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Journal articles on the topic 'Plurisens'
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Nyabola, A. O., P. G. Kareru, E. S. Madivoli, S. I. Wanakai, and Ernest Gachui Maina. "Formation of Silver Nanoparticles via Aspilia pluriseta Extracts Their Antimicrobial and Catalytic Activity." Journal of Inorganic and Organometallic Polymers and Materials 30, no. 9 (2020): 3493–501. http://dx.doi.org/10.1007/s10904-020-01497-7.
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Njeru, Sospeter N., and Jackson M. Muema. "Antimicrobial activity, phytochemical characterization and gas chromatography-mass spectrometry analysis of Aspilia pluriseta Schweinf. extracts." Heliyon 6, no. 10 (2020): e05195. http://dx.doi.org/10.1016/j.heliyon.2020.e05195.
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Nyabola, A. O., P. G. Kareru, E. S. Madivoli, E. G. Maina, and I. S. Wanakai. "Assessment of the Anti-microbial Action of Zero Valent Iron Nanoparticle Synthesized by Aspilia pluriseta Extracts." Chemical Science International Journal 25, no. 3 (2018): 1–10. http://dx.doi.org/10.9734/csji/2018/45799.
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OHTAKA, AKIFUMI. "Taxonomical study of Japanese Aulodrilus Bretscher (Annelida, Clitellata, Tubificinae) with descriptions of two new species." Zootaxa 4952, no. 1 (2021): 1–32. http://dx.doi.org/10.11646/zootaxa.4952.1.1.
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Seven species of the genus Aulodrilus (Annelida, Clitellata, Tubificinae) are studied, based on new material from Japan. Aulodrilus dentosus sp. nov. is characterized as having tubular atrium, forked or bifid distal ends of dorsal crotchets, but no genital chaetae, and A. aestivus sp. nov. is characterized as having crescent-shaped atrium, median male bursa, and bifid chaetae in the dorsal bundles. Five other congeners are redescribed: A. limnobius Bretscher, A. pluriseta (Piguet), A. pigueti Kowalewski, A. japonicus Yamaguchi, and A. americanus Brinkhurst & Cook. Comparison of taxonomic characters among the 14 species recognized in the genus to date shows that three species differ from other congeners in several features: A. paucichaeta Brinkhurst & Barbour, A. adetus (du Bois-Reymond Marcus), and A. apeniatus Cui & Wang. They are provisionally maintained in the genus.
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Nemo, François, and Binène Horchani. "Accounting for transcategorial morphemes." Cognitive Linguistic Studies 5, no. 1 (2018): 77–105. http://dx.doi.org/10.1075/cogls.00014.nem.
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Abstract The article presents a morphemic account of transcategoriality, with detailed illustrations (e.g. English but and even, French encore, tout, meme, Latin to French morpheme /tant/) of the approach. After making explicit the paradigmatic differences between exoskeletal and endoskeletal approaches, and showing that ultimately it can be summarized in terms of existence or not of grammar-free morphemes becoming lexemes through grammatical and contextual insertion, it turns to the issue of knowing what an exoskeletal non-categorial meaning can be. It introduces at this stage the notion of fractality, before making explicit and detailing the method which allows isolation of a morpheme’s indicational semantics. The whole approach is finally illustrated with the study of the whole distribution of French /tant/, first semantically in synchrony before extending the tests to Latin data, showing that polysemy, transcategoriality and plurisemy are various forms of the same issue.
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Martín, Miguel Ángel, Miguel Reyes, and F. Javier Taguas. "Intermittent Plurisink Model and the Emergence of Complex Heterogeneity Patterns: A Simple Paradigm for Explaining Complexity in Soil Chemical Distributions." Journal of Chemistry 2015 (2015): 1–5. http://dx.doi.org/10.1155/2015/138202.
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The spatial complexity of the distribution of organic matter, chemicals, nutrients, and pollutants has been demonstrated to have multifractal nature. This fact supports the possibility of existence of some emergent heterogeneity structure built under the evolution of the system. The aim of this paper is providing a consistent explanation of the mentioned results via an extremely simple model.
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Østrup, O., F. Strejcek, I. Petrovicova, et al. "37 LIMITATION OF BOVINE OOPLASM IN REPROGRAMMING OF PORCINE SOMATIC CELLS." Reproduction, Fertility and Development 23, no. 1 (2011): 125. http://dx.doi.org/10.1071/rdv23n1ab37.
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Ooplasm posses several, undefined factors allowing for reprogramming of somatic genome into pluripotent state. However, it is presently unknown if and to what extent the ooplasm is able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). The 1-cell stage embryos were processed at different time points (2, 4, 8, and 12 h post-activation) for detailed nuclear and nucleolar analysis by TEM, and immunofluorescence for visualisation of nucleolar proteins related to transcription (UBF) and processing (fibrillarin). The embryos produced by SCNT of porcine fibroblast into non-activated bovine ooplasm were compared to bovine parthenogenetic counterparts. In the absence of morphological remodelling features (premature chromatin condensation, nuclear envelope break-down), intergeneric embryos showed bovine nuclear and nucleolar precursor body (NPB) morphology. The somatic cell introduced into the bovine ooplasm displayed intranuclear activity at the post-translational level signalling a universal function of ooplasmic factors. The lack of distinct UBF localization in intergeneric embryos, however, suggests failures in sequence-specific interactions between the ooplasm and transferred cell from another genus. We conclude that sperm or a somatic cell of the same species is required for successful embryonic development. Clonet MRTN-CT-2006-035468; MedRat LSHG-CT-2006-518240; PluriSys FP7-HEALTH-2007-223485; DFG, Alexander von Humboldt fellowship, VEGA 1/0057/08; VEGA 1/0012/10.
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TIMM, TARMO. "Observations on the life cycles of aquatic Oligochaeta in aquaria." Zoosymposia 17, no. 1 (2020): 102–20. http://dx.doi.org/10.11646/zoosymposia.17.1.11.
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Observations on the life cycles of aquatic oligochaetes were made in the period 1962–2017 at the Võrtsjärv Limnological Station (Estonia) using small aquaria with sieved profundal mud covered with unaerated water. The aquaria were mostly inseminated with 10 juvenile worms and checked four times a year, changing the mud and eliminating the progeny, until the natural death of the original worms. Besides, mass cultures were kept in bigger aquaria. Many individuals of Tubifex tubifex, T. newaensis, Limnodrilus hoffmeisteri, L. udekemianus, Ilyodrilus templetoni, Psammoryctides barbatus, Spirosperma ferox, Potamothrix moldaviensis, P. vejdovskyi, P. bavaricus, Stylodrilus heringianus and Rhynchelmis tetratheca survived for several years, reproduced repeatedly, and died out one by one during the observation period. In some cases, the most longevous individuals reached an age of up to 8 years (I. templetoni), 10–12 years (T. tubifex), 15–17 years (L. hoffmeisteri, P. barbatus, S. heringianus), or even more than 20 years (L. udekemianus, S. ferox, T. newaensis). Criodrilus lacuum did not reproduce in aquaria, although the oldest individual spent 46 years there. Potamothrix hammoniensis, Lophochaeta ignota, Lamprodrilus isoporus, most naidines and some others did not thrive in aquaria and usually died without reproducing. In a cellar, where temperature conditions imitated seasonal fluctuations in lakes, or when the aquaria were maintained at continuously low temperature, the lifetime of worms was often longer than at room temperature. At elevated temperatures (+25° to +35°C) T. tubifex and L. hoffmeisteri formed cocoons mostly with only 1–2 eggs while their life span was then shorter. Architomic clones of Potamothrix bedoti, Bothrioneurum vejdovskyanum, Aulodrilus pluriseta and A. japonicus survived and propagated for years. The architomic Lumbriculus variegatus was thriving only when fed, e.g., with yeast. Uniparental reproduction by parthenogenesis was observed in T. tubifex, L. hoffmeisteri and S. heringianus kept or reared single. Two special races(?) were noted both within T. tubifex and L. udekemianus.
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ARSLAN, NAIME, and DENIZ MERCAN. "Long-term macrobenthic community structure changes in the Upper Sakarya River System (1995–2015)." Zoosymposia 17, no. 1 (2020): 89–101. http://dx.doi.org/10.11646/zoosymposia.17.1.10.
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The Upper Sakarya River System (USRS) is one of the most important river systems in Turkey. Its primary drainage is provided by the Porsuk Seydi and Bardakçı Rivers and their tributaries. Long term benthic invertebrate community structure in the USRS was investigated from 1995–2015 (with sampling conducted every five years) in order to assess changes in their composition and in relation to water quality. Oligochaete specimens sorted from samples were identified to the species level when possible; all other invertebrate specimens sorted from samples were identified to order and family level. In addition, some environmental parameters (e.g., dissolved oxygen, temperature, and hydrogen ion concentration as pH) were measured in situ. Although Ephemeroptera-Plecoptera-Trichoptera fauna were the most abundant group in fauna of USRS during the years 1995, 2000 and (in part) 2005 (18.80, 17.69, and 14.07%, respectively), this ratio decreased to 7.90% during the more recent years of monitoring. In 1995, 2000 and 2005, Nais bretscheri, Chaetogaster diastrophus, Chaetogaster langi, Pristinella jenkinae, Aulodrilus pigueti, Aulodrilus pluriseta, Potamothrix hammoniensis, and Psammoryctides albicola were the dominant oligochaete taxa. After 2005, tubificine species became more prevalent in samples. While 6 stations had high BMWP (Biological Monitoring Working Party) value in 1995, 2000 and 2005, only 1 station had high value after 2005. Values of Shannon Diversity Indices ranged from 2.00 to 3.05 for the years 1995–2000, 1.87 to 2.24 for the years 2000–2005, 1.06 to 1.85 for the years 2005–2010, and 0.97 to 1.80 for the years 2010–2015. In USRS, while values of dissolved oxygen were measured as 8.00 mg/l and 9.00 mg/l in 1995 and 2000, this high value was measured only at one station in 2015. It was found that numerical and proportional distributions of benthic invertebrates in the USRS have changed considerably between 1995 and 2015. It is obvious that these changes are the result of anthropogenic habitat degradation.
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Lagutina, I., R. Duchi, S. Colleoni, G. Lazzari, and C. Galli. "43 SHORT- AND LONG-LASTING EFFECTS OF TRICHOSTATIN A TREATMENT OF SCNT EMBRYOS IN CATTLE." Reproduction, Fertility and Development 23, no. 1 (2011): 127. http://dx.doi.org/10.1071/rdv23n1ab43.
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Both preimplantation and full-term development of mouse somatic cell nuclear transfer (SCNT) embryos are significantly enhanced by treatment with trichostatin A (TSA), an inhibitor of histone deacetylase (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). The present study was designed to examine the effect of TSA treatment on preimplantation and full-term development of bovine cloned embryos. To investigate the effect of TSA on bovine NT embryos development, we treated them with 50 nM TSA during the first 10 h after activation. Bovine NT-embryos were reconstructed using adult fibroblasts of 2 female donors (A and B) with significantly different in vitro cloning efficiency (respectively, 84/245; 34.3% v. 155/298; 52.1% blastocyst D7, P ≤ 0.05, chi-square test). TSA treatment significantly improved blastocyst rate in A, however did not affect development in B (56.3% and 50.5%, respectively). The level of acetylated histone H3K9 10 h after activation detected by anti-acH3K9 antibody was significantly increased after TSA-treatment in A (P ≤ 0.05, Student’s t-test) but did not change in B, thus demonstrating that the levels of histone acetylation in cloned embryos correlate with their in vitro developmental potential. To evaluate the long-lasting effect of TSA-treatment on the full-term development of cloned embryos, SCNT embryos derived from 4 female donor animals were reconstructed. 196 TSA-treated embryos at the blastocyst stage were transferred into 98 recipients and 2 calves (2%) were born. In the control group, 167 embryos were transferred into 141 recipients and 3 calves (2.1%) were born. Our data show that cell lines demonstrate different susceptibility to TSA that may affect reprogramming of the somatic genome with low level of acetylation resulting in higher in vitro embryo development. However, TSA does not improve overall cloning efficiency in cattle, measured as full-term development. Project partly supported by EU grants Plurisys (n 22348), Xenome (LSHB-CT-2006-037377) and Regione Lombardia.
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Dinnyes, A., M. K. Pirity, E. Gocza, et al. "GENERATION OF RABBIT PLURIPOTENT STEM CELL LINES." Reproduction, Fertility and Development 24, no. 1 (2012): 286. http://dx.doi.org/10.1071/rdv24n1ab246.
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Pluripotent stem cells have the capacity to divide indefinitely and to differentiate to all the somatic tissues. They can be genetically manipulated in vitro by knocking in and out genes, therefore they serve as an excellent tool for gene-function studies and for the generation of models for human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, several attempts have been made to generate pluripotent stem cells from other species as it would help us to understand the differences and similarities of signaling pathways involved in pluripotency and differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved among different species. This review gives an overlook of embryonic and induced pluripotent stem cell (iPSCs) research in the rabbit which is one of the most relevant non-rodent species for animal models. To date, several lines of putative ESCs and iPSCs have been described in the rabbit. All expressed stem cell-associated markers and exhibited longevity and pluripotency in vitro, but none have been proven to exhibit full pluripotency in vivo. Moreover, similarly to several domestic species, markers used to characterize the putative ESCs are not fully adequate because studies in domestic species have revealed that they are not specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a reliable panel of molecular markers specific to pluripotent cells of the developing rabbit embryo. The status of isolation and characterization of the putative pluripotency genes in rabbit will be discussed. Using rabbit specific pluripotency genes we might be able to reprogram somatic cells and generate induced pluripotent stem cells more efficiently thus overcome some of the challenges towards harnessing the potential of this technology. This study was financed by EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; RabPstem, PERG07-GA-2010-268422; PluriSys, HEALTH-2007-B-223485; AniStem, PIAP-GA-2011-286264), NKTH-OTKA-EU-7KP HUMAN-MB08-C-80-205; Plurabbit, OMFB-00130-00131/2010 ANR-NKTH/09-GENM-010-01.
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Rungarunlert, S., N. Klincumhom, C. Nemes, M. Techakumphu, M. K. Pirity, and A. Dinnyes. "293 MASS PRODUCTION OF Nkx2.5-POSITIVE CARDIAC PROGENITOR CELLS DERIVED FROM MOUSE EMBRYONIC STEM CELLS IN SLOW-TURNING LATERAL VESSEL FOR CELL TRANSPLANTATION AND DRUG TESTING." Reproduction, Fertility and Development 23, no. 1 (2011): 244. http://dx.doi.org/10.1071/rdv23n1ab293.
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Regenerative cell therapy against cardiovascular disease would require mass production and purification of specific cell types before transplantation. To enable large-scale production of embryonic stem (ES)-derived pure cardiomyocytes, we developed an animal model for a single-step scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled slow-turning lateral vessel (STLV, Synthecon, Inc., Houston, TX, USA) bioreactor following inoculation with a single cell suspension of mouse ES cells. To enhance the yield of cardiac progenitor cells, mouse ES cells (HM1; 129Sv/Ola, Magin et al. 1992 Nucl. Acids Res. 20, 3795–3796) were targeted with the cardiac-specific mouse Nkx2.5 promoter driven enhanced fluorescent green protein (EGFP). Among 15 targeted colonies, which were characterised based on morphology, the ability to form EB, EGFP expression, and in vitro differentiation ability toward cardiomyocytes, 3 lines were further evaluated for the efficiency of cardiomyocyte production. The 3 lines were cultured in STLV bioreactor and compared with classical hanging drop (HD) and static suspension culture methods. Embryonic bodies at day 3 to 8 were collected and analysed by using fluorescence-activated cell sorting for markers of pluripotency (e.g. Oct-4, SSEA1, Nanog) and cardiac (e.g. Nkx2.5, Troponin T) lineage commitments. Data was analysed by one-way ANOVA and t-tests. The results showed that both level and kinetics of Nkx2.5 expression was dependent on culture conditions. The STLV and static suspension culture methods produced higher rates of Nkx2.5-positive cells on day 5 than that of HD (70 and 54 v. 30%, respectively). The STLV method produced a highly uniform population of efficiently differentiating EB in large quantities and resulted in the highest, 108 yield of cardiomyocytes in a single 110-mL STLV on day 4. In conclusion, the STLV method provides a technological platform for controlled large-scale generation of ES-cell-derived cardiomyocytes for clinical and industrial applications. In vivo transplantation tests of cardiomyocytes produced via STLV are currently underway. This study was financed by EU FP6 (CLONET, MRTN-CT-2006-035468), EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduStem, PIAP-GA-2008-230675; PluriSys, HEALTH-2007-B-223485); NKTH-OTKA-EU FP7-HUMAN-2009-MB08-C 80205 and NKTH/KPI (NKFP_07_1-ES2HEART-HU OM-00202-2007), CHE-TRF senior scholarship, No. RTA 5080010 (M.T.), and the Thailand Commission on Higher Education [CHE-PhD-SW-2005-100 (S.R.), CHE-PhD-SW-RG-2007 (N.K.)].
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Muenthaisong, S., O. Ujhelly, E. Varga, et al. "292 GENERATION OF MOUSE INDUCED PLURIPOTENT STEM CELLS FROM VARIOUS GENETIC BACKGROUND BY SLEEPING BEAUTY TRANSPOSON-MEDIATED GENE TRANSFER." Reproduction, Fertility and Development 23, no. 1 (2011): 243. http://dx.doi.org/10.1071/rdv23n1ab292.
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Induced pluripotent stem (iPS) cell technology allows the reprogramming of somatic cells to a pluripotent state; however, it requires viral gene transduction and permanent existence of the exogenous genes in the genome, which is a potential risk for abnormalities in the derived iPS cells. Recently, there was report that iPS cells have been made with piggyBack transposon. Here, we first reported that nonviral transfection of a Sleeping Beauty transposon, which comprises c-Myc, Klf-4, Oct3/4 (Pou5f1), and Sox-2, can reprogram mouse fibroblasts from 3 different genetic backgrounds: ICR (outbred), C57BL/6 (inbred), and F1 hybrid (C57BL/6 × DBA/2J), with parallel robust expression of all exogenous (c-Myc, Klf-4, Oct3/4, and Sox-2) and endogenous (e.g. Nanog) pluripotency genes. The iPS cells were cultured under standard conditions with promotion of differentiate by withdrawal of leukemia inhibitory factor. We chose 6 cloned of each line that exhibited characteristics typical for undifferentiated embryonic stem (ES) cell: ES-cell-like morphology, alkaline phosphatase positivity, and gene expression pattern [quantitative real-time PCR and immunofluorescence of ES cell markers (e.g. Oct-4, SSEA1, Nanog]. Furthermore, cells were able to form embryoid bodies and beat rhythmically and expressed cardiac markers assayed by immunofluorescence (e.g. cardiac Troponin T, desmin). In vivo testing of iPS cell lines for their developmental potential (diploid and tetraploid embryo complementation assay) is currently underway. The iPS cell lines generated from the ICR strain appeared the earliest in time (ICR-d11, F1 day-2 and Bl6-d12), with higher efficiency than colonies from the other 2 backgrounds. The differentiation potential of the iPS lines derived from the 3 genetic backgrounds was similar. Interestingly, the ICR-iPS lines had higher differentiation potential than did the ICR-ES cell lines: the rate of embryoid bodies forming rhythmically beating cardiomyocytes was 4% in ICR-ES and 79% in ICR-iPS cells, respectively. Our results suggest that the iPS technology provide a new tool to generate pluripotent stem cells from genetic backgrounds where good-quality ES cell generation is difficult. These studies provide new insights into virus-free iPS technology and contribute to defining future cell-based therapies, drug screening methods, and production of transgenic animals with genetically modified iPS cells. This study was financed by EU FP6 (CLONET, MRTN-CT-2006-035468), EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; InduStem, PIAP-GA-2008-230675; PluriSys, HEALTH-2007-B-223485); NKTH-OTKA-EU FP7-HUMAN-2009-MB08-C 80205, and NKTH/KPI (Jedlik NKFP_07_1-ES2HEART-HU OM-00202-2007).
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Tancos, Z., O. Ujhelly, M. K. Pirity, and A. Dinnyes. "223 ISOLATION OF RABBIT PLURIPOTENCY GENES TO GENERATE RABBIT INDUCED PLURIPOTENT STEM CELLS." Reproduction, Fertility and Development 24, no. 1 (2012): 223. http://dx.doi.org/10.1071/rdv24n1ab223.
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Induced pluripotent stem cells (iPSC) technology, which allows direct reprogramming of somatic cells to a pluripotent state, is a promising tool for gene-function studies disease modelling, drug screening, toxicology tests and to generate knockout animal models. The goal of the current work was to close the gap in knowledge with regard to the molecular biological background for rabbit iPS work by isolating the putative pluripotency genes from the rabbit, based on the sequences published for other species. The sequence of known pluripotency genes (Oct4, Sox2, Klf4, c-Myc, Nanog) were analysed and primers designed based on the similarity of sequences. Sequences of each individual rabbit pluripotency gene was compared to other vertebrates (e.g. human, mouse, bovine) phylogenetically. Rabbit ESCs and blastocyst stage embryos were collected from superovulated rabbits to isolate total RNA. Genes of interest were amplified using RT-PCR and electrophoretically separated for cDNA fragment isolation. Isolated and subcloned cDNA fragments were sequenced and analysed. Our results showed that after restriction digestion the size of amplified and cloned rabbit Oct4, Sox2, Klf4, c-Myc and Nanog gene fragments correspond to the expected amplicon size. Furthermore, sequence confirmation by DNA sequencing has been completed in the case of Oct4, c-Myc, Klf4 and Nanog. The homology of these genes to that of their mouse and human orthologs were as follows: Oct4: at Na level 79% homologue to mouse, 85% homologue to human, at Aa level 81% homologue to mouse, 90% homologue to human; Klf4: at Na level 98% homologue to mouse, 85% homologue to human, at Aa level 95% homologue to mouse, 84% homologue to human; c-myc: at Na level 88% homologue to mouse, 92% homologue to human, at Aa level 91% homologue to mouse and 94% homologue to human; Nanog: at Na level 71% homologue to mouse, 78% homologue to human, at Aa level 55% homologue to mouse, 66% homologue to human. In conclusion, we have revealed differences at both Na and Aa level in all four major rabbit pluripotency gene sequences in comparison to their mammalian orthologs which might partially explain difficulties in generation of rabbit iPSC capable of germline transmission. Our further goal is to apply rabbit specific pluripotency genes to reprogram somatic cells and generate iPSC more efficiently than by using mouse or human genes. This work was supported by grants from Plurabbit, OMFB-00130/2010 ANR-NKTH; NKTH-OTKA-EU-7KP HUMAN-MB08-C-80-205; EU FP7 (AniStem, PIAP-GA-2011-286264PartnErS, PIAP-GA-2008-218205; InduStem, PIAP-GA-2008-230675; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; PluriSys, HEALTH-2007-B-223485).
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Rasmussen, M. A., V. J. Hall, S. G. Petkov, et al. "REPROGRAMMING OF PORCINE EPIBLAST-DERIVED NEURAL PROGENITOR CELLS TO PLURIPOTENCY." Reproduction, Fertility and Development 24, no. 1 (2012): 289. http://dx.doi.org/10.1071/rdv24n1ab252.
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Human induced pluripotent stem cells (iPSC) and neural progenitor cells (NPC) are envisioned to play a vital role in future cell replacement therapy. In this context, porcine iPSC and NPC would be highly useful for pre-clinical safety testing by autologous transplantation in a porcine biomedical model. The objective of this study was to establish iPSC from porcine epiblast-derived NPC by use of a tetracycline-inducible Tet-ON approach. A total of 1.5 × 105 porcine NPC at passage 6 (Rasmussen et al. 2011) were transduced O/N with 0.5 ml active virus containing the following porcine pluripotency genes: pOCT4 (pO); pOCT4 and pKLF4 (pOK); pOCT4 and pC-MYC (pOM); pOCT4, pC-MYC, and pKLF4 (pOMK) or polycistronic pOCT4, pSOX2, pC-MYC, and pKLF4 (pOSMK); all including 0.25 ml transactivator (rtTA). After 3 days, the cells were trypsinized and passaged to MEF feeder cells and cultured in iPSC medium containing DMEM/F12, 20% KSR, 1% NEAA, 10 μM β-Me, 20 ng mL–1 human bFGF and 2 μg mL–1 doxycycline. On Day 8, tightly packed colonies of cells presenting an embryonic stem cell-like morphology were visible in the pOM, pOMK, and pOSMK combinations. In contrast, colonies were not observed with the pO and pOK combination. On Day 14, several iPSC-like colonies were manually picked and sub-cultured on MEF feeder cells in iPSC medium. Two lines from the pOSMK combination were capable of prolonged clonal propagation while maintaining an ESC-like morphology. However, when doxycycline was removed from the culture medium, growth arrest and spontaneous differentiation occurred. The iPSC-like lines expressed OCT4, SOX2, C-MYC, and KLF4, as evaluated by immunocytochemistry, and expression of NANOG, SSEA-1, and SSEA-4 was also confirmed, demonstrating activation of endogenous pluripotency genes. The iPSC-like lines were capable of forming embryoid bodies (EB) without addition of doxycycline and in vitro differentiation of EB in medium containing DMEM and 15% FCS confirmed the presence of meso- (SMA) and endodermal (AFP) derivatives by immunocytochemistry. Furthermore, co-culture experiments with MS5 stromal cells in medium containing DMEM, 15% KSR, and 150 ng mL–1 human Noggin resulted in differentiation into neuroectoderm (NESTIN and SOX2), as well as more mature neurons (TUJI and GFAP). The latter resulted in establishment of new NPC lines. The system can be used to study mechanisms involved in the early transition from pluripotency to multipotency in the pig and the reversal of the process caused by reprogramming. The Danish Agency for Science, Technology and Innovation, the Danish National Advanced Technology Foundation as well as the EU projects, EU FP7 Stem Cell Project “PartnErS” (218205; 204, 523) and EU FP7 Stem Cell Project “PluriSys” (223485).
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Dieci, C., F. Franciosi, V. Lodde, et al. "198 CILOSTAMIDE SUSTAINS GAP JUNCTION-MEDIATED COMMUNICATION AND CHROMATIN REMODELLING IN PIG OOCYTES." Reproduction, Fertility and Development 24, no. 1 (2012): 211. http://dx.doi.org/10.1071/rdv24n1ab198.
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In the pig, the efficiency of in vitro embryo production procedures is still limited. It has been suggested that prematuration treatments could improve the developmental capability of oocytes. In particular, recent studies conducted in the bovine (Luciano, 2011, BOR, in press) indicate that the prolongation of a patent bidirectional crosstalk between the oocyte and the surrounding cumulus cells, together with the maintenance of a proper level of cAMP during the prematuration culture, could be beneficial to oocytes that have not yet acquired full meiotic and developmental capability. The aim of the present study was to assess the effect of treatment with cilostamide, an inhibitor of the phosphodiesterase 3 (PDE3), which degrades cAMP, on the functional status of gap junction-mediated communication (GJC) in pig cumulus–oocyte complexes (COC). Moreover, since chromatin configuration represents a marker of oocyte differentiation and competence, the effect of cilostamide on the process of chromatin remodeling was also evaluated during the culture period. To this aim, COC were collected from 3- to 6-mm antral follicles and cultured for up to 24 h in defined culture medium supplemented with 0.1 IU mL–1 of FSH in the presence or absence of 1 μM cilostamide. The GJC functionality was assessed by Lucifer Yellow fluorescent dye microinjection at the time of collection (0 h) and after 12, 18, or 24 h of culture. Chromatin configuration was evaluated by fluorescence microscopy after removal of cumulus cells and DNA staining with Hoechst and oocytes were classified according to Bui et al. (2004 BOR 70, 1843–1851) as SC, (with stringy chromatin within the germinal vesicle), GVI (with chromatin condensed in a rim around the nucleolus), GVII-IV (where the beginning of formation of chromatin strands is typical), ProMI (prometaphase I) and MI (metaphase I). The administration of cilostamide sustained functional coupling for up to 24 h of culture as the percentage of COC with open GJC was significantly higher when compared with the control group (62.2% vs 30%; P < 0.05) and not significantly different from the time 0 h (80%). The maintenance of the coupling during the culture period was accompanied by a delay of the meiotic resumption as only 26.3% of cilostamide-treated oocytes underwent germinal-vesicle breakdown and reached ProMI stage compared to the control group (62.1%; P < 0.05). Moreover the transition towards advanced stages of differentiation, as judged by the chromatin configuration, was slowed down in the presence of cilostamide. In conclusion, our study indicates that the maintenance of elevated cAMP levels through the inhibition of PDE3 sustains a functional bidirectional communication between the oocyte and cumulus cells and delays meiotic resumption in the pig oocyte. This could be a useful approach for the development of prematuration treatments aimed at improving the embryonic developmental potential of pig oocytes. Experiments are in progress in our laboratories to confirm this hypothesis. This study has been supported by EU FP6 grant n LSHB-CT-2006-037377 (Xenome) EU FP7- n°223485 (Plurisys).
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Rasmussen, M. A., V. J. Hall, S. G. Petkov, et al. "211 REPROGRAMMING OF PORCINE NEURAL PROGENITOR CELLS TO INDUCED PLURIPOTENT STEM CELL-LIKE CELLS." Reproduction, Fertility and Development 24, no. 1 (2012): 217. http://dx.doi.org/10.1071/rdv24n1ab211.
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Human-induced pluripotent stem cells (iPSC) are envisioned to play a vital role in future cell replacement therapy. In this context, porcine iPSC would be highly useful for pre-clinical safety testing by autologous transplantation in a porcine biomedical model. However, a major impediment is that currently, continuous expression of reprogramming factors is required to maintain the pluripotent state of porcine iPSC. In the mouse, neural progenitor cells (NPC) have proved to be highly amenable to reprogramming due to their partly stem-like epigenetic state and expression of pluripotency-related genes such as SOX2. The objective of this study was to establish iPSC from porcine epiblast-derived NPC by use of a tetracycline-inducible Tet-ON approach. A total of 1.5 × 105 porcine NPC at passage 6 (Rasmussen et al. 2011) were transduced O/N with 0.5 mL of active virus containing the following porcine pluripotency genes: pOCT4 (pO); pOCT4 and pKLF4 (pOK); pOCT4 and pC-MYC (pOM); pOCT4, pC-MYC and pKLF4 (pOMK) or polycistronic pOCT4, pSOX2, pC-MYC and pKLF4 (pOSMK); all including 0.25 mL of transactivator (rtTA). After 3 days, the cells were trypsinized and passaged to MEF feeder cells and cultured in iPSC medium containing DMEM/F12, 20% KSR, 1% NEAA, 10 μM β-Me, 20 ng mL–1 human bFGF and 2 μg mL–1 doxycycline. On Day 8, tightly packed colonies of cells presenting an embryonic stem cell-like morphology were visible in the pOM, pOMK and pOSMK combinations. In contrast, colonies were not observed with the pO and pOK combination. On Day 14, several iPSC-like colonies were manually picked and subcultured on MEF feeder cells in iPSC medium. Two lines from the pOSMK combination were capable of prolonged clonal propagation while maintaining an ESC-like morphology. However, when doxycycline was removed from the culture medium, growth arrest and spontaneous differentiation occurred. The iPSC-like lines expressed OCT4, SOX2, C-MYC and KLF4, as evaluated by immunocytochemistry and expression of NANOG, SSEA-1 and SSEA-4 was also confirmed, demonstrating activation of endogenous pluripotency genes. The iPSC-like lines were capable of forming embryoid bodies (EB) without addition of doxycycline and in vitro differentiation of EB in medium containing DMEM and 15% FCS confirmed the presence of meso- (SMA) and endodermal (AFP) derivatives by immunocytochemistry. Furthermore, co-culture experiments with MS5 stromal cells in medium containing DMEM, 15% KSR and 150 ng mL–1 human Noggin resulted in differentiation into neuroectoderm (NESTIN and SOX2), as well as more mature neurons (TUJI and GFAP). The porcine iPSC-like lines could serve as an excellent platform for optimizing culture conditions, which may sustain the pluripotency network in the pig and could be applied for autologous stem cell transplantation in a porcine model for evaluation of safety and efficacy. The Danish Agency for Science, Technology and Innovation, the Danish National Advanced Technology Foundation as well as the EU projects, EU FP7 (PartnErS, PIAP-GA-2008-218205; PluriSys, HEALTH-2007-B-223485).
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Njeru, Sospeter N., and Jackson M. Muema. "In vitro cytotoxicity of Aspilia pluriseta Schweinf. extract fractions." BMC Research Notes 14, no. 1 (2021). http://dx.doi.org/10.1186/s13104-021-05472-4.
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Abstract Objectives We and others have shown that Aspilia pluriseta is associated with various biological activities. However, there is a lack of information on its cytotoxicity. This has created an information gap about the safety of A. pluriseta extracts. As an extension to our recent publication on the antimicrobial activity and the phytochemical characterization of A. pluriseta root extracts, here we report on cytotoxicity of tested solvent fractions. We evaluated the potential cytotoxicity of these root extract fractions on Vero cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results We show that all solvent extract fractions (except methanolic solvent fractions) had cytotoxic concentration values that killed 50% of the Vero cells (CC50) greater than 20 µg/mL and selectivity index (SI) greater than 1.0. Taken together, we demonstrate that, A. pluriseta extract fractions’ earlier reported bioactivities are within the acceptable cytotoxicity and selective index limits. This finding scientifically validates the potential use of A. pluriseta in the discovery of safe therapeutics agents.
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"Pluripotent cell–specific inhibitors (PluriSIns) to reduce the tumorigenic risk of stem cell–based therapies." Science-Business eXchange 6, no. 5 (2013): 126. http://dx.doi.org/10.1038/scibx.2013.126.
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WAWERU, PETER, ELIPHAS GITONGA MAKUNYI, and FREDERICK BUKACHI. "Investigation of the hemostatic effects of freeze-dried extracts of selected Kenyan plants." Asian Journal of Natural Product Biochemistry 17, no. 1 (2019). http://dx.doi.org/10.13057/biofar/f170104.
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Abstract. Makunyi EG, Bukachi F, Waweru P. 2019. Investigation of the hemostatic effects of freeze-dried extracts of selected Kenyan plants. Biofarmasi J Nat Prod Biochem 17: 39-46. This study aims to investigate the effect and mechanism of action of freeze-dried extracts of Tridax procumbens, Terminalia brownii, Euphorbia tirucalli, and Asphillia africana on hemostasis. Freeze-dried extract of the selected plants was prepared and dose determined for the study. Twelve male New Zealand white rabbits were randomly allocated to two groups (control and test). Blood was collected under standard procedures. Duke's method was used for the bleeding time while the capillary method was used for clotting time. ACL Elitepro machine was used to do prothrombin time and activated partial thromboplastin time. Thromboelastography was done for the most potent extracts. Data were analyzed using independent t-test and results were presented as mean± standard error of means. Differences were considered to be significant if P < 0.05. The results showed that the percentage yield of the extract was; Tridax procumbens (0.8%), Terminalia brownii (0.5%), Euphorbia tirucalli (0.2%) and Aspillia pluriseta (1.3%). Bleeding and clotting time: The bleeding time was reduced by freeze-dried leaf extract of Tridax procumbens (p = 0.0068) and by freeze-dried bark extract of Terminalia brownii (p=0.0068). Freeze-dried leaf extract of Asphillia africana increased the bleeding time (p=0.01). The clotting time was reduced by freeze-dried leaf extract of Tridax procumbens (p = 0.038), the freeze-dried bark extract of Terminalia brownii (p=0.043) and by freeze-dried stem extract of Euphorbia tirucalli (p=0.01). Prothrombin and activated partial thromboplastin time: The prothrombin time was reduced by freeze-dried leaf extract of Tridax procumbens (p = 0.004), the freeze-dried bark extract of Terminalia brownii (p<0.001) and Freeze-dried stem extract of Euphorbia tirucalli (p=0.001). Activated partial thromboplastin time was reduced by freeze-dried leaf extract of Tridax procumbens (p< 0.001), the freeze-dried bark extract of Terminalia brownii (p<0.001) and by freeze-dried stem extract of Euphorbia tirucalli (p<0.001). The results for thromboelastogrpahy showed that four parameters of thromboelastography were tested. Freeze-dried leaf extract of Tridax procumbens reduced the r time (p =0.04), k time (p=0.04) and maximum amplitude (p =0.026) but increased the alpha angle (p =0.01). The freeze-dried bark extract of Terminalia brownii did not have statistically significant differences in thromboelastography variables.