The preterm lung is particularly vulnerable to ventilator-induced lung injury (VILI) as a result of mechanical ventilation. However the developmental and pathological cellular mechanisms influencing the changing patterns of VILI have not been comprehensively delineated, preventing the advancement of targeted lung protective therapies. This study aimed to use SWATH-MS to comprehensively map the plasma proteome alterations associated with the initiation of VILI following 60 minutes of standardized mechanical ventilation from birth in three distinctly different developmental lung states; the extremely preterm, preterm and term lung using the ventilated lamb model. Across these gestations, 34 proteins were differentially altered in matched plasma samples taken at birth and 60 minutes. Multivariate analysis of the plasma proteomes confirmed a gestation-specific response to mechanical ventilation with 79% of differentially-expressed proteins altered in a single gestation group only. Six cellular and molecular functions and two physiological functions were uniquely enriched in either the extremely preterm or preterm group. Correlation analysis supported gestation-specific protein-function associations within each group. In identifying the gestation-specific proteome and functional responses to ventilation we provide the founding evidence required for the potential development of individualized respiratory support approaches tailored to both the developmental and pathological state of the lung. Fundamentally, the multi-organ problems of prematurity relate to exposing developmentally immature systems to ex-uterine life. Of these, the respiratory system is the most acutely compromised in early life, being structurally immature, surfactant deficient and prone to collapse2. These defects often necessitate the application of mechanical respiratory support in the form of positive pressure ventilation to sustain life. However, assisted ventilation is an unnatural process, and when applied to the fragile, under-developed alveolar structure of the preterm lung, causes ventilator-induced lung injury (VILI) and subsequent bronchopulmonary dysplasia (BPD)4. Even a single large lung inflation can initiate VILI in the preterm lung5. The consequences of VILI development in the immediate postnatal period include inhibition of surfactant function7, impaired angiogenesis8 and lung growth9 and increased oxygen need10, thereby committing the infant to continuing respiratory support which perpetuates lung injury. In the long-term, VILI-related lung abnormalities manifest as chronic lung disease in infancy11, with reduced pulmonary function13 into adult life14.With advances in perinatal care, preterm birth has now become a major public health issue15, antenatal steroids16, better nutritional support17 and non-invasive ventilation18, have significantly improved outcomes, more than 50% of extremely preterm infants ) still develop BPD19. VILI is a multi-factorial process involving a complex interplay between multiple pathophysiological and patient-specific factors. In the preterm infant, VILI pathogenesis is specifically linked to immaturity of lung tissue, direct mechanical injury, oxidant injury, and proinflammatory mediators15. Therefore, identification of VILI-associated mediators and pathways will be paramount in improving respiratory outcomes for those born prematurely. Proteomics, with its ability to elucidate pathological pathways and determine protein alterations associated with specific development and/or disease states, is perfectly suited to uncovering the intermediaries associated with VILI development within the premature lung.Minimizing VILI remains a key goal of neonatal intensive care. Whilst major advances, such as exogenous surfactant20. Proteomic studies of plasma using traditional label-dependent methods have been challenging, with labels themselves often co-selected21 and difficulties in identifying and quantifying low abundance proteins from samples in which albumin, α2-macroglobulin, transferrin, and immunoglobulins, may represent as much as 80% of the total serum protein22. In contrast, sequential window acquisition of all theoretical mass spectra (SWATH-MS) is a label-free data acquisition method which generates, in a single measurement, a complete recording of the fragment ion spectra of all the analytes in a biological sample23 and has been shown to outperform alternative methods in plasma quantification, reducing single variation, and markedly increasing the number of precisely quantified peptides21.Plasma is increasingly used in proteomic studies of lung disease as it is easily accessible and contains measureable tissue-derived proteins with the potential to delineate disease-associated mechanisms and pathophysiologyIn this study we aimed to use SWATH-MS to (1) characterize alterations in the plasma proteome associated with the application of a uniform mechanical ventilation strategy to extremely preterm, preterm or term lambs and (2) identify associations between observed proteome alterations and key functional outcomes. We present here the first comprehensive map of the ventilation-induced plasma proteome after preterm birth, and reveal a GA-specific response to mechanical ventilation. Furthermore, using integrated bionformatic analysis we have identified unique GA-specific protein-function associations.A total of 22 lambs delivered from 15 ewes were studied. No ewes had evidence of sepsis or chorioamnionitis. Birth weight increased with gestational age. Drained lung fluid volumes and measures of fetal wellbeing were comparable between groups (Supplementary Table 2) in extremely preterm and preterm animals when compared with term animals  and alveolar-arterial oxygen difference  was 3-fold higher in extremely preterm and preterm lambs when compared with term lambs, whilst the number of detchaced epithelial cells was increased 4-fold in extremely preterm lambs when compared to term lambs. Gene expression of interleukin-6 (IL6) was increased five-fold in non-dependent lung tissue from extremely preterm animals compared to term animals, conversely gene expression of connective tissue growth factor (CTGF) was decreased two-fold in gravity-dependent lung tissue from either extremely preterm or preterm animals when compared to term.A total of 125 proteins were identified via SWATH-MS with identification confidence ≥99% and false discovery rate ≤1% (Supplementary Table 40/125 proteins (32%) were differentially expressed in response to ventilation, including eight up-regulated Fig.  and 32 dMirroring the multi-variate analysis, 83% of differentially-expressed proteins were altered only in a single GA-group with the largest number of altered processes observed in the preterm group 20/40) when compared with the extremely preterm group (8/40) and term group (5/40), respectively. The hemoglobin proteins HBA2, HBB and HBBF were increased in all three GA-groups with increasing abundance of plasma HBB over-time in all GA-groups confirmed via ELISA  qualified for inclusion in IPA-based analyses using a human homology threshold of ≥65%  or negatively  associated with gene expression of inflammatory makrers in the non-dependent and gravity-dependent lung Fig. . Static 2/FiO2, SpO2, OI, minute ventilation and tidal ventilation were positively associated with altered expression of proteins COL1A2 and CFHR3-like in term aniamls . Whilst the cause of VILI has been well studied and documented, the impact of gestational age (GA) at ventilation on VILI development has not been comprehensively studied. In the ventilated preterm lamb model 83% of differentially-expressed proteins identified by SWATH-MS were confined to a single GA-group, with the largest number of alterations observed within the preterm plasma proteome. Furthermore, correlation of differential protein expression against key measures of lung function and injury confirmed associstion between changes in protein expression and GA-specific biomarkers of the early lung injury response. Such tools may contribute to the development of individualized treatment strategies aimed at protecting the preterm lung and minimizing damage.24. We have previously shown that the preterm group exhibits better dependent lung aeration in early life than the extremely preterm and term group25. The results of this study, in which the largest proteome alterations were observed in lambs in whom the dependent hemithorax was preferentially aerated suggest additional studies characterizing regional proteome alterations within lung tissue are warranted.Consistent with clinical experience, increasing prematurity was associated with reduced oxygenation, impaired lung compliance and increasing evidence of lung injury. Paradoxically however, the greatest magnitude of differentially-expressed proteins were observed in the preterm group rather than the extremely preterm group. The answer to this paradox may lie in significantly different physiological responses to the same ventilation strategy. Amongst the functional parameters measured, only differences in gas exchange differentiated extremely preterm and preterm lambs. Lung injury is rarely consistent throughout the lung, exhibiting a regional gravitational behavior that is postulated to be initiated by ventilation and aeration homogeneity26 and regulating airway hyperactivity27. In the current study we observed a significant decrease in APOE protein expression following ventilation, concurrent with morphological evidence of lung injury and increased IL1B gene expression. Our results parallel those of the ApoE null mouse in which ApoE ablation is associated with hypersensitive pulmonary inflammatory responses28 and increased susceptibility to VILI development30. Similarly reduced levels of complement precursor proteins and functional activity have previously been observed in preterm infants with RDS31, however this is the first study to associate complement cascade disturbances with mechanical ventilation of the preterm lung.In pretern lambs, several lines of evidence confirmed the influence of ventilation on the inflammatory response including (i) increased abundance of the inflammatory-mediator apolipoprotein E (APOE), (ii) reduced abundance of key members of the complement cascade including complement components 3 (C3) and 5 (C5), complement factor I (CFI), and the C5 amplification mediator vitamin D binding protein (GC), (iii) results from the integrative analysis in which the ventilation-associated abundance of seven proteins  was strongly correlated with the gene expression of inflammatory markers IL1B, IL6 and IL8 in both non-dependent and gravity-dependent lung tissue. APOE is expressed by alveolar epithelial cell type I cells and plays an important role in facilitating lipoprotein transport33 where under pathologic conditions, such as following initiation of mechanical ventilation, the hemostatic balance favours thrombosis35 and fibrin deposition37. Fibrin deposition may then exacerbate lung injury via surfactant inactivation and reduced surfactant synthesis, ultimately disturbing gas exchange.The coagulation pathway was also highly perturbed in ventilated preterm lambs which is suggestive of a prothrombotic response to ventilation in this GA cohort. Our observation of decreased levels of coagulant Factor II (F2) and Factor IX (F9) together with decreased levels of fibrinolysis mediators FGB, FGG, FBLN1 and PLG concur with anticoagulant hypofunction previously described in infants with respiratory distress syndrome (RDS)39 has not previously been associated with either VILI or mechanical ventilation. We postulate that increased expression of APOA1 in the ventilated extremely preterm lamb model may represent a lung protective mechanism, as increased levels of APOA1 in mouse models of respiratory disease are associated with anti-inflammatory activity41, anti-fibrotic properties43 and the promotion of tight junction recovery44.Similar to the preterm ventilated proteome, extremely preterm lambs exhibited evidence of hemostatic dysregulation with increased expression of plasma protease C1 inhibitor precursor (SERPINC1) and fibrinogen A (FGA) and decreased expression of antithrombin III and apolipoprotein H (APOH). In addition, our novel identification of increased APOA1 in ventilated extremely preterm lambs is also of interest. APOA1, a lipid transport proteinet al.45 of comparable fold changes in endotracheal aspirate expression of HBB, HBA2 and HBBF in a cohort of 37 infants with chronic lung disease, our evidence of plasma alteration of hemoglobin proteins warrants further investigation as a potential non-invasive monitoring tool of preterm lung injury.In addition to identifying proteins that uniquely defined the response to ventilation in each GA, we also identified within our study increases in the differential expression of fetal hemoglobin (HBBF), hemoglobin beta (HBB) and hemoglobin subunit alpha-1/2 (HBA2) across GA groups. HBB expression during ventilation was further validated via ELISA and shown to steadily increase with time on ventilation in all three GA-groups. The primary function of all three forms of hemoglobin is the transport of oxygen from the lung to various peripheral tissues, and therefore their comparable increase across all GA groups may predominantly reflect a response to oxygen delivery via mechanical ventilation, indicative of critical hemoglobin achievement within the ventilation parameters applied. Given recent reports by Ballard 46. As such, a major finding with significant clinical implications is our observation of reduced protein abundance of retinol binding protein 4 (RBP4) in ventilated extremely preterm and preterm lambs. Vitamin A supplementation has been proposed as a lung protective therapy in preterm infants but results of clinical trials to date have been conflicting47. RBP4 transports retinol with a 1:1 ratio48, thus our finding of reduced RBP4 in ventilated preterm lambs may provide a biological rationale for the relatively modest improvement in respiratory outcomes observed following vitamin A delivery in very preterm infants.Despite advances in neonatal care and continued research into therapeutic strategies aimed at prevention of BPD, the incidence of this condition has remained unchanged54 and high sheep-human protein homology (82% of identified proteins exhibit high homology) is well suited to address this challenge, with some limitations. The ventilated preterm lamb model is labor-intensive and expensive, limiting the study to group sizes of 7–8 lambs/group and a single ventilation strategy. However, the techniques and workflow used in the current study provide the platform for larger studies focusing on longer periods of different mechanical ventilation strategies. Antenatal betamethasone exposure was limited to the two premature groups only, consistent with current clinical practice16 and the lack of steroid exposure in the term group may have influenced our results, specifically suppression of IGF255 and COL1A256 proteins. Whilst these studies suggest a limited influence of betamethasone on the observed proteome sets, confirmation via direct comparison of betamethasone treated and untreated samples may be warranted. Similarly the influence of anaesthesia has not been formally delineated, however up-stream analysis within IPA was unable to identify any association between the anaesthtic agents used  and the proteins identified. The inability to maintain survival in preterm lambs without any respiratory support limits the ability to determine whether the proteome differences were solely related to a ventilation response. Also, our investigation of the plasma proteome, whilst allowing for rapid clinical translation, is reflective of a whole body response and is therefore not confined to ventilation-associated alterations. To overcome this we excluded any proteins known to be related to the birth response or antenatal steroid exposure. Nevertheless, follow up studies in a sample type such as BALF, which directly reflects ventilation-induced damage within the lung, will be important.Gaps in knowledge regarding the mechanisms underlying VILI are related, in part, to the challenges associated with studying an extremely preterm or preterm cohort within the confines of the Neonatal Intensive Care Unit. The preterm lamb model, with its ability to mirror the clinical setting of preterm birth57. If we are to improve the respiratory outcomes of those born prematurely, we must increase our understanding of the basic pathophysiology underlying VILI development. This is the first study to use proteomics and integrated bioinformatics to (i) identify the injury mechanisms and pathways which underlie VILI development and (ii) define the protein-function associations underlying VILI. By characterizing the extremely preterm and preterm VILI proteome, this study importantly broadens our understanding of how prematurity-specific factors, such as the developmental state of the lung, shape the cellular response to ventilation. Furthermore, our demonstration of clear differentiation between the ventilation-associated proteome of extremely preterm, preterm and term lambs in response to a uniform ventilation strategy supports a need for the development of individualized respiratory support approaches which are tailored to both the gestational age of the infant and their underlying pathology.Respiratory support is necessary to support preterm life, however, even brief periods of mechanical ventilation when applied to the immature lung can initiate lung injury. As a consequence, whilst advances in respiratory care have significantly altered survival, rates of chronic lung disease have not changed for 30 years58 the 119–120 d and 127–128 d cohorts were exposed to maternal 11.4 mg IM betamethasone 24 and 48 h before delivery. Whilst on placental support the carotid arterial and external jugular vein were cannulated, the trachea intubated with an appropriately sized cuffed endotracheal tube, and lung fluid passively drained24. After delivery, animals were weighed and placed supine, bedside monitoring commenced, intravenous fluids commenced  and mechanical respiratory support initiated. Continuous infusions of ketamine and midazolam were used to maintain anesthesia/analgesia and suppress spontaneous respiratory effort throughout the ventilatory period.Date-mated Border-Leicester/Suffolk lambs, mated from the same flock over one winter, were delivered via caesarean section under general anesthesia at one of three gestational age (GA) cohorts: 119–120 d (extremely preterm), 127–128 d (preterm) or 139–140 d (term). These GAs are consistent with lung morphology in human infants at approximately 23 weeks, 27–28 weeks, and term gestation. Consistent with clinical practice61. Initially a sustained lung inflation was delivered at 40 cmH2O using a Neopuff Infant Resuscitator  as per our previous reports60. Thereafter the lung was supported with positive pressure ventilation  in a volume-targeted ventilation mode . To monitor physiological stability, preductal peripheral oxygen saturation (SpO2), heart rate, arterial blood pressure, and rectal temperature  were recorded continuously from birth, and arterial blood gas analysis  performed at 5 min of age and then 15 minutely from the first inflation. Parameters of oxygenation, including oxygenation index (OI) and alveolar-arterial oxygen difference (AaDO2), were calculated using standard formulae. FiO2, tidal volume (5.5 to 8 ml/kg) and ventilator rate (40 to 60) were adjusted as appropriate using a standardized protocol to maintain SpO2 between 88–95% and partial arterial pressure of carbon dioxide between 45–60 mmHg. At 60 minutes, all lambs were ventilated with an FiO2 of 1.0 for 3 min and disconnected to atmosphere for 2 min. An in vivo super syringe pressure–volume (PV) curve was obtained using a maximum pressure of 35 cmH2O to determine static respiratory system compliance (ml/kg/cmH2O). At the end of the study period all lambs received a lethal IV dose of pentobarbitone.All lambs were ventilated for 60 min using the same strategy. Previous studies of this animal model have demonstrated alterations in gene expression and histological evidence of VILI after 15–30 min of ventilation2O and then paraffin embedded.Arterial blood samples for SWATH-MS proteomic analysis were collected at study commencement and following 60 minutes of ventilation in S-Monvette® tubes (Sarstedt), and were centrifuged at 3000 rpm, 10 minutes, 10 °C. Plasma was stored at −80 °C until required. Lambs were euthanized following mapping of the PV curve and the whole lung removed and weighed. Bronchoalveolar lavage fluid (BALF) was collected by repetitive saline lavage of the isolated left lung. Tissue samples from standardized non-dependent  regions and gravity-dependent  regions of the right lower lung lobe were snap-frozen for RNA isolation. The right upper lobe was inflation fixed with 4% paraformaldehyde at 20 cmH2) determined in 8-bit converted images using the threshold function to optically determine areas of tissue (grey) versus airway space (red). Measurements of alveolar morphology were measured in five alveolar spaces/micrograph  as predetermined by a standardized grid overlay. The total number of detached epithelial cells/micrograph  were counted using the ImageJ picker tool.Histological examination was performed on hematoxylin and eosin stained 4 μm formalin-fixed lung sections from three standardized lung regions. Post-staining, five fields of view (FOV)/region totalling 15 FOV/lamb were photographed (Leica Microsystems) and injury measurements performed using Image J software with % lung tissue, % airway space and amount of lung tissue . Primers were designed using the Roche Universal ProbeLibrary Assay Design Center .Briefly, 25 μL of plasma sample was diluted in 475 μL of 50 mM ammonium bicarbonate solution before reducing with dithiothreitol (5 mM DTT) and alkylating with iodoacetamide (10 mM IAA). One fifth of the reduced and alkylated sample (121 μL) was digested with 10 μL trypsin (20 μg) for 16 hours at 37 °C. The digested sample was diluted (X 10.5) in 0.1% formic acid prior to all mass spectrometry analysis.A 5600 TripleTOF mass spectrometer (AB Sciex) coupled to an Eksigent Ultra nanoLC-1D system (Eksigent) was employed for both IDA and SWATH-MS analysis. For 1D IDA data, 10 μl of digested sample from each group was pooled, making three pooled groups  and 5 μl duplicates of each pooled sample was injected. For 2D IDA 20 μl of each group pooled-sample was pooled , then dried and fractionated in eight fractions using a High pH Reversed-Phase Peptide Fractionation kit (Pierce). 10 μl of each fraction was injected for 2D IDA.Protein identification from IDA data was performed with ProteinPilot  based on a NCBI database, filtered for sheep and Ovis aries and containing 117933 protein entries. SWATH data were extracted using PeakView (v2.1) with peptides (max 100 peptides per protein) with confidence ≥99% and false discovery rate (FDR) ≤ 1% included in the quantitation and shared and modified peptides excluded. A local peptide assay library was derived from the 1D IDA and contained 163 identified proteins. An extended peptide assay library was constructed from the combination of the 1D and 2D IDA and contained 179 identified proteins.For SWATH-MS experiments 5 μl of each digested sample  was injected in random order, with two technical replicates for each injection. SWATH data were acquired with one blank run between every sample.SWATH peaks were extracted using PeakView . Shared and modified peptides were excluded. Peak extraction parameters were set as the following: 100 peptides per protein, 6 transition ions per peptide, peptide confidence threshold 99%, FDR extraction threshold 1%, XIC (Extract Ion Chromatogram) retention time window 10 min and mass tolerance 75 ppm. The extracted transition ion peak areas, peptide peak areas and protein peak areas were exported in Excel for further statistical analysis.HBB was detected in plasma samples obtained prior to ventilation and at 5, 30 and 60 minutes post ventilation via ELISA .Shapiro-Wilk normality testing confirmed the normality of data distribution, with results represented as either mean values with their standard deviation (SD) or median and interquartile range. Significant differences between gestation groups were sought using parametric and non-parametric ANOVA as appropriate  with post hoc testing to identify intergroup differences as necessary . All statistical analysis was performed with GraphPad PRISM 6  and P < 0.05 considered significant. Unless otherwise stated P values refer to ANOVA.To assess whether extremely preterm, preterm and term groups exhibited compositional proteome differences following 60 minutes of ventilation, we firstly used an unsupervised clustering approach  based on scaled protein abundances (ie. difference between post- and pre-ventilation protein abundance). To further investigate which proteins potentially contribute most to differences between GA-groups, we used partial least squares discriminant analysis (PLS-DA) in the R package MixOmics (version 3.1.1).2-transformed expression. P values of <0.05 were considered significant.To identify significant alterations in protein abundance paired 0 and 60 minute plasma samples were compared first by a paired t-test and represented within the results as logTo address the requirement for human, mouse or rat data in IPA, we first used the NCBI Basic Local Alignment Search Tool (BLAST) to identify those sheep proteins with ≥65% homology to human protein sequences. These highly homologous protein identities were then uploaded into Qiagen’s IPA system for core analysis and down-stream processing to identify canonical pathways, disease and functions and protein networks that were most significant within each GA group.Correlation between differential protein abundance and functional parameters: To test how differential protein abundance and clinical and functional parameters co-vary within the gestation groups, distance matrices using Manhattan distances were calculated for proteins (60 minute abundance − 0 minute abundance) and functional parameters separately, and then Pearson correlation coefficients between both distance matrices were calculated. Significance of correlations was calculated using a Mantel test with 1000 permutations.All experiments were conducted according to the guidelines of the National Health and Medical Research Council , with prior approval from the Animal Ethics Committee of the Murdoch Children’s Research Institute .Table 1 and 2


Microbial communities associated with indoor dust abound in the built environment. The transmission of sunlight through windows is a key building design consideration, but the effects of light exposure on dust communities remain unclear. We report results of an experiment and computational models designed to assess the effects of light exposure and wavelengths on the structure of the dust microbiome. Specifically, we placed household dust in replicate model “rooms” with windows that transmitted visible, ultraviolet, or no light and measured taxonomic compositions, absolute abundances, and viabilities of the resulting bacterial communities.Light exposure per se led to lower abundances of viable bacteria and communities that were compositionally distinct from dark rooms, suggesting preferential inactivation of some microbes over others under daylighting conditions. Differences between communities experiencing visible and ultraviolet light wavelengths were relatively minor, manifesting primarily in abundances of dead human-derived taxa. Daylighting was associated with the loss of a few numerically dominant groups of related microorganisms and apparent increases in the abundances of some rare groups, suggesting that a small number of microorganisms may have exhibited modest population growth under lighting conditions. Although biological processes like population growth on dust could have generated these patterns, we also present an alternate statistical explanation using sampling models from ecology; simulations indicate that artefactual, apparent increases in the abundances of very rare taxa may be a null expectation following the selective inactivation of dominant microorganisms in a community.Our experimental and simulation-based results indicate that dust contains living bacterial taxa that can be inactivated following changes in local abiotic conditions and suggest that the bactericidal potential of ordinary window-filtered sunlight may be similar to ultraviolet wavelengths across dosages that are relevant to real buildings.The online version of this article (10.1186/s40168-018-0559-4) contains supplementary material, which is available to authorized users. Humans spend most of their time in the built environment , exposedblock most ultraviolet wavelengths [Sunlight is a central component of architectural design  and has elengths . ChangesOne impediment to a comprehensive understanding of indoor microbiome community structure is that controlled and manipulable built environment experiments are logistically challenging and rarely achievable. As a result, indoor microbiome research has primarily relied on non-invasive in situ observational sampling. These studies have revealed associations between abiotic features like humidity, temperature, and ventilation, and the structure of indoor microbial communities , 27–29. Microcosms—small artificial habitats—have been central in experimentally testing otherwise intractable community-level hypotheses in ecology and microbiome research –32, due 1We constructed eleven identical built environment microcosms that simulated lighting, reflectance, temperature, and humidity conditions in a typical indoor room. These boxes were 1:32 scale models of a room measuring 4.3 m wide, 7.9 m deep, and 3.3 m tall, with a single 3.5 m × 1.2 m view window and 1 m sill—dimensions and proportions that are well within those of typical residential and non-residential rooms. These microcosms were positioned in south-facing building openings with very little solar obstruction such that the window of each microcosm was exposed to the outside and were sealed to their base plates with rubber gasketing to prevent air exchange. The microcosm floors were demarcated by a 3 × 5 grid  visible, (ii) ultraviolet, or (iii) no light . The visible-transmitting glass has a spectral profile intended to represent ordinary architectural glass used in buildings , blockin11∘C for the duration of the experiment, typical of conditions in buildings, with an average of 20.28 ∘C. We confirmed that neither maximum nor minimum daily temperatures varied significantly between rooms, regardless of light treatment, using linear mixed effects models  [Two additional microcosms were outfitted with light sensors within each cell of the 3 × 5 grid: one microcosm for visible  and UV wavelengths , in order to measure hourly visible and UV light dosages throughout the microcosms. One additional sensor of each type was placed on the roof of the building to monitor total exterior light. Temperature sensors  were installed in the ceiling of each microcosm to monitor ambient conditions to confirm that they held within ranges observed in buildings. Microcosms were placed in plywood enclosures with thermostatically controlled climate systems and small fans for air mixing to provide additional temperature regulation. Temperatures were maintained between 18.19 and 22.34 ctively) . Relativctively) .ca. 2.7% over the course of the experiment. As a reference, schools and classrooms are often designed for a ratio of 2 to 4%, whereas buildings like warehouses typically range from 2 to 10% [Microcosms produced an average visible light ratio of interior to available exterior light of 2 to 10% . Thus, tDust was collected from seven volunteer residential single family homes in Eugene, OR, USA. Residents were instructed to use personal vacuum cleaners to collect and pool dust from every room of their homes. The collected dust was mixed and homogenized using scissors in a dark laboratory. Six replicate dust samples weighing 0.25 g were collected from the homogenized dust pool and applied in a thin layer to individual sterile petri dishes for each microcosm. We demonstrate that repeated samples from this homogenized dust pool produce relatively similar bacterial communities in Additional file 1μL of 20 mM PMA  based on the manufacturer’s instructions, was vortexed for 5 s, placed in the dark for 5 min, and finally placed on a bed of ice for photo activation. PMA was activated using two 500-W halogen lamps placed above the samples for 15 min. At the 5- and 10-min marks, tubes were vortexed and placed back on the bed of ice. After PMA activation by light treatment, an additional 2-mL of PBS was added to each sample. The samples were then centrifuged (Eppendorf 5810R) at 3000 rpm for 10 min and the supernatant removed; the remaining bolus of dust was extracted from the tube and transferred to a MoBio PowerLyzer Glass Bead Tube for DNA extraction.After a 90-day exposure period, the dust samples were collected from all microcosms and subdivided into two equal aliquots of 0.125 g. A 90-day period was chosen based on estimated residence times for dust particles in real buildings with normal cleaning frequencies  and becaBoth PMA- and non-PMA-treated DNA were amplified in a PCR enrichment of the V3 and V4 (319F-806R) regions of the 16S rRNA gene following the protocol described by Kembel et al. : PCRs weμL) contained ABS PowerUp SYBR Green PCR Master Mix (25 μL), 10 μM Total Bacteria F SYBR Primer 5 ′-gtgStgcaYggYtgtcgtca-3 ′ (2 μL), 10 μM Total Bacteria R SYBR Primer 5 ′-acgtcRtccMcaccttcctc-3 ′ (2 μL), PCR grade water (16 μL), and 5 μL of 1:10 diluted DNA template [∘C, 2 min at 95 ∘C; 40 cycles of 15 s at 95 ∘C, 15 s at 60 ∘C, and 60 sec at 72 ∘C; followed by a melt curve in the range of 60 ∘C to 95 ∘C. Standard curves were generated using serial-dilutions of synthetic 167 bp gBlocks Gene Fragments  with known gene sequence copy numbers.We estimated the total counts of 16S rRNA gene copies per milligram of dust  of living and total communities using real-time quantitative PCR . The reaction mixture (50 template . The pla−1 dust) by scaling relative normalized RSV counts in each community by estimates of total bacterial abundance per milligram dust generated by qPCR assays [Raw Illumina sequence data were filtered, trimmed, and denoised using the DADA2 v1.7.0 statistical inference algorithm , 44, whiR assays . To remoR assays  and subtβ-diversities, were calculated using the Canberra distance measure [x-transformed absolute RSV abundances. The effects of different light treatments on the community compositions of dust were quantified using a permutational multivariate analysis of variance (PERMANOVA). Pairwise contrasts between treatment groups were accomplished by performing PERMANOVA analyses with 10,000 matrix permutations for each pair of factor levels and adjusting P values for multiple comparisons using the Benjamini-Hochberg procedure [Quantitative bacterial community dissimilarities, or  measure  and log1rocedure . Differerocedure ) with ANrocedure .β-diversities were visualized with unconstrained principal coordinates analysis (PCoA). We accomplished t-SNE visualization by initializing the Barnes-Hut implementation of the algorithm [Community dissimilarities were visualized using t-distributed Stochastic Neighbor Embedding (t-SNE) , 53. t-SN=94) from local volunteers and a subset of fecal communities from the American Gut Project’s [N=83). Environmental microbiome training data included outdoor air settling dishes (N=27) placed outside local residential homes and a set of soil cores (N=21) collected from an Oregon forest for the Earth Microbiome Project [We classified the types of living and dead microbial communities that remained in dust following the 90-day exposures using a Bayesian source tracking classifier . Our goroject’s  Oregon r Project . DetailsTo account for variation in sample collection, processing, and sequencing depth among individual studies and sequencing runs, the final collated training dataset used for source tracking was aggregated at the level of bacterial genus and rarefied to a depth of 2500 sequences per sample; taxa whose genus-level classification did not meet the 75% bootstrap threshold against the Silva version 128 reference database were aggregated at the next highest taxonomic level. The trained model was then tested on experimental samples that were aggregated using the same procedure, generating coarser-grained predictions than RSV-level analyses.x-transformed absolute abundances of each RSV leaf, and another comprising each node in the tree. For the latter set, values associated with each node were log101+x-transformed summed abundances of all descending RSV leaves. These were scaled and used as input to the implementation of sLDA in the sparseLDA package; the optimal number of model predictors and sparsity parameter were determined by five repeats of fivefold cross-validation. This approach ignores branch lengths and instead incorporates phylogenetic information by employing a sparsity constraint that allows the simultaneous modeling and selection of leaf and node features with strongly covarying feature values [We used phylogenetic tree-based sparse linear discriminant analysis (sLDA) as a feature selection tool, to identify whether individual RSVs or groups of related RSVs discriminated between experimental dust communities under different lighting regimes. The details of this analysis are described by Fukuyama et al.  and summe values .apparent abundances, of taxa in pairs of nearly identical communities where one has lost a small number of abundant community members. These changes are said to be apparent because the underlying communities are otherwise identical; differences in RSV abundances only seem to occur as a result of the loss of highly abundant taxa, which relaxes limitations on the detection rates of all others [We build upon theory developed by Klein et al.  and devel others . The priS bacterial taxa in a community. We assumed a lognormal distribution for this SAD, which is commonly used in ecological models [μ,σ). Second, we assumed that the fraction of the χi gene copies which originate from living cells is given by the logistic function Our model is derived from two community scale patterns. Analogous to the species abundance distribution in ecology , we firsl models , whence ϕ and λ are the minimum and maximum viabilities, k is a parameter describing the steepness of the curve, and χ0 is a half-saturation constant. Thus, ϕ,λ,χ0, and k) to evaluate a wide range of community structures and dependencies between total DNA amounts and viabilities.where 4 iterations of this simulation procedure, independently drawing parameter values from uniform distributions  communities varied significantly with treatment type  indicating that patterns in bacterial abundances were in part determined by exposure to light and variation in particular wavelengths  or ultraviolet  light wavelengths. We detected minor but significant differences between total communities experiencing visible and UV light , suggesting that different light wavelengths effected only minor changes in community RSV membership and abundance distributions for living and dead taxa.Absolute abundance-weighted β-diversity patterns , indicating that differences between bacterial dust communities experiencing visible and ultraviolet light wavelengths manifested primarily in abundances of dead members of those communities. Living dust communities were distinct from their combined living and dead counterparts on average, regardless of light treatment . A multivariate dispersion analysis  revealed that quantitative community compositions in the dark were more variable than in either visible or UV light microcosms  portion of each dust community exhibited similar quantitative ns Fig. 2; we did P=0.051) and ultraviolet  and UV light  were marginally lower in visible  and instead contained a plurality of outdoor air-derived genera  would indeed change exposures raising the possibility of linkages between the spatial context of buildings, design decisions that impact the transmittance of light, geographical or seasonal variation in sunlight availability, and the structure of indoor dust microbial communities.Our experiment suggests that the use of ultraviolet-filtering glazing, that is found in many if not most buildings, may not be a significant shaper of indoor dust communities as originally anticipated, in comparison to glazing that transmits ultraviolet wavelengths. It also suggests that architects and lighting professionals designing building facades and rooms with more or less access to daylight may play a role in influencing the microbial communities of indoor dust. However, the impacts of daylight exposure on the dust microbiome uncovered here, relative to other factors like building occupancy, geography, ventilation, and humidity , 64, 75,Additional file 1Supplementary Information. Additional details on training data used for microbial source tracking and dust homogenization techniques. (PDF 68 kb)Additional file 2Table S1. Table describing the range of parameter values considered for sampling model simulations. (XLSX 29 kb)Additional file 3Table S2. Table describing the taxonomy of RSVs identified by sLDA analysis. (XLSX 10 kb)


The aim of the present study was to identify genetic variants based on RNA-seq data, obtained via transcriptome sequencing of muscle tissue of pigs differing in muscle histological structure, and to verify the variants’ effect on histological microstructure and production traits in a larger pig population.RNA-seq data was used to identify the panel of single nucleotide polymorphisms (SNPs) significantly related with percentage and diameter of each fiber type . Detected polymorphisms were mapped to quantitative trait loci (QTLs) regions. Next, the association study was performed on 944 animals representing five breeds  in order to evaluate the relationship of selected SNPs and histological characteristics, meat quality and carcasses traits.longissimus muscle samples confirmed a significant positive effect of transforming acidic coiled-coil-containing protein 2 (TACC2) polymorphisms on fiber diameter, while SNP within forkhead box O1 (FOXO1) locus was associated with decrease of diameter of fiber types IIA and IIB. Moreover, subsequent general linear model analysis showed significant relationship of FOXO1, delta 4-desaturase, sphingolipid 1 (DEGS1), and troponin T2 (TNNT2) genes with loin ‘eye’ area, FOXO1 with loin weight, as well as FOXO1 and TACC2 with lean meat percentage. Furthermore, the intramuscular fat content was positively associated (p<0.01) with occurrence of polymorphisms within DEGS1, TNNT2 genes and negatively with occurrence of TACC2 polymorphism.Mapping of detected genetic variants to QTL regions showed that chromosome 14 was the most overrepresented with the identification of four QTLs related to percentage of fiber types I and IIA. The association study performed on a 293 This study’s results indicate that the SNP calling analysis based on RNA-seq data can be used to search candidate genes and establish the genetic basis of phenotypic traits. The presented results can be used for future studies evaluating the use of selected SNPs as genetic markers related to muscle histological profile and production traits in pig breeding. Analyzing transcriptomes using next generation sequencing methods (RNA-seq) has been useful in various applications: estimation of gene expression; identification of novel genes or transcripts; and detection of genomic variants, gene fusion and RNA editing ,2. AccorIn pigs, an RNA-seq approach enabled Jung et al  to detecIt has been well-established that percentage number of fiber types in muscle determines metabolic properties and water holding capacity of muscle tissue, as well as marbling, intramuscular fat (IMF) level, meat color and texture –12. In olongissimus lumborum samples collected from 11 Large White pigs that differed in the histological structure of this muscle. Pigs (unrelated sows) were maintained under the same housing and feeding conditions and slaughtered at final body weight of 100 kg (±2.5 kg). Immediately after slaughter, muscle samples were collected and stored at −20°C in RNAlater solution  for RNA-seq application and at −80°C for histological analysis. The details of the animals used, the RNA-seq experiment and histological analysis were described in a previous study focused on differential expression analysis ; forkhead box O1 [FOXO1]). Two additional polymorphisms within two genes related to analyzed histological properties  were selected as significant based on the transcriptome-wide association study. For all genes, genotypes were assessed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method  and polymerase chain reaction-artificially created restriction site (PCR-ACRS) method (FOXO1). The details of the analyses were presented in For an association study, four genes were selected based on high gene expression differences during RNA-seq analysis and their location within QTLs related to percentage of fibers I, IIA, and IIB . The analysis was performed in two measurement points for each sample.For the downstream association study, a larger pig population comprising 944 animals representing five breeds  was used. All pigs (sows) were unrelated at least three generations back and were maintained under the same housing and feeding conditions according to Pig Test Station procedures. In the Test Station pigs are slaughtered, dissected and after carcass evaluation, meat is standard intended for consumption, thus research does not require the approval of Animal Experimentation committee. Pigs were fattened from 30 kg up to 100 (±2.5) kg according to test procedures described previously by Ropka-Molik et al  and werelongissimus muscle samples were collected immediately after slaughter and histological profile—percentage and diameter of the I, IIA, and IIB types of fibers—were estimated according to the procedure described previously by Ropka-Molik et al [For selected animals, in total 293 , ik et al . The aniik et al . The obtThe statistical analysis for all selected genes was performed using the general linear model (GLM) procedure in SAS software  and the model used was:ijk, the trait measured; μ, the overall mean for the trait; fi, the fixed effect of j genotype group of analyzed gene; hj, the fixed effect of k breed; α(xij), covariate with weight of right half-carcass for selected slaughter traits; eijk, random error.Where, YThe correlation coefficients between histological features and carcass characteristics and meat quality traits were calculated using Pearson correlation method .Based on RNA-seq data, 65,535 SNPs were identified. After filtering according to the criteria outlined above, 51,590 SNPs were selected for use in the association study. The significant association of SNPs and percentage of I, IIA, and IIB fiber types were identified  . We idenTACC2, DEGS1, and FOXO1 genes, which were related to multiple histological properties.Several polymorphisms/genes influenced more than one analyzed histological trait, simultaneously affecting the percentage and the diameter of the fibers. The identified panel of genes was the basis for selecting significant SNPs for subsequent GLM analysis performed on a larger population. From such genes, we selected the Based on the pig QTL database (pigQTLdb), DEGs (n = 355) identified by the RNA-seq, were located within QTLs related with fiber percentage and diameter . The most interesting locus seems to be on chromosome 14, at which 23 genes located in regions affecting percentage of I and IIA fibers were identified. DEGs localized in QTLs associated with percentage of fiber type IIB were identified mainly on chromosome 2 (SSC2) and SSC10 . The mosAfter the transcriptome-wide association study, 149 genes were selected (222 SNPs) and mapped to QTL regions. Results showed that chromosome 14, on which four QTLs were identified , was the most overrepresented . Also, oTNNT2; FOXO1; TACC2; DEGS1. These genes were previously identified as differentially expressed between muscle characterized by different histological properties; were localized within QTL regions associated with percentage of fiber types , and contained SNPs potentially related with muscle microstructure. Moreover, subsequent GLM analysis showed significant relationships of FOXO1, DEGS1, and TNNT2 genes with loin ‘eye’ area, FOXO1 with loin weight, as well as FOXO1 and TACC2 with lean meat percentage. Furthermore, the significant (p<0.01) effect of polymorphisms within DEGS1, TACC2, and TNNT2 genes on IMF content was detected  confirmed significant effect of FOXO1 and TACC2 genes on histological characteristics in pigs. FOXO1 was associated with diameter of fiber types IIA and IIB (p<0.01)  . The lowtype IIB .Furthermore, our result confirmed also significant correlation between selected histological traits and carcass characteristic. The diameters of all fiber types were positively, significant correlated with weight of loin. Diameters of fibers I and IIA was also correlated with lean meat percentage, while diameter of fibers I and IIB with meat color parameter, yellowness (b*) . PercentCurrently, much focus is placed on improving meat quality traits in pig breeding programs while maintaining satisfactory levels of meat and fatness characteristics . ResearcTNNT2, FOXO1, myosin XVIIIB, FERM domain containing 4B, and scavenger receptor class a member 5 [The present study is a continuation of previous research concerning identification of genes determining muscle fiber structure in pigs . The difmember 5 . The SNPMapping both DEGs and SNPs significantly correlated with muscle microstructure to the known QTLs showed that a high number of genes were localized on SSC14. The most overrepresented were QTL regions associated with percentage of fiber type I (ID#2822 and #2824), IIB (ID#2823 and #7024), and diameter of fibers IIA (ID#7038) and IIB (ID#7018). These results were consistent with previous findings of Wimmers et al   due to the highly potential results on protein structure, as well as biological function.Genome location was one of the selection criteria of genes for the subsequent GLM analysis. The association study carried out on a larger group of animals confirmed the significant effect of scrutinized polymorphisms in or study  indicateTACC2 gene affected diameter of both fiber types I and IIA and percentage amount of fiber IIB. The same polymorphism was also significantly related with lean meat percentage as well as IMF content. In the cell cycle, TACC2 protein is involved in G2/M progression [TACC2 gene is a critical factor controlling mitotic spindle dynamics during the normal course of the mitosis process [TACC2 gene in fiber type specification or muscle growth, even though this gene is highly expressed in skeletal and cardiac muscle. Our study suggested that transforming acidic coiled-coil protein 2 could play an important role during muscle fiber differentiation, resulting in modification of the number and size of muscle fibers. In turn, the differences in histological features are related to muscle mass and/or meat quality traits. The fiber composition in muscle is related with fatty acid and glycogen content due to the type of energy sources. The fiber type I is characterized by high fatty acid storage and low glycogen content, whereas the opposite trend is observed for the fiber type IIB. The observed missense variant changing arginine to cysteine in two different TACC2 isoforms  may affect function of synthesized proteins. Our results confirmed previous findings that percentage amount of type fiber IIB is inversely related with IMF [TACC2 missense variant, pigs with the AA genotype had significantly lower numbers of fiber type IIB and higher IMF content.Our results show that investigated SNP in gression  by playi process . To datewith IMF . AccordiFOXO1 mutation and diameter of fibers IIA and IIB and carcass characteristics such as weight of loin, loin ‘eye’ area and lean meat percentage. Furthermore, the significant correlation was observed between diameter of all fiber types and weight of loin. A previous study [FOXO1 gene depends on histological microstructure of muscle tissue. Moreover, our current research, which confirmed a significant relationship of a 3′UTR variant in the FOXO1 gene with fiber diameter, suggests that the analyzed SNP can affect FOXO1 transcript levels and histological profile as a result. Schiaffino and Mammucari [FOXO1 gene leads to muscle atrophy and increases transcript level of cathepsin L [FOXO1 was characterized by decrease size of both fiber types I and II, likewise reducing the number of fiber type I. Subsequently, the up-regulation of the cathepsin L gene can indicate increasing protein degradation processes in muscle tissues. The FOXO1 gene has been considered as a main genetic factor closely associated with muscle fiber type specification and skeletal muscle differentiation [FOXO1 gene is related to muscle mass, as well as size of fiber types IIA and IIB, and should be investigated in the future for relation with important production traits.Conversely, our results indicated significant association between a us study  showed dammucari  showed thepsin L . Researcntiation . Our resDEGS1 and TNNT2 genes with histological traits. However, polymorphisms identified in both genes affected loin ‘eye’ area and lean meat content in carcasses. This suggests that detected SNPs can be related with muscle growth processes but do not affect histological structure of muscle tissue at the same time. Furthermore, there is also the possibility of occurrence of another polymorphism within the DEGS1 and TNNT2 loci (in promoter or intron regions), which are associated with gene expression, and as a result, with cell size and fiber type distribution.Our results did not show the significant association of The use of RNA-seq data enabled us to identify a panel of SNPs within coding and UTRs regions with potential effects on histological microstructure. The genes detected by differential expression and the transcriptome-wide association analyses enabled us to select candidate genes potentially related to distribution of fiber types in porcine muscle tissue. These results can inform future studies evaluating the use of selected SNPs as genetic markers related to muscle histological profile and production traits in pig breeding.


Human-induced rapid environmental changes challenge individuals by creating evolutionarily novel scenarios, where species encounter novel enemies, the new species sometimes being humans themselves. However, little is known about how individuals react to human presence, specifically whether they are able to habituate to human presence, as frequently assumed, or are selected based on their fear of humans. We tested whether fear of humans  is reduced through habituation to human presence (plasticity) or whether it remains unchanged throughout the individuals' life. Results show an unusually high level of individual consistency in fear of humans throughout the adult lifespan of both rural (r = 0.96) and urban (r = 0.90) birds, lending no support to habituation. Further research should assess the role of inter-individual variability in fear of humans in shaping the distribution of individuals and species in an increasingly humanized world. The two habitats greatly differ in their degree of human presence, with marked differences in fear of humans among individuals occupying themParus major), suggesting that individual variations in some behaviours such as exploration may largely be shaped by mechanisms acting within populations20Here, we investigated long-term individual variation in fear of humans taking advantage of a large data set of individually marked burrowing owls , most individuals (n = 202) were tested for FID in only one year, while inter-annual repeated measures were available for 136 birds . FIDs greatly varied among individuals, ranging from 3.5 to 130 m.REML = 2.68, 2(df = 2) = 2.84, p = 0.2417). Thus, even when adult birds varied in their average level of fear of humans (Vi), their long-term responses were similar (no IxE effect). However, our data structure  yields an estimated statistical power of 1 to detect Vi but just 0.18 to detect IxE  to explain variability in FID indicated that sex of birds, the habitat where they lived and the successive years birds were tested affected this behaviour , significantly exceeding the value obtained in simulations . This reehaviour . Owls br1,24 = 9.59, p = 0.0049) but not for urban  birds. The variance among rural territories seems to have had a reduced effect on individual FIDs since models including the individual as random terms were much better supported than models fitting individual nested within territory  but no sexual  or age  effects. Although the two latter effects might have lost statistical significance due to the smaller sample size, the lack of a significant age effect may also result from a bias in sample size towards urban birds , whose FID did not change across years. Regardless, it is worth noting that individuals showed no signals of habituation to humans from their first reproduction through their adult lifespan (The subsample of adults banded as chicks and tested for FIDs since their first reproduction (n = 38) showed significant differences between rural and urban birds  in the FID of rural burrowing owls26Differences in fear of humans among populations have been traditionally explained through habituation1617282961719It is worth noting that our study focused on the adult lifetime of individuals, and thus we cannot discard parental or early experience effects3536Capture, banding and FID measures of Burrowing owls were conducted under permits from the Argentinean wildlife agency (22500-4102/09), the Ethic Committee of CSIC (CEBA-EBD-11-28), and the owners of private properties.Athene cunicularia) is found across American open landscapes, showing diurnal activity and nesting in burrows excavated by the owls or by mammalsThe burrowing owl , a young city reaching 293,000 inhabitants in recent yearsWe annually GPS-located up to 480 nests of burrowing owls in a 4,000 kmWe trapped >1,000 adults and chicks since 2006 using bow nets and ribbon carpets to mark them with a plastic colour-numbered ring readable at a distance. Pair members were sexed based on plumage patterns and colourationWe investigated intra-individual consistency in FID (log-transformed) as well as factors influencing its inter-individual variability using Generalized Linear Mixed Models . Whenever FIDs of some individuals were measured in different years and these inter-annual trials were measured in different contexts (territories), we first examined the optimal random effect structure testing whether or not individuals were nested within territoriesbeyond optimal model) to find the optimal structure of the random component using Restricted Maximum Likelihood (REML) estimators and comparing AICREML values. Once the optimal random structure was found, we modelled the fixed term and compared models using Maximum Likelihood (ML) estimators. Final models and repeatabilities are presented using REML estimatorsi”) while taking into account differences in behavioural plasticity . For each individual, average annual FIDs were sequentially numbered, and the test number was used as the environmental variable, which reflects the accumulated exposure of individuals to humans with years. In the case of known-age birds tested from their first-year onwards (see below), the test number equals their age in years. Evidence for the presence of a reaction norm (I × E) was assessed by comparing through a likelihood-ratio test2). We calculated the power of our data structure to detect Vi and IxE, as well as the sample size necessary to properly test them using simulationsWe started with a model where the fixed component contained all explanatory variables (the 2α / (σ2α + σ2ϵ), where σ2α and σ2ϵ can be extracted directly from the output of mixed modelsp value was the proportion of randomizations that yielded rrandomized ≥ robserved. 95% CI obtained in simulations are provided.Repeatability of FID was estimated as r = σ


Canis familiaris), most likely as a variant of the feline panleucopaenia virus. Relatively recently, canine parvovirus-2a and canine parvovirus-2b infections have been identified in both symptomatic and asymptomatic domestic cats, while canine parvovirus infections have also been demonstrated in wild felids. This report documents the first known case of canine parvovirus-2b detected in unvaccinated serval  from South Africa. The serval presented with clinical signs of vomiting, anorexia and diarrhoea that responded to symptomatic treatment. Two weeks later, severe leucopaenia, thrombocytopenia and death occurred. Typical enteric histological lesions of parvovirus infection were not observed on histopathological examination of the small intestine; however, histological lesions consistent with septicaemia were present. Canine parvovirus was detected in formalin-fixed paraffin-embedded small intestine using polymerase chain reaction. Phylogenetic analysis of the sequence of the canine parvovirus viral capsid protein gene showed similarities between the sample from the serval and canine parvovirus-2b isolates from domestic dogs in Argentina and South Africa. A case of canine parvovirus-2b in a domestic dog from South Africa in 2012 that fell within the same clade as the serval sample appears distantly related because of the long branch length. The significance of these findings is explored. More extensive surveys of canine parvovirus in domestic and wild felids and canids are needed to understand the epidemiology of canine parvovirus in non-domestic felids in South Africa.Canine parvovirus first emerged in domestic dogs ( Parvoviridae) and their associated diseases affect various carnivores including felids and canids , during the 1930s and 1940s, and also infects non-domestic felids (Parrish Mustela vison), and within 15 years, it was observed on mink ranches throughout Canada, the United States, Europe and Scandinavia , most likely as a variant of FPLV or a closely related parvovirus. Although the exact mechanism of emergence is unclear, amplification of parvovirus DNA sequences intermediate between FPLV and CPV from red foxes (Vulpes vulpes) suggests that wildlife may have played a role in the adaptation of the virus in a new host  first emerged in domestic dogs , Siberian tiger , African wildcat (Felis lybica), puma (Puma concolor), ocelot  and spotted cat (Leopardus tigrinus) , mink (Neovison vison) and arctic fox (Vulpes lagopus) , grey wolf (Canis lupus), maned wolf (Chrysocyon brachyurus), crab-eating fox (Cerdocyon thous), bush dog (Speothos venaticus), dingo (Canis lupus dingo), raccoon dog (Nyctereutes procyonoides) and African wild dog (Lycaon pictus)  with histological lesions typical for parvoviral disease and in another with salmonellosis , severe leucopaenia and thrombocytopenia (subjectively evident on a blood smear). Treatment was initiated but the animal died the following day. No evidence of warfarin or other anticoagulant exposure was reported. No further clinical tests or culture were performed, and no bone marrow was submitted for evaluation or further analysis.A necropsy showed that the serval was in good condition with mild icterus, a slightly enlarged liver and mild fluid accumulation in the body cavities. The cause of death was suspected to be because of aspiration of blood from the epistaxis. Selected tissue samples preserved in 10% buffered formalin were processed routinely and stained with haematoxylin–eosin stain. The histopathological examination confirmed septicaemia . Hepatic macrophages contained bile pigment in the cytoplasm. The urinary bladder mucosa showed multifocal mucosal ulceration and haemorrhage with mild submucosal lymphoplasmacytic interstitial infiltration and fibroplasia. An unspecified lymph node was mildly active and cortical follicular structures contained central hyalinisation of the stroma and possible fibrin deposition. The small intestine did not reveal any enteric lesions including changes characteristic of parvovirus-induced necrosis.The molecular diagnostics and phylogenetic analyses formed part of a larger project at the National Zoological Garden, South African National Biodiversity Institute , which investigates the molecular identification and genetic diversity of FPLV in both wild and domestic felids  following the manufacturer’s specifications for FFPE tissue, and deparaffinisation of FFPE samples was performed using xylene.Nucleic acids from a formalin-fixed, paraffin-embedded (FFPE) intestinal sample (PV19) were extracted using the Epicentre MasterPure™ Complete DNA & RNA Purification Kit (Whitehead ScientificµL nuclease-free water (supplied with DreamTaq™), 12.5 µL DreamTaq master mix, 1 µL of each primer  and 2 µL DNA sample standardised to a concentration of 50 ng/µL. Cycling conditions for tissue isolates using primer sets 2 to 5 were as follows: initial denaturation (94 °C for 5 minutes), 30 cycles of denaturation (94 °C for 30 seconds), annealing (58 °C for 50 s) and extension (72 °C for 1 min). A final extension step (72 °C for 10 min) concluded the cycling. Cycling conditions for primer sets 1 and 6 were identical except annealing occurred at 54 °C. Conditions were adjusted for FFPE isolates by decreasing the annealing temperatures to 54 °C for primer sets 2 to 5, and 50 °C for primer sets 1 and 6. Polymerase chain reaction setup was performed in a DNA-free hood and a DNA negative control was included.Molecular analysis of parvovirus from the isolate was achieved by amplification of a ~1400 bp region of the viral capsid protein (VP2) gene  using Dr®, Boehringer Ingelheim; Felocell®, Zoetis), six CPV positive case samples and 42 parvovirus reference strains (https://www.ncbi.nlm.nih.gov/). Identical field isolate sequences were selectively removed, to reduce the phylogenetic tree size, so that only representative isolates remained.All positive amplicons were purified using an exonuclease I and alkaline phosphatase PCR purification protocol, sequenced using BigDye V3.1 (Applied Biosystems) chemistry and analysed on an ABI3500 genetic analyser. Sequences were aligned using the ClustalX function incorporated in MEGA6 .Results from the NJ analysis (1000 bootstrap replicates) illustrate a clear separation between FPLV and CPV isolates, as indicated in This study reports the first detection of CPV-2b in a captive serval from South Africa, while the study by Lane et al.  previousThe significance of the presence of CPV and FPLV in tissues from servals with septicaemia is uncertain. Parvovirus is known to cause lymphoid necrosis and atrophy in lymphoid organs such as the thymus, gut-associated lymphoid tissue and lymph nodes as well as intestinal epithelial necrosis. The resultant immunosuppression and breach of intestinal integrity make affected animals susceptible to secondary systemic bacterial infection. Typically, domestic dogs and cats that die as a result of septicaemia secondary to parvoviral infection show histological lesions of intestinal epithelial necrosis. Detection of FPLV in a serval (PV15) with typical intestinal necrosis suggests that the disease, in FPLV at least, may show a similar course in servals. However, as domestic cats have been shown to be asymptomatic carriers of CPV-2a, -2b and -2c . Information on the contact or not between domestic dogs and this serval was not available. Further investigation of samples PV36, PV38 and PV40 may reveal whether or not these isolates represent unique variants of CPV-2a. The case of CPV-2c isolated from a domestic dog sample collected in Namibia is most likely an artefact of shortening the sequences to the shortest sequence in the data set, as this virus variant has not been reported in Africa or Australia (Decaro et al. Canine parvoviruses have spread widely across the globe since the 1970s because of contact with infected animals, inanimate fomites, flies or other reservoirs (Bagshaw et al.


ER/PR index to measure gene expression microarray probe sets that were correlated with hormone receptors (ESR1 and PGR) and robust to preanalytical and analytical influences. We tested SETER/PR index in biopsies of metastastic HR+/HER2− breast cancer against the treatment outcomes in 140 patients. Then we customized the SETER/PR assay to measure 18 informative, 10 reference transcripts, and sequence the ligand-binding domain (LBD) of ESR1 using droplet-based targeted RNAseq, and tested that in residual RNA from 53 patients. Higher SETER/PR index in metastatic samples predicted longer PFS and OS when patients received endocrine therapy as next treatment, even after adjustment for clinical-pathologic risk factors . Mutated ESR1 LBD was detected in 8/53 (15%) of metastases, involving 1−98% of ESR1 transcripts . A signature based on probe sets with good preanalytical and analytical performance facilitated our customization of an accurate targeted RNAseq assay to measure both phenotype and genotype of ER-related transcription. Elevated SETER/PR was associated with prolonged sensitivity to endocrine therapy in patients with metastatic HR+/HER2− breast cancer, especially in the absence of mutated ESR1 transcript.There is a clinical need to predict sensitivity of metastatic hormone receptor-positive and HER2-negative (HR+/HER2−) breast cancer to endocrine therapy, and targeted RNA sequencing (RNAseq) offers diagnostic potential to measure both transcriptional activity and functional mutation. We developed the SET Notably, the onset, rate and mechanisms of molecular progression vary for each patient.Endocrine therapy is the principal treatment for metastatic HR+/HER2− breast cancer until resistance becomes clinically manifest.9 combine this with another targeted therapy, or switch to chemotherapy-based treatment. Furthermore, it might inform a secondary concern: when in the course of therapies for metastatic breast cancer it might be optimal to add a cdk4/6 or PI3kinase/mTOR inhibitor to endocrine therapy. For example, it is still unclear whether addition of currently approved targeted agents to endocrine therapy in advanced disease improves progression-free survival (PFS) by reversing endocrine resistance or augmenting partial endocrine sensitivity.Clinically, endocrine treatment resistance is recognized from short disease-free interval in the adjuvant or metastatic setting of endocrine treatment, development of visceral disease, or loss of ER or PR in metastatic breast cancer. However, these criteria are inexact. A quantitative biomarker of sensitivity to endocrine therapy (SET) in metastatic cancer might potentially contribute clinically useful information to address a clinical conundrum: whether to continue with endocrine therapy,ESR1-related transcripts in early breast cancer,10 we hypothesized that a combination of genes with expression related to both estrogen and progesterone receptors (gene symbols ESR1 and PGR), but not proliferation, might predict sensitivity to endocrine therapy in metastatic breast cancer.11 We also considered preanalytical and analytical effects on measurement of gene expression in our approach to select transcripts for our signature, in order to develop a technically robust signature of a few genes that we could then translate to a customized assay with strong analytical validity. In addition, we considered that elevated hormone receptor-related transcription might represent natural activity (and indicate sensitivity to endocrine therapy) or perversely result from constitutive activating mutation of ESR1 transcripts . Overall, we felt that the current evidence for altered biology of progressive breast cancer after relapse requires a more specialized approach to risk stratification than adoption of multi-gene assays that were developed for the earliest stages of hormone receptor-positive breast cancer.15 Hence, we aimed to combine both genotypic and phenotypic information, using a customized RNA sequencing (RNAseq) assay to measure sensitivity to endocrine therapy (SET).Based on our previous development of a signature of ESR1 and PGR and without obvious association with proliferation) and ten reference transcripts were selected for inclusion in the SETER/PR index , intratumoral sampling (ICC = 0.953), type of cancer sample , and type of microarray platform . Score values obtained from Plus2.0 arrays had a slight bias towards higher values when compared to U133A microarrays . SETER/PR was stable over relevant ranges of contamination with liver or normal breast tissue with negative score values regressing more rapidly to the baseline levels from normal liver or normal breast tissues. Categories of high vs. low SETER/PR index (relative to median of 0.82) were consistent  over a range of 0−90% RNA added from normal liver or breast. There was no statistically significant effect of time delay (ex vivo ischemic time) and sample preservation method (RNAlater versus snap frozen) on SETER/PR measurements  and prior clinical history of endocrine sensitivity .The characteristics of 140 patients with hormone receptor-positive, HER2-negative metastatic breast cancers are summarized in Table ER/PR index was prognostic for PFS and OS in patients receiving endocrine-based therapy (PFS: hazard ratio (HR) 0.51 (0.41−0.74), p < 0.001; OS: 0.40 (0.26–0.62), but not in patients receiving chemotherapy (PFS: HR 0.76 (0.45−1.27), p = 0.290). We selected the median value (0.82) as threshold to dichotomize SETER/PR index. Dichotomized SETER/PR was independently prognostic for PFS  and the proportion of ESR1 transcript reads with activating LBD mutation. SETER/PR index was calibrated between microarray and customized RNAseq assays in 40 breast cancer samples analyzed in duplicate with both assays  in an independent test of 23 breast cancer samples.The customized RNAseq assay integrates measurements of ER and PR-related transcriptional activity  may be intermediate between those with low SETER/PR index and high SETER/PR index with wild-type ESR1  and analytical conditions . We also describe how it was customized into an assay that also integrates measurement of mutated ESR1 transcripts.SETER/PR is the first multigene expression assay to be developed specifically for metastatic breast cancer. Higher SETER/PR index was associated with longer PFS and OS for patients treated by endocrine therapy, particularly for those who had previously demonstrated clinical sensitivity to hormonal therapy. Although we observed that SETER/PR was not associated with outcome in patients treated with chemotherapy, that cohort was too small to be able to make any conclusion. Additionally, the observation might be confounded because chemotherapy is usually offered when there is already clinical evidence for endocrine resistance. We also note that high expression of SLC39A6 is observed in the SETER/PR index  treatment, for example fulvestrant. In the FERGI and PALOMA-3 trials, ESR1 mutations had no effect on PFS in patients receiving fulvestrant with or without a PI3K inhibition or cdk4/6 inhibition, respectively.19 In the BOLERO-2 trial, patients with ESR1 mutations had shorter PFS under exemestane with or without everolimus.20 In the SoFEA trial, patients with ESR1 mutations had a longer PFS after a regimen containing fulvestrant as compared to anastrozole.19 While available data are inconclusive, there appears to be a trend toward associations of ESR1 mutation and endocrine resistance that might be reversed by SERD treatment.ESR1 mutations were associated with higher values of SETER/PR (presumably because these mutations are constitutively activating), but only some cancers with higher values of SETER/PR index contained an ESR1 mutation. Indeed, we observed three main groups in our data: (1) high SETER/PR index with wild-type ESR1 (better prognosis with endocrine therapy); (2) low SETER/PR index with wild-type ESR1 (worse prognosis with endocrine therapy); and (3) high SETER/PR index with activating mutation of ESR1 (possibly intermediate prognosis with endocrine therapy). This potentially highlights the importance of integrating both transcriptional measurements (phenotype) with mutation status (genotype) to understand genomic effects on sensitivity to endocrine therapy. A future challenge will involve accurate combination of SETER/PR index with the percent of mutated ESR1 transcripts, since this RNAseq assay precisely measures that fraction even to minimal values because the number of transcripts per cell and the depth of sequencing are both high. Our results are in line with the assumption that transcriptional activation by ER in metastatic disease could be pertinent to endocrine sensitivity in the context of wild-type ESR1, but could be active yet resistant to endocrine treatment if predominantly due to mutant ESR1. However, a far larger experience of samples data and treatment outcomes will be necessary to understand whether this is a real observation and whether the proportion of mutant ESR1 is relevant to outcomes. Clinical utility will depend on further clinical validation and how the information might inform treatment options.We observed that PGR expression. The presence of progesterone receptor is considered an indicator of estrogen-dependence and better differentiation of a tumor with the most favorable prognosis being associated with the phenotype or ER- and PR-expressing tumors.21 Recently, it has been shown that PR can directly remodulate ER-associated transcriptional profiles by altering its chromatin-binding characteristics, indicating complex interaction between ER and PR.22 In early-stage breast cancer, estrogen receptor-related transcriptional profiles can predict prognosis following endocrine therapy10 and PR might be prognostic rather than predictive for endocrine response.23 However, PR holds greater interest for endocrine prediction in stage IV disease.11 Thus, even if we did not have semiquantitative data on PR expression for all patients, it is important that the SETER/PR index remained prognostic for endocrine therapy even after adjustment for PR immunohistochemistry status (≥10% nuclear staining) and the other relevant clinical risk factors , include an SERD agent to target emergent mutated ESR1 clone, or switch to a different treatment strategy . Of course, any definitive statement on such clinical utility would require testing the SETER/PR index using samples from randomized trials and goes beyond the scope of this first description of the assay. But even within those trials, we might gain insight as to whether the addition of different targeted therapies might augment sensitivity, or reverse resistance to endocrine therapy—questions that are difficult to answer without a biomarker for endocrine sensitivity.Treatment of stage IV HR+/HER2− breast cancer typically relies on available endocrine treatmentsER/PR index and percent mutated ESR1 transcripts. Another limitation is the lack of an independent clinically annotated cohort to validate the findings that would also allow the definition and validation of an optimized cut-point for patient stratification.There are several important caveats to the interpretation and generalizability of our results. Despite an overall sample size of 140 prospective biopsies of relapsed metastatic disease, the clinical and treatment subsets are small, requiring cautious interpretation of these results. This is a limitation of the combined analysis of SETOverall, this manuscript introduces a novel approach to assay development and this assay appears to be analytically valid. The promising clinical performance is still exploratory, and further independent clinical validation studies of the assay and its cut-point will still be required.29All patients gave informed written consent to take part in the study and for the use of tissue material for research purposes. Protocols were approved by the MD Anderson Institutional Review Board (IRB). The microarray and accompanying data are available on NCBI GEO and summarized under a figshare metadata record.N = 389) from invasive hormone receptor-positive breast cancers included 242 cases from our published dataset10 and 147 additional samples (GSE129551), all derived from fresh tissue or FNA biopsy samples obtained prior to any systemic therapy and stored frozen at −80 °C in RNAlater . Receptor status, tumor stage and type of tumor samples are described in Supplementary Table The discovery cohort of Affymetrix U133A microarrays  by fluorescence in situ hybridization.30 Target probe sets for gene transcripts in the 389 cases of the discovery cohort were identified based on Spearman’s rank correlation coefficient for coexpression with ESR1 and PGR (probe sets 205225_at and 208305_at) in hormone receptor-positive breast cancer samples. Reference probe sets were selected based on consistency and range of expression values. This manuscript follows REMARK guidelines.31RNA was extracted, processed and hybridized to Affymetrix human genome U133A microarrays  as described previously. In brief, the raw intensity files were processed using the MAS5.0 algorithm to generate probe set-level intensities, normalized to a median array intensity of 600, log2-transformed and scaled using the expression of 1322 breast cancer reference genes within each sample.ER/PR gene expression index in other studies: 11 breast cancers contaminated with increasing known amounts of liver RNA (GSE33116); 10 other breast cancers diluted with increasing known amounts of normal breast RNA (GSE124648); 17 other breast cancers with increasing duration of ischemic delay at room temperature, testing two sample preservation methods (GSE25011)32; matched U133A and Plus2 arrays in two different laboratories ; and technical replicates using U133A arrays in another 63 breast cancers from MDACC (GSE129560).We conducted a series of studies to evaluate the reproducibility of gene expression measurements in breast cancer samples according to replication of technical, intratumoral, interplatform, and inter-sample type conditions . These included 6 technical  replicates from 20 breast cancers GSE129558), 3 tumor samples from each of 51 breast cancers (GSE129557), inter-sample type comparisons of 116 matched cytology and tissue samples (GSE129559) that were collected from multiple institutions, and interplatform comparisons of Affymetrix U133A and Plus2 array platforms from 88 breast cancers (GSE129556). Figure 29558, 3 ESR1 transcript were analyzed for single nucleotide variants and reported as the percent of ESR1 reads and type of mutation.We employed a digital PCR-based RNAseq strategy with three steps: (1) droplet-generation using RainDance Source system  and one-step RT-PCR reaction (first PCR) to target the regions of interest with our custom multiplex primer set; (2) second PCR to incorporate RainDance DirecSeq primers for sample indexing and Illumina specific adapters for cluster generation/sequencing; (3) library quantification, QC, and llumina MiSeq sequencing . We perform pooled sequencing of up to 40 sequence libraries per flow cell. The read count of each targeted sequence was log2 transformed, and the sequence reads of the LBD of the 33 The clinical variable of prior endocrine sensitivity was defined as a history of at least 6 months of freedom from progression while on endocrine therapy for metastatic disease or 5 years adjuvant endocrine therapy for primary breast cancer without recurrence. A subset of 53 cases was available for analysis of ESR1 gene mutations by RNAseq.Patients with metastatic HR+ breast cancer were offered participation in a prospective research protocol to obtain a research sample at the time of their clinical biopsy of metastasis at MD Anderson (protocol LAB04-0093) between 2004 and 2013, obtained as fine-needle aspiration (FNA) or core biopsy (CBX). Their next treatment was recorded and was at the discretion of their oncologist. A total of 234 samples were profiled using Affymetrix U133A gene expression microarrays, 212 microarrays passed our quality control analysis. We excluded 32 HER2-positive and 26 hormone receptor-negative cases based on immunohistochemistry and (where appropriate) HER2 in situ hybridization testing of the metastatic samples. Fourteen additional cases were excluded for other reasons , resulting in 140 eligible cases with quality microarray data in this study (GSE124647). Median PFS and OS were 5.5 and 24.0 months, respectively (Table ρP) was used to compare cross-platform and cross-tissue reproducibility of each candidate probe set on the array. The intraclass correlation coefficient (ICC) was used to evaluate intra-assay and intratumoral reproducibility. A linear mixed-effects model (LME) with random within-group intercept was used to estimate the effect of sample preservation method (RNAlater vs. fresh frozen) and time delay (0 vs. 40 min)32 using the r package lme4. The effect of sample stabilization delay (cold ischemic time) was assessed using a similar model with fixed slope (for the cold ischemic time effect) and random intercept . The statistical significance of the coefficients was evaluated by using the likelihood ratio test to compare the full model with a reduced model that did not include the term of interest. To examine the impact of contamination with normal breast tissue and liver tissue, SETER/PR values were plotted against the percentage of contaminant. Fleiss’ κ statistic for multiple raters was used to evaluate the reproducibility of risk class assignment. We used the R package survival for survival analyses. PFS was defined as the time from the start of new treatment after the biopsy of relapsed disease, until disease progression or death from any cause. The endpoint definition for overall survival was death from any cause. We used Cox regression to model relationship between the continuous SETER/PR and survival outcomes. The Kaplan−Meier method and log-rank test were used to evaluate survival outcomes using the dichotomized score. All statistical analyses and computations were performed in R v. 3.1.2 34 and Bioconductor.35Pearson’s correlation coefficient (Further information on experimental design is available in the Nature Research Supplementary InformationReporting Summary Checklist


The two quantification approaches were compared. We also evaluated changes in the measures derived from diffusion tensor imaging and arterial spin labeling.We included 18 relapsing-remitting MS patients and 12 healthy controls. Each subject performed a hybrid [18F]florbetapir DVRs decreased from normal-appearing white matter to the centre of T2 lesion (P < 0.001), correlated with fractional anisotropy and with mean, axial and radial diffusivity within T2 lesions . Cerebral blood flow was reduced in white matter damaged areas compared to white matter in healthy controls . SUV70–90 and DVR are equally able to discriminate between intact and damaged myelin .[18F]florbetapir PET imaging can measure in-vivo myelin damage in patients with MS. Demyelination in MS is not restricted to lesions detected through conventional MRI but also involves the normal appearing white matter. Although longitudinal studies are needed, [18F]florbetapir PET imaging may have a role in clinical settings in the management of MS patients.Our findings demonstrate that [ Multiple Sclerosis (MS) usually presents with episodes of transient neurological deficits, called ‘relapses’ . Relapse11C]PIB, [18F]florbetaben and [18F]florbetapir PIB, 1Fflorbetarbetapir  also shorbetapir . In factrbetapir . Thereforbetapir .11C]PIB PET to image myelin using dynamic acquisitions that has a short half-life (about 20 min) that limits its clinical viability, whereas Fluorine-18 labelled PET tracers have longer half-life (about 120 min), which allows for a clinical use in patients MS. However, by combining PET and MRI Bodini and colleagues demonstrated that myelin changes, detected with amyloid PET imaging, within lesions was associated with physical disability florbetapir PET to quantify myelin content in MS patients. We investigated the relationship between [18F]florbetapir uptake and MRI measures, e.g. diffusion tensor imaging and arterial spin labelling together with standard measures of cortical grey matter volume. Finally, we compared SUV70–90 analyses with the DVR kinetic modelling to estimate the clinical applicability of PET imaging through simplified semi-quantitative PET measures.In this study we adopted a hybrid PET-MR approach using florbetapir, we performed a dynamic 90-min scan. PET dynamic images included 27 frames of data . MRI sequences included pre- and post-gadolinium three-dimensional magnetization-prepared rapid gradient-echo T1 , fast fluid-attenuated inversion recovery  and turbo spin-echo T2-weighed images . For each subject a diffusion tensor echo-planar imaging sequence was registered, with generalized auto-calibrating partial parallel acquisition, at 2-fold acceleration  and a single-shot gradient echo-planar imaging sequence was used to assess cerebral blood perfusion through pseudo-continuous arterial spin labelling .PET images were acquired using a 3-Tesla Magnetom Biograph mMR PET/MR hybrid scanner  with a spatial resolution of 4.3 mm and a FOV of 258 mm at St Thomas PET centre. After the intravenous bolus injection of approximately 300 MBq of florbetapir was implemented following procedures already applied to [11C]PIB [18F]florbetapir were defined for the normalized PET dynamic sequence using the twelve healthy controls, who received [18F]florbetapir PET in this study: normal appearing grey matter (class 1), blood pool (class 2) and white matter (class 3). Class 1 and 3 were calculated from the average of normalized time activity curve for [18F]florbetapir in the grey matter and white matter mask respectively. Class 2 was defined by extracting the 30 voxels with the highest [18F]florbetapir radioactivity concentration over the first 60 s of PET acquisition, corresponding to the big blood vessels. The supervised cluster algorithm performs multiple linear regressions for each voxel in the grey matter in order to model its kinetic as a linear combination of the predefined kinetic classes for [18F]florbetapir. For each voxel the weight from each class was calculated. Voxels with a class 1 weighted ratio higher than 0.9 were selected as reference regions and the time activity curve in this area was calculated as the average of the non-normalized voxel time activity curve.The supervised clustering algorithm for Fflorbeta18F]florbetapir binding measured as the DVR [Logan graphical reference method was applied at a voxel level on PET scans in native space to produce a parametric maps of [ the DVR .18F]florbetapir PET was also quantified using standardized uptake value (SUV) in the 70–90 min after injection. SUV70–90 map was generated by correcting absolute radioactivity concentrations  for subject body weight  and injected dose : SUV=C/(ID/BW). [18F]florbetapir SUV70–90 in the cerebellar cortex was compared between MS patients and healthy controls; if no differences were detected, the SUVR map would be calculated.florbetapir DVR between MS patients and healthy controls were tested using a general linear model including the group as factor of interest, age and gender as covariates and DVR as the dependent variable. Differences in [18F]florbetapir DVR between MS patients’ normal appearing, healthy controls’ white matter, and each defined region of interest were assessed using a mixed-effect linear model including regions of interest as the factor of interest, age and gender as covariates, the subject as the random effect and the DVR as the dependent variable. The relationship between lesion intensity on conventional T1 and T2 MRI images and DTI measures and [18F]florbetapir DVR values was further explored using a mixed-effect linear model including each lesion’s intensity or DTI measures as the factor of interest, age and gender as covariates, the subject as the random effect, and each lesion’s DVR as the dependent variable. The potential additional value of DVR over fractional anisotropy as measured through DTI in differentiating normal appearing white matter, outer T2 perilesion layer and inner T2 lesion layer was investigated via hierarchical linear regression analyses. In particular, age, gender and fractional anisotropy were entered in a first step using the brain region  as dependent variable. We chose the fractional anisotropy measure, as it is the most widely used measure from DTI to assess microstructural changes in white matter. The second step added DVR in order to evaluate the additional value of such measure in distinguish between the aforementioned brain regions. The association between [18F]florbetapir DVR and cortical and NAWM normalized volume was assessed through a mixed-effect linear model including mean T1 and T2 lesions DVR as the factor of interest, age and gender as covariates, the subject as the random effect, and the cortical and NAWM normalized volume as the dependent variable.Differences in white and grey matter florbetapir PET is a valid tool to detect the pattern of demyelination in MS, with a comparable accuracy between SUV70–90 PET measure and the computational DVR analysis methods. Combining PET with advanced MRI techniques, we showed that myelin damage was correlated with microstructural white matter changes and lower perfusion rate, providing additional information.In this study, we demonstrated that [18F]florbetapir binding from dynamic DVR and static SUV measures have comparable accuracy in differentiating myelin damaged tissue from NAWM. [18F]florbetapir quantification with SUV has a number of advantages including short scan-time, with acquisition within a predefined time window and lower costs compared to the dynamic continuous acquisition. Bodini and colleagues demonstrated that longitudinal [11C]PIB PET imaging scans quantified through the DVR, might depict not only demyelination but also remyelination over time [18F]florbetapir PET might deserve further studies to be confirmed.We showed that [ver time . Hence S18F]florbetapir binding was decreased in focal white matter lesions of the MS patients, defined on conventional MRI, compared to the tracer uptake in the white matter of the healthy controls. T1 lesions showed the lowest value, confirming that they are characterized by extensive myelin destruction [18F]florbetapir PET imaging can measure the extent of demyelination without including biases from other pathological changes, such as gliosis or axonal loss, and we demonstrated that DVR provides additional information on tissue damage compared to DTI measures. Further studies including other advanced MRI techniques such as magnetization transfer imaging might also evaluate the specificity of PET imaging over MRI in detecting demyelination.We showed that florbetapir binding was similar for MS patients and healthy controls in NAWM. The absence of difference in myelin content of grey and white matter that we found for the two groups appears not to be in line with previous studies that have shown tissue changes within both grey and white matter outside the lesions detected by conventional MRI through diffusion tensor imaging, magnetisation transfer imaging and spectroscopy [11C]PIB PET imaging study [18F]florbetapir PET imaging might detect with a single scan a larger areas of damaged tissue compared to conventional MRI and, thus, might be considered a better predictor of disease progression. Moreover, the increased size of a lesion may also depend on an ineffective and incomplete process of remyelination and does not necessarily implies the reactivation of the lesion. Interestingly, longitudinal [18F]florbetapir PET scans might shed further light in the biological bases of the remyelinating process around the lesions.florbetapir binding between MS patients and healthy controls, because cortical demyelinating lesions are becoming a hallmark in MS pathology [18F]florbetapir PET imaging within MS lesions per-se is not the cause of brain atrophy. Supposedly, the balance between remyelination, preserved axonal integrity and the wallerian degeneration of axons traversing lesions might impact the total brain volume and the cortical thickness [11C]flumazenil PET, specifically measuring neurodegeneration [11C]flumazenil and amyloid-β PET imaging study might also shed further light on the interplay between demyelination, eventually remyelination and axonal degeneration. A recent paper, using a complex approach involving optical coherence tomography, conventional and advanced MRI, reported that demyelination over a specific white matter tract (i. e. optic radiation) precedes neurodegeneration florbetapir DVR measures are unlikely to be significantly influenced by cerebral arterial blood perfusion.We observed a reduced cerebral arterial blood perfusion in white matter regions with a higher extent of demyelination. MS lesions and normal appearing white matter are characterized by cerebral hemodynamic impairment even in absence of blood-brain-barrier damage , resultiWe did not find any correlations between the extent of white matter lesions demyelination and the disease severity, assessed through the MSSS, disease duration and annualized relapse rate and physical disability assessed through the EDSS. However, we found a correlation between physical disability assessed through the Timed 25-ft walk and the extent of myelin loss within T2 lesions. Bodini and colleagues showed that both the EDSS and the MSSS were not related to the tracer DVR in T2 lesions at a cross-sectional evaluation . Physica18F]florbetapir PET imaging might be able to assess in vivo myelin pathology with a higher extent of specificity compared to MRI. The static SUV70–90 PET imaging evaluation provides similar information to the dynamic PET. Dynamic PET imaging requires long dynamic scans and, hence, high costs. Conversely, the SUV might be calculated through a static acquisition lasting for twenty minutes. This makes the technique highly appealing to implement in clinical settings. Clinicians could use the PET imaging as a tool to measure the subjective remyelinating potential, with the ultimate goal of personalizing treatment. They could also rely on its quantitative measures when assessing the effect of a drug targeting remyelination, e.g. opicinumab [In conclusion, our study reported that [icinumab . Finally