A Cartan-Eilenberg approach to Homotopical Algebra

In this paper we propose an approach to homotopical algebra where the basic ingredient is a category with two classes of distinguished morphisms: strong and weak equivalences. These data determine the cofibrant objects by an extension property analogous to the classical lifting property of projective modules. We define a Cartan-Eilenberg category as a category with strong and weak equivalences such that there is an equivalence of categories between its localisation with respect to weak equivalences and the relative localisation of the subcategory of cofibrant objects with respect to strong equivalences. This equivalence of categories allows us to extend the classical theory of derived additive functors to this non additive setting. The main examples include Quillen model categories and categories of functors defined on a category endowed with a cotriple (comonad) and taking values on a category of complexes of an abelian category. In the latter case there are examples in which the class of strong equivalences is not determined by a homotopy relation. Among other applications of our theory, we establish a very general acyclic models theorem.

Comparação entre métodos de amostragem do solo para recomendação de calagem e adubação do cafeeiro conilon

O objetivo deste trabalho foi comparar a metodologia convencional de amostragem de solo com a análise espacial para a recomendação de calagem e adubação de nitrogênio, fósforo e potássio em solo cultivado com café conilon. O experimento foi realizado nas safras de 2004/2005 e 2005/2006, em área de 1,0 ha, com as amostras retiradas na profundidade de 0-0,20 m. No método convencional, coletaram-se 15 subamostras em caminhamento ziguezague, constituindo uma amostra composta, e, no método espacial, construiu-se uma malha amostral de 109 pontos georreferenciados. Com os resultados das análises, foi calculada a necessidade de calagem e adubação em função do teor do elemento no solo e da produtividade esperada das plantas. Os dados foram analisados por meio da estatística clássica (descritiva e exploratória) e pela análise espacial, utilizando técnicas de geoestatística (modelagem da estrutura de variabilidade espacial e realização de inferências) e geoprocessamento (álgebra de mapas). Com exceção da recomendação de fósforo em 2005 e potássio em 2006, todas as demais recomendações apresentaram dependência espacial. A análise dos dados pelo método espacial possibilitou identificar zonas de déficit ou excesso de calagem e de adubação na área, que não poderiam ser definidas com o método convencional de amostragem (ziguezague).

Uso da geotecnologia para caracterizar os cafezais no município de Londrina-PR, em relação à altimetria, declividade e tipo de solo

O sensoriamento remoto e o geoprocessamento são ferramentas importantes no apoio aos levantamentos de dados da agricultura. O estudo teve por objetivo relacionar as variáveis físicas, como altimetria, declividade e tipo de solo com o agrossistema cafeeiro, no município de Londrina-PR, por meio de imagem do sensor Thematic Mapper (TM) do satélite Landsat-5, aliado às técnicas de geoprocessamento. Foi criado um banco de dados com informações de altimetria, declividade e classes de solos, e através de álgebra de mapa, realizado o cruzamento destas informações com a localização das lavouras cafeeiras, a qual foi obtida por meio do classificador digital Bhattacharya aplicado na imagem. A imagem do TM possibilitou o mapeamento de 79% das lavouras cafeeiras e verificou-se que 86% destas lavouras estão em altitudes superiores a 540 m, e 50% estão localizadas sobre áreas de 8 a 20% de declividade. Em relação aos solos, observou-se que 53% dos cafeeiros estão localizados em Nitossolo Vermelho eutroférrico. Todas estas tarefas foram executadas por meio do programa SPRING 4.3.3, o qual foi uma adequada ferramenta para obter dados da agricultura a partir de imagens de satélites.

Immunogenicity of an inactivated bovine herpesvirus type 5 strain defective in thymidine kinase and glycoprotein E

The immunogenicity of an inactivated, experimental vaccine based on a bovine herpesvirus type 5 strain defective in thymidine kinase and glycoprotein E (BoHV-5 gE/TKΔ) was evaluated in cattle and the results were compared with a vaccine containing the parental BoHV-5 strain (SV507/99). To formulate the vaccines, each virus (wildtype SV507/99 and BoHV-5 gE/TK∆) was multiplied in cell culture and inactivated with binary ethyleneimine (BEI). Each vaccine dose contained approximately of 10(7.5) TCID50 of inactivated virus mixed with an oil-based adjuvant (46:54). Forty calves, 6 to 9-months-old, were allocated into two groups of 20 animals each and vaccinated twice (days 0 and 22pv) by the subcutaneous route with either vaccine. Serum samples collected at day 0 and at different intervals after vaccination were tested for virus neutralizing (VN) antibodies against the parental virus and against heterologous BoHV-5 and BoHV-1 isolates. The VN assays demonstrated seroconversion to the respective homologous viruses in all vaccinated animals after the second vaccine dose (mean titers of 17.5 for the wildtype vaccine; 24.1 for the recombinant virus). All animals remained reagents up to day 116 pv, yet showing a gradual reduction in VN titers. Animals from both vaccine groups reacted in similar VN titers to different BoHV-1 and BoHV-5 isolates, yet the magnitude of serological response of both groups was higher against BoHV-5 field isolates. Calves vaccinated with the recombinant virus did not develop antibodies to gE as verified by negative results in a gE-specific ELISA, what would allow serological differentiation from naturally infected animals. Taken together, these results indicate that inactivated antigens of BoHV-5 gE/TK recombinant virus induced an adequate serological response against BoHV-5 and BoHV-1 and thus can be used as an alternative, differential vaccine candidate.

A recombinant bovine herpesvirus 5 defective in thymidine kinase and glycoprotein E is attenuated and immunogenic for calves

Bovine herpesvirus 5 (BoHV-5) is an important pathogen of cattle in South America and efforts have been made to produce safer and more effective vaccines. In addition to afford protection, herpesvirus vaccines should allow serological differentiation of vaccinated from naturally, latently infected animals. We previously reported the construction and characterization in vitro of a double mutant BoHV-5 (BoHV-5gE/TK Δ) lacking the genes encoding thymidine kinase (tk) for attenuation, and glycoprotein E (gE) as the antigenic marker, as a vaccine candidate strain (Brum et al. 2010a). The present article reports an investigation on the attenuation and immunogenicity of this recombinant in calves. In a first experiment, 80 to 90-day-old seronegative calves (n=6) inoculated intranasally with the recombinant (titer of 10(7.5)TCID50) shed virus in low to moderate titers in nasal secretions for up to 6 days, yet did not develop any respiratory, systemic or neurological signs of infection. At day 30 post-infection (pi) all calves had BoHV-5 specific neutralizing (VN) antibodies in titers of 4 to 8 and were negative for anti-gE antibodies in a commercial ELISA test. Administration of dexamethasone (0.1mg/kg/day during 5 days) to four of these calves at day 42 pi did not result in virus shedding or increase in VN titers, indicating lack of viral reactivation. Secondly, a group of 8-month-old calves (n=9) vaccinated intramuscularly (IM) with the recombinant virus (10(7.5)TCID50/animal) did not shed virus in nasal secretions, remained healthy and developed VN titers from 2 to 8 at day 42 post-vaccination (pv), remaining negative for gE antibodies. Lastly, 21 calves (around 10 months old) maintained under field conditions were vaccinated IM with the recombinant virus (titer of 10(7.3)TCID50). All vaccinated animals developed VN titers from 2 to 16 at day 30 pv. A boost vaccination performed at day 240 pv resulted in a rapid and strong anamnestic antibody response, with VN titers reaching from 16 to 256 at day 14 post-booster. Again, serum samples remained negative for gE antibodies. Selected serum samples from vaccinated animals showed a broad VN activity against nine BoHV-5 and eight BoHV-1 field isolates. These results show that the recombinant virus is attenuated, immunogenic for calves and induces an antibody response differentiable from that induced by natural infection. Thus, the recombinant BoHV-5gE/TKΔ is an adequate candidate strain for a modified live vaccine.

A thymidine kinase-negative bovine herpesvirus 5 is highly attenuated for rabbits, but is neuroinvasive and establishes latent infection

Mutant viral strains deleted in non-essential genes represent useful tools to study the function of specific gene products in the biology of the virus. We herein describe an investigation on the phenotype of a bovine herpesvirus 5 (BoHV-5) recombinant deleted in the gene encoding the enzyme thymidine kinase (TK) in rabbits, with special emphasis to neuroinvasiveness and the ability to establish and reactivate latent infection. Rabbits inoculated with the parental virus (SV-507/99) (n=18) at a low titer (10(5.5)TCID50) shed virus in nasal secretions in titers up to 10(4.5)TCID50 for up to 12 days (average: 9.8 days [5-12]) and 5/ 16 developed neurological disease and were euthanized in extremis. Rabbits inoculated with the recombinant BoHV-5TKΔ at a high dose (10(7.1)TCID50) also shed virus in nasal secretions, yet to lower titers (maximum: 10(2.3)TCID50) and for a shorter period (average: 6.6 days [2-11]) and remained healthy. PCR examination of brain sections of inoculated rabbits at day 6 post-infection (pi) revealed a widespread distribution of the parental virus, whereas DNA of the recombinant BoHV-5TKΔ-was detected only in the trigeminal ganglia [TG] and olfactory bulbs [OB]. Nevertheless, during latent infection (52pi), DNA of the recombinant virus was detected in the TGs, OBs and also in other areas of the brain, demonstrating the ability of the virus to invade the brain. Dexamethasone (Dx) administration at day 65 pi was followed by virus reactivation and shedding by 5/8 rabbits inoculated with the parental strain (mean duration of 4.2 days [1 - 9]) and by none of seven rabbits inoculated with the recombinant virus. Again, PCR examination at day 30 post-Dx treatment revealed the presence of latent DNA in the TGs, OBs and in other areas of the brain of both groups. Taken together, these results confirm that the recombinant BoHV-5TKΔ is highly attenuated for rabbits. It shows a reduced ability to replicate in the nose but retains the ability to invade the brain and to establish latent infection. Additional studies are underway to determine the biological and molecular mechanisms underlying the inability of BoHV-5TKΔ to reactivate from latency.

A thymidine kinase-deleted bovine herpesvirus 5 establishes latent infection but reactivates poorly in a sheep model

The ability of thymidine kinase (tk)-deleted recombinant bovine herpesvirus 5 (BoHV-5tkΔ) to establish and reactivate latent infection was investigated in lambs. During acute infection, the recombinant virus replicated moderately in the nasal mucosa, yet to lower titers than the parental strain. At day 40 post-infection (pi), latent viral DNA was detected in trigeminal ganglia (TG) of all lambs in both groups. However, the amount of recombinant viral DNA in TGs was lower (9.7-fold less) than that of the parental virus as determined by quantitative real time PCR. Thus, tk deletion had no apparent effect on the frequency of latent infection but reduced colonization of TG. Upon dexamethasone (Dx) administration at day 40 pi, lambs inoculated with parental virus shed infectious virus in nasal secretions, contrasting with lack of infectivity in secretions of lambs inoculated with the recombinant virus. Nevertheless, some nasal swabs from the recombinant virus group were positive for viral DNA by PCR, indicating low levels of reactivation. Thus, BoHV-5 TK activity is not required for establishment of latency, but seems critical for efficient virus reactivation upon Dx treatment.

Mapping the sites of latency and reactivation by bovine herpesvirus 5 (BoHV-5) and a thymidine kinase-deleted BoHV-5 in lambs

A thymidine kinase (tk)-deleted bovine herpesvirus 5 (BoHV-5tkΔ) was previously shown to establish latent infection and reactivate - even poorly - in a sheep model (Cadore et al. 2013). As TK-negative alphaherpesviruses are unlike to reactivate in neural tissue, this study investigated the sites of latency and reactivation by this recombinant in lambs. For this, groups of lambs were inoculated intranasally with the parental BoHV-5 strain (SV-507/99) or with the recombinant BoHV-5tkΔ. During latent infection (40 days post-inoculation, pi), the distribution of recombinant virus DNA in neural and non-neural tissues was similar to that of the parental virus. Parental and recombinant virus DNA was consistently detected by PCR in trigeminal ganglia (TGs); frequently in palatine and pharyngeal tonsils and, less frequently in the retropharyngeal lymph nodes. In addition, latent DNA of both viruses was detected in several areas of the brain. After dexamethasone (Dx) administration (day 40pi), the recombinant virus was barely detected in nasal secretions contrasting with marked shedding of the parental virus. In tissues of lambs euthanized at day 3 post-Dx treatment (pDx), reverse-transcription-PCR (RT-PCR) for a late viral mRNA (glycoprotein D gene) demonstrated reactivation of parental virus in neural (TGs) and lymphoid tissues (tonsils, lymph node). In contrast, recombinant virus mRNA was detected only in lymphoid tissues. These results demonstrate that BoHV-5 and the recombinant BoHV-5tkΔ do establish latent infection in neural and non-neural sites. Reactivation of the recombinant BoHV-5tkΔ, however, appeared to occur only in non-neural sites. In anyway, the ability of a tk-deleted strain to reactivate latent infection deserves attention in the context of vaccine safety.

Avaliação do desempenho de bicos para aplicação de herbicidas

A avaliação do desempenho de barras de pulverização pode ser feita a partir do padrão de distribuição ou deposição do líquido de cada bico individual em mesa de prova. Neste trabalho comparam-se os padrões de deposição produzidos por diversos bicos em várias alturas. Os resultados mostraram melhores desempenho para os bicos TF e XR, seguidos pelos bicos TJ60-8010, TJ60-8006 e FL. Os bicos TJ60-11006 e os TQ mostraram problemas localizados e os bicos TK foram considerados os piores entre os testados. Relações entre o espaçamento entre bicos e altura do alvo para as quais os coeficientes de variação apresentam valores satisfatórios foram propostas.

Uniformidade de deposição da calda de pulverização de herbicidas em barra lateral protegida com diferentes combinações de pontas de pulverização

O presente trabalho objetivou estudar a uniformidade de distribuição da calda de pulverização contendo herbicidas, em culturas perenes arbustivas, utilizando combinações de pontas de pulverização em barra lateral protegida, conduzida a pequena distância do alvo, na linha de culturas perenes arbustivas. Para isso, foi desenvolvido um programa computacional que permite simular a sobreposição do leque de pulverização, da porção protegida da barra e do leque formado pela ponta de pulverização do bico mais extremo da barra, de modo diferente dos demais programas. Após a seleção das melhores combinações de pontas de pulverização por meio de simulação dos padrões de deposição da pulverização das pontas individuais e dos coeficientes de variação menores que 10%, algumas dessas combinações foram testadas em campo, aplicando-se um herbicida sistêmico (glyphosate) e outro com ação de contato (paraquat). Os resultados indicaram que o programa computacional desenvolvido pode constituir-se em um auxiliar valioso para a seleção das melhores combinações de pontas de pulverização. Em aplicações tanto do herbicida glyphosate quanto do paraquat, com volumes de calda mais reduzidos,abaixo de 100 L ha-1, destacaram-se como arranjos mais eficientes: a) pontas TT110015 distanciadas de 52,5 cm entre si, combinadas com a ponta TK-0,5 na extremidade da barra a 50 cm do último bico, operando na velocidade de 5 km h-1 e pressão de 103 kPa (15 lbf pol-2), com distância de caminhamento do tronco da árvore de 20 cm ; b) pontas SMCE2 distanciadas de 15 cm entre si, combinadas com a ponta TK-0,5 na extremidade da barra de 20 cm do último bico, operando na velocidade de 4 km h-1 e pressão de 414 kPa (60 lbf pol-2), com distância de caminhamento do tronco da árvore de 30 cm ; e c) pontas TLX-2 distanciadas de 15 cm entre si, combinadas com a ponta TK-0,5 na extremidade da barra de 20 cm do último bico, operando à velocidade de 5 km h-1 e pressão de 414 kPa (60 lbf pol-2), com distância de caminhamento do tronco da árvore de 30 cm. A velocidade de deslocamento do pulverizador de 5 km h-1 proporcionou melhores condições para que os herbicidas estudados apresentassem melhor controle de plantas daninhas, quando comparada com a velocidade de deslocamento do pulverizador de 4 km h-1.

Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE) or thymidine kinase (TK) gene or both (gE/TK) from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99). A gE-deleted recombinant virus (BoHV-5 gE∆) was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK∆) was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric β-galactosidase gene. Subsequently, using the BoHV-5 gE∆ virus as backbone, a double gene-deleted (TK plus gE) BoHV-5 recombinant (BoHV-5 gE/TK∆) was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK) cells, the mutants lacking gE (BoHV-5 gE∆) and TK + gE (BoHV-5 gE/TK∆) produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE∆, BoHV-5 TK∆ and BoHV-5 gE/TK∆) were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK∆) produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

Etelä-Savon maaseudun kehittämissuunnitelman 2014–2020 ennakkoarviointi

Ennakkoarvioinnin keskeisenä tavoitteena oli selvittää Etelä-Savon maaseudun kehittämissuunnitelman 214-2020 vastaavuutta Etelä-Savon maaseutustrategiaan, Manner-Suomen maaseudun kehittämisohjelmaan sekä päättyneen ohjelmakauden kehittämissuunnitelman väliarvioinnin havaintoihin. Kehittämissuunnitelma perustuu alueen maaseutustrategiaan, jota toteutetaan valtakunnallisessa kehittämisohjelmassa määriteltyjen toimenpiteiden avulla. Etelä-Savon ELY-keskuksen tilaaman maaseudun kehittämissuunnitelman ennakkoarvioinnin toteuttivat Suomen Aluetutkimus FAR ja TK-Eval Ay. Ennakkoarvioinnissa todetaan, että kehittämissuunnitelman tavoitteet ovat sisällöltään hyvin yhdensuuntaisia alueen maaseutustrategian kanssa. Maaseutustrategiassa on kolme painopistettä: 1) vahvistetaan maaseudun elinvoimaisuutta edistäviä rakenteita, 2) parannetaan ihmisten hyvinvointia maaseudulla ja 3) kehitetään maaseudun elinkeinoja. Arvioinnin mukaan suunnitelmassa on eniten toimenpiteitä maaseudun elinkeinojen kehittämiseksi, erityisesti yritysten kilpailukyvyn parantamiseksi. Kehittämissuunnitelmassa on otettu käyttöön kaikki Manner-Suomen maaseudun kehittämisohjelmaan kuuluvat toimenpiteet ja vastaavuutta löytyy valtakunnallisen ohjelman tavoitteisiin. Näin ollen edellytykset kehittämissuunnitelman toteutuksen vaikuttavuudelle ovat olemassa. Vaikuttavuuden parantaminen on puolestaan kiinni siitä, mihin tavoitteisiin alueella panostetaan ja miten eri kehittämistoimenpiteitä otetaan käyttöön. Arviointitehtävään sisältyi myös ohjelmakauden 2007–2013 väliarvioinnin keskeisten tulosten ja havaintojen huomioiminen kehittämissuunnitelmassa. Ennakkoarvioinnin mukaan ne suositukset, joihin kehittämissuunnitelman kautta voitiin vaikuttaa, oli huomioitu hyvin. Arvioinnin lopussa tarkasteltiin Etelä-Savon maaseudun kehittämissuunnitelman tarve- ja asiakaslähtöisyyttä, viestintää ja tiedottamista, yhteensovitusta muiden ohjelmien ja toimijoiden kanssa sekä suunnitelman ohjelmallisuutta.

Electrical properties of some single crystal dodecaborides /|nby Jyoti Burte. -- 260 St. Catharines [Ont. : s. n.],

PreVi011.3 ':i or~ : indicat e('. tk~t ho t~)rE's sed ~-Al B 12 1i~2, ~' a semiconductor. r:Toreove r , the s i mpl.(~ electronic t heory also indi cates that ~ -AIB1 2 should be a semico nductor, since thf're is one nonbonding e 'Le ctrofl per AlB12- uni t. JPor these reasons, we decided to measure t he electrical n ropert i ~ s of ~ -AlB1 2 single crystal s . Singl e crystal s of¥- AIB 12 ab ou t 1 x 1 r1n1 . size were grown from a copper mel t at 12500 C. The melt technique coupled. 1,vi th slow cooling vilas used because of i ts advantages such as : siTYInle set- up of the expe rimon t ; only e ;l.sil y available c hemi cals are required and it i s a c omparatively strair::bt forvvard y,le t hod still yielding crystal s big enouGh for OtU' purpose . Copper rms used as a solvent , i nst8ad of previOl.wly used aluminum , because it allows c.l.'ystal growth at hig he r t emneratures. HovlGver, the cry s tals of ] -AlB12 shm'red very hi gh res i s t ance a t r oom temperature . From our neasureJ'lents we conclude that the r esistivity of j3- Al B12 is, at least, given as ~ = 4. x 107 oblD .em •• Those results are inc ons i s t ent wi 'uh the ones .. reported by IIiss Khin fo r bot- pressed j3-AlB12 g i ven a s = 7600 ohm . em . or I e s s . ' Since tbe hot pressing was done at about 800 - ' 9000C i n ~ rap hi te moul ds 1,7i th 97% AlB12- p oVJder, vie thi nk there is pas s ib i 1 i ty th a.t lower borides or borot] carbide are , being formed, ':.Jhich are k11 own to be good semiconductors . v7e tried to ro-pe r-AlB12 by addi'J,'?: agents s uch as l:Ig , IG.-InO 4. ' HgS04 , KI12PO 4·' etc. to t he melt .. However , all these re age 11 t eel either reduced the yield and size of t lJe crystals or r;ave crystals of high r esis'can ce again. We think tba t molten copper keeps t he i mpurities off . There is also a pos s i bil i ty t hc:!,t these doping agents get oxidi~::;ed at '1 250°C • Hence, we co ~ clud e that J -AIB12 has v~ ry high r es i stance at r oom temperature . This was a l s o C011 - fi rmed by checki ng the siYlgle and. polycrystals of .~-AIB12 from Norton Co., Ontario and Cooper Nletallurgical Association. Boron carbide has been reported to be a semiconductor with ~ - 0.3 to 0.8 ohm . cm. for hotpres sed s araples. Boron carbide b e inq: struct urally related to ¥-AIB12 , we de cided to study the electrical prone rties of it~ Single crystals. These crystals were cut from a Single melt grovvn crystal a t Norton Co., Ontario. The resistivity of th," se crystal s was measured by the Van der Pam-v' s ~ nethod, which \vas very c onvenient fo r our crystal sha-pp.s. Some of the crystals showed resistivity ~ == 0.50 ob,Tn.cr] . i n agreement with the previously reported results . However , a few crystals showed lower resistivity e.g . 0 .13 and 0.20 ohm.cra • • The Hall mobility could .not be measured and th8reiore i s lower than 0 .16 em 2 v - 1 sec -1 • This is in agreement \vith t he re1)orted Hall mobility for pyrolytic boron . _ 2 -1 -1 carbide as 0.13 cm v sec • We also studied the orientation of the boron carbide crystals by the Jjaue-method. The inclination of c-axis with res pect to x-ray be81Il was det ermined . This was found to be 100 t o 20° f or normal resistivity sarnples (0.5 ohm . cm.) and 27 - 30° for t he lower r esistivity samples (0.1 ~5 to 0.20 ohm.cm .). This indica tes the possibility that th.e r es if.1tivity of B13C3 i s orientation dependent.

Engineering an improved adenovirus packaging cell line

Recombinant human adenovirus (Ad) vectors are being extensively explored for their use in gene therapy and recombinant vaccines. Ad vectors are attractive for many reasons, including the fact that (1) they are relatively safe, based on their use as live oral vaccines, (2) they can accept large transgene inserts, (3) they can infect dividing and postmitotic cells, and (4) they can be produced to high titers. However, there are also a number of major problems associated with Ad vectors, including transient foreign gene expression due to host cellular immune responses, problems with humoral immunity, and the creation of replication competent adenoviruses (RCA). Most Ad vectors contain deletions in the E1 region that allow for insertion of a transgene. However, the E1 gene products are required for replication and thus must be supplied in trans by a helper ceillille that will allow for the growth and packaging of the defective virus. For this purpose the 293 cell line (Graham et al., 1977) is used most often; however, homologous recombination between the vector and the cell line often results in the generation of RCA. The presence of RCA in batches of adenoviral vectors for clinical use is a safety risk because tlley . may result in the mobilization and spread of the replication-defective vector viruses, and in significant tissue damage and pathogenicity. The present research focused on the alteration of the 293 cell line such that RCA formation can be eliminated. The strategy to modify the 293 cells involved the removal of the first 380 bp of the adenovirus genome through the process of homologous recombination. The first step towards this goal involved identifying and cloning the left-end cellular-viral jUl1ction from 293 cells to assemble sequences required for homologous recombination. Polymerase chain reaction (PCR) was performed to clone the junction, and the clone was verified through sequencing. The plasn1id PAM2 was then constructed, which served as the targeting cassette used to modify the 293 cells. The cassette consisted of (1) the cellular-viral junction as the left-end region of homology, (2) the neo gene to use for positive selection upon tranfection into 293 cells, (3) the adenoviral genome from bp 380 to bp 3438 as the right-end region of homology, and (4) the HSV-tk gene to use for negative selection. The plasmid PAM2 was linearized to produce a double strand break outside the region of homology, and transfected into 293 cells using the calcium-phosphate technique. Cells were first selected for their resistance to the drug G418, and subsequently for their resistance to the drug Gancyclovir (GANC). From 17 transfections, 100 pools of G418f and GANCf cells were picked using cloning lings and expanded for screening. Genomic DNA was isolated from the pools and screened for the presence of the 380 bps using PCR. Ten of the most promising pools were diluted to single cells and expanded in order to isolate homogeneous cell lines. From these, an additional 100 G41Sf and GANef foci were screened. These preliminary screening results appear promising for the detection of the desired cell line. Future work would include further cloning and purification of the promising cell lines that have potentially undergone homologous recombination, in order to isolate a homogeneous cell line of interest.

Functional characterization on invertebrate and vertebrate tissues of tachykinin peptides from octopus venoms

It has been previously shown that octopus venoms contain novel tachykinin peptides that despite being isolated from an invertebrate, contain the motifs characteristic of vertebrate tachykinin peptides rather than being more like conventional invertebrate tachykinin peptides. Therefore, in this study we examined the effect of three variants of octopus venom tachykinin peptides on invertebrate and vertebrate tissues. While there were differential potencies between the three peptides, their relative effects were uniquely consistent between invertebrate and vertebrae tissue assays. The most potent form (OCT-TK-III) was not only the most anionically charged but also was the most structurally stable. These results not only reveal that the interaction of tachykinin peptides is more complex than previous structure–function theories envisioned, but also reinforce the fundamental premise that animal venoms are rich resources of novel bioactive molecules, which are useful investigational ligands and some of which may be useful as lead compounds for drug design and development.