Solid modeling interactive interface for design drafting

Providing certain Solid Modeling software systems with Computer-Aided Design Drafting facilities allows these systems to produce industry acceptable engineering drawing displays. A method is put forth to provide these facilities through the design and implemention of a two-dimensional interactive graphics package that interfaces with these systems. This allows other graphical and textual elements to be added to displays produced by the Solid Modeling systems

Location as Vocation: An Urban College\u27s Engagement with Their Somali Neighbors

This article focuses on the significance of an urban college’s location and mission in guiding disciplines within the college to engage with its immigrant neighbors through service-learning.The authors describe how social connectedness theory and transformational learning theory were used to facilitate learning and increase students’ awareness of their sense of vocation.Student reflections on the service learning experience in both disciplines, religion and social work, are used to illustrate the application of each theory

L'eau comme élément symbolique dans La Chartreuse de Parme de Stendhal, Les Années de Virginia Woolf et dans Le Guépard de Tomasi di Lampedusa

[EN] In the novels The Chartehouse of Parme by Stendhal, The Years by Virginia Woolf and The Leopard by Tomasi di Lampedusa, water is defined as a symbolic element imbued with profound meanings. In The Chartehouse of Parme water refers to the nostalgia of Italy an idealized country. In particular the memory of Lake Como’s view, when the writer leaves Italy, is often mentioned in the novel. This lake is « sublime » to Fabrice’s eyes for whom « nothing so beautiful can be seen in the world ». As demonstrated by Gaston Bachalard, in the work of Standhal water has two different natures, the maternal one and the reverie one. In The Years by looking the water the characters seem to experience again the same feelings which the writers had in life and described in her private diary. Rosa’s images of the past emerge from the contemplation of water. This long-buried past is still painful to her. In the same way all the other characters are enchanted and hyptonized coming across the sight of this natural element. In The Leopard water is embodied in symbolic functions and in the level of style. As Prince Salina is dying in a hotel room on the seashore his inward death is depicted with aquatic images « Now it was not a river erupting over him but an ocean, tempestuous, all foam and raging white-flecked waves [...] ». Through this comparative reading of Stendhal, Virginia Woolf and Tomasi di Lampedusa it will be demonstrated how nostalgia, the detph of soul, life and death are depicted with the image of water[ES] En La Cartuja de Parma de Stendhal, Los Años de Virginia Woolf y en El Guepardo de Tomasi di Lampedusa, el agua está presente como elemento simbólico y se carga de sentidos profundos. En La Cartuja de Parma el agua remite a la nostalgia de la patria idealizada, Italia. En particular el recuerdo de la vista del lago de Como, antes de que el escritor abandonara Italia en 1821, aparece recurrentemente en el texto. Este lago es « sublime » a los ojos de Fabricio, para quien « nada tan bello se puede ver en el mundo ». Como muestra Gaston Bachelard, el agua puede asumir un doble sentido, maternal y de ensueño. La obra de Stendhal es la perfecta ilustración de ello. En Los Años, los personajes, mirando el agua, parecen revivir las mismas experiencias interiores que la escritora había vivido y descrito en su diario íntimo. Del agua emergen las imágenes del pasado de Rosa. Se trata de un pasado enterrado aún doloroso. De esa manera el agua fascina a los personajes que se paran a contemplar, como hipnotizados, este elemento natural. En El Guepardo, el agua, dotada de gran valor simbólico, también está presente a nivel estilístico. Cuando el príncipe de Salina muere en su habitación del hotel situada al borde del mar, su muerte es descrita con imágenes acuáticas: « ya no era un río que se desplegaba de él, sino un océano, una tempestad, erizada de espuma y grandes olas desatadas [...] ». En esta lectura comparada veremos como Stendhal, Tomasi di Lampedusa y Virginia Woolf, han representado en sus novelas la nostalgia, la profundidad del alma y el binomio vida-muerte a través del agua.[FR] Dans La Chartreuse de Parme de Stendhal, Les Années de Virginia Woolf et dans Le Guépard de Tomasi di Lampedusa, l’eau est présente en tant qu’élément symbolique et se charge de significations profondes. Dans La Chartreuse de Parme l’eau renvoie à la nostalgie de la patrie idéalisée, l’Italie. En particulier le souvenir de la vue du lac de Côme, avant que l’écrivain quitte l’Italie en 1821, revient dans le texte à plusieurs reprises. Ce lac est « sublime » aux yeux de Fabrice, pour qui « rien d’aussi beau ne peut se voir au monde ». Comme l’a montré Gaston Bachelard, l’eau, peut assumer un double caractère, maternel et de rêverie. L’œuvre de Stendhal en est la parfaite illustration. Dans Les Années, les personnages, en regardant l’eau, semblent revivre les mêmes expériences intérieures que l’écrivaine avait vécues et décrites dans son journal intime. De l’eau émergent les images du passé de Rose. Il s’agit d’un passé enseveli mais toujours douloureux. Ainsi, l’eau fascine les personnages qui s’arrêtent et contemplent, comme hypnotisés, cet élément naturel. Dans Le Guépard, l’eau se charge de fonctions symboliques mais elle est ainsi présente au niveau du style. Lorsque le prince Salina meurt dans sa chambre d’hôtel située au bord de la mer, la description intérieure de sa mort se fait par des images aquatiques : « Ce n’était plus un fleuve qui déferlait de lui, mais un océan, en tempête, hérissé d’écume et de grosses vagues déchaînées [...] ». À travers une lecture comparée, nous verrons comment Stendhal, Tomasi di Lampedusa et Virginia Woolf, ont représenté dans leurs romans la nostalgie, la profondeur de l’âme ainsi que le binôme vie-mort à travers l’eau.Maruggi, M. (2017). L'eau comme élément symbolique dans La Chartreuse de Parme de Stendhal, Les Années de Virginia Woolf et dans Le Guépard de Tomasi di Lampedusa. En Palabras e imaginarios del agua. Les mots et les imaginaires de léau. XXV coloquio AFUE. Editorial Universitat Politècnica de València. 513-520. https://doi.org/10.4995/XXVColloqueAFUE.2016.3132OCS51352

811. Correction of Laminin-5 β3 Chain Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors

Mutations in any of the genes encoding the laminin 5 heterotrimer (|[alpha]|3, |[beta]|3 and |[gamma]|2) cause junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. We and others have shown that expression of a retrovirally transferred |[beta]|3-chain cDNA in keratinocytes from affected patients reconstitutes normal synthesis, assembly and secretion of laminin 5, and corrects the adhesion defect in vitro and in vivo. We have recently started a phase-I clinical trial of gene therapy of JEB based on transplantation of cultured skin derived from autologous epidermal stem cells transduced with a MLV-derived retroviral vector. Since gamma- retroviral vectors have raised safety concerns for the genotoxic risk associated with the insertion of LTR elements into the human genome, we developed an alternative gene transfer strategy based on LTR- modified, HIV-derived lentiviral vectors. Two self-inactivating (SIN) lentiviral vectors were built, in which expression of either GFP or a LAMB3 cDNA is under the control of either a constitutive promoter (PGK) or the keratinocyte-specific, 2.2-kb promoter-enhancer of keratin 14 (K14). In a third construct, expression of the transgene is under the control of the viral LTR, modified by replacing the U3 region with two K14 enhancer elements. Analysis in human keratinocyte cultures and in full-thickness human skin equivalents reconstituted onto immunodeficient mice showed that GFP expression directed by the K14 elements is tissue-specific and restricted to the basal layer of the epidermis. Expression of laminin5 from the three alternative vectors was evaluated in keratinocyte cultures derived from skin biopsies of JEB patients. Biochemical and cell kinetics assays demonstrated transduction of epidermal clonogenic stem/progenitor cells and full phenotypic correction of JEB keratinocytes with all vectors. Southern blot analysis of individual cell clones showed that LTR-modified lentiviral vectors are genetically stable and integrate in multiple copies in the human genome. This study shows that the use of lentiviral vectors transcriptionally targeted to the basal keratinocytes by the insertion of restricted enhancer elements is an effective, and potentially safer, alternative for gene therapy of JEB

Advances in mRNA Vaccines for Infectious Diseases

During the last two decades, there has been broad interest in RNA-based technologies for the development of prophylactic and therapeutic vaccines. Preclinical and clinical trials have shown that mRNA vaccines provide a safe and long-lasting immune response in animal models and humans. In this review, we summarize current research progress on mRNA vaccines, which have the potential to be quick-manufactured and to become powerful tools against infectious disease and we highlight the bright future of their design and applications

NadA3 structures reveal undecad coiled coils and LOX1 binding regions competed by meningococcus B vaccine-elicited human antibodies

Neisseria meningitidis serogroup B (MenB) is a major cause of sepsis and invasive meningococcal disease. A multicomponent vaccine, 4CMenB, is approved for protection against MenB. Neisserial adhesin A (NadA) is one of the main vaccine antigens, acts in host cell adhesion, and may influence colonization and invasion. Six major genetic variants of NadA exist and can be classified into immunologically distinct groups I and II. Knowledge of the crystal structure of the 4CMenB vaccine component NadA3 (group I) would improve understanding of its immunogenicity, folding, and functional properties and might aid antigen design. Here, X-ray crystallography, biochemical, and cellular studies were used to deeply characterize NadA3. The NadA3 crystal structure is reported; it revealed two unexpected regions of undecad coiled-coil motifs and other conformational differences from NadA5 (group II) not predicted by previous analyses. Structure-guided engineering was performed to increase NadA3 thermostability, and a second crystal structure confirmed the improved packing. Functional NadA3 residues mediating interactions with human receptor LOX-1 were identified. Also, for two protective vaccine-elicited human monoclonal antibodies (5D11, 12H11), we mapped key NadA3 epitopes. These vaccine-elicited human MAbs competed binding of NadA3 to LOX-1, suggesting their potential to inhibit host-pathogen colonizing interactions. The data presented provide a significant advance in the understanding of the structure, immunogenicity and function of NadA, one of the main antigens of the multicomponent meningococcus B vaccine.IMPORTANCE The bacterial microbe Neisseria meningitidis serogroup B (MenB) is a major cause of devastating meningococcal disease. An approved multicomponent vaccine, 4CMenB, protects against MenB. Neisserial adhesin A (NadA) is a key vaccine antigen and acts in host cell-pathogen interactions. We investigated the 4CMenB vaccine component NadA3 in order to improve the understanding of its immunogenicity, structure, and function and to aid antigen design. We report crystal structures of NadA3, revealing unexpected structural motifs, and other conformational differences from the NadA5 orthologue studied previously. We performed structure-based antigen design to engineer increased NadA3 thermostability. Functional NadA3 residues mediating interactions with the human receptor LOX-1 and vaccine-elicited human antibodies were identified. These antibodies competed binding of NadA3 to LOX-1, suggesting their potential to inhibit host-pathogen colonizing interactions. Our data provide a significant advance in the overall understanding of the 4CMenB vaccine antigen NadA

725. Correction of Laminin-5-Deficient Junctional Epidermolysis Bullosa by Transplantation of Genetically Modified Epidermal Stem Cells. A Phase-I Clinical Trial

Mutations in genes encoding the laminin-5 heterotrimer, a key component of the epidermal-dermal junction, cause junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Epidermal stem cells isolated from patients affected by |[beta]|3 chain-deficient JEB were transduced with a retroviral vector expressing a |[beta]|3 cDNA, and used to generate uniformly transduced cultured skin implants. The transgene was steadily expressed for >160 cell doublings in culture, leading to restoration of normal laminin 5 levels, assembly of functional hemidesmosomes, and full phenotypic correction. Cloning and sequencing of vector integrations showed that <20 stem cells are responsible for long-term maintenance of a transplantable skin culture. A phase-I clinical trial started in October 2005, aimed at proving the safety of the transduction/transplantation procedure, and analyzing persistence of transgene expression and long-term survival of transduced stem cells. The first patient was a 30-yr-old male affected by non-lethal JEB, carrying a null mutation in one LAMB3 allele and a point mutation (E212K) in the other one. The mutation affects the assembly of the laminin-5 heterotrimer, present at residual levels (<5%) in vitro and in vivo. Six genetically modified, cultured epidermal sheets of 100 sq cm were transplanted on both legs after removal of the outer skin layer using a minimally invasive technique. The procedure was well tolerated, and the patient discharged after five days. Engraftment was completed after 10 days, and transplanted skin remained stable on both legs in the absence of blistering or inflammation for the duration of the follow-up (4 months at the time of writing). 3-mm punch biopsies were taken 1 and 3 months after transplantation, and analyzed for vector presence by quantitative PCR and for protein expression by immunohistochemistry. A vector signal compatible with full transduction of the transplanted epidermis was observed at both time points. Synthesis and assembly of normal levels of heterotrimeric laminin-5 and |[alpha]|6|[beta]|4 integrin was observed at the level of the basal lamina in all biopsies, together with the development of a firmly adherent, fully differentiated epidermis. Epidermal stem cells (p64+) were detected in the basal layer of the transplanted skin in normal numbers. These data show that gene therapy of JEB by transplantation of genetically corrected stem cells is feasible, and leads to full phenotypic correction of the adhesion defect in vivo. Safety studies are under way, which include detection or humoral or cytotoxic immune responses against laminin-5, and ex vivo cloning and sequencing of the integrated proviruses

Targeted Gene Addition in Human Epithelial Stem Cells by Zinc-finger Nuclease-mediated Homologous Recombination

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a “safe harbor” locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of >20% in a human keratinocyte cell line, >10% in immortalized keratinocytes, and <1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs

High-Definition Mapping of Retroviral Integration Sites Defines the Fate of Allogeneic T Cells After Donor Lymphocyte Infusion

The infusion of donor lymphocytes transduced with a retroviral vector expressing the HSV-TK suicide gene in patients undergoing hematopoietic stem cell transplantation for leukemia/lymphoma promotes immune reconstitution and prevents infections and graft-versus-host disease. Analysis of the clonal dynamics of genetically modified lymphocytes in vivo is of crucial importance to understand the potential genotoxic risk of this therapeutic approach. We used linear amplification-mediated PCR and pyrosequencing to build a genome-wide, high-definition map of retroviral integration sites in the genome of peripheral blood T cells from two different donors and used gene expression profiling and bioinformatics to associate integration clusters to transcriptional activity and to genetic and epigenetic features of the T cell genome. Comparison with matched random controls and with integrations obtained from CD34+ hematopoietic stem/progenitor cells showed that integration clusters occur within chromatin regions bearing epigenetic marks associated with active promoters and regulatory elements in a cell-specific fashion. Analysis of integration sites in T cells obtained ex vivo two months after infusion showed no evidence of integration-related clonal expansion or dominance, but rather loss of cells harboring integration events interfering with RNA post-transcriptional processing. The study shows that high-definition maps of retroviral integration sites are a powerful tool to analyze the fate of genetically modified T cells in patients and the biological consequences of retroviral transduction