Search

DNA double-strand break (DSB) repair is mediated by multiple pathways, including classical non-homologous end-joining pathway (NHEJ) and several homology-driven repair pathways. This is particularly important for Cas9-mediated genome editing, where the outcome critically depends on the pathway that repairs the break. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a newly developed multiplexed reporter assay in combination with Cas9 cutting, we systematically measured the relative activities of three DSB repair pathways as function of chromatin context in >1,000 genomic locations. This revealed that NHEJ is broadly biased towards euchromatin, while microhomology-mediated end-joining (MMEJ) is more efficient in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 shifts the balance towards NHEJ. Single-strand templated repair (SSTR), often used for precise CRISPR editing, competes with MMEJ, and this competition is weakly associated with chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance, and guidance for the design of Cas9-mediated genome editing experiments.

Lamin proteins not only form the nuclear lamina, but some are also found in the nuclear interior. In this issue of Developmental Cell, Ikegami et al. describe that phosphorylated Lamin C in the nuclear interior interacts with enhancer-like elements and link this to deregulated transcription in progeria.

G quadruplexes (G4s) and R loops are noncanonical DNA structures that can regulate basic nuclear processes and trigger DNA damage, genome instability, and cell killing. By different technical approaches, we here establish that specific G4 ligands stabilize G4s and simultaneously increase R-loop levels within minutes in human cancer cells. Genome-wide mapping of R loops showed that the studied G4 ligands likely cause the spreading of R loops to adjacent regions containing G4 structures, preferentially at 3′-end regions of expressed genes, which are partially ligand-specific. Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4 ligands in BRCA2 -proficient and BRCA2 -silenced cancer cells. Moreover, even if the studied G4 ligands increased noncanonical DNA structures at similar levels in nuclear chromatin, their cellular effects were different in relation to cell-killing activity and stimulation of micronuclei, a hallmark of genome instability. Our findings therefore establish that G4 ligands can induce DNA damage by an R loop-dependent mechanism that can eventually lead to different cellular consequences depending on the chemical nature of the ligands.

Summary DNA double-strand break (DSB) repair is mediated by multiple pathways. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a multiplexed reporter assay in combination with Cas9 cutting, we systematically measure the relative activities of three DSB repair pathways as a function of chromatin context in >1,000 genomic locations. This reveals that non-homologous end-joining (NHEJ) is broadly biased toward euchromatin, while the contribution of microhomology-mediated end-joining (MMEJ) is higher in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 reverts the balance toward NHEJ. Single-stranded template repair (SSTR), often used for precise CRISPR editing, competes with MMEJ and is moderately linked to chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance and guidance for the design of Cas9-mediated genome editing experiments., Graphical abstract, Highlights • Sequencing-based reporter detects activities of multiple DSB repair pathways • Multiplexing approach to integrate reporter in >1,000 genomic locations • Overlay with epigenome data reveals effects of chromatin context on pathway balance • Pathway balance differs between heterochromatin and euchromatin, Schep et al. designed a reporter to probe the activities of three DNA double-strand break repair pathways. Integration of this reporter in >1,000 genomic locations in a human cell line provided a detailed view of the impact of local chromatin context on the balance between the repair pathways.

DNA topoisomerase I (Top1) inhibition by camptothecin derivatives can impair the hypoxia-induced cell transcriptional response. In the present work, we determined molecular aspects of the mechanism of camptothecin's effects on hypoxia-inducible factor-1α (HIF-1α) activity in human cancer cells. In particular, we provide evidence that low concentrations of camptothecin, without interfering with HIF-1α mRNA levels, can reduce HIF-1α protein expression and activity. As luciferase assays demonstrated the involvement of the HIF-1α mRNA 3′ untranslated region in camptothecin-induced impairment of HIF-1α protein regulation, we performed microarray analysis to identify camptothecin-induced modification of microRNAs (miRNA) targeting HIF-1α mRNA under hypoxic-mimetic conditions. The selected miRNAs were then further analyzed, demonstrating a role for miR-17-5p and miR-155 in HIF-1α protein expression after camptothecin treatments. The present findings establish miRNAs as key factors in a molecular pathway connecting Top1 inhibition and human HIF-1α protein regulation and activity, widening the biologic and molecular activity of camptothecin derivatives and the perspective for novel clinical interventions. Mol Cancer Ther; 13(1); 239–48. ©2013 AACR.

Resumen Introduccion Actualmente se ha incrementado la ureteronefroscopia flexible para el tratamiento de calculos renales, existiendo gran variacion en la tecnica quirurgica e indicaciones a traves de todo el mundo. Objetivos Conocer la practica actual, variaciones en la tecnica, uso e indicaciones de la ureteronefroscopia flexible para tratamiento de calculos renales en Latinoamerica. Metodos Enviamos un cuestionario anonimo de 30 preguntas sobre ureteronefroscopia flexible para el tratamiento de calculos renales, via correo electronico y enlace Web a urologos de Latinoamerica de enero de 2015 a julio de 2015. Recolectamos las respuestas a traves del sistema Survey Monkey. Resultados Participaron 283 urologos de 15 paises latinoamericanos (tasa de respuesta del 10,8%); 254 contestaron completamente el cuestionario; 52,8% son urologos de Mexico y 11% de Argentina, 11,8% realizan > 100 casos por ano, 15,2% consideran la ureteronefroscopia como tratamiento de eleccion para calculos > 2 cm y 19,6% realiza ureteronefroscopia en etapas solo para calculos > 2,5 cm. El 78,4% utiliza fluoroscopia, el 69,1% utiliza camisa ureteral en todos sus casos, el 55,8% deja el cateter doble J al final de la cirugia, el 37,3% considera estado libre de lito con 0 fragmentos y el 41,2% utiliza radiografia simple para evaluar el estado libre de calculos. Conclusiones La mayoria de urologos participantes considera la ureteronefroscopia flexible como el tratamiento de primera eleccion para calculos 100 ureteronefroscopias por ano. Mas de la mitad utiliza fluoroscopia y camisa de acceso ureteral rutinariamente, el metodo mas frecuente para la evaluacion del estado libre de calculos es la radiografia simple de abdomen.

Introduction The use of flexible ureterorenoscopy for treating kidney stones has increased in recent years, with considerable worldwide variation in the surgical technique and indications. Objectives To determine the current practice, technique variations, use and indications of flexible ureterorenoscopy for treating kidney stones in Latin American. Methods We sent (by email and web link) an anonymous questionnaire with 30 questions on flexible ureterorenoscopy for treating kidney stones to Latin American urologists from January 2015 to July 2015. We collected the responses through the Survey Monkey system. Results A total of 283 urologists in 15 Latin American countries participated (response rate, 10.8%); 254 answered the questionnaire completely; 52.8% were urologists from Mexico and 11% were from Argentina; 11.8% of the responders stated that they performed >100 cases per year; 15.2% considered ureterorenoscopy as the treatment of choice for stones >2 cm, and 19.6% performed ureterorenoscopy in single stages for calculi measuring >2.5 cm. Some 78.4% use fluoroscopy, 69.1% use a ureteral sheath in all cases, 55.8% place double-J catheters at the end of surgery, 37.3% considered a stone-free state to be 0 fragments, and 41.2% use plain radiography to assess the stone-free condition. Conclusions Most participating urologists consider flexible ureterorenoscopy as the first-choice treatment for stones 100 ureterorenoscopies per year. More than half of the urologists routinely used fluoroscopy and ureteral access sheath; the most common method for determining the stone-free state is plain abdominal radiography.

Background Co-transcriptional R-loops are abundant non-B DNA structures in mammalian genomes. DNA Topoisomerase I (Top1) is often thought to regulate R-loop formation owing to its ability to resolve both positive and negative supercoils. How Top1 regulates R-loop structures at a global level is unknown. Results Here, we perform high-resolution strand-specific R-loop mapping in human cells depleted for Top1 and find that Top1 depletion results in both R-loop gains and losses at thousands of transcribed loci, delineating two distinct gene classes. R-loop gains are characteristic for long, highly transcribed, genes located in gene-poor regions anchored to Lamin B1 domains and in proximity to H3K9me3-marked heterochromatic patches. R-loop losses, by contrast, occur in gene-rich regions overlapping H3K27me3-marked active replication initiation regions. Interestingly, Top1 depletion coincides with a block of the cell cycle in G0/G1 phase and a trend towards replication delay. Conclusions Our findings reveal new properties of Top1 in regulating R-loop homeostasis in a context-dependent manner and suggest a potential role for Top1 in modulating the replication process via R-loop formation. Electronic supplementary material The online version of this article (10.1186/s13059-018-1478-1) contains supplementary material, which is available to authorized users.

DNA Topoisomerase I (Top1) is required to relax DNA supercoils generated by RNA polymerases (RNAPs). Top1 is inhibited with high specificity by camptothecin (CPT), an effective anticancer agent, and by oxidative base damage and ribonucleotides in DNA strands, resulting into Top1-DNA cleavage complexes (Top1ccs). To understand how Top1ccs affect genome stability, we have investigated the global transcriptional response to CPT-induced Top1ccs. Top1ccs trigger an accumulation of antisense RNAPII transcripts specifically at active divergent CpG-island promoters in a replication-independent and Top1-dependent manner. As CPT increases antisense transcript levels in the presence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, a transcription inhibitor, Top1ccs likely impair antisense RNA degradation. Time-course data showed a burst of Top1ccs increased by CPT at promoter sites and along transcribed regions, causing a transient block of RNAPII at the promoter. Moreover, cell immunofluorescence analyses showed that Top1ccs induce a transient increase of R-loops specifically at highly transcribed regions such as nucleoli in a Top1-dependent manner. Thus, a specific and highly dynamic transcriptional response to Top1ccs occurs at divergent active CpG-island promoters, which may include a transient stabilization of R-loops. The results clarify molecular features of a response pathway leading to transcription-dependent genome instability and altered transcription regulation.

Triptolide is a bioactive ingredient in traditional Chinese medicine that exhibits diverse biologic properties, including anticancer properties. Among its many putative targets, this compound has been reported to bind to XPB, the largest subunit of general transcription factor TFIIH, and to cause degradation of the largest subunit Rpb1 of RNA polymerase II (RNAPII). In this study, we clarify multiple important questions concerning the significance and basis for triptolide action at this core target. Triptolide decreased Rpb1 levels in cancer cells in a manner that was correlated tightly with its cytotoxic activity. Compound exposure blocked RNAPII at promoters and decreased chromatin-bound RNAPII, both upstream and within all genes that were examined, also leading to Ser-5 hyperphosphorylation and increased ubiqutination within the Rbp1 carboxy-terminal domain. Notably, cotreatment with inhibitors of the proteasome or the cyclin-dependent kinase CDK7 inhibitors abolished the ability of triptolide to ablate Rpb1. Together, our results show that triptolide triggers a CDK7-mediated degradation of RNAPII that may offer an explanation to many of its therapeutic properties, including its robust and promising anticancer properties. Cancer Res; 72(20); 5363–73. ©2012 AACR.

Here we propose a straightforward method to functionalize gold nanostructures by using an appropriate peptide sequence already selected toward gold surfaces and derivatized with another sequence for the capture of a molecular target. Large scale 3D-plasmonic devices with different nanostructures were fabricated by means of direct nanoimprint technique. The present work is aimed to address different innovative aspects related to the fabrication of large-area 3D plasmonic arrays, their direct and easy functionalization with capture elements, and their spectroscopic verifications through enhanced Raman and enhanced fluorescence techniques.

ResumenEl divertículo congénito de la uretra es una entidad extremadamente rara, al parecer a la fecha sólo se han descrito alrededor de 260 casos. La mayoría de los casos publicados han sido diagnosticados en la infancia, debido a infecciones urinarias a repetición, síntomas obstructivos urinarios bajos, aumento del tamaño uretral en la exploración física y vejiga de esfuerzo con o sin reflujo secundario.Se presenta el caso de un niño de 5 años de edad, con incontinencia urinaria y aumento de tamaño importante en la parte ventral del pene, diagnosticado con un divertículo uretral congénito por uretrocistografía, sin vejiga de esfuerzo, ni reflujo vesicoureteral secundario, quien fue tratado con uretroplastía en un solo tiempo (técnica abierta), sin complicaciones, cursando un posquirúrgico adecuado. Se realiza seguimiento periódico, resolviendo la incontinencia urinaria, completamente asintomático, sin infecciones de vías urinarias, con adecuado flujo de orina y un pene estéticamente normal a 2 años de seguimiento.AbstractCongenital urethral diverticulum is an extremely rare entity and only an approximate 260 cases have been reported to date. The majority of published cases were diagnosed at infancy due to recurrent urinary infections, symptoms of lower urinary tract obstruction, an enlarged urethra found upon physical examination, and a decompensated bladder with or without secondary reflux.We present herein the case of a 5-year-old boy with urinary incontinence and an important increase in size in the ventral part of the penis. Diagnosis of a congenital urethral diverticulum was made through urethrocystography; there was no bladder decompensation or secondary vesicoureteral reflux. The patient was treated with single-stage urethroplasty (open technique) with no complications and the postoperative period was adequate. His urinary incontinence was resolved and he was completely asymptomatic at periodic follow-ups with no further urinary tract infections. Two years after treatment, he has satisfactory urine flow and an esthetically normal penis.

OBJECTIVE: To investigate the expression of thymosin beta10 - a small conserved acidic protein involved in the inhibition of actin polymerization - in human and experimental thyroid goiters as well as the regulation exerted by TSH on thymosin beta10 expression in thyroid follicular cells both in vivo and in vitro. DESIGN: To this aim, we have used 5 bioptic specimens from patients affected by thyroid goiter, a well known experimental model of thyroid goitrogenesis (rat fed with the drug propylthiouracil) and a cultured rat thyroid cell line (PC Cl 3 cells) as a model system. RESULTS: We report that the mRNA expression of thymosin beta10 is markedly enhanced in human goiters compared with normal thyroid. In vivo results showed that the steady-state level of thymosin beta10 mRNA is up-regulated in the thyroid gland of propylthiouracil-fed rats in parallel with follicular cell proliferation: iodide administration to goitrous rats, which induced a marked involution of thyroid hyperplasia, reduced the mRNA level of thymosin beta10. Finally, in vitro studies showed that in cultured rat thyrocytes, the expression of thymosin beta10 mRNA is induced in a time- and dose-dependent manner by the activation of pathways which are mitogenic for thyroid cells (i.e. the protein kinase (PK) A and PKC pathways). CONCLUSION: Taken together, the findings reported here demonstrate that thymosin beta10 expression is regulated by extracellular signals that stimulate growth of thyroid cells both in vitro and in vivo, and suggest a role for this protein in thyroid diseases characterized by proliferation of follicular cells.

G-quadruplexes (G4s) and R-loops are non-B DNA structures that can regulate basic processes such as transcription and replication. Unscheduled formation of R-loops is regarded as highly deleterious to cells, as R loops can induce replicative stress and DNA damage leading to genome instability. G4s are formed by four guanine residues held together by Hoogsteen hydrogen bonds and stabilized by monovalent cations. R loops are triple-stranded structures that contain an RNA-DNA hybrid duplex and a displaced single-stranded DNA. We have recently shown that Topoisomerase I (Top1) can affect genome-wide levels of R loops in human cancer cells consistently with the knowledge that DNA superhelical tension is a main driving force allowing non-B DNA structure formation. As G4 ligands were suggested to synergize with Top1 inhibitors, we asked the question of whether G4 ligands can affect R loops. We have thus investigated by immunofluorescence microscopy the effects of two different G4 binders, Pyridostatin (PDS) and FG (compound 1 in Amato et al J Med Chem 2016), on R-loops in human cancer cells. These G4 binders can increase both G4s and R loops while an inactive derivative of FG cannot induce them. Interestingly, the induction of G4s and R loops well correlate to each other in time and intensity with PDS being more effective than FG. After 24 hours, G4 binders induce a cell cycle arrest at the G2/M phase associated to double-stranded DNA cleavage as detected by γH2AX and 53BP1 foci formation and to activation of checkpoint response as shown by ATM phosphorylation. Interestingly, overexpression of RNaseH1 reduces both R loops and γH2AX foci induced by G4 binders showing that the ligands induce DNA cleavage via an R loop-mediated mechanism. Then, we silenced the BRCA2 gene by RNAi in U2OS cells and the results show that BRCA2 depletion increased γH2AX foci induced by PDS while overexpression of RNaseH1 rescue DNA cleavage induction. We also determined genomic R loop maps by DRIP-seq, and bioinformatic analyses of the specific location of G4-stabilized R loops provided information consistent with a model in which a G4 opposite to a DNA:RNA hybrid can stabilize R loops. Our study establishes for the first time that G4 binders stabilize either G4s and R loops in human cancer cells, and that they induce genome instability with a mechanism dependent on R loop formation. Partially supported by AIRC, Milan. Citation Format: Alessio De Magis, Stefano Giustino Manzo, Marco Russo, Olivier Sordet, Rita Morigi, Giovanni Capranico. R loop-driven genome instability by G-quadruplex binders in BRCA2-silenced human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4838.

The use of flexible ureterorenoscopy for treating kidney stones has increased in recent years, with considerable worldwide variation in the surgical technique and indications.To determine the current practice, technique variations, use and indications of flexible ureterorenoscopy for treating kidney stones in Latin American.We sent (by email and web link) an anonymous questionnaire with 30 questions on flexible ureterorenoscopy for treating kidney stones to Latin American urologists from January 2015 to July 2015. We collected the responses through the Survey Monkey system.A total of 283 urologists in 15 Latin American countries participated (response rate, 10.8%); 254 answered the questionnaire completely; 52.8% were urologists from Mexico and 11% were from Argentina; 11.8% of the responders stated that they performed100 cases per year; 15.2% considered ureterorenoscopy as the treatment of choice for stones2cm, and 19.6% performed ureterorenoscopy in single stages for calculi measuring2.5cm. Some 78.4% use fluoroscopy, 69.1% use a ureteral sheath in all cases, 55.8% place double-J catheters at the end of surgery, 37.3% considered a stone-free state to be 0 fragments, and 41.2% use plain radiography to assess the stone-free condition.Most participating urologists consider flexible ureterorenoscopy as the first-choice treatment for stones2cm; a small percentage of these urologists perform100 ureterorenoscopies per year. More than half of the urologists routinely used fluoroscopy and ureteral access sheath; the most common method for determining the stone-free state is plain abdominal radiography.

Wafer thinning process before dicing, die attach, wire bonding, and in resin molding package operations requires the protection of the silicon wafer surface by means of a sticking grinding tape. The adhesive layer between this tape and wafer front side can leave glue residuals both in terms of ''macroscopic'' particles and organic ultra-thin layer. The organic contamination (OC) in these features is potentially detrimental for a good final passivation to plastic molding compound adhesion, leading to local package de-lamination, especially at the silicon die corners, where stress effects are known to be maximized. As a consequence, reliability issues are enhanced right from the interface original weakness. Different empirical and analytical techniques are available in order to characterize the starting conditions of final passivation surface and an oxygen plasma cleaning process has been chosen as most reliable and applicable eventually in a manufacturing environment to overcome such a kind of constraints. As a matter of fact, oxygen burns out the organic nature particles and ''mono-layer'', eventually left on the wafer front side after back grinding tape peeling off operation. This paper, deals with the direct adhesion study of the package resin to silicon nitride and PSG final passivation films correlated to water droplet contact angle, ToF-SIMS measurements, and packaging resin shear tests, revealing the effectiveness of a downstream oxygen plasma cleaning treatment in increasing plastic package to die mechanical coupling.

The insertion of Argentina into the capitalist world system was not performed in the nineteenth and twentieth centuries from a fully linear homogeneous process. The country thus has a mosaic of agricultural practices and differentiated realities rationalities. The eco-Catamarca-La Rioja region has historically been structured through a social configuration in which prevail microfundios and a traditional economy oriented to the satisfaction of the family or the local market needs. In this context, the wine sector has a prominent place with good projection in the future if it is noted that, in recent years, most dynamic productive agents eco-region have incorporated new technologies and production techniques How these changes have impact on the world of small traditional producers? This paper, from the theoretical categories of Bourdieu, seeks to provide elements that help to elucidate this question by analyzing the “production strategies” chilecitenos small wine producers. This analysis is performed using as a frame of reference empirical 67 interviews in Anguinan, Malligasta and Tilimuqui between 2010 and 2013 as part of a research project of the UNdeC. The observed results warn of the possible existence of a group of traditional producers with a fledgling business rationale that merits a differentiated intervention.

Expectancy and efficacy theories provide the foundation for the development of a measure of sport confidence. The Carolina Sport Confidence Inventory (CSCI) was conceptualized as a three-factor model represented by dispositional optimism, perceived competence, and perceived control. Two independent studies supported a two factor (dispositional optimism and perceived competence) 13- item model for the CSCI. The first study utilized exploratory factor analytic techniques, concurrent validity, social desirability assessment, internal consistency, and test-retest reliability to establish the psychometric properties of the CSCI. Study II employed confirmatory factor analytic techniques with a sample of 123 intercollegiate varsity athletes along with measures of convergent validity to provide further support for the two factor model and the psychometric properties of the instrument.

To evaluate the stability of an extemporaneously prepared 7% chloral hydrate syrup under different conditions of storage and dispensing.Three batches of 7% chloral hydrate syrup were prepared. Each batch was stored in 50 light-resistant glass containers of 60 mL with child-resistant caps and in two bottles of 1000 mL to simulate two forms of dispensing, mono and multi-dose, respectively. Twenty five mono-dose bottles and a multi-dose bottle of each batch were stored under room conditions (20 ± 1 °C) and the rest of the samples were stored in the fridge (5 ± 2 °C). The physical, chemical and microbiological stability was evaluated for 180 days. Stability was defined as retention of at least 95% of the initial concentration of chloral hydrate, the absence of both visible particulate matter, or color and/or odor changes and the compliance with microbiological attributes of non-sterile pharmaceutical products.At least 98% of the initial chloral hydrate concentration remained throughout the 180-day study period. There were no detectable changes in color, odor, specific gravity and pH and no visible microbial growth. These results were not affected by storage, room or refrigeration conditions or by the frequent opening or closing of the multi-dose containers.Extemporaneously compounded 7% chloral hydrate syrup was stable for at least 180 days when stored in mono or multi-dose light-resistant glass containers at room temperature and under refrigeration.

DNA topoisomerases are enzymes responsible for the relaxation of DNA torsional strain, as well as for the untangling of DNA duplexes after replication, and are important cancer drug targets. One class of topoisomerase inhibitors, "poisons", binds to the transient enzyme-DNA complex which occurs during the mechanism of action, and inhibits the religation of DNA. This ultimately leads to the accumulation of DNA double strand breaks and cell death. Different types of topoisomerases occur in human cells and several poisons of topoisomerase I and II are widely used clinically. However, their use is compromised by a variety of side effects. Recent studies confirm that the inhibition of the α-isoform of topoisomerase II is responsible for the cytotoxic effect, whereas the inhibition of the β-isoform leads to development of adverse drug reactions. Thus, the discovery of agents selective for topoisomerase IIα is an important strategy for the development of topoisomerase II poisons with improved clinical profiles. Here, we present a computer-aided drug design study leading to the identification of structurally novel topoisomerase IIα poisons. The study combines ligand- and structure-based drug design methods including pharmacophore models, homology modelling, docking, and virtual screening of the National Cancer Institute compound database. From the 8 compounds identified from the computational work, 6 were tested for their capacity to poison topoisomerase II in vitro: 4 showed selective inhibitory activity for the α- over the β-isoform and 3 of these exhibited cytotoxic activity. Thus, our study confirms the applicability of computer-aided methods for the discovery of novel topoisomerase II poisons, and presents compounds which could be investigated further as selective topoisomerase IIα inhibitors.

The dbl oncogene, generated by the truncation of the amino-terminal portion of the proto-oncogene sequence, encodes a guanine-nucleotide-releasing factor. The transforming activity of this oncogene has never been demonstrated in vivo or in vitro except in the NIH 3T3 mouse fibroblast cell line. The expression of the proto-dbl transcript is confined to tissues and tumors of neuroectodermal derivation. Therefore, to study the transforming activity of the dbl oncogene in vivo, we have generated transgenic mice that express this oncogene in neuroepithelial tissues. Mice carrying the dbl oncogene did not develop a tumor. Successively, to establish whether dbl interacts with the tumor suppressor gene p53 in tumorigenesis, we have used a p53 deficient mouse strain. The results reported here indicate that dbl is capable of causing tumor formation in vivo when its expression is driven in an appropriate cellular and genetic environment. The dbl oncogene, generated by the truncation of the amino-terminal portion of the proto-oncogene sequence, encodes a guanine-nucleotide-releasing factor. The transforming activity of this oncogene has never been demonstrated in vivo or in vitro except in the NIH 3T3 mouse fibroblast cell line. The expression of the proto-dbl transcript is confined to tissues and tumors of neuroectodermal derivation. Therefore, to study the transforming activity of the dbl oncogene in vivo. we have generated transgenic mice that express this oncogene in neuroepithelial tissues. Mice carrying the dbl oncogene did not develop a tumor. Successively, to establish whether dbl interacts with the tumor suppressor gene p53 in tumorigenesis, we have used a p53 deficient mouse strain. The results reported here indicate that dbl is capable of causing tumor formation in vivo when its expression is driven in an appropriate cellular and genetic environment. (C) 1995 Academic Press, Inc.

Stem tumour cells would be paraembryonal cells, major histocompatibility complex lacking and able of inducing adjoining cells to become tumour differentiated cells with major histocompatibility complex; thus, they would generate a histological tumour organization into paraembryonal cell clusters surrounded by tumour-differentiated cells. Such an organization, actually found in recent studies, might be one factor responsible for the limits of the immunotherapies carried out so far. But, there might be another, perhaps more important, factor. Analysis of ontogenetic aspects of MHC-nonrestricted immunity would lead, indeed, to the prediction that natural killer clones reactive for stem tumour cells (paraembryonal cells) would be missing in the adult organism, since they would be deleted in particular ontogenetic phases. This might explain why natural killer cells locate only in certain organs and nearly not at all in tumour sites. By such an analysis, possible evolutive and functional correlations between natural killer cells and heat shock proteins, hemopoietic histocompatibility, major histocompatibility complex molecules are suggested. From here, explanations and implications for grafts, pregnancy and tumour immunoprophylaxis arise.

In the following paper we describe an event position reconstruction and energy correction technique that is being implemented on the High Pressure Gas Scintillation Proportional Counter (HPGSPC), that will be flown aboard the Italian Satellite for X-ray astronomy, SAX. We discuss how this technique will greatly enhance the spectroscopic performances of the detector and we report some results regarding the full area energy resolution of the scientific model of the HPGSPC.

We report a measurement of the total photoelectron yield of gold as a function of the incident x‐ray polarization. Polarized x rays with energies of 2.5, 2.7, and 4.0 keV were used to excite a gold photocathode at a glancing incidence angle between 5° and 50°. Contrary to a previous report, we found no measurable polarization dependence for all three x‐ray energies. We conclude that the modulation factor, i.e., the fractional change of the total yield when the polarization state is varied, is less than 4%.

Neurofibromatosis 1 is an autosomal dominant disease with an estimated incidence 1:2500 to 1:3000 live newborns. The disease presents with multiple cutaneous and non cutaneous lesions. NF1 occurs with equal frequency in males and females and has been identified in all ethnic group. The morbidity and the mortality caused by NF1 are the result of complications that may involve any of the body systems. This disease has been linked with mutations of the NF1 gene which encodes tumor suppressor neurofibromin. At least half of patients with NF1 will have only cutaneous involvement that is not considered to be a major medical problem, even though it can be a source of psychologic burden as a result of cosmetic disfigurement. The cardinal features of the disorder are cafè-au-lait spots, axillary freckling, cutaneous neurofibromas and Lisch nodules, but there are a lot of wide variety of complications affecting almost every system of the body, including the eyes (optic glioma), the nervous system (intracranial tumors), the skeleton (short stature, scoliosis), the endocrine and cardiovascular system (hypertension). Manifestations of NF1 vary at different times in an individual's life. Substantial variability exists among affected members of a single family. This variability confounds clinical management and the severity of the disease cannot be predicted. We present a case in young woman 24 years-old treated by reiterative plastic surgery.

The Stellar X-Ray Polarimeter SXRP will be flown at the focus of the SODART X-ray telescope aboard the Russian satellite SPECTRUM-X-Gamma by the end of 1995. Four imaging proportional counters will detect photons diffracted by a graphite crystal (2.6 and 5.2 keV) and scattered from a lithium rod enclosed in a beryllium case (from 5 to 20 KeV). The counters are position-sensitive by the Wedge and Strip (WS) readout method. The gas mixture is 80% xenon, 10% argon and 10% methane. In this contribution we resume the results of the measurements performed on the engineering models of the counters. Position resolution of about 1.5 mm is attained with an energy resolution of about 25%. The pulse shape discrimination offers more than 98%60Co events rejection, with at least 90% X-ray acceptance. Some improvements are foreseen for the flight units in terms of sensitivity and uniformity of response.

Stellar X-ray polarimetry has for a long time been indicated as a very powerful diagnostic tool. In spite of this widely recognised interest, positive results are limited to just one: the detection, in the now far 1978, of the polarisation of the X-ray emission from the Crab Nebula. Novel-generation experiments promise a wider and richer amount mess of results, at least for strong galactic sources. Polarimetry however, remains an unusually delicate technique that requires a very tight control of systematic effects (at the level of 1% or better) over the very long observing period (105 seconds or more) needed for obtaining a reasonable sensitivity.

The Stellar X-ray Polarimeter (SXRP) is a focal plane instrument which will be flown on the SPECTRUM-X-Gamma mission in 1993. The polarimeter is composed of two separate instruments: the first exploits the dependence on the polarization of the Bragg reflection from a graphite crystal, and of the Thomson scattering from a metallic lithium target. The second instrument makes use of the recently discovered polarization dependence of X-ray photoemission from CsI. The SXRP will permit sensitive measurements of several classes of galactic X-ray sources, such as X-ray pulsars, black-hole candidates and supernova remnants. Moreover, and for the first time, SXRP will be able to perform highly sensitive measurements of the brightest extragalactic sources.

We report results from multifrequency observations of the X-ray source 4U1700+24/HD154791. This source was observed simultaneously with the EXOSAT and IUE satellites on March 25, 1985, and it is continuously monitored in the optical band at the Bologna Astronomical Observatory since July 1984. Our observations show source features which are different from those measured during previous observations. The X-ray spectrum is significantly softer than in previous measurements, and much more absorbed; high excitation lines in UV spectra (C IV and N V) have disappeared; the optical spectrum, despite a fairly good photometric stability of the M giant HD154791 in V and B bands also shows differences with respect to previous observations. We discuss our results in the framework of both a binary model and a coronal model. We point out that our observations strengthen the hypothesis that the X-ray emission is due to mass accretion onto a compact object orbiting around the M giant star HD154791.

A description of one of the experiments on board the Italian X-ray Astronomy Satellite SAX is presented. The principle of operation and the technological problems are discussed in some detail.

Placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) represent two closely related angiogenic growth factors active as homodimers or heterodimers. Since goiters of the thyroid gland are extremely hypervascular, we investigated the expression of PlGF, VEGF and their receptors, Flt-1 and Flk-1/KDR, in a small panel of human goiters from patients with Graves's disease, in an animal model of thyroid goitrogenesis and in in vitro cultured thyroid cells. Here we report that the mRNA expression of PlGF, VEGF and their receptors is markedly enhanced in biopsies of goiters resected from Graves's patients. in vivo studies demonstrated that in the thyroid gland of thiouracil-fed rats, increased mRNA and protein expression of PlGF, VEGF, Flt-1 and Flk-1/KDR occurred subsequent to the rise in the serum thyroid stimulating hormone (TSH) levels and in parallel with thyroid capillary proliferation. In vitro studies confirmed the existence of such TSH-dependent paracrine communication between thyroid epithelial cells and endothelium since the conditioned medium collected from TSH-stimulated thyrocytes acquired mitogenic activity for human umbilical vein endothelial (HUVE) cells, Altogether, these data suggest that PlGF and VEGF, released by thyrocytes in response to the chronic activation of the TSH receptor pathway, may act through a paracrine mechanism on thyroid endothelium.

I have previously assumed that in tumours there are stem cells, that owing to the morphophysiologic properties shared with embryonal cells I have defined as ‘para-embryonal’ cells (PECs). Owing to a blocking mutation, PECs might be able to express only the genic program upstream from the block, but not that downstream. As a consequence, PECs might lack in genic differentiated products, such as MHC molecules, and might be very rich in primitive genic products, such as HSP70 molecules. Like embryonal cells, PECs might carry out induction on adjoining hyperplastic cells, thus transforming them, only phenotypically, into ‘differentiated para-embryonal cells’ (DPECs), endowed with both MHC and HSP70 molecules. In such a way, nuclei of MHC-non-expressing/HSP70-high expressing stem tumour cells might be surrounded by layers of MHC-expressing/HSP70-expressing non-stem tumour cells. Such a structural tumour organization, actually found by C. Cordon Cardo et al. with regard to the MHC molecule expression, might be responsible for interference phenomena versus the MHC-non-restricted immune cells, such as macrophages and NK cells. So, these cells, the only potentially able to recognize and eliminate MHC-non-expressing stem tumour cells (PECs), might spare them, thus rendering cancer a biological process without any natural immunological solution. Now, I would like to theorically demonstrate that cancer might be a process without immunological solution from the very beginning: the first stem tumour cell might be tolerated as a sort of ‘primordial self’ because of its MHC-null/HSP70-very high phenotype, recognizable by neither the MHC-restricted nor the MHC-non-restricted immunesurveillance systems of the host. Possible biological roles of HSP70 molecules might account for the immunesurveillance escape of stem tumour cells. Existence of these cells appears to be confirmed by the recent experiments of immunotherapy with autologous tumour-specific HSPs carried out by P. K. Srivastava; moreover, their ‘self’ nature appears to be confirmed by the most recent experiments of compatible bone marrow allograft carried out by A. M. Carella. On this ground, the main steps for a resolutive antitumour immunotherapy are proposed.

The close association of heat shock protein (HSP), haemopoietic histocompatibility (Hh), major histocompatibility complex (MHC), and complement genes on the same chromosomal region, and the fact that all these genes are inherited on the whole in each haplotype of an individual, might indicate some evolutionary and functional correlations among them. Several data suggest for HSP70 molecules a possible role as a molecular target recognizable by natural killer (NK) cells. HSP70 sequences from both prokaryotic and eukaryotic organisms reveal that about half of the amino acid residues are identical and many of the remaining residues are similar. I here assume that NK reactivity might start, early in the immunogenesis process, as a effect of the interaction between HSP70 molecules and a hypothetical HSP receptor of yet immature non-cytolytic NK cells. To this receptor, an HSP molecule might act as an activator or an inhibitor depending on whether its amino acid residues are reactive or not with it, respectively. Later in the immunogenesis process, murine Hh or human equivalent molecules, dominantly expressed in bone marrow target cells, might select the non-reactive NK clones of an individual, inducing them to mature and express a lytic machinery. As a consequence of the NK maturation, proliferating hemopoietic target cells expressing only or mainly activator HSPs on their surface might undergo NK cytolysis. This might explain the NK lysis of apparently normal cells found in human foetal marrow; moreover, this might explain in some way the F1 hybrid resistance phenomenon. The NK reactivity of an individual would be further modulated by the expression on the NK surface of particular receptors (CD94, p58) specific for defined MHC molecules (Cw1, Cw3, Bw6, B7) on the target cells. Such a specific interaction would induce an 'NK effector inhibition'. The NK reactivity mechanism might have been further evolutionarily modified and adapted by the involvement of other NK receptors, such as CD11b (specific for the C3b factor of the complement) and CD16 (specific for the IgG Fc piece). Cooperation among HSP, MHC, CD11b, CD16, C3b and Fc allows us to propose original models of the activation and cytolysis mechanisms in the NK cytotoxicity and antibody-dependent cell cytotoxicity phenomena.

I have previously assumed that stem tumour cells are ‘para-embryonal cells’ (PECs) poor or missing in major histocompatibility complex (MHC) antigens. PECs might induce adjoining differentiated hyperplastic cells to also express tumoral phenotype and properties, thus transforming them into ‘differentiated para-embryonal cells’ (DPECs), MHC endowed. In such a way, PECs, MHC-lacking, would be automatically surrounded by DPECs, MHC-endowed: this tumour organization was experimentally found by Cordon-Cardo et al in a variety of cancers. Now, I suggest that such a tumour histology might preferentially induce an anti-DPEC T cell immune response which, sparing PECs, might release increasing amounts of DPEC antigens in the peritumour site. DPEC antigens might increase synthesis of specific antibodies and subsequent immunocomplex formation at the peritumour site. Here, abundant immunocomplexes might react through their Fc pieces with CD16 receptors of antibody-dependent cell cytotoxicity (ADCC)-endowed immune cells (natural killer (NK) cells, macrophages, polymorphonuclear cells). These cells would thus be stimulated to secrete their lytic factors before and without their coming into contact with target tumour cells. On the other hand, abundant immunocomplexes at the peritumour site might massively activate the complement system, thus generating large amounts of C3b. C3b might react with CD11b receptors of NK cells, stimulating them to also secrete their lytic factors in an ectopic way at the peritumour site, thus impairing NK cytotoxicity. In such a way, in the absence of ADCC and NK cytotoxicity, a tumour cell enhancement might easily occur. In the light of these ideas, a strategy for antitumour immunotherapy and vaccine is then proposed.

DNA Topoisomerase I (Top1) is specifically inhibited by camptothecin (CPT), a natural product with effective anticancer activity. CPT can stabilize a DNA-Top1 DNA cleavage complex that can lead to irreversible DNA breakage at replication forks, and transcription-dependent genome instability. The bulk of cellular Top1 activity is required at transcribing regions to modulate DNA supercoils generated by elongating RNA polymerases. However, the molecular effects of CPT inhibition of Top1 at transcriptional levels are not fully established. By next generation sequencing analyses of bisulfite-treated RNAs extracted from human HCT116 cancer cells, we here show that CPT promoted high levels of antisense tags specifically at active promoters containing CpG islands (CGI). We found several not annotated transcripts close to Refseq genes, the majority of which were promoter-associated antisense transcripts increased by CPT. We identified 256 and 84 of such transcripts in HCT116 cells and Top1-silenced HCT116-siRNATop1 cells, respectively. We validated CPT-stimulated antisense transcripts by PCR in both cell lines, and found that several of them could form R-loops at corresponding genomic regions by affinity purification with a recombinant inactive mutant RNaseH1. Drug effects were independent from replication, and required both Top1 and ongoing transcription. In addition, CPT induced an immediate burst and a subsequent rapid reduction of Top1-DNA cleavage complexes at active, but not inactive, promoters indicating a partial, time-dependent removal of Top1 from chromatin. Our findings demonstrate that Top1 activity prevents accumulation of antisense R-loops and RNAs at human active CGI promoters by relaxing negative DNA supercoils. The transcriptional CPT effects can contribute to drug therapeutic activity in cancer and other diseases. We will discuss our findings in relation to transcription regulation and genome instability caused by interference with Top1 activity. Citation Format: Jessica Marinello, Giovanni Chillemi, Susana Bueno, Stefano G. Manzo, Giovanni Capranico. Antisense transcripts and R-loops caused by DNA topoisomerase I inhibition by camptothecin at human active CpG island promoters. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 637. doi:10.1158/1538-7445.AM2013-637

JEM-X will extend the energy range of the gamma ray instruments on ESA's INTEGRAL mission (SPI, IBIS) to include the x-ray band. JEM-X will provide images with arcminute angular resolution in the 2 - 60 keV band. The baseline photon detection system consists of two identical, high pressure, imaging microstrip gas chambers, each with a collecting area of 500 cm2. They view the sky through a coded aperture mask (0.5 mm tungsten) at a separation of 3.4 m. The two detector boxes are formed from 2 mm thick stainless steel plate and are filled with 5 bar Xe. The field of view is defined by the collimator mounted on top of the detector. Each collimator consists of an array of bonded square tubes of Mo. The internal surface of these tubes is covered by a graded shield. The collimator provide also the support for the detector windows which are made out of 250 micrometer thick beryllium foils. The detector sensor elements consists of microstrip plates shaped as regular octagons with a diameter of 292 mm. The basic microstrip pattern is similar to the one chosen for the HEPC/LEPC detector system on SRG. The detector position resolution will be sufficient to ensure an angular resolution for JEM-X of better than 3 arcmin throughout the 2 - 60 keV band.© (1996) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Neoangiogenesis is a prerequisite for tumor growth and metastasis. In germ cell cancer patients with the disease limited to the testicle (stage A), tumor-associated neovascularization is predictive of metastatic disease (stage B). To investigate the molecular mechanisms underlying neovascularization in human germ cell tumors (GCTs), we analysed the expression of two angiogenic growth factors, vascular endothelial growth factor (VEGF) and placenta growth factor (P1GF), and of their receptors (FLT-1) and Flk-1/KDR) in a panel of testicular tumors. In this study we show a marked increase in VEGF expression in 36/44 (81.8%) primary testicular-derived GCTs, as compared to normal testis, that significantly correlates with a high density of intratumor microvessels (r = 0.72461, P0.001; n = 24). As determined by RT - PCR and/or Western blot, the predominant VEGF isoforms expressed in GCTs are the VEGF121 and VEGF165, which are more efficiently secreted by the cells, and thus more active in eliciting angiogenesis. Conversely, in the case of PIGF, only a weak correlation with the vascular density of tumors is observed (r = 0.26599, P0.05; n = 24). Northern blot analysis also revealed significant up-regulation of VEGF/ PIGF receptors in highly vascularized germ cell tumors, compared to normal testes. These findings suggest that VEGF may act in a paracrine manner to induce neovascularization, oedema extravasation and cyst formation in human germ cell tumors. The correlation between VEGF expression and the vascular density of tumors, suggest that the evaluation of VEGF expression may be of help in predicting patients at risk for metastatic diseases. Finally, we demonstrate that VEGF up-regulation may occur at the RNA level since no gene amplification is observed; conversely, in in vitro models such as the embryonal stem cell line NTERA-2 and the choricarcinoma JEG-3 cell line, VEGF (but not PIGF) mRNA expression is regulated by hypoxic stress.

Gene rearrangements activating the RET proto-oncogene are frequently associated with human thyroid carcinomas belonging to the papillary subtype. These arrangements cause the fusion of the tyrosine-kinase domain of RET to the 5'-terminal region of different genes creating the RET/PTC chimeric oncogenes. Here we report the generation of transgenic mice lines expressing the RET/PTC1 oncogene under the control of the thyroid-specific rat thyroglobulin promoter. RET/PTC1-transgenic mice developed thyroid tumors displaying the histological aspect of papillary carcinomas. These tumors were slowly progressive and did not cause premature death of the animals. Two additional mice developed areas of thyroid hyperplasia. Immunohistochemical and reverse-transcriptase polymerase chain reaction analyses confirmed the thyroid-specific expression of the transgene. Given the frequency of activating rearrangements of RET in human papillary thyroid carcinomas we conclude that this animal system could be a good model for studying the neoplastic progression of thyroid carcinomas.

The high pressure gas scintillation proportional counter (HPGSPC) is one of the narrow field instruments onboard the Italian Dutch mission for x-ray astronomy SAX. Sensitive in the 4 - 120 keV band the HPGSPC will investigate all categories of astrophysical sources emitting in the not yet well studied hard x-ray domain with a special emphasis on the cyclotron line features that are present in the hard x-ray spectrum of many celestial sources. The on-ground calibration of the flight model of the HPGSPC was carried out at Laben, Italy during October/November 1994. In this paper after describing the flight model of the HPGSPC instrument, we report preliminary results on its spectral capabilities and background rejection efficiency.

Several recent experiments were conducted to study the x-ray induced photoemission from solids by polarized x rays for its potential application to stellar x-ray polarimetry. However, it was shown recently that the earlier observed polarization dependence of the secondary photoelectron yield was affected by spurious effects and no polarization dependence could be observed after these spurious effects had been eliminated. Here, we report our first measurement of the primary photoelectrons, i.e., photoelectrons with energy higher than 50 eV. We measured the primary photoelectron yield of a silicon sample as a function of the polarization state of the incident 2.69 keV x-ray beam. We observed that the change of photoelectron yield for different polarization states is especially pronounced around the no-loss photoelectron peaks. This polarization dependency can be explained by the angular distribution of photoelectrons from free atoms excited by polarized x rays. We discuss the experimental results and their implications.© (1994) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The performance of the engineering prototype Stellar X-Ray Polarimeter (SXRP) has been evaluated. One hundred percent polarized monochromatic x rays at 2.6 keV and 9.7 keV were used to measure the response of the instrument in the energy bands of the graphite and lithium polarizing elements, respectively. On-line analysis showed that the respective depths of modulation are 96% ad 70% as expected. Irradiating SXRP with broadband unpolarized x rays in the energy band 2 - 17 keV demonstrated that the level of spurious modulation inherent in the instrument is less than 3%. Up-to-date results are presented and compared to the predictions of Monte Carlo simulations.

The Stellar X-Ray Polarimeter (SXRP) uses the polarization sensitivity of a graphite Bragg crystal and a lithium Thomsom scattering target to measure the polarization of X-rays from astrophysical sources. The SXRP is a focal plane detector for the Soviet-Danish SODART telescopes which will be launched on the Soviet Spectrum-X-Gamma mission. The SXRP will be the third orbiting stellar X-ray polarimeter, and should provide an order of magnitude increase in polarization sensitivity over its predecessors.

Transgenic mice have been generated bearing three fusion genes consisting of: (a) a 900-base pair rat thyroglobulin promoter followed by a gene coding for a chloramphenicol acetyl transferase activity; (b) the same promoter followed by the complementary DNA of the human activated Ki-ras oncogene; (c) a 2000-base pair rat thyroglobulin promoter followed by the complementary DNA of the human activated Ki-ras. We have shown that the 900-base pair rat thyroglobulin promoter is able to direct the expression of the reporter gene specifically in the thyroid gland of transgenic mice. The mice bearing the two Ki-ras constructs, which express the transgene in thyroid glands, show thyroid abnormalities, although at very low incidence. These lesions appear after a long latency and with a benign aspect, thus suggest that, in agreement with literature data on naturally occurring human thyroid tumors, the action of an activated ras gene is not sufficient to attain a complete malignant conversion of thyroid glands in vivo. However, ras expression in thyroid follicular cells represents a favorable ground for tumor development, as shown by the fact that goitrogen stimulation experiments increase the occurrence of tumors.