Solid tumors residing in tissues and organs leave footprints in circulation through circulating tumor cells (CTCs) and circulating tumor DNAs (ctDNA). Characterization of the ctDNA portraits and comparison with tumor DNA mutational portraits may reveal clinically actionable information on solid tumors that is traditionally achieved through more invasive approaches.We isolated ctDNAs from plasma of patients of 103 lung cancer and 74 other solid tumors of different tissue origins. Deep sequencing using the Guardant360 test was performed to identify mutations in 73 clinically actionable genes, and the results were associated with clinical characteristics of the patient. The mutation profiles of 37 lung cancer cases with paired ctDNA and tumor genomic DNA sequencing were used to evaluate clonal representation of tumor in circulation. Five lung cancer cases with longitudinal ctDNA sampling were monitored for cancer progression or response to treatments.TP53, EGFR, and KRAS genes are most prevalent in our cohort. Mutation rates of ctDNA are similar in early (I and II) and late stage (III and IV) cancers. Mutation in DNA repair genes BRCA1, BRCA2, and ATM are found in 18.1% (32/177) of cases. Patients with higher mutation rates had significantly higher mortality rates. Lung cancer of never smokers exhibited significantly higher ctDNA mutation rates as well as higher EGFR and ERBB2 mutations than ever smokers. Comparative analysis of ctDNA and tumor DNA mutation data from the same patients showed that key driver mutations could be detected in plasma even when they were present at a minor clonal population in the tumor. Mutations of key genes found in the tumor tissue could remain in circulation even after frontline radiotherapy and chemotherapy suggesting these mutations represented resistance mechanisms. Longitudinal sampling of five lung cancer cases showed distinct changes in ctDNA mutation portraits that are consistent with cancer progression or response to EGFR drug treatment.Mutations in This study demonstrates that ctDNA mutation rates in the key tumor-associated genes are clinical parameters relevant to smoking status and mortality. Mutations in ctDNA may serve as an early detection tool for cancer. This study quantitatively confirms the hypothesis that ctDNAs in circulation is the result of dissemination of aggressive tumor clones and survival of resistant clones. This study supports the use of ctDNA profiling as a less-invasive approach to monitor cancer progression and selection of appropriate drugs during cancer evolution.The online version of this article (doi:10.1186/s13045-017-0468-1) contains supplementary material, which is available to authorized users. Tumors have been broadly classified as either hematopoietic or solid types. Hematopoietic cancers are derived from neoplastic cells of blood cell origin, and solid tumors are normally associated with a specific organ or tissue type. In the molecular and genomic era of medicine, there are two seemingly opposite trajectories in the characterization of cancer. One trajectory is the zoom-in approach, which divides each cancer type further into subtypes. In this effort, a traditionally recognized cancer type, such as lung cancer, can be further divided into multiple subtypes based on the molecular and genomic signature . The oppIt has long been recognized that tumor cells can release protein molecules into circulation, e.g., tumor markers such as PSA, CEA, and CA125 –15. ThisThere are, however, many unanswered questions regarding this emerging approach. First, will mutation rates detected in ctDNA be as valuable as mutation profiling in tumor tissues? Second, is there a specific mechanism to disseminate tumor tissue mutations into circulation? Third, would these metastasis-related events serve as more effective targets for intervention?In this study, we evaluated the clinical value of assessing ctDNA using the Guardant360 platform in 177 patients with solid tumors, the majority of whom were treated for lung cancer. The results allow us to evaluate mutational portraits of solid tumors in circulation. We also present the initial success in application of this new genomics platform in cancer monitoring and treatment.We retrospectively reviewed the liquid biopsy results of 177 consecutive de-identified patients with diverse cancers who were seen at Wake Forest Baptist Comprehensive Cancer Center. Blood samples were collected between June 2015 and September 2016. Sociodemographic and clinical data including gender , age , body mass index , smoking history , race , stage , metastasis , tumor type, and vital status  of each patient were collected. Smoking status was defined by self-reported smoking history obtained from Cancer Registry and/or Epic Electronic Medical Record. Never smokers were defined as respondents who smoked less than 100 cigarettes in their lifetime. Based on evidence that smoking cessation reduces cancer risk by half at 5 years, active smokers at the time of clinical data collection and those who had quit smoking within the previous 5 years were considered current/recent smokers . We defig within 4 h of the blood draw. The supernatant containing the plasma was further centrifuged at 14,000 g for 10 min at room temperature and was stored at −80 °C until analysis. Initially, the plasma samples were selected on the basis of their availability, and then, consecutively. DNA was extracted from plasma using the QIAamp DSP Virus Kit (Qiagen), according to the manufacturer’s instructions. A real-time quantitative PCR TaqMan Assay targeting GAPDH was used to measure plasma DNA concentration. tDNA was extracted from the fresh frozen biopsy sample using the AllPrep DNA/RNA Mini Kit (Qiagen) and was quantified with Qubit 2.0 (Life Technologies).Blood samples (3–10 ml) were collected in EDTA tubes  and centrifuged for 10 min at 1000 www.guardanthealth.com), a Clinical Laboratory Improvement Amendments (CLIA)-certified and College of American Pathologists (CAP)-accredited clinical laboratory . At the time of this study, this test identifies potential tumor-related genomic alterations via complete exon sequencing of 73 cancer-related genes in ctDNA extracted from plasma. ctDNA was extracted from plasma, and the amount of ctDNA was quantified using electrophoretic separation in a massively parallel capillary array system allowing for post-extraction high-throughput, high-resolution fragment size-specific data acquisition for each sample processed. The ctDNA was then analyzed by paired-end sequencing by synthesis utilizing an Illumina Hi-Seq 2000 platform and hg19 as the reference genome as described [Next generation digital sequencing was performed using the Guardant360 test by Guardant Health., . Point mutations were identified by a Bayesian algorithm; short insertions and deletions determined by local assembly; gene copy number alterations identified by comparison to process-matched normal controls; and gene fusions/rearrangements determined by clustering chimeric reads mapped to targeted introns [Thirty-seven lung tissue samples were processed  by Foundation Medicine  using the FoundationOne NGS panel. For the FoundationOne test, DNA was extracted from one or more 40-μm sections of FFPE tissue using the Maxwell 16 FFPE Plus LEV DNA Purification kit (Promega) and was quantified using a standardized PicoGreen fluorescence assay (Invitrogen). Library construction was performed using 50–200 ng of DNA sheared by sonication to approximately 100–400 bp before end-repair, dA addition and ligation of indexed, Illumina sequencing adaptors. Enrichment of target sequences was achieved by solution-based hybrid capture with custom biotinylated oligonucleotide bases. Enriched libraries were sequenced to an average median depth of >500× with 99% of bases covered >100× (IlluminaHiSeq 2000 platform using 49 × 49 paired-end reads) and were mapped to the reference human genome (hg19) using the Burrows-Wheeler Aligner and the publicly available SAM tools, Picard, and Genome Analysis Toolkit. Genomic alterations detected include base substitutions, insertions, deletions, copy number alterations, and selected gene fusions  available were extracted from the medical reports and were analyzed. Non-synonymous somatic mutation calls were quantified. Patients were assigned to low or high mutation load groups based on the cohort median mutation number. We applied a Mann-Whitney-Wilcoxon rank sum  and a KrAll lung cancer patients with available ctDNA and tDNA results were included in the main clonality analysis. Mutations called from ctDNA sequencing (plasma) were compared with mutations from tissue biopsy DNA sequencing (tumor). Tumor clonality was analyzed with R package “SciClone” . Non-synThe sample was comprised of 177 patients with diverse cancers who had a liquid biopsy next-generation sequencing ctDNA test performed on their plasma. Patients’ median age was 65 years old (range 26–90). Most were white (83.6%) with a history of smoking . There was a predominance of advanced-stage (84.7%) versus early-stage (14.2%) cancers, and the most commonly represented cancers were lung adenocarcinoma (29.4%), non-small cell lung cancer (13.5%), and lung squamous carcinoma (11.9%) followed by head/neck cancer (9.0%), colorectal and cancer of unknown primary (CUP) (6.8%), pancreatic cancer (3.9%), other GI (3.4%), small cell lung cancer (3.4%), and other cancers. Table TP53, KRAS, EGFR, PIK3CA, ERBB2, MYC, and BRCA1. The distribution of the top 30 mutated genes in the major cancer types is shown in Additional file We detected 628 non-synonymous alterations in 61 of the 73 cancer-related genes from the Guardant360 ctDNA tests with a mean of 3.55 mutations per patient. Twelve genes were not found to be mutated in the whole sample. Eighteen patient plasma samples did not reveal mutations in the test  exhibited similar levels in early (I and II) and later (III and IV) stages   Fig. . We alsoior Fig. . The patA recent study reported that a mutation rate of three in the plasma using the Guardant360 platform was associated with survival in a pan-cancer study similar to ours . We analp = 0.02, Mann-Whitney-Wilcoxon rank sum). None of the 18 ctDNA mutation-free plasma samples, many from late stage cancer patients, had metastasized to more than two different distant organ sites , but not in the multivariable test  , squamous carcinoma (LUSC), non-small cell-not otherwise specified (NSCLC-NOS), and small cell lung cancer (SCLC) .There are 34 current/recent smokers and 53 former (>5 years) smokers, and only 16 never smokers among the lung cancer sample. We detect a higher mutation rate in never smokers than in current/recent and former smokers Fig. . FurtherTP53, EGFR, and KRAS were detected in both early and late stages lung cancers , TP53 and ERBB2 mutations were found in a minor allele in the tumors with VAF less than 20%. Both mutations were detected in the plasma. For the stage III patients (n = 4), TP53 and KRAS mutations were found in either minor (<25%) and major (>25%) populations in tumor tissues, and both could be detected in the plasma. For the stage IV patients (n = 20). Two cases had both tumor and blood collected in the same day. Both cases were positive for ALK fusion in both tests, and ALK fusions were present in the major population in the tumor. Four cases had blood drawn before surgery. Mutations of known driving mutations were found in minor populations in some cases, and these mutations  were detected in plasma  and BRAF (in patient 131) found in the minor populations in the tumors were still present in the blood even after adjuvant therapy post-surgery suggesting they represent resistant clones  and started on erlotinib. She had stable systemic disease but experienced progression of CNS metastases despite erlotinib treatment. She received both GKRS as well as whole brain radiation therapy. She was judged to have meningeal carcinomatosis, and treatment was switched to pulse-dose erlotinib (1500 mg weekly) with concurrent pemetrexed. This provided some control of her disease for 10 months. She then experienced progressive neurologic decline and had Guardant360 testing, which revealed EGFR T790M mutation. After starting osimertinib, she experienced an improvement in her leg strength and concentration. She has been able to return to work part-time and continues to have control of her disease with osimertinib for 6 months.Patient 5’s lung cancer had metastasized to the brain. The rapid advancement of deep sequencing has allowed development of clinical tests that sequence cell-free tumor DNA in circulation. Our understanding that solid tumor cells enter circulation or shed DNA into circulation created a pathway for a non-invasive approach to detect cancer and to monitor cancer progression over time. This approach is clinically valuable because tumor biopsy is often not possible or at least not feasible for multiple sampling during disease progression and management. As a part of the Precision Oncology Initiative at the Wake Forest Baptist Comprehensive Cancer Center in partnership with Guardant Health, we have processed plasma ctDNAs from 177 patients in several solid tumor types, the majority of whom have lung cancer. Our patient population is enriched with ever smokers because of the unique geographical and historical reasons that Winston-Salem, North Carolina serves as a major headquarters for tobacco manufacturing.TP53, KRAS, and EGFR are found in both early and late stages of cancer. This is highly clinically significant and suggests that ctDNA mutation detection can be a non-invasive early detection approach that could potentially be applied to screening of high risk populations.Analysis of the sequencing results has revealed several important insights. First, we found that the ctDNA mutation patterns are independent of tumor stage, regardless of whether we examined the total population of solid tumor patients or a subsample of lung cancer patients. This suggests that circulating tumor cells may enter circulation during an early stage of cancer progression, and the dissemination of tumor DNA is an early event. The best known tumor-associated mutations such as mutations in Second, mutation rates in ctDNA are a robust predictor of survival, which have not previously been reported using tumor tissues. This suggests that the ctDNA mutations, which may reflect a transition from local to systemic disease, represent a key clinical transformation. In other words, the ability of tumor cells to enter circulation may determine the disease outcome.TP53 mutations are frequently found in plasma even when the mutations were found in a minor clone in the tumor tissues. Other gene mutations with similar behavior include EGFR, BRAF, CTNNB1, ARID1A, ERBB2, and PDGFRA. The results demonstrate that mutations in these genes are likely driver for metastasis through dissemination into circulation from circulating tumor cells.The finding of tumor stage-independent mutation patterns in plasma led us to hypothesize that the key for tumor heterogeneity investigations is to determine the clinically aggressive clones or clones that are resistant to therapies. To test this hypothesis, we have identified the lung cancer cases that had both tumor tissue DNA mutation detection using FoundationOne test and Guardant360 liquid biopsy test. More than 400 genes are included in FoundationOne test, clonality analysis can be carried out to exhibit clonalities based on allele frequencies of the mutated genes. Although such analysis is not feasible with Guardant360 test due to the lower mutation rate in general, we could match the mutations in plasma to these found in the tumor tissues. The results of our analysis showed that known mutations like BRCA1, BRAF, and PDGFBA were still detected in the plasma suggesting that tumor cells harboring these mutations were not eliminated by the therapy and that these mutations may actually confer a resistance mechanism. An intriguing observation is that BRCA2 mutations are more commonly found in tumor tissue exome sequencing but rarely in the plasma ctDNA mutation detection. This is consistent with the notion that BRCA2 is a key double strand DNA repair gene product and mutations of which render the tumor cells sensitive to DNA damaging chemotherapy and radiation treatment [BRCA1 is also involved in DNA repair, BRCA1 is involved in a broader scope of cellular functions and mutations of which have been suggested to contribute to metastasis. These results suggest that comparative analysis of tumor tissue DNA mutation detection and plasma ctDNA mutation results can reveal mutational events that may sensitize or resist chemotherapy and radiation therapy. These mutational events may be better suited for targeted therapy. Given that, there are specific drugs that target BRAF [PDGFRA [ARID1A [Detailed examination of gene mutations in the cases where plasma ctDNA test was obtained after surgery and tumor DNA sequencing showed a large fraction of which had been treated by chemotherapy and radiation therapy. Nevertheless, mutations in genes like reatment , 50. Altget BRAF –53, PDGF [PDGFRA –56, and  [ARID1A  mutants,EGFR activating mutations, 90% of which consist of in-frame deletions in exon 19 and the L858R point mutation in exon 21. First-line treatment of patients harboring EGFR-activating mutations with EGFR TKIs gefitinib, erlotinib, or afatinib has resulted in superior overall response rates, progression-free survival, and quality of life compared to chemotherapy [EGFR-activating mutations in plasma, which had reappeared together with the T790M resistance mutation. Our study has validated the reports by others. We join investigators from other studies to propose the routine use of the longitudinal ctDNA testing as an effective way to monitor cancer progression, which can potentially lead to real-time changes in treatment.The emergence of clinical resistance to previously effective anti-neoplastic therapy results from the acquisition of molecular alterations in genes or pathways that govern resistance mechanisms. Defining these mechanisms of resistance to targeted agents is challenging because it is difficult to acquire serial tumor biopsies in patients with advanced disease. Therefore, we performed serial plasma ctDNA collections at two time points to detect the emergence of genomic alterations and acquired resistance to targeted therapy. At present, the most widely known tyrosine kinase inhibitor (TKI) targets in NSCLC-NOS are otherapy . The acqIn summary, our liquid biopsy project provides supporting evidence that ctDNA mutation detection constitutes less-invasive real-time surrogates for early diagnosis, prognosis, therapeutic tailoring, and resistance monitoring and mitigates needle biopsy sampling errors related to intra- or inter-tumor heterogeneity.Additional file 1: Table S1.Table S2. Description of mutation load in total cohort (n = 177). Table S3. All mutations detected in patient ctDNA compared with other studies. (DOC 625 kb)Clinical and treatment characteristics of the 18 cases with no ctDNA alterations detected. Additional file 2: Figure S1.Figure S2. ctDNA mutational landscape of patients’ stage known for the top 30 genes in lung cancer cohort. Figure S3. Higher mutation numbers in the ctDNA are associated with decreased survival in lung cancers. Higher mutation numbers in ctDNA are associated with poor survival. “n” defines the number of mutations and survival plots are separated by mutation numbers: n = 1, 2, 3, 4, 5, and 6 mutations. Blue lines indicate more than “n” mutations and the pink lines indicate equal to or less than “n” mutations. P-values were derived using the log-rank test. Figure S4. The concordance of top mutated genes in the ctDNA and tDNA of 37 lung cancer patients. (DOC 1098 kb)ctDNA mutational landscape for major cancer groups for the top 30 genes in total cohort.


Nrl-/- mouse retina in which all photoreceptor precursor cells commit to a cone fate including those which would have become rods in wild-type retinas. The dendritic distribution of OFF bipolar cell types was investigated by confocal and electron microscopic imaging of immunolabeled tissue sections. The cells’ dendrites formed basal contacts with cone terminals and expressed the corresponding glutamate receptor subunits at those sites, indicating putative synapses. All of the four analyzed cell populations showed distinctive patterns of vertical dendritic invasion through the OPL. This disparate behavior of dendritic extension in an environment containing only cone terminals demonstrates type-dependent specificity for dendritic outgrowth in OFF bipolar cells: rod terminals are not required for inducing dendritic extension into distal areas of the OPL.Retinal OFF bipolar cells show distinct connectivity patterns with photoreceptors in the wild-type mouse retina. Some types are cone-specific while others penetrate further through the outer plexiform layer (OPL) to contact rods in addition to cones. To explore dendritic stratification of OFF bipolar cells in the absence of rods, we made use of the ‘cone-full’  Retinal bipolar cells are a diverse class of excitatory interneurons, passing the photoreceptor signals on to amacrine and ganglion cells (reviewed in ). ON bipSubstantial evidence has accumulated for OFF bipolar cells contacting rods as well as cones in different mammalian species –13. OFF Nrl-/-) is an excellent model to explore dendritic patterning of OFF bipolar cells in this regard. The Nrl gene encodes a transcription factor which in turn regulates several genes required for rod development from photoreceptor precursor cells [Nrl-/- mouse have been shown to consist of only cone-like photoreceptors. Despite the formation of ‘rosettes’ in the outer nuclear layer and some other morphological abnormalities at the cellular level, cone-like photoreceptors in the Nrl-/- mouse are fully functional and co-express varying ratios of middle-wavelength sensitive (M-) and S-opsins [Nrl-/- mouse has therefore been used previously as a mouse model for a ‘cone-full’ retina in the absence of rods [Nrl-/- mouse model to eliminate a putative extrinsic signal dictating the different wiring patterns of OFF bipolar cells.It remains unclear, however, if the presence of rods is required to elicit dendritic outgrowth into more distal areas of the OPL, where rod terminals are typically located in wild-type retinas. The neural retina leucine zipper knockout mouse . Euthanasia of mice for organ harvesting (retinas) used in this study has been approved by the animal welfare officer of the Max-Planck-Institute for Brain Research and reported to the local authorities (Regierungspräsidium Darmstadt).All procedures were approved by the University of California at Santa Barbara Institutional Animal Care and Use Committee (IACUC). These were in full accordance with institutional guidelines of the Max-Planck-Institute for Brain Research, Frankfurt, following the standards described by the German animal protection law  were injected with a lethal dose of sodium pentobarbital (120mg/kg). The eyes were dissected, the cornea and lens were removed and the remaining eyecups were then fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for 30 min for light microscopy (LM) or 60 min for electron microscopy (EM). After fixation, each retina was dissected from the eyecup and cryoprotected in graded sucrose solutions . For light microscopy, the retina was embedded in freezing medium . Vertical sections of 14 μm thickness were cut on a cryostat , collected on glass slides, and stored at -20°C. For electron microscopy, the retina was embedded in 4% agar after freezing and thawing the tissue. The agar block was mounted on a vibratome , and vertical sections of 60 μm thickness were cut. Vibratome sections were processed free-floating in 0.01 M phosphate buffered saline (PBS), pH 7.4.Adult mice  and afterward in a solution containing the primary antibodies  overnight at room temperature. After extensive washing in PBS, sections were incubated with secondary donkey antibodies conjugated either to Cy3 or Cy5 (Dianova), or Alexa Fluor 488 (Molecular Probes) for 1 hour at room temperature. Bipolar cells were labeled with antibodies against NK3R  , HCN4 . The position of the dendritic tips was measured and then expressed as percent vertical penetration relative to the borders of the CtBP2-positive area (‘synaptic region’) across the length of several sections of retinas from at least two different animals per cell type. Experiments were performed in age-matched mice at postnatal weeks 5 (n = 3) and 10 (n = 2). A difference between these two ages was not observed. Areas of Nrl-/- mouse specific rosettes were excluded from the measurements. The borders of the synaptic region were defined by the inner (0%) and outer (100%) margins of CtBP2 (ribbon) staining at the location of each dendritic tip. The data failed the Shapiro-Wilk normality test. Therefore, the Kruskal-Wallis one-way ANOVA with post-hoc tests (Dunn’s Multiple Comparison) were applied to determine if cell types were significantly different from each other.To quantify the vertical penetration of the OPL by bipolar cell dendrites in the 2O2 for 5–10 min. Afterward, the sections were rinsed in Tris-HCl, subsequently in 0.1 M cacodylate buffer, pH 7.4, and then postfixed in 2.5% glutaraldehyde in cacodylate buffer for 1 h. After several washes in distilled water, the DAB reaction product was silver-intensified by incubating the sections in a solution containing 3% hexamethylene tetramine, 5% silver nitrate, 2,5% borax, 2,5% sodium thiosulfate and 0,05% gold chloride for 10 min at 60°C. Subsequently the sections were postfixed with 0.5% OsO4 in cacodylate buffer for 30 min and dehydrated in a graded series of ethanol (30–100%) followed by propylene oxide. They were embedded in Epon and serial ultrathin sections were cut, stained with uranyl acetate and lead citrate, and examined with a Zeiss EM10 electron microscope.Vibratome sections were incubated for 4 d at 4°C in a primary antibody solution containing 3% normal goat serum (NGS), 1% bovine serum albumin, and 0.05% sodium azide in PBS. After rinsing in PBS, biotinylated goat anti-rabbit IgG or goat anti-mouse IgG was applied , and a peroxidase-based enzymatic detection system  was used according to the manufacturer’s instructions to visualize antibody binding. The sections were rinsed again in PBS, subsequently in 0.05 M Tris-HCl, pH 7.6, and then treated with 3,3-diaminobenzidine  with 0.01% HNrl-/- retina, however, the dendrites of all four investigated populations of bipolar cells were found to also extend further into the OPL . These findings were quantified by measuring the positions of dendritic tips relative to the local borders of the synaptic region . CtBP2 wNrl-/- retinas form synaptic contacts with photoreceptors  were labeled. The dominant receptor subunits were previously shown to be GluA1 (AMPA) and GluK1 (kainate) [Nrl-/- mice, where the two GluR subunits were no longer clustered as bands. The labeling occurred as individual puncta and showed a much broader vertical spread throughout the OPL  and type 4 cells (Csen). To obtain a more complete picture of putative synapse formation between photoreceptors and OFF bipolar cells in kainate) . Double-kainate) –31 and o the OPL  correspo subunit . Taken tNrl-/- mouse retina where all photoreceptor precursor cells develop as cone photoreceptors. Instead of a single row of cone terminals as in wild-type retinas, the synaptic region of cone terminals is greatly increased across the depth of the OPL [Nrl-/- mouse retinas allowed a quantification of the vertical extension of OFF bipolar cell dendrites into the outer parts of the OPL. Our experiments revealed that the stratification patterns of OFF bipolar cells differ significantly between cell types in Nrl-/- retinas and that they form putative synapses with cone terminals in these outer portions of the OPL.To analyze dendritic stratification patterns of OFF bipolar cells, we took advantage of the  the OPL . This in the OPL , whereas the OPL . Here, tInterestingly, OFF bipolar cell types 3a, 3b, and 4 have previously been shown to contact rods as well as cones , 6, 8 anOne possible explanation for the distinctive behavior of OFF bipolar cell dendrites is that those differences reflect distinct times of differentiation and colonization of cone pedicles , 35. It Nrl-/- retinas. In line with this hypothesis, a comparison of dendritic connectivity patterns from two ON bipolar cells during retinal development has shown that different strategies (targeted or exploratory) are employed to form contacts with cone photoreceptors [Yet another possibility is that this distinctive patterning occurs for exclusively cell-intrinsic reasons, independent of any spatial or temporal variation in presynaptic specializations associated with differences between rods and cones (this study), or when synaptic transmission is strongly attenuated . By thiseceptors . Still tNrl-/- and the highest amount of rod contacts in wildtype).Furthermore, our findings directly relate to the cells’ dendritic connectivity patterns in wild-type retina. A quantitative analysis of rod and cone contacts among mouse OFF bipolar cells via reconstruction in a serial block-face electron microscopic sample  has reveIt remains a challenge for future studies to reveal if this differential extension of OFF bipolar cell dendrites is regulated via intrinsic mechanisms which act differently depending on cell type or if the timing of synaptogenesis is the key factor for the type-specific differences observed here. Nevertheless, the consistency of the cells’ distinct dendritic stratification and connectivity patterns between cell types in the aforementioned mouse models suggests that the rod contacts of certain OFF bipolar cell types are facilitated via individual levels of enhanced dendritic extension into the OPL that is independent of any extrinsic signal offered by the rod terminals.


Pparg2 and Leptin in gonadal adipose tissue (GAT) and subcutaneous adipose tissue (SAT), at baseline and after 6, 12 and 24 weeks of HFD exposure in adult mice. HFD induced hypermethylation of both the Leptin promoter  and the Pparg2 promoter in GAT . The differential methylation was independent of immune cell infiltration upon HFD exposure. In contrast, no differential methylation in the Pparg2 and Leptin promoter was observed in SAT. Leptin and Pparg2 DNA methylation were correlated with gene expression in GAT. Our study shows that prolonged exposure to HFD in adulthood is associated with a gradually increasing DNA methylation level at the Leptin and Pparg2 promoters in a depot-specific manner.High-fat diets (HFD) are thought to contribute to the development of metabolism-related diseases. The long-term impact of HFD may be mediated by epigenetic mechanisms, and indeed, HFD has been reported to induce DNA methylation changes in white adipose tissue (WAT) near metabolism related genes. However, previous studies were limited to a single WAT depot, a single time-point and primarily examined the pre-pubertal period. To define dynamic DNA methylation patterns specific for WAT depots, we investigated DNA methylation of  Leptin and Pparg2 (the adipocyte-specific isoform 2 of Pparg)67Leptin is a critical signalling component regulating food intake, energy homeostasis, and exhibits potent immunomodulatory functions912Pparg2 is a master regulator of adipogenesis and is involved in adipocyte differentiation and maturation as well as fat storage and glucose metabolism. Its activation it thought to have insulin sensitizing effects615Obesity induced by exposure to a high content of saturated fat diet (HFD) is characterized by hypertrophy and hyperplasia of adipocytes in white adipose tissue (WAT). It is followed by a chronic state of mild inflammation and changes in adipokine secretion, a phenomenon particularly apparent in metabolically active visceral WAT depots, including gonadal adipose tissue (GAT), rather than subcutaneous adipose tissue (SAT)1234Leptin and Pparg2 have primarily focused on HFD exposure in utero or in early life182021226202123242525Until now, studies investigating promoter DNA methylation of Pparg2 and Leptin promoters during prolonged HFD exposure  in adult male C57BL/6J mice in two different WAT depots, GAT and SAT. In addition, we analysed the impact of immune cell infiltration on DNA methylation and investigated the association between DNA methylation and expression of Leptin and Pparg2.Here, we report on dynamic changes in DNA methylation of the P < 2.2·10−16; P = 4.4·10−12), while SAT mass increased more gradually up to 89% after 24 weeks of HFD exposure (P = 3.2·10−11). The chow control group showed minor and non-significant increases over time confirming that the changes observed were due to the HFD exposure and not an age-related effect (We investigated the effect of HFD on bodyweight and mass of GAT and SAT depots as compared with chow after 0 (baseline), 6, 12, or 24 weeks of exposure (n = 11/12 per group). Food intake in HFD and chow groups was isocaloric. Body weight increased with 34.3% after 24 weeks of exposure  over time (Pdynamic = 8.46·10−3). Analysis of the individual CpG sites . In-depth analyses of individual CpG sites in GAT revealed a hypermethylated state of 2 out of 3 measured CpG sites .DNA methylation of the pG sites , where CPparg2 and Leptin promoter region . Moreover, the effect of HFD exposure on DNA methylation remained significant after correction for CLS in both genes . Together, these results indicate that CLS formation did not drive the changes in DNA methylation associated with exposure to a HFD.The increasing storage of lipids in adipose tissue is associated with an increased number of crown-like structures, which are thought to constitute of dead adipocytes that are surrounded by macrophagesr region . SubsequPparg2 and Leptin, we compared the observed DNA methylation to gene expression of Pparg2 and Leptin in GAT. Leptin promoter DNA methylation was positively correlated with gene expression of Leptin  studies should be performed that focus on the more acute effects of dietary constituents on DNA methylation and how these effects translate into changed expression of target genes and the functional effects (e.g. effect on protein translation or indirect measures such as insulin sensitivity or adipose differentiation).Both Pparg2 and Leptin gene expression in GAT and SAT. This corresponds with previous literature showing that primarily GAT is responsible for the development of metabolic imbalance232535243839The inter-depot differences in DNA methylation we report, indicate differential regulation of the Leptin and Pparg2 genes in adipocytes.It is well-known that saturated GAT accumulates CLS2041Leptin and Pparg2 genes in GAT. Our study demonstrates that DNA methylation changes induced by prolonged HFD exposure are fat depot specific and may primarily occur in metabolically active depots that become saturated such as GAT, while DNA methylation in SAT remains unaffected. The depot-specific changes in epigenetic regulation may be a starting-point in unravelling the putatively adverse adipokine profiles produced by saturated fat depots that contribute to obesity-induced metabolic imbalance.In conclusion, we show that prolonged HFD exposure during adulthood dynamically changes DNA methylation and expression of 2 asphyxiation after 0, 6, 12 and 24 (n = 12) weeks of exposure to the diet. After termination, GAT and SAT tissue were collected and partly fixed in formalin for histological analysis as reportedTissues and plasma were obtained from a subset of mice of a large time-resolved cohort study in C57BL/6J miceLeptin and Pparg2 genes to target with region-specific DNA methylation assay were taken from previous experimental studies on dietary exposures in rodents7Leptin, Pparg2). Locus-specific DNA methylation was measured using the mass-spectrometry method Epityper . Primers were designed, assays run and data processed as described previously® EpiDesigner BETA software (http://www.epidesigner.com/) based on the sequences obtained from UCSC genome browser mm10 (http://genome.ucsc.edu/). In silico mass spectrometry using R package RSeqMeth was performed to determine the CpG coverage and base mass of the expected PCR products. Details of the primers are shown in ®, USA). To account for possible batch effects the plate was designed with a similar distribution in HFD and chow exposed samples. The bisulfite treated samples were amplified by performing touchdown PCRs using selected PCR primers containing a T7-promotor tag and a 10-mer tag on the reverse and forward primer, respectively  and analysed by EpiTYPER software 1.2. All measurements were performed in triplicate and individual CpG measurement with ≤2 success rate or with a standard deviation ≥0.1 were discarded. For statistical analysis the average of the triplicate measurements was used.DNA methylation ratios were determined by the ratio of C and G spectral peaks using the mass spectrometry–based method Epityper MassARRAY compact MALDITOF . The Illumina® TotalPrep™ RNA Amplification Kit  was used to synthesize biotin labeled cRNA starting with 500 ng total RNA. The biotinylated cRNA was then hybridized onto the MouseRef-8 Expression BeadChip was 750 ng. Lastly, the default setting of Illumina’s Genomestudio v1.1.1 was used for Gene Expression analysis. All the quality control data of this BeadChip were within specifications of the microarray service provider . The microarray gene expression data were validated using quantitative real-time PCR for Leptin using established protocols and primer/probe setsTotal RNA was extracted from adipose tissues using glass beads and RNAbee . RNA integrity was examined using the RNA 6000 Nano Lab-on-a-Chip kit and a Bioanalyzer 2100 .Plasma leptin levels were determined by a Quantikine ELISA for murine leptin , age (Time ∗ b3) and individual variation of the mice (Mouse ID ∗ b2). To test for time specificity, the interaction between time and exposure was added to the model  with the packages: ggplot2 and lme4.All analyses were performed using R statistics version 3.1.2 .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


This suggests that diffuse white matter injury begins in utero for a significant proportion of preterm infants, which focuses attention on the development of methods for detecting fetuses and placentas at risk as a means of reducing preterm brain injury.Preterm infants are susceptible to inflammation-induced white matter injury but the exposures that lead to this are uncertain. Histologic chorioamnionitis (HCA) reflects intrauterine inflammation, can trigger a fetal inflammatory response, and is closely associated with premature birth. In a cohort of 90 preterm infants with detailed placental histology and neonatal brain magnetic resonance imaging (MRI) data at term equivalent age, we used Tract-based Spatial Statistics (TBSS) to perform voxel-wise statistical comparison of fractional anisotropy (FA) data and computational morphometry analysis to compute the volumes of whole brain, tissue compartments and cerebrospinal fluid, to test the hypothesis that HCA is an independent antenatal risk factor for preterm brain injury. Twenty-six (29%) infants had HCA and this was associated with decreased FA in the genu, cingulum cingulate gyri, centrum semiovale, inferior longitudinal fasciculi, limbs of the internal capsule, external capsule and cerebellum ( Globally, preterm birth affects around 10% of deliveries and is a leading cause of neurodevelopmental impairment234Chorioamnionitis  affects around 40–80% of very preterm deliveries and it can initiate a fetal inflammatory response that is injurious to the developing brain and other organs11Diffusion MRI (dMRI) provides objective measures of white matter microstructure in the newborn that are altered in association with preterm birth: a consistent finding is that fractional anisotropy (FA), a biomarker linked to white matter microstructure, is reduced in white matter tracts of preterm infants at term equivalent age compared with term-born matched controls142820We combined histologic classification of chorioamnionitis with neonatal brain MRI, and used TBSS and computational morphometry to test the hypothesis that HCA is associated with altered white matter microstructure and brain volume in preterm infants at term equivalent age.Placental histopathology and brain dMRI were acquired from 90 infants born preterm: 26 (29%) had histologic chorioamnionitis and 64 (71%) had no evidence of chorioamnionitis. p = 0.001), and the prevalence of prolonged rupture of membranes (>24 hours before delivery) was higher in this group (69% versus 8%). The proportion of infants exposed to antenatal MgSO4 for the purpose of fetal neuroprotection was greater in the HCA group: 69% versus 44% (p = 0.037). There was no significant difference in antenatal steroid exposure between the groups. Five out of 90 participants were treated for necrotizing enterocolitis, and three required treatment for retinopathy of prematurity. Postnatal somatic growth, measured as the difference between birth weight z-score and weight at scan z-scores, did not differ significantly between the two groups: the mean difference (SD) in weight z-score for the HCA group was −0.98 (1.07) versus −0.67 (1.06) for preterm infants without HCA exposure .Preterm infants with HCA had a lower GA at birth (26 of 90 (29%) placental plates showed patterns of inflammatory reaction on the maternal side, and 20 of these showed an inflammatory response of the cord and/or the umbilical vessels. The patterns of maternal/fetal inflammation are shown in the et al.49 infants (54%) had abnormal white matter using the classification system described by Woodward p < 0.05 corrected. Skeleton-wide FA was 5.3% lower in preterm infants with HCA compared to infants born without this exposure; p = 0.006 , , inferior longitudinal fasciculi (ILF), left arcuate, anterior and posterior limbs of internal capsule, external capsule and cerebellum ; p < 0.0tively), .Structural MRI data were available for 81 of the 90 participants; nine structural MRI data were not useable due to movement artefact. Whole brain tissue volume increased over the range of gestational age at image acquisition , but thein utero contributes to altered microstructure in major white matter tracts of preterm infants at term equivalent age. The effect was independent of age at image acquisition and known predictors of preterm brain injury and poor neurodevelopmental outcome including degree of prematurity, BPD and postnatal sepsis.By combining histological diagnosis of chorioamnionitis with quantitative brain MRI, we have shown that inflammation Meta-analysis has shown that HCA is a significant predictor of cerebral palsy (CP)26antenatal exposure to systemic inflammation contributes to the prevailing form of preterm brain injury. This is consistent with a previous observation that elevated cytokines and CD45RO+ T lymphocytes in umbilical cord blood are associated with overt cerebral lesions on MRI very soon after birth3940414243The fetal inflammatory response is characterized by increased concentration of pro-inflammatory cytokines in plasma36We used TBSS to survey the white matter skeleton for group-wise differences that might be associated with HCA because of its sensitivity for detecting alterations in FA in the newborn period, and because of its predictive value for later outcome219846Our study has some limitations. Firstly, although 77% of the HCA group had fetal vasculitis, which is indicative of FIRS, we did not analyze cord blood so further study is required to uncover the specific immune mediators that might link HCA with white matter injury, and also to investigate susceptibility in infants without FIRS. Secondly, the study was not powered to investigate the role of multiple placental lesions, which could act synergistically to increase fetal risk. Thirdly, the diagnosis of neonatal sepsis is imprecise; we overcame this by using a rigorous clinical protocol that meets international bench-marking standards (Vermont Oxford Network) but it is possible that some cases were misclassified. In future, integrating quantitative imaging data with information about the host response to infection/inflammation provided by pathway analysis may offer new insights into the timing and drivers of inflammation-induced perinatal brain injury.in utero for a significant proportion of preterm infants exposed to HCA during fetal life. Future research could focus on the development of antenatal tests to identify HCA and fetuses at risk of neuroinflammation; and placental histopathology may have a role in risk stratification for trials of immune-modulatory therapies designed to improve outcome.In conclusion, these results suggest that the pathway to white matter injury originates The study was conducted according to the principles of the Declaration of Helsinki, and ethical approval was obtained from the UK National Research Ethics Service (South East Scotland Research Ethics Committee). Written parental informed consent was obtained for all subjects. The cohort consisted of preterm infants born at <33 completed weeks’ gestational age (GA) who received care at the Royal Infirmary of Edinburgh between July 2012 and May 2015, who underwent brain MRI at term equivalent age and whose placentas were available for histologic examination. Exclusion criteria were infants with congenital infection, chromosomal abnormalities, post-haemorrhagic ventricular dilatation, porencephalic cysts and cystic periventricular leucomalacia.or 2) blood cultures negative or positive for coagulase negative staphylococcus (CoNS) plus generalized signs of infection plus physician decision to treat with antibiotics for 5 days or more. BPD was defined by the requirement for supplemental oxygen at 36 weeks’ GA.Postnatal sepsis was defined by: 1) positive blood culture growing pathogenic bacteria; st reference standards for preterm infantsPostnatal somatic growth was described in terms of change in weight z-score between birth and scan, calculated using Intergrowth-21After delivery, placentae were formalin fixed and stored at 4 °C before sampling. The placentae were sampled according to a standardized protocol; distal and proximal sections of cord , a roll of extraplacental membranes starting at the point of rupture and 4 full thickness sections from each quadrant. All were stained with Haematoxylin and Eosin. Slides were examined by a single experienced placenta pathologist (ME) with no knowledge of the MRI findings. Placental reaction patterns were reported according to site and degree of inflammation, using the structure proposed by Redline and colleagues3 resolution), T2-weighted STIR (~0.9 mm3 resolution), T2-weighted FLAIR (~1 mm3 resolution), and dMRI (11 T2- and 64 diffusion encoding direction (b = 750 s/mm2) single-shot spin-echo echo planar imaging (EPI) volumes with 2 mm isotropic voxels data, TE = 106 ms and TR = 7300 ms) data. Images were reported by a radiologist with experience in neonatal MRI (AGW).MRI was performed on a Siemens Magnetom Verio 3 T system  using a 12-channel matrix phased array head coil. All infants were scanned axially to acquire: 3D T1-weighted MPRAGE volume  were used.et al.et al.et al.Structural MRI scans were scored using the system described by Woodward http://www.ndcn.ox.ac.uk/divisions/fmrib). This included brain extraction, and removal of bulk infant motion and eddy current induced artifacts by registering the diffusion-weighted volumes to the first T2-weighted EPI volume for each subject. Using DTIFIT, FA maps were generated for every subject.dMRI data were preprocessed using FSL tools . All images from the dataset were non-linearly aligned to the 40 weeks’ GA template from a neonatal brain 4D atlasThe brain tissue was separated from non-brain tissue using an atlas-based approachStudent’s t-test or the Mann-Whitney test was used to investigate differences in clinical and demographic variables between infants with (n = 26) and without chorioamnionitis (n = 64), and chi-square test or Fisher’s exact test was used to compare proportions. Statistical analysis was performed using SPSS v21.0 .How to cite this article: Anblagan, D. et al. Association between preterm brain injury and exposure to chorioamnionitis during fetal life. Sci. Rep.6, 37932; doi: 10.1038/srep37932 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


This network approach also permitted us to identify additional residues in the network of the known hotspot residues of different anti-Ebola antibodies that would impact antibody-epitope interactions.Recently, progress has been made in the development of vaccines and monoclonal antibody cocktails that target the Ebola coat glycoprotein (GP). Based on the mutation rates for Ebola virus given its natural sequence evolution, these treatment strategies are likely to impose additional selection pressure to drive acquisition of mutations in GP that escape neutralization. Given the high degree of sequence conservation among GP of Ebola viruses, it would be challenging to determine the propensity of acquiring mutations in response to vaccine or treatment with one or a cocktail of monoclonal antibodies. In this study, we analyzed the mutability of each residue using an approach that captures the structural constraints on mutability based on the extent of its inter-residue interaction network within the three-dimensional structure of the trimeric GP. This analysis showed two distinct clusters of highly networked residues along the GP Filoviridae. Infection with these viruses causes a severe hemorrhagic fever in humans, with mortality rates as high as 90%. Since the first outbreaks of Sudan ebolavirus (SUDV) and Zaire ebolavirus (EBOV) in 1976, additional strains have been identified from subsequent incidents and outbreaks: Tai Forest (TAFV), Bundibugyo (BDBV), and Reston (RESTV), though SUDV and EBOV remain the most prevalentEbola virus (EBOV) is a negative-strand RNA virus that originated in equatorial Africa, and together with the Marburg virus, comprises the viral family 1 and GP2. GP1 contains both the glycan cap and mucin domains, which are heavily glycosylated and important for receptor binding, while GP2 is involved in mediating fusion and viral entry351 and GP2 subunits, and then by cathepsins which remove glycan cap and mucin domain in the endosome of the targeted cell to allow for viral fusion and entry89111213141516The Ebola virus surface glycoprotein (GP) is a homotrimer that plays a key role in viral entry, and it also presents the predominant surface epitopes for neutralization by therapeutic antibodies, vaccines and host immune response. The mature virion surface GP has two subunits, GP−3 substitutions per site per yearThe evolutionary pressure of EBOV has not resulted in significant antigenic drift in terms of sequence variation of GP given the localized outbreaks and long time frame between large outbreaks. As a result, there is a high degree of sequence conservation across reported EBOV GP . However1 and GP2 on the surface of the mature virion. This approach permitted us to capture the structural constraints on the mutability of solvent-exposed surface residues on the GP trimer imposed by the extent of their inter-residue interaction network. Using this approach, we identified two distinct clusters along the interface of GP1-GP2 on the trimeric GP surface that are comprised of highly networked residues that are constrained to mutate. These regions may present new epitope surfaces for potent neutralization by one or more therapeutic antibodies. The network map of GP surface residues also presented new insights on the epitope regions that are targeted by known anti-GP antibodies such as 2G4, 4G7, KZ52, etc. Specifically, we identified additional residues in the network of known hotspots on epitopes that could significantly impact antibody binding. Taken together, our approach provides a rational way to structurally define constraints on mutability of viral surface proteins and also provides new insights into epitope regions on these proteins from a network perspective.Towards this goal, we employed an approach we had developed previouslyTo define constraints on mutability of residues in EBOV GP, we computed the inter-residue interaction network from the available X-ray crystal structures. Briefly, this residue interaction network is a method for numerically describing the interconnectedness of various residues found within a protein crystal structure. Network score attempts to capture not only the directly bonded residue pairs, but those pairs of residues whose connection is mediated by a common bonded residue or series of residues. In this way, the network score describes more than the bond strength of a residue and its neighbors but the full bond environment of a residue. Taking the sum total of the network scores between a given residue and all of those residues within its environment produces a score which can be thought of as a measure of total connectedness for the given residue. These network scores are then normalized to scale of 0–1, where 1 represents maximum connectivity in terms of inter-residue interactions. A low score indicates that a residue is not as connected and hence is structurally less constrained to mutate.1-GP2 interface, and is below the cathepsin-cleavage site (which cleaves just below the glycan cap). These clusters comprise of residues that are highly constrained to mutate either through natural evolution or through selection pressure by therapeutic antibodies designed to target these regions. Though there is limited information on the functional importance of these clusters, both C1 and C2 contain residues that have been described as being part of the viral fusion loop for EBOV GP , we computed the network scores for all residues, with and without including the binding of KZ52 . We obseWe observed that for the each of the mutants tested, few of the mutants experienced a decrease in 13C6 binding greater than 3-fold compared to wild type. We used 13C6 as a control, since it binds an epitope on the GP surface away from the mutated regions. We observed that in addition to the known residue hotspots, 511, 550, 552, 553, and 556, the L547A and N506A mutations had drastic effects on the binding of these antibodies without drastically affecting 13C6 affinity. The L547A mutation primarily affected the binding of 4G7 (>100-fold reduction in affinity) while also having noticeable impacts (>10-fold) on KZ52 and 2G4 binding. Conversely, the N506A mutation primarily affected KZ52 and 2G4 binding (>100-fold) while only slightly affecting 4G7 binding (>3-fold).The outbreak of EBOV is unique (when compared to other pathogens) in that it is highly localized, the mortality rate is very high, and there are no approved therapeutic or vaccine strategies. As a consequence, the sequence evolution of GP is slower owing to limited evolutionary and host selection pressure – although the mutation frequency is similar when compared to other pathogens, such as influenza A virus. Therefore there is a need to look beyond sequence evolution to understand constraints on mutability of amino acids in GP.a priori two distinct clusters C1 and C2 that comprised of residues that are highly constrained to mutate and hence present new targets for neutralization by therapeutic antibodies. In fact, our findings were independently validated by studies that identified antibodies that targeted novel neutralizing epitope regions that significantly overlapped with the C2 cluster11We have presented in this study a new approach to define constraints on amino acid mutability of GP based on their inter-residue interactions in three-dimensional space, which are mapped into a two-dimensional network. Our approach permits overcoming the limitation of understanding amino acid changes based on sequence evolution of EBOV. The strength of our approach is highlighted by our ability to identify 1-GP2 interface, but primarily interfaces with residues that are relatively poorly networked in the GP structure. These antibodies are contrasted to the constrained binding networks of the KZ52, 2G4, 4G7 and #3327 antibodies, the latter of which seems to engage with the highly constrained C2 cluster. Finally we have also expanded the hotspot regions based on the network analysis to include residues in addition to those identified by Davidson et al. Specifically the N506 residue appears to be a hotspot for the binding of KZ52 and 2G4, and the L547 residue appears to be a hotspot for 4G7. Compared to the method used in Davidson et al., we use a soluble expression of GPΔTMΔmuc, a construct missing the transmembrane and mucin domains, which could have affected antibody binding. Additionally, in Davidson et al., the authors used a flow cytometry approach and did not list data for the additional residues we identified. Given the reduced expression of some of these mutants, it is possible that they were discarded from their analysis.In addition to identifying highly networked clusters, our approach also provided new insights into the known epitope regions of existing antibodies. The antibodies that bound the glycan cap region of GP  had low network scores and that the residues within the networks were not highly interconnected. Given our observations relating residue network score to filovirus sequence conservation , it is pTaken together, our analyses here have demonstrated that an inter-residue network approach is a useful methodology to gain insights about viral antigens from x-ray crystal structure data, without needing extensive evolutionary history or prior knowledge of antibody epitopes.A pcDNA 3.3 expression vector containing a sequence encoding for GPΔTMΔmuc (missing transmembrane and mucin domain) was transiently transfected into HEK 293 F cells. After 6 days, supernatant was harvested and purified using a 1 mL HisTrap HP column on an AKTA FPLC . Fractions were collected and analyzed using Native PAGE. Fractions containing trimeric GP species were combined and buffer exchanged into PBS using Amicon Ultra Centrifugation Filters (Millipore). Protein concentration was determined using a BCA assay (Pierce) and GP was assessed again using Native PAGE to confirm purity.Similarly to the method for expressing GP, pcDNA 3.3 expression vectors encoding for anti-GP mAbs c13C6, c2G4 or c4G7 were transiently transfected into HEK 293 F cells. After 6 days, supernatant was harvested and purified using a 1 mL HiTrap Protein A column using an AKTA FPLC . Fractions containing antibody were collected and buffer exchanged into PBS. Protein concentration was determined using BCA assay.GPΔTMΔmuc point mutants were created using site-directed mutagenesis. Mutagenesis primers were designed corresponding to the mutant sequence, and a PCR amplification reaction was carried out using a QuikChange Mutagenesis Kit (Agilent). PCR reactions were then digested using Dpn1 for 3 hours, transformed into One Shot TOP10 Chemically Competent cells (Thermo) and then plated onto LB agar plates containing ampicillin. Plasmid DNA was generated by growing colonies in LB broth containing ampicillin overnight and then by purifying using plasmid DNA preparation kits (Invitrogen). Positive colonies were confirmed using Sanger sequencing (Genewiz).2SO4. Plates were read using a Spectramax S5e plate reader at 450 nm.WT GPΔTMΔmuc or mutants were plated onto clear 96-well Maxisorp plates (Nunc) at a concentration of 1 μg/mL and left at 4 °C overnight. Plates were then washed with PBST (PBS with 0.05% tween) and incubated with 100 μL of 1% BSA in PBST for 1 hour at room temperature. Plates were then washed with PBST and incubated with 100 μL of anti-Ebola mAbs  at concentrations following a 3-fold dilution scheme for 2 hours. Plates were washed again with PBST and 100 μL of secondary antibody, Rabbit anti-Human IgG at a 1:5000 dilution for 1 hour. Finally, plates were washed, and incubated with 100 μL of TMB Substrate (KPL) for 5 minutes and quenched with 100 μL of 1 N Hhttps://www.ncbi.nlm.nih.gov/genome/viruses/variation/). For the EBOV GP conservation analysis, analyzed sequences included human Zaire ebolavirus GP that were collected prior to August 2015, for a total of 955 unique protein sequences. For filovirus conservation analysis, representative protein sequences of filovirus coat glycoproteins were chosen: Zaire ebolavirus (genbank: AAB81004.1), Sudan ebolavirus (genbank: AGL73446.1), Reston ebolavirus (genbank: BAB69006.1), Tai Forest ebolavirus (genbank: ALT19763.1), Bundibugyo ebolavirus (genbank: AGL73474.1) and Marburg marburgvirus (genbank: ACT79243.1). Downloaded sequences were then aligned using MAFFT (www.ebi.ac.uk/Tools/msa/mafft/). Conservation percentages were obtained by importing the alignment into Jalview, and percentages were then mapped to the surface of EBOV GP (PDB: 3CSY) in PyMol for visualization. Coloring cutoffs were chosen from either 95–100% or 70–100% to highlight the most relevant surface trends.To analyze Ebola virus protein sequence conservation, GP sequences were downloaded from the NCBI Virus Variation database (http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html) to determine residues located at the various intra-domain interfaces (between GP1 and GP2) and epitope-paratope interfaces. Further, the solvent accessibility of the residues was calculated using DSSP server (http://www.cmbi.ru.nl/dssp.html). For each residue the inter-residue interactions were calculated using a custom MATLAB script that incorporates putative hydrogen bonds (including water-bridged ones), disulfide bonds, pi-bonds, polar interactions, salt bridges, and Van der Waals interactions (non-hydrogen) occurring between pairs of residues within a threshold distance and has been described previouslyi, j is the sum of the path scores of all paths between residues i and j. The degree of networking score for each residue was computed by summing across the rows of the matrix, which was meant to correspond to the extent of “networking” for each residue. This interactional relationship is represented using a two-dimensional network diagram. The degree of networking score was normalized with the maximum score for each protein so that the scores varied from 0 (absence of any network) to 1 (most networked). This entire process is depicted graphically in The coordinates of EBOV GP trimer-antibody co-crystal structures  were uploaded into the PDBePISA server .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Cancer development and progression are characterized by complex molecular events. The acquisition of these events is primarily believed to result from alterations in gene and protein expression/function. Recent studies have also suggested the role of metabolic alterations, or “metabolic reprogramming,” that may similarly contribute to these events. Indeed, our previous investigations in osteosarcoma (OS) identified metabolic changes uniquely linked to metastasis. Based on those findings, here we sought to build a more detailed understanding of the specific alterations in metabolites or metabolic pathways that may be responsible for the observed metastasis-associated metabolic alterations, suggested by gene expression data. This was pursued using a combination of high-throughput liquid- and gas-chromatography-based mass spectrometry (LC/MS and GC/MS) for a global metabolic profiling/subtraction of four pairs of high/low metastatic OS cell lines. By comparing the identity and level of the metabolites between high/low metastatic cells, several metabolic pathways were identified to be differentially activated, such as arginine, glutathione, inositol and fatty acid metabolic pathways. To further interrogate these results, we investigated the effects of inositol pathway dysregulation, through the exposure of metastatic OS cells to IP6 (inositol hexaphosphate). Although IP6 exposures had modest to minimal effects on cell proliferation, we observed reduced cellular glycolysis, down-regulation of PI3K/Akt signaling and suppression of OS metastatic progression. Collectively these data supported further investigation of metabolic sensitivities as anti-metastatic strategies in a clinical setting as well as investigation of altered metabolomics associated with metastatic progression. Osteosarcoma is a malignant bone tumor that often develops during the period of rapid growth associated with adolescence . Treatmein vitro and have highly similar in vivo characteristics of primary tumor development when grown in mice; however, these cells are fully distinguished based on metastatic behavior, ex vivo and in mouse models of metastasis. Collectively, these findings now suggest the hypothesis that the metastatic behavior of OS cells is in part the result of metabolic alterations.There has been recent and significant interest in defining metabolic alterations that explain the development of cancer, and metabolic differences between normal cells and cancer cells. Distinct from these studies on cancer phenotype and carcinogenesis, we previously identified changes in gene expression that suggested that alterations in metabolic pathways may be linked to metastasis . In thisIn the present study, we have begun to define the cellular metabolic profiles of highly metastatic OS cell lines  compared to their clonally related, low metastatic parental cell lines . Our current studies were conducted to address the hypothesis that specific alterations in metabolites, or their associated pathways, are present between high and low metastatic cells and that these metabolites/pathways may be causally linked to the metastatic proclivity of the highly metastatic cells. Our findings indicate that arginine metabolism, glutathione metabolism, fatty acid and the inositol metabolic pathways were most consistently altered in highly metastatic OS cells compared to the parental control cells. In this report, we present our studies on the inositol pathway . Our results demonstrated that dysregulation of the inositol pathway through inositol hexaphosphate (IP6) exposure dramatically inhibits the metastatic phenotype, with only minimal effects on cell survival and growth. It is critical to emphasize that IP6 has minimal effects on cell survival and growth, but that these IP6 exposures have dramatic and much more exaggerated effects on metastatic progression, collectively suggesting that the effects on cell growth and survival alone do not fully explain the observed anti-metastatic effects.IP6 is present in almost all plant and mammalian cells and is widely recognized as a natural antioxidant . The antIn our studies, the addition of IP6 to OS models reduced their glucose metabolism (ECAR), and suppressed tumor metastasis in mouse xenograft models. These anti-metastatic effects were observed without significant effects on cancer cell growth/proliferation and with no apparent impact on normal cell or organ function in mice.Collectively our data indicate that dysregulation of the inositol metabolic pathway disrupts the metabolic advantage of the highly metastatic cells and likely increases their sensitivity to apoptosis and growth inhibition which is disproportionately observed in the setting of metastasis and its associated stress on cells .t-test was used for the mouse dataset. A summary of the numbers of biochemicals that achieved statistical significance (p≤0.05), as well as those approaching significance (0.05<p<0.10), are shown in Figure Global metabolomics profiling was conducted using a combination of high-throughput LC- and GC-based MS on a total of 4 pairs (three human and one mouse) of clonally related high/low metastatic OS cell lines  15]. Fr. Fr15]. One of the main goals of this analysis was to identify the most consistent alterations in metabolic pathways that were conserved across both panels of high versus low metastatic OS cell line pairs in a metastasis-specific context, but also across species (mouse and human). In highly metastatic human OS cells, arginine metabolism, glutathione metabolism, membrane remodeling, and the inositol pathway were significantly altered compared to low metastatic control cells. A similar trend was also observed in the mouse OS cell lines analyzed. In contrast, nucleotide catabolism differed between species. Furthermore, cell line-specific changes were also observed between the biochemical signatures of the human OS cell lines .myo-inositol 1-phosphate and myo-inositol) was significantly decreased in human and mouse highly metastatic OS cells compared to their clonally related low metastatic  counterparts. Additionally, a reduction in scyllo-inositol was also observed (except for HOS-MNNG cells). Thus, alterations in inositol metabolism were commonly observed in our comparison of high and low metastatic cells, and prompted us to focus on this pathway first and examine whether alterations in this pathway may contribute to OS metastasis. We nonetheless have identified other metabolomic candidates and candidate pathways that will also be examined for causal associations with metastasis.Among these pathways, the inositol pathway was significantly altered across all the high/low metastatic pairs of OS lines. As shown in Figure myo-inositol or IP6) can inhibit cancer cell growth, at specific exposures [Inositols are an essential structural component for the generation of a number of secondary messengers including inositol phosphates, phosphatidylinositol (PI) and phosphatidylinositol phosphate (PIP). The reduction of inositol levels in various malignancies such as lung cancer and cerebral astrocytomas  has beenxposures .To assess the impact of IP6 exposure on OS cell proliferation, we first performed SRB proliferation assays on two highly metastatic OS cell lines, MG63.3 (human) and K7M2 (mouse). As shown in the dose-response curve in Figure ex vivo lung culture assay (Pulmonary Metastatic Assay or PuMA) revealed a unique and disproportionate effect of IP6 pathway dysregulation on the metastatic phenotype compared to our observations of the effects on simple cell growth (above). Fluorescent micro-metastases in the cultured lung slides were enumerated and analyzed quantitatively [ex vivo fluorescence whole mount images taken from the PuMA at study endpoint are shown in Figure Given the modest effects of IP6 exposure on cell growth, we next asked if similar exposures of IP6 would have a greater effect on the metastatic phenotype of OS cells in lung tissue. The tatively . As showin vitro at 0.1 mM . As predicted, and consistent with our hypothesis, the OS primary tumor growth rate was not changed with IP6 treatment  and mitochondrial oxygen consumption rate (OCR) were measured with a Seahorse Bioscience XFTo further identify the cell-signaling pathways that were altered by dysregulating inositol metabolism, kinase array assays were performed. Cell lysates from MG63.3/GFP cells treated with 5 mM IP6 or vehicle were used in the assays. Significantly reduced phosphorylation of kinases in both PI3K and MAPK signaling pathways  was observed with IP6 supplementation Figure . The effThe study of cancer cell metabolism has garnered recent attention, both as a means to understand carcinogenesis and to develop therapeutic approaches, for the most important remaining area of unmet need in the field , 27. In In cancer cells, metabolism is dramatically reprogrammed to support accelerated cell proliferation and adaption to the tumor microenvironment . The oppIn this study, we have identified numerous metabolic differences between human OS low metastatic cell lines and their highly metastatic clonal relatives. In highly metastatic cells, arginine metabolism, glutathione metabolism, lipid metabolism and the inositol pathway were significantly altered compared to parental control cells. A similar trend was also observed in the high/low metastatic mouse OS model comparisons. Given the biological complexity of metastasis and our heterogeneous set of experimental samples taken from high and low metastatic OS, it is not surprising that a simple bioinformatics and statistical approach would not be able to give us a clear method for prioritizing one pathway or alteration over another. We are currently investigating the glutathione metabolism , lipid mInositols are essential structural components for the generation of a number of secondary messengers including inositol phosphates, phosphatidylinositol (PI) and phosphatidylinositol phosphate (PIP). The inositol phospholipids in the plasma membrane have received much attention in cancer research because of their biological significance for the signal transduction system. Several reports have shown decreased inositol pathway metabolites with tumor progression , 31. Conmyo-inositol, to alter the inositol metabolic pathway of highly metastatic OS cells. As previously reported, exogenously administered IP6 is rapidly taken up into cells and dephosphorylated to inositol and lower inositol phosphates (IP1-5) in most mammalian cell systems. The rate and pattern at which IP6 is metabolized by cancer cells varies depending on the cell type [in vitro or within the primary tumors, but IP6 treatment suppressed the metastatic phenotype of highly metastatic OS cell ex vivo and in vivo. The ex vivo study showed significant reduction of OS cell colonization in lung tissue with IP6 treatment. Furthermore, IP6 also inhibited OS cell lung metastasis in a mouse model of spontaneous OS metastasis. It did so at exposures that had limited effects on cell viability in vitro in either high or low metastatic cells, suggesting that these inhibitory effects on metastasis could not be simply the result of cell growth inhibition or cell survival, and that IP6 exposure is reasonably affecting metastatic progression in other ways, potentially linked to the adjustments to metabolic reprogramming, demonstrated by our functional metabolomics studies.We used inositol hexaphosphate (IP6), a primary naturally occurring source of ell type . In our These effects on metastatic progression may also be consistent with our hypothesis that cell stress adaptation is a critical feature of the metastatic phenotype, and provides a rationale to consider the role of metabolic reprogramming as an adaptation to the presumed stress faced by cells involved in highly energy consuming and dynamic events (such as metastasis). Our data may suggest that highly metastatic cells are capable of meeting these metabolic needs, and that disruption of the adaptive metabolic machinery leads to a profound effect on metastasis, whereas a similar disruption outside the context of metastasis (and in the absence of cell stress) is less significant. Collectively, this may explain the apparent effect of inositol pathway dysregulation only in the setting of metastasis.ex vivo and in vivo. The correction of the dysregulated inositol pathway, through IP6 supplementation, suppressed metastatic progression. Although an alternative approach to augment the inositol pathway in low metastatic cells may be interesting, the outcome would not influence the preclinical data and translational imperative of studying IP6 to safely alter the metastatic phenotype of this highly metastatic cancer.Cancer metastasis is a multi-step process, as such many distinct cellular processes are involved and are necessary for successful metastasis. A common approach taken to study single step biological phenotypes, is to ask if a process is “necessary” or “sufficient” for the given biological phenotype. In the setting of the multi-step process of metastasis this approach to defining a single process as being “necessary” or “sufficient” is less relevant and less informative. However, as this is common practice, we have asked the question of “necessary” as we have examined the causal association of IP6 on metastasis. We have not emphasized, or viewed as informative the “sufficient” question, which would involve the correction of the IP6 defect in the low metastatic cells with the goal of conversion to a highly metastatic phenotype. In our studies of altering the metastatic phenotype of highly metastatic cells, we observed consistent results across species The molecular mechanisms of the IP6 mediated antitumor activity have not been fully specified. Anti-oxidative properties , inhibitIn conclusion, this report is the first investigation of the causal link between OS cancer cell metabolic changes and their metastatic potential. Differential metabolites involved in arginine metabolism, glutathione metabolism, lipid metabolism and the inositol pathway were identified in highly metastatic OS cells compared to their low metastatic parental cells. To evaluate our metabolic profiling, IP6 (inositol hexaphosphates) supplementation was used to alter inositol metabolism. We propose that IP6 treatment may have increased the sensitivity of highly metastatic OS cells to cell death, in the context of metastasis and limited their ability to adapt to and resist cell stresses known to exist during metastatic progression and in-so-doing suppressed metastatic progression in a mouse spontaneous metastasis model. The findings of this study contribute to our knowledge of cancer metastasis and may lead to the development of potential anti-metastatic cancer therapy.in vivo selection using an experimental metastasis assay [7 cells were collected from each cell line and frozen at -80 for metabolic profiling. Quadruplicate samples were collected for each cell line. A recent characterization of these cell lines both in vitro and in vivo has shown that although these cell lines are fully distinct in terms of metastatic proclivity, they share and are fundamentally indistinguishable from the perspective of cell growth/proliferation [Murine OS cell lines K12 and K7M2 were developed in our lab and described previously . Human His assay . All theferation .The sample preparation process was carried out using the automated MicroLab STAR® system from Hamilton Company. Recovery standards were added prior to the first step in the extraction process for QC purposes. Sample preparation was conducted using a proprietary series of organic and aqueous extractions to remove the protein fraction while allowing maximum recovery of small molecules . The extracted samples were split into equal parts for analysis on the GC/MS and LC/MS/MS platforms. Also included were several technical replicate samples created from a homogeneous pool containing a small amount of all study samples (“Client Matrix”).The LC/MS portion of the platform was based on a Waters ACQUITY UPLC and a Thermo-Finnigan LTQ mass spectrometer, which consisted of an electrospray ionization (ESI) source and linear ion-trap (LIT) mass analyzer. The sample extract was split into two aliquots, dried, then reconstituted in acidic or basic LC-compatible solvents, each of which contained 11 or more injection standards at fixed concentrations. One aliquot was analyzed using acidic positive ion optimized conditions and the other using basic negative ion optimized conditions in two independent injections using separate dedicated columns. Extracts reconstituted in acidic conditions were gradient eluted using water and methanol both containing 0.1% Formic acid, while the basic extracts, which also used water/methanol, contained 6.5mM Ammonium Bicarbonate.The samples destined for GC/MS analysis were re-dried under vacuum desiccation for a minimum of 24 hours prior to being derivatized under dried nitrogen using bistrimethyl-silyl-triflour- oacetamide (BSTFA). The GC column was 5% phenyl and the temperature ramp was from 40u to 300uC in a 16-minute period. Samples were analyzed on a Thermo-Finnigan Trace DSQ fast- scanning single-quadrupole mass spectrometer using electron impact ionization.The informatics system consisted of four major components, the Laboratory Information Management System (LIMS), the data extraction and peak-identification software, data processing tools for QC and compound identification, and a collection of information interpretation and visualization tools for use by data analysts. The hardware and software foundations for these informatics components were the LAN backbone, and a database server running Oracle 10.2.0.1 Enterprise Edition.Compounds were identified by comparison to library entries of purified standards or recurrent unknown entities. Identification of known chemical entities was based on comparison to metabolomic library entries of purified standards. The combination of chromatographic properties and mass spectra gave an indication of a match to the specific compound or an isobaric entity.Cells were plated in a 96-well plate at a density of 5000 cells per well in 100μl of complete media and incubated overnight. Cells were then treated with vehicle or IP6 as indicated and processed after 48 hours as previously described .Cell proliferation assay was performed with Cell Counting Kit-8 (CCK-8) from Dojindo Molecular Technologies, Inc. . K7M2 or MG63.3 cells were seeded in a 96-well tissue culture plate at 2000 cells per well and treated with 0-5 mM IP6 for 1-4 days. At each time point, the CCK-8 reagent (10 μl) was added to each well and incubated at 37°C for 4 hours. The absorbance at 450 nm was measured using the microplate reader . Each data point represents the mean reading from sextuplicate analyses.Caspase 3/7 activity was measured with Caspase-Glo 3/7 Assay kit from Promega Corporation . MG63.3 cells were seeded in a white 96-well tissue culture plate at 5000 cells per well and treated with 0-10 mM IP6 for 2 or 6 hours. The caspase3/7 reagent was added to each well and the chemiluminescent signal was measured with a Victor 3 plate reader  after 30 minute incubation.5) or MG63.3 (105) were delivered by tail-vein injection to Balb/c or SCID/Beige  mice. Within 15 minutes of tumor injection, the mice were euthanized by CO2 inhalation. A well-mixed medium/agarose solution (40°C) was introduced by endotracheal insufflation. Following cooling at 4°C, complete transverse sections (1-2 mm in thickness) were made from each lobe, yielding 16-20 lung sections. Then 4-6 lung sections were placed in a well of a 6-well plate. Lung sections were incubated at 37°C in humidified conditions of 5% CO2. IP6 was added to the culture media on day 0 of the culture period and the IP6-containing medium was changed every day. A LEICA-DM IRB fluorescent inverted microscope  and Retiga-EXi Fast 1394 Mono Cooled CCD camera  were used to capture images of GFP-positive tumor cells within the lung sections at 25 X magnification. Fluorescent events were acquired using OpenLab software . Metastatic burden was quantified by measuring the fluorescent area of metastatic cells in each lung section at each time point and was expressed as mean percentage of tumor burden vs. whole lung section. The lung sections were processed for histology evaluation after the fluorescent imaging.Pulmonary Metastasis Assay (PuMA) was performed as previously described . BrieflyscidLyst bgPrkdc/Crl and Balb/c mouse strains were used for these experiments. Primary tumor growth was examined by orthotopic injection of K7M2 2×106 cells/ 0.1 ml of HBSS into a paraosseous site deep to the left caudal gastrocnemius of 4-week-old female Balb/C mice as described previously [2)/6 × 3.12 (where D = the maximum diameter and d = the minimum diameter) as previously reported [3 were surgically removed and examined histologically. Following surgery, mice were observed for spontaneous lung metastases using PAAM [Animal studies were performed in accordance with the guidelines of the NIH Animal Care and Use Committee. Fox Chase SCID-Beige mouse CB17.B6-eviously –20. Oncereported . Tumor-bing PAAM  method. 7 IP6 treated and vehicle treated cells. Total protein of 200 μg was used for the immunoblotting with human phospho-kinase array. The experiment was performed following the manufacturer's instructions . Image J was used to quantify the intensity of the Western blot signals.MG63.3 cells were treated with 5 mM IP6 for 6 hours. Cell lysates were collected from 10Western blot analysis was performed as described previously . MG63.3 e96 Extracellular Flux Analyzer. Cells were plated at 10,000 cells/well in XF96 cell culture microplates . Following attachment, cells were treated with IP6 or vehicle control and incubated overnight at 37°C. Just prior to performance of the Seahorse assay, growth media was replaced with 180 μL of Seahorse XF Base Media (Seahorse Bioscience) supplemented with glucose, glutamine, and sodium pyruvate, and the plate was incubated in a 37°C incubator lacking CO2 for 45 to 60 minutes. OCR and ECAR were determined by performing the Cell Mito Stress Test (Seahorse Bioscience) according to the manufacturer's specifications, as previously described [Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured using a Seahorse Bioscience XFescribed .


Cannabis sativa L.) is increasing due to the development of a new range of industrial applications based on bast fibers. However the variability of bast fiber yield and quality represents an important barrier to further exploitation. Primary and secondary fiber content was examined in two commercial hemp varieties  grown under contrasted sowing density and irrigation conditions. Both growing conditions and hemp varieties impact stem tissue architecture with a large effect on the proportion of secondary fibers but not primary fibers. Attenuated total reflectance infrared spectroscopy allowed the discrimination of manually-isolated native primary fibers and secondary fibers but did not reveal any clustering according to growing conditions and variety. Infrared data were confirmed by wet chemistry analyses that revealed slight but significant differences between primary and secondary fiber cell wall composition. Infrared spectroscopy of technical fibers obtained after mechanical defibering revealed differences with native primary, but not secondary fibers and also discriminated samples obtained from plants grown under different conditions. Altogether the results suggested that the observed variability of hemp technical fibers could be partially explained by i) differences in secondary fiber production and ii) differential behavior during mechanical defibering resulting in unequal separation of primary and secondary fibers.Interest in hemp ( Hemp fibers have been traditionally used for centuries in the textile and paper industries . Over thHemp fibers are located in the outer stem tissues of the plant and are therefore referred to as bast fibers. Hemp bast fibers are in fact composed of two fiber types with different origins, primary bast fibers are sclerenchyma cells derived from the procambium whereas secondary bast fibers are produced by the vascular cambium . These tin planta, bast fiber quality and yield can be greatly modified by biological and/or physicochemical processing  [In addition to the fiber variability  fibers) , 18–21.  fibers) , 23 and  fibers) –26. Anot fibers) . The dev fibers) .The aim of this work was to evaluate the main sources of variability in fibers obtained from two hemp cultivars grown under different growing conditions using attenuated total reflectance—based infrared spectroscopy as a rapid and non-destructive tool.Cannabis sativa L.) were grown at Marigny-le-Châtel, France  by professional hemp producers. Field trials were conducted by the French Hemp Technical Institute ‘Terres Inovia’ (http://www.terresinovia.fr) that possesses all legal authorization and expertise necessary for hemp field studies. Two monoecïous fiber varieties (Fedora 17: F17 and Santhica 27: S27) were sown early April and cultivated under 3 different growing conditions on a chalky soil: Condition 1 (standard conditions): sowing density = 50 kg.ha-1, no irrigation; Condition 2 (irrigation): sowing density = 50 kg.ha-1 and irrigation corresponding to 50% additional water supply calculated on the base of average rainfall in this region  and provided every 10 days during 3 months before flowering stage; Condition 3 (100 kg.ha-1): sowing density = 100 kg.ha-1, no irrigation. For all conditions, 120 U N.ha-1 ammonium nitrate was added under solid form at crop planting (one unit nitrogen correspond to 1 kg nitrogen provided by ammonium nitrate). Average rainfall was 319 mm for the cultivation year. Plants were harvested at the end of the flowering stage .Hemp plants  were isolated from 4 individual plants for each growing condition and variety. Small discs (2.5 cm thick) were then removed and rapidly frozen in liquid nitrogen and stored at– 20°C. Frozen samples were gently thawed in deionized water at 4°C before hand-cutting thick transverse sections. The average thicknesses of bark (outer part of the stem) and wood  obtained were calculated from measurements made on thick transverse sections examined under a stereomicroscope . The surface areas of primary and secondary fibers were determined using UV fluorescence microscopy . Square images (715 X 715 μm) per section (4 samples per growing condition) were extracted and images analyzed with image-processing software .Fibers were manually isolated from the basal stem region (between 15 and 45 cm above ground level) of individual plants. The bark of three plants per growing condition was separated from the inner core and fiber bundles were then peeled out and the epidermis discarded as previously described . The sec-1 at a resolution of 4 cm-1, and corrected for background absorbance by subtraction of the spectrum of the empty ATR crystal. FTIR spectra were baseline corrected and vector-normalized in the range of 1850–675 cm-1.Manually-isolated and technical fibers were then characterized without grinding by infrared spectroscopy. Spectra were recorded by attenuated total reflectance (ATR) using a Nicolet 6700 spectrometer (ThermoScientific) equipped with a SmartPerformer ATR accessory. Measurements were made in triplicate on 3 samples per growing condition for each variety Spectra average were obtained from 64 scans recorded from 4,000 to 400 cm2SO4 for 2 h at 100°C. The released monosaccharides were separated by high performance anion-exchange chromatography  and analyses performed in triplicate as previously described [Fiber bundles were ground with a ball crusher  and extractives were removed using 80% ethanol at 40°C , then dried. Complete acid hydrolysis of cell wall polysaccharides into sugar monomers was performed using 1 M Hescribed . Lignin escribed .Statistical analysis was performed with Statgraphics software. Equality of group variances was checked with a Barlett Test. The normality assumption was assessed by Kolmogrov Smirnov Test. Differences between treatments were evaluated using Analysis of Variance (ANOVA) and the HSD Tukey test used to separate averages at p < 0.005. When the normal curve of distribution was not verified or the number of samples was reduced, the non-parametric Kruskal Wallis test was applied. Simple regression analysis was used to show the correlation between certain variables. Multivariate statistical analysis of infrared spectra was performed by principal component analysis using the Unscrambler software .Stem morphological features including bark and wood thickness and bast fiber distribution were determined by microscopy observations of hemp stem transverse sections. Examination of basal stem regions indicates significant variation in the thicknesses of stem outer tissues (designated as bark) and stem inner tissues  in transverse cross-sections . For botExamination of the sections by UV fluorescence microscopy was then used to observe fiber distribution. UV autofluorescence which indicates the presence of lignin is essentially restricted to the middle lamella-primary cell wall region in both primary and secondary bast fibers, whereas autofluorescence is also detected in the secondary cell walls of xylem tissue , 9, 32 . Microsc2) of primary fibers  structure of esterified pectin [Our results showed that the amount of secondary fibers produced depends upon both the genotype and the growing conditions thereby affecting the overall quality of the harvested material. To confirm that ATR-FTIR spectroscopy could be used as a potential tool for rapidly characterizing the proportions of primary and secondary fibers in hemp plants we firstly recorded spectra on manually-dissected primary and secondary fibers from both varieties grown under different conditions . Since tsorption , 35–37. d pectin . OverallWhen analyzed separately, no clear clustering of the spectra recorded on primary  and seco-1  showed that spectra were separated along PC1 . This co samples . These b samples .-1 in the PC loading of ATR FTIR spectra of technical fibers obtained from plants grown under additional irrigation conditions would suggest that few/no secondary fibers are present. These results are in clear contradiction with our microscopic observations showing that greater amounts of secondary fibers are present in plants grown under additional irrigation conditions as compared to plants grown under high density conditions  amounts of secondary fibers . The resVariability in technical fiber quality is generally considered as one of the major disadvantages of natural (plant) fibers and represents an important obstacle to their widespread use in composite materials. A rapid and reliable method of evaluating technical fiber quality would therefore represent a valuable help to industry. In this paper we have shown that ATR FTIR spectroscopy represents an efficient and powerful technique for evaluating variability in technical fibers obtained from different sources. Both growing conditions and genetic differences (variety effect) impact on hemp stem tissue architecture with a major effect on i) secondary (but not primary) fiber production and ii) primary and secondary fiber separation during mechanical defibering. Our results also indicate that a major source of variability in technical fibers is related to the presence of secondary fibers. This information represents an important contribution to our understanding of the biological reasons underlining the variability of natural fibers and should lead to the improvement of production and defibering protocols necessary for a better control of fiber quality. The use of ATR infrared analysis also provides chemical information about the surfaces of technical fibers. Such information is of great importance for a better understanding of bast fiber cohesion in agrocomposites and is complementary to information about the mechanical properties of technical fibers.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.