Most bladder tumors have complex genomes characterized by a high mutation burden as well as frequent copy number alterations and chromosomal rearrangements. Alterations in DNA repair pathways—including the double-strand break (DSB) and nucleotide excision repair (NER) pathways—are present in bladder tumors and may contribute to genomic instability and drive the tumor phenotype. DNA damaging such as cisplatin, mitomycin C, and radiation are commonly used in the treatment of muscle-invasive or metastatic bladder cancer, and several recent studies have linked specific DNA repair pathway defects with sensitivity to DNA damaging-based therapy. In addition, tumor DNA repair defects have important implications for use of immunotherapy and other targeted agents in bladder cancer. Therefore, efforts to further understand the landscape of DNA repair alterations in bladder cancer will be critical in advancing treatment for bladder cancer. This review summarizes the current understanding of the role of DNA repair pathway alterations in bladder tumor biology and response to therapy. An estimated 75,000 cases of bladder cancer will be diagnosed in the USA in 2017, making it the fifth most common cancer among adults . In addiIn the USA, the majority of bladder cancer cases are diagnosed at an early stage and can be managed with cystoscopic tumor removal followed by close surveillance. However, nearly one in four patients will present with muscle-invasive bladder cancer (MIBC), which requires aggressive multimodality therapy to provide an opportunity for cure. Approximately 5% of patients present with metastatic disease at diagnosis, and outcomes are poor in this population, with an average survival of approximately one year despite aggressive therapy.The majority of bladder tumors are urothelial  carcinomas. Although early-stage (non-MIBC) can demonstrate low- or high-grade histologic features, nearly all MIBCs are high-grade. Less common histologic variants also occur, and can be associated with unique clinical features and genetic properties that are reviewed elsewhere ,5.BRCA1, BRCA2, or other HR genes—also plays an important role in other settings such as prostate, gastric, and endometrial cancers . In. In32]. patients . Interespatients ,35. It icibility ,37,38,39BRCA1 and BRCA2 alterations are relatively common in some tumors settings and have important implications for treatment selection, somatic alterations or expression changes (resulting from BRCA1 promoter methylation or other epigenetic events) are relatively uncommon in bladder tumors [Differences in expression levels of genes in DNA repair pathways beyond NER have also been associated with bladder tumor response to DNA damaging agents. The homologous recombination (HR) pathway plays a critical role in the repair of double-strand breaks (DSBs) resulting from damage caused by ionizing radiation, inter-strand cross-linkers such as cisplatin and mitomycin C, and intercalating agents such as doxorubicin. Although r tumors .In an attempt to determine if changes in other DSB repair genes were associated with treatment response in MIBC, Choudhury and colleagues used an IHC-based approach to measure levels of five DSB repair proteins  in pre-treatment biopsies from bladder cancer patients treated with radiation . In the MRE11A do not vary significantly and somatic events (mutations or epigenetic silencing) are rare, suggesting that cellular MRE11A protein levels may be controlled post-transcriptionally [The association between MRE11 and radiation response in these studies is perhaps counter-intuitive, as it could be anticipated that decreased expression of a DSB repair gene such as MRE11 would be associated with decreased repair capacity and thus increased radiation sensitivity. However, the opposite effect was observed: lower MRE11A levels were associated with worse outcomes in each study. It is possible that low MRE11A levels may fail to trigger a DNA damage response sufficient to signal growth arrest and thus allow tumor cells to continue to proliferate despite the presence of unrepaired DNA damage; however, additional studies are needed to understand the molecular underpinnings of this association. It is interesting that in the TCGA bladder tumor cohort, transcript levels of tionally .As discussed previously, works by multiple groups have used gene expression profiling to identify and characterize several distinct MIBC subtypes. Similar to breast cancers, MIBCs can be broadly categorized into luminal and basal subtypes . In addiTaken together, these findings underscore the ability of expression profiling to provide important prognostic and predictive information. As evidenced by their differential sensitivity to DNA damaging regimens, MIBC subtypes harbor important differences in DNA damage signaling and repair capacity. Unraveling the mechanisms that drive differences in DNA repair capacity across histopathologic and expression-based tumor subtypes could have important implications for the management of bladder cancer as well as other tumor types with similar transcriptional subtypes.Although the use of cisplatin-based chemotherapy is associated with an overall survival benefit in large randomized studies, tumor response varies widely across patients. Given an uncertain benefit in the face of the known cisplatin-related toxicities, only a fraction of eligible MIBC patients receive cisplatin-based regimens . Thus, rTP53, RB1, KDM6A, and ARID1A—all of which have been implicated in MIBC biology. In addition, the authors performed an enrichment analysis to identify genes that were more frequently mutated in responders compared to non-responders. This analysis yielded only one significant gene—ERCC2. Nine of 25 responders (36%) had a somatic missense mutation in ERCC2 compared to 0 of 25 non-responders . In addition, in both the discovery and validation cohorts, patients with ERCC2-mutated tumors who received cisplatin-based neoadjuvant chemotherapy lived significantly longer than patients with WT ERCC2 tumors. A similar survival difference between mutant and WT ERCC2 cases was not observed in the TCGA cohort—a cohort in which no patients received neoadjuvant chemotherapy. Thus, ERCC2 mutations may be predictive of cisplatin response rather than simply prognostic of better outcomes in the absence of chemotherapy.Recently, the association between somatic C cohort . Eight oERCC2 missense mutations were present in approximately 15% of tumors in the TCGA cohort. Interestingly, bladder cancer is the only solid tumor type identified to date in which ERCC2 is significantly mutated  [ERCC2 mutations were significantly associated with reduced metastatic recurrence rate, but no association between MRE11 protein expression levels and outcomes was noted.In a similar study reported by researchers from the University of Chicago, whole exome or targeted sequencing of tumors from MIBC patients receiving perioperative chemotherapy revealed improved recurrence-free survival among patients with tumors harboring a mutation in one or more of six DNA repair genes (d BRCA2) . Similard BRCA2) . Finallyd BRCA2) . In the ERCC2) on treatment response remains to be determined. In addition, as the number of queried genes in a “DNA repair gene” list grows, the event rate  may begin to reflect overall tumor mutation burden rather than gene- or pathway-specific processes , and tumor mutation burden has also been associated with response to DNA damaging agents in several of these (and other) studies [Taken together, these studies provide compelling evidence for an interaction between DNA repair gene alterations and treatment response in bladder cancer patients receiving DNA damaging-based therapy. However, in these studies, events in multiple DNA repair genes (often representing more than one repair pathway) were considered together, so the impact of individual genes  and is associated with an increased risk of multiple cancer types [MSH2 mutation carriers. However, germline alterations in other well-known DNA repair genes such as BRCA1, BRCA2, or ATM have not been clearly linked to bladder cancer risk.In addition to the well-known associations linking DNA damaging agents such as cigarette smoke and analine dyes with bladder cancer risk, attempts have also been made to identify germline DNA repair alterations that impact the risk of developing bladder cancer and/or predict response to treatment. Lynch syndrome is characterized by inherited mutations in one of the mismatch repair (MMR) genes  into routine clinical management, although significant logistical challenges regarding timing, payment, and interpretation remain .The impact of DNA repair pathway alterations on sensitivity to DNA damaging agents has been demonstrated in several retrospective analyses (discussed above) and is also supported by preclinical data. However, in order to shift clinical practice, prospective data are needed. To this end, several prospective trials are planned that stratify MIBC patients by mutation status of one or more DNA repair genes. Given that the presence of a DNA repair gene mutation is associated with improved response to DNA damaging-based treatment, it is exciting to think that treatment decisions could be tailored based on the DNA repair features of a tumor. In this regard, MIBC patients with tumor DNA repair alterations may be ideal candidates for organ-sparing approaches that utilize DNA damaging-based chemotherapy and radiation.ERCC1 and ERCC2; In addition to increasing sensitivity to DNA damaging agents such as cisplatin, loss of DNA repair capacity can also be associated with sensitivity to DNA repair targeted therapies. PARP inhibitors are the most well-developed DNA repair targeted agents and are now approved in several DNA repair deficient clinical settings . HoweverOne of the most exciting advances in bladder cancer is the recent approval of several immune checkpoint inhibitors (ICIs) for treatment of advanced bladder cancer patients. In the past year, three different agents have been approved for use in patients with metastatic disease, including in both cisplatin-naïve and cisplatin-refractory settings. ICIs agents block inhibitory immune signaling molecules, allowing the patient’s immune system to mount a more effective anti-tumor immune response. Although response to ICIs varies widely within and across tumor types, the most robust activity has been observed in carcinogen-associated tumors with high mutation burdens such as bladder cancer, non-small cell lung cancer, and melanoma. Somatic point mutations resulting from carcinogen-induced damage or from an underlying tumor DNA repair deficiency can lead to production of neoantigenic peptides which are recognized as non-self by the host immune system. Although there is a correlation between somatic mutation burden and ICI sensitivity across tumor types, the relationship among somatic mutation burden, tumor neoantigens, host immune activation, and ICI response is clearly multifaceted and is the subject of intense ongoing study.The first ICI approved for use in bladder cancer was the anti-PD-L1 agent atezoluzimab, and exploratory biomarker analyses from the Phase 2 trial revealed associations between TCGA expression subtype and response rate as well as between mutation load and response rate . AlthougThese findings underscore the impressive activity of ICIs in bladder cancer and also highlight the importance of integrating clinical, pathologic, and genomic biomarkers to provide the best opportunity to guide clinical ICI use. Numerous additional trials are ongoing, and it is very likely that the scope of ICIs in bladder cancer will expand in the coming years. As ICIs are incorporated earlier in treatment regimens, one important challenge that is particularly relevant to bladder cancer will be to understand the interplay between ICIs and DNA damaging agents. Although DNA damage can lead to somatic mutations that ultimately result in tumor-specific neoantigens, DNA damaging agents can also have immune suppressive effects that may dampen the anti-tumor immune response. These interactions are complex and multi-faceted, and further clinical and pre-clinical work is needed. It is possible that the optimal integration of immune checkpoint blockade with DNA damaging agents may vary significantly across tumor types and clinical contexts. DNA repair pathway alterations have the potential to be important biomarkers and therapeutic targets in bladder cancer, and additional studies may reveal that the true scope of DNA repair pathway dysfunction in bladder cancer is even broader than is currently appreciated. Integrative approaches that combine genomic, proteomic, and functional approaches will be required to more fully understand the contribution of DNA repair dysfunction to bladder tumor biology and treatment response.


Neural connectivity was reduced in a left-hemisphere pre-language region, and the degree to which connectivity of this left language region extended to right-hemisphere homologs was positively associated with the time elapsed between fMRI assessment and delivery. These results provide the first evidence that altered functional connectivity in the preterm brain is identifiable before birth. They suggest that neurodevelopmental disorders associated with preterm birth may result from neurological insults that begin in utero.It has been suggested that neurological problems more frequent in those born preterm are expressed prior to birth, but owing to technical limitations, this has been difficult to test in humans. We applied novel fetal resting-state functional MRI to measure brain function in 32 human fetuses  Developmental problems are significantly more common in children born preterm. Epidemiological and meta-analytic studies indicate children born preterm are three times more likely to develop autism, attention deficit/hyperactivity disorders, and emotional disorders1578911131510in utero, the untoward influences of extrauterine factors cannot be the source of those differences. The recent development of resting-state fMRI (rs-fMRI) methodology for the human fetus offers the first opportunity to investigate altered functional connectivity prior to birth44A critical target for current research is to separate intrauterine from extrauterine influences on injurious brain development, and to isolate the earliest indicators of change in connectional architecture of the preterm brain. While much has been gained from examination of the postnatal preterm brain, examinations of human brain networks at or after preterm birth are confounded by potential insults conferred both by the absence of neuroprotective elements4042in utero in 32 human fetuses, 14 of which were subsequently born preterm. We employed an intrinsic connectivity distribution analysis of rs-fMRI data to map synchrony in MRI signals over time across the fetal brain. We then investigated the relationship between connectivity and gestational age at fMRI assessment and delivery. Lastly, post-hoc seed analysis was performed to explore the regions in which specific connections were most responsible for changes intrinsic connectivity distribution (ICD)In this study we utilized rs-fMRI to measure neural connectivity Thirty-six women, mean age 25.3 years, SD, 5.6, underwent MRI between their 22nd and 36th week of pregnancy. Half of participating pregnant women were high-risk for early delivery and gave birth prior to the 37th week of pregnancy, mean GA preterm birth = 32 weeks, range = 24–35 weeks. The comparison group was case control matched based on GA at time of MRI and gender of the fetus . Four hiStudies that demonstrate postnatal differences in brain development as a function of sexin utero.ICD comparisons of preterm- and term-born fetuses revealed a substantial area of the left hemisphere, proximal to what will later become Broca’s area, where connectivity was greater in term-born fetuses . In cont11125354We reasoned that if this fetal pre-language region is a critical hub of connectional group differences, then variation in connectivity of this region may relate to individual differences in the preterm-born sample. Correspondence between functional connectivity, or ICD, of this region, and both gestational age at the time of scan and gestational age at delivery were evaluated. Generally, postnatal evaluation of the human premature brain has pointed to early developmental delays, some of which improve with age, either through restorative or compensatory processesTo better understand differences between groups, we performed follow-up region of interest seed connectivity analysis using this putative pre-language, ICD peak region as an area of interest. Signal from this region was correlated with signal intensity in every other grey matter voxel to identify brain regions that demonstrated similar signal properties over time. In the preterm-born fetal group, we observed diminished signal correlation in ipsilateral posterior superior temporal gyrus, extending into the posterior pre-language brain network  = 8.089, p = 0.007. These clinical variables may relate to neural functional connectivity, but due to small sample size and potential for type II error, it was not appropriate to examine those potential associations here.Conditions that increase risk for prematurity may also be those that enhance risk for deficient neural functional connectivity of the fetus. Intra-amniotic infection increases risk for both preterm delivery and white matter injury58Defining the nascent architecture of the preterm brain provides a basis for understanding the underlying etiology of neurological impairments that can accompany prematurity. Using recent developments in fetal resting-state fMRI we examined neural functional connectivity in 32 human fetuses and found reduced connectional integrity in fetuses that would subsequently be born preterm, particularly in regions of the left hemisphere that later support language processing. Strength of functional connectivity in this region was related to gestational age at delivery, such that those born closer to expected due date demonstrated connectivity profiles more similar to those in the case-matched control group. Thus, observed differences in neural functional connectivity were related to proximal health outcomes in the preterm group.in utero, prior to the potentially injurious experiences of early delivery. These constitute the first human data to suggest that disabilities frequently accompanying extreme prematurity, such as autism and ADHD, may derive from pre-existing intrauterine neurological conditions, especially given that these disorders have neuroconnectional bases6162636567These results demonstrate that neurological connectivity differences associated with human preterm birth begin human fetuses. This group exposed fetuses to varied quantities of word-like sounds and found that, after birth, those exposed to intrauterine stimuli showed behavioral learning and enhanced brain activity that scaled with quantity of exposurein utero. Understanding that extrauterine environmental stimuli not only induce behavioral response in the fetus, but also change brain function in lasting ways, supports the possibility that prenatal behavioral intervention may serve as an effective therapy for those at risk.Knowledge that functional connectivity differs before birth in those born preterm encourages evaluation of prenatal intervention. Potential efficacy of prenatal neurobehavioral therapy is supported by growing evidence from non-human animals and from humans that learning occurs prior to birth. Markham and colleagues have demonstrated that birds learn about the calls of conspecifics while still in the egg, and that this embryonic programming alters neural cellular activation after birth and also characteristics of later vocalizationsfetal fMRI, as partial volume effects, physiological noise, motion, and image registration are broad concerns for the larger MR field, and many strategies exist for addressing these72737457While this study presents a novel comparison of human brain function before birth in fetuses destined either for term or preterm delivery, considerations about nascent fetal fMRI methodology merit discussion. Challenges inherent in the methodology include small fetal head volume, influences of physiological signals originating from mother and fetus, limited constraints over motion, and variation in orientationAnother topic requiring careful consideration in fetal fMRI is that we possess limited understanding of the physiological basis of blood-oxygen level dependent (BOLD) fMRI signals in the fetal brain. Current understanding is derived from assumptions regarding neurovascular coupling that stem from neurophysiological research performed in animals8183In conclusion, we provide the first evidence that neural pathways are likely to be altered prior to preterm delivery. This discovery suggests that factors influencing early delivery may also impact development of the human brain, which has implications for life-long health. Future work will address sources such as infection and inflammation that may play a causal role in altering these parallel pathways, bringing us closer to understanding both the primary neurological injury and the optimal timing for early intervention.Singleton pregnancies with normal brain anatomy assessed by ultrasound and MRI examination reporting no contraindications for MRI were eligible to participate. MRI T1 weighted images revealed no brain injury in any fetal cases at the time of prenatal MRI examination. Despite that no areas suggestive of brain lesions were observed, the presence of micro bleeds, or other more subtle forms of injury, cannot be completely excluded. The mean age of fetuses at the time of MRI was 29.9 weeks, SD, 3.4, post-menstrual gestational age (GA). Ultrasound (US) examination administered by study physician (E.H.-A.) was performed within 1 week of MRI examination to determine fetal GA. All women were native English speakers. All women provided written informed consent before undergoing MRI examination. Participation was approved by the Institutional Review Board of the National Institute of Child Health and Human Development (NICHD) and by the Human Investigation Committee of Wayne State University. All experiments were performed in accordance with relevant guidelines and regulations.Fetal MR exams were performed with a Siemens Verio 70-cm open-bore 3-T scanner with a light-weight (~550 g) abdominal 4-Channel Siemens Flex Coil. The MRI examination lasted 45 min. Images were collected with the following parameters and computed SAR values: (i) localizer ; (ii) T2 anatomical ; (iii) echo planar imaging (EPI) BOLD ; (iv) susceptibility weighted imaging (SWI) .Fetal fMRI data were censoreddegree was calculated. ICD models the entire distribution of correlation thresholds using a Wiebull distribution avoiding the need for choosing an arbitrary connectivity threshold. This parameterization is akin to modeling the change in network theory metric degree, as the threshold used to calculate degree is increased, with a stretched exponential. Specifically, the time-course for any gray matter voxel was correlated with every other voxel in the gray matter. A histogram of these correlations was constructed to estimate the distribution of connections to the current voxel. This distribution was converted to a survival function and the survival function was fitted with a stretched exponential with unknown variance. As variance controls the spread of the distribution of connections, a larger variance indicates a greater number of high correlation connections. Finally, this process is repeated for all voxels in the gray matter resulting in a whole-brain parametric image summarizing the connectivity of each tissue element. ICD was computed in each study group  connectivity of the node where ICD connectivity differed most significantly between groups. Signal intensity was extracted from voxels centered on the ICD peak at location 27 mm left, 18 mm anterior, 5 mm inferior of the center of image data registered to a common 32-week fetal brain templateFor imaging data, voxel-wise two sample independent t-tests were used to compare the connectivity data between groups. Voxel-wise Pearson’s correlation was used to assess association between connectivity and GA. Spearman rank correlation and partial Spearman rank correlation were used to assess the association between extracted connectivity values and GA. Results are shown at a cluster-level threshold of p < 0.05 family-wise error (FWE) correction as determined by AFNI’s 3dClustSim program. The cluster threshold was determined using cluster-forming threshold of p = 0.001, 10,000 iteration, smoothness estimated using a mixture of Exponential and Gaussian distributions (i.e. the –acf option), and 16.0.09 release of AFNI. For non-imaging data, two sample independent t-tests and chi-square tests were used with significances assessed at p < 0.05.How to cite this article: Thomason, M. E. et al. Weak functional connectivity in the human fetal brain prior to preterm birth. Sci. Rep.7, 39286; doi: 10.1038/srep39286 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Whole tumor ADC volumes were analyzed using a histogram-based approach. Several ADC parameters showed significant correlations with immunohistopathological parameters. Most importantly, ADC histogram skewness and ADC histogram kurtosis were able to differentiate between nodal negative and nodal positive thyroid carcinoma. Conclusions: histogram analysis of whole ADC tumor volumes has the potential to provide valuable information on tumor biology in thyroid carcinoma. However, further studies are warranted.Pre-surgical diffusion weighted imaging (DWI) is increasingly important in the context of thyroid cancer for identification of the optimal treatment strategy. It has exemplarily been shown that DWI at 3T can distinguish undifferentiated from well-differentiated thyroid carcinoma, which has decisive implications for the magnitude of surgery. This study used DWI histogram analysis of whole tumor apparent diffusion coefficient (ADC) maps. The primary aim was to discriminate thyroid carcinomas which had already gained the capacity to metastasize lymphatically from those not yet being able to spread via the lymphatic system. The secondary aim was to reflect prognostically important tumor-biological features like cellularity and proliferative activity with ADC histogram analysis. Fifteen patients with follicular-cell derived thyroid cancer were enrolled. Lymph node status, extent of infiltration of surrounding tissue, and Ki-67 and p53 expression were assessed in these patients. DWI was obtained in a 3T system using  The incidence of thyroid cancer, being the most abundant endocrine malignancy, is rapidly increasing . The vasA variety of therapeutic options is available for thyroid cancer , but surConsidering the broad spectrum of aggressiveness in thyroid cancer and the resulting necessity for customized treatment, employing presurgical imaging is of great importance, as it allows the thyroid surgeon to identify disease subtypes being associated with increased mortality and morbidity such as metastasizing and undifferentiated thyroid cancer.Diffusion-weighted magnetic resonance imaging (DWI) has the potential to reveal tumor architectural details like cellular density and proliferative activity in different malignant entities ,12. UsinHowever, in the clinical setting, obtained DWI data is commonly analyzed using a two-dimensional region of interest in the slice of the apparent diffusion coefficient (ADC) map representing the maximum diameter of the tumor. This approach does not account for the regularly encountered heterogeneity of whole tumors and certainly does not reflect the complex micro-architectural properties of malignantly transformed tissue.An enhanced approach using every voxel of the tumor to compute a histogram of intensity levels could help to further increase prediction of histological features of tumors by magnetic resonance imaging (MRI) . This waTo the best of the authors’ knowledge, only one study used ADC histogram analysis in thyroid cancer to differentiate benign from malignant nodules and furthermore reveal extra-thyroidal growth of papillary thyroid cancer . So far,For reasons of clarity and comprehensibility, results of MRI and histopathology were organized in tables. mean with p53 , ADCmax with Ki67  and p53 , ADCp75 with p53 , ADCp90 with Ki67  and p53 , ADCmedian with p53 , ADCmodus with p53 , and kurtosis with cell count . skewness (p = 0.015). For all remaining ADC derived histogram parameters, Levene’s Test showed heterogeneity of variance when comparing the nodal-negative and the nodal-positive group. Hence, group comparisons were performed using unpaired t-test for ADCskewness and Mann-Whitney-U Test for all remaining parameters. The corresponding p-values are given in p = 0.031) and kurtosis (p = 0.028). No other significant differences or trends were delineable when comparing thyroid carcinoma patients with restricted vs. advanced infiltration pattern (results not presented).Histogram analysis derived ADC values are compared between the nodal negative and the nodal positive group in This study aimed to investigate the potential of 3T RESOLVE DWI using an ADC histogram analysis approach to distinguish between limited and advanced thyroid cancer with reference to the status of lymphatic metastatic dissemination. To the author’s best knowledge, this work is the first to show differences in ADC histogram parameters between nodal-positive and nodal-negative thyroid cancer.In detail, skewness and kurtosis of the ADC histograms were significantly increased in nodal-positive compared to nodal-negative thyroid cancer. This finding corresponds to previous studies in other malignant tumors, exemplarily clear cell renal cell carcinoma, and rectal cancer, which revealed that increased skewness of ADC histograms is associated with a more advanced disease stage ,20. FurtAdditionally, this study found significant correlations between ADC histogram analysis derived values of thyroid cancer and corresponding immune-reactivity for p53. p53 has great importance as tumor suppressor and controls cell fate via induction of apoptosis, cell cycle arrest and senescence . Under nIncreased proliferation of cells, as indicated by increased expression of Ki-67, consecutively decreases the corresponding extracellular space in a given volume of tissue and thereupon reduces water diffusibility, which is reflected by decreased ADC values . Thus—inmean and ADCmin were used to investigate histopathological features like cellularity of tumors in vivo [mean and ADCmin are strongly scanner-dependent and cannot be used to compare patients investigated in different MRI devices without normalization. In contrary, histogram parameters estimate characteristics of the ADC distribution, which is not scanner-dependent like the absolute ADC values. Therefore, ADC derived histogram parameters  might be superior when investigating histopathological features in vivo using more than one MRI scanner in a singular study.This study furthermore identified a significant inverse correlation between cell count and kurtosis. Only few studies investigated the potential of ADC kurtosis to reflect histological properties, for example Chandarana and colleagues were able to differentiate clear cell from papillary subtype of renal cell cancer by means of ADC kurtosis . It is t in vivo . HoweverThis study suffers from few limitations. The major limitation is the small number of patients included in this study. Furthermore, this study did not include all clinically relevant subtypes of thyroid cancer, exemplarily medullary thyroid carcinomas were not investigated. Therefore, future works including greater cohorts with different histopathological subtypes have to confirm these findings and further elucidate the relationship between histopathological findings and ADC alterations. Also, ADC histogram analysis was performed by a single, experienced reader. The suitability of histogram analysis for the clinical routine necessitates assessment of inter-reader and intra-reader variability including readers with different levels of experience. A future work needs to investigate these phenomena in a larger cohort.ADC histogram analysis can provide more detailed information on diffusion characteristics of tumors than commonly obtained ADC parameters. For example, a previously published study demonstrated that common ADC parameters  did not reflect histopathological features like cellularity and proliferative activity in thyroid carcinoma . In contThis retrospective study was approved (No. 2014-99) by the local research ethics committee of the Martin-Luther-University Halle-Wittenberg.The radiological database for thyroid carcinoma was reviewed. In total, 20 patients were identified, but only 15 patients with histopathologically confirmed thyroid carcinoma had received proper DWI (using the RESOLVE sequence) and were therefore enrolled in our study.n = 4, papillary thyroid carcinoma: n = 5, anaplastic thyroid carcinoma: n = 6. Five patients were diagnosed with nodal negative thyroid cancer, and 10 patients had pathologically confirmed lymph node metastases. One patient was diagnosed with distant metastatic disease . Infiltration pattern ranged from restriction to the thyroid gland to advanced infiltration including infiltration of the trachea, esophagus, and internal jugular vein. An overview of demographic, clinical and pathological information is given in The patient group was comprised of one male and 14 female patients. The mean age was 67 years (with a standard deviation of 12.9 years). The distribution of histopathological subtypes was as follows; follicular thyroid carcinoma: axial T2 weighted (T2w) turbo spin echo (TSE) sequence ;®, Bayer Schering Pharma, Leverkusen, Germany);axial T1 weighted (T1w) turbo spin echo (TSE ) sequences  before and after intravenous application of contrast medium  with b values of 0, 400 and 800 s/mmeviously .MRI of the neck was performed for all patients using a 3T device . The imaging protocol included the following sequences:All images were available in digital form and were analyzed by an experienced radiologist without knowledge of the histopathological diagnosis on a PACS workstation . 3), mean (ADCmean), maximum (ADCmax), minimum (ADCmin), median (ADCmedian), modus (ADCmodus), and the following percentils: 10th (ADCp10), 25th (ADCp25), 75th (ADCp75), and 90th (ADCp90). Additionally, histogram-based characteristics of the VOI—kurtosis, skewness, and entropy—were computed. All calculations were performed using in-build Matlab functions.DWI data was transferred in DICOM format and processed offline with a custom-made Matlab-based application  on a standard windows operated system. The ADC maps were displayed within a graphical user interface (GUI) that enables the reader to scroll through the slices and draw a volume of interest (VOI) at the tumor’s boundary. The VOI was created by manually drawing regions of interest (ROIs) along the margin of the tumor using all slices displaying the tumor (whole lesion measure). All measures were performed by one author (AS). The ROIs were modified in the GUI and saved (in Matlab-specific format) for later processing. After setting the ROIs, the following parameters were calculated and given in a spreadsheet format: ROI volume  were analysed. The area with the highest number of positive nuclei was selected. All thyroid carcinomas were surgically resected and histopathologically analysed. In every case, the proliferation index was estimated on Ki-67 antigen stained specimens using MIB-1 monoclonal antibody  as reported previously . FurtherU test was performed to compare values among the different groups. Group comparisons were performed for nodal negative vs. nodal positive patients and patients with restricted (thyroid gland and trachea) vs. advanced  infiltration pattern. Since only one patient with distant metastatic disease was included, a sufficient group comparison between M0 and M1 patients could not be performed. p-Values ≤ 0.05 were considered as statistically significant.Statistical analysis was performed using IBM SPSS 23™ . Collected data was first evaluated by means of descriptive statistics. Correlative analysis was then performed using Spearman’s correlation coefficient in order to analyze associations between histogram analysis derived values of ADC and (immuno-) histopathological parameters. Subsequently, Levene’s Test for homogeneity of variance was performed to assess the equality of variances of ADC derived histogram parameters between different groups of thyroid carcinoma patients in order to identify the suitable test for group comparisons. In case of homoscedasticity, unpaired t test was performed to compare values among different  groups. In case of heteroscedasticity, Mann-Whitney-This exploratory study revealed significant differences in ADC histogram skewness and kurtosis comparing nodal negative and nodal positive thyroid cancer. Significant correlations between different ADC parameters were identified with p53, Ki-67, and cell count, substantiating the potential of ADC as an important prognostic imaging biomarker. This information certainly has the potential to aid thyroid surgeons in identifying the optimal treatment strategy for patients with thyroid cancer. Further studies investigating a greater cohort of patients are necessary to confirm these findings.


Intramedullary fixation of comminuted mid-shaft clavicle fractures has traditionally been employed with satisfactory clinical outcomes; however, pins with smooth surfaces may protrude from the bone and are prone to migration, while some threaded pins are difficult to remove post-operatively. The aim of this proof-of-concept study was to develop and evaluate the biomechanical strength of a novel intramedullary Echidna pin device designed to maintain fracture reduction, resist migration and facilitate ease of post-operative removal.Thirty human clavicle specimens were harvested and fractured in a comminuted mid-shaft butterfly configuration. Each specimen was randomly allocated to three surgical repair groups including intramedullary fixation using the Echidna pin and Herbert Cannulated Bone Screw System, as well as plate fixation using bi-cortical locking screws. Using a biomechanical testing apparatus, construct bending and torsional stiffness were measured, as well as ultimate bending strength.p > 0.05); however, the Echidna pin construct demonstrated a significantly greater bending stiffness compared to that of the Herbert screw construct . The plate construct demonstrated significantly greater torsional stiffness, bending stiffness and ultimate bending moment compared to those of the Herbert screw and Echidna pin (p < 0.05).There was no significant difference in torsional stiffness and ultimate bending moment between the Echidna pin and Herbert screw repair constructs (An intramedullary Echidna pin device was designed to stabilize comminuted fractures of the clavicle, maintain fracture compression and provide ease of removal post-operatively. Since the results suggest equivalent or superior torsional and bending stability in the Echidna pin compared to that of the Herbert screw, the Echidna pin concept may represent an alternative fixation device to conventional intramedullary screws, nails and pins; however, superior plating using bi-cortical locking screws provides substantially higher construct structural rigidity than intramedullary devices, and may therefore be useful in cases of osteoporotic bone, or where high fracture stability is required. Middle-third clavicle fractures represent approximately 80% of all clavicle fractures, with over half of these fractures displaced , 2. FracIndications for surgical treatment of acute mid-third clavicle fractures include non-union with concomitant pain, open fracture, severe malpositioning, skin tenting and a fracture gap greater than half of one clavicle diameter . Open rePlate fixation includes the use of dynamic compression plates, tubular plates or reconstruction plates , and mayKirschner wires, Steinmann pins and Knowles and Hagie pins have been employed with good clinical and function outcomes , 19–21; Thirty embalmed clavicle specimens  were harvested from 17 cadavers. Based on previously reported biomechanical studies evaluating fixation strength of mid-shaft fracture repair constructs, we proposed a sample size of 30, with 10 specimens per group to obtain a statistical power of 0.8 for a bending stiffness effect , 27. AllA mid-shaft osteotomy was first made on the middle third of each specimen to simulate a ‘B-type’ wedge segmental fracture. A template was used to outline a standardized 20-mm-long and 8-mm-wide diamond shape on the inferior surface of the clavicle in line with the mid-shaft of the clavicle. Using an oscillating bone saw, a wedge-shaped osteotomy was performed by removing the portion of outlined bone to a depth of 3 mm. A transverse mid-shaft cut through the entire clavicle was then performed.Each clavicle was then randomly assigned to one of three surgical repair groups which included intramedullary fixation using an Echidna pin and Herbert screw  , 25, andThe testing protocol employed was based on a previously reported procedure . All repEach repair construct was then loaded in cantilever bending following the torsional testing. To orient the specimen horizontally, the acromial potting fixture was released from the upper Instron crosshead, while the sternal potting fixture was rotated by 90°. A vertical support was placed just medial to the clavicle fracture, to ensure that peak bending moments occurred at the fracture site and not at the sternal end of the clavicle. The acromial end of the clavicle was displaced downward at a constant rate of 0.5 mm/s by lowering the upper crosshead of the Instron , 30. Loap < 0.05. All statistical data analyses were performed using the Statistical Package for the Social Sciences .A single-way analysis of variance (ANOVA) was used to evaluate between-group differences in mean bending stiffness, ultimate bending moment and torsional stiffness for the three repair groups, with Games-Howell post hoc tests for unequal variances used to compute mean differences between groups. Standard deviation was used as a measure of the dispersion of results. Level of significance was set at p < 0.001) and Herbert screw constructs  during clockwise twisting . The torsional stiffness of the Herbert screw during counter-clockwise twisting was significantly lower than that during clockwise twisting , whereas there was no significant direction-dependent torsional stiffness in the Echidna pin construct .Plate constructs were significantly stiffer in torsion than Echidna pin constructs  and the Herbert screw construct . The Echidna pin construct demonstrated a significantly greater bending stiffness than that of the Herbert screw construct . Similar between-group trends were observed in ultimate bending strength, with the plate construct exhibiting a significantly greater ultimate bending moment than that of the Echidna pin construct  and Herbert screw construct . While a greater ultimate bending moment was observed in the Echidna pin construct compared to that of the Herbert screw construct (mean difference 2.56 Nm), this difference was not significant (p > 0.05).The plate construct was significantly stiffer in bending than the Echidna pin construct (mean difference 2.76 Nm/deg, Open reduction together with internal plate fixation and intramedullary fixation are two of the most common surgical techniques for the treatment of displaced mid-shaft clavicle fractures. A prospective comparison of Knowles pinning and plate fixation suggested that plating has been associated with longer operation time, larger wound incision, higher pain levels, more analgesic use and more symptomatic hardware complications . While iDespite significantly lower torsional stiffness, bending stiffness and ultimate bending moment observed in intramedullary fixation constructs compared to those of superior plating, clinical studies suggest no difference in outcome, and even reduced post-operative complication rates compared to that of plate fixation constructs for displaced mid-third clavicle fractures , 31, 32.A clinical and radiographic review of symptomatic non-unions of the clavicle treated with open reduction and intramedullary fixation using the Herbert screw revealed satisfactory union with no loosening after 13 months . Since tOne shortcoming of clavicular fracture surgery is the requirement for implants to be removed in a second operation , 36, whiThere are a number of limitations of this study that ought to be considered. First, torsion of specimens may have weakened the repair construct and subsequently adversely influenced the behaviour of the construct under bending; however, since each specimen was tested in an identical manner, we do not anticipate this to significantly influence the relative differences in bending stiffness and ultimate strength between the three repair groups evaluated. Second, the cadaveric clavicle specimens harvested were embalmed and from elderly donors, and these specimens are likely to have lower strength and structural integrity compared to those from younger or fresh-frozen specimens. However, our specimens were of a similar age group, and we anticipate these effects would not significantly influence between-group differences in construct functional performance. Finally, the present study was based on a mechanical model which neglects the effects of bone remodelling and fracture healing. Therefore, conclusions about optimal fracture fixation and bone healing cannot be explicitly reported.The present study reports the biomechanical performance of a novel intramedullary Echidna pin design for mid-shaft clavicle fracture fixation. The results suggest equivalent or superior torsional and bending stability in the Echidna pin compared to that of the Herbert screw. Since the Echidna pin was developed to be stable within the intramedullary canal, maintain fracture compression and be easily removed post-operatively, it may provide a suitable alternative to conventional intramedullary screws, nails and pins; however, superior plating using bi-cortical locking screws provides substantially higher construct structural rigidity than intramedullary devices, and may therefore be useful in cases of osteoporotic bone, or where high fracture stability is required.


Zinc (Zn) is an essential trace element which has favorable antioxidant, anti-inflammatory, and apoptotic effects. The liver mainly plays a crucial role in maintaining systemic Zn homeostasis. Therefore, the occurrence of chronic liver diseases, such as chronic hepatitis, liver cirrhosis, or fatty liver, results in the impairment of Zn metabolism, and subsequently Zn deficiency. Zn deficiency causes plenty of metabolic abnormalities, including insulin resistance, hepatic steatosis and hepatic encephalopathy. Inversely, metabolic abnormalities like hypoalbuminemia in patients with liver cirrhosis often result in Zn deficiency. Recent studies have revealed the putative mechanisms by which Zn deficiency evokes a variety of metabolic abnormalities in chronic liver disease. Zn supplementation has shown beneficial effects on such metabolic abnormalities in experimental models and actual patients with chronic liver disease. This review summarizes the pathogenesis of metabolic abnormalities deriving from Zn deficiency and the favorable effects of Zn administration in patients with chronic liver disease. In addition, we also highlight the interactions between Zn and other trace elements, vitamins, amino acids, or hormones in such patients. Zinc (Zn) is an essential trace element that plays pivotal roles in cellular integrity and biological functions related to cell division, growth and development ,2. Zn acZn homeostasis is primarily regulated in the liver. Therefore, chronic liver damage results in the impairment of zinc homeostasis, and eventually zinc deficiency ,9,10,11.Here, we mainly focus on the metabolic abnormalities deriving from Zn deficiency in patients with chronic liver disease and the beneficial effects resulting from zinc administration in such patients. Finally, the interactions between Zn deficiency and levels of other trace elements, vitamins, amino acids, or hormones are described.Liver cirrhosis is considered the most advanced stage of chronic liver disease. An unfavorable hepatic reserve results in numerous metabolic disorders, such as hypoalbuminemia. Consequently, a relative increase in α2 macroglobulin, which more strongly binds to Zn , caused Hepatic encephalopathy (HE) is one of the most common neuropsychiatric complications in patients with liver cirrhosis. The pathogenesis of HE has not been completely understood, although ammonia is considered to play a key role. Approximately 90% of patients with liver cirrhosis and HE have elevated ammonia levels in the plasma . Poor ZnHepatocellular carcinoma (HCC) is the fifth most common form of cancer worldwide . Liver cThe roles of trace elements underlying hepatocarcinogenesis have not been fully understood yet, although previous studies have identified a number of signal transduction pathways that are involved in this process. Elevated serum copper (Cu) levels were associated with the development of HCC ,26,27,28Based on the observations of intracellular Zn concentrations in different carcinomas, it has been postulated that these changes in Zn levels may contribute to the development of tumors by affecting a wide variety of molecular structures, such as receptors, kinases, caspases, phosphatases and transcriptional factors .The decisive role of Zn in malignant transformation may derive from change in the expression of Zn transporters such as Zip4, Zip6, Zip7 and Zip10, as shown by the previous studies on certain types of carcinomas ,37,38. NSerum Zn status may be recognized as a prognostic serological hallmark after initial hepatectomy in HCV-related HCC patients. Imai and colleagues elucidated that HCC patients with lower serum Zn levels at the preoperative stage had significantly lower overall survival than those with a normal range of Zn levels .Hepatitis C virus (HCV) is known to induce a spectrum of chronic liver diseases from chronic hepatitis to liver cirrhosis, and ultimately to HCC. More than 184 million people worldwide are estimated to be infected with HCV worldwide at present . PersistZn deficiency may trigger oxidative stress in patients with HCV-related chronic hepatitis and liver cirrhosis, termed HCV-related chronic liver disease (CLD) . A decrePreviously, NS3 proteinase, which is involved in the process of HCV replication, proved to be a zinc-containing enzyme . In addiNonalcoholic fatty liver disease (NAFLD) is currently the most prevalent liver disease worldwide, characterized by the accumulation of triglycerides in the liver, and the absence of excessive alcohol consumption. NAFLD covers a spectrum of liver diseases that range from simple steatosis called nonalcoholic fatty liver (NAFL) through nonalcoholic steatohepatitis (NASH), which is associated with hepatic inflammation and fibrosis in addition to simple steatosis .Previous studies elucidated that dietary habits are likely to affect Zn metabolism in patients with NAFLD. Lower oral intake of Zn was observed in patients with NAFLD than in normal healthy controls . NASH paZn and zinc transporters play crucial roles in the attenuation of endoplasmic reticulum (ER) stress-related signaling and the unfolded protein response (UPR) . TherefoZn plays a pivotal role in the secretion and activation of insulin. Specifically, Zn participates as a potent physiological regulator of insulin signal transduction through its inhibitory effect on protein tyrosine phosphatase 1b, the key phosphatase that dephosphorylates the insulin receptor . TherefoInsulin resistance and/or concurrent type 2 DM are often associated with the occurrence of HCV-related CLD . PreviouDepressed serum Zn levels were found in some patients with NAFLD (unpublished data). Zn deficiency is likely to evoke insulin resistance in such patients by way of almost the same mechanism as chronic HCV infection, including hyperferritinemia and/or lower circulating free IGF-1 levels .Zn deficiency is also observed in patients with primary biliary cholangitis (PBC), an autoimmune liver disease characterized by progressive destruction of the intrahepatic bile ducts, leading to cholestasis . Serum ZPersistent HCV infection and nonalcoholic fatty liver disease (NAFLD) are well recognized cofactors affecting body iron storage . A patieIt has been well established that Zn and iron may compete for access to transporters. Formigari and colleagues suggested that divalent metal transporter 1 (DMT1) is a possible candidate zinc-iron transporter . It is oIron often influences glucose metabolism. The amount of iron storage in the body was found to be significantly associated with the development of glucose intolerance or type 2 DM in a general population .In addition, iron overload reciprocally seems to affect insulin action. Iron storage interferes with the production of glucose by the liver. Hepatic extraction of insulin and the body’s metabolism of insulin is reduced with increasing iron storage, leading to peripheral hyperinsulinemia.This status is called “iron overload-related insulin resistance” . Iron ovInsulin-like growth factor-1 (IGF-1), a liver-derived humoral growth factor, has crucial anabolic and metabolic actions. It is secreted from hepatocytes and has a negative feedback control on growth hormone release from the pituitary gland. The biological activity of IGF-1 is primarily dependent on IGF-binding proteins (IGFBPs). The binding of IGFBP-3 to IGF-1 has been shown to inhibit the bioavailability of IGF-1 . TherefoZn is likely to participate in the stabilization of IGF-1 transcripts . Thus, tA previous study demonstrated that Zn deficiency was linked to hepatic steatosis in an experimental animal model of fatty liver induced by tetracycline . We confZn has been considered to participate in the enhancement of peroxisome proliferator-activated receptor-α (PPAR-α), a regulator of lipid homeostasis . Zn is nWe also revealed that a lower IGF-1/IGFBP-3 ratio might cause more advanced hepatic steatosis in patients with HCV-related CLD . A signiIron overload is also believed to cause lipid peroxidation, and subsequently to lead to hepatic steatosis in patients with HCV-related CLD . We confWilson’s disease is an autosomal recessive, hereditary disorder of Cu metabolism due to defective Cu-transporting ATPase activity, leading to the accumulation of Cu in the liver and basal ganglia of the brain . The accPatients with hereditary hemochromatosis, characterized by HFE point mutations , are morZinc administration has shown favorable effects on metabolic abnormalities in patients with chronic liver disease. The effects of zinc supplementation and possible mechanisms of these effects are summarized in l-carnosine, showed anti-inflammatory effects in the liver by attenuating hepatic iron storage in such patients [Zn has beneficial effects on hepatic inflammation in patients with HCV-related CLD. We previously documented that the administration of polaprezinc, a complex of Zn and patients . Howeverpatients . Likewispatients .Zn supplementation is likely to have favorable effects on impaired glucose tolerance. Yoshikawa and colleagues demonstrated that oral administration of Zn (II)-dithiocarbamate complex improved hyperglycemia, glucose intolerance and insulin resistance in an experimental animal model of type 2 DM . It is oOral Zn administrations, using zinc sulfate or zinc acetate, have been prescribed in patients with T2DM. A systemic review by Jayawardena documented that Zn supplementation had beneficial effects on glycemic control in such patients . The autIt is of interest that long-term Zn treatment improved impaired glucose tolerance in patients with advanced liver cirrhosis . FurtherSugino and colleagues investigated the effect of Zn (polaprezinc) in a mouse model of NASH. Zn supplementation did not affect the steatosis, but visibly attenuated fibrosis in the liver . AnotherUnfortunately, the effects of Zn administration on hepatic steatosis in patients with CLD-C or NAFLD has not been fully verified yet. Hence, Zn supplementation should be evaluated as an optional treatment for such patients .Long-term Zn supplementation significantly improved the grade of HE and blood ammonia levels in patients with liver cirrhosis ,86,87,88l-carnosine, had a significantly lower incidence of HCC than those without administration of polaprezinc [Zn administration appears to have a preventive effect on the development of HCC. Matsumura and colleagues documented that chronic hepatitis C patients treated with polaprezinc, a complex of zinc and aprezinc . MaintenInsulin resistance seems to be one of the risk factors for the development of HCC . Zn suppIt has been widely established that oral Zn administration often provides Wilson’s disease patients with favorable effects. Zn administration induced the synthesis of metallothionein (MT), a copper-binding protein , in the It has been widely recognized that some trace elements, including Cu and iron, compete with Zn, while selenium and vitamin A are positively associated with Zn status. It is well known that Zn competes with Cu . The risThe ratio of serum Cu to Zn levels has been utilized as a clinical assessment in certain diseases . Among cZn also competes with iron. We previously showed a weakly inverse correlation between serum zinc and ferritin levels in patients with HCV-related chronic liver disease . The invInterestingly, Zn supplementation caused an increased iron concentration in the duodenal mucosa of patients with Wilson’s disease, although the mechanism of this change remains unclear . FurtherWe previously revealed that serum selenium levels were positively associated with serum Zn levels in patients with HCV-related CLD . We alsoIt is of interest that the administration of selenium modulated serum Zn levels in diabetic rats fed a zinc-deficient diet .A previous study elucidated that significant correlations between serum Zn and vitamin A or retinol-binding protein (RBP) levels were found in patients with liver cirrhosis . Zn defiSerum 25-hydroxy vitamin D (25-OH vitamin D) levels are also decreased in proportion to the severity of hepatic fibrosis as well as serum Zn levels in patients with HCV-related CLD . SurprisMoriyama and colleagues documented that there was not a significant correlation between serum Zn and BCAA levels in patients with HCV-related CLD, but that an inverse correlation between serum Zn and tyrosine levels did exist . TherefoSurprisingly, oral administration of BCAA granules caused a decrease in serum Zn levels of patients with liver cirrhosis . The autIt has been well established that Zn plays a fundamental role in the stability of IGF-1 transcripts . TherefoZn seems to participate in modulating serum testosterone levels in normal men. Therefore, hypogonadism has been linked to Zn deficiency . In contThis review complied overwhelming evidence that Zn deficiency is common in patients with chronic liver diseases such as chronic hepatitis and NASH, as well as liver cirrhosis. In such patients, Zn deficiency causes various types of metabolic abnormalities, including insulin resistance, hepatic steatosis, iron overload and hepatic encephalopathy. As expected, these metabolic abnormalities may be recovered by Zn supplementation. Further trials will be required to verify the beneficial effects of Zn administration on theses metabolic abnormalities especially in NASH patients.


The Bayesian estimation algorithm improves the precision of bolus tracking perfusion imaging. However, this algorithm cannot directly calculate Tmax, the time scale widely used to identify ischemic penumbra, because Tmax is a non-physiological, artificial index that reflects the tracer arrival delay (TD) and other parameters. We calculated Tmax from the TD and mean transit time (MTT) obtained by the Bayesian algorithm and determined its accuracy in comparison with Tmax obtained by singular value decomposition (SVD) algorithms.The TD and MTT maps were generated by the Bayesian algorithm applied to digital phantoms with time-concentration curves that reflected a range of values for various perfusion metrics using a global arterial input function. Tmax was calculated from the TD and MTT using constants obtained by a linear least-squares fit to Tmax obtained from the two SVD algorithms that showed the best benchmarks in a previous study. Correlations between the Tmax values obtained by the Bayesian and SVD methods were examined.The Bayesian algorithm yielded accurate TD and MTT values relative to the true values of the digital phantom. Tmax calculated from the TD and MTT values with the least-squares fit constants showed excellent correlation (Pearson’s correlation coefficient = 0.99) and agreement (intraclass correlation coefficient = 0.99) with Tmax obtained from SVD algorithms.Quantitative analyses of Tmax values calculated from Bayesian-estimation algorithm-derived TD and MTT from a digital phantom correlated and agreed well with Tmax values determined using SVD algorithms. Therefore, this technique can robustly estimate individual hemodynamic parameters, including CBF, MTT, and TD, without delay sensitivity.10 However, the Bayesian method cannot calculate Tmax directly, as it is a nonphysiological index of the distorted R(t) profile obtained by SVD algorithms, which reflects combined information of the TD and MTT.6 Hence, in this study, we attempted to establish a computational method to generate Tmax from the Bayesian method derived TD and MTT using correlations with Tmax values obtained by SVD algorithms.Various metrics generated by bolus tracking perfusion imaging such as magnetic resonance (MR) perfusion-weighted imaging (PWI) or perfusion computed tomography (PCT) have been used to evaluate areas of the ischemic penumbra in acute stroke patients, although these techniques are considered non-mandatory for guiding reperfusion therapies mainly because being time-consuming.11 (available here: http://amrc.iwatemed.ac.jp/ibms-en/projects/cmri/download.html). This Digital Imaging and Communication in Medicine (DICOM) format phantom includes timedensity curves of brain tissue embedded in 7 × 7 quadratic tiles, as well as the global AIF and venous output function (VOF) of real brain computed tomography (CT) images at 16 slice locations taken every 2 s over a 60-s duration (R(t) . These parameter sequences were accompanied with Gaussian noise typical of PCT data scanned with 80 kVp at 200 mAs. In this phantom, the dispersion was incorporated into the MTT values using the global AIF.We used a digital phantom introduced in a previous studyduration . The tim8 (2) Perfusion Mismatch Analyzer (PMA)  with the block-circulant SVD (bSVD) algorithm,12 and 3) CBP Study Ph8  with the reformulated SVD (rSVD).13 The last two software packages with their associated algorithms provided the best benchmarks in a previous digital phantom validation study.11 The TD and MTT maps were generated by the Bayesian algorithm, while Tmax maps were generated by the bSVD and rSVD algorithms  Olea Sphere  with the Bayesian estimation algorithm,gorithms . These m6p and q are best-fit parameters determined for each SVD algorithm and TD(Bayesian) and MTT(Bayesian) are the TD and MTT values, respectively, obtained by the Bayesian method. The p and q values specific to each SVD algorithm were found by a linear least square method that minimized differences between Tmax(Bayesian) and the Tmax values of the SVD algorithms, Tmax(bSVD), and Tmax(rSVD). Pearson’s correlation coefficient (r) was calculated to evaluate correlations between Tmax(Bayesian) and Tmax(bSVD) or Tmax(rSVD) values. In addition, the slopes and intercepts of the linear regression lines, as well as intraclass correlation coefficients (ICCs), were also calculated to evaluate agreement between the Tmax values. Correlations and agreement of TD and MTT values obtained by the Bayesian method with ground truth values of the digital phantom were also evaluated using r and ICC. These analyses were performed using SPSS Ver. 19 .Tmax values determined from Bayesian algorithm metrics, Tmax(Bayesian), were calculated by the following equation, which was simplified from a previous study:r = 0.98 and 1.00, respectively) and agreement  with the true values of the digital phantom  or Tmax(rSVD)   calculated with the each set of ax(rSVD) . Linear or both) , Fig. 3.or both) , Fig. 3.or both) . These r16 Quantitative automated detection of hypoperfused areas with prolonged Tmax over certain cut-off values, e.g., 6 s, has been commonly adopted to determine perfusion-diffusion mismatch areas reflecting ischemic penumbra.17 Although Tmax is a wellestablished and reliable metric, it is also non-physiological and artificial, and includes biological and algorithmic information. A recent study showed that Tmax depends mainly on TD, dispersion, and, to a lesser extent, MTT, and can be expressed as a complex function of these parameters.6 However, in this study, we used a simple equation to calculate accurate Tmax values from only TD and MTT values obtained using a Bayesian estimation algorithm. Theoretically, under the global AIF, MTT values include tracer dispersion information,7 indicating that precise MTT measurements are required to calculate Tmax. A recent phantom study revealed that the Bayesian estimation method generated more accurate MTT values than did SVD methods.9 In addition, as demonstrated in this study, the Bayesian method can generate accurate TD values, which are largely inaccessible by other deconvolution or non-deconvolution algorithms. Therefore, the Bayesian method should be able to indirectly but readily generate Tmax comparable to those generated by SVD methods.Tmax has been used to determine ischemic penumbral regions that were potential targets of reperfusion therapies in recent major clinical trials.11 When calculating Tmax from TD and MTT obtained by the Bayesian algorithm according to p and q should be optimized for the individual SVD algorithms. Least squares fits to the Tmax of bSVD and rSVD returned different sets of optimizing p and q. Various software programs using different types of the SVD family, such as standard SVD (sSVD), bSVD, and oscillation-index regularized SVD (oSVD), have been used to obtain Tmax.11 In order to reproduce these SVD-derived Tmax values by the Bayesian method, the digital phantom we used could provide constants p and q specific to each algorithm. Other advantages of the Bayesian method against SVD algorithms include robustness to the noise, truncation, and oscillation,8 which can improve precision of the Tmax, although postprocessing time cannot be shortened.One of the advantages of the Bayesian method against the SVD methods is the capability to simulate Tmax values of other various algorithms, which can help compare the results with those in previous studies or other institutions. One inherent issue with a Tmax calculated by SVD is its dependence on the specific deconvolution algorithm.R(t) is unknown in normal and hypoperfused brain tissues; hence, the shape used may have been non-realistic. We did not investigate the dependence of our results and analyses on the signal-to-noise ratio (SNR), which can affect perfusion analysis accuracy, although the PCT phantom we used had a low SNR. We did not perform a similar investigation using the PWI phantom because the SNR was higher than that of the PCT phantom and one of the PWI software programs showing the best benchmark was unavailable in this study; however, we assume that the method we introduced can readily regenerate Tmax on PWI by the Bayesian algorithm. We could not demonstrate comparisons of Tmax images obtained by the Bayesian and SVD methods in phantoms or clinical cases because the software was still unavailable to generate the Bayesian Tmax images, which will be overcome by modifying the program in the near future. We performed no comparisons between Tmax and other physiological parameters in acute stroke patients, because this issue is beyond the scope of this study. Thus, potential advantages of Tmax against other parameters obtained by the Bayesian method remain unclear. Further studies using MR and CT data from patients with acute stroke and revised software program will allow us to determine whether indirect estimation of Tmax from TD and MTT by the Bayesian method is accurate and can readily determine penumbral areas, relative to major SVD methods or major physiological parameters.There are several limitations in this study. First, the phantom we used included limited perfusion information. Tracer dispersion was inexplicit, but instead incorporated into the MTT data by the global AIF. The shape of Quantitative analyses of a digital phantom with a global AIF revealed that the Bayesian estimation algorithm readily calculated Tmax from TD and MTT. The Bayesian method Tmax values were nearly identical to the bSVD and rSVD algorithm Tmax values when calculated using individually optimized constants for each algorithm.


Data from ChIP-seq experiments can derive the genome-wide binding specificities of transcription factors (TFs) and other regulatory proteins. We analyzed 765 ENCODE ChIP-seq peak datasets of 207 human TFs with a novel motif discovery pipeline based on recursive, thresholded entropy minimization. This approach, while obviating the need to compensate for skewed nucleotide composition, distinguishes true binding motifs from noise, quantifies the strengths of individual binding sites based on computed affinity and detects adjacent cofactor binding sites that coordinate with the targets of primary, immunoprecipitated TFs. We obtained contiguous and bipartite information theory-based position weight matrices (iPWMs) for 93 sequence-specific TFs, discovered 23 cofactor motifs for 127 TFs and revealed six high-confidence novel motifs. The reliability and accuracy of these iPWMs were determined via four independent validation methods, including the detection of experimentally proven binding sites, explanation of effects of characterized SNPs, comparison with previously published motifs and statistical analyses. We also predict previously unreported TF coregulatory interactions (e.g. TF complexes). These iPWMs constitute a powerful tool for predicting the effects of sequence variants in known binding sites, performing mutation analysis on regulatory SNPs and predicting previously unrecognized binding sites and target genes. Transcription factors (TFs) interact with regulatory elements in genes to mediate positive or negative regulation of tissue- and stage-specific expression ,2. TFs eiR) of a TF-bound sequence, which represents the affinity of the TF–DNA interaction, is the dot product between the binary matrix of the sequence and an iPWM of the TF  consisting of 207 human TFs. 93 primary and 23 cofactor binding motifs were successfully recovered and refined for 127 TFs. Reanalysis of the same data using the masking and thresholding techniques revealed many known and previously unreported TF cofactors; however, frequently our approach revealed cofactor motifs directly. These primary motifs were validated by comparing predicted with experimentally-detected true binding sites, explaining effects of characterized SNPs on binding site strengths, and through comparisons to an independent motif database.The ENCODE Consortium conducted ChIP-seq assays for human TFs and generated initial peak datasets for each replicate of each assay using a uniform peak calling pipeline ,17. For We started with the IDR-thresholded peak datasets, because we found that these data are more likely to produce primary or cofactor motifs than the initial (i.e. unprocessed) datasets; they contain greater numbers of ChIP-seq peaks (and thus more binding sites) than the truncated SPP datasets. The initial, unfiltered datasets were examined if neither IDR-thresholded nor SPP datasets were available.Initially, iPWMs from ChIP-seq reads were derived by entropy minimization with Bipad . HoweverThis masking technique, which contrasts with the likelihood approach used by MEME , provideTo eliminate noisy patterns that suppress the expected TF binding motifs due to ChIP-seq peaks with low signal strengths (i.e. read counts), the dataset is truncated based on signal strengths as follows Figure . First, iR value of each site;To detect experimentally proven binding sites in known target genes, derived iPWMs were used to evaluate the To predict changes in binding site strength, characterized variants were evaluated with the corresponding iPWMs. The predicted changes were compared with experimentally supported effects on TF binding or gene expression;The iPWMs were compared with the corresponding annotated motifs in the CIS-BP database  based oniR values of binding sites discovered by the iPWM and their corresponding binding energy   . PrimaryThe methods used to evaluate the accuracy of our iPWMs include:The derived iPWMs displayed primary motifs for 93 TFs , as wellThe initial iPWMs directly exhibited primary motifs for 76 TFs and 18 cofactor motifs for 107 primary TFs. Thresholding the datasets revealed 31 primary motifs and 14 cofactors for 38 primary TFs. We used the masking technique to discover an additional 4 primary motifs; 7 cofactor motifs were also found in 21 datasets .n = 367), we determined the false positive detection rate from the null iR distribution, which is approximately Gaussian , <10% of the information present in the iPWM is dependent, and for 62 TFs (∼66.7%), <5% is dependent. Neglecting the interactions between positions introduces a minimal error into the calculation of iR values of binding sites, and would be expected to have little impact on assessment of the mutations in these sequences.Similarly, the independence of contributions of each position in a binding site to the overall information content was analyzed for one iPWM of each primary motif. The total mutual information, which measures the interdependence between individual positions in the same binding site, was determined by summing the pairwise mutual information at each position . Then, tin vivo and are able to regulate gene expression , and recognize two types of bipartite palindromic motifs, defined by the predominant binding sites TGCTGA(C)TCAGCA and TGCTGA(CG)TCAGCA . The preFor 19 TFs, bipartite iPWMs were successfully derived and were in agreement with previously reported motifs. The following examples illustrate key insights that can be taken from bipartite modeling:Cell-type-specific differences between iPWMs of the same TF were evident for certain contiguous and bipartite motifs. For instance, among the three contiguous iPWMs of ESR1 derived from the ECC1 steroid-responsive endometrial cell line, conservation levels in the respective half sites are asymmetric, whereas the average information of these half sites are much more symmetric in iPWMs derived from T47D, a breast tumor cell line Figure . For thecis-regulatory effects. De novo motif discovery from ChIP-seq datasets provides an effective approach for confirming or predicting statistically significant TF interactions on a genome-wide scale; by contrast, the abundant, existing literature overwhelmingly documents gene-by-gene evidence about such interactions which constrains arguments supporting their generalizability. Figure Discovery of the binding motif of a cofactor in the same ChIP-seq dataset for a primary TF implies that the two TFs transcriptionally co-regulate this set of common target genes. This could be accomplished either by formation of a physical complex on the promoter, or by synergistic or antagonistic et al.  for 46 primary TFs . The SOX2–OCT4 complex was used as a primary negative control, as it is primarily expressed in the H1-hESC cell line and is unlikely to be a cofactor for primary TFs in other cell lines. A large percentage of peaks have short intersite distances between the primary TF and the corresponding cofactor binding sites (e.g. <20 nt), whereas there is no such a trend for the negative control sequences and the primary TF. The same difference is observed between the distribution for the documented TEAD4–AP1 binding site pair and for the negative control set. Consistent with previous reports  in which YY was revealed as a cofactor were from K562 cells, with one exception  cell lines, with four exceptions  contains a stronger binding site of STAT1 than the one starting at GRCh38 (chr14:94612291)  detected by the bipartite iPWM is indeed 22.13 (or 4.38)-fold stronger than the other site  detected in this promoter.A total of 803 experimentally-confirmed, previously published binding sites were verified for the 93 TFs whose primary binding motifs had been identified . We dete4612291)  115); t; tRi valiR value of a binding site, the effect of a SNP on the binding site strength can be predicted with iPWMs . The F-values are plotted as a histogram to illustrate probability density distributions . We note that only primary and cofactor motifs exhibit F-values > 1000, which comprise 37.2% (251 of 674) of all iPWMs. The iPWMs with F-values < 1000 remain valid based on the other criteria described above.To distinguish true binding motifs from noise motifs, the relationship between s Figure . The hisIn this study, we derived and validated TF binding motifs from ChIP-seq datasets using an information theory-based approach, also revealing TF cofactor binding sites and other novel motifs. The primary TF motifs were validated by comparison with motifs derived independently from binding studies, by analysis of gene variants known to alter TF binding affinities, and by comparing the locations of binding sites predicted by iPWMs with those of true sites previously determined in published binding and expression studies. In addition to contiguous iPWMs, bipartite iPWMs with variable-length spacers were also derived. These iPWMs more precisely reflect the binding behavior of dimeric TFs, as they incorporate intermediate and often weak binding sites that are often excluded from consensus sequence-based (strong) binding site sets . This enet al.  were successfully detected by the iPWMs (iR > 0). By contrast, the FLR identified 35–85% of the verified binding sites  investigated by both tools, Maskminent and MEME-ChIP discovered primary motifs for 80 (∼81.6%) and 92 (∼93.9%) TFs, respectively. Among the 59 TF datasets analyzed by Maskminent, MEME-ChIP, SeqGL and HOMER . For 11 primary TFs, the binding site sequences were extremely variable; that is, the overall conservation levels of their binding motifs contain less information than noisy, low complexity sequences or cofactor motifs. For 18 primary TFs associated with cofactors, which themselves physically contact DNA, the primary TF motif was not enriched. The inability of the software to discover such primary motifs is a limitation of this approach. Interactions between the primary TFs and a subset of the cofactors which are known to cooperate with them were detected, since the association has to occur with a prevalence sufficient to produce a recognizable motif . Nevertheless, the algorithm may not find cofactors with weakly conserved motifs or those that overlap with other conserved motifs.et al. (et al. (While unable to discover cofactors nor identify bipartite motifs of variable spacing, the oligonucleotide microarray technique adopted by Weirauch et al.  and Jolm (et al.  theoretiThe Maskminent pipeline can be applied to other ChIP-seq data not included in ENCODE. The quality control criteria we described are capable of ensuring that the user-built iPWMs are accurate and can be used for binding site detection. The first and second criteria are particularly important, because they provide a straightforward assessment of iPWM performance. The recursively thresholded feature is crucial for guaranteeing that the discovered cofactors do not appear by chance, because the greater the number of peaks from which a cofactor is derived, the higher the confidence that the cofactor indeed interacts with the primary factor.In summary, we comprehensively investigated and implemented a new approach to define TF binding specificities based on the ChIP-seq TF data that ENCODE has released. This allowed us to mine and quantify both known and previously unrecognized TF binding motifs and cofactor interactions on a genome scale. This information expands the granularity of the current knowledge on TF interaction with DNA and points out potential directions for future experimental study on interaction between TFs.http://dx.doi.org/10.5281/zenodo.49234 and https://www.mutationforecaster.com.Supplementary DataClick here for additional data file.


Older adults are disproportionately targeted by fraud schemes that advertise unlikely but large returns (positively skewed risks). We examined adult age differences in choice and neural activity as individuals considered risky gambles. Gambles were symmetric (50% chance of modest win or loss), positively skewed (25% chance of large gain), or negatively skewed (25% chance of large loss). The willingness to accept positively skewed relative to symmetric gambles increased with age, and this effect replicated in an independent behavioral study. Whole-brain functional magnetic resonance imaging analyses comparing positively (vs. negatively) skewed trials revealed that relative to younger adults, older adults showed increased anticipatory activity for negatively skewed gambles but reduced activity for positively skewed gambles in the anterior cingulate and lateral prefrontal regions. Individuals who were more biased toward positively skewed gambles showed increased activity in a network of regions including the nucleus accumbens. These results reveal age biases toward positively skewed gambles and age differences in corticostriatal regions during skewed risk-taking, and have implications for identifying financial decision biases across adulthood.The online version of this article (10.3758/s13415-017-0545-5) contains supplementary material, which is available to authorized users. Given an uncertain future, nearly all long-term financial choices involve some degree of risk. Although research on financial risk-taking has explored the effects of expected value (mean) and risk (variance) on financial choice , researchers have begun to investigate how skewed gambles influence affective responses and brain activity, which modulate choice , it has produced mixed results  and neural activity (Study 1) before individuals chose to accept or reject symmetric, positively skewed, or negatively skewed gambles as they underwent fMRI scanning. Our first aim was to determine whether age influenced the choice of risky skewed gambles. Based on an anticipatory affect account of financial risk-taking  were recruited from the San Francisco Bay Area community to complete a study at the Stanford Center for Cognitive and Neurobiological Imaging. Five were excluded from analyses because of head motion >2 mm during the fMRI scan, leaving a total sample of 32. Our a priori sample size calculation suggested that we needed to collect data from at least 27 subjects in order to have a power level of at least .8, to observe an effect at least as large as the linear effect of age on brain activity  reported in the most similar published study of financial risk-taking across adulthood . Participants completed 24 trials of each gamble type, presented in pseudorandom order. In other words, the three gambles described above were repeated 24 times in a mixed order, for a total of 72 trials.Three types of gambles were included: symmetric, positively skewed, and negatively skewed. Symmetric gambles featured an equal probability (50%) of winning or losing a moderate amount of money ($3.05). Positively skewed gambles featured a low probability (25%) of winning a large amount ($5.25) paired with a high probability (75%) of losing a small amount ($1.75). Negatively skewed gambles were the opposite, and featured a low probability (25%) of losing a large amount ($5.25) paired with a high probability of gaining a small amount ($1.75). Critically, the expected value of each gamble was set to $0, making it equivalent to both the alternative conditions and to a “no gamble” option, which was to accept $0 for certain, and variance was also equated across all gamble types  extending from the mid-pons to the top of the skull were acquired with an axial interleaved scheme. Functional scans were acquired using a T2*-weighted gradient pulse sequence . Anatomical scans, which were used for localization and coregistration of functional data, were acquired using T1-weighted spoiled grass sequence .Analysis of functional neuroimaging data was conducted using Analysis of Functional Neuroimages (AFNI) software Cox, . PreprocMultilevel binary logistic regressions were carried out using the lme4 package (Version 1.11.1)  and its interactions, were included in the model  skewed versus symmetric , and (2) positive versus negative skew . Before inclusion in the regression models, these contrasts were convolved with a single gamma function to model the hemodynamic response. Subject-level regression models also included nine covariates of noninterest: residual motion (in six dimensions), white matter and cerebral spinal fluid time series, and polynomial trends across the experiment. For each contrast of interest, T-statistic maps were transformed to Z scores and spatially normalized by warping into Talairach space. The residual error time series from these subject-level models were used to estimate the noise smoothness values for cluster estimation. Statistical maps were then generated to examine three covariates of interest across the entire sample: (1) age, (2) positive-skew bias, and (3) the interaction between these two covariates. For follow-up verification of these analyses, regions of interest were specified around the significant clusters of activation that emerged in group analyses. Follow-up activity time-course analyses examined whether activation prior to choice, or anticipatory activation,  differed by gamble condition. Bootstrapped statistics with 2,000 replications were conducted to verify each significant effect to confirm that outliers were not overly influencing the analysis.Analyses focused on changes in brain activation during anticipation . Based on extensive prior research that suggests that there are no adult age differences in neural activation following the receipt of gains versus losses  to evaluate the reliability of the age effects on choice. Detailed methods and results are presented in the A separate behavioral study was conducted with a much larger sample , but did not confirm that older adults were more likely to reject negatively skewed than symmetric gambles . To ensure that these age effects were not driven by outliers, we compared the difference between the proportion of positively skewed and negatively skewed gambles chosen by each participant. This individual difference measure—skew bias—varied from 1 to −1, with those who chose a higher proportion of positively skewed gambles (positive-skew bias) having positive scores and those who chose a higher proportion of negatively skewed gambles (negative-skew bias) having negative scores. While young participants varied widely on this measure, with similar numbers of young individuals showing negative and positive skew biases, almost all of the older participants displayed a positive skew bias .First-level general linear models quantified differences between skewed versus symmetric trials  and positively skewed versus negatively skewed trials  across the brain. Second-level regression analyses examined individual differences by age and behavioral bias scores on the effects of general skew and valenced skew. Both main and interaction effects of age and behavioral bias were included together in second-level models because of the behavioral interaction between age and skew bias. Whole-brain analyses revealed significant age differences in anticipatory neural activity related to both skew and valence  and a bias-related decrease in anticipatory activation for negatively skewed gambles .Individual differences in the behavioral measure of positive skew bias from the task were also related to individual differences in anticipatory brain activity related to skew valence. In clusters along the cingulate gyrus, as well as bilateral regions of the caudate and posterior insula, a behavioral positive-skew bias was associated with see Fig. . The revb = 0.01, 95% CI ). Within older adults, individuals showed increased activity in the right inferior frontal gyrus on trials where they were more likely to accept the gamble: those with a positive-skew bias showed greater activity on positively skewed trials, while those with a negative-skew bias showed greater activity on negatively skewed trials. Follow-up bootstrapped correlations of bias on anticipatory activity in right inferior frontal gyrus VOI in older adults confirmed that this interaction was driven by a bias-related increase in activation in anticipation of negatively skewed gambles . These associations were not present in younger adults  gambles and more likely to reject negatively skewed (compared to symmetric) gambles , where again we found an age-related increase in acceptance of positively skewed gambles relative to symmetric gambles , AIns, and anterior cingulate cortex (ACC), regions commonly associated with executive function and cognitive control  is a significant and growing problem for people of all ages. While some evidence suggests that older adults might not necessarily be more susceptible to fraud