MicroRNAs (miRNAs) play important roles in the fibrosis of systemic sclerosis (SSc). However, the underlying miRNA-mRNA regulatory network is not fully understood. A systemic investigation of the role of miRNAs would be very valuable for increasing our knowledge of the pathogenesis of SSc. Here, we combined miRNA and mRNA expression profiles and bioinformatics analyses and then performed validation experiments. we identified 21 miRNAs and 2698 mRNAs that were differentially expressed in SSc. Among these, 17 miRNAs and their 33 target mRNAs (55 miRNA-mRNA pairs) were involved in Toll-like receptor, transforming growth factor β and Wnt signalling pathways. Validation experiments revealed that miR-146b, miR-130b, miR-21, miR-31 and miR-34a levels were higher whereas miR-145 levels were lower in SSc skin tissues and fibroblasts, normal fibroblasts and endothelial cells that were stimulated with SSc serum. ACVR2B, FZD2, FZD5 and SOX2 levels were increased in SSc skin fibroblasts, normal fibroblasts and endothelial cells that were stimulated with SSc serum. We did not identify any negative correlations among these miRNA-mRNA pairs. miR-21 was specifically expressed at higher levels in SSc serum. Six miRNAs and 4 mRNAs appear to play important roles in the pathogenesis of SSc are worth investigating in future functional studies. Systemic sclerosis (SSc) is a complex heterogeneous autoimmune disease that is characterized by inflammation, vasculopathy, and extensive fibrosis1The exact cause of SSc currently remains elusive but is likely to involve the effects of environmental factors on genetically primed individuals578in vitro experiments, such as transfection or luciferase activity assays, which may not reflect their real in vivo effectsmiRNAs are non-coding RNAs that are ~22 nucleotides in length and function as intracellular regulators of gene expression. miRNAs play key biological roles by modulating both gene and protein levels by destabilizing transcripts and inhibiting protein translation, respectively10In our previous miRNA array experiments, we found aberrantly expressed miRNAs in dSSc and lSSc lesioned skin and 21 miRNAs were altered in both types of tissuesIn our previous study, we used a custom microarray platform to evaluate the miRNA expression profiles of skin tissues obtained from SSc patients. This microarray set included nine biologically independent samples, including three normal skin samples, four dSSc skin samples, and two lSSc skin samples. Each skin sample was derived from a single specimen. We identified a total of 21 miRNAs that were similarly altered in both dSSc and lSSc (13 miRNAs were upregulated and eight miRNAs were downregulated)We identified 13689 predicted conserved target mRNAs for the 21 previously identified miRNAs using the TargetScan database. Among these, 326 of the target mRNAs (2.38%) were involved in the TLR, TGF-β and Wnt signalling pathways, including 178 mRNAs that might be targeted by 10 upregulated miRNAs  and 148 mRNAs that might be targeted by 7 downregulated miRNAs   and C. Ahttp://www.ncbi.nlm.nih.gov/geo/GSE accession number GSE9285), which contains 31 SSc skin tissues and 9 normal skin tissues that were obtained from forearm skin biopsiesWe analysed the gene expression data using the Gene Expression Omnibus  serum; miR-10a, miR146b, miR-1246 and miR-130b were specifically decreased in systemic lupus erythematosus (SLE) serum; and miR-34a was increased in SSc, SLE, and IIM serum .To our knowledge, this is the first study to integrate the effects of the dysregulation of miRNA and mRNA expression profiles in SSc. In this study, we used TargetScan to predict miRNA target mRNAs, and we combined these target mRNAs with genes that we found in other microarray data to be significantly altered. A small portion of the target genes  were differentially expressed in SSc skin tissues, and these were selected for further analysis. The expression levels of six of the miRNAs  were higher, the expression level of miR-145 was lower, and the expression levels of 4 genes  were identified in SSc skin tissues and fibroblasts and in normal skin fibroblasts and endothelial cells that were stimulated with SSc serum. These markers might play fundamental roles in SSc pathogenesis. However, our validation experiments did not reveal any miRNA-mRNA pairs that were negatively correlated and it’s complicated to explain. Recently, progress has been made in comparative genomics analyses and high-throughput experimental studies. The underlying miRNA-mRNA interaction networks are large, complex and only partially explored2223Previous studies have reported that the expressions of some miRNAs, such as miR-2930The data in this study suggest that stimulation with SSc serum may activate the Wnt/β-catenin pathway in both fibroblasts and endothelial cells. A pathway analysis of miRNA targets showed that the Wnt signalling pathway was the most affected pathway. Aberrant Wnt/β-catenin signalling has been demonstrated in SSc tissues. Canonical Wnt signalling is necessary for TGF-β-mediated fibrosis because it induces the activation and differentiation of and excessive collagen release from fibroblasts3235We also found that there was no difference in the miRNA and mRNA expression profiles of SSc serum-stimulated fibroblasts and endothelial cells. We hypothesized that the components of SSc serum are complex and that they may contain circulating miRNAs enclosed in exosomes that could be transferred from the serum into the cells37In summary, these novel findings regarding miRNA and mRNA expression profiles provide hints about the mechanisms underlying SSc pathology and are therefore worth further functional analysis. The complexity of these regulatory networks is worth further study.The limitations of this study include a lack of phenotypic information, such as modified Rodnan skin scores or data regarding the systemic involvement of SSc. In addition, we detected serum miRNAs that might act as biomarkers, but these data may not reproducible in plasma. The difference between serum and plasma miRNA concentration profiles showed that there were some associations between miRNAs obtained from platelets, potentially indicating that the coagulatory process may affect the profile of extracellular miRNAs in the bloodThe collection and use of human samples for this study was approved by the Ethics Committee of Central South University , and the study was carried out according to the medical research regulations of China. Informed consent was obtained from all patients.Biopsy specimens were obtained from 14 subjects during one of our former studies, as previously described2 in Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated foetal bovine serum . Cells in passages 3–5 were used in the experiments. The cells were used when they had reached 80–90% confluence.Primary skin fibroblasts were expanded by culturing the cells at 37 °C in a humidified atmosphere containing 95% air and 5% CO2 in DMEM supplemented with 10% heat-inactivated foetal bovine serum. The cells were used when they had been cultured to 80–90% confluence.HMVECs were obtained from normal adult skin  and cultured at 37 °C in an atmosphere containing 95% air and 5% COWhole blood samples were obtained from 66 SSc patients, 37 SLE patients, 39 IIM samples and 46 normal controls. All SLE patients met the American College of Rheumatology (ACR) classification criteria for SLEData for 21 miRNAs were obtained from our previous miRNA array studyhttp://www.ingenuity.com/) tool and Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 were used to identify the networks, functions, and canonical pathways of these gene45The predicted target genes of the miRNAs were obtained using TargetScan v7.0. All of the target genes with conserved sites were selected. For the differentially expressed miRNAs, all of their predicted targets that were human genes and those that overlapped with differentially expressed genes were used to perform a KEGG pathway enrichment analysis. The ingenuity pathway analysis (IPA: http://www.ncbi.nlm.nih.gov/geo/GSE accession number GSE9285)The whole human genome oligo microarray (G4112A) data set was used to evaluate 31 SSc skin tissues and 9 normal skin tissues that were obtained from forearm skin biopsies. These data were downloaded from the Gene Expression Omnibus  for 24 h and then stimulated with 20% SSc serum for 48 h. HMVECs were cultured for 24 h in serum-free DMEM (starvation medium), and then stimulated with 20% SSc serum for 48 h.Caenorhabditis elegans miRNA-238  was used as an exogenous control to normalize the serum miRNA data. To detect mRNA, cDNA was prepared using a Reverse Transcription System  and amplified using real-time PCR with SYBR Green  using the primers shown in Total RNA was isolated from skin biopsy specimens, fibroblasts and endothelial cells using an miRNeasy mini kit (Qiagen). Serum miRNAs were isolated using a PAXgene blood miRNA system (Qiagen). An miScript reverse transcription kit (Qiagen) and miScript SYBR Green PCR kit (Qiagen) were used to measure the expression levels of selected miRNAs in a model 7500 real-time PCR system analyzer (Applied Biosystems). miRNA-specific primers and the miScript Universal Primer (Qiagen) were used. The expression of the U6B small nuclear RNA (RNU6B) was used as the endogenous control to normalize the sample data. The spiked GraphPad Prism software was used for all statistical analyses (not including the microarray data). Numerical variables with a normal distribution were compared using unpaired t-tests. Data with a non-normal distribution were compared using the Wilcoxon rank sum test. All data are expressed as the mean ± standard deviation .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Genetic selection for body size during domestication of animal species can inadvertently affect a number of physiological and behavioural traits. The pace-of-life syndrome (POLS) hypothesis predicts that domestication in an artificial environment lacking predators and providing abundant resources prefers proactive individuals with strong feeding motivation, high levels of aggression and risk taking, with low hypothalamus–pituitary–adrenal (HPA) axis responsiveness. In the present experiment we weaned fingerling pike-perch from live feed and habituated them to formulated feed. We recorded the number of weeks needed for the fish to accept pellets, their body length at the age of 100 days, their boldness in a novel object test and their HPI axis responsiveness. Individuals accepting the artificial feed within the first week grew larger than fish habituating later; therefore early weaners would be kept and bred in routine aquaculture procedures. Contrary to predictions of POLS hypothesis, fish weaning earlier and thus growing faster were less bold and had higher HPI axis responsiveness than fish accepting the pellets later or never. As live feed is preferred to artificial pellets, less competitive individuals may switch to pellets earlier. Inadvertent selection for stress sensitive fish may have an effect on production in aquaculture as well as on natural population after intensive restocking. Foraging in a habitat rich in food may be at trade-off with high predatory pressure and intraspecific competition. An alternative adaptive response to such challenges can be to forage at patches with less food, competitors and predators, as there might be more than a single optimal solution, alternative strategy can be present in a population ., defined as joint behavioural and physiological stress responses that are stable over time and which are characteristic to a certain group of individuals  RIA kit  and gamma counter [From the Early, Normal and Late weaning groups 17-17-17 individuals were subjected to confinement stress by keeping them in a net (15x20 cm) for 5 min. After the confinement the fish were anesthetized with clove oil (10 drops in 10 L water). Blood samples were taken 12–15 minutes after the start of the confinement, from the caudal vasculature to heparin-coated tubes, centrifuged (10000G/2 min) and the plasma was stored frozen (-80°C) until analysed. Blood serum cortisol concentrations were measured by radioimmunoassay (RIA) method with Kortizol [ counter . Intra aShapiro-Wilk test showed the data was not normally distributed therefore nonparametric statistics were used. A k-means cluster analysis  was performed on the novel-object test variables to categorise the individuals as explorative, average or retractile. The distribution of the clusters in the five feeding groups  was analysed by Mann-Whitney U test. Bonferroni correction was applied in all multiple comparisons of the five groups by setting critical P value at 0.01 (0.05 divided by 5). Individual cortisol levels in the three pellet feeding groups  were analysed by One-way ANOVA using post-hoc Tukey test. Statistical analyses were conducted using SPSS Statistics 11.5 (2002).Tubifex, therefore fish accepting pellets earlier grew faster. Early weaners (104.8±6.5mm) had significantly higher body length compared to the Late-weaning group (78.9±8.3 mm) which differed from Non-weaners (91.1±13.6 mm) as well. Normal (85.0±8.0 mm) and Cannibal (85.2±9.1 mm) groups showed intermediate size  within groups on all three variables reducing the complexity of the data to three clusters of higher, average or lower exploration variables  = 4.628, P = 0.015). Post-hoc Tukey test revealed significant difference between Early and Late weaners  whereas Normal weaners had intermediate cortisol levels (217.4 ±96.8 ng/ml) which are statistically not different from the two other groups.Among the three investigated factors  only the feeding type proved to have significant effect on the standard length .Tubifex, fish accepting the artificial feed earlier grow larger, while individuals rejecting the pellets would not survive in intensive aquaculture practice.Cross-context analysis of the behaviour of juvenile pikeperch  assessedSalmo trutta) learn to reduce its search time for cryptic prey compared to wild which performs higher predator vigilance [In case of percid fish the ontogenetic changes in foraging (switching to piscivory) occur when the net energy intake is higher on the new diet and the potential prey (fry or larvae) is available in an adequate density . Naïve pigilance . Less exigilance .Although we did not estimate dominance hierarchy during transition, the pattern of competition clearly suggests that pellets were accepted earlier by the less competitive, opportunistic feeders. The early transition group tended to be shyer and had higher stress induced cortisol level than fish that accepted the pellets later. Since the landmark study of Huntingford  on threeAnother unexpected result is that cannibalistic fish were as shy as Early weaners in the novel object test. The present study did not focus on cannibalism; fish were labelled as cannibals upon a single event of intraspecific predation. In accordance with aquaculture practice, cannibalistic fish were removed as soon as recognised but instead of discarding them they were treated as an additional experimental group. Intraspecific predation is generally linked to aggression   in an enDicentrarchus labrax fish reared under unpredictable food supply showed bolder behaviour and realized lower growth rate compared to individuals reared on predictable food supply [Selective breeding causes targeted changes in the genetic composition of farmed populations  as well as inadvertent changes as natural selective pressures are reduced in the artificial environment  . Severald supply . Early wGenetic selection of artificially bred pikeperch would favour less bold and more stress sensitive early pellet eaters. Further studies (including growth studies over longer period of time) are needed to confirm our findings which may have yet unexpected consequences on the breeding and conservation of perch-pike. Production of fish with high stress sensitivity would be disadvantageous in aquaculture production as well as in nature where the large number of artificially selected fish might interbreed with the native population.S1 Table(XLS)Click here for additional data file.


Highly monodisperse sequence-defined spherical nucleic acids (12–SNAsHE) for delivery of small-molecule anticancer drugs. In vitro studies show that BKM120-loaded nanoparticles promote apoptosis in primary patient CLL lymphocytes, and act as sensitizers of other antitumor drugs, without causing non-specific inflammation. Evaluation of this drug delivery system in vivo shows long circulation times up to 24 hours, full body distribution, accumulation at tumor sites and minimal leakage through the blood–brain barrier. Our results demonstrate the great potential of these delivery vehicles as a general platform for chemotherapeutic drug delivery.We report a spherical nucleic acid (SNA) system for the delivery of BKM120, an anticancer drug for treatment of chronic lymphocytic leukemia (CLL). While promising for cancer treatment, this drug crosses the blood–brain barrier causing significant side-effects in patients. The DNA nanoparticle encapsulates BKM120 in high efficiency, and is unparalleled in its monodispersity, ease of synthesis and stability in different biological media and in serum. These DNA nanostructures demonstrate efficient uptake in human cervical cancer (HeLa) cells, and increased internalization of cargo.  This holds true for antitumor chemotherapeutic drugs, where much of their success has been hampered by off-target side-effects, poor pharmacokinetics and systemic toxicity.16 In particular, amphiphilic block copolymers that contain a water-soluble block, and a hydrophobic block, have been extensively used as building blocks for chemotherapeutic drug delivery. These molecules phase-separate into micelles that contain a hydrophobic core which can accommodate lipophilic drug molecules and alter their kinetics both in vitro and in vivo.18 In recent years, a new class of amphiphilic block copolymers has also emerged which contains a hydrophobic synthetic polymer attached to a hydrophilic DNA segment, called DNA–polymer hybrids.19 These molecules can self-assemble into a wide-range of morphologies,20 including spherical micellar particles that expose a hydrophilic DNA shell and a hydrophobic core.22 A particularly successful example of such assemblies are spherical nucleic acids (SNAs).23 These structures are composed of a gold nanoparticle core and a corona of tightly packed DNA strands. SNAs have shown efficient cellular penetration and gene silencing ability both in vitro and in vivo.26Polymers are the most commonly used material for developing nanoparticle-based drug carriers.via solid-phase synthesis.27 Unlike conventional synthetic polymer chemistry, this method yields DNA–polymer conjugates that are fully monodisperse and sequence-defined. This class of material self-assembles spontaneously to generate highly monodisperse spherical micellar DNA nanoparticles in aqueous solution. Several examples in recent years have emerged demonstrating the suitability of DNA nanostructures in mimicking biological systems,29 construction of nanoelectronics30 and nanophotonics,31 and delivery of cancer therapeutics.32 Compared to DNA nanostructures, such as DNA origami,33 which require a large number of unique DNA strands to generate the designed structure,32 limiting their use in large-scale applications, micellar DNA nanoparticles are composed of only a single DNA–polymer conjugate strand. This type of hybrid strand also offers advantages over other block copolymers in that the DNA portion can be highly functional and programmable in the final structure.34 Additionally, the poly-phosphodiester units in both the oligonucleotide and hydrophobic portions of the DNA–polymer strands are biocompatible and biodegradable making them suitable for biological applications.35 Particles of self-assembled DNA–polymer conjugates expose a ssDNA corona, and have been used in ligand targeting,37 delivery of anti-sense oligonucleotides,40 DNA detection,41 formation of higher order assemblies,43 and templating organic reactions.45 In particular, these DNA particles have shown great potential in cancer therapy.46 However, the exploration of these structures for cancer therapy has only been limited to in vitro cell studies. Our interest also focuses on cancer therapy, specifically, the development of a DNA nanoparticle delivery system for BKM120, an anticancer drug towards the treatment of Chronic Lymphocytic Leukemia (CLL).Recently, we reported a highly efficient and versatile method to generate DNA–polymer conjugates 47 Current treatments of CLL include chemotherapeutic agents such as alkylating agents , purine analogs (fludarabine) and immunotherapeutics .49 The current gold standard for treatment is through chemoimmunotherapy; a combination of fludarabine, cyclophosphamide and rituximab (FCR).53 Unfortunately, none of these treatments results in curative therapy providing strong justification for investigating new therapeutic approaches for CLL. The phosphoinositide 3-kinase (PI3K) pathway has been shown to be a critical component of CLL survival and proliferation.56 The expression of PI3K triggers downstream cellular events that inhibit cell death by inactivating pro-apoptotic proteins.60 This makes the selective inhibition of PI3K a promising approach for the treatment of CLL and a focus of many efforts to develop novel inhibitors targeting this pathway.Chronic Lymphocytic Leukemia (CLL) remains the most common type of leukemia with an incidence rate of approx. 4/100 000 people in the United States.61 This molecule has shown high selectivity and potency against class I PI3Ks.62 BKM120 has demonstrated high cytotoxicity in B-chronic lymphocytic leukemia cells in vitro, and significant antitumor activity in human tumor xenograft models.63 Currently, this drug is under clinical investigation in advanced solid tumor and CLL patients.64 However, BKM120 can cross the blood–brain barrier and inhibit PI3K in the central nervous system (CNS), inducing anxiety, low serotonin levels, schizophrenia, and hindering its success for translation into the market. Hence, a strategy to effectively deliver BKM120 to its intended biological target without deleterious side-effects in the CNS would be a major goal for therapy with this small-molecule drug.Buparlisib (codenamed BKM120) is one such example of a pyrimidine-derived selective pan class I PI3K inhibitor.in vivo shows long circulation times up to 24 hours, full body distribution, high accumulation at tumor sites and minimal leakage through the blood–brain barrier. Our results demonstrate the great potential of these delivery vehicles as a general platform for chemotherapeutic drug delivery. We have previously shown that the DNA component of these structures is able to silence gene expression to a greater extent than DNA antisense structures alone, highlighting the promise of these DNA nanoparticles as combination small molecule and oligonucleotide therapeutics.38In this article, we report the development of a DNA nanoparticle platform for the delivery of BKM120. The drug-loaded structures are unique in their monodispersity, can be readily prepared and are stable in different biological media and in serum. We show the increased cellular uptake of these structures in HeLa cells, and the internalization of their cargo. The structures show minimal non-specific interaction with human serum albumin (HSA), a major protein component of blood. Moreover, BKM120-loaded DNA particles promote apoptosis in primary patient CLL lymphocytes and induce cell death when co-administered with doxorubicin in HeLa cells, without eliciting inflammation. Evaluation of this drug delivery system 12–DNAHE conjugate, 12 units were appended to DNA by automated solid-phase synthesis using phosphoramidite chemistry.27 This approach offers monodisperse DNA–polymer conjugates in high yields and provides control over the length and sequence of the monomer units in the final structure. In our previous work, we showed that 12–DNAHE conjugates self-assemble into highly monodisperse spherical nucleic acid particles (12–SNAsHE) in aqueous media containing divalent cations. These structures consist of an exterior DNA corona, and a hydrophobic HE12 core which provides a favourable environment for the entrapment of hydrophobic guest molecules. We also showed the encapsulation of a dye molecule, Nile Red, in the hydrophobic core of DNA nanoparticles.27 In this current study, we sought to test the encapsulation of a small-molecule protein kinase inhibitor, BKM120. We were interested in BKM120 because (1) despite its high potency, it suffers from deleterious side-effects in the CNS of patients. (2) The drug dimensions are compatible with the core size of the DNA nanoparticle system and (3) BKM120 exhibits an aqueous solubility of <1 mg ml–1, which makes it a suitable guest for our system.In order to construct a scalable and highly monodisperse drug delivery system, we generated a single type of DNA–polymer conjugates that self-assemble in aqueous buffer to form micellar DNA particles. These conjugates consist of a 19-mer DNA sequence attached to 12 dodecane  units . Thermal annealing was shown to yield less size-variability compared to overnight shaking at room temperature. Following the encapsulation process, the products were purified by size-exclusion chromatography and analyzed by reversed-phase HPLC (RP-HPLC) (To prepare BKM120-loaded RP-HPLC) . The suc12–SNAsHE in comparison to ssDNA and buffer controls. Traces were obtained at two different channels: a channel selective for DNA at 260 nm, and a BKM120-optimal channel at 320 nm. The co-elution of the DNA and BKM120 was only observed in 12–SNAHE solutions, indicating the association of the drug with the structure. In the case of ssDNA, only a DNA peak was observed at 260 nm, reflecting the efficiency of the purification method at removing free drug in solution. The drug loading capacity of DNA nanoparticles was calculated from RP-HPLC data and separately confirmed by UV-Vis spectroscopy , dynamic light scattering (DLS), atomic force microscopy (AFM) and transmission electron microscopy (TEM) . Data fr67 Deviations from this window of buffer conditions can have devastating effects on the structures, causing shape distortion, aggregation or total collapse of structure. This limits the use of DNA nanostructures for biological applications. In our case, the assembly of 12–SNAsHE was also shown to be dependent on the presence of divalent metal cations, however, the main driving force of assembly is hydrophobic interactions rather than Watson–Crick base-pairing. With this in mind, we sought to test whether our system can withstand variations in ionic concentrations and preserve structural identity in physiologically relevant environments. Evaluation of the nanoparticle stability in different buffer conditions was carried out by DLS measurements (2+ and Ca2+) in Tris buffer (2+ and Ca2+), a buffer used in cell culture , no structural aggregation was observed. Additionally, at Mg2+ concentrations as low as 0.25 mM (in 0.5× DPBS), the structures maintained their natural morphology with no observed disassembly. The structures were also compatible with a calcium-containing Tris buffer at concentrations similar to physiological plasma concentrations (∼1.2–1.5 mM).68 Only upon total depletion of divalent cations did the structures disassemble into monomeric 12–DNAHE units  in order to mask the electrostatic repulsion between DNA strands.urements . We tests buffer  and diffin vitro culture environment.66 We measured a half-life of 2.2 h for 12–SNAsHE, which was 4.6-fold higher than the results obtained for ssDNA (28 min). This demonstrated the enhanced stability of our system against nuclease degradation and could be due to the dense packing of DNA creating a steric barrier. We further assessed the serum stability in phosphorothioated DNA nanoparticles, where the nonbridging oxygen in the phosphate backbone of the DNA strand was replaced with a sulfur. This simple modification resulted in nuclease resistance for over 72 hours  solution at 37 °C . This waFig. SF19, and couin vitro cellular uptake and internalization of 12–SNAsHE were studied by confocal fluorescence microscopy. As a first step, we generated Cy3-labeled DNA nanoparticles  and unlabeled 12–DNAHE conjugates in 25 : 75 molar ratios, followed by thermal annealing, 95–4 °C over 4 hours  cells. Cytotoxicity was evaluated by comparing a dose-dependent administration of BKM120 in DNA nanoparticles with naked BKM120 and DNA particles as controls . Our platform acting as a sensitizer in HeLa cells also highlights the versatility of our system as a general drug delivery system for anticancer drugs. For this study, HeLa cells were initially sensitized with three different concentrations of BKM120-loaded particles prior to incubation with various Dox dosages  staining assay in primary patient Chronic Lymphocytic Leukemia (CLL) lymphocytes  protected CLL lymphocytes from spontaneous apoptosis as seen with the untreated controls  and a synthetic dsRNA (PolyIC) which have been reported to induce the expression TNF-alpha.73 It has also been reported that the time course of TNF-alpha induction shows a rise-and decline profile with peak elevation at 2–6 hours post exposure.75 As illustrated in 12–SNAsHE exhibited no systemic inflammation with very low levels of TNF-alpha induction compared to LPS and PolyIC. The effect becomes less pronounced at the 12 and 24 hour mark. This result supports the non-immunogenic nature of 12–SNAsHE.To evaluate the potential immunogenicity of this delivery system, we investigated the effect of in vivo opsonisation with serum proteins, mainly human serum albumin (HSA).76 This not only prevents the rapid recognition of structures by the reticuloendothelial system (RES), but also provides prolonged blood circulation of nanostructures and higher accumulation at targeted sites.79 In our case, we hypothesized that the dense hydrophilic DNA outer shell would provide a surface unfavorable for binding HSA protein. 12–SNAsHE were pre-assembled then incubated with a 5× molar excess of HSA for 2 hours at room temperature, and analyzed by agarose gel electrophoresis (AGE) (Previous reports have demonstrated that inert polymers such as hydrophilic polyethylene glycol chains improve the efficacy of encapsulated drugs by reducing is (AGE) . Since His (AGE) , an assois (AGE) . In thisis (AGE) .in vivo behaviour of polymeric DNA nanoparticles has not been previously studied. With that in mind, we proceeded with an in vivo screening via optical imaging which would allow for real-time tracking and overall biodistribution profiles. For this purpose, highly monodisperse Cy5.5–12–DNAHE nanoparticles were prepared which contained the dye molecule in their core. This was achieved by mixing Cy5.5–12–DNAHE (where Cy5.5 was attached to the HE12 polymer at the opposite end of the DNA) and unlabeled 12–DNAHE conjugates in 25 : 75 molar ratios, followed by thermal annealing (95–4 °C over 4 hours)  . Notably81 In our case, we measured the accumulation of nanoparticles at the tumor site over time following intraperitoneal injection  into rag2–/–γc–/–mice. Compared to CLL, colon cancer xenografts formed solid tumors much more efficiently in our mice model. Therefore, HCT116 colon cancer xenografts were used as a model system to test the biodistribution of our DNA nanostructures. Previous reports have demonstrated that nanoparticles tend to accumulate in higher levels in tumor tissue, a phenomenon known as the enhanced permeation and retention (EPR) effect.njection . Cy5.5-lnjection . High acnjection . The steIn vitro studies show the ability of BKM120-loaded particles to induce cellular death and apoptosis, including synergistic effects between BKM120 and antitumor drugs, without causing non-specific inflammation. Moreover, the absence of interaction with HSA can possibly protect the structures from RES uptake. Further investigation of the in vivo biodistribution of DNA nanoparticles demonstrates full-body distribution and long circulation times of these structures. Furthermore, the particles are not observed to cross the blood–brain barrier an important feature towards limiting the side-effects of BKM120 or any drug molecule with CNS off-target activity. The structures also show high tumor accumulation in xenograft models highlighting their potential for targeted cancer therapy.We have developed a highly monodisperse DNA nanoparticle delivery platform for small-molecule chemotherapeutics. Our structures show effective loading and slow release of BKM120, and have a long shelf-life. The DNA nanoparticles are made of monodisperse, sequence-defined polymers units, they are stable under physiological ionic concentrations, and exhibit increased resistance to nucleases in biological environments. Furthermore, these structures demonstrate efficient uptake in cancer cells, and increased internalization of cargo. 12–DNAHE nanoparticles show great promise as delivery vehicles for chemotherapeutics. This initial work has demonstrated the ability to load drugs and protect them in different biological conditions, achieve in vitro activity in primary patient cell lines, and monitor the in vivo biodistribution of these structures in mice to understand their real-time trafficking and stability. Future studies on this platform will focus on adapting cross-linking strategies to enhance drug loading capacity, retention and structural stability in vivo. Additionally, taking advantage of the DNA shell, surface modifications such as targeting ligands and oligonucleotide therapeutics will be implemented. We envisage this system to see applications in targeted cancer therapy and delivery of combinational small-molecule and oligonucleotide therapeutics.Given our findings,


Novel therapeutic targets against which we can develop medications are urgently needed in the field of Alzheimer Disease (AD) research. While immense progress has been made in the description and diagnosis of AD, no effective medication to prevent or treat AD exists. Yet, 47 million individuals suffer from AD worldwide. The cost to care for AD and other dementias is $259 billion dollars/year in the USA alone.The current anti-cholinesterase and NMDA receptor antagonist treatments offered to patients have a modest therapeutic effect. In the last 30 years, research to improve treatments has mainly focused on eliminating amyloid, a short peptide that accumulates in AD brains and forms the extracellular amyloid plaques, a hallmark pathology of AD. Unfortunately, despite a multitude of approaches, clinical trials have failed to show efficacy with anti-amyloid treatments. An emerging current indicates more efforts to be directed to target Tau, a conformationally modified and hyperphosphorylated intraneuronal protein that forms the neurofibrillary tangles, the other quintessential neuropathological lesion of AD brains.While time will tell if these approaches will eventually yield an effective drug, it may be prudent to pursue additional therapeutic targets to increase our chances of developing efficient treatments against AD. Focusing on the identification of tractable earlier molecular mechanisms of neurodegeneration in human age-dependent cognitive impairment and AD, my laboratory has identified a potential novel therapeutic target in Caspase-6 (Casp6).Casp6, a cysteinal protease belonging to the family of caspase proteins involved in inflammation and programmed cell death, is associated with AD pathology and memory impairment . Active At a molecular level, Casp6 activity induces parallel pathways involved in AD neurodegeneration and pathology Figure  1]. Str. Str1]. In human neurons, Casp6 is activated through the inflammatory Nlrp1-Casp1 pathway and causes neuritic degeneration , 4. NlrpCasp6 is likely a good drug target. Adult human brains express the lowest levels of Casp6 zymogen protein compared to other human tissues, suggesting that Casp6 may not normally play an important role in the brain. Casp6 null mice do not have obvious deficits although further probing indicates a role for Casp6 in B cell activation and differentiation into plasma cells . Casp6 aOur latest paper shows that Casp6-mediated memory impairment can be reversed in old mice . A vinylIn conclusion, finding a selective non-toxic and blood brain barrier permeable Casp6 inhibitor is essential to validate Casp6 as an early therapeutic target of neurodegeneration in mouse AD models, and to assess if inhibiting Casp6 can prevent the progressive cognitive impairment and the ensuing dementia in AD.


PHYTOENE SYNTHASE (PSY1) gene results in yellow fruits with wild type red sectors forming via HR-mediated DSB repair. We also show that in heterozygote plants containing one psy1 allele immune and one sensitive to CRISPR, repair of the broken allele using the unbroken allele sequence template is a common outcome. In another assay, we show evidence of a somatically induced DSB in a cross between a psy1 edible tomato mutant and wild type Solanum pimpinellifolium, targeting only the S. pimpinellifolium allele. This enables characterization of germinally transmitted targeted somatic HR events, demonstrating that somatically induced DSBs can be exploited for precise breeding of crops.Homologous recombination (HR) between parental chromosomes occurs stochastically. Here, we report on targeted recombination between homologous chromosomes upon somatic induction of DNA double-strand breaks (DSBs) via CRISPR-Cas9. We demonstrate this via a visual and molecular assay whereby DSB induction between two alleles carrying different mutations in the  et al. show that CRISPR-Cas9 can be used to induce DNA double strand breaks in somatic tissue and achieve targeted recombination between homologs at an endogenous locus in tomato.Targeted homologous recombination between parental chromosomes could facilitate precision breeding of crop plants. Here, Filler Hayut  DNA double-strand breaks (DSBs) are one of the powerful forces that shape plant genomes. These DSBs may occur throughout the plant life cycle, in somatic or meiotic cells, spontaneously during the movement of replication forks or developmentally controlled as in the early stages of first meiosis12DNA DSB repair mechanisms have been widely studied in many organisms, including plants67891718192225Arabidopsis, using transgenic assays that tested repair mechanisms such as intrachromosomal recombination, unequal crossover and so on. In all cases, DSB induction enhanced HR-repair rates2714Several studies that addressed the mechanism of DSB repair via HR in somatic tissues were done in Activator (Ac)Mutator elementscis, between repeats flanking the transposon in somatic tissues. By contrast, germinal Ac activity did not stimulate the rate of meiotic recombination between homologous chromosomes at the maize bronze locus35DSB induction of HR between endogenous (non-transgenic) recombination partners was shown in maize upon excision of the In this study, we tested whether targeted recombination between homologous chromosomes can be achieved in plants. For this purpose, we combined a series of new technologies, such as CRISPR-Cas, whole genome sequences, High-Throughput sequencing and a variety of mutants in tomato fruit colour, enabling both identification and quantification of resulting recombination products. We provide evidence for the occurrence of induced allele-dependent DSB repair in tomato, including the characterization of germinally transmitted HR events.To estimate the rate of somatic NHEJ versus HR based DSB repair at an endogenous plant locus, we designed a series of fruit colour assays. These assays enabled us to measure only those events of NHEJ that are error-prone as we cannot detect events of precise end-joining which might occur2129Phytoene synthase 1 (PSY1) gene. The yellow flesh e3756  allele is an EMS mutant with a premature stop codon in PSY1 leading to a yellow fruit phenotypebicolorcc383 allele  is a mutant with a 3.7 Kb deletion in the promoter of PSY1 leading to a yellow–red moulted fruit phenotype . This u6-26:Ps#1-sgRNA was designed to induce a DNA DSB between the bicolorcc383 and yellow flesh e3756 mutations, on both alleles . The outcome of DSB repair by HR based mechanisms (inter-homologues crossover or non-crossover events), could be a red fruit in case of an HR event that occurred early in development, or fruits with red spots or sectors in a yellow or bicolour background in case of late events , an average of 88% of the reads of yellow flesh e3756 35S:Cas9 × bicolorcc383 u6-26:Ps#1-sgRNA plants contained a mutation at the CRISPR DSB site, while only 2% of the illumina reads of yellow flesh e3756 × bicolorcc383 plants deviated from the WT sequence, presumably due to PCR and sequencing errors , enabled to distinguish parental from recombinant molecules  at the CRISPR-Cas9 DSB site and crossed it with the wild tomato accession. In this assay, the S. pimpinellifoliumLA1578 is the only target for DNA DSB due to the +A insertion, in the M82 psy1 allele, that disrupts the protospacer adjacent motif (PAM) and prevents Cas9 cleavage. The +A mutation of M82 allele is recessive and therefore F1 plants are expected to have small red fruits. DSB repair in PSY1 by NHEJ, or HR (crossover or non-crossover) leads to yellow fruits or red fruits with yellow sectors, depending on the developmental fruit stage when the repair occurred. NHEJ repair events are expected to leave small indels at the DSB site, while crossover and non-crossover events can be identified by the difference in SNPs patterns on both sides of the DNA DSB (The above cross between  DNA DSB .S. pimpinellifoliumLA1578) and from five F1 plants  parent, supporting the designed allele-specificity of the gRNA .For the analysis of somatic DSB repair, we PCR-amplified and sequenced by Illumina HiSeq 2500 paired-end sequencing the DSB DNA area from leaves of both parents  than in the heterozygote where only one target is available )/2. The occurrence of a new +A mutation in the WT allele, when the second allele contains the +A mutation (in M82-WT PSY1/M82 psy1+A heterozygote plants) is calculated by taking the percentage of +A reads in the M82-WT PSY1/M82 psy1+A plants and deducing 50% . We used the following equation for the observed rate of +A mutation in heterozygote M82-WT PSY1/M82 psy1+A plants: Observed=%(+A reads)T=4 weeks, –50%. When we compared the expected to the observed +A footprint, we found a significantly higher than expected rate of novel +A mutations in the heterozygote population . Considering that the two populations are isogenic, this suggests that the repair at the site of DSB is dependent on the sequence of its homologous allele , an event seen as red spots  is a germinal HR event that might be either a crossover event or a non-crossover event—this could not be demonstrated due to the plant death. Finally, trying to quantify the ratio of HR versus NHEJ, we designed a sgRNA for allele-specific DSB induction in the S. lycopersicum background. This experimental set-up enabled to measure an excess of repair footprints originating from the homologous allele compared to expectation, suggesting that out of all the detectable DSB repair events at least 14% are allele-dependent and the rest is non-homologous.Earlier studies on somatic DSB-induced HR repair were done mostly with transgenic assays for intrachromosomal recombination27ed spots  and chared spots . In thisf 5–6 Kb . The thiIt is interesting to compare HR-mediated repair in somatic versus meiotic cells. Overall little is known on inter-homologues recombination in somatic tissues probably owing to the low frequency of such events, to the lack of phenotypic markers and to the difficulty to retrieve germinal events. We could not detect any red sector in the absence of DSBs, and the presence of intragenic recombinant molecules was null or negligible. This is consistent with earlier studies in tobacco showing that the occurrence of somatic HR is very low in the absence of DSB induction for both reciprocal and non-reciprocal HR events41et al.The rate of HR DSBs repair that we report here  is apparently higher than that reported during meiosis. Indeed, only a small fraction of meiotic breaks (∼3–5%) evolves into crossovers1243S. pimpinellifolium SNPs at positions 4324510, 4326379, 4329658 and 4329832 appeared within the M82 homozygous patch at the boundaries of the tract (21Arabidopsis) or between meiotic and somatic cells or it might be due to the experimental set-up (Spo11 versus Cas9 DSB induction).We found a rather long (∼5 kb) conversion tract where homozygous Finally, we showed that it is possible to induce germinally transmitted targeted HR between endogenous homologous chromosomes in somatic cells. It remains to be shown if and how efficiently targeted HR can occur during meiosis.Our results suggest that custom-designed nucleases, such as CRISPR-Cas, can be used for precise reshuffling of chromosomal segments between homologous chromosomes in somatic cells. For example, it might be possible to transfer a disease resistance gene from a wild relative to the crop, without a long process of backcrossing which not only takes several generations in order to achieve isogenic lines, but also drags large segments of undesirable DNA flanking the desirable gene. In other words, we showed that somatic HR can be used for allelic replacement. Another potential application of DSB-induced somatic HR is ‘targeted crossover', that is, the reciprocal exchange of large chromosomal segments at a precise site. Crossing over between homologous chromosomes during meiosis is the engine of breeding in sexually reproducing crops, but it takes place at uncontrolled and unpredictable sites in the genome. Obtaining rare recombination events—such as shown here for intra-allelic crossover—is a major hindrance in most breeding programs. We propose to exploit targeted somatic HR to segregate between undesirable genetic linkages. Somatic crossover does occur in plants2728293031S. lycopersicum) mutant line of yellow flesh e3756, Bicolorcc383, M82 and S. pimpinellifoliumLA1578 were kindly provided by the labs of Prof. Joseph Hirschberg and Prof. Daniel Zamir at the Hebrew university of Jerusalem (http://zamir.sgn.cornell.edu/mutants/)All tomato plants were grown in greenhouse conditions with temperature ranging between 18 and 25 °C. The tomato . For each plant 300 ng of DNA from leaves sample or control plasmid were treated separately: first, they were incubated over night with 10 × FD buffer, ApaLI (ThermoFisher scientific) and HindIII-HF (New England BioLabs). After 20 min of 80 °C inactivation, 150 ng of the digested fragments were blunted with T4 DNA polymerase (New England BioLabs) for 2 hs at room temperature. The T4 DNA polymerase was inactivated at 75 °C for 10 min and the linear DNA was self-ligated with Quick T4 DNA ligase (New England BioLabs) for 30 min at room temperature. Control plasmids were diluted 1:10,000 with DDW and mixed together for mimicking ‘heterozygosity'. Then all samples were amplified by 18 cycles of PCR with Phusion High-Fidelity DNA polymerase (New England BioLabs) (for primers see primers list). The primers for this assay were designed for allele-specific amplification. Samples were pooled and sequenced by high-throughput sequencing.yellow flesh e3756 and Bicolorcc383 DNA samples using Phusion. High-Fidelity DNA polymerase (New England BioLabs) (for primers sequence see primers list) and then cloned using the GoldenBraid cloning systemhttps://gbcloning.upv.es/). First, each of the four amplicons was cloned into the pUPD plasmid. Then pUPD2 plasmid with ‘ps' segment from yellow flesh e3756 was cloned with pUPD2 plasmid with ‘y1' segment from Bicolorcc383 into pDGB3_alpha1 plasmid. In parallel, pUPD2 plasmid with ‘ps' segment from Bicolorcc383 was cloned with pUPD2 plasmid with ‘y1' segment from yellow flesh e3756 into pDGB3_alpha1 plasmid. These two ‘synthetic allele' plasmids were pooled together and were subjected to inverse PCR and sequencing. For the specific sequence of synthetic plasmids see R1 and R2 in For the cloning of the synthetic crossover-control plasmids, two PCR fragments were amplified from et al.DNA samples for high-throughput sequencing were amplified using Phusion. High-Fidelity DNA polymerase (New England BioLabs) and 18 PCR cycles (for specific primers of each experiment see primers list). Libraries were prepared as Blecher-Gonen DNA samples for Sanger sequencing were amplified using REDTaq (SIGMA-ALDRICH) with 35 PCR cycles .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Table and Supplementary Note


Endoglin (CD105) is an auxiliary membrane receptor of transforming growth factor beta (TGF-β) that interacts with type I and type II TGF-β receptors and modulates TGF-β signaling. Endoglin is overexpressed in the tumor-associated vascular endothelium, where it modulates angiogenesis. This feature makes endoglin a promising target for antiangiogenic cancer therapy. In addition, recent studies on human and experimental models of carcinogenesis point to an important tumor cell–autonomous role of endoglin by regulating proliferation, migration, invasion, and metastasis. These studies suggest that endoglin behaves as a suppressor of malignancy in experimental and human epithelial carcinogenesis, although it can also promote metastasis in other types of cancer. In this review, we evaluate the implication of endoglin in tumor development underlying studies developed in our laboratories in recent years.


Epstein-Barr virus (EBV) was identified as the first human virus to be associated with a human malignancy, Burkitt’s lymphoma (BL), a pediatric cancer endemic in sub-Saharan Africa. The exact mechanism of how EBV contributes to the process of lymphomagenesis is not fully understood. Recent studies have highlighted a genetic difference between endemic (EBV+) and sporadic (EBV−) BL, with the endemic variant showing a lower somatic mutation load, which suggests the involvement of an alternative virally-driven process of transformation in the pathogenesis of endemic BL. We tested the hypothesis that a global change in DNA methylation may be induced by infection with EBV, possibly thereby accounting for the lower mutation load observed in endemic BL. Our comparative analysis of the methylation profiles of a panel of BL derived cell lines, naturally infected or not with EBV, revealed that the presence of the virus is associated with a specific pattern of DNA methylation resulting in altered expression of cellular genes with a known or potential role in lymphomagenesis. These included ID3, a gene often found to be mutated in sporadic BL. In summary this study provides evidence that EBV may contribute to the pathogenesis of BL through an epigenetic mechanism. However, more than 50 years ago, EBV particles were found in Burkitt’s lymphoma derived cultures2. This discovery resulted in the virus being recognized as the first human tumor virus. Since then, several epidemiological studies have shown that EBV is an etiological factor for endemic Burkitt’s lymphoma (BL) in Africa as well as of other human malignancies 1. Nevertheless, the majority of individuals infected with EBV do not develop EBV-associated cancers, which suggests the involvement of additional genetic or environmental factors in the development of Burkitt’s lymphoma and other EBV-related cancers7. Well-recognized co-factors of EBV-induced malignancies include insect-borne parasitic infections like malaria, young age at first infection, immune suppression, and dietary factors. In the endemic variant of BL (eBL), EBV is found in each cancer cell, suggesting a direct role of EBV in the process of lymphomagenesis. However, other events, such as c-myc translocation are also required8. To date understanding of Burkitt’s lymphoma and the mechanistic role of EBV infection in the pathogenesis of this disease remain incomplete.Epstein-Barr virus (EBV) is a double-stranded DNA herpesvirus, which infects 90% of the adult population worldwide with no adverse consequence for health in the majority of the cases10. The new technology has confirmed some of the previous findings, such as c-myc translocation being a hallmark of all Burkitt’s lymphomas, independent of the clinical variant or EBV-status. However, they also helped reveal novel BL-associated genetic alterations, such as protein-damaging sequence mutations affecting the ID3-TCF3 regulatory loop. It was shown that mutations that impair the inhibitory function of ID3 on proteins of the TCF family, leads to constitutive activation of B-cell signaling and to a cMYC-independent lymphoid proliferation12. Moreover, ID3 knock-out mice showed a predisposition for lymphomagenesis in comparison to wild type mice13. Abate and colleagues recently showed that the ID3-TCF3 loop genes carry fewer mutations in the endemic (EBV+) than in the sporadic (EBV−) BL variant14. Overall, their data on RNA sequencing of eBL primary tumors revealed a lower rate of cellular mutations in genes previously found altered in sporadic BL (sBL) such as MYC and TP53. This highlights a potential role for non-genetic virally-driven events in the pathogenesis of EBV+ eBL.The development of omics technologies has enabled a fresh approach to the molecular characterization of EBV-induced malignancies and to further delineate the role of the virus in the process of transformation17.Epigenetic modifications are important in cancer development and several lines of evidence suggest that certain oncogenic viruses have the ability to hijack enzymes that govern epigenetic modification, thereby altering the structure and function of the host genome18.Recent studies have reported epigenetic changes occurring in B cells during the process of EBV-driven transformation. A profound epigenetic remodeling was also shown in EBV-driven epithelial cancers, such as Gastric Cancer (GC)In the present study, we tested the hypothesis that the lower load of somatic mutation observed in eBL compared to the sBL variant can be explained by abnormal DNA methylation induced by infection with EBV. Our results show that EBV modifies the epigenetic profile of the B cell genome and as a consequence alters the expression of genes with a known or potential role in lymphomagenesis, supporting a direct role of the virus in the pathogenesis of eBL.To identify a potential impact of EBV on DNA methylation patterns in Burkitt Lymphoma (BL), we first profiled the DNA methylome of 10 EBV (+) and 9 EBV (−) BL-derived human cell lines. The EBV (−) BL cell lines derived from BL samples from individuals of Caucasian origin and display a very low number of EBV copies (from 0.02 to 0 copies per cell) when analysed by Taqman PCR, while the EBV (+) BL were almost all derived from BL samples from individuals of African origin and displayed at least 1 copy of EBV genome per cell  as a significant change in mean methylation of at least 40% between the two conditions . 4712 DMPs were found using these criteria, with 453 hypomethylated and 4259 hypermethylated in EBV+ samples  content was calculated for each set of probes . On average, hypermethylated sites were low in GC content, while hypomethylated sites displayed a similar GC content as the whole probe set represented in the HM450 arrays Fig. . Hypo an19, as well as other cancer related genes, such as the Telomerase Reverse Transcriptase TERT  definition of at least 2 differentially methylated sites and a gap of less than 1000 bp, we identified 966 DMRs  BL, we performed pathway analysis of genes localised within or in proximity of the DMR Fig. . Hypomet14 and revealed lower frequencies of mutations in ID3 and TCF3 in the endemic BL variant compared to the sporadic variant. Therefore, we examined whether in eBL, EBV may target mutational drivers by epigenetic silencing. To this end, we first drew heat-maps for each of the driver subsets, sBL  and eBL . Our BL samples were hierarchically classified according to EBV status when selecting the sBL mutational signature ure Fig. , while ture Fig. . Indeed,ure Fig. . Moreoveure Fig.  appearedure Fig. . In lineure Fig. , ID3 andure Fig. , whereasure Fig. .Figure 4The analysis of the methylome profiles of EBV (+) and EBV (−) BL derived cell lines led to identification of other genes with a potential role in transformation  and that could be differentially regulated in the two groups of BL. Q PCR analysis of the mRNA levels of these genes showed that they were significantly down regulated in all the analyzed BLs (14 lines) compared to primary B cells  independently of the EBV status  is well known to contribute to EBV-associated oncogenesis. Therefore we infected primary B cells from 2 independent donors with WT or ΔLMP1 EBV genome. Two days after infection cells were collected and analyzed by qPCR for the levels of EBER and LMP1. WT and ΔLMP1 EBV infected B cells showed similar levels of EBER, indicating that the two infections worked with similar efficiency  but often altered in lymphoproliferative disease35. Moreover many of the EBV associated DMPs were found in proximity to SUZ and EZH2 binding sites in B cells. This is relevant considering that deregulation of EZH2 activity, due to gain of function mutations, is a key event in lymphomagenesis37. One could speculate that the changes of DNA methylation levels in proximity to the EZH2 binding region observed in EBV+ BLs, could be due to virus-induced changes in the amount or activity of PC2 enzymes and/or of DNMT1 that is known to interact with EZH238. Furthermore, we observed that in EBV+ BLs, the hypomethylated DMPs were common in promoter regions . On the contrary, hypermethylated sites were in average significantly more distant from TSS, with a trend to be enriched in enhancer regions. This different scenario between EBV (+) and (−) BLs, could also be the consequence of the viral-mediated deregulation or redistribution of the epigenetic modifiers. The biological meaning of this hypermethylation at the enhancer sites will be the object of further investigations.Previous studies have focused on the characterization of the genetic landscape of the different BL clinical variants14. For the first time we show that EBV infection in B cells leads to increased methylation of ID3 promoter and silencing of ID3 expression by a LMP1-mediated mechanism. LMP1 is known to modulate the levels of DNMTs in GC B cells39, indeed our data indicate that during EBV infection LMP1 increases the recruitment of the DNMTs to the promoter of ID3; this activity of LMP1, that appears to require its ability to induce NFκB, could then result in increased methylation of ID3 promoter and silencing of its gene expression; further studies are needed to validate this hypothesis.To assess whether EBV-associated differential methylation of specific gene sites led to altered gene expression and to gain insight into the biological relevance of the identified epigenetic changes, we analyzed the RNA levels of a panel of genes in proximity to DMPs, in EBV (+) and EBV (−) BL cell lines compared to primary B cells from different donors. Some genes were differentially expressed in the two BL groups and appear to be regulated by DNA methylation in EBV (+) BLs. Among them, we found ID3 and TCF3 often mutated in sporadic BLin vitro. This implies that additional events may occur in vivo to regulate TCF3 levels, possibly independent of the presence of EBV, as supported by the observation that DNA demethylation by Aza leads to a significant rescue of TCF3 mRNA levels in both EBV(+) and EBV(−) BLs; this also indicates that methylation at different CpG positions could be one of the mechanisms responsible for TCF3 deregulated expression in EBV− BL, additional to the frequent mutation rate reported in sporadic BL. In line with an epigenetic modulation of the ID3-TCF3 axes in EBV (+) BLs, we also found DMRs in proximity of ID2 and TWIST1; the latter is known to interact with TCF3 in presence of low levels of ID proteins41. We also found other genes differentially methylated and expressed in EBV (+) BLs that were not previously associated with endemic BL, but that are known to play a role in many cancers. This includes RUNX1 (data not shown), known to be involved in B cell and lymphoid development, and whose deregulation can accelerate Myc-induced lymphomagenesis42, or the lysine (K)-specific demethylase 2B (KDM2B), an important mediator of hematopoietic cell development that has opposing roles in tumor progression depending on the cellular contest43. Altered activity of different KDMs has been associated with cellular transformation. Anderton and collaborators reported the EBV-mediated deregulation of KDM6B and its role in Hodgkin’s lymphoma, however no previous study has assessed the role of KDM2B in the process of EBV-mediated B cells transformation. It has been shown that recognition of demethylated CpG island by KDM2B targets them for polycomb-mediated silencing44. Altered levels of KDM2B could then affect both host and virus chromatin structure and gene expression. Therefore further functional characterization of KDM2B promoter methylation in EBV+ BLs is warranted. DNA demethylation by Aza treatment and direct pyrosequencing confirmed the EBV-mediated epigenetic regulation of these genes.Despite the strong difference in levels of methylation of TCF3 promoter in EBV (+) BL versus EBV (−) BL cell lines, TCF3 mRNA levels are not directly altered by EBV infection or expression of LMP1 ex vivo samples.Some of the analyzed genes appeared to be repressed in all BL cell lines when compared to primary B cells, independently of the EBV status. This was confirmed by whole expression profiles in EBV (+) and (−) BLs, which showed that despite the profound difference in their epigenetic profiles, EBV (+) and (−) BLs appear to be phenotypically very similar as they exhibited few differences in RNA levels. In our view this result confirms that similar events occur in the process of lymphomagenesis, but the molecular mechanisms and the selective pressure through which the cells pass during BL pathogenesis are different in the viral and non-viral-BL variants. Future studies will be needed to assess if these methylation patterns identified in BL-derived cell lines are also found in BL In summary, this study describes the methylome signatures and the expression profile of different EBV (+) and EBV (−) BL derived cell lines and shows the EBV-mediated epigenetic silencing of drivers in B cell transformation therefore demonstrating an active role of the virus in the process of lymphomagenesis.45. The myeloma-derived RPMI-8226 cells (http://web.expasy.org/cellosaurus/CVCL_0014) and the Burkitt lymphomas cell lines (BL), including the BL EBV(−) cell line Louckes (http://web.expasy.org/cellosaurus/CVCL_8259), were obtained from the IARC biobank. The EBV genome copy number determined by Taqman PCR and the geographical origin of BL used in the present study are described in Supplementary Table Peripheral B cells were purified from blood samples as previously describedTo demethylate the DNA cells were treated with 5-Aza-2′-deoxycytidine ≥97%,  at the final concentration of 10 μM for 48 h and/or 96 h. To inhibit the canonical NFκB pathway, cells were treated with the IκBα kinase inhibitor Bay11-7082 (10–20 μM)  for 2 h.45. For each primer set the qPCR was performed in duplicate and the mRNA levels obtained were normalized on the average mRNA levels of three housekeeping genes , measured in the same samples.Extraction of total RNA, reverse transcription to cDNA and quantitative PCR (qPCR) were performed as previously describedet al.45. The PCR primer sequences are indicated in Supplementary Table EBV genome copy number per cell was measured by Taqman PCR, primers and probes described in Accardi Chromatin immunoprecipitation (ChIP) was performed with Diagenode Shearing ChIP and OneDay ChIP kits according to the manufacturer’s protocols, by using the following antibodies: DNMT1 (Abnova MAB0079), DNMT3A (Abcam ab13888), DNMT3B (Abcam ab13604) and IgG (Diogenode). The eluted DNA was used as template for qPCR with primers designed on the promoter region of ID3 (5′-GCCACTGACTGACCCCTAAG-3′ and 5′-CCCGGTTCCTTCCTTCCTT-3′).46. For samples processed with Illumina 450 K Infinium bead arrays (see below), 600 ng of DNA was converted using the EZ DNA methylation Kit (Zymo Research) and modified DNA was eluted in 16 ul of water. Quality of modification was checked by PCR using modified and unmodified primers for GAPDH gene.Cells were pelleted and resuspended in lysis buffer  with Proteinase K (500ug/ml) and incubated for 2 hours at 55 °C. DNA was saturated with NaCl (6 M), precipitated with isopropanol, and cleaned with 70% ethanol. Extracted DNA was finally resuspended in water. Quantity and quality of the extracted DNA were assessed with a ND-8000 spectrophotometer . To quantify the percentage of methylated cytosine in individual CpG sites, we performed bisulfite pyrosequencing, as previously described47. The analysis on the bead array was conducted following the recommended protocols for amplification, labelling, hybridization and scanning.Methylation profiles of the different samples were analyzed using the 450 K Infinium methylation bead arrays . Briefly, the Infinium Humanmethylation450 beadchip interrogates more than 480,000 methylation sites49. Data quality was inspected using boxplots for the distribution of methylated and unmethylated signals, and inter-sample relationship using multidimensional scaling plots and unsupervised clustering. Probes were filtered for low quality  and known cross-reactive probes50. The remaining dataset was background subtracted, and normalized using intra-array beta-mixture quantile normalization51. Methylation beta values were logarithmically transformed to M values before parametric statistical analyses, as recommended52. To define differentially methylated positions (DMPs) and differentially methylated regions (DMRs), we modelled the EBV status as a categorical variable in a linear regression using an empirical Bayesian approach53. DMPs were selected based on a differential methylation (delta beta) of at least 40% when comparing the two EBV categories. DMRs were identified with the DMRcate package using the recommended proximity-based criteria54. A DMR was defined by the presence of at least 2 differentially methylated CpG sites with a maximum gap of 1000 bp. Differentially methylated genes (DMPs and DMRs) were further analyzed to determine functional pathways and ontology enrichment using Enrichr55. All methylation data have been deposited to the Gene Expression Omnibus repository (GEO accession number GSE92378).Raw methylation data was imported and processed using R/Bioconductor packages56 and 500 ng total RNA isolated with the TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. Raw expression bead array data , with no normalization and no background subtraction was exported from Genome Studio  into BRB-ArrayTools software  < 0.05 and a fold-change of at least 1.5 were considered differentially expressed.Differential expression analysis was performed using Human HT-12 Expression BeadChips (Illumina) as previously describedStatistical significance was determined by Student T test. The p value of each experiment is indicated in the corresponding Figure legend. Error bars in the graphs represent the standard deviation.supplementary figures and tables


The genetic reprogramming technology allows generation of induced pluripotent stem cells (iPSCs) from somatic cells   Specifications TableValue of the data●For the first time, data on DNA methylation of isogenic iPSCs obtained by integrating and non-integrating methods are reported.●There is little data on genome-wide studies of isogenic iPSС lines. DNA methylation data of isogenic iPSCs lines, obtained by lentiviruses and Sendai virus, allow comparison of the results obtained by different reprogramming methods, including data on rare diseased iPSCs, and to combine them into large data sets.●Epigenome-wide analysis of iPSCs often require comparison of the lines, not only with different genetic backgrounds, but also obtained in various ways. Our data will allow understanding, whether the way of reprogramming makes significant changes in the DNA methylation landscape.1The data presented here originates from four iPSC lines of a healthy donor generated via integrating  and isogenic cell lines generated by non-integrating  methods at different passages Genomic DNA was isolated from iPSCs and differentiated neurons, converted with sodium bisulfite, and applied onto a chip according to the manufacturer's instructions. Data from the Infinium Human Methylation BeadChip 450K chip for 12 samples were deposited into the NCBI GEO database under the accession number GSE105093. Raw data with signal intensities of methylated and non-methylated samples were processed using the RnBeads package Correlation analysis of the data on genome wide methylation of non-differentiated iPSC lines obtained from two different donors by two methods and their differentiated derivatives (TH-positive neurons) showed that correlation between differentiated neurons and their parent iPSCs was the least. iPSC lines grouped into two clusters according to the donor of parental fibroblasts but not reprogramming method .Fig. 1A 22.1Generation of integration free iPSC lines was described in Ref. 2.2iPSCs were grown in the medium for neuronal differentiation  for 14 days. Then, cells were cultured for 24 more days in maturation Neurobasal Medium (Invitrogen) containing B27 supplement (Invitrogen) 2 mM L-glutamine, 10 ng/ml BDNF, and 10 ng/ml GDNF .2.3To isolate DNA, iPSCs and neurons differentiated thereof enriched with TH-positive cells (day 38 of differentiation) were used. Both cell types were removed from the support with 0.05% trypsin solution. Trypsin was inactivated with two volumes of DMEM supplemented with 10% FBS and cells were centrifuged at 300 g for 5 min. Cell pellet was washed with PBS and centrifuged under the same conditions. DNA from the cells was isolated by phenol–chloroform extraction. The isolated DNA was subjected to bisulfite conversion, hybridization, and scanning on an iScan.2.4To analyze the Illumina BeadChip 450K platform data, the RnBeads package was used


Sutterella genus. MP provided superior DNA yield and quality, MO BIO depleted Gram positive organisms while using QIAGEN with OMNIgene.GUT resulted in greatest variability compared to other two kits. MiSeq and IT platforms in their supplier recommended setups provided comparable reproducibility of donor faecal microbiota. The differences included higher diversity observed with MiSeq and increased capacity of MiSeq to detect Akkermansia muciniphila, [Odoribacteraceae], Erysipelotrichaceae and Ruminococcaceae . The results of our study could assist the investigators using NGS technologies to make informed decisions on appropriate tools for their experimental pipelines.The information on microbiota composition in the human gastrointestinal tract predominantly originates from the analyses of human faeces by application of next generation sequencing (NGS). However, the detected composition of the faecal bacterial community can be affected by various factors including experimental design and procedures. This study evaluated the performance of different protocols for collection and storage of faecal samples (native and OMNIgene.GUT system) and bacterial DNA extraction , using two NGS platforms for 16S rRNA gene sequencing (Ilumina MiSeq and Ion Torrent PGM). OMNIgene.GUT proved as a reliable and convenient system for collection and storage of faecal samples although favouring  The main source of the information regarding human gut microbiota originated from the analyses of the faeces. The importance of this easily available metabolic waste comes from its microbial content found to hold the potential in diagnosis, disease prediction and therapeutic intervention. This justifies it being considered a “virtual organ”3 or a human tissue5. The analyses of human intestinal microorganisms were, until recently, performed by culture-dependent methodologies, limiting the outputs to the cultivable species. The availability of novel tools, primarily next-generation sequencing (NGS), enabled the assessment of genes and genomes contained within complex microbial communities. The most widely used NGS method for the taxonomic and phylogenetic evaluation of bacterial community composition relies on 16S rRNA gene amplicon analysis9. However, detected composition of the faecal bacterial community can be affected by experimental design and procedures, including sampling and storage protocol as well as DNA extraction method.The scientific interest and mounting knowledge on human gut microbiota have led to many important findings associating the composition of bacterial taxa in the human gastrointestinal tract with disorders from the neurologic, psychiatric, respiratory, cardiovascular, gastrointestinal, hepatic, autoimmune, metabolic and oncologic spectra10, subsequent reports showed importance of the collection procedure14 with accuracy increased by prompt sample downstream processing (within 2–3 hours14), immediate stabilization15 and appropriate storage conditions17. Faeces represents one of the most complex biological materials for bacterial DNA isolation as it also contains remnants of human DNA, food DNA and many inhibitors hampering subsequent PCR amplification and NGS procedures18. DNA extraction from faeces represents the essential step in obtaining good quality DNA and ensuring accurate identification of microbial composition and relative abundance19. It is therefore critical to evaluate the available methodologies for bacterial DNA extraction from faeces and optimize protocols and procedures that would provide sufficient amount, purity and integrity of the DNA, thus obtaining high quality sample for further studies.Sample collection procedures represent one of the first crucial steps that ensure integrity and stability of the collected material. Although an  early study claimed no difference in community structure upon collection under field conditions20 and currently available technologies with sequencers from Pacific Bioscience, Roche, Thermo Fischer Scientific and Illumina, have all been successfully applied for the analyses of complex biological samples22. NGS is used for determining bacterial composition in a sample based on DNA fragment detection from the total DNA isolate. Although shotgun sequencing provides the most complete information on the entire gene pool within the sample, the high amount of generated data requires substantial bioinformatic efforts in sequence assembly, mapping and analyses. In many studies, both clinical and environmental, sequencing of 16S rRNA gene amplicons, covering variable regions of the gene, is the method of choice for the analyses of bacterial community composition, providing cost-effectiveness, sufficient resolution and sequencing depth20. The 16S rRNA gene is approximately 1500 bp in size and consists of nine variable regions separated by conserved regions23. Illumina and Thermo platforms with their MiSeq and Ion Torrent (IT) Personal Genome Machine (PGM) benchtop sequencers are increasingly being used for 16S rRNA-based analyses of diverse bacterial populations22. Although both Illumina and IT platforms sequence DNA by detecting the nucleotide addition during DNA synthesis, the two platforms function on different principles, which could affect their performance and ultimately result in variation of the obtained data27.During the last decade sequencing tools have gradually shifted from conventional Sanger sequencing technology to NGSAs each step in the experimental pipeline introduces variation influencing the final output, there is an unmet need for standardization of methodology which would enable reliable and reproducible analysis of valuable human biological samples for studying gut microbiota. This study evaluated the performance of different protocols for collection and storage of faeces (native versus OMNIgene.GUT system) as well as bacterial DNA extraction  using the manufacturer recommended methodology for 16S rRNA gene sequencing on two commercially available platforms (Ilumina MiSeq and IT PGM).Stool samples from 4 individuals were collected and processed as described in Fig. 260/A280 absorbance ratio was obtained with MP and QIA, with mean values of 2.00 and 1.91, respectively, while mean ratio for MO BIO was significantly lower at 1.55  and Ion Torrent PGM . Different primer sets were used to enable comparison of supplier recommended protocols.Clustering and annotation of OTUs was performed by the same pipeline for both sequencing platforms. Average number of counts per sample was 257.806 ± 23.076 for MiSeq and 110.512 ± 7.529 for IT. A comparison of OTU assignment on each taxonomic level (from species to phylum) revealed that, all OTUs were assigned from phylum to order level for both platforms. At lower taxonomic levels, however, higher proportion of the assigned OTUs was obtained for samples sequenced with MiSeq than those sequenced with IT  , displaying comparable abundance ratios between platforms   and less Lachnospiraceae (17.3%), compared to MP (33.4% and 25.2%) and QIA extraction kit . Comparing the frozen native samples (native 14) with their corresponding reference points revealed the increase in Bacteoridaceae/Lachnospiraceae ratio and identification of slightly less Prevotellaceae across all extraction kits. Collecting the samples with OMNIgene.GUT stabilizing system  tended to increase the Bacteoridaceae/Lachnospiraceae ratio for QIA and MO BIO extracted samples. Altogether, application of MP and MO BIO resulted in fewer discrepancies across all tested parameters  and genus levels . Omni 0 was found different from both native 0 and native 14 groups of samples only at the genus level .In order to test the effect of each covariate on taxon composition, two tests were performed on clr-transformed data: Kruskal Wallis rank test testing each pair of covariates, and Wilcoxon rank test on Monte-Carlo simulated data testing a covariate vs. all others, e.g. one kit vs. other two. Table Actinobacteria, Firmicutes, Proteobacteria and Tenericutes were found more abundant on IT while Lentisphaerae and Verrucomicrobia were found more abundant on MiSeq. These differences were statistically significant but the effect sizes were not substantial to support genuine relevance, except Verrucomicrobia, which was both highly significant and had a high effect size (a measure quantifying the magnitude of difference between two groups). Even though Verrucomicrobia was not a highly abundant phylum, MiSeq detected 10 fold more Verrucomicrobia than IT . It is worth mentioning that the only contribution to Verrucomicrobia phylum stems from Akkermansia muciniphila species. At the family level, there were ten families that contributed to platform differences, all more abundant on MiSeq, but only four [Odoribacteraceae], Erysipelotrichaceae, Ruminococcaceae and Verrucomicrobiaceae have demonstrated high significance and effect sizes. Covariance biplots for organisms with statistical significance (p < 0.05) on family and genus levels (showing highest platform discrepancy) are available in Supplementary Information  between kits were found in MP versus other kits  show significance which stems from lower abundance (by a factor of 2–3.8) these taxa achieve with MOBIO kit   than the other two kits , the results of the MP protocol from the same study (protocol #14) demonstrated high DNA quantity and quality, as well as community diversity and G+ recovery, positioning this extraction method high on the performance scale.The evaluated three DNA extraction kits successfully extracted bacterial DNA from faecal samples. MP kit was the most efficient, extracting approximately three times more DNA than MO BIO and QIA kits per mg faeces. The use of MP and QIA kits resulted in high quality of the extracted DNA, while MO BIO provided DNA of inferior quality, consistent with literature datats Table . Statist27. While most of the published work assessed the performance of NGS approaches using the same primer set(s) across platforms, this study focused on investigating the comparability of taxonomic composition obtained with supplier recommended sequencing protocols, including the 16S rRNA gene regions and suggested primers.The influence of using different NGS platforms, MiSeq and IT, on microbial community composition analyses was already compared using different experimental setups and conditions27. Additionally, the mock community revealed that both sequencing technologies have the ability to detect all bacteria present in the mock sample with >99% of detected OTUs annotated correctly down to family level. In contrast to previous platform comparison using even and uneven mock community designed for that study9, we identified all 17 genera present in our mock community sample (HM-782D). The detected false positive genera comprised <1% of total OTU counts, resulting in overall substantial correspondence to theoretical composition unlike the abovementioned study. The different capacity of platforms to capture mock community diversity in these two studies could be related to the differences in community composition, tested 16S gene region and primer sets used. The relative abundances of taxa in our study were not fully reproduced and MiSeq in general had closer proportions and improved detection of Actinobacteria (Actinomycetaceae (Actinomyces odontolyticus) and Propionibacteriaceae (Propionibacterium acnes)), when compared to the expected values. This is in accordance with previous work on the same mock sample7 that also found marked differences with these organisms and attributed them to premature read truncation observed on the IT platform. In general, we found more deviations from theoretical abundance with IT than with MiSeq sequencing of mock community . We considered these taxa as the most relevant drivers of platform differences. The notable finding of this study, and not reported so far, was the increased capacity of MiSeq to detect Verrucomicrobia (Akkermansia muciniphila species was the only representative of this phylum) as well as Faecalibacterium genus with abundances being 10 and 5 fold higher than with IT, respectively. This finding may be of special interest to researchers of health promoting bacteria since both Faecalibacterium prausnitzii, a butyrate-producing species in the Ruminococcaceae family, and Akkermansia muciniphila, a mucus-colonizing and degrading bacteria, showed sufficient efficacies to be considered next generation probiotics40. Other taxa of interest that were statistically significant and 2 fold more abundant on MiSeq were [Odoribacteraceae], Erysipelotrichaceae and Ruminococcaceae  or to the lower required matching sequence for MiSeq (V3-V4 region) to assign taxonomy to an OTU, than for IT .The assessment of relative abundances did not reveal marked differences between platforms  with respect to DNA amount and quality, except when QIA kit was subsequently used for DNA extraction. Out of three kits used, MP provided most efficient DNA extraction resulting in significantly higher quantity and quality of the DNA, as well as higher richness of faecal microbiota content. Despite the fact that this study investigated optimized NGS platform technologies which differ in multiple parameters (16S rRNA gene region(s), primers, amplicon lengths, sequencing technology (single vs. pair-end)), when analysed using the same processing pipeline, both MiSeq and IT adequately reproduce donor faecal microbiota specificity. However, all the technology-related bias described here suggests that particular care should be given to appropriate methodology choices suitable for the microbial profiling in a specific study.The outline of the study design is presented in Fig. Genomic DNA from Microbial Mock Community B , v5.1 L, for 16S rRNA Gene Sequencing (#HM-782D) was acquired from BEI Resources. It is comprised of DNA from 20 bacterial strains containing equimolar ribosomal RNA operon counts (100.000 copies per organism per μL) and the complete list of organisms can be found in Supplementary Table Informed consent was obtained from all participants and all procedures were in accordance with the approved study protocol . A fraction of faeces sample (1–2 g) was collected fresh in its native state in a screw-cap sterile container. An aliquot was processed within a few hours of collection while the remaining aliquot was frozen at −20 °C for 14 days and processed after thawing. The other fraction of the same faeces sample (400–500 mg) was collected in OMNIgene.GUT kit , a system for self-collection and liquid stabilization of microbial DNA from faeces for gut microbiome profiling, according to manufacturer’s protocol. Briefly, the faeces sample was thoroughly mixed with 2 mL of stabilization liquid in the provided tube. Again, two aliquots of this faeces sample were processed, one within a few hours of collection and the other at day 14, after two-week storage at room temperature.42. Homogenization of samples was performed using Minilys homogenizer (Bertin Corp) in tubes prefilled with high quality beads which ensures disruption of the cell walls and release of the DNA molecules in solution. MP kit contains tubes with beads (Lysing matrix E) and for QIA and MO BIO kit homogenization was performed using Soil grinding kit SK38 , with 0.1 mm glass beads, 1.4 mm ceramic beads and 1 glass bead of 4.0 mm. After extraction, DNA integrity was assessed by 1% agarose gel electrophoresis.Three commercial faecal DNA extraction kits were evaluated in this study: Power Fecal DNA Isolation Kit , QIAamp Fast DNA Stool Mini Kit  and Fast DNA SPIN Kit for Feces . The manufacturers’ protocols were followed for each kit, and recommendations for application to hard-to-lyse organisms were incorporated (detailed protocols for each DNA extraction procedure are supplied in Supplementary Methods). All extractions were performed by the same experimenter. The sample size for DNA extraction from native faeces ranged from 50–250 mg, while for the OMNIgene.GUT system 250 µL was used. Prior to extraction, samples were homogenized by bead-beating260/A280 absorbance ratio determined with Nanodrop was used to estimate the quality and purity of the extracted DNA while concentration was measured with Qubit 3.0.The DNA quantity and quality measurements were performed on two spectrophotometric devices, Nanodrop 2000 and Qubit 3.0 (Thermo Fisher Scientific), based on the absorbance and fluorescence readouts, respectively. The A260/A280 are reported as mean values ± SEM (16 samples per group). P-values are reported after applying Bonferroni correction on unpaired two-sided t-test, p < 0.001 is reported as ***.DNA concentration and absorbance ratio at AFaecal bacterial communities were profiled by 16S rRNA amplicon sequencing using two NGS platforms: Illumina MiSeq (MiSeq) and Ion Torrent PGM (IT). Manufacturer recommended primers, reagents and protocols were applied.43. After PCR amplification and PCR product clean up with Agencourt AMPure XP , Illumina sequencing adapters and dual-index barcodes were added to the amplicon target with Nextera XT Index Kit . Sequencing was performed on MiSeq platform (Illumina) with MiSeq Reagent Kit v3 , according to the manufacturer’s instructions.For MiSeq sequencing Nextera XT DNA Sample Preparation Kit  was used for construction of 16S libraries. Hypervariable regions V3-V4 of 16S rRNA gene were amplified using primers with overhanging adapter sequences for compatibility with Illumina index and sequencing adapters needed for paired-end sequencing, resulting in a single amplicon with mean length of 464 bpFor IT sequencing 16S libraries from faecal DNA samples were constructed by amplifying seven regions V2, V4, V8 and V3, V6-V7, V9 of bacterial 16S rRNA using primers from Ion 16S™ Metagenomics Kit . Amplicons were further processed using manufacturer recommended protocols and ION PGM™ Template OT2 400 Kit . Final DNA library concentration was quantified using Qubit dsDNA BR Assay Kit . Following library quantification the Ion OneTouch™ 2  system was used to prepare template positive ion sphere particles containing the clonally amplified DNA libraries using the ION PGM™ Template OT2 400 Kit. Template positive sphere density was within the optimal range of 10–30%. Sequencing was performed on Ion Personal Genome Machine (PGM) System (Thermo Fisher Scientific) with Ion PGM Hi-Q Sequencing Kit  and Ion 316 Chip Kit v2  according to the manufacturer’s instructions.44. For MiSeq, fastq files containing paired end reads were first merged, allowing overlap between mates , using FLASh45. Then for both platforms,.fastq files were trimmed, filtered by quality and chimera removed as described in the default QIIME pipeline. Operational Taxonomic Units (OTUs) were assigned using the vsearch46 algorithm and PyNast alignment47 against the GreenGenes database . Cumulative OTU counts for each taxonomy level from phylum to genus were extracted into separate tables from the resulting assignment.Raw sequencing files from both platforms were processed using QIIME pipeline48, using ALDEx2 R package30 with additions of multivariate generalized linear models for testing the significance of multiple factors and their interactions.Subsequent processing and analysis was done for each taxonomy level separately according to the Gloor CoDa Microbiome tutorial49, and major contributions to the differences in beta diversity were presented as principle coordinate analysis, as implemented in the QIIME pipeline.The compositional diversity within each sample was ascertained using several alpha diversity indices: observed OTUs, followed by the PD whole tree and Chao1, as implemented in the QIIME pipeline. Sequences were rarefied from 0 to 30 000 range. Beta diversity was reported using weighted UniFrac distance measure2 - the goodness of fit of the line to the data.To assess the reproducibility between relative taxa abundances across time points and collection/storage parameters within a DNA extraction kit, abundance values were plotted against reference point (native 0) abundance from the same kit. Linear regression analysis was used to plot the linear regression line and determine RAggregate relative abundances at different taxonomic ranks as well as precision analysis were displayed on raw counts.50 and the statistical analyses were performed as previously described30. Briefly, the approach incorporates Bayesian multiplicative replacement of count zeros and centred log-ratio (clr) OTU count data transformation30. The centred log-ratio (clr) transformation28 ensures the data are scalable and sub-compositionally coherent thus making them amenable for both univariate and multivariate analyses. The raw OTU counts on taxonomic levels from phylum to genus were transformed to compositional values using the ALDEx2  R package.The obtained sequencing data were evaluated as compositional dataPrincipal component analysis (PCA) is shown on zero-imputed and clr transformed counts using ‘prcomp’ function in R.Kruskal-Wallis multivariate test was performed to detect overall significance for each variable per OTU. P-values were considered significant if p < 0.05 after Benjamini-Hochberg (BH) correction.The transformed data were further assessed for significant differences among the group of samples collected/processed using Wilcoxon rank test and Welch’s t-test, as implemented in ALDEx2 pipeline. Tests were performed on 50 Monte Carlo simulated instances of Dirichlet-distributed original datasets to estimate per-feature technical variation within each sample. All p-values are reported after adjusting for multiple testing with BH correction. Pairwise comparisons of transformed data were performed for pairs of donors, time points, kits and faecal collection type for each taxonomic level (phylum to genus).Supplementary information