Molecular profiling of lung adenosquamous carcinoma: hybrid or genuine type?

Lung adenosquamous carcinoma is a particular subtype of non-small cell lung carcinoma that is defined by the coexistence of adenocarcinoma and squamous cell carcinoma components. The aim of this study was to assess the mutational profile in each component of 16 adenosquamous carcinoma samples from a Caucasian population by a combination of next generation sequencing using the cancer hotspot panel as well as the colon and lung cancer panel and FISH. Identified mutations were confirmed by Sanger sequencing of DNA from cancer cells of each component collected by Laser Capture microdissection. Mutations typical for adenocarcinoma as well as squamous cell carcinoma were identified. Driver mutations were predominantly in the trunk suggesting a monoclonal origin of adenosquamous carcinoma. Most remarkably, EGFR mutations and mutations in the PI3K signaling pathway, which accounted for 30% and 25% of tumors respectively, were more prevalent while KRAS mutations were less prevalent than expected for a Caucasian population. Surprisingly, expression of classifier miR-205 was intermediate between that of classical adenocarcinoma and squamous cell carcinoma suggesting that adenosquamous carcinoma is a transitional stage between these tumor types. The high prevalence of therapy-relevant targets opens new options of therapeutic intervention for adenosquamous carcinoma patients.

Elucidation of the gas-phase structure of a sugar-modified DNA analogue

Antisense oligonucleotides are medical agents for the treatment of genetic diseases that are designed to interact specifically with mRNA. This interaction either induces enzymatic degradation of the targeted RNA or modifies processing pathways, e.g. by inducing alternative splicing of the pre-mRNA. The latter mechanism applies to the treatment of Duchenne muscular dystrophy with a sugar-modified DNA analogue called tricyclo-DNA (tcDNA). In tcDNA the ribose sugar-moiety is extended to a three-membered ring system, which augments the binding affinity and the selectivity of the antisense oligonucleotide for its target. The advent of chemically modified nucleic acids for antisense therapy presents a challenge to diagnostic tools, which must be able to cope with a variety of structural analogues. Mass spectrometry meets this demand for non-enzyme based sequencing methods ideally, because the technique is largely unaffected by structural modifications of the analyte. Sequence coverage of a fully modified tcDNA 15mer can be obtained in a single tandem mass spectrometric experiment. Beyond sequencing experiments, tandem mass spectrometry was applied to elucidate the gas-phase structure and stability of tcDNA:DNA and tcDNA:RNA hybrid duplexes. Most remarkable is the formation of truncated duplexes upon collision-induced dissociation of these structures. Our data suggest that the cleavage site within the duplex is directed by the modified sugar-moiety. Moreover, the formation of truncated duplexes manifests the exceptional stability of the hybrid duplexes in the gas-phase. This stability arises from the modified sugar-moiety, which locks the tcDNA single strand into a conformation that is similar to RNA in A-form duplexes. The conformational particularity of tcDNA in the gas-phase was confirmed by ion mobility-mass spectrometry experiments on tcDNA, DNA, and RNA.

Structural elucidation and antisense properties of a sugar-modified DNA analogue

Antisense oligonucleotides deserve great attention as potential drug candidates for the treatment of genetic disorders. For example, muscle dystrophy can be treated successfully in mice by antisense-induced exon skipping in the pre-mRNA coding for the structural protein dystrophin in muscle cells. For this purpose a sugar- and backbone-modified DNA analogue was designed, in which a tricyclic ring system substitutes the deoxyribose. These chemical modifications stabilize the dimers formed with the targeted RNA relative to native nucleic acid duplexes and increase the biostability of the antisense oligonucleotide. While evading enzymatic degradation constitutes an essential property of antisense oligonucleotides for therapeutic application, it renders the oligonucleotide inaccessible to biochemical sequencing techniques and requires the development of alternative methods based on mass spectrometry. The set of sequences studied includes tcDNA oligonucleotides ranging from 10 to 15 nucleotides in length as well as their hybrid duplexes with DNA and RNA complements. All samples were analyzed on a LTQ Orbitrap XL instrument equipped with a nano-electrospray source. For tandem mass spectrometric experiments collision-induced dissociation was performed, using helium as collision gas. Mass spectrometric sequencing of tcDNA oligomers manifests the applicability of the technique to substrates beyond the scope of enzyme-based methods. Sequencing requires the formation of characteristic backbone fragments, which take the form of a-B- and w-ions in the product ion spectra of tcDNA. These types of product ions are typically associated with unmodified DNA, which suggests a DNA-like fragmentation mechanism in tcDNA. The loss of nucleobases constitutes the second prevalent dissociation pathway observed in tcDNA. Comparison of partially and fully modified oligonucleotides indicates a pronounced impact of the sugar-moiety on the base loss. As this event initiates cleavage of the backbone, the presented results provide new mechanistic insights into the fragmentation of DNA in the gas-phase. The influence of the sugar-moiety on the dissociation extends to tcDNA:DNA and tcDNA:RNA hybrid duplexes, where base loss was found to be much more prominent from sugar-modified oligonucleotides than from their natural complements. Further prominent dissociation channels are strand separation and backbone cleavage of the single strands, as well as the ejection of backbone fragments from the intact duplex. The latter pathway depends noticeably on the base sequence. Moreover, it gives evidence of the high stability of the hybrid dimers, and thus directly reflects the affinity of tcDNA for its target in the cell. As the cellular target of tcDNA is a pre-mRNA, the structure was designed to discriminate RNA from DNA complements, which could be demonstrated by mass spectrometric experiments.

Supramolecular Polymers in DNA Nanotechnology

Creation of biocompatible functional materials is an important task in supramolecular chemistry. In this contribution, we report on noncovalent synthesis of DNA-grafted supramolecular polymers (SPs). DNA-grafted SPs enable programmed arrangement of oligonucleotides in a regular, tightly packed one-dimensional array. Further interactions of DNA-grafted SPs with complementary DNA strands leads to the formation of networks through highly cooperative G-C blunt-end stacking interactions. The structural changes in the polymeric core enable to monitor spectroscopically the stepwise formation of networks. Such stimuli-responsive supramolecular networks may lead to the development of DNA-based smart materials.

REV7 counteracts DNA double-strand break resection and affects PARP inhibition

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

Organisation of the equine immunoglobulin constant heavy chain genes. II. Equine cgamma genes.

The number of immunoglobulin G constant heavy chain genes (cgamma genes) varies broadly among mammalian species, reflecting structural and functional differences between expressed immunoglobulin G (IgG) isotypes and allotypes. Up to now equine IgG isotypes have been defined only at the biochemical and serological level. It is still not clear how many IgG isotypes exist in horses and whether there are any allotypes. Here, we describe the isolation and characterisation of equine cgamma genes. An equine genomic lambda phage library was screened with a human cgamma4 probe. Cross-hybridising equine cgamma sequences were cloned twice and characterised by restriction mapping with the human cgamma4 and a murine sgamma1 probe. Genomic equine DNA probes for both, cgamma genes and corresponding switch regions (sgamma), were isolated and used for a more detailed BamHI restriction analysis, comparing genomic DNA of various horses. This analysis reveals the existence of at least five, or probably six cgamma genes in the equine haploid genome. Beside the porcine system, this is the highest number of cgamma genes described for any mammalian species. Moreover, for two of these cgamma genes, BamHI restriction fragment length polymorphism became evident.

Carbohydrate Estimation by a Mobile Phone-Based System Versus Self-Estimations of Individuals With Type 1 Diabetes Mellitus: A Comparative Study

Background: Diabetes mellitus is spreading throughout the world and diabetic individuals have been shown to often assess their food intake inaccurately; therefore, it is a matter of urgency to develop automated diet assessment tools. The recent availability of mobile phones with enhanced capabilities, together with the advances in computer vision, have permitted the development of image analysis apps for the automated assessment of meals. GoCARB is a mobile phone-based system designed to support individuals with type 1 diabetes during daily carbohydrate estimation. In a typical scenario, the user places a reference card next to the dish and acquires two images using a mobile phone. A series of computer vision modules detect the plate and automatically segment and recognize the different food items, while their 3D shape is reconstructed. Finally, the carbohydrate content is calculated by combining the volume of each food item with the nutritional information provided by the USDA Nutrient Database for Standard Reference. Objective: The main objective of this study is to assess the accuracy of the GoCARB prototype when used by individuals with type 1 diabetes and to compare it to their own performance in carbohydrate counting. In addition, the user experience and usability of the system is evaluated by questionnaires. Methods: The study was conducted at the Bern University Hospital, “Inselspital” (Bern, Switzerland) and involved 19 adult volunteers with type 1 diabetes, each participating once. Each study day, a total of six meals of broad diversity were taken from the hospital’s restaurant and presented to the participants. The food items were weighed on a standard balance and the true amount of carbohydrate was calculated from the USDA nutrient database. Participants were asked to count the carbohydrate content of each meal independently and then by using GoCARB. At the end of each session, a questionnaire was completed to assess the user’s experience with GoCARB. Results: The mean absolute error was 27.89 (SD 38.20) grams of carbohydrate for the estimation of participants, whereas the corresponding value for the GoCARB system was 12.28 (SD 9.56) grams of carbohydrate, which was a significantly better performance ( P=.001). In 75.4% (86/114) of the meals, the GoCARB automatic segmentation was successful and 85.1% (291/342) of individual food items were successfully recognized. Most participants found GoCARB easy to use. Conclusions: This study indicates that the system is able to estimate, on average, the carbohydrate content of meals with higher accuracy than individuals with type 1 diabetes can. The participants thought the app was useful and easy to use. GoCARB seems to be a well-accepted supportive mHealth tool for the assessment of served-on-a-plate meals.

Assessing the Phylogenetic Utility of DNA Barcoding Using the New Zealand Cicada Genus Kikihia

DNA Barcoding (Hebert et al. 2003) has the potential to revolutionize the process of identifying and cataloguing biodiversity; however, significant controversy surrounds some of the proposed applications. In the seven years since DNA barcoding was introduced, the Web of Science records more than 600 studies that have weighed the pros and cons of this procedure. Unfortunately, the scientific community has been unable to come to any consensus on what threshold to use to differentiate species or even whether the barcoding region provides enough information to serve as an accurate species identification tool. The purpose of my thesis is to analyze mitochondrial DNA (mtDNA) barcoding’s potential to identify known species and provide a well-resolved phylogeny for the New Zealand cicada genus Kikihia. In order to do this, I created a phylogenetic tree for species in the genus Kikihia based solely on the barcoding region and compared it to a phylogeny previously created by Marshall et al. (2008) that benefits from information from other mtDNA and nuclear genes as well as species-specific song data. I determined how well the barcoding region delimits species that have been recognized based on morphology and song. In addition, I looked at the effect of sampling on the success of barcoding studies. I analyzed subsets of a larger, more densely sampled dataset for the Kikihia Muta Group to determine which aspects of my sampling strategy led to the most accurate identifications. Since DNA barcoding would by definition have problems in diagnosing hybrid individuals, I studied two species (K. “murihikua” and K. angusta) that are known to hybridize. Individuals that were not obvious hybrids (determined by morphology) were selected for the case study. Phylogenetic analysis of the barcoding region revealed insights into the reasons these two species could not be successfully differentiated using barcoding alone.

Elucidation of the role of 53BP1 in DNA double strand break (DSB) repair and tumor suppression

The protein p53 binding protein one (53BP1) was discovered in a yeast two-hybrid screen that used the DNA binding domain of p53 as bait. Cloning of full-length 53BP1 showed that this protein contains several protein domains which help make up the protein, which include two tandem BRCT domains and a amino-terminal serine/glutamine cluster domain (SCD). These are two protein domains are often seen in factors that are involved in the cellular response to DNA damage and control of cell cycle checkpoints and we hypothesize that 53BP1 is involved in the cellular response to DNA damage. In support of this hypothesis we observe that 53BP1 is phosphorylated and undergoes a dramatic nuclear re-localization in response to DNA damaging agents. 53BP1 also interacts with several factors that are important in the cellular response to DNA damage, such as the BRCA1 tumor suppressor, ATM and Rad3 related (ATR), and the phosphorylated version of the histone variant H2AX. Mice deficient in 53BP1 display increased sensitivity ionizing radiation (IR), a DNA damaging agent that introduces DNA double strand breaks (DSBs). In addition, 53BP1-deficient mice do not properly undergo the process of class switch recombination (CSR). We also observe that when a defect in 53BP1 is combined with a defect in p53; the resulting mice have an increased rate of formation of spontaneous tumors, notably the formation of B and T lineage lymphomas. The T lineage tumors arise by two distinct mechanisms: one driven by defects in cell cycle regulation and a second driven by defects in the ability to repair DNA DSBs. The B lineage tumors arise by the inability to repair DNA damage and over-expression of the oncogene c-myc. ^ With these observations, we conclude that not only does 53BP1 function in the cellular response to DNA damage, but it also works in concert with p53 to suppress tumor formation. ^

Role of telomere dysfunction, DNA damage response and p53 mutations in tumorigenesis and aging

The ends of eukaryotic chromosomes are protected by specialized ribonucleoprotein structures termed telomeres. Telomeres protect chromosomes from end-to-end fusions, inappropriate repair and degradation. Disruption of this complex activates an ATM/ATR DNA damage response (DDR) pathway. One component of the complex is the Protection Of Telomeres 1 (POT1) protein, an evolutionarily conserved protein which binds single-stranded 3' overhang and is required for both chromosomal end protection and telomere length regulation. The mouse contains two POT1 orthologs, Pot1a and Pot1b. Here we show that both proteins colocalize with telomeres through interaction with the adapter protein TPP1. In addition, compared to Pot1a, the OB-folds of Pot1b possess less sequence specificity for telomeres. Disruption of POT1 proteins result in telomere dysfunction and activation of an ATR-dependent DDR at telomeres, suggesting that this response is normally suppressed by POT1 binding to the single-stranded G-overhang. ^ Telomeres are maintained by telomerase, and its absence in somatic cells results in telomere progressive loss that triggers the activation of p53. Telomere dysfunction initiates genomic instability and induces both p53-dependent replicative senescence and apoptosis to suppress tumorigenesis. In the absence of functional p53, this genomic instability promotes cancer. It was previously not known which aspect of the p53 dependent DNA damage response is important to suppress tumorigenesis initiated by dysfunctional telomeres. The p53R172P knock-in mouse, which is unable to induce apoptosis but retains intact cell cycle arrest/cellular senescence pathways, allowed us to examine whether p53-dependent apoptosis is a major tumor suppression pathway initiated in the setting of telomere dysfunction. Spontaneous tumorigenesis remains potently suppressed in late generation telomerase null mice possessing the p53P/P mutation. These results suggest that suppression of spontaneous tumorigenesis initiated by dysfunctional telomeres requires activation of a p53-dependent senescence pathway. In addition, we used another knock-in mouse model with a p53R172H (p53H) point mutation to test the hypothesis that telomere dysfunction promotes chromosomal instability and accelerates the onset of tumorigenesis in vivo in the setting of this most common gain-of-function mutation in the human Li Fraumeni cancer syndrome. We unexpectedly observed that telomerase null mice possessing dysfunctional telomeres in the setting of the p53H/+ mutation develop significantly fewer tumors, die prematurely and exhibit higher level of cellular senescence, apoptosis and elevated genomic instability compared to telomerase intact p53H/+ and telomerase null p53+/+ mice. These contrasting results thus link cancer and aging to the functional status of telomeres and the integrity of the p53 pathway. ^

AN INVESTIGATION OF THE POSSIBLE RELATIONSHIP BETWEEN CERTAIN PHYSIOLOGICAL AND PSYCHOLOGICAL MEASURES AND SMOKING CESSATION IN A SELF - SELECTED POPULATION

This study focused on the possible relationship between certain physiological and psychological variables and the cessation of smoking. The population studied was employees enrolled in a multimodality smoking cessation program at the local offices of a major American corporation. In order to be eligible to participate, each individual must have become a non-smoker by the end of the smoking cessation program.^ Three physiological measures were taken on each individual while performing a relaxation exercise; (1) Electromyogram (EMG), (2) Galvanic Skin Response (GSR), and (3) Skin Temperature. The psychological measure consisted of the variable "anxiety" in the Cattell 16-PF personality inventory. Individual's self report of their smoking status was verified through a test for expired carbon monoxide levels.^ For the total population (N-31) no significant relationships were found between the physiological and psychological variable measured and cessation; however, with the removal of two cases discovered during the post-test interview to be influenced by external factors of high caffeine level and a severe family crisis, the measure of EMG, attained significance in discriminating between the successful and unsuccessful in Smoking Cessation. ^

A novel DNA damage inducible inhibitor of p53

p53 is required for the maintenance of the genomic stability of cells. Mutations in the p53 tumor-suppressor gene occur in more than 50% of human cancers of diverse types. In addition, 70% of families with Li-Fraumeni syndrome have a germline mutation in p53, predisposing these individuals to multiple forms of cancer. In response to DNA damage, p53 becomes stabilized and activated. However the exact mechanism by which DNA damage signals the stabilization and activation of p53 still remains elusive. The biochemical activity of p53 that is required for tumor suppression, and presumably the cellular response to DNA damage, involves the ability of the protein to bind to specific DNA sequences and to function as a transcription factor. For the downstream targets, p53 transactivates many genes involved in growth arrest, apoptosis and DNA repair such as p21, Bax and GADD45, respectively. An open question in the field is how cells can determine the downstream effects of p53. ^ We hypothesize that, through its associated proteins, p53 can differentially transactivate its target genes, which determine its downstream effect. Additionally, p53 interacting proteins may be involved in signaling for the stabilization and activation of p53. Therefore, a key aspect to understanding p53 function is the identification and analysis of proteins that interact with it. We have employed the Sos recruitment system (SRS), a cytoplasmic yeast two-hybrid screen to identify p53 interacting proteins. The SRS is based on the ability of Sos to activate Ras when it becomes localized to the plasma membrane. The system takes advantage of an S. cerevisiae strain, cdc25-2 temperature sensitive mutant, harboring a mutation in Sos. In this strain, fusion proteins containing a truncated Sos will only localize to the membrane by protein-protein interaction, which allows growth at non-permissive temperature. This system allows the use of intact transcriptional activators such as p53. ^ To date, using a modified SRS library screen to identify p53 interacting proteins, I have identified p53 (known to interact with itself) and a novel p53-interacting protein (PIP). PIP is a specific p53 interacting protein in the SRS. The interaction of p53 and PIP was further confirmed by performing in vitro and in vivo binding assays. In the in vivo binding study, the interaction can only be detected in the presence of ionizing radiation suggesting that this interaction might be involved in DNA-damage induced p53-signalling pathway. After screening cDNA and genomic libraries, a full-length PIP-cDNA clone ( ∼ 3kb) was obtained which encodes a protein of 429 amino acids with calculated molecular weight of 46 kDa. The results of genebank search indicated that the PIP is an unidentified gene and contains a conserved ring-finger domain, which is present in a diverse family of regulatory proteins involved in different aspects of cellular function. Northern blot analysis revealed that the size of its messenge is approximately 3 kb preferentially expressed in brain, heart, liver and kidney. The PIP protein is mainly located in the cytoplasm as determined by the cellular localization of a green fluorescence fusion protein. Preliminary functional analysis revealed that PIP downregulated the transactivation activity of p53 on both p21 and mdm2 promoters. Thus, PIP may be a novel negative regulator of p53 subsequent to DNA damage. ^

Safety door for the unloading mechanism of self-unloading wagons

A safety door for the discharge opening of a selfunloading wagon is disclosed herein. The wagon includes a material receiving box having a first conveyor therein for conveying the material towards one end thereof. A cross conveyor is mounted at the one end of the box for conveying the material outwardly from the box through the lower end of a discharge opening formed in the side of the box. A material beater means is rotatably mounted in the box above the cross conveyor. The safety door extends across the discharge opening to prevent the operator from reaching inwardly through the discharge opening. The safety door is self-closing and is opened by the material being discharged outwardly through the discharge opening.

Diversity of zooplankton assemblages at Station L4, Western English Channel, measured using next generation DNA sequencing

Background: Zooplankton play an important role in our oceans, in biogeochemical cycling and providing a food source for commercially important fish larvae. However, difficulties in correctly identifying zooplankton hinder our understanding of their roles in marine ecosystem functioning, and can prevent detection of long term changes in their community structure. The advent of massively parallel Next Generation Sequencing technology allows DNA sequence data to be recovered directly from whole community samples. Here we assess the ability of such sequencing to quantify the richness and diversity of a mixed zooplankton assemblage from a productive monitoring site in the Western English Channel. Methodology/Principle Findings: Plankton WP2 replicate net hauls (200 µm) were taken at the Western Channel Observatory long-term monitoring station L4 in September 2010 and January 2011. These samples were analysed by microscopy and metagenetic analysis of the 18S nuclear small subunit ribosomal RNA gene using the 454 pyrosequencing platform. Following quality control a total of 419,042 sequences were obtained for all samples. The sequences clustered in to 205 operational taxonomic units using a 97% similarity cut-off. Allocation of taxonomy by comparison with the National Centre for Biotechnology Information database identified 138 OTUs to species level, 11 to genus level and 1 to order, <2.5% of sequences were classified as unknowns. By comparison a skilled microscopic analyst was able to routinely enumerate only 75 taxonomic groups. Conclusions: The percentage of OTUs assigned to major eukaryotic taxonomic groups broadly aligns between the metagenetic and morphological analysis and are dominated by Copepoda. However, the metagenetics reveals a previously hidden taxonomic richness, especially for Copepoda and meroplankton such as Bivalvia, Gastropoda and Polychaeta. It also reveals rare species and parasites. We conclude that Next Generation Sequencing of 18S amplicons is a powerful tool for estimating diversity and species richness of zooplankton communities.