Novel blood-based screening tests are strongly desirable for early detection of colorectal cancer (CRC). We aimed to identify and evaluate autoantibodies against tumor-associated antigens as biomarkers for early detection of CRC. 380 clinically identified CRC patients and samples of participants with selected findings from a cohort of screening colonoscopy participants in 2005–2013 (N=6826) were included in this analysis. Sixty-four serum autoantibody markers were measured by multiplex bead-based serological assays. A two-step approach with selection of biomarkers in a training set, and validation of findings in a validation set, the latter exclusively including participants from the screening setting, was applied. Anti-MAGEA4 exhibited the highest sensitivity for detecting early stage CRC and advanced adenoma. Multi-marker combinations substantially increased sensitivity at the price of a moderate loss of specificity. Anti-TP53, anti-IMPDH2, anti-MDM2 and anti-MAGEA4 were consistently included in the best-performing 4-, 5-, and 6-marker combinations. This four-marker panel yielded a sensitivity of 26%  for early stage CRC at a specificity of 90%  in the validation set. Notably, it also detected 20%  of advanced adenomas. Taken together, the identified biomarkers could contribute to the development of a useful multi-marker blood-based test for CRC early detection. With approximately 1.4 million new cases and 700,000 deaths occurring in 2012, colorectal cancer (CRC) is the third most cancer and the fourth most common cause of death from cancer worldwide . RandomiAn alternative approach for cancer screening might be blood-based screening tests. Due to their minimally invasive nature and straightforward implementation in routine medical examinations, blood tests might achieve high levels of adherence when applied in population-based screening , 9. For Autoantibodies as potential biomarkers for early detection of cancer have been intensively studied in previous studies -14. AlthIn this study, we evaluated the individual and joint diagnostic performance for CRC and its precursors through antibodies against a panel of 64 predefined autoantigens, and we validated the most promising marker combinations in independent samples of participants recruited in a true CRC screening setting.Figure Table A head-to-head comparison of the diagnostic performance of all 64 autoantibody markers for detecting colorectal neoplasms is shown in Overall, there were only 9 autoantibody markers whose sensitivities for detecting early stage CRC were higher than 5%, ranging from 7% to 11% in the validation set see . Anti-MAA systematic search for the best n-marker combination was conducted among all the 64 autoantibody markers with the purpose to enhance the diagnostic performance. Results for the best performing 2-, 3-, 4-, 5- and 6-marker combinations are shown in Table We further evaluated additional seven 6-marker combinations, whose Youden indices were only slightly lower than the best-performing 6-marker combination in the training set. Details of the diagnostic performance of these combinations are shown in Table In this study, we first identified autoantibody markers and their combinations for early detection of CRC and its precursors in a large set of samples, which were then independently validated in prospectively collected samples from a true screening population. Overall, most of the tested autoantibodies showed relatively low sensitivity at cutoff levels yielding high specificity. Among all single markers, anti-MAGEA4 showed the highest sensitivity for detecting early stage CRC  and advanced adenomas  at a specificity of 96% . We also explored the potential of multi-marker combinations to enhance diagnostic performance. Sensitivity increased and specificity decreased with increasing numbers of markers included in multi-marker combinations. All marker-combinations had higher sensitivities for early stage than for advanced stage CRC even in the validation set. Four markers, including anti-TP53, anti-MAGEA4, anti-MDM2 and anti-IMPDH2, were consistently included in best performing 4-, 5-, and 6-marker combinations. A combination of these four markers could detect 26% of early stage CRC  and 20% of advanced adenomas  at a specificity of 90% .Many single autoantibody markers tested in our study were also evaluated in previous studies, mostly using retrospective case-control designs. Apparently substantially better discriminative ability between CRC and healthy controls were reported for some markers. For instance, Kanojia and colleagues  measuredThe apparent discrepancies could be explained by different design issues and different study settings adopted in different studies. Most previous studies recruited participants in clinical settings, i.e., using clinically detected cases and convenience controls or healthy donors who often substantially differed from cases with respect to major characteristics that may be related to the autoantibody levels, such as age and sex -21. UndeDefinition of cutoff directly affects the sensitivity and specificity of respective markers. In our analysis, the cutoffs of 64 autoantibody markers were set at relatively high levels to ensure very high specificities of each single marker (≥ 98% in the training set). This may explain the somewhat lower sensitivity for the single markers compared to previous reports, but it ensured maintenance of reasonably high specificity of multi-marker combinations. For instance, anti-TP53 is a widely evaluated antibody marker for cancer detection. In the current study, ROC (receiver operating characteristic) analysis showed that the AUC (area under the curve) of anti-TP53 for detecting CRC was 0.60 in the validation set (data not shown in the results). This result was quite similar to results from other studies evaluating anti-TP53 for early detection of CRC in large prospective studies , 17.Among tested single autoantibody markers, anti-MAGEA4 was found to have good diagnostic potential for detecting early stage CRC  and advanced adenomas  in our analysis. MAGEA4 is a member of cancer/testis (CT) antigens, which is expressed in malignant cells and in immune-privileged germ-line cells . It has As genetic or epigenetic alterations leading to aberrantly expression of antigens, such as TP53  and CTAG35,36 and any of other suggested blood tests that were validated in screening settings. For instance, a recent study by Church and colleagues [The sensitivities for detecting CRC of the included autoantibodies are clearly inferior to detection of CRC by currently recommended tests for early detection of CRC, i.e., faecal immunochemical tests (FITs) for hemoglobin , 34, andlleagues  tested mlleagues , which olleagues , 39. Earlleagues .It should be stated that the multi-marker panels identified in this study cannot be regarded as a final solution for CRC screening due to their overall limited sensitivity. Nevertheless, given their potential for detecting early stage CRC and especially its most important precursor – advanced adenomas , at high specificities, combining these panels with other promising biomarkers or tests might contribute to the development of clinically useful multi-marker panel in the future. In particular, further research should also explore the possibility of enhanced detection of advanced adenomas by combining FIT with a panel of autoantibody markers. However, additional issues such as compliance to specimen sampling (blood and stool), complexity of conducting multiple tests and cost, need to be taken into consideration for such potential test combinations.Specific strengths and limitations deserve careful consideration when interpreting our study. Strengths include that we adopted a two-step approach to minimize overestimation of indicators of diagnostic performance. The validation of biomarkers was conducted in a large subset sample of participants of screening colonoscopy, which represent the target population for CRC screening in which the diagnostic performance of biomarkers should be evaluated. Furthermore, we applied the state-of-art technique to measure a large number of autoantibodies simultaneously, enabling a head-to-head comparison of the diagnostic performance of these biomarkers possible. Limitations of our study include the relatively small number of CRC cases in the validation samples, despite the very large screening population recruited, which reflects the very low prevalence of CRC in a true screening population. Moreover, the target antigens evaluated in our measurement were mostly native full-length proteins. Other types of TAAs, such mutated neo-antigens or proteins with abnormal posttranslational modifications were not tested in our study. In addition, the cutoffs of respective autoantibody markers were defined based on MFI values of controls with no significant findings at screening colonoscopy in the training set. Further determination of optimal cutoffs of respective autoantibody markers needs to be validated in larger screening populations.In summary, autoantibodies against TAAs carry potential for early detection of CRC. The multi-marker combinations developed in our study exhibited promising diagnostic performance for detecting a relevant proportion of early stage CRC and its relevant precursors. The combination of the most promising biomarkers identified in our study with additional promising biomarkers or tests might contribute to the development of a useful multi-marker blood-based test for early detection of CRC and its precursors in the future.1Our analysis is based on the ongoing BliTz study , which has been described in detail elsewhere , 41. BriFor this analysis, the following exclusion criteria were applied to exclude participants without adequate blood samples, participants not representing a true screening setting, and participants with potentially inadequate colonoscopy results: blood samples taken after screening colonoscopy, history of CRC or inflammatory bowel disease, incomplete colonoscopy or insufficient bowel preparation (latter only for controls). From the remaining participants of the BliTz study recruited in 2005-2013 (N=5680), all 50 newly detected CRC cases, as well as random samples of participants with advanced colorectal adenomas , non-advanced colorectal adenomas  and with no colorectal neoplasms  were included in the measurement. Because in a true screening population like ours CRC patients are expected to be on average slightly older and to include a somewhat larger proportion of men, age and sex were not matched between different disease groups to avoid biased estimates of specificity [Given the limited number of CRC cases even in a screening study as large as the BliTz study, we also included 380 CRC cases recruited after diagnosis but before treatment at four hospitals in Southern Germany for the discovery phase of this study. All studies were approved by the ethics committees of the medical faculty of the University of Heidelberg and of the respective state medical boards. Written informed consent was obtained from each participant.2th version). In addition, advanced adenomas were subclassified into high grade dysplasia (HGD), villous architecture without HGD, and large adenomas (≥10 mm) with neither HGD nor villous architecture. Relevant information was extracted by two research assistants independently who were blind to the blood test results.Based on the colonoscopy reports or hospital records of all participants, invasive CRC was classified into stages Tis (carcinoma in situ) and I–IV according to the UICC  TNM (tumor-node-metastasis) stage classification (733.1Blood samples were collected in Sarstedt S-Monovette (Product No. 02.1388) tubes and Vacutainer SST II (Product No. 367953) tubes before bowel preparation for colonoscopy (BliTz study), or prior to large bowel surgery or neoadjuvant therapy . After completion of blood clotting, blood samples were immediately centrifuged at 2000−2500g for 10 minutes and stored at −80°C until analyses. Details on the standard operating procedures have also been described previously .3.2We selected 64 candidate TAAs encoded by 59 genes based on previous autoantibody measurements  and two Sera were sent on dry ice to the Division of Molecular Diagnostics of Oncogenic Infections, DKFZ, Heidelberg and analyzed in a 1:1000 dilution by laboratory staff that was blinded for the case-control status.Antigen-loading of the beads was controlled via detection of the C-terminal tag. Identity of the antigen loaded on the beads was verified by identifying the encoding plasmids via PCR and sequencing. Serum samples were denominated as invalid due to either insufficient amount of serum or too high GST-background values (>300 MFI). Variation between different assay-plates was controlled by pipetting three control sera on each plate as replicates. For overall 19 replicates, coefficients of variation (CV) were calculated for every antigen with a mean reactivity above 30 MFI. The median (range) CVs for the three samples were 16% (10-24%), 14% (11-21%), and 18% (11-25%), respectively.4We used a two-step approach with selection of markers and marker combinations in a training set, and validation of the results in an independent validation set. The validation set was defined in such a way that it represents a true screening setting, i.e., only participants from the BliTz study were included. CRC cases recruited in the clinical setting and randomly selected controls from the BliTz study were used for the training set.Characteristics of the study population were first described and compared between the training set and the validation set. The strategy for selection of autoantibodies and their cutoff levels for predicting presence of colorectal neoplasm were based on the particular character of single autoantibody markers, which typically have rather low sensitivity at very high specificity , 14. CutAll statistical analyses were performed with the statistical software R version 3.1.1 . All tes


In our previous study of 3-year-old children in a dioxin contamination hot spot in Vietnam, the high total dioxin toxic equivalent (TEQ-PCDDs/Fs)-exposed group during the perinatal period displayed lower Bayley III neurodevelopmental scores, whereas the high 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-exposed group displayed increased autistic traits. In autistic children, urinary amino acid profiles have revealed metabolic alterations in the amino acids that serve as neurotransmitters in the developing brain. Therefore, our present study aimed to investigate the use of alterations in urinary amino acid excretion as biomarkers of dioxin exposure-induced neurodevelopmental deficits in highly exposed 3-year-old children in Vietnam. A nested case-control study of urinary analyses was performed for 26 children who were selected from 111 3-year-old children whose perinatal dioxin exposure levels and neurodevelopmental status were examined in follow-up surveys conducted in a dioxin contaminated hot spot. We compared urinary amino acid levels between the following 4 groups: (1) a high TEQ-PCDDs/Fs and high TCDD-exposed group; (2) a high TEQ-PCDDs/Fs but low TCDD-exposed group; (3) a low TEQ-PCDDs/Fs exposed and poorly developed group; and (4) a low TEQ-PCDDs/Fs exposed and well-developed group. Urinary levels of histidine and tryptophan were significantly decreased in the high TEQ-PCDDs/Fs and high TCDD group, as well as in the high TEQ-PCDDs/Fs but low TCDD group, compared with the low TEQ-PCDDs/Fs and well-developed group. However, the ratio of histidine to glycine was significantly lower only in the high TEQ-PCDDs/Fs and high TCDD group. Furthermore, urinary histidine levels and the ratio of histidine to glycine were significantly correlated with neurodevelopmental scores, particularly for language and fine motor skills. These results indicate that urinary histidine is specifically associated with dioxin exposure-induced neurodevelopmental deficits, suggesting that urinary histidine may be a useful marker of dioxin-induced neurodevelopmental deficits and that histaminergic neurotransmission may be an important pathological contributor to dioxin-mediated neurotoxicity. We previously followed up on infant cohorts in dioxin contamination hot spots in Vietnam and reported considerable neurodevelopmental impacts of perinatal dioxin exposure including postnatal exposure by breast feeding, which were examined using the Bayley Scales of Infant and Toddler Development, Version 3 (Bayley III) when the infants were 4 months old –3. RecenAutism spectrum disorders (ASD) are neurodevelopmental disorders that are characterized by impairments in socioemotional interactions, communication, and repetitive or restricted behaviors. Typically, patients with autism are suspected to have a genetic predisposition. In a recent report, genomic sequencing of six children with autism revealed mutations in a gene that prevents several essential amino acids from being depleted, leading to the development of autism-related neurological problems . PreviouTherefore, in the present study, we investigated possible differences in urinary amino acid levels in 3-year-old children with high perinatal dioxin exposure and lower neurodevelopmental scores compared to well-developed children with low exposure levels, which were estimated based on dioxin concentrations in the breast milk of mothers residing in the most highly polluted areas of Vietnam. In our analysis, we also performed dietary surveys and compared amino acid levels between the high—and low-exposure groups after adjusting for food intake.The Thanh Khe district wards in Da Nang City, which are located within 3 km of Da Nang airbase, have been characterized as dioxin-contaminated areas due to the presence of Agent Orange and other herbicides that were stored and spilled there during the Vietnam War between 1961 and 1971 , 11 and Pregnant mothers were recruited for the present study by obstetricians upon arriving to give birth at the district hospital in 2008. The criteria for recruitment were as follows: (i) The mothers must have resided in one of the study areas for a period encompassing at least the duration of their pregnancy, and (ii) the mothers must have given birth to full-term babies without complications during childbirth. These mothers had no pregnancy-induced hypertension or pregnancy complication. A total of 159 mother-infant pairs were enrolled at baseline [Follow-up examinations were conducted 4 months, 1 year and 3 years after birth to assess the development of the infants. In the 3 year follow-up survey, a total of 122 pairs participated, and 111 children underwent neurodevelopmental testing. Urinary analyses using a gas chromatograph equipped with a mass spectrometer (GC-MS) were performed for 26 children who were selected from children whose neurodevelopmental status had been examined; twelve poorly-developed  children with higher TEQ-PCDDs/Fs levels in their breast milk (TEQ-PCDDs/Fs ≥ 20.0 pg-TEQ/g-lipid), including 6 children with higher TCDD levels and 6 children with lower TCDD levels, and 14 children with low dioxin exposure (TEQ-PCDDs/Fs < 11.5 pg-TEQ/g-lipid), including 10 well-developed children and 4 poorly-developed children, who served as controls. The TCDD cut-off value was 3.5 pg/g of lipids, which was reported as the threshold level for increased autistic traits and lower developmental scores in our previous study [Body size parameters for the infants, including weight and height, were measured at the survey site. The food intake of the children during the day prior to conducting the survey was obtained using a 24-hour recall method from the mothers or caretakers, and the estimated daily intake (g) of the main food groups, including milk and milk products, cereals, meat, fish, and eggs, was recorded. The Bayley III is a neurodevelopmental assessment measure that was used to assess the children’s development of cognition, language (including receptive and expressive communication), and motor skills, which were further divided into fine and gross motor skills. The children were tested by one examiner in the presence of each child’s caregiver at community health centers.A breast milk sample was collected from each nursing mother 1 month after birth by hand expression with the assistance of a midwife or medical worker. Samples were placed in clean polyethylene containers and temporarily stored in freezers set at −4°C in the local health centers. The samples were frozen in dry ice for transport to Japan by airplane and were analyzed at the Center for High Technology, Kanazawa Medical University . Approximately 10 ml of breast milk from each sample was used to quantify the levels of 17 different 2,3,7,8-substitued polychlorodibenzo-p-dioxin (PCDD) and dibenzofuran (PCDF) congeners using high resolution GC-MS. The established method of analysis was previously described in detail . The conApproximately 10 ml of spot urine was collected from each child after examination. The urine specimens were transported in cool containers and frozen at −4°C in the local health centers. The samples were transported to Kanazawa Medical University, Japan in containers with dry ice and kept frozen at −20°C prior to analysis. The urea in the urine was decomposed by pretreating the samples with 30 units of urease  per 100 μL sample. Stable isotope-labeled compounds were used as internal standards for the quantification analysis to demonstrate the accuracy of the measurements. After the protein in each urine sample was precipitated, the residue was derivatized by adding 100 μL of N,O-bis-(trimethylsilyl) trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS). The metabolites in the derivatized samples were analyzed using GC-MS with a 7890A gas chromatograph and a DB-5MS GC column  coupled with a 5975A inert XL-mass selective detector . The conditions used for GC-MS were as previously described . The masThe SPSS  software package for Windows  was used for the statistical analyses of the data. The concentrations of dioxins in breast milk and urinary amino acids were base 10 logarithmically transformed to improve normality. A generalized linear model was used to compare the urinary amino acid levels in the four children groups at different dioxin levels after adjustment for covariates including maternal education (years). To analyze the relationships between neurodevelopmental scores and urinary amino acids, a linear regression model was performed with covariates, excluding the 4 poorly-developed children with low dioxin exposure. The results were considered to be significant if P < 0.05.To investigate the relationships between urinary amino acids and dioxin exposure, differences in five urinary amino acid concentrations and their ratios to glycine (/G) at 3 years of age were investigated among the four groups of children: 1) The high TEQ-PCDDs/Fs and high TCDD-exposed children (high TEQ-high TCDD group), 2) the high TEQ-PCDDs/Fs and low TCDD-exposed children (high TEQ-low TCDD group), 3) the low TEQ-PCDDs/Fs-exposed and poorly-developed children (low TEQ/PD group), and 4) the low TEQ-PCDDs/Fs-exposed and well-developed children (low TEQ/WD group). Because the high TEQ-PCDDs/Fs-exposed children displayed lower communication scores on the Bayley III, we attempted to compare their urinary amino acid levels not only with the well-developed children who had been exposed to lower dioxin levels but also with the poorly-developed children who had been exposed to lower levels of dioxins.Body sizes, levels of TCDD and TEQ-PCDDs/Fs, and Bayley III scores are shown in Generally, Bayley III scores were lower in the high TEQ-high TCDD, high TEQ-low TCDD, and low TEQ/PD groups. However, all of the composite scores of cognitive, language, and motor functions were lower in the low TEQ/PD group, while the motor composite scores were not significantly lower in the two high TEQ-exposed groups. The language composite score was not significantly lower in the high TEQ-high TCDD group due to the wide standard deviation .Urinary concentrations of glycine, lysine, glutamine, histidine, tryptophan, and taurine following correction by creatinine were compared among the four groups of children . The uriTo confirm the specific alteration of the urinary excretion of histidine and tryptophan, the ratios of five amino acids to glycine  were compared among the four groups of children . BecauseOnly the histidine/G ratio was significantly lower in the high TEQ-high TCDD group compared to the low TEQ/WD group. The ratios of histidine/G were also compared among the four groups after adjusting for age, gender and body weight. After adjusting for the covariates, the mean ratios of histidine/G in the high TEQ-high TCDD and high TEQ-low TCDD groups were significantly lower than those in the low TEQ/WD group . MoreoveThe multiple regression analysis (with adjustment for the covariates) between the urinary amino acids and the Bayley III scores of each function  with subscales is illustrated in The present study demonstrated that perinatal dioxin exposure to TCDD and TEQ-PCDDs/Fs, as indicated by levels in breast milk, significantly decreased urinary excretion of histidine and tryptophan in 3-year-old Vietnamese children with lower neurodevelopmental scores in dioxin contamination hot spots. Urinary histidine and tryptophan were significantly decreased in children exposed to high levels of both total dioxin and TCDD with lower Bayley III scores. Moreover, children exposed to high levels of total dioxin but not TCDD, who displayed slightly lower neurodevelopmental scores on the Bayley III, also displayed decreased levels of histidine and tryptophan, although the impact was weaker in the children in this group than in the children who had been exposed to high levels of both TEQ-PCDDs/Fs and TCDD. In contrast, decreased histidine levels were not significant in children with poor neurodevelopment and low dioxin exposure compared to well-developed children exposed to low dioxin levels, suggesting that poor development itself is not a primary factor contributing to altered histidine metabolism. These results suggest that alterations of urinary histidine and tryptophan are specific to neurodevelopmental deficits resulting from dioxin exposure before and after birth when neurogenesis, especially myelination, continues .In the present study, urinary levels of histidine were decreased in 3-year-old Vietnamese children who had been exposed to high dioxin levels. Furthermore, urinary histidine and the histidine/G ratio were associated with Bayley III scores in 3-year-old children, particularly with fine motor skill scores and not gross motor skill scores. These results suggest that low levels of urinary histidine are significantly associated with disturbances in the development of motor functions. Because histamine is synthesized from histidine, urinary depletion of histidine suggests histamine alteration in the brain. Consistent with this idea, TCDD administration induced histamine level alterations in the hypothalami of rats . HistamiIn contrast, decreased urinary histidine levels in 3-year-old Vietnamese children who had been exposed to high dioxin levels may have been caused by alterations of histamine metabolism in the whole body. Lower urinary levels of histidine were observed in untreated patients with allergies whose histamine synthesis from histidine increased even as their plasma histidine levels were maintained at constant levels . This fi1 receptor binding sites were decreased [Recently, genetic variations in the histidine decarboxylase gene (the key enzyme that is responsible for the biosynthesis of histamine) that overlapped with those observed in ASD  were repecreased . Methampecreased . These pUrinary tryptophan concentrations were also significantly decreased in the two groups of children who were exposed to high TEQ-PCDDs/Fs and were significantly associated with language and fine motor skill scores in the present study. Tryptophan is a precursor of serotonin that functions as a neurotransmitter and is related to the control of mood and emotional behavior. Several previous studies indicated that dysregulation of serotonin function may profoundly influence the severity of symptoms in certain individuals with autism , 29. CroAlthough no significant differences in urinary taurine levels or the taurine/G ratio were detected among the groups that had been exposed to different levels of dioxin, inverse associations between the taurine/G ratio and neurodevelopment scores were observed for almost all functions (with the exception of fine motor skills) in the present study. These results suggest that the urinary taurine/G ratio, rather than taurine concentration, is a good marker of poor neurodevelopment, independent of dioxin exposure. Exogenous taurine has been reported to play a more important role in the developing brains of infants and children compared to the adult brain , and aniIt has been suggested that dioxin toxicity is mediated partly through aryl hydrocarbon receptor (AhR) , which uMany studies have been performed to discover and develop biomarkers that contribute to the early and accurate diagnosis of neurodevelopmental disorders , 43–45, The validity of the results and conclusions of the present study must be interpreted within the context of the study’s strengths and limitations. The neurodevelopmental test battery that was used (Bayley III) has some limitations with respect to the present study because this test was developed and standardized for infants and children in the United States rather than for Vietnamese infants; therefore, we could not judge the developmental levels of individual children based on the scores obtained using this test. However, the test was performed by a single well-trained examiner; thus, the associations between dioxin levels and test scores within this population should be reliable. Another limitation is that the neurodevelopmental status of those children with lower Bayley III scores was not clinically diagnosed.Urinary amino acid measurements were shown to be useful in detecting metabolic changes related to neurodevelopmental alterations in 3-year-old children who were perinatally exposed to dioxins. In particular, urinary histidine was suggested to be a candidate biomarker of neurodevelopmental deficits following dioxin exposure, and histaminergic neurotransmission may be an important pathological feature of dioxin-mediated neurotoxicity.S1 File(XLSX)Click here for additional data file.


The role of microRNAs (miRNAs) as small non-coding RNAs in regulation of gene expression has been recognized. They appear to be involved in regulation of a wide range of cellular pathways that affect several biological processes such as development, the immune system, survival, metabolism and host-pathogen interactions. Arthropod-borne viruses impose great economic and health risks around the world. Recent advances in miRNA biology have shed some light on the role of these small RNAs in vector-virus interactions. In this review, I will reflect on our current knowledge on the role of miRNAs in arbovirus-vector interactions and the potential avenues for their utilization in limiting virus replication and/or transmission. Flaviviridae (+ssRNA), Togaviridae (+ssRNA), Bunyaviridae (-ssRNA), Rhabdoviridae (-ssRNA) and Reoviridae (dsRNA) . In. InAe. aed cells .Ago1 gene resulted in no change in virus replication suggesting that the miRNA pathway may not have any effect on virus replication. For example, silencing of Ago1 gene in An. gambiae mosquitoes did not change O'nyong-nyong virus replication [Ago1 in Ae. aegypti Aag2 cells had no effect on Semliki Forest virus [Ago2 or Ago3, known to be involved in the RNAi and piRNA pathways, respectively, had significant impacts on virus replication. Given that recent evidence suggests that miRNAs could also be loaded into Ago2 [Drosha or Dicer1 gene, which are not yet known to be involved in the RNAi or piRNA pathways, has any effect on alphavirus replication.In a number of alphaviruses transmitted by mosquitoes, knockdown of lication . Similarst virus  or CHIKVst virus . Howevernto Ago2 ,8,9,10, nto Ago2 . It willAedes taeniorhynchus. However, it is not known how these binding site sequences facilitate virus infection in mosquitoes. Given that miR-142-3p is not produced in insects, it is possible that a mosquito-specific miRNA may interact with the sequences resulting in strong selection for the sequences; although there could be other explanations such as the presence of RNA secondary/tertiary structures. Of relevance, a recent study that analyzed the secondary structures in the 3’UTR region of a number of flaviviruses showed the presence of duplicated RNA structures and numerous repeated sequences in the region that could impede the insect immune response by serving as molecular sponges to sequester host proteins or miRNAs [Viruses can also utilize host miRNAs for their own benefit. For example, in American eastern equine encephalitis virus transmitted by mosquitoes, binding of the mammalian host miR-142-3p to the 3’ UTR of the virus genome is important for tissue tropism and suppression of the host innate immune system . The miRr miRNAs . TherefoAedes mosquitoes when infected with DENV-2 and CHIKV. They may be part of a common response to viral infection or alternatively both manipulated by the viruses. Future research is needed to elucidate the role cellular miRNAs play in arbovirus-vector interactions.So far, we only have a very small number of examples of the impact of arbovirus infections on the miRNA of arthropod vectors and even these mainly provide preliminary information in regards to differential expression of miRNAs which await experimental validations to demonstrate their role in arbovirus-vector interactions. Furthermore, it remains to be determined if the differentially abundant miRNAs following infection are the result of host exploitation by the virus or part of the host response to infection. Another area of interest is to find out how common or different are miRNA changes in a particular vector to different viruses that it transmits and in turn whether the same virus elicits similar changes in different vectors. From the little we know, it appears that miR-2b and miR-1000 are both repressed in The skepticism in regards to the production of miRNAs by RNA viruses, as detailed above, has meant that investigations in this area have been very limited. However, since several studies have shown that the production of mature miRNAs (endogenous or exogenous) from RNA viruses is possible, interest in looking for miRNAs or miRNA-like viral small RNAs from RNA viruses has increased. Arboviruses being mainly RNA viruses fall into this group. So far, there have only been two reports demonstrating production of functional miRNA-like small RNAs from arboviruses, WNV and DENV , althougFlaviviridae family and are exclusively transmitted by mosquitoes. Their genomes consist of a single positive strand RNA of around 11 kb with one long open reading frame encoding three structural proteins involved in the formation of virions and seven non-structural (NS) proteins mainly involved in virus replication but also in virus assembly and suppression of the host antiviral response . Theiewed in , for somiewed in ,57). Froiewed in ,59. In aiewed in ; althougiewed in . FurtherBioinformatics analysis of the WNV sfRNA followed by Northern blot detection and molecular cloning led to the identification of a 21 nt miRNA, KUN-miR-1, from the terminal stem-loop structure (3’SL) in the Kunjin strain of WNV . KUN-miRAe. albopictus) cells prior to virus exposure led to significant increases in DENV-2 replication only in the case of vsRNA-5. This 23-nucleotide vsRNA mapped to the very first stem-loop in the 3’UTR. These small RNAs, including vsRNA-5, appeared in low copy numbers in deep sequencing; hence, vsRNA-5’s significance was questioned suggesting that it is an RNAi degradation product [ in vitro synthesized single stranded precursor stem-loop RNA transfected into mosquito cells, or when the precursor was overexpressed from a plasmid in the cells independent of the virus. In contrast to the effect of the vsRNA inhibitor in improving virus replication, application of the vsRNA-5 mimic led to reductions in DENV-2 replication. Using bioinformatics and deep sequencing analysis, six miRNA-like viral small RNAs (vsRNAs) were identified in DENV-2 infected mosquitoes that mapped to the stem-loops in the 5’UTR (one vsRNA) and 3’UTR (five vsRNAs) of the virus genome . Transfe product . However product . For exa product . FurtherBioinformatics analysis followed by experimental validation showed the presence of target sequences of vsRNA-5 in the non-structural protein 1 (NS1) region. Considering that one long transcript is produced during DENV replication, which consists of structural as well as non-structural proteins, targeting NS1 by vsRNA-5 basically means that the virus genome is targeted. A follow up bioinformatics analysis revealed that vsRNA-5 and its NS1 target sequences might be mainly conserved in DENV serotype 2 and not in the other serotypes .Heliothis virescens ascovirus [–H2-30 and miR-H6 from Heliothis zea nudivirus 1 [In all instances found so far in which virus genes are the targets of virus-encoded miRNAs, the miRNA-virus interaction leads to regulation of virus replication. For example, miR-BART2 from Epstein Barr virus , HvAV-miscovirus , and miRivirus 1  all targDrosophila melanogaster flies and Aag2 cells. Taking a blood meal by an arthropod vector results in massive changes in the expression of a large number of genes, including miRNAs, that are involved in reproduction and blood-meal processes  and 3-fold reduced REL1 levels (negative regulation) as compared with the control mimic-injected mosquitoes. Interestingly, DENV-2 replicated more efficiently in the presence of aae-miR-375 mimic as compared to controls, which suggested that alterations in miRNA levels following blood feeding might have an indirect impact on replication of arboviruses.As demonstrated in gulation . Overallgulation . These c aegypti . Similar aegypti . These wo midgut . The dowlication . In fact-miR-375   that contribute towards this anti-viral property; reviewed recently by Rainey et al. [Wolbachia on virus replication may vary from inhibition [Wolbachia strain, the host , and the virus. Strikingly, a recent study showed that the wAlbB Wolbachia strain enhanced WNV replication in transiently transinfected Culex tarsalis mosquitoes [Ae. aegypti Aag2 cells infected with wMelPop strain of Wolbachia, in which replication of WNV genomic RNA was enhanced as compared to non-infected cells, however, virus secretion and/or assembly in this host (cell line and whole mosquitoes) was inhibited leading to less virion secretion [Considering the lack of available vaccines and drugs against most arboviruses, management of the diseases caused by arboviruses has heavily concentrated on controlling vector populations or inhibiting virus-transmission by the vectors. Issues with chemical insecticides such as the development of resistance in vector populations, affecting non-target insects and environmental contamination have made non-chemical control alternatives more attractive. These include application of microbial control agents such as entomopathogenic bacteria and fungi, or utilization of symbionts.  species ,88. Due pulation . In two nogaster ,91. Thisexplored . Notablynhibited . Due to iewed in ). Althouy et al. . It is whibition ,91,93, nhibition  to even hibition  dependinsquitoes . This isecretion . Ae. aegypti mosquitoes with and without wMelPop Wolbachia infection revealed that about 13 miRNAs were differentially expressed in Wolbachia-infected mosquitoes. The mosquito-specific miRNA, aae-miR-2940, was highly induced and exclusively detected  in Wolbachia-infected mosquitoes. Initially, MetP was determined as one of the targets of the miRNA and was shown to be important for replication/maintenance of Wolbachia [Wolbachia density. Interestingly, in a subsequent study it was found that MetP enhances WNV replication in Aag2 cells [Wolbachia-infected mosquitoes and mosquito cell lines [Wolbachia induces MetP expression via induction of aae-miR-2940  undergo rapid, positive and diversifying selection, and that refractoriness of mosquitoes to DENV infection positively correlates with nucleotide diversity indices in the Dicer-2 gene [Based on evolutionary studies carried out in ath rate . New miRath rate . Althougr-2 gene . This maArboviruses of medical and veterinary importance continue to impose large numbers of deaths, health risks and financial losses around the world. In many instances, there are no effective drugs or vaccines available for diseases caused by arboviruses. Despite many groups around the world working on arboviruses, there are still many gaps in our knowledge in regards to the interaction of arboviruses with their hosts, in particular, the arthropod vector. Deeper understanding of molecular mechanisms regulating interplaying events such as blood-feeding, reproduction and anti-viral responses may lead to development of new approaches in arbovirus control through blocking/limiting transmission of arboviruses by arthropod vectors. MiRNAs have appeared as key molecules in the regulation of gene expression at both transcriptional and post-transcriptional levels, playing important roles in development and response of animals to environmental stimuli. Current knowledge, although limited, points to the fact that miRNAs are involved in the regulation of various aspects of arbovirus-vector interactions. However, we have only scratched the surface. Many differentially expressed miRNAs upon blood feeding, exposure to arbovirus infections, and at different developmental stages, have been identified, but their significance in those events or interactions have not been experimentally tested. Considering the short length of miRNAs and the absence of host immune responses to miRNA, they are ideal for incorporation into innovative strategies to control arboviruses; for example, generating genetically modified vectors that are refractory to virus infection by overexpressing miRNAs that boost the host anti-viral response or directly target arboviruses. Wolbachia in arthropod vectors has provided a unique opportunity to explore the endosymbiont in control of arboviruses and in fact other arthropod-borne pathogens [The discovery of the virus blocking property of athogens ,104; altathogens . Explora


Early parental separation may be a stress factor causing a long-term alteration in the hypothalamic-pituitary-adrenal-axis activity possibly impacting on the susceptibility to develop overweight and obesity in offspring. We aimed to examine the body mass index (BMI) and the risk of overweight and obesity in children whose parents lived separately before the child was born.A follow-up study was conducted using data from the Aarhus Birth Cohort in Denmark and included 2876 children with measurements of height and weight at 9-11-years-of-age, and self-reported information on parental cohabitation status at child birth and at 9-11-years-of-age. Quantile regression was used to estimate the difference in median BMI between children whose parents lived separately (n = 124) or together (n = 2752) before the birth. We used multiple logistic regression to calculate odds ratio (OR) for overweight and obesity, adjusted for gender, parity, breast feeding status, and maternal pre-pregnancy BMI, weight gain during pregnancy, age and educational level at child birth; with and without possible intermediate factors birth weight and maternal smoking during pregnancy. Due to a limited number of obese children, OR for obesity was adjusted for the a priori confounder maternal pre-pregnancy BMI only.2 : 0.10; 0.98) between children whose parents lived separately before birth and children whose parents lived together. The risk of overweight and obesity was statistically significantly increased in children whose parents lived separately before the birth of the child; OR 2.29  and OR 2.81 , respectively. Additional, adjustment for possible intermediate factors did not substantially change the estimates.The difference in median BMI was 0.54 kg/mParental separation before child birth was associated with higher BMI, and increased risk of overweight and obesity in 9-11-year-old children; this may suggest a fetal programming effect or unmeasured difference in psychosocial factors between separated and non-separated parents. Exposure to stress during fetal life presumably impacts on the susceptibility to develop overweight and obesity ,2. The sWe have earlier found that schoolchildren born to mothers who experienced loss of a close relative during the prenatal time period had an increased risk of overweight . BereaveThe effect of early parental separation may lead to detectable, though mild, long-term alterations in the hypothalamic-pituitary-adrenal axis activity . We hypoWe conducted a follow-up study using data from the Aarhus Birth Cohort (ABC). A total of 8719 Danish-speaking pregnant women were included in the ABC at their first antenatal visit between 1 August 1989 and 30 September 1991 at the Department of Obstetrics and Gynaecology, Aarhus University Hospital in Denmark . Around In 2001 when the children were 9 to11years old, invitations were sent to participate in a follow up study. A total of 7953 mother-child pairs received a child health questionnaire including a question on whether the biological parents of the child lived together. The parents were also asked to give written consent to collect historical data on the child; permission was granted by 6504 mother-child pairs. In the period from 2010 to March 2012 information on height and weight of 5421 children was collected by general practitioners and health visitors. The number of measurements varied but all children had a least one measurement and two children had 28 measurements collected from birth to the age of 22 years. Finally, we had 2876 singletons for analysis with one measurement under standardized conditions at 9 to 11 years-of-age, and with complete self-reported information on parental cohabitation at the time of birth and at the age of 9 to11 years. A flow chart of selection of the study population is shown in parental cohabitation was dichotomized into children whose biological parents lived together before birth  and children whose biological parents lived separately before birth  with Cohabiting as the reference group.The exposure variable The outcome variable was defined by BMI. Further, BMI was categorized as normal weight, overweight including obesity, and obesity, according to the standardized age and gender-specific cut-off points defined by the International Obesity Task Force . BMI z-s2), maternal weight gain during pregnancy , self-reported maternal educational level , maternal age at birth, parity , and breastfeeding status (mean months). Information of the possible intermediate factors, birth weight  and self-reported maternal smoking during pregnancy , was also collected.Information on potential confounders and covariates was collected from medical records or questionnaire: gender (boy/girl), self-reported maternal pre-pregnancy BMI .Separate and Cohabiting were tested using chi-square test. The distribution of the BMI data is positively skewed and therefore quantile regression analysis was used to estimate the median BMI with corresponding 95% confidence intervals (CI) in 9 to11 years old children whose parents lived together or separately before the time of birth. The difference in median BMI between Separate and Cohabiting was tested by Wilcoxon rank-sum test. As BMI z-scores often are used in pediatric studies, we also estimated the mean BMI z-scores and the difference was tested by two-sample t-test with unequal variance in Separate and Cohabiting. We found no indication of effect modification by gender, and the two genders were thus analyzed together. Further, we found no statistical interaction between parental separation and maternal educational level or smoking during pregnancy; thus, no stratified analyses were made. Significance of interaction was tested using the likelihood-ratio test.Descriptive characteristics between Multiple logistic regression analysis was performed estimating the odds ratio (OR) with corresponding 95% CI for overweight and obesity in children whose parents lived separately before birth. As birth weight and smoking were considered possible intermediate factors rather than confounders, we performed adjusted analyses with and without birth weight and maternal smoking during pregnancy. The OR for overweight was adjusted for gender, maternal pre-pregnancy BMI, maternal weight gain during pregnancy, maternal age and educational level at birth, parity, and breastfeeding status. The OR for obesity was adjusted for the a priori selected confounder maternal BMI only due to a limited number of obese children. A p value of < 0.05 was considered statistically significant.The follow-up study in 2001 was approved by the Danish Central Ethical Committee .Separate group was characterized by younger maternal age and a higher pre-pregnancy BMI, more smokers, lower maternal educational level, and more first-time mothers than the Cohabiting group.The descriptive characteristics of the study population are presented in Cohabiting and Separate groups, respectively are shown in 2, 95% CI: 17.35; 18.43) than in children whose parents lived together  before birth of the child, p = 0.02. The mean BMI z-scores were insignificantly higher in the Separate group  than the Cohabiting group , p = 0.18.The median and mean BMI z-scores in the We found significantly higher median BMI in 9 to11years old children whose parents lived separately before the time of birth compared with children whose parents lived together before time of birth. We found that parental separation before birth was significantly associated with increased risk of overweight and obesity in the offspring.Our finding of a higher median BMI in children whose parents lived separately before childbirth could reflect a change in the right tail of the BMI distribution. Therefore, we also focused on the risk of overweight and obesity where our findings are in line with other studies ,13. Two We have previously found that schoolchildren born to mothers who experienced bereavement during the prenatal time period had an increased risk of childhood overweight at the age of 12 years  [Two previous Danish studies have examined self-reported maternal distress  either during pregnancy or postpartum and risk of overweight in 7 years old children ,11. One Separate group begin puberty growth spurt earlier, we may have overestimated the association. Moreover, maternal smoking during pregnancy may be considered a confounder rather than an intermediate factor. However, the association between parental separation before birth and the risk of overweight and obesity slightly changed, and it did not alter the conclusion. Finally, biological parents to an unborn child may have chosen to live separately for practical reason. This could be the case in situations where one of the parents has a long-distance job that geographically requires them to live apart or a single mother having a child with an anonymous donor. We had no available data on such cases, but if they caused less stress we may have underestimated the association between prenatal stress exposure due to parental separation and risk of overweight and obesity in offspring.We believe the use of height and weight measured under standardized conditions, and the rating of parental separation as a moderately stressful life event , are strExamining the exposure during pregnancy may predominantly be exploring a potential programming effect rather than interfering in children’s active coping skills that may change through childhood. A biological mechanism prenatally may be that the placenta enzyme 11β-hydroxysteroid dehydrogenase type 2 during normal conditions acts as a fetal-placental barrier preventing the majority (80–90%) of maternal glucocorticoid cortisol from crossing the placenta in an active form ,23. As tSocietal mechanism may be an alternative explanation. The impact of separation is potentially complicated by a multitude of potential environmental factors during the process . In adulIn conclusion, parental separation before the birth of the child was associated with higher BMI, as well as increased risk of overweight and obesity in 9 to11 years old children. These findings may indicate a fetal programming effect due to prenatal moderate stress exposure. However, future studies are to estimate whether these programming alterations will persist into adulthood. Alternative, the association may reflect the difference in psychosocial characteristics of separated parents.


WNT signaling is a complex process comprising multiple pathways: the canonical β-catenin-dependent pathway and several alternative non-canonical pathways that act in a β-catenin-independent manner. Representing these intricate signaling mechanisms through bioinformatic approaches is challenging. Nevertheless, a simplified but reliable bioinformatic WNT pathway model is needed, which can be further utilized to decipher specific WNT activation states within e.g. high-throughput data.In order to build such a model, we collected, parsed, and curated available WNT signaling knowledge from different pathway databases. The data were assembled to construct computationally suitable models of different WNT signaling cascades in the form of directed signaling graphs. This resulted in four networks representing canonical WNT signaling, non-canonical WNT signaling, the inhibition of canonical WNT signaling and the regulation of WNT signaling pathways, respectively. Furthermore, these networks were integrated with microarray and RNA sequencing data to gain deeper insight into the underlying biology of gene expression differences between MCF-7 and MDA-MB-231 breast cancer cell lines, representing weakly and highly invasive breast carcinomas, respectively. Differential genes up-regulated in the MDA-MB-231 compared to the MCF-7 cell line were found to display enrichment in the gene set originating from the non-canonical network. Moreover, we identified and validated differentially regulated modules representing canonical and non-canonical WNT pathway components specific for the aggressive basal-like breast cancer subtype.In conclusion, we demonstrated that these newly constructed WNT networks reliably reflect distinct WNT signaling processes. Using transcriptomic data, we shaped these networks into comprehensive modules of the genes implicated in the aggressive basal-like breast cancer subtype and demonstrated that non-canonical WNT signaling is important in this context. The topology of these networks can be further refined in the future by integration with complementary data such as protein-protein interactions, in order to gain greater insight into signaling processes. The WNT signaling pathway plays an important role not only in embryonic development processes but also in adult tissue homeostasis and repair after injury. Furthermore, its aberrant regulation is involved in a range of human pathologies such as cancer and degenerative diseases . The widCurrently, there are a number of WNT signaling pathways in public databases, such as BioCarta , ReactomIn brief, the canonical WNT pathway is defined by the nuclear translocation of β-catenin, in which it functions as a co-activator of transcription. In the absence of WNT signaling, β-catenin is targeted by a destruction complex composed of AXIN, APC, and GSK3β leading to its degradation by the proteasome. Binding of a WNT ligand to a frizzled (Fz) receptor and its co-receptors LRP5/6 activates intracellular dishevelled (DVL), leading to inactivation of the β-catenin destruction complex. This in turn leads to the stabilization of β-catenin, its relocation into the nucleus, and triggers the expression of target genes facilitated by TCF/LEF1 transcription factors ,11. This2+ signaling, representing the non-canonical WNT pathway. On the one hand this pathway can result in the activation of small GTPases such as Rho, Rac, and Cdc42, which regulate cytoskeleton rearrangement and PCP. On the other hand, it can also regulate transcription by activating the transcription factors NFAT or AP-1. Generally speaking, the non-canonical WNT pathway is linked to changes in cell motility, cytoskeletal reorganization processes, and invasiveness [However, within the WNT signaling pathway, several other β-catenin-independent mechanisms exist that are generally defined by a lack of β-catenin-mediated transcription. Broadly, these cascades can be grouped into planar cell polarity (PCP) and WNT/Casiveness .While the separation between these β-catenin-dependent and -independent pathways has proven valuable in understanding the signaling output, there is a high degree of interaction between these different signaling components on several levels of the cascades: The WNT protein family in humans comprises 19 members, all of which appear to be neither intrinsically canonical nor non-canonical. It is rather the multitude of possible combinations between WNT ligands and the respective Fz receptors and non-Fz (co)-receptors such as ROR1, ROR2, and RYK that channel WNT signals into one of the WNT sub-pathways . ApproprThis unclear distinction of WNT signals and their consequences are illustrated by the example of breast cancer. The inappropriate activity of both canonical and non-canonical WNT pathways has been implicated in breast cancer initiation as well as progression . Breast The particular WNT pathway involved in this enrichment has so far eluded detection. On the one hand, there is evidence pointing to the activation of β-catenin-dependent WNT signaling in basal-like breast cancer primaries ,20. On tPhenotypic differences such as invasiveness are often attributed to differences in the transcriptome between the investigated populations. By comparing their expression profiles differentially expressed genes (DEGs) are identified, which can result in fairly long lists. To reduce the complexity of these lists of DEGs and unravel the underlying biological processes, gene-set enrichment or overrepresentation analysis is a widely utilized bioinformatic approach, which allows the identification of the most affected pathways ,25. AnotPrior knowledge representing the pathway is required in order to test for enrichment of differential genes in a certain pathway or to integrate expression data into a pathway network . SeveralThus the aims of this study can be considered as four-fold: 1) We seek to collect available WNT signaling data across different pathway databases in the computer-readable BioPAX format to allow semi-automatized processing. 2) We aim to assemble this knowledge into directed graphs representing signaling networks of distinct WNT sub-pathways. 3) To demonstrate that these newly constructed WNT networks reliably reflect distinct WNT signaling processes, we evaluate them in an application example in the context of WNT regulation in breast cancer. Based on our previous work  we generIn order to assemble public pathway knowledge into network models of WNT signaling pathways three steps were taken: 1) Pathway data were gathered across several databases and parsed. 2) Gene IDs were mapped on pathway graphs. 3) The graphs were sorted and merged into four networks.rBiopaxParser R-package [Five pathway databases were used to collect WNT signaling data: BioCarta , Reactom-package . Pathway-package  Fig 1A)rBiopaxPaThe pathway databases use different identifications to annotate pathway molecules. In most cases Entrez Gene or UniProt IDs were available as reference annotations for a node and these were converted into HUGO Gene Nomenclature Committee (HGNC) gene symbols. However, whenever no official ID was available, we chose the instance name property defined within the BioPAX model to represent a node label. Pathway entities that could not be mapped into gene expression data, such as small molecules, DNA, RNA, or nested pathways, were filtered out, while connectivity of the graphs was preserved by introducing new edges connecting the genes that were thus indirectly connected. Further, if multiple gene symbols were mapped onto a single node, such a node was split into multiple nodes and each one was assigned with a single gene symbol. Vice versa, if several nodes were annotated with the same gene symbol, these nodes were merged into one.Wnt signaling pathway we exported from the KEGG database contained canonical as well as non-canonical sub-pathways and therefore the corresponding connected components of the pathway graph were sorted into canonical WNT signaling and non-canonical WNT signaling groups, respectively  data repository . Three MThe human breast cancer cell line MCF-7 was purchased from the German Collection of Microorganisms and Cell Cultures  and the MDA-MB-231 breast cancer cell war purchased from the American Type Culture Collection . Both cell lines were maintained in RPMI-1640 medium  supplemented with 10% heat-inactivated fetal calf serum . For gene expression studies, cells were seeded on extracellular matrix -coated tissue culture wells. Total RNA was isolated using TRIZOL reagent according to the manufacturer’s instructions . RNA integrity for each sample was confirmed with the Agilent 2100 Bioanalyzer .Library preparation for RNA-Seq was performed using the TruSeq RNA Sample Preparation Kit  starting from 1000 ng of total RNA according to Illumina's instructions. Accurate quantitation of cDNA libraries was performed by using the QuantiFluor™ dsDNA System (Promega). The size range of final cDNA libraries was determined applying the DNA 1000 chip on the Bioanalyzer 2100 from Agilent (280 bp). The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the HiSeq2000 following the manufacturer's protocols. Illumina Casava 1.8.2 software used for base-calling.edgeR R-package [edgeR. Multiple testing correction was done using the Bonferroni method for the family wise error rate (FWER) [Quality checking of RNA-Seq data was done via FastQC . Alignment to the human transcriptome based on the ENSEMBL genome (version GRCh38 release 78) was performed using the STAR tool version 2.4.0 . Lowly e-package . Differee (FWER) . RNA-SeqGene sets corresponding to the four WNT networks were created as lists of node labels represented by HUGO gene symbols. Furthermore, additional gene subsets were produced as: 1) the difference of canonical and non-canonical sets, containing only unique canonical genes (can. \ non-can.), 2) the difference of non-canonical and canonical sets, containing genes belonging exclusively to the non-canonical gene set (non-can. \ can.), and 3) the intersection of these two sets (can. ∩non-can.). 4) The last gene set was created as a union of canonical and non-canonical sets (can. ∪non-can.).Fisher's exact test was performed in order to test for over-representation. When testing for enrichment of a gene set in a list of differential genes a ranking based analysis was performed using Wilcoxon rank-sum test .tkplot R-function from the igraph R-package. For visualization purposes the range for color coding of fold-changes was limited to ±3 and ±4 for the modules based on microarray expression data and on RNA-Seq expression data, respectively.In order to identify differentially regulated WNT modules the up- and down-regulated DEGs were mapped onto the WNT network nodes. In the next step, nodes induced by DEGs were used as terminal nodes for the Steiner tree algorithm as implemented in the SteinerNet R-package ,42. In tcalcGraphOverlap R-function from the rBiopaxParser R-package was utilized and the modules based on microarray data were used as reference graphs.The differentially regulated modules of WNT networks originating from the public microarray expression data and the newly generated RNA-Seq data were investigated for graph overlaps. Therefore, the Several publicly accessible databases provide WNT pathways to differing extents. We collected 26 pathways linked to WNT signaling and sorted them into four conceptual groups . We defiWithin each group, the pathway graphs were merged into a singular signaling network resulting in four distinct networks  Figs: cabeta-catenin independent WNT signaling pathway graph.The size of the non-canonical network  exceeded that of the canonical network , even though the number of scientific publications seems to be imbalanced in favor of canonical WNT signaling knowledge . The greIn order to create a computationally suitable signaling network model in an automatized and reproducible way, the used pathways were limited to the databases using the BioPAX model. We focus on defining a graphical model of the WNT signaling pathways that can be used in network-integration analysis of static expression data. Our model does not contain any kinetic or mechanistic information. Alternative modeling approaches such as kinetic WNT models ,46 basedOwing to their considerable size, systematic manual inspection of the networks was challenging. However, before we began integrating the new networks with any experimental data we wanted to assess the quality of these networks. To evaluate their biological soundness, we took two approaches: First, we examined the interconnections of well-established WNT pathway members within our networks. Second, we identified key nodes in the networks which represent central signaling genes.In order to ascertain the biological relevance of the newly constructed networks, we were interested in surveying how well-known WNT pathway genes are represented therein. We collected an unbiased list of 121 genes of different WNT sub-pathways reviewed in several publications ,15,47,482+ pathway , are not included. Therefore, we do not expect the networks to be the complete representation of cell signaling processes. For a more complex view on the signaling pathways these networks could be further combined with complementary information, such as PPI data.In conclusion, this subnetwork revealed the relevant topology of the core members of WNT signaling. Moreover, its densely clustered communities and their interconnections reflect established biological models of WNT signaling. Although we reused knowledge from the literature within the subnetwork construction, our approach allowed us to acquire relevant insight into the WNT networks in general.To further examine the features of the four newly constructed WNT networks we identified their key nodes  between the cell lines, from which 1037 genes were up-regulated and 732 genes were down-regulated  in MDA-MOur next step was to integrate the differential genes between the MDA-MB-231 and MCF-7 cell lines with the graph representations of canonical and non-canonical signaling pathways.Differentially regulated modules, containing nodes of DEGs and Steiner nodes for graph connectivity, were extracted from canonical and non-canonical networks. In these modules, the DEGs are placed into a context of their signaling interaction partners representing aggressive breast-cancer-specific WNT pathway networks.As expected from the sizes of the original WNT networks and the results of the enrichment analysis, the non-canonical module was greater in size and more richly interconnected than that of the canonical module. The non-canonical module comprised 267 edges and 82 nodes, 68 of which were induced by DEGs and 14 were connecting Steiner nodes . The can2+ signaling [Both the canonical and non-canonical WNT modules demonstrate the importance of Steiner nodes to maintain connectivity of the network. Either these genes are not differentially expressed under the different conditions or are not measured within the microarray experiment at all. Key cancer drivers are often not detected through the analysis of differential expression, but they play central role in networks by interconnecting many genes, which are expression-responsive . In the ignaling  towards ignaling . While tThus, the identified WNT modules highlight intriguing events as well as several genes, for which it might prove interesting to test for their biological relevance within the aggressive basal-like breast cancer subtype.Although we are aware of the absence of some connections in the WNT networks, it is not possible to infer any new edges when adopting this integration approach. However, there are other bioinformatic methods, e.g. nested effect models  or BooleIn order to confirm the findings of our integrative analysis we generated an independent dataset by performing transcriptome sequencing of MCF-7 and MDA-MB-231 breast cancer cell lines. Differential analysis of the RNA-Seq data yielded 6638 DEGs between the cell lines , from whWe further performed network integration using the RNA-Seq data in the same manner as previously in the microarray application–including enrichment analysis and the identification of differentially regulated modules.For the purpose of enrichment analysis the Fisher's exact test was not the most suitable in a setting with such a high number of differential genes. Therefore, we preferred the ranking-based enrichment approach utilizing the Wilcoxon rank-sum test. The non-canonical WNT gene set was significantly enriched (p = 0.0198) while the canonical WNT set did not show enrichment (p = 0.3302) in MDA-MB-231 cells. This falls in line with our formulated hypothesis about non-canonical WNT signaling having a predominant role over the canonical WNT pathway in the more aggressive basal-like breast cancer cells In contrast to the microarray expression data, no split of the gene sets into unique subsets was required to recognize this distinction.validation modules, while their counterparts–non-canonical and canonical WNT modules identified based on microarray data–are referred to as original modules.Next, the differentially regulated modules of non-canonical and canonical networks were revealed by integration of the WNT networks with the RNA-Seq expression data . We refeThe non-canonical validation module  by evaluating the graph overlaps on node and edge levels while using the original modules as reference, 2) by assessing the correlation of folds-changes between the overlap genes of the corresponding modules and 3) by inspecting the consistency of Steiner node statuses.Non-canonical modules showed a node overlap of 75.6% and an edge overlap of 59.5% and a correlation of 0.81  for the non-canonical gene fold-changes. Canonical modules exhibited overlaps of 78.4% at the node-level and 70.8% at the edge-level. Their correlation was 0.81 .Prominent agreement was further found when focusing on the connecting Steiner nodes of the original modules. In case of the 14 non-canonical Steiner nodes, 10 of them were found in the corresponding validation module: 8 as Steiner nodes  and 2 were detected as significantly differentially expressed genes (FURIN and PPARGC1A). From the 10 Steiner nodes of the original canonical module, 5 were confirmed as Steiner nodes in the validation module  and 4 were identified as DEGs .Within the validated Steiner node genes, several have already been investigated in the context of aggressive breast cancer cells or tumors. For instance, NFATC2 was reported to promote breast cancer cell migration and to induce invasion . SimilarIn summary, the validation modules showed high level of topological as well as transcription concordance with the original differentially regulated WNT modules from the microarray analysis. Moreover, the validated nodes underline the importance of the Steiner nodes within the modules' topology and point to genes with a substantial role in invasiveness of breast cancer cells. The relevance of these nodes would have been neglected by a standard differential analysis without using the signaling network information.We constructed computationally suitable network models of WNT signaling cascades in a form of directed signaling graphs and demonstrated in the application example that these WNT networks reasonably reflect distinct WNT sub-pathways. We subsequently integrated the networks with microarray data to better understand the underlying biology of gene expression differences between different breast cancer cell lines. We identified differentially regulated modules representing canonical and non-canonical breast-cancer-specific WNT pathway components, validated these modules in an independent transcriptomic dataset, and highlighted relevant function of topologically-essential elements of the modules.Following our approach, the intricate WNT networks were shaped into a comprehensive graphical overview of the genes implicated in the aggressive basal-like breast cancer subtype. Utilizing these models as gene sets, we revealed that non-canonical WNT signaling is of importance in this system. Further enhancement of the network topology may be achieved by integration with complementary data types, such as PPIs, to gain even greater insight into pathway signaling processes.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S1 File(EPS)Click here for additional data file.S1 TableThe table contains pathway names, source databases and assigned groups for the network construction.(XLS)Click here for additional data file.S2 TableThe directed edges are weighted as 1 for activation and as -1 for inhibition interaction.(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 TableUp- and down- regulated significantly differentially expressed genes in the MDA-MB-231 cell line compared to the MCF-7 cell line.(XLS)Click here for additional data file.S5 TableSignificantly differentially expressed genes in the MDA-MB-231 cell line compared to the MCF-7 cell line.(XLS)Click here for additional data file.S6 Table(XLS)Click here for additional data file.


The anxiety-reducing effect of long-term administration of serotonin reuptake inhibitors is usually seen only in subjects with anxiety disorders, and such patients are also abnormally inclined to experience a paradoxical anxiety-enhancing effect of acute serotonin reuptake inhibition. These unique responses to serotonin reuptake inhibitors in anxiety-prone subjects suggest, as do genetic association studies, that inter-individual differences in anxiety may be associated with differences in serotonergic transmission.The one-third of the animals within a batch of Wistar rats most inclined to spend time on open arms in the elevated plus maze were compared with the one-third most inclined to avoid them with respect to indices of brain serotonergic transmission and how their behavior was influenced by serotonin-modulating drugs.p-chlorophenylalanine eliminated them by reducing anxiety in “anxious” but not “non-anxious” rats. Acute administration of a serotonin reuptake inhibitor, paroxetine, exerted an anxiety-enhancing effect in “anxious” but not “non-anxious” rats, which was eliminated by long-term pretreatment with another serotonin reuptake inhibitor, escitalopram.“Anxious” rats displayed higher expression of the tryptophan hydroxylase-2 gene and higher levels of the tryptophan hydroxylase-2 protein in raphe and also higher levels of serotonin in amygdala. Supporting these differences to be important for the behavioral differences, serotonin depletion obtained by the tryptophan hydroxylase-2 inhibitor Differences in an anxiogenic impact of serotonin, which is enhanced by acute serotonin reuptake inhibitor administration, may contribute to differences in anxiety-like behavior amongst Wistar rats. Many authors have suggested inter-individual differences in serotonergic transmission to be the cause of inter-individual differences in proneness for anxiety . The speIn the same vein, genetic studies have revealed serotonin-related genes to be associated with anxiety-related personality traits  and anxiNumerous animal studies suggest that manipulation of brain serotonergic neurotransmission, obtained by drugs  or genetpromoting or anxiety reducing, and 3) is the influence of SRIs, administered acutely or on a continuous basis, respectively, best described as a facilitation or a dampening of a serotonergic influence on anxiety-generating circuits.Previous studies of EPM performance in outbred Wistar rats support the existence of relatively stable (test vs retest) inter-individual variations that are sufficiently robust to justify the characterization of the rats as more or less “anxious” . These dam) and with standard chow and water available ad libitum. The animals were allowed 1 week of acclimatization after arrival before being subjected to any behavioral tests. In all experiments, animals were subdivided on the basis of how they performed in the EPM, the one-third most prone to spend time on the open arm constituting one group, presumably characterized by low anxiety  and the one-third most prone to avoid it constituting the other, tentatively more anxious group . The middle group was thus excluded from all further analyses in order to avoid misclassification of animals belonging to this group but being close to one of the extreme groups.Male Wistar rats , aged 10 to 11 weeks at arrival, were housed with a 12-h-light/-dark cycle  and LO (one-third) rats 2 weeks before being subjected to a test of unconditioned noise burst-elicited startle  followed 1 week later by a forced-swim test (comprising two 10-minute sessions separated by 24 hours). One week after the forced-swim test, the animals were killed and their brains extracted for assessment of gene expression using real-time PCR (TaqMan), tryptophan hydroxylase 2 (TPH2) levels using Western blot, and serotonin and 5-hydroxyindoeacetic acid (5-HIAA) levels using high-pressure liquid chromatography (HPLC) .Sixty animals were pretested in the EPM for categorization into HO (one-third) and LO (one-third) rats. After 3 weeks, treatment with the tryptophan hydroxylase inhibitor para-chlorophenylalanine (p-CPA) was initiated. Animals were again tested in the EPM 24 hours after the last injection. The following day, the animals were sacrificed; the brains from one-third of them were extracted for validation of serotonin depletion.One hundred twenty animals were pretested in the EPM for categorization into HO (one-third) and LO (one-third), whereupon one-half of the animals in each group received escitalopram p.o. in food pellets and the other one-half control pellets. After 5 weeks of treatment, the animals received one injection of paroxetine or vehicle 1 hour before a second EPM session was undertaken.p-CPA  was dissolved in 0.9% saline and administered i.p. as one injection of 300mg/kg per day for 3 days with the last injection being given 24 hours before the EPM test. Escitalopram oxalate  was admixed into food pellets  at a concentration (0.65g/kg) aimed at providing a daily dose of 25 to 30mg/kg . ParoxetA standard black acrylic plastic rat EPM (Med Associates) was placed in a quiet room with a light level in the center of the maze of 35 lx. All tests lasted for 5 minutes. In the experiments comprising 2 tests, the EPM apparatus was placed in a room new to the animals at session 2 in order to minimize habituation effects .See supplementary Materials and Methods.t test was used to compare groups with respect to biochemical data. Due to non-normal distribution of the relevant behavioral parameters in drug-treated animals, EPM data were log-transformed according to the formula log10(x+1), with x being the behavioral parameter in question, before being analyzed using ANOVA followed by LSD posthoc test. Interactions were tested using 2-way ANOVA when appropriate. Pearson correlation was used to calculate correlations.Student’s P=.003; experiment III, n=20, r=0.68, P=.001). In line with this, 18 of 20 (experiment II) and 16 of 20 (experiment III) animals in the nontreated groups were found to be in the same HO/LO group in the second EPM session as in the first.Justifying the categorization of rats into more or less anxious, we could, by examining the test-retest stability with respect to EPM performance in animals that had obtained no active pharmacological treatment between the tests, confirm the previous observation  of relatP=.9) or with immobility in the forced-swim test ; in the same vein, no differences between HO and LO animals with respect to startle response  or FST performance  were found.Experiment I revealed that EPM behavior (time spent on open arms) does not correlate with average startle amplitude  and 5-HIAA  levels (n=9–10/group) in p-CPA–treated animals (n=9–10/group) with no tendencies for any differences between LO and HO animals in this regard.Administration of the tryptophan hydroxylase inhibitor (p-CPA) led to an increase in time spent on open arms  and entrAcute administration of paroxetine exerted an anxiogenic effect, that is, decreased the time spent on, as well as entries onto, open arms in otherwise untreated LO animals. No such effect was seen in the corresponding HO group, albeit a trend towards decreased entries to open arms was observed . HO rats given subchronic escitalopram displayed reduced time on open arms; like otherwise untreated HO rats and escitalopram-treated LO rats, they did not, however, display an anxiogenic-like response to acute administration of paroxetine. Subchronic escitalopram administration hence eliminated all differences with respect to open arm duration and entries between LO and HO animals  for 5 weeks caused a modest and nonsignificant reduction in time spent on open arms in otherwise untreated LO rats but totally prevented the anxiogenic-like effect of acute administration of another SRI, paroxetine, in these animals, the interaction between escitalopram and paroxetine being significant (P<.01). Administration of paroxetine to escitalopram-treated LO rats, however, counteracted this effect of escitalopram (4.1±0.67); this group hence differed neither from HO rats given escitalopram plus NaCl nor from HO rats given escitalopram plus paroxetine, but did differ from LO rats given escitalopram plus NaCl (P<.05).To evaluate if the effect of acute SRI administration on anxiety as assessed using EPM could be secondary to an effect on locomotion in general, we also measured entries into closed arms. This parameter was not different in untreated HO rats (3.9±0.7) compared with untreated LO animals . Administration of paroxetine did not cause a significant reduction in the number of entries into closed arms in either HO (3.4±0.4: n.s.) or LO rats ; hence, there were no significant differences between any of the groups displayed in This study suggests that inter-individual differences in anxiety in a batch of male Wistar rats may be partly explained by differences in an anxiogenic influence of serotonin. While animals prone to anxiety-related behavior (LO) hence displayed indices of enhanced serotonergic transmission, including enhanced TPH2 expression, arresting serotonergic transmission by inhibiting this enzyme reduced anxiety-like behavior selectively in these rats, thereby making the differences in behavior between LO and HO animals disappear. Further, similar to the human situation and in line with the assumption that nonanxious and anxious animals differ with respect to the magnitude of an anxiogenic-like serotonergic influence, we found animals with high baseline anxiety-like behavior to display a greater anxiogenic-like response to acute administration of an indirect serotonin agonist, the SRI paroxetine. Finally, long-term administration of an SRI, which has previously been suggested to cause an adaptive downregulation of the anxiogenic influence of serotonin , was fouOur claim that LO animals are characterized by enhanced serotonergic activity is based mainly on the observation that they display enhanced expression of the gene encoding the rate-limiting enzyme for the synthesis of serotonin, TPH2, in the region where serotonergic cell bodies are situated, the raphe nuclei, and that this increase in TPH2 expression was confirmed by means of Western-blot assessment of the TPH2 protein. Our findings are in line with previous studies showing that raphe TPH2 expression is enhanced in rodents rendered anxious by genetic modulation of nonserotonergic genes  or by suThe apparent enhancement of serotonergic transmission in animals displaying enhanced anxiety-like behavior could be regarded either as support for an anxiety-promoting role of serotonin or explained in terms of compensatory mechanisms aiming to dampen the influence of various anxiety-promoting circuits. In the present study, we show a TPH2 inhibitor, p-CPA, to reduce anxiety-like behavior in anxious LO rats but not in nonanxious HO animals, hence eliminating the behavioral difference between the 2 groups. While the anxiolytic-like effect of p-CPA in this paradigm, which supports an anxiogenic-like influence of this transmitter, is well known , this isIt should also be underlined that the observed effects of p-CPA in LO and HO rats, respectively, refute the possibility that the apparent increase in serotonergic transmission in LO rats should be regarded as a compensatory mechanism aiming to dampen anxiety enhanced by other mechanisms; thus, had this been the case, p-CPA should enhance anxiety-like behavior in LO animals and amplify rather than counteract the difference between LO and HO animals. Likewise, had serotonin exerted an anxiety-dampening influence, administration of paroxetine should have enhanced rather than further reduced open arm activity in LO rats.When interpreting the TPH2 elevation observed in the present study, it should be noted that LO rats displayed enhanced expression not only of TPH2 but also of other genes encoding proteins expressed by serotonergic neurons, such as the monoamine oxidase subtype expressed by serotonergic neurons (MAO-B), the serotonin transporter, aromatic L-amino acid decarboxylase, and a transcription factor of importance for the development of these neurons, fev. LO rats hence seem characterized either by a more developed serotonergic network or by serotonergic neurons displaying a more active transcription machinery compared with less anxious animals. The notion that LO rats are characterized by a stronger serotonergic innervation gains indirect support from a report by Acute administration of an SRI to rodents causes elevated extracellular levels of serotonin in many  of the brain regions innervated by serotonergic nerve terminals , which ienhanced expression of the serotonin transporter, given that anxiety-related traits in humans have been associated with the s allele of a polymorphism in the promoter of the serotonin transporter gene causing reduced expression of this protein  have failed to reveal consistent results, some but not all showing a modest anxiogenic-like effect . In the It hence seems as if subchronic administration of an SRI in animals displaying enhanced baseline anxiety-like behavior, as well as indices of enhanced serotonergic neurotransmission, does lead to a downregulation of the anxiogenic-like influence of serotonin as reflected by the enhanced anxiety-like behavior elicited by acute SRI administration. In line with this, When evaluating the inability of subchronic escitalopram to reduce baseline anxiety in LO animals, it should be considered that all animals, when retested under the influence of escitalopram, had been exposed to the EPM at an earlier occasion . Previous studies thus suggest that prior experience of the test may abolish the anxiolytic effect of drugs such as benzodizapines . AlthougWhile long-term escitalopram treatment thus reduced the anxiety elicited by acute paroxetine in LO rats but failed to alter baseline anxiety-like behavior in these animals, HO animals exposed to the same treatment displayed enhanced baseline anxiety-like behavior, hence eliminating also this difference between HO and LO rats. Tentatively, the dominating net effect of long-term administration of SRIs on the serotonergic synapses regulating the studied behavior in these animals is to exert a modest enhancement from a low starting point rather than to downregulate an excessive influence.The aim of experiment I was not only to assess the possible association between EPM performance and brain serotonergic activity but also to explore to what extent HO and LO rats differ with respect to another behavior tentatively reflecting human anxiety, unconditioned acoustic startle. While a study on the above-mentioned HAB and LAB rats, bred on the basis of EPM performance, suggests rats with high anxiety, as assessed using EPM, to display lower unconditioned (as well as fear-sensitized) startle than LAB animals , we obseThis study has certain limitations. First, while the EPM paradigm having bearing on human anxiety gains support from the fact that GABA A-receptor-activating anxiolytics reliably reduce anxiety-like behavior in this model  while drassociated with enhanced anxiety-like behavior but also seems to be an important causal factor underlying inter-individual differences in this regard, 3) the anxiogenic effect of acute administration of an SRI in Wistar rats is more pronounced in animals with enhanced baseline anxiety-like behavior than in those with low anxiety (hence corresponding to the human situation), and 4) subchronic administration of an SRI blunts the anxiogenic effect of the acute administration of another SRI in animals with high anxiety-like behaviour at baseline. Our data support the view that proneness for anxiety in humans may also be partly caused by an enhanced anxiogenic influence of serotonin and that this is the reason why acute administration of an SRI may elicit anxiety in susceptible individuals while subchronic administration of the same agents often have the opposite effect.The major novel conclusions of the present data are that: 1) inter-individual differences within a batch of Wistar rats can be utilized to study the influence of serotonin on anxiety-like behavior, 2) enhanced serotonergic activity is not only None.


In this study, the spin coating of template-assisted method is used to synthesize poly (PFO-DBT) nanorod bundles. The morphological, structural, and optical properties of PFO-DBT nanorod bundles are enhanced by varying the spin coating rate  of the common spin coater. The denser morphological distributions of PFO-DBT nanorod bundles are favorably yielded at the low spin coating rate of 100 rpm, while at high spin coating rate, it is shown otherwise. The auspicious morphologies of highly dense PFO-DBT nanorod bundles are supported by the augmented absorption and photoluminescence. In recent years, poly (PFO-DBT) has attracted numerous attention due to its exceptional optical properties. Applications in electronic devices such as solar cells and light-emitting diodes have elevated PFO-DBT thin films to be one of the most promising materials  in accorOne of the mutual approaches in fabricating the nanostructures is template-assisted method. Template-assisted method has been generally used to produce the unique nanostructured materials ,10,14-16Spin coating of solution into the porous template can possibly enhance the infiltration. On the planar substrate, the thickness of macroporous polymers can be easily tuned by varying the spin coating rate , in whicThe commercially existing copolymer of PFO-DBT from Lum-Tec  was utilized without further purification. A 5-mg/ml solution concentration of PFO-DBT was dissolved in chloroform. Commercially available porous alumina template from Whatman Anodisc Inorganic Membrane  with nominal pore diameter of 20 nm and a thickness of 60 μm was cleaned by sonicating it in water and acetone for 10 min prior to the deposition of PFO-DBT solution. The PFO-DBT solution was dropped onto the porous alumina template prior to the spin coating process. The spin coating rate was varied to 100, 500, and 1000 rpm at a constant spin time of 30 s, by using a standard spin coater model WS-650MZ-23NPP . In order to dissolve the template, 3 M of sodium hydroxide (NaOH) was used, leaving the PFO-DBT nanorods. The PFO-DBT nanorods were purified in deionized water prior to its characterization. The characterizations of PFO-DBT nanorods were performed using a field emission scanning electron microscope (FESEM) , transmission electron microscope (TEM) , X-ray diffraction spectroscope , UV-vis spectroscope , and photoluminescence spectroscope (Renishaw).A common practice in producing nanostructured materials via template-assisted method is by drop casting the solution on the template. However, the drop casting alone without the assistance of a spin coating technique would not efficiently allow the solution to infiltrate into the template. Infiltration of PFO-DBT solution into the cavity of an alumina template can be done by varying the spin coating rate. The FESEM images of the PFO-DBT nanorod bundles are shown in Figure At the low spin coating rate of 100 rpm, the denser PFO-DBT nanorod bundles are synthesized. Looking at the top of the bundles, the tips of the nanorods are tending to join with one another which could be due to the van der Waals force interaction. Apart of that, the high aspect ratio of the PFO-DBT nanorods obtained at low spin coating rate can be one of the contributions as well. However, the main contribution to the distinct morphological distribution is merely the different behaviors exhibited by PFO-DBT during the spin coating. The smallest diameter recorded at 100, 500, and 1,000 rpm is 370, 200, and 100 nm, respectively. An analysis of nanorods' length is depicted in Figure Depending on the applications, the morphological distributions of the PFO-DBT nanorods can be simply tuned via the spin coating of template-assisted method. Further corroboration on the effect of spin coating rate can be confirmed by the ability of the PFO-DBT solution to occupy the cavity of the template. At the intermediate spin coating rate (500 rpm), the gaps between the nanorod bundles started to form. The formation of these gaps may be due to the infirmity of PFO-DBT solution to occupy the cavity. In other words, the gap corresponded to the unoccupied cavity that will be dissolved with NaOH. Auxiliary increase of centrifugal force in spin coating rate will create an intense gap between the nanorod bundles which is identical to the scattered islands. Rapid evaporation of the PFO-DBT solution at 1,000 rpm has caused the formation of scattered islands. The top view images of the PFO-DBT nanorod bundles are illustrated in Figure The morphologies of the PFO-DBT nanorod bundles are further supported by the TEM images  spectra of the PFO-DBT nanorod bundles synthesized at different spin coating rates are shown in Figure In the present study, the effect of different spin coating rates on the morphological, structural, and optical properties of PFO-DBT nanorod bundles is reported. Polymer solution has been demonstrated to have different characteristics and abilities to infiltrate into the cavities at different spin coating rates. Highly dense PFO-DBT nanorod bundles are obtained at low spin coating rate with enhancement of structural and optical properties.FESEM: field emission scanning electron microscopy; FWHM: full-width half maximum; NaOH: sodium hydroxide; PFO-DBT: poly; PL: photoluminescence; rpm: rotation per minute; TEM: transmission electron microscopy; XRD: X-ray diffraction.The authors declare that they have no competing interests.MSF carried out the experiment, participated in the sequence alignment, and drafted the manuscript. AS participated in the design of the study, performed the analysis, and helped draft the manuscript. KS conceived of the study and helped draft the manuscript. All authors read and approved the final manuscript.MSF is currently doing his Ph.D. at the University of Malaya. AS and KS are senior lecturers at the Department of Physics, University of Malaya. AS's and KS's research interests include the synthesis of nanostructured materials via template-assisted method and applications in organic electronic devices such as sensors and photovoltaic cells.