Cancer metastasis is a disease of extreme clinical relevance, as it is responsible for more than 90% of cancer-associated mortality. The molecular mechanism and critical regulators involved in this complex multi-stage process of metastasis is poorly deciphered in soft tissue sarcomas (STS), a heterogeneous group of rare tumors with high metastatic potential. Therefore, we aimed at identifying miRNA and transcription factor (TF) regulatory networks and paths in STS metastasis. We integrated mRNA and miRNA expression profiles with curated regulations  from different databases and constructed a potentially active regulatory sub-network in STS metastasis. From functional and topological analysis, we found nine novel regulators of Notch signaling sub-network which are conjectured to play critical role in metastasis of STS. This illustrated that the sub-network is promising for identification of critical regulators. Further analysis deploying our developed tool ‘RiNAcyc’ and computing coverage ratio of known STS associated genes and miRNAs identified a 15 node active path. This potential path highlights the crucial role of BMP2, hsa-miR-24, AP2 and MYC as the up-stream regulators of the path and hsa-miR-215 and TYMS as potential indicator of chemotherapeutic benefit in STS metastasis. Soft tissue sarcomas (STS) are rare form of cancer comprising of a heterogeneous group of more than 50 histological subtypes derived from mesenchymal tissuesMetastasis is a complex multi-stage process of extreme clinical relevance which is responsible for more than 90% of cancer-associated mortality5Transcriptional and post-transcriptional regulations are the critical components of tumor progression and metastasis which have increasingly garnered the attention of cancer investigators in recent years. The deregulation of transcription factors (TFs), the major regulators controlling expression of different sets of RNAs at transcriptional level, whereas miRNAs mainly acting at post-transcriptional level modulate target mRNA expression influencing multiple steps of the metastatic cascade. miRNAs are known to be involved in a wide array of biological processes including cell differentiation, development, cell death, homeostasis, and fine-tuning their regulationConsistent Active Subnetworks) was developed later by Gaire et al. to extract ASN by integrating difference in gene expression13Gene expression profile has been utilized largely for identification of underlying mechanism of a disease through interactome and network studies. To identify and understand the signaling and regulatory interactions that are operational in a system due to gene expression changes, the concept of active sub-network (ASN) was devisedhttp://vvekslab.in/tools.html can be exploited to perform similar studies in other disease systems. In addition to finding acylic paths, we generated a sub-network from the components of a core pathway, the Notch signaling pathway and identified novel regulatory components which might participate in STS metastasis-specific Notch signaling. This signaling is more alluring for investigation as this highly conserved pathway is reported to be involved in developmental processes, proliferation, apoptosis and play critical role in modulating other oncogenic signaling besides its role in sarcoma invasion and metastasis1617In this study, we contemplated differentially expressed (DE) miRNAs and mRNAs of different STS types for metastasis analysis taking into consideration of the fact that most of the human cancer types share small number of cellular, molecular and biochemical traits which form the basis of hallmarks of cancers. We amalgamated expression profiles of different STSs, curated data on various regulatory interactions  from different databases to identify potentially active sub-network (PASN) from a larger bio-molecular network which might be active during metastasis of STS. Integrating these with known STS related genes and miRNAs to improve prediction accuracy followed by network topology and functional studies, we were able to find potentially significant STS metastasis specific paths, hubs and regulators from the network. The curated regulations considered in our study to generate ASNs and paths are more discerning as compared to predicted regulations adopted in other methods to identify key modulators. Furthermore, we developed a standalone application called RiNAcyc (RNA-interacting Nodes in Acyclic paths) for identification of all acyclic paths from a network which would be enriched with DE genes/miRNAs and will provide coverage ratio of a list of entities that are known STS associated genes/miRNAs in our study. This tool available at our website, Thus, our study provided a pool of genes and miRNAs with an unexplored potential to be critical regulators during STS metastasis progression. This miRNA-TF regulatory network generation and path analysis, first of its kind in STS will provide a framework for further understanding and targeting crucial step in STS progression to harness these for therapeutics.Microarray analysis of the two data sets using GeneSpring GX 12.6 (Agilent Technologies) resulted in 343 common DE mRNAs and 250 common DE miRNAs in STS, with p < 0.05 and fold change ≥2.0. Functional analysis of the DE genes and miRNAs using Metacore ™ resulted in 47 genes and 11 miRNAs to be involved in metastasis of cancers.−10).A curated TF-miRNA regulatory network was constructed using Cytoscape by integrating curated data from databases . The generated network included 300 miRNAs, 414 TFs and 2470 target genes that formed 3184 nodes and 6153 interactions/regulations . A STS mTo have a general view of this regulatory active sub network, we calculated degrees (connectivity) and their distributions, which are basic topological network measures. In this network, the degree values of the genes, miRNAs and TFs ranged from 1 to 18, 1 to 80 and 1 to 78 respectively. The degree distribution for genes, miRNAs and TFs were found to be strongly right-skewed, indicating that most nodes had a low degree, while only a small portion of nodes had a high degree called as hubs .We screened nodes with greater than 10 degrees as potential hubs which are assessed for their potential roles in STS metastasis . We obta141920Functional analysis of active sub-network for enriched pathways yielded many curated pathways which might be active in STS metastasis . From 23From CPDB pathway analysis, we found 5 genes  from the active sub-network to be part of Notch signaling. We generated a sub-network from these 5 genes by mapping them as seed nodes to the active sub-network involved in STS metastasis and extracted the network they formed with their neighboring nodes . We obtaFrom the potentially active TF-miRNA regulatory sub-network in STS metastasis, we extracted all the acyclic pathways from zero-indegree to zero-outdegree using RiNAcyc tool developed by us. 1565 acyclic paths with 3 or more nodes which could be potentially active paths in STS metastasis were obtained. These paths consisted of 127 genes and miRNAs. The maximum length of path we obtained was 14. To identify the significant active paths from these potential active paths, we mapped the known STS genes and miRNAs obtained from Malacard, Human MicroRNA Disease Database (HMDD) database and Metacore. 221 STS associated genes and 16 miRNAs were obtained which were associated with STS pathogenesis. Of these 237 STS associated genes and miRNAs, 27 were found to be present in 1565 potential paths. We then calculated the coverage ratio as described in materials and methods section for each path to measure the probability of occurrence of the path in STS metastasis. We obtained 12 paths with p < 0.001  that were considered significantly active TF-miRNA regulatory paths in STS metastasis . Due to We also carried out topology measure/degree connectivity of the nodes  forming The 12 significant paths obtained are inferred to be potentially involved in STS metastasis. Firstly, while correlating the extent to which the components of all significant active paths appeared in the set of known STS associated genes and miRNAs, we obtained 27 STS associated entities from the list of 127 genes/miRNAs forming 1565 potential active regulatory paths. Further, the 12 active paths consisted of 15 genes/miRNAs of which 9 were reported to be associated with STS. Using hypergeometric test, it was seen that entities in all of the active pathways significantly enriched (p = 0.00053) the set of known STS-associated genes and miRNAs. Secondly, Metacore™ analysis was performed for remaining 6 genes/miRNAs  where all were found to be related to STS indirectly by their involvement in other mesenchymal tumors/sarcomas except hsa-miR-302c.De-regulation at the transcriptional and post-transcriptional level has a crucial impact on the oncogenesis of STS. In this study, we established a TF-miRNA regulatory network which might be playing a critical role in the muti-stage process of metastasis of STS at both transcripts and protein level. There is vast literature dedicated to the study of carcinomas, but not much information available on critical regulators of the metastatic pathway in STS. Elucidating the regulators controlling metastasis is vital for improving patient outcome and developing novel therapeutics. Therefore, we were impelled to construct an active TF-miRNA regulatory network in STS metastasis with the help of curated regulations and deduced critical regulators, novel notch signaling components and significant active paths which have illuminated the most significant regulatory nodes in metastatic cascades operating in STS. This work represents the first TF-miRNA regulatory network in STS.We obtained 16 pathways from the functional analysis of the active sub-network analysis. Among them, a number of pathways have role reported in metastasis of cancers, such as TGFβ receptor, EGFR1, androgen receptor, Notch signaling pathways and few more. We selected Notch signaling to assess its role in STS metastasis because of its reported crosstalks with other oncogenic signaling pathways like EGFR, NF-κB, Akt, Sonic hedgehog (Shh), mammalian target of rapamycin (mTOR), Ras, Wnt, platelet-derived growth factor (PDGF), TGFβ and androgen receptor signaling which pave tumor aggressiveness2324The STS metastasis specific Notch signaling network analysed consisted of 28 nodes. Of these, 2 TFs , 3 miRNAs  and 1 target gene (IL6) are reported to be associated with STS pathogenesis141920The most interesting part of the study is the deduction of acyclic paths from the potential active TF-miRNA regulatory network. We obtained 12 statistically significant paths (p < 0.001) which when integrated formed a 15 node path composed of different TFs, miRNAs and target genes. This composite path might be active in STS metastasis and plays a crucial role in the disease progression. All the regulators of the paths except miR-302c have considerable evidences to support their direct involvement in sarcoma . However, hsa-miR-302c has been observed to act as a tumor suppressor in hepatocellular carcinoma by inhibiting angiogenesis, an important event in metastasisComparative genomic hybridization (CGH) studies on copy number in dedifferentiated and pleomorphic liposarcomas have revealed gain in copy number of oncogenes, notably ESR1 (Estrogen receptor 1), a receptor in estrogen signalingde novo source of thymidylate, an essential precursor for DNA biosynthesis and a target of the most widely used chemotherapeutic agents like TDX against gastrointestinal malignanciesTumors included in STS are relatively more chemotherapeutic resistant than other sarcomasFrom the predicted regulatory cascades of 15 nodes , it was 39Our method of uncovering paths can be implemented to delineate critical regulatory cascades in other disease systems to unveil the complex TF-miRNA-gene regulatory relationships. However, further validation by biological experiments of the potential regulations is warranted for confirming their role in other diseases and more specifically in metastasis of STS of this study. Even though our study provides a comprehensive landscape of TF-miRNA regulatory network in STS metastasis, recent reports about novel cross-talks among the diverse RNA species like mRNAs and other non-coding RNAs indicate that there remain more dynamic regulations to be considered. One such promising intricacy involves interplay among competing endogenous RNAs (ceRNAs) and target RNAs, wherein ceRNAs regulate them by competing for shared miRNA response elements (MREs). Incorporating this novel crosstalk among the varied classes of RNAs in generating regulatory network will lead to significant gain in understanding of their role in development of STS and other cancers.Human STSs are a heterogeneous group of aggressive mesenchymal cancers where metastasis related death occurs in 50% of the patients with high grade STS. Despite their heterogeneous nature, these must be sharing common features in their metastatic process. Therefore, we performed a comprehensive analysis on five major subtypes of STS for gene expression studies and six different sub-types of STS for miRNA expression studies to obtain a common set of DE genes and miRNAs. We retrieved primary high grade STS gene expression profile (GSE21122) of Affymetrix's platform (Human Genome U133A technology) and miRNA expression profile (GSE36982) of Illumina platform from Gene expression omnibus database (GEO)Functional analysis of DE mRNAs and miRNAs obtained from microarray data processing was carried out to check for their involvement in different biological processes using Metacore™ (Thomson Reuters). The DE genes and miRNAs with established role in the multi-stage process of metastasis of cancer were screened out for further analysis.The genes and miRNAs associated with STS pathogenesis were collected from two different databases. Genes were collected from the STS specific annotations in MalaCards human disease database version 1.05The regulatory relationships among TFs, miRNAs and target genes  were obtained from five different curated databases: TransmiR (version 1.2)A potentially active TF-miRNA sub-network in STS metastasis was generated by mapping entities which are the common DE genes and miRNAs with a role in metastasis obtained from Metacore™ analysis, as active seed nodes. Any neighboring undirected node connected to the active seed node was extracted from the network to form a potential active TF-miRNA sub-network in STS metastasis.With an aim of finding critical regulatory elements, we performed network topology and functional analysis by ignoring direction of the nodes. The PASN in STS metastasis was analysed considering degree (connectivity), the basic topological measure and their distribution to assess the network characteristics. We determined the degrees of nodes and degree distribution of our network to identify hubs (nodes having large number of connections) in the network which are likely to play important regulatory roles.We used NetPath of CPDB, a manually curated pathway resource in humans to perform gene set enrichment analysis and identify signal transduction pathways overrepresented with genes involved in STS metastasis-specific regulatory networksIn our study, paths means a series of connected molecules  of a minimum length of 3 nodes and maximum of 30, which represents a signaling cascade occurring in a cell. The paths which connected multiple TFs, miRNAs and target mRNAs were identified from the active sub-network to uncover the regulatory cascades involved in transcriptional and post transcriptional regulations playing important role in STS metastasis pathogenesis. For identification of active paths from the active sub-network, all the acyclic paths with zero in-degree to zero out-degree nodes were identified using RiNAcyc, the tool developed by our group. It is a freely available standalone tool written in python using DFS algorithm that can be implemented in any disease system for generating acyclic paths. We selected paths enriched with 50% DE genes and miRNAs and evaluated to identify the significant potential active regulatory paths operating in STS metastasis. Evaluation was based on finding the coverage ratio of known STS genes and miRNAs for each path. Coverage ratio is defined as the probability of path occurring in STS; higher the coverage ratio of a path, higher its chances of occurring in STS metastasis. It was calculated as number of known genes/miRNAs in STS divided by path length . The statistical significance was calculated using hypergeometric test by the tool.B.M. and D.S. conceived the idea. D.S., M.S. and B.M. performed the experiment and data analysis. B.B.M. and J.R. assisted in analysis and fine-tuning the interpretations. C.B. developed the RiNAcyc. D.S., B.M. and J.R. have written the manuscript. All authors reviewed and edited the manuscript. B.M. supervised the project.How to cite this article: Samantarrai, D. et al. Unraveling novel TF-miRNA regulatory crosstalk in metastasis of Soft Tissue Sarcoma. Sci. Rep. 5, 9742; DOI:10.1038/srep09742 (2015).Supplementary Figures & Tables


As currently used, the Alzheimer’s Disease Assessment Scale-Cognitive subscale (ADAS-Cog) has low sensitivity for measuring Alzheimer’s disease progression in clinical trials. A major reason behind the low sensitivity is its sub-optimal scoring methodology, which can be improved to obtain better sensitivity.Using item response theory, we developed a new scoring methodology (ADAS-CogIRT) for the ADAS-Cog, which addresses several major limitations of the current scoring methodology. The sensitivity of the ADAS-CogIRT methodology was evaluated using clinical trial simulations as well as a negative clinical trial, which had shown an evidence of a treatment effect.The ADAS-Cog was found to measure impairment in three cognitive domains of memory, language, and praxis. The ADAS-CogIRT methodology required significantly fewer patients and shorter trial durations as compared to the current scoring methodology when both were evaluated in simulated clinical trials. When validated on data from a real clinical trial, the ADAS-CogIRT methodology had higher sensitivity than the current scoring methodology in detecting the treatment effect.The proposed scoring methodology significantly improves the sensitivity of the ADAS-Cog in measuring progression of cognitive impairment in clinical trials focused in the mild-to-moderate Alzheimer’s disease stage. This provides a boost to the efficiency of clinical trials requiring fewer patients and shorter durations for investigating disease-modifying treatments.The online version of this article (doi:10.1186/s13195-015-0151-0) contains supplementary material, which is available to authorized users. The Alzheimer’s Disease Assessment Scale’s cognitive subscale (ADAS-Cog) is the standard primary cognitive outcome measure for evaluating treatments in clinical trials of mild-to-moderate Alzheimer’s disease. In patients, the ADAS-Cog measures impairment across several cognitive domains that are considered to be affected early and characteristically in Alzheimer’s disease . HoweverThe low sensitivity of the ADAS-Cog is primarily due to most of its items suffering from either floor or ceiling effects in different stages of Alzheimer’s disease , 4, 5, 8Another major reason behind the low sensitivity of the ADAS-Cog is its suboptimal scoring methodology, which suffers from low accuracy in measuring cognitive impairment. Currently, cognitive impairment is estimated by simply summing scores across the ADAS-Cog items. This methodology suffers from several limitations. Firstly, the current scoring methodology makes an implicit assumption that a single patient trait is measured by the ADAS-Cog. However, psychometric analysis of the ADAS-Cog has suggested that its items measure impairment in multiple cognitive domains –13. The Secondly, the current scoring methodology also implicitly assumes that the levels of cognitive impairment required for answering the ADAS-Cog items incorrectly are uniformly ordered. However, the difficulty levels of the ADAS-Cog items are not uniform –4 and moThirdly, the current scoring methodology violates core assumptions of the statistical methods typically employed in clinical trials. The primary efficacy analysis of treatments typically involves linear modeling of serial determinations of the total ADAS-Cog scores of patients using an analysis-of-covariance (ANCOVA) methodology –22. It iFourthly, the current scoring methodology lacks a proper definition for the measurement scale of cognitive impairment. This makes comparison and interpretation of cognitive impairment across patients challenging when different variants of the ADAS-Cog are used. In theory, the administration of additional items should only improve measurement precision. However, the current scoring methodology also changes the scale of measurement, with a wider range of scores possible when additional items are administered. The current scoring methodology is also sensitive to missing item responses, scoring errors and variability in the administration of the ADAS-Cog, which are common in clinical trials , 26.In this study, we investigated the hypothesis that addressing these limitations associated with the current scoring methodology would improve the sensitivity of the ADAS-Cog in clinical trials. This resulted in a new scoring methodology for the ADAS-Cog based on a comprehensive psychometric analysis using item response theory (ADAS-CogIRT). Some prior studies have investigated the potential of item response theory for scoring the ADAS-Cog and reported very promising preliminary results , 27. TheThe data for this study were assembled from three major public cohorts to ensure that the developed scoring methodology is robust against heterogeneity in patients and study designs. The three cohorts are the Alzheimer’s Disease Neuroimaging Initiative (ADNI), the Coalition against Major Diseases (CAMD), and the Alzheimer’s Disease Cooperative Study (ADCS). These cohorts are briefly described in the Additional file The data consist of longitudinal ADAS-Cog responses over the duration of trial, basic demographics, apolipoprotein-E (APOE) genotype, and details on concomitant treatments of patients. The most common version of the ADAS-Cog, which contains a ‘Delayed word recall’ item in addition to the original 11 items, was used in this study . Table 1The patient data from the ADNI, ADCS, and CAMD cohorts were deidentified before transfer to The University of Texas at Austin. A study specific protocol for the collection and the analysis of deidentified data in this research was approved by the Institutional Review Board of The University of Texas at Austin. Since the data are publicly available and deidentified, ethics approval was not necessary from all the participating institutions in the ADNI, ADCS, and the CAMD cohorts for conducting this research. However, as part of the study protocols for data collection in the three cohorts, all participating institutions obtained ethics approval from their respective institutional review boards in accordance with the Good Clinical Practice guidelines, the Declaration of Helsinki, US 21CFR Part 50-Protection of Human Subjects, and Part 56-Institutional Review Boards, and pursuant to state and federal HIPAA regulations. Written informed consents were obtained from all subjects and authorized study partners in accordance with local institutional review board guidelines before data collection and study-specific procedures were conducted. A list of participating institutions that obtained ethics approval is provided in the Acknowledgements section.We used multidimensional item response theory (IRT) to evaluate the psychometric properties of the ADAS-Cog for measuring cognitive impairment over the course of disease progression. Traditionally, IRT has been employed for investigating psychometric properties of scales in social and educational research that measured a single latent trait in respondents. However, with advances in estimation theory –32 and cModel fit: The latent trait structure should have good global and item-level fits to the ADAS-Cog responses. Global fit was assessed using the two standard statistics of root mean squared error of approximation (RMSEA) [S-X2 statistic, which effectively controls type-I error rates [ (RMSEA)  and Tuck (RMSEA) . The cri (RMSEA) . Item-leor rates , 44.Local item independence: The local item independence assumption was tested using the recommended G2 statistic, which has high sensitivity in detecting local item dependence [pendence .Clinical relevance: The individual latent traits should be clinically meaningful constructs that are worth measuring separately. The latent trait structure should be in agreement with the motivation behind the design of the ADAS-Cog items in the original study [al study . MoreoveThe evaluation of the cognitive domains assessed by the ADAS-Cog in Alzheimer’s patients is important not only for its associated clinical significance but also for ensuring the validity of IRT analysis. IRT makes a strong assumption of local item independence, i.e., patients’ responses to the ADAS-Cog items are determined solely by their underlying extents of cognitive impairment. The use of an inappropriate set of latent traits violates this key assumption, which severely compromises the validity of inferences from IRT analysis . More imAfter determining the most appropriate latent trait structure, a confirmatory IRT model was estimated with a restricted item-trait loadings structure. From the exploratory IRT model corresponding to the most appropriate latent trait structure, only the item-trait loadings greater than 0.2 were allowed in the confirmatory IRT model . Furtherε4 genotype . The ADNI, CAMD, and ADCS cohorts contain predominantly non-Hispanic Caucasian patients, which did not allow DIF analysis due to racial and ethnic factors. In the DIF analyses, the ADAS-Cog item parameters were estimated separately for patient groups and compared using the Lord’s Wald test [The ADAS-Cog items should show measurement invariance across patients, despite their characteristics. We performed differential item functioning (DIF)  analysesald test  with theald test . The Lorald test  due to tald test .We additionally investigated the invariance properties of the ADAS-Cog item characteristic functions with respect to the status of concomitant symptomatic therapy using AChEI drugs (presence/absence). In clinical trials involving heterogeneous patient samples with respect to the status of AChEI drugs, the effects of AChEI drugs should be accounted for during statistical analysis to isolate and accurately evaluate the effects of investigative treatments. If AChEI drugs uniformly affect all the ADAS-Cog items assessing a cognitive domain (such as memory), the inclusion of an interaction term with the corresponding progression rate is sufficient to account for the effects of symptomatic therapy. However, an item-level analysis may become necessary if AChEI drugs affect only a subset of the ADAS-Cog items that measure a cognitive domain. In the absence of an item-level analysis, the treatment effects of the AChEI drugs may be inaccurately modeled leading to a biased evaluation of the investigative treatments in clinical trials. While measurement invariance of the ADAS-Cog item characteristic functions is investigated using the same DIF methodology as discussed earlier, violation of measurement invariance should not be interpreted as measurement bias of the ADAS-Cog items but rather as treatment effects of the AChEI drugs on specific subdomains within the cognitive domains.Longitudinal invariance of the item characteristic functions across different disease stages was also investigated by comparing item parameters estimated using baseline responses of patients versus using their responses at the 24-month visit, when the disease has significantly progressed. We additionally investigated the extent of sample bias and variance in the ADAS-Cog item characteristic functions due to different patient samples considered for estimation. Sample bias was assessed as the goodness-of-fit of the item characteristic functions to the ADAS-Cog response data from the treatment arms of ADCS studies, which were not used for parameter estimation. Sample variance was evaluated by conducting 1,000 bootstrap replications of estimation of the item characteristic functions with sample replacement.We propose a new ADAS-Cog scoring methodology based on psychometric modeling using IRT (ADAS-CogIRT) for more accurate measurement of cognitive impairment. Given a patient’s responses to the ADAS-Cog items, the ADAS-CogIRT methodology collectively uses the ADAS-Cog item characteristic functions to measure cognitive impairment via maximum-likelihood estimation, i.e., the latent trait values that have the highest likelihood of producing the observed set of item-wise responses. Based on the DIF analysis, appropriate adjustments were included in the item slopes and the intercepts to ensure measurement invariance across patient characteristics. By default, the latent traits in IRT are estimated with means of zero and standard deviations of one. We defined appropriate measurement scales for cognitive impairment by linearly scaling the latent traits obtained from the maximum likelihood estimation, a technique commonly used in educational testing. The values of the scaling parameters were determined based on fulfilling the following two criteria: (1) scores of cognitive impairment in mild-to-moderate Alzheimer’s patients should be non-negative; and (2) scores of cognitive impairment can be rounded off to the nearest integers without loss of precision.ADAS) between the observed and the predicted total ADAS-Cog scores at the 24-month visit. The RMSEADAS of the ADAS-CogIRT methodology was compared to the RMSEADAS achieved by using the total ADAS-Cog scores as estimates of cognitive impairment in the initial visits.Since the ground truth cognitive impairment is unknown, the accuracy of the ADAS-CogIRT methodology for measuring cognitive impairment cannot be directly evaluated. Therefore, we indirectly evaluated the ADAS-CogIRT methodology by assessing its accuracy to predict future ADAS-Cog responses of patients based on their responses in a few initial visits. Specifically, we used the ADAS-Cog responses at the baseline, 6-month, and 12-month visits of patients belonging to the treatment arms of the five ADCS studies to obtain longitudinal estimates of their cognitive impairment. These estimates were used to predict cognitive impairment and the corresponding total ADAS-Cog scores at the 24-month visit. The accuracy of the ADAS-CogIRT methodology was calculated using the root mean squared error  and treatment effects, random variability across patients is also substantial. Therefore, we modeled baseline cognitive impairment and progression rates as mixed-effects in the model to ensure validity of the key assumptions of efficacy analysis. While the fixed effects modeled systematic variability due to patient factors and treatment effects, the random effects accounted for random variability across patients. We evaluated the sensitivity of the ADAS-CogIRT methodology for detecting treatment effects in clinical trials using simulation experiments and a real clinical trial, which had been reported as negative but which showed some evidence of a treatment effect [We propose a generalized mixed-effects approach for using the ADAS-CogIRT methodology in clinical trials. Besides estimating baseline cognitive impairment, this approach estimates the rates of progression in cognitive impairment based on patients’ longitudinal ADAS-Cog responses. We assumed linear progression of cognitive impairment in patients because the durations of clinical trials are typically too short (~2-3 years) to observe any complex patterns of disease progression. Significant inter-patient variability in baseline cognitive impairment and progression rates is typically observed in clinical trials. While some variability is systematic due to patient-level factors , 0.2 (mild effect), 0.5 (moderate effect), and 0.8 (large effect) simulated in the treatment arms of clinical trials [The statistical power of the newly proposed (ADAS-CogIRT) and the standard ADAS-Cog scoring methodologies for detecting treatment effects was evaluated through two simulation experiments. In the first experiment, their power was evaluated for different sample sizes of 200, 400, 600, 800, and 1,000 patients considered in clinical trials of fixed 24 months duration. For the second experiment, the sample size was fixed as 400 patients and the statistical power was evaluated for different durations of 12, 24, 36, and 48 months. These fixed values were selected based on the average characteristics of past clinical trials. Both experiments were repeated for four hypothetical treatment effects of Cohen’s l trials . The casz-statistic with a Bonferroni correction for multiple comparisons. While a significance level of α = 0.05 was considered for the ANCOVA methodology, the significance level for investigating treatments effects using the ADAS-CogIRT scoring methodology was pre-specified as α = 0.05/m, where m denotes the number of cognitive domains assessed by the ADAS-Cog. In both experiments, 500 clinical trials were simulated for every possible combination of treatment effect, sample size, and trial duration. The statistical power was evaluated as the proportion of clinical trials in which a statistically significant treatment effect on patients’ progression rates was detected.The simulated ADAS-Cog responses were analyzed using the proposed ADAS-CogIRT and the currently employed analysis-of-covariance (ANCOVA) methodologies. The treatments effects in both of the scoring methodologies were assessed using the μg had a marginal effect  on patients’ cognitive functioning after 16 weeks [μg huperzine A treatment arms using the ADAS-CogIRT methodology. The sample size was 141 patients across the two arms in the 16-week long trial. Besides statistical significance, we also calculated the size of treatment effects estimated by the ANCOVA and the ADAS-CogIRT methodologies for a comparison of sensitivities.Besides simulations, we additionally evaluated the sensitivity of the ADAS-CogIRT methodology in a real clinical trial study of huperzine A . In the 16 weeks . Given tAll data analyses in this study were performed using the R 3.2.1 software environment for statistical computing. A more detailed description of our Methods is provided in the Additional file From the parallel analysis we estimated that the upper limit on the required number of latent traits is seven. By comparing the latent trait structures for up to seven latent traits using the criteria defined in the 'Cognitive domains assessed by the ADAS-Cog' section, the three-dimensional latent trait structure was found to be the most appropriate one. All models with the number of latent traits greater than or equal to three showed a good global and item-level fit to the ADAS-Cog response data. Local item dependence (LID) between a set of items typically indicates that the item set measures additional latent traits besides the traits already considered in the model. The three-dimensional trait structure had LID only between a few subitems, which belong to the same ADAS-Cog items. Since subitems within items tend to share item-specific contexts, such LID is expected. To eliminate all LID, seven traits were required, where several traits were measured only by single items indicating an overfit to the ADAS-Cog response data. We investigated the effect of the presence of LID on the item parameter estimates of the three-dimensional latent trait structure. The item parameter estimates of IRT models with three and seven latent traits were very similar, which suggests that LID in the three-dimensional model is negligible and does not affect item parameter estimates. While lower asymptotes were considered in the item characteristic functions of all the dichotomous ADAS-Cog items, only the constructional praxis subitem assessing a patient’s ability to draw a cube was found to have a statistically significant lower asymptote of 20.6 %. Drawing a cube is related to the patients’ spatial visualization ability, which is known to deteriorate with aging . TherefoThe three-dimensional trait structure also provides a clinically meaningful interpretation. The pattern of dominant item-trait loadings suggests that the three traits basically represent impairment in the memory, language, and praxis cognitive domains Fig. . In the The three-dimensional latent trait structure is also supported by the underlying neurodegenerative profile of Alzheimer’s disease. The classic topography of brain tissue loss in Alzheimer’s disease starts early in the medial temporal lobe, which deals with memory functions, followed by involvement of the parietal, frontal, and occipital lobes, which have functions in language processing and praxis –56. A fa2 insignificant for all the ADAS-Cog items) and low levels of local item dependence as observed in the exploratory IRT model.The confirmatory IRT model using the item-trait loading structure in Fig. ε4 genotype. AChEI drugs showed treatment effects only on a subset of the ADAS-Cog items that assess the memory domain. Specifically, the item slopes of the ‘Word recall’, ‘Delayed word recall’, and ‘Word recognition’ items were significantly smaller in patients receiving AChEI drugs. This indicates that patients receiving AChEI drugs have much slower deterioration in their ability to recall and recognize words, which probe short-term working memory. However, other memory-related items (such as ‘Orientation’) assessing other subdomains of the memory domain were not affected by the use of AChEI drugs.Table S-X2 was not statistically significant) to response data from the treatment arms of the ADCS clinical trials. The item characteristics functions also showed little variance across different patient samples with a tight agreement observed across 1,000 bootstrap replicates  and item-level fit  non-negative cognitive impairment scores for mild-to-moderate Alzheimer’s patients, and (2) standard errors of magnitude ~ 1 point in the mild-to-moderate Alzheimer’s stage. Similar approaches have been previously utilized with different scaling factors in the educational and social domains. Instead of scaling the cognitive impairment scores post-estimation, we performed an equivalent linearly scaling of the ADAS-Cog item parameters to enforce these measurement scales for estimation of memory, language, and praxis impairment. An additional advantage of the defined measurement scales is the fractional interpretation of the cognitive impairment scores. Severe Alzheimer’s patients have cognitive impairment scores close to 100 points and, therefore, a patient’s extent of cognitive impairment can be interpreted fractionally relative to severe Alzheimer’s disease patients, who have lost the ability to independently function in daily life activities.ADAS = 1.82 points. In comparison, the current scoring methodology resulted in an error of RMSEADAS = 6.05 points, which is similar in magnitude to the annual change of 5-10 points in the total ADAS-Cog scores of mild-to-moderate Alzheimer’s patients [The ADAS-CogIRT methodology illustrated good accuracy in predicting total ADAS-Cog scores at the 24-month visit with RMSEpatients , 59. FigWhile the memory items of the ADAS-Cog are informative over the whole range of memory impairment, language and praxis items hold information only for pronounced levels of language and praxis impairment Fig. . The ADAα = 0.05/3 since treatments are evaluated in the three domains of memory, language, and praxis simultaneously.The treatment effects in the simulated trials and the huperzine A trial were investigated using the ADAS-CogIRT scoring methodology at a significance level of ~300 patients) and shorter trial duration (~18 months) than the ANCOVA methodology, which requires ~ 1,000 patients in a 24-month trial to achieve 80 % power . The effects of huperzine A on progression rates of memory  and language impairment  were not statistically significant. The size of the treatment effect detected by the ADAS-CogIRT methodology (d = 1.97) was significantly higher than that detected by the ANCOVA methodology (d = 0.35). Since praxis items contribute the least to the total ADAS-Cog scores (15/70 points), the ANCOVA methodology detects a much smaller treatment effect in comparison to the ADAS-CogIRT methodology. When only the praxis ADAS-Cog items are used in the current scoring methodology, the ANCOVA analysis detects a significantly larger treatment effect size (d = 0.68), which is statistically significant .The analysis of the huperzine A trial data using the ADAS-CogIRT methodology revealed that 400 The proposed ADAS-CogIRT scoring methodology addresses several limitations associated with the current scoring methodology. An in-depth psychometric analysis showed that the ADAS-Cog measures impairment in the three distinct cognitive domains of memory, language, and praxis in patients. This is in agreement with the design of items in the original ADAS-Cog study  and findThe ADAS-CogIRT methodology estimates cognitive impairment based on patients’ response patterns across the ADAS-Cog items. Such an item-level analysis also allows adjustment for measurement bias of the ADAS-Cog items due to gender. Gender differences in item difficulty are likely due to socio-cultural factors that expose one gender to certain objects and tasks more often than the other gender experiences them. AChEI drugs have a treatment effect on patients’ performance on word recall and recognition items. Since these items contribute heavily to the total ADAS-Cog scores (32/80 points), this may be the reason behind the slower cognitive deterioration observed in patients undergoing AChEI drugs, as assessed by the current methodology , 63. SinInspired by the application of IRT in educational testing, we defined a clinically meaningful scale to measure cognitive impairment. In mild-to-moderate Alzheimer’s patients, the scale allows estimates to be rounded off to the nearest integers without loss of precision. The scale also facilitates a fractional interpretation of cognitive impairment in study patients, relative to severely impaired patients, who have a cognitive impairment score of 100 points. The parameters of the ADAS-CogIRT methodology are scale independent. Therefore, items can be easily added or removed from the ADAS-CogIRT methodology without having to re-estimate parameters or redefine properties (such as range) of the measurement scale. This is relevant because active research towards improving the ADAS-Cog items is already underway . Since tBy addressing limitations of the current scoring methodology, the ADAS-CogIRT methodology measures cognitive impairment more accurately Fig.  and makePrior work on the application of IRT to the ADAS-Cog mostly focused on evaluating its measurement properties , 60, 66.While our study addressed several limitations of the current scoring methodology, some limitations persist. Firstly, we could not investigate measurement invariance of the proposed scoring methodology across all patient-level factors (such as race and ethnicity) due to a lack of heterogeneity in the data. This limitation should be noted in future work in order to avoid biased estimates of cognitive impairment using the ADAS-CogIRT methodology with patient groups not considered in this study. Secondly, when compared to the current scoring methodology, the ADAS-CogIRT methodology requires the use of a computer or a handheld device for measuring cognitive impairment in patients. However, this limitation is less relevant for clinical trials than for routine practice because computing is already required for efficacy analysis of investigative treatments. For routine practice, a specialized application  could be developed to help providers use the ADAS-CogIRT methodology. Thirdly, the precision of the ADAS-CogIRT methodology for measuring language and praxis impairment is affected by the inherent limitations of the ADAS-Cog items Fig. . As a reDespite these limitations, the ADAS-CogIRT methodology holds great significance for clinical trials of Alzheimer’s treatments. A significant proportion of clinical trials still focus on the mild-to-moderate disease stages due to the inability to detect Alzheimer’s disease early with high specificity. The proposed scoring methodology significantly improves the efficiency of clinical trials focused on the mild-to-moderate stages of Alzheimer’s disease. Such an improvement in efficiency of clinical trials is highly desirable for rapid testing of future treatments in the critical quest for a disease-modifying treatment. The ADAS-CogIRT methodology also allows separate evaluation of treatment effects in the memory, language, and praxis domains, which can potentially provide additional information on the pharmacological properties of treatments and facilitate development of improved therapies. Future clinical trials of Alzheimer’s treatments should consider the proposed ADAS-CogIRT scoring methodology as part of their secondary efficacy analysis to further evaluate and establish the significance of the proposed methodology in comparison to the current scoring methodology.The sensitivity of the Alzheimer’s disease assessment scale-cognitive subscale (ADAS-Cog) in its current form can be significantly improved by addressing limitations associated with its scoring methodology. In this study, we described a new scoring methodology for the ADAS-Cog called the ADAS-CogIRT, which addresses several major limitations of the current scoring methodology and significantly improves the sensitivity of the ADAS-Cog in measuring progression in cognitive impairment in clinical trials. Future clinical trials of Alzheimer’s disease-modifying treatments should consider the application of the described scoring methodology as part of their secondary efficacy analysis to further validate its significance in comparison to the currently employed scoring methodology.Additional file 1:Supplementary materials: A supplementary document provides details on the methods required for reproducing the results reported in this paper. The supplementary material also contains some additional statistical results, which have not been included in the paper. (PDF 2056 kb)


E. coli isolates by their animal sources. The novel method is readily deployable to any cultivable bacteria, and may be used for several fields of study such as environmental microbiology, applied microbiology, and clinical microbiology.Shotgun proteomics is an emerging tool for bacterial identification and differentiation. However, the identification of the mass spectra of peptides to genome-derived peptide sequences remains a key issue that limits the use of shotgun proteomics to bacteria with genome sequences available. In this proof-of-concept study, we report a novel bacterial fingerprinting method that enjoys the resolving power and accuracy of mass spectrometry without the burden of peptide identification (i.e. genome sequence-independent). This method uses a similarity-clustering algorithm to search for mass spectra that are derived from the same peptide and merge them into a unique consensus spectrum as the basis to generate proteomic fingerprints of bacterial isolates. In comparison to a traditional peptide identification-based shotgun proteomics workflow and a PCR-based DNA fingerprinting method targeting the repetitive extragenic palindromes elements in bacterial genomes, the novel method generated fingerprints that were richer in information and more discriminative in differentiating  Sensitive and accurate differentiation of bacteria is important not only to microbiology but also many disciplines of basic and applied sciences. DNA fingerprinting has been one of the most commonly used strategies, as it offers a good balance between detection performance and operational convenienceProteomic fingerprints can be generated using MS analysis of intact proteins (top-down) or digested peptides (bottom-up). Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS is a top-down technique widely used for rapid identification of clinically relevant bacteria at the genus and species levels3Helicobacter pylori as well as pathogenic strains of E. coli and Yersinia pestis from non-pathogenic ones2Contrarily, shotgun proteomics is a bottom-up technique that involves tryptic digestion of the total proteins extracted from cell lysate, followed by liquid chromatography (LC) coupled with tandem mass (MS/MS) spectrometry analysis of the peptide mixture57LC-MS/MS offers high detection sensitivity and high dynamic range, resolving thousands of peptide species that vary widely in abundance within a sample11Ixodes scapularis), and tracing the blood meal to the vertebrate host that a tick had last fed on.Recently, a novel peptide identification-free, and thus genome sequence-independent, shotgun proteomic workflow has been developedE. coli isolates pertaining to four species of animal hosts of contrasting diet and habitat. E. coli is prevalent in the enteric environments of mammalian hosts. Nonetheless, each animal species features a unique combination of diet, gut anatomy, physiology, and behavior that shape the conditions of its enteric environment, which in turn, enriches for a different diversity of E. coli populationsE. coli isolates to animal sources has important implications to epidemiology and the control of food and water hygieneE. coli isolates in reference to those of a conventional proteomic workflow that involved peptide identification19In this study, we tested this peptide identification-free proteomics workflow as a novel fingerprinting method for bacterial differentiation and identification (referred to as UNID-proteomic fingerprinting hereafter). The test model was comprised of E. coli isolates were obtained from four sources  , and the genes that encode the enzymes whose activities would lead to the blue coloration of E. coli colonies on CHROMagarTM ECC E. coliEighty rm pigs) . Sewage E. coli isolates were cultured to early stationary phase and washed to obtain cell pellets for DNA and protein extraction (traction  as descrtraction .−1) were 0.1% FA in water and 0.1% FA in acetonitrile, respectively. The chromatographic conditions and data acquisition method are described in the Each cell pellet was lysed and it had protein digested with trypsin as detailed in the E. coli isolate  pairwise dot product similarity >0.7 were regarded as replicate spectra derived from multiple observations of the same peptide ion species, and were hence merged into a unique consensus spectrum (UNID-consensus spectrum hereafter) after peak alignment and noise-filtering  of the replicate spectra constituting each of them. Then, for each isolate, the numbers of replicate spectra that constituted its individual UNID-consensus spectra were normalized using the total number of MS/MS spectra acquired for the isolate. The collection of UNID-consensus spectra (i.e. presumably each represented a different peptide ion species) and the normalized abundance of the constituting replicate spectra  associated with each isolate were used as the proteomic fingerprint (UNID-proteomic fingerprint hereafter) of the isolate  (DNA was extracted from cell pellet as described in the d Maths) .E. coli isolates were compared pairwise for similarity in each fingerprinting type using Pearson correlation coefficient. The similarity matrices were visualized using multidimensional scaling (MDS). Differences among the four source groups  were tested using the analysis of similarity (ANOSIM). Jackknife analysis was used as an internal measure of the accuracy of a fingerprint library in classifying a query isolate to the correct sourceAll analyses were conducted in RE. coli isolates by source groupde novo sequencing to deduce a peptide’s amino acid sequence from its MS/MS spectra without searching a protein database37De novo sequencing was conducted with PEAKs studio Since UNID-proteomic fingerprinting did not involve peptide identification, selected UNID-consensus spectra were computationally evaluated for their likelihood of being derived from peptides. Briefly, partial least squares discriminant analysis (PLS-DA) was conducted to rank the UNID-consensus spectra for their relative importance (referred to as “loading” in PLS-DA terminology) in differentiating The mass spectrometry proteomics data have been deposited to the ProteomeXchange ConsortiumE. coli isolates. Using the similarity-clustering algorithm, 61%  of the MS/MS spectra were merged into 19,950 UNID-consensus spectra; the remaining ones were discarded for being regarded as low-quality or non-peptide derived. On the contrary, the conventional workflow of peptide identification could made use of only 11.9%  of the MS/MS spectra and merged them into 5,025 ID-consensus spectra. The consensus spectra (both UNID and ID) varied in ubiquity, from being associated with only one isolate to being associated with all isolates tested  but also the ability of LC-MS/MS to survey its analytes in greater depth. In spite of the substantial differences in information richness, the analysis of the two proteomic (UNID and ID) fingerprints and the REP-PCR fingerprints consistently indicated that E. coli isolates of the same source group were generally closer related to each other than were isolates between groups  resulted in the raise of ARCC to 95.9% . In furtE. coli isolates by source groups  remains a key issue that limits the use of shotgun proteomics to bacteria that are genome-sequenced. Recently, shotgun proteomics has been used in conjunction with metagenomics for strain-level characterization of uncultivated bacterial communitiesE. coli genome is relatively well sequenced, the similarity-clustering workflow yielded a much larger number of consensus spectra than the peptide identification workflow did MADLI-TOF is also an identification-free proteomic fingerprinting method. As a method that differentiates intact proteins by mass, MADLI-TOF has been used for the identification of protein markers to distinguish Shiga-toxin producing E. coli isolate, and the throughput was one sample at a time for each machine. To construct UNID-proteomic fingerprints, MS/MS spectra were compiled into consensus spectra using spectral matching but not peptide identification. The reliability of this fingerprinting method ultimately depends on the effectiveness of the quality filtering algorithm to detect and eliminate MS/MS spectra derived from non-peptides, and also the sensitivity and specificity of the clustering algorithm to find the MS/MS spectra of one peptide and distinguish them from those of another. The clustering algorithm used in this study was previously demonstrated as being able to merge MS/MS spectra that originated from the same peptide  with 95% accuracy. In this study, the performance of the clustering algorithm in handling the MS/MS spectra of bacterial peptides was indicated by the prominent sequence tags that were inferred for the top 10 most discriminating UNID-consensus spectra of each source group, suggesting the likelihood of those UNID-consensus spectra being peptide-derived  offers very high data reproducibility and portability, due to the unambiguous nature of DNA sequence dataIn this study, shotgun proteomics was tested for the first time for its utility in bacterial differentiation without involving peptide identification. The similarity-clustering algorithm used was previously shown to be able to generate proteomic fingerprints that allowed the blood meal of a tick to be traced to one of the 24 species of phylogenetically diverse vertebrate hosts tested E. coli isolates by animal sources were traceable to putative peptide sequences through de novo sequencing .


Strombus gigas, commonly known as the giant pink queen conch shell, exhibits outstanding mechanical properties, especially a high fracture toughness. It is known that the basic building block of conch shell contains a high density of growth twins with average thickness of several nanometres, but their effects on the mechanical properties of the shell remain mysterious. Here we reveal a toughening mechanism governed by nanoscale twins in the conch shell. A combination of in situ fracture experiments inside a transmission electron microscope, large-scale atomistic simulations and finite element modelling show that the twin boundaries can effectively block crack propagation by inducing phase transformation and delocalization of deformation around the crack tip. This mechanism leads to an increase in fracture energy of the basic building block by one order of magnitude, and contributes significantly to that of the overall structure via structural hierarchy.As a natural biocomposite,  As a natural biocomposite, Strombus gigas, commonly known as the giant pink queen conch shell, exhibits outstanding mechanical properties such as fracture toughness. Here, the authors show that these properties can be partially attributed to nanoscale twin boundaries in the basic building block of the shell. Strombus gigas conch shell contains a high density of nanoscale {110} growth twins in its third-order aragonite lamellae, the basic building block of the material. Although the existence of nanotwinned aragonite has been known for decades89123456732As an important class of natural biocomposite materials, mollusk shells possess remarkable mechanical strength and toughness as a consequence of their hierarchical structuring of soft organic and hard mineral constituents through biomineralization123456in situ fracture experiments are conducted inside a transmission electron microscope (TEM)  and only a tiny fraction of soft organics. They typically exhibit a fracture toughness several orders of magnitude higher than the corresponding single crystal of pure mineral214389pe (TEM)  to invesStrombus gigas is its third-order lamellae made of nanotwinned aragonite platelets 100∼400 nm long, ∼75 nm thick and ∼200 nm wide. Parallel stacking of the third-order lamellae forms a second-order lamella with width of 5–60 μm and thickness of 5–30 μm, and many second-order lamellae aggregate into a first-order lamella that is several micrometres wide and 5–60 μm thick  and its single-crystalline (twin-free) counterpart, and to quantitatively determine the fracture toughness by measuring crack tip opening displacement (CTOD). Specially designed nanomechanical testing specimens for CTOD measurement are shown in −1 to initiate a crack from a pre-introduced notch. Details of the TEM sample design and preparation using a focused ion beam (FIB) are given in the ‘Methods' section and 5 definitionin situ TEM measurement of CTOD are given in To quantify the fracture resistance in these two specimens, we measured the CTOD based on the δin situ TEM observations and analysis in suggesting that the nanoscale growth twins are critical in allowing the conch shell to achieve an extraordinarily high fracture toughness with minimal organic content.Furthermore, we investigated the crack propagation path in twin-free biogenic aragonite specimen prepared from nacre, and found that the nanotwinned aragonite from conch shell has much higher fracture resistance . This is2in situ TEM fracture experiments. In addition, dedicated in situ TEM nanoindentation experiments were carried out to observe the deformation mechanisms in real-time . Two simplified model patterns of nanocracks were considered, one perpendicular (referred to as y-nanocracks) and the other parallel (x-nanocracks) to the primary crack (161718y-nanocracks (s/2a=2.0) is very close to that observed in the current in situ TEM experiments  simulations of crack propagation in both nanotwinned and twin-free aragonites. The nanotwinned sample used in our simulations has the TB spacing of 10 nm as in the conch shell . The samn domain , and then domain , which aIn the twin-free aragonite with T- and M-orientations, deformation is localized in the vicinity of the crack tip, and the crack propagates smoothly, as evidenced by 3) over the transformed region driven by the crack-tip stress field, as can be seen from the typical atomic configurations of aragonite before and after the transformation  play a common and crucial role in maintaining sufficient toughness to prevent fracture, but the difference in microscopic deformation mechanisms essentially reflects that nature has evolved different energy-dissipating strategies to synthesize bio-composites with unique mechanical properties.Notably, there are many different species of mollusk shells in nature that exhibit different toughening mechanisms. Some of the bivalve shells, such as the brachiopod shells and in situ TEM studyDuring the past decade, it has been demonstrated that nanoscale twins embedded in micron- or sub-micron-sized grains of polycrystalline metals induce an exceptional combination of mechanical and physical properties including ultra-high strength and hardness, good tensile ductility and strain hardening, superior fatigue and wear resistance2134353637392134353637Strombus gigas conch shell play a critical role in the mechanical properties of the material. In situ nanoindentation-based fracture experiments on both nanotwinned and twin-free specimens from Strombus gigas, nacre and single-crystalline aragonite reveal that the TBs of nanoscale growth twins can effectively block crack propagation and induce phase transformation in the vicinity of the crack-tip, leading to the delocalization of deformation and efficient energy dissipation that delay catastrophic fracture, which is corroborated by atomistic simulations and FEM modelling. The results from both experiments and simulations show that the nanotwinned microstructure improves the fracture toughness of aragonite by roughly an order of magnitude. These findings provide a fundamental understanding of the contribution of hard basic building blocks at nanoscale to fracture resistance of the overall structure, which complement our previous knowledge about the roles of soft organic content and bio-composite structure in the toughness of bio-materials. The present study may also have profound impact on the design of strong and tough biomimetic materials with minimum organic content.In summary, the present study clearly indicates that nanoscale growth twins in Strombus gigas conch shells were cut normal to the growth line (shell axis) to expose three macroscopic layers in the cross-section of the outer whorl. The sectioned sample was sliced and polished down to 30 μm  at 45° with respect to the shell surface to expose the end face and the large face of third-order lamellae in an edge-on projection , it was further thinned for electron transparency at liquid nitrogen (LN2) temperature. To remove any surface damage layer that might be produced by the ion milling, low energy Ar+ ion milling was carried out at 250 V for 10 min using Gentle Mill (Technoorg Linda). Finally, thin layers of carbon were coated on both sides of the TEM sample to make it conductive to the electron beam. The TEM specimens of nacre and single-crystalline aragonite were prepared following the same method. A field-emission TEM  operated at 200 kV and a high voltage TEM  operated at 1.25 MV were used for the conventional microstructure analysis and the atomic structure of nanoscale twins and the fractured surfaces, respectively.Dry and externally cleaned ojection . The samojection . The wedojection . Using ain situ TEM nanomechanical testing, the samples cut from Strombus gigas conch shell and single crystal aragonite were polished to expose a specific crystallographic orientation before FIB processes. In the case of Strombus gigas conch shell, the polishing was conducted along the large face of the third-order lamellae to expose the + ion beam in FIB and lifted out from the polished surface and attached to a Cu grid through Pt deposition. Subsequently, the sample design shown in + ion beam. The details on sample preparation are summarized in In situ fracture tests with CTOD measurement were carried out in a TEM  operated at 200 kV by using a single tilt nanoindentation holder (Nanofactory). The holder is equipped with a flat-ended diamond indenter and a piezo-stage to which a TEM specimen is mounted for precise positioning and quantitative manipulation of displacement. The diamond indenter was machined using FIB to fit into the notch of the TEM sample. The sample was mounted to the piezo-stage with great care to reduce the misalignment between the tip and the sample. All the mechanical tests were performed in displacement control mode at a displacement rate of 3 nm s−1. Dynamic events of crack initiation and propagation were recorded in real-time by using a charge-coupled device camera  at 25 frames per second. For in situ nanoindentation tests in TEM, the wedge-polished TEM samples and a sharp diamond indenter were used. The TEM specimen was bonded to a Au wire using silver epoxy and assembled into the piezo-stage of the nanoindentation holder. After making contact between the sample edge and the indenter tip, the indentation test was conducted at a typical displacement rate of 10 nm s−1.Notched TEM samples for CTOD measurement were prepared using a focused ion beam . To precisely control the loading axis for in situ TEM experiments. Aragonite was modelled as a homogeneous elastic material with Young's modulus of 120 GPa and Poisson's ration of 0.3. The pre-existing crack was set to different lengths according the CTOD measurements. The J-integral value was calculated after loading until the CTOD in FEM matched with the experiment data.Two-dimensional samples  were conT-orientation, a twin-free aragonite in M-orientation and a nanotwinned aragonite with alternating T- and M-orientations (referred to as nanotwinned aragonite) in which TBs are slanted with respect to an advancing crack along a cleavage plane. Each sample is about 50 nm wide, 100 nm long and 1.1 nm thick and contains about 0.5 million atoms. All the MD simulations were performed using software package LAMMPS8 s−1. During loading, an NVT ensemble with Nosé–Hoover thermostatThree aragonite samples with controlled twin orientations were used for the simulations: a twin-free single-crystalline aragonite in How to cite this article: Shin, Y. A. et al. Nanotwin-governed toughening mechanism in hierarchically structured biological materials. Nat. Commun. 7:10772 doi: 10.1038/ncomms10772 (2016).Supplementary Figures 1-21, Supplementary Table 1, Supplementary Notes 1-8, Supplementary Methods and Supplementary ReferencesIn-situ TEM nanoscale CTOD measurements of conch shell. The results of two separate tests are displayed in series. The TEM movies were recorded at 25 fps and played at 37.5 fps (1.5 times faster playback speed) for the first movie  and at 25 fps for the second movie (Conch B). The mechanical testing was conducted at a constant displacement (3 nm/s) of the FIB-machined diamond indenter normal to the In-situ TEM nanoscale CTOD measurements of single crystal aragonite. The results of two separate tests are displayed in series. The TEM movies were recorded at 25 fps and played at the same playback speed for the first movie (Aragonite A), and at 37.5 fps (1.5 faster playback speed) for the second movie . The mechanical testing was conducted at a constant displacement (3 nm/s) of the FIB-machined diamond indenter along the Deformation twinning during crack propagation in single crystal aragonite. Deformation twinning occurs ahead of crack tip as a major plastic deformation induced by the crack tip stress fields. The deformation twins form and propagate along the Formation of nanocracks and their interaction with main crack in the conch shell. The advancing main crack was blocked frequently by the slanted TBs. While the main crack is trapped at the TBs for a while, nanocracks (marked by yellow arrows in the movie) were nucleated ahead of the main crack. The main crack propagated through repeated deflection and coalescence with the nanocracks. This observation inspired coarse-grained simulations to reveal the optimum spacing and size of nanocracks (Supplementary Method 5). The observed crack pattern is very close to one that yields the highest fracture toughness in the simulation (Supplementary Discussion 7).Crack propagation paths in conch shell, nacre, and single crystal aragonite. The plot of crack length versus loading time is displayed in sync with the corresponding TEM movie. In the conch shell, the main crack propagated through crack deflection and coalescence with nanocracks . In the case of nacre, a single crack propagated through the aragonite plates without hindrance (shown as steep ramps of the curve), while being trapped or deflected at the organic interfaces between adjacent plates (plateau regions in the curve). In contrast, crack propagated in single crystal aragonite in a brittle manner along the In-situ TEM nanoindentation. Deformation twinning occurred under the indenter as a major plastic deformation driven by the stress fields. The loading axis was parallel to the Deformation twinning in aragonite during In-situ TEM nanoindentation. The stress fields under the indenter induced a type of phase transformation (or amorphization) of nanotwinned aragonite with no indication of dislocation slip or deformation twinning. The loading axis was normal to the Phase transformation in conch shell during MD simulation of crack propagation in nanotwinned aragonite (with twin boundary spacing of 10nm). Under increasing loading, an edge crack starts to propagate toward an array of TBs. When the crack reaches a TB, it is trapped by the TB over a time period. The crack tip becomes blunted due to sliding on the TB. Plastic deformation via structural transformation ahead of the crack tip is confined within the nanoscale twins. As the applied loading continues, the crack eventually penetrates through the TB.MD simulation of crack propagation in nanotwinned aragonite (with twin boundary spacing of 20nm). The results are qualitatively similar to those in Supplementary Movie 8.MD simulation of crack propagation in single-crystalline aragonite in T-orientation. Structural transformation occurs ahead of the crack tip while the crack propagates in a straight path.MD simulation of crack propagation in single-crystalline aragonite in M-orientation. The crack is slightly deflected along a


To overcome the common problem of donor-site morbidity of standard autologous bone grafts, we successfully combined tissue engineering techniques for the first time with the arteriovenous loop model to generate vascularized large bone grafts. We present two cases of large bone defects after debridement of an osteomyelitis. One of the defects was localized in the radius and one in the tibia. For osseus reconstruction, arteriovenous loops were created as vascular axis, which were placed in the bony defects. In case 1, the bone generation was achieved using cancellous bone from the iliac crest and fibrin glue and in case 2 using a clinically approved β-tricalciumphosphate/hydroxyapatite (HA), fibrin glue and directly auto-transplanted bone marrow aspirate from the iliac crest. The following post-operative courses were uneventful. The final examinations took place after 36 and 72 months after the initial operations. Computer tomogrphy (CT), membrane resonance imaging (MRI) and doppler ultrasound revealed patent arterio-venous (AV) loops in the bone grafts as well as completely healed bone defects. The patients were pain-free with normal ranges of motion. This is the first study demonstrating successfully axially vascularized  Tissue defects in poorly vascularized recipient areas require the transplantation of replacement tissue that creates a donor-site defect elsewhere in the body. This has challenged the search for alternative means and has fostered the development of new scientific areas such as Tissue Engineering. Treatment of large bone defects after trauma, tumour or infection remains such a major challenge in different surgical fields such as orthopaedic surgery, neurosurgery, head and neck surgery or plastic surgery. Autologous bone grafting still represents the gold standard for osteogenic bone replacement, although availability is limited in quality and quantity . Furtherin vitro to the in vivo environment as nutrition and oxygen supply via diffusion is limited to a maximum range of 200 μm [et al. showed a clinical application of a custom-designed axially vascularized bone graft using the latissimus dorsi muscle as a vascular carrier leaving a significant muscle donor-site defect [A multitude of different synthetic bone substitutes have been developed for bone replacement to overcome the inherent shortcomings and donor-site morbidities of autologous bone grafting. Adequate vascularization plays a crucial role for transferring tissue engineering models from the f 200 μm . The maje defect .To overcome the problem of donor-site morbidity (leaving neither bone nor muscle defect), Erol and Spira developed the rat arteriovenous ‘arterio-venous (AV)’ loop model using only a vein graft as a vascular axis . Over thde novo bone generation in large bone defects in a clinical scenario using the arteriovenous loop model without creation of a significant donor-site defect in patients.To verify this promising technique from bench to bedside in the human recipient, this is the first study demonstrating Between 2006 and 2009, two patients presented with large bone defects after debridement of an osteomyelitis. One of the defects was localized in the radius and one in the tibia. In all two cases, the osteomyelitis caused extensive poorly vascularized bony defects, which required vascularized bone grafts. To prevent any kind of donor-site morbidity but to achieve a vascularization inside the bone grafts in the poorly vascularized recipient areas, only a vein graft as a vascular axis was implanted into the bone graft using microsurgical techniques.©, Baxter, Vienna, Austria). Finally, the moulded mass in the defect was covered and held together using a vicryl mesh with a corresponding hole for the vascular pedicle for the arterial and the venous side each.A 39-year-old patient sustained an open tibial fracture in 1991 in a motorcycle accident. A multitude of operative interventions resulted in an instable scar of the lower leg. The patient was referred to our department in November 2006 with a suspicion of an osteomyelitis of the tibia, which was noticed in membrane resonance imaging (MRI) after the patient hit the leg another time. An angiography of the left leg revealed that the lower leg was perfused only by collaterals. After surgical debridement and prolonged antibiotic treatment, the bony and soft tissue defects were reconstructed using an arteriovenous loop as a vascular connection for a free gracilis muscle flap transfer in one stage. Post-operatively the bypass showed an insufficient perfusion because of arteriovenous loop failure. After flap debridement and loop revision, a vacuum-assisted closure (VAC) therapy was applied temporarily and was followed by a second arterial bypass reconstruction and free rectus abdominis muscle flap with skin grafts. Initially, the post-operative course was then uneventful. However, 2 years post-operatively, the patient was readmitted with redness and swelling of the lower leg. She was found to suffer again from a severe osteomyelitis of the tibia. After multiple radical osseus and periosteum debridements and repeated application of VAC therapy, a bony defect of the tibia of 8 × 2.5 cm remained. As the patient refused another free flap reconstruction such as with a free vascularized tibia or free vascularized partial scapula despite the large bony defect, we discussed an attempt at minimally invasively transplanting cancellous bone chips augmented with an arteriovenous axis for osseus reconstruction. Therefore, an arteriovenous loop was created using a vein graft from the lower contralateral leg interposed between the popliteal artery  and the popliteal vein (end-to-side) penetrating the tibial bone defect. The bone defect was then filled with a custom-made bone substitute using small cancellous bone chips from the iliac crest and fibrin glue , fibrin glue  and directly auto-transplanted bone marrow aspirate from the right iliac crest , the patient was selected for the generation of an axially vascularized tissue engineered bone construct. The vascularization was achieved using an arteriovenous loop created by a vein graft from the lower arm interposed between the radial artery on the volar site  and the cephalic vein (end-to-end) at the dorsal site of the lower arm penetrating the bone construct. The bone construct was composed of a clinically approved β-tricalciumphosphate/HA  imaging revealed that the tibial bone defect was healed without any signs of osteomyelitis. The final review took place 2 years after the bone reconstruction. The patient remained pain-free and started water skiing until now, which is 5 ½ years after this treatment.The following post-operative course was uneventful. Patency of the AV loop was verified using doppler ultrasound. In the follow-up examination, the range of active and passive motion was normal and the patient was pain-free. At 14 and at 32 months follow-up, CT and MRI revealed a patent arteriovenous loop as well as a completely healed bone defect in the radius Figs . She is in vitro and in vivo in experimental settings, but have widely failed to enter the clinical routine when it comes to large defects or major organ functional replacements [in vitro or in vivo using an extrinsic vascularization in a small animal model [et al. [de novo axially vascularized tissue formation was achieved. Pre-fabricated bone grafts can be generated using two different methods: Either a bone graft is transplanted in an intrinsically vascularized tissue  and after the graft is vascularized in an extrinsic pathway by the donor tissue, the whole construct can be transferred to the recipient site using the vascular pedicle of the host tissue. This technique has already been applied in a clinical setting by Terheyden et al. in 2004 [Until now, Tissue Engineering techniques have frequently been shown to be promising acements . One of al model . Over thal model ,16. Thisal model . Autologal model –4. Althoal model . Further [et al. ,19. Orig in 2004 . Here, a in 2004 ,22. Beca in 2004 ,9. Furth in 2004 ,25. Base in 2004 ,26. We w in 2004 . Recentl in 2004 . This isi) extracellular matrix; (ii) osteogenic cells; and (iii) growth factors. A vast variety of different scaffolds have been described for bone tissue engineering applications such as HA and β-tricalcium phosphate (TCP) or processed bovine cancellous bone [Beside vascularization of bone grafts, bioartificial bone substitutes consist of mainly three more components, which should be addressed in any successful tissue engineering strategy:  with subsequent microsurgical transfer to the distant defect site using the vascular pedicle.In the long-term follow-up examination, over 3 1/2-year post-operatively patent arteriovenous loops were detectable as well as a completely healed bone defects showing successful osseus reconstruction. The patients are performing daily tasks and sporty activities proving stable bony reconstructions without any osteosynthetic fixation. In cases of irradiated or traumatized defect sites perhaps a two-stage procedure would meet the demands of a vascularized bone graft more appropriately. In these cases, the construct would be first implanted into a site of high vascularization potential (in situ tissue engineered bone generation in large bone defects in a clinical scenario using the arteriovenous loop model without the creation of a significant donor-site defect. This technique will potentially expand the frontiers of reconstructive surgery towards custom-designed vascularized bone grafting without donor-site morbidity.This is the first clinical study demonstrating axially vascularized


The cerebellum is known to play a critical role in learning relevant patterns of activity for adaptive motor control, but the underlying network mechanisms are only partly understood. The classical long-term synaptic plasticity between parallel fibers (PFs) and Purkinje cells (PCs), which is driven by the inferior olive (IO), can only account for limited aspects of learning. Recently, the role of additional forms of plasticity in the granular layer, molecular layer and deep cerebellar nuclei (DCN) has been considered. In particular, learning at DCN synapses allows for generalization, but convergence to a stable state requires hundreds of repetitions. In this paper we have explored the putative role of the IO-DCN connection by endowing it with adaptable weights and exploring its implications in a closed-loop robotic manipulation task. Our results show that IO-DCN plasticity accelerates convergence of learning by up to two orders of magnitude without conflicting with the generalization properties conferred by DCN plasticity. Thus, this model suggests that multiple distributed learning mechanisms provide a key for explaining the complex properties of procedural learning and open up new experimental questions for synaptic plasticity in the cerebellar network. Biological motor learning is characterized by several attributes: it usually proceeds through a rapid convergence toward a stable state, it can become consolidated into persistent memory, it can be generalized to analogous cases, it can proceed along multiple consolidation steps, and it can be saved. The cerebellum is known to play a critical role in learning relevant patterns of activity for adaptive motor control, but the underlying network mechanisms are only partly understood.The cerebellum is also widely assumed to act as a control module which is embedded in a feedforward control loop , and Purkinje cells (PCs), which drives learning under inferior olive (IO) action, thus regulating the background activity of PCs and supplying a teaching signal  and MFs as well as by inhibitory synapses from PCs. The effect that these excitatory and inhibitory connections produce still remains uncertain   represents the average firing rate of the PCs associated to the ith muscle. Our robot arm-plant presents 3 agonist-antagonist pairs of muscles, representing a total of 6 muscles. fi defines the function which matches each granular layer state (active PF) with a particular output firing rate at each PC. This function is modified during the learning process of a particular movement. In this model the output activity at different cell layers  has been normalized between 0 (representing the absence of activity) and 1 (representing the maximum firing rate of the cell layer).Purkinje-cell layer: The activity at PCs is defined in Equation (1):Mossy fibers: Our cerebellar model assumes that mossy fibers (MFs) transmit a baseline neural activity during the trajectory execution according to studies of eye blink conditioning experiments  represents the average firing rate of the DCN cells associated to the ith muscular group and WMF − DCN,i is the synaptic strength of the MF-DCN connection at the muscular group i, WPC − DCNi represents the synaptic strength of the PC-DCN connection of the ith muscle. Finally iIO(t) represents the average firing rate of the CFs associated to the ith muscle where WIO − DCNi represents the synaptic strength of the IO-DCN connection of the ith muscle.DCN cells: The activity of these nuclei cells has traditionally been related with both the excitatory-activity integration coming from MFs and the inhibitory-activity integration from PCs, neglecting the impact of IO-DCN connections. Due to the low number of MFs and climbing fibers (CFs) in comparison to granule cells (GrCs), the capacity of these fibers for generating a sparse representation of different cerebellar states seems to be very limited . This fact shows that the reported synaptic plasticity at MF-DCN synapses  represents the weight change between the jth PF and the target PC associated with the ith muscle. iIO (t) stands for the current activity coming from the associated climbing fiber , MaxLTP and MaxLTD are the maximum long term potentiation/long terms depression (LTP/LTD) values, and α is the LTP decaying factor. In the experiments α is set to 1000 in order to ensure a fast LTP action decreasing  represents the weight change between the active MF and the target DCN associated with the ith muscle, iPC(t) is the current activity coming from the associated PCs, MaxLTP, and MaxLTD are the maximum LTP/LTD values, and α is the LTP decaying factor; α is set to 1000 in order to ensure a fast LTP action decreasing  is the synaptic weight adjustment at the PC-DCN connection reaching the DCN cell associated with the ith muscle, iPC(t) is the current activity coming from the associated PCs and finally DCN is the current activity regarding DCN cells present. Again, α is set to 1000 in order to ensure a fast LTP action decreasing  this IO-DCN action decays and its control action is assumed and improved by the two pathways whose actions have been previously described; the internal MF-GrC-PC-DCN and MF-DCN. The assumption involving a relevant action of this connection (IO-DCN) at early stages of the learning process implies that the initial synaptic strength set by this learning law must be in the same range as the one assumed in subsequent learning stages by the other two control pathways  represents the differential synaptic weight factor related to the active connection at time t [whose associated activity state corresponds to iIO(t)]. The connection corresponds to the DCN cell associated to the ith muscle. This weight can be seen as a fast modulation adaptation term, MaxMTP and MaxMTD (modulating term plasticity) are both the maximum MTP/MTD values to be applied at any time. Both terms present an enormous value in comparison to LTP/LTD values previously described, thus ensuring a fast response and a negligible contribution to the learning process in the long term. iIO is the normalized current activity in the range  coming from the associated climbing fiber , and finally α defines the MTD decaying factor (set to 1000 in our simulations ensuring a fast MTD action decreasing).where ΔW to be applied is high and in a very few time-steps this weighting factor is adapted. Although from the beginning the action of the other long-term synaptic mechanisms is active, it still requires some time to arrive at stable weights (due to its slower dynamics). When the error is low, the activity of the IO is closed to 0 and the depression term dominates, which means that the weight factor is quickly decreased. As we can see the potentiation/depression action compensates each other. What it is quickly learnt due to an action is quickly forgotten due to the opposite action.For instance, in a scenario with a significant mismatch between the inverse model and the robot-arm plant, there is a high activity at IO , which means a high ongoing error value. In this situation, the potentiation term dominates the expression, the incremental difference ΔFigure 6), the normalized mean absolute error (MAE) convergence speed defined in Luque et al.  arm within our feedforward control loop. The simulated-robot-plant physical characteristics can be dynamically modified to match different contexts . This LWR  attached to the robot end effector producing dynamic deviations when they are manipulated (light-2 kg-payload and heavy-10 kg-payload).An external variable force  which is applied to the end effector as described by Equation (8).This external force has 10 times the period of the trajectory movement and is in the same range than the needed torque values to operate the robot plant. This new scenario demands not just fast adjustment in agonist-antagonist pair of muscles  but also a fast control due to the quick changeable working point that the robot-arm undergoes. This set up including external forces is often used to evaluate potential roles of the cerebellum in control tasks  repetitions Figures . Thus, tIn order to evaluate the effectiveness of the IO-DCN connection in controlling adaptation during the initial learning stages, the IO-DCN synaptic weights were pre-calculated to handle different masses with the same values . Then the IO-DCN synaptic weights were kept fixed and the MF-DCN and PC-DCN weights were allowed to self-adapt during the learning process composed of 1 s trial trajectories repeated 5000 times using either light or heavy payloads.Using pre-calculated synaptic weights allowed the IO-DCN connection to operate over the whole learning process providing a rough control facilitating the DCN to operate in its pseudo-optimal firing rate from the very beginning Figures . Pre-calIn order to further evaluate its impact on the initial learning stage, the IO-DCN connection was made self-adaptive. We evaluated how these adaptive IO-DCN synaptic weights optimize payload manipulation. The IO-DCN weights were allowed to self-adapt during a learning process composed of 1 s trial trajectories repeated 5000 times using either light or heavy payloads.During the initial learning stage, the IO-DCN corrective action dominated Figures , bottom.In this configuration, all the learning sites were working complementing each other, generating an effective distributed learning network Figure . The addmodulating term (MTP/MTD) (see Equation 6). The impact of the modulating term was assessed on manipulation of a 2 kg payload. In the meantime, MF-DCN and PC-DCN weights were also allowed to self-adapt during the learning process composed of 1 s trial trajectories 5000 times.In simulations shown in Figures windup effect appeared. Wind-up occurred when the IO-DCN connection control command exceeded the physical limits of the arm-robot-system .Low MTP/MTD values (from 0.001 to 1) caused a smooth self-regulated IO-DCN action or, in other words, an IO-DCN optimal corrective action was not obtained at the very first trial Figure . High MTIn other words, the IO-DCN connection control momentum was incapable of responding instantaneously to changes in the next-integration-step-incoming error. In this case, the rate of IO-DCN synaptic weight evolution was faster than the error-speed of the robot-arm-system. Thus, the IO-DCN corrective action control exceeded by far the optimal control value but it kept on growing in the very same integration step. When the next-integration-step-incoming error reached the cerebellar system, the sign of the error was then reversed, thus causing the IO-DCN control action to start “winding” down.  in the cerebellar response as shown in Figure The effect of the modulated term MTP/MTD is shown in Figure To demonstrate whether the IO-DCN connection contributed to cerebellar control in more demanding operative scenarios, the system was exposed to an external force field. The force field was made up of an external set of variables and periodical forces  applied to the robot arm end-effector (see Equation 8). In these simulations, all adaptation sites at PF-PC, MF-DCN, PC-DCN, and IO-DCN synapses were enabled. The learning process occurred over 1 s trial trajectories repeated 5000 times and the MTP/MTD value was set to 10.The actual torque values operating the robot-arm-end effector under a variable periodic force field are shown in Figure The contribution of the IO-DCN Figure  connectiBy using closed-loop robotic simulations, this paper reveals the internal dynamics of long-term plasticity and neuronal firing in a cerebellar network model incorporated into a system control platform. The cerebellar network exploits the IO-DCN connection to implement an internal feed-back loop and embeds multiple forms of synaptic plasticity . Since the exact function of these structures is not fully resolved, an assessment of the assumption may come from a reverse engineering approach. Electrophysiological analysis has revealed complex properties in cerebellar neurons and plasticity mechanisms  and the motor cortex  provides a giant switchboard for associative learning” (Ohtsuki et al., an embedded feedback controller ensuring stability in the first stages of the learning process. These results also imply that degradation or malfunctioning of the IO would affect fast adaptation in the early stages of the learning of a control task. The predicted role of the IO-DCN pathway for fast cerebellar adaptation could be tested by using genetically modified animals (for review see Ito, Whilst it has been claimed that 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.