Pets represent a large reservoir for human infection. Bartonella spp., pets represent a large reservoir for human infection because most Bartonella spp. infecting them are zoonotic. Cats are the main reservoir for Bartonella henselae, B. clarridgeiae, and B. koehlerae. Dogs can be infected with B. vinsonii subsp. berkhoffii, B. henselae, B. clarridgeiae, B. washoensis, B. elizabethae, and B. quintana. The role of dogs as an important reservoir of Bartonella spp. is less clear than for cats because domestic dogs are more likely to be accidental hosts, at least in nontropical regions. Nevertheless, dogs are excellent sentinels for human infections because a similar disease spectrum develops in dogs. Transmission of B. henselae by cat fleas is better understood, although new potential vectors (ticks and biting flies) have been identified. We review current knowledge on the etiologic agents, clinical features, and epidemiologic characteristics of these emerging zoonoses.Among the many mammals infected with  Bartonella spp. are fastidious, hemotropic, gram-negative bacteria that are mainly transmitted by vectors. Among the 11 species or subspecies known or suspected to be pathogenic for humans, 6 have been isolated from pet dogs and cats ; B. clarridgeiae, which has been suspected in a few cases of CSD; and B. koehlerae, recently reported as the cause of human endocarditis  because as it can cause prolonged bacteremia in this species . As with human disease, the clinical spectrum of Bartonella infection in dogs is expanding , and Australia, whereas type Houston-1 is dominant in Asia (Japan and the Philippines) . Prevaliewed in ). Howeveiewed in ). Howeve,,B. henselae in healthy persons has ranged from 3.6% to 6% , B. vinsonii berkhoffii (1 case), B. vinsonii arupensis (1 case), B. koehlerae (1 case), B. washoensis (1 case), and B. alsatica (1 case) (On the basis of serologic testing or polymerase chain reaction (PCR), several recent publications have associated (1 case) .B. henselae infection can cause prolonged fever, prolonged bacteremia, or both , clinical signs were minimal, and gross necropsy results were unremarkable, but histopathologic examination showed peripheral lymph node hyperplasia, splenic follicular hyperplasia, lymphocytic cholangitis/pericholangitis, lymphocytic hepatitis, lymphoplasmacytic myocarditis, and interstitial lymphocytic nephritis  of 724 patients with lymphadenopathy but in none of 100 healthy controls. However, substantial cross-reactivity between B. henselae and B. clarridgeiae detected by indirect fluorescence antibody assay was noted in human sera in a recent study from Japan (iewed in ). FurtheB. koehlerae is a Bartonella sp. that has rarely been isolated from domestic cats worldwide, as it is a very fastidious bacterium  infections have been reported in cats from Illinois and Utah in the United States  appear to be a reservoir of this pathogen, as 35% of the coyotes tested in California were seropositive, and 28% of the coyotes tested within a highly disease-endemic region of California were bacteremic , which suggests a possible reservoir role for this Bartonella sp. in Africa . Two recent studies reported a B. henselae antibody prevalence of 10% in healthy dogs in the eastern United States  in dogs examined at a university teaching hospital in northern California  suggested an association between the seropositive status and lameness, arthritis-related lameness, splenomegaly, and nasal discharge/epistaxis .Bartonella spp. identified in sick dogs are also pathogenic or potentially pathogenic in humans.Unlike the domestic cat, for which clinical manifestations of natural infection is rarely documented, a wide range of clinical and pathologic abnormalities develop in dogs that are very similar to those observed in humans  is essential for the maintenance of the infection within the cat population . ExperiBartonella DNA, including B. henselae, has been detected in Ixodes ricinus ticks collected on humans . B. quiiewed in ). Tick eBartonella species identified in the last 15 years has increased considerably. Pets have been identified as a notable reservoir of Bartonella species  and may play an important role as source for human infection. Furthermore, domestic dogs may represent excellent sentinels for Bartonella infection because of the wide diversity of the Bartonella spp. identified in canines, all of which are human pathogens. A better understanding of the modes of transmission and vectors involved in dog bartonellosis is an urgent priority to implement appropriate parasite control measures for pets.The number of zoonotic


Conjugated 1,3-dienes are important building blocks in organic and polymer chemistry. Enyne metathesis is a powerful catalytic reaction to access such structural domains. Recent advances and developments in ene–yne cross-metathesis (EYCM) leading to various compounds of interest and their intermediates, that can directly be transformed in tandem procedures, are reviewed in this article. In addition, the use of bio-resourced olefinic substrates is presented. The interaction of alkyne triple bonds with metal carbenes or metal vinylidene species was already known before the discovery of the very efficient molybdenum and ruthenium metathesis catalysts. In 1980, the polymerization of alkynes initiated by tungsten carbene was demonstrated by Katz  who propIn this review, we will focus on recent developments in EYCM transformations with ruthenium carbene catalysts –32. ThisThe EYCM is an attractive bimolecular transformation as it is an atom economical reaction which results in formal cleavage of a double bond and introduction of the two generated alkylidene fragments to the triple bond with formation of a conjugated 1,3-diene. However, this metathesis reaction is associated with some difficulties due to possible formation of several regio- and stereoisomers, as well as possible olefin self-metathesis (SM) and even secondary EYCM . Though,I, II) and Hoveyda  catalysts  in terms of reactivity has been shown, whereas a reverse effect was obtained, when the heteroatom was located in homopropargylic position ..86].III, which led to 93% conversion of the starting oleate and selective production of dec-1-ene and methyl undec-9-enoate with trace amounts of octadec-9-ene and dimethyl octadec-9-enedioate resulting from secondary self metathesis of the generated terminal olefins. The cross-metathesis with a terminal propargylic carbonate was then carried out in the same pot with catalyst II and led to the formation of dienes. It was not detected that the hindered diene with the carbonate group and the aliphatic chain connected to the same double bond gave a trisubstituted olefin, which indicated a regioselective cross-coupling reaction. On the other hand, the stereoselectivity of the 1,2-disubstituted double bond was not controlled. Under the conditions for the second catalytic step mentioned above, a two-fold excess of olefin was used in a way that only half of the olefin could be transformed by EYCM.The ethenolysis cleavage was performed in the presence of catalyst The utilization of a symmetrical fatty acid diester arising from self metathesis or selective oxidation of oleate leads to improved experimental conditions as only one terminal olefin was produced upon ethenolysis, and the subsequent EYCM also yielded only one diene . We haveThe EYCM is a very efficient transformation which creates conjugated diene structures with atom economy. It has not been developed as rapidly as the olefin cross-metathesis because it suffers from some reactivity and selectivity issues that have not yet been solved. Regioselectivity of the cross-coupling reaction is usually good as no problem is encountered starting from symmetrical alkynes; and in the case of unsymmetrical alkynes, the products featuring less steric hindrance are formed. The stereoselectivity of the newly formed trisubstituted double bonds is still not controlled. It must be noted that no EYCM starting from acetylene has been described. On the contrary, polymerization of acetylene in the presence of ruthenium carbene complexes has been reported . A chall


EGLN1 and EPAS1, encode major components of the hypoxia-inducible factor transcriptional system, which has a central role in oxygen sensing and coordinating an organism's response to hypoxia, as evidenced by studies in humans and mice. An association between genetic variants within these genes and hemoglobin concentration in Tibetans at high altitude was demonstrated in some of the studies (The Tibetan Plateau is one of the highest regions on Earth. Tibetan highlanders are adapted to life and reproduction in a hypoxic environment and possess a suite of distinctive physiological traits. Recent studies have identified genomic loci that have undergone natural selection in Tibetans. Two of these loci,  studies , 80, 96. As such, it is likely to have acted as an agent of natural selection. Three human populations have lived at high altitude for millennia: Andeans on the Andean Altiplano, Tibetans on the Tibetan Plateau, and Ethiopians on the Semian Plateau. Of these, Tibetans appear to have lived at high altitude the longest. According to archeological and other data, e.g., genetic studies, humans have lived on the Tibetan Plateau, one of the highest regions on Earth with an average elevation of ∼4,000 m, a barometric pressure of <500 mmHg, and an inspired partial pressure of oxygen of ∼80 Torr, for at least 25,000 yr  upon which natural selection has acted. Finally, we consider how the evolutionary paradigm of the Tibetan high-altitude adaptation provides insights and raises questions on the role of the hypoxia-inducible factor (HIF) system in humans who live at sea level.The human response to hypoxia is characterized by systemic changes in cardiovascular, respiratory, and hematopoietic physiology, which affect convective oxygen transport. Central to the Tibetan high-altitude phenotype is the fact that certain components of convective oxygen transport are markedly different in Tibetans compared with other humans living at high altitude. In this review, most comparisons are made between Tibetans and Andeans, as the physiology of these two populations has been extensively studied. In contrast, significantly less is known about the physiology of the Ethiopian highlanders.An immediate rise in ventilation is the most obvious response of human lowlanders exposed to acute high-altitude hypoxia, followed by more complex time-dependent changes with prolonged exposure . TibetanHypoxia constricts rather than dilates the human pulmonary vasculature . RegionaA hallmark of high-altitude hypoxic exposure in humans is the rise in red cell mass accompanied by elevated hemoglobin (Hb) concentration and hematocrit, which is associated with a rise in blood erythropoietin (Epo) levels . Perhaps2 saturation < 83%, a lower prevalence was reported in Tibetans (0.9%) at 4,300 m (Madu) (Chronic mountain sickness (CMS) is a high-altitude disorder linked with the above components of oxygen transport. It is a syndrome of adaptive failure to chronic hypoxia characterized by excessive erythrocytosis with abnormally high Hb and hematocrit levels, hypoventilation, pulmonary hypertension, and eventually right heart failure , 53. It m (Madu)  comparedAnother characteristic of the Tibetan population is a relative protection against the occurrence of intrauterine growth restriction (IUGR), which is associated with low birth weight at high altitude. It is well recognized that reproductive success is more difficult at high than low altitude, especially among nonnatives . Birth wThe phenotypic characteristics of Ethiopian highlanders have not been examined as extensively. Amhara Ethiopians at 3,500 m were reported to have low Hb concentrations comparable with those of lowlanders , resemblThe distinctive oxygen-transport traits found in Tibetans compared with other high-altitude populations, together with reports of intrapopulation heritability, have been interpreted as demonstrating the presence of genetic influences on high-altitude adaptation. Moreover, the association of these traits to disorders such as CMS and IUGR, which have the potential to influence reproduction and survival, suggests that some of these phenotypic characteristics, alone or in combination, may be offering an evolutionary survival benefit to Tibetans.Recent genome-wide analyses in Tibetans provided the first lines of evidence that this population has undergone genetic adaptation to high altitude. These studies used different methodologies to compare the Tibetan genome with genomes of closely related ethnic backgrounds, such as the Han Chinese , 95, 96.EPAS1, EGLN1, and PPARA, with EGLN1 identified by both genomic statistics. In addition, in a cohort of 29 Tibetans, genetic variants in the latter two genes were associated with Hb concentration such that the selected haplotype in each gene was correlated with low Hb concentration. However, people with excessive erythrocytosis or anemia were not excluded from this genotype-phenotype analysis.Simonson et al.  performeP values ranging from 2.81 × 10−7 to 1.49 × 10−9) in terms of their divergence between the two ethnic groups, which clustered within ∼235 kb on chromosome 2, just downstream of EPAS1. These SNPs were found to be in high linkage disequilibrium (LD) with one another: i.e., they were associated with each other in a nonrandom way such that they occurred together more often than expected by chance. This extended haplotype was present at high frequencies in Tibetans (46%) and low frequencies (2%) in Han Chinese. In addition, through candidate gene approaches in two further separate cohorts of Tibetans, they identified ∼30 SNPs in EPAS1 in high LD (consistent with a dominant variant hypothesis) that correlated significantly with Hb concentration. The correlation was such that major allele  homozygotes averaged a Hb concentration of ∼1 g/dl lower than the heterozygotes in each separate cohort. These EPAS1 SNPs were found at higher frequencies in Tibetans than in Han Chinese and were in high LD with the signal from the genome-wide allelic differentiation scan, thus providing evidence for selection on EPAS1 associated with low Hb concentration in Tibetan highlanders.Beall et al.  used a gEPAS1 as the strongest candidate gene for natural selection. In addition, the most differentiated  EPAS1 variant was correlated with erythrocyte count within a larger Tibetan cohort. This was an intronic EPAS1 SNP, which was captured by this exome-targeted approach and was found at 87% frequency in Tibetans compared with 9% in Han Chinese. Interestingly, no coding genetic variants were identified to be highly differentiated between the populations. This leads to suggest that adaptation to high altitude has not proceeded by way of selection on coding variants that might be expected to alter protein structure and function. Based on their analyses, they estimated a Tibetan-Han divergence time of 2,750 yr, which is far more recent than previously estimated based on archeological data or other analyses . They went on to identify positive selective sweeps and dominant haplotypes in EPAS1 and EGLN1 genes in Tibetans and also showed a correlation of the prevalence of the dominant haplotypes for these genes with altitude of residence.Xu et al.  comparedEPAS1 as one of the genes that underwent a selective sweep in Tibetans. They also sequenced the full length of the EPAS1 gene in these individuals and showed marked allelic differentiation at this locus between Tibetans and Han Chinese. They constructed haplotypes, and further analysis allowed an estimation of a divergence time for Tibetans that was sixfold greater than that of Yi et al.  and EGLN1 [which codes for prolyl hydroxylase domain (PHD) 2], both key components of the HIF pathway, thus implicating these genes in the genetic adaptation of Tibetans to high altitude.Additional genes that may have undergone selection in Tibetans with rather weaker signals of selection do exist and have been reported in the various studies , 80, 96.HIFs are a family of transcription factors that coordinate oxygen sensing and intracellular responses to hypoxia through regulation of expression of hundreds of genes belonging to biological pathways such as energy metabolism, angiogenesis, erythropoiesis, iron homeostasis, apoptosis, and others .EPAS1 encodes HIF-2α (EGLN1 gene), and PHD3, in an oxygen-dependent manner (EPAS1 (HIF-2α) and EGLN1 (PHD2), in systemic erythropoiesis, but also in other aspects of integrative physiology related to oxygen homeostasis, comes from studying patients with genetic abnormalities in the HIF system and from genetic mouse models. The most extensively studied patients are those with Chuvash polycythemia, who are homozygous for a hypomorphic allele of the VHL tumor suppressor, which impairs HIF-1α and HIF-2α degradation  upon which natural selection may have acted.EPAS1 and EGLN1 as loci that have undergone recent positive selection in Tibetans .N. Petousi was funded by a No conflicts of interest, financial or otherwise, are declared by the author(s).Author contributions: N.P. drafted manuscript; N.P. and P.A.R. edited and revised manuscript; N.P. and P.A.R. approved final version of manuscript.


Attention Deficit Hyperactivity Disorder (ADHD) is a neurodevelopmental disorder, but diagnosed by subjective clinical and rating measures. The study’s aim was to apply Gaussian process classification (GPC) to grey matter (GM) volumetric data, to assess whether individual ADHD adolescents can be accurately differentiated from healthy controls based on objective, brain structure measures and whether this is disorder-specific relative to autism spectrum disorder (ASD).Twenty-nine adolescent ADHD boys and 29 age-matched healthy and 19 boys with ASD were scanned. GPC was applied to make disorder-specific predictions of ADHD diagnostic status based on individual brain structure patterns. In addition, voxel-based morphometry (VBM) analysis tested for traditional univariate group level differences in GM.The pattern of GM correctly classified 75.9% of patients and 82.8% of controls, achieving an overall classification accuracy of 79.3%. Furthermore, classification was disorder-specific relative to ASD. The discriminating GM patterns showed higher classification weights for ADHD in earlier developing ventrolateral/premotor fronto-temporo-limbic and stronger classification weights for healthy controls in later developing dorsolateral fronto-striato-parieto-cerebellar networks. Several regions were also decreased in GM in ADHD relative to healthy controls in the univariate VBM analysis, suggesting they are GM deficit areas.The study provides evidence that pattern recognition analysis can provide significant individual diagnostic classification of ADHD patients and healthy controls based on distributed GM patterns with 79.3% accuracy and that this is disorder-specific relative to ASD. Findings are a promising first step towards finding an objective differential diagnostic tool based on brain imaging measures to aid with the subjective clinical diagnosis of ADHD. Attention Deficit Hyperactivity Disorder (ADHD) is the most commonly diagnosed childhood disorder, defined by age-inappropriate problems with inattention, impulsivity and hyperactivity Despite these neurobiological underpinnings, accurate diagnosis for ADHD is a challenge and based solely on subjective clinical and rating measures, which are often unreliable with diagnostic variability between clinicians, cultures and countries patterns) and are ideally suited to make predictions for individual subjects based on brain imaging patterns, as opposed to group-level inferences. These methods can provide sensitive and specific diagnostic indicators for individual patients with other psychiatric disorders such as autism, depression and schizophrenia Recent multivariate pattern classification or regression analysis (MVPA) methods for imaging data, however, take into account interactions between regions (i.e. brain structure/function http://fcon_1000.projects.nitrc.org/indi/adhd200/). The published studies applied a range of classification approaches including random forests, gradient boosting, multi-kernel learning and support vector machines However, to date, few imaging studies have used multivariate analysis techniques to classify ADHD patients. An early study employing discriminative features derived from resting state fMRI reported promising accuracy of 85%, but the extremely small sample (9 ADHD patients) makes the generalizability of this result uncertain The aim of the present study was to address these challenges in three ways: First, to test the hypothesis that GPCs applied to grey matter (GM) images from ADHD adolescents and healthy controls acquired in the same scanner and protocol can identify distributed structural neuroimaging patterns that will provide sensitive and specific diagnostic predictors of ADHD. Second, to demonstrate that these predictors are disorder-specific to ADHD when compared to another childhood disorder, of ASD. Third, to provide patterns of predictive weights to characterize the discriminating brain regions underlying the predictions. Fourth, to carry out univariate voxel-based morphometry (VBM) case-control comparisons to investigate whether regions that accurately classify ADHD patients or controls overlap with regions that can be identified in conventional group comparisons and to replicate previous structural imaging deficit findings in frontal, temporo-parietal, striatal and cerebellar regions in ADHD.Twenty-nine, mostly medication-naïve, right-handed adolescent boys with a clinical diagnosis of inattentive/hyperactive-impulsive combined ADHD between 10–18 years were recruited from clinics . DiagnosNineteen right-handed age-matched medication-naïve adolescent boys with ASD were recruited through clinics. ASD diagnosis was made using ICD-10 research diagnostic criteria Participants were excluded if they had comorbid psychiatric disorders including learning disability, reading, speech or language disorder as assessed with the Maudsley diagnostic interview, neurological abnormalities, epilepsy, drugs or substance abuse and IQ <70 on the Wechsler Abbreviated Scale of Intelligence (WASI) Images were acquired using a 3 T GE Signa HDx system  at the Centre for Neuroimaging Sciences, Institute of Psychiatry, King’s College London, UK. The body coil was used for RF transmission and an eight channel head coil for RF reception. High resolution structural 3D T1-weighted SPGR images were acquired. Full brain and skull coverage was required for each subject and detailed quality control carried out on all MR images according to previously published quality control criteria http://www.fil.ion.ucl.ac.uk/spm). The T1-weighted volumetric images were preprocessed using the VBM protocol with modulation The images were first visually inspected for artifacts and structural abnormalities. Next, VBM We used a binary Gaussian Process Classifier (GPC) http://www.mlnl.cs.ucl.ac.uk/pronto) (for details see We used a linear binary GPC to discriminate between ADHD and control subjects as implemented in the Pattern Recognition for Neuroimaging Toolbox (PRoNTo) software . We computed the sensitivity and specificity as the proportion of ADHD patients or controls respectively having the correct label, averaged across all test splits. Finally we computed the  accuracy as the mean of sensitivity and specificity, which quantifies the overall categorical classification performance of the classifier in a way that accommodates potential class imbalance in the data.We also employed an additional metric for the binary classifiers, known as “target information”. This measure quantifies the information gain obtained by the classifier and is measured in bits. A simple baseline classifier that always makes probabilistic predictions based on the proportion of samples from each class in the training set will obtain a target information of zero and a classifier that obtains perfect accuracy and perfect confidence will obtain a target information of one. See For the multi-class classifier, we primarily assessed performance using the obvious generalizations of sensitivity, positive predictive value and balanced accuracy, because the other metrics do not generalize naturally to the multi-class setting.p-value to determine if the balanced accuracy exceeded chance levels (50%). This is preferred to a parametric test, as it does not require distributional assumptions. To achieve this, we permuted class labels 1000 times, each time randomly assigning patient and control labels to each image and repeating the entire cross-validation procedure. We then counted the number of times the permuted test accuracy was higher than the one obtained for the true labels. This number was then divided by 1000 to give a p-value for the classification.Permutation testing was used to derive a We also correlated the GPC predictive probabilities with age, IQ, SDQ hyperactivity-impulsive/inattention subscale and the CPRS ADHD T-scores.To establish the degree of clinical specificity of the classification algorithm to ADHD, we wanted to assess whether the clinical specificity of the classifier remained high, when we added another disorder-group to the control group, i.e. we aimed to discriminate ADHD from “non-ADHD”, including another disorder group.For this purpose, we included another sample of 19 patients with ASD and trained a linear binary GPC to discriminate ADHD from a combined group of ASD and healthy controls (i.e. “not ADHD”). In this case, we employed a LOO-CV approach where we excluded a single subject per cross-validation fold, and assessed classifier performance by the same metrics noted above. Note, however, that since this dataset is unbalanced, we employed a categorical decision threshold defined by the frequency of classes in the training set.To further establish the degree of clinical specificity of the classification algorithm to ADHD relative to ASD specifically, we trained another linear binary GPC to discriminate ADHD from the group of 19 ASD patients. As above, we employed a LOO-CV approach where we excluded a single subject per cross-validation fold, and assessed classifier performance by the same metrics noted above. Since this dataset is unbalanced, we employed a categorical decision threshold defined by the frequency of classes in the training set.In addition, we also performed a 3-class GPC classification which aims to simultaneously discriminate each group  from one another. As above, we employed a LOO-CV approach where we excluded a single subject per cross-validation fold, and assessed classifier performance primarily in terms of balanced classification accuracy. In contrast to the binary classifiers described above, we use the Laplace approximation to the posterior predictive distribution  corrected at the cluster level. Cluster sizes were adjusted for smoothness non-uniformity by means of the VBM5 toolbox p<0.001 uncorrected for multiple comparisons to identify regions that may have contributed to the classification but did not survive multiple comparison correction.Group differences were evaluated for GM, WM, CSF volumes and total intracranial volume (TIV)  obtained in the tissue segmentation step of the VBM-DARTEL preprocessing. The normalized modulated and smoothed GM images in each group were entered into a voxel-wise two-sample Groups did not significantly differ in age, but in IQ see . ADHD boGPC based on whole brain analysis differentiated ADHD patients from healthy controls with 79.3% accuracy (p<0.001). The sensitivity of classification for the ADHD group was 75.9%, while the specificity of the classification for controls was 82.8%. The positive and negative predictive values (PPV/NPV) for the classifier were 81.5% and 77.4%, respectively . The areRegions in the discriminating patterns predictive of controls included predominantly bilateral hemispheres and cerebellar vermis, middle temporal, inferior and dorsolateral prefrontal cortices (IFC/DLPFC), caudate and thalamus, precuneus/cuneus and inferior and superior parietal regions as well as left ventromedial frontal cortex, including anterior cingulate (ACC) and supplementary motor area (SMA) .Regions in the discriminating pattern predictive of ADHD patients were most predominantly in earlier developing ventral brain regions relative to the more dorsal counterparts that classified healthy controls, such as bilateral ventrolateral, premotor and ventral temporal cortices, limbic regions including hippocampus, amygdala, hypothalamus, ventral striatum/putamen, insula, posterior cingulate and brain stem. There was also a small cluster in the inferior vermis that had greater classification weights for ADHD, as opposed to the rest of the cerebellum that showed greater weights for classifying controls (see above) .No correlations were observed between GPC probability measures and clinical measures within each group.To test for potential effects of IQ or age on the classification patterns, we also correlated these within each group. No significant correlations were observed.The binary classifier trained to discriminate ADHD patients from the group of healthy controls and ASD patients (i.e. non-ADHD) achieved a balanced accuracy of 77.1% (p<0.001). The sensitivity of this disorder-specific classification for the ADHD group was 79.3% and the specificity was 75.0%. The PPV and NPV were 65.7% and 85.7% respectively, the AUC was 0.83 and the classifier delivered 0.26 bits of information per test case. No significant correlations were observed between GPC and either age, IQ or clinical measures within either group.The binary classifier trained to discriminate ADHD patients from ASD patients achieved a balanced accuracy of 85.2% (p<0.001). The sensitivity of this disorder-specific classification for the ADHD group was 86.2% and the specificity was 84.2%. The PPV and NPV were 89.3% and 80.0% respectively, the AUC was 0.91 and the classifier delivered 0.33 bits of information per test case. No significant correlations were observed between GPC and either age, IQ or clinical measures within each group.The 3-class classifier trained to discriminate among ADHD, healthy controls and ASD adolescents achieved a balanced accuracy of 68.2% (p<0.001) which easily exceeded the 33.33% accuracy that would be predicted for a 3-class classifier by chance. The sensitivity of this classification for the ADHD, healthy controls and ASD groups were 75.9%, 65.5%, and 63.2%, respectively; and the PPV were 62.9%, 73.1% and 75%, respectively  (p<0.0001) and in left inferior parietal lobule (p<0.03)  . At an u(p<0.03) , Table 3We show that it is possible to correctly classify mostly medication-naïve ADHD boys from controls based on their GM patterns with an overall accuracy of 79.3%, providing a sensitivity of 81.5% and a specificity of 77.4%. Crucially, this classifier also demonstrated excellent disorder-specificity relative to ASD with a sensitivity of 86.2% and a specificity of 84.2%, showing that 1) the patterns of structural abnormality predictive of ADHD are not attributable to psychopathology in general, such as ASD, which shares symptoms and is frequent comorbid with ADHD The regions of the discriminative pattern most predictive of ADHD were mostly in earlier developing ventral frontal, premotor, temporal, limbic and brain stem regions. The regions of the discriminative pattern most predictive of controls included typically later developing more dorsolateral and inferior prefrontal regions, ACC/SMA, dorsal striatum, thalamus and inferior parietal areas that form fronto-striato-parieto-cerebellar networks mediating the higher level cognitive control, attention and timing functions that are impaired in ADHDThe classification accuracy findings of over 79% based on ADHD brain structure measures and its disorder-specificity relative to ASD with a 85% of accuracy are promising and, if replicated, suggest that it may be possible in the future to use machine learning based pattern recognition analyses to aid in the differential diagnostic classification of ADHD with a more objective and reliable measure such as a short structural MRI scan.The pattern classification analysis revealed relatively high prediction accuracy for ADHD, with a classification accuracy of 79.3%. The overall accuracy obtained in this study is thus not only higher than all studies derived from the ADHD-200 competition using resting state and structural MRI data (ranging between 67% and 76%)Interestingly, the distributed GM patterns that showed the highest weights for classifying the ADHD boys were in predominantly subcortical areas, including most parts of the limbic system  and in brain stem, as well as in more ventral frontal, premotor and temporal regions, which contrasted with the distributed network of more dorsal DLPFC and IFC, dorsal striatum and inferior parietal areas that correctly classified controls. Furthermore, the predictive probabilities for ADHD patients were correlated with ADHD severity ratings, reinforcing their diagnostic classification utility. Subcortical as well as ventrolateral frontal and striatal regions that classified ADHD develop earlier than the more dorsal cortical and striatal brain regions that were characteristic for the classification of controls All brain regions that were reduced in ADHD patients in their GM relative to controls in the univariate analyses overlapped with brain regions that showed a higher weight for classifying controls, suggesting that the regions in the multivariate patterns predictive of controls reflect GM deficit areas in ADHD. The findings of reduced GM in the lateral parts and vermis of the cerebellum, in bilateral inferior frontal, left parietal, and right temporal cortices replicates previous findings of reduced GM in these regions in whole brain Importantly, we demonstrated that the achieved classification is disorder-specific to ADHD, as similar and even increased accuracy was achieved when we added a psychiatric control group of ASD adolescents and when we compared ADHD and ASD alone with each other, where we achieved a sensitivity of 86.2% and a specificity of 84.2%. The findings extend the previous literature of multivariate pattern analyses of ADHD by showing for the first time that the patterns that classify ADHD patients are disorder-specific and do not classify ASD patients. The findings extend previous findings of disorder-specific classification in adults with ASD, where the classifier was not suitable to classify adult patients with ADHD The pattern of brain regions that discriminated the ASD from the ADHD group is largely in areas that have been found to be different in ASD patients relative to controls such as in cerebellar hemispheres and cerebellar vermis, ACC caudate/thalamus, inferior frontal cortex, middle and superior temporal and right inferior parietal regions Together, the findings suggest that it is possible to use multivariate pattern recognition analyses for disorder-specific classification of ADHD based on structural imaging data. If replicated this may have future implications as a possible aid in differential diagnosis, in particular for difficult to diagnose patients.The strength of the study is the use of probabilistic GPC methods that confers multiple benefits for clinical studies. Another strength is the inclusion of mostly (73%) medication-naïve ADHD patients, since long-term stimulant medication has been associated with more normal GM and cortical thickness of fronto-cingulate, parietal, cerebellar and striatal regionsA limitation is a relatively modest sample size. While the study advances neuroimaging towards providing useful diagnostic markers for ADHD, it does not definitively quantify their discriminative value. Also, patients differed in IQ, which is typical for this population In conclusion, this is the first study demonstrating probabilistic GPC methods for accurately classifying ADHD patients based on their brain structure patterns. We achieved a considerable overall classification accuracy of 79.3% based on distributed and clearly distinct GM brain structure patterns, with later developing dorsolateral fronto-striato-parieto-cerebellar networks discriminating controls and earlier developing ventrolateral/premotor fronto-limbic–brain stem networks discriminating ADHD. Importantly, the classifier was both sensitive and specific for ADHD and was also disorder-specific relative to ASD. The findings are a promising step towards finding an objective differential diagnostic tool based on brain imaging measures to aid with the subjective clinical diagnosis of ADHD.Figure S1Non-thresholded three-classmultivariate discrimination weight maps. A. Multivariate discrimination weight map for ADHD (orange) vs. Controls and ASD (light blue). B. Multivariate discrimination weight map for Controls (orange) vs. ADHD and ASD (light blue). C) Multivariate discrimination weight map for ASD (orange) vs. ADHD and Controls (light blue). The intensity values of the multivariate discrimination weight-maps illustrate the relative positive weight distributions (orange) and negative weight distributions (cyan).(TIF)Click here for additional data file.


Background Children with attention deficit hyperactivity disorder (ADHD) are characterised by developmentally inappropriate levels of hyperactivity, impulsivity and/or inattention and are particularly impaired when performing tasks that require a high level of cognitive control. Methylphenidate (MPH) and motivational incentives may help improve cognitive control by enhancing the ability to monitor response accuracy and regulate performance accordingly.Methods Twenty-eight children with DSM-IV ADHD (combined type) aged 9–15 years and pairwise-matched typically developing children (CTRL) performed a go/no-go task in which the incentives attached to performance on no-go trials were manipulated. The ADHD group performed the task off and on their usual dose of MPH. CTRL children performed the task twice but were never medicated. EEG data were recorded simultaneously and two electrophysiological indices of error monitoring, the error-related negativity (ERN) and error positivity (Pe) were measured. Amplitudes of each ERP were compared between diagnostic groups , medication days  and motivational conditions .Results Error rates were lower in the reward and response cost conditions compared with baseline across diagnostic groups and medication days. ERN and Pe amplitudes were significantly reduced in ADHD compared with CTRL, and were significantly enhanced by MPH. Incentives significantly increased ERN and Pe amplitudes in the ADHD group but had no effect in CTRL. The effects of incentives did not interact with the effects of MPH on either ERP. Effect sizes were computed and revealed larger effects of MPH than incentives on ERN and Pe amplitudes.Conclusions The findings reveal independent effects of motivational incentives and MPH on two electrophysiological markers of error monitoring in children with ADHD, suggesting that each may be important tools for enhancing or restoring cognitive control in these children. Children with attention deficit hyperactivity disorder (ADHD) are characterised by developmentally inappropriate levels of hyperactivity, impulsivity and/or inattention. Although early research posited ‘weak’ inhibitory control as the single factor underlying cognitive deficits in ADHD , evidencTo optimise performance in cognitive control tasks, it is essential to monitor one’s actions and adjust behaviour when necessary. Two electrophysiological markers thought to index these functions are the error-related negativity (ERN), a response-locked fronto-central event-related potential (ERP) peaks around 100 ms after a response; and the error positivity (Pe); a centro-parietal ERP with a latency of around 300 ms postresponse. The ERN and Pe follow different developmental trajectories, the ERN tending to increase in amplitude over development, whilst the Pe remains stable . AnatomiIn ADHD, there is evidence of reduced ERN  and Pe , 2009 amTo address these questions, we compared ERN and Pe amplitudes in children with and without ADHD during a go/no-go task. Children performed the task under three different motivational conditions in which the points awarded/deducted for accuracy on no-go trials relative to speed on go trials were manipulated between conditions. In a baseline (low incentive) condition the points awarded were equal. In two high-incentive conditions, the number of points awarded for successfully inhibiting the response on no-go trials (Reward condition) or deducted for errors on no-go trials (Response Cost condition) were greater than the points awarded for timely responses on go trials. We predicted that ERN and Pe amplitudes would be significantly greater in the high-incentive conditions when the motivation to exert inhibitory control on no-go trials and, consequently, the salience of errors, was greatest. We also examined interactions between the effects of motivational incentives and the presence/absence of ADHD diagnosis, hypothesising that if incentives ameliorate an underlying deficit in error monitoring in children with ADHD, this would be reflected in an interaction between diagnostic group and motivational condition, arising from a greater effect of incentives in the ADHD group than controls.To investigate the effects of the indirect dopamine agonist methylphenidate (MPH) on error monitoring in ADHD, the children completed the task once on and once off their usual dose of stimulant medication. Based on evidence that dopamine agonists enhance error monitoring in healthy adults  and ADHDParticipants were 28 children with DSM-IV ADHD (combined type)  aged 9–15 years and 28 typically developing controls matched on age (±6 months), gender, handedness [assessed using the Annett Handedness Scale ] and socDetails of recruitment have been previously described ; brieflyT scores ≥60 on the Conners ADHD index) were excluded.Right-handed children with no known psychiatric diagnosis were identified from local schools and matched pairwise to a member of the ADHD group on age, gender and SES. Exclusion criteria were the same as those listed for the ADHD group. In addition, any participants with characteristics indicative of ADHD  and conduct disorder (CD) did not warrant exclusion from either group.A visual go/no-go task was programmed using E-Prime . Stimuli were presented centrally on a 17-inch colour monitor positioned approximately 57 cm in front of participants. Interstimulus interval was randomly jittered between 2.8 and 3.8 s; stimulus duration was 100 ms. Participants were instructed to fixate on a central point and press a response button (Cedrus Superlab button box) each time a frequent ‘go’ stimulus appeared and to refrain from responding to an infrequent ‘no-go’ stimulus. There were 600 trials with 150 (25%) no-go trials, presented in three motivational conditions , in the context of a ‘space’ theme; go stimuli were green aliens measuring 43 mm height by 40 mm width; no-go stimuli were black aliens of the same size. A time limit (‘RT cap’) was imposed on go trials: participants lost one point for slow or missed responses and gained one point for timely responses. Visual feedback was given 1000 ms poststimulus on slow or missed go trials.To minimise between-subject variance in error rates the RT cap was dynamically altered by a tracking algorithm. An initial practice session comprising 20 Go trials used a stair-case procedure to identify the shortest time within which each participant could respond. This value became the lower bound of the tracking algorithm during the main experiment. The initial value of the tracking algorithm for each motivational condition was set at this value plus 200 ms. The cap then adjusted dynamically based on performance on no-go trials; following errors, the cap increased by 25 ms to improve the chance of success on the next no-go trial, whilst each successfully inhibited response resulted in a 25 ms decrease. There was an upper limit of 900 ms.Participants performed the task in three motivational conditions: baseline (low incentive); reward; response cost. Points awarded and deducted for response speed on go trials remained the same in all conditions whereas those awarded for correct/error responses on no-go trials were manipulated between conditions. In the baseline condition, participants gained one point for each successfully withheld response on a no-go trial, and lost one point for each commission error. In the reward condition, five points were given for each correctly withheld response with no penalty for errors and in the response cost condition, five credits were deducted for each error with no reward for successful inhibition. A visual stimulus was displayed in the top right corner of the monitor to remind participants which motivational block they were performing.Stimuli were presented in five sets of three blocks , with each motivational condition randomly presented once within each set. Visual feedback at the end of each block summarised the numbers of points lost and gained. At the end of each set of three blocks, instructions emphasised either the need to withhold responses to the no-go stimulus or make timely responses to the go stimulus, depending upon whether participants achieved 50% successful inhibitions during that set.SD = 0.42) mg/kg/day. Symptoms were assessed on each day using the DSM-IV Rating scale  with 128-channel montage (including the standard 10–20 coordinates) of silver/silver-chloride (Ag/AgCl) electrodes. Additional electrodes were placed at the inner-orbital ridge and the outer canthi of each eye to record eye movements. During data collection, voltage signals were referenced to an electrode left of Cz.Analysis was performed using Brain Vision Analyzer (BVA), version 2.0 . After removal of persistently noisy/flat channels, data were re-referenced to the average of all electrodes and filtered with 0.5 Hz high-pass and 30 Hz low-pass zero-phase-shift Butterworth filters with slopes of 24 decibels/octave. Ocular artefacts were corrected using a linear regression method . As the To improve measurement of peak ERN amplitude, the data were filtered between 2 and 15 Hz prior to segmenting into epochs . Previous research has shown that the ERN is predominantly composed of theta (4–7 Hz) and alpha (8–13 Hz) power ; these fA paired analysis was conducted in which the off and on medication days of each ADHD participant were paired with the equivalent day (1 or 2) of their matched control. Of 28 CTRL-ADHD pairs recruited to the study, two ADHD participants did not complete the EEG session when un-medicated and one CTRL did not complete testing on the day equivalent to the on medication day of their matched ADHD participant, leaving 25 fully paired datasets for analysis.p <.05 were analysed further. Significant effects of Motivation were analysed with planned orthogonal contrasts comparing, first, the Baseline condition with the mean of the two motivational incentive conditions (Reward and Response Cost), and second the Reward and Response Cost conditions. Effect sizes  are reported.Repeated measures ANOVAs were conducted on error rates and on the amplitudes of each ERP . Each ANOVA consisted of three within-subjects factors: Diagnosis , Day  and Motivation . This fully paired design capitalised on the careful pairwise matching of the CTRL and ADHD groups on age, gender and SES. Off MPH and On MPH refer to the un-medicated and medicated testing days of participants in the ADHD group, respectively, and to the equivalent day (1 or 2) in their matched CTRL. Interactions significant at Two participants  had too few trials for ERP averaging (<20) in one or more motivational conditions and were excluded from analysis. For the ERN, one CTRL participant was a multivariate outlier . The matched partners of these excluded participants were also removed from all analyses involving both groups, but not from analyses involving single groups or days.n = 4) or depressive (n = 1) disorder as these might be expected to influence ERN amplitude. The pattern of significant effects was unchanged. Finally, to determine whether the high prevalence of CD/ODD diagnosis in the ADHD group (n = 21) influenced the ERP findings, Pearson’s correlation coefficients were computed between CD/ODD symptom ratings from the teacher Conners scale and ERP amplitudes. All were nonsignificant (p >.1). The results are therefore reported without adjustment for IQ and comorbidities.The ERP analyses were re-run to determine whether main effects of Diagnosis were influenced by group differences in IQ. IQ was not a significant predictor of either ERN or Pe amplitude and did not alter the Diagnosis effects. The analyses were also re-run after excluding children with comorbid anxiety (p < 1). A main effect of Motivation  = 18.76, p <.001) revealed significantly lower error rates in the Reward and Response Cost conditions compared with Baseline  = 32.78, p <.001), indicating that participants adjusted their performance according to motivational incentives. Motivation did not interact with Diagnosis or Day (p >.1).Analysis of error rates confirmed that the tracking algorithm was effective in minimising differences between subjects and testing sessions . There wp >.1), but a significant Diagnosis-by-Day interaction  = 10.03, p =.005, ηp2 =.32). There was also a significant Diagnosis-by-Motivation interaction  = 4.24, p =.02, ηp2 =.17).There was no significant main effect of Diagnosis  = 5.49, p =.03, ηp2 =.21) with smaller amplitude in the ADHD group. Next, the two groups were compared using data from the On MPH day. No significant difference was found (p >.1). Finally, an ANOVA was performed on data from the ADHD group only and revealed a main effect of medication Day  = 24.35, p <.001, ηp2 =.5) with greater ERN amplitude On than Off MPH, shown in To clarify the effects indicated by the Diagnosis*Day interaction, three repeated-measures ANOVAs were conducted. First, the two diagnostic groups (ADHD and CTRL) were compared using data from the Off MPH day. This yielded a significant effect of Diagnosis (p >.1). In the ADHD group, there was a main effect of Motivation  = 3.52, p =.04, ηp2 =.13), which did not interact with medication Day (p >.1)  = 4.54, p =.04, ηp2 =.16), and a trend for greater amplitude for Response Cost than Reward  = 2.49, p =.1, ηp2 =.09). The pattern of effects was further explored using a polynomial contrast to determine whether there was a significant relationship between ERN amplitude and the magnitude of the penalty for errors on no-go trials. The Baseline condition yielded the smallest penalty (loss of one point), followed by slightly greater penalty in the Reward condition (missed opportunity to gain five points), and the greatest penalty in the Response Cost condition . The linear term was significant  = 8.23, p =.008, ηp2 =.27).To clarify the effects indicated by the Diagnosis-by-Motivation interaction, repeated-measure ANOVAs were performed on each group separately. In the CTRL group, there was no effect of Motivation (>.1) see C1 The plF = 6.17, p =.02, ηp2 =.22). There was also a main effect of Motivation  = 4.27, p =.02, ηp2 =.16) that interacted significantly with Diagnosis  = 3.99, p =.03, ηp2 =.15).There was no main effect of Diagnosis but a Diagnosis-by-Day interaction  = 4.13, p =.05) with greater amplitude for CTRL. There was no significant group difference for the On MPH day (p >.1). For the ADHD group, there was a main effect of medication Day  = 35.29, p <.001, ηp2 =.60) with greater amplitude On MPH than Off MPH  = 8.50, p =.001, ηp2 =.26), but no Motivation-by-Day interaction (p >.1). In the CTRL group, as with the ERN analysis, there was no significant effect of Motivation (p >.1). The planned contrasts in the ADHD group revealed significantly greater Pe amplitude for Reward than Response Cost  = 9.96, p =.004, ηp2 =.29)t-tests were conducted. These revealed significantly greater amplitude in the Reward condition than the Response Cost (p =.02) and Baseline (p =.02) conditions (Bonferroni corrected), which did not differ from one another (p >.1)  C.This study investigated the effects of motivational incentives and stimulant medication on electrophysiological correlates of performance monitoring in children with ADHD. ERN and Pe amplitudes in the ADHD group were reduced when withdrawn from MPH compared with pairwise-matched typically developing controls. The amplitudes of both ERPs were significantly increased by MPH and by motivational incentives in the ADHD group, and these effects did not interact.The finding of reduced ERN and Pe amplitudes in the ADHD group is consistent with previous research , 2009, aThe effects of motivation in the ADHD group differed between the ERN and Pe. ERN amplitude followed a linear pattern related to the size of the penalty for error commission, indicating that children’s error detection processes were influenced to a greater extent by the possibility of losing credits gained on previous trials than by a missed opportunity to gain further credits. Conversely, Pe amplitude, and thus response evaluation, was significantly greater in the reward condition than either the response cost or baseline conditions. Previous studies have reported insensitivity to penalties in children with ADHD compared with oversensitivity to loss of anticipated rewards, although others have found the opposite pattern [reviewed in ]. FurtheIn one influential theory of reinforcement learning , the ERNOne important difference between the findings of the stimulus-locked  and respIt is known that performance monitoring during challenging cognitive tasks is impaired in children with ADHD. This is reflected in reduced amplitude of two electrophysiological markers of error monitoring.We found that stimulant medication (methylphenidate), when administered at the usual and therefore clinically optimal dose of children with ADHD, enhances the amplitudes of these markers. We also found that performance-based motivational incentives enhanced amplitudes, although the effect size for incentives was smaller than for medication.The clinical implications of this study are that medication and incentives are effective in ameliorating deficits in this aspect of cognition in children with ADHD.


However, the magnitude and the underlying mechanisms for CH4 uptake in response to mowing remain uncertain.Mowing is a widely adopted management practice for the semiarid steppe in China and affects CH4 uptake in a steppe community. Vegetation was mowed to 2 cm (M2), 5 cm (M5), 10 cm (M10), 15 cm (M15) above soil surface, respectively, and control was set as non-mowing (NM). Compared with control, CH4 uptake was substantially enhanced at almost all the mowing treatments except for M15 plots of 2009. CH4 uptake was significantly correlated with soil microbial biomass carbon, microbial biomass nitrogen, and soil moisture. Mowing affects CH4 uptake primarily through its effect on some biotic factors, such as net primary productivity, soil microbial C\N supply and soil microbial activities, while soil temperature and moisture were less important.In two consecutive growing seasons, we measured the effect of mowing on CH4 in the semiarid temperate steppe of north China.This study found that mowing affects the fluxes of CH Ex Prokh., and Agropyron cristatum (L.) Gaertn.The field experiment was conducted in a typical temperate steppe in Duolun County , Inner Mongolia, North China. This area has a continental monsoon climate, being semiarid and temperate in summer. Mean annual temperature is about 2.1°C with monthly mean extreme temperatures of 18.9°C in July and −17.5°C in January. Mean annual precipitation is approximately 385 mm with about 80% occurring from mid-June to late September. The study site's soil is chestnut soil (Chinese classification) or Haplic Calcisols according to the FAO classification, with sand, silt and clay being 62.8%, 20.3% and 16.9% respectively. Mean soil bulk density is 1.31 g cmStipa krylovii community was enclosed, in which mowing (including collection of the hay) plots were established. We used a Latin square design with control and four levels of mowing treatments. Each treatment had five replicates. Twenty-five 10×20 m plots were arranged in a 5×5 matrix. The buffer distance between plots was 4 m. We used mowing height as a surrogate for mowing intensity. Vegetation was mowed at heights of 2 cm (M2), 5 cm (M5), 10 cm (M10),15 cm (M15) above soil surface and the control had non-mowing . A machine was used to mow the plots once annually in late August since 2003.The study site has been fenced to exclude grazing since 2001. From 2003, a 10-ha area in the 4 flux. One stainless steel base (50×50 cm) was installed into the soil of each plot. The steel base had a groove on top to ensure airtight connection with the chamber (50×50×50 cm) 4 was conducted using a gas chromatograph , which was equipped with flame ionization detector (FID) using 60–80 mesh 13 XMS column (2 mm inner diameter and 2 m long), with an oven temperature of 55°C. Nitrogen was used as the carrier gas with a flow rate of 30 mL min−1, and the CH4 flux was determined from changes in the slope of the mixing ratio of four samples taken at 0, 10, 20 and 30 min after chamber closure. Corrections were made for air temperature and pressure. The correlation coefficient of the regression was validated . CH4 flux was measured weekly in 2008 from June to September and every two weeks in 2009 from May to September. Meanwhile soil (5 cm) temperature and moisture were measured by the Long-Stem Thermometer 6310 (Made in US) and portable soil moisture measuring kit ML2x  were collected using soil corers (5 cm diameter) every month during the growing season in 2009. Three soil samples were taken randomly in each plot and mixed evenly. The mixed sample was then divided into two sub-samples, one stored at 4°C for microbial analysis and the other air-dried for soil total C, N and phosphorus (P) analyses. We collected a total of 250 soil samples (5 treatments×5 replicates×2 sub-samples×5 months). Soil microbial biomass carbon (MBC) and nitrogen (MBN) were determined using the chloroform fumigation–extraction method 4 uptake was calculated from the monthly mean values which were averaged by month. Seasonal cumulative CH4 uptake was calculated using a simple linear interpolation, by which the arithmetical mean of the two temporally closest observations was extrapolated to represent the flux of each duration. Differences in seasonal cumulative CH4 uptake, average ST, SM, soil MBC, and MBN among treatments were determined by analysis of variance (ANOVA) followed by multiple comparisons (Duncan test). Because the effect of mowing was different between 2008 and 2009, repeated-measures ANOVAs were applied to determine the main and interactive effects of measurement time and mowing treatment on CH4 uptake rate, ST, SM, soil MBC and MBN in the two growing seasons, respectively. The linear regression was used to determine the seasonal variation of CH4 uptake responses to ST, SM, soil MBC and MBN. Stepwise multiple linear analyses were used to examine post-mowing ecosystem CH4 uptake as a function of ST, SM, soil MBC, and MBN. All statistical analyses were conducted with SAS software .Seasonal mean CH2 = 0.74, p<0.001). Only 15 cm and 2 cm mowing height treatments significantly affected soil temperature  showed strong seasonal fluctuations with peak values  between June and July 2009 . Mostly,4 uptake for control and the mowing treatments in both 2008 and 2009 , and for M15 and M2 in 2008 , suggesting that SM is the dominant environmental factor controlling CH4 uptake in the study area. Previous studies reported that the activity of methanotrophs can be greatly inhibited by small variation in soil moisture 4 oxidation in dry soils is likely to be limited due to low microbial activity occurring during periods of low levels of soil moisture 4 uptake rate  and soil moisture (SM) slightly improved the correlation between CHake rate .4 uptake. Variations in soil MBC and MBN explained 34.9% (p = 0.002) and 20.7% (p = 0.022) of variations in CH4 uptake, respectively  resulted in less CH4 uptake than the M10 height, whereas no significant effects were found for other treatments (4 are complex and possibly mediated by: (1) changes to soil moisture; 2) changes to soil C/N supply possibly as a result of altered NPP; and 3) affects on soil microbial C and N.Our results show that effects of mowing on CHg height . Moderateatments . Our stu4 uptake, mowing treatments generally had no effect on soil moisture except for two mowing treatments   resulted in lower soil temperature and was associated with changes in community composition such as reduced forbs. This might explain the reduced CH4 uptake in M15  . This sue in M15  and 3, s4 uptake was negatively correlated with net above ground primary productivity (ANPP)  . This co4 uptake may be mediated by changes in MBC and MBN (4 oxidation 4 uptake rates in soil cores in New Zealand where type I methanotrophs are dominant 4 uptake with mowing could also result from changes in methanotrophy community structure and activity 4 uptake, such as variation of root/shoot ratios In our study, mowing-induced increases in CH and MBN . It has 4 uptake in the semiarid steppe ecosystem. Long-term mowing increased CH4 uptake mainly due to its effect on soil biotic factors. 10 cm appeared to be the optimal mowing height. The substantial inter-annual variations in CH4 uptake indicate that it is necessary to conduct long-term observations in grasslands in the future to accurately determine the optimal mowing height for enhancing CH4 uptake.In general, our study demonstrates that moderate mowing can substantially enhance CH


RSAD2, is an interferon-inducible protein that potently restricts a broad range of different viruses such as influenza, hepatitis C virus, human cytomegalovirus and West Nile virus. Viperin is thought to affect virus budding by modification of the lipid environment within the cell. Since HIV-1 and other retroviruses depend on lipid domains of the host cell for budding and infectivity, we investigated the possibility that Viperin also restricts human immunodeficiency virus and other retroviruses.Viperin, also known as viperin has also been evolving under positive selection in primates. The pattern of positive selection is indicative of Viperin's escape from multiple viral antagonists over the course of primate evolution. Furthermore, we find that Viperin is interferon-induced in HIV primary target cells. We show that exogenous expression of Viperin restricts the LAI strain of HIV-1 at the stage of virus release from the cell. Nonetheless, the effect of Viperin restriction is highly strain-specific and does not affect most HIV-1 strains or other retroviruses tested. Moreover, knockdown of endogenous Viperin in a lymphocytic cell line did not significantly affect the spreading infection of HIV-1.Like other host restriction factors that have a broad antiviral range, we find that viperin among primates.Despite positive selection having acted on Viperin throughout primate evolution, our findings indicate that Viperin is not a major restriction factor against HIV-1 and other retroviruses. Therefore, other viral lineages are likely responsible for the evolutionary signatures of positive selection in  Antiviral proteins engaged in virus-host interactions are often locked in evolutionary "arms-races", which have been referred to as "Red Queen” conflicts. Viral infections continuously exert immense selective pressures on the host antiviral proteins to evolve adaptively. The signatures of these evolutionary conflicts can be inferred by observing signals of adaptive evolution  in antiviral genes that result from repeated episodes of Darwinian selection due to past viral infections . Often, Virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible, also known as RSAD2) is a host protein with broad antiviral activity (reviewed in [viperin knockout mice demonstrate the importance of this protein in controlling West Nile Virus pathogenesis in vivo[Viperin (iewed in -7). Vipeiewed in , Hepatitiewed in -11, and iewed in . Moreoves in vivo. In the s in vivo but alsos in vivo.The precise mechanism of the broad-spectrum antiviral function of Viperin remains unclear. However, one model for Viperin antiviral activity links lipid raft disruption to the restriction of Influenza virus release . Lipid rviperin, like other host restriction factors against viruses, has evolved under positive selection in primates. We find that Viperin inhibits the release of the LAI strain of HIV-1. However, we show that HIV-1 and SIV strains have intrinsic differences in their sensitivity to Viperin, and most are unaffected by over-expression of Viperin. Furthermore, we did not see an effect of Viperin knockdown on HIV-1 growth. Collectively, our findings suggest that Viperin is not a major restriction factor against HIV-1 and retroviruses, and thus its positive selection must have been driven by other viral pathogens.We find that viperin might also be evolving under positive selection. To investigate this possibility, we sequenced the viperin gene from 18 species of primates and obtained 2 sequences of prosimian viperin from Genbank . Whiles Figure D. In pars Figure C, validaGiven the broad antiviral range of Viperin, we wished to investigate whether Viperin might also be relevant to restricting HIV-1 infection. We first studied whether Viperin is expressed at the protein level in HIV-1 target cells. Primary CD4+ T cells and monocytes were isolated from peripheral blood mononuclear cells of two donors and treated with interferon β for twenty hours. We found that both primary CD4+ T cells and monocytes express endogenous Viperin after induction with interferon Figure A, but exviperin gene into 293T cells. We found that untransfected 293T cells express undetectable levels of endogenous Viperin. However, the transient expression of Viperin in 293T cells transfected with between 0.3 and 1 μg of DNA bracketed the amount of endogenous Viperin expression in primary CD4+ T cells and U937 cells when induced with interferon . Consistent with other studies, we found that wildtype HIV virus was more infectious than HIVΔNef virus Figure C. HoweveSince Viperin seemed to affect virus release, we compared Viperin restriction to that of Tetherin, a well-characterized host restriction factor that inhibits virus release ,34. Virunef gene to exclude the possible confounding effects of Nef specificity. Consistent with the earlier experiments . In addition to their f Figure D, indicaThe difference in restriction profiles between Lai and NL4-3 strains was unexpected since NL4-3 is a recombinant virus of NY5 and Lai strains . We atteA more critical test of the physiological role of Viperin on HIV-1 replication is to change levels of endogenous Viperin in cells where spreading infections can be performed. To determine the effect of endogenous Viperin on HIV-1, we sought to knock-down Viperin expression with shRNAs. Unfortunately, we were unable to obtain suitable stable or transient knockdown in primary T cells (data not shown). Many T cell lines such as SupT1 cells also do not express Viperin after interferon induction (data not shown). However, CD4+ U937 monocytic cells do express Viperin after interferon induction Figure B. Using viperin allele. However, since viperin evolves rapidly under positive selection, we might not be accurately capturing the potential ability of Viperin proteins to restrict lentiviruses. The species-specificity of action is one of the key features that have emerged from the study of rapidly evolving restriction factors. To address the possibility that the human Viperin might not accurately capture the restrictive potential of Viperin, we carried out two experiments to measure any functional divergence between primate Viperin orthologs that may have arisen from the positive selection.Most of the experiments that we have carried out were using the human viperin does not manifest a functional difference in the degree of restriction of HIVLaiΔNef. Second, we compared the human and rhesus orthologs against a panel of viruses to assess whether we could discern any key restriction differences between these two Viperin orthologs . One possible explanation is that LTR promoter efficiency and the presence of Nef may affect viral Gag production in a manner that renders it sensitive to Viperin. Alternatively, we believe it is more likely that less infectious viral combinations are more sensitive to perturbations caused by exogenous expression of Viperin. Nonetheless, considering that most strains of HIV-1, SIVs (excluding SIVmac239), tested are resistant to Viperin, we favor the more parsimonious conclusion that Viperin is not a significant player in the immune defense against lentiviruses. Moreover, the results describe here serve as an important caution that over-expression systems with single isolates cannot be relied on to functionally identify and characterize restriction factors.Our findings of Viperin restriction of the Lai strain of HIV-1 but not the NL4-3 strains are unexpected since NL4-3 is a recombinant virus of NY5 and Lai strains . We haveviperin has been evolving under positive selection in primates. The signatures of rapid evolution in viperin may provide valuable information about the mechanism by which it restricts this broad repertoire of viruses and likely avoids viral antagonism. This is analogous to the dynamics of the host restriction factor Tetherin, where the highest recurrent signal of positive selection corresponds to the amino acid that is a determinant for antagonism by Nef [Antagonistic genetic conflicts between hosts and viruses have driven rapid adaptive evolution of antiviral protein which is characteristic of many retroviral restriction factors  as well m by Nef . In the m by Nef ,41 by fim by Nef .viperin was likely driven by selective pressures imposed by virus families other than the lentiviruses.We document an ancient, episodic and recurrent history of adaptive evolution in Viperin over primate evolution. However, despite the fact that Viperin restricts other a wide range of other virus families , it doesATGAAAGACTCCTACCTTATTAGTGAAGCCACAGATGTAATAAGGTAGGAGTCTTTCATCTTGCCTACTGCCTCGGA) were purchased from FHCRC RNAi core facility.Human Viperin was cloned from human cDNA derived from 293T cells, and inserted into a retroviral expression vector pLPCX as an untagged construct. The five primate Viperin orthologs were similarly cloned from cDNA into the pLPCX retroviral expression vector as untagged constructs. HIVLai, HIVLaiΔNef and HIVLaiΔVpuΔNef, SIVagmTANΔEnvΔNef were described previously . HIVNL4-5 cells/ml in 12-well plates, and DNA was transfected with TransIT LT-1 (Mirius) according to the manufacturer's recommendations. The total amount of DNA in all transfections was maintained constant with appropriate empty vectors. Forty-eight hours after transfection, supernatant was collected, filtered through a 0.2μM filter and serially diluted for the following infectivity assay. SupT1 cells at 2.5 x 105 cells/ml in 96-well plates were as described previously [5 cells/ml in 96-well plate were as described previously [293T cells were seeded at 1.67 x 10eviously . The β-GVirus were serially diluted and measured by HIV-1 p24 antigen capture assay (Advanced BioScience Lab Inc) and detected with QuantaRed enhanced chemifluorescent HRP substrate (Thermo Scientific) according to the manufacturer’s protocol.Western blot analysis was performed as described previously ,42 with Patient pall filters were obtained from Puget Sound Blood Center. PBMCs were isolated by standard ficoll histopaque gradient methods. Monocytes and CD4+ T cells were isolated by Human CD14 selection and CD4+ magnetic bead isolation (EasySep), and the isolation purity (>97-99%) was confirmed by flow cytometry staining (BD Pharmingen). Monocytes were maintained in RPMI containing 10% FBS. CD4+ T cells were activated with 2.5μg/ml PHA and 20U/ml IL-2 for 3 days before interferon treatment. Monocytes and CD4+ T cells were treated with 500 IU/ml human interferon β1b for 20 hours, followed by western blot analysis on total cell lysates.U937 cells stably transduced with either a Viperin-targeting shRNA or control shRNA constructs were infected with a wild type HIV-1Lai virus or HIV-1LaiΔNef at a moi of 0.5. Cells were washed with PBS three times and maintained in media containing 500 IU/ml human interferon β1b throughout the course of the experiment. Supernatant was collected at indicated time points and virus was quantified by p24 ELISA.viperin genes from the following primates were amplified from RNA isolated from cell lines obtained from Coriell Cell Repositories : chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), Sumatran orangutan (Pongo pygmaeus), Siamang gibbon (Hylobates syndactylus), agile gibbon (Hylobates agilis), rhesus macaque (Macaca mulatta), greater white-nosed monkey (Cercopithecus nictitans), kikuyu colobus (Colobus guereza kikuyuensis), Francois' leaf monkey (FLM) (Trachypithecus francoisi), spider monkey (Ateles geoffroyi), owl monkey (Aotus trivirgatus), dusty titi monkey  and woolly monkey (Lagothrix lagotricha). Human (Homo sapiens), African green monkey (Chlorocebus aethiops) and Baboon (Papio anubis) Viperin were amplified by reverse transcription-PCR (RT-PCR) from an RNA extract of 293T cells, COS-7 cells and B-LCL cells respectively. Viperin was amplified by RT-PCR with a OneStep RT-PCR kit (Qiagen), and the cDNA derived was directly sequenced. Viperin was amplified with "forward" primer (5'-ATGTGGGGTGCTTACACCTGCTGCTTTTGCTG-3') or (5'-ATGTGGGTACTCACGCCTGCTGCTTTTGCTG-3') in combination with "reverse" primer (5'-CTACCAATCCAGCTTCAGATCAGCCTTACTC-3') or (5'-CTACCAATCCAGCTTCAGATCAGCCTTACTC-3'). Sequences for prosimian grey mouse lemur (Microcebus murinus) and tarsier (Tarsius syrichta) viperin gene were obtained by tblastx search on the NCBI database from cont1.216710 (ABDC01216711.1) and contig1.93320 (ABRT010093321.1) respectively.The viperin genes was constructed from DNA sequences with ClustalX by the neighbor-joining method using the Jukes Cantor method of correction and with PhyML [Viperin sequences were fitted to NSsites models that disallowed (NSsites model 1 and 7) or permitted (NSsites model 2 and 8) positive selection. Likelihood ratio tests were performed to evaluate whether permitting codons to evolve under positive selection gave a better fit to the data. A cutoff of posterior probability of p > 0.95 was implemented in these analyses (M8) to identify amino acid residues having evolved under positive selection. Analyses were also validated with REL from the HyPhy package [viperin genes between M0  and M1 (different dN/dS ratio for each branch). The degree of freedom is equal to one less than the total number of branches in the phylogeny.DNA sequences were aligned by ClustalX  and wereth PhyML  by the mth PhyML  as previth PhyML ,3. Seque package . Free raviperin genes have been entered into the GenBank database under accession numbers NM_080657, JQ437821 to JQ437837.The sequences of the 18 primate The authors declare that they have no competing interests.ESL performed all experiments and except for Figure Figure 1. Amino acid alignment of 20 primate viperin genes sequenced for the evolutionary analyses. The protein domain structure is shown above the alignment, and residues under positive selection are highlighted in bold. The amino acid positions are annotated in reference to the human Viperin sequence.Click here for file


However useful and successful this product is, its presence in the public eye is tainted by predominantly negative press about gene patenting and business practices.The development of Herceptin® story - as a corporate good citizen, Myriad as a ruthless mercenary. Since patents undergird both products yet the narratives are so different, the stories raise the question: why have patents taken the fall as the scapegoat in current biotechnology policy debate?While retrospection invites a sharp contrast between Genentech's triumphal narrative of scientific achievement and Myriad's public image as a controversial monopolist, a comparative history of these companies' products reveals two striking consistencies: patents and public discontent. Despite these similarities, time has reduced the narrative to that of hero versus villain: Genentech is lauded - at least for the final outcome of the HerceptinA widely publicized lawsuit and accompanying bad press have cast Myriad as a villain in the evolving narrative of biotechnology. While the lawsuit suggests that this villainy is attributable to Myriad's intellectual property, we suggest through a comparative case study that, at least in the Myriad case, it is not simply about the patents but also other business strategies the company chose to pursue. Patents were a necessary but not sufficient cause of controversy. Association for Molecular Pathology v. US Patent and Trademark Office (the 'Myriad' case). He ruled that Myriad Genetics' patents on the BRCA1 and BRCA2 genes claimed non-patentable DNA molecules and methods . The BRCA1 and BRCA2 genes continue a long-standing pattern of granting similar patents in the United States . Whi patents . Even be patents , and puboverage) .BRCA testing service. Should the focus be on whether Myriad should have gotten patents at all, or also on what Myriad did with them?Why have these particular patents aroused such intense controversy? As patent scholar Rebecca Eisenberg noted, 'Significant opposition to gene patenting within the medical and scientific communities did not arise until the patentability of DNA had long been established' ,14. It m®. While not directly comparable because Herceptin® is a therapeutic and BRACAnalysis® is a diagnostic, the development of Herceptin® nonetheless resembles that of BRACAnalysis® in several respects. Both are novel breakthroughs in managing breast cancer. Both were brought to market by biotechnology companies. Both products were mainly developed in the 1990s. Most importantly, for the purposes of this analysis, both inventions were patented. Among the differing elements of these two stories is profoundly different public reception. Instead of a pubic outcry in the form of a very public lawsuit, Genentech was celebrated with a corporate leadership award from the National Breast Cancer Coalition AgaAgaBRCA1/to FORCE .® testing if needed (pricing quoted by a Myriad customer service representative to one of the authors [ALB] on 7 August 2012), which some define as the 'standard of care'  genetic testing" [Cancer Research recommending BART® rearrangement testing as standard of care for high-risk women, including those outside of Myriad's screening criteria [Test cost and efficacy are also a source of concern to advocacy groups. In its statement to the USPTO, FORCE cited limited financial assistance and an increase in test cost over time, despite dramatic drops in the cost of DNA sequencing. The Myriad test 'list' price out-of-pocket is in the range of $3,300, somewhat higher than its price a decade ago, with an additional $700 for expanded BARTof care' . In her testing" . She alscriteria .®. This group posted a public open letter imploring the company to incorporate the BART re-arrangement screening into its comprehensive BRACAnalysis® test, instead of treating it as an extra-cost test conducted only when BRACAnalysis® is negative and yet suspicion of a BRCA mutation persists [®, and NBCC applauded FDA's July 2010 withdrawal of approval for its use in breast cancer, an indication Genentech fought hard to keep) [The Yale Society of Genetic Counselors, an advocacy group of physicians, nurses, and genetic counselors associated with Yale, has been particularly outraged by the additional cost associated with BARTpersists . CNN covpersists . Yale geto keep) . The int® started in the wake of the gene's discovery, before the test had even been fully developed. In 1996, the American Society of Clinical Oncology released a set of guiding principles regarding the management of BRCA testing [BRCA testing should, therefore, take place at least initially only in the context of research, along the lines of OncorMed's policy. Visco advocated a direct research program to examine treatment options for women who test positive for deleterious BRCA mutations, to study what counseling is necessary and how to avoid discrimination in health insurance and employment. She optimistically recognized that:This poor communication with relevant advocacy groups over BRACAnalysis testing . In resp testing . BRCA te"This is a time when we, as breast cancer activists, need the medical community to stand with us and recognize the need to do less, rather than more... We have a wonderful opportunity here: we can form a partnership between patient and physician and thoughtfully respond to this discovery... We ask that you take advantage of this standard of care, one that recognizes how little we know and is designed to get the answers." Frances Visco, on behalf of the National Breast Cancer Coalition .BRCA testing.In this statement, NBCC, as the pre-eminent organization for breast cancer activism, asserted its right to a place at the table with the scientists, policy makers, and corporate heads charged with implementing Journal of Clinical Oncology. His arguments directly countered Visco's, stating that to withhold the launch of the test, even to make time for the study recommended by Visco, would be to, "turn back the clock and ignore the ability to provide knowledge to women who seek it; to me this is unethical." Skolnick directly opposed the sentiments of NBCC and announced a release plan for BRCA testing that flaunted NBCC's suggestions (confirmed in an interview between the authors and Frances Visco). In the wake of Myriad's lawsuit, NBCC has gone on record in opposition to Myriad's patents, although neither NBCC nor FORCE is a direct party in the ongoing lawsuit .Mark Skolnick offered conflicting commentary in the same issue of the BRCA patents, had committed to a research program to build evidence for the utility of BRCA testing; Myriad made a different strategic choice. Since both firms had patents, it is clear patents did not drive the differing choices, although Myriad's business model was enabled by its patents and subsequent emergence as sole provider of BRCA testing with OncorMed's demise. It is hard to imagine Myriad could have been as successful in becoming the dominant US testing service, even in clinical research, without its patent rights, so patent exclusivity is a contributing cause. But the fact that other models for deploying patented genetic diagnostics can avoid the controversies associated with BRACAnalysis® shows that business decisions about how to use exclusivity require separate attention in the causal network. Patents were necessary but not sufficient to construct the narrative of villainy associated with Myriad.The role of the patents in these stories is ambiguous: the monopoly gave Myriad strong exclusive rights that bolstered its legal authority, and may have emboldened it. Or perhaps the patents had little to do with the degree to which the company abided by professional recommendations and related to disease advocacy organizations. By heeding Visco's recommendations in the beginning, Myriad might have mitigated some of the public outcry by forging alliances instead of fostering enmity. OncorMed, the firm that had licensed Mary-Claire King's genetic linkage patent and Michael Stratton's BRCA1/2 and went on to turn a profit, while OncorMed did not survive. OncorMed's strategy may have bred less controversy, but Myriad is the firm that went on to survive as a business. Figure Myriad emerged from its patent battles with OncorMed with patent rights to BRCA testing service despite its better relations with health professional organizations and advocacy organizations as a 'good citizen' vowing compliance with health professional standards; Myriad emerged with patent rights but poor relations with organizations representing its customer base of physicians and families at risk of breast and ovarian cancer. It is clear that from 2001 to 2012, Myriad's was the more successful business strategy, mainly because of its patent position.The settlement between the companies may well have been driven by the perceived likely outcomes of patent litigation or the outcome of a patent interference proceeding. Outcomes from the private settlement suggest Myriad had the upper hand. OncorMed failed in its BRCA1/2. New competitors might sequence not only the two BRCA genes but also find mutations in other genes associated with breast and ovarian cancer [BRCA genes. Its claims on short DNA fragments  are likely also invalid [BRCA genetic testing could be undermined by the plummeting cost of multi-gene deep sequencing, all-exome sequencing, and whole-genome sequencing. Depending on the outcome of the pending case, Myriad could end up with only patents on specific mutations associated with cancer risk, or with no patents on naturally occurring sequences at all.  Even iast 2028 .BRCA mutations. In this way, like patented brand-name drugs, Myriad's blockbuster model is vulnerable to introduction of competition with patent expiry or technological competition. OncorMed's strategy might have produced a loyal customer base of those ordering and seeking BRCA testing; if Myriad's customers are only using its services grudgingly, and jump to competitors as soon as they can, then Myriad's business success could be evanescent. Whether Myriad's long-term business success depends on its exclusive patent rights will become more apparent over the next few years.Myriad's 'diagnostic blockbuster' model has proven to be a $400 million per year business success. It does not follow, however, that it will withstand technological competition, patent expiration and litigation or determined public efforts to improve interpretation of ® . Recognnate use . As toldrogram'" . With thIn April 1995, advocates from many groups, including NBCC, were invited to help design the phase III trials. This new model of collaboration resulted in a markedly successful trial. Visco noted in retrospect that Genentech was the first pharmaceutical company that agreed to partner with breast cancer advocates, commenting that, "Genentech worked with us on all aspects of the trial, from protocol design, to outreach, to oversight. In particular, the company's agreeing to our request that investigators in specific communities partner with trained activists in facilitating accrual to the trial helped lead to record accrual". In OctoA critical difference between Genentech and Myriad was Genentech's open and systematic cultivation of collaborative and amicable relationships with advocacy groups representing their client base in response to early conflict. Both companies encountered conflict; Genentech made it productive while Myriad persisted with its initial business plan. The populations served by Genentech and Myriad overlapped to a significant extent. Media coverage of both conflicts includes very similar negative language alluding to companies favoring profit over patient access. The narratives differ at the point of resolution: Genentech made a deliberate effort to repair a poor relationship with BCA and turned to NBCC as an active partner, whereas Myriad did little to reconcile with FORCE or NBCC. Myriad has made efforts to create its own advocacy organizations, but these lack credibility precisely because they are corporate-affiliated, rather than collaborations with established, credible, national organizations.®. But no organization sued Genentech over its Herceptin® patent, or even over Avastin®. BCA did join the suit against Myriad's patent, and NBCC and FORCE have made statements opposing Myriad's management of its BRCA patents [Amicable and meaningful relationships with advocacy organizations dedicated to both patients and clinicians can matter to a company's success. This is not a simple matter; NBCC is not at all happy with Genentech's pursuit of the breast cancer indication for the highly expensive Avastin patents ,63 even Genentech was wise, and ultimately more successful, to recognize the detrimental effect of a poor corporate image and the very practical benefit of an engaged constituency that could mobilize clinical research participation. Genentech moved forward with a collaborative approach to clinical trial implementation. Myriad's unapologetic decisions to test outside health professional recommendations and to risk alienating national organizations representing breast cancer advocates is where these stories fundamentally diverge .Take Back your Genes public campaign, "I take back my genes so that profit will not be at the expense of women's lives" [® or any other particular product; neither is it confined to products and services covered by patents. 'Patents over patients' is a verse of the general 'profit' refrain. It is nonetheless instructive that when Genentech encountered opposition from activists, it recalibrated its strategy for developing Herceptin®; when Myriad encountered similar opposition, it largely ignored it, countered it, or attributed it to misinformation or lack of understanding. What Myriad did not do was change its plan, or even its rhetoric.Considering that both Genentech and Myriad had patent protection, we can conclude that the attribution of cause and the construction of venal narratives are to some degree misdirected at patents. In health care, company strategy is inherently bound to the consumers that it serves, regardless of patent protection. When the product being sold is so directly related to health and wellbeing, the stakes and standards for benevolent corporate behavior are raised. As Zoe Christopher, a resource liaison for BCA stated in her contribution to the American Civil Liberties Union's s lives" . We specAmicus curiae and famous co-discoverer of the DNA double helix James Watson concluded his amicus brief by quoting his own words: "The Human Genome Project... is as precious a body of knowledge as humankind will ever acquire, with a potential to speak to our most basic philosophical questions about human nature, for purposes of good and mischief alike" [f alike" . In bothSeveral strategic decisions contributed to Myriad's abysmal corporate image. By comparing Myriad's decisions to those of Genentech and OncorMed, it becomes apparent that the story is not just about patents. There are many points of contention, and the full weight of Myriad's controversial business model should not be attributed to patents alone. Arguments about patentable subject matter are playing out between the American Civil Liberties Union and its plaintiffs and Myriad and the defendants in the case before the US Supreme Court, but our purpose here is to stress that the Myriad case is an exceptional one, and not the rule. The legal debate has focused solely on patents, but Myriad was largely sued because of its business model: patents provided a legal scapegoat. While many would agree that Myriad should be held accountable for the practices that patents did indeed allow them to pursue, the legal consequences are ultimately not only aimed at the vehicle driving these practices, but also at the public road this vehicle is utilizing, the patent system. In short, this comparative case study shows it is not only - or even mainly - about the patents.BRCA lawsuit.The risk of a patent-centered narrative is that the crucial nuances of this story will be lost in monomaniacal attention to whether genes can be patented, to the neglect of business practices that are equally to blame. The social issues that have accompanied the vigorous, often acrimonious, debate about gene patents have both legal and moral elements, and that debate will go on, regardless of court decisions in the ongoing BRCA testing is about business decisions that did indeed rest on enforcing exclusive rights in the US market - but the story is also about failing to include advocacy organizations and disregarding health professional standards. Patents matter, but so do business practices.The public debate has been impoverished in two respects, by assuming: (1) that the cause of the problems underlying the debate are inherently due to patents; and (2) that eliminating gene patents will solve those problems. Patents contribute and enable business practices, but many genes have been patented that did not lead to the same problems. The story of BCA: Breast Cancer Action; BIC: Breast Cancer Information Core; CAFC: Court of Appeals for the Federal Circuit; FDA: Food and Drug Administration; FORCE: Facing our Risk of Cancer Empowered; NBCC: National Breast Cancer Coalition; NCI: National Cancer Institute; SEC: Securities and Exchange Commission; USPTO: US Patent and Trademark Office.BMJ list of experts who agree they do not have "any financial support in any form from pharmaceutical or medical device manufacturers during the past five years and that they do not have other affiliations or financial involvements that would present a conflict of interest. A three member board decides whether to accept applicants" [LB declares no competing interests. RC-D is on the licants" . He accoRC-D conceived of the paper, helped to draft and edit the manuscript, and participated in its design. ALB helped to draft and edit the manuscript, and participated in its design. Both authors read and approved the final manuscript.RC-D is research professor and director of Genome, Ethics, Law & Policy, Institute for Genome Sciences & Policy and Sanford School of Public Policy at Duke University, Durham, NC, USA. ALB is a research aide in Genome, Ethics, Law & Policy at Duke University, where she is part of the Center for Public Genomics.


To the Editor:Bartonella are fastidious hemotropic gram-negative bacteria with a worldwide distribution. In Thailand, Bartonella species have been demonstrated in mammalian hosts, including rodents, cats and dogs, and in potential vectors, including fleas  and 2 in Khon Kaen (northeastern Thailand). We enrolled patients Bartonella spp. by immunofluorescent antibody assay at the Bartonella Laboratory of the Centers for Disease Control and Prevention, Fort Collins, CO, USA. Strains used for antigen production were: B. elizabethae (F9251), B. henselae (Houston-1), B. quintana (Fuller), and B. vinsonii subsp. vinsonii (Baker). Homologous hyperimmune serum specimens were produced in BALB/c mice as previously described  a 4-fold antibody titer rise but convalescent-phase titers of 64, or 2) high and stable titers , or 3) acute-phase titer >512 with a >4-fold titer fall. Paired serum samples from febrile patients were also tested for serologic evidence of other common causes of febrile illness in Southeast Asia.Serum samples were tested for immunoglobulin (Ig) G antibodies to Bartonella infection; we compared demographic and clinical characteristics of these patients to Bartonella-infected patients. To evaluate potential risk factors, we compared Bartonella-infected case-patients >14 years of age without serologic evidence of other infections (n = 20) to nonfebrile controls with IgG to Bartonella <128 (n = 70). Age adjusted odds ratios (AORs) with 95% confidence intervals (CIs) were calculated.Febrile patients with acute-phase and convalescent-phase IgG antibody titers <128 were considered not to have Bartonella infections. Thirty-five (38%) of these 92 had serologic evidence of infection with another pathogen. The remaining 57 Bartonella-infected case-patients  had a median age of 19 years (range 7–72 years); 65% were males, 47% were students, and 35% were rice farmers. Common clinical characteristics of Bartonella-infected patients included myalgias (83%), chills (79%), and headache (77%). Thirty (60%) patients had anemia (hemoglobin level <13 mg/dL); 18 (32%) had a hemoglobin level <12 mg/dL, and 4 (7%) had <11 mg/dL. When compared with 193 febrile patients without Bartonella infection, the 57 Bartonella-infected patients were similar in age and sex but were more likely to be rice farmers and were more likely to have leukocytosis  and outdoor activities  during the 2 weeks before illness onset. Prevalence of reported rat exposure and animal ownership  was similar among case-patients and controls.Serologic testing was completed on paired serum samples for 336 (46%) of 732 febrile patients enrolled; 92 (27%) had serologically confirmed (50) or probable (42) ocytosis . CompareBartonella infection among febrile patients in Thailand. Over 25% of patients with undifferentiated febrile illness had serologic evidence of Bartonella infection . Our findings indicate that Bartonella infections may be common and underrecognized causes of acute febrile illness in rural Thailand. Although our results are limited by lack of culture confirmation, we used conservative case definitions for serologic diagnosis and therefore believe that most cases represent true Bartonella infections. The common clinical features of anemia and leukocytosis and the frequent tick exposure and outdoor activity are consistent with known features of Bartonella infections and lend support to serologic findings. Because of the potential for serologic cross-reactivity between Bartonella species, we did not attempt species identification. The case-control study was therefore limited by grouping case-patients that were likely infected with different Bartonella species for which risk factors may differ. Such studies could lead to meaningful recommendations for prevention and control of Bartonella infections. Additional epidemiologic and transmission studies are needed to improve understanding of risk factors, identify key animal reservoirs and vectors, and ascertain transmission dynamics.We describe the frequency and clinical characteristics of acute