Primary Lines Research Articles

BI-27 * ANALYSIS OF THE BIOCHEMICAL PROFILE OF LOW GRADE GLIOMA WITH DIFFERENT IDH1 MUTATION STATUS USING VIBRATIONAL SPECTROSCOPY

Mutations in human cytosolic isocitrate dehydrogenases 1 (IDH1) are a common feature of low grade gliomas (LGG) and cause profound changes of the metabolites of the Krebs cycle. Vibrational spectroscopy comprises label-free techniques that probe a sample's molecular composition. Therefore, we investigated the ability of fourier-transform infrared (FT-IR) spectroscopy to analyze the IDH1-mutation status in cell culture systems and human brain tumor samples. Cell lines (U87-MG, SVG p12 and the primary glioblastoma cell lines HT7606, HT12346 and HT12347) were transduced with either IDH1 wild-type or mutated IDH1. Clusteranalysis and principal-component-analysis were able to detect differences in the respective FT-IR spectral datasets in regions assigned to saccharides (1050 and 1120 cm−1) and proteins (1236, 1545 and 1651 cm−1). Brain tumor samples of human LGG were obtained during routine surgery and the IDH1 mutation status was determined by DNA sequencing (n = 17). Difference spectra (IDH1-mut vs. IDH1-wt) showed changes in the region around 1100 cm−1 which is attributed to saccharides. Additionally, amide I and amide II spectral bands (around 1550 and 1650 cm−1, respectively) were reduced in the IDH1-mut dataset whereas the band at 1740 cm−1 assigned to C = O stretching vibrations was more pronounced. Principal-component-analysis confirmed differences between the two groups based on spectral regions assigned to saccharides, proteins and to C = O stretching vibrations, as identified previously in the difference spectra. Supervised classification recognized relevant spectral regions at 974, 1005, 1038-1044, 1078-1082, 1267-1275, 1342, 1417-1421, 1479 cm−1 and was able to assign 16 of the 17 tumor samples to the correct group. On the basis of FT-IR spectroscopy samples of LGG carrying IDH1 mutations can be discerned from IDH1 wildtype tumors. Relevant spectral regions assigned to saccharides and C = O stretching vibrations can be explained by changes in the Krebs cycle and in particular by the accumulation of 2-hydroxyglutarate (rich in C = O bonds).

QTL Detection and Epistasis Analysis for Heading Date Using Single Segment Substitution Lines in Rice (Oryza sativa L.)

Heading date of rice is a key agronomic trait determining cultivated areas and seasons and affecting yield. In the present study, five primary single segment substitution lines with the same genetic background were used to detect quantitative trait loci (QTLs) for heading date in rice. Two QTLs, qHD3 and qHD6 on the short arm of chromosome 3 and the short arm of chromosome 6, respectively, were identified under natural long-day (NLD). Nineteen secondary single segment substitution lines (SSSLs) and seven double segments pyramiding lines were designed to map the two QTLs and to evaluate their epistatic interaction between them. By overlapping mapping, qHD3 was mapped in a 791-kb interval between SSR markers RM3894 and RM569 and qHD6 in a 1125-kb interval between RM587 and RM225. Results revealed the existence of epistatic interaction between qHD3 and qHD6 under natural long-day (NLD). It was also found that qHD3 and qHD6 had significant effects on plant height and yield traits, indicating that both of the QTLs have pleiotropic effects.

PM-20 * COMPARATIVE PROTEOME PROFILING OF IN VIVO AND IN VITRO MODELS OF DIPG

BACKGROUND: Diffuse intrinsic pontine glioma is one of the least understood pediatric tumors. We have recently identified two molecular subtypes of DIPGs (sonic hedgehog and N-Myc). Despite the existence of in vitro and in vivo models of the disease, molecular characterization of these models and their proximity to representing the disease has not been established. Here, we have done comparative protein profiling of a number of DIPG primary cells as well as protein profiles of a single specimen and specimens derived from its' in vivo and in vitro models. METHODS: Human primary DIPG (n = 4) and normal human cells derived from hindbrain (n = 2) were processed for protein profiling. Total protein was extracted and analyzed using SDS PAGE, and analyzed using Q Exactive mass spectrometer (ThermoFisher, CA, USA). Data and pathway analysis were done using the SequestHT and Ingeniuty Pathway Analysis software respectively. Protein was also extracted from a single human DIPG specimen, and its' in vitro and in vivo derived cells. Protein profiling of this specimens was performed as described above. RESULTS: In this study, we first investigated the protein profiles of four widely used DIPG primary lines, all H3.3K27M mutant. We show the overlap of the protein profile of these cells with established human DIPG subtypes. We further investigated the protein profile of a single DIPG tissue to its in vitro and in vivo models. We describe the fidelity of each model with respect to the molecular entity of the original tumor. The largest number of proteins were shared between primary tumor and murine model (846 proteins, 44%), where the primary tumor and cultured cells shared 30% (721) proteins. CONCLUSIONS: Established human DIPG cells represent tumor biology at the protein level. However, differences in molecular pathway of in vitro and in vivo models representing the disease exist.

Abstract 1379: Identification of global DNA methylation signatures in glioblastoma-derived cancer stem cells

Abstract Glioblastoma (GBM) is the most common and most aggressive brain tumor in adults. Its frequent recurrence after resection and dismal prognosis are thought to be due to a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy. In this study, we performed an in depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary GBM tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified two groups hyper- and hypomethylated of genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also be found in primary GBM-derived xenograft tumors suggesting that they were not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down regulated in GBMs such as SPINT2, NEFM, and PENK. The comparison between the NSC line and the normal brain tissue sample leads to the identification of a group of NSC-specific differentially methylated regions (nsDMRs). Cluster analysis using nsDMRs revealed the presence of stem cell-specific DNA methylation signatures in both primary GBM and GSC lines. Higher expression of genes associated with stem cell-specific DNA methylation signatures such as DNMT3A, STAT5A, CSTB, PMEPA1 and G6PD in GBM patients was found to be associated with poor patient survival. The results from this study demonstrate the utility of using cancer stem cell models for advancing understanding of the pathobiology of gliomas. Citation Format: Eun Joon Lee, Prakash Rath, Jimei Liu, Dungsheng Ryu, Alan Free, Lirong Pei, Douglas C Anthony, Suash Sharma, Mark D Kirk, John J. Laterra, Duck Hwan Ryu, Jeong-Hyeon Choi, Huidong Shi, Douglas C. Miller, N. Scott Litofsky, Qi Feng. Identification of global DNA methylation signatures in glioblastoma-derived cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1379. doi:10.1158/1538-7445.AM2014-1379

Abstract 4483: IO125, a novel Pt-based supramolecular therapeutic exhibits increased anti-cancer efficacy compared with oxaliplatin

Abstract Despite the widespread use of platinum chemotherapy, only few drugs have been approved in this class of molecules. Here, we describe a novel supramolecular chemistry-based platform technology to engineer more potent oxaliplatin analogues. In IO125, a unique platinum (II) is tethered to a cholesterol backbone via a monocarboxylato bond and an N→Pt coordination environment, releasing diaminocyclohexane (DACH)-platinum in a sustained pH-dependent manner. The molecule facilitates assembly into defined nanoscale supramolecular structures of uniform size distribution as measured using dynamic light scatter and electron microscopy. Such a supramolecular therapeutic demonstrates enhanced activity compared with oxaliplatin in 4T1 and MDA-MB-231 triple negative breast cancer lines. The uptake of IO125-based supramolecular therapeutic was 10-20 fold greater than free oxaliplatin in these cells. In addition, the nanoparticles exhibited significantly lower IC50 values in comparison to oxaliplatin in primary colorectal carcinoma lines generated from patient tumor sample. Nanostructures have been shown to exhibit superior pharmacokinetics profile and biodistribution to tumors due to an enhanced permeability and retention (EPR) effect. Based on these observations, we anticipate an IO125-based supramolecular therapeutic may emerge as a novel next-generation platinum-based potent anti-cancer agent. Citation Format: Monideepa Roy, Sk Samad Hossain, Arindam Sarkar, Aniruddha Sengupta, Nimish Gupta, Sajid Hussain, Aasif Ansari, Sanghamitra Mylavarapu, Shiladitya Sengupta. IO125, a novel Pt-based supramolecular therapeutic exhibits increased anti-cancer efficacy compared with oxaliplatin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4483. doi:10.1158/1538-7445.AM2014-4483

Abstract LB-207: mTORC2/Akt signaling is modulated by noncanonical mitochondrial Notch1/PINK1 interaction in myc-amplified medulloblastoma tumorigenesis

Abstract Medulloblastoma is known to be the most malignant pediatric brain tumor. The armamentarium of targeted therapies to currently treat medulloblastoma and similar pediatric central nervous system malignancies is extremely limited, often necessitating the need to combat such tumors with modified regimens of therapeutic options designed originally to target adult neoplasms. Given such limited therapies, a budding focus on the role of mitochondrial dysregulation in the tumorigenesis of such pathologies merits consideration. Mitochondria are known to play fundamental roles in multiple processes conserved across eukaryotic species. Aside from their key role in energy production through oxidative phosphorylation, the organelles also serve as the sites of essential metabolic pathways, redox regulation, calcium homeostasis, apoptosis, and cell fate determination and differentiation. Recently, we documented the role of noncanonical Notch signaling and mitochondrial involvement in adult glioblastoma brain tumor-initiating cells (Lee KS et al., Genes and Development, 2013). Although the canonical Notch pathway and is generally well-characterized, involving the ligand-induced cleavage of Notch for transcriptional regulation, only more recently has credible evidence surfaced documenting the role of a second noncanonical pathway, where Notch can function independently of ligand and transcription through a mechanism that remains to be fully elucidated. The regulatory self-renewal versus differentiation choice of Drosophila and mammalian human neural stem cells requires Notch signaling, and in our work, we found noncanonical Notch pathway interaction with PTEN-induced putative kinase 1 (PINK1) to influence mitochondrial function, activating mTORC2/Akt signaling. siRNA-induced knockdown preferentially impaired the maintenance of Drosophila and human glioblastoma cancer stem cell-like tumor-forming cells to a far greater degree than normal stem cell counterparts. Further experiments have elucidated similar findings of increased Notch1/PINK1 mitochondrial interaction and mTORC2/Akt activity in patient-derived Group 3 (myc-amplified) medulloblastoma primary lines to levels greater than normal or glioblastoma samples. Additionally these medulloblastoma samples appear to be more susceptible to siRNA-induced knockdown of PINK1 than their counterparts. In vivo experiments are ongoing to address the role of mitochondrial Notch1/Pink1 interaction in tumor initiation and its targeting by small molecule inhibitors. Such results underscore the importance of mitochondria in both normal and cancer stem cell biology, a highly conserved mechanism across species, with exciting implications for the treatment of pediatric central nervous system malignancies. Citation Format: Abdullah H. Feroze, Kyu-Sun Lee, Sharareh Gholamin, Zhihao Wu, Irving Weissman, Bingwei Lu, Siddhartha S. Mitra, Samuel Cheshier. mTORC2/Akt signaling is modulated by noncanonical mitochondrial Notch1/PINK1 interaction in myc-amplified medulloblastoma tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-207. doi:10.1158/1538-7445.AM2014-LB-207

The role of reproductive hormones in postpartum depression.

Despite decades of research aimed at identifying the causes of postpartum depression (PPD), PPD remains common, and the causes are poorly understood. Many have attributed the onset of PPD to the rapid perinatal change in reproductive hormones. Although a number of human and nonhuman animal studies support the role of reproductive hormones in PPD, several studies have failed to detect an association between hormone concentrations and PPD. The purpose of this review is to examine the hypothesis that fluctuations in reproductive hormone levels during pregnancy and the postpartum period trigger PPD in susceptible women. We discuss and integrate the literature on animal models of PPD and human studies of reproductive hormones and PPD. We also discuss alternative biological models of PPD to demonstrate the potential for multiple PPD phenotypes and to describe the complex interplay of changing reproductive hormones and alterations in thyroid function, immune function, hypothalamic-pituitary-adrenal (HPA) axis function, lactogenic hormones, and genetic expression that may contribute to affective dysfunction. There are 3 primary lines of inquiry that have addressed the role of reproductive hormones in PPD: nonhuman animal studies, correlational studies of postpartum hormone levels and mood symptoms, and hormone manipulation studies. Reproductive hormones influence virtually every biological system implicated in PPD, and a subgroup of women seem to be particularly sensitive to the effects of perinatal changes in hormone levels. We propose that these women constitute a "hormone-sensitive" PPD phenotype, which should be studied independent of other PPD phenotypes to identify underlying pathophysiology and develop novel treatment targets.

P17.68 * CHARACTERISATION OF A NOVEL ASPIRIN ANALOGUE IN MALIGNANT GLIOMA

Gliomas are the most common intracranial brain tumours and are associated with poor prognosis and median survival time of less than 15 months from first diagnosis. Clinical and preclinical research has suggested a role for aspirin in the treatment or prevention of cancer, with a recent focus on the development of novel aspirin analogues. Established (U87 MG, 1321N1, GOS-3& SVG-p12) and primary cell lines (BTNW911 & BTNW914) were treated with the novel aspirin analogue PN517, aspirin and cisplatin for 24 and 48 hr at 0.1 and 1mM. Cells were subsequently harvested and cell cycle distribution determined by flow cytometry following PI (50 mg/ml) and RNAse (100 mg/ml) treatment, and apoptosis examined by flow cytometry following staining with annexin-V and PI (50 mg/ml), or the mitochondrial membrane potential marker JC-1 (5 mM). Migration was examined over 18 hr using scratch assay and Boyden chamber technique. A two-way ANOVA test was used to analyse the significance between treatments with significance set at p < 0.05. PN517 induced apoptosis in the primary and established cell lines with a similar efficacy to cisplatin following 24 hours treatment at 0.1 mM. The induction of apoptosis was also assessed over a period of 48 hours and it was found to be the predominant mode of cell death at both 0.1 and 1mM concentrations and at early time points, with necrosis being observed at higher drug concentrations and after 24 hours of drug treatment. Following 48 hours of treatment PN517 (1 mM) significantly increased the sub G1 phase apoptotic population as determined by cell cycle analysis and decreased both the G1 and G2/M phase populations when compared to control. Treatment with the aspirin analogue (0.1 mM) also reduced migration significantly when compared to control. The novel aspirin analogue PN517 induced apoptosis, caused cell cycle arrest and reduced migration in both primary and established glioma cell lines. These results show that PN517 has therapeutic potential in the treatment of glioma.

Molecular characterization of immortalized normal and dysplastic oral cell lines.

Cell lines have been developed for modeling cancer and cancer progression. The molecular background of these cell lines is often unknown to those using them to model disease behaviors. As molecular alterations are the ultimate drivers of cell phenotypes, having an understanding of the molecular make-up of these systems is critical for understanding the disease biology modeled. Six immortalized normal, one immortalized dysplasia, one self-immortalized dysplasia, and two primary normal cell lines derived from oral tissues were analyzed for DNA copy number changes and changes in both mRNA and miRNA expression using SMRT-v.2 genome-wide tiling comparative genomic hybridization arrays, Agilent Whole Genome 4x44k expression arrays, and Exiqon V2.M-RT-PCR microRNA Human panels. DNA copy number alterations were detected in both normal and dysplastic immortalized cell lines-as well as in the single non-immortalized dysplastic cell line. These lines were found to have changes in expression of genes related to cell cycle control as well as alterations in miRNAs that are deregulated in clinical oral squamous cell carcinoma tissues. Immortal lines-whether normal or dysplastic-had increased disruption in expression relative to primary lines. All data are available as a public resource. Molecular profiling experiments have identified DNA, mRNA, and miRNA alterations for a panel of normal and dysplastic oral tissue cell lines. These data are a valuable resource to those modeling diseases of the oral mucosa, and give insight into the selection of model cell lines and the interpretation of data from those lines.

Synthetic hexaploid wheat enhances variation and adaptive evolution of bread wheat in breeding processes

AbstractSynthetic hexaploid wheat (SHW) that combines novel and elite genes from the tetraploid wheat Triticum turgidum L. and wild ancestor Aegilops tauschii Coss., has been used to genetically improve hexaploid common wheat. The abundant genetic diversity in SHW can effectively make breakthroughs in wheat genetic improvement through the inclusion of increased variation. In this paper, we reviewed the current advances in research and utilization of the primary SHW lines and SHW‐derived wheat varieties that have enhanced evolution of modern wheat under conditions of natural and artificial selection in southwestern China. Using primary SHW lines, four high‐yielding wheat varieties have been developed. In addition, using the SHW‐derived varieties as breeding parents, 12 new wheat varieties were also developed. Results of genotype–phenotype and fingerprint analysis showed that the introgressed alleles from SHW lines have contributed a great number of elite characters to the new wheat varieties, and these elite characters include disease resistance, more spikes per plant, more grains per spike, larger grains, and higher grain‐yield potential. We found that the primary SHW lines and SHW‐derived varieties have identifiable effects to enhance genetic variation and adaptive evolution of modern hexaploid wheat, which significantly increased the grain yields of hexaploid wheat in recent years. These findings have significant implications in the breeding of high‐yielding wheat varieties resistant to biotic and abiotic stresses using SHW as genetic resources.

Foxa1 and Foxa2 regulate α-cell differentiation, glucagon biosynthesis, and secretion.

The Forkhead box A transcription factors are major regulators of glucose homeostasis. They show both distinct and redundant roles during pancreas development and in adult mouse β-cells. In vivo ablation studies have revealed critical implications of Foxa1 on glucagon biosynthesis and requirement of Foxa2 in α-cell terminal differentiation. In order to examine the respective role of these factors in mature α-cells, we used small interfering RNA (siRNA) directed against Foxa1 and Foxa2 in rat primary pancreatic α-cells and rodent α-cell lines leading to marked decreases in Foxa1 and Foxa2 mRNA levels and proteins. Both Foxa1 and Foxa2 control glucagon gene expression specifically through the G2 element. Although we found that Foxa2 controls the expression of the glucagon, MafB, Pou3f4, Pcsk2, Nkx2.2, Kir6.2, and Sur1 genes, Foxa1 only regulates glucagon gene expression. Interestingly, the Isl1 and Gipr genes were not controlled by either Foxa1 or Foxa2 alone but by their combination. Foxa1 and Foxa2 directly activate and bind the promoter region the Nkx2.2, Kir6.2 and Sur1, Gipr, Isl1, and Pou3f4 genes. We also demonstrated that glucagon secretion is affected by the combined effects of Foxa1 and Foxa2 but not by either one alone. Our results indicate that Foxa1 and Foxa2 control glucagon biosynthesis and secretion as well as α-cell differentiation with both common and unique target genes.

COMBINED DRUG SCREENING AND PHOSPHOPROTEOMICS IDENTIFIES CANDIDATE BRAIN TUMOR THERAPEUTICS AND NOVEL TARGETS IN PRIMARY HUMAN BRAIN TUMOR-INITIATING CELLS

BACKGROUND: “Therapeutic Targeting of Glioblastoma” is a new pan-Canadian research team of the Terry Fox Research Institute and the Canadian Stem Cell Network funded to discover efficacious therapeutics for GBM. The team's goals are also to discover novel signaling pathways regulating GBM cell survival and genetic alterations that mediate drug resistance. As a platform, we use our collection of over 100 primary GBM tumor-initiating lines (BTIC) that are subjected to drug screening by over 1400 compounds, and to genetic and phosphoproteomic analysis. METHODS: We performed drug screening of 21 BTIC lines that represent the molecular heterogeneity of GBM patients, with a library of 110 clinically-relevant kinase inhibitors at three concentrations. We have also completed a phosphotyrosine characterization of 14 BTIC lines using phosphoproteomics. RESULTS: Multiple compounds that exhibit nanomolar cytotoxicity towards all of the lines were prioritized based upon their potency, novelty for GBM, ability to accumulate to relevant concentrations in brain, clinical status and cytotoxicity towards normal cells. Of the several hit compounds that fulfilled all of these criteria, the CDK inhibitor dinaciclib was chosen as our lead compound. We are currently investigating its mechanism of action using phosphoproteomics and RNAi as well as testing its efficacy in vivo in an orthotopic xenograft model as a single agent and in combination with TMZ. Furthermore, we have identified several compounds that exhibit selective cytotoxicity towards only a specific set of BTIC lines, suggesting that genetic differences between the lines account for differential sensitivities. The sensitivity of BTICs to one such compound, EMD-1214063, a selective MET inhibitor, may correlate with Met amplification status. Finally, phosphoproteomics results show activation of RTKs (EGFR, FGFR, PDGFRA, Eph receptor family) and non-receptor kinases (FYN, FAK1, GSK3b, MAPK, CDKs, DYRK) in the majority of BTIC lines, and of adaptor/scaffold proteins and proteins important in adhesion and migration. We are now (1) determining whether drugs that inhibit the activity of identified phosphoproteins suppress BTIC survival in culture and in orthotopic models, (2) validating the importance of those phosphoproteins in GBM survival, and (3) asking whether the prioritized drug hits from our drug screens suppress the phosphorylation of the identified phosphoproteins. CONCLUSIONS: We anticipate that our drug screening approach will generate several clinical candidates that will enable us to proceed to the clinic in the next several years. We also believe that our phosphoproteomics results will lead to identification of novel therapeutic targets for GBM. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

Stabilization of magnet assemblies of permanent magnet sodium flowmeters used in fast breeder reactors

Permanent magnet flow meters (PMFMs) are used to measure the sodium flow in sodium cooled Fast Breeder Reactor Circuits. Prototype fast breeder reactor (PFBR) which is under construction at Kalpakkam is a 500MWe, sodium cooled, pool type reactor. Sodium flow measurement in various loops of the reactor is of prime importance from operational and safety point of view. To measure the flow of electrically conducting liquid sodium, in primary and secondary circuit pipe lines of PFBR, permanent magnet flow meters are used. PMFM is a non-invasive device, which works on the principle of generation of motional EMF by magnetic forces exerted on the charges in a moving conductor. Flowmeters of different pipe sizes ranging from 10mm to 200mm pipe diameter are required for PFBR. Long term performance of the flowmeters mainly depends on stability of permanent magnets used in flowmeters to generate constant magnetic field in stainless steel (SS) pipes. This paper describes the effects of time and temperature on permanent magnet assemblies made of ALNICO-V used in PFBR flowmeters. The stabilization methodology for ALNICO-V permanent magnet assemblies is evolved and established. Loss of magnetic field strength with respect to time and temperatures is determined by experiments and found negligible.

Relation between skin micro-topography, roughness, and skin age.

The topography of the skin surface consists of lines, wrinkles, and scales. Primary and secondary lines form a network like structure that may be identified as polygons. Skin surface roughness measurements are widely applied in dermatological research and practice but the relation between roughness parameters and their anatomical equivalents are unclear. This study aimed to investigate whether the number of closed polygons (NCP) per measurement field can be used as a reliable parameter to measure skin surface topography. For this purpose, we analysed the relation between skin surface roughness parameters and NCP in different age groups. Images of the volar forearm skin of 38 subjects (14 children, 12 younger, and 12 older adults) were obtained with the VisioScan VC98. The NCP was counted by three independent researchers and selected roughness parameters were measured. Interrater reliability of counting the number of closed polygons and correlations between NCP, roughness parameters, and age were calculated. The mean NCP/mm² in children was 3.1 (SD 1.1), in younger adults 1.0 (SD 0.7), and in older adults 1.0 (SD 0.9). The interrater reliability was 0.9. A negative correlation of NCP/mm² with age was observed, whereas measured roughness parameters were positively associated with age. NCP/mm² was weakly related to skin roughness. The NCP/mm² is a reproducible parameter for characterizing the skin surface topography. It is proposed as an additional parameter in dermatological research and practice because it represents distinct aspects of the cutaneous profile not covered by established roughness parameters.

On How Many Fundamental Kinds of Cells are Present on Earth: Looking for Phylogenetic Traits that Would Allow the Identification of the Primary Lines of Descent

The phylogenetic analyses as far as the identification of the number of domains of life is concerned have not reached a clear conclusion. In the attempt to improve this circumstance, I introduce the concept that the amino acids codified in the genetic code might be of markers with outstanding phylogenetic power. In particular, I hypothesise the existence of a biosphere populated, for instance, by three groups of organisms having different genetic codes because codifying at least a different amino acid. Evidently, these amino acids would mark the proteins that are present in the three groups of organisms in an unambiguous way. Therefore, in essence, this mark would not be other than the one that we usually try to make in the phylogenetic analyses in which we transform the protein sequences in phylogenetic trees, for the purpose to identify, for example, the domains of life. Indeed, this mark would allow to classify proteins without performing phylogenetic analyses because proteins belonging to a group of organisms would be recognisable as marked in a natural way by at least a different amino acid among the diverse groups of organisms. This conceptualisation answers the question of how many fundamental kinds of cells have evolved from the Last Universal Common Ancestor (LUCA), as the genetic code has unique proprieties that make the codified amino acids excellent phylogenetic markers. The presence of the formyl-methionine only in proteins of bacteria would mark them and would identify these as domain of life. On the other hand, the presence of pyrrolysine in the genetic code of the euryarchaeota would identify them such as another fundamental kind of cell evolved from the LUCA. Overall, the phylogenetic distribution of formyl-methionine and pyrrolysine would identify at least two domains of life--Bacteria and Archaea--but their number might be actually four; that is to say, Bacteria, Euryarchaeota, archeobacteria that are not euryarchaeota and Eukarya. The usually accepted domains of life represented by Bacteria, Archaea and Eukarya are not compatible with the phylogenetic distribution of these two amino acids and therefore this last classification might be mistaken.

Liberalism and Constitutionalism in Latin America in the 19th Century

AbstractFor generations, 19th century Latin American history has been portrayed as a period of anarchic turmoil, wasted energy, and pointless division. It was the age of caudillos and of political breakup. But recent evidence paints a very different picture. More and more historians see the period from 1821 (when the wars of independence ended) to 1860s as an era of experimentation and innovation, of building new institutions to replace the old. This essay reprises some of the primary lines of investigation. It explores the ways in which Latin Americans built a hybrid model of constitutionalism, adapted from elements circulating around the Atlantic world. It explores how people charted new meanings of citizenship by applying new legal tools and practices of public organization in the civic sphere. It examines the experiments in political organizing and vertical alliances. Finally, it suggests that older intellectual and ideological coordinates of secular vs religious, liberal vs conservative, fail to capture the very strong emphasis on pragmatic republican strains in 19th century statebuilders.

Contribution of human skin topography to the characterization of dynamic skin tension during senescence: morpho-mechanical approach

The structuring of the dermis with a network of collagen and elastic fibres gives a three-dimensional structure to the skin network with directions perpendicular and parallel to the skin surface. This three-dimensional morphology prints on the surface of the stratum corneum a three dimensional network of lines which express the mechanical tension of the skin at rest. To evaluate the changes of skin morphology, we used a three-dimensional confocal microscopy and characterization of skin imaging of volar forearm microrelief. We have accurately characterize the role of skin line network during chronological aging with the identification of depth scales on the network of lines (z ≤ 60μm) and the network of lines covering Langer's lines (z > 60 microns). During aging has been highlighted lower rows for elastic fibres, the decrease weakened the tension and results in enlargement of the plates of the microrelief, which gives us a geometric pertinent indicator to quantify the loss of skin tension and assess the stage of aging. The study of 120 Caucasian women shows that ageing in the volar forearm zone results in changes in the morphology of the line network organisation. The decrease in secondary lines (z ≤ 60 μm) is counterbalanced by an increase in the depth of the primary lines (z > 60 μm) and an accentuation of the anisotropy index.

Multiple lesions in receptor tyrosine kinase pathway determine glioblastoma response to pan-ERBB inhibitor PF-00299804 and PI3K/mTOR dual inhibitor PF-05212384

A novel pan ERBB inhibitor PF-00299804 (dacomitinib) is currently in phase II clinical trials in glioblastoma multiforme (GBM) patients; however its pre-clinical efficacy in GBMs has not been tested. In this study, we evaluated the efficacy of dacomitinib alone or in combination with PI3K/mTOR dual inhibitor PF-05212384 in GBM and assessed the mechanisms of resistance and the molecular determinants of response. A panel of established and patient derived primary GBM lines that present different molecular profiles and also the GBM lines engineered to express EGFRvIII mutant or PTEN were treated with either dacomitinib, PF-05212384, or combination and assessed for their viability and changes in EGFR/PI3K/mTOR signaling. We show that dacomitinib significantly reduced phosphorylated EGFR in all the GBM lines but did not show a dose-dependent response on cell viability in a majority of the lines tested. Multiple lesions in the receptor tyrosine kinases (RTKs) pathway including PTEN mutation, co-activation of RTKs, and EGFRvIII mutation resulted in unaltered active status of PI3K/mTOR in the GBM lines even in the presence of EGFR inhibition. Blocking PI3K/mTOR dramatically inhibited cell proliferation in most GBM lines and enhanced dacomitinib induction of apoptosis in a GBM line that has both EGFR amplification and EGFR-independent PI3K activation. These data suggest molecular profiling of EGFR/PI3K/PTEN status to select GBM patients for EGFR or/and PI3K/mTOR targeted therapies.

Proliferation of ASC-derived endothelial cells in a 3D electrospun mesh: Impact of bone-biomimetic nanocomposite and co-culture with ASC-derived osteoblasts

BackgroundFractures with a critical size bone defect are associated with high rates of delayed- and non-union. The treatment of such complications remains a serious issue in orthopaedic surgery. Adipose derived stem cells (ASCs) combined with biomimetic materials can potentially be used to increase fracture healing. Nevertheless, a number of requirements have to be fulfilled; in particular, the insufficient vascularisation of the bone constructs. Here, the objectives were to study the impact of ASC-derived osteoblasts on ASC-derived endothelial cells in a 3D co-culture and the effect of 40wt% of amorphous calcium phosphate nanoparticles on the proliferation and differentiation of ASC-derived endothelial cells when present in PLGA. Materials and methodsFive primary ASC lines were differentiated towards osteoblasts (OBs) and endothelial cells (ECs) and two of them were chosen based on quantitative PCR results. Either a mono-culture of ASC-derived EC or a co-culture of ASC-derived EC with ASC-derived OB (1:1) was seeded on an electrospun nanocomposite of poly-(lactic-co-glycolic acid) and amorphous calcium phosphate nanoparticles (PLGA/a-CaP; reference: PLGA). The proliferation behaviour was determined histomorphometrically in different zones and the expression of von Willebrand Factor (vWF) was quantified. ResultsIndependently of the fat source (biologic variability), ASC-derived osteoblasts decelerated the proliferation behaviour of ASC-derived endothelial cells in the co-culture compared to the mono-culture. However, expression of vWF was clearly stronger in the co-culture, indicating further differentiation of the ASC-derived EC into the EC lineage. Moreover, the presence of a-CaP nanoparticles in the scaffold slowed the proliferation behaviour of the co-culture cells, too, going along with a further differentiation of the ASC-derived OB, when compared to pure PLGA scaffolds. ConclusionsThis study revealed significant findings for bone tissue-engineering. Co-cultures of ASC-derived EC and ASC-derived OB stimulate each other's further differentiation. A nanocomposite with a-CaP nanoparticles offers higher mechanical stability, bioactivity and osteoconductivity compared to mere PLGA and can easily be seeded with pre-differentiated EC and OB.

Changes in actin dynamics are involved in salicylic acid signaling pathway

Changes in actin cytoskeleton dynamics are one of the crucial players in many physiological as well as non-physiological processes in plant cells. Positioning of actin filament arrays is necessary for successful establishment of primary lines of defense toward pathogen attack, depolymerization leads very often to the enhanced susceptibility to the invading pathogen. On the other hand it was also shown that the disruption of actin cytoskeleton leads to the induction of defense response leading to the expression of PATHOGENESIS RELATED proteins (PR). In this study we show that pharmacological actin depolymerization leads to the specific induction of genes in salicylic acid pathway but not that involved in jasmonic acid signaling. Life imaging of leafs of Arabidopsis thaliana with GFP-tagged fimbrin (GFP-fABD2) treated with 1mM salicylic acid revealed rapid disruption of actin filaments resembling the pattern viewed after treatment with 200nM latrunculin B. The effect of salicylic acid on actin filament fragmentation was prevented by exogenous addition of phosphatidic acid, which binds to the capping protein and thus promotes actin polymerization. The quantitative evaluation of actin filament dynamics is also presented.