after 16 h growth at 37°C were selected, resuspended in PBS and respread on new plates. Phase transition frequencies were calculated using the formula (M/N)/g where M is the number of cells that underwent a phase transition, N the total number of cells, and g the total number of generations that gave rise to the colony.Phase transition rates were estimated as described by Eisenstein 5 in 24-well plates and grown in DMEM medium supplemented with 10% (v/v) fetal bovine serum at 37°C, 5% CO2. Bacteria were added to the wells at macrophage-to-bacteria ratio of 1∶10. Phagocytosis was allowed to proceed for 30 min before washing three times with sterile PBS and adding fresh DMEM media supplemented with 20 µg/ml gentamicin. Macrophages were lysed by using 1% Triton X-100, and the number of viable bacteria that survived the gentamicin treatment was determined by subsequent plating onto LB agar plates. The replication rate was determined as the ratio between the number of bacteria at time 18 h and the number of internalized bacteria after 30 min phagocytosis. The Student's t test was used to determine if the differences in replication rates observed in different backgrounds were statistically significant.The rate of intramacrophage replication after 18 h infection was performed in J774 mouse macrophages as described in S. enterica were serially diluted in PBS + 2 mM MgCl2 to 2×104 cfu/ml. Guinea pig serum was added to 30% final concentration and the mixtures were incubated at 37°C without shaking. Samples were taken at 30 min intervals by plating on nutrient agar, and viable counts were expressed as a percentage of the initial concentration . The Student's t test was used to determine if the differences in survival to serum observed in different backgrounds were statistically significant.Survival in guinea pig serum (Sigma-Aldrich) was analyzed as described in STM2209 and STM2208 genes were PCR amplified using oligonucleotides pKT25-STM2209-PstI-5 and pKT25-STM2209-BamHI-3 (STM2209 cloned into pKT25), pUT18C-STM2209-PstI-5 and pKT25-STM2209-BamHI-3 (STM2209 cloned into pUT18C), pUT18C-STM2208-PstI-5 and pUT18C-STM2208-BamHI-3 (STM2208 cloned into pUT18C), pKT25-STM2208-PstI-5 and pUT18C-STM2208-BamHI-3 (STM2208 cloned into pKT25), and cloned onto plasmids pUT18C and pKT25 using the PstI and BamHI sites. Recombinant plasmids carrying STM2209 and STM2208 were sequenced using oligonucleotides pKT25-seq5 and pKT25-seq3 and co-transformed into an E. coli CyaA− strain (BTH101). Transformants were plated on LB + ampicillin + kanamycin + X-gal medium at 30°C for 30 h. To quantify the interaction between hybrid proteins, bacteria were grown overnight at 30°C in LB + Ap + Km liquid medium supplemented with 0.5 mM isopropyl-ß-D-1-thiogalactopyranoside (IPTG). ß-galactosidase assays were carried out as described above. A level of ß-galactosidase activity at least five fold higher than that measured for vectors alone indicates a positive interaction.The Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system TM2209 and STM2208 are contiguous loci annotated as putative protein-coding genes in the chromosome of Salmonella enterica. The STM2209 and STM2208 ORFs are conserved in Salmonella enterica serovar Typhimurium strains ATCC 14028, SL1344, and LT2 , in the vicinity of the sugar transport gene setBSTM2209 and STM2208 ORFs are also conserved in other Salmonella enterica serovars but not in Salmonella bongori nor in the genera Escherichia and Shigella. Alignment of the predicted amino acid sequences of STM2209 and STM2208 using BLASTP STM2209-STM2208 outside Salmonella enterica. A diagram of the chromosome region in Salmonella enterica and related Enterobacteriaceae is shown in SSTM2209 and STM2208 have low G+C content (37% for STM2209 and 38% for STM2208) compared to both the average of the region (53%) and that of the Salmonella enterica genome (52%) STM2209-STM2208 may have been acquired by horizontal gene transfer. The organization of the STM2209 and STM2208 ORFs suggests that they may be part of a single transcriptional unit: both coding sequences are on the same DNA strand, and are separated by only one nucleotide. Genome sequence analysis in silico predicts that STM2209 may encode a small peptide of 40 amino acids, while STM2208 may be a larger protein product of 221 amino acids. In silico analysis of protein structure using the TMHMM transmembrane prediction software In silico analysis also indicates that STM2208 shares a domain with proteins belonging to the Wzz superfamily of O-antigen chain length regulators. This family includes proteins involved in lipopolysaccharide biosynthesis that confer a modal distribution of chain length on the O-antigen component of lipopolysaccharide Both STM2209 and STM2208 are expressed at higher levels (13 fold for STM2209 and 8 fold for STM2208) in a S. enterica Dam− mutant STM2209-STM2208 transcriptional unit might be repressed by Dam methylation. To confirm Dam-dependent regulation, transcriptional and translational lac fusions were constructed in both loci. Protein variants tagged with the 3xFLAG epitope were also constructed. The effect of Dam methylation on STM2209-STM2208 expression was monitored by ß-galactosidase assays, qRT-PCR, and Western blotting in isogenic Dam+ and Dam− strains. Higher level of ß-galactosidase activity, higher amount of retrotranscribed STM2209-STM2208 mRNA, and increased level of the STM2208-3xFLAG product were detected in the Dam− background . Although the extent of derepression differed slightly depending on the method, expression of STM2209-STM2208 was significantly higher in a Dam− background in all experiments. These results confirm that Dam methylation represses STM2209-STM2208. Furthermore, our ability to detect Dam-dependent regulation with both transcriptional lac fusions and qRT-PCR suggests that Dam-dependent regulation of STM2209-STM2208 may be transcriptional.A previous study showed that ckground . The STMSTM2209-STM2208 transcriptional unit, we mapped the 5′ terminus of the putative STM2209-STM2208 transcript using primer extension (STM2208 and STM2209 are expressed at low levels in Dam+S. entericaSTM2209-STM2208 and part of the coding sequence of STM2209-STM2208 was cloned on the pGEMT multicopy vector to obtain higher amounts of transcript(s). The resulting plasmids (pIZ1758 and pIZ1759) were introduced in the wild type strain, and two primer extension reactions were performed. One reaction was primed by an oligonucleotide complementary to STM2209 (PE2209), and the second reaction by an oligonucleotide complementary to STM2208 (PE2208). Both reactions yielded extension products with identical 3′ ends (STM2209 proposed in the XBASE (http://www.xbase.ac.uk/) and NCBI (http://www.ncbi.nlm.nih.gov/) databases. A DNA sequence reminiscent of a canonical ribosome-binding site is however missing in this putative mRNA organization. For this reason, we propose that translation of STM2209 may be actually initiated at position +25, 10 nucleotides downstream a putative ribosome binding site (5′ TGTGG 3′). This hypothesis is supported by additional evidence: a translational lac fusion constructed upstream +25 proved to be non functional: ß-galactosidase activity was not detected in a Dam− background (data not shown). Altogether, these observations may indicate that STM2209 consists of 34 amino acids and not 40 amino acids as described in the Salmonella enterica ATCC 14028 genome annotation.To characterize the xtension . Because 3′ ends , indicatIn silico analysis of the DNA sequence upstream of the +1 site identified DNA sequences with features similar to those of canonical, sigma70-dependent promoters STM2209 and STM2208 are co-transcribed from this promoter, an hypothesis consistent with the observation that the STM2209 and STM2208 products are co-expressed  was used. This strain forms deep blue colonies on LB supplemented with X-gal. Isolates carrying T-POP3 insertions were selected on LB + tetracycline + kanamycin + X-gal, and white colonies were sought. Only a small white colony was obtained in the screen. Cloning and sequencing of T-POP3 boundaries indicated that T-POP3 had inserted in the oxyR gene. OxyR− mutants are severely impaired to form colonies on LB plates − mutants oxyR loss-of-function abolished STM2209-STM2208 expression in a Dam− background, the oxyR gene was disrupted using lambda Red recombineering. The resulting strain (SV5925), which carries a null oxyR allele, was used in further experiments.A genetic screen based on the T-POP3 transposon STM2209-STM2208 is virtually abolished in an OxyR− background .Analyses of ß-galactosidase activity and Western blotting showed that expression of ckground . As abovckground , high leSTM2209-STM2208 regulation. One was that an H2O2 concentration sufficient to promote the expression of genes belonging to the classical OxyR regulon (genes activated by oxidative damage) showed no effect on the expression of STM2209-STM2208 (data not shown). Furthermore, the spacing between the half sites in the putative OxyR binding sites described below is consistent with specific binding of the reduced form of OxyR STM2209-STM2208 expression, we constructed a point mutant version of the oxyR gene (strain SV6397). The resulting OxyRC199S protein is locked in the reduced form as it cannot form the disulfide bond required for oxidation + and Dam− strains harboring this mutation showed levels of STM2209-STM2208 expression similar to those described above for strains carrying the wild type oxyR allele (data not shown). These observations suggest that oxidation of OxyR is not necessary for STM2209-STM2208 expression.OxyR is a global transcription factor that can sense oxidative stress by direct oxidation. In the oxidized state, OxyR activates the expression of oxidative-stress-responding genes STM2209::lac or STM2208::lac fusions in a wild type background, we detected phenotypic heterogeneity when culture aliquots were spread on plates containing X-gal. These strains formed white colonies that later turned pale blue, indicating low expression of STM2209 and STM2208. However, deep blue colonies were also seen, especially on plates that contained high numbers of colonies . Whenever a blue colony was isolated and streaked out for single colonies, a mixture of white and blue colonies was obtained. This observation suggested that STM2209-STM2208 expression might undergo phase variation, and that switching from OFF to ON might occur at lower frequencies than switching from ON to OFF.In the course of our experiments with strains carrying STM2209-STM2208 locus were calculated using the formula (M/N)/g where M is the number of cells that underwent a phase transition, N the total number of cells, and g the total number of generations that gave rise to the colony STM2208::lac translational fusion was used for these experiments. The frequency of OFF→ON transition was estimated to be 6.1±1.7×10−5 per cell and generation. The ON→OFF switching rate was around 1,000-fold higher: 3.7±0.1×10−2 per cell and generation. Phase variation of STM2209-STM2208 expression was also unaffected by the oxidation state of OxyR (data not shown).Phase variation frequencies in the − and OxyR− mutants . Furthermore, the four base pair substitutions introduced in the STM2209-STM2208 UAS do not destroy known critical regions of the OxyR binding sequence Because of the pleiotropy of STM2209-STM2208 was ≥2 fold higher in the GATC-less mutant (SV7031) than the Dam− mutant (SV5683) (STM2209-STM2208 expression was locked in the ON state in both strains (STM2209-STM2208 constitutive expression avoiding the pleiotropic effects of dam mutations (see below).ß-galactosidase activity assays and Western blotting analysis proved that regulation by Dam methylation was abolished when the GATC sites were eliminated . Express(SV5683) , but STM strains . ConstruS. enterica inner (cytoplasmic) membrane  was performed, and Western analysis of the separated protein preparations was carried out with a commercial anti-FLAG antibody. The results unambiguously showed that STM2209 and STM2208 are located in the membrane .in vivo, we used the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) assay, a procedure that permits the detection of specific interactions between inner membrane proteins STM2209 and STM2208 were independently cloned on plasmids pUT18C and pKT25. Four plasmid constructs were obtained , and their interaction was tested in an E. coli CyaA− mutant (BTH101). Functional complementation was determined by measuring ß-galactosidase activity. High levels of ß-galactosidase activity were obtained with both plasmid pairs, compared with the basal activities of the plasmid vectors or with the activity of one fusion protein only  was used as a recipient. This observation, combined with the fact that STM2209 and STM2208 are components of the cell envelope, raised the possibility that constitutive synthesis of STM2209 and STM2208 might impair adsorption of bacteriophage P22. To test this hypothesis, we compared the kinetics of P22 adsorption to the wild type strain, to a strain that constitutively expresses STM2209-STM2208 (SV6401), and to a strain that harbors a deletion of STM2209-STM2208 (SV6013). Suspensions of P22 bacteriophage and S. enterica were mixed, and samples were taken every two minutes, and centrifuged. The supernatant was subsequently titrated to monitor the presence of unattached phages (S. enterica cells was found to be severely impaired in the strain that constitutively expressed STM2209-STM2208 (SV6401), which proved to be largely refractory to phage P22 attachment. In contrast, P22 adsorption remained unaltered in a strain carrying a STM2209-STM2208 deletion (SV6013) regardless of the presence of the mutated GATCs (SV6976). These experiments suggest that phase variation of STM2209-STM2208 may split clonal populations of S. enterica into two subpopulations, one of which is P22-sensitive while the other is P22-resistant.During strain construction experiments by P22 HT transduction, we obtained reduced numbers of transductants whenever the strain that constitutively expresses d phages . AdsorptSalmonella enterica to initiate infection STM2209-STM2208 (SV6401) showed LPS alterations. Migration of the LPS in polyacrylamide gel is known to be affected by the number and size of repeating oligosaccharide units in long-chain LPS, such that bands in the profile represent progressively larger concatemers of the repeating oligosaccharide units Salmonella LPS shows a bimodal distribution typical of many Enterobacteriaceae, with higher amounts of bands with 16–35 and >100 repeats STM2209-STM2208 deletion (SV6013), in agreement with its ability to permit a normal level of P22 adsorption  growth in various media at different temperatures and different osmolarities; (ii) resistance to acidic pH, cationic peptides, bile, and hydrogen peroxide; (iii) motility; (iv) biofilm formation; (v) and invasion of and proliferation in epithelial and macrophage cell lines. Most trials did not show differences associated either to loss or constitutive expression of STM2209-STM2208. A remarkable exception was that constitutive expression of STM2209-STM2208 impaired intracellular proliferation within macrophages , suggests acquisition by horizontal transfer.bacteria . This asSTM2209 and STM2208 are part of a single transcriptional unit, and are transcribed from a promoter upstream STM2209  while STM2208 encodes a larger hydrophobic protein . Both STM2209 and STM2208 are located in the cytoplasmic membrane ( STM2209 . The STMmembrane . Certainmembrane . The funSTM2209-STM2208 locus is subjected to phase variation (−5vs. 3.7×10−2 per cell and generation). Skewed frequencies of switching are also found in other phase variation loci: for instance, in the E. coli pap operon, the OFF→ON switching frequency is 5.54×10−4 per cell and generation, while the ON→OFF switching frequency is 2.34×10−2 per cell and generation pap, the subpopulation of cells that express STM2209-STM2208 in LB is smaller than the population of cells that do not express STM2209-STM2208. However, the switching frequencies detected under laboratory conditions can be different from those occurring in natural environments pap operon, for instance, the switching frequencies are skewed by environmental inputs involving global regulators like Crp and H-NS and the stress-responsive system CpxRA Expression of the ariation , and theSTM2209-STM2208 in the ON state  and WzzfepE (>100 repeats): a common protein structure consisting of two transmembrane domains and a hydrophilic periplasmic domain, relative richness in proline residues in the second transmembrane segment STM2209-STM2208 leads to short modal length of the O-antigen  may be too low to produce a bacterial subpopulation of relevant size in animal tissues, especially in macrophages which typically host very low numbers of Salmonella cells STM2209-STM2208 might thus occur at different rates in different environments. Formation of a phage-resistant subpopulation, however, can be expected to have selective value regardless of the subpopulation size.Formation of a phage-resistant subpopulation upon STM2209-STM2208 locus is renamed opv (for O-antigen phase variation) so that the STM2209 gene is henceforth known as opvA, and the STM2208 gene as opvB.We propose that the Table S1Oligonucleotides.(DOC)Click here for additional data file.