Modelling of pathological processes in cells is one of the most sought-after technologies of the 21st century. Using models of such processes may help to study the pathogenetic mechanisms of various diseases. The aim of the present study was to analyse the literature, dedicated to obtaining and investigating cybrid models. Besides, the possibility of modeling pathological processes in cells and treatment of different diseases using the models was evaluated. Methods of obtaining Rho0 cell cultures showed that, during their creation, mainly a standard technique, based on the use of mtDNA replication inhibitors (ethidium bromide), was applied. Cybrid lines were usually obtained by PEG fusion. Most frequently, platelets acted as donors of mitochondria. According to the analysis of the literature data, cybrid cell cultures can be modeled to study the dysfunction of the mitochondrial genome and molecular cellular pathological processes. Such models can be very promising for the development of therapeutic approaches to the treatment of various human diseases. Modelling of pathological processes in the cells is one of the most sought-after technologies of the 21st century. With the use of such models, it may become possible to study the pathogenetic mechanisms of various diseases. In addition, efforts to transfer genes, which do not have mutations, in human cells and tissues for the treatment of certain diseases, were made by the scientists throughout the world. Great success has been achieved in the implementation of genetic constructs, containing nuclear gene mutations associated with pathologies, into cell cultures. Afterwards, such structures were embedded into the nuclear genome. The final result of the transfer of genetically engineered constructs into the nuclear genome was the expression of mutant RNA in these cultures and synthesis of mutant proteins. Concerning the mitochondrial genome, such progress is not found. For example, experimental data of the members of our laboratory suggest that the transduction of genetic constructs or synthetic RNA in the mitochondria of monocytic origin cell line THP-1 was not possible. Scientists began to pay more and more attention to cybrid cultures that are more suitable models for the detection of pathological mechanisms and the development of approaches to the treatment of mitochondrial cytopathies and also other diseases associated with mutations in mtDNA.Cytoplasmatic hybrids (cybrids) are created by fusion of mitochondria-deficient cell lines (Rho0) and cell donors of mitochondria. Since in the mitochondria with deactivated mtDNA, any fission processes do not occur because of the dysfunction of these organelles, the cell gradually loses them. Therefore, some researchers conventionally call Rho0 cells mitochondria deficient.As a material for creating cybrid cultures, most often cell line 43B –8 is useμg/ml) partially or completely inhibit the replication of the mitochondrial genome, but they do not affect the replication of nuclear DNA [The possibility of Rho0 human cell creation is based on the use of mtDNA replication inhibitors, such as DNA intercalating dye ethidium bromide . Relatively low concentrations of ethidium bromide (0.1 to 2 lear DNA .μg/ml of sodium pyruvate).The verification of possibility of mitochondria-deficient cell obtaining. The native cell line is cultured in a growth medium supplemented with ethidium bromide and uridine in one embodiment and with only the addition of ethidium bromide in another embodiment. In this case, the growth medium should contain glucose and pyruvate in concentration of complete medium DMEM  standard, the methodology of King and Attardi is used, which includes two stages:After obtaining the cell lines, stably living in previously described conditions, a cell analysis is performed in order to confirm the absence of mitochondria in these lines. The scheme of this method is shown in μg/ml uridine [μg/ml uridine and after 120 days of cultivation, with increasing addition of ethidium bromide up to a concentration of 500 ng/ml within 3-4 months [μg/ml uridine were added to the medium. Despite the assertion that the research team used the technique of King and Attardi, ethidium bromide concentration exceeded 10 times, and uridine exceeded 2 times the concentration of these additives in the original paper [It is noteworthy that while checking the possibility of certain cell lines to receive Rho0 cell cultures, it is necessary to choose favorable conditions of cultivation, namely, the concentration of various additives, especially ethidium bromide and uridine which may considerably vary. For example, King and Attardi for obtaining Rho0 cells from the cell line 143B.TK− cultivated the cells on a growth medium supplemented with 50 ng/ml ethidium bromide and 50 4 months . Muellertrans retinoic acid and arsenic trioxide on differentiation and cell survival in human leukemic cell line HL60 and mitochondria-deficient HL60rho0. As a result, HL60rho0 cells possessed a lower differentiation ability but a higher respiratory activity than parental cell HL60. HL60rho0 cells were also significantly more resistant to apoptosis [Herst et al. analyzed the efficiency of the impact of all-poptosis .Shen et al. conducted a comparative analysis of oxygen consumption levels in cultures of human lymphoblast Molt-4 of wild type and in Rho0 cultures. The rate of oxygen consumption in mitochondria-deficient culture was significantly lower as compared to that in the native culture (7 times). The level of oxygen consumption equally changed in the native and in the mitochondria-deficient cell line Molt-4 in both the inhibition of parachloromercurobenzoate (PCMB) level and during the stimulation of sodium bisulfite by menadione (MSB). At the same time, potassium cyanide reduced and carbonyl cyanide m-chlorophenylhydrazone (CCCP) increased the oxygen consumption rate only in native culture Molt-4. The authors suggest that this can be associated with the absence of important protein subunits encoded by the mitochondrial genome, which in its turn leads to disruption of the electron transport in the respiratory chain .Brar et al. compared the ultrastructural morphology of mitochondria of human epithelial cell line A549 of wild type and Rho0 cells. The mitochondria-deficient culture contained irregularly shaped mitochondria with a translucent matrix in contrast to the native cell line. In addition, the morphology of the cristae Rho0 cells was more heterogeneous: for example, both short and circular growths of the inner membrane of the mitochondria occurred. The authors analyzed the effect of bleomycin on these cell cultures, which is an antitumor agent, and in clinical use, it causes damage to the lungs, and in 1-2% of patients, it causes severe progressive pulmonary fibrosis. It was shown that Rho0-A549 cell lines are resistant to bleomycin, which, according to the authors, may be associated with the deterioration of mitochondria-dependent apoptosis .Cheon et al. analyzed the role of mtDNA mutations in carcinogenesis. This research group investigated Rho0 cells obtained from cell line SK-Hep1 . Native cell culture SK-Hep1 was used as a control culture. It was revealed that in mitochondria-deficient cells, an expression of the gene and protein level of hypoxia-inducible factor- (HIF-) 2 alpha has increased. Korean researchers suggested that mitochondrial dysfunction caused by the inhibition of mtDNA by ethidium bromide increases the angiogenic potential of tumor cells. In addition, an increase in the expression of vascular endothelial growth factor (VEGF), a key angiogenic molecule induced by HIF, was observed. Increased expression of genes associated with invasion, such as genes of urokinase-type plasminogen activator (uPA) and metalloproteinases of matrix (MMPs) in Rho0-cells of SK-Hep1 can also, according to the authors, indicate mitochondrial dysfunction, which in its turn leads to increased invasive potential of tumor cells .Our research team found that Rho0 cells of THP-1 culture have lower levels of cellular and mitochondrial respiration, compared to cells of native lines of monocytic origin THP-1.ρ0 with lines BALB/cJ and C57BL/6J [There are two fundamentally different fusion methods of mitochondria-deficient lines with cell donors of mitochondria. The transfer of mitochondria into Rho0 cells can only occur during the formation of pores in the cytoplasmic membranes of cells resulting from the influence of certain chemical or physical agents on them. One of the methods involves culturing Rho0 cells with enucleated cytoplasts under the influence of a flow of direct current (electrofusion) . ScientiC57BL/6J , the creC57BL/6J , in the C57BL/6J  and fibrC57BL/6J . The secC57BL/6J , 3. It sC57BL/6J , cell liC57BL/6J –25 and nC57BL/6J .Cybrids are used as cellular models for studying mitochondrial dysfunction and its association with a variety of human diseases such as Parkinson's disease, Alzheimer's disease, HIV, human herpes virus type 8, MELAS syndrome, Leber's optic atrophy, and Leigh syndrome , 27–29. Several research groups discovered an association of certain mutations and mitochondrial genome haplogroups with cell dysfunction. For example, Mueller et al. evaluated the survival index of cell line HEK293. Cybrids carrying in one case haplogroup T and haplogroup N in the other were obtained from this cell culture. During the treatment with hydrogen peroxide, cybrids with haplogroup T had a higher survival index than the other cell line [Trounce et al. found an association between mtDNA mutation m.8993T>G and dysfunction of mitochondria isolated from lymphoblasts of patients with Leigh syndrome. This association is characterized by a decrease in oxidative phosphorylation processes in the studied cells. In their investigations, a decrease in the activity to 26–50% of 3 complexes of respiratory chain and also a 30% reduction of ADP/oxygen ratio have been shown [A research about creating mitochondria-deficient cell lines from cells of patients with mitochondrial encephalopathy and MERRF syndrome, having a reduced activity of cytochrome C oxidase, is rather interesting. The obtained Rho0 cells were fused with enucleate cells, HeLaCOT. As a result, cybrids with restored oxidase activity of cytochrome C were obtained .Some researchers study the association of mitochondrial dysfunction with oncological diseases, using various cybrid lines.For example, Cruz-Bermúdez et al. created cytoplasmic hybrids based on cell line 143B. Their cybrids had mitochondrial genome mutations “with varying degrees of pathogenicity”: one line was with a severe pathogenicity (mutation m.8363G>A in gene tRNA-Lys) and 3 lines with a mild pathogenicity, namely, Leber's optic atrophy (LHON). The recent cell lines carried one mtDNA mutation in each cybrid culture . The study results showed that these cybrid lines have mild pathogenicity and have OXPHOS dysfunction. However, they were not associated with tumorigenicity and do not cause tumors when they were used with “naked” mice .Yoshii et al. determined mitochondrial dysfunction in cybrid lines based on cell line 143B carrying mutant mitochondria in patients with MELAS syndrome. In this article, radioactive Cu-diacetyl-bis (N4-methythiosemicarbazone) (Cu-ATSM) was used, which is a potential marker for the visualization of hypoxic tumors for positron emission tomography (PET). Cybrid cells with MELAS had an increased absorption of Cu-ATSM under normoxia compared to wild-type cells. It also showed that Cu-ATSM absorption correlates with the change in the level of NADH and NADPH .At the predementia stage of Alzheimer's disease (AD) may occur MCI (mild cognitive impairment). During this stage of disease, a decrease in cognitive abilities may be observed, which often represents a transition between AD dementia and normal cognition. A group of researchers transferred mitochondria from MCI, AD, and platelets of control subjects to mtDNA-depleted SHSY5Y cells to model bioenergetic dysfunction of AD and MCI. Bioenergetics-related infrastructures and bioenergetic fluxes were characterized in the obtained cybrid cell lines. Compared to cybrids of control subjects, AD and MCI cybrids had changes in respiratory coupling, consumption of oxygen, and utilization of glucose. The level of ADP/ATP in AD and MCI cybrids was higher and the ratios of NAD1/NADH were lower , 34.Trimmer and Bennett created and analyzed cybrid neuroblastoma SH-SY5Y and NT2 with mitochondria of patients with Parkinson's disease. The research of cells in cybrid cultures revealed an increase in oxidative stress and the frequency of apoptosis compared to normal cells. SH-SY5Y and NT2 in case of applying the light therapy, characteristic for the treatment of neurological diseases, normalization in the mitochondrial respiratory activity cybrid cells SH-SY5Y and NT2 was observed .In another investigation, Trimmer and colleagues studied that the possibilities of the treatment of Parkinson's disease (PD) on a cybrid model were created with the use of platelets from patients with this pathology. It should be noted that ATP, necessary for the support of axonal transport and many other cellular functions essential for the survival of neurons, is produced by mitochondrial respiratory chain. Scientists exposed cybrids to low level, near-infrared laser light and found that in these cells, mitochondrial metabolism enhanced, the level of oxidative phosphorylation increased, and redox capacity improved. The results of this investigation show the opportunity of low-level light therapy to improve defects in neuronal cells in individuals which have Parkinson's disease .The analysis of literature dedicated to the methods of obtaining Rho0 cell cultures showed that during their creation, mainly a standard technique, based on the use of mtDNA replication inhibitors (ethidium bromide), is used. Cybrid lines are usually obtained by PEG fusion. Most frequently, platelets act as donors of mitochondria.It should be emphasized that cybrid cell cultures can serve as models for studying the dysfunction of the mitochondrial genome and molecular cellular pathological processes. Such models can be very promising for the development of therapeutic approaches to the treatment of various human diseases.


Recent advances in sequencing technologies have enabled parallel assays of chromatin accessibility and gene expression for major human cell lines. Such innovation provides a great opportunity to decode phenotypic consequences of genetic variation via the construction of predictive gene regulatory network models. However, there still lacks a computational method to systematically integrate chromatin accessibility information with gene expression data to recover complicated regulatory relationships between genes in a tissue-specific manner.http://bioinfo.au.tsinghua.edu.cn/jianglab/CSNETS/.We propose a Markov random field (MRF) model for constructing tissue-specific transcriptional regulatory networks via integrative analysis of DNase-seq and RNA-seq data. Our method, named CSNets (cell-line specific regulatory networks), first infers regulatory networks for individual cell lines using chromatin accessibility information, and then fine-tunes these networks using the MRF based on pairwise similarity between cell lines derived from gene expression data. Using this method, we constructed regulatory networks specific to 110 human cell lines and 13 major tissues with the use of ENCODE data. We demonstrated the high quality of these networks via comprehensive statistical analysis based on ChIP-seq profiles, functional annotations, taxonomic analysis, and literature surveys. We further applied these networks to analyze GWAS data of Crohn’s disease and prostate cancer. Results were either consistent with the literature or provided biological insights into regulatory mechanisms of these two complex diseases. The website of CSNets is freely available at CSNets demonstrated the power of joint analysis on epigenomic and transcriptomic data towards the accurate construction of gene regulatory network. Our work provides not only a useful resource of regulatory networks to the community, but also valuable experiences in methodology development for multi-omics data integration.The online version of this article (10.1186/s12864-018-5277-6) contains supplementary material, which is available to authorized users. The complicated process of transcription in eukaryotes largely attributes to the collaboration among DNA regulatory elements, RNA polymerases, mediator and cohesion complexes, and sequence-specific transcription factors (TFs). Such collaboration is encoded in a comprehensive gene regulatory network that determines how the expression of a gene is regulated, what responses a cell would adopt to answer external stimuli, and which phenotypic consequences a genetic variation could result in . In the A gene regulatory network is often inferred based on high-throughput assays about interactions among transcription factors and their target genes. RNA-seq, as a means of capturing a snapshot of the whole transcriptome, has provided the most abundant data in such studies. For example, Hu and Chen constructed a transcriptional regulatory network in memory CD8+ T cells with gene expression profiles and predicted TF information, and then identified the core TFs . Li et aWith the promise of detecting TF binding sites at high resolution, ChIP-seq has been used with RNA-seq data to infer gene regulatory networks. For example, Roy et al. constructed a mixed regulatory network that combines transcriptional regulation by TFs from ChIP experiments and posttranscriptional regulation by miRNAs . Chen etThere are several difficulties that restrict large-scale applications of the ChIP-seq technology. Besides the restriction of suitable antibodies for TFs, the number and cost of experiments required by a large number of TFs also limit the feasibility to construct gene regulatory networks for a variety of phenotypes and species via ChIP-seq. To overcome these limitations, DNase-seq has been developed to enable the capture of chromatin accessibility in whole-genome scale , 20. TakMotivated by the above understanding, we propose in this paper a Markov random field (MRF) model, named CSNets (Cell-line Specific regulatory Networks), that integrates DNase-seq data with RNA-seq data towards large-scale inference of gene regulatory networks. In this method, we first roughly infer regulatory networks for individual cell lines using DNase-seq data alone. Then, we fine-tune these networks using an MRF model, based on pairwise similarity between cell lines derived from RNA-seq data. Focusing on data released by the ENCOODE project, we constructed regulatory networks specific to 110 cell lines and 13 major tissues for human. Using ChIP-seq experimental data as a gold standard, we showed the superior quality of our networks over that obtained by existing methods. Through functional enrichment analysis, we demonstrated that TFs and their predicted targets tend to share similar biological functions. Besides, integrative analysis of our networks with GWAS data of Crohn’s disease and prostate cancer both suggested genes and genetic variants that were either consistent with the literature or provided biological insights into regulatory mechanisms of these two complex diseases.We extracted DNase-seq profiles for 110 human cell lines, representing 70 diverse cell types and 13 unique tissue lineages, from the ENCODE project . We collWe proposed to construct a transcriptional regulatory network specific to a cell line by integrating DNase-seq data, transcription factor binding motif information, and gene expression data, as illustrated in Fig. Homo sapiens (GRCh37), and position weighted matrix of motif for the 368 transcription factors. Focusing on predicted binding sites in promoter regions (TSS ± 2 kb), we linked transcription factors to their target genes, and thus obtained preliminary regulatory networks specific to the 110 human cell lines. Second, we incorporated gene expression data and adopted a rigorous Markov random field model to fine-tune these preliminary networks. The basic assumption behind this model is that similar cell lines tend to share similar regulatory patterns. With this understanding, we used gene expression data to measure the similarity between cell lines, and then connect regulatory relationships of different cell lines by using a Markov random field (MRF) model based on the similarity. Detailed explanations of components in our method are given below.We first followed a computational method called Protein Interaction Quantitation (PIQ)  to perfoq-value = 0) in the 110 cell lines. Then, we applied the singular value decomposition (SVD) to expression data of these genes to perform a dimensionality reduction. In detail, expression values of a gene across the 110 cell lines was first normalized to zero mean and standard deviation one. The resulting expression matrix regarding the 592 genes and 110 cell lines were then decomposed into USVT, where columns of U were called eigenarrays, diagonals in S singular values, and rows of VT right singular vectors. Finally, we characterized the similarity between two cell lines as the Pearson’s correlation coefficient of the first 16 dimensions of the eigenarrays in the SVD decomposition. Here, we calculated CSI values based on different numbers of dimensions and found that they were robust and slightly changed. We name such a similarity measure as the Cell-line Similarity Index (CSI).We adopted a measure, called TSI (Tissue Similarity Index) , to charA regulates target gene B), we constructed an MRF network G = {VAB, EAB}, where a node vABi ∈ VAB,  indicates the regulation of TF A on target gene B in cell line i, and an edge  ∈ EAB denotes the regulation coherence for TF A and gene B between cell line i and j. We introduced an indicator variable xi ∈ X for the node vABi, indicating whether the regulation exists (xi = 1) or not (xi = 0) in cell line i. Suppose that the higher the degree of similarity between cell line i and j, the stronger the positive correlation of variable xi and xj, we define a criterion called the TF non-specific index (TNI) as the proportion of common targets for the TF involved in cell line i and j. The larger the TNI value, the more similar of the regulatory mechanism corresponding to the concerned TF. We define the edge weight, wij ∈ W, as the average of CSI and TNI. We set a threshold c with default value 0.5, and regarded cell line i and j as connective if and only if wij > c.We proposed a Markov random field model to fine-tune the preliminary networks. Specifically, for a given regulatory relationship  or failure (xi = 0) of the given regulatory relationship in cell line i, respectively. We used the probability of TF binding inferred from PIQ as the probability of Pi) is named as pseudo-energy function. With this formulation, we transformed the problem of maximizing the joint distribution Pr(X) into that of minimizing the pseudo-energy function [With the definition of two types of potential functions, the joint distribution of all indicator variables X can be denoted asfunction , 34. We function  to furthαi(xi) = ln ϕi(xi)and βij = ln ψi, j). When the value of xi is not consistent with its probability distribution (i.e. Pi), the value of αi(xi) is 0, rather than ∣ ln ϕi(1) − ln ϕi(0)∣. When cell lines i and j are connective and xi and xj are of differential values, the value of βij is 0 rather than  + TNI)/2. It is verified that we can transform the problem of minimizing the pseudo-energy function into that of summing total losses of αi(xi)and βij when the following inequality satisfies [In detail, firstly we define atisfies .\documenThis equation suggests that the problem of minimizing the pseudo-energy function is transferred into the maximum flow problem of networks. We finally applied the loopy belief propagation algorithm  to calcuWe collected 353 ChIP-seq experiments, regarding 108 TFs in 59 cell lines, from the ENCODE project. We then evaluated the contribution of the MRF model as follows.M) of three gene sets, which include candidate target genes according to ChIP-seq data, target genes of the TF according to the network constructed by the MRF model for the given cell line, and target genes of the TF according to the preliminary network for the given cell line. Finally, we ranked candidate target genes according to their scores and used those ranked among top M as the gold standard of target genes for the TF in the given cell line.We first generated a gold standard of target genes for a TF in a cell line from the corresponding ChIP-seq experiment. To achieve this objective, we mapped binding sites identified in the experiment to promoter regions (TSS ± 2Kbps) of protein coding genes and assigned experimental scores of the binding sites to the mapped genes, which were used as candidate target genes. To further reduce false positives in these genes, we identified the median size  curve (sensitivity versus 1-specificity) and calculated the AUC score as the area under this curve. In a similar way, we obtain the AUC score of for the preliminary network. The relative change of these two AUC scores is then used to compare the performance of these two networks, for the given TF in the give cell line.M and then counting the number of genes shared in the remaining target genes. Using a similar strategy, we obtained an overlapping score for the preliminary network. The relative change of these two scores can then be used to compare the performance of these two networks, for the given TF in the give cell line.We further evaluated quality of the constructed networks by checking the overlap between target genes in the networks and those identified by ChIP-seq experiments. This was done by filtering out low confidence target genes that were ranked below the threshold We constructed regulatory networks specific to 110 human cell lines, and we further merged networks specific to cell lines belonging to the same tissues to obtain 13 tissue-specific regulatory networks, as summarized in Table We then extracted sub-networks regarding transcription factors only from the 13 networks and illustrated 6 of such networks in Fig. We evaluated contributions of the MRF model using 353 ChIP-seq experiments collected from the ENCODE project. Briefly, we first identified a gold standard of target genes for a TF in a cell line from the corresponding ChIP-seq experiment. Then, we evaluated the improvement of a network constructed by using the MRF model over the corresponding preliminary one in terms of relative changes in the AUC score and the overlapping score, as detailed in Methods.p-value = 8.4e-08). In terms of the relative change in the overlapping score, as shown in Fig. p-value< 2.2e-16).As shown in Fig. p-value = 1.1e-05). In terms of the relative change in the overlapping score, networks constructed by using the MRF model show higher consistency with ChIP-seq data for 88 out of the 108 (81.5%) TFs. Again, he positive contribution of the MRF model is supported by the one-sided binomial exact test .We further performed a TF level evaluation by aggregating ChIP-seq experiments according to TFs and averaging a criterion over corresponding cell lines. As shown in Fig. We testified the rationality of the tissue similarity measured by gene expression data and observed the consistency between regulatory networks and the human cell hierarchical taxonomy graph. After extracting the directed acyclic subgraph of the human cell hierarchical taxonomy graph from the Foundational Model of Anatomy Database  in the UFirst, we performed hierarchical clustering of tissues according to gene expression profiles. Figure Further, hierarchical clustering was performed for the 110 cell lines based on their similarity index, with results shown in Fig. Next, we inspected the consistency between regulatory networks and the human cell hierarchical taxonomy graph to evaluate the rationality of the tissue similarity measured by regulatory networks. For this objective, we merged regulatory networks specific to cell lines belonging to the same tissue, used the Jaccard coefficient of tissue-specific regulatory networks to measure the similarity between tissues, and then perform hierarchical clustering on the 13 tissues, as shown in Fig. Finally, we performed hierarchical clustering on the 110 cell lines based on their specific regulatory networks. The results, in Fig. p-values < 2.2e-16 for all these cell lines). These results suggest that the networks constructed by using our method can well characterize the regulatory relationship between transcription factors and their target genes.We evaluated whether expression of TFs exhibited positive correlation with their predicted target genes in a cell line-specific manner. For this objective, we collected gene expression data for the GM12878, K562, MCF-7, and SK-N-SH cell lines. For each of these cell lines, we calculated Pearson’s correlation coefficient between a TF and each of its target genes, and we plot the statistical significance of the correlation coefficient in Fig. With the assumption that a TF and its target genes tend to share common biological functions, we performed a functional enrichment analysis on the target genes of a TF. To achieve this objective, we collected 1454 gene sets from the MSigDB database, covering 825 biological process terms, 396 molecular function terms, and 233 cellular component terms in GO. We then derived two criteria to characterize the degree that a TF and its target genes share a common function, as follows.Given a TF, a cell line and a GO term, we identified target genes of the TF in the network specific to the cell line, and we performed a Fisher’s exact test to see whether the target genes were enriched in the function corresponding to the GO term. With results of such an analysis for every TF, in every cell line, and for every GO term collected, we were able to derive a statistic to indicate consistency between functions of TFs and their target genes. Particularly, we defined such a statistic as the proportion of significant tests (FDR ≤ 0.2) over all tests performed, and we calculated a statistic for each of the three GO categories, biological process, molecular function, and cellular component, separately. Because all the 1454 GO terms were used in the above analysis, we referred to such a statistic as the total enrichment score. By contrast, we repeated the above enrichment analysis with GO terms not relevant to a TF discarded, and we referred to this formulation as positive enrichment analysis.p-values are as small as 5.60 × 10− 7, 7.95 × 10− 3 and 7.80 × 10−4for the cellular component, biological process and molecular function, respectively.As shown in Fig. p-values in Additional file We further analyzed whether target genes of a TF exhibit different functions in different cell lines, especially in normal and cancer cell lines. To achieve this objective, we identified 65 normal cell lines and 25 cancer cell lines. For each TF, we collected its target genes, and we performed functional enrichment analysis to see whether functions of the TF were enriched in its target genes, for the normal and cancer cell lines, separately. We showed TFs and GO terms with most significant enrichment For example, EP300 plays an important role in regulating cell growth and blocking the promotion of cancerous tumors. The targets of EP300 are enriched in normal cells rather than cancer cells in two GO terms, corresponding to apoptotic process and programmed cell death respectively, indicating the function of EP300 is altered in cancer cells Fig. . As for We applied the constructed regulatory networks to analyze a GWAS data set of Crohn’s disease, demonstrating the potential of these networks in identifying disease-related TFs and their regulatory mechanisms. We first select a regulatory network that is specific to Crohn’s disease from the 110 networks. On one hand, it is generally thought that the inflammatory reaction in Crohn’s disease is driven by the activated type 1 helper T cells (Th1) . On the p-value that indicates the strength of association between the gene and this disease, using the tool Pascal [p-values, we obtained a gene list, in which top ranked genes can be treated as candidate disease genes. To avoid determining true disease genes based on a hard cut-off of the p-value, we resorted to GSEA [We then collected GWAS data for this disease from the literature  and calcl Pascal . By rank to GSEA  to assig to GSEA –50 have We further explored the importance of the cell line specificity of a regulatory network in identifying the NFκB family in the above analysis. Briefly, we extracted target genes of the NFκB family for each of the 110 networks and used Fisher’s exact test to measure the degree of enrichment of these target sets in the candidate genes identified above for Crohn’s disease. Results show that the target gene set of the NFκB family in Th1 cell line is ranked 2nd, and specifically, the target gene set of NFKB1 in Th1 cell line is ranked 1st, among the 110 cell lines


Oxaliplatin (OHP) treatment of colorectal cancer (CRC) frequently leads to resistance. OHP resistance was induced in CRC cell lines LoVo-92 and LoVo-Li and a platinum-sensitive ovarian cancer cell line, A2780, and related to cellular platinum accumulation, platinum-DNA adducts, transporter expression, DNA repair genes, gene expression arrays, and array-CGH profiling. Pulse  and continuous exposure  resulted in 4.0 to 7.9-fold and 5.0 to 11.8-fold drug resistance, respectively. Cellular oxaliplatin accumulation and DNA-adduct formation were decreased and related to OCT1-3 and ATP7A expression. Gene expression profiling and pathway analysis showed significantly altered p53 signaling, xenobiotic metabolism, role of BRCA1 in DNA damage response, and aryl hydrocarbon receptor signaling pathways, were related to decreased ALDH1L2, Bax, and BBC3 (PUMA) and increased aldo-keto reductases C1 and C3. The array-CGH profiles showed focal aberrations. In conclusion, OHP resistance was correlated with total platinum accumulation and OCT1-3 expression, decreased proapoptotic, and increased anti-apoptosis and homologous repair genes. Oxaliplatin is a third generation analogue of cisplatin and has shown remarkable clinical activity in colon cancer with intrinsic resistance to cisplatin. Oxaliplatin is used in combination with other drugs including 5-fluorouracil with leucovorin (FOLFOX), resulting in response rates of up to ~60%  and for Clearly, subtle differences in determinants of cisplatin and oxaliplatin resistance exist which may be tissue specific and possibly influenced by p53 expression. To further dissect out mechanisms of oxaliplatin resistance we induced resistance in colon cancer cells with different p53 functional status and compared this to ovarian cancer cells. Resistance towards oxaliplatin was studied in relation to common mechanisms of drug resistance including cellular drug accumulation; expression status of relevant uptake and efflux transporters; and formation, retention, and repair of platinum DNA-adducts. Since the underlying pathways that mediate resistance towards oxaliplatin are poorly described, we also investigated alterations in gene expression in the drug-resistant cell lines and explored whether or not these changes are a result of aberrations at the genomic level.The results of the growth inhibition experiments aimed at determining the degree of drug resistance induced by exposure to gradually increasing concentrations of oxaliplatin over seven months . Since o50 value of 0.21 μM. Induction of resistance after four hours of oxaliplatin pulses resulted in a 7.9-fold resistance and after continuous drug exposure resulted in a 11.8-fold resistance. The LoVo-Li cells that express inactive p53 were 3.5-fold less sensitive to oxaliplatin and the resistant variants showed a similar pattern of resistance towards oxaliplatin as the wt p53 parental cell line LoVo-92 with resistance factors of 4.0 and 5.6 for LoVo-Li/4OHP and LoVo-Li/cOHP, respectively. Remarkably, these LoVo-92 and LoVo-Li variants retained parental cell sensitivity to cisplatin. Moreover, the oxaliplatin-resistant sublines of the ovarian cancer cell line A2780 displayed higher resistance levels after continuous exposure to oxaliplatin rather than after four hours of pulses, hence being 11.1- and 5.3-fold, respectively. In contrast to the colon cancer cell lines, these oxaliplatin-resistant ovarian cancer cells were also cross-resistant to cisplatin; 7.0- and 9.3-fold for A2780/4OHP and A2780/cOHP, respectively. However, resistance to l-OHP was lower than that of CDDP in a CDDP-resistant variant, ADDP, which was included for comparison. In order to determine the stability of the resistance, we also cultured the cells for several months without l-OHP and determined the sensitivity to l-OHP, but no loss of resistance was found. Although we did not clone our cells, this result indicates that induction of resistance led to a homogenous, resistant cell population.The colon cancer cell line, LoVo-92, was most sensitive to oxaliplatin with an ICIn order to determine whether or not oxaliplatin resistance is mediated by reduced drug uptake or increased efflux, which might result in decreased formation of platinum DNA adducts, cellular drug accumulation and DNA adduct formation were determined . EarlierConsistent with the decreased oxaliplatin accumulation, formation of platinum-DNA adducts was also decreased in LoVo-92/4OHP and LoVo-92/cOHP B. RemovaThe net cellular accumulation of oxaliplatin and consequent formation of platinum DNA adducts might be affected by influx and efflux transporters as well as DNA repair. Therefore, mRNA expression of the copper transporter, hCTR1; the organic cation transporters OCT1, 2, and 3 (SLC22A1-3); as well as the efflux transporters ATP7A and ATP7B were determined by Q-PCR. The expression of nucleotide excision repair gene ERCC1 was also evaluated . The resLoVo-Li cells displayed lower gene expression levels of influx transporters corresponding with lower drug accumulation and formation of oxaliplatin DNA adducts in LoVo-Li cells as compared to LoVo-92. The LoVo-Li/4OHP cells showed decreased expression of all influx and efflux transporters, as well as the DNA repair gene ERCC1. The hCTR1, OCT1, OCT2, and ERCC1 were decreased by 40%, whereas, OCT3 showed a decreased expression of 80%. ATP7A and ATP7B were decreased by 60% and 40%, respectively. The LoVo-Li/cOHP OCT1 and ERCC1 genes were unchanged, whereas, the other genes showed a decreased expression. Expression of the influx transporters hCTR, OCT2, and OCT3 were decreased by 15%, 40%, and 95%, respectively. The efflux transporters, ATP7A and ATP7B, showed a decreased expression of 45% and 30%, respectively. The drug-resistant ovarian cancer cell lines exhibited decreased expression of all transporter genes except for OCT2 and OCT3 which were not detectable in the parental cells, as well in the resistant cells. Expression of OCT1 in A2780/4OHP and A2780/cOHP was decreased by 20% and 60%, respectively.ATP7A was decreased by 25% in A2780/4OHP but unchanged in A2780/cOHP. ATP7B was decreased by 35% in both A2780/4OHP and A2780/cOHP. The nucleotide excision repair gene ERCC1 showed an increased expression in A2780/4OHP but was decreased by 80% in A2780/cOHP. The Pearson correlation analysis  of the QAlthough in LoVo-92 variants, total platinum accumulation, DNA adducts formation, and transporter gene expression appear to correlate and provide adequate explanation for drug resistance, however, the picture for LoVo-Li and A2780 variants is less clear. We, hence, performed gene expression arrays to gain better insight into the mechanisms underlying oxaliplatin resistance. The gene expression array data were significantly correlated with the quantitative gene expression data obtained.The Q-PCR data for the genes are described above. Ingenuity pathway analysis (IPA) of normalized log2 ratios of the resistant cell lines and corresponding parental cell line (gene expression omnibus: GSE19992) was used to pinpoint the canonical pathways that were most significantly enriched. The most frequently and significantly enriched pathways are shown in The axonal guidance signaling and aryl hydrocarbon receptor signaling pathways were significantly enriched for four out of six resistant cell lines. The expression of genes of the ALDH family was most frequently altered in the aryl hydrocarbon receptor signaling pathway. ALDH1L2 was decreased in all cell lines with log2 ratios from −2.26 for A2780/4OHP to −3.45 for LoVo-Li/4OHP. The ALDH1A2 gene was increased in all cell lines except for the LoVo-Li/cOHP. Unexpectedly, resistant ovarian cancer cell lines showed a decreased expression of the glutathione-S-transferase family of genes. However, microsomal glutathione-S-transferase 1 showed an increased expression in these resistant variants and to some extent also in drug-resistant colorectal cancer cell line variants.Various signaling pathways showed major changes in the expression of their pertaining genes, which may, in general, point to an altered, mostly reduced capacity to induce cell death, as summarized in Less frequently observed yet highly significant for the drug-resistant variants of LoVo-Li is the role of BCRA1 in DNA damage response, metabolism of xenobiotics by cytochrome p450, xenobiotic metabolism signaling pathways, and G1/S checkpoint regulation. The role of BRCA1 in DNA damage response was most prominent in LoVo-92/cOHP and LoVo-Li/4OHP, but the ovarian cancer cell lines also showed altered expression of genes in this pathway. Excluding E2F5, all genes in this pathway were increased in LoVo-Li/4OHP, whereas, in LoVo-92/cOHP increased expression of BLM, CHEK1, E2F1, Rad51, and RFC2 was observed. In the ovarian cancer cell lines A2780/4OHP and A2780/cOHP the highest increase was observed for FANCA.Both xenobiotics metabolism pathways were only significantly enriched for LoVo-Li/4OHP but included genes that were found to be increased in other resistant variants as well. Most prominent was the above described ALDH family of genes. Other frequently increased genes were aldo-keto reductase family members 1C1 and 1C3 which were increased in four out of six and all drug-resistant variants, respectively. The expression of the multidrug efflux transporter, ABCC2 (MRP2), was increased in oxaliplatin-resistant ovarian cancer cell lines, however, decreased gene expression was observed in resistant LoVo-Li variants. However, resistant LoVo-Li variants showed increased expression of another MDR efflux transporter, ABCB1 (Pgp). The G1/S checkpoint regulation was only significant for LoVo-Li/4OHP and showed increased expression of cyclins D1 and D3; cyclin dependent kinases 2 and 4; E2F family members 1, 3, and 4 and also RBL1; and the transcription factor TFDP1.To determine whether alterations in gene expression could be the result of gene copy number variations, we performed aCGH. The chromosomal profiles  are shown in Analysis of aCGH data for common aberrations revealed that eight out of 10 observed common or overlapping aberrations are found in drug-resistant cell lines derived from the same parental cell line. Focal gains observed in Chr 15 and Chr 16 showed that similar aberrations are also found in both resistant colon and ovarian cancer drug-resistant cell lines. Remarkably, parental LoVo-92 cells displayed a gain at Chr15 which was completely lost in LoVo-92/4OHP and partly lost in LoVo-92/cOHP cells.Oxaliplatin resistance is a major clinical hurdle for curative chemotherapy since it is a crucial component of combination regimens for colorectal cancer. Although much knowledge has been gained regarding mechanisms underlying resistance to the first-generation platinum compound, cisplatin, those relating to oxaliplatin resistance are still not well characterized.Since the pharmacological activity or resistance to oxaliplatin might be affected by tissue type and treatment schedule as well as by other factors including p53 status, we established oxaliplatin-resistant cell lines representing these parameters. The colorectal cancer cell lines LoVo-92 and its variant LoVo-Li, as well as the ovarian cancer cell line, A2780, were made resistant via short-term drug pulses to reflect the clinical bolus administration and compared the latter to cell lines which acquired resistance after continuous exposure to oxaliplatin. The data show moderate levels of resistance  ranging from four- to 12-fold as compared with parental cells. Remarkably, low level cross-resistance towards cisplatin was observed in the colorectal cell lines, whereas, oxaliplatin-resistant ovarian cancer cell lines showed similar resistance levels for both cisplatin and oxaliplatin. This observation may be explained by the intrinsic cisplatin resistance of both parental colorectal cancer cell lines which are deficient in mismatch repair and an established mechanism of resistance to cisplatin but not oxaliplatin ,18,19,29Although both cisplatin and oxaliplatin are believed to exert their cytotoxic activity via formation of DNA adducts, involvement of mismatch DNA repair is different for each drug, although for both cisplatin and l-OHP a decreased accumulation was found in resistant cells . MoreoveThe lack of correlation between sensitivity to oxaliplatin and the formation of platinum DNA adducts may possibly be the result of cellular sequestration of the drug away from its target. Previously, it has been shown that the ATPases ATP7A and ATP7B play a role in intracellular trafficking and efflux of the drug . The effTo gain insight into the alternative mechanisms of oxaliplatin resistance, we also performed gene expression arrays and subjected the data to ingenuity pathway analysis (IPA) to search for canonical pathways that were significantly affected. Several pathways were frequently and significantly affected and might add to the resistance mechanisms described above. The most frequently altered gene in the aryl hydrocarbon receptor (AHR) signaling pathway was ALDH1L2; this gene was decreased in all oxaliplatin resistant cell lines and is the mitochondrial homolog of ALDH1L1 which has been implicated in cell proliferation and apoptosis ,38. AnotIn the p53 signaling pathway, the most frequently decreased genes were the proapoptotic genes, BAX and BBC3 (PUMA), which were reported previously to play a role in oxaliplatin resistance ,23 and iSince the expression of ERCC1 is unaltered or even decreased in the drug-resistant cell lines, it is unlikely that nucleotide excision repair is involved in the resistance towards oxaliplatin in these cell lines. Decreased ERCC1 mRNA and protein expression in oxaliplatin resistant cells has been previously demonstrated  and was The xenobiotic metabolism pathways, highly significant in LoVo-Li/cOHP, suggested a possible contribution to oxaliplatin resistance of aldo-keto reductase family members, 1C1 and 1C3. Aldo-keto reductase family members have been implicated previously in poor prognosis and resistance towards platinum and other drugs ,48,49. CChanges in gene expression observed in the drug-resistant cell lines as discussed above could be mediated by altered gene expression but might also be a result of gains and losses at the genomic level. Primarily, focal gains and losses were observed in the oxaliplatin resistant variants. The parental cell line LoVo-92 has been characterized by trisomy of chromosome (Chr) 5, 7 and 15 . The majOne of the main findings of the present study is that sensitivity to oxaliplatin is highly correlated with total platinum accumulation which was associated with expression status of the organic cation transporters OCT1-3, hence highlighting the role of these influx transporters in acquisition of oxaliplatin resistance. Since formation of DNA adducts, which was highly correlated to ATP7A, was not related to oxaliplatin sensitivity, it can be concluded that mechanisms, other than DNA damage signaling, may contribute to the pharmacological activity of oxaliplatin. Decreased expression of proapoptotic genes such as Bax and BBC3(PUMA) and increased expression of inhibitors of apoptosis, as well as genes involved in homologous repair, warrants combination studies with drugs that target these proteins, to overcome resistance to oxaliplatin and other platinum drugs.®) was obtained from Bristol-Myers Squib B.V.  in a concentration of 0.5 mg/mL. The RPMI and DMEM culture medium were obtained from BioWhittaker  and fetal bovine serum (FBS) was obtained from GIBCO . All other chemicals were of analytical grade. Solutions were made in water purified by a Millipore Reagent Q system .Oxaliplatin (l-OHP) was kindly provided by Sanofi-Synthelabo . Cisplatin . Periodic assays were performed to check for mycoplasma contamination.The colon carcinoma cell lines, LoVo-92 and LoVo-Li, were kindly provided by Dr. Poupon . LoVo-92Resistance to oxaliplatin was induced by exposing LoVo-92, LoVo-Li, and A2780 cells to increasing concentrations of oxaliplatin using two different schedules for a period of 7 months. In the first schedule, cells were exposed to 4 h pulses every passage to mimic the clinical bolus treatment. In parallel, cells were exposed every passage for 72 h to mimic a continuous infusion. Platinum-resistant cell lines were termed after the parental cell line with 4OHP and cOHP extensions reflecting the resistance induction scheme. To maintain the resistance phenotype, cells were regularly exposed to oxaliplatin during routine culturing. For all mechanistic experiments as described below , cells were cultured without drug for at least one week.50) was determined from the growth curves.Growth inhibition experiments were performed at least three times using the sulforhodamine B (SRB) assay as previously described . BrieflyTo determine cellular oxaliplatin accumulation and formation of DNA adducts, cells were exposed to either 20 μM l-OHP for 4 h or to 200 µM l-OHP for 24 h. To determine retention of the accumulated l-OHP and the formed DNA adducts, indicative for alterations in efflux or repair, cells were subsequently incubated for 3 h in a drug-free medium as described previously . Since 2To determine gene expression, RNA was isolated from cell pellets using Trizol  according to the manufacturer’s protocol. After RNA quantification (nanodrop ND-1000), 500 ng to 1500 ng of RNA were used for cDNA synthesis, as described previously . SubsequParental cell lines and their oxaliplatin-resistant sublines were subjected to microarray analysis. Both array comparative genomic hybridization (aCGH) and mRNA expression arrays were performed. The isolation of genomic DNA (gDNA) for aCGH analysis was performed using the Wizard Genomic DNA Purification kit  according to the manufacturer’s protocol. Total RNA was isolated using Trizol extraction, as described above. The gDNA and RNA quality was checked (OD 260/280 > 1.8). Subsequently, aCGH was performed using the Agilent Human Genome CGH 44 K array platform . Samples and human universal reference were hybridized using a cross array hybridization as described previously [After hybridization and scanning of aCGH slides the raw data were extracted from the images using Agilent Feature Extraction (version 9.5.1.1). Subsequently, data were normalized and smoothened with the R-script “nowave” to increase accurate detection of breakpoints . Genomicp < 0.05 were analyzed using ingenuity pathway analysis .After hybridization and scanning of slides for genome wide gene expression, the raw data were extracted from the images using Agilent feature extraction and subsequently processed using the LIMMA package in Bioconductor to determine Lowess normalized Log2 ratios. For determination of enrichment of genes in canonical pathways, normalized Log2 ratios >1 or <−1 and t-test for paired and unpaired samples, provided by the statistical option of Excel. p-Values below 0.05 were considered as statistically significant. Correlation studies, both the parametric Pearson correlation coefficient (r) for linear correlation and the nonparametric Spearman’s rank correlation coefficient (rho), were performed using SPSS 16.0. Data analysis of the microarray data were performed with specific programs as described in the microarray sections.Differences between cell lines regarding sensitivity, drug accumulation, adduct formation, and gene expression were evaluated using the Student


Phyllomedusa tarsius by the combination of molecular cloning and LC-MS analysis. Chemically synthesised DPT9 was broadly effective against the tested microorganisms through the disruption of cell membranes and showed weak haemolytic activity towards horse erythrocytes. It also exhibited anti-proliferative effect against various human cancer cells. Moreover, an analogue with enhanced cationicity, K8, 23-DPT9, in which Asp8 and Glu23 were substituted by lysine residues, had a markedly increased antimicrobial effect against all tested microorganisms and disrupted microbial cell membranes. This analogue also showed no haemolysis at its effective antimicrobial concentrations. In addition, K8, 23-DPT9 displayed an enhanced anti-proliferative effect against cancer cells, while displayed weak activity against the normal human cell line, HMEC-1.A novel dermaseptin peptide, dermaseptin-PT9 (DPT9), was isolated and identified from  Antimicrobial peptides have gained much attention as novel antimicrobial agents. Most of them are cationic that they are able to kill the microbes through the electrostatic interaction with negatively charged lipid membrane of microorganisms, causing irreversible and potent membrane disruption and cellular dysfunction ,2,3. TheDermaseptins are a group of antimicrobial peptides that have been predominantly isolated from the frogs belonging to Phyllomedusinae subfamily, and they exhibit antimicrobial activity against an array of pathogens such as bacteria, yeasts, protozoa and filamentous fungi ,6. ThesePhyllomedusa tarsius by the combination of cloning and mass spectrometry, namely dermaseptin-PT9 (DPT9). It demonstrates a high degree of similarity to the dermaseptin-5.1TR/5.2TR, while the biological functions have not been studied yet [Herein, a novel dermaseptin peptide was identified from the skin secretion of died yet . TherefoPhyllomedusa tarsius (n = 3) were commercially purchased  and maintained in our purpose-designed amphibian facility. The skin secretion was obtained by mild transdermal electrical stimulation and hand massaging [Specimens of the assaging . The proassaging . The stuThe precursor-encoding cDNA of the peptide from was isolated from a skin secretion-derived cDNA library as in a previous study . The 3′-The process of isolating the mature peptide from crude skin secretion by reverse-phase (RP)-HPLC  and the primary structure analysis of the novel peptide by tandem mass spectrometric (MS/MS) fragmentation sequencing used an LCQ-fleet electrospray ion-trap mass spectrometer  as previously described .8, 23-DTP9 were chemically synthesized via solid-phase Fmoc chemistry using a Tribute automated peptide synthesizer  with Rink amide resin. The synthesised peptides cleaved from resin by a cleavage cocktail containing trifluoroacetic acid (TFA), ethanedithiol (EDT), thioanisole and water (v/v/v/v = 94:2:2:2). The peptides were precipitated and washed by ice-cold diethyl ether, and further dissolved for lyophilisation. The final synthetic peptides were purified and identified by using RP-HPLC and matrix-assisted laser desorption/ionization, time of flight (MALDI-TOF) MS , and then lyophilised as TFA salts.DPT9 and its analogue, KPhysico-chemical properties of the peptides were predicted by Heliquest and the helical wheel plots of the secondary structures were obtained from the helical wheel projections . The secStaphylococcus aureus (S. aureus) (NCTC 10788), methicillin-resistant Staphylococcus aureus (MRSA) (NCTC12493), and Enterococcus faecalis  (NCTC 12697); Gram-negative bacteria Escherichia coli (E. coli) (NCTC 10418), Pseudomonas aeruginosa (P. aeruginosa) (ATCC27853), and Klebsiella pneumoniae (K. pneumoniae) (ATCC 43816), and the yeast Candida albicans  (NCYC 1467). The determination of MICs and MBCs of the peptides was described previously [The minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the synthetic peptides were assessed using different microorganisms including Gram-positive bacterium RSA) NCTC493, and S. aureus, MRSA and E. coli as previously performed [Anti-biofilm activity of the peptides was assessed using the minimum biofilm inhibitory concentration (MBIC) and the minimum biofilm eradication concentration (MBEC) assays against erformed .C. albicans, respectively.Gentamicin (5 µg/mL), vancomycin (5 µg/mL) and amphotericin B (10 µg/mL) were employed as positive controls for Gram-negative bacteria, Gram-positive bacteria and v/v) erythrocytes were incubated with an equal volume of peptide solution in a range of final concentration from 1 to 512 μM in a 96 well plate as performed previously [Defibrinated horse erythrocytes  were used to evaluate the haemolytic activity of the peptides. A total of 4%  as performed in previously . The micThe human neuronal glioblastoma cell lines, U251MG (ECACC-09063001), the human breast cancer cell lines MCF-7 (ATCC-HTB-22), the human pancreatic cancer cell lines, PANC-1, the non-small cell lung cancer cell line NCl-H157 (ATCC-CRL-5802) and the human prostate carcinoma cell line PC-3 (ATCC-CRL-1435) were selected to test the cytotoxicity of the peptides. The human microvascular endothelial cell line, HMEC-1 (ATCC-CRL-3243) was utilized to evaluate the cytotoxicity of the peptides on normal human cells. The cell lines were cultured as described previously .The cell proliferation inhibitory rate was assessed by MTT assay as previously performed  with minThe cell lines were cultured as previous section. After the cells were seeded in the 96-well plate for 12 h, different peptide doses were subjected to the cells for 6 h. The LDH released in the cell supernatant was detected using Pierce LDH cytotoxicity assay  .Phyllomedusa tarsius. The translated open reading frame consists of 71 residues , exhibited 4-32-fold potency against microbes [5, 17-DPS3, K8, 23-DPT9 was also designed by replacing two Lys and it exhibited similar fold change of antimicrobial activity. Furthermore, from the antimicrobial potency of the other dermaseptins in Charge is one of main parameters for optimizing the bioactivity of antimicrobial peptides . In our microbes . In addi8, 23-DPT9 processes more helical content than DPT-9 in a membrane-mimetic environment. Although, the algorisms for calculating the helical contents of peptides via experimental CD spectra might not be precise enough, the helical contents of dermaseptins are not closely correlated with their activities as observed form Following initial membrane binding, the key event is the process of conformational transformation of the peptides . When atAlthough it is proposed that membrane lysis can result via various mechanisms including membrane perforation, destruction, or solubilization, the microbicidal potential is thought to result from the capacity of the antimicrobial peptides to permeate the target cell membrane ,29. The 8, 23-DPT9 displayed significant membrane permeability at their two-fold MICs and four-fold MICs, which indicated that the peptides kill microorganisms by cell membrane disruption. However, in fact, K8, 23-DPT9 induced the same membrane permeability as DPT9 at lower concentration that is consistent with the consideration of enhanced binding affinity by net positive charges.In our previous study, increasing cationicity and hydrophobicity can enhance membranolytic activity, while hydrophibicity makes dermaseptin rather nonspecifically towards both bacterial and mammalian cell membrane . It coul8, 23-DPT9 also displayed anti-proliferative effects against tested human cancer cells. According to the results of LDH, both peptides could induce significantly release of LDH in 6 h against cancer cells, suggesting that the anticancer action of the peptides might be related to necrosis by cell membrane damage at the high concentration. Previous research has demonstrated that cancer cell membranes are generally negatively-charged, containing more phosphatidyserine and other anionic molecules including heparin sulphates and O-glycosylated mucins than normal cells [8, 23-DPT9 is more effective against cancer cell membranes and thus exert increased anticancer activity, comparing to DPT9. Whilst, both peptides could induce further cell death after 6 h when comparing the MTT and LDH results, indicating more mechanisms could be involved along with cell necrosis. Dermaseptin B2 can bind to the plasma membrane of human PC3 cancer cells, aggregate on and penetrate the cells, resulting in a necrotic-like pathway which ruptures the cancer cell membrane due to alteration in the mitochondrial membrane potential and stimulation of caspase-3 [DPT9 and Kal cells ,33. Elecaspase-3 . Furtheraspase-3 . Therefo


Steroid-refractory graft-versus-host disease (SR-GvHD) is a leading cause for late non-relapse mortality and contributes to high morbidity rates and poor quality of life post allogeneic stem cell transplantation . Although numerous treatment approaches have been proposed and studied in clinical trials , SR-GvHD management still remains suboptimal , 2. ReceHerein, we present the clinical course and outcome in a patient who developed SR-cGvHD, which demonstrated refractoriness when bortezomib or ruxolitinib were administered as single agents but experienced a meaningful response to the bortezomib plus ruxolitinib combination, suggesting a potential synergistic effect of these two agents. Patient had previously signed an informed consent for the anonymous use of the medical data, including photos, for scientific purposes.2  every 35 days plus MMF plus steroids were administered but unfortunately after 2 cycles, the patient showed stable GvHD findings, while maintaining steroid dependency. Because of their different mechanism of action, we next decided to combine bortezomib (in the previous administered dose) plus ruxolitinib at the dose of 5 mg bid along with MMF and steroids (dose 1.5 mg/kg daily). After 3 months of treatment with the above combination, marked skin improvement was noticed; lichenoid lesions disappeared, sclerodermatosis was improved (mRSS:12), and liver enzymes became almost normal  methotrexate was administered as GvHD prophylaxis. The early course post transplant was uneventful; however, 2 months post allograft, the immunosuppression was tapered rigorously because of molecularly detectable residual disease. Two months later, he achieved complete molecular remission with 100% of donor origin hematopoietic cells, but at the cost of induced-GvHD with severe extensive skin involvement : 25), buccal mucosal, and liver involvement. Chronic-GVHD was treated with CNIs  plus steroids. No improvement was noticed, so MMF was added, however, only minimal response was achieved. Since ibrutinib, which has been approved by the FDA for the treatment of SR-GvHD [mal Fig. . NoticeaDespite the improvements in its prophylaxis and treatment, SR-cGvHD, continues to pose a major hurdle to ultimately improve outcome of alloSCT . EspeciaJAK signaling has been proven to be a key regulator of inflammation and tissue damage in GvHD, transducing inflammatory signaling downstream of cytokines and regulating development and function of immune cells including antigen-presenting cells (APC) and T cells. Recently JAK-pathway inhibition was proposed as a potential target for the treatment of SR-GvHD . Zeiser The proteasome-inhibitor bortezomib, through inhibition of nuclear factor kappa-B (NF-kB), results in depletion of proliferating alloreactive T cells, decrease of type-1 helper T-cell cytokine production, and inhibition of APC function, while demonstrating limited influence on the number and function of regulatory T cells, the important mediators for GvHD control . In a liInterestingly, the combination of ruxolitinib plus bortezomib resulted in almost normalization of liver function tests and a 50% improvement in the skin sclerotic lesions. Moreover, it was feasible to discontinue MMF while steroids dosing was reduced approximately by 75%.So far, there are no published data regarding the combination of ruxolitinib plus bortezomib as treatment for SR-GvHD. However, the above combination has already been tested in multiple myeloma cell lines and exhibited significant anti-myeloma activity .It is well known that ruxolitinib affects directly the JAK/STAT pathway and indirectly the PI3K/Akt and Ras/Raf/MAPK pathways, while bortezomib inhibits the NF-kB pathway. We assume that the inhibition of these four major signaling pathways by this drug combination finally resulted in the effective activity against this refractory and steroid-depended GvHD.In this heavily pretreated patient, the combination of ruxolitinib plus bortezomib proved to be highly effective, also demonstrating no toxicity. Undoubtedly, this is a single case and also other immunosuppressive agents additionally to bortezomib and ruxolitinib were used and therefore, it is  inaccurate to draw definite conclusions regarding the real impact of these agents on the treatment response. However, these promising results indicate that the combination of bortezomib plus ruxolitinib could be an alternative approach for patients with SR-cGvHD and merits further investigation in future prospective clinical trials.


Pistacia genus on human tumor cell lines. In particular, the cytotoxic activity of essential oils obtained from P. lentiscus, P. lentiscus var. chia (mastic gum), P. terebinthus, P. vera, and P. integerrima, was screened on three human adenocarcinoma cell lines: MCF-7 (breast), 2008 (ovarian), and LoVo (colon). The results indicate that all the Pistacia phytocomplexes, with the exception of mastic gum oil, induce cytotoxic effects on one or more of the three cell lines. PCA highlighted the presence of different cooperating clusters of bioactive molecules. Cluster variability among species, and even within the same species, could explain some of the differences seen among samples suggesting the presence of both common and species-specific mechanisms. Single molecules from one of the most significant clusters were tested, but only bornyl-acetate presented cytotoxic activity, although at much higher concentrations (IC50 = 138.5 µg/mL) than those present in the essential oils, indicating that understanding of the full biological effect requires a holistic vision of the phytocomplexes with all its constituents.Principal component analysis (PCA) multivariate analysis was applied to study the cytotoxic activity of essential oils from various species of the  Herbal medicine is emerging as a holistic tool in novel personalized medicine approaches, especially in primary and secondary prevention . In addid-Limonene, one of the most widespread natural monoterpenes, exerts in vitro anti-tumor activity against several cancer types: neuroblastoma, leukemia, breast, skin, liver, lung, and stomach cancer [p-cymene, 1,4-terpineol, α-terpinene, α-copaene, α-elemene, α-humulene, and α-caryophyllene, among others, have shown significant cytotoxic activity in different cancer cell lines [Plants are a major source of chemopreventive phytochemicals . Among th cancer ,13,14,15ll lines ,19,20,21Pistacia, a genus of flowering plants from the family Anacardacee, includes several species largely distributed and rich in terpenoids [Pistacia genus comprises more than twenty species and, among them, P. lentiscus, P. terebinthus, P. vera, P. integerrima, and P. lentiscus L. var. chia are more popular and have been extensively used in traditional medicine for their tonic, antiseptic, antihypertensive, and gastro-protective properties [rpenoids ,22, whicoperties ,22,23. Moperties .P. lentiscus L. var. chia (known as mastic gum) which has been reported to exert cytotoxic activity on tumor cells and inhibition of cancer cell proliferation, angiogenesis, and inflammatory response [Most published data are related to the resin of response ,29,30,31Pistacia species have been carried out, mostly using extracts and not terpenes-rich essential oils [P. lentiscus, P. lentiscus L. var. chia, P. terebinthus, P. integerrima, and P. vera) on three human adenocarcinoma cell lines: MCF-7 (breast), 2008 (ovarian), and LoVo (colon). A multivariate analysis approach was then used to investigate the statistical correlations among the different phytocomplex components of the essential oils, allowing the identification of clusters of molecules cooperating in the cytotoxic activity.A number of studies concerning the cytotoxic activity of other ial oils ,36,37,38ial oils . In the P. lentiscus L. , P. terebinthus L., P. vera L., and P. integerrima J.L. Stewart ex Brandis, as identified by the Wiley mass spectra library. A total of 32 samples from different aerial parts of the plants were analysed. Although most terpenes were present in both P. lentiscus and P. terebinthus samples, the oil composition varied considerably, with limonene and cis-ocimene being more meaningfully present in samples from P. terebinthus, while β-phellandrene, 1,4- and α-terpineol, α- and γ-terpinene, and T and α-cadinol were relatively more abundant in samples from P. lentiscus. Collectively, the two species show similar amounts of α- and β-pinene, β-caryophyllene, and butylated hydroxy toluene. Among the samples from other species, those from P. integerrima showed similarities to P. terebinthus in the content of α- and β-pinene and of β-caryophyllene, and to P. lentiscus in the amounts of 1,4- and α-terpineol, endoborneol, and bornyl acetate. Finally, a significant amount of δ-carene appears to be characteristic of all the three species. Oil samples from P. lentiscus var. chia (resin) and from P. vera (nuts) are the ones with the least similarities with the others. The composition of the essential oils indicates consistency with the major terpenes known to be present in these species [Essential oils were obtained by hydrodistillation of aerial parts of the plants, or obtained from producers, and oil composition was determined using GC-MS.  species .Pistacia species was evaluated in vitro on three human adenocarcinoma cell lines: MCF-7 (breast adenocarcinoma); 2008 (ovarian adenocarcinoma); and LoVo (colon adenocarcinoma). Results are shown in The cytotoxic activity of the phytocomplexes from the different P. lentiscus were active, with the exception of the one from P. lentiscus var. chia. Samples from P. terebinthus displayed a high frequency of activity, above 70%, while both samples from P. integerrima were active and the one from P. vera was not. The sample with the most activity was one essential oil from P. lentiscus leaves (sample A: IC50 = 173.9 µg/mL).Most oil samples displayed some cytotoxic activity on one or more cell lines with different potencies. In particular, looking at the results obtained with LoVo cells, all the samples from P. lentiscus on LoVo cells, none of the species was active on all the other cell lines. In particular the activity towards 2008 cells was higher for the samples from P. terebinthus (71%), while P. lentiscus displayed a significant activity in 55% of samples, while the two samples from P. integerrima were active and those from P. vera and P. lentiscus var. chia were not. The two most active samples were both from P. lentiscus leaves (sample A: IC50 = 181.5 µg/mL and sample C: IC50 = 220.4 µg/mL).Apart from P. lentiscus (73%), and it was the only cell line sensitive to the essential oil from P. vera. Essential oils from P. terebinthus were active in 44% of cases, while the two oils from P. integerrima had a weak effect or were inactive. The two most active samples were from P. lentiscus leaves (sample B: IC50 = 239.6 µg/mL), from P. terebinthus galls (sample AB: IC50 = 254.9 µg/mL) and from P. vera nuts (sample A3: IC50 = 290.6 µg/mL).The activity on MCF-7 cells was higher for the samples from Pistacia species phytocomplexes on all the three cell lines tested, in most cases with IC50 values below 500 µg/mL, suggesting that this effect might be a shared feature of the Pistacia genus. There were wide differences in biological activity between samples, regardless of the species and even the anatomical part, likely related to the high variability in oil composition even within the same species, as determined by GC/MS fingerprinting.Taken together the results indicate a dose dependent cytotoxic effect of different The oils did not show a consistent order of potency from one cell line to the other, and in some case the same sample could display high activity on one cell line, while being inactive on the others . Such diIn general, the biological activity of a phytocomplex is related to its chemical composition, with each single compound acting synergistically with the others. Thus, given the high heterogeneous compositions of essential oils, likely due to genetic diversity of the samples , it is dPistacia genus, but most studies have focused on different types of extracts and not on the terpene-rich essential oils, suggesting other types of chemicals and mechanisms were involved [Pistacia species have been shown to exert cytotoxic activity on tumoral cell lines. Pistacia vera and Pistacia khinjuk resin essential oils were found to be active against retinoblastoma cells (Y79) [P. palestina Boiss. Although inactive on MCF-7 cells, displayed cytotoxic activity towards amelanotic melanoma cells (C32) and renal cell adenocarcinoma (ACHN) [P. lentiscus var. chia, although in most cases not with essential oil [P. lentiscus var. chia resin (mastic), we did not observe any cytotoxic activity in our experimental conditions. This discrepancy could be related to the different experimental models used, to the different treatment with the essential oil, or to the relatively stringent threshold we used for the cytotoxic effectiveness (500 µg/mL). To eliminate nonspecific interferences with adhesion of cells to the experimental substrate, a “pre-incubation” strategy was also used. Unlike the other published data, cell cultures were exposed to essential oils only for three hours and then allowed to recover for the following 21 h. The limitation of unwanted effects on cell adhesion, by improving the focus on cytotoxicity, might explain the different results.Terpenes and terpene-rich essential oils have been shown to exert specific cytotoxic activity on several tumoral cell lines ,43,44,45involved ,32,34,38ls Y79) . Essentia (ACHN) . Our res9 . Essential oil ,31. In stial oil ,26. Althn = 7), while LoVo and 2008 cells had six and five, respectively. All the compounds showing a high level of correlation with bioactivity were included at least in one cluster, with the exception of sabinene and α-humulene, the latter a known anticancer agent [P. lentiscus was the species with more clusters of bioactivity-associated molecules in all cell lines. P. lentiscus was also the species with higher principal component scores, with L2 being the cluster with the highest values .PCA analysis was performed with the aim of identifying synergic clusters of compounds present in the phytocomplexes, with the best correlation with biological activity . Four prer agent . P. lentP. integerrima on LoVo cells  is also the core part of I1a and I1b and correlates with activity in all cell lines and, is shared by all L1s P. lentiscus clusters, thus suggesting a common mechanism of P. lentiscus and P. integerrima phytocomplexes throughout the cell lines. Although the aim of the work was not to identify biological activity in single terpenes, given that this particular cluster is shared by all cell lines and at the same time by more than one Pistacia species, we tested the cytotoxic activity of the pure compounds on LoVo cells, where the activity of the phytocomplexes seemed to be stronger. Bornyl acetate was the only tested molecule showing a cytotoxic activity when used alone (IC50 = 138.5 (130.8 to 146.7 µg/mL), although its potency was not as strong as that corresponding to the actual concentration range in the oils. The other compounds displayed no activity in the pre-treatment paradigm used (data not shown), although p-cymene and spathulenol had been previously shown to have anti-tumoral activity [P. lentiscus): α-copaene, α-cadinol, τ-cadinol, cadina-1,4-diene, α-elemene, α-amorphene, germacrene A, α-muurolene, β-elemene, and alloaromandrene. In this cluster α-copaene and β-elemene are known cytotoxic compounds for tumoral cells [P. lentiscus and P. terebinthus . Ten more compounds are differentially cooperating in clusters of cytotoxic activity towards all cell lines, some in the same group, some in totally different contexts , the last one being the only clustered molecule shared by P. vera with other species. Other compounds’ bioactivity are not shared by all three cell lines, thus suggesting a different cell sensitivity and cell-specific mechanisms. Two such molecules, β-caryophyllene and d-limonene, previously shown to have cytotoxic activity and considered promising potential anti-tumorals [The active cluster identified for activity ,42. Thisal cells . α-Terpial cells ,17. Otheal cells ,17. α-Hual cells , is prestumorals ,15,42,45In summary, PCA allowed the identification of 46 compounds in the phytocomplexes correlated with potential biological activity, most of which had not been previously reported for their in vitro cytotoxic activity on tumoral cells (80%). The compounds are distributed within different clusters of molecules potentially cooperating to achieve the cytotoxic activity on the cell lines.P. lentiscus is the species with the most clusters, which is in line with its higher potency and frequency of activity with respect to the other species tested. Only two of such clusters are common for all the cell lines, they are characteristic of P. lentiscus, and one is also shared with P. integerrima. In other cases clusters are shared only for one or two cell lines. In the case of P. vera, only one cluster could be identified. These similarities and differences suggest that the cytotoxic effect is complex. Some are common for all three cell lines which suggests a shared mechanism for all three adenocarcynomas, but some are not and P. lentiscus seems to display the most variability, while samples from the other species seem more consistent throughout the different cell lines. This behaviour points to different mechanisms exerted in a cell-specific fashion.Taken together, the PCA results indicate that the cytotoxic activity of the phytocomplexes can be highlighted in several clusters of likely-synergizing compounds. Keeping in mind that the results with PCA do not necessarily explain the full activity of the whole phytocomplex, the analysis allowed the identification of substances, mostly terpenes, sesquiterpenes, and fatty acids, present in the essential oils likely playing a central role in the in vitro anti-tumoral effect. Of these, some had been previously shown to have such activity when used as single compounds. Almost all the substances identified cooperate with others, thus forming clusters of activity, often different for one or more components in terms of cell sensitivity.Finally, although all the compounds identified had a positive correlation with the activity, none of the single clusters or the single components are, by themselves, sufficient to justify the whole activity, and at least all the molecules identified contribute to the biological effects.Methanol, ethyl acetate, formic acid, acetonitrile, and HPLC-grade solvents were purchase from Carlo Erba Reagents Srl . The solvents were of analytical grade. Double distilled water was used in the HPLC mobile phase. All other reagents, including single purified terpenes, were obtained from Sigma Aldrich srl. .P. terebinthus flowers  which were collected in Provence , aerial parts  of the plants were collected in three different Italian regions: Pistacia terebinthus L.: Veneto, Colli Euganei (45°21′50.01″ N 11°40′13.51″ E) ; Pistacia lentiscus L.: Tuscany, San Giuliano Terme (43°45′45″ N 10°26′29″ E), Pisa , Tuscany, Montepescali, Maremma (42°52′58.04″ N/11°05′11.97″ E/42.882788° N) ; Pistacia vera L.: Sicily, Mount Etna (37°45′04.45″ N 14°59′38.49″ E) (sample M). Other essential oils were purchased: P. lentiscus essential oil from Portugal (sample A) was obtained from Lifetree Aromatix, John Steel, 3949 Longridge Ave. Sherman Oaks, CA, USA; P. integerrima J.L. Stewart ex Brandis essential oil was obtained from Bluebell Herbal Products, Bhaktapur, Nepal ; P. lentiscus var. chia resin (mastic) essential oil, was obtained from “Anemos” Benetos John Galatoulas George Co, Chios, Karfas RD, Kontari, Chios, Greece (sample L).With the exception of P. lentiscus, P. terebinthus, and P. vera) were collected, rinsed, dried, and frozen at −21 °C within three hours from collection to preserve their phytochemical composition until the moment of the distillation. The material was then hydrodistilled within a few months. All the samples, before distillation, were minced to obtain the maximum extraction yield and make the process of diffusion of the essence easier [Plant parts were recognized by one of the authors (G.I.), University of Padua, Italy. Voucher specimens were deposited at the Department of Pharmaceutical and Pharmacological Sciences of the University of Padova . Plant parts , branches, galls or nuts (250 g), berries (100 g), and flowers (270 g). The yield for the various parts of the plant, express as percent of initial wet weight was: leaves (0.05%), branches (0.06%), nuts (0.02%), berries (0.11%), flowers (0.08%), and galls (0.40%).P. lentiscus, P. terebinthus, P. vera, P. integerrima, and P. lentiscus var. chia was determined by gas chromatography analysis coupled with a mass spectrometer detector (GC/MS), with a Hewlett-Packard 6890-5973 system .The chemical composition of the essential oils obtained from The identification of compounds by GC-MS was performed considering not only the matching of mass spectra fragmentation but also the retention index. These two parameters allow the identification of phytoconstituents of the essential oil. In particular, the separation was achieved using a HP-5 MS capillary column (30 m × 0.25 mm i.d. × 0.25 film thickness), with a stationary phase of 95%polydimethylsiloxane. GC-MS analysis carried out under the following conditions: carried gas, He; flow rate, 0.8 mL/min; split ratio, 1:10; injection volume, 1 µL; injector temperature, 200 °C. The temperature programme was as follows: 60 °C with 3 min initial hold, and then to 280 °C at a rate of 3 °C/min, and finally held isothermally for 5 min. The ionization mode was electronic impact at 70 Ev. Identification of the components was achieved by comparison of their mass spectral fragmentation patterns with those stored in the data bank (Wiley Library) ,49,50,51Pistacia oils were dissolved in DMSO and then diluted in culture medium to obtain stock solutions which were stored at −20 °C. Each final stock solution contained 1% DMSO, 9% essential oil, and 90% culture medium. All the procedures were carried out under sterile conditions. Before each experiment the stock solutions were diluted with growth medium and used immediately. The final DMSO concentration was 1%.2) 5%, at 37 °C).Cytotoxicity was evaluated on three human adenocarcinoma cell lines: ovarian (2008) breast (MCF-7), and colon (LoVo). The cell line 2008 was kindly supplied by Prof. G. Marverti  and was maintained in RPMI 1640 medium . MCF-7 cells were supplied by the Experimental Zooprophylactic Institute of Lombardy and Emilia  and were grown in Eagle’s Minimum Essential Medium . LoVo cells were supplied by Dr. G. Toffoli , and were grown in HAM’s F12 (Lonza). L929 fibroblasts  and HFFF2 cells, a human Caucasian foetal foreskin fibroblast derived from a 14–18 old human foetus , were used as healthy controls to verify the specificity of the cytotoxic effects for cancer cells (data not shown). L929 and HFFF2 cells were grown in RPMI 1640 medium  and in DMEM  respectively, and 1% penicillin/streptomycin  (Seromed-Biochrom KG). All cultured media were supplemented with 10% heat-inactivated FCS  (20% for HFFF2 cells), 1% antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) (Seromed-Biochrom KG), and 1% 200 mM glutamine . Cells were maintained under humidified conditions  were seeded in 96-well tissue plates  and treated for 3 h with different concentrations of Pistacia essential oils, with single compounds, or with vehicle . The cells were then rinsed with sterile PBS and incubated in culture medium for 21 h. The cytotoxic effect was evaluated by MTT reduction assay after 21 h of incubation. An amount of 20 µL of MTT solution (5 mg/mL in PBS) was added to each well, and plates were incubated for 4 h at 37 °C. DMSO (150 µL) was added to all wells and mixed thoroughly to dissolve the dark-blue crystals. The absorbance was measured on a micro-culture plate reader  using a test wavelength of 570 nm and a reference wavelength of 630 nm. The 3 h pre-incubation followed by recovery model was chosen after some preliminary tests aimed at identifying the best conditions to assay for specific cytotoxic effects. Cisplatin was used as the positive control. IC50 values below 500 µg/mL were taken into consideration for the cytotoxic activity of each phytocomplex tested. This value was chosen as threshold in line with recent literature on the subject and other works on cytotoxicity of Pistacia essential oils, although some authors have used different thresholds [Cells . IC50, dose-response curve best-fit, Pearson correlation, and linear regression analysis were performed using GraphPad Prism Version 3.0. Principal component analysis (PCA) was performed using XLSTAT® software . PCA was performed on a correlation matrix (Pearson (n)), computed with the contribution (%) of each compound to the IC50 of each oil sample, calculated with the following formula: The results were statistically evaluated by Student’s 50 value takes into consideration the intrinsic contribution of the compound to the cytotoxic effect of the phytocomplex and allows the evaluation of its relative potency in the context of the phytocomplex. The resulting values were used as variables in the matrix, and are the vectors in the biplot graphs of principal components, while the samples of the different Pistacia species represent the observations in the matrix and the points in the graph. The clusters of molecules in a given species were identified in the graphs according to four vector characteristics: correlation with the Pistacia species, positive distribution with respect to one or more given principal component, distance from the origin, and angle widths between vectors.Correlating the amount of each compound with the relative ICPistacia. The phytocomplexes in essential oils from four different species of Pistacia  consistently, though differently, induced cytotoxicity on the three cell lines of human adenocarcinoma, with variable potency and tropism depending on the species, and no significant difference among samples from different aerial parts. In general, this suggests that the in vitro anti-tumoral effect is a shared feature of the Pistacia genus, although there are differences that also indicate patterns of activity characteristic for each species and for each adenocarcinoma.This is the largest study conducted to investigate the in vitro anti-tumoral effect of different species from the genus 50 of the considered essential oils. Merging the chemical composition data and the results of the biological assay by a multivariate approach offers a new approach in the evaluation of the bioactivity of these complex mixture. This approach allows the identification of clusters of compounds that, when present together in the mixture, are correlated with increased bioactivity.PCA was used to investigate the correlations between the molecular components of the phytocomplexes, most of them terpenes, sesquiterpenes, and fatty acids, and the observed biological activity. This approach appears to be favourable because it takes into consideration the relative contribution of each compound of the phytocomplex to the final effect on the tumoral cells creating a correlation with the measured ICMolecular cooperation is the most important aspect of the biological activity found in natural products, where a holistic approach is needed to understand their therapeutic potential. This was confirmed in our PCA study and we found several clusters of bioactivity-associated molecules in each species, likely to specifically synergize on each cell line. Two such clusters were shared for the three adenocarcinomas, indicating the presence of at least two common pathways of cytotoxic activity, while all the others were different, thus suggesting specific cell line sensitivity and different species-specific mechanisms observed in vitro. Among the molecules identified only about 20% were compounds already known for their anti-tumoral effect as single drugs, while the others were novel findings. Among the new molecules identified, mostly terpenes, bornyl acetate showed significant cytotoxic activity even when used alone, but to a much lesser extent if compared to the dose range present in the essential oil, thus highlighting the importance of cooperation between different molecules in the phytocomplex to achieve the final effect.


The ability of an organism to respond to physical stresses and return to homeostasis (i.e. resilience) has been suggested to correlate with longevity. Here, we investigated whether this extends to resilience at a cellular level using primary fibroblasts isolated from tail skin of genetically heterogeneous young adult UM-HET3 mice. Cells isolated from each individual mouse (cell line) were tested in their response to concentrations of agents or conditions predicted to induce a cellular challenge, including paraquat, hydrogen peroxide, antimycin a, cadmium chloride, mdivi-1, thapsigargin, and nutrient starvation. Cell viability was monitored in real-time using an incucyte S3 live cell analysis system and we addressed the response following challenge as a marker of resilience. Cellular uptake of ethidium homodimer-1 was used to determine the loss of viability. Cellular bioenergetics were assessed using a seahorse XF24. We found that cell lines that were resistant to paraquat were also resistant to antimycin a, and hydrogen peroxide. Cell lines that were resistant to nutrient starvation were also resistant to mdivi-1. Indices of cellular bioenergetics status including ATP production rate and cell respiratory control ratio, revealed potential relationships with resiliency. Taken together, our data indicate that skin fibroblasts retain individual physiological programs that may in part explain the patterns of resiliency or sensitivity to a stressor at the organismal level. Since the cell lines tested in this study were obtained from living mice, future work will investigate whether these patterns of resiliency change with age and elucidate their utility in predicting future health outcome.