In vitro studies showed that treatment of cells lacking TMPRSS4 expression with a demethylating agent significantly increased TMPRSS4 levels. In conclusion, TMPRSS4 is a novel independent prognostic biomarker regulated by epigenetic changes in SCC and a potential therapeutic target in this tumor type, where targeted therapy is still underdeveloped.Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, which highlights the need of innovative therapeutic options. Although targeted therapies can be successfully used in a subset of patients with lung adenocarcinomas (ADC), they are not appropriate for patients with squamous cell carcinomas (SCC). In addition, there is an unmet need for the identification of prognostic biomarkers that can select patients at risk of relapse in early stages. Here, we have used several cohorts of NSCLC patients to analyze the prognostic value of both protein expression and DNA promoter methylation status of the prometastatic serine protease TMPRSS4. Moreover, expression and promoter methylation was evaluated in a panel of 46 lung cancer cell lines. We have demonstrated that a high TMPRSS4 expression is an independent prognostic factor in SCC. Similarly, aberrant hypomethylation in tumors, which correlates with high TMPRSS4 expression, is an independent prognostic predictor in SCC. The inverse correlation between expression and methylation status was also observed in cell lines.  Lung cancer still remains as the most common cause of cancer death worldwide and presents a 5-year relative survival of around 18% . Among tThe implementation of targeted therapies aimed to inhibit specific mutations is mainly beneficial for a small group of patients with advanced NSCLC displaying adenocarcinoma histology , but notGenetic abnormalities associated with SCC that are under-represented in ADC include PI3KCA, SOX2, and FGFR1 amplification, PTEN loss, and DDR2 and TP53 mutations [In a previous study, we demonstrated that TMPRSS4 was highly upregulated in NSCLC, particularly in SCC, in comparison to non-malignant lung . MoreoveEpigenetic control of gene expression acts as a switch to either induce or repress the transcriptional activity of multiple genes implicated in cancer development. Specific epigenetic mechanisms have been identified as responsible to regulate the expression of certain EMT-related genes , 16. OneIn vitro experiments were performed as well to demonstrate that TMPRSS4 expression is regulated by promoter methylation. We have found that TMPRSS4 overexpression correlates with poor prognosis in NSCLC patients with squamous histology. Abnormal promoter hypomethylation was found in tissue samples from patients but not in non-malignant tissues and was inversely correlated with TMPRSS4 expression. Moreover, hypomethylation was associated with reduced relapse free survival in patients.In this study, we have evaluated the prognostic value of both TMPRSS4 protein levels and promoter methylation status in NSCLC patients. We first performed an immunohistochemical study on tissue microarrays (TMAs) containing NSCLC  and matched non-malignant (n=66) samples. The clinical and pathological characteristics of the NSCLC patients are shown in We then evaluated the relationship between TMPRSS4 protein expression and clinicopathological characteristics of the patients. No association between TMPRSS4 expression and any of the clinicopathological characteristics analyzed was found except for the histological type p=0.017; . RemarkaRegarding prognosis, patients with high levels of TMPRSS4 showed significantly shorter relapse free survival  and overall survival  than those with low levels Figure . We nextWhen considering only the cases corresponding to stage I patients (n=52), we found that high TMPRSS4 protein expression was significantly correlated with worse OS (p=0.001) and borderline for RFS (p=0.058) . SimilarTo rule out the possibility that post-surgery treatment with chemotherapy or radiotherapy could influence the results on survival, we performed logRank tests excluding patients treated with post-surgery adjuvant therapy. As shown in We next evaluated if TMPRSS4 could be considered as an independent prognostic factor in NSCLC using Cox regression analyses Table . In the All these data indicate that TMPRSS4 is an independent prognostic factor at early stages in NSCLC, associated with poor prognosis in SCC patients.In order to ascertain the possible cause of TMPRSS4 overexpression in lung cancer patients, we evaluated through bioinformatic analyses using public databases  possible genetic alterations  of TMPRSS4 in NSCLC. Analysis showed that these changes were very infrequent and unlikely to explain TMPRSS4 upregulation (data not shown). We then hypothesized that increased TMPRSS4 expression could be due to epigenetic modifications. The TMPRSS4 promoter does not contain a canonical CpG island, but gene overexpression through hypomethylation in non-CpG islands has been previously described . Based oTo validate this result, DNA samples from another cohort of 88 NSCLC patients . Data on OS was not available in this cohort.Table Methylation patterns regarding prognosis observed for SCC were different in ADC tumors. In the multivariate analyses, TMPRSS4 promoter hypomethylation was not an independent prognostic factor for any of the CpGs analyzed in ADC Table . In the When considering all NSCLC samples, hypomethylation was an independent predictor of reduced RFS only for probe CG27300950 (p=0.015). The logRank tests showed statistical differences in the same CpGs that were obtained for ADC, plus probe CG25116503. These results highlight the differences in hypomethylation patterns related to prognosis found for SCC and ADC. Taking into account results from both multivariate analyses and longRank tests we conclude that loss of TMPRSS4 promoter methylation in CpGs spanning from −116 bp to +271 bp relative to the TSS (CG03634928 to CG05416223 probes) consistently predicts poor outcome in SCC.We then retrieved data from both TMPRSS4 expression and promoter methylation status available at TCGA  to perform correlation analyses with those CpGs that were localized between −116 bp to +271 bp relative to the TSS and were significantly associated with poor prognosis in the multivariate analysis.Figure We also evaluated both expression and promoter methylation status in a panel of 46 lung cancer cell lines. Firstly, analysis of the methylome using the 450k methylation array in these cells showed different methylation patterns of TMPRSS4 promoter Figure . ClassifFor analysis using bisulfite sequencing we selected 16 cell lines representative of the 3 different clusters previously identified in the 450k methylation array Figure . SequencMethylation status was next validated by pyrosequencing of the CpGs located at −116 bp and −99 bp and results were correlated with those obtained in the array. As shown in We then quantified TMPRSS4 mRNA levels by real time PCR in the whole set of cell lines and performed Spearman's test to correlate expression with degree of methylation . Figure To test whether demethylation of TMPRSS4 promoter would result in increased TMPRSS4 levels, we treated cell lines lacking TMPRSS4 expression with the demethylating agent 5-aza-2′-deoxycytidine. After 48 hours of treatment with 2 or 4 μM, overexpression of TMPRSS4 mRNA was observed in all cell lines . PyroseqTaken together, all these results suggest that TMPRSS4 expression is controlled by promoter methylation, and that abnormal hypomethylation results in TMPRSS4 overexpression.Expression of the serine protease TMPRSS4 is highly increased in a variety of solid tumors and is associated with poor prognosis in some cancer types such as breast, colon, cervix, thyroid and liver , 11. Ouret al., have shown that TMPRSS4 is upregulated in NSCLC by epigenetic silencing of the tissue factor pathway inhibitor-2 [Thus far, the molecular mechanisms that may explain why TMPRSS4 is highly expressed in lung tumors are largely underexplored. Hamamoto ibitor-2 . We now Results from the 450k methylation array, and both bisulfite sequencing and pyrosequencing in our study show that, although TMPRSS4 promoter does not contain canonical CpG islands, there are relevant methylation regions in the TMPRSS4 promoter, which include CpGs located at positions −116 bp to +271 bp relative to the TSS. Analyses of ENCODE and transcription factor binding sites prediction databases (cREMaG and MotifScanner) suggest a possible involvement of transcription factors related to proliferation, EMT and inflammation , whose binding in the vicinity of those CpGs could be mediated by hypomethylation. Future studies should determine whether binding of these factors is epigenetically regulated and whether this would have an effect on TMPRSS4 expression.DNA hypomethylation is being increasingly acknowledged as a recurrent event in cancer, a mechanism that frequently affects oncogenes and tumor-promoting genes involved in proliferation, migration and metastasis . In a geet al., have recently developed a biological tool that links ZF proteins with DNMT3A to methylate the DNA promoter of the SOX2 oncogene, which results in efficient gene expression silencing and antitumor effects in vivo [Our study shows that both TMPRSS4 hypomethylation and high protein expression predict shorter RFS in patients with SCC, but results are not so consistent for patients with ADC. Over the past few years, different studies have described genomic alterations associated with SCC that are under-represented in ADC, although targeted therapy based on these alterations is still to be developed . Deciphe in vivo .In conclusion, we show for the first time that there is an epigenetic DNA hypomethylation of the TMPRSS4 promoter, a mechanism that may explain the TMPRSS4 overexpression found in NSCLC. We also demonstrate that both TMPRSS4 levels and promoter methylation status are useful biomarkers to predict poor prognosis in patients with lung SCC at early stages.To detect TMPRSS4 by immunohistochemistry, tissue microarrays (TMAs) containing samples from NSCLC (n=79) and matched non-malignant lung tissues (n=66) were used. Tumor samples were obtained from patients diagnosed at stage I or II who did not receive neoadjuvant therapy prior to surgery. Detailed histopathological and clinical characteristics of the patients are summarized in For promoter methylation analysis, two cohorts of patients were used. We first evaluated our previously published FP7 CURELUNG discovery cohort of lung tumors (n=444 patients) and non-tumor lung tissue samples (n=25)  with theThis work followed REMARK (REporting recommendations for tumour MARKer prognostic studies) criteria for tumor marker studies.A panel of 46 cell lines corresponding to different lung cancer histological types was included in this study . Of thesFor treatments with 5-aza-2′deoxycitidine , A549, H82, H23, H520 and H460 cells were cultured in RPMI and exposed to the drug at 2 μM and 4 μM concentrations. After 48h, cells were trypsinized and pellets were washed with PBS and maintained at −80°C for RNA and DNA extraction.® DNA Sample Preparation Kit, and RNA was obtained from frozen pellets using the Nucleospin RNA kit. Real time PCR was performed with SYBR green using standard protocols and primer sequences are shown in Genomic DNA was extracted from FFPET  using Cobas® Gel and PCR Clean-up (Macherey-Nagel) and then cloned into the pGEMT Easy Vector System (Promega) according to the manufacturer's protocol. In all the samples, 10 colonies were randomly chosen, and DNA was purified using NucleoSpin® 96 Plasmid (Macherey-Nagel) and sequenced. After sequencing analysis, results were transformed into percentages of CpGs showing methylation.The Methyl Primer Express v1.0 software (Applied Biosystems) was used to identify the TMPRSS4 promoter CpGs and to design specific primers for the methylation analysis . None ofQuantitative DNA methylation analysis of the TMPRSS4 promoter was performed by bisulfite pyrosequencing of two consecutive potentially methylated cytosines located at −116 bp and −99 bp relative to the TSS. Bisulfite conversion of 500 ng of each DNA sample was performed with EZ DNA Methylation-Gold Kit (Zymo Research) according to the manufacturer's recommendations. Primer sequences  were desDifferent statistical methods were applied depending on the type of experimental data. The processed transcriptomic (RNA-Seq experiments) and DNA methylation data generated by the 450k methylation array for lung cancer datasets were downloaded from the public TCGA project portal . BioinfoCorrelation analysis between TMPRSS4 expression and promoter methylation in cell lines was assessed with Spearman's test and cells were clustered based on their methylation status by hierarchical clustering analysis using Euclidean distance and complete linkage analysis.Normality of the data was assessed with the Shapiro-Wilk test. The association between TMPRSS4 expression and clinicopathological features of patients was analyzed by Pearson's chi-square test. Relapse free survival (RFS) and overall survival (OS) were evaluated with Kaplan-Meier curves and significant differences among groups were assessed by the logRank test. Overall survival was defined as the time from study enrollment to death. To evaluate the prognostic value of TMPRSS4, univariate and multivariate Cox proportional hazard analyses were used. To assess statistical differences between groups when comparing TMPRSS4 promoter methylation status in tumor versus non-malignant samples, the Mann-Whitney U test was used. Comparison of H-score for TMPRSS4 expression between non-malignant and malignant samples was assessed with T-test.th percentile for control samples. Data was analyzed with the SPSS statistical software (version 17.0 for Windows SPSS) and GraphPad Prism 5 software (GraphPad). Statistical significance was defined as p<0.05 (*), p<0.01 (**), and p<0.001 (***).To classify patients with respect to their methylation levels in specific CpGs, we set a threshold β-value of 0.5 to define non-methylation (β<0.5) vs. methylation based on averaged 95


Columbid alphaherpesvirus 1 was detected from a powerful owl (Ninox strenua) with inclusion body hepatitis, and an avian paramyxovirus most similar to Avian avulavirus 5 was detected from a musk lorikeet (Glossopsitta concinna). Two distinct novel avian alphaherpesviruses were detected in samples from a sulphur-crested cockatoo  and a tawny frogmouth (Podargus strigoides). Avian influenza viruses and avian coronaviruses were not detected. The clinical significance of the newly detected viruses remains undetermined. Further studies are needed to assess the host specificity, epidemiology, pathogenicity and host-pathogen relationships of these novel viruses. Further genome characterization is also indicated, and would be required before these viruses can be formally classified taxonomically. The detection of these viruses contributes to our knowledge on avian virodiversity. The low level of avian virus detection, and the absence of any viruses with zoonotic potential, suggests low risk to biosecurity and human health.Viruses in avian hosts can pose threats to avian health and some have zoonotic potential. Hospitals that provide veterinary care for avian patients may serve as a site of exposure of other birds and human staff in the facility to these viruses. They can also provide a useful location to collect samples from avian patients in order to examine the viruses present in wild birds. This study aimed to investigate viruses of biosecurity and/or zoonotic significance in Australian birds by screening samples collected from 409 birds presented to the Australian Wildlife Health Centre at Zoos Victoria’s Healesville Sanctuary for veterinary care between December 2014 and December 2015. Samples were tested for avian influenza viruses, herpesviruses, paramyxoviruses and coronaviruses, using genus- or family-wide polymerase chain reaction methods coupled with sequencing and phylogenetic analyses for detection and identification of both known and novel viruses. A very low prevalence of viruses was detected.  Avian species serve as hosts for a diversity of viruses that pose both biosecurity and zoonotic risks. Numerous outbreaks of disease in collections of caged birds and commercial domestic poultry have been attributed to viruses introduced by direct or indirect contact with wild birds –7. Some In Australia, the Australian Wildlife Health Centre (AWHC) at Zoos Victoria’s Healesville Sanctuary functions as a large primary care and referral centre for sick, injured and orphaned native wildlife, including providing veterinary care for approximately 700–900 wild birds each year, as well as Healesville Sanctuary’s collection of captive animals. Patients may be presented to the AWHC by members of the public, wildlife carers, keepers, government wildlife officers or by referral from other veterinary clinics for veterinary diagnosis and treatment. During this period in captivity there is an elevated risk of stress-induced shedding of pathogens, presenting a risk for both the veterinary staff and other animals in care, including the Sanctuary’s collection animals and captive breeding programs. Close contact between infected and susceptible animals can facilitate the transmission of pathogens between species, particularly when the hosts are taxonomically related. The large number of avian patients passing through the AWHC provides an opportunity to study the viruses present in Australian birds and better understand the potential risks these viruses present.Neophema chrysogaster), which is facing imminent extinction in the wild [Psittacid alphaherpesvirus 1 (PsHV1) is the aetiologic agent of both Pacheco’s disease and mucosal papillomas in Psittaciformes [Ara chloropterus) imported from the United Kingdom in the 1990s [Neophema chrysogaster recovery program, would be potentially devastating. Avian paramyxoviruses (APMVs) also pose a threat to Australian Psittaciformes. Avian avulavirus 3  has been documented causing mortality rates of up to 70% in captive collections of Neophema species and other Australian psittacines overseas [Neophema is highly susceptible to APMV-3, with infection frequently resulting in fatal neurological disease [Avian avulavirus 5  has caused outbreaks of severe disease with up to 100% mortality in captive budgerigars (Melopsittacus undulatus), including an outbreak in a captive budgerigar flock in Queensland in 1972 [Of particular importance and conservation value at Healesville Sanctuary are the captive breeding and insurance programs for threatened species. This includes a captive breeding program for the critically endangered orange-bellied parrot  can threaten human health , 11–13. This study aimed to collect samples from birds presenting to the AWHC and use them for the molecular detection and characterization of viruses with potential significance to biosecurity, human health or animal health. Four virus families were targeted in this study: avian influenza viruses, herpesviruses, paramyxoviruses and coronaviruses. Samples were screened using a combination of genus- or family-wide polymerase chain reaction methods coupled with sequencing and phylogenetic analyses for detection and identification of both known and novel viruses. Two novel viruses were detected in this study, hence contributing to our knowledge of avian virodiversity, while the overall low level of virus detection adds to our understanding about the presence or absence of viruses of birds in Australia and their potential risk to avian and human health.® (Ambion) and stored at -20°C. For the 210 birds sampled at post-mortem , sterile dry swabs were used to separately swab the trachea and intestine/caecum, cut into 500 μL of RNAlater® and stored at -20°C. A piece of liver was also aseptically collected and stored at either -20°C or -80°C. The collection of these liver samples, and their subsequent testing for BFDV, has been previously reported in a separate study [Samples were collected from 409 birds presented to the AWHC at Healesville Sanctuary  for veterinary diagnosis and treatment between December 2014 and December 2015 see . This st® were vortexed for 10 seconds and then centrifuged briefly before 100 μL aliquots of each choanal/tracheal and cloacal/intestinal swab in RNAlater® from the same bird were pooled to produce 200 μL samples for genomic nucleic acid extraction. Nucleic acid extraction was performed manually using a QIAamp Viral RNA Mini Kit (Qiagen), using the spin protocol according to the manufacturer’s instructions, eluting into two lots of 40 μL of Buffer AVE, and robotically using VX Universal Liquid Sample DNA Extraction Kits (Qiagen) and a Corbett X-tractor Gene Robot (Corbett Robotics), according to the manufacturer’s instructions, with products being eluted in 70 μL of elution buffer. Samples of liver tissue collected at post-mortem were pulverised with a swab and the swab placed into 500 μL RNAlater®. Liver swabs in RNAlater® were vortexed for 10 seconds then centrifuged briefly before taking 200 μL aliquots from which nucleic acid was extracted as above. Whenever pooled clinical samples tested positive by PCR, nucleic acid was extracted from 140 μL aliquots of the corresponding original choanal/tracheal and cloacal/intestinal swab samples separately using the QIAamp Viral RNA Mini Kit spin protocol, and eluted in 60 μL of Buffer AVE. Laboratory stocks of Influenza A/Memphis/1/1971 (H3N2), Chlamydia felis, Macropodid alphaherpesvirus 1 3076/08 and Newcastle disease virus vaccine strain (NDV.V4) were pooled to give 200 μL positive extraction control samples. Negative extraction control samples utilised 200 μL aliquots of RNAlater®.Swabs in RNAlaterMacropodid alphaherpesvirus 1 or Equid alphaherpesvirus 4 DNA were used as template for positive control reactions, and sterile H2O for negative control reactions. When herpesvirus DNA was detected, additional viral DNA sequence information was obtained by first repeating the first round of the nested universal herpesvirus PCR assay, using DNA extracted from separate choanal/tracheal and cloacal/intestinal swab samples as template. Further PCRs using forward primer TGV paired with reverse primer KG1, and forward primer DFA paired with reverse primer IYG, were then performed using 5 μL aliquots of the first round reaction as template.Samples were tested for the presence of herpesvirus DNA using a universal herpesvirus PCR assay utilising degenerate, deoxyinosine-substituted oligonucleotide primers specifically targeting a highly conserved region of the herpesvirus DNA polymerase gene, as previously described . MacropoSuperScript™ III Reverse Transcriptase (Invitrogen) was used to synthesise cDNA from aliquots of extracted nucleic acid in preparation for screening of samples for the presence of paramyxoviruses and coronaviruses by PCR. The reverse transcriptase (RT) reaction was performed according to the manufacturer’s instructions using 100 ng random primer oligonucleotides (Invitrogen), and 10 μL of nucleic acid extract as template. All assays included a no template control with sterile water, and the appropriate positive control as described for each assay.Paramyxoviridae RNA was detected by RT-PCR, forward and reverse primers PAR-F1 and PAR-R [Paramyxoviridae [® Flexi buffer, 3 mM MgCl2 and 1 U GoTaq® Flexi DNA Polymerase in a 25 μL reaction volume. PCR mixtures were incubated at 94°C for 2 minutes prior to 40 cycles of 94°C for 15 seconds, 41°C for 30 seconds and 72°C for 30 seconds, followed by a final extension at 72°C for 7 minutes.Samples were tested for the presence of paramyxovirus RNA using a family-wide RT-PCR assay utilising consensus degenerate and inosine-containing oligonucleotide primers annealing to conserved motifs in domain III of the RNA-dependent RNA polymerase gene, as described previously . cDNA synd PAR-R  were usend PAR-R  to obtaioviridae . PAR-F1 ® dual-labelled probe [® GTXpress™ Master Mix (Life Technologies) according to the manufacturer’s instructions, with forward and reverse primers to a final concentration of 0.5 μM each, 5-carboxyfluorescein (FAM)-labelled probe with 3’-BHQ1-quencher (GeneWorks) to a final concentration of 0.1 μM, and 5 μL of cDNA template. cDNA synthesised from RNA extracted from laboratory stocks of infectious bronchitis virus (strain VIC-S) was used as the template for positive controls reactions. Reactions were conducted in a Stratagene Mx3000P™ quantitative PCR thermocycler, with initial enzyme activation at 95°C for 20 seconds, then 40 cycles of a denature step at 94°C for 1 second followed by 60°C for 60 seconds for annealing and extension. Results were analysed using Stratagene MxPro software, with CT values above 40 considered negative.A real-time RT-PCR assay using primers targeting conserved sequences within the 5’-untranslated region (UTR) gene and a TaqManed probe  was usedSamples were tested for the presence of influenza A virus RNA using a TaqMan Real-Time matrix gene-specific RT-PCT assay  within aThroughout this study 95% confidence intervals (95% C. I.) for sample proportions and prevalence estimates were calculated using the Jeffreys method , while s® 9.1.8 bioinformatics software  [® database  using the NCBI Nucleotide Basic Local Alignment Search Tool (BLASTN®) online algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and ClustalW2 alignments with their closest matches were generated within Geneious R9 to determine nucleotide identities for the region of available sequence. PhyML 2.2.0 maximum likelihood phylogenetic trees for the herpesviruses were generated from ClustalW2 [Paramyxoviridae, a ClustalW2 alignment [PCR products were purified from PCR reaction mixtures and sequenced as described previously . GeneiouZealand)  was usedlustalW2  nucleotilustalW2 . For thelignment  was usedlignment . For eacNinox strenua) swabbed at the very end of the sampling period were not included. No influenza A viruses or avian coronaviruses were detected from any of these 408 birds , while a single avian paramyxovirus was detected from a choanal swab from a wild musk lorikeet (Glossopsitta concinna) using the pan-paramyxovirus RT-PCR assay   . While nCacatua galerita), a tawny frogmouth (Podargus strigoides) and a powerful owl  with 100% nucleotide identity [st May 2015 in St Kilda, Victoria , and died shortly after admission to the AWHC. This same powerful owl has been the subject of previously reported diagnostic investigations which revealed the presence of BFDV DNA in a sample of liver [The 181 bp nucleotide sequence obtained from the powerful owl aligned with the DNA polymerase genes of strains of identity –67. Of tidentity . The groidentity . Histopaof liver .Herpesviridae were detected, one from DNA extracted from a choanal swab from a tawny frogmouth, and the other from DNA extracted from a tracheal swab collected from a sulphur-crested cockatoo. The closest matches to the 485 bp nucleotide sequence obtained from the tawny frogmouth swab were avian herpesviruses within the subfamily Alphaherpesvirinae, with highest (76%) nucleotide identity with the DNA polymerase genes of strains of CoHV1 [Podargid alphaherpesvirus 1 (PodHV1). The nucleotide sequence of the DNA polymerase gene fragment from this novel herpesvirus has been deposited in GenBank (accession number MF576272).Two novel members of the family of CoHV1 –65, 67. th August 2015, but after a two week rehabilitation went on to fly off when released back where found in Badger Creek, Victoria . The virus was not detected in DNA extracted from pooled choanal/tracheal and cloacal/intestinal swab samples from any of the other 34 tawny frogmouths tested , nor any of the other 82 avian species in this study. The clinical significance of this newly identified alphaherpesvirus is undetermined, but the tawny frogmouth showed no expression of clinical disease during its period of rehabilitation.The tawny frogmouth had presented at the AWHC with a minor laceration and clinical signs suggestive of head trauma  on 27Psittacid alphaherpesvirus 2 (PsHV2), which has been detected in African grey parrots (Psittacus erithacus) and a blue and gold macaw (Ara ararauna) in the USA [Alphaherpesvirinae, and we tentatively propose the name Cacatuid alphaherpesvirus 1 (CacHV1) after the host family in which it was found. The nucleotide sequence of the DNA polymerase gene fragment from this novel herpesvirus has been deposited in GenBank (accession number MF576271).The closest match to the 489 bp nucleotide sequence from the sulphur-crested cockatoo swab was the DNA polymerase gene of PsHV1 (GenBank accession number AY372243), with 66.5% nucleotide identity . This se the USA . The phyth September 2015, presenting emaciated and weak with haemorrhagic enteritis and clinical signs suggestive of beak and feather disease virus (BFDV) infection. This cockatoo tested positive for BFDV when DNA extracted from a liver sample collected at post-mortem was used as template in a BFDV PCR assay [Macrorhabdus ornithogaster in the ventriculus. It is presumed that the protozoan and Macrorhabdus overgrowth were secondary to immunosuppression caused by BFDV infection, but it is unknown what role the newly identified alphaherpesvirus may have been playing in this emaciated and very unwell bird. No inclusion bodies, inflammation in the lungs nor any other sign of herpesvirus infection were seen in any of the tissues examined histologically. CacHV1 was only detected from one of the 27 sulphur-crested cockatoos tested in this study , and was not detected from any of the other 54 Cacatuids tested .The female sulphur-crested cockatoo had been found unable to fly in Lilydale, Victoria  on 17CR assay , 70. HisAvian avulavirus 5 (GenBank accession numbers GU206351 and LC168750) [Avian avulavirus 5, and perhaps represents a novel genotype or genetic subgroup within this serotype  in December 2014, and presented to the AWHC with an old fracture of the right coracoid, on the day of sample collection. After a successful four-week rehabilitation, during which the lorikeet ate well, gained bodyweight and showed no signs of ill health, the bird was released back where it was found. On BLAST analysis, the 603 bp sequence fragment of the paramyxovirus RNA-dependent RNA polymerase (L protein) gene obtained from this musk lorikeet did not match any currently known paramyxovirus, but had highest (77.4–77.6%) nucleotide sequence identity with strains of C168750) , 71. Theserotype . ParamyxA very low prevalence of viruses was detected in the birds examined in this study. Sampling birds presented to veterinary hospitals, such as the AWHC, is a useful approach to studying viruses in wild birds. However, this approach does involve an inherently biased population sample  which should be considered when these results are interpreted, or compared to other studies. It is likely that the sample population in this present study, particularly the low numbers of Anseriformes and Charadriiformes (28 of the 408 birds tested) contributed to the absence or low prevalence of detection of AIVs, APMVs and AvCoVs, as previous studies have shown that these viruses are present mainly in the orders Anseriformes and Charadriiformes , 72–74. Coronaviridae PCR assay to test Australian wild bird samples for AvCoVs would be useful for gaining a more general understanding of AvCoVs in Australia. Future studies focused on elucidating the origin of variant IBV in Australian poultry may be best focused on species of wild birds known to be in closest contact with commercial poultry, such as introduced  passerine birds that commonly live in close proximity to humans and domestic animal production facilities, like European starlings (Sturnus vulgaris) and house sparrows (Passer domesticus) [Studies of avian coronaviruses in wild birds in other countries, using the same primers  as the pesticus) , 78.Avian avulavirus 5, which had previously only been isolated from budgerigars [Our results showing a very low prevalence 0.25%) of APMVs in wild birds in Victoria are consistent with the results of other similar Australian studies , 79, 80.% of APMVgerigars –34, 71, gerigars –34. Of tAvian avulavirus 5, but this putative classification can only be confirmed if the virus can be isolated in the future for antigen-based testing and more extensive genome sequencing. Formal classification of APMV strain musk lorikeet/Melbourne/ML22-141263/2014 will require more genomic sequence information than the 603 bp of the RNA polymerase gene that was obtained in the current study. The clinical significance of this possible novel APMV-5 genotype in other Psittaciformes, such as those in involved in threatened species recovery programs, is also undetermined.It is possible that the APMV strain detected in this study represents an avirulent genotype of APMV-5 that may be circulating amongst wild psittacines in Australia. The complete genome sequences of strains of other avian paramyxoviruses have provided evidence for the existence of genetic subgroups within their respective serotypes , 81, 82.Avian avulavirus 3 nor Psittacid alphaherpesvirus 1 were detected. Many species of Australian parrots are susceptible to APMV-3 and/or PsHV1 infection and disease [Despite 193 birds belonging to the order Psittaciformes being tested in this study, which included seven orange-bellied parrots from Healesville Sanctuary’s collection, neither  disease –28, 83,  disease , particu disease –28, 83.  disease –31, whil disease . However disease . Ongoing disease .Columbid alphaherpesvirus 1 in a powerful owl and two novel viruses in a sulphur-crested cockatoo and a tawny frogmouth, respectively. CoHV1 infection had previously been documented in three species of native birds of prey in Australia: powerful owls, barking owls (Ninox connivens) and an Australian hobby  [Columba livia)) [Three avian herpesviruses were detected in the present study: ipennis) . The vir livia)) , 85, 86. livia)) . This re livia)) . The preEquid alphaherpesvirus 1 and 4 , but there is 100% nucleotide identity at this same region between different isolates of EHV1 and 100% identity between different isolates of EHV4 [Alphaherpesvirinae  in Australian wild birds and the level of potential risk to animal and human health, and to threatened avian species in Australia. Whilst it is interesting that no avian herpesviruses nor paramyxoviruses were detected from the captive birds tested in this study, there was no significant difference in the prevalence of detection of avian viruses between the wild and captive bird study populations. The detection of two previously unknown viruses has contributed to our knowledge of avian viral communities and their diversity in Australia. Future studies incorporating deep sequencing methods could be expected to possibly uncover more avian viruses and make further contributions to our understanding of avian virodiversity.S1 TableWild birds were presented to the AWHC at Healesville Sanctuary for veterinary care. Captive birds were part of the Healesville Sanctuary collection.(DOCX)Click here for additional data file.


Stat2-/- mice are highly susceptible to ZIKV infection, recapitulate virus spread to the central nervous system (CNS), gonads and other visceral organs, and display neurological symptoms. Further, we exploit this model to compare ZIKV pathogenesis caused by a panel of ZIKV strains of a range of spatiotemporal history of isolation and representing African and Asian lineages. We observed that African ZIKV strains induce short episodes of severe neurological symptoms followed by lethality. In comparison, Asian strains manifest prolonged signs of neuronal malfunctions, occasionally causing death of the Stat2-/- mice. African ZIKV strains induced higher levels of inflammatory cytokines and markers associated with cellular infiltration in the infected brain in mice, which may explain exacerbated pathogenesis in comparison to those of the Asian lineage. Interestingly, viral RNA levels in different organs did not correlate with the pathogenicity of the different strains. Taken together, we have established a new murine model that supports ZIKV infection and demonstrate its utility in highlighting intrinsic differences in the inflammatory response induced by different ZIKV strains leading to severity of disease. This study paves the way for the future interrogation of strain-specific changes in the ZIKV genome and their contribution to viral pathogenesis.Zika virus (ZIKV) is a mosquito borne flavivirus, which was a neglected tropical pathogen until it emerged and spread across the Pacific Area and the Americas, causing large human outbreaks associated with fetal abnormalities and neurological disease in adults. The factors that contributed to the emergence, spread and change in pathogenesis of ZIKV are not understood. We previously reported that ZIKV evades cellular antiviral responses by targeting STAT2 for degradation in human cells. In this study, we demonstrate that  Stat2-/- mice are highly susceptible to ZIKV infection and recapitulate aspects of ZIKV pathogenesis and disease. We use this model to delineate whether strain specific differences in ZIKV pathogenesis exist, using diverse strains representing both African and Asian lineages. We show that African strains in general are more virulent than Asian strains and their pathogenicity associates closely with the degree of inflammatory immune response in the CNS of infected mice, and does not necessarily correlate with viral RNA levels. Thus, we establish Stat2-/- mice as new model to study ZIKV pathogenesis and use it to characterize inherent differences in the virulence among ZIKV strains. More importantly, we also highlight a potential role of the host inflammatory immune response in mediating differential pathogenesis among ZIKV strains.Zika virus is the most recent viral pathogen to cause a global public health emergency. It is distinct from other flaviviruses in its ability to cause transplacental infection, fetal abnormalities and vector independent transmission through body fluids in humans. Over the last year, there has been rapid progress in the development of animal models, which can be used to study ZIKV pathogenesis. In this study, we demonstrate for the first time that  Flavivirus of the family Flaviviridae includes important vector-borne human pathogens such as dengue (DENV), yellow fever (YFV), West Nile (WNV), Japanese encephalitis (JEV), and tick-borne encephalitis viruses (TBEV). Many of these viruses are prevalent in the equatorial region of the globe that harbors the arthropod vectors required for their transmission [Aedes africanus mosquitoes [The genus smission –3. Zika smission –3. ZIKV squitoes . Subsequsquitoes –3. Here squitoes –3. Imporsquitoes .in vitro, ex vivo and in vivo models to study ZIKV infection and pathogenesis [Ex vivo human placental tissue models have shown transplacental infection of ZIKV, its preference for trophoblasts and sensitivity to type III IFN [et al tested ZIKV infection in several animal species including mice, cotton rats, guinea pigs and rhesus macaques, and showed that only neonatal mice were prone to symptomatic ZIKV infections [Phylogenetic analysis of ZIKV genomes reveals African and Asian as two distinct lineages. It has been postulated that ZIKV originated in East Africa, spread to West Africa and Asia nearly 50~100 years ago, before making its appearance in the Western hemisphere in 2014 , 7. The ogenesis . Studiesogenesis . Ex vivo III IFN . In the  III IFN . At the fections . Later, fections , 13–17. fections . Althougfections –28. Immufections .Stat2-/- mice results in ZIKV infection and spread to CNS, gonads and other vital organs, with manifestation of neurological symptoms. Further we have used the Stat2-/- mouse model to compare the virulence and pathogenesis of a panel of ZIKV strains representing different lineages and spatio-temporal isolation histories.In an earlier report, we showed that ZIKV targets human STAT2 for degradation, thereby reducing induction of IFN stimulated genes (ISGs) and subsequent antiviral responses in human cells, but not in mouse cells . These fStat2-/- C57BL/6 mice with Uganda ZIKV . We used a subcutaneous route to mimic the natural route of infection and recorded ZIKV replication and pathogenesis. Specifically we injected 1,000 plaque formation units (PFU) of Uganda ZIKV in the footpad of 5–6 week old female Stat2-/- mice. To compare the sensitivity of these mice to ZIKV with other previously used mouse strains, Ifnar1-/- and wild type (WT) C57BL/6 mice were also infected using the same protocol [Stat2-/- mice started losing weight 3 days post infection, and became morbid, displayed limited movement and succumbed to infection between day 6 to 7  as early as 2 days post infection and showed slightly reduced levels by day 6  and Ifnar1-/- mice   caused 100% lethality in Ifnar1-/- mice by day 10 at dose of 100 PFUs, which is different from our observations with Asian strains. It could’ve resulted due amino acid differences between different Asian strains. However for African lineage Senegal strain (Dakar 41519) they also observed 100% lethality in 4 week old Ifnar1-/- mice, which is consistent with our findings. The WT mice did not show any clear symptoms of disease with either Asian or African ZIKV strains. Taken together these data show inherent differences in morbidity and lethality induced by African and Asian ZIKV strains in Stat2-/- and Ifnar1-/- mice.Although there have been several studies showing the utility of different mouse models to study ZIKV pathogenesis, a comprehensive comparative analysis of different ZIKV strains ried out . To addr-/- mice . In comp-/- mice . In term-/- mice . Among A-/- mice . Interes-/- mice . Only thrtality) . All othrtality) . In the Stat2-/- mice and measured ZIKV viral RNA levels in the serum, CNS and gonads. Clinical signs of ZIKV disease started with reduced motility and hunched posture followed by partial or complete paralysis in hind limbs, front limbs and lethality. In most cases mice suffering front limb paralysis succumbed to the disease the same or next day. We observed that African lineage strains induced higher mortality and severe neurological symptoms in a short period as compared to Asian lineage strains  and cytolytic response , monocyte/macrophage infiltration , and neutrophil migration (CXCL1 and CXCL2). Additional cytokines with known antiviral activity were also induced, such as IL-1b and IL15 [CD4, CD8, CCR5, CXCR3), monocytes  and NK cells (CCR5) were also differentially upregulated, while microglial activation appeared to be uniformly induced irrespective of the ZIKV strain as measured by CX3CR1 expression  [Stat2-/- mice were highly susceptible to ZIKV infection and disease. In comparison Ifnar1-/- mice showed in general delayed disease symptoms and less body weight loss due to ZIKV infection than Stat2-/- mice. While Ifnar1-/- mice lack type I IFN signaling, Stat2-/- mice lack both type I and type III IFN signaling [et al [Stat2-/- and Ifnar1-/- mice, we cannot exclude the possible role of STAT2 independent, IFNAR1 dependent, type I IFN signaling. Immunocompromised mouse models are useful options when WT mice are refractory to viral infection. In our study Stat2-/- mice recapitulated ZIKV tissue tropism to CNS and gonads and displayed neurological symptoms of various degrees, which are consistent with human disease. It will be interesting to test ZIKV infection of Stat2-/- as well as Stat2-/+ heterozygous mice in pregnancy models to see whether they can simulate the transplacental infection and neuroteratogenic properties of ZIKV.In November 2016 WHO declared that ZIKV is no longer a public health emergency, however it remains a serious public health concern, especially in the light of recent studies showing fetal microcephaly and other long-term cognitive disorders resulting from ZIKV infection . From a esponse) , which pesponse) . This knignaling . This mag [et al  who showvitro stem cell derived models have been very useful in confirming the neurotropic nature and teratogenic potential of ZIKV [Stat2-/- mice [In  of ZIKV , 37–39. -/- mice . HoweverStat2-/- mice with all ZIKV strains led to inflammatory immune responses in the brain. Importantly, the differences in ZIKV strain-specific pathogenesis were found to be closely correlated with the level of induction of inflammatory cytokines and possible recruitment of immune effector cells to the CNS of infected mice. These findings highlight that disease severity induced by ZIKV infection is more likely a product of the host response, rather than the level of viral replication, at least in immunocompromised mouse models. We also observed that, other than Uganda, all ZIKV strains achieved similar viral RNA levels in the mouse brain, however this led to variable induction of IFNα, IFNβ, and IFNγ. This could have resulted from differences in the ability of ZIKV strains to antagonize the induction of IFN. As these mice lacked STAT2, the NS5 mediated IFN antagonism through degradation of STAT2 does not account for these differences. For ZIKV and other flaviviruses, IFN antagonism has also been attributed non-structural proteins other than NS5 [Consistent with earlier reports showing CNS inflammation in ZIKV infected neonatal mice and Rhesus macaques , 25, infthan NS5 , 41. It Aedes sp. mosquitoes, increased global transportation of ZIKV infected individuals and immunological naivety of human populations in new areas may have facilitated these events. However it is also plausible that during the course of its evolution ZIKV acquired changes in its genome [It is critical to understand the factors which facilitated sudden emergence and spread of ZIKV, to take preventive measures in the future. It is likely that global warming, deforestation, increased prevalence of s genome  that mays genome . InteresAedes mosquitos for transmission [Stat2-/- mouse model described here could be a useful tool to evaluate the pathogenesis of recombinant ZIKV carrying different combinations of these genetic changes.ZIKV is not the first arbovirus to show sudden emergence and global spread. In fact, ZIKV has followed a trajectory displayed earlier by chikungunya virus, which in the course of evolution acquired a small number of mutations allowing it to use additional species of smission . Interessmission , 7. ThesStat2-/- C57BL/6 mice were kindly provided by Dr. Christian Schindler. Ifnar1-/- C57BL/6 mice deficient in IFN α/β receptor -/- were kindly provided by Dr. Thomas Moran. WT C57BL/6 mice were obtained from Jackson Laboratories.The following ZIKV strains were used in this study: Uganda 1947 , Puerto Rico 2015 , Cambodia 2010 , Malaysia 1966  and Senegal 1984 . The Uganda strain was obtained from American type culture collection (ATCC). All virus stocks were grown in Vero cells and quantified by plaque assay. A summary of isolation history of all ZIKV strains and related references is provided in Aedes albopictus cells), which were obtained from ATCC. Virus titers of the stocks were measured by plaque formation assay on Vero cells. Specifically, 100 μl of 10 fold virus stock serial dilution was incubated for 2 hour on Vero cell monolayer in 12 well plate format. Finally cells were overlaid with 1 ml DMEM (Invitrogen) supplemented with 0.8% methyl cellulose, 2% FBS, and 50 μg/ml penicillin and streptomycin. Cells were incubated for 5 days at 37°C, fixed with 4% PFA, and stained with crystal violet for plaque visualization.Virus stocks were grown in Vero (African green monkey kidney) cells and in some cases passaged in C6/36  and xylazine (5 μg per gram). Afterwards, 1,000 PFUs of the indicated ZIKV strain were injected subcutaneously in the mouse footpad in 25 μl of phosphate-buffered saline (PBS). Mice were monitored daily for weight loss and clinical signs. Examination included appearance, posture, motility, hind and front limb paralysis. Animals that showed more than 25% weight loss or complete paralysis were humanely euthanized.2 asphyxiation and organs were harvested aseptically. Tissue homogenates were prepared in PBS supplemented with 0.3% BSA, using metal beads and a FastPrep24 system . ZIKV RNA quantification was conducted as previously described. Briefly, total RNA was isolated from 200 μl tissue homogenate using RNeasy tissue Kit (Qiagen) or 50 μl plasma using QIAamp viral RNA kit (Qiagen). In total, 10ng of RNA or 5 μl of plasma RNA was used with ZIKV-specific primers (5′-TTGGTCATGATACTGCTGATTGC-3′ and 5′-CCYTCCACRAAGTCYCTATTGC-3′) and probe (5′-6FAM-CGGCATACAGYATCAGGTGCATWGGAG-MGBNFQ-3′) (ThermoFisher) and the LightCycler 480 Master Hydrolysis Probes kit  using the LightCycler 480 II Real Time PCR System (Roche Applied Science). Sequence of the primers and probe targeting ZIKV have been modified from previously published sequences [in vitro transcribed RNA standard.Infected mice were euthanized by COequences  and desiTotal RNA was isolated from cells using Trizol reagent (Invitrogen). Reverse transcription was performed with the high-capacity cDNA reverse transcription kit (Applied Biosystems). qPCR was done in duplicates using SYBR green I master mix (Roche) in a Roche Light Cycler 480. Relative mRNA values were calculated using ΔΔCt method with 18s RNA as an internal control, and shown as fold change by normalizing to mock control. The sequences of primers used in the study are listed in Protein levels of cytokines/chemokines in clarified tissue homogenates were evaluated using a multiplex bead array assay. All antibodies and cytokine standards were purchased as antibody pairs from R&D Systems or PeproTech. Individual magnetic bead regions (Luminex) were coupled to cytokine-specific capture antibodies according to the manufacturer’s recommendations and as previously described . Conjugawww.viprbrc.org/). From these genomes we manually selected a subset of sequences representative of the complete diversity to build the phylogenetic tree. The final selection contained one viral genome from each country and year available, including the 5 strains used in this study. Viral sequences were aligned using the MUSCLE algorithm in MEGA7 (version 7.0.20) and a neighbor joining tree was built using the program Geneious (version 9.1.5). The tree was visualized with the program FigTree and rooted at the midpoint.To prepare a phylogenetic tree presenting the global diversity of the Zika virus strains, we downloaded all the available complete genomes from the Virus Pathogen Resource database (nsp>o.o5 (not significant). Data are given as mean ± SD or mean ± SEM as indicated; ‘n’ refers to the sample size.All data were analyzed with GraphPad Prism software . For survival analysis, Kaplan-Meier survival curves were analyzed by the log-rank test. An unpaired Student's t-test was used to determine significant differences in viral RNA levels. Values were considered statistically significant when *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001, This study was carried out in strict accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All mouse procedures were approved by Institutional Animal Care and Use Committee (IACUC) of the Icahn School of Medicine at Mount Sinai and performed in accordance with the IACUC guidelines .S1 FigStat2-/- B6 mice (n = 5) were injected with 1,000 PFU of ZIKV strain MR 766 by the subcutaneous route in the footpad. Indicated organs were harvested on indicated days  and ZIKV RNA levels were measured by qRT PCR as described in methods. D6 group is the same set of animals used to measure ZIKV RNA in Three groups of five to six week old female (TIF)Click here for additional data file.S2 Fig Clinical signs of ZIKV infection were monitored every day in the group of animals used for survival analysis in (TIF)Click here for additional data file.S3 FigStat2-/- C57BL/6 mice  were injected with 1,000 PFU of the indicated ZIKV strain by the subcutaneous route in the footpad. Mice were weighed daily and weights are expressed as percentage of body weight prior to infection. Results shown are the mean ± SEM. Data are censored at 6 days after infection, as mice were euthanized for brain harvesting. (B) ZIKV RNA levels were measured in the brain homogenates by qRT PCR as described in methods. Error bars represent mean ±standard deviation (SD). ZIKV strains are abbreviated as SN , CB (Cambodia) and PR (Puerto Rico). (C) Mouse cytokine mRNA expression in brain was measured by qRT PCR as described in the methods section. Fold change of the gene expression over the mock was calculated and plotted on the graphs. Data are presented as log 2 fold increase over mock controls mean ±standard deviation (SD).(A) Six week old female (TIF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 TableAedes pseudoscutellaris cells; BHK = baby hamster kidney epithelial cells; C6/36 = Aedes albopictus cells; SM = suckling mouse; Vero = African green monkey kidney cells.AP61 = (XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


MYCN whose amplification is known to contribute to the pathogenesis of neuroblastoma. MYCN is known to negatively regulate NMYC downstream-regulated gene 1 (NDRG1) in neuroblastomas. MYCN down-regulation induced by CDDO correlated with increased expression of NDRG1. CDDO decreased Anaplastic Lymphoma Kinase (ALK) mRNA expression without affecting its protein level, while ATRA significantly down-regulated ALK. Antagonism of PPARγ receptor by T0070907 meddled with differentiation inducing effects of CDDO as observed by stunted neurite growth, increased viability and decreased expression of differentiation markers. Our findings indicate that IMR32 differentiation induced by CDDO in combination with ATRA enhances, differentiation followed by cell death via cAMP-response-element binding protein (CREB) independent and PPARγ dependent signaling mechanisms.Neuroblastoma is the most common solid extra cranial tumor in infants. Improving the clinical outcome of children with aggressive tumors undergoing one of the multiple treatment options has been a major concern. Differentiating neuroblastoma cells holds promise in inducing tumor growth arrest and treating minimal residual disease. In this study, we investigated the effect of partial PPARγ agonist 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) on human neuroblastoma IMR32 cells. Our results demonstrate that treatment with low concentration of CDDO and particularly in combination with all trans retinoic acid (ATRA) induced neurite outgrowth, increased the percentage of more than two neurites bearing cells, and decreased viability in IMR32 cells. These morphological changes were associated with an increase in expression of bonafide differentiation markers like β3-tubulin and Neuron Specific Enolase (NSE). The differentiation was accompanied by a decrease in the expression of  Cell fate determination and differentiation are processes that are crucial in developmental biology, which assigns cells their functional roles and gives them unique identity. Cell differentiation is an essential process. It is a result of differential gene expression, epigenetic modifications, ceased proliferation, and morphological changes converting the precursor cells to specialized ones committed to a specific lineage Slack, . The difIn the first decade of twentieth century, three pioneers: James Homer Wright, William Pepper, and Robert Grieve Hutchison, contributed in delineating the pathology of neuroblastoma in pediatric patients with distinctive clinical presentations  and ursolic acid (UA) are known to have relatively weak anti-inflammatory and anti-carcinogenic activities. In order to increase these activities, new synthetic derivatives have been created . Media was supplemented with 10% fetal bovine serum , 1X antibiotic-antimycotic (HiMedia) and 1X glutamax (Gibco). CDDO and PPARγ antagonist (T0070907) were purchased from Cayman. ATRA was purchased from Sigma Aldrich. Stocks were prepared in DMSO.IMR32, a human neuroblastoma cell line was purchased from NCCS (Pune) and was maintained at 37°C in 5% humidified COFor inducing differentiation, IMR32 cells were seeded in a 6-well plate at a density of 50,000 cells per well and treated with CDDO and ATRA alone and in combination for 5 days and the differentiation inducers were replenished on day 3 by changing the media. Cells were pre-incubated with T0070907 for 2 h prior to addition of CDDO. To observe the cell morphology and neurites, cells were washed with DPBS, prefixed for 2 min in media containing 3.7% formaldehyde and further fixed with DPBS containing 3.7% formaldehyde at −20°C. Permeabilization was done using absolute methanol at −20°C and finally cells were stained with methylene blue solution (0.2% methylene blue in methanol). Cells were imaged on EVOS FLoid Imaging Station (Thermo Fisher Scientific) equipped with a monochrome CCD camera. Neurites were traced using ImageJ software with neuron growth plug-in and represented as average total neurite length (μm). For neurite length analysis, detectable neurites were measured from different focuses and cells without neurites were excluded from analyses. The total number and number of cells bearing more than two neurites were counted in 10 random focuses from independent experiments and reported as a percentage. Control cells received the vehicle alone.Cell viability was estimated by measuring cellular ATP levels using CellTiter Glo luminescent assay (Promega)   the cultured cells were isolated using RNAiso Plus (Takara). RNA quality and quantity were assessed by Nanodrop UV-VIS spectrophotometer (Thermo Fisher Scientific). Two micrograms of total RNA from each sample was reverse transcribed to cDNA using Prime Script RT reagent kit (Takara). Using SYBR Premix Ex Taq , RT-qPCR was performed on Applied Biosystems Step one plus PCR machine. The RNA 18S gene was amplified as an internal standard reference gene (invariant control). Fold changes in the target gene expression were normalized to 18S ribosomal RNA gene expression using comparative CT method  and anti-Neuron Specific Enolase (NSE) . Alexa Fluor® 594 Conjugate goat anti rabbit  was used as secondary antibody. Every step was followed according to manufacturer's instructions. The cells were counterstained with DAPI (HiMedia) in phosphate buffered saline for 5 min and the coverslips were mounted using fluoroshield . Fluorescent images were acquired on EVOS FLoid Imaging Station using 20X fluorite objective and LED light cubes containing hard coated filters (blue and red), (Thermo Fisher Scientific). Imaging parameters like brightness and contrast were kept constant throughout the experimental conditions for particular protein expression. No image modifications were done post imaging.2EDTA, 1 mM EGTA, 1% NP-40, 1% Sodium deoxycholate) supplemented with 1 mM PMSF, Protease inhibitor cocktail (Sigma Aldrich) and Phosphatase inhibitor cocktail . Cell lysates were passed several times through insulin syringe and incubated on ice for 30 min. Total protein was estimated by Lowry's method . Figure legends include mean ± SEM and p values for the statistical analysis performed.Statistical tests were performed using Graph Pad Prism6 software. Methylene blue staining and immunocytochemistry has been performed several times and reported as a representative image. All other experiments have been done twice and data is represented as an average of two independent experiments performed in triplicates except for trypan blue viability assay that was performed twice in duplicates. One-way ANOVA followed by Tukey's test was applied to compare more than two groups. Two-way ANOVA followed by Tukey's To test our hypothesis that synthetic triterpenoid CDDO could induce neuroblastoma differentiation; we initially performed a dose response experiment by treating IMR32 cells with various concentrations of CDDO  and observed for neurite outgrowth. We observed neurite outgrowth in treated cells at 0.5 and 0.7 μM of CDDO whereas slightly higher concentrations of (1–2 μM) induced cell death without displaying neuritogenesis in IMR32 cells. Similarly, a dose response experiment was performed with various concentrations of a known differentiation inducer, i.e., ATRA  and it revealed that ATRA 10 and 15 μM induced neurite outgrowth. Subsequently, IMR32 cells were treated with 0.7 μM CDDO individually, and also in combination with 10 μM ATRA for 5 days to check the combinatorial effect. For comparison, we limited the ATRA treatment period to 5 days. CDDO treatment as a single agent exhibited delayed morphological changes. However, in combination with ATRA, initial sprouting of neurite was observed as early as day 3 and eventually, a branched neurite network between cells became apparent on day 5 for both the treatment conditions. A comparatively high rate of differentiation was observed in the cells that received the CDDO and ATRA treatment in combination compared to the cells that were treated only with CDDO. Vehicle control cells failed to exhibit aforementioned morphological features and continued to proliferate. At first, methylene blue staining was performed to determine the differentiation features Figure .Neurite lengths were then semi-automatically traced on methylene blue stained images using ImageJ software  with neuron growth plug-in   to check the effect of serum mitogens on IMR32 differentiation. In the absence of serum mitogens, control cells, CDDO, and ATRA treated cells lost the viability in 48 h. In case of medium containing 0.5% serum, CDDO treated cells struggled to produce neurites and maximum cells morphologically appeared dead after 5 days. ATRA induced certain neurite formation in low serum conditions but not as effective as in 10% serum containing media. In addition, reduced proliferation and unhealthy morphology were observed in treated as well as non-treated conditions in low and no serum conditions  and NSE (encoded by ENO2) by RT-qPCR. Although not exclusive, the expression of β3-tubulin, a neuron associated tubulin isotype and NSE, a neuronal glycolytic isoenzyme is considered to be regulated during neuronal differentiation  expression was studied. NDRG1 is repressed in cell lines with MYCN amplification and this down-regulation is known to be associated with MYCN effects in neuroblastomas  in IMR32 cells. MYCN was observed to be amplified by 50-folds in IMR32 cells relative to normal placental DNA  is a tyrosine kinase receptor and a MYCN targeted oncogene. ALK can be mutated or amplified in neuroblastomas and contributes to the neuroblastomas progression. Also, ALK inhibition has been shown to decrease the biological effects of MYCN amplified cells/tumor growth in xenograft models  activation was studied to identify the signaling mechanisms independent of receptor binding. CREB phosphorylation at Ser133 is considered important for the binding of its co-activator CREB binding protein (CBP) to form a transcriptionally active complex  are key ligands for members of nuclear hormone superfamily which regulate retinoic acid mediated signaling -C cell lines have been earlier reported to promote cell survival during the differentiation process, suggesting a potential mechanism for neuroblastoma resistance to retinoic acid therapy (Qiao et al., in vivo and therefore combination therapy holds a chance in the treatment of neuroblastoma. The results contribute to the current knowledge on neuroblastoma differentiation by showing that CDDO induced neurite outgrowth, decreased viability, up-regulation of differentiation markers and down-regulation of active CREB were PPARγ dependent events in IMR32 cells. The highlight of this research in our opinion is that CDDO and ATRA together are capable of triggering events that help the IMR32 cells to differentiate and die. The in vivo efficacy of this combination in neuroblastoma xenograft models is yet to be studied.Differentiation therapy aims to reprogram cells instead of directly killing them and thus it is received as a treatment option that is mild on young children. Our concern was to focus on evaluating CDDO from a perspective of differentiation therapy instead of chemotherapy, which is known to have various side effects. Because retinoic acid is in clinical practice, we attempted testing the effect of CDDO in combination with ATRA on neuroblastoma differentiation. In conclusion, our findings provide early evidence that combination treatment would be a more effective choice than a single drug treatment in differentiating and killing aggressive cells like IMR32. Moreover, differentiated tumors are associated with favorable outcome. Additionally, many neuroblastomas are found refractive to retinoic acid therapy PR conceptualized and designed the work. NC performed the experiments. PR and NC wrote the manuscript. PT and CL contributed in the discussion.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aging-related tau astrogliopathy (ARTAG) describes tau pathology in astrocytes in different locations and anatomical regions. In the present study we addressed the question of whether sequential distribution patterns can be recognized for ARTAG or astroglial tau pathologies in both primary FTLD-tauopathies and non-FTLD-tauopathy cases. By evaluating 687 postmortem brains with diverse disorders we identified ARTAG in 455. We evaluated frequencies and hierarchical clustering of anatomical involvement and used conditional probability and logistic regression to model the sequential distribution of ARTAG and astroglial tau pathologies across different brain regions. For subpial and white matter ARTAG we recognize three and two patterns, respectively, each with three stages initiated or ending in the amygdala. Subependymal ARTAG does not show a clear sequential pattern. For grey matter (GM) ARTAG we recognize four stages including a striatal pathway of spreading towards the cortex and/or amygdala, and the brainstem, and an amygdala pathway, which precedes the involvement of the striatum and/or cortex and proceeds towards the brainstem. GM ARTAG and astrocytic plaque pathology in corticobasal degeneration follows a predominantly frontal-parietal cortical to temporal-occipital cortical, to subcortical, to brainstem pathway (four stages). GM ARTAG and tufted astrocyte pathology in progressive supranuclear palsy shows a striatum to frontal-parietal cortical to temporal to occipital, to amygdala, and to brainstem sequence (four stages). In Pick’s disease cases with astroglial tau pathology an overlapping pattern with PSP can be appreciated. We conclude that tau-astrogliopathy type-specific sequential patterns cannot be simplified as neuron-based staging systems. The proposed cytopathological and hierarchical stages provide a conceptual approach to identify the initial steps of the pathogenesis of tau pathologies in ARTAG and primary FTLD-tauopathies.The online version of this article (10.1186/s40478-018-0552-y) contains supplementary material, which is available to authorized users. Deposition of pathologically altered proteins in astrocytes has been increasingly detected in neurodegenerative diseases (NDD) leading to the concept of protein astrogliopathies . One maiThe concept of cell-to-cell propagation of neurodegeneration-related proteins was a significant step in our understanding of NDDs . In the n = 40), PSP (n = 97), PiD (n = 22), only PART (n = 70), only Alzheimer disease (AD) pathology (n = 243), and other disorders (n = 215) including multiple system atrophy (n = 34), Lewy body disorders (n = 120), and amyotrophic lateral sclerosis (n = 61). This pooled cohort of AD plus PART plus other disorders (n = 528) is termed here as non-FTLD-tauopathy group. Thus for better overview we did not include PART or the presence of AGD in the group of primary FTLD-tauopathies.This study includes 687 individuals from the collection of brains from longitudinally followed subjects in the Center Neurodegenerative Disease Research (CNDR) Brain Bank at the University of Pennsylvania, Philadelphia, PA Table  2, 55]., 55.2, 5Formalin fixed, paraffin-embedded tissue blocks from the investigated cases were evaluated. Immunostaining for tau was performed with anti-tau PHF-1 . The Vectashield ABC detection kit, peroxidase/DAB, rabbit/mouse/rat  was used for the visualization of antibody reaction.Following the consensus recommendations  we evaluFor the subpial and WM ARTAG we stratified the regions into three groups: brainstem, basal brain regions  and lobar. For lobar regions we performed a separate analysis for frontal, temporal, parietal and occipital lobes. For subependymal ARTAG we distinguished three regions: brainstem (aqueduct), 3rd ventricle and midline of the lateral ventricle together, and the inferior horn of the lateral ventricle. For GM ARTAG and primary FTLD-tauopathy related astroglial tau pathologies, we first stratified as brainstem, subcortical , MTL and lobar. Next, we performed a detailed comparison of nine regions . In the same regions we evaluated the tau pathologies using a semiquantitative score ; the median of these in each region was used to generate heatmaps , 35.Our approach contains 5 steps: 1) We select cases showing a specific type of ARTAG; then 2) describe patterns of involvements of different anatomical regions; followed by 3) comparison of different anatomical regions to calculate conditional probabilities, which region might precede another one; complemented by 4) logistic regression to correct for influencing factors; and finally 5) evaluating clusters of cases showing different patterns of ARTAG involvement.A being positive while region B being negative and the region A being negative and region B being positive is equally likely, thus A and B region is affected at the same time . McNemar’s test was used to assess the evidence against the null hypothesis. We used p < 0.01 to determine whether we can reject the null hypothesis. We generated a matrix for different ARTAG types involving various anatomical regions where each cell in the matrix corresponds to a conditional probability that one region is involved before another one. Conditional probability was calculated using crosstab function of SPSS.We applied conditional probability analysis as reported recently for the staging of TDP-43 pathology in AD . Accordip value we interpret this as high likelihood that two regions are affected together. In case OR < 1 with a significant p value we interpret this as low likelihood that the two regions are affected together, eventually meaning that they are affected independently from each other. For details on the methodological approach see Additional file If the conditional probability for one region is significantly higher than for the other region then we interpret that that this region is most likely affected before the other. However, this is interpreted analogously to the measurement of observer agreement for categorical data , 38. BinWe performed the statistical evaluation in six diagnostic groups (see case cohort). We report results for primary FTLD-tauopathies  and, in case there were no differences, the pooled cases of non-FTLD-tauopathies . We mention AD or PART cases separately only if a particular pattern is seen. We applied a significance level of 0.01 for McNemar’s test and 0.05 for logistic regression with multiple independent variables. We chose a lower significance level than the traditional 0.05 for McNemar’s test in order to reduce the likelihood of false positive findings. We provide the detailed tables of conditional probabilities and OR in the Additional file In addition, we performed a hierarchical cluster analysis using the nearest neighbour approach to evaluate how cases within disease groups cluster together based on the 1) the patterns of primary FTLD-tauopathy related astroglial tau pathologies; 2) GM ARTAG with astroglial tau pathologies; and 3) subpial, WM and GM ARTAG in three major regions , lobar and brainstem (any location for both) in different case-cohorts. In cases showing subpial ARTAG the highest frequency was observed in basal brain areas except for CBD and PiD (data not shown) where the lobar location was more frequent . This is followed by a bidirectional sequence rostrally  or caudally , which two, however, are usually affected together (stage 3). A second pattern  or in brainstem (stage 1b) or appear together (stage 2) and precede that in basal brain regions (stage 3). These two patterns are seen in the pooled cohort of non-FTLD-tauopathies. The third pattern  and both are followed by the involvement of the brainstem, representing stage 2. PSP cases show overlapping features of these patterns. Heatmap of severity scores in the cohort of non-FTLD tauopathies reveals also a MTL to temporal and frontal lobe to parietal to occipital lobe and parallel also to the brainstem  followed by lobar regions  or brainstem  and then all regions are involved (Stage 3)  is followed by the involvement of the basal brain regions (stage 2a) or occasionally the brainstem (stage 2b) and then all regions are involved (stage 3)  Fig. . However 3) Fig. . Evaluat 3) Fig. .Fig. 4FrNext we were interested in whether lobar WM ARTAG shows a sequential involvement pattern or not. We focused only on AD cases, since these show a high frequency of lobar WM ARTAG thereby rendering this analysis feasible. Briefly, lobar WM ARTAG is usually present in frontal, parietal, or temporal lobe usually in combination of these Fig. . Thus noFirst we evaluated the frequency and constellations of GM ARTAG in four major regions: MTL, lobar , subcortical  and brainstem (any location) in different case-cohorts. We observed different patterns  proceeds either towards the amygdala (stage 2a), cortex (stage 2b), or rarely to brainstem (stage 2c) followed by stage 3a  or stage 3b  and eventually involving all regions (stage 4)  precedes the involvement of the striatum (stage 2a), the cortex (stage 2b) or very rarely the brainstem (stage 2c). This is followed by three combinations of stage 3 , and eventually followed by the involvement of all regions (stage 4) . The striatal pathway  Fig. . Note thies Fig. . If we e 4) Fig. . This paPresence of GFA-like morphologies reveals different sequences in primary tauopathies. CBD is characterized by a fronto-parietal to temporal to occipital and to amygdala and to brainstem sequence represented by substantial to high  conditional probability values  is followed by temporal and occipital cortex (stage 2) parallel moving into subcortical areas including either or both the striatum and the amygdala (stage 3) followed by the brainstem (stage 4) including the substantia nigra followed by pons and medulla oblongata . The amygdala and striatum clearly precedes brainstem regions. Comparison of the striatum and amygdala show high values for both indicating that sequential involvement cannot be clearly defined for these two regions. It is important to recognize combined sequential patterns for GFA-like morphologies and mature astroglial tau pathologies. A four-staged sequence can be proposed: frontal (including premotor) and parietal cortex  to cortical  areas  to amygdala (stage 3) and to brainstem (stage 4), including the substantia nigra followed by pons and medulla oblongata, sequence can be recognized  when compared to the presence of grains as vice versa (0.009) Fig. . This phFinally, we were interested how many cases show primary-FTLD-tauopathy related astroglial tau pathology or GFAs without any neuronal tau pathology in the same region. Interestingly, cortical areas in primary FTLD-tauopathy cases not infrequently show astroglial tau depositions Table . In the First we evaluated clustering of cases based on the presence of primary FTLD-tauopathy-related astroglial tau pathologies  in three major anatomical regions . Two major clusters are seen in primary tauopathies  or dorsolateral parts of the brainstem. A second pattern, however, most likely begins in the convexity of the brain paralleled by the brainstem involvement but preceding the basal brain regions. Two aspects need to be considered for the interpretation: a pathogenesis related to the circulation of the CSF or local mechanical factors. During the circulation of the CSF from the lateral ventricles it enters the aqueduct and then the cerebellomedullary cistern at the brainstem level (i.e. via the foramen of Magendie and foramina of Luschka). The fluid then circulates in the subarachnoid space and reaches the basal areas before proceeding to the convexity. During the pulsatile flow of the CSF, the vascular expansion following cardiac systole occurs first at the base of the brain reversing then the flow of cisternal CSF superimposed by a circadian or diurnal rhythm . Thus, sThe existence of a second pattern of subpial and WM ARTAG raises the possibility of further pathogenic events such as a history of repeated mild traumatic brain injury (TBI) or a single severe TBI, where diffuse axonal injury across the WM is thought to be an important pathological feature . Regardistage 1) identified in CBD could be termed as “masked” bidirectional. This means that the basal brain regions-to-convexity and bidirectional to brainstem sequence seen in non-FTLD-tauopathies with the typical subpial TSA morphologies representing typical subpial ARTAG are masked by the predominant end-feet tau immunoreactivity.Importantly, subpial astroglial tau pathology is different in CBD, in particular in lobar areas, i.e., the convexity of the brain, where the morphology of subpial TSAs is different in CBD. These are mostly the astrocytic feet immunostained and less the cell body as in typical TSAs, although a few TSAs can be recognized as well. Lobar subpial TSA always associates with tau pathology in the GM and WM contrasting non-FTLD tauopathies where lobar subpial ARTAG can be present alone. The pathogenesis of subpial astrocyte feet tau immunoreactivity in CBD is most likely different from subpial lobar ARTAG. Therefore the sequence  the sequential stages can be better recognized Additional file 2:Pairwise conditional probability matrix and odds ratios of different ARTAG types. (PDF 876 kb)Additional file 3:Pairwise conditional probability matrix and odds ratios of grey matter ARTAG and primary FTLD-tauopathy associated astroglial tau immunoreactivities. (PDF 437 kb)Additional file 4:Hierarchical cluster analysis of astroglial tau pathologies in different disorders. (PDF 1463 kb)


Vicia faba, 2n = 12), a long-established model for cytogenetic studies in plants, contains a diverse set of satellite repeats, most of which remained concealed until their present investigation. Using next-generation sequencing combined with novel bioinformatics tools, we reconstructed consensus sequences of 23 novel satellite repeats representing 0.008–2.700% of the genome and mapped their distribution on chromosomes. We found that in addition to typical satellites with monomers hundreds of nucleotides long, V. faba contains a large number of satellite repeats with unusually long monomers (687–2033 bp), which are predominantly localized in pericentromeric regions. Using chromatin immunoprecipitation with CenH3 antibody, we revealed an extraordinary diversity of centromeric satellites, consisting of seven repeats with chromosome-specific distribution. We also found that in spite of their different nucleotide sequences, all centromeric repeats are replicated during mid-S phase, while most other satellites are replicated in the first part of late S phase, followed by a single family of FokI repeats representing the latest replicating chromatin.Satellite DNA, a class of repetitive sequences forming long arrays of tandemly repeated units, represents substantial portions of many plant genomes yet remains poorly characterized due to various methodological obstacles. Here we show that the genome of the field bean ( Except for the specific types of tandem repeats including rRNA gene arrays and telomeric motifs that have coding or structural roles5, the function of satDNA in the genome is still a matter of debate. It has been proposed that satellite repeats may have a structural role in the genome6 and that they affect expression of nearby genes by epigenetic modifications induced by specific changes in the environment7. Perhaps best documented is the frequent association of satellite repeats with centromeres, implying their importance for centromere determination or function8. On the other hand, it has been shown that neocentromeres may arise at satDNA-free regions9, and some established centromeres may be free of satellite repeats3. Thus, it is yet to be established whether satDNA is a key functional component of centromeric regions or whether centromeres merely provide favorable conditions for satDNA accumulation.Satellite DNA (satDNA) is a class of repetitive DNA characterized by its genomic organization into long arrays of tandemly arranged units called monomers. It is best distinguished from other tandemly repeated sequences by its formation of much larger arrays spanning up to megabases in length and often forming blocks of heterochromatin that appear as nuclear chromocenters and chromosomal bands. Although monomer sizes of 135–195 bp and 315–375 bp, corresponding to the length of DNA wrapped around mono- and di-nucleosome particles, were found to be predominant1. However, precise molecular mechanisms leading to this rapid turnover and their eventual regulation in individual species have not yet been elucidated. A variety of mechanisms have been proposed to generate short arrays of tandem repeats that may provide a template for further expansion. Such mechanisms include unequal crossing over of random sequences10, slipped-strand mispairing11, and sequence-directed mutagenesis12. Tandem duplications of varying length can also result from aberrant replication and replication stress15. In addition, satellite repeat arrays were found to originate from amplification of short tandemly repeated regions present in rDNA intergenic spacers and 3′ untranslated regions of Ty3/gypsy LTR-retrotransposons17. Regardless of the primary origin of short tandemly repeated loci, it is supposed that there are additional mechanisms that mediate their expansion into long arrays and subsequent concerted evolution of monomer sequences resulting in their genome-wide homogenization20. One of the potential mechanisms mediating amplification and sequence homogenization of satellite DNA is recombination-based formation of extrachromosomal circular DNA (eccDNA) from tandem repeats which in turn could serve as a template for their rolling circle replication and subsequent re-integration of the products. Although populations of eccDNA molecules derived from satellite repeats were successfully detected in a number of plant species22, the evidence for their amplification and re-integration into the genome is still missing. Other potential mechanisms of satDNA amplification include unequal chromatid exchange10 and segmental duplication23.SatDNA belongs to the most dynamic components of eukaryotic genomes, and its high evolutionary rate results in considerable sequence diversification. Therefore, most satellite repeat families are species- or genus-specific24. Alternatively, satellite repeats were often identified based on the presence of conserved restriction sites in their monomer sequences25. Consequently, the satellites lacking these features and those with small proportions in the genome were, in principle, hard to identify. On the other hand, novel sequencing technologies provide deep information about sequence composition of complex genomes of eukaryotes via generation of unprecedented amounts of sequence data. These data can then be utilized by bioinformatic pipelines specifically tailored to the identification of satellite repeats from NGS reads without the need for their assembly29. These approaches have proved to be very efficient and revealed surprising diversity of satellite repeat families in some plant and animal species31.To gain better insight into the biology of satellite repeats, comprehensive analysis of sequence diversity, abundance, and homogenization of satDNA families within and between species is needed. In spite of the relatively long history of satDNA investigation, such knowledge is still limited in several ways. Recently introduced methodologies utilizing a combination of next-generation sequencing (NGS) with appropriate bioinformatics tools revealed that previously used experimental approaches suffered from relatively low sensitivity, resulting in efficient identification of only the most amplified satellite repeats with specific properties of their sequences. For example, the very discovery of satDNA was achieved by density gradient centrifugation, whereby it was revealed as satellite bands formed due to the different buoyant density of satellite repeats compared to the bulk of genomic DNAVicia faba, a species that has long served as a model for cytogenetic studies in plants34. Owing to its relatively large genome (1 C = 13.41 Gbp) and small chromosome number (2n = 12), V. faba chromosomes are large and easy to investigate with cytogenetic techniques. Consequently, a number of features like bands corresponding to different types of chromatin and epigenetic modifications have been revealed; however, only a few are associated with specific genomic sequences36. In our previous study of the repeat composition of species from the legume tribe Fabeae31, V. faba was found to carry a large number of satellite repeats that together constituted 935 Mbp (7%) of its genome. Putative satellite repeats were identified based on the properties of cluster graphs obtained by similarity-based clustering of low-pass genome sequencing Illumina reads, as implemented in the RepeatExplorer pipeline27. These graphs represent the reads and their sequence similarities as nodes and connecting edges, respectively, and form globular or ring-like shapes in the case of tandem repeats. Such shapes, combined with other properties of the clusters, are reliable indicators of satellite repeats, regardless of their monomer lengths28. Over 30 putative families of satDNA were identified and partially characterized by these bioinformatics approaches28, in contrast with only four satellites  that had been previously reported in this species40. Here, we provide experimental verification for most of the satellite repeat families predicted by the bioinformatic analysis, their localization on chromosomes, and information about replication timing and association with different types of chromatin. Moreover, we employed ChIP-seq analysis using CenH3 antibody to identify centromeric satellites, revealing their surprising diversity.In this work, we focus on the characterization of the satellite DNA population in the genome of V. faba genome that were classified as putative satellites in our previous study31 were inspected manually, as well as by the TAREAN pipeline28, to reconstruct consensus monomer sequences from tandem repeats. All novel satellite repeats with an abundance exceeding 0.1% of the V. faba genome and selected representatives of less abundant satellites (Table in situ hybridization (FISH) on mitotic chromosomes. The selected families differed in their nucleotide sequences, and their genomic abundance ranged from 0.008 up to 2.72% of the genome, corresponding to a physical size between 1.1 and 365.1 Mb/1 C. Besides repeat families with a typical monomer length of hundreds of base pairs, there were some with substantially smaller monomers and an unexpectedly large number of 17 families with long monomers ranging from 687 up to 2033 bp  satellite TR-9 6, which probably originated from a degenerated simple sequence repeat, (GTT)n. Due to its simple sequence, this satellite showed high strand asymmetry values  had estimated monomer sizes between 687 and 2033 bp, thus being significantly larger than the previously reported preferred monomer length of 135–375 bpSelected clones were labeled and used as probes for FISH. Most of these long monomer satellites produced only a single hybridization signal, except for VfSat9 . The resulting reads were mapped to repeat clusters based on their sequence similarities, as were the reads obtained by sequencing DNA fragments extracted from chromatin preparations prior to ChIP (input control). A total of 21.3 million ChIP and 10.7 million input reads were mapped to reference clusters, and normalized ratios of ChIP to input reads were evaluated for the 500 largest clusters representing highly and moderately repeated sequences with genomic proportions of at least 0.002%. There were seven clusters that showed elevated ratios of ChIP/input reads (41- to 149-fold enrichment), indicative of their association with centromeric chromatin, whereas all other analyzed clusters showed ratios close to or below 1 . Depending on the time elapsed since the labeling pulse, the fixed material displayed labeling of early-, middle-, or late-replicating chromatin. Examples of labeled chromosomes are shown in Fig. V. faba satellites is not uniform  with similarities to satellite repeats from Pisum sativum, and VfSat4, which was related to VicTR-B repeats highly amplified in several Vicia species44 47. Long monomer satellites were also reported in Solanum species, including the Sobo satellite (4.7 kb monomer) of S. bulbocastanum48 and a diverse group of centromeric satellites with monomers up to 5.4 kb from S. tuberosum3 and S. verrucosum49. Most of these satellite sequences display similarities to various retrotransposons or have a complex structure indicating their origin from different genomic repeats49. These features indicate that the rye and Solanum long monomer satellites might be evolutionary young and mostly originate at specific genomic regions represented by dispensable B chromosomes and centromeres. However, none of the repeats reported here for V. faba had detectable sequence similarity to other genomic sequences, and only a small fraction was located in centromeres. On the other hand, most V. faba long monomer satellites displayed a preference for pericentric regions, the significance of which is yet to be investigated.Satellites with long monomers (0.7–2.0 kb) were found to be surprisingly numerous in the V. faba centromeric repeats, as the other three centromeric satellites had extremely short monomers, ranging from 44 to 50 bp. There is mounting evidence that most eukaryotic centromeres are determined epigenetically, independent of the underlying DNA sequence51. The frequent accumulation of satDNA in centromeric regions is then explained by its positive role in stabilizing centromeres, promoting deposition of inner kinetochore proteins, or simply by passive accumulation due to the absence of recombination-based elimination mechanisms53. Most higher plant species investigated so far have a single or only a few centromeric satellites with monomers hundreds of nucleotides long that are shared by all chromosomes54, an observation that is explained by their coevolution with kinetochore proteins55. Thus, the monomer length diversity as well as the overall number of different centromeric satellites, all of which are specific to a single V. faba chromosome, are unusual. Similar diversity has been reported in potato (Solanum tuberosum), where part of the centromeres contains chromosome-specific satellites, while the rest is free of satellite DNA3. Comparison of homeologous centromeres between potato and its wild relative S. verrucosum revealed that all but one of the centromeric satellites differ in their sequences, which, together with the absence of centromeric satellites on some chromosomes, indicates that centromeres in these species are evolutionarily novel and still undergo rapid cycles of satellite DNA expansion and contraction/elimination that precede fixation of a single satellite in most centromeres49. Even higher intra-specific diversity of centromeric satellites has been described in the pea (Pisum sativum), which was found to carry 13 sequence families differing in their genomic abundance and distribution on chromosomes41. The genus Pisum is closely related to Vicia, but chromosomes in Pisum and its sister genus Lathyrus exhibit a unique morphology of their centromeres, which are composed of multiple separated CenH3 loci arranged along extended primary constrictions56. While it was tempting to speculate that the extraordinary diversity of pea satellites originated from the evolutionary shift to its complex centromere structure, the diverse repeat composition of a simple V. faba centromere calls for the investigation of additional species from both genera to get more representative insight into evolution of their centromeres.The four satellite repeats with long monomers (1.7–2.0 kb) that were localized in primary constrictions of metaphase chromosomes were proved to be associated with centromeric chromatin using ChIP-seq with the CenH3 antibody. However, the long monomer size was not a universal feature of V. faba satellites during the late and the centromeric repeats in mid-S phase is in agreement with observations from other plant species57. Our data also suggest that, although the replication patterns were found to be conserved for specific satellite sequences regardless of their chromosomal location, the nucleotide sequences alone are not likely determinant of the replication timing, as we did not identify any conserved sequence features among synchronously replicating satellite repeats. Thus, other features like epigenetic modifications of chromatin proteins that were found to correlate with replication timing may be more important58. Indeed, FokI repeats were previously shown to be distinguishable from the rest of the V. faba genome by a specific combination of epigenetic marks36 which could also explain their partially different replication timing compared to other satellites.The replication of most V. faba chromosomes to investigate their eventual correlation with sequence composition and chromosomal distribution of novel satellite repeats. For example, previous studies revealed that the region located proximally on the NOR-bearing arm of chromosome 1 exhibits extreme sensitivity regarding misrepair of DNA damage, in particular after exposure to the mutagens mitomycin C and maleic hydracid, but also to other genotoxins60. This S phase-dependent misrepair yielded a high frequency of chromatid-type structural aberrations, such as isochromatid breaks, interstitial deletions, duplication deletions, and reciprocal translocations. The clustering of aberration breakpoints was likely due to misrepair of DNA double-strand breaks, which arise from the overlap of excision repair with replication during S phase. The misrepair results from the ligation of the wrong strand ends, favored by sequence homology with the break ends and/or by strand discontinuities at the border between regions with different replication timing61. The present study discovered that this aberration hotspot correlates with a region of unusual clustering of diverse satellite repeats, including five repeat families  mapped to two adjacent DAPI- and Giemsa-positive chromatin bands and one (VfSat2) located between them in a DAPI-negative area . Total genomic DNA was isolated from young leaves as described by Dellaporta et al.63.Seeds of the 31 via graph-based clustering of V. faba genomic shotgun reads using the RepeatExplorer pipeline27. Reconstruction of monomer sequences of selected satellites was performed using TAREAN28. Reconstructed sequences were used to design oligonucleotide probes for hybridization  using nick translation66. The oligo-probes were labeled with biotin or fluorescein at their 5′ ends during synthesis . FISH was performed according to Macas et al.67 with hybridization and washing temperatures adjusted to account for AT/GC content and hybridization stringency allowing for 10–20% mismatches. The slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), mounted in Vectashield mounting medium , and examined using a Zeiss AxioImager.Z2 microscope with an Axiocam 506 mono camera. Images were captured and processed using ZEN pro 2012 software (Carl Zeiss GmbH).Mitotic chromosomes were prepared from root tip meristems synchronized using 2.5 mM hydroxyurea and 2.5 μM amiprophos-methyl as described previously41 using a custom-made antibody raised against a peptide designed according to the CenH3 protein sequence identified in Vicia faba (CenH3-2_VF)56. ChIPed DNA and input DNA control were sequenced on the Illumina platform  in a single-end, 101 nt read mode. The resulting reads were trimmed to 100 nt by removing the first base and quality filtered to exceed the cutoff quality score of 10 over at least 95 nucleotides. Quality-filtered reads were mapped to reference contigs assembled from clusters of genome shotgun sequencing reads representing V. faba repetitive sequences produced and characterized in our previous work31. Similarity-based mapping of reads to repeat contigs was done using BLASTn68 with the parameters “-m 8 -b 1 -e 1e-20 -W 9 -r 2 -q -3 -G 5 -E 2 -F F” and was followed by output parsing to ensure that each read was mapped to a maximum of one repeat cluster with the highest similarity score. The proportion of ChIP and input reads mapped to individual clusters was evaluated to identify repeats with a ChIP/input ratio >10, which were considered to represent repeats enriched in the ChIP sample.Chromatin immunoprecipitation was performed with nuclei isolated from fresh leaves as described previouslyV. faba S phase was reported to last 7.5 h, followed by 5 h of G2 phase before entering mitosis69, this time sampling enabled observation of metaphase chromosomes with their DNA labeled at various stages of the S phase (late replicating chromatin was labeled in samples collected 1–3 h after EdU treatment while collecting tissues after 6–9 h provided information about early replicating chromatin). Tissue fixation was performed with methanol-acetic acid (3:1) and chromosome preparations were done as described above for FISH experiments. EdU detection was performed using EdU HTS kit  according to the manufacturer’s protocol, except for the washing procedure, which was done at 35 °C in 2× saline sodium-citrate (SSC) buffer for 5 min, followed by 5 min in 50% formamide/2× SSC, 10 min in 2× SSC, and finally 5 min in 1× BT buffer  at room temperature.Root tip meristems were treated with thymidine analog 5-Ethynyl-2′-deoxyuridine (EdU) by submersing the roots of three-day-old seedlings for 15 or 30 min in a 10 μM EdU solution in Hoagland medium. The EdU treatment was followed by incubating the seedlings in Hoagland medium for various time intervals ranging from 1 to 9 hours . Since Cloned sequences of satellite repeats were deposited in GenBank under accession numbers MF796528-MF796546. Raw Illumina reads from ChIP-seq experiment will be available from European Nucleotide Archive under the study PRJEB5241.Supplementary Information S1-S10