Histological grading of choroid plexus tumors (CPTs) remains the best prognostic tool to distinguish between aggressive choroid plexus carcinoma (CPC) and the more benign choroid plexus papilloma (CPP) or atypical choroid plexus papilloma (aCPP); however, these distinctions can be challenging. Standard treatment of CPC is very aggressive and often leads to severe damage to the young child’s brain. Therefore, it is crucial to distinguish between CPC and less aggressive entities (CPP or aCPP) to avoid unnecessary exposure of the young patient to neurotoxic therapy. To better stratify CPTs, we utilized DNA methylation (DNAm) to identify prognostic epigenetic biomarkers for CPCs.We obtained DNA methylation profiles of 34 CPTs using the HumanMethylation450 BeadChip from Illumina, and the data was analyzed using the Illumina Genome Studio analysis software. Validation of differentially methylated CpG sites chosen as biomarkers was performed using pyrosequencing analysis on additional 22 CPTs. Sensitivity testing of the CPC DNAm signature was performed on a replication cohort of 61 CPT tumors obtained from Neuropathology, University Hospital Münster, Germany.TP53. CPCs with homozygous TP53 mutations clustered as a group separate from those carrying a heterozygous TP53 mutation or CPCs with wild type TP53 (TP53-wt) and showed the worst survival outcome. Specific DNAm signatures for CPCs revealed AK1, PER2, and PLSCR4 as potential biomarkers for CPC that can be used to improve molecular stratification for diagnosis and treatment.Generated genome-wide DNAm profiles of CPTs showed significant differences in DNAm between CPCs and the CPPs or aCPPs. The prediction of clinical outcome could be improved by combining the DNAm profile with the mutational status of We demonstrate that combining specific DNAm signature for CPCs with histological approaches better differentiate aggressive tumors from those that are not life threatening. These findings have important implications for future prognostic risk prediction in clinical disease management.The online version of this article (10.1186/s13148-019-0708-z) contains supplementary material, which is available to authorized users. Choroid plexus tumors (CPTs) are rare neoplasms of the central nervous system. Within this family of tumors, choroid plexus carcinoma (CPC) is a malignant neoplasm, categorized as a grade III tumor by the World Health Organization (WHO). In contrast, choroid plexus papilloma (CPP) is benign and classified as a grade I tumor, while atypical choroid plexus papilloma (aCPP) is a grade II tumor . CPTs acDistinction between these tumor subtypes for accurate diagnosis is essential but can be challenging. Current diagnosis relies on assessment of histopathological features, i.e., mitotic activity, cellularity, and nuclear pleomorphism as well as the presence of necrosis and blurring of the papillary growth pattern . AlthougTP53 tumor suppressor gene, and TP53 mutant CPCs have been associated with increased genome instability and poor prognosis [TP53 mutations have also been observed in ~ 50% of children with CPC which is now considered a component tumor of Li–Fraumeni syndrome (LFS) [Over 50% of CPC tumors carry somatic mutations in the rognosis . Germlinme (LFS) . We haveme (LFS) . In factTo improve outcome prediction, more accurate molecular distinction among CPTs is needed. The power of DNAm to identify novel, more molecularly defined tumor subtypes has been established and led to improved stratification and specific tailoring of therapy for patients with a wide range of cancers –12. ReceAK1, PER2, and PLSCR4 as prospective diagnostic biomarkers for CPC and potentially tractable therapeutic targets.In this study, we performed a comprehensive analysis of DNAm in CPTs and discovered a highly sensitive and specific DNAm signature for CPCs which is able to segregate CPC from other CPT tumors as well as other brain tumors. This signature includes n = 53) or formalin-fixed paraffin-embedded  or optimal cutting temperature (OCT) compound (n=1) tumor samples. Tumor DNA was extracted using standard phenol–chloroform extraction from fresh frozen samples and the RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Ambion) from FFPE samples.Institutional Research Ethics Board approval was obtained for the study. Clinical data and tumor samples were collected from many sources, including two large pediatric neuro-oncology centers: the Hospital for Sick Children (SickKids), Toronto, Ontario, and the Children’s Hospital of Los Angeles (CHLA), Los Angeles, CA. The other contributing centers were as follows: St. Jude Children’s Research Hospital, Memphis, TN; the Collaborative Human Tissue Network (CHTN) in Columbus, OH; Schneider Children’s Medical Center, Tel-Aviv, Israel; Montreal Children’s Hospital, Montreal, Quebec; and University of Colorado Health Sciences Center, Denver, CO. Informed consent was obtained from the parents/legal guardians of all patients. Pathologic review of CPTs was conducted by Dr. C. Hawkins. In all other institutions, expert neuropathologists critically examined each case. All samples were processed as described in detail in our previous work . DNA wasOur primary cohort comprised 34 samples, including 15 CPPs, 5 aCPPs, and 14 CPCs. An additional validation cohort comprised 22 samples, including 4 CPPs, 3aCPPs, and 15 CPCs. We also used a replication cohort of 61 CPTs from Neuropathology, University Hospital Münster, Germany, for sensitivity testing of the CPC-specific DNAm signature . PatientTP53 (exons 2–11 as well as up to 50 bases into spanning introns of the TP53 gene) was performed in the molecular diagnostic laboratory at The Hospital for Sick Children in Toronto by direct Sanger sequencing of genomic DNA as previously described [Sequencing of the coding region of escribed .Genomic DNA ~ 1 μg) from CPT patients ). Mapping of CpG sites to regulatory genomic regions was done using the Illumina annotation.The data generated from the HumanMethylation450 BeadChip arrays were analyzed using the Illumina Genome Studio analysis software and included normalization, quality control measurements, and background correction. We removed probes targeting the X or Y chromosomes, probes near polymorphic sites , internal control probes, and non-specific cross-reactive probes . This fiU test at each CpG probe. For all subsequent investigations except the biomarker discovery, we applied the statistical significance level at a p value < 0.05 (after FDR correction) and an additional cutoff of at least 30% average methylation difference (Δβ) between the groups.To determine the sites that are differentially methylated between the carcinoma and papilloma groups, we used a non-parametric Mann–Whitney p value < 0.001 and average methylation difference Δβ ≥ 40%. Data were visualized in Qlucore Omics Explorer 3.3 and Partek Genomics Suite 6.6 software using heatmaps and principal component analysis (PCA) plots.For biomarker discovery, more stringent criteria were used, with a All differentially methylated genes identified in this study were analyzed using Ingenuity Pathway Analysis (IPA) software to identify pathways that are involved in the etiology of the carcinomas.www.cs.waikato.ac.nz/ml/weka) and R package caret [CfsSubsetEval–BestFirst to search for the most predictive non-redundant subset of CpGs using the correlation-based feature selection method. For the purposes of predictive analysis, all 34 CPT data samples were labeled as either “carcinoma” (CPC) or “papilloma” (including CPP and aCPP). Leave-one-out (LOO) cross-validation was applied where one sample was withheld; a new epigenetic signature was identified, and a predictive model was built on the remaining 33 samples and then applied to the withheld sample to predict its status as “carcinoma” or “papilloma.” Repeating this process for each of the 34 CPTs gave the measure of the predictive accuracy.Predictive analysis was performed using the Weka machine learning suite , we performed multivariate factor analysis of the DNAm levels along with other sample genotype and phenotype attributes to identify possible correspondence between them. Beta values in each of the 59 signature CpGs were analyzed jointly with attributes such as age, AK1 (cg14578146), PER2 (cg11903188), and PLSCR4 (cg07038342). All targeted assays were designed using the PyroMark Assay Design Software 1.0 (Qiagen). All primer sets are listed in Additional file Three genes were chosen as biomarkers that demonstrate the greatest degree of segregation between CPCs and CPPs, and the corresponding CpG sites were validated for differential methylation between these tumor groups using both the initial discovery panel of 34 samples and a validation set of 22 samples. Quantitative sodium bisulfite pyrosequencing was performed for Survival analysis was performed using the Kaplan–Meier method, and curves were compared using both log-rank and Wilcoxon–Gehan chi-square tests. Overall survival (OS) measured time from initial diagnosis to death from any cause or last follow-up.p value ≥ 0.55 after FDR correction in all 485577 CpG probes) but there was a significant difference in the CpG methylation profile between CPCs and CPPs or aCPPs . Although there were no significant differences in genome-wide DNAm between CPPs and aCPPs, there was a CPC-specific signature in comparison to CPP.Analysis of the genome-wide DNAm of primary CPTs did not show significant differences between CPPs and aCPPs  in the CPCs as compared to the CPPs  were hypomethylated and the remaining 1973 CpG sites (or 59%) were hypermethylated in CPCs compared to CPPs. Two-way clustering performed using Pearson’s correlation and average linkage for both the sample tree and the gene tree revealed segregation between the majority (86%) of CPCs and CPPs or aCPPs , than for the methylation group 3 that contained predominantly CPPs or aCPPs and had no death events. Methylation group 1 comprising only CPCs with homozygous TP53 mutation had a particularly poor prognosis compared to methylation group 2 which contained CPCs with heterozygous TP53 mutation or the TP53-wt .As shown in Fig. PTs Fig.  showed sp value < 0.05, with between 14 and 29 differentially methylated genes in each pathway) in comparison to CPPs  receptor subunits alpha 4-5 and gamma 3, as well as hypermethylation in the promoter region of subunit gamma 2 in CPCs compared to CPPs  software on a 1328 gene set overlapping the 3361 CpG probes. We identified nine canonical pathways that were significantly enriched in CPCs  in CPCs. The most significantly enriched categories in CPCs  were associated with cellular development, cellular growth and proliferation, cancer, cell death and survival, and cellular assembly and organization  (which are associated with open chromatin and hence active transcription), cancer-specific differentially methylated regions (cDMR), reprogrammed-specific differentially methylated regions (rDMR), and non-CpG island sites. However, we did not find any enrichment at CpG island shores and shelves , the 5′UTR region, and the first exon), which was inversely correlated with their expression in CPCs , which allowed us to extract 59 high-confidence differentially methylated CpG sites encompassing 33 candidate genes  for the validation of their differentially methylated status by targeted quantitative pyrosequencing. The selection was based on satisfying the following criteria: (1) CpG sites methylated in the promoter region, (2) inverse correlation between methylation and expression, and (3) known biological association with cancer. Results obtained by pyrosequencing using the discovery subset of 34 CPT tumors as well as the validation subset of 22 CPT samples are presented in Additional file We chose CpG sites representing 3 genes  cross-validation was used to determine the predictive accuracy.AK1 (cg14578146), PER2 (cg11903188), and PLSCR4 (cg07038342). Even with this minimal data representation, several predictive model types, such as logistic regression, multilayer perceptron neural network, and Naïve Bayes model, were able to predict carcinoma status on all left-out samples with perfect accuracy (area under ROC = 100% in each case). The logistic regression model showed that cg11903188 (gene PER2) was the most predictive of the three, with an odds ratio = 7.33, followed by cg07038342  and cg14578146 .First, we used only three CpGs as data attributes representing the minimal signature genes: p value < 0.001 after FDR correction and Δβ > 0.4 by magnitude). This signature was then used to build predictive models of several types, both without feature selection  and with feature selection to optimize model accuracy by using only the most predictive non-redundant CpGs. We applied several types of machine learning models available through the R package caret, such as logistic regression, support vector machines, and random forests, to detect carcinoma cases. In all such results, we achieved perfect 100% specificity, i.e., no false positives. However, the carcinoma sample CPC146-T was persistently misclassified by all models and occasionally the models also failed to detect another sample CPC153-T, thereby giving a sensitivity estimate between 85.7 and 92.9% . These observations match well with the results shown in Fig. Next, we explored the predictive accuracy and robustness of the full epigenetic signature using LOO cross-validation. A full new epigenetic signature was identified for each of the 34 cross-validation folds using the same parameters  of adult origin and analyzed them jointly with our CPT methylation data. The brain tumor DNAm datasets derived from the Illumina HumanMethylation450 array consisted of 28 diffuse intrinsic pontine gliomas (DIPG) (GSE50022) and 67 pilocytic astrocytomas (GSE44684), 12 embryonal tumors with multilayered rosettes (ETMR) and 28 primitive neuroectodermal tumors (PNET) (GSE52556) from GEO, and 24 glioma and 30 low grade glioma (LGG) from TCGA. The extracted data were combined with our CPT data, all samples were restricted to the CpGs comprising the CPC-specific signature, and hierarchical clustering was used to examine the separation of CPTs from other brain tumors  Data Portal  and some aCPPs as expected. The second cluster is divided into two subclusters cluster 2A and 2B) where one is enriched in mostly CPCs (2B) and the other one (2A) includes a histologically defined mix of CPCs, CPPs, and aCPPs Fig. b. HierarA and 2B  and someWe generated a genome-wide DNAm profile in CPTs to address our hypothesis that epigenetic alterations would be associated with specific phenotypes. We undertook a detailed approach to define the differences in DNAm signature for different groups of CPTs as one of the epigenetic factors responsible for CPC development and progression.TP53 mutation status. CPCs with homozygous TP53 mutations segregated from CPCs with heterozygous TP53 mutations or CPCs with TP53-wt and showed significantly worse survival outcome compared to CPPs and aCPPs or CPCs with heterozygous TP53 mutations or TP53-wt. This is consistent with our previous observation of the worst outcome for CPC tumors carrying homozygous TP53 mutations [TP53-wt CPCs cluster within group 2 (together with heterozygous mutated TP53) and in our cohort one of the TP53-wt patients did not survive, but two TP53-wt CPC patients cluster out of the CPC clusters (1 or 2), with CPPs in cluster 3 showing excellent survival. It is hard to predict, based on TP53 mutational status alone, which CPC patient with TP53-wt would have a better prognosis. By combining DNAm and TP53 mutational status, we show that we can distinguish between TP53-wt cases and predict the outcome.We showed significant genome-wide hypermethylation in CPCs compared to either CPPs or aCPPs, but no difference between CPPs and aCPPs. This could be in part due to the fact that we had a smaller number of aCPP cases. A total of 3361 differentially methylated probes showed segregation between the majority of CPCs and CPPs or aCPPs, thus distinguishing the aggressive form of CPTs, that is, CPCs from benign CPTs (CPPs or aCPPs). Unsupervised hierarchical clustering revealed two main clusters within CPCs based on utations . Some TPPathway analysis using IPA identified nine canonical pathways with GABA receptor at the top of the list that were significantly enriched in CPCs in comparison with CPPs or aCPPs. GABA neurotransmitter in the mammalian central nervous system (CNS) acts at either ionotropic (GABA(A)Rs) or metabotropic (GABA(B)Rs) receptors.Intriguingly, GABA has also emerged as a tumor signaling molecule in the brain and periphery that controls tumor cell proliferation , 18. RecAK1, PER2, and PLSCR4. This signature is highly specific when compared to other brain tumors highlighting its potential clinical diagnostic utility. When the CPC-specific signature was applied to a replication cohort of 39 samples (18 CPPs and 21 CPCs), CPCs were clustered together, separate from CPPs, but 2 histologically defined CPPs (CPP80g and CPP36g) clustered with CPCs. Thus, we are able to distinguish two distinct molecular subgroups, one comprised of a mixed population of histopathologically defined samples with a majority of one histologically defined subtype and one homogenous subgroup entirely comprised of CPPs.In this study, we demonstrate that DNAm profiling can distinguish aggressive forms of CPTs from benign forms of CPTs. We have generated a CPC-specific DNAm signature which includes several important genes such as Also, checking sensitivity of the CPC-specific DNAm signature in classifying the aCPP in the replication cohort of 61 samples , we found that aCPPs are distributed across different molecular subgroups. These data strongly suggest that our CPC-specific DNAm signature is more accurate in classifying CPT tumors based on their methylation profiles compared to histopathological classification alone and that the WHO classification of the CPT based on CPP, aCPP, and CPC can benefit from the addition of the molecular signature to provide a more accurate diagnosis that could impact patient clinical management.AK1, PER2, and PLSCR4. Methylation of the CpG sites in the promoter regions of these genes was more than 30% higher in CPC than in CPPs or aCPPs. This was correlated with significant downregulation at the mRNA level indicating that the transcription of these genes is epigenetically regulated in CPCs.The CPC-specific DNAm signature we described here comprised several genes including Emerging evidence has also revealed tight links between the regulation of cellular metabolism  and the Per1, Per2, and Per3 are reported in a spectrum of human cancers [The mechanism of dysregulation of circadian genes in cancers includes epigenetic silencing by promoter methylation. We suggest that loss of circadian homeostasis through circadian disruption may be a novel risk factor in choroid plexus tumorigenesis. Decreased expression through hypermethylation of CpG islands or aberrant acetylation in the promoters of the core circadian genes  cancers . More stPER2 is correlated with significantly lower expression of PER2 in CPCs compared to CPPs or aCPPs. PER1 and Cryptochrome-2 (CRY2) genes belonging to the core clock family of genes showed significantly lower expression in CPCs as well [In our study, hypermethylation of CpG sites in the promoter region of  as well .PER1 and PER2 genes are currently considered to be true tumor suppressor genes, as decreased expression of either (or both) has been reported in several types of human cancers [ cancers –35 and h cancers .Per2 is correlated with increased levels of Cyclin D and Cyclin E and accelerated tumor growth in vivo [Per1 or Per2 has been shown to inhibit the growth of cancer cells and increase their apoptotic rate [Cyclin A2 as well as Cyclin E1 and E2 in CPCs compared to CPPs or aCPPs further confirming a correlation between downregulation of PER2 and increased levels of Cyclin A and Cyclin E as one of the factors involved in CPCs tumorigenesis.Downregulation of  in vivo  whereas tic rate . In our tic rate , we obseThe findings generated from this study provide a framework for improved molecular stratification for diagnosis and treatment as well as the development of potential prognostic markers to better differentiate aggressive CPT tumors from those that are not life threatening. As well, some of the markers might be predictors of response to particular chemotherapeutic agents as previously reported .Furthermore, our study confirms the relationship between the circadian clock, cancer, and DNAm at clock genes and suggests that improper DNAm may alter clock gene expression, contributing to the etiology of CPCs. The distinct proliferation rhythm between tumor cells and normal cells  providesIn conclusion, we discovered a highly sensitive and specific DNAm signature for CPCs which is able to segregate CPC not only from other CPTs but also from other brain tumors. However, the study is not able to differentiate the proportion of the non-tumor cells. We demonstrate that heterogeneity in the clinical outcome of CPT patients cannot be predicted by histopathological assessment alone, but implementation of the specific DNAm signature for CPCs in association with the morphology of the CPT tumor can significantly improve diagnosis. This signature needs to be validated with larger sample numbers before being used in the clinical setting; however, it shows its usefulness in identifying cases which would otherwise be undertreated or perhaps some which would be overtreated. The intention of making this signature accessible to the scientific–medical community raises awareness of the existence of this potentially more accurate classification.CPC-specific DNAm signature into existing survival prediction based solely on histopathological criteria can significantly improve the estimation of disease outcome. Seven percent of patients could be classified as lower risk compared to 0% when using histopathological criteria alone, while the recurrence frequency measured by current criteria was underestimated when compared to frequency assessment based on specific DNAm signature for CPCs.Incorporation of DNA methylation profiling enables the subclassification of CPTs into 3 disease subgroups in routinely collected material, and the integration of CPC-specific DNAm signature can significantly improve prognostic risk prediction allowing for informed treatment decision, protecting some young patients from devastating and permanent neurological impairment due to aggressive treatment.Additional file 1:Table S1. Patient information for samples used in DNA methylation experiment. Table S2. Patient information for samples in replication cohort used in sensitivity testing of the CPC DNA methylation signature. Table S3. Multivariate factor analysis. Table S4. Primer sets for quantitative sodium bisulfite pyrosequencing. Table S5. GABA receptor DNA methylation status in CPTs. Table S6. Differentially methylated genes in cpc vs cpp showing differential expression and reverse correlation with DNA methylation in the promoter region of the gene. Table S7. Differentially methylated genes in cpc vs cpp showing differential expression and positive correlation with DNA methylation in the body of the gene. Table S8. 39 variables (encompassing 33 single genes) segregating CPCs and CPPs. Table S9. Comparison between CPC-specific DNAm signature classification and the Heidelberg classifier. (XLSX 108 kb)Additional file 2:Figure S1. Volcano plot showing significantly differentially methylated CpGs (yellow and blue) between CPCs (carcinomas) and CPPs (papilomas). The X-axis shows the difference between average DNAm levels in carcinomas and in papilomas, whereas the Y-axis shows the significance as the Mann–Whitney U p value . Each point represents a CpG position from the Illumina HumanMethylation450 BeadChip. CPC tumors show an overall predominance of hypermethylation (yellow) compared with CPPs across the signature CpGs, which were identified using the significance level p < 0.05 (or log10(p) > 1.30) and DNA methylation difference of at least 30%. Figure S2. Functional genomic distribution of CpG sites in CPCs. Genomic enrichment of the CPC DNA methylation signature is presented as percentage of all CpG sites on HumanMethylation450 BeadChip from Illumina (green) or of CpG sites derived from differential analysis using corrected Mann–Whitney U p value < 0.05 and at least 30% difference in average beta between CPCs and CPPs (orange). Figure S3. BoxPlot showing differential methylation of tested CpG sites between CPCs and CPPs or aCPPs. (A) Ak1-cg14578146, (B) PER2-cg11903188, (C) PLSCR4-cg07038342; the number of samples in discovery set was 34 and in validation set 22; Y-axis shows average beta values and X-axis tumor type: CPC = red, aCPP = pink, and CPP = turquoise. p value was generated using two group comparison (t test) and represents significance of the difference in methylation between CPCs and CPPs or aCPPs. ***p < 0.001. Figure S4. CPC-specific minimal DNA methylation signature. Heatmap (A) and PCA (B and C) of 3 differentially methylated CpG sites encompassing 3 candidate genes from the dataset of 59 CpG sites of CPC-specific signature. This minimal signature shows segregation between CPCs and CPPs or aCPPs. Hierarchical clustering was done using Euclidean metric. High methylation = yellow, low methylation = blue, discovery set (Illumina HumanMethylation450 BeadChip) on 34 discovery samples = orange, validation set (targeted quantitative sodium bisulfite pyrosequencing) on 34 discovery, and 22 validation samples = green; diagnosis: cpc = red, acpp = pink, and cpp = turquoise. The numbers 1, 2, and 3 in PCA plots represent component 1, component 2, and component 3. Figure S5. Correlation plot of DNA methylation values (%) in 34 DNA samples for each tested CpG obtained using the Illumina HumanMethylation450 BeadChip and pyrosequencing. High correlation between the two methodologies was observed with an r2 value of ≥ 0.9; r2—Pearson’s correlation coefficient. Figure S6. Hierarchical clustering of DNA methylation profile performed separately for each histologically defined group of 18 CPPs (6A) and 22 aCPPs (6B) from replication cohort. Hierarchical clustering was done using Euclidean metric. High methylation = yellow, low methylation = blue; diagnosis: cpp = turquoise and acpp = pink. Figure S7. Factor analysis of DNAm signature along with phenotype and genotype sample attributes. The heatmap shows the magnitude of factor loadings for n = 12 factors in each of the data attribute. The DNAm beta values at 59 CpG sites contribute strongly to factors 1–3 and 6–12, for which no other available data attributes contribute. Factor 4 is associated with recurrence status, death status, and P53 mutation status, and factor 5 with age. Several CpGs also show an association with these phenotype attributes, but generally less so that with the DNAm-related factors 1–3. Figure S8. Factor analysis of DNAm signature along with phenotype and genotype sample attributes, with recurrence attribute removed. The heatmap shows the magnitude of factor loadings for n = 12 factors in each of the data attribute. Factor 4 is associated with age, and factor 6 with death status and P53 mutation status. The DNAm beta values at 59 CpG sites contribute strongly to the rest of the factors, with the strongest association being in the top three factors. (DOCX 15 kb)Additional file 3:Multivariate factor analysis. (DOCX 16 kb)


In vitro selection with long random RNA libraries has been used as a powerful method to generate novel functional RNAs, although it often requires laborious structural analysis of isolated RNA molecules. Rational RNA design is an attractive alternative to avoid this laborious step, but rational design of catalytic modules is still a challenging task. A hybrid strategy of in vitro selection and rational design has been proposed. With this strategy termed “design and selection,” new ribozymes can be generated through installation of catalytic modules onto RNA scaffolds with defined 3D structures. This approach, the concept of which was inspired by the modular architecture of naturally occurring ribozymes, allows prediction of the overall architectures of the resulting ribozymes, and the structural modularity of the resulting ribozymes allows modification of their structures and functions. In this review, we summarize the design, generation, properties, and engineering of four classes of ligase ribozyme generated by design and selection. The 3D structures of these biopolymers often have a modular organization, in which the whole structure is divided into several structural domains ,2. In thD structuofactors  indicatein vitro selection as this technique enables identification of particular RNA sequences with desired functions from structurally unconstrained RNAs containing random sequences ranging from 30 to 200 nucleotides with 1014–1016 different nucleotide sequences .,20.19,20election , right ,3920,39]14–1016 different sequences, four distinct classes of artificial ribozymes with 3',5'-ligase activity were isolated through the selection procedure developed by Bartel and Szostak . T. Tin vitn system A, left aCoevolution experiments were performed using two distinct classes of ligase ribozymes (the class-DSL ribozyme and the class I ligase ribozyme) capable of undergoing continuous evolution . In the in vitro selection system to isolate novel RNA-RNA interacting modules that functionally substitute the GNRA/receptor module. Ohuchi et al. generated a novel RNA sequence that recognizes the C-loop motif found in ribosomal RNA (cis-DSL-1S in which the L1 GAAA loop and the R(11nt) motif were replaced with the C-loop motif and a random sequence of 19 nucleotides  receptor in P3 ,43. Thisomal RNA B 55]. U. Uin vitleotides B, left. isolated B, right in vitro selection. The design and selection would be applicable not only to generate novel ribozymes but also to construct novel aptamers and riboswitches because naturally occurring riboswitches often possess modular architectures in which RNA structural motifs such as GNRA-receptor interactions are used as key motifs to establish their 3D structures [Four classes of RNA ligase ribozyme have been generated using the design and selection strategy, the concept of which was inspired by the modular architectures of naturally occurring ribozymes and also protein antibodies. Modular architectures of these RNA ligase ribozymes allowed us to engineer their structures and functions more readily and quickly than ribozymes generated by ordinary ructures ,57. This


Our aims were to identify the phylogeographic pattern and mtDNA structure of the population and to evaluate the role of environmental conditions in shaping the spatial pattern of mtDNA diversity. We found out that three European haplogroups occurred sympatrically in north-eastern Poland. Inferences based on mtDNA haplotype distribution and frequency defined five subpopulations. The mtDNA-based structure of mice significantly correlated with winter temperature: frequency of Haplogroup 1 was positively, and that of Haplogroup 3 negatively correlated to mean temperature of January in the year of trapping. Synthesis of the published pan-European data on the species phylogeography also showed that the possibly 'thermophilous' Haplogroup 1 has the westernmost occurrence, whereas the more 'cold-resistant' Haplogroup 3 occurs much further to north-east than the other haplogroups. The observed patter may be a byproduct of the tight coevolution with nuclear genes, as we have earlier found that − in mice population in NE Poland − the spatial pattern of nuclear DNA was best explained by January temperature. Alternatively, the observed association of mitochondrial genetic variation with temperature is possible to be adaptive as cytochrome b is involved in the process of ATP production via oxidative phosphorylation.We analysed a fragment (247 bp) of cytochrome  Historical events such as isolation in glacial refugia during the Last Glacial Maximum (LGM), landscape discontinuities created by ice sheets and recolonization routes at the end of Pleistocene are still detectable in genomes of contemporary living animals. It is commonly believed that during the glacial maxima the ranges of temperate and boreal species were restricted to Mediterranean refugia –4. HowevApodemus flavicollis  one of the most common species of forest small mammals of temperate zone in Europe ..9% is sl2), productivity, and mean age of tree stands. The most common tree species were Scots pine (Pinus silvestris), Norway spruce (Picea abies), common oak (Quercus robur), silver birch (Betula pendula), white birch (B. pubescens), and common alder (Alnus glutinosa). The Białowieża Forest (BIAL) is the oldest and the best preserved woodland in the north-eastern Poland [The study area  spanned over a maximum distance of 230 km in the S–N and 180 km in the E–W direction. Majority of the sampling sites were located in managed forests, several in the national parks in BIAL and AUG forests and in small nature reserves in all forests. The woodlands differed in size  from mid June until early September in 2004–2006 and in four woodlands  and along three transects  from early July to late September in 2007–2008. Trapping and sampling protocols are described in details in Czarnomska et al. . In totaApodemus: wood mouse A. sylvaticus, striped field mouse A. agrarius and yellow–necked mouse [Tissue samples were stored in tubes with alcohol in –20°C upon DNA extraction. Genomic DNA was extracted using DNeasy Blood & Tissue Kit (QIAGEN) according to the manufacturer’s protocol. Quantity and quality control was performed using spectrophotometer NanoDrop ND–1000. A cytochrome b gene fragment (247 bp) of mitochondrial DNA (mtDNA) was amplified using primers designed to discriminate three sympatric species of ed mouse . AmplifiSequences of analysed mtDNA fragment were aligned and visually inspected in program BioEdit ver.7.0.9.1 . Obtaineet al., unpubl.; Guenther et al., unpubl.; Splettstoesser et al., unpubl.) available at NCBI GenBank website. Sequences deposited in NCBI database differed in length, therefore for a purpose of phylogenetic tree and haplotype network construction only a fragment of 247 bp used in this project was selected. It resulted in 52 additional sequences of A. flavicollis from Europe and Near East . The program implements the algorithm developed for MODELTEST [To determine the model of nucleotide substitution that best fitted our data, FINDMODEL software was used  were estimated using DnaSP ver. 5 . While hEach mtDNA haplotype was assigned to one of three confirmed haplogroups. For each study site (the forest or the transect) and trapping year, which had at least 5 assigned haplotypes, we calculated the proportion of haplotypes from each of the haplogroups as well as mean temperatures of January and July calculated for the year of sampling and the percentage cover of deciduous-mixed forests (preferred habitat of yellow-necked mouse). The cover of deciduous-mix ed forest was measured in 1-km buffer zone around trapping siteson the basis of the Corine Land Cover 2006 database  using the ArcView GIS software by ESRI (version 9.3.1).The mean temperatures of January and July were calculated by averaging 4 measurements per each month of land surface temperature with high spatial (grid 1×1 km) resolution , all variables  were included in the global model. We applied GLMM with observation-level random effect instead of generalized linear model (GLM) because results of the latter indicated an excess of variation in the data which caused considerable overdispersion.We applied an information theoretic approach with a second-order correction for small sample size  for binomial data using the “glmer” function implemented in the lme4 package . We set ze (AICc ) implemeze  obtained from the WorldClime database (resolution ~ 1 km2 ).A total of 24 mtDNA haplotypes (H1–H24), defined at 22 polymorphic sites (in which eight were parsimony informative), were recorded in 353 analysed samples, with only six of these reported previously in other studies . The remPhylogenetic relationship among 24 haplotypes found in NE Poland was expressed in the form of a Neighbour Joining phylogenetic tree and median-joining network . There wThe median-joining network  has a stet al. [et al. [et al. [et al. [Haplotypes derived from this project were assigned to three mtDNA subclades described by Michaux et al. . Haploty [et al. ), Haplog [et al. ) being i [et al. ). ComparSpatial distribution of identified haplotypes  showed tCT value occurred between K = 3 and K = 5 and all parameters of the Φ–statistics stabilized at K = 5 . The primal division (for two groups) corresponded to separation of PIS from the remaining regions. The highest increase in Φat K = 5 . Thus, 5at K = 5 . Mice frat K = 5 . This grAlthough there was no clear pattern in spatial distribution of single haplotypes, such pattern was visible at the level of haplogroups. Haplotypes belonging to Haplogroup 1 dominated in the southeastern part of the study area . Haplotypes of Haplogroup 2  made up 27% of all identified haplotypes in the whole study area and were most common in S4–34% . HaplotyST values calculated among five subpopulations (S1–S5) were moderate but highly significant  but haplotype diversity did not show such correlation .Pairwise Фnificant . Definednificant . The higc scores  and negative effect on the probability of haplotype assignment to Haplogroup 3 . With increasing mean temperature of January from -14.8°C to 0.1°C the probability of haplotype assignment to Haplogroup 1 increased from 7 to 56%, while the probability of haplotype assignment to Haplogroup 3 decreased from 70% to 10%  among either mean January or mean July temperatures in study sites, where different mtDNA haplogroups were found . However, the study sites, where haplogroup 3 occurred, indeed spanned over colder January temperatures <-13.1oC, 3.4oC> than those of the two other haplogroups  and might be related to high habitat fragmentation observed in that forest [Haplotypes found in north–eastern Poland were not geographically fixed but substantially differed in frequency among the studied forests. Inferences in program SAMOVA defined five genetic groups with subpopulation inhabiting Pisz Forest being genetically most distinct from the remaining samples. Such distinction of Pisz Forest was supported furthermore by genetic differentiation indices  was found in Belarus [Twenty-four mitochondrial cyt  Belarus . It migh Belarus , 13.Low level of differentiation between mtDNA haplotypes recorded in yellow-necked mouse in north–eastern Poland resulted in star–like shape of the median–joining networks, which is in high concordance with published data , 13: theThe network shape showed that the two haplogroups (1 and 3) stem from H7 and H2, respectively. H7 occurs widely in Europe, whereas H2 has mostly been recorded in eastern Europe see .et al. [Apodemus sp. in Mediterranean refugia, southern France and Black Sea area, but additionally suggested the southern parts of Russian plains as an area with suitable climatic conditions for temperate species during the glacial maximum. Results of our study also suggest the additional, eastern source of the contemporary European population. It might indicate better adaptation to harsh winter conditions in mice which exhibit cytochrome b gene variants of Haplogroup 3, presumably of eastern origin.It was commonly believed that during the glacial maxima, the occurrence of temperate and boreal species was restricted to the Mediterranean refuge areas , 60. Baset al.  supporteMean January temperature in the field work period in NE Poland correlated with the frequency of mtDNA haplotypes belonging to Haplogroup 3 (negative correlation) and Haplogroup 1 (positive correlation with temperature). Such a strong regional pattern may reflect a genuine genetic structure of the examined population only, as the pan-European spatial ranges of possibly 'thermophilous' Haplogroups 1 and 2 and more 'cold-resistant' Haplogroup 3 largely overlap see .b gene encodes an integral membrane protein component of the cytochrome bc1 complex, which catalyzes the redox transfer of electrons from ubiquinone to cytochrome c in the mitochondrial electron transport chain. Several investigators have already suggested that functional modifications of enzyme involved in mitochondrial oxidative phosphorylation (OXPHOS), such as cyt b, may be involved in physiological adaptation to different thermal environments [Mustela nivalis it was found that climate significantly affect the current distribution of the Carpathian lineage [The mitochondrial cytochrome ronments –24. Mostronments –23, 62 oronments , 25. For lineage  showed tImportantly, ATP production via OXPHOS complexes critically depends on interaction between mitochondrial and nuclear genomes (mitonuclear interactions), which evidences that the evolutionary trajectories of the two genomes are tightly interlaced –64. InteAlthough further studies are needed to make firm conclusions, one may suggest that low January temperatures may have impact on contemporary populations of mice by inducing higher survival rates of individuals that belong to mtDNA Haplogroup 3 and microsatellite Cluster 1. It was already found by Wójcik  that − ib haplotypes (including 18 new ones) were identified in NE Poland. They exhibited an overall low level of differentiation and were assigned to three known European haplogroups. The results suggested presence of additional, eastern source of contemporary European population of yellow-necked mouse, apart from the recognized Balkan LGM refugium. The majority of haplotypes occurred over the whole study area but they considerably differed in frequency among regions. SAMOVA defined five genetic groups with subpopulation inhabiting Pisz Forest found to be the most distinct and separated from the remaining samples. Individuals from Borki, Rominta and Białowieża Forests formed three distinct subpopulations. Mice from Augustów, Knyszyn, Mielnik Forests and the three studied transects were clustered in one, yet geographically disjunct, subpopulation. SAMOVA results suggested a high gene flow among local populations and among the defined groups. Mean January temperature was the most relevant environmental predictor of the prevalence of mtDNA haplotypes belonging to Haplogroups 1 and 3. However, to confirm the hypothesis that mice with haplotypes from Haplogroup 3 (presumably of eastern origin) have better adaptation to survive harsh winter conditions, further studies are needed.A total of 24 mitochondrial cytochrome S1 TableNumbers of identified mtDNA haplotypes that belong to each defined mtDNA haplogroup.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableThe numbers of haplogroup of each of the haplotypes are in brackets.(DOCX)Click here for additional data file.S4 TableSee (DOCX)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 FigSC−proportion of the variance among local populations within groups. ΦST−proportion of the variance among local populations within the total population. ΦCT−proportion of the total variance explained by the grouping.Φ(PDF)Click here for additional data file.


LoxP conditional knockout animals have become a prominent tool to understand gene function in discrete cell-types and neural circuits. However, this technology has significant limitations including off target cre-dependent recombination. The Rgs9cre strain has been used to generate a conditional knockout in striatal medium spiny neurons, but, as presented in the current study, off target recombination in the germline results in nonconditional deletion of LoxP alleles. Using a Rem2 conditional allele, germline deletion (GD) was observed in a sex dependent manner. When Cre and LoxP alleles were co-inherited from the female parent, 27 of 29 LoxP alleles were recombined, but when co-inherited from the male parent, 5 of 36 LoxP alleles were recombined. Rem2 expression measured by RT-qPCR confirmed nonconditional recombination in extrastriatal nuclei. Cre-LoxP is a powerful technique to modify genomic DNA (gDNA), however careful characterization of these mice is required to confirm control of conditional recombination.Cre- LoxP technology have been instrumental in dissecting the function of proteins in neurophysiology by allowing gene deletion or gene expression with temporal, tissue and cell-type specificity. This technology exploits the function of a bacteriophage P1 topoisomerase, Cre-recombinase (Cre), to recombine genomic DNA (gDNA) that is flanked by a pair of Cre recognition sequences call LoxP sites. In many experimental designs, mutant mice containing a floxed  gene of interest are bred with mice expressing Cre driven by a tissue or cell-type specific promoter. These Cre-driver lines are generated by identifying a gene with advantageous expression patterns and inserting the Cre coding sequence into either the endogenous gene locus or a bacterial artificial chromosome (BAC) harboring the gene promoter of interest Arte; Liput et al., LoxP sequences flanking exons 2 and 3  or the entire floxed DNA sequence containing exons two and three  and PCR control primers .gDNA was isolated from ear biopsies using the DNeasy kit (Qiagen) and polymerase chain reaction (PCR) was performed using GoTaq DNA polymerase (Promega). For genotyping the Rem2 mRNA quantification, striata and hippocampi were dissected and placed in Trizol regent for isolation of total RNA using the RNeasy Lipid Tissue kit (Qiagen). For Cre mRNA quantification, total RNA was purified from ovaries and testis according to the RNeasy Mini kit (Qiagen). After purification, 500 ng (brain tissues) or 1 μg (ovaries and testis) of RNA was reverse transcribed using the QuantiTect Reverse Transcription kit (Qiagen) and diluted to an estimated 5 ng/μL (cDNA was not diluted for Cre reactions). qPCR was performed in triplicate on a StepOnePlus thermocycler using 2 μL (5 μL for Cre reactions) cDNA template, Universal Master Mix II, and predesigned TaqMan Gene Expression Assays  for Rem2 , Cre (Mr00635245_cn), and actb (4352933E). Amplification was quantified using the 2−ΔCT method with actb serving as the normalization control.For RT-qPCR data was plotted as mean ± SEM and analyzed by two-way ANOVA. For all tests α was set at 0.05.Rem2 floxed allele  or the entire floxed region . However, none of the 115 mice genotyped for the current study were genotyped GD homozygous, which indicates Cre expression and recombination occurred in the germline, prior to fertilization. Additionally, some mice had a GD allele, but were Cre negative, further indicating that recombination occurred during gametogenesis prior to generation of the final haploid gametes.The current report demonstrates germline recombination of Cre expression patterns are validated by crossing a Cre-driver mouse with a floxed reporter  mouse and observing reporter expression in mice from the F1 generation . Lastly, it is important to report detailed breeding schemes used for generation of experimental animals and methods for validating conditional knockouts so that the possibility of GD deletion can be assessed when interpreting the presented data (Song and Palmiter, As put forth by others (Schmidt-Supprian and Rajewsky, e Figure . Second,DL designed the experiments, collected and analyzed data and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Patients with HIV and opportunistic infections (OIs), such as tuberculosis and cryptococcosis, have been recognised to have immune reconstitution inflammatory syndrome (IRIS) following initiation of highly active antiretroviral therapy (HAART). The initiation of HAART boosts CD4 T cells, which result in exacerbations of OIs. We have identified deterioration of neural lesion in imaging after the initiation of HAART in a patient with HIV/cryptococcal infection, although patient's clinical conditions have not deteriorated. It is evident that IRIS is in subtlety. This has been possible because of prompt treatment with antifungal drugs for the cryptococcal infection, followed by early initiation of HAART. Patients with HIV and opportunistic infections, such as tuberculosis and cryptococcosis, have been recognised to have immune reconstitution inflammatory syndrome following initiation of highly active antiretroviral therapy (HAART). The neurological manifestations following HAART may be devastating. Cryptococcal meningitis-immune reconstitution inflammatory syndrome (IRIS) reportedly has an incidence rate of 13%-30% in patients with HIV coinfections . These fA 32-year-old HAART-naive HIV seropositive male weighing 48 kg presented with forgetfulness, irritability and incontinence of urine, along with fever and headache for the last two months. His CD4+ cell counts were 36/μl, and he was seronegative for hepatitis B and C. He did not experience seizure or took anti-epileptic medication. Clinical examination revealed neurocognitive impairment with a mini-mental status score of 14/30. There was no focal neurological deficit. A computed tomography (CT) of the brain Figure  showed aFluconazole (200 mg per day) was continued along with HAART. At the end of six months, CD4+ cell counts were up to 246/μl and a further CT image Figure  showed aThe hypodense lesion Figure  is the rCattelan et al.  have repIn a retrospective study between 2005 and 2014, the imaging pattern in patients receiving HAART is found to be different from neuroimages in patients without HAART. All the patients on HAART show leptomeningeal and parenchymal enhancement in cortical and subcortical region of brain under immune reconstitution, while those without HAART have pseudocysts and lacunar infarcts .About one-third cases of cryptococcal meningitis experience mild to devastating form of IRIS after the initiation of HAART. In our case, neuroimaging enhancement in the left frontal lobe of the brain without any clinical exacerbation following HAART has been observed. This subtle form of IRIS is the result of rapid treatment of cryptococcal infection with antifungal drugs and prompt initiation of HAART.


Purpose: Application of Mummy material for treatment of different diseases such as bone fracture, cutaneous wounds and joint inflammation has been advised since hundred years ago in Persian traditional medicine. Due to the claims of indigenous people and advice of traditional medicine for application of this material in healing of bone fractures, this study has been designed to evaluate whether Mummy material can promote the differentiation of mesenchymal stem cells into osteoblasts and enhance the expression of bone specific genes and proteins.Methods: Adipose derived stem cells (ASCs) at fourth cell passage were divided into control, osteogenesis group (received osteogenic medium), Mummy group (received Mummy at concentration of 500 µg/ml). ASCs in the fourth group were treated with both osteogenic medium and Mummy (500µg/ml). Cells in all groups were harvested on days 7, 14 and 21 days for further evaluation through Real time RT-PCR, Von kossa staining, Immunocytochemistry and flowcytometery.Results: Treatment of ASCs with Mummy at concentration of 500µg/ml promotes the expression level of Osteocalcin, RUNX-2 and β1-integrin genes in different time points but that of the Osterix did not changed. Furthermore the expression of Osteocalcin protein enhanced significantly in ASCs treated with Mummy detected by Immunocytochemistry and flowcytometery technique compared to the control groups. The results of this study also showed that treatment of ASCs with Mummy resulted in formation of mineral deposits which was evaluated by Von Kossa staining method.Conclusion: Obtained data from this study reveals that Mummy is a potent enhancer for differentiation of ASCs into osteoblasts in in vitro system, probably through increasing the level of bone specific genes and proteins. On the other hand bone defects can also occur due to trauma, surgery, tumor metastasis and metabolic diseases, so finding efficient therapeutic methods for bone defects is of great importance.Bone is a specialized type of connective tissue which is composed of two different cells named as osteoblasts and osteoclasts.in vitro production of bone constructs produced by tissue engineering technique has been introduced as a new strategy for treatment of bone disorders.5 Successful tissue engineering can be achieved through application of biodegradable scaffolds seeded by adequate cells such as mesenchymal stem cells or osteoblasts. Mesenchymal stem cells are multipotent and undifferentiated cells which exhibits potential for differentiation into variety of cells including chondrocytes, adipocytes and osteoblasts.6 These cells can be harvested from different sources such as bone marrow, umbilical cord Wharton's jelly, muscular and also adipose tissue.7 Among different sources, adipose tissue has been introduced as a suitable candidate because of its wide distribution through the body, easy access, less morbidity and great number of stem cells which can be extracted from it.8 During embryonic development, osteoblasts are derived from mesenchymal stem cells. Osteogenic differentiation of these cells is regulated by different specific transcription factors such as osterix and RUNX-2(Runt-related transcription factor-2).4 These key factors modulate the mesenchymal stem cells commitment into osteoblasts through expression of bone marker genes such as Alkaline phosphatase (Alp), collagen type, osteocalcin and osteopontin.12Recently, 13 For improving these cell-based therapeutic methods, growth factors and cytokines have been applied to enhance bone repair.15 Despite of advantages, considering to the high costs and rapid degradation the clinical application of these factors is limited.16 Therefore there is a continuing and urgent need to develop alternative agents with higher efficacy, less side effects and lower costs. In recent decades, researcher's focus has switched on application of natural compounds derived from several plants with positive effects on bone repair.17 Over hundreds of years, in Persian traditional medicine, Mummy has been used as a healer for different diseases as bone fractures, joint inflammation, cutaneous wounds and gastric ulcers.18 Mummy which is named also Mumnaye by local people is a dark brown, semi-solid and pitch like material found in some cracks of rare caves and secreted due to oil oxidation.19 Chemical analysis revealed the presence of calcium, phosphate, carbonate, magnesium, oxygen, nitrogen and also polysaccharide in this material. A clinical investigation performed by Dehghan et al.(2012) showed that Mummy can enhance the healing process of tibial and femoral fractures and reduce the complications.18As it has been previously noted, in recent decades bone tissue engineering has been emerged as a successful technique for treatment of bone defects.In consideration to the probable effects of Mummy in acceleration of bone healing claimed by indigeneous people, the present study was designed to evaluate whether Mummy can enhance the differentiation of Adipose derived mesenchymal stem cells (ASCs) into osteoblast through expression of major transcription factors as RUNX-2 and osterix and also osteogenesis-related marker named osteocalcin.20 After transferring to the culture lab, samples were washed ascetically using phosphate buffered saline (PBS) containing 1% Penicillin/streptomycin (P/S) for 3 times. Mechanical digestion was performed by mincing the adipose tissue samples using sterile scalpel, and then followed by enzymatical digestion. Through incubation of samples in collagenase type I (0.2% in free DMEM) per each gram while shaking in water bath for about 60-90 minutes. After digestion of main pieces, DMEM medium containing 10% fetal bovine serum(FBS) was added for neutralizing the enzyme activity, and then filtered through 70µm cell strainer for isolation the undigested particles. Obtained cell suspension was centrifuged for 10 minutes at 1500 rpm and then cells were plated at density of 5×105/ T75 flasks in 5% co2 and 37°c.After reached about 70% confluency, cells were trypsinized and expanded. Adipose derived stem cells (ASCs) at fourth passage were used for this study.Adipose tissue samples were obtained from patients undergoing laparotomy. Isolation of mesenchymal stem cells from adipose tissue was performed as described previously by Fathi et al. (2017).21 Mummy can be solved completely in water, so it was solved in DMEM culture medium and then passed through syringe filter (0.22µm) for sterilization.Mummy was purchased from local botanical markets in Kermanshah, Iran. Mummy was used at concentration of 500µg/ml in this study; it should be noted that the selection of Mummy material concentration was done based on the previous evaluations has been performed by our research team.th passage were divided into different groups as: control (group I), which received serum DMEM(0/05% FBS), osteogenesis group (group II) which treated with osteogenic induction medium containing DMEM base medium, 10-7 mol dexamethasone , 10mmol β-glycerophosphate  and 50µmol ascorbake-2-phosphate , ASCs in third group (group III) were treated with osteogenic medium and Mummy at concentration of 500µg/ml and in fourth group (group IV), ASCs received Mummy (500µg/ml). Cells were harvested on 7, 14 and 21 days for further evaluation through Real time RT-PCR, Von kossa staining, Immunocytochemistry and flowcytometery.To evaluate whether Mummy can affect osteogenesis in ASCs, cells at 4The gene expression profile for Osterix, RUNX-2, Osteocalcin and β-1 integrin in different groups was detected by Real time RT-PCR technique.2O was added to samples to obtain RNA. Approximately 1000µg/ml of total obtained RNA was used for cDNA synthesis using reverse transcription kit .In the next step using PCR Rotorgene 6000.  and SYBR Green PCR master mix ,Real time RT-PCR reactions were performed. For performing this method, total cellular RNA was extracted using YTA mini kit  according to instructions. Briefly cell lysis was done using RB buffer and then mixture was transferred to the collection tube. After centrifugation, 70% ethanol was added to each sample. Subsequently RNase-free HMonolayer cultivated ASCs were stained with Von kossa for observing the mineralized depositions. For performing the staining protocol, cells in different groups were fixed by ethanol and then washed 3 times with distilled water (D/W). Then incubation with silver nitrate was performed and followed by incubation in pyrogalic acid 1% for 5 minutes. After washing with D/W, cells were fixed with sodium thiosulfate (5%) and finally counterstained with nuclear fast red solution (0/1%) for observing the nuclei.3 cells were seeded in an 8-wells cell culture chamber slide. After one day of culture, the cells were washed with PBS and then fixed in 4% paraformaldehyde for 30 minutes at room temperature. After fixation, the cells were washed twice with PBS, and once with PBS and 3% FBS. The cells were incubated overnight at 4ºC with a 1:100 dilution of mAbs against osteocalcin  in PBS. The cells were then washed with PBS, and incubated with a 1:500 dilution of the biotin-conjugated mouse monoclonal IgG1 antibody against rats in PBS and 1% BSA overnight. After washing with PBS, a 1:500 dilution of secondary antibody  was added for 2 hours at 37°C. The cells were washed three times with PBS and the nuclei were stained with DAPI for 30 seconds. After three washes with PBS, the fluorescent cells were visualized under the fluorescence microscop.22A total of 200 × 106 cells were washed twice with PBS containing 3% FBS (washing buffer), were blocked by blocking reagents, were fixed with FCM Fixation Buffer and were incubated with osteocalcin primary antibody  for 30 minutes on ice. After terminating the incubation time, the cells were then washed with washing buffer, and incubated with a PE-conjugated gout monoclonal IgG1 antibody against mouse for 30 min on ice. hADSCs were washed with washing buffer again and FACSCalibur system  was used for determing the osteocalcin protein expression as intracellular staining. Data were analyzed using with the FlowJo software (version 6.2).Approximately 10Statistical difference between different groups was determined by Two-Way ANOVA method and followed by t-test post test. The results are shown as the mean ± SD of a representative experiment performed three times. Data shown are representative of three independent experiments.1-integrin gene expression was evaluated to examine whether Mummy can enhance the cell-cell and cell-matrix adhesion which is crucial for cell survival. The effect of Mummy on differentiation of ASCs to osteoblasts were evaluated through expression of osteocalcin, RUNX-2 and osterix genes, also βIn this study the expression level of osterix as an essential marker for bone formation also was evaluated to find whether treatment of ASCs with Mummy can enhance its expression. The expression level of β1-integrin in different groups at different time points has shown in Von kossa staining method was performed to evaluate mineral deposition in different groups after 7 days of culture. As it can be found in As shown in In the present investigation we examined whether Mummy can enhance the differentiation of ASCs into osteoblasts. The result of this study showed that: Treatment of ASCs by Mummy at dose of 500µg/ml increased the expression level of RUNX-2, osteocalcin and β1-integrin in different time points, in the presence of Mummy the expression of osteocalcin protein detected by Immunocytochemistry technique was increased, and treatment of ASCs with Mummy (500µg/ml) resulted in formation of mineral deposition which was detected using Von Kossa staining protocol.23 For performing a successful tissue engineering on this field focused on finding an ideal cell source.24 Identifying mesenchymal stem cells (MSCs) opened a new frontier for skeletal tissue engineering approaches. Adipose derived mesenchymal stem cells(ASCs) with potential for differentiating into chondrocytes, adipocyte and osteoblasts are introduced as a suitable candidate for bone regenerative techniques.23 Another factor for enhancing bone regeneration and promoting the differentiation of MSCs into osteoblasts is the use of anabolic growth factors.14 Despite of many advantages, application of this factor for tissue engineering approaches has its own limitations such as production costs and short half life, so finding other enhancers with more effectiveness and less cost is of great importance.Recently cell-based bone tissue engineering technique has been emerged as a new therapeutic method for bone reconstruction.25 Since hundreds of years Mummy has been advised for treatment of bone fracture in Persian traditional medicine. Due to unclear underlying mechanisms of this material in fracture healing, we designed this study to investigate whether Mummy can enhance the differentiation of Mesenchymal stem cells into osteoblasts through upregulation of specific bone transcription factors and ECM components.In most Asian countries application of natural-based products hold great promise for treatment of different diseases such as bone deformities and fractures.In this study we understood that treatment of ASCs with Mummy at concentration of 500µg/ml resulted in a significant increase in expression level of RUNX-2, osteocalcin and β1-integrin but that of the osterix didn’t changed.26 This transcription factor plays a crucial role in both intramembraneos and endochondral ossification and loss of its function results in severe skeletal deformities.27 This knowledge about importance of RUNX-2 led us to examine whether Mummy material can alter the expression of this transcription factor in ASCs. In fact we found that Mummy at concentration of 500µg/ml can promote the expression of RUNX-2 gene significantly on 7 and 21 days. These finding suggest probable involvement of RUNX-2 signaling in differentiation of osteoblasts from ASCs in the presence of Mummy material.Runt-related transcription factor 2(RUNX-2) is a key transcription factor that regulates osteoblast differentiation from mesenchymal stem cells and also controls the expression of bone extracellular matrix proteins during osteogenesis.28 This transcription factor has an essential role in differentiation of osteoblast precursors into mature cells and furthermore is expressed during bone repair fracture sites. In this study we investigated whether treatment of ASCs with Mummy can promote the expression level of osterix mRNA.Another transcription factor involved in bone formation is osterix.Obtained data showed that on the evaluated time points the level of osterix gene decreased compared to the control cells.Additionally in this investigation we found that in the presence of Mummy the expression level of osteocalcin and β1-integrin mRNA increased significantly compared to the control and osteogenic-induced groups. Furthermore using ICC method we found that the expression of osteocalcin protein in Mummy treated ASCs elevated compared to other groups.30 This protein is produced by osteoblasts, odontoblasts and hypertrophic chondrocytes. Having role in regulating the mineralization of bone matrix, osteocalcin is used as a marker for evaluation of osteoblast activity.Osteocalcin is the most abundant glycoprotein in bone extracellular matrix which can bind to calcium ions.Moreover we understood that Mummy can increase significantly the expression level of β1-integrin gene compared to other groups. This transmembrane protein mediates cell-cell and cell-matrix interactions and also involves in maintenance of cell phenotype. Furthermore it has been reported that integrin molecules are involved in regulation of collagen type I synthesis.Taking toghether, the results of this investigation showed that Mummy material can upregulate the expression of RUNX-2, osteocalcin and β1-integrin mRNA levels in ASCs. More extensive investigation is necessary for better understanding the underlying mechanisms of this material in differentiation of ASCs into osteoblasts.As a summary, the results of this study showed for the first time that treatment of ASCs with Mummy material can enhance the expression level of bone-specific markers and promote the deposition of mineral components by ASCs. Further investigation for better understanding the precise mechanisms of this material on intracellular signaling pathways in adipose derived stem cells is necessary.This article is resulted from the research proposal leading to thesis of Maryam Eyvazi, M.Sc student of Anatomical Sciences and approved by Stem Cell Research center, Tabriz University of Medical sciences, Tabriz, Iran. The authors gratefully acknowledge the research deputy of Tabriz University of Medical Sciences for financial support.Written consent was taken from all patients and the study protocol was approved by medical ethics committee of Tabriz University of Medical sciences .The authors declare no conflict of interests.


Typha capensis (TC) and its lignin isolated by milling (MWL), ionosolv (ILL) and organosolv (EOL) methods were investigated for acetyl group content using FT-Raman, 1H NMR, 2D-NMR, back-titration, and Zemplén transesterification analytical methods. The study revealed that TC is a highly acetylated grass; extractive free TC (TCextr) and TC MWL exhibited similar values of acetyl content: 6 wt % and 8 wt % by Zemplén transesterification, respectively, and 11 wt % by back-titration. In contrast, lignin extracted from organosolv and [EMIm][OAc] pulping lost 80% of the original acetyl groups. With a high acetyl content in the natural state, TC could be an interesting raw material in biorefinery in which acetic acid could become an important by-product.During biomass fractionation, any native acetylation of lignin and heteropolysaccharide may affect the process and the resulting lignin structure. In this study,  Miscanthus x giganteus, however, after pulping with 1-butylimidazolium hydrogen sulfate, acetyl groups were not detected in the isolated lignin [OAc] and with ethanol. We further assess the value of the Zemplén transesterification method to quantify covalently bound acetates to lignin and TC biomass [OAc] treatment. The observation that cellulose rich residue has been acetylated during [EMIm][OAc] treatment, together with the previously observed cellulose acetylation by this ionic liquid [OAc] treatment, part of the acetyl groups in Typha capensis was relocated from lignin to cellulose and hemicelluloses, while another part of lignin acetyl groups was released during ionic liquid treatment.Acetyl values of the  the MWL , indicatus study  showed tc liquid ,9 suggesTo the best of our knowledge, the Zemplén transesterification method is demonstrated for the first time to accurately monitor the fate of lignocellulose acetyl groups during pulping. The Zemplén method correlates well with the back-titration method and also reveals similar trends than those hinted by NMR and Raman spectroscopies. The Zemplén method is advantageous due to its high sensitivity and small sample requirement. While the back-titration method quantifies total acetyl content in TC grass, the Zemplén method discriminates for covalently bound acetyl groups and is therefore particularly sensitive for grasses and their pulping products. TC exhibits higher acetyl content than other lignocellulosic materials reported in literature. The HSQC further indicated that the acetyl groups are linked on both lignin and hemicelluloses moieties with acetate in lignin exclusively connected through the γ-side chain. Organosolv and [EMim][OAc] pulping remove the majority of acetyl groups in lignin compared to MWL. Several dynamics occurred during [EMIm][OAc] treatment in which deacetylation and translocation of acetate from lignin to hemicelluloses moieties as well as to a cellulose-rich residue were observed.


Clostridium difficile (CD) is the leading cause of infectious health-care associated diarrhea. However, little is known regarding CD carriage and transmission amongst asymptomatic colonizers. We evaluated carriage, characterized strains and examined epidemiologic linkages in asymptomatic colonized CD patients.Rectal swabs from asymptomatic patients admitted to the general medicine ward from April 1-June 30 2012 were collected. PCR-confirmed CD colonies were ribotyped and characterized by Modified-Multi Locus Variable Number Tandem Repeat Analysis (MMLVA).1549-swabs were collected from 474-patients. Overall, 50/474(10.6%) were CD PCR-positive, 24/50 were colonized at admission, while 26/50 were first identified > = 72 hours after admission. Amongst the 50 CD PCR-positive patients, 90% were asymptomatically colonized and 80% of individuals carried toxigenic CD-strains, including ribotype-027 (5/45:11%). MMLVA revealed five-clusters involving 15-patients harboring toxigenic (4/5) and non-toxigenic CD strains (1/5). In two clusters, patients were CD positive on admission while in the other three clusters involving 10 patients, we observed CD transmission from asymptomatically colonized patients to 8 previously CD-negative patients.We identified increasing rates of colonization during admission to medical wards. MMLVA typing effectively discriminated between strains and suggests that 20% of patients with CD colonization acquired their strain(s) from asymptomatically colonized individuals in hospital. Clostridium difficile (CD) is the leading causes of infectious healthcare-associated diarrhea [diarrhea . CD-disediarrhea , 3. CD idiarrhea . Asymptodiarrhea –7. Severdiarrhea –13.Although active CD disease poses an obvious organism source in institutional settings, the impact, contribution and transmission of CD from asymptomatic carriers in institutional settings is less clear. Asymptomatic colonization has been observed in 3–5% of healthy adults, 15 and Here we present data on the rates of asymptomatic carriage of CD in patients admitted to a tertiary care hospital in Toronto, Canada. Using both ribotyping and MMLVA in addition to epidemiologic investigations we identified several clusters of asymptomatic CD carriers that harbored both toxigenic and non-toxigenic CD strains and investigated the role that asymptomatic patients play as an underappreciated source for CD.st and June 30th 2012. This study was approved by the research ethics board at Mount Sinai Hospital.A prospective study evaluating the role of asymptomatic carriage of CD in patients hospitalized on one of two 24 bed general medical wards at Mount Sinai Hospital, a tertiary care hospital in Toronto, Canada between April 1Rectal swabs, both e-swabs  and charcoal swabs  were collected for all patients as part of admission screening for antimicrobial resistant organisms (ARO) into the general medicine ward. The frequency of repeat swabbing was related to additional screening to monitor a VRE outbreak on one of the general medicine wards. Swabs were assigned unique study codes and stripped of identifiers prior to transfer to the research laboratory.C. difficile assay . Hospital infection control staff identified clinical specimens obtained from study patients for CD testing from February 1 to September 30, 2012 and provided coded information to study staff after all study analyses were complete.During the study period CDI was diagnosed clinically and microbiologically confirmed by the detection of the gene for toxin B in stool specimens using the Xpert 2O, vortexed, and heat inactivated at 95°C for 10 minutes[20. The plate was then sealed and DNA extracted using a thermocycler programmed for 10 minutes at 98°C. The PCR plate was centrifuged for 5 minutes at 3000g to collect insoluble debris, and 2 μl of supernatant was used in the real-time PCR reaction [forward primers; 5’-TTGAGCGATTTACTTCGGTAAAGA-3’ and reverse primers; 5’-CCATCCTGTACTGGCTCACCT-3’) that amplifies a 157bp fragment of a gene of C. difficile and visualized using a 6-carboxyfluorescein-labelled Taqman probe (TaqMan Probe CGGCGGACGGGTGAGTAACG)[All swabs were alcohol shocked and re-suspended in 1mL of dH0 minutes. The susreaction , 28 on tGAGTAACG).PCR-confirmed CD isolates were then ribotyped and further analyzed using Modified-Multi Locus Variable Number Tandem Repeat Analysis (MMLVA). CD isolCombining MMLVA (with a higher weight of 3X) with ribotyping (with a weight of 1X) ensures against homoplasy seen in some cases when only MMLVA is used. This allows generating results in a single experiment (ribo-MMLVA) when DNA from an isolate is tested in 4 PCR reactions in the same run . BioNumerics Manhattan distance for clustering was selected over absolute number of tandem repeats for two reasons: (1) widely used assumption that any change in number of repeats per locus should be counted as a difference is not supported by our data on multiple isolates per specimen/patient when minor variation per locus are normal due to a high sensitivity of the method.; (2) significance of increased/decreased number of repeats varies–Cd_A and Cd_C are the most variable, followed by Cd_B and Cd_G and Cd_E being the most stable.Hospital infection control staff investigated groups of patients with similar MMLVA- and ribotypes retrospectively using patient charts and clinical notes. Information related to time and/or location on the units in the hospital was noted and compared between patients with related strains. Residential postal codes were used to assess the geographical proximity of patients in the community.st and June 30th 2012 were included in the study  admitted to two general medicine wards between April 1he study ; S1 DataOverall 50/474 patients (10.6%)  were posFive of 50 CD positive participants  were clinically diagnosed as having symptomatic CD disease within 3 days of study/hospital admission using a toxigenic-CD specific PCR  performed on their stool specimens in the clinical microbiology laboratory at Mt Sinai hospital. In addition, 5 patients had negative screening swabs but a diagnosis of CD infection: 3 individuals had negative rectal swabs obtained on the same day as their positive stool specimen; one individual had a negative swab obtained the day after a positive stool specimen (where antibiotic initiation might have reduced CD concentration), and one individual’s only rectal swab was collected 3 days before the stool specimen positive for CD was obtained.As this study focuses on asymptomatic carriers of CD, the 5 patients with active CD disease on admission were excluded from the study analysis. Data in Ribotyping analysis was performed on 72 isolates from 40/45 colonized patients, and revealed that 31/40 (69%) were colonized with non-ribotype-027 toxigenic strains including ribotype-449 (n = 4), ribotype-015 (n = 6), and ribotype-081 (n = 3) .Of thoseMMLVA identified 5 CD clusters with a total of 15 patients harboring highly related CD strains . AmongstMMLVA and ribotyping analysis confirmed that 3/45 (6.7%) colonized patients  harbored multiple CD strains , S2 DataEpidemiologic investigations of two cluster, one with three individuals  and the other consisting of four individual  harboring identical CD strains (< 1% divergence) found that all of the patients within each cluster had overlapping hospital stays and patients were in adjoining rooms on the ward. In the case of one of the clusters, MS067 was positive on admission, while MS207 and MS363 were CD positive post admission, suggesting that transmission may have occurred between these patients. In contrast, the 4 individuals  with ribotype-027 strains that were identical by MMLVA were identified as colonized on admission to the hospital, and only one had been previously admitted to our hospital. The three other patients were transferred from 2 different long-term care facilities. Additional investigations of patient movements within the hospital and outside of the hospital based on their residential postal codes identified that 8/15 individuals in five clusters acquired their CD nosocomially at a time when other individuals colonized with the same strain were present on the same ward, while the remaining 7 individuals were colonized on admission to the hospital. Of the five clusters representative data is shown on three of the clusters . Of the Understanding the role asymptomatic carriers play on perpetuating CD transmission in healthcare settings is particularly challenging to examine, because most facilities do not screen for CD colonization. In patients with active CDI, contact precautions are used to manage and minimize CD transmission to other patients, however asymptomatically colonized individuals remain an unrecognized source of CD in the hospital environment. A recenTo determine how related the CD isolates from asymptomatic shedders were, we ribotyped the majority of CD isolates and then used MMLVA to distinguish between CD isolates of the same ribotype. Since MMLVA is sensitive to changes between strains and have been observed to change within the same CD isolates over time, we ensured that the changes observed were not related to instability in the MMLVA method but attributable to differences between CD strains. Stability of the MMLVA methodology was examined by testing multiple specimens collected over time from the same patient . In 34 pEyre et al. reported that in over a third of patients that acquired CD nosocomially and became carriers of CD had no exposures to patients with CDI in the community or hospital [Although, the specific role of asymptomatic carriers remains unclear, this study and others suggest that asymptomatic carriage plays a central role in the dissemination of CD in institutional settings. In a large multi-institutional study, hospital  suggestihospital . This CDhospital  and was hospital .It remains unclear what factors are associated with asymptomatic carriage of CD, however in human and muriThe ribotype-027 strain was detected in four asymptomatically colonized patients on admission, three of four patients were admitted to our hospital from 2 different long term care facilities, and the fourth had multiple previous admissions to our hospital; thus, although these patients did not acquired the strain directly from each other, it is likely that their CD was acquired in a hospital or long term care facility from either a patient or the environment.Ribotype analysis of CD isolates revealed that multiple CD strains were found in a small proportion of asymptomatic carriers . CarriagOur study has several limitations including the identification of a fairly limited number of asymptomatic CD carriers. The study likely underestimates the rates of CD carriage as rectal swabs would have significantly less organism burden compared to stool specimens as has been observed with VRE. The stuIn summary, both ribotyping and MMLVA analysis of CD stains colonizing hospitalized patients identified that asymptomatic carriers of CD can carry and shed multiple unrelated CD strains, including toxigenic and non-toxigenic strains . AlthougS1 Data(XLSX)Click here for additional data file.S2 Data(ZIP)Click here for additional data file.


Under NIR irradiation a temperature increase was observed leading to a reduction of the IC50 value to 1 µM, with total tumor cell death at 100 µM.A gold therapeutic nanoplatform with the same molecule used as reductant, coating and therapeutic agent has been developed in a one-pot, one-phase process using alendronate, a drug from the bisphosphonate family known for its antitumor effects. In addition, the core made of gold nanoparticles (NPs) brings thermal functionalities under irradiation within the first biological window (650–900 nm). The Au@alendronate nanoplatform thus provided a combined antitumor activity through drug delivery and photothermal therapy. Au@alendronate NPs inhibited in vitro the proliferation of prostate cancer cells (PC3) in a dose-dependent manner, with an IC The BP affinity for calcium is improved by adding a hydroxy (–OH) group, for instance in HMBP (hydroxylmethylene bisphosphonate), allowing for a tridentate coordination to Ca2+ ions (10(PO4)6(OH)2)) tissue [®) in the treatment of prostate and breast metastatic cancers [Bisphosphonates (BPs) are used in the treatment of a variety of bone diseases, such as osteoporosis, solid tumor bone metastases and myeloma bone disease –4. BPs c) tissue –6. We fo cancers . Nitroge cancers –9. HowevHMBPs are also effective ligands to stabilize nanoparticles under biological conditions –15. MoreUsing a one-pot synthesis strategy, we developed Au@alendronate NPs for a combined application of the antitumor activity of alendronate and an efficient gold-mediated PTT. We further evaluated their combined chemo-photothermal antitumor activity.Gold NPs were synthesized in water by using alendronate both as reducing agent and chelating ligand. The gold solution is simply added to an alendronate solution at boiling temperature see . The exc2 for a 30.5 nm spherical gold NP. Remarkably, the alendronate density is much higher than estimated values for other NPs coated with HMBP molecules  [Under similar synthesis conditions, gold NPs obtained with (1-hydroxy-1-phosphonopent-4-enyl)phosphonic acid (HMBPene), replacing alendronate were characterized by a smaller NPs size around 10 nm . This inene/nm2) . Indeed,−1). The free alendronate spectrum exhibits two sharp peaks at 1211 and 957 cm−1, assigned to P=O and P–OH, respectively [−1 is characteristic for the vibrational mode for the PO3 group [−1 (red curve), which is characteristic of the chelation of phosphorus species on a metallic surface [−1) and bending vibration bands (1675 cm−1) for alendronate were slightly shifted in the Au@alendronate NPs confirming the multilayer formation through anion/cation interactions due to the alendronate zwitterion form. These results suggest that alendronate was grafted onto the nanocrystal surface through the phosphonate groups and could be activated for further biocoupling [The chemisorption of alendronate was qualitatively assessed  by Fouriectively . The broO3 group . For Au@ surface ,33, suggcoupling .The excellent coating density led to a good colloidal stability, as confirmed by dynamic light scattering (DLS) measurements at physiological pH values. A hydrodynamic diameter equal to 31.5 ± 13.9 nm  was dete2 (2 (2 laser power was efficient for inducing tumor growth inhibition in vivo without exhibit nonspecific phototoxicity [2).Since gold NPs bring their own therapeutic asset, in the form of PTT, we first evaluated the specific photothermal properties of Au@alendronate NPs. In cancer therapy, it is desirable to use NPs that are active in the near-infrared (NIR) region to minimize light absorption of the laser radiation by surrounding tissues ,34. The 2  at low concentrations. These photothermal properties are in good agreement with those of other thermal agents [The temperature elevation clearly increased with the gold concentration and reached Δl agents  and showThe colloidal stability of Au@alendronate NPs at physiological pH values and their photothermal properties within the NIR first biological window allowed us to further consider their study in a biological environment.50 value) can be determined. This value is a good indicator of the effectiveness of a compound for inhibiting biological or biochemical functions. Free alendronate and Au@alendronate gold NPs reduced cell viability in a concentration-dependent manner , while at intermediate concentration of 100 µM, cell death was total. It clearly evidences the efficiency of the combined drug delivery and photothermal treatment of Au@alendronate NPs.At extracellular concentrations of alendronate below 1 µM, similar cell viability was observed in absence or presence of laser irradiation. This could be related to the low dose of internalized gold NPs and indicates that the laser power is sufficiently low to avoid nonspecific biological damage. At extracellular concentrations of alendronate over 1 µM, cell viability was considerably lowered in the presence of laser irradiation. The ICIn summary, we developed a one-pot synthesis by simply mixing, in water, gold ions and alendronate molecules as reductant, coating and therapeutic agent. The synthesized Au@alendronate NPs maintain the alendronate antitumor activity, which is greatly improved under NIR laser radiation. These results pave the way for an efficient antitumor activity of Au@alendronate NPs through combining drug delivery in the form of a nanoplatform carrying alendronate and photothermal therapy. Indeed, Au@alendronate NPs will accumulate within cells because of the enhanced permeability retention effect: An enhanced permeability of blood vessels near the tumor allows for the penetration of nanoparticles into the tumor. The impaired lymphatic function within the tumor will not be able to clear those nanoparticles efficiently . This prFile 1Materials and methods and supplementary figures.


The goal of this research work was to determine widespread impact kiln-produced hardwood biochar has upon temperate agricultural soil characteristics in a long-term field experiment. This dataset is supplementary to the submitted research by [1] and presents select physical and chemical characteristics of the biochar and field plots amended with hardwood biochar. Data on soil gravimetric moisture content (GMC), soil acidity and soil nitrate-N concentration at lower depth of soil under different biochar application rates is presented. Fourier Transform Infrared (FTIR) spectroscopy is provided to demonstrate the difference between fresh and aged biochar in terms of surface functional group content. DiffereNitrate content in soils during years 2 and 3 were determined at 0–20, 20–40, and total 0–40 cm depth , Fig. 3 3+ concentration corresponded to lower application rate of biochar  were recorded from pellets containing 2 mg of the air-dried biochar. The surface functional groups of fresh and aged biochar samples were identified using a PerkinElmer Spectum One spectrometer with ATR attachment. IR spectra were collected from 4000 to 650 cmferences .All data were analyzed by one-way ANOVA using the GLM Procedure in SAS 9.4 (SAS Institute Inc., Cary, NC;