Extending and generalizing the computational concept of analog series-based (ASB) scaffolds.Methodological modifications were introduced to further increase the coverage of analog series (ASs) and compounds by ASB scaffolds. From bioactive compounds, ASs were systematically extracted and second-generation ASB scaffolds isolated.More than 20,000 second-generation ASB scaffolds with single or multiple substitution sites were extracted from active compounds, achieving more than 90% coverage of ASs.Generalization of the ASB scaffold approach has yielded a large knowledge base of scaffold-capturing compound series and target information. ‘Scaffolds’ represent core structures of small molecules. A series of structural analogs contains the same scaffold (core) and different functional groups attached to the core. Scaffolds are often used to organize active compounds into different structural families. They are also used to aid in compound design in medicinal chemistry. Scaffolds can be extracted computationally from single molecules or, following a recently introduced method, from analog series. Herein, an extension of the series-based scaffold methodology is presented to further increase the number of informative scaffolds that can be obtained and represent as many analog series and compounds as possible. The concept of analog series-based scaffolds was further extended and taking multiple substitution sites into account. The figure shows an exemplary second-generation analog series-based scaffold. Analog series (AS):Series of related compounds sharing the same core structure and having different substituents at one or more sites.Analog series-based (ASB) scaffold:Scaffolds derived from analog series instead of individual compounds taking chemical reaction information into account.Matched molecular pair (MMP):A pair of compounds that are only distinguished by a chemical modification at a single site. Hence, an MMP represents two structural analogs.Matching molecular series (MMS):Extension of the MMP definition to any series of two or more analogs that are only distinguished by a chemical modification at a single site.Scaffold:A term describing the core structure of a compound or series.Substitution site:Position (site) of chemical variation in a core structure carrying different substituents  in different analogs.In chemistry, the scaffold concept is applied to represent core structures of small molecules . ScaffolThe first formal and generally applicable scaffold definition was introduced by Bemis and Murcko two decades ago . The BemRecently, a different scaffold definition has been introduced to represent core structures of analog series (ASs), rather than individual compounds, and thereby further increase the utility of scaffolds for capturing core structure information from compound series . So-callIn this work, we extend and generalize the ASB scaffold methodology by introducing second-generation ASB scaffolds that are derived from ASs with varying numbers of substitution sites. Extracting ASB scaffolds from ASs with multiple substitution sites maximizes the coverage of ASs and compounds belonging to different series and further increases the SAR information content of ASB scaffolds.In the following, the two-stage ASB scaffold approach originally introduced in  is summaRMMPs are then organized in a network in which nodes represent compounds and edges pairwise RMMP relationships. Each disjoint cluster in this network contains a unique AS . In stagThe generation of ASB scaffolds can also be rationalized by considering an extension of the MMP concept termed matching molecular series (MMS). An MMS is defined as a series of two or more analogs that are distinguished by chemical modifications at a single site . Thus, aIn the original study introducing ASB scaffolds , more thAlthough a large number of ASB scaffolds were obtained, ASs and compound coverage were limited. The original method extracted ASB scaffolds from approximately 70% of all ASs in ChEMBL (version 21). The composition of the remaining 30% of the series prohibited the generation of first-generation ASB scaffolds that were confined to ASs with single substitution sites , as discAn MMP core modification operation was implemented to reduce RMMP cores with structural extensions at a given substitution site to the smallest possible core and eliminate redundant cores for a given site. Nonredundant RMMP cores represented second-generation ASB scaffolds with single substitution sites.One or more analogs shared by different MMSs forming an AS were identified. Then, the substitution sites of the corresponding nonredundant RMMP cores were transferred to the shared analogs, thereby accounting for multiple substitution sites. From shared analogs with mapped substitution sites, a second-generation ASB scaffold with multiple substitution sites was extracted.Two methodological extensions were introduced to further increase the coverage of ASs:From ChEMBL (version 22), compounds with available high-confidence activity data were selected using a standardized data curation protocol . From seThe extended ASB scaffold method and routines for compound selection and activity data analysis were implemented using in-house Perl and Python scripts and JAVA programs with the aid of KNIME  protocolThe ASB scaffold approach was extended to generalize ASB scaffolds, in other words, derive them from ASs containing single as well as multiple substitution sites. Generalization of ASB scaffolds aimed to maximize ASs and compound coverage and further increase the SAR information content of ASB scaffolds.If an AS contains a single substitution site, it represents – by definition – an MMS. The presence of multiple substitution sites in an AS results from several MMSs forming the series. In this case, each MMS must overlap with another at least once. This means that two MMSs must share at least one analog with structural modifications at two distinct sites, in other words, one substitution site per MMS, which therefore participates in two RMMPs. Accordingly, such a relationship between two MMSs gives rise to the presence of two substitution sites in the AS. Each MMS overlap in an AS then yields an additional substitution site.To extend the ASB scaffold methodology, first, redundant RMMP cores were identified and removed, thereby reducing an AS to one or multiple nonredundant cores, depending on the number of MMSs forming the AS. Series yielding multiple qualifying RMMP cores including redundant cores were originally excluded from first-generation ASB scaffold generation.In addition, ASs consisting of multiple MMSs yield multiple nonredundant RMMP cores. In these cases, analogs shared between MMSs are identified and substitution sites are transferred from nonredundant cores to shared analogs. From shared compounds with mapped substitution sites, a second-generation ASB scaffold of a given AS is derived that captures multiple substitution sites. This is illustrated in From ChEMBL (version 22), a total of 22,015 ASs were extracted containing 133,441 compounds with available high-confidence activity data. ASs statistics are provided in The methodological extensions detailed above made it possible to extract ASB scaffolds from ASs that were on average larger than those yielding first-generation ASB scaffolds. As reported in Two basic ideas provided the foundation for the development of the ASB scaffold concept. First, scaffolds representing ASs contain more SAR and target information than compound-based scaffolds. Second, an AS should contain one and only one formally defined scaffold. Compounds forming an AS might be active against single or multiple targets. This information is captured at the level of ASB scaffolds. In addition, different from compound-based scaffolds, an ASB scaffold contains the structural framework conserved within an AS. Importantly, the ASB scaffold concept does not distinguish between a core structure and substituents and thereby covers all structural elements conserved in an AS that are relevant for bioactivity. Thus, ASB scaffolds are not restricted structurally by a predefined molecular hierarchy. In addition, taking synthetic information into account further increases the utility of computationally generated scaffolds for the practice of medicinal chemistry, for example, scaffold-based compound design.First-generation ASB scaffolds were confined to ASs having single substitution sites, which represent 71% of currently available ASs but only less than 40% of the compounds participating in ASs. Therefore, we have attempted to generalize the ASB scaffold approach by making it applicable to ASs containing multiple substitution sites. To achieve this goal, methodological extensions of the ASB scaffold approach were designed and implemented. Applying the further extended methodology, ASs and compound coverage of second-generation ASB scaffolds exceeded 90 and 70%, respectively.Given these enhancements, the potential of ASB scaffolds for medicinal chemistry applications further increases. We also anticipate that ASB scaffolds representing analogs with single- or multitarget activity will provide interesting templates for the design of focused compound libraries. This is also supported by applying retrosynthetic rules to obtain ASB scaffolds instead of random bond fragmentation, as originally used for MMP generation.In the near future, we intend to compute second-generation ASB scaffolds from all currently available bioactive compounds, including new database releases, and make a comprehensive collection of ASB scaffolds with target annotations freely available for drug discovery and design applications.Extensions of the analog series-based (ASB) scaffold methodology are introduced to capture multiple substitution sites and maximize analog series (ASs) and compound coverage.Only nonredundant RECAP-matched molecular pair cores are retained and substitution sites from multiple matching molecular series forming an AS are combined.More than 20,000 second-generation ASB scaffolds were extracted from compounds with available high-confidence activity data.Generalized ASB scaffolds approach complete ASs and compound coverage.Methodological modifications are designed to generalize the ASB scaffold methodology.Second-generation ASB scaffolds are expected to have further increased utility for medicinal chemistry.


Neonatal sepsis is associated with high mortality and morbidity, yet challenges with available diagnostic approaches can lead to delays in therapy. Our study assessed whether newborn screening analytes could be utilized to identify associations with neonatal sepsis. We linked a newborn screening registry with health databases to identify cases of sepsis among infants born in Ontario from 2010–2015. Correlations between sepsis and screening analytes were examined within three gestational age groups , using multivariable logistic regression models. We started with a model containing only clinical factors, then added groups of screening analytes. Among 793,128 infants, 4,794 were diagnosed with sepsis during the neonatal period. Clinical variables alone or in combination with hemoglobin values were not strongly predictive of neonatal sepsis among infants born at term or late preterm. However, model fit improved considerably after adding markers of thyroid and adrenal function, acyl-carnitines, and amino acids. Among infants born at early preterm gestation, neither clinical variables alone nor models incorporating screening analytes adequately predicted neonatal sepsis. The combination of clinical variables and newborn screening analytes may have utility in identifying term or late preterm infants at risk for neonatal sepsis. Even in high-resource countries where the incidence of confirmed neonatal sepsis is relatively low  and all such infants would receive treatment, approximately 40% will either die or suffer major developmental disabilities13. Although reported incidence rates of neonatal sepsis from low-resource countries are generally higher16, cases are likely underascertained owing to many factors, including a lack of access to care, poor quality care, and a lack of adequate laboratory services17. In one review of community-based studies from low- and middle-income countries, incidence rates ranged from 49 to 170 cases per 1,000 live births across 11 studies16.Neonatal sepsis, broadly defined as a systemic infection of predominantly bacterial origin in neonates21. To prevent negative health outcomes in the face of these diagnostic challenges, antibiotic therapy is generally initiated in all clinically-suspected cases of neonatal sepsis22. However, concerns are being increasingly raised about this recommended approach23, as it contributes to unnecessary or prolonged antibiotic therapy, antimicrobial resistance, and heightened risk of nosocomial infection due to increased length of hospital stay while the infant is monitored24.The successful clinical management of neonatal sepsis is predicated on early detection and prompt initiation of antibiotic therapy; however, this is difficult to achieve in practice owing to the non-specific signs and symptoms of sepsis, low sensitivity of the gold standard blood culture test (particularly following intrapartum antibiotic prophylaxis), and delayed availability of culture results (approximately 48–72 hours after blood collection)23 and many have important limitations that preclude their utility for neonatal sepsis diagnosis25. High-dimensional biology, including the study of the metabolome, is increasingly being used to help provide insights on complex biological processes and disorders26. Newborn screening measures a diverse panel of analytes, including amino acids, acyl-carnitines, hemoglobin variants, and enzymatic and endocrine markers to identify infants with rare, treatable conditions. We hypothesized that newborn screening analytes — either individually or in combination27 — could be utilized beyond their traditional application to identify neonates at high risk for sepsis. For example, differences in these analytes could reflect underlying susceptibility to neonatal sepsis through suboptimal metabolic responses to the physiologic stress associated with infection. Alternatively, any differences could also represent the sequelae of early subclinical infection as a manifestation of associated catabolic stress. Our primary objective, therefore, was to assess whether there was any association between newborn screening analyte profiles and neonatal sepsis in a large, population-based cohort of infants.Given current challenges with diagnosis, and the high mortality and morbidity associated with neonatal sepsis, particularly in low-income countries, there is a need to develop novel approaches for identifying neonates at greatest risk. Numerous biomarkers have been studied to assess their ability to identify neonatal sepsis, but thus far no individual biomarkers have demonstrated adequate sensitivity and specificityOverall, there were 793,128 infants in the full study cohort following exclusions  did not improve the model fit; however, the optimism-adjusted c-statistic increased when 17-OHP and TSH were added (from 0.685 in Model 2 to 0.725 in Model 3) and again when the additional screening analytes/analyte ratios were added to the final model (adjusted c-statistic increased to 0.782). The IDI also increased significantly in Models 2, 3, and 4 ; however, to a lesser magnitude than the incremental improvements observed among the term infants . The c-statistic adjusted for optimism in the final model (Model 4) was 0.667, only minimally different from the baseline model (optimism-adjusted c-statistic 0.65) and of a magnitude indicating poor predictive ability. When we reanalyzed these models including early preterm infants who had received a transfusion, the adjusted c-statistic showed little change across the models (0.74 in Model 1 and 0.75 in Model 4).Supplementary Figs 28. In this large, population-based study we assessed whether analyte values from newborn screening samples taken close to the time of birth demonstrated the potential to identify infants at higher likelihood of developing sepsis during the neonatal period. Our results indicate that the newborn screening profiles of infants with neonatal sepsis are different from those of non-septic infants. In particular, a combination of baseline clinical variables and newborn screening analytes performed well in identifying neonatal sepsis among late preterm and term infants. In these two gestational age groups, clinical variables alone or in combination with hemoglobin values were not strongly predictive of developing neonatal sepsis. However, the model fit improved considerably after adding markers of thyroid and adrenal dysfunction, along with acyl-carnitines, enzyme makers, and amino acids, with adjusted c-statistic values indicating a strong model for identifying cases of neonatal sepsis. However, among infants born at early preterm gestational ages , clinical variables alone were markedly less predictive of neonatal sepsis and the addition of newborn screening analytes minimally altered the predictive ability of the model. These results provide insights into the pathophysiology of neonatal sepsis and may provide guidance for the identification of early biomarkers.Reducing the burden and harms associated with neonatal sepsis is an international priority warranting further exploration of the potential for early detection of this condition31. Tighter oxygen binding capacity and thus inferior oxygen delivery to tissues may play a role in susceptibility to sepsis. However, in our models, the addition of fetal hemoglobin to baseline clinical characteristics had no impact on the model’s ability to identify infants with neonatal sepsis in any of the gestational age groups. We also hypothesized a priori that measures of thyroid and adrenal function  could be associated with neonatal sepsis. A low level of cortisol is a marker of inadequate adrenal response to illness33, and links have previously been identified between levels of TSH and neonatal sepsis34. We found the addition of markers of thyroid and adrenal function did improve the model fit, but not as substantially as the addition of the acyl-carnitines and amino acids.Different analyte patterns associated with neonatal sepsis in infants could reflect differences in underlying physiology that may predispose infants towards the condition. For example,various studies have suggested that elevated fetal hemoglobin levels may predispose infants to adverse health outcomes, including neonatal sepsis36 and positive correlations being reported between certain acyl-carnitine levels and 28-day mortality37. High acyl-carnitine levels have also been reported in other cases of injury35 and catabolic stress, which is likely the mechanism of their association with neonatal sepsis40. In cases of stress, fatty acid oxidation is disrupted, resulting in higher levels of circulating acyl-carnitines36. Abnormal carnitine metabolism can then contribute to the development of septic shock and multi-system organ damage36. Our hypothesis that catabolic stress mediated the associations we observed also explains the model performing less well among early preterm infants, many of whom experience catabolic stress even in the absence of sepsis41. However, the changes in acyl-carnitines and amino acids we observed may also reflect differences in metabolic processes that could predispose infants towards neonatal sepsis. In several of the conditions screened for with newborn screening, such as medium-chain acyl-CoA dehydrogenase deficiency, an infectious stressor can result in metabolic decompensation42. The subtle alterations we observed may reflect milder changes in the same pathways that are altered in these conditions.Alternatively, different analyte patterns associated with neonatal sepsis in infants could reflect the stress response to sepsis. Our analyses demonstrate that in late preterm and term infants, acyl-carnitines, enzyme markers, and amino acids are the newborn screening analytes most strongly associated with neonatal sepsis. Associations between acyl-carnitines and sepsis have been previously documented, with heightened analyte levels occurring among septic infants3, it is possible that newborn screening analytes could also display distinct patterns according to the timing of onset. In addition, we did not capture diagnoses of late-onset sepsis after 28 days of age. Finally, the use of diagnostic codes in administrative databases to identify cases of neonatal sepsis is expected to generate some measurement error due to low sensitivity of the codes28. Although it is likely that many of the infants who received an ICD-10-CA code for sepsis (P36) in the hospitalization database had a confirmed diagnosis of sepsis based on blood culture, we could not distinguish these confirmed diagnoses from infants classified on the basis of possible sepsis. False-negative classification of neonatal sepsis owing to low sensitivity would be expected to dilute the strength of our findings. The low prevalence of neonatal sepsis could also result in more false-positive results despite the high specificity; however, we expect any false-positive classification of sepsis is likely identifying other serious neonatal illness also causing catabolic stress. To our knowledge, the diagnostic codes for sepsis have not been validated against medical record data, and this would be informative for future studies43.A strength of our study was the size of the cohort — after exclusions, we were able to analyze data from nearly 800,000 newborns, 4,800 of whom received a diagnosis of sepsis during the neonatal period. This large, population-based cohort provided substantial statistical power to perform the complex analyses needed to develop our models. Nevertheless, our study also had several important limitations. The exact timing of onset of clinical signs of neonatal sepsis was not documented in our data, and could have occurred prior to blood spot collection, which typically occurs between 24 and 72 hours after birth. In the hospitalization database, we were also unable to reliably distinguish between early- and late-onset sepsis. Since early- and late-onset disease may differ in their clinical presentation and etiology44, whose study found substantial differences in the metabolic profiles of urine samples from septic and non-septic infants. As with our study, they also noted significantly heightened levels of several amino acids among late-onset neonatal sepsis cases, which the authors hypothesized may suggest disturbances in protein metabolism44.Our findings have value in identifying future directions for the early detection of neonatal sepsis. Future studies should examine whether measurement of markers of catabolic stress soon after birth can help identify neonatal sepsis in infants who would otherwise not be considered at risk for the condition. Our findings complement those recently published by Sarafidis and colleaguesIn this study, we sought to assess whether there was any association between newborn screening analyte profiles and neonatal sepsis. Our models suggest potential utility in using a combination of clinical variables and newborn screening analytes to identify neonatal sepsis among infants born at term or late preterm gestational ages, among whom illness may not otherwise be suspected. Indications of catabolic stress in newborn screening analyte values can serve to prompt clinicians to further explore the possibility of sub-clinical or impending illness, potentially resulting in earlier diagnosis and treatment, and ultimately improved health outcomes.www.ices.on.ca/DAS. The full dataset creation plan is available from the authors upon request.This study was conducted at the Institute for Clinical Evaluative Sciences (ICES), a non-profit research organization with Prescribed Entity status under provincial privacy legislation. ICES acts as a secure repository for Ontario’s newborn screening database and provincial health administrative databases, with the ability to deterministically link individual patient health information across databases using unique encoded identifiers to protect privacy and confidentiality. This study received approval from the institutional review board at Sunnybrook Health Sciences Centre, the ICES Privacy Office, the Ottawa Health Science Network Research Ethics Board, and the Children’s Hospital of Eastern Ontario Research Ethics Board. All procedures were performed in accordance with these institutions’ relevant guidelines and regulations. Given the Prescribed Entity status of ICES under Ontario’s privacy legislation, express consent was not required for the use of the anonymized health administrative data used in this study. The dataset from this study is held securely in coded form at ICES. While data sharing agreements prohibit ICES from making the dataset publicly available, access may be granted to those who meet pre-specified criteria for confidential access, available at th, 2015 with completed newborn screening. Infants were identified using electronic screening records from Newborn Screening Ontario (NSO), a province-wide program that screens all infants for rare conditions using a panel of screening analytes obtained via heel prick blood spot, typically between 24 and 72 hours following birth. Each infant’s electronic screening record contained information on sex, gestational age (completed weeks), mode of feeding , timing of blood spot collection, transfusion of blood or blood products, and specific analyte values.We conducted a population-based retrospective cohort study of all live births in the province of Ontario between January 1, 2010 and December 3045), medical interventions received, length of hospital stay and other data elements. Only hospitalizations with a date of admission within the neonatal period were included.We excluded infants who screened positive for one or more disorders on the newborn screening panel, those with unsatisfactory sample quality, and those who were transfused prior to blood spot collection, as donor blood interferes with hemoglobin and galactosemia-related analytes. We additionally excluded those without continuous eligibility for provincial health care benefits between birth and the end of the neonatal period . To ascertain the study outcome, we linked the study cohort to hospitalization records from the Canadian Institute for Health Information’s Discharge Abstract Database (DAD) using unique encrypted identifiers at ICES. For each admission of an infant to any Ontario hospital , the DAD contains information on up to 25 medical diagnoses  with the analyte value at those percentile levels. We computed analyte ratio combinations  to capture the relative balance/imbalance between individual analytes after replacing any analyte value of zero with the next lowest positive value in the study population in order to have a mathematically valid denominator. We standardized all analytes to the standard normal distribution to render a unit change in each analyte comparable regardless of different measurement scales. This standardization was conducted by calendar week of birth of the screened infant to minimize variation due to any external factors, including changes in assays, seasonal effects, or other non-biological factors over the study period.All newborn screening samples in Ontario are analyzed in one central laboratory using standardized biochemical assays and reported in specific measurement units for each analyte. The 49 individual analytes used in our study  recorded during any hospitalization initiated within the neonatal period , but we were unable to determine the exact time of onset or distinguish between early- or late-onset cases. A Canadian validation study of diagnostic codes for neonatal sepsis using administrative health data found sensitivity and specificity values of 38.4% and 99.7%, respectively, for the P36 code when validated against a clinical perinatal database49, we made an a priori decision to stratify all remaining analyses into subgroups defined by gestational age: <34 weeks (early preterm), 34–36 weeks (late preterm), and ≥37 weeks (term). To improve the statistical efficiency of predictive modelling within the large group of term infants, we subset the data by randomly selecting 10 non-septic infants for every infant diagnosed with sepsis.We described baseline characteristics of the study population using frequency distributions and descriptive statistics, and calculated crude rates of neonatal sepsis per 1,000 screened infants. As gestational age is strongly correlated with both incidence of neonatal sepsis and with newborn screening analyte valuesa priori, we determined the maximum number of model parameters that could be accommodated in the analysis for each gestational age group, based on a requirement of 10 sepsis cases per parameter. We then used several steps to reduce the number of analytes and analyte ratios prior to modeling. First, we computed crude Spearman’s rank correlation coefficients between sepsis and each individual standardized analyte value and standardized analyte ratio. We then computed partial Spearman’s correlations between sepsis and all individual standardized analytes, as well as the top 100 ranked standardized analyte ratios based on crude Spearman’s correlation , adjusted for infant sex, gestational age, birth weight, plurality, and mode of feeding. We then prioritized variables for inclusion in multivariable modelling in order of magnitude of partial Spearman’s correlation, and the constraint requiring at least 10 sepsis cases for every parameter included in the model. Variables modelled categorically, or using restricted cubic splines required additional degrees of freedom and thus represented more than one regression parameter.To avoid over-fitting and conserve degrees of freedom in our gestational age-specific models, post-hoc by inspection of the ranked partial Spearman’s correlations, and using splines to model a small number of the top analytes if there was a sharp drop-off in correlation after the first few ranked analytes. In a sensitivity analysis of the early preterm subgroup, we re-ran the models while including infants with a documented transfusion of blood or blood products, and adjusted for transfusion in the first nested model containing only clinical variables (Model 1) as well as all subsequent models.The models were developed using multivariable logistic regression with sepsis as the binary dependent variable. We chose models based on both pathophysiological parameters as well as mechanism of measurement. We began with a baseline model (Model 1) containing only the following clinical factors: infant sex , continuous gestational age (weeks), continuous birth weight (grams), plurality (singleton vs. twin or higher-order multiple), and mode of feeding . To this baseline clinical model, we then added the relative fetal-to-adult hemoglobin (Hb) level (Model 2), which represents a marker of oxygen delivery to tissues and is measured using high performance liquid chromatography. Next, markers of endocrine function and analytes measurable using non-mass spectrometry methods were added in Model 3 (thyroid stimulating hormone [TSH] and 17-hydroxyprogesterone [17-OHP]). In the final model (Model 4), we added the remaining analytes/analyte ratios  according to rank order based on Spearmans’s correlation until the maximum number of parameters was reached. We used restricted cubic spline terms to model the top five ranked analytes/analyte ratios to capture non-linear associations. The decision of how many analytes to model using splines was made 50 to quantify the impact of additional groups of analytes on the average sensitivity of the preceding nested model. We also computed the Net Reclassification Improvement (NRI) to quantify the net increase/decrease in predicted values for the outcome compared to the preceding nested model50. Since we were unable to partition our dataset into random subsamples for validation purposes owing to the low prevalence of sepsis, we internally validated our model performance using a bootstrap approach as described by Harrell et al.51. We drew repeated samples with replacement to produce 200 bootstrap samples, each the same size as the original dataset. The average difference in c-statistic values between the original model and each bootstrap sample was used to calculate an optimism-adjusted c-statistic51 and we additionally report the Akaike Information Criterion (AIC) for each model, which is a metric that rewards model goodness of fit, but includes a penalty against a large number of parameters to reduce overfitting. Lower AIC values are indicative of improved model fit, relative to preceding nested models. We generated correlation heat maps to visualize the relationships between analytes and differences in correlation patterns between infants with and without neonatal sepsis. Analyses were conducted with SAS version 9.4  and with R/R Studio using the RMS and HMISC packages.We assessed model discrimination using the area under the receiver operating characteristic curve (AUC) and used the Integrated Discrimination Improvement (IDI)Supplementary Information


Chromosome ends in mammalian cells are protected by a specialized 6 subunit complex called shelterin. Mutations in one component of this complex, POT1, have been associated with a variety of cancers including solid and lymphoid tumors . SurprisTo address the physiological implication of our findings we generated a mouse model in which POT1 was inactivated in the common progenitor lymphoid (CLPs) cells. We chose this compartment because of the predominance of POT1 mutations in cancer of lymphoid origin. Analysis of these mice revealed that, when combined with p53 inactivation, POT1 inhibition resulted in highly aggressive thymic lymphomas. Tumor cells derived from these mice showed increased levels of telomere dysfunction with many end-to-end chromosomal fusions and signs of replication defects at telomeres .in vivo. The impairment of the ATR-pathway in POT1-mutated tumors at first seems counterintuitive, given that increased ATR signaling has been suggested to allow tumor cells to cope with oncogene-induced replication stress [The existence of telomere dysfunction in POT1- depleted tumors created a paradox. How do POT1 mutated cells survive despite the high level of telomere dysfunction and the resultant DNA damage signaling? To get insight into this problem, we interrogated the cancer genomes for clues. Specifically, we compared by NGS the transcription profile of tumors derived from POT1-proficient and POT1-deficient tumors. This analysis revealed that the ATR pathway is significantly downregulated in tumors with POT1 mutations. This observation was confirmed in tissue culture based experiments as well as n stress . Howevern stress . In addin stress .Several questions remain to be addressed to understand the link between replication stress at telomeres and cancer development. First of all, we still do not understand how telomere replication defects and fragility leads to chromosome fusions. Additionally, from a mechanistic point of view, how POT1 mutations impact telomere replication remains unknown. Our data implicate the CST complex in this process. However, additional biochemical and structural analysis are required to elucidate how mutations in POT1 compromise CST function. Next, it will be of interest to define whether other sources of replication stress, particularly other genetic alterations known to affect telomere replication, promote a similar telomere dysfunction-induced genomic instability and increased tumor formation. Finally, and most importantly, it will be crucial to decipher the mechanism(s) by which cancer cells harboring POT1 mutations bypass telomere-driven crisis. Our data suggest that tumors derived from POT1 deficient mice display attenuated ATR signaling and have a high degree of genome instability. But how is ATR signaling attenuated? The Costanzo lab recently showed that supercoiling of repetitive centromeric DNA by Topoisomerase I precludes loading of RPA, and suppresses ATR signaling . It is t


Leishmania has a plastic genome, and drug pressure can select for gene copy number variation (CNV). CNVs can apply either to whole chromosomes, leading to aneuploidy, or to specific genomic regions. For the latter, the amplification of chromosomal regions occurs at the level of homologous direct or inverted repeated sequences leading to extrachromosomal circular or linear amplified DNAs. This ability ofLeishmania to respond to drug pressure by CNVs has led to the development of genomic screens such as Cos-Seq, which has the potential of expediting the discovery of drug targets for novel promising drug candidates. Leishmania are dimorphic parasites living as extracellular promastigotes in the digestive tract ofPhlebotomus orLutzomyia sandflies and as intracellular amastigotes within phagocytic cells (mainly macrophages) of the vertebrate hosts.Leishmania species cause leishmaniasis, the second largest parasite killer; there are 1.3 million new cases annually, 12 million people are affected worldwide, and 350 million people are currently at risk1. TheLeishmania genus includes several species, among which more than 20 are pathogenic to humans2.Leishmania can be divided into two subgenera: theLeishmania Leishmania subgenus responsible for visceral and cutaneous leishmaniasis and theLeishmania Viannia subgenus often associated with either cutaneous or muco-cutaneous forms of the disease2. Visceral leishmaniasis is mainly caused byL. donovani andL. infantum and is characterized by fever, hepatosplenomegaly, and pancytopenia3, making it the most severe and deadly form of the disease when compared with the self-healing but nonetheless debilitating skin lesions of cutaneous leishmaniasis5.Leishmania (a canineLeishmania vaccine was recently registered in Europe6), and control measures mainly involve chemotherapy9. Pentavalent antimony (Sb) has been the standard drug for 70 years and remains the mainstay in many endemic regions, apart from Northern India, where antimonial formulations have been rendered obsolete because of widespread parasite resistance. Other first-line therapies include the polyene antibiotic amphotericin B (AMB) for which a single dose was shown to be 95% effective against visceral leishmaniasis in India10. Liposomal AMB has become a standard treatment in many countries11 but requires administration by the intravenous route. Geographical differences in the response to liposomal AMB were reported, and visceral leishmaniasis cases in India were more responsive than those from East Africa or South America12. Clinical AMB resistance is scarce and parasites remained susceptible even after multiple rounds of treatment in the same patient13. The alkyl-lysophospholipid analogue miltefosine (MTF) is the only oral drug againstLeishmania15. It has been successfully used for the treatment of visceral leishmaniasis since its registration in 2002 in India16. However, relapse rates are on the rise in India18 and Nepal20, making MTF resistance a likely problem. The aminoglycoside paromomycin (PMM) is also approved for the treatment of visceral leishmaniasis in India22. So far, the scarce use of PMM has limited the emergence of resistance, but geographical variations in PMM efficacy against visceral leishmaniasis were noted between East Africa  and India24. Lastly, pentamidine (PTD) has been abandoned for the treatment of visceral leishmaniasis because of serious toxicities and is mainly restricted to patients with cutaneous leishmaniasis in South America27.No effective human vaccine is currently available against31, the depletion of trypanothione32, and apoptosis-like death36, but an exact MOA is still awaited. The same applies for MTF, AMB, PTD, and PMM, with the possible exception of AMB, which killsLeishmania by forming pores in ergosterol-containing membranes. New molecules with well-defined drug targets are clearly needed.Despite six decades of use, the mode of action (MOA) of antimonials is not known. It has been shown to lead to the production of reactive oxygen speciesLeishmania genome is around 32 Mb and displays over 8,300 coding genes38. Within theLeishmania genus, gene synteny is conserved for more than 99% of genes betweenL. major,L. infantum, andL. braziliensis, and only few species-specific genes were found38.Leishmania species have between 34 and 36 chromosomes ranging in size from 0.3 to 2.8 Mb41. One unique feature characterizing trypanosomatid parasites lies in their genome architecture, their protein-coding genes being organized as large polycistronic units43. In the absence of defined RNA polymerase II promoters, transcription of the long polycistronic units occurs in a bidirectional fashion from transcriptional start sites located at strand switch regions44. Processing into individual messenger RNAs (mRNAs) occurs by the addition throughtrans-splicing of a spliced leader RNA (39 nt) to the 5′ ends of each mRNA, coupled to 3′ end polyadenylation46. Because of its lack of transcriptional control,Leishmania uses several adaptive mechanisms to regulate gene expression when facing changing environmental conditions during its development. 3′ untranslated regions (3′ UTRs) were shown to be major players in monitoring mRNA stability and translation rates in this parasite53. To overcome stressful conditions like drug pressure,Leishmania also often relies on DNA copy number variations (CNVs)  for regulating the expression of drug targets, drug transporters, or other determinants of resistance. This is not restricted toLeishmania, however, and variations in gene dosage or chromosome copy numbers also influence drug susceptibility, adaptability, and proliferation in fungi and cancer cells57. In addition to CNVs, single-nucleotide polymorphisms (SNPs) in drug targets or in transporters can lead to drug resistance without the need for altering gene expression.TheLeishmania parasites were considered to be essentially diploid but recent data have shown that aneuploidy seems to be the norm64. Within populations ofLeishmania parasites, distinct aneuploidy patterns were shown to occur at the level of individual cells. This phenomenon was called mosaic aneuploidy and can translate into a seemingly average diploid population when the cumulative ploidy is derived from next-generation DNA sequencing data but in which few parasites actually share the same ploidy for individual chromosomes65. Interestingly, variations in the size and content of chromosomes have also been observed between different strains of the related trypanosomatid parasiteTrypanosoma cruzi67. In the case ofLeishmania, circumstantial links between the presence of supernumerary chromosomes or chromosomal losses and drug resistance have been observed71, suggesting that a particular group of genes on the variant chromosomes may possibly act together in establishing resistance, but this has yet to be demonstrated.During the last few decades,Leishmania uses aneuploidy as a lifestyle. This is raising a number of questions about aneuploidy generation, stability, transmission, and biological significance (reviewed elsewhere72). In the absence of transcription initiation control, increases (or decreases) in chromosome copy number may serve as a strategy for regulating expression under environmental cues. This can happen at the level of whole chromosomes, and indeed there was a good correlation between chromosome ploidy and the level of DNA and RNA expression59. Increasing the copy numbers of entire chromosomes may lead to the overexpression of toxic genes, but theLeishmania genome (32 Mb) is spread in 34 to 36 chromosomes, thus reducing the co-expression of many genes. However, as explained in detail below,Leishmania also has the ability to amplify (or delete) specific smaller regions of DNA as part of extrachromosomal elements by recombination/rearrangements at the level of homologous repeated sequences (RSs). RNA levels derived from these amplifications are correlated to DNA copy number.Aneuploidy is generally linked to developmental abnormalities as best exemplified by the trisomy 21 syndrome in humans. However,Leishmania is populated with repeated DNA sequences. A recent study highlighted the entire set of RSs in differentLeishmania species, and it was found that the wholeLeishmania genome has the potential to be rearranged at the level of those RSs for generating extrachromosomal elements73. Indeed, almost 2,000 RSs are distributed over the genome ofL. infantum and these potentially support the formation of more than 3,000 extrachromosomal DNA elements73. Short interspersed degenerate retroposons (SIDERs) account for up to 65% of all RSs. SIDERs are truncated versions (~0.55 kb) of formerly active retroposons that are predominantly located in 3′ UTRs and have been associated with post-transcriptional regulation at the levels of both mRNA stability and translation75. Because SIDERs are degenerated, they were found in different RS groups. Remarkably, SIDER elements would have dual roles: one functional by regulating gene expression and a second one structural, providing the backbone to facilitate gene rearrangements for changing copy number of chromosomal DNA regions.The genome ofLeishmania parasites challenged with drugs or other stressful conditions87. The episomes are amplified as either circular or linear extrachromosomal DNA and formed through rearrangements at the level of direct or inverted homologous RSs, respectively 91. Inactivating RAD51 led to viable parasites unable to generate circular extrachromosomal elements but still capable of producing linear amplicons upon drug pressure73.Leishmania has three RAD51 paralogs  that were shown to work as a complex in promoting HR through their capacity to stimulate RAD51 activity92. Inactivation of RAD51-4 was also shown to prevent the formation of circular amplicons inL. infantum exposed to drugs, but not of linear amplicons92.Since gene rearrangements through RSs are primary responses to drug pressure, a reasonable hypothesis was that identifying recombinase proteins involved in these rearrangements could lead to strategies to prevent the emergence of resistance. A first candidate was the RAD51 DNA repair protein, a key protein involved in homologous recombination (HR), a mechanism evolutionarily conserved in trypanosomatids93. MRE11 is a DNA repair nuclease that interacts with RAD50 and NBS1 to form the MRN complex95 and is important for DSB repair by HR97 or for non-homologous end joining89 101 or in its Ros3 subunit102. This was further confirmed in additional mutants using whole genome sequencing104 or by deep sequencing of MT105. Mutations detected in the MT gene ofL. infantum isolates serially collected from an MTF-treated patient who had multiple relapses were shown to correlate with resistance106, suggesting that MTF resistance could become a clinical reality in the near future.Although CNVs are important contributors of drug resistance, SNPs and small nucleotide insertions or deletions (indels) can also contribute to resistance. This was proven with experimental drugsL. donovani isolates from the Indian subcontinent supported the notion that resistance to antimonials emerged on several distinct occasions107. Isolates could also be clustered on the basis of their genetic structure and haplotypes, with some groups being enriched for non-responsive strains108. Interestingly, a particular group of highly resistant isolates that clustered together were found to share genomic features associated with resistance107. Among these were a higher copy number for the H-locus, coding for the well-characterized ABC transporter MRPA109, and a homozygous two-base-pair insertion in the aquaglyceroporin 1 (AQP1) gene involved in Sb uptake and whose inactivation or downregulation is strongly correlated with resistance117. The potential for these genomic variants in predicting treatment outcome is exciting, given the lack of molecular markers for Sb resistance, but will need to be thoroughly evaluated by using larger and geographically diversified sets of well-defined isolates.Genome-wide surveys of genetic variations in99. Since CNVs are often associated with resistance, forward genetic tools can experimentally mimic this. One such gain-of-function screen was based on functional cloning whereLeishmania cosmid libraries were electroporated intoLeishmania and these transfectants were selected for a specific phenotype118. Selection is possible because of the high copy number (and gene expression) of the cosmids. This screen was successfully applied while selecting for drug resistance or susceptibility123. This technique selects for cosmids conferring dominant phenotypes (leaving out less enriched cosmids) and is not easily amenable to high-throughput screening. The sensitivity of cosmid-based functional screening was enhanced by its recent coupling to next-generation sequencing in an approach termed Cos-Seq124. The proportion of parasites with cosmids providing a selective advantage is expected to rise with increasing drug pressure, and these can be tracked and quantified at each drug increment by Illumina sequencing125. Thus, the dynamics of cosmid enrichment can be followed over the entire course of selection instead of being monitored only at endpoint. Published or ongoing Cos-Seq screens using experimental drugs with known targets  confirmed the recovery of the relevant target genes by Cos-Seq124. Interestingly, Cos-Seq supported the hypothesis that the current anti-leishmanials  may not act via specific major protein targets124. Indeed, although an unprecedented number of resistance genes (known and novel) were isolated using Cos-Seq, none emerged as a clear target candidate and it is conceivable that these antiquated drugs are broadly cytotoxic by disrupting multiple minor targets. Whether some of the genes are genuine drug targets remains to be established, and non-protein targets represent another possibility as these would not be detected by Cos-Seq.Target-based assays and phenotypic whole-cell-based assays are the cornerstones of drug discovery. The current trend for anti-parasitic agents is for whole cell assays. A drawback of phenotype-based assays is the lack of knowledge about the targets of hit compounds. Although the molecules could be brought to the clinic without further knowledge about their MOA, a clear understanding of the molecular targets will facilitate the improvement of a candidate drug through lead optimization. Characterization of drug-resistant mutants, which often revealed mutations or CNVs in drug targets or in proteins responsible for drug transport, is one strategy to pinpoint drug targets. However, it is salient to point out that this strategy has not yet led to targets against the current anti-leishmanials, although amplification of gene targets was observed in mutants made resistant to experimental drugsL. donovani amastigotes126 is also key in the search for novel molecules having favourable anti-leishmanial properties directly on the intracellular stage of the parasite. This allowed the discovery of 192 new leads against visceral leishmaniasis from an initial set of 1.8 million compounds from GlaxoSmithKline127. An MOA could be hypothesized for 80 of the lead compounds using prior proprietary knowledge and bioinformatics analyses of TriTryp genomes, which revealed an over-representation of putative kinase inhibitors127. Cos-Seq was initially carried out with the insect form of the parasite but this could easily be adapted to intracellular parasites and this technique could be used to find the targets of these promising novel molecules or of other drugs repurposed againstLeishmania128.The advent of high-content screening for intracellular129. Although RNA interference is absent from theL. Leishmania subgenus, it is active in species of theL. Viannia subgenus130. The lack of inducible expression inLeishmania was also a limitation of this technique, but two recent reports have shown the feasibility of inducible expression inLeishmania132. Thus, it is theoretically possible to develop a technology similar to RIT-Seq inL. Viannia parasites. An alternative approach to RIT-Seq would be to rely on RNA-guided nuclease systems using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) enzymes, as these have proven very efficient for achieving targeted genomic modifications in a wide range of genomes135. In trypanosomatid parasites, the CRISPR/Cas9 system derived fromStreptococcus pyogenes has been used for disrupting genes inL. major136,L. donovani137, andT. cruzi138 and in principle could be used for generating whole-genome Cas9-mediated gene deletion libraries.The Cos-Seq technique does not allow the isolation of loss-of-function mutations such as those found in the aquaglyceroporin AQP1 or in the MT transporter genes (see above). These require high-throughput dominant negative screening approaches, like inducible RNA interference target sequencing (RIT-Seq), which proved instrumental in elucidating mechanisms of drug uptake in trypanosomesLeishmania is expanding. With new promising drug candidates in the pipeline and further technological developments, it should now be possible to find new targets which should further help in the control of this important neglected tropical disease.The toolkit for drug target discovery and resistance mechanism elucidation for3′ UTR, 3′ untranslated region; AMB, amphotericin B; AQP1, aquaglyceroporin 1; Cas, clustered regularly interspaced short palindromic repeat-associated; CNV, copy number variation; CRISPR, clustered regularly interspaced short palindromic repeat; DSB, double-strand break; HR, homologous recombination; MOA, mode of action; mRNA, messenger RNA; MT, miltefosine transporter; MTF, miltefosine; PMM, paromomycin; PTD, pentamidine; RIT-Seq, RNA interference target sequencing; RS, repeated sequence; Sb, antimony; SIDER, short interspersed degenerate retroposon; SNP, single-nucleotide polymorphism.


Functional brain-imaging techniques used in humans and animals, such as functional MRI and intrinsic optical signal (IOS) imaging, are thought to largely rely on neurovascular coupling and hemodynamic responses. Here, taking advantage of the well-described micro-architecture of the mouse olfactory bulb, we dissected the nature of odor-evoked IOSs. Using in vivo pharmacology in transgenic mouse lines reporting activity in different cell types, we show that parenchymal IOSs are largely independent of neurotransmitter release and neurovascular coupling. Furthermore, our results suggest that odor-evoked parenchymal IOSs originate from changes in light scattering of olfactory sensory neuron axons, mostly due to water movement following action potential propagation. Our study sheds light on a direct correlate of neuronal activity, which may be used for large-scale functional brain imaging. In the last decades, imaging techniques have allowed us to watch the brain at work with extraordinary details and have provided an in-depth understanding of how neural networks function. In humans, non-invasive imaging techniques do not directly measure electrical signals but rather measure correlates of neuronal activity. Functional MRI (fMRI) of blood-oxygen-level-dependent (BOLD) contrast relies on changes in blood oxygenation in active brain regions . IntrinsIn animal models, IOSs have been used as a surrogate of BOLD-fMRI to study neurovascular coupling . AdditioIn the present study, we unexpectedly found that stimulus-evoked parenchymal IOSs in the OB are independent of neurovascular coupling. We present evidence that parenchymal IOSs in the OB mainly come from the activity of olfactory sensory neuron (OSN) axons and are independent of neurotransmitter release. Our findings represent a significant step forward in understanding the origin of IOSs and provide crucial information about the different physiological correlates of neuronal activity that can be monitored by large-scale non-invasive functional imaging techniques.To study the origin of in vivo stimulus-evoked intrinsic signals, we first assessed whether odor-evoked IOSs were dependent on hemodynamic changes in awake head-restrained mice . We recoIn order to further pharmacologically dissect out the origin of IOSs in the OB, we planned to carry out experiments in anesthetized mice. Knowing that a number of anesthetics affect neurovascular coupling , we firsA direct measurement of blood flow changes (by injection of fluorescein dextran in the bloodstream) gave us the same latency values, with hemodynamics lagging odor onset by more than 1 s . This deHowever, one has to note that the delay in vascular IOSs does not necessarily mean that vascular changes do not contribute at all to glomerular IOSs. Indeed, it is possible that a late component of glomerular IOSs stems from hemodynamics. Given the difference in wavelength dependency between IOSs originating from light scattering and from hemodynamics , we compOxyhemoglobin (HbO)  has a different absorption spectrum than deoxyhemoglobin (Hb) . It is tAs a conclusion, neither blood flow nor blood oxygenation changes can explain the first second of the glomerular intrinsic signals. Given the wavelength invariance of the response waveform, we conclude that vascular and parenchymal IOSs have different origins and need to be treated separately. In the OB, parenchymal IOSs do not result from hemodynamic features such as blood volume and oxygenation and thus must originate from variations in light scattering. In order to investigate which cellular components of the OB network give rise to light scattering changes, we pharmacologically dissected out the origin of in vivo stimulus-evoked IOSs.2 and GABAB receptors , in OMP-expressing cells , and in GFAP-expressing cells . We additionally used a mouse line reporting synaptic vesicle fusion in OSNs  and a heterogeneous population of interneurons called periglomerular (PG) cells  Figure . Glutamaeceptors . All synB  did not decrease but rather increased IOS amplitude  did indeed decrease astrocyte Ca2+ signals  channels, which is key in preventing epileptic activity  channels with 2 mM CD2+. The application of this broad Cav antagonist reduced significantly vesicle fusion but had a minimal effect on intrinsic signals  along with glutamate. The receptor of such transmitter would have been missed in our initial pharmacological dissection. We first measured SpH and IOS odor-evoked activity in the same OMP-SpH animals before and after blocking voltage-gated Ca signals . These rerminals . Its remerminals . In thiserminals .Altogether, these data suggest that odor-evoked parenchymal IOSs in the OB do not depend on the release of neurotransmitter or other messenger molecule by vesicle fusion from OSNs. Parenchymal IOSs are thus purely presynaptic and reflect changes in light-scattering properties of OSN axons in the OB.What could be the nature of changes in OSN axons leading to intrinsic signals? Activity-induced axonal swelling has been described in vitro , 1988 anIn conclusion, odor-evoked parenchymal IOSs are most likely generated by morphological changes caused by activity-induced solute and water movements across the membrane of OSN axons in the OB.Our study assesses the nature of in vivo IOSs in the mouse OB. With a wide and complementary set of approaches, we show that odor-evoked IOSs recorded in glomeruli are unrelated to hemodynamics  and 2 anIntrinsic optical imaging has been used in the rodent olfactory system for more than a decade and has been shown to give functionally relevant “glomerular odor maps” . Indeed,2+ did indeed reduce vesicle fusion with little effect on IOSs. Likewise, we could measure odor-evoked signals in mice lacking Nav channels in OSN terminals. Though we cannot completely rule out a small contribution of other cell types, like astrocytes and bulbar neurons, our data suggest that most of odor-evoked parenchymal intrinsic signals we recorded arise from OSN axons.Our pharmacological approach aimed at exhaustively blocking channels, receptors, and transporters sensing OSN activity. None of the pharmacological manipulations significantly altered IOSs without affecting OSN activity . AnotherOne explanation for the predominant contribution of OSN axons to parenchymal IOSs is their massive convergence. It is estimated that 11,000 OSNs project in a given glomerulus in rodents . MoreoveIt is well known that neuronal activity precedes hemodynamic changes . As earlAltogether, our data strongly argue against a significant impact of hemodynamics on parenchymal IOSs in the OB. Although the contribution of hemodynamics to IOSs may vary between brain areas, our data highlight the contribution of activity-dependent light-scattering signals, even at wavelengths usually considered reporting only hemodynamics. The interpretation of IOSs collected at such wavelengths might then not be straightforward and may explain some controversial IOS-based results failing to link local neuronal activity and hemodynamics .+ clearance , glutamate reuptake, and vasodilation to shuttle nutrients to and metabolites from active brain regions  nor glutamate reuptake (blocked by DHK) affected glomerular intrinsic signals, and the block of metabotropic glutamate receptors almost completely abolished odor-induced Ca2+ transients in astrocytes (Mom (Omptm4cre).All experiments were performed on 12- to 20-week-old male and female mice. We used C57BL/6J  and hemizygous om  was made over the OB, leaving the dura intact. The craniotomy was filled with ACSF and covered with a glass coverslip (5 mm of diameter). See also 2+ and Ba2+ (purchased from Sigma Aldrich) were purchased from Tocris Bioscience. Drugs were dissolved in ACSF containing in mM 125 NaCl, 10 glucose, 10 HEPES, 5 KCl, 2 CaCl2, and 2 MgCl2. Ten microliters of ACSF with or without drugs were topically applied to the surface of one OB. After removing the cover glass, the dura mater was dried by gently puffing air. Drugs were applied on the dried dura, and a cover glass was placed back on the craniotomy. This procedure allows the dura membrane to be more permeable to pharmacological agents.All the drugs used, except for CD2O) or sterile isotonic solution  was injected i.p.  were from Sigma-Aldrich. See also All analyses were performed with custom Matlab (MathWorks) scripts and Statistica. See also http://dx.doi.org/10.1016/j.celrep.2015.06.016.Supplemental Information includes Supplemental Experimental Procedures and five figures and can be found with this article online at Supplemental Information


However, relevant study results could be missing from a meta-analysis because of selective publication and inadequate dissemination. If missing outcome data differ systematically from published ones, a meta-analysis will be biased with an inaccurate assessment of the intervention effect. As part of the EU-funded OPEN project ((i) compared pooled effect estimates of meta-analyses of health care interventions according to publication status of data or (ii) examined whether the inclusion of unpublished or grey literature data impacts the result of a meta-analysis.Systematic review of published literature . Four bibliographic databases were searched up to February 2016 without restriction of publication year or language. Methodological research projects were considered eligible for inclusion if they reviewed a cohort of meta-analyses which Seven methodological research projects including 187 meta-analyses comparing pooled treatment effect estimates according to different publication status were identified. Two research projects showed that published data showed larger pooled treatment effects in favour of the intervention than unpublished or grey literature data . In the remaining research projects pooled effect estimates and/or overall findings were not significantly changed by the inclusion of unpublished and/or grey literature data. The precision of the pooled estimate was increased with narrower 95% confidence interval.Although we may anticipate that systematic reviews and meta-analyses not including unpublished or grey literature study results are likely to overestimate the treatment effects, current empirical research shows that this is only the case in a minority of reviews. Therefore, currently, a meta-analyst should particularly consider time, effort and costs when adding such data to their analysis. Future research is needed to identify which reviews may benefit most from including unpublished or grey data. A meta-analysis as part of a systematic review aims to provide a thorough, comprehensive and unbiased statistical summary of data from the literature. However,There is some evidence that indicates that published randomized controlled trials tend to be larger and show an overall greater treatment effect in favor of the intervention than grey literature trials or unpublished data.–8 HoweveWe investigated the impact of study data that were not published in full text articles in scientific journals on pooled effect estimates and the overall interpretation of meta-analyses.www.open-project.eu) which aimed to investigate non-publication of study data and related dissemination bias through a series of systematic reviews –36 (i) Ianalyses. Selectivanalyses., 37, 38 ent care. (iii) Maials.gov. 41] [42 [42(i) Iials.gov. (iv) OurS1 Search Strategy(DOCX)Click here for additional data file.


Abnormal thickened lesions of the gastric wall are usually covered with normal mucosa. Conventional endoscopic biopsies often do not yield sufficient positive histological results for clinical treatment. To increase the rate of diagnosis of conventional endoscopic biopsy-negative gastric wall thickening, we used an endoscopic submucosal dissection (ESD)-like sampling method under endoscopic ultrasound (EUS) guidance to obtain tissue of gastric wall-thickening lesions. Between 2012 and 2016, patients with gastric wall thickening (as identified by computed tomography (CT), EUS or other imaging methods that showed no positive findings in repeating conventional endoscopic biopsy) underwent via mucosa incision EUS-guided sampling. Final diagnosis was determined after surgical or biopsy pathology. A total of 10 patients with gastric wall thickening were included in this study. Eight cases received definite results, whereas in two cases the biopsy results were ambiguous and in these two patients poorly differentiated adenocarcinoma was determined by postoperative pathology. The results of the cases presented in this study demonstrated that via mucosa incision EUS-guided sampling provided a complementary option for the diagnosis of conventional endoscopic biopsy-negative gastric wall thickening. Gastric wall thickening can also be caused by benign lesions, including eosinophilic infiltration or Ménétrier’s disease. Localized gastric wall thickening has also been observed in stromal tumors growing outward or in ectopic pancreas. Therefore, it is of utmost importance to sample the lesions for pathological examination to complete a definitive diagnosis.Abnormal thickening of the gastric wall with or without gastric stenosis is often identified by a computed tomography (CT) scan or endoscopic ultrasound (EUS). The most common cause is subepithelial infiltrating malignance, such as linitis plastica2. Unfortunately, it is not rare that patients with malignancies, especially linitis plastica, cannot be preoperatively diagnosed by multiple endoscopic biopsies. In the digestive tract, EUS-guided fine needle aspiration (EUS-FNA) is often used for cytological examination of lesions beneath the mucosa. However, inadequate sampling also causes negative or vague results3. Endoscopic submucosa dissection (ESD) is another approach for taking biopsies of the submucosal tumor. However, submucosal lesions are not clearly visible in the endoscopic view, which increases the risk of uncertain outcomes and potential gastric wall perforation5. In our clinical practice, we use via mucosa incision EUS-guided sampling as an ESD-like sampling means under EUS guidance. The advantage of this sampling approach is that once the submucosal lesion is located by convex EUS, a small mucosa incision is made similar as in the ESD-like technique, then forceps are inserted through the mucosal incision and a submucosal tunnel is created to sample the target lesion under EUS guidance. Adequacy of the tissue sample is guaranteed and the risk of perforation or injury of large submucosal vessels is prevented.Because the thickened lesions are usually covered with normal mucosa, a standard endoscopic biopsy often yields negative results. Bite-on-bite biopsy, an endoscopic technique commonly used in clinical settings, is not highly targeted and is associated with uncertain outcomes and a potential risk of perforationA total of 10 patients who visited our hospital between 2012 and 2016 were enrolled in this study. Patients included 3 females and 7 males with an average age of 51.4 ± 15.2 years (range: 33–77 years)  technique for biopsy of subepithelial tumors12. When lesions show no obvious submucosal uplift lesions, the ESD-like biopsy may miss the lesion in the submucosal layer, leading to unexpected damage to the gastric wall.In the digestive tract, EUS-FNA can be used for cytological examination of lesions beneath the mucosaIn most cases, thickened walls vary in thickness, the thickest site being where the diseased tissue is concentrated. Mucosa incision EUS-guided sampling allows for taking samples in areas where the diseased tissue is most concentrated, which ensures sampling adequacy. In addition, unlike direct endoscopic imaging with submucosal endoscopy, mucosa incision EUS-guided sampling only requires a minimal mucosal incision of ~5 mm long in which the forceps are inserted. This is relatively safe and reliable under real-time EUS observation. The mucosa incision EUS-guided forceps biopsies could obtain adequate samples see Fig. . In this2 can be used for insufflation, which may have a potential impact on post-procedure pain. None of the patients experienced massive gastric hemorrhage or perforation within the 5-day observation period.Our data showed that via mucosa incision EUS-guided sampling is a feasible technique for obtaining gastric submucosal tissue and is considered safe and reliable with low complication rates. Via mucosa incision EUS-guided forceps biopsies benefits from the expertise of endoscopists who are familiar with both EUS-FNA skills and ESD skills. The post-procedure pain may relate to gastroenteric gas caused by endoscope introduction for which we used air for insufflation, and could be completely alleviated within 2 h after the EUS-guidance sampling procedure. In future studies, COTherefore, our study established a potential method and strategy to diagnose the cases of conventional endoscopic biopsy-negative gastric wall thickening. However, this primary study was established in a single cancer center unit, and the results were limited to the target population who suffered from gastric wall thickening but presented with negative endoscopic biopsy results. The diagnostic efficiency of mucosa incision EUS-guided sampling for the diagnosis of biopsy-negative gastric wall thickening is promising but needs to be confirmed a larger scale.In conclusion, for endoscopic biopsy-negative gastric wall thickening, via mucosa incision EUS-guided sampling can successfully be used to help obtain more samples. Therefore, it provides a powerful complementary means for making a definitive pathological diagnosis of patients with gastric wall thickening, especially for patients with suspicious malignancies.Patients who met the following criteria were included in this study: (1) patients who visited the Cancer Center of Sun Yat-sen University  between 2012 and 2016, (2) patients who had gastric wall thickening as identified by CT, EUS, or other imaging methods and were suspected to have tumor lesions, especially malignancies, (3) patients who showed no positive findings in more than one conventional endoscopic biopsy, and (4) patients were classified as class 1 or 2 using the American Society of Anesthesiologists (ASA) physical status classification system.Patients who met any of the following criteria were excluded from this study: (1) patients who had severe cardiopulmonary dysfunction or serious disease of the liver or other vital organs, (2) patients who refused to be examined, (3) patients who underwent radiotherapy, chemotherapy or therapeutic surgery before examination. The study was approved by the Ethical Committee of Sun Yat-sen University Cancer Center, and written informed consent was provided by the patients (RDDA2017000271). All experiments were performed in accordance with relevant guidelines and regulations.Patients were fasted at least 6 h prior to the operation. A total of 10 mg of raceanisodamine was injected intramuscularly 10–15 min before the procedure, and 10 mg of diazepam was given to induce sedation. A total of 10 ml of dyclonine was administered orally to induce pharyngeal anesthesia.Longitudinal convex EUS  was performed to facilitate access to the site of wall thickening. The mucosal incision was performed by using an ERBE 200D electrosurgical system  and an Olympus Dual knife . Firstly, submucosal saline injection was performed following a standard ESD-like procedure through the EUS channel. After a about 5-mm mucosal opening cut was made, a shallow submucosal tunnel was created. Then, submucosal tissue sampling was performed by using forceps through the submucosal tunnel at an appropriate depth under the EUS guidance. The incision was closed by clips after submucosal tissue sampling.Figures After performing the via mucosa incision EUS-guided sampling, patients were hospitalized for 2 h for observation purposes. At any sign of massive gastric hemorrhage or perforation, the patient would be transferred to the hospital for emergency treatment. Patients received remote follow-up to observe any signs of tardive massive gastric hemorrhage or perforation. The total observation period was 5 days.Gastric tissue samples were embedded in paraffin, sectioned in 4um, and stained with hematoxylin and eosin (HE). All biopsy specimens were evaluated by pathologists of the Sun Yat-sen University Cancer Center .The stained sections were evaluated for (1) positivity , (2) ambiguous , and (3) negativity .In cases of resectable malignancy tumor, patients underwent surgery and a postoperative pathological report was prepared to verify the diagnosis. Ambiguous or negative cases that were highly suspicious of malignancies also underwent surgical resection to obtain a pathology report. Cases in which resection of the tumor was not an option, received chemotherapy. In benign cases, such as mass-like ectopic pancreas or lipoma, after full informed consent, patients could choose surveillance by using endoscopy/EUS for half a year to prevent potential malignancy of the disease or complete lesion resection.


Recognition of noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP) that distinguishes them from invasive malignant encapsulated follicular variant of papillary thyroid carcinoma (EFVPTC) can prevent overtreatment of NIFTP patients. We and others have previously reported that programmed death-ligand 1 (PD-L1) is a useful biomarker in thyroid tumors; however, all reports to date have relied on manual scoring that is time consuming as well as subject to individual bias. Consequently, we developed a digital image analysis (DIA) protocol for cytoplasmic and membranous stain quantitation (ThyApp) and evaluated three tumor sampling methods . A patient cohort of 153 cases consisting of 48 NIFTP, 44 EFVPTC, 26 benign nodules and 35 encapsulated follicular lesions/neoplasms with lymphocytic thyroiditis (LT) was studied. ThyApp quantitation of PD-L1 expression revealed a significant difference between invasive EFVPTC and NIFTP; but none between NIFTP and benign nodules. ThyApp integrated with hotspot nucleus tumor sampling method demonstrated to be most clinically relevant, consumed least processing time, and eliminated interobserver variance. In conclusion, the fully automatic DIA algorithm developed using a histomorphological approach objectively quantitated PD-L1 expression in encapsulated thyroid neoplasms and outperformed manual scoring in reproducibility and higher efficiency. The icurrence . Fu et aof NIFTP , 14 evenof NIFTP . HoweverConventional DIA IHC stain quantitation requires defining an area surrounding each nucleus as cytoplasm from the nucleus border –21. WithHigh efficiency DIA can be achieved through strategically sampling a tumor. For pixel-precision in feature extraction, DIA quantification is usually conducted at high resolution (20X). Analyzing the whole lesion at high resolution is time consuming, whereas analyzing at low resolution reduces accuracy. One approach to circumvent this problem is to screen the tumor at low resolution to extract a sampling subset and then within these subROIs to perform DIA quantitation at high resolution. In the initial semi-manual DIA, the user defines subROIs in the tumor where the DIA protocol package (ThyApp), a systematic image processing operation algorithm developed in our lab using the Visiopharm platform, is applied. Moving towards minimizing inter- and intra-observer variability, automatic subROI selection methods are applied. We evaluated tumor sampling methods including uniform systematic random sampling (SURS) and a combination of nuclear density and 3,3′-Diaminobenzidine (DAB) intensity hotspots. The aim of this study was to develop a fully automatic DIA algorithm that provides an efficient, objective, and accurate quantitation of PD-L1 expression. A standardized technical and interpretational method can be used as a useful aid to histopathological diagnosis to effectively distinguish NIFTP from invasive EFVPTC and avoid overdiagnosis and overtreatment of NIFTP.The clinicopathological characteristics of the study cohort are summarized in Table + or PD-L1- area respectively, Figure p < 0.001), and to benign nodules (p < 0.001); but none was observed between NIFTP and benign nodules (p = 0.113). The coexisting predominant lymphocytic/Hashimoto's thyroiditis (LT) enhanced PD-L1 expression in tumor cells and follicular cells within the inflammatory infiltrated area. Therefore, the PD-L1 staining in the presence of increased lymphocytic infiltration needs to be interpreted with caution. Using a similar but not identical patient cohort from a previously published paper [With the reclassification of NIFTP and the emergence of PD-L1 as a protein biomarker to differentiate NIFTP from invasive EFVPTC, we examined whether the DIA application protocol developed, ThyApp, could be used to assess PD-L1 expression and to distinguish the subgroups. Representative images for each subgroup are shown in Figure s p = 0.1. The coeThyroid App was developed to quantify PD-L1 expression in follicular cells. Outlined in Figure The robustness of ThyApp's histomorphological approach to segment thyroid tissue components was examined. DIA was applied to thyroid neoplasms, with a variety of distinctive histomorphologies ranging from adenoma to follicular thyroid carcinoma (FTC), and to typical papillary thyroid carcinoma (PTC), representing the complexity of the surgical specimens analyzed in the clinics. Shown in Figure p < 0.001 for all groups, Table To examine whether the DIA quantitation accurately assessed the IHC staining, photomicrographs of visually apparent low to high DAB intensity were analyzed by ThyApp Figure . The expIn addition to ANOVA analysis that revealed the relationship between PD-L1 expression score mean averages in the subgroups, distribution of the expression scores in each of the subgroups was examined. The scores were from either manual scoring or DIA. With a large sample size, normal distribution of the frequency histogram for individual subgroup could be an indicator for objective assessment and evidence supporting true differentiation of the groups. Normal distribution was observed in all fully automatic DIAed subgroups (except LT), shown by the Shapiro-Wilk normality test with significance above 0.05; whereas, in manual scoring and manual subROI, NIFTP scores failed the normality test Table . Digital6 um2, > 1000 cells analyzed) took ∼2 min each. The sample fraction study was also carried out on the complete set of cases to assess its ability to differentiate NIFTP from invasive EFVPTC. It was observed through sensitivity and specificity study that increasing the sample fraction from 3 to 5 or 10 % did not increase the area under curve (AUC), shown in Moving towards DIA full-automation, upon user-defined tumor ROI, four tumour sampling methods  in Figure p-values, larger AUC and smaller proportion of misclassified cases for all the subROI methods examined , the best performing fully-automated tumor sampling method was found to be Hotspot Nucleus , Table The PD-L1 expression parameters extracted from each subROI method were examined for its effectiveness to differentiate NIFTP from invasive EFVPTC. Mean intensity  was superior to percent positivity (binary with each pixel coded as 1 or 0 for color intensity above or below a threshold) with lower Distinguishing between invasive EFVPTC and NIFTP is clinically important and has gained increasing recognition. Given the wealth of insights that can be drawn from sectioned tissues, precise assessment of the protein biomarker expression could yield crucial ancillary diagnostic or prognostic information that have implications for personalized medicine and guiding treatment decisions .Variability between expert assessments of IHC-stained tissue was reported to be high, reflecting the unevenness in histopathologists availability, difference in the level of training, and human factors (interobserver variability) –36. Intret al. indicated in a retrospective study that 3% of NIFTP had lymph node metastasis [et al. reported 6% of NIFTP showed evidence of malignant behavior [in situ risk or undetected lymphocytic infiltration and the need for cautious follow-up monitoring. We are cognizant that the limited follow up period (1.5–5 years) did not permit a clear statement regarding the relationship of the observed high PD-L1 expression in some NIFTP and their aggressiveness and/or recurrence. Nevertheless, the differential expression of PD-L1 in the different subgroups was significant and with further studies may support its future use in clinical management as an ancillary aid to a histopathologist report [In this study, we developed and implemented a DIA protein biomarker IHC staining assessment algorithm in thyroid tissue using a histomorphological approach for PD-L1 expression. The ThyApp first processed the image as geometrical tissue components (colloid and stroma), and then applied operators to specifically select a spatially relevant element (the follicular cell). The software then performed quantitation and thresholding in this specified region. It consistently segmented thyroid variants with diverse histomorphology, extracted parameters reflecting stain variability, and effectively differentiated invasive EFVPTC from NIFTP and benign nodules by accurately quantitating PD-L1 expression in the follicular cells. The PD-L1 expression was significantly increased in EFVPTC with invasion comparing to NIFTP subgroup. Furthermore, the low PD-L1 expression in NIFTP comparable to benign nodules supports the concept that NIFTP are low risk thyroid cancers. However, we observed NIFTP PD-L1 expression spectrum distribution was positively skewed with a long tail and noted a few NIFTP cases with high PD-L1 expression scores. The close oncogenetic similarity and expression distribution overlap between the two encapsulated subgroups could contribute to the low sensitivity and specificity reported. Cho tastasis  and Parebehavior . These rt report . With cot report , 42.5 um2) analyzed a minimum of 1000 viable tumor cells. The circular subROI was chosen (over square) for best conformability to the irregular tumor tissue delineated. The size of the subROI has shown in literature to impact DIA parameter output [The ThyApp was performed in five circular subROI regions distributed pentatonically, sampling the tumor tissue. Each subROI . In extension to the ThyApp developed, here we also report the first tumor sampling methods evaluation study for DIA thyroid tissue. Upon user delineation of the tumor tissue, ThyApp was conducted in subROIs selected based on: expert guided manual subROI selection that relied heavily on the individual's knowledge and experience and three fully-automatic tumor sampling methods . Based on the effectiveness, reproducibly, and efficiently to distinguish invasive EFVPTC from NIFTP, in this patient cohort, Hotspot Nucleus outperformed SURS and Hotspot Nucleus and DAB. In addition, it showed the highest concordance correlation coefficient to manual scoring suggesting the Hotspot Nucleus offered a simulated pathologist approach to focus on target areas where tumor cells were evaluated. Though Hotspot Nucleus and manual scoring showed similar AUC and proportion of misclassified cases, DIA is advantageous in providing reproducible data. The successful implementation of an objective and fully automatic DIA algorithm that reliably quantitates thyroid protein biomarker expression could be added to the pathologist's report.No training set is required for the ThyApp used in this study. Global thresholding values used for segmentation were selected gearing the App to be less sensitive to background staining variation across image. Consistent preservation and staining processes applied to tissue provides reliable information. The assumption holds relatively true in patient cohort where specimens were processed and stained in the same institution and facility; however, as specimen preparation becomes dynamic  of Sinai Heath System, Mount Sinai Hospital (MSH) in Toronto, Canada. From a total of 1859 cases of surgically removed thyroid nodules at MSH from Jan 2010 to May 2015, 153 cases were selected based on analysis of clinical charts, surgical pathology reports and histologic slides. Inclusion criteria for the study include: the presence of a recorded chart with the gender and age of the patient at the time of diagnosis, clinical history such as recurrence episodes, size of the nodule (more than 1 cm), focality (mono), and surgical pathology diagnosis of adenomatous nodules or EFVPTC as well as the existence of enough biological material in the paraffin blocks. It has been shown in the literature that NIFTP and EFVPTC share the same types of mutation  and NIFTet al. [Immunohistochemistry staining procedures were performed and described in detail by Fu et al. . BrieflyImmunostaining scores were based on the percentage positivity and staining intensity as described previously . PercentNano Zoomer 2.0 RS , whole slide images were captured with a 20X objective (Achroplan 40X/0.65 ∞/0.17) with a Coolsnap digital camera . Digitalized images have a resolution of 1392 × 1040 pixels with RGB 24 True Color format and were saved in NDP format. The same range of illumination values was used to ensure the greatest reproducibility.ThyApp, a systematic image processing operation algorithm was developed in our lab using the Visiopharm integrator system for Windows 7, version 5.0.1.1122 . DIA analysis were carried out on a standard off-the-shelf desktop computer.The Visiopharm integrator system software runs on an image processing pipeline. Specific add-on modules implemented are listed below. In preprocessing, median filter were applied to perform background subtraction and noise reduction. Color deconvolution filters (Hematoxylin and DAB) were applied to separate the color space. Next image pixels were segmented, defining different tissue subcomponents (colloid and non-colloid). The elementary image is no longer individual pixels but connected sets of pixels belonging to the same group. In the post processing step, the segmented layer labels were refined based on the morphological and contextual of the surrounding elements. The cell to cell spatial relationship that drives the structural organization of the thyroid tissue was explored. Interface between parafollicular space and colloid was specified as the follicular cells for IHC staining quantitation. Nuclei of these cells were excluded for the subcellular staining assessment. Second thresholding on the pixel intensity in the IHC quantitation area was performed. Lastly, features were extracted and exported as DIA parameter values including percentage of positively IHC labelled cells  and digital immunostaining intensity in the subROIs quantified . Both are continuous numerical values given equal weight to every pixel analyzed.The WSI was DIA analyzed using a multi-scale feature extraction approach that mimics the human eye when examining a slide. First, an operator delineated the tumor tissue exclusion of CIS, ink artifacts, tumor satellites and all tumor areas with intense inflammation, fibrosis, necrosis or poor fixation and termed it ROI. Next, areas within the ROI, termed subROIs, where the software performs the quantitation at high resolution were selected. One semi-manual and three fully automatic subROI selection methods were evaluated. In semi-manual, subROIs for DIA were manually selected by a histopathologist. Three fully automatic subROI selection methods: Systematic Uniform Random Sampling (SURS) (a specified sample fraction of tumor); hotspot nucleus ; hotspot nucleus and DAB  were individually implemented.t-test for quantitative variables. PD-L1 scores were compared among the different diagnostic groups using one-way ANOVA test. The results were considered significant when the obtained values were p < 0.05. Hypothesis tests were 2-sided. For measurement of linear relationship between manual and DIA scoring of PD-L1 expression, Pearson covariance and correlation coefficient were computed. In addition, for measurement of concordance, Lin's correlation coefficient was computed. Shapiro-Wilk test was performed to determine normality of the scores in each subgroup.The data collected in a database designed for this study were analyzed using the SPSS version 20  program. A descriptive analysis of the population was performed. The qualitative variables were described with frequencies, and the quantitative variables were described with central tendency measures: means, standard error and dispersion standard deviation, range to 95%. A bivariate analysis was performed using independent samples