Cln3−/− mouse model of a juvenile form of NCL. We profiled and compared the molecular composition of anatomically-distinct, differentially-affected pre-synaptic populations from the Cln3−/− mouse brain using proteomics followed by bioinformatic analyses. Identified protein candidates were then tested using a Drosophila CLN3 model to study their ability to modify the CLN3-neurodegenerative phenotype in vivo. We identified differential perturbations in a range of molecular cascades correlating with synaptic vulnerability, including valine catabolism and rho signalling pathways. Genetic and pharmacological targeting of key ‘hub’ proteins in such pathways was sufficient to modulate phenotypic presentation in a Drosophila CLN3 model. We propose that such a workflow provides a target rich method for the identification of novel disease regulators which could be applicable to the study of other conditions where appropriate models exist.Synapses are an early pathological target in many neurodegenerative diseases ranging from well-known adult onset conditions such as Alzheimer and Parkinson disease to neurodegenerative conditions of childhood such as spinal muscular atrophy (SMA) and neuronal ceroid lipofuscinosis (NCLs). However, the reasons why synapses are particularly vulnerable to such a broad range of neurodegeneration inducing stimuli remains unknown. To identify molecular modulators of synaptic stability and degeneration, we have used the  Alzheimer (AD)4 and Parkinson disease6), neurodevelopmental conditions 8), protein misfolding/accumulation diseases (e.g. Huntington disease (HD)9), prion diseases10, spinocerebellar ataxias (SCA)11 and lysosomal storage disorders 16. However, our understanding of the reasons why specific synaptic populations are so vulnerable to such a broad range of neurodegenerative stimuli, and the mechanisms that govern their stability, remains in its infancy18.Synapses are an early pathological target in a range of diseases19. Incidence in the USA is estimated at 1.6–2.4/100,000 whereas in Scandinavian countries it is 2–7/100,00021. The term “NCL” currently encompasses up to 14 disease subtypes, which are grouped together due the lysosomal accumulation of autofluorescent storage material, distinct ultrastructural properties, broadly similar pathology and clinical features and a severe neurodegenerative phenotype22. Crucially for the purposes of this study, pre-synaptic disruption is a key early event in NCL, accurately predicting the distribution of subsequent neuronal loss16.In this study, we sought to define the molecular regulators of synaptic stability, using animal models of CLN3 disease . The NCLs, are the most frequent autosomal-recessive neurodegenerative disease and form of dementia in childhood25. However, the pathways and molecular cascades leading to neurodegeneration in NCL, as in many other neruodegenerative diseases, are still unknown.The knowledge of the underlying genetic cause and/or storage material composition have provided a base for the basic understandying of the pathogenesis and their correlation to the clinical progression of the disease, the design of gene replacement therapies and the development of animal modelsCLN3 underlie a juvenile form of NCL, the most prevalent form worldwide26. The CLN3 gene encodes a putative transmembrane protein whose function is not completely understood. However, experiments in yeast and in vitro studies have revealed possible functions relating to vacuolar pH regulation27 and endocytic membrane trafficking28. Although CLN3 is ubiquitously expressed throughout the body, the most obviously affected tissues are neurologic based. This feature is shared by other monogenetic neurodegenerative conditions such as SMA29. Yet, the reasons why neurons appear to be particularly vulnerable to defects in such broadly expressed proteins is not understood.Mutations in 30. Unlike other more complex neurodegenerative diseases, the growing knowledge of the NCL-causative genes has facilitated the development of powerful animal models in recent years. These models have significatively improved our understanding of the progressive nature of the different forms of NCL. Cln3 null mice (Cln3−/−) reproduce various aspects of the human disorder32. Although neuronal loss is widespread in terminal disease33, differential vulnerability can be detected across brain regions and their respective resident cell types/subcellular compartments at specific time-points. This differential degenerative progression follows the same pattern in the vast majority of NCL murine models: during pre-symptomatic stages there is an early selectivity for relay neurons within the thalamic nuclei followed by the corresponding cortical areas35, as well as GABAergic hippocampal interneurons and Purkinje neurons32. These reports in murine models regarding the vulnerability of the thalamus correlates with MRI studies in human patients showing alterations in thalamic areas at “pre-clinical” stages37. Furthermore, differential regional/neuronal vulnerability is also a shared event in a great number of neurodegenerative diseases in which some brain regions seem affected earlier than others, such as in Alzheimer40, Parkinson41, Huntington diseases43.In humans, the onset of CLN3 disease occurs typically between 4–7 years of age, when loss of visual acuity is identified. Learning deficits are followed by speech and motor difficulties and seizures. As no treatment is currently available, premature death results at a mean age of 24 years oldCln3−/− mice31 across two different time-points. This enabled the subsequent application of high-throughput proteomics in order to map the molecular fingerprint of differentially vulnerable biochemically-isolated pre-synaptic populations. We identify multiple cascades correlating with synaptic vulnerability and describe valine degradation and rho signalling pathways as two major regulators of synaptic vulnerability. The direct contribution of these pathways to neurodegeneration was confirmed in vivo using a Drosophila CLN3 model. Finally, we are confident that regulators of synaptic vulnerability and degeneration identified in the context of NCL are also conserved in their expression across other neurodegenerative conditions, in which pre-synaptic alterations are an early event2. This research therefore opens a window for further investigation into common molecular therapeutic targets and strategies for novel interventions across a range of neurodegenerative conditions during early disease onset.In this study, we aimed to define the molecular regulators of both synaptic stability and vulnerability using animal models of NCL. We initially characterized differential patterns of pre-synaptic pathology in Cln3−/− mice has already been studied33, little is known about the progression of synaptic pathology across differentially vulnerable brain regions. Studies in other NCLs using Ppt1−/−, Cln6−/− or CathD−/− mice have revealed progressive synaptic loss starting in the thalamus and followed later in corresponding cortical areas14. Hippocampal structures seem to be also affected early, although to a lesser extent than seen in the thalamus33. To investigate whether a similar pattern of synaptic pathology might be present in Cln3−/− mice we studied the expression of the presynaptic marker synaptophysin  in three brain regions that exhibit different degrees of neuronal vulnerability using quantitative immunohistochemistry  showed no statistical difference between genotypes in Syp immunoreactivity at 13 months. Immunohistochemistry using antibodies against VAMP2 and SNAP-25 (two more pre-synaptic markers) also hint at pre-synaptic alterations in the thalamus .Thus, the pre-synaptic alterations seen here (as determined with the markers detailed above) appears to follow a similar pattern to that previously reported for neuronal loss in mouse models of other forms of NCLCln3−/− mice and controls at 13 months of age where we characterise the pre-synaptic pathology as thalamus > hippocampus > cortex.Despite advances in the characterization of the mammalian synaptic proteome, little is known about how these proteins interact and the molecular mechanisms that govern synaptic vulnerability. After confirming the synaptic vulnerability pattern across three distinct brain regions, we wanted to examine if the onset of pre-synaptic pathology correlated with distinct molecular alterations in the proteome. To address this, we examined biochemically isolated pre-synaptically enriched fractions (through the generation of “crude” synaptosomes – see methods) produced from microdissected brain regions from After synaptosome production and protein extraction, iTRAQ proteomics was carried out as detailed in Fig. Next, these pre-filtered proteins from the three pre-synaptic populations were aligned to look for overlapping proteins. 709 proteins were identified in all three brain regions and these were taken forward for comparative analysis Fig. .Ingenuity pathway analysis (IPA) . Simplistically, BioLayout is a complex pattern recognition software which generates a visual representation of the data based on protein abundance44. In this case the software interrogates a regional expression profile generated by arranging the individual proteomic sets in order of increasing pre-synaptic pathology/vulnerability . The outcome of this is that proteins were found to cluster into 14 different groups according to similarities in their relative expression across these three regions , 4 (68 proteins), 6 (51 proteins) and 7 (50 proteins) comprise proteins whose expression can be grouped (or clustered) together as having either a gradual upregulation or downregulation, therefore correlating (directly or inversely with) with pre-synaptic vulnerability . In the thalamic dataset, 1536 total proteins were identified following iTRAQ processing. Further DAVID enrichment analysis was applied to the unfiltered thalamic data to confirm that the starting material was suitably enriched for synaptic proteins  software. A total of 374 proteins met these criteria  to elucidate which molecular pathways and biological networks were disrupted in “affected” thalamic pre-synaptic compartments. Here we input the total pre-filtered thalamic data into IPA and applied the 1.2 fold-change cut-off in the software. Of the three-hundred and seventy-four proteins recognised by IPA, 68.3% have previously been reported in the literature as being associated with neurological disease . The contribution of synaptic mitochondria in neurodegeneration has been highlighted in recent years46, and mitochondrial abnormalities have also been related to CLN3 disease and other NCLs50, and our data is therefore consistent with these previous findings. However, in this study we wanted to consider pathways that have not previously been related to the NCLs before. Within the top canonical pathways identified with IPA, we also detect perturbations in ROCK signalling and valine degradation pathways . ROCK2 signaling and valine degradation pathways and specifically proteins such as ROCK2 and HIBCH, could therefore represent potentially novel regulators of synaptic stability in an NCL disease context, which have not been investigated yet. So we next sought to determine if such candidates could have the ability to moderate NCL disease progression in vivo.The “Canonical pathways” function in ays Fig. . Examini51. Mutations in HIBCH cause a progressive infantile neurodegeneration in humans, characterized by hypotonia, motor delay and neurological regression53. Moreover, manipulation of HIBCH has been suggested to modulate injury-induced axonal degeneration2. However, despite this apparently restricted function, very little is known about its role (if any) in synaptic compartments or its binding/interaction partners , and its deficiency causes a metabolic disease that presents with Reye-like syndrome, with cardiomyopathy, neuromyopathy and sudden death in infancy55. More recent reports have indicated that HADHA deficiency results in a pigmentary retinopathy leading to vision loss56.From comparing synaptic vulnerability Fig. , HIBCH bin vivo in order to assess their influence on synaptic and neuronal stability in an NCL-disease specific context. To do so, we used a CLN3 gain of function fly model previously characterized by Tuxworth et al. in which the Drosophila eye undergoes degeneration 57. Although this is an accepted model for investigations into the cascades underpinning CLN3, the human disease is normally caused by loss of function. As with the human disease where dose is crucial to phenotype (i.e. heterozygous humans are not obviously affected) we can also demonstrate that degeneration ocuring in the Drosophila eye is CLN3 dose dependent  we first established a recombinant fly containing the GMR-GAL4 eye driver and the UAS-CLN3 gene in the second chromosome , eyes photographed and total surface area measured with Image J (see Methods). As expected, experimental suppression of either HIBCH or HADHA increased the neurodegenerative phenotypes observed (i.e. acting as enhancers of the CLN3-dependent phenotype)  Fig. . Interespe) Fig. . Interespe) Fig. . This suin silico analysis) to be activated when compared to wildtype controls  were non-significant with respect the control eyes and therefore the phenotype was fully-recued. Qualitatively, there was also an obvious reduction/absence of black patches (indicative of cell death), and clear recovery of the gross structure of the Drosophila compound eye. With the RNAi GD line the eye size was also improved (25.22% of recue)  Fig. . Thus, eDrosophila.Following the rescue of the CLN3-dependent eye degeneration by means of genetic tools we therefore wanted to test the possibility of targeting ROCK2 pharmacologically in 67. Importantly, fasudil has been demonstrated to be safe and effective in clinical trials of cerebral vasospasm, pulmonary hypertension, Raynaud phenomenon and cancer73. Moreover, fasudil has previously been applied to murine models of SMA showing an improvement of the phenotype and lifespan of these mice66. However, fasudil also targets other kinases such as MAP4K4, PKC, PRKAA1 or PRKAA274, which are predicted to be also activated by IPA . The drug was added into the fly food in order to obtain 1.5 mM of fasudil in the food and the Drosophila were raised as normal (see Methods). Pharmacological inhibition with fasudil caused a significant, albeit modest suppression of the CLN3-derived eye phenotype. Quantification of eye surface areas confirmed a 17% rescue of the CLN3 eye phenotype in the treated animals , followed by hippocampal pre-synaptic compartments, while cortical synaptic populations remain relatively unaffected . Its genetic and/or pharmacological downregulation has also been shown to ameliorate the phenotype of several conditions including SMA, another relatively early onset neurodegenerative disease66. In this study we demonstrate for the first time that downregulation of ROCK2 also recues the phenotype in a CLN3 in vivo model when manipulated genetically, and to a lesser degree with available pharmacological agents  which is likely goverened by mechanisms which may indeed be conserved across multiple conditions ranging from injury through to chronic neurodegenerative conditions (i.e. Huntington disease and spinocerebellar ataxia) and diseases of childhood (i.e. SMA and the NCLs).We suggest that common mechanisms regulating the process of neurodegeneration and/or vulnerability of synapses may occur across a range of neurodegenerative conditions triggered by different insults. Unlike more common adult-onset complex polygenetic diseases such as Alzheimer or Parkinson disease for which the genetic insult triggering the neurodegeneration is not well understood, the NCLs are monogenetic diseases for which there are available well-characterized mice models that more accurately replicate the human diseasein silico candidate identification and in vivo phenotypic assessment in Drosophila is an efficient pipeline for elucidating the mechanistic cascades governing neurodegenerative processes, and a “target-rich” way of identifying factors which are capable of modulating neurodegeneration, and therefore may be therapeutically targetable.The workflows highlighted here should not be viewed as a mass screening “stamp collecting” exercise. Instead it should be clear that combining mammalian model “–omic” screening of differentially vulnerable tissues with All animal experiments were approved by a University of Edinburgh internal ethics committee and were performed under license by the UK Home Office (project license numbers 70/6567 and 70/7364).Cln3−/− mice31 inbred on a C57BL/6 background for at least 10 generations and control (+/+) littermates resulting from heterozygous crosses were used in this study. Mice were genotyped as described in31. Five Cln3−/− and five wild-type control mice (WT) at 6.5 months and 12–13 months of age were used for immunohistochemistry experiment. Four Cln3−/− and four control mice (WT) at 12–13 months were used for the proteomics and biochemical experiments.Human brain samples were obtained from the Human Brain and Spinal Fluid Resource Center, Los Angeles and from The MRC London Neurodegenerative Disease Brain Bank, Institute of Psychiatry, Psychology & Neuroscience, King’s College London. Samples were obtained at routine autopsy with fully informed written consent by the families. Study protocols for the use of this human material were approved by the Ethical Research Committees of the Institute of Psychiatry under the approval numbers 223/00 and 181/02. All experiments using human tissue samples and/or derivatives of, were performed in accordance with the relevant guidelines and regulations governing their storage, handling and disposal. Details about the samples are described in Supplementary Table Cln3−/− and five wild-type control mice (WT) at 6.5 months and 12-13 months of age were terminated and brains rapidly removed and immersed for fixation in 4% paraformaldhyde (pH 7.4). The IHC protocol used allows quantitative and qualitative comparisons between animal tissues as previously described in90. Please refer to Supplemental Experimental Procedures for more details.Five Cln3−/− and wild-type (WT) controls mice at 13 months old and briefly chilled in ice cold ACSF  before regional microdissection. Thalamus, cortex  and hippocampus and were microdissected bilaterally91 pooled by mouse and immediately processed for “crude” synaptosome production as previously described92. Please refer to Supplemental Experimental Procedures for more details.Brains were harvested from six post mortem human samples, a portion of the total sample was obtained from frozen tissue (stored at −80 °C) and transferred in an eppendorf tube containing cold isotonic sucrose solution. Please refer to Supplemental Experimental Procedures for more details.For the et al.93. Please refer to Supplemental Experimental Procedures for more details.For iTRAQ proteomics, protein extraction from “crude” synaptosomes was carried out as previously described in Fuller “crude” synaptosomes were resuspended and homogenized in a solution containing 100 mM Tris-HCl, 4% (w/v) SDS and 1% protease inhibitor cocktail (Roche). Samples were spun at 20,000 g and 4 °C for 20 minutes. Supernatant was aspirated and collected as extracted sample. Pellets and extracted samples were stored at −80 °C for QWB. Extracted samples concentration was determined using a BCA assay (Thermo).et al.93. Please refer to Supplemental Experimental Procedures for more details.Sample preparation and protein identification and quantification analysis by mass spectrometry was carried out as previously described in Fuller 95. Briefly, samples were denatured in NuPage® LDS Sample buffer 4X  at 98 °C and 15ug of protein loaded and run on 4–12% Bis-Tris gel (Invitrogen). Accuracy of loading and protein estimation was confirmed by total protein analysis of Instant Blue (Expedeon) stained gels as previously described94. Protein transfer to a polyvinylidene fluoride (PDVF) membrane was carried out using the I-Blot® transfer system . Membranes were incubated with Odyssey blocking buffer (Li-Cor) for 30 minutes. Next, membranes were incubated in primary antibodies overnight at 4 °C and secondary antibodies for 1 h at room temperature. Please refer to Supplemental Experimental Procedures for more details.QWB was performed as previously described in refs44. Please refer to Supplemental Experimental Procedures for more details.BioLayout software incorporates a complex pattern recognition algorithm which groups protein data based only on expression profile. It allows the visualization and graphing of expression trends of co-expressed proteins to allow better characterization and understanding of complex large datasetsDatabase for Annotation, Visualization and Integrated Discovery (DAVID) software (available at http://david.abcc.ncifcrf.gov). DAVID provides a relatively comprehensive set of functional annotation tools for large data set interpretation97. DAVID was also used to characterize the functions associated to the protein clusters correlating to vulnerability mapped using BioLayout. For analysis to confirm synaptic protein enrichment we have applied a cut off of four fold as previously described in98  application (Ingenuity Systems) was used, as previously described99. Please refer to Supplemental Experimental Procedures for more details.To obtain further insight into potential cellular pathways that may be perturbed in the Drosophila mouse orthologs for candidate proteins were identified using DRSC Integrative Ortholog Prediction Tool (DIOPT)100 and RNAi lines were chosen from Flybase and obtained from Vienna Drosophila stock centre (VDSC) and from Bloomington Drosophila Stock Centre (BDSC) . RNAi candidate lines were crossed to this line and the F1 progeny was assessed for suppression or enhancement of the CLN3-derived small and rough eye phenotype. To have a quantitative read out of the severity of the phenotypes obtained in our experiments we measured the eye surface area, as previously described101. Stocks were maintained on standard cornmeal food at room temperature. For all the crosses, flies were raised at 25 °C in a circadiarian light incubator. Details of the lines used can be found in Table F-4660), which was previously used in mice models of SMA improving survival and skeletal muscle development65. GMR-GAL4 x UAS-CLN3 crosses were raised in Nutri-Fly instant food  containing 1.5 mM of Fasudil Monohydrochloride Salt and blue food colouring diluted in water, as described in102. Eyes of the offspring were photographed and total surface area was measured with Image J. Suppression of the CLN3-degenerative eye were calculated from 3 independent experiments as “(x- average eye surface area of the disease model)/(average of eye surface area of the control − average eye surface area of disease model) × 100” for each experiment.The rock inhibitor used was Fasudil, Monohydrochloride Salt , USA), as previously described103. Thirty non-overlapping images (x40) were taken, on three consecutive sections, through the brain areas described above. All parameters including lamp intensity, video camera setup and calibration were kept constant throughout image capturing.Micrographs were taken with Leica DMRB × 5/0.12 objective and AxioCam HRC Zeiss from the following brain regions: 1. The thalamic nuclei ventral posterior medial/ventral posterior lateral thalamic nucleus (VPM/VPL) and the dorsal lateral geniculate nucleus (LGNd); 2. Their respective cortical projections in the primary somatosensory cortex (S1BF) and primary visual cortex (V1) respectively, and 3; the hippocampal stratum radiatum and stratum oriens See Fig. . ImmunorFor the HIBCH and HADHA experiments fruit fly eye images were photographed with a Nikon D5100 camera attached to a SZX9 Nikon stereomicroscope. For the ROCK2 experiments eye images were taken with an AxioCam ERc 5 s Rev.2 attached to a Zeiss Stemi 305 trino stereomicroscope. Images were analyzed with Image J software  as previously described in101.Odyssey software as per manufactures guidelines and as previously described104. Data was graphed and statistical comparisons for IHC thresholding, QWB protein intensity and Drosophila eye surface area quantification were carried out using GraphPad Prizm as previously described101. For QWB and IHC Student’s T test was applied. For Drosophila eye quantifications one-way ANOVA was carried out followed by Tukey’s multiple comparison test as a post-hoc when a significant difference was found in the ANOVA. P values < 0.05 were considered to be statistically significant for all analyses . Statistical analysis of proteomic data was automatically carried out by MASCOT. P & Benjamini values to determine enrichment were automatically calculated by DAVID software (see above and http://david.abcc.ncifcrf.gov). P values/Fishers exact tests for pathway and networking analysis were automatically determined by IPA (see above and http://www.ingenuity.com/). Heatmap representing the differentially expressed proteins and pathways across brain regions was produced using “R” (http://www.R-project.org)105.QWB data was analysed using The raw datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary Information fileSupplementary Dataset 1


Optimal perinatal care of infants born less than 24 weeks gestation remains contentious due to uncertainty about the long-term neurodevelopment of resuscitated infants. Our aim was to determine the short-term mortality and major morbidity outcomes from a cohort of inborn infants born at 23 and 24 weeks gestation and to assess if these parameters differed significantly between infants born at 23 vs. 24 weeks gestation. We report survival rates at 2-year follow-up of 22/38 (58%) at 23 weeks gestation and 36/60 (60%) at 24 weeks gestation. Neuroanatomical injury at the time of discharge (IVH ≥ Grade 3 and/or PVL) occurred in in 3/23 (13%) and 1/40 (3%) of surviving 23 and 24 weeks gestation infants respectively. Rates of disability at 2 years corrected postnatal age were not different between infants born at 23 and 24 weeks gestation. We show evidence that with maximal perinatal care in a tertiary setting it is possible to achieve comparable rates of survival free of significant neuroanatomical injury or severe disability at age 2 in infants born at 23-week and 24-weeks gestation. As technological capacity increases, the need to define the threshold of infant viability and agree on the ethical boundaries of neonatal intensive care becomes more urgent4. Despite improved overall survival rates the burden of neurocognitive morbidity associated with extreme prematurity remains high7. However, outcomes in previously published cohort studies reflect heterogeneous patient groups combining in-born and out-born infants and those pooled from several institutions where practice variation may influence outcomes9. Additionally, most outcome data focus on neurodevelopmental morbidity which, whilst important, does not necessarily reflect the overall wellbeing of the child.Advances in perinatal medicine have led to a dramatic improvement in overall survival rates for extremely preterm infants11. Clinicians are therefore faced with the dilemma as to whether it is ethical to initiate or withhold resuscitation when the outcomes are so uncertain12. Professional advisory bodies17 recognise this dilemma and recommend that discussion between health professionals and the infants’ family must underpin any clinical decision to withhold or instigate resuscitation of the periviable infant18. Ideally this discussion should be informed by clear short- and long-term morbidity and mortality data that is pertinent to the local tertiary institution.It remains extremely difficult to accurately predict the clinical course of an individual infant born in the periviable period of 23 weeks gestationIn the current study we sought to address, in a population of 23 and 24 weeks gestation infants, rates of death and major morbidity at time of discharge home, and rates of disability at 2 years corrected postnatal age. The secondary aim was to assess whether the above parameters differed significantly between infants born at 23 or 24 weeks gestation.We report our primary outcome of survival at 2 years corrected postnatal age free of moderate or severe disability. Our secondary outcomes include 1: survival to (i) NICU admission, (ii) discharge home, (iii) 2 years corrected postnatal age; 2: Maternal and neonatal wellbeing at the time of birth; 3: Major neonatal morbidity.+0 and 24+6 weeks of gestation in Wellington Regional Hospital, between 1st January 2003 and 31st December 2012. Cases were retrieved from our institutional database and clinical details cross-checked against the paper clinical record. Gestational age was defined by first trimester dating ultrasound, or by date of last menstrual period if no early ultrasound scan was available. Infants were excluded if out-born, the parents declined resuscitation, or significant congenital anomalies were present. Seven infants were excluded due to congenital abnormalities including major cardiac and abdominal wall anomalies. Resuscitation was not initiated in 11 infants due to wishes of parents in agreement with the medical team. Key data were extracted including decisions to initiate or withhold newborn resuscitation, the infants’ medical course, health status at the time of discharge home and during follow-up assessments at 2–3 years corrected postnatal age.Cases were identified from the birth registration of infants born between 23Maternal age, ethnicity and health status were obtained from patient records. Chorioamnionitis was determined by the attending obstetrician and confirmed following placental histology. Prolonged rupture of membranes was defined as rupture of membranes >24 hrs before delivery. Pre-eclampsia was defined by the obstetricians’ clinical interpretation of maternal symptoms, blood pressure, blood and urinary tests. Incomplete course of steroid was defined as one dose of betamethasone given <12 hours prior to delivery. Complete course of steroid was defined as two doses of betamethasone given 12 hours apart with the second dose <7 days prior to delivery. Mode of delivery and its indication were obtained from the maternal delivery record.Birth weight was measured in the NICU using a portable scale with accuracy of +/−2 grams  or an in-built incubator scale with accuracy of +/−10 grams . Birth weight centile was derived from the New Zealand – World Health Organisation growth charts . Neonatal sepsis was defined by presence of a positive blood, urine or CSF culture within 48 hours of sample collection. Presence of patent ductus arteriosus (PDA) was determined by an echocardiogram performed within 24hrs of birth for all infants, and in this cohort prophylactic indomethacin  was given routinely if a PDA was present unless a contraindication was identified . Diagnosis of necrotising enterocolitis (NEC) was based on the Bell classification and includes stages I to III. It is customary practice in our Unit to complete 7 days of intravenous triple antibiotics  and nil by mouth for infants with NEC (Bell stage I-III). Therefore, all cases significant enough to warrant treatment have been included in subsequent analysis as a measure of potentially serious gastrointestinal morbidity. Presence of intraventricular haemorrhage (IVH) and/or periventricular leucomalacia (PVL) was determined by serial cranial ultrasonography performed and interpreted by paediatric radiologists. Reported grade of IVH/PVL (Papile classification) reflects the most significant abnormality found at any time during the infants’ admission. Diagnosis of bronchopulmonary dysplasia (BPD) was made if supplementary oxygen was required at 28 days of life, and was defined as moderate if infants required <30% oxygen at 36 weeks corrected postnatal age and severe if infants required >30% oxygen at 36 weeks corrected postnatal age. Presence of retinopathy of prematurity (ROP) and need for laser therapy was determined by serial paediatric ophthalmology examination commenced between 30 and 31 weeks corrected postnatal age. Of note, in our Unit, administration of exogenous surfactant to infants born at 23 and 24 weeks gestation was universal practice; all infants therefore received at least one dose in the newborn period. Similarly, administration of probiotics only commenced in our Unit after 2013; none of the infants included in this cohort therefore received probiotic treatment.19. This allows validated reporting of morbidity outcomes in children unable to access a standardised post-discharge developmental surveillance program. Many of the children in this cohort were domiciled in rural communities where follow-up is with a Paediatrician but may not include specialist neurodevelopmental assessment using Bayley or Griffith neurodevelopmental scales. Health outcomes were obtained by review of clinical records at 2 years corrected postnatal age. All records were independently assessed by at least 2 paediatric-trained investigators and reconciled by a third senior, paediatric-trained investigator if necessary. Wellbeing was assessed in domains of: neurodevelopmental , respiratory, renal and gastrointestinal. One or more impairment in any ‘severe disability’ domain was classified as the child having severe disability. Similarly, one or more impairment in the ‘moderate’ disability domain was required for classification as moderately disabled  was used for statistical analyses. χ2 analysis was used to compare outcomes between those born at 23 vs. 24 weeks gestation. Associations between maternal characteristics and infant outcome were explored using multivariate regression analysis. The level of statistical significance for all analyses was set at p ≤ 0.05 using two-tailed comparisons. Data are presented as the mean ± SEM or median (range) as indicated in the text. Percentage survival rate was determined using the number of live born infants at each gestational age as the denominator for all outcomes.Institutional ethics approval to review the health records was prospectively obtained from the Wellington Hospital Clinical Audit and Research Committee. All health records were anonymised and de-identified prior to analysis. All methods and patient information were performed in accordance with the relevant guidelines and regulations outlined by the Wellington Hospital Clinical Audit and Research Committee.+0−6 gestation and 60 were born at 24+0−6 gestation. There were more males (n = 60) than females (n = 38) . Apgar score at 5 minutes was significantly lower in 23-week infants when compared to 24-week infants  . No difference in rates of NEC, ROP or any other medical or surgical complications were observed between or with groups (Table Twenty-three infants in the 23-week cohort 61%) and 40 infants in the 24-week cohort (67%) survived to discharge home  in the 23-week cohort and 6/40 infants (15%) in the 24-week cohort. 11/23 infants (48%) and 14/40 infants (35%) in the 23-week cohort and 24-week cohort respectively were discharged home with supplementary low-flow oxygen. Twenty out of 23 surviving 23-week gestation infants (87%) and 39/40 surviving 24-week gestation infants (97%) had no major neuroanatomical abnormalities (IVH ≥ 3 or PVL) detected at any time during their clinical course.The majority of NICU deaths occurred within the first week Fig. . Of the Following discharge from the neonatal units, one 23 weeks gestation infant and four 24 weeks gestation infants died before 2 years corrected age. Overall survival rates of liveborn infants is therefore 58% (22/38) and 60% (36/60) in 23-week and 24-week cohorts respectively at 2 years corrected postnatal age Fig. .Of the surviving children at 2 years corrected postnatal age for whom health status data were available , 11/19 (58%) and 23/31 (74%) were free of any moderate or severe health impairments, in the 23- and 24-week cohorts respectively  in surviving 23-week gestation infants and 1/40 (3%) in surviving 24-week gestation infants) and rates of moderate to severe disability were comparable between those born at 23 and 24 weeks gestation at 2 years corrected age. A recently published study suggests that a higher rate of survival without impairment in 23 weeks gestation infants is found in those institutions with higher rate of active resuscitation for extremely preterm infantsOur study has a number of limitations that need to be acknowledged. This is a retrospective study where the data derive from a single tertiary centre with materno-fetal medicine and neonatal expertise available on site. The type of resource available to women and their babies is therefore not representative of the majority of birthing centres where access to tertiary services will necessarily be delayed. Similarly, as we wished to describe outcomes where it was perceived that maximal perinatal support was provided we did not include out-born infants requiring postnatal retrieval to tertiary care as this is recognised to significantly increase the risk of morbidity and mortality. Outcomes at 2 years corrected postnatal age are derived as a composite of formal developmental and, or, paediatric assessment. While this is not as robust as a universal standardised health and developmental assessment, it does provide a valuable, holistic overview of the child’s wellbeing while reflecting the limitations of routine clinical follow-up.We have shown survival free of moderate or severe disability can be achieved in infants born at 23 weeks gestation when maximal perinatal care is provided in a publically-funded tertiary setting. We also report that, in the current study, there were no clear perinatal factors that reliably predicted infant outcome. Importantly, our high rate of resuscitation in infants born at 23 weeks did not result in a disproportionately high burden of severely neurologically compromised infants. Furthermore, given that most death occurs in the first week after birth our practice of active intervention does not have major adverse implications for resource management. We therefore recommend that dialogue in this area should focus on improving equitable access of high-risk women to maximal perinatal care settings, rather than focus on whether or not to withhold potentially lifesaving treatment in infants born before 24 weeks of gestation.Supplementary Table 1


Here, we present evidence to support the role of common drug interactions and low vitamin B12 levels among pediatric leukemia patients during therapy.We review the published evidence and highlight key potential drug interactions as well as present clinical evidence of severe methotrexate neurotoxicity in conjunction with nitrous oxide anesthesia and measurements of vitamin B12 levels and hence are likely to increase the toxicity of methotrexate in these patients. We also present a brief case study supporting out hypothesis that nitrous oxide contributes to methotrexate neurotoxicity and a nutritional study, showing that vitamin B12 deficiency is common in pediatric leukemia patients.We describe a very plausible mechanism for methotrexate neurotoxicity in pediatric leukemia patients involving reduction in methionine and consequential disruption of myelin production. We provide evidence that a number of commonly prescribed drugs in pediatric leukemia management interact with the same folate biosynthetic pathways and/or reduce functional vitamin B12 in patients suspected of having methotrexate-induced neurotoxic effects.Use of nitrous oxide in pediatric leukemia patients at the same time as methotrexate use should be avoided especially as many suitable alternative anesthetic agents exist. Clinicians should consider monitoring levels of vitamin B Adverse neurological events are very common during treatment for pediatric acute lymphoblastic leukemia (ALL) and include seizures, stroke-like syndrome and leukoencephalopathy. Recent trials report neurological adverse events in 4–20 % of patients , 2. AddiAdditionally, although rare, neurotoxicity with similar radiological features of leukoencephalopathy has occasionally been reported following oral methotrexate used in patients with autoimmune and inflammatory disorders , 6, wher12  levels, which lead to elevated methotrexate levels in cerebrospinal fluid (CSF) or result in synergistic or additive effects on convergent metabolic pathways. A particularly important and under-appreciated drug interaction may be the concomitant use of inhaled nitrous oxide (N2O) and methotrexate, which is common practice in many pediatric hematology centers where lumbar punctures to administer intrathecal (IT) methotrexate are performed under general anesthesia.We propose that methotrexate-induced neurotoxicity may be potentiated by common drug interactions, and/or the presence of low vitamin B2O and methotrexate [2O and oxygen. Other concomitant drugs included the proton-pump inhibitor (PPI) omeprazole. Four days after the 5th dose of IT methotrexate, she was presented with focal seizures rapidly progressing to generalized tonic–clonic seizures, disinhibition, severe agitation and left upper limb (LUL) weakness. MRI (T2/FLAIR and diffusion weighted) imaging on day 2 showed widespread hyperintense subcortical white matter lesions in the frontal and parietal regions with areas of restricted diffusion consistent with leukoencephalopathy. Seizure activity continued for 5 days despite maximal anticonvulsant doses of benzodiazapines and levetiracetam and the radiological changes worsened with increasing cerebral edema and pressure effects requiring dexamethasone. She made a clinical recovery over the next 7 days but with some residual LUL weakness. Serum vitamin B12 was measured on recovery (3 weeks from the last IT methotrexate) and was low at 154 ng/L .A recent case report highlighted the risk of severe neurotoxicity with the combination of Notrexate . Here, w12 as a co-factor. Methionine is then converted to S-adenosyl methionine (SAM), a methyl donor which has a critical role in regulating myelin sheath formation and lipid production [Methotrexate exerts its anti-leukemic action via inhibition of the enzyme dihydrofolate reductase, ultimately reducing the amount of tetrahydrofolate available for DNA synthesis leading to cell death 12 as a c12  with ALL. Three out of four patients where levels were low experienced severe gastrointestinal enteropathy and 1 patient experienced a severe neurological event. In addition, the same study revealed that 9/17 of the same cohort tested for zinc had clinically low levels at some point during their treatment. Zinc is also a critical co-factor for methionine synthase  a direct drug interaction leading to increased methotrexate plasma (and/or CSF) concentrations or (2) interference with the same metabolic pathways as methotrexate. Examples of (1) include fluoroquinolone antibiotics, piperacillin (the most commonly prescribed antibiotic for episodes of febrile neutropenia during pediatric leukemia treatment in the UK) and proton-pump inhibitors (PPIs). The latter delay plasma elimination of methotrexate leading to renal and liver toxicity . A numbe a direct(Fig. 12  and anti12 levels, recent general anesthesia with nitrous oxide or introduction of additional drugs interacting with the same pathways, may be responsible for the idiosyncratic occurrence of methotrexate neurotoxicity in pediatric leukemia patients undergoing treatment with high doses. We aim to raise awareness globally of this potential interaction and particularly ensure that nitrous oxide anesthesia is avoided in all patients on methotrexate as advised by the British National Formulary [The evidence presented above suggests that low Bormulary .In the era where personalized medicine integrated with genomic approaches is seen as the ultimate goal, it is important not to overlook ‘old-fashioned’ drug interactions, especially if such risks can easily be minimized by a change in drug scheduling or use of suitable alternative agents.


Faecalibacterium prausnitzii and Ruminococcus bromii from controls to CP non-diabetics to CP diabetics. On the other hand, there was increase in LPS (endotoxin) synthetic pathways (KEGG orthology) in the groups. Faecalibacterium prausnitzii abundance correlated negatively with plasma endotoxin and glycemic status; while plasma endotoxin correlated positively with blood glucose and negatively with plasma insulin. Our results have important implications for future studies exploring mechanistic insights on secondary diabetes in CP.Intestinal dysbiosis and its functional implications in chronic pancreatitis (CP) have not been elaborately studied. We evaluated the taxonomic and functional alterations in intestinal microbiota in 30 well-characterised patients with CP  and 10 healthy controls. The patients with CP and diabetes had significantly longer disease duration and greater degree of malnutrition. There was increase in plasma endotoxin concentrations from controls to CP non-diabetics to CP diabetics. We observed significant differences in richness and alpha diversity between the groups. We also observed increase in the Firmicutes:Bacteroidetes ratio in CP patients without and with diabetes. There was reduction in abundance of  Chronic pancreatitis (CP) is characterised by abdominal pain, reduction in digestive enzyme secretion (pancreatic exocrine insufficiency) and endocrine dysfunction/diabetes (DM)235As shown in PCA showed distinct clustering of healthy controls, CP patients without and those with diabetes at the species level taxa  . This waFaecalibacterium prausnitzii [p(corr) = 0.001] and Ruminococcus bromii [p(corr.) = 0.001] abundances from controls to non-diabetic CP to diabetic CP patients , Bacteroidetes (31.0%), Actinobacteria (3.8%) and Proteobacteria (2.9%). As shown in patients .Next, we evaluated the functional pathways using Kyoto Enclyopaedia of genes and genomes (KEGG) orthology (KO) in the three groups and their correlations with bacterial abundance. Faecalibacterium prausnitzii with plasma endotoxin levels , fasting blood glucose  and post-prandial blood glucose   . There wn levels . However glucose . On the  glucose .Other clinically relevant correlations were : negativIn this study we have shown significant association of intestinal dysbiosis with host metabolic functions, including diabetes, in patients with CP.The CP patients with DM had a significantly longer disease duration and higher degree of malnutrition compared to the CP patients without diabetes. This indicates that the changes in the intestinal microbiota in our patients were dependent on disease duration and severity. We included healthy family members of enrolled patients with CP as controls so that the dietary, environmental, and genetic confounders789Faecalibacterium prausnitzii from healthy controls to non-diabetic CP to CP with diabetes. There was also a negative correlation between the abundance of this organism with circulating endotoxin levels. We further observed an increase in the LPS (endotoxin) synthetic pathways in the patients without and with DM. Faecalibacterium prausnitzii is among the most abundant commensals in the human intestineFaecalibacterium prausnitzii could improve gut barrier function by stimulating the synthesis of mucin and tight-junction proteins1214Faecalibacterium prausnitzii in our patients with CP was likely to have impaired the intestinal mucosal barrier integrity in a duration dependent manner.One of our key findings was a reduction in the abundance of Ruminococcus bromii in patients with non-diabetic and diabetic CP patients. Ruminococcus bromii bears a unique capability to degrade especially enzymatic digestion resistant starch, a function that is poorly seen in other starch degradersRuminococcus bromii was likely to have contributed to the disruption of gut mucosal barrier and altered bacterial metabolism within the intestinal epithelial-bacterial ecological system.We also observed reduction in abundance of the amylolytic organism Abundance of the LPS synthetic pathways and plasma endotoxin levels were higher in our patients without and with DM, despite the reduction in abundance of the phylum Bacteroidetes, which includes primarily Gram-negative organisms (source of LPS). It is likely that the majority of the unclassified phyla and genera derived from bacteria were Gram negative, resulting in increasing LPS synthetic pathways and plasma endotoxin as a function of duration and severity of the disease.Faecalibacterium prausnitzii with fasting and postprandial blood glucose. The validity of this result was substantiated by the significant positive correlation of the abundance of Faecalibacterium prausnitzii with plasma insulin levels. There was also significant positive correlation of plasma endotoxin with fasting and postprandial blood glucose. It is known that LPS could induce inflammation in beta cells via Toll like receptors (TLRs) and nuclear factor of kappa-B (NFk-B) thereby resulting in beta cell dysfunctionThe other key finding in our study was a significant negative correlation of the abundance of It has been shown that diabetesEven though our results demonstrated discrete changes in the intestinal microbiota that were associated with metabolic changes of CP, there were few limitations in the study. The sample size was small and we could not perform any formal sample size calculation since there were no previous quantitative data on the intestinal microbiota in patients with CP. However, our selection criteria were strict and we also characterised the disease phenotype in detail. We believe these steps had circumvented the sample size limitation. We also did not perform any metabolome assessment (including short chain fatty acids) in this study. However, functional analyses based on KEGG orthology using the metagenomic data provided plausible results that explain the observed association between the intestinal dysbiosis and phenotypic alterations. Furthermore, we did not perform a pancreatic polypeptide response assay, which could have conclusively confirmed that our patients had pancreatogenic diabetes. We believe these patients did have pancreatogenic diabetes since diabetes developed at a younger age (mean 27.7 yrs), with a consistent lag period after the onset of CP; and the C-peptide levels were low. Of note, CP and secondary diabetes develops earlier in India compared to that seen in the west2324Treatment of CP is currently restricted to palliation of pain, supplementation of pancreatic enzymes, and control of diabetes1We initiated this study after approval from the Institutional Review Board of the Asian Institute of Gastroenterology ; and obtained written informed consent from all enrolled cases and controls. All experimental procedures were performed according to standard guidelines and procedures that were approved by the above mentioned Institutional Review Board.We screened adult patients of both genders with CP who presented to the Pancreas Clinic at the Asian Institute of Gastroenterology, Hyderabad, India for eligibility over a 6-month period (April-September 2014). Detailed clinical history from the cases and controls was recorded and disease morphology was evaluated by cross sectional imaging [contrast enhanced computed tomography (CECT) or magnetic resonance cholangiopancreatography (MRCP)] or endoscopic retrograde cholangiography (ERCP). All controls underwent transabdominal ultrasonography for evaluating the pancreas and the liver.All subjects were subjected to routine biochemical evaluation and nutritional assessment including dietary history, anthropometric measurements, serum transferrin, serum prealbumin, serum vitamin B12, serum vitamin D, serum calcium, and haemoglobin levels. Oral glucose tolerance test (OGTT), plasma C-peptide, plasma insulin and glycosylated haemoglobin (HbA1c) were assessed to define the glycemic status of the subjects. Composite nutritional status was expressed according to the Subjective Global Assessment scaleOGTT was performed with a 75 g glucose load and blood glucose was measured using the Glucose Oxidase-Peroxidase (GOD-POD) method employing a commercial kit . HbA1c was measured by high performance liquid chromatography (HPLC) using a National Glycohemoglobin Standardised Program (NGSP)Plasma insulin and C-peptide estimation were performed using a Sandwiched Electrochemical Immunoassay (ECLIA) technique in the ROCHE Cobas‘e’ 601 Immunoassay Analyzer. Briefly, the samples were incubated initially with 20 μl of biotinylated monoclonal antibodies (anti-insulin and anti C-peptide), followed by incubation with streptavidin coated microparticles. The bound microparticles were then magnetically captured onto the surface of the electrode that resulted in induction of chemiluminescent emission which was measured on a photomultiplier and quantified by 2-point calibration with a master curve.Serum concentrations of vitamins D and B12 were estimated by ECLIA method in the ROCHE Cobas’e’ 601instrument; serum transferrin and pre-albumin were analyzed by immunoturbidimetry on the Random Imola fully automated analyzer; and serum calcium contents were determined using the metallochromatic indicator Arsenazo III on the fully automated UniCelDxC 800 (Beckman Coulter Inc.) analyser. All assays were performed as per manufacturer’s instructions.Plasma endotoxin levels were estimated using the Pierce LAL Chromogenic Endotoxin Quantitation Kit  as per manufacturer’s instructionsThe enzyme was assayed in fresh fecal samples using the solid phase double-sandwich ELISA based kit  as per manufacturer’s instructions260/A280. After quantity and quality check, the isolated DNA was stored at −80 °C till sequencing.Fresh fecal specimens, collected in sterile containers, were immediately processed for DNA extraction. Genomic DNA was isolated from the stool specimen (200 mg) using QIAGEN (Germany) mini stool DNA isolation kit (Cat. No 81504) as per the manufacturer’s instruction. DNA concentration was determined using the Nano drop 2000 spectrophotometer  and the purity was recorded based on absorbance at AWe subjected metagenomic DNA from the 40 samples to Next Generation Sequencing (NGS) at Xcelris Genomics . The V3-V4 region of 16S rDNA amplicon was sequenced on the Illumina MiSeq platform to analyse the bacterial diversity. The NexteraXT Index kit  was used to generate a MiSeq amplicon library as per the 16S metagenomic sequencing library preparation protocol (Part # 15044223 Rev. B). Two primers (V3-forward: 5′CCTACGGGNGGCWGCAG3′ and V4-reverse: 5′GACTACHVGGGTATCTAATCC3′) were designed and synthesized for the amplification of V3–V4 region of 16S rDNA gene. Amplicons were then ligated with Illumina adaptors and i5 and i7 primers were used to amplify them. The amplicon libraries were purified using 1X Ampure XP beads and checked on Agilent DNA 1000 chip on bioanalyzer 2100 and further quantified on fluorometer by Qubit DNA HS Assay kit . After obtaining the Qubit concentration for the library and the mean peak size (~600 to ~630 bp) from bioanalyser profile, 600 μl of 10 ρM pooled libraries (spiked with 5% 12.5 ρM PhiX Control) was loaded into MiSeq reagent cartridge for cluster generation involving hybridization of template molecules onto the oligonucleotide-coated surface of the flow cell. Immobilized template copies were amplified by bridge amplification to generate clonal clusters. Barcode and sequencing primers were trimmed from sequences. The trimmed sequences were saved as FASTQ files, which were then uploaded to Metagenomic RAST server Bacterial richness, alpha diversity and beta diversity was expressed as Chao 1, Shannon index and Whitaker index respectivelyHow to cite this article: Manasa Jandhyala, S. et al. Altered intestinal microbiota in patients with chronic pancreatitis: implications in diabetes and metabolic abnormalities. Sci. Rep.7, 43640; doi: 10.1038/srep43640 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We then used this approach to identify an effective treatment strategy within the clinical toxicity limits of lapatinib, and developed a partial differential equation modeling approach to study the in vivo GBM treatment response by taking into account the heterogeneous and diffusive nature of the disease. Despite the inability of lapatinib to induce tumor regressions with a continuous daily schedule, our modeling approach consistently predicts that continuous dosing remains the best clinically feasible strategy for slowing down tumor growth and lowering overall tumor burden, compared to pulsatile schedules currently known to be tolerated, even when considering drug resistance, reduced lapatinib tumor concentrations due to the blood brain barrier, and the phenotypic switch from proliferative to migratory cell phenotypes that occurs in hypoxic microenvironments. Our mathematical modeling and statistical analysis platform provides a rational method for comparing treatment schedules in search for optimal dosing strategies for glioblastoma and other cancer types.Human primary glioblastomas (GBM) often harbor mutations within the epidermal growth factor receptor (EGFR). Treatment of EGFR-mutant GBM cell lines with the EGFR/HER2 tyrosine kinase inhibitor lapatinib can effectively induce cell death in these models. However, EGFR inhibitors have shown little efficacy in the clinic, partly because of inappropriate dosing. Here, we developed a computational approach to model the  In vivo inhibition of tumor expansion requires a sufficient amount of therapeutic agent to be present in the tumor tissue. A number of factors affect drug concentrations including the maximum tolerated dose, pharmacokinetics and pharmacodynamics profiles. We present a computational modeling platform incorporating both in vitro data and published clinical trial data to investigate the efficacy of lapatinib as a function of different dosing schedules for inhibiting glioblastoma tumor cell growth. The goal of our method is to find the best dosing schedule balancing both toxicity and efficacy. Our modeling approach identifies continuous dosing as the best clinically feasible strategy for slowing down tumor growth even when taking into consideration intratumor heterogeneity, drug resistance and reduced lapatinib concentrations in the tumor due to the blood brain barrier. Glioblastoma is the most common and aggressive form of brain tumors in adults, characterized by short survival and poor treatment response . CurrentIn the last decade, several molecularly targeted agents that inhibit recurrently mutated proteins have been investigated as a therapeutic strategy in glioblastoma. These have included several inhibitors of the epidermal growth factor receptor (EFGR), which is mutationally activated in approximately 50% of adult GBMs. Lapatinib is a small molecule tyrosine kinase inhibitor of human epidermal receptor 2 (HER2) and EGFR, which currently has regulatory approval for the treatment of HER2-positive advanced or metastatic breast cancer –14 incluThe use of mathematical models for treatment optimization is part of a growing effort to improve clinical trial design for cancer patients. Many models have been developed to investigate the relationship between toxicity and dose in an attempt to better characterize the toxicity profile of the therapeutic products under investigation. For instance, Huitema et al. reviewed several differential equation-based models of chemotherapy-induced myelosuppression, cardiovascular events and other ordinal adverse events . AmanteaIn addition to understanding toxicity profiles, mathematical models have also been used to model pre-clinical data from animal or in vitro systems in order to bypass the difficulty associated with directly observing tumor progression in humans. Translational models incorporating in vitro or animal data have also be developed to investigate the relationship between efficacy endpoints and exposure. Mould et al. provided a comprehensive review of how the use of mathematical models can aid early development of anti-cancer therapy, in particular for describing tumor volume as a function of drug exposure . Most paHere we present a computational modeling approach incorporating both toxicity data from phase I clinical trials and efficacy data from pre-clinical research to examine the effects of various lapatinib dosing schedules on tumor growth . The goaSF268 cells were obtained from the National Cancer Institute (NIH) and routinely grown in DMEM supplemented with 10% fetal bovine serum. To assess the effects of drug treatment, 150,000 cells were seeded on 6-cm dishes and allowed to attach overnight. Cells were then switched to growth media supplemented with 1% fetal bovine serum and the indicated concentrations of lapatinib or DMSO (vehicle). Each treatment condition was done in triplicate. For treatment discontinuation, cells were washed 3 times with media containing vehicle, and then allowed to continue growth in media containing vehicle. Vehicle-treated cells were also washed 3 times to control for the effects of washing. Cell viability and cell death were evaluated by the trypan blue exclusion assay using a ViCell cell viability analyzer (Beckman Coulter).in vitro cell dynamics, we designed a differential equation model of cell growth given byY1 and Y2 denote the numbers of viable and dead cells, respectively; the parameters b and d denote the birth and death rates of viable cells; K denotes the carrying capacity of the in vitro assay system; and c denotes the clearance rate of dead cells.To describe We implemented a grid search algorithm, given byb0 and b1 are the intercept and slope parameters, respectively, describing the birth rate, and d0 and d1 are the intercept and slope parameters, respectively, describing the death rate.We used an exponential model to describe the relationship between birth and death rates and lapatinib dose given byydose denotes the lapatinib intake per treatment day and xdays is the number of treatment days per treatment cycle.To characterize the relationship between the maximally tolerated lapatinib dose per treatment and number of treatment days, we fit a linear model given byxoral is the dose of oral lapatinib in miligrams and yserum is the concentration of lapatinib in the serum in ng/mL units.We fit a logarithmic function to model lapatinib absorption into the blood stream, according toyserum is the serum concentration in ng/mL, ytumor is the concentration of lapatinib in the tumor, and 0.61 was the mean tumor/serum ratio reported in Vivanco et al. . T. T69]. Tσ0 = 2.068 × 10−9 mol mm−3; proliferative to migratory switching parameter λ1 = 4.134 nmol mm−3 ; migratory to proliferative switching paramter λ2 = 0.5, 1, and 2; oxygen diffusion rate Dσ = 1.51 × 102 mm2 day−1; oxygen supply rate h1 = 3.37 × 10−1 day−1; and oxygen consumption rate h2 = 10−2 mm cell−1 day−1 . When iin vitro tumor cell data measured based on the SF268 GBM cell line during lapatinib exposure. Our data demonstrate that lapatinib concentrations are negatively correlated with cell birth rates, but positively correlated with death rates. However, the necessary lapatinib concentration needed to arrest further tumor expansion is calculated to be 2,180nM in vitro, a concentration that cannot be reached in GBM patients based on current toxicity limits and oral absorption profiles. To achieve this concentration, our absorption model predicts that an oral dose of 34,000mg is required—5 times greater than the highest dose ever tested. The therapeutic potential seems to be restricted by the transport of lapatinib from the gastrointestinal tract to tumor sites. Due to the logarithmic relationship between oral intake and serum concentration, a higher than tolerated dose is required to achieve tumor reduction. Other methods of delivery, such as intravenous injection, may need to be considered to bypass the current delivery problem. This result agrees with the conclusions from early clinical trials that lapatinib failed to reduce tumor sizes . D. Din vitin vitro tumor dynamics and in vivo lapatinib dose and schedule, taking into account the blood brain barrier, GBM motility, heterogeneity, and phenotypic changes that occur in hypoxic microenvironments. Our approach allows for the systematic comparison between different treatment strategies and their effects on tumor growth. Although none of the tolerable doses and schedules evaluated were found capable of arresting tumor expansion, our approach predicts that continuous treatment within these parameters is likely to be more successful at slowing down tumor growth. Further optimization of effective dosing strategies using our computational platform will benefit from additional dose-limiting toxicity studies that incorporate multiple pulsatile schedules.Optimization of dosing strategies for cancer treatments is a complex problem. Our mathematical model provides a quantitative relationship bridging S1 FigA-C: There is no obvious association between birth and death rates with lapatinib concentrations when clearance rates are allowed to vary. D-F: Constraining the clearance rates to be zero, birth rates decrease with increasing lapatinib concentrations and death rates increase lapatinib concentrations. The solid black lines show the relationships between birth and death rates and lapatinib concentration assuming an exponential function. The exponential function is selected to ensure that birth and death rates are always positive for all concentrations of lapatinib.(TIF)Click here for additional data file.S2 FigA-D: Predicted long-term growth trajectories (20 treatment cycles) for the five MTD schedules with 0%, 1%, 5%, and 10% pre-existing resistance based on the logistic diffusion PDE model.(TIF)Click here for additional data file.S3 FigA-D: Predicted long-term growth trajectories (20 treatment cycles) for the five MTD schedules with 50%, 75%, 90%, and 100% of serum lapatinib concentrations penetrating the blood brain barrier and entering into the tumor based on the logistic diffusion PDE model.(TIF)Click here for additional data file.S4 Fig2/day based on the logistic diffusion PDE model.A-D: Predicted long-term growth trajectories (20 treatment cycles) for the five MTD schedules with diffusion parameters equal to 0.0183, 0.033, 0.067, 0.1, 0.133, 0.167 mm(TIF)Click here for additional data file.S5 Fig2 = 0.5, 1, 2.A-C: Predicted long-term growth trajectories (20 treatment cycles) for the control and five MTD schedules with migratory to proliferative switching parameter λ(TIF)Click here for additional data file.


DNA methylation of regulatory and growth-related genes contributes to fetal programming which is important for maintaining the correct development of three germ layers of the embryo that develope into different tissues and organs, and which persists into adult life. In this study, a preliminary epigenetic screen was performed to define genomic regions that are involved in fetal epigenome remodeling. Embryonic ectodermic tissues (origin of nervous tissue), mesenchymal tissues (origin of connective and muscular tissues), and foregut endoderm tissues , from day 28 sheep fetuses were collected and the distribution of methylated CpGs was analyzed using whole-genome bisulfite sequencing. Patterns of methylation among the three tissues showed a high level of conservation of hypo-methylated CpG islands CGIs, and a consistent level of methylation in regulatory genetic elements. Analysis of tissue specific differentially methylated regions, revealed that 20% of the total CGIs differed between tissues. A proportion of the methylome was remodeled in gene bodies, 5′ UTRs and 3′ UTRs . Genes with overlapping differentially methylated regions in gene bodies and CGIs showed a significant enrichment for tissue morphogenesis and development pathways. The data presented here provides a “reference” for the epigenetic status of genes potentially involved in the maintenance and regulation of fetal developmental during early life, a period expected to be particularly prone to epigenetic alterations induced by environmental and nutritional stressors. Methylation of CpG dinucleotides in the mammalian genome is a heritable epigenetic mark and also serves as an important mediator between the environment and genome function. Epigenetic modifications in regulatory and growth-related genes influence fetal development, with effects on important traits later in life . DNA metIn this work, variations in methylation among tissues during early mammalian development was assessed in all three germ layers of sheep fetuses by whole-genome bisulfite sequencing (WGBS).The objective was to identify genomic regions with specific epigenetic signatures that differ between tissues. Sheep fetuses were used because of their similarity to human fetuses and potential for high external stressor exposure on farms. In particular we selected sheep since the developmental biology of the ovine fetus resembles closely the human situation displaying a comparable birth weight, organogenesis, and growth rate . MoreoveSardinian ewes obtained from local breeders were housed at the Istituto Zooprofilattico Abruzzo  authorized experimental farm. The ewes were fed and kept under the best sheep housing standards. All animal experiments were performed in accordance with Italian animal experimentation legislation  in concordance with European Community regulation 86/609 and were ethically approved by CEISA .Figure 1): Ectodermal tissue EC ; Mesodermal tissue M (somites and heart); foregut Endodermal tissue EN (liver). Samples were snap frozen in liquid nitrogen and stored at -80°C for subsequent analysis. Examples of fetal tissues were fixed in 4% paraformaldehyde, embedded in Paraplast and hematoxylin-and-eosin and stained as described by Sheep were synchronized for ovulation with 25 mg Crono-gests sponges  and naturally mated. Fetal measurement and tissues collection: three fetuses were surgically collected at 28 day of gestation by paramedian laparatomy under general anesthesia. Conceptuses were immediately immersed in pre-warmed phosphate-buffered saline (PBS) and examined under a stereomicroscope to verify their vitality by the presence of a heartbeat. For each fetus, crown-rump measurement was recorded in triplicate using Image J software, an open source image-processing program designed for scientific multidimensional images. Fetuses were transferred to clean Petri dishes containing ice cold PBS and the Amniotic sac was removed to expose the fetus. Tissues were collected under a dissection microscope using forceps to stabilize the fetus and micro-scissors or a blade to isolate the three germ layer tissues , following the manufacturer’s instructions. DNA concentration was estimated by PicoGreen® . Libraries were generated using the TruSeq® DNA PCR-Free Library Preparation Kit (Illumina) including a step of bisulfite treatment. After ligation of adapters, samples were converted with EpiTect Bisulfite Kits (Qiagen) and finally PCR amplified with KAPA HiFi Uracil+ (Kapa Biosystems). WGBS was performed on an Illumina Hiseq 2000  to generate 100-base paired-end reads.Genomic DNA from each tissue was isolated using the NucleoSpin1. Illumina raw sequences were then filtered with Trimmomatic software to remove adapters and low quality bases at the ends of sequence, using a sliding window approach. Data are available in the Sequence Reads Archive (SRA), BioProject accession number, PRJNA385562.Preliminary quality control of raw reads was carried out with FastQCBismarkmethylation_extractor function. Seqmonk software (version 0.34.1)2 was used for visualization and analysis of the Bismark output. Methylated regions (MRs) were detected by dividing the genome in 100 bp tiles and analyzing average methylation for tiles containing at least four mutually covered Cs in CpG context per position. Only regions sequenced in all tissues for at least two samples were retained to investigate enrichment for different features, 5′ UTR, 3′ UTR and gene bodies, in CpG islands, shores and shelves. 5′ and 3′ UTRs were defined as 2000 bp upstream or downstream of a gene. Differentially methylated regions (DMRs) were calculated for each tissue (n = 3) vs. the others two tissues  using the logistic regression filter in R to assess differential methylation . Hierarchical clustering was calculated for DMRs present in CGIs, genes (FDR < 10exp-7), 5′ UTRs and 3′ UTRs. The level of methylation was normalized between samples and methylation percentage from a selection of DMRs showing the highest differences in methylation was used for clustering using the Genesis software  with option -N 1, and methylation calls were extracted using software .A list of DMRs found for at least one tissue vs. the other two tissues (DMRs EC + DMRs M + DMRs EN) for CGIs and different genomic features was created for pathway analysis. Gene ontology (GO) classification of the DMRs was performed according to classical GO categories, using the Cytoscape plug-in ClueGO which integrates GO  and enhaFigure 1).Fetuses collected at 28 day of gestation were measured and crown-rump length was 12.60 ± 0.179 mm (Mean ± SEM) (n = 3) and at least two in the other tissues (n = 4–6) were selected to compare the DNA cytosine methylation profile.The average number of reads per sample was 38.7 M (ranging from 25.2 to 77.3 M) with a high mapping efficiency on the sheep reference genome for all samples (range between 75.6 and 79.8%). After calculating cytosine methylation conversion rate, the sequencing coverage for each cytosine was estimated to be about 2.5X . The CpG methylation level in the CpG island shores  and shelves  increased moving away from CGIs. A lower level of methylation was observed in the EC tissue . CpG methylation at 1,459 MRs located in CGIs was examined in the three tissues: EC had 870 MRs, M 868 MRs and EN 915 MRs with low CpG methylation status (defined as a region with DNA methylation 0 ≤ α ≤ 0.2). Intriguingly, 797 low methylated regions (about 84.4%) were shared among the three tissues, whereas hypermethylated CGIs (0.8 ≤ α ≤ 1.0) were less conserved (about 32.0%)  and 2,942 downstream (+2 Kb) of genes, and 1,459 were located within the 19,450 CpG islands (CGIs) mapped. Considering CpG methylation frequency, genes and 3′ UTR of genes were prevalently hyper-methylated, whereas 5′ UTR, and CGIs showed a higher proportion of hypo-methylated regions (Data Sheet 4). Hierarchical analysis of the most significant DMRs found in CGIs, in gene bodies, 5′ UTR and 3′ UTR discriminated among samples from different tissues  were differentially methylated across the three fetal tissues. Many of these (28.8%) were located near (±2 Kb) the TSSs and more than 46.1% were within well-characterized genes. A genome-wide analysis including genes and regulatory elements revealed that variation in CpG methylation occurred within genes, 5′ UTRs and 3′ UTRs regions  respectively  in gene bodies . Variation in CpG methylation in gene bodies was predominantly found in pathways that were related to tissue morphogenesis and development such as multicellular organism signaling, cell–cell junction assembly and organization, cardiac muscle development and axon guidance and synapse organization. CpG methylation in CGIs occurred most frequently in genes related to nervous system regulation as shown in Table 1. Pathway analysis of DMRs in CGIs and gene bodies identified several related genes (members of the same gene family), or subunits of the same protein. These genes mapped at distant chromosomal locations . 535; 321 and 295 DMRs located near 5′ UTR, 3′ UTRs and CGIs, were close (±2 Kb) to 407; 284 and 90 DMGs respectively. Pathway analysis was performed on DMGs found in 5′ UTR, 3′ UTRs and CGIs, and a selection of DMGs  with higher level of methylation in CGIs was conserved among the three tissues studied. About 20% of the CGIs were differentially methylated among the three tissues. In a study of nearly 800 genes, Each of the tissues examinated in the present study was composed of several cell types and the observed methylation profile was therefore an average. Assessment of ELISA-based global DNA methylation quantification in fetal tissues at the first trimester showed variations in the methylation level in the different tissues . In the Although tissue-specific methylation of CGIs was observed at many loci that are essential for development including HOX and PAX family members , it has ache, chrnA4, chrnB2, chrnB4. This pathway seems to be involved in nervous system development and behavior: analysis of methylation variation in dorsolateral prefrontal cortex from individuals ranging from fetal to 84 years old, showed that methyl-CpG levels of chrnB4 change over the human life span  and neural development such as nrxn2, has been shown to be sensitive to environmental condition such as prenatal exposure to infectious or inflammatory insults (Comparison between fetal tissue in the present study revealed variation of CpG methylation in the gene bodies of several cell adhesion proteins, including; and FZD7 . Adhesioand FZD7 . CpGs me insults .MYO18B, MEF2A, MYH11. Deficiency of MYO18B in mice results in disruption of myofibrillar structures in embryonic cardiac myocytes (MEF2A deficiency is associated with myofibrillar disarray in embryonic heart development (MYP18B and MEF2A with expression in fetal tissues. Hypomethylation of MYH11 has been observed in muscle tissue from pigs that have undergone constant heat stress and were affected in muscle development (MYH11 expression seems to be regulated by chromatin remodeling of histone acetylation rather than methylation status (In the present study differences in CpG methylation were also found in genes related to cardiac muscle tissue development; myocytes  while MEelopment . Howeverelopment , althougn status .The WGBS profiling of ovine fetal tissues presented here provides a catalog of DNA methylation patterns across the sheep genome related to three developmental layers of the embryo. Many DMRs were layer specific and enriched in genes often closely related to specific developmental functions. Knowledge of the epigenetic status of genes potentially involved in the maintenance and regulation of normal developmental and differentiation provides a starting reference point for studies on the effects of environmental and nutritional stressors on development.PL, JW, PA-M, and AS conceived the study and revised the manuscript. PT and PS isolated fetal tissue. EC performed DNA extraction, libraries preparation and sequencing. MDC and BL carried out the bioinformatic analysis. EC and PT wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The relationship between viewer individual differences and gaze control has been largely neglected in the scene perception literature. Recently we have shown a robust association between individual differences in viewer cognitive capacity and scan patterns during scene viewing. These findings suggest other viewer individual differences may also be associated with scene gaze control. Here we expand our findings to quantify the relationship between individual differences in clinical traits and scene viewing behavior in a normative sample. The present study used Successor Representation Scanpath Analysis (SRSA) to quantify the strength of the association between individual differences in scan patterns during real-world scene viewing and individual differences in viewer attention-deficit disorder, autism spectrum disorder, and dyslexia scores. The SRSA results revealed individual differences in vertical scan patterns that explained more than half of the variance in attention-deficit scores, a third of the variance in autism quotient scores, and about a quarter of the variance in dyslexia scores. These results suggest that individual differences in attention-deficit disorder, autism spectrum disorder, and dyslexia scores are most strongly associated with vertical scanning behaviors when viewing real-world scenes. More importantly, our results suggest scene scan patterns have promise as potential diagnostic tools and provide insight into the types of vertical scan patterns that are most diagnostic. How we direct our attention in complex visual scenes has been studied since the pioneering eye tracking work of Buswell  and YarbWe  recentlyAttention-deficit disorder (ADD), dyslexia, and autism spectrum disorder (ASD) are pervasive conditions that have each been linked to atypical attentional control (for review see ). For exThis previous body of work predominantly uses simple, controlled experimental stimuli to study atypical attentional control in ADD, ASD, and dyslexia. However, recent work  took a nIn the present study, we examined the association between individual differences in clinical traits and individual differences in real-world scene scan patterns in a normative sample. We had two goals in the study. The first goal was to quantify the strength of the association between individual differences in clinical traits and scan patterns during real-world scene viewing in a normative sample. Achieving this goal will serve as a proof of concept that scan patterns can be used to identify individuals with higher/lower clinical trait scores. Second, we sought to define and visualize the scene scan patterns associated with higher and lower clinical trait scores. In particular, creating a representation of the scan patterns that are most diagnostic of high clinical scores might provide potential diagnostic markers for future clinical studies.Successor Representation Scanpath Analysis  to  to Succes  that caOur results revealed that individual differences in clinical traits are associated with individual differences in scene scanning behavior in a normative sample. Specifically, SRSA identified individual differences in scan patterns during scene viewing that explained more than half of the variance in attention-deficit scores, a third of the variance in autism quotient scores, and just under a quarter of the variance in dyslexia scores. An analysis of the state spaces and scan pattern representations that were most predictive suggested that individual differences in clinical traits were most strongly associated with how viewers guided their attention vertically across the scenes.Seventy-nine University of South Carolina undergraduate students with normal or corrected-to-normal vision participated in the study. Data in the full study was collected from June 2013 to November 2013 and no participants dropped out (0/79). Our previous work  reportedEye movements were recorded with a SR Research EyeLink 1000 plus tower mount eye tracker  sampling at 1000 Hz . ParticiStimuli consisted of 40 digitized photographs of real-world scenes. The real-world scene stimuli included a variety of indoor and outdoor environments. Participants were instructed to memorize each scene in preparation for a later memory test that was not administered. Each trial began with a fixation on a cross at the center of the display for 300 msec. Following fixation, each scene was presented for 12 seconds while eye movements were recorded. Scenes were presented in the same order across all participants. After completing the scene memorization task, participants completed a series of clinical individual difference measures including pre-diagnostic screens for attention-deficit disorder, autism spectrum disorder, and dyslexia. Eye movements were not recorded for the clinical individual difference measures.Individual differences in attention-deficit score were measured using the Jasper/Goldberg adult ADD (ADDAT) screen , 29. TheRadiating state space consisted of a series of radiating rectangular areas of interest (AOIs) from the scene center to the periphery, and was used to represent observers’ tendencies to shift their overt attention between more central and peripheral scene information. The Vertical state space consisted of 4 equal rectangular horizontal AOIs, and was used to represent observers’ tendencies to shift their overt attention vertically across each scene. The Horizontal state space consisted of 4 equal rectangular vertical AOIs, and was used to represent observers’ tendencies to shift their overt attention horizontally across each scene. Note each of these 3 state spaces contained an outside border that reflected the central fixation bias, the commonly observed phenomenon in which participants concentrate their fixations more centrally and rarely fixate the outside border of a scene [Following Hayes and Henderson (2017), three different state spaces were defined a priori to capture simple scene viewing tendencies and applied to eye movements to produce scan pattern sequences across each scene See . Each st a scene . A state a scene , 23, becA 13-point calibration procedure was performed at the start of each session to map eye position to screen coordinates. Successful calibration required an average error of less than 0.49° and a maximum error of less than 0.99°. Fixations and saccades were segmented with EyeLink’s standard algorithm using velocity and acceleration thresholds . The eysuccessor representation , and γ is a temporal discount factor (0 < γ < 1). The learning rate parameter α controls the incremental updating and γ controls the amount of temporal discounting. The γ parameter is the key to extending the temporal step size to encompass both immediate and long-range transitions—it includes the discounted future states in the prediction from the current state. After traversing a scan pattern for a given scene, the resulting successor representation can be conceptualized as having extracted the statistical regularities in temporally extended scan patterns. Specifically, an SR matrix contains, for each state, the temporally discounted number of expected future fixations to all states [Successor Representation Scanpath Analysis SRSA, , 22, 23), 2322, 2ion . The keion  to predict participant clinical individual difference scores.In the present study, SRSA was used to quantify the relationship between individual differences in clinical trait measures  and scan patterns during real-world scene viewing. Briefly summarized, for each state space definition shown in f-fit R2 –40. The One of the major advantages of SRSA is that the prediction weight matrices and principal components are interpretable . The maiα and γ) were used to convert their trial scan patterns into trial SRs. The correlation between the trial SRs from the 10 highest/lowest scoring participants were then correlated with the mean cross-validation prediction weight matrix from their respective SRSA model. These correlations represented on a trial-by-trial basis the association strength between the SRSA prediction weights and the trial scan patterns, where positive correlations were indicative of higher clinical trait scores and negative correlations were indicative of lower clinical trait scores. The trial with the greatest difference between the highest/lowest participants trial SR correlations was selected to help visualize high and low scoring scan patterns for each clinical trait measure within a common scene trial.A simple procedure was used to search for the most illustrative scan patterns for each clinical trait SRSA model. For each clinical trait measure, the participants with the 10 highest and 10 lowest scores were selected and the optimal SRSA parameters  fixations per participant. The mean participant fixation duration across all scene trials was 281 msec (SD = 41.2 msec). The mean participant saccade amplitude was 3.6° (SD = 0.38°) and the mean participant fixation number per scene trial was 37.4 (SD = 4.4). Recall that our first goal was to quantify the strength of the association between individual differences in clinical trait measures and scene scan patterns. The goodnesss-of-fit and cross-validated SRSA prediction performance is shown in With an association established, our second goal was to try to gain insight into the scan pattern differences during scene viewing that distinguish between individuals’ clinical trait scores. Fortunately, the SRSA prediction weights and principal components can be used for this purpose , 22, 23.γ = 0.67. Recall that as γ moves toward 1, the relevant scan patterns become more temporally extended across multiple state transitions. The ADD prediction weight matrix in Attention-deficit scores were the best predicted by scene encoding scan patterns. Vertical scene scan patterns accounted for more than half the variance in ADD scores Rcv2=0.3. The beγ of 0.21. ASD had the most readily interpretable prediction weights of the 3 clinical trait measures. Higher ASD scores were predicted by a strong scan pattern bias to move between and/or remain within states 2 and 3. In Autism quotient scores were also well predicted, with the vertical cross-validated SRSA model accounting for a third of the variance  suggesting the relevant scan patterns were occurring across nearly the entire scene scan pattern. A comparison of the dyslexia and attention-deficit prediction weights shows a similar pattern. The correlation between them (R = 0.41) confirmed the visual overlap.Dyslexia had the weakest prediction of our clinical trait measures Click here for additional data file.S2 Appendix(PDF)Click here for additional data file.S3 Appendix(PDF)Click here for additional data file.S1 TableR2) between each clinical and cognitive measure followed by the number of subjects in parentheses. The abbreviated measures Ospan and Rspan indicate operation span and reading span respectively.Clinical trait and cognitive capacity squared correlation matrix. The matrix shows the squared correlation ((PDF)Click here for additional data file.S2 TableGoodness-of-fit and leave-one-out cross-validated performance for predicting clinical individual difference measures using traditional eye metrics. The traditional eye metric model included the mean and standard deviation of fixation duration, saccade amplitude, and fixation number as predictors in a multiple regression model to predict each participant’s clinical trait score. The results revealed that traditional eye metrics are poor predictors of individual differences in the clinical traits we measured.(PDF)Click here for additional data file.S3 TableR2 and leave-one-out cross-validated (The Goodness-of-fit (PDF)Click here for additional data file.