Discovering and differentiating new and emerging clonal populations of Chlamydia trachomatis with a novel shotgun cell culture harvest assay.

Science.gov (United States)

Somboonna, Naraporn; Mead, Sally; Liu, Jessica; Dean, Deborah

2008-03-01

Chlamydia trachomatis is the leading cause of preventable blindness and bacterial sexually transmitted diseases worldwide. Plaque assays have been used to clonally segregate laboratory-adapted C. trachomatis strains from mixed infections, but no assays have been reported to segregate clones from recent clinical samples. We developed a novel shotgun cell culture harvest assay for this purpose because we found that recent clinical samples do not form plaques. Clones were strain-typed by using outer membrane protein A and 16S rRNA sequences. Surprisingly, ocular trachoma reference strain A/SA-1 contained clones of Chlamydophila abortus. C. abortus primarily infects ruminants and pigs and has never been identified in populations where trachoma is endemic. Three clonal variants of reference strain Ba/Apache-2 were also identified. Our findings reflect the importance of clonal isolation in identifying constituents of mixed infections containing new or emerging strains and of viable clones for research to more fully understand the dynamics of in vivo strain-mixing, evolution, and disease pathogenesis.

21 CFR 876.5885 - Tissue culture media for human ex vivo tissue and cell culture processing applications.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Tissue culture media for human ex vivo tissue and cell culture processing applications. 876.5885 Section 876.5885 Food and Drugs FOOD AND DRUG... DEVICES Therapeutic Devices § 876.5885 Tissue culture media for human ex vivo tissue and cell culture...

Cloning crops in a CELSS via tissue culture: Prospects and problems

Science.gov (United States)

Carman, John G.; Hess, J. Richard

1990-01-01

Micropropagation is currently used to clone fruits, nuts, and vegetables and involves controlling the outgrowth in vitro of basal, axillary, or adventitious buds. Following clonal multiplication, shoots are divided and rooted. This process has greatly reduced space and energy requirements in greenhouses and field nurseries and has increased multiplication rates by greater than 20 fold for some vegetatively propagated crops and breeding lines. Cereal and legume crops can also be cloned by tissue culture through somatic embryogenesis. Somatic embryos can be used to produce 'synthetic seed', which can tolerate desiccation and germinate upon rehydration. Synthetic seed of hybrid wheat, rice, soybean and other crops could be produced in a controlled ecological life support system. Thus, yield advantages of hybreds over inbreds (10 to 20 percent) could be exploited without having to provide additional facilities and energy for parental-line and hybrid seed nurseries.

Selection and Clonal Propagation of High Artemisinin Genotypes of Artemisia annua

Science.gov (United States)

Wetzstein, Hazel Y.; Porter, Justin A.; Janick, Jules; Ferreira, Jorge F. S.; Mutui, Theophilus M.

2018-01-01

Artemisinin, produced in the glandular trichomes of Artemisia annua L. is a vital antimalarial drug effective against Plasmodium falciparum resistant to quinine-derived medicines. Although work has progressed on the semi-synthetic production of artemisinin, field production of A. annua remains the principal commercial source of the compound. Crop production of artemisia must be increased to meet the growing worldwide demand for artemisinin combination therapies (ACTs) to treat malaria. Grower artemisinin yields rely on plants generated from seeds from open-pollinated parents. Although selection has considerably increased plant artemisinin concentration in the past 15 years, seed-generated plants have highly variable artemisinin content that lowers artemisinin yield per hectare. Breeding efforts to produce improved F1 hybrids have been hampered by the inability to produce inbred lines due to self-incompatibility. An approach combining conventional hybridization and selection with clonal propagation of superior genotypes is proposed as a means to enhance crop yield and artemisinin production. Typical seed-propagated artemisia plants produce less than 1% (dry weight) artemisinin with yields below 25 kg/ha. Genotypes were identified producing high artemisinin levels of over 2% and possessing improved agronomic characteristics such as high leaf area and shoot biomass production. Field studies of clonally-propagated high-artemisinin plants verified enhanced plant uniformity and an estimated gross primary productivity of up to 70 kg/ha artemisinin, with a crop density of one plant m-2. Tissue culture and cutting protocols for the mass clonal propagation of A. annua were developed for shoot regeneration, rooting, acclimatization, and field cultivation. Proof of concept studies showed that both tissue culture-regenerated plants and rooted cutting performed better than plants derived from seed in terms of uniformity, yield, and consistently high artemisinin content. Use of

Propagation of jarrah (Eucalyptus marginata) by organ and tissue culture

Energy Technology Data Exchange (ETDEWEB)

Bennett, M.J.; McComb, J.A.

1982-01-01

Micropropagation methods are described for the production of clonal lines from Eucalyptus marginata (jarrah) seedlings. Nodal explants from mature trees can also yield shoot cultures, but a high frequency of contamination occurs among such explants. Uncontaminated callus cultures can be produced from mature trees by culturing stamen filaments and shoots can subsequently be regenerated from this callus. The rooting percentage of shoot cultures from either nodes or stamen callus of mature trees is low compared with that from seedling explants. Considerable variation was observed between trees in the ability of stamen callus to regenerate shoots and in the frequency of rooting. (Refs. 27)

Clonal analysis of stem cells in differentiation and disease.

Science.gov (United States)

Colom, Bartomeu; Jones, Philip H

2016-12-01

Tracking the fate of individual cells and their progeny by clonal analysis has redefined the concept of stem cells and their role in health and disease. The maintenance of cell turnover in adult tissues is achieved by the collective action of populations of stem cells with an equal likelihood of self-renewal or differentiation. Following injury stem cells exhibit striking plasticity, switching from homeostatic behavior in order to repair damaged tissues. The effects of disease states on stem cells are also being uncovered, with new insights into how somatic mutations trigger clonal expansion in early neoplasia. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Assessment of clonal fidelity of Tylophora indica (Burm. f.) Merrill "in vitro" plantlets by ISSR molecular markers.

Science.gov (United States)

Sharma, Madan Mohan; Verma, Roop Narayan; Singh, Abhijeet; Batra, Amla

2014-01-01

Tylophora indica Burm F. Merrill. is widely used against various diseases owing to the presence an array of medicinally important secondary metabolites. Its stem is bitter, stomachic, stimulates bile secretion, enriches the blood and cures diseases like diabetes, fever, flatulence, hypertension, jaundice, leucorrhoea, urinary disease and upper respiratory tract infection. It is neglected for tissue culture work because of deciduous nature of climbing shrub, facing problems for micropropagation. Hence, in vitro regeneration of complete plantlets was done through indirect organogenesis in Tylophora indica. Calli were produced from in vivo leaves of T. indica on MS medium supplemented with 6-Benzylaminopurine (BAP: 2.0 mg l(-1)) and Indole-3-butyric acid (IBA: 0.5 mg l(-1)). The multiple shoots (12.00 ± 1.50) emerged and elongated on MS medium fortified with Thidiazuron (TDZ: 0.1 mg l(-1)). They were rooted on half strength MS medium having IBA (0.5 mg l(-1)) (7.75 ± 0.25) after 20 days of sub-culturing followed by hardening and acclimatization. During indirect regeneration of plants, chances of somaclonal variations may arise. These variations should be identified to produce true to type plants. Plantlets raised through tissue culture were used to validate the clonal fidelity through Inter simple sequence repeat markers (ISSR). Clonal fidelity is a major consideration in commercial micropropagation using in vitro tissue culture methods. During the study, total 71 clear and distinct bands were produced using 6 primers. The banding pattern of each primer was uniform and comparable to mother plant and showed about 93% homology using un-weighted pair group method with arithmetic averaging (UPGMA). ISSR analysis confirmed the genetic stability of in vitro raised plants.

Comparison of bacterial culture and 16S rRNA community profiling by clonal analysis and and pyrosequencing for the characterisation of the caries-associated microbiome

Directory of Open Access Journals (Sweden)

Kathrin eSchulze-Schweifing

2014-11-01

Full Text Available Culture-independent analyses have greatly expanded knowledge regarding the composition of complex bacterial communities including those associated with oral diseases. A consistent finding from such studies, however, has been the under-reporting of members of the phylum Actinobacteria. In this study, five pairs of broad range primers targeting 16S rRNA genes were used in clonal analysis of 6 samples collected from tooth lesions involving dentine in subjects with active caries. Samples were also subjected to cultural analysis and pyrosequencing by means of the 454 platform. A diverse bacterial community of 229 species-level taxa was revealed by culture and clonal analysis, dominated by representatives of the genera Prevotella, Lactobacillus, Selenomonas and Streptococcus. The five most abundant species were: Lactobacillus gasseri, Prevotella denticola, Alloprevotella tannerae, S. mutans and Streptococcus sp. HOT 070, which together made up 31.6 % of the sequences. Two samples were dominated by lactobacilli, while the remaining samples had low numbers of lactobacilli but significantly higher numbers of Prevotella species. The different primer pairs produced broadly similar data but proportions of the phylum Bacteroidetes were significantly higher when primer 1387R was used. All of the primer sets underestimated the proportion of Actinobacteria compared to culture. Pyrosequencing analysis of the samples was performed to a depth of sequencing of 4293 sequences per sample which were identified to 264 species-level taxa, and resulted in significantly higher coverage estimates than the clonal analysis. Pyrosequencing, however, also underestimated the relative abundance of Actinobacteria compared to culture.

Improved clonality detection in B-cell lymphoma using a semi-nested modification of the BIOMED-2 PCR assay for IGH rearrangement: A paraffin-embedded tissue study.

Science.gov (United States)

Sakamoto, Yuma; Masaki, Ayako; Aoyama, Satsuki; Han, Shusen; Saida, Kosuke; Fujii, Kana; Takino, Hisashi; Murase, Takayuki; Iida, Shinsuke; Inagaki, Hiroshi

2017-09-01

The BIOMED-2 PCR protocol for targeting the IGH gene is widely employed for detecting clonality in B-cell malignancies. Unfortunately, the detection of clonality with this method is not very sensitive when paraffin sections are used as a DNA source. To increase the sensitivity, we devised a semi-nested modification of a JH consensus primer. The clonality detection rates of three assays were compared: the standard BIOMED-2, BIOMED-2 assay followed by BIOMED-2 re-amplification, and BIOMED-2 assay followed by semi-nested BIOMED-2. We tested more than 100 cases using paraffin-embedded tissues of various B-cell lymphomas, and found that the clonality detection rates with the above three assays were 63.9%, 79.6%, and 88.0%, respectively. While BIOMED-2 re-amplification was significantly more sensitive than the standard BIOMED-2, the semi-nested BIOMED-2 was significantly more sensitive than both the standard BIOMED-2 and BIOMED-2 re-amplification. An increase in sensitivity was observed in all lymphoma subtypes examined. In conclusion, tumor clonality may be detected in nearly 90% of B-cell lymphoma cases with semi-nested BIOMED-2. This ancillary assay may be useful when the standard BIOMED-2 fails to detect clonality in histopathologically suspected B-cell lymphomas. © 2017 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

A computational clonal analysis of the developing mouse limb bud.

Directory of Open Access Journals (Sweden)

Luciano Marcon

Full Text Available A comprehensive spatio-temporal description of the tissue movements underlying organogenesis would be an extremely useful resource to developmental biology. Clonal analysis and fate mappings are popular experiments to study tissue movement during morphogenesis. Such experiments allow cell populations to be labeled at an early stage of development and to follow their spatial evolution over time. However, disentangling the cumulative effects of the multiple events responsible for the expansion of the labeled cell population is not always straightforward. To overcome this problem, we develop a novel computational method that combines accurate quantification of 2D limb bud morphologies and growth modeling to analyze mouse clonal data of early limb development. Firstly, we explore various tissue movements that match experimental limb bud shape changes. Secondly, by comparing computational clones with newly generated mouse clonal data we are able to choose and characterize the tissue movement map that better matches experimental data. Our computational analysis produces for the first time a two dimensional model of limb growth based on experimental data that can be used to better characterize limb tissue movement in space and time. The model shows that the distribution and shapes of clones can be described as a combination of anisotropic growth with isotropic cell mixing, without the need for lineage compartmentalization along the AP and PD axis. Lastly, we show that this comprehensive description can be used to reassess spatio-temporal gene regulations taking tissue movement into account and to investigate PD patterning hypothesis.

Discarded human fetal tissue and cell cultures for transplantation research

International Nuclear Information System (INIS)

Hay, R.J.; Phillips, T.; Thompson, A.; Vilner, L.; Cleland, M.; Tchaw-ren Chen; Zabrenetzky, V.

1999-01-01

A feasibility study has been performed to explore the utility of various tissues from discarded human abortuses for transplantation and related research. Specifically, aborted fetuses plus parental blood samples and all relevant clinical data were obtained through a local hospital complex. Whenever possible, pancreas, skin and skeletal muscle, heart, liver, kidney, cartilage and lung tissues were removed, dissociated and subfractionated for cryopreservation, characterization and cultivation trials in vitro. Existing protocols for these manipulations were compared and improved upon as required. Clonal culture, cell aggregate maintenance techniques and use of feeder cell populations have been utilized where appropriate to develop quantitative comparative data. Histological and biochemical assays were applied both to evaluate separation/cultivation methods and to identify optimal culture conditions for maintaining functional cells. Immunochemical and molecular biological procedures were applied to study expression of Major Histocompatibility Vomplex (MHC) class 1 and 11 molecules on cell lines derived. Tissue and cell culture populations were examined for infections with bacteria, ftingi, mycoplasma, HIV, CMV, hepatitis B and other viruses. Only 1% of the abortuses tested were virally infected. Cytogenetic analyses confin-ned the normal diploid status in the vast majority (>98%) of lines tested. A total of over 250 abortuses have been obtained and processed. Only 25 were found to be contaminated with bacteria or fungi and unsuitable for further cultivation trials. A total of over 200 cell populations were isolated, characterized and cryopreserved for further study. Included were kidney, lung, liver and epidermal epithelia: cartilage-derived cells from the spine and epiphyses plus myogenic myoblasts. Selected lines have been immortalized using HPV I 6E6/E7 sequences. Epithelia from the liver and pancreas and cardiac myocytes were the most problematic in that initial

The plant tissue culture

International Nuclear Information System (INIS)

Crocomo, O.J.; Sharp, W.R.

1973-01-01

Progress in the field of plant tissue culture at the Plant Biochemistry Sector, Centro de Energia na Agricultura (CENA), Piracicaba, S.P., Brazil, pertains to the simplification of development in 'Phaseolus vulgaris' by dividing the organism into its component organs, tissues, and cells and the maintenance of these components on defined culture media 'in vitro'. This achievement has set the stage for probing the basis for the stability of the differentiated states and/or the reentry of mature differentiated cells into the mitotic cell cycle and their subsequent redifferentiation. Data from such studies at the cytological and biochemical level have been invaluable in the elucidation of the control mechanisms responsible for expression of the cellular phenotype. Unlimited possibilities exist for the application of tissue culture in the vegetative propagation of 'Phaseolus' and other important cultivars in providing genocopies or a large scale and/or readily obtaining plantlets from haploid cell lines or from protoplast (wall-less cells) hybridization products following genetic manipulation. These tools are being applied in this laboratory for the development and selection of high protein synthesizing 'Phaseolus' cultivars

Clonal dominance among T-lymphocyte infiltrates in arthritis

International Nuclear Information System (INIS)

Stamenkovic, I.; Stegagno, M.; Wright, K.A.; Krane, S.M.; Amento, E.P.; Colvin, R.B.; Duquesnoy, R.J.; Kurnick, J.T.

1988-01-01

Synovial membranes in patients with rheumatoid arthritis as well as other types of chronic destructive inflammatory arthritis contain infiltrates of activated T lymphocytes that probably contribute to the pathogenesis of the disease. In an effort to elucidate the nature of these infiltrates, interleukin 2 (IL-2)-responsive T lymphocytes were grown out of synovial fragments from 14 patients undergoing surgery for advanced destructive inflammatory joint disease. Eleven of the samples examined were from patients with classical rheumatoid arthritis, while three others were obtained from individuals with clinical osteoarthritis. Southern blot analysis of T-cell receptor (TCR) β-chain genes in 13 of 14 cultures showed distinct rearrangements, indicating that each culture was characterized by the predominance of a limited number of clones. T-cell populations from peripheral blood stimulated with a variety of activators and expanded with IL-2 did not demonstrate evidence of similar clonality in long-term culture. These results suggest that a limited number of activated T-cell clones predominate at the site of tissue injury in rheumatoid synovial membranes as well as in other types of destructive inflammatory joint disease. Further characterization of these T-cell clones may aid our understanding of the pathogenesis of these rheumatic disorders

Laboratory Workflow Analysis of Culture of Periprosthetic Tissues in Blood Culture Bottles.

Science.gov (United States)

Peel, Trisha N; Sedarski, John A; Dylla, Brenda L; Shannon, Samantha K; Amirahmadi, Fazlollaah; Hughes, John G; Cheng, Allen C; Patel, Robin

2017-09-01

Culture of periprosthetic tissue specimens in blood culture bottles is more sensitive than conventional techniques, but the impact on laboratory workflow has yet to be addressed. Herein, we examined the impact of culture of periprosthetic tissues in blood culture bottles on laboratory workflow and cost. The workflow was process mapped, decision tree models were constructed using probabilities of positive and negative cultures drawn from our published study (T. N. Peel, B. L. Dylla, J. G. Hughes, D. T. Lynch, K. E. Greenwood-Quaintance, A. C. Cheng, J. N. Mandrekar, and R. Patel, mBio 7:e01776-15, 2016, https://doi.org/10.1128/mBio.01776-15), and the processing times and resource costs from the laboratory staff time viewpoint were used to compare periprosthetic tissues culture processes using conventional techniques with culture in blood culture bottles. Sensitivity analysis was performed using various rates of positive cultures. Annualized labor savings were estimated based on salary costs from the U.S. Labor Bureau for Laboratory staff. The model demonstrated a 60.1% reduction in mean total staff time with the adoption of tissue inoculation into blood culture bottles compared to conventional techniques (mean ± standard deviation, 30.7 ± 27.6 versus 77.0 ± 35.3 h per month, respectively; P < 0.001). The estimated annualized labor cost savings of culture using blood culture bottles was $10,876.83 (±$337.16). Sensitivity analysis was performed using various rates of culture positivity (5 to 50%). Culture in blood culture bottles was cost-effective, based on the estimated labor cost savings of $2,132.71 for each percent increase in test accuracy. In conclusion, culture of periprosthetic tissue in blood culture bottles is not only more accurate than but is also cost-saving compared to conventional culture methods. Copyright © 2017 American Society for Microbiology.

Improved clonality detection in Hodgkin lymphoma using a semi-nested modification of the BIOMED-2 PCR assay for IGH and IGK rearrangements: A paraffin-embedded tissue study.

Science.gov (United States)

Han, Shusen; Masaki, Ayako; Sakamoto, Yuma; Takino, Hisashi; Murase, Takayuki; Iida, Shinsuke; Inagaki, Hiroshi

2018-05-01

The BIOMED-2 PCR protocols targeting IGH and IGK genes may be useful for detecting clonality in Hodgkin lymphoma (HL). The clonality detection rates, however, have not been very high with these methods using paraffin-embedded tumor sections. We previously described the usefulness of the semi-nested BIOMED-2 IGH assay in B-cell malignancies. In this study, we devised a novel semi-nested BIOMED-2 IGK assay. Employing 58 cases of classical HL, we carried out the standard BIOMED-2, BIOMED-2 followed by BIOMED-2 re-amplification, and BIOMED-2 followed by semi-nested BIOMED-2, all targeting IGH and IGK, using paraffin-embedded tissues. In both IGH and IGK assays, semi-nested assays yielded significantly higher clonality detection rates than the standard assays and re-amplification assays. Clonality was detected in 13/58 (22.4%) classical HL cases using the standard IGH/IGK assays while it was detected in 38/58 (65.5%) cases using semi-nested IGH/IGK assays. The detection rates were not associated with the HL subtypes, CD30-positive cell density, CD20-positive cell density, or Epstein-Barr virus (EBV) positivity. In conclusion, tumor clonality was detected in nearly two-thirds of classical HL cases using semi-nested BIOMED-2 IGH/IGK assays using paraffin tumor sections. These semi-nested assays may be useful when the standard IGH/IGK assays fail to detect clonality in histopathologically suspected HLs. © 2018 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

Uncovering the Number and Clonal Dynamics of Mesp1 Progenitors during Heart Morphogenesis

Directory of Open Access Journals (Sweden)

Samira Chabab

2016-01-01

Full Text Available The heart arises from distinct sources of cardiac progenitors that independently express Mesp1 during gastrulation. The precise number of Mesp1 progenitors that are specified during the early stage of gastrulation, and their clonal behavior during heart morphogenesis, is currently unknown. Here, we used clonal and mosaic tracing of Mesp1-expressing cells combined with quantitative biophysical analysis of the clonal data to define the number of cardiac progenitors and their mode of growth during heart development. Our data indicate that the myocardial layer of the heart derive from ∼250 Mesp1-expressing cardiac progenitors born during gastrulation. Despite arising at different time points and contributing to different heart regions, the temporally distinct cardiac progenitors present very similar clonal dynamics. These results provide insights into the number of cardiac progenitors and their mode of growth and open up avenues to decipher the clonal dynamics of progenitors in other organs and tissues.

Progress in planta transformation without tissue culture

International Nuclear Information System (INIS)

Gu Yunhong; Chinese Academy of Sciences, Hefei; Qin Guangyong; Huo Yuping; Yu Zengliang

2004-01-01

With the development of planta genetic engineering, more emphases have been laid on convenient and high efficient genetic transformation methods. And transformation without tissue culture is a prospective direction of it. In this paper, traditional transformation methods and the methods of non-tissue culture were summarized. With the exploration and application of Arabidopsis transformation mechanism, with the use of ion beam-mediated transformation invented by Chinese scientists and the development of other transformation methods, transformation methods without tissue culture and planta genetic engineering could be improved rapidly. (authors)

Cloning higher plants from aseptically cultured tissues and cells

Science.gov (United States)

Krikorian, A. D.

1982-01-01

A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.

Clonal status and clinicopathological observation of cervical minimal deviation adenocarcinoma

Directory of Open Access Journals (Sweden)

Lan Miao

2010-04-01

Full Text Available Abstract Background Minimal deviation adenocarcinoma (MDA of the uterine cervix is defined as an extremely well differentiated variant of cervical adenocarcinoma, with well-formed glands that resemble benign glands but show distinct nuclear anaplasia or evidence of stromal invasion. Thus, MDA is difficult to differentiate from other cervical hyperplastic lesions. Monoclonality is a major characteristic of most tumors, whereas normal tissue and reactive hyperplasia are polyclonal. Methods The clinicopathological features and clonality of MDA were investigated using laser microdissection and a clonality assay based on the polymorphism of androgen receptor (AR and X-chromosomal inactivation mosaicism in female somatic tissues. Results The results demonstrated that the glands were positive for CEA, Ki-67, and p53 and negative for estrogen receptor (ER, progesterone receptor (PR, and high-risk human papilloma virus (HPV DNA. The index of proliferation for Ki-67 was more than 50%. However, the stromal cells were positive for ER, PR, vimentin, and SM-actin. The clonal assay showed that MDA was monoclonal. Thus, our findings indicate that MDA is a true neoplasm but is not associated with high-risk HPV. Conclusions Diagnosis of MDA depends mainly on its clinical manifestations, the pathological feature that MDA glands are located deeper than the lower level of normal endocervical glands, and immunostaining.

Bacterial clonal diagnostics as a tool for evidence-based empiric antibiotic selection.

Science.gov (United States)

Tchesnokova, Veronika; Avagyan, Hovhannes; Rechkina, Elena; Chan, Diana; Muradova, Mariya; Haile, Helen Ghirmai; Radey, Matthew; Weissman, Scott; Riddell, Kim; Scholes, Delia; Johnson, James R; Sokurenko, Evgeni V

2017-01-01

Despite the known clonal distribution of antibiotic resistance in many bacteria, empiric (pre-culture) antibiotic selection still relies heavily on species-level cumulative antibiograms, resulting in overuse of broad-spectrum agents and excessive antibiotic/pathogen mismatch. Urinary tract infections (UTIs), which account for a large share of antibiotic use, are caused predominantly by Escherichia coli, a highly clonal pathogen. In an observational clinical cohort study of urgent care patients with suspected UTI, we assessed the potential for E. coli clonal-level antibiograms to improve empiric antibiotic selection. A novel PCR-based clonotyping assay was applied to fresh urine samples to rapidly detect E. coli and the urine strain's clonotype. Based on a database of clonotype-specific antibiograms, the acceptability of various antibiotics for empiric therapy was inferred using a 20%, 10%, and 30% allowed resistance threshold. The test's performance characteristics and possible effects on prescribing were assessed. The rapid test identified E. coli clonotypes directly in patients' urine within 25-35 minutes, with high specificity and sensitivity compared to culture. Antibiotic selection based on a clonotype-specific antibiogram could reduce the relative likelihood of antibiotic/pathogen mismatch by ≥ 60%. Compared to observed prescribing patterns, clonal diagnostics-guided antibiotic selection could safely double the use of trimethoprim/sulfamethoxazole and minimize fluoroquinolone use. In summary, a rapid clonotyping test showed promise for improving empiric antibiotic prescribing for E. coli UTI, including reversing preferential use of fluoroquinolones over trimethoprim/sulfamethoxazole. The clonal diagnostics approach merges epidemiologic surveillance, antimicrobial stewardship, and molecular diagnostics to bring evidence-based medicine directly to the point of care.

Effect of lunar materials on plant tissue culture.

Science.gov (United States)

Walkinshaw, C. H.; Venketeswaran, S.; Baur, P. S.; Croley, T. E.; Scholes, V. E.; Weete, J. D.; Halliwell, R. S.; Hall, R. H.

1973-01-01

Lunar material collected during the Apollo 11, 12, 14, and 15 missions has been used to treat 12 species of higher plant tissue cultures. Biochemical and morphological studies have been conducted on several of these species. Tobacco tissue cultures treated with 0.22 g of lunar material exhibited increased greening more complex chloroplasts, less cytoplasmic vacuolation and greater vesiculation. Pine tissue cultures reacted to treatment by an increased deposition of tannin-like materials. The percentage of dry weight and soluble protein was increased in cultures treated with either lunar or terrestrial rock materials.

Clonal expansion of CD4+ Cytotoxic T Lymphocytes in IgG4-related disease

Science.gov (United States)

Mattoo, Hamid; Mahajan, Vinay S.; Maehara, Takashi; Deshpande, Vikram; Della-Torre, Emanuel; Wallace, Zachary S.; Kulikova, Maria; Drijvers, Jefte M.; Daccache, Joe; Carruthers, Mollie N.; Castellino, Flavia; Stone, James R.; Stone, John H.; Pillai, Shiv

2016-01-01

Background IgG4-related disease (IgG4-RD) is a systemic condition of unknown etiology, characterized by highly fibrotic lesions with dense lymphoplasmacytic infiltrates. CD4+ T cells constitute the major inflammatory cell population in IgG4-RD lesions. Objective We used an unbiased approach to characterize CD4+ T cell subsets in IgG4-RD subjects based on their clonal expansion and their ability to infiltrate affected tissue sites. Methods We used flow cytometry to identify CD4+ effector/memory T cells (TEM) in a cohort of 101 IgG4-related disease (IgG4-RD) patients. These expanded cells were characterized by gene expression analysis and flow cytometry. Next-generation sequencing of the T cell receptor β chain gene was performed on CD4+SLAMF7+ CTLs and CD4+GATA3+ TH2 cells in a subset of patients to identify their clonality. Tissue infiltration by specific T cells was examined using quantitative multi-color imaging. Results CD4+ effector/memory T cells with a cytolytic phenotype were expanded in IgG4-RD patients. Next-generation sequencing revealed prominent clonal expansions of these CD4+CTLs but not CD4+GATA3+ memory TH2 cells in subjects with IgG4-RD. The dominant T cells infiltrating a range of inflamed IgG4-RD tissue sites were clonally-expanded CD4+CTLs that expressed SLAMF7, granzyme A, IL-1β, and TGF-β1. Clinical remission induced by rituximab-mediated B cell depletion was associated with a reduction in disease-associated CD4+ CTLs Conclusions IgG4-RD is prominently linked to clonally-expanded, IL-1β, and TGF- β1 secreting, CD4+ CTLs in peripheral blood as well as in inflammatory tissue lesions. These active, terminally-differentiated, cytokine-secreting effector CD4+ T cells are now linked to a human disease characterized by chronic inflammation and fibrosis. PMID:26971690

Smallholder adoption and economic impacts of tissue culture ...

African Journals Online (AJOL)

This study was conducted with an objective of determining the correlates of adoption of tissue culture banana technology and its impacts on household incomes in Kenya. The results show that while some households have opted not to adopt tissue culture banana biotechnology, almost all the adopters are growing tissue ...

Walnut tissue culture: research and field applications

Science.gov (United States)

2004-01-01

Vitrotech Biotecnologia Vegetal began researching propagating Juglans regia (English walnut) and various Juglans hybrids by tissue culture in 1993 and has operated on a commercial scale since 1996. Since this time, more than one and a half million walnuts of different species have been propagated and field planted. Tissue cultured...

Biotransformations with plant tissue cultures.

Science.gov (United States)

Carew, D P; Bainbridge, T

1976-01-01

Suspension cultures of Catharanthus roseus, Apocynum cannabinum and Conium maculatum were examined for their capacity to transform aniline, anisole, acetanilide, benzoic acid and coumarin. None of the cultures transformed acetanilide but each produced acetanilide when fed aniline. All three cultures converted benzoic acid to its para-hydroxy derivative. Coumarin was selectively hydroxylated at the 7-position by Catharanthus and Conium and anisole was O-demethylated only by older Catharanthus tissue.

Bacterial clonal diagnostics as a tool for evidence-based empiric antibiotic selection.

Directory of Open Access Journals (Sweden)

Veronika Tchesnokova

Full Text Available Despite the known clonal distribution of antibiotic resistance in many bacteria, empiric (pre-culture antibiotic selection still relies heavily on species-level cumulative antibiograms, resulting in overuse of broad-spectrum agents and excessive antibiotic/pathogen mismatch. Urinary tract infections (UTIs, which account for a large share of antibiotic use, are caused predominantly by Escherichia coli, a highly clonal pathogen. In an observational clinical cohort study of urgent care patients with suspected UTI, we assessed the potential for E. coli clonal-level antibiograms to improve empiric antibiotic selection. A novel PCR-based clonotyping assay was applied to fresh urine samples to rapidly detect E. coli and the urine strain's clonotype. Based on a database of clonotype-specific antibiograms, the acceptability of various antibiotics for empiric therapy was inferred using a 20%, 10%, and 30% allowed resistance threshold. The test's performance characteristics and possible effects on prescribing were assessed. The rapid test identified E. coli clonotypes directly in patients' urine within 25-35 minutes, with high specificity and sensitivity compared to culture. Antibiotic selection based on a clonotype-specific antibiogram could reduce the relative likelihood of antibiotic/pathogen mismatch by ≥ 60%. Compared to observed prescribing patterns, clonal diagnostics-guided antibiotic selection could safely double the use of trimethoprim/sulfamethoxazole and minimize fluoroquinolone use. In summary, a rapid clonotyping test showed promise for improving empiric antibiotic prescribing for E. coli UTI, including reversing preferential use of fluoroquinolones over trimethoprim/sulfamethoxazole. The clonal diagnostics approach merges epidemiologic surveillance, antimicrobial stewardship, and molecular diagnostics to bring evidence-based medicine directly to the point of care.

Tissue culture as a plant production technique for horticultural crops ...

African Journals Online (AJOL)

Over 100 years ago, Haberlandt envisioned the concept of plant tissue culture and provided the groundwork for the cultivation of plant cells, tissues and organs in culture. Initially plant tissue cultures arose as a research tool and focused on attempts to culture and study the development of small, isolated cells and segments ...

Application of Hanging Drop Technique for Kidney Tissue Culture.

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Wang, Shaohui; Wang, Ximing; Boone, Jasmine; Wie, Jin; Yip, Kay-Pong; Zhang, Jie; Wang, Lei; Liu, Ruisheng

2017-01-01

The hanging drop technique is a well-established method used in culture of animal tissues. However, this method has not been used in adult kidney tissue culture yet. This study was to explore the feasibility of using this technique for culturing adult kidney cortex to study the time course of RNA viability in the tubules and vasculature, as well as the tissue structural integrity. In each Petri dish with the plate covered with sterile buffer, a section of mouse renal cortex was cultured within a drop of DMEM culture medium on the inner surface of the lip facing downward. The tissue were then harvested at each specific time points for Real-time PCR analysis and histological studies. The results showed that the mRNA level of most Na+ related transporters and cotransporters were stably maintained within 6 hours in culture, and that the mRNA level of most receptors found in the vasculature and glomeruli were stably maintained for up to 9 days in culture. Paraffin sections of the cultured renal cortex indicated that the tubules began to lose tubular integrity after 6 hours, but the glomeruli and vasculatures were still recognizable up to 9 days in culture. We concluded that adult kidney tissue culture by hanging drop method can be used to study gene expressions in vasculature and glomeruli. © 2017 The Author(s). Published by S. Karger AG, Basel.

Application of Hanging Drop Technique for Kidney Tissue Culture

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Shaohui Wang

2017-05-01

Full Text Available Background/Aims: The hanging drop technique is a well-established method used in culture of animal tissues. However, this method has not been used in adult kidney tissue culture yet. This study was to explore the feasibility of using this technique for culturing adult kidney cortex to study the time course of RNA viability in the tubules and vasculature, as well as the tissue structural integrity. Methods: In each Petri dish with the plate covered with sterile buffer, a section of mouse renal cortex was cultured within a drop of DMEM culture medium on the inner surface of the lip facing downward. The tissue were then harvested at each specific time points for Real-time PCR analysis and histological studies. Results: The results showed that the mRNA level of most Na+ related transporters and cotransporters were stably maintained within 6 hours in culture, and that the mRNA level of most receptors found in the vasculature and glomeruli were stably maintained for up to 9 days in culture. Paraffin sections of the cultured renal cortex indicated that the tubules began to lose tubular integrity after 6 hours, but the glomeruli and vasculatures were still recognizable up to 9 days in culture. Conclusions: We concluded that adult kidney tissue culture by hanging drop method can be used to study gene expressions in vasculature and glomeruli.

The use of animal tissues alongside human tissue: Cultural and ethical considerations.

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Kaw, Anu; Jones, D Gareth; Zhang, Ming

2016-01-01

Teaching and research facilities often use cadaveric material alongside animal tissues, although there appear to be differences in the way we handle, treat, and dispose of human cadaveric material compared to animal tissue. This study sought to analyze cultural and ethical considerations and provides policy recommendations on the use of animal tissues alongside human tissue. The status of human and animal remains and the respect because of human and animal tissues were compared and analyzed from ethical, legal, and cultural perspectives. The use of animal organs and tissues is carried out within the context of understanding human anatomy and function. Consequently, the interests of human donors are to be pre-eminent in any policies that are enunciated, so that if any donors find the presence of animal remains unacceptable, the latter should not be employed. The major differences appear to lie in differences in our perceptions of their respective intrinsic and instrumental values. Animals are considered to have lesser intrinsic value and greater instrumental value than humans. These differences stem from the role played by culture and ethical considerations, and are manifested in the resulting legal frameworks. In light of this discussion, six policy recommendations are proposed, encompassing the nature of consent, respect for animal tissues as well as human remains, and appropriate separation of both sets of tissues in preparation and display. © 2015 Wiley Periodicals, Inc.

Developing a Plant Culture Medium Composed of Vinasse Originating from Haematococcus Pluvialis Culture

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Gollo, A. L.; Silva, A. L. L. D.; Lima, K. K. D. D.; Camara, M. C.; Rodrigues, C.; Vandenberghe, L. P. D. S.; Soccol, V. T.; Soccol, C. R.; Biasi, L. A.

2016-01-01

The mineral nutrients in vinasse provide support for algal and plant growth. Algal culture releases organic compounds into its liquid culture medium. These organic and inorganic substances can be useful for formulating a plant tissue culture medium, because tissue culture medium is composed of organic and inorganic components. Therefore, the aims of this study were to develop a plant culture medium by using the vinasse that is employed for Haematococcus pluvialis culture (algal filtrate); to investigate the possible beneficial effects of the biocompounds in the micropropagation of Nidularium procerum (Bromeliaceae), to evaluate quercetin content, total phenolics content in vinasse and to evaluate the cytotoxicity of the media by performing a bioassay with Artemia salina. The vinasse that originated from H. pluvialis culture can be used to formulate plant tissue culture at a 3% dilution, and its mineral nutrients can support In vitro plant growth, but some nutrients must be supplemented to enhance its efficiency. An efficient micropropagation protocol was developed for N. procerum. The micropropagated plants were suitable for transfer to the field (they were acclimatized). This culture medium provides a way to reuse wastewater, gives a rational alternative to vinasse disposal and adds value to what is currently considered to be an undesirable residue. Moreover, this process can reduce the production costs of clonal seedlings and/or bioactive compounds in biofactories. There was no apparent biostimulatory effect by the algal filtrate on morphogenesis; however, it did increase quercetin production. The H. pluvialis culture that was grown in the vinasse decreased the cytotoxicity and phenolic compound contents, which prevented explant tissue necrosis and represented a treatment for this residue for safer disposal in the environment. (author)

Characterization of single cell derived cultures of periosteal progenitor cells to ensure the cell quality for clinical application.

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Stefan Stich

Full Text Available For clinical applications of cells and tissue engineering products it is of importance to characterize the quality of the used cells in detail. Progenitor cells from the periosteum are already routinely applied in the clinics for the regeneration of the maxillary bone. Periosteal cells have, in addition to their potential to differentiate into bone, the ability to develop into cartilage and fat. However, the question arises whether all cells isolated from periosteal biopsies are able to differentiate into all three tissue types, or whether there are subpopulations. For an efficient and approved application in bone or cartilage regeneration the clarification of this question is of interest. Therefore, 83 different clonal cultures of freshly isolated human periosteal cells derived from mastoid periosteum biopsies of 4 donors were generated and growth rates calculated. Differentiation capacities of 51 clonal cultures towards the osteogenic, the chondrogenic, and the adipogenic lineage were investigated. Histological and immunochemical stainings showed that 100% of the clonal cultures differentiated towards the osteogenic lineage, while 94.1% demonstrated chondrogenesis, and 52.9% could be stimulated to adipogenesis. For osteogenesis real-time polymerase chain reaction (PCR of BGLAP and RUNX2 and for adipogenesis of FABP4 and PPARG confirmed the results. Overall, 49% of the cells exhibited a tripotent potential, 45.1% showed a bipotent potential (without adipogenic differentiation, 3.9% bipotent (without chondrogenic differentiation, and 2% possessed a unipotent osteogenic potential. In FACS analyses, no differences in the marker profile of undifferentiated clonal cultures with bi- and tripotent differentiation capacity were found. Genome-wide microarray analysis revealed 52 differentially expressed genes for clonal subpopulations with or without chondrogenic differentiation capacity, among them DCN, NEDD9, TGFBR3, and TSLP. For clinical

Advances in tissue engineering through stem cell-based co-culture.

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Paschos, Nikolaos K; Brown, Wendy E; Eswaramoorthy, Rajalakshmanan; Hu, Jerry C; Athanasiou, Kyriacos A

2015-05-01

Stem cells are the future in tissue engineering and regeneration. In a co-culture, stem cells not only provide a target cell source with multipotent differentiation capacity, but can also act as assisting cells that promote tissue homeostasis, metabolism, growth and repair. Their incorporation into co-culture systems seems to be important in the creation of complex tissues or organs. In this review, critical aspects of stem cell use in co-culture systems are discussed. Direct and indirect co-culture methodologies used in tissue engineering are described, along with various characteristics of cellular interactions in these systems. Direct cell-cell contact, cell-extracellular matrix interaction and signalling via soluble factors are presented. The advantages of stem cell co-culture strategies and their applications in tissue engineering and regenerative medicine are portrayed through specific examples for several tissues, including orthopaedic soft tissues, bone, heart, vasculature, lung, kidney, liver and nerve. A concise review of the progress and the lessons learned are provided, with a focus on recent developments and their implications. It is hoped that knowledge developed from one tissue can be translated to other tissues. Finally, we address challenges in tissue engineering and regenerative medicine that can potentially be overcome via employing strategies for stem cell co-culture use. Copyright © 2014 John Wiley & Sons, Ltd.

Study on tissue culture for Gelidium seedling

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Pei, Lu-Qing; Luo, Qi-Jun; Fei, Zhi-Qing; Ma, Bin

1996-06-01

As seedling culture is a crucial factor for successful cultivation of Gelidium, the authors researched tissue culture technology for producing seedlings. The morphogeny and experimental ecology were observed and studied fully in 2 5 mm isolated tissue fragments. Regeneration, appearance of branching creepers and attaching structure and new erect seedlings production and development were studied. Fragments were sown on bamboo slice and vinylon rope. The seedlings were cultured 20 30 days indoor, then cultured in the sea, where the density of erect seedlings was 3 19 seedlings/cm2, growth rate was 3.84% day. The frond arising from seedlings directly was up to 10 cm per year. The ecological conditions for regenerated seedlings are similar to the natural ones. The regenerated seedlings are suitable for raft culture in various sea areas.

Clonal expansion of CD4(+) cytotoxic T lymphocytes in patients with IgG4-related disease.

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Mattoo, Hamid; Mahajan, Vinay S; Maehara, Takashi; Deshpande, Vikram; Della-Torre, Emanuel; Wallace, Zachary S; Kulikova, Maria; Drijvers, Jefte M; Daccache, Joe; Carruthers, Mollie N; Castelino, Flavia V; Stone, James R; Stone, John H; Pillai, Shiv

2016-09-01

IgG4-related disease (IgG4-RD) is a systemic condition of unknown cause characterized by highly fibrotic lesions with dense lymphoplasmacytic infiltrates. CD4(+) T cells constitute the major inflammatory cell population in IgG4-RD lesions. We used an unbiased approach to characterize CD4(+) T-cell subsets in patients with IgG4-RD based on their clonal expansion and ability to infiltrate affected tissue sites. We used flow cytometry to identify CD4(+) effector/memory T cells in a cohort of 101 patients with IgG4-RD. These expanded cells were characterized by means of gene expression analysis and flow cytometry. Next-generation sequencing of the T-cell receptor β chain gene was performed on CD4(+)SLAMF7(+) cytotoxic T lymphocytes (CTLs) and CD4(+)GATA3(+) TH2 cells in a subset of patients to identify their clonality. Tissue infiltration by specific T cells was examined by using quantitative multicolor imaging. CD4(+) effector/memory T cells with a cytolytic phenotype were expanded in patients with IgG4-RD. Next-generation sequencing revealed prominent clonal expansions of these CD4(+) CTLs but not CD4(+)GATA3(+) memory TH2 cells in patients with IgG4-RD. The dominant T cells infiltrating a range of inflamed IgG4-RD tissue sites were clonally expanded CD4(+) CTLs that expressed SLAMF7, granzyme A, IL-1β, and TGF-β1. Clinical remission induced by rituximab-mediated B-cell depletion was associated with a reduction in numbers of disease-associated CD4(+) CTLs. IgG4-RD is prominently linked to clonally expanded IL-1β- and TGF-β1-secreting CD4(+) CTLs in both peripheral blood and inflammatory tissue lesions. These active, terminally differentiated, cytokine-secreting effector CD4(+) T cells are now linked to a human disease characterized by chronic inflammation and fibrosis. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Oxygen and tissue culture affect placental gene expression.

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Brew, O; Sullivan, M H F

2017-07-01

Placental explant culture is an important model for studying placental development and functions. We investigated the differences in placental gene expression in response to tissue culture, atmospheric and physiologic oxygen concentrations. Placental explants were collected from normal term (38-39 weeks of gestation) placentae with no previous uterine contractile activity. Placental transcriptomic expressions were evaluated with GeneChip ® Human Genome U133 Plus 2.0 arrays (Affymetrix). We uncovered sub-sets of genes that regulate response to stress, induction of apoptosis programmed cell death, mis-regulation of cell growth, proliferation, cell morphogenesis, tissue viability, and protection from apoptosis in cultured placental explants. We also identified a sub-set of genes with highly unstable pattern of expression after exposure to tissue culture. Tissue culture irrespective of oxygen concentration induced dichotomous increase in significant gene expression and increased enrichment of significant pathways and transcription factor targets (TFTs) including HIF1A. The effect was exacerbated by culture at atmospheric oxygen concentration, where further up-regulation of TFTs including PPARA, CEBPD, HOXA9 and down-regulated TFTs such as JUND/FOS suggest intrinsic heightened key biological and metabolic mechanisms such as glucose use, lipid biosynthesis, protein metabolism; apoptosis, inflammatory responses; and diminished trophoblast proliferation, differentiation, invasion, regeneration, and viability. These findings demonstrate that gene expression patterns differ between pre-culture and cultured explants, and the gene expression of explants cultured at atmospheric oxygen concentration favours stressed, pro-inflammatory and increased apoptotic transcriptomic response. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of ionizing radiation on plant tissue cultures

International Nuclear Information System (INIS)

Hell, K.G.

1978-01-01

A short review is done of the biological effects of ionizing radiations on plant tissues kept in culture, from the work of Gladys King, in 1949, with X-ray irradiated tobacco. The role of plant hormones is discussed in the processes of growth inhibition and growth restoration of irradiated tissues, as well as morphogenesis. Radioresistance of cells kept in culture and the use of ionizing radiations as mutagens are also commented. Some aspects of the biological effects of ionizing radiations that need to be investigated are discussed, and the problem of genome instability of plant tissues kept in culture is pointed out. (M.A.) [pt

Clonal chromosomal and genomic instability during human multipotent mesenchymal stromal cells long-term culture.

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Victoria Nikitina

Full Text Available Spontaneous mutagenesis often leads to appearance of genetic changes in cells. Although human multipotent mesenchymal stromal cells (hMSC are considered as genetically stable, there is a risk of genomic and structural chromosome instability and, therefore, side effects of cell therapy associated with long-term effects. In this study, the karyotype, genetic variability and clone formation analyses have been carried out in the long-term culture MSC from human gingival mucosa.The immunophenotype of MSC has been examined using flow cytofluorometry and short tandem repeat (STR analysis has been carried out for authentication. The karyotype has been examined using GTG staining and mFISH, while the assessment of the aneuploidy 8 frequency has been performed using centromere specific chromosome FISH probes in interphase cells.The immunophenotype and STR loci combination did not change during the process of cultivation. From passage 23 the proliferative activity of cultured MSCs was significantly reduced. From passage 12 of cultivation, clones of cells with stable chromosome aberrations have been identified and the biggest of these (12% are tetrasomy of chromosome 8. The random genetic and structural chromosomal aberrations and the spontaneous level of chromosomal aberrations in the hMSC long-term cultures were also described.The spectrum of spontaneous chromosomal aberrations in MSC long-term cultivation has been described. Clonal chromosomal aberrations have been identified. A clone of cells with tetrasomy 8 has been detected in passage 12 and has reached the maximum size by passage 18 before and decreased along with the reduction of proliferative activity of cell line by passage 26. At later passages, the MSC line exhibited a set of cells with structural variants of the karyotype with a preponderance of normal diploid cells. The results of our study strongly suggest a need for rigorous genetic analyses of the clone formation in cultured MSCs before

Evaluating Clonal Expansion of HIV-Infected Cells: Optimization of PCR Strategies to Predict Clonality

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Laskey, Sarah B.; Pohlmeyer, Christopher W.; Bruner, Katherine M.; Siliciano, Robert F.

2016-01-01

In HIV-infected individuals receiving suppressive antiretroviral therapy, the virus persists indefinitely in a reservoir of latently infected cells. The proliferation of these cells may contribute to the stability of the reservoir and thus to the lifelong persistence of HIV-1 in infected individuals. Because the HIV-1 replication process is highly error-prone, the detection of identical viral genomes in distinct host cells provides evidence for the clonal expansion of infected cells. We evaluated alignments of unique, near-full-length HIV-1 sequences to determine the relationship between clonality in a short region and clonality in the full genome. Although it is common to amplify and sequence short, subgenomic regions of the viral genome for phylogenetic analysis, we show that sequence identity of these amplicons does not guarantee clonality across the full viral genome. We show that although longer amplicons capture more diversity, no subgenomic region can recapitulate the diversity of full viral genomes. Consequently, some identical subgenomic amplicons should be expected even from the analysis of completely unique viral genomes, and the presence of identical amplicons alone is not proof of clonally expanded HIV-1. We present a method for evaluating evidence of clonal expansion in the context of these findings. PMID:27494508

Evaluating Clonal Expansion of HIV-Infected Cells: Optimization of PCR Strategies to Predict Clonality.

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Sarah B Laskey

2016-08-01

Full Text Available In HIV-infected individuals receiving suppressive antiretroviral therapy, the virus persists indefinitely in a reservoir of latently infected cells. The proliferation of these cells may contribute to the stability of the reservoir and thus to the lifelong persistence of HIV-1 in infected individuals. Because the HIV-1 replication process is highly error-prone, the detection of identical viral genomes in distinct host cells provides evidence for the clonal expansion of infected cells. We evaluated alignments of unique, near-full-length HIV-1 sequences to determine the relationship between clonality in a short region and clonality in the full genome. Although it is common to amplify and sequence short, subgenomic regions of the viral genome for phylogenetic analysis, we show that sequence identity of these amplicons does not guarantee clonality across the full viral genome. We show that although longer amplicons capture more diversity, no subgenomic region can recapitulate the diversity of full viral genomes. Consequently, some identical subgenomic amplicons should be expected even from the analysis of completely unique viral genomes, and the presence of identical amplicons alone is not proof of clonally expanded HIV-1. We present a method for evaluating evidence of clonal expansion in the context of these findings.

Clonality analysis of lymphoid proliferations using the BIOMED-2 clonality assays: a single institution experience

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Kokovic, Ira; Novakovic, Barbara Jezersek; Cerkovnik, Petra; Novakovic, Srdjan

2014-01-01

Background Clonality determination in patients with lymphoproliferative disorders can improve the final diagnosis. The aim of our study was to evaluate the applicative value of standardized BIOMED-2 gene clonality assay protocols for the analysis of clonality of lymphocytes in a group of different lymphoid proliferations. Materials and methods. With this purpose, 121 specimens from 91 patients with suspected lymphoproliferations submitted for routine diagnostics from January to December 2011 were retrospectively analyzed. According to the final diagnosis, our series comprised 32 cases of B-cell lymphomas, 38 cases of non-Hodgkin’s T-cell lymphomas and 51 cases of reactive lymphoid proliferations. Clonality testing was performed using the BIOMED-2 clonality assays. Results The determined sensitivity of the TCR assay was 91.9%, while the sensitivity of the IGH assay was 74.2%. The determined specificity of the IGH assay was 73.3% in the group of lymphomas and 87.2% in the group of reactive lesions. The determined specificity of the TCR assay was 62.5% in the group of lymphomas and 54.3% in the group of reactive lesions. Conclusions In the present study, we confirmed the utility of standardized BIOMED-2 clonality assays for the detection of clonality in a routine diagnostical setting of non-Hodgkin’s lymphomas. Reactions for the detection of the complete IGH rearrangements and reactions for the detection of the TCR rearrangements are a good choice for clonality testing of a wide range of lymphoid proliferations and specimen types while the reactions for the detection of incomplete IGH rearrangements have not shown any additional diagnostic value. PMID:24991205

Multiplexing clonality: combining RGB marking and genetic barcoding

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Cornils, Kerstin; Thielecke, Lars; Hüser, Svenja; Forgber, Michael; Thomaschewski, Michael; Kleist, Nadja; Hussein, Kais; Riecken, Kristoffer; Volz, Tassilo; Gerdes, Sebastian; Glauche, Ingmar; Dahl, Andreas; Dandri, Maura; Roeder, Ingo; Fehse, Boris

2014-01-01

RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies. PMID:24476916

[Chromosome variability in the tissue culture of rare Gentiana species].

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Tvardovs'ka, M O; Strashniuk, N M; Mel'nyk, V M; Adonin, V I; Kunakh, V A

2008-01-01

Cytogenetic analysis of plants and tissue culture of Gentiana lutea, G. punctata, G. acaulis has been carried out. Culturing in vitro was found to result in the changes of chromosome number in the calluses of the species involved. Species specificity for variation of the cultured cell genomes was shown. Contribution of the original plant genotypes to the cytogenetic structure of the tissue culture was established. Gentiana callus tissues (except for in vitro culture of G. punctata, derived from plant of Breskul'ska population) were found to exhibit modal class with the cells of diploid and nearly diploid chromosome sets.

Effect of clonal integration on nitrogen cycling in rhizosphere of rhizomatous clonal plant, Phyllostachys bissetii, under heterogeneous light.

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Li, Yang; Chen, Jing-Song; Xue, Ge; Peng, Yuanying; Song, Hui-Xing

2018-07-01

Clonal integration plays an important role in clonal plant adapting to heterogeneous habitats. It was postulated that clonal integration could exhibit positive effects on nitrogen cycling in the rhizosphere of clonal plant subjected to heterogeneous light conditions. An in-situ experiment was conducted using clonal fragments of Phyllostachys bissetii with two successive ramets. Shading treatments were applied to offspring or mother ramets, respectively, whereas counterparts were treated to full sunlight. Rhizomes between two successive ramets were either severed or connected. Extracellular enzyme activities and nitrogen turnover were measured, as well as soil properties. Abundance of functional genes (archaeal or bacterial amoA, nifH) in the rhizosphere of shaded, offspring or mother ramets were determined using quantitative polymerase chain reaction. Carbon or nitrogen availabilities were significantly influenced by clonal integration in the rhizosphere of shaded ramets. Clonal integration significantly increased extracellular enzyme activities and abundance of functional genes in the rhizosphere of shaded ramets. When rhizomes were connected, higher nitrogen turnover (nitrogen mineralization or nitrification rates) was exhibited in the rhizosphere of shaded offspring ramets. However, nitrogen turnover was significantly decreased by clonal integration in the rhizosphere of shaded mother ramets. Path analysis indicated that nitrogen turnover in the rhizosphere of shaded, offspring or mother ramets were primarily driven by the response of soil microorganisms to dissolved organic carbon or nitrogen. This unique in-situ experiment provided insights into the mechanism of nutrient recycling mediated by clonal integration. It was suggested that effects of clonal integration on the rhizosphere microbial processes were dependent on direction of photosynthates transport in clonal plant subjected to heterogeneous light conditions. Copyright © 2018 Elsevier B.V. All rights

Universality of clone dynamics during tissue development

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Rulands, Steffen; Lescroart, Fabienne; Chabab, Samira; Hindley, Christopher J.; Prior, Nicole; Sznurkowska, Magdalena K.; Huch, Meritxell; Philpott, Anna; Blanpain, Cedric; Simons, Benjamin D.

2018-05-01

The emergence of complex organs is driven by the coordinated proliferation, migration and differentiation of precursor cells. The fate behaviour of these cells is reflected in the time evolution of their progeny, termed clones, which serve as a key experimental observable. In adult tissues, where cell dynamics is constrained by the condition of homeostasis, clonal tracing studies based on transgenic animal models have advanced our understanding of cell fate behaviour and its dysregulation in disease1,2. But what can be learnt from clonal dynamics in development, where the spatial cohesiveness of clones is impaired by tissue deformations during tissue growth? Drawing on the results of clonal tracing studies, we show that, despite the complexity of organ development, clonal dynamics may converge to a critical state characterized by universal scaling behaviour of clone sizes. By mapping clonal dynamics onto a generalization of the classical theory of aerosols, we elucidate the origin and range of scaling behaviours and show how the identification of universal scaling dependences may allow lineage-specific information to be distilled from experiments. Our study shows the emergence of core concepts of statistical physics in an unexpected context, identifying cellular systems as a laboratory to study non-equilibrium statistical physics.

Effects of artemisinin and Artemisia annua extracts on xenic bacteria isolated from clonal cultures of Histomonas meleagridis

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Thøfner, I.C.N.; Hess, C.; Liebhart, D.

Infection with the protozoa Histomonas meleagridis in poultry has re-emerged since the ban of effective drugs. Consequently efforts are set to find alternatives to chemotherapeutics to combat histomonosis. At present histomonads need accompanying bacteria when cultured in vitro, probably serving...... nutrient supply due to their appearance in parasitic food vacuoles. However, the relationship of the parasite and the bacteria is not fully clear. Six previously established clonal cultures of H. meleagridis were used to evaluate the effect of five Artemisia annua derived materials (i.e. dry leaves...... with the antibacterial tests, it is reasonable to assume that the observed inhibitory effect of the tested materials is attributed to a direct effect on the protozoa. However, the potential of these materials on histomonosis has been tested in vivo in chickens and in turkeys without success....

Simple method for clonal selection of hepatitis A virus based on recovery of virus from radioimmunofocus overlays

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Lemon, S M; Jansen, R W

1985-06-01

Hepatitis A virus (HAV), has been quantitated in cell culture by autoradiographic detection of foci of viral replication developing beneath an agarose overlay following fixation and 'staining' of the cell sheet with radiolabelled antibody (radioimmunofocus assay). Using a modification of this basic technique, a clonal variant of HM-175 strain HAV was isolated from agarose overlying individual radioimmunofoci. Virus recovered from the agarose was amplified in small volume cultures of BS-C-1 cells and identified in supernatant culture fluids by cDNA-RNA hybridizaton. No virus was recovered from agarose which did not overlie a focus of viral replication. This method offers a simple, yet relatively rapid and certain means of selecting clonal variants of non-plaquing viruses such as hepatitis A virus.

Variations on metabolic activities of legume tissues through radiation in tissue culture

International Nuclear Information System (INIS)

Batra, Amla

1977-01-01

Cell cultures from Arachis hypogaea L. cultivated in a modified medium developed by Murashige and Skoog (1962) showed vigorous qrowth after radiation treatment. Investigations on the effect of various sugars on the chlorophyll formation and growth of the irradiated tissues showed that sucrose was superior to maltose, glucose or fructose as a carbon source. Lactose and mannitol supported growth and development of chlorophyll to a less degree. On prolonging the cultures on a sugar free medium, the tissues failed to regain either growth or chlorophyll content. (author)

Variations on metabolic activities of legume tissues through radiation in tissue culture

Energy Technology Data Exchange (ETDEWEB)

Batra, A [Rajasthan Univ., Jaipur (India). Dept. of Botany

1977-12-01

Cell cultures from Arachis hypogaea L. cultivated in a modified medium developed by Murashige and Skoog (1962) showed vigorous qrowth after radiation treatment. Investigations on the effect of various sugars on the chlorophyll formation and growth of the irradiated tissues showed that sucrose was superior to maltose, glucose or fructose as a carbon source. Lactose and mannitol supported growth and development of chlorophyll to a less degree. On prolonging the cultures on a sugar free medium, the tissues failed to regain either growth or chlorophyll content.

An Invasive Clonal Plant Benefits from Clonal Integration More than a Co-Occurring Native Plant in Nutrient-Patchy and Competitive Environments

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You, Wenhua; Fan, Shufeng; Yu, Dan; Xie, Dong; Liu, Chunhua

2014-01-01

Many notorious invasive plants are clonal, however, little is known about the different roles of clonal integration effects between invasive and native plants. Here, we hypothesize that clonal integration affect growth, photosynthetic performance, biomass allocation and thus competitive ability of invasive and native clonal plants, and invasive clonal plants benefit from clonal integration more than co-occurring native plants in heterogeneous habitats. To test these hypotheses, two stoloniferous clonal plants, Alternanthera philoxeroides (invasive), Jussiaea repens (native) were studied in China. The apical parts of both species were grown either with or without neighboring vegetation and the basal parts without competitors were in nutrient- rich or -poor habitats, with stolon connections were either severed or kept intact. Competition significantly reduced growth and photosynthetic performance of the apical ramets in both species, but not the biomass of neighboring vegetation. Without competition, clonal integration greatly improved the growth and photosynthetic performance of both species, especially when the basal parts were in nutrient-rich habitats. When grown with neighboring vegetation, growth of J. repens and photosynthetic performance of both species were significantly enhanced by clonal integration with the basal parts in both nutrient-rich and -poor habitats, while growth and relative neighbor effect (RNE) of A. philoxeroides were greatly improved by clonal integration only when the basal parts were in nutrient-rich habitats. Moreover, clonal integration increased A. philoxeroides's biomass allocation to roots without competition, but decreased it with competition, especially when the basal ramets were in nutrient-rich sections. Effects of clonal integration on biomass allocation of J. repens was similar to that of A. philoxeroides but with less significance. These results supported our hypothesis that invasive clonal plants A. philoxeroides benefits

Co-culture systems-based strategies for articular cartilage tissue engineering.

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Zhang, Yu; Guo, Weimin; Wang, Mingjie; Hao, Chunxiang; Lu, Liang; Gao, Shuang; Zhang, Xueliang; Li, Xu; Chen, Mingxue; Li, Penghao; Jiang, Peng; Lu, Shibi; Liu, Shuyun; Guo, Quanyi

2018-03-01

Cartilage engineering facilitates repair and regeneration of damaged cartilage using engineered tissue that restores the functional properties of the impaired joint. The seed cells used most frequently in tissue engineering, are chondrocytes and mesenchymal stem cells. Seed cells activity plays a key role in the regeneration of functional cartilage tissue. However, seed cells undergo undesirable changes after in vitro processing procedures, such as degeneration of cartilage cells and induced hypertrophy of mesenchymal stem cells, which hinder cartilage tissue engineering. Compared to monoculture, which does not mimic the in vivo cellular environment, co-culture technology provides a more realistic microenvironment in terms of various physical, chemical, and biological factors. Co-culture technology is used in cartilage tissue engineering to overcome obstacles related to the degeneration of seed cells, and shows promise for cartilage regeneration and repair. In this review, we focus first on existing co-culture systems for cartilage tissue engineering and related fields, and discuss the conditions and mechanisms thereof. This is followed by methods for optimizing seed cell co-culture conditions to generate functional neo-cartilage tissue, which will lead to a new era in cartilage tissue engineering. © 2017 Wiley Periodicals, Inc.

Gastric tissue biopsy and culture

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... symptoms may include: Loss of appetite or weight loss Nausea and vomiting Pain in the upper part of the belly Black stools Vomiting blood or coffee ground-like material A gastric tissue biopsy and culture can help detect: Cancer Infections, most commonly Helicobacter ...

Improved Diagnosis of Prosthetic Joint Infection by Culturing Periprosthetic Tissue Specimens in Blood Culture Bottles

Directory of Open Access Journals (Sweden)

Trisha N. Peel

2016-01-01

Full Text Available Despite known low sensitivity, culture of periprosthetic tissue specimens on agars and in broths is routine. Culture of periprosthetic tissue samples in blood culture bottles (BCBs is potentially more convenient, but it has been evaluated in a limited way and has not been widely adopted. The aim of this study was to compare the sensitivity and specificity of inoculation of periprosthetic tissue specimens into blood culture bottles with standard agar and thioglycolate broth culture, applying Bayesian latent class modeling (LCM in addition to applying the Infectious Diseases Society of America (IDSA criteria for prosthetic joint infection. This prospective cohort study was conducted over a 9-month period (August 2013 to April 2014 at the Mayo Clinic, Rochester, MN, and included all consecutive patients undergoing revision arthroplasty. Overall, 369 subjects were studied; 117 (32% met IDSA criteria for prosthetic joint infection, and 82% had late chronic infection. Applying LCM, inoculation of tissues into BCBs was associated with a 47% improvement in sensitivity compared to the sensitivity of conventional agar and broth cultures (92.1 versus 62.6%, respectively; this magnitude of change was similar when IDSA criteria were applied (60.7 versus 44.4%, respectively; P = 0.003. The time to microorganism detection was shorter with BCBs than with standard media (P < 0.0001, with aerobic and anaerobic BCBs yielding positive results within a median of 21 and 23 h, respectively. Results of our study demonstrate that the semiautomated method of periprosthetic tissue culture in blood culture bottles is more sensitive than and as specific as agar and thioglycolate broth cultures and yields results faster.

A high density of tertiary lymphoid structure B cells in lung tumors is associated with increased CD4+ T cell receptor repertoire clonality.

Science.gov (United States)

Zhu, Wei; Germain, Claire; Liu, Zheng; Sebastian, Yinong; Devi, Priyanka; Knockaert, Samantha; Brohawn, Philip; Lehmann, Kim; Damotte, Diane; Validire, Pierre; Yao, Yihong; Valge-Archer, Viia; Hammond, Scott A; Dieu-Nosjean, Marie-Caroline; Higgs, Brandon W

2015-12-01

T and B cell receptor (TCR and BCR, respectively) Vβ or immunoglobulin heavy chain complementarity-determining region 3 sequencing allows monitoring of repertoire changes through recognition, clonal expansion, affinity maturation, and T or B cell activation in response to antigen. TCR and BCR repertoire analysis can advance understanding of antitumor immune responses in the tumor microenvironment. TCR and BCR repertoires of sorted CD4 + , CD8 + or CD19 + cells in tumor, non-tumoral distant tissue (NT), and peripheral compartments (blood/draining lymph node [P]) from 47 non-small cell lung cancer (NSCLC) patients (age median = 68 y) were sequenced. The clonotype spectra were assessed among different tissues and correlated with clinical and immunological parameters. In all tissues, CD4 + and CD8 + TCR repertoires had greater clonality relative to CD19 + BCR. CD4 + T cells exhibited greater clonality in NT compared to tumor ( p = 0.002) and P ( p 68). Younger patients exhibited greater CD4 + T cell diversity in P compared to older patients ( p = 0.05), and greater CD4 + T cell clonality in tumor relative to P ( p cell clonality in tumor and P, respectively (both p = 0.05), correlated with high density of tumor-associated tertiary lymphoid structure (TLS) B cells, a biomarker of higher overall survival in NSCLC. Results indicate distinct adaptive immune responses in NSCLC, where peripheral T cell diversity is modulated by age, and tumor T cell clonal expansion is favored by the presence of TLSs in the tumor microenvironment.

Versatile electrochemial sensor for tissue culturing and sample handling

DEFF Research Database (Denmark)

Bakmand, Tanya; Kwasny, Dorota; Al Atraktchi, Fatima Al-Zahraa

2014-01-01

Culturing of organtypic brain tissues is a routine procedure in neural research. The visual inspection of the medium is the only way of determining the state of the tissue. At the end of culturing, post-processing techniques such as HPLC can be used to measure the concentration of the secreted...

Epigenetics in plant tissue culture

NARCIS (Netherlands)

Smulders, M.J.M.; Klerk, de G.J.M.

2011-01-01

Plants produced vegetatively in tissue culture may differ from the plants from which they have been derived. Two major classes of off-types occur: genetic ones and epigenetic ones. This review is about epigenetic aberrations. We discuss recent studies that have uncovered epigenetic modifications at

A simple method for clonal selection of hepatitis A virus based on recovery of virus from radioimmunofocus overlays

International Nuclear Information System (INIS)

Lemon, S.M.; Jansen, R.W.

1985-01-01

Hepatitis A virus (HAV), has been quantitated in cell culture by autoradiographic detection of foci of viral replication developing beneath an agarose overlay following fixation and 'staining' of the cell sheet with radiolabelled antibody (radioimmunofocus assay). Using a modification of this basic technique, a clonal variant of HM-175 strain HAV was isolated from agarose overlying individual radioimmunofoci. Virus recovered from the agarose was amplified in small volume cultures of BS-C-1 cells and identified in supernatant culture fluids by cDNA-RNA hybridizaton. No virus was recovered from agarose which did not overlie a focus of viral replication. This method offers a simple, yet relatively rapid and certain means of selecting clonal variants of non-plaquing viruses such as hepatitis A virus. (Auth.)

Populations in clonal plants

Directory of Open Access Journals (Sweden)

Jussi Tammisola

1986-12-01

Full Text Available Population phenomena in higher plants are reviewed critically, particularly in relation to clonality. An array of concepts used in the field are discussed. In contrast to animals, higher plants are modular in structure. Plant populations show hierarchy at two levels: ramets and genets. In addition, their demography is far more complicated, since even the direction of development of a ramet may change by rejuvenation. Therefore, formulae concerning animal populations often require modification for plants. Furthermore, at the zygotic stage, higher plants are generally less mobile than animals. Accordingly, their population processes tend to be more local. Most populations of plants have a genetic structure: alleles and genotypes are spatially aggregated. Due to the short-ranged foraging behaviour of pollinators, genetically non-random pollination prevails. A generalized formula for parent-offspring dispersal variance is derived. It is used to analyze the effect of clonality on genetic patchiness in populations. In self-compatible species, an increase in clonality will tend to increase the degree of patchiness, while in self-incompatible species a decrease may result. Examples of population structure studies in different species are presented. A considerable degree of genetic variation appears to be found also in the populations of species with a strong allocation of resources to clonal growth or apomictic seed production. Some consequences of clonality are considered from the point of view of genetic conservation and plant breeding.

Clonal growth and plant species abundance.

Science.gov (United States)

Herben, Tomáš; Nováková, Zuzana; Klimešová, Jitka

2014-08-01

Both regional and local plant abundances are driven by species' dispersal capacities and their abilities to exploit new habitats and persist there. These processes are affected by clonal growth, which is difficult to evaluate and compare across large numbers of species. This study assessed the influence of clonal reproduction on local and regional abundances of a large set of species and compared the predictive power of morphologically defined traits of clonal growth with data on actual clonal growth from a botanical garden. The role of clonal growth was compared with the effects of seed reproduction, habitat requirements and growth, proxied both by LHS (leaf-height-seed) traits and by actual performance in the botanical garden. Morphological parameters of clonal growth, actual clonal reproduction in the garden and LHS traits (leaf-specific area - height - seed mass) were used as predictors of species abundance, both regional (number of species records in the Czech Republic) and local (mean species cover in vegetation records) for 836 perennial herbaceous species. Species differences in habitat requirements were accounted for by classifying the dataset by habitat type and also by using Ellenberg indicator values as covariates. After habitat differences were accounted for, clonal growth parameters explained an important part of variation in species abundance, both at regional and at local levels. At both levels, both greater vegetative growth in cultivation and greater lateral expansion trait values were correlated with higher abundance. Seed reproduction had weaker effects, being positive at the regional level and negative at the local level. Morphologically defined traits are predictive of species abundance, and it is concluded that simultaneous investigation of several such traits can help develop hypotheses on specific processes (e.g. avoidance of self-competition, support of offspring) potentially underlying clonal growth effects on abundance. Garden

Clonal sets of a binary relation

Science.gov (United States)

Zedam, Lemnaouar; Pérez-Fernández, Raúl; Bouremel, Hassane; De Baets, Bernard

2018-05-01

In a recent paper, we have introduced the notion of clone relation of a given binary relation. Intuitively, two elements are said to be "clones" if they are related in the same way w.r.t. every other element. In this paper, we generalize this notion from pairs of elements to sets of elements of any cardinality, resulting in the introduction of clonal sets. We investigate the most important properties of clonal sets, paying particular attention to the introduction of the clonal closure operator, to the analysis of the (lattice) structure of the set of clonal sets and to a distance metric expressing how close two elements are to being clones.

Culture methods of allograft musculoskeletal tissue samples in Australian bacteriology laboratories.

Science.gov (United States)

Varettas, Kerry

2013-12-01

Samples of allograft musculoskeletal tissue are cultured by bacteriology laboratories to determine the presence of bacteria and fungi. In Australia, this testing is performed by 6 TGA-licensed clinical bacteriology laboratories with samples received from 10 tissue banks. Culture methods of swab and tissue samples employ a combination of solid agar and/or broth media to enhance micro-organism growth and maximise recovery. All six Australian laboratories receive Amies transport swabs and, except for one laboratory, a corresponding biopsy sample for testing. Three of the 6 laboratories culture at least one allograft sample directly onto solid agar. Only one laboratory did not use a broth culture for any sample received. An international literature review found that a similar combination of musculoskeletal tissue samples were cultured onto solid agar and/or broth media. Although variations of allograft musculoskeletal tissue samples, culture media and methods are used in Australian and international bacteriology laboratories, validation studies and method evaluations have challenged and supported their use in recovering fungi and aerobic and anaerobic bacteria.

Metabolic Profile of Pancreatic Acinar and Islet Tissue in Culture

Science.gov (United States)

Suszynski, Thomas M.; Mueller, Kathryn; Gruessner, Angelika C.; Papas, Klearchos K.

2016-01-01

The amount and condition of exocrine impurities may affect the quality of islet preparations especially during culture. In this study, the objective was to determine the oxygen demandand viability of islet and acinar tissue post-isolation and whether they change disproportionately while in culture. We compare the OCR normalized to DNA (OCR/DNA, a measure of fractional viability in units nmol/min/mg DNA), and percent change in OCR and DNA recoveries between adult porcine islet and acinar tissue from the same preparation (paired) over a 6-9 days of standard culture. Paired comparisons were done to quantify differences in OCR/DNA between islet and acinar tissue from the same preparation, at specified time points during culture; the mean (± standard error) OCR/DNA was 74.0 (±11.7) units higher for acinar (vs. islet) tissue on the day of isolation (n=16, p<0.0001), but 25.7 (±9.4) units lower after 1 day (n=8, p=0.03), 56.6 (±11.5) units lower after 2 days (n=12, p=0.0004), and 65.9 (±28.7) units lower after 8 days (n=4, p=0.2) in culture. DNA and OCR recoveries decreased at different rates for acinar versus islet tissue over 6-9 days in culture (n=6). DNA recovery decreased to 24±7% for acinar and 75±8% for islets (p=0.002). Similarly, OCR recovery decreased to 16±3% for acinar and remained virtually constant for islets (p=0.005). Differences in the metabolic profile of acinarand islet tissue should be considered when culturing impure islet preparations. OCR-based measurements may help optimize pre-IT culture protocols. PMID:25131082

Clonal hematopoiesis in acquired aplastic anemia

OpenAIRE

Ogawa, Seishi

2016-01-01

Clonal hematopoiesis (CH) in aplastic anemia (AA) has been closely linked to the evolution of late clonal disorders, including paroxysmal nocturnal hemoglobinuria and myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML), which are common complications after successful immunosuppressive therapy (IST). With the advent of high-throughput sequencing of recent years, the molecular aspect of CH in AA has been clarified by comprehensive detection of somatic mutations that drive clonal evolut...

Revision washout decreases implant capsule tissue culture positivity: a multicenter study.

Science.gov (United States)

Henry, Gerard D; Carson, Culley C; Wilson, Steven K; Wiygul, Jeremy; Tornehl, Chris; Cleves, Mario A; Simmons, Caroline J; Donatucci, Craig F

2008-01-01

Positive cultures, visible biofilm and confocal micrography confirm bacterial presence on clinically uninfected inflatable penile prostheses at revision surgery. Salvage irrigation has been proved to rescue patients with clinically infected inflatable penile prostheses. Similar washout at revision for noninfectious reasons significantly lowers subsequent infection rates. We investigated a larger series of patients for positive culture rates and evaluated implant capsule tissue culture rates before and after revision washout. At 4 institutions a total of 148 patients with inflatable penile prostheses underwent revision surgery for noninfectious reasons between June 2001 and September 2005. Swab cultures of the fluid around the pump and visible biofilm were obtained. Also, in 65 patients a wedge of tissue from the capsule that forms around the pump was cultured. After implant removal revision washout of the implant spaces was performed and a second wedge of tissue was cultured. Of the 148 patients 97 (66%) had positive bacterial swab cultures of the fluid around the pump or biofilm. A total of 124 isolates were cultured. Of the 65 implant capsule tissue cultures obtained before washout 28 (43%) were positive for bacteria, while 16 (25%) obtained after revision washout were positive. Positive cultures and visible bacterial biofilm are present on clinically uninfected inflatable penile prostheses at revision surgery in most patients. Revision washout appears to decrease the bacterial load on implant capsule tissue at revision surgery of inflatable penile prostheses for noninfectious reasons.

The evolutionary ecology of clonally propagated domesticated plants.

Science.gov (United States)

McKey, Doyle; Elias, Marianne; Pujol, Benoît; Duputié, Anne

2010-04-01

While seed-propagated crops have contributed many evolutionary insights, evolutionary biologists have often neglected clonally propagated crops. We argue that widespread notions about their evolution under domestication are oversimplified, and that they offer rich material for evolutionary studies. The diversity of their wild ancestors, the diverse ecologies of the crop populations themselves, and the intricate mix of selection pressures, acting not only on the parts harvested but also on the parts used by humans to make clonal propagules, result in complex and diverse evolutionary trajectories under domestication. We examine why farmers propagate some plants clonally, and discuss the evolutionary dynamics of sexual reproduction in clonal crops. We explore how their mixed clonal/sexual reproductive systems function, based on the sole example studied in detail, cassava (Manihot esculenta). Biotechnology is now expanding the number of clonal crops, continuing the 10 000-yr-old trend to increase crop yields by propagating elite genotypes. In an era of rapid global change, it is more important than ever to understand how the adaptive potential of clonal crops can be maintained. A key component of strategies for preserving this adaptive potential is the maintenance of mixed clonal/sexual systems, which can be achieved by encouraging and valuing farmer knowledge about the sexual reproductive biology of their clonal crops.

From gene manipulation to forest establishment: shoot cultures of woody plants can be a central tool

Energy Technology Data Exchange (ETDEWEB)

McCown, B.H.

1985-05-01

Establishing germplasm of woody plants in microculture as shoot cultures has proved to be an effective method of overcoming many of the obstacles in working with these crops. Shoot cultures eliminate the changes associated with seasonal growth cycles and phase change and put large plants into a more manageable form. Well-established shoot cultures are central to successful clonal propagation systems for forest trees as well as to genetic improvement based on the use of cellular techniques such as protoplast manipulation. The physiological basis as to why tissues from shoot cultures are so readily manipulated is not well understood.

Extensive clonal spread and extreme longevity in saw palmetto, a foundation clonal plant.

Science.gov (United States)

Takahashi, Mizuki K; Horner, Liana M; Kubota, Toshiro; Keller, Nathan A; Abrahamson, Warren G

2011-09-01

The lack of effective tools has hampered out ability to assess the size, growth and ages of clonal plants. With Serenoa repens (saw palmetto) as a model, we introduce a novel analytical framework that integrates DNA fingerprinting and mathematical modelling to simulate growth and estimate ages of clonal plants. We also demonstrate the application of such life-history information of clonal plants to provide insight into management plans. Serenoa is an ecologically important foundation species in many Southeastern United States ecosystems; yet, many land managers consider Serenoa a troublesome invasive plant. Accordingly, management plans have been developed to reduce or eliminate Serenoa with little understanding of its life history. Using Amplified Fragment Length Polymorphisms, we genotyped 263 Serenoa and 134 Sabal etonia (a sympatric non-clonal palmetto) samples collected from a 20 × 20 m study plot in Florida scrub. Sabal samples were used to assign small field-unidentifiable palmettos to Serenoa or Sabal and also as a negative control for clone detection. We then mathematically modelled clonal networks to estimate genet ages. Our results suggest that Serenoa predominantly propagate via vegetative sprouts and 10,000-year-old genets may be common, while showing no evidence of clone formation by Sabal. The results of this and our previous studies suggest that: (i) Serenoa has been part of scrub associations for thousands of years, (ii) Serenoa invasion are unlikely and (ii) once Serenoa is eliminated from local communities, its restoration will be difficult. Reevaluation of the current management tools and plans is an urgent task. © 2011 Blackwell Publishing Ltd.

The autologus graft of epithelial tissue culture

Directory of Open Access Journals (Sweden)

Minaee B

1999-08-01

Full Text Available With the intention of research about culture and autologus graft of epithelial tissue we used 4 french Albino Rabbits with an average age of 2 months. After reproduction on the support in EMEM (Eagle's Minimum Essential Medium we used this for graft after 4 weeks. This region which grafted total replaced. After fixation of this sample and passing them through various process, histological sections were prepared. These sections were stained with H & E and masson's trichrome and studied by light microscope. We succeeded in graft. We hope in the near future by using the method of epithelium tissue culture improving to treat burned patients.

Ribosomal DNA sequence analysis of different geographically distributed Aloe Vera plants: Comparison with clonally regenerated plants

International Nuclear Information System (INIS)

Yagi, A.; Sato, Y.; Miwa, Y.; Kabbash, A.; Moustafa, S.; Shimomura, K.; El-Bassuony, A.

2006-01-01

A comparison of the sequences in an internally transcribed spacer (ITS) 1 region of rDNA between clonally regenerated A.vera and same species in Japan, USA and Egypt revealed the presence of two types of nucleotide sequences, 252 and 254 bps. Based on the findings in the ITS 1 region, A.vera having 252 and 254 bps clearly showed a stable sequence similarity, suggesting high conversation of the base peak sequence in the ITS 1 region. However, frequent base substitutions in the 252 bps samples leaves that came from callus tissue and micropropagated plants were observed around the regions of nucleotide positions 66, 99 and 199-201. The minor deviation in clonally regenerated A.vera may be due to the stage of regeneration and cell specification in cases of the callus tissue. In the present study, the base peak sequence of the Its 1 region of rDNA was adopted as a molecular marker for differentiating A.vera plants from geographically distributed and clonally regenerated A.vera plants and it was suggested that the base peak substitutions in the ITS 1 region may arise from the different nutritional and environmental factors in cultivation and plant growth stages. (author)

[Research progress of co-culture system for constructing vascularized tissue engineered bone].

Science.gov (United States)

Fu, Weili; Xiang, Zhou

2014-02-01

To review the research progress of the co-culture system for constructing vascularized tissue engineered bone. The recent literature concerning the co-culture system for constructing vascularized tissue engineered bone was reviewed, including the selection of osteogenic and endothelial lineages, the design and surface modification of scaffolds, the models and dimensions of the co-culture system, the mechanism, the culture conditions, and their application progress. The construction of vascularized tissue engineered bone is the prerequisite for their survival and further clinical application in vivo. Mesenchymal stem cells (owning the excellent osteogenic potential) and endothelial progenitor cells (capable of directional differentiation into endothelial cell) are considered as attractive cell types for the co-culture system to construct vascularized tissue engineered bone. The culture conditions need to be further optimized. Furthermore, how to achieve the clinical goals of minimal invasion and autologous transplantation also need to be further studied. The strategy of the co-culture system for constructing vascularized tissue engineered bone would have a very broad prospects for clinical application in future.

Improved Diagnosis of Prosthetic Joint Infection by Culturing Periprosthetic Tissue Specimens in Blood Culture Bottles.

Science.gov (United States)

Peel, Trisha N; Dylla, Brenda L; Hughes, John G; Lynch, David T; Greenwood-Quaintance, Kerryl E; Cheng, Allen C; Mandrekar, Jayawant N; Patel, Robin

2016-01-05

Despite known low sensitivity, culture of periprosthetic tissue specimens on agars and in broths is routine. Culture of periprosthetic tissue samples in blood culture bottles (BCBs) is potentially more convenient, but it has been evaluated in a limited way and has not been widely adopted. The aim of this study was to compare the sensitivity and specificity of inoculation of periprosthetic tissue specimens into blood culture bottles with standard agar and thioglycolate broth culture, applying Bayesian latent class modeling (LCM) in addition to applying the Infectious Diseases Society of America (IDSA) criteria for prosthetic joint infection. This prospective cohort study was conducted over a 9-month period (August 2013 to April 2014) at the Mayo Clinic, Rochester, MN, and included all consecutive patients undergoing revision arthroplasty. Overall, 369 subjects were studied; 117 (32%) met IDSA criteria for prosthetic joint infection, and 82% had late chronic infection. Applying LCM, inoculation of tissues into BCBs was associated with a 47% improvement in sensitivity compared to the sensitivity of conventional agar and broth cultures (92.1 versus 62.6%, respectively); this magnitude of change was similar when IDSA criteria were applied (60.7 versus 44.4%, respectively; P = 0.003). The time to microorganism detection was shorter with BCBs than with standard media (P Prosthetic joint infections are a devastating complication of arthroplasty surgery. Despite this, current microbiological techniques to detect and diagnose infections are imperfect. This study examined a new approach to diagnosing infections, through the inoculation of tissue samples from around the prosthetic joint into blood culture bottles. This study demonstrated that, compared to current laboratory practices, this new technique increased the detection of infection. These findings are important for patient care to allow timely and accurate diagnosis of infection. Copyright © 2016 Peel et al.

Tumor tissue slice cultures as a platform for analyzing tissue-penetration and biological activities of nanoparticles.

Science.gov (United States)

Merz, Lea; Höbel, Sabrina; Kallendrusch, Sonja; Ewe, Alexander; Bechmann, Ingo; Franke, Heike; Merz, Felicitas; Aigner, Achim

2017-03-01

The success of therapeutic nanoparticles depends, among others, on their ability to penetrate a tissue for actually reaching the target cells, and their efficient cellular uptake in the context of intact tissue and stroma. Various nanoparticle modifications have been implemented for altering physicochemical and biological properties. Their analysis, however, so far mainly relies on cell culture experiments which only poorly reflect the in vivo situation, or is based on in vivo experiments that are often complicated by whole-body pharmacokinetics and are rather tedious especially when analyzing larger nanoparticle sets. For the more precise analysis of nanoparticle properties at their desired site of action, efficient ex vivo systems closely mimicking in vivo tissue properties are needed. In this paper, we describe the setup of organotypic tumor tissue slice cultures for the analysis of tissue-penetrating properties and biological activities of nanoparticles. As a model system, we employ 350μm thick slice cultures from different tumor xenograft tissues, and analyze modified or non-modified polyethylenimine (PEI) complexes as well as their lipopolyplex derivatives for siRNA delivery. The described conditions for tissue slice preparation and culture ensure excellent tissue preservation for at least 14days, thus allowing for prolonged experimentation and analysis. When using fluorescently labeled siRNA for complex visualization, fluorescence microscopy of cryo-sectioned tissue slices reveals different degrees of nanoparticle tissue penetration, dependent on their surface charge. More importantly, the determination of siRNA-mediated knockdown efficacies of an endogenous target gene, the oncogenic survival factor Survivin, reveals the possibility to accurately assess biological nanoparticle activities in situ, i.e. in living cells in their original environment. Taken together, we establish tumor (xenograft) tissue slices for the accurate and facile ex vivo assessment of

Gastrointestinal Epithelial Organoid Cultures from Postsurgical Tissues.

Science.gov (United States)

Hahn, Soojung; Yoo, Jongman

2017-08-17

An organoid is a cellular structure three-dimensionally (3D) cultured from self-organizing stem cells in vitro, which has a cell population, architectures, and organ specific functions like the originating organs. Recent advances in the 3D culture of isolated intestinal crypts or gastric glands have enabled the generation of human gastrointestinal epithelial organoids. Gastrointestinal organoids recapitulate the human in vivo physiology because of all the intestinal epithelial cell types that differentiated and proliferated from tissue resident stem cells. Thus far, gastrointestinal organoids have been extensively used for generating gastrointestinal disease models. This protocol describes the method of isolating a gland or crypt using stomach or colon tissue after surgery and establishing them into gastroids or colonoids.

Tissue culture of surgically prepared temporalis fascia.

Science.gov (United States)

Walby, A P; Kerr, A G; Nevin, N C; Woods, G

1982-10-01

Temporalis fascia which is used to graft the tympanic membrane has been shown to be viable in tissue culture by a previous pilot study. This present study reports the effect on the viability of the fascia by scraping loose connective tissue from it and allowing it to dry. Pieces of fascia from 30 patients were each divided in 4 and prepared to give explants, fresh, fresh and scraped, dried, and dried and scraped. The fascia grew from 17 patients when cultured fresh, 5 when fresh and scraped, 1 when dried, and none when dried and scraped. These results are significantly different and show that the fascia is devitilized when prepared by the normal method for use in tympanoplasty.

Mary Jane Hogue (1883-1962): A pioneer in human brain tissue culture.

Science.gov (United States)

Zottoli, Steven J; Seyfarth, Ernst-August

2018-05-16

The ability to maintain human brain explants in tissue culture was a critical step in the use of these cells for the study of central nervous system disorders. Ross G. Harrison (1870-1959) was the first to successfully maintain frog medullary tissue in culture in 1907, but it took another 38 years before successful culture of human brain tissue was accomplished. One of the pioneers in this achievement was Mary Jane Hogue (1883-1962). Hogue was born into a Quaker family in 1883 in West Chester, Pennsylvania, and received her undergraduate degree from Goucher College in Baltimore, Maryland. Research with the developmental biologist Theodor Boveri (1862-1915) in Würzburg, Germany, resulted in her Ph.D. (1909). Hogue transitioned from studying protozoa to the culture of human brain tissue in the 1940s and 1950s, when she was one of the first to culture cells from human fetal, infant, and adult brain explants. We review Hogue's pioneering contributions to the study of human brain cells in culture, her putative identification of progenitor neuroblast and/or glioblast cells, and her use of the cultures to study the cytopathogenic effects of poliovirus. We also put Hogue's work in perspective by discussing how other women pioneers in tissue culture influenced Hogue and her research.

Establishment of primary keratinocyte culture from horse tissue biopsates

Directory of Open Access Journals (Sweden)

Jernej OGOREVC

2015-12-01

Full Text Available Primary cell lines established from skin tissue can be used in immunological, proteomic and genomic studies as in vitro skin models. The goal of our study was to establish a primary keratinocyte cell culture from tissue biopsates of two horses. The primary keratinocyte cell culture was obtained by mechanical and enzymatic dissociation and with explant culture method. The result was a heterogeneous primary culture comprised of keratinocytes and fibroblasts. To distinguish epithelial and mesenchymal cells immunofluorescent characterisation was performed, using antibodies against cytokeratin 14 and vimentin. We successfully at attained a primary cell line of keratinocytes, which could potentially be used to study equine skin diseases, as an animal model for human diseases, and for cosmetic and therapeutic product testing.

Media Compositions for Three-Dimensional Mammalian Tissue Growth under Microgravity Culture Conditions

Science.gov (United States)

Goodwin, Thomas J. (Inventor)

1998-01-01

Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue.The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

Media Compositions for Three Dimensional Mammalian Tissue Growth Under Microgravity Culture Conditions

Science.gov (United States)

Goodwin, Thomas J. (Inventor)

1998-01-01

Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

Micro fluidic System for Culturing and Monitoring of Neuronal Cells and Tissue

DEFF Research Database (Denmark)

Bakmand, Tanya; Waagepetersen, Helle S.

The aim of this Ph.D. project was to combine experience within cell and tissue culturing, electrochemistry and microfabrication in order to develop an in vivo-like fluidic culturing platform, challenging the traditional culturing methods. The first goal was to develope a fluidic system for cultur...... with mass production. The last part of this thesis also includes perspectives on how to expand the latest designed device to facilitate culturing of tissue and co-culturing of cells....

Ex vivo culture of patient tissue & examination of gene delivery.

LENUS (Irish Health Repository)

Rajendran, Simon

2012-01-31

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

Yield improvement strategies for the production of secondary metabolites in plant tissue culture: silymarin from Silybum marianum tissue culture.

Science.gov (United States)

AbouZid, S

2014-01-01

Plant cell culture can be a potential source for the production of important secondary metabolites. This technology bears many advantages over conventional agricultural methods. The main problem to arrive at a cost-effective process is the low productivity. This is mainly due to lack of differentiation in the cultured cells. Many approaches have been used to maximise the yield of secondary metabolites produced by cultured plant cells. Among these approaches: choosing a plant with a high biosynthetic capacity, obtaining efficient cell line for growth and production of metabolite of interest, manipulating culture conditions, elicitation, metabolic engineering and organ culture. This article gives an overview of the various approaches used to maximise the production of pharmaceutically important secondary metabolites in plant cell cultures. Examples of using these different approaches are shown for the production of silymarin from Silybum marianum tissue culture.

Tissue culture of ornamental cacti

Directory of Open Access Journals (Sweden)

Eugenio Pérez-Molphe-Balch

2015-12-01

Full Text Available Cacti species are plants that are well adapted to growing in arid and semiarid regions where the main problem is water availability. Cacti have developed a series of adaptations to cope with water scarcity, such as reduced leaf surface via morphological modifications including spines, cereous cuticles, extended root systems and stem tissue modifications to increase water storage, and crassulacean acid metabolism to reduce transpiration and water loss. Furthermore, seeds of these plants very often exhibit dormancy, a phenomenon that helps to prevent germination when the availability of water is reduced. In general, cactus species exhibit a low growth rate that makes their rapid propagation difficult. Cacti are much appreciated as ornamental plants due to their great variety and diversity of forms and their beautiful short-life flowers; however, due to difficulties in propagating them rapidly to meet market demand, they are very often over-collected in their natural habitats, which leads to numerous species being threatened, endangered or becoming extinct. Therefore, plant tissue culture techniques may facilitate their propagation over a shorter time period than conventional techniques used for commercial purposes; or may help to recover populations of endangered or threatened species for their re-introduction in the wild; or may also be of value to the preservation and conservation of the genetic resources of this important family. Herein we present the state-of-the-art of tissue culture techniques used for ornamental cacti and selected suggestions for solving a number of the problems faced by members of the Cactaceae family.

21 CFR 864.2240 - Cell and tissue culture supplies and equipment.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cell and tissue culture supplies and equipment. 864.2240 Section 864.2240 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products...

Biomaterials in co-culture systems: towards optimizing tissue integration and cell signaling within scaffolds.

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Battiston, Kyle G; Cheung, Jane W C; Jain, Devika; Santerre, J Paul

2014-05-01

Most natural tissues consist of multi-cellular systems made up of two or more cell types. However, some of these tissues may not regenerate themselves following tissue injury or disease without some form of intervention, such as from the use of tissue engineered constructs. Recent studies have increasingly used co-cultures in tissue engineering applications as these systems better model the natural tissues, both physically and biologically. This review aims to identify the challenges of using co-culture systems and to highlight different approaches with respect to the use of biomaterials in the use of such systems. The application of co-culture systems to stimulate a desired biological response and examples of studies within particular tissue engineering disciplines are summarized. A description of different analytical co-culture systems is also discussed and the role of biomaterials in the future of co-culture research are elaborated on. Understanding the complex cell-cell and cell-biomaterial interactions involved in co-culture systems will ultimately lead the field towards biomaterial concepts and designs with specific biochemical, electrical, and mechanical characteristics that are tailored towards the needs of distinct co-culture systems. Copyright © 2014 Elsevier Ltd. All rights reserved.

T cell receptor sequencing of early-stage breast cancer tumors identifies altered clonal structure of the T cell repertoire.

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Beausang, John F; Wheeler, Amanda J; Chan, Natalie H; Hanft, Violet R; Dirbas, Frederick M; Jeffrey, Stefanie S; Quake, Stephen R

2017-11-28

Tumor-infiltrating T cells play an important role in many cancers, and can improve prognosis and yield therapeutic targets. We characterized T cells infiltrating both breast cancer tumors and the surrounding normal breast tissue to identify T cells specific to each, as well as their abundance in peripheral blood. Using immune profiling of the T cell beta-chain repertoire in 16 patients with early-stage breast cancer, we show that the clonal structure of the tumor is significantly different from adjacent breast tissue, with the tumor containing ∼2.5-fold greater density of T cells and higher clonality compared with normal breast. The clonal structure of T cells in blood and normal breast is more similar than between blood and tumor, and could be used to distinguish tumor from normal breast tissue in 14 of 16 patients. Many T cell sequences overlap between tissue and blood from the same patient, including ∼50% of T cells between tumor and normal breast. Both tumor and normal breast contain high-abundance "enriched" sequences that are absent or of low abundance in the other tissue. Many of these T cells are either not detected or detected with very low frequency in the blood, suggesting the existence of separate compartments of T cells in both tumor and normal breast. Enriched T cell sequences are typically unique to each patient, but a subset is shared between many different patients. We show that many of these are commonly generated sequences, and thus unlikely to play an important role in the tumor microenvironment. Copyright © 2017 the Author(s). Published by PNAS.

Changes of heterogeneous cell populations in the Ishikawa cell line during long-term culture: Proposal for an in vitro clonal evolution model of tumor cells.

Science.gov (United States)

Kasai, Fumio; Hirayama, Noriko; Ozawa, Midori; Iemura, Masashi; Kohara, Arihiro

2016-06-01

Genomic changes in tumor cell lines can occur during culture, leading to differences between cell lines carrying the same name. In this study, genome profiles between low and high passages were investigated in the Ishikawa 3-H-12 cell line (JCRB1505). Cells contained between 43 and 46 chromosomes and the modal number changed from 46 to 45 during culture. Cytogenetic analysis revealed that a translocation t(9;14), observed in all metaphases, is a robust marker for this cell line. Single-nucleotide polymorphism microarrays showed a heterogeneous copy number in the early passages and distinct profiles at late passages. These results demonstrate that cell culture can lead to elimination of ancestral clones by sequential selection, resulting in extensive replacement with a novel clone. Our observations on Ishikawa cells in vitro are different from the in vivo heterogeneity in which ancestral clones are often retained during tumor evolution and suggest a model for in vitro clonal evolution. Copyright © 2016 Elsevier Inc. All rights reserved.

Propagule pressure, habitat conditions and clonal integration influence the establishment and growth of an invasive clonal plant, Alternanthera philoxeroides

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Wen-Hua eYou

2016-05-01

Full Text Available Many notorious invasive plants are clonal, spreading mainly by vegetative propagules. Propagule pressure (the number of propagules may affect the establishment, growth and thus invasion success of these clonal plants, and such effects may also depend on habitat conditions. To understand how propagule pressure, habitat conditions and clonal integration affect the establishment and growth of the invasive clonal plants, an 8-week greenhouse with an invasive clonal plant, Alternanthera philoxeroides was conducted. High (five fragments or low (one fragment propagule pressure was established either in bare soil (open habitat or dense native vegetation of Jussiaea repens (vegetative habitat, with the stolon connections either severed from or connected to the relatively older ramets. High propagule pressure greatly increased the establishment and growth of A. philoxeroides, especially when it grew in vegetative habitats. Surprisingly, high propagule pressure significantly reduced the growth of individual plants of A. philoxeroides in open habitats, whereas it did not affect the individual growth in vegetative habitats. A shift in the intraspecific interaction on A. philoxeroides from competition in open habitats to facilitation in vegetative habitats may be the main reason. Moreover, clonal integration significantly improved the growth of A. philoxeroides only in open habitats, especially with low propagule pressure, whereas it had no effects on the growth and competitive ability of A. philoxeroides in vegetative habitats, suggesting that clonal integration may be of most important for A. philoxeroides to explore new open space and spread. These findings suggest that propagule pressure may be crucial for the invasion success of A. philoxeroides, and such an effect also depends on habitat conditions.

Propagation of Aquilaria malaccensis seedlings through tissue culture techniques

International Nuclear Information System (INIS)

Salahbiah Abdul Majid; Zaiton Ahmad; Mohd Rafaie Abdul Salam; Nurhayati Irwan; Affrida Abu Hassan; Rusli Ibrahim

2010-01-01

Aquilaria malaccensis or karas is the principal source of gaharu resin, which is used in many cultures for incense, perfumes and traditional medicines. The species is mainly propagated conventionally through seeds, cuttings and graftings. Propagation by seeds is usually a reliable method for other forest species, but for karas, this technique is inadequate to meet the current demand of seedling supplies. This is principally due to its low seed viability, low germination rate, delayed rooting of seedlings, long life-cycle and rare seed production. Tissue culture has several advantages over conventional propagation, especially for obtaining large number of uniform and high-yielding plantlets or clones. This paper presents the current progress on mass-propagation of Aquilaria malaccensis seedlings through tissue culture technique at Nuclear Malaysia. (author)

Bridging the gap between cell culture and live tissue

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Stefan Przyborski

2017-11-01

Full Text Available Traditional in vitro two-dimensional (2-D culture systems only partly imitate the physiological and biochemical features of cells in their original tissue. In vivo, in organs and tissues, cells are surrounded by a three-dimensional (3-D organization of supporting matrix and neighbouring cells, and a gradient of chemical and mechanical signals. Furthermore, the presence of blood flow and mechanical movement provides a dynamic environment (Jong et al., 2011. In contrast, traditional in vitro culture, carried out on 2-D plastic or glass substrates, typically provides a static environment, which, however is the base of the present understanding of many biological processes, tissue homeostasis as well as disease. It is clear that this is not an exact representation of what is happening in vivo and the microenvironment provided by in vitro cell culture models are significantly different and can cause deviations in cell response and behaviour from those distinctive of in vivo tissues. In order to translate the present basic knowledge in cell control, cell repair and regeneration from the laboratory bench to the clinical application, we need a better understanding of the cell and tissue interactions. This implies a detailed comprehension of the natural tissue environment, with its organization and local signals, in order to more closely mimic what happens in vivo, developing more physiological models for efficient in vitro systems. In particular, it is imperative to understand the role of the environmental cues which can be mainly divided into those of a chemical and mechanical nature.

ClonEvol: clonal ordering and visualization in cancer sequencing.

Science.gov (United States)

Dang, H X; White, B S; Foltz, S M; Miller, C A; Luo, J; Fields, R C; Maher, C A

2017-12-01

Reconstruction of clonal evolution is critical for understanding tumor progression and implementing personalized therapies. This is often done by clustering somatic variants based on their cellular prevalence estimated via bulk tumor sequencing of multiple samples. The clusters, consisting of the clonal marker variants, are then ordered based on their estimated cellular prevalence to reconstruct clonal evolution trees, a process referred to as 'clonal ordering'. However, cellular prevalence estimate is confounded by statistical variability and errors in sequencing/data analysis, and therefore inhibits accurate reconstruction of the clonal evolution. This problem is further complicated by intra- and inter-tumor heterogeneity. Furthermore, the field lacks a comprehensive visualization tool to facilitate the interpretation of complex clonal relationships. To address these challenges we developed ClonEvol, a unified software tool for clonal ordering, visualization, and interpretation. ClonEvol uses a bootstrap resampling technique to estimate the cellular fraction of the clones and probabilistically models the clonal ordering constraints to account for statistical variability. The bootstrapping allows identification of the sample founding- and sub-clones, thus enabling interpretation of clonal seeding. ClonEvol automates the generation of multiple widely used visualizations for reconstructing and interpreting clonal evolution. ClonEvol outperformed three of the state of the art tools (LICHeE, Canopy and PhyloWGS) for clonal evolution inference, showing more robust error tolerance and producing more accurate trees in a simulation. Building upon multiple recent publications that utilized ClonEvol to study metastasis and drug resistance in solid cancers, here we show that ClonEvol rediscovered relapsed subclones in two published acute myeloid leukemia patients. Furthermore, we demonstrated that through noninvasive monitoring ClonEvol recapitulated the emerging subclones

Culture-Independent Identification of Mycobacterium avium Subspecies paratuberculosis in Ovine Tissues: Comparison with Bacterial Culture and Histopathological Lesions

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Kamal R. Acharya

2017-12-01

Full Text Available Johne’s disease is a chronic debilitating enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP. Current abattoir surveillance programs detect disease via examination of gross lesions and confirmation by histopathological and/or tissue culture, which is time-consuming and has relatively low sensitivity. This study aimed to investigate whether a high-throughput quantitative PCR (qPCR test is a viable alternative for tissue testing. Intestine and mesenteric lymph nodes were sourced from sheep experimentally infected with MAP and the DNA extracted using a protocol developed for tissues, comprised enzymatic digestion of the tissue homogenate, chemical and mechanical lysis, and magnetic bead-based DNA purification. The extracted DNA was tested by adapting a previously validated qPCR for fecal samples, and the results were compared with culture and histopathology results of the corresponding tissues. The MAP tissue qPCR confirmed infection in the majority of sheep with gross lesions on postmortem (37/38. Likewise, almost all tissue culture (61/64 or histopathology (52/58 positives were detected with good to moderate agreement (Cohen’s kappa statistic and no significant difference to the reference tests (McNemar’s Chi-square test. Higher MAP DNA quantities corresponded to animals with more severe histopathology (odds ratio: 1.82; 95% confidence interval: 1.60, 2.07. Culture-independent strain typing on tissue DNA was successfully performed. This MAP tissue qPCR method had a sensitivity equivalent to the reference tests and is thus a viable replacement for gross- and histopathological examination of tissue samples in abattoirs. In addition, the test could be validated for testing tissue samples intended for human consumption.

Long-Term Overgrazing-Induced Memory Decreases Photosynthesis of Clonal Offspring in a Perennial Grassland Plant.

Science.gov (United States)

Ren, Weibo; Hu, Ningning; Hou, Xiangyang; Zhang, Jize; Guo, Huiqin; Liu, Zhiying; Kong, Lingqi; Wu, Zinian; Wang, Hui; Li, Xiliang

2017-01-01

Previous studies of transgenerational plasticity have demonstrated that long-term overgrazing experienced by Leymus chinensis , an ecologically dominant, rhizomatous grass species in eastern Eurasian temperate grassland, significantly affects its clonal growth in subsequent generations. However, there is a dearth of information on the reasons underlying this overgrazing-induced memory effect in plant morphological plasticity. We characterized the relationship between a dwarf phenotype and photosynthesis function decline of L. chinensis from the perspective of leaf photosynthesis by using both field measurement and rhizome buds culture cultivated in a greenhouse. Leaf photosynthetic functions (net photosynthetic rate, stomatal conductance, intercellular carbon dioxide concentration, and transpiration rate) were significantly decreased in smaller L. chinensis individuals that were induced to have a dwarf phenotype by being heavily grazed in the field. This decreased photosynthetic function was maintained a generation after greenhouse tests in which grazing was excluded. Both the response of L. chinensis morphological traits and photosynthetic functions in greenhouse were deceased relative to those in the field experiment. Further, there were significant decreases in leaf chlorophyll content and Rubisco enzyme activities of leaves between bud-cultured dwarf and non-dwarf L. chinensis in the greenhouse. Moreover, gene expression patterns showed that the bud-cultured dwarf L. chinensis significantly down-regulated (by 1.86- to 5.33-fold) a series of key genes that regulate photosynthetic efficiency, stomata opening, and chloroplast development compared with the non-dwarf L. chinensis . This is among the first studies revealing a linkage between long-term overgrazing affecting the transgenerational morphological plasticity of clonal plants and physiologically adaptive photosynthesis function. Overall, clonal transgenerational effects in L. chinensis phenotypic traits

Long-Term Overgrazing-Induced Memory Decreases Photosynthesis of Clonal Offspring in a Perennial Grassland Plant

Directory of Open Access Journals (Sweden)

Xiangyang Hou

2017-04-01

Full Text Available Previous studies of transgenerational plasticity have demonstrated that long-term overgrazing experienced by Leymus chinensis, an ecologically dominant, rhizomatous grass species in eastern Eurasian temperate grassland, significantly affects its clonal growth in subsequent generations. However, there is a dearth of information on the reasons underlying this overgrazing-induced memory effect in plant morphological plasticity. We characterized the relationship between a dwarf phenotype and photosynthesis function decline of L. chinensis from the perspective of leaf photosynthesis by using both field measurement and rhizome buds culture cultivated in a greenhouse. Leaf photosynthetic functions (net photosynthetic rate, stomatal conductance, intercellular carbon dioxide concentration, and transpiration rate were significantly decreased in smaller L. chinensis individuals that were induced to have a dwarf phenotype by being heavily grazed in the field. This decreased photosynthetic function was maintained a generation after greenhouse tests in which grazing was excluded. Both the response of L. chinensis morphological traits and photosynthetic functions in greenhouse were deceased relative to those in the field experiment. Further, there were significant decreases in leaf chlorophyll content and Rubisco enzyme activities of leaves between bud-cultured dwarf and non-dwarf L. chinensis in the greenhouse. Moreover, gene expression patterns showed that the bud-cultured dwarf L. chinensis significantly down-regulated (by 1.86- to 5.33-fold a series of key genes that regulate photosynthetic efficiency, stomata opening, and chloroplast development compared with the non-dwarf L. chinensis. This is among the first studies revealing a linkage between long-term overgrazing affecting the transgenerational morphological plasticity of clonal plants and physiologically adaptive photosynthesis function. Overall, clonal transgenerational effects in L. chinensis

The role of activated charcoal in plant tissue culture.

Science.gov (United States)

Thomas, T Dennis

2008-01-01

Activated charcoal has a very fine network of pores with large inner surface area on which many substances can be adsorbed. Activated charcoal is often used in tissue culture to improve cell growth and development. It plays a critical role in micropropagation, orchid seed germination, somatic embryogenesis, anther culture, synthetic seed production, protoplast culture, rooting, stem elongation, bulb formation etc. The promotary effects of AC on morphogenesis may be mainly due to its irreversible adsorption of inhibitory compounds in the culture medium and substancially decreasing the toxic metabolites, phenolic exudation and brown exudate accumulation. In addition to this activated charcoal is involved in a number of stimulatory and inhibitory activities including the release of substances naturally present in AC which promote growth, alteration and darkening of culture media, and adsorption of vitamins, metal ions and plant growth regulators, including abscisic acid and gaseous ethylene. The effect of AC on growth regulator uptake is still unclear but some workers believe that AC may gradually release certain adsorbed products, such as nutrients and growth regulators which become available to plants. This review focuses on the various roles of activated charcoal in plant tissue culture and the recent developments in this area.

Mathematical modelling of tissue formation in chondrocyte filter cultures.

Science.gov (United States)

Catt, C J; Schuurman, W; Sengers, B G; van Weeren, P R; Dhert, W J A; Please, C P; Malda, J

2011-12-17

In the field of cartilage tissue engineering, filter cultures are a frequently used three-dimensional differentiation model. However, understanding of the governing processes of in vitro growth and development of tissue in these models is limited. Therefore, this study aimed to further characterise these processes by means of an approach combining both experimental and applied mathematical methods. A mathematical model was constructed, consisting of partial differential equations predicting the distribution of cells and glycosaminoglycans (GAGs), as well as the overall thickness of the tissue. Experimental data was collected to allow comparison with the predictions of the simulation and refinement of the initial models. Healthy mature equine chondrocytes were expanded and subsequently seeded on collagen-coated filters and cultured for up to 7 weeks. Resulting samples were characterised biochemically, as well as histologically. The simulations showed a good representation of the experimentally obtained cell and matrix distribution within the cultures. The mathematical results indicate that the experimental GAG and cell distribution is critically dependent on the rate at which the cell differentiation process takes place, which has important implications for interpreting experimental results. This study demonstrates that large regions of the tissue are inactive in terms of proliferation and growth of the layer. In particular, this would imply that higher seeding densities will not significantly affect the growth rate. A simple mathematical model was developed to predict the observed experimental data and enable interpretation of the principal underlying mechanisms controlling growth-related changes in tissue composition.

Effects of Cu Pollution on the Expansion of an Amphibious Clonal Herb in Aquatic-Terrestrial Ecotones.

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Liang Xu

Full Text Available Physiological integration can enhance the performance of clonal plants in aquatic and terrestrial heterogeneous habitats and associated ecotones. Similar to nutrients, pollutants may be transported among connected ramets via physiological integration. Few studies have examined the expansion of amphibious clonal plants from terrestrial to aquatic environments, particularly when the local water supply is polluted with heavy metals. A greenhouse experiment was conducted using the amphibious plant Alternanthera philoxeroides to determine whether Cu can spread among clonal plants and examine the corresponding effects of this pollution on the expansion of clonal plants in aquatic-terrestrial ecotones. Ramets from the same clonal fragments were rooted in unpolluted soil and polluted water at five different levels. The responses of the ramets in terrestrial and aquatic habitats were quantified via traits associated with growth, morphology and Cu accumulation. The results indicated that ramets in soil and water significantly differed in nearly all of these traits. The expansion of populations from terrestrial to polluted aquatic habitats was facilitated by stem elongation rather than new ramet production. The accumulated Cu in polluted ramets can be horizontally transported to other ramets in soil via connected stolons. In terms of clonal growth patterns, variations in Cu pollution intensity were negatively correlated with variations in the morphological and growth traits of ramets in polluted aquatic habitats and unpolluted soil. We concluded that Cu ions are distributed among the clones and accumulated in different ramet tissues in heterogeneous habitats. Therefore, we suggest that Cu pollution of aquatic-terrestrial ecotones, especially at high levels, can affect the growth and expansion of the whole clones because Cu ions are shared between integrated ramets.

Effects of Cu Pollution on the Expansion of an Amphibious Clonal Herb in Aquatic-Terrestrial Ecotones.

Science.gov (United States)

Xu, Liang; Zhou, Zhen-Feng

2016-01-01

Physiological integration can enhance the performance of clonal plants in aquatic and terrestrial heterogeneous habitats and associated ecotones. Similar to nutrients, pollutants may be transported among connected ramets via physiological integration. Few studies have examined the expansion of amphibious clonal plants from terrestrial to aquatic environments, particularly when the local water supply is polluted with heavy metals. A greenhouse experiment was conducted using the amphibious plant Alternanthera philoxeroides to determine whether Cu can spread among clonal plants and examine the corresponding effects of this pollution on the expansion of clonal plants in aquatic-terrestrial ecotones. Ramets from the same clonal fragments were rooted in unpolluted soil and polluted water at five different levels. The responses of the ramets in terrestrial and aquatic habitats were quantified via traits associated with growth, morphology and Cu accumulation. The results indicated that ramets in soil and water significantly differed in nearly all of these traits. The expansion of populations from terrestrial to polluted aquatic habitats was facilitated by stem elongation rather than new ramet production. The accumulated Cu in polluted ramets can be horizontally transported to other ramets in soil via connected stolons. In terms of clonal growth patterns, variations in Cu pollution intensity were negatively correlated with variations in the morphological and growth traits of ramets in polluted aquatic habitats and unpolluted soil. We concluded that Cu ions are distributed among the clones and accumulated in different ramet tissues in heterogeneous habitats. Therefore, we suggest that Cu pollution of aquatic-terrestrial ecotones, especially at high levels, can affect the growth and expansion of the whole clones because Cu ions are shared between integrated ramets.

Gamma irradiation effect on the formation of Clonal variation from catharantus roseus plant

International Nuclear Information System (INIS)

Syukur, Sumaryati

2000-01-01

Clonal variation have been found in Catharantus roseus plant after gamma irradiation. Several doses have been used to produce clonal variation. The most effective doses used to perform better clonal variation was 20 krad. About 103 seeds irradiated for every radiation treatment, but only several clones were grown better than wild type. We have success to get (M) seeds the expected mutant. The seeds from selected mutant are bigger when compare to the wild type and growth better on medium containing 5-methyl Tryptophan (5-MT). The chlorophyll content is higher (almost twice) as compared to the wild type. Fulther experiment continue to do in vitro culture in order to develop embryonic callus from leaf tip and leaf base. Several manipulation of auxin and cytokini have been used to differentiate the callus formation. Modified MS medium with kinetin and cytokinin (10:1) can induce globular embryo like structure. Dragendrof alkaloid reagent were used to determine high alkaloid clones from the expected mutant. TLC analysis from callus mutant shows 3 clear bands with subsequence Rf about 0.22, 0.58 while control shows two smearing bands at 0.21 and 0.52

Recent advances in understanding clonal haematopoiesis in aplastic anaemia.

Science.gov (United States)

Stanley, Natasha; Olson, Timothy S; Babushok, Daria V

2017-05-01

Acquired aplastic anaemia (AA) is an immune-mediated bone marrow failure disorder inextricably linked to clonal haematopoiesis. The majority of AA patients have somatic mutations and/or structural chromosomal abnormalities detected as early as at diagnosis. In contrast to other conditions linked to clonal haematopoiesis, the clonal signature of AA reflects its immune pathophysiology. The most common alterations are clonal expansions of cells lacking glycophosphotidylinositol-anchored proteins, loss of human leucocyte antigen alleles, and mutations in BCOR/BCORL1, ASXL1 and DNMT3A. Here, we present the current knowledge of clonal haematopoiesis in AA as it relates to aging, inherited bone marrow failure, and the grey-zone overlap of AA and myelodysplastic syndrome (MDS). We conclude by discussing the significance of clonal haematopoiesis both for improved diagnosis of AA, as well as for a more precise, personalized approach to prognostication of outcomes and therapy choices. © 2017 John Wiley & Sons Ltd.

Production of immunoglobulins in gingival tissue explant cultures from juvenile periodontitis patients

International Nuclear Information System (INIS)

Hall, E.R.; Falkler, W.A. Jr.; Suzuki, J.B.

1990-01-01

B lymphocytes and plasma cells are histologically observed in granulomatous periodontal tissues of juvenile periodontitis (JP) patients. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. An in vitro explant culture system was utilized to demonstrate the production of immunoglobulins by diseased JP tissues. Immunodiffusion studies using goat anti-human gamma, alpha, or mu chain serum revealed IgG to be the major immunoglobulin present in 92% of the day 1 supernatant fluids (SF) of the 47 JP gingival tissue explant cultures. IgA was present in 15% of the SF; however, no IgM was detected. Staph Protein A isolated 14C-labeled IgG from the SF, when allowed to react with goat anti-human gamma chain serum, formed lines of precipitation. Positive autoradiographs confirmed the biosynthesis of IgG by the explant cultures. The in vitro gingival tissue explant culture system described provides a useful model for the study of localized immunoglobulins produced by diseased tissues of JP patients

Production of immunoglobulins in gingival tissue explant cultures from juvenile periodontitis patients

Energy Technology Data Exchange (ETDEWEB)

Hall, E.R.; Falkler, W.A. Jr.; Suzuki, J.B. (Univ. of Maryland Dental School, Baltimore (USA))

1990-10-01

B lymphocytes and plasma cells are histologically observed in granulomatous periodontal tissues of juvenile periodontitis (JP) patients. Local immune processes may participate in protective or immunopathologic roles in the pathogenesis of this disease. An in vitro explant culture system was utilized to demonstrate the production of immunoglobulins by diseased JP tissues. Immunodiffusion studies using goat anti-human gamma, alpha, or mu chain serum revealed IgG to be the major immunoglobulin present in 92% of the day 1 supernatant fluids (SF) of the 47 JP gingival tissue explant cultures. IgA was present in 15% of the SF; however, no IgM was detected. Staph Protein A isolated 14C-labeled IgG from the SF, when allowed to react with goat anti-human gamma chain serum, formed lines of precipitation. Positive autoradiographs confirmed the biosynthesis of IgG by the explant cultures. The in vitro gingival tissue explant culture system described provides a useful model for the study of localized immunoglobulins produced by diseased tissues of JP patients.

Insights in Anaphylaxis and Clonal Mast Cell Disorders

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David González-de-Olano

2017-07-01

Full Text Available The prevalence of anaphylaxis among patients with clonal mast cell disorders (MCD is clearly higher comparing to the general population. Due to a lower frequency of symptoms outside of acute episodes, clonal MCD in the absence of skin lesions might sometimes be difficult to identify which may lead to underdiagnosis, and anaphylaxis is commonly the presenting symptom in these patients. Although the release of mast cell (MC mediators upon MC activation might present with a wide variety of symptoms, particular clinical features typically characterize MC mediator release episodes in patients with clonal MCD without skin involvement. Final diagnosis requires a bone marrow study, and it is recommended that this should be done in reference centers. In this article, we address the main triggers for anaphylaxis, risk factors, clinical presentation, diagnosis, and management of patients with MC activation syndromes (MCASs, with special emphasis on clonal MCAS [systemic mastocytosis and mono(clonal MC activations syndromes].

Novel avian oropharyngeal trichomonads isolated from European turtle doves (Streptopelia turtur) and racing pigeons (Columba livia): genetic and morphometric characterisation of clonal cultures.

Science.gov (United States)

Martínez-Herrero, M C; Garijo-Toledo, M M; Liebhart, D; Ganas, P; Martínez-Díaz, R A; Ponce-Gordo, F; Carrero-Ruiz, A; Hess, M; Gómez-Muñoz, M T

2017-11-01

Extensive diversity has been described within the avian oropharyngeal trichomonad complex in recent years. In this study we developed clonal cultures from four isolates selected by their different ITS1/5.8S/ITS2 (ITS) genotype and their association with gross lesions of avian trichomonosis. Isolates were obtained from an adult racing pigeon and a nestling of Eurasian eagle owl with macroscopic lesions, and from a juvenile wood pigeon and an European turtle dove without clinical signs. Multi-locus sequence typing analysis of the ITS, small subunit of ribosomal rRNA (SSUrRNA) and Fe-hydrogenase (Fe-hyd) genes together with a morphological study by optical and scanning electron microscopy was performed. No significant differences in the structures were observed with scanning electron microscopy. However, the genetic characterisation revealed novel sequence types for the SSUrRNA region and Fe-hyd gene. Two clones were identified as Trichomonas gallinae in the MLST analysis, but the clones from the racing pigeon and European turtle dove showed higher similarity with Trichomonas tenax and Trichomonas canistomae than with T. gallinae at their ITS region, respectively. SSUrRNA sequences grouped all the clones in a clade that includes T. gallinae, T. tenax and T. canistomae. Further diversity was detected within the Fe-hyd locus, with a clear separation from T. gallinae of the clones obtained from the racing pigeon and the European turtle dove. In addition, morphometric comparison by optical microscopy with clonal cultures of T. gallinae revealed significant statistical differences on axostyle projection length in the clone from the European turtle dove. Morphometric and genetic data indicate that possible new species within the Trichomonas genus were detected. Taking in consideration the diversity in Trichomonas species present in the oral cavity of birds, a proper genetic analysis is highly recommended when outbreaks occur. Copyright © 2017 Elsevier B.V. All rights

Clonal mutations in primary human glial tumors: evidence in support of the mutator hypothesis

International Nuclear Information System (INIS)

Misra, Anjan; Chattopadhyay, Parthaprasad; Chosdol, Kunzang; Sarkar, Chitra; Mahapatra, Ashok K; Sinha, Subrata

2007-01-01

A verifiable consequence of the mutator hypothesis is that even low grade neoplasms would accumulate a large number of mutations that do not influence the tumor phenotype (clonal mutations). In this study, we have attempted to quantify the number of clonal mutations in primary human gliomas of astrocytic cell origin. These alterations were identified in tumor tissue, microscopically confirmed to have over 70% neoplastic cells. Random Amplified Polymorphic DNA (RAPD) analysis was performed using a set of fifteen 10-mer primers of arbitrary but definite sequences in 17 WHO grade II astrocytomas (low grade diffuse astrocytoma or DA) and 16 WHO grade IV astrocytomas (Glioblastoma Multiforme or GBM). The RAPD profile of the tumor tissue was compared with that of the leucocyte DNA of the same patient and alteration(s) scored. A quantitative estimate of the overall genomic changes in these tumors was obtained by 2 different modes of calculation. The overall change in the tumors was estimated to be 4.24% in DA and 2.29% in GBM by one method and 11.96% and 6.03% in DA and GBM respectively by the other. The difference between high and lower grade tumors was statistically significant by both methods. This study demonstrates the presence of extensive clonal mutations in gliomas, more in lower grade. This is consistent with our earlier work demonstrating that technique like RAPD analysis, unbiased for locus, is able to demonstrate more intra-tumor genetic heterogeneity in lower grade gliomas compared to higher grade. The results support the mutator hypothesis proposed by Loeb

Low technology tissue culture materials for initiation and ...

African Journals Online (AJOL)

Low technology tissue culture materials for initiation and multiplication of banana plants. ... African Crop Science Journal ... locally available macronutrients, micronutrients, sugar, equipment and facility reduced the cost of consumable material

Human meniscal proteoglycan metabolism in long-term tissue culture

NARCIS (Netherlands)

Verbruggen, G.; Verdonk, R.; Veys, E. M.; van Daele, P.; de Smet, P.; van den Abbeele, K.; Claus, B.; Baeten, D.

1996-01-01

For the purpose of human meniscal allografting, menisci have been maintained viable in in vitro culture. The influence of long-term tissue culture on the extracellular matrix metabolism of the meniscus has been studied. Fetal calf serum (FCS) was used as a supplement for the growth factors necessary

Renal cell carcinoma primary cultures maintain genomic and phenotypic profile of parental tumor tissues

International Nuclear Information System (INIS)

Cifola, Ingrid; Magni, Fulvio; Signorini, Stefano; Battaglia, Cristina; Perego, Roberto A; Bianchi, Cristina; Mangano, Eleonora; Bombelli, Silvia; Frascati, Fabio; Fasoli, Ester; Ferrero, Stefano; Di Stefano, Vitalba; Zipeto, Maria A

2011-01-01

Clear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues. We established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation). A very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed. ccRCC primary cultures are a reliable in vitro model, well-reproducing original tumor genetics and phenotype, potentially useful for future functional approaches

Substituted Indoleacetic Acids Tested in Tissue Cultures

DEFF Research Database (Denmark)

Engvild, Kjeld Christensen

1978-01-01

Monochloro substituted IAA inhibited shoot induction in tobacco tissue cultures about as much as IAA. Dichloro substituted IAA inhibited shoot formation less. Other substituted IAA except 5-fluoro- and 5-bromoindole-3-acetic acid were less active than IAA. Callus growth was quite variable...

21 CFR 864.2220 - Synthetic cell and tissue culture media and components.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Synthetic cell and tissue culture media and components. 864.2220 Section 864.2220 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture...

A Common Origin for B-1a and B-2 Lymphocytes in Clonal Pre- Hematopoietic Stem Cells

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Brandon K. Hadland

2017-06-01

Full Text Available Recent evidence points to the embryonic emergence of some tissue-resident innate immune cells, such as B-1a lymphocytes, prior to and independently of hematopoietic stem cells (HSCs. However, whether the full hematopoietic repertoire of embryonic HSCs initially includes these unique lineages of innate immune cells has been difficult to assess due to lack of clonal assays that identify and assess HSC precursor (pre-HSC potential. Here, by combining index sorting of single embryonic hemogenic precursors with in vitro HSC maturation and transplantation assays, we analyze emerging pre-HSCs at the single-cell level, revealing their unique stage-specific properties and clonal lineage potential. Remarkably, clonal pre-HSCs detected between E9.5 and E11.5 contribute to the complete B cell repertoire, including B-1a lymphocytes, revealing a previously unappreciated common precursor for all B cell lineages at the pre-HSC stage and a second embryonic origin for B-1a lymphocytes.

Sugarcane in vitro culture technology: Opportunities for Kenya's ...

African Journals Online (AJOL)

free clonal materials. Successful protocols for shoot tip culture, callus culture, embryo culture, virus free plant production and somatic embryogenesis have already been established. Thus, in vitro technology can be used to enhance ...

Self-mating in the definitive host potentiates clonal outbreaks of the apicomplexan parasites Sarcocystis neurona and Toxoplasma gondii.

Directory of Open Access Journals (Sweden)

Jered M Wendte

2010-12-01

Full Text Available Tissue-encysting coccidia, including Toxoplasma gondii and Sarcocystis neurona, are heterogamous parasites with sexual and asexual life stages in definitive and intermediate hosts, respectively. During its sexual life stage, T. gondii reproduces either by genetic out-crossing or via clonal amplification of a single strain through self-mating. Out-crossing has been experimentally verified as a potent mechanism capable of producing offspring possessing a range of adaptive and virulence potentials. In contrast, selfing and other life history traits, such as asexual expansion of tissue-cysts by oral transmission among intermediate hosts, have been proposed to explain the genetic basis for the clonal population structure of T. gondii. In this study, we investigated the contributing roles self-mating and sexual recombination play in nature to maintain clonal population structures and produce or expand parasite clones capable of causing disease epidemics for two tissue encysting parasites. We applied high-resolution genotyping against strains isolated from a T. gondii waterborne outbreak that caused symptomatic disease in 155 immune-competent people in Brazil and a S. neurona outbreak that resulted in a mass mortality event in Southern sea otters. In both cases, a single, genetically distinct clone was found infecting outbreak-exposed individuals. Furthermore, the T. gondii outbreak clone was one of several apparently recombinant progeny recovered from the local environment. Since oocysts or sporocysts were the infectious form implicated in each outbreak, the expansion of the epidemic clone can be explained by self-mating. The results also show that out-crossing preceded selfing to produce the virulent T. gondii clone. For the tissue encysting coccidia, self-mating exists as a key adaptation potentiating the epidemic expansion and transmission of newly emerged parasite clones that can profoundly shape parasite population genetic structures or cause

Self-mating in the definitive host potentiates clonal outbreaks of the apicomplexan parasites Sarcocystis neurona and Toxoplasma gondii.

Science.gov (United States)

Wendte, Jered M; Miller, Melissa A; Lambourn, Dyanna M; Magargal, Spencer L; Jessup, David A; Grigg, Michael E

2010-12-23

Tissue-encysting coccidia, including Toxoplasma gondii and Sarcocystis neurona, are heterogamous parasites with sexual and asexual life stages in definitive and intermediate hosts, respectively. During its sexual life stage, T. gondii reproduces either by genetic out-crossing or via clonal amplification of a single strain through self-mating. Out-crossing has been experimentally verified as a potent mechanism capable of producing offspring possessing a range of adaptive and virulence potentials. In contrast, selfing and other life history traits, such as asexual expansion of tissue-cysts by oral transmission among intermediate hosts, have been proposed to explain the genetic basis for the clonal population structure of T. gondii. In this study, we investigated the contributing roles self-mating and sexual recombination play in nature to maintain clonal population structures and produce or expand parasite clones capable of causing disease epidemics for two tissue encysting parasites. We applied high-resolution genotyping against strains isolated from a T. gondii waterborne outbreak that caused symptomatic disease in 155 immune-competent people in Brazil and a S. neurona outbreak that resulted in a mass mortality event in Southern sea otters. In both cases, a single, genetically distinct clone was found infecting outbreak-exposed individuals. Furthermore, the T. gondii outbreak clone was one of several apparently recombinant progeny recovered from the local environment. Since oocysts or sporocysts were the infectious form implicated in each outbreak, the expansion of the epidemic clone can be explained by self-mating. The results also show that out-crossing preceded selfing to produce the virulent T. gondii clone. For the tissue encysting coccidia, self-mating exists as a key adaptation potentiating the epidemic expansion and transmission of newly emerged parasite clones that can profoundly shape parasite population genetic structures or cause devastating disease

Identification of QTLs Associated with Callogenesis and Embryogenesis in Oil Palm Using Genetic Linkage Maps Improved with SSR Markers.

NARCIS (Netherlands)

Ting, N.C.; Jansen, J.; Nagappan, J.; Ishak, Z.; Chin, C.W.; Tan, S.G.; Cheah, S.C.; Singh, R.

2013-01-01

Clonal reproduction of oil palm by means of tissue culture is a very inefficient process. Tissue culturability is known to be genotype dependent with some genotypes being more amenable to tissue culture than others. In this study, genetic linkage maps enriched with simple sequence repeat (SSR)

Renal cell carcinoma primary cultures maintain genomic and phenotypic profile of parental tumor tissues.

Science.gov (United States)

Cifola, Ingrid; Bianchi, Cristina; Mangano, Eleonora; Bombelli, Silvia; Frascati, Fabio; Fasoli, Ester; Ferrero, Stefano; Di Stefano, Vitalba; Zipeto, Maria A; Magni, Fulvio; Signorini, Stefano; Battaglia, Cristina; Perego, Roberto A

2011-06-13

Clear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues. We established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation). A very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed. ccRCC primary cultures are a reliable in vitro model, well-reproducing original tumor genetics and phenotype, potentially useful for future functional approaches

Addressing the instability of DNA nanostructures in tissue culture.

Science.gov (United States)

Hahn, Jaeseung; Wickham, Shelley F J; Shih, William M; Perrault, Steven D

2014-09-23

DNA nanotechnology is an advanced technique that could contribute diagnostic, therapeutic, and biomedical research devices to nanomedicine. Although such devices are often developed and demonstrated using in vitro tissue culture models, these conditions may not be compatible with DNA nanostructure integrity and function. The purpose of this study was to characterize the sensitivity of 3D DNA nanostructures produced via the origami method to the in vitro tissue culture environment and identify solutions to prevent loss of nanostructure integrity. We examined whether the physiological cation concentrations of cell culture medium and the nucleases present in fetal bovine serum (FBS) used as a medium supplement result in denaturation and digestion, respectively. DNA nanostructure denaturation due to cation depletion was design- and time-dependent, with one of four tested designs remaining intact after 24 h at 37 °C. Adjustment of medium by addition of MgSO4 prevented denaturation. Digestion of nanostructures by FBS nucleases in Mg(2+)-adjusted medium did not appear design-dependent and became significant within 24 h and when medium was supplemented with greater than 5% FBS. We estimated that medium supplemented with 10% FBS contains greater than 256 U/L equivalent of DNase I activity in digestion of DNA nanostructures. Heat inactivation at 75 °C and inclusion of actin protein in medium inactivated and inhibited nuclease activity, respectively. We examined the impact of medium adjustments on cell growth, viability, and phenotype. Adjustment of Mg(2+) to 6 mM did not appear to have a detrimental impact on cells. Heat inactivation was found to be incompatible with in vitro tissue culture, whereas inclusion of actin had no observable effect on growth and viability. In two in vitro assays, immune cell activation and nanoparticle endocytosis, we show that using conditions compatible with cell phenotype and nanostructure integrity is critical for obtaining reliable

The basic design and requirement for plant tissue culture laboratory in MINT

International Nuclear Information System (INIS)

Azraf Azman; Rosli Darmawan; Rusli Ibrahim; Mohd Nazir Basiran; Azhar Mohamad; Mohamed Najli Mohamed Yasin; Shuhaimi Shamsuddin

2005-01-01

The production of multiple species plantlets involves a relatively complex process and it is a highly specialized operation. Tissue culture technology is rapidly becoming a commercialized method for propagating new cultivars, rare species and difficult-to-propagate plant. Not only are skills and knowledge essential but the laboratory itself also plays an important role to ensure the successful growth of the plantlets. To produce quality plantlets, plant tissue culture laboratories should fulfill the basic requirements. The laboratory should have proper building and layout which comprise of media preparation and washing room, sterilization or autoclave room, transfer room and culture or growth room. The scope of this paper is to compare these fundamental requirements with the plant tissue culture laboratory in MINT. All the basic needs and differences will be discussed and the proposal for corrective actions will be presented. (Author)

Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues.

Science.gov (United States)

Eiraku, Mototsugu; Sasai, Yoshiki

2011-12-15

Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9-10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.

Cellular lead toxicity and metabolism in primary and clonal osteoblastic bone cells

International Nuclear Information System (INIS)

Long, G.J.; Rosen, J.F.; Pounds, J.G.

1990-01-01

A knowledge of bone lead metabolism is critical for understanding the toxicological importance of bone lead, as a toxicant both to bone cells and to soft tissues of the body, as lead is mobilized from large reservoirs in hard tissues. To further understand the processes that mediate metabolism of lead in bone, it is necessary to determine lead metabolism at the cellular level. Experiments were conducted to determine the intracellular steady-state 210 Pb kinetics in cultures of primary and clonal osteoblastic bone cells. Osteoblastic bone cells obtained by sequential collagenase digestion of mouse calvaria or rat osteosarcoma (ROS 17/2.8) cells were labeled with 210 Pb as 5 microM lead acetate for 20 hr, and kinetic parameters were determined by measuring the efflux of 210 Pb from the cells over a 210 -min period. The intracellular metabolism of 210 Pb was characterized by three kinetic pools of 210 Pb in both cell types. Although the values of these parameters differed between the primary osteoblastic cells and ROS cells, the profile of 210 Pb was remarkably similar in both cell types. Both types exhibited one large, slowly exchanging pool (S3), indicative of mitochondrial lead. These data show that primary osteoblastic bone cells and ROS cells exhibit similar steady-state lead kinetics, and intracellular lead distribution. These data also establish a working model of lead kinetics in osteoblastic bone cells and now permit an integrated view of lead kinetics in bone

Clonal proliferation of cultured nonmalignant and malignant human breast epithelia

International Nuclear Information System (INIS)

Smith, H.S.; Lan, S.; Ceriani, R.; Hackett, A.J.; Stampfer, M.R.

1981-01-01

We have developed a method for clonal growth of human mammary epithelial cells of both nonmalignant and malignant origin. Plating efficiencies of 1 to 50% were obtained by seeding second-passage mammary epithelial cells on fibroblast feeder layers in an enriched medium composed of various hormones and growth factors, as well as conditioned media from three specific human cell lines. Single mammary epithelial cells seeded sparsely onto the fibroblasts underwent at least eight population doublings to form large, readily visible colonies. Optimal colony formation required both feeder cells and the enriched medium. Epithelial colonies containing at least 16 cells were visible 5 days postseeding, and these colonies continued to grow progressively. Plating efficiency and colony size were similar on ultraviolet-irradiated or nonirradiated fibroblasts. The number of colonies formed was proportional to the number of epithelial cells plated. The colonies were identified as epithelial by the presence of human mammary epithelial antigens

Structure and component alteration of rabbit Achilles tendon in tissue culture.

Science.gov (United States)

Hosaka, Yoshinao; Ueda, Hiromi; Yamasaki, Tadatsugu; Suzuki, Daisuke; Matsuda, Naoya; Takehana, Kazushige

2005-12-01

The aim of this study was to investigate alterations of cultured tendon tissues to determine whether tissue culture is a useful method for biological analyses of the tendon. Tendon tissues for tissue culture were isolated from Achilles tendons of rabbits. The tendon segments were placed one segment per well and incubated in growth medium consisting of Dullbecco's modified Eagle's medium supplemented with 5% fetal bovine serum at 37 degrees C in a humidified atmosphere with 5% CO(2) for various periods. The alignment of collagen fibrils was preserved for 48 h, but tendon structure has disintegrated at 96 h. Alcian blue staining and gelatine zymography revealed that proteoglycan markedly diminished and that matrix metalloproteinase (MMPs) activity was upregulated sharply at 72 and 96 h. The ratio of collagen fibrils with large diameter had increased and the mean diameter and mass average diameter value had reached maximum at 48 h. The values then decreased and mean diameters at 72 and 96 h were significantly different from that at 48 h. At 96 h, the ratio of collagen fibrils with small diameters had increased and collagen fibrils with large diameters had disappeared. These findings indicate that structural alteration is possible to be induced by disintegration of collagen fibrils and disappearance of glycosaminoglycans from extracellular matrix (ECM), subsequent of upregulation of MMPs activity. Although the study period is limited, the tissue culture method is available for investigating cell-ECM interaction in tendons.

Microfluidic 3D cell culture: potential application for tissue-based bioassays

Science.gov (United States)

Li, XiuJun (James); Valadez, Alejandra V.; Zuo, Peng; Nie, Zhihong

2014-01-01

Current fundamental investigations of human biology and the development of therapeutic drugs, commonly rely on two-dimensional (2D) monolayer cell culture systems. However, 2D cell culture systems do not accurately recapitulate the structure, function, physiology of living tissues, as well as highly complex and dynamic three-dimensional (3D) environments in vivo. The microfluidic technology can provide micro-scale complex structures and well-controlled parameters to mimic the in vivo environment of cells. The combination of microfluidic technology with 3D cell culture offers great potential for in vivo-like tissue-based applications, such as the emerging organ-on-a-chip system. This article will review recent advances in microfluidic technology for 3D cell culture and their biological applications. PMID:22793034

Clonal mutations in primary human glial tumors: evidence in support of the mutator hypothesis

Directory of Open Access Journals (Sweden)

Sarkar Chitra

2007-10-01

Full Text Available Abstract Background A verifiable consequence of the mutator hypothesis is that even low grade neoplasms would accumulate a large number of mutations that do not influence the tumor phenotype (clonal mutations. In this study, we have attempted to quantify the number of clonal mutations in primary human gliomas of astrocytic cell origin. These alterations were identified in tumor tissue, microscopically confirmed to have over 70% neoplastic cells. Methods Random Amplified Polymorphic DNA (RAPD analysis was performed using a set of fifteen 10-mer primers of arbitrary but definite sequences in 17 WHO grade II astrocytomas (low grade diffuse astrocytoma or DA and 16 WHO grade IV astrocytomas (Glioblastoma Multiforme or GBM. The RAPD profile of the tumor tissue was compared with that of the leucocyte DNA of the same patient and alteration(s scored. A quantitative estimate of the overall genomic changes in these tumors was obtained by 2 different modes of calculation. Results The overall change in the tumors was estimated to be 4.24% in DA and 2.29% in GBM by one method and 11.96% and 6.03% in DA and GBM respectively by the other. The difference between high and lower grade tumors was statistically significant by both methods. Conclusion This study demonstrates the presence of extensive clonal mutations in gliomas, more in lower grade. This is consistent with our earlier work demonstrating that technique like RAPD analysis, unbiased for locus, is able to demonstrate more intra-tumor genetic heterogeneity in lower grade gliomas compared to higher grade. The results support the mutator hypothesis proposed by Loeb.

Aging in a long-lived clonal tree.

Directory of Open Access Journals (Sweden)

Dilara Ally

2010-08-01

Full Text Available From bacteria to multicellular animals, most organisms exhibit declines in survivorship or reproductive performance with increasing age ("senescence". Evidence for senescence in clonal plants, however, is scant. During asexual growth, we expect that somatic mutations, which negatively impact sexual fitness, should accumulate and contribute to senescence, especially among long-lived clonal plants. We tested whether older clones of Populus tremuloides (trembling aspen from natural stands in British Columbia exhibited significantly reduced reproductive performance. Coupling molecular-based estimates of clone age with male fertility data, we observed a significant decline in the average number of viable pollen grains per catkin per ramet with increasing clone age in trembling aspen. We found that mutations reduced relative male fertility in clonal aspen populations by about 5.8 x 10(-5 to 1.6 x 10(-3 per year, leading to an 8% reduction in the number of viable pollen grains, on average, among the clones studied. The probability that an aspen lineage ultimately goes extinct rises as its male sexual fitness declines, suggesting that even long-lived clonal organisms are vulnerable to senescence.

Clonal origins of cells in the pigmented retina of the zebrafish eye

International Nuclear Information System (INIS)

Streisinger, G.; Coale, F.; Taggart, C.; Walker, C.; Grunwald, D.J.

1989-01-01

Mosaic analysis has been used to study the clonal basis of the development of the pigmented retina of the zebrafish, Brachydanio rerio. Zebrafish embryos heterozygous for a recessive mutation at the gol-1 locus were exposed to gamma-irradiation at various developmental stages to create mosaic individuals consisting of wild-type pigmented cells and a clone of pigmentless (golden) cells in the eye. The contribution of individual embryonic cells to the pigmented retina was measured and the total number of cells in the embryo that contributed descendants to this tissue was determined. Until the 32-cell stage, almost every blastomere has some descendants that participate in the formation of the pigmented retina of the zebrafish. During subsequent cell divisions, up to the several thousand-cell stage, the number of ancestral cells is constant: approximately 40 cells are present that will give rise to progeny in the pigmented retina. Analysis of the size of clones in the pigmented retina indicates that the cells of this tissue do not arise through a rigid series of cell divisions originating in the early embryo. The findings that each cleavage stage cell contributes to the pigmented retina and yet the contribution of such cells is highly variable are consistent with the interpretation that clonal descendants of different blastomeres normally intermix extensively prior to formation of the pigmented retina

Three-dimensional hydrogel cell culture systems for modeling neural tissue

Science.gov (United States)

Frampton, John

Two-dimensional (2-D) neural cell culture systems have served as physiological models for understanding the cellular and molecular events that underlie responses to physical and chemical stimuli, control sensory and motor function, and lead to the development of neurological diseases. However, the development of three-dimensional (3-D) cell culture systems will be essential for the advancement of experimental research in a variety of fields including tissue engineering, chemical transport and delivery, cell growth, and cell-cell communication. In 3-D cell culture, cells are provided with an environment similar to tissue, in which they are surrounded on all sides by other cells, structural molecules and adhesion ligands. Cells grown in 3-D culture systems display morphologies and functions more similar to those observed in vivo, and can be cultured in such a way as to recapitulate the structural organization and biological properties of tissue. This thesis describes a hydrogel-based culture system, capable of supporting the growth and function of several neural cell types in 3-D. Alginate hydrogels were characterized in terms of their biomechanical and biochemical properties and were functionalized by covalent attachment of whole proteins and peptide epitopes. Methods were developed for rapid cross-linking of alginate hydrogels, thus permitting the incorporation of cells into 3-D scaffolds without adversely affecting cell viability or function. A variety of neural cell types were tested including astrocytes, microglia, and neurons. Cells remained viable and functional for longer than two weeks in culture and displayed process outgrowth in 3-D. Cell constructs were created that varied in cell density, type and organization, providing experimental flexibility for studying cell interactions and behavior. In one set of experiments, 3-D glial-endothelial cell co-cultures were used to model blood-brain barrier (BBB) structure and function. This co-culture system was

Smallholder adoption and economic impacts of tissue culture ...

African Journals Online (AJOL)

PRECIOUS

2009-12-01

Dec 1, 2009 ... ISSN 1684–5315 © 2009 Academic Journals. Full Length ... Key words: Biotechnology, adoption, tissue culture bananas, Kenya. INTRODUCTION ... Recent studies about the agronomic and economic impacts of biotech- ..... accused scientist for 'playing God', others have supported biotechnologies.

Clonal hematopoiesis in acquired aplastic anemia.

Science.gov (United States)

Ogawa, Seishi

2016-07-21

Clonal hematopoiesis (CH) in aplastic anemia (AA) has been closely linked to the evolution of late clonal disorders, including paroxysmal nocturnal hemoglobinuria and myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML), which are common complications after successful immunosuppressive therapy (IST). With the advent of high-throughput sequencing of recent years, the molecular aspect of CH in AA has been clarified by comprehensive detection of somatic mutations that drive clonal evolution. Genetic abnormalities are found in ∼50% of patients with AA and, except for PIGA mutations and copy-neutral loss-of-heterozygosity, or uniparental disomy (UPD) in 6p (6pUPD), are most frequently represented by mutations involving genes commonly mutated in myeloid malignancies, including DNMT3A, ASXL1, and BCOR/BCORL1 Mutations exhibit distinct chronological profiles and clinical impacts. BCOR/BCORL1 and PIGA mutations tend to disappear or show stable clone size and predict a better response to IST and a significantly better clinical outcome compared with mutations in DNMT3A, ASXL1, and other genes, which are likely to increase their clone size, are associated with a faster progression to MDS/AML, and predict an unfavorable survival. High frequency of 6pUPD and overrepresentation of PIGA and BCOR/BCORL1 mutations are unique to AA, suggesting the role of autoimmunity in clonal selection. By contrast, DNMT3A and ASXL1 mutations, also commonly seen in CH in the general population, indicate a close link to CH in the aged bone marrow, in terms of the mechanism for selection. Detection and close monitoring of somatic mutations/evolution may help with prediction and diagnosis of clonal evolution of MDS/AML and better management of patients with AA. © 2016 by The American Society of Hematology.

Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

Science.gov (United States)

Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

2013-01-01

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

Directory of Open Access Journals (Sweden)

Anja Marciniak

Full Text Available Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

A Common Origin for B-1a and B-2 Lymphocytes in Clonal Pre- Hematopoietic Stem Cells.

Science.gov (United States)

Hadland, Brandon K; Varnum-Finney, Barbara; Mandal, Pankaj K; Rossi, Derrick J; Poulos, Michael G; Butler, Jason M; Rafii, Shahin; Yoder, Mervin C; Yoshimoto, Momoko; Bernstein, Irwin D

2017-06-06

Recent evidence points to the embryonic emergence of some tissue-resident innate immune cells, such as B-1a lymphocytes, prior to and independently of hematopoietic stem cells (HSCs). However, whether the full hematopoietic repertoire of embryonic HSCs initially includes these unique lineages of innate immune cells has been difficult to assess due to lack of clonal assays that identify and assess HSC precursor (pre-HSC) potential. Here, by combining index sorting of single embryonic hemogenic precursors with in vitro HSC maturation and transplantation assays, we analyze emerging pre-HSCs at the single-cell level, revealing their unique stage-specific properties and clonal lineage potential. Remarkably, clonal pre-HSCs detected between E9.5 and E11.5 contribute to the complete B cell repertoire, including B-1a lymphocytes, revealing a previously unappreciated common precursor for all B cell lineages at the pre-HSC stage and a second embryonic origin for B-1a lymphocytes. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Culture of three-dimensional tissue model and its application in bystander-effect research

International Nuclear Information System (INIS)

Wu Ruqun; Xu An; Wu Lijun; Hu Burong

2012-01-01

Compared with the cultured monolayer (2D) cells, three-dimensional (3D) tissue could be more similar to the environment in vivo including the physical support, chemical factors, cell-cell and cell-matrix interaction and so on. With the development of three-dimensional cell culture techniques (TDCC), 3D tissue is widely used in the areas of bystander effect research. This review focuses on introducing the TDCC method and its application in bystander-effect research. First, the development process of 3D tissue culture method was introduced. Secondly, the induction of radiation induced bystander effects both in 2D cell and 3D tissue and its mechanisms were reviewed. Finally, because heavy ion (carbon ion beam) has been developed as a useful tool to cure solid cancer, and the 3D tissue model is an ideal material to study the damages on body after being irradiated and to understand the underlying mechanisms, future study about heavy ion radiation inducing bystander effect in 3D tissue was discussed. (authors)

Demonstration of the economic feasibility of plant tissue culture for jojoba (Simmondsia chinensis) and Euphorbia spp

Energy Technology Data Exchange (ETDEWEB)

Sluis, C.

1980-09-01

The economic feasibility of plant tissue culture was demonstrated as applied to two plants: jojoba (Simmondsia chinensis) and Euphorbia spp. The gopher weed (Euphorbia lathyris) was selected as the species of Euphorbia to research due to the interest in this plant as a potential source of hydrocarbon-like compounds. High yield female selections of jojoba were chosen from native stands and were researched to determine the economic feasibility of mass producing these plants via a tissue culture micropropagation program. The female jojoba selection was successfully mass produced through tissue culture. Modifications in initiation techniques, as well as in multiplication media and rooting parameters, were necessary to apply the tissue culture system, which had been developed for juvenile seedling tissue, to mature jojobas. Since prior attempts at transfer of tissue cultured plantlets were unsuccessful, transfer research was a major part of the project and has resulted in a system for transfer of rooted jojoba plantlets to soil. Euphorbia lathyris was successfully cultured using shoot tip cultures. Media and procedures were established for culture initiation, multiplication of shoots, callus induction and growth, and root initiation. Well-developed root systems were not attained and root initiation percentages should be increased if the system is to become commercially feasible.

Stomatal characteristics of Eucalyptus grandis clonal hybrids in ...

African Journals Online (AJOL)

This study describes the stomatal response occurring during water stress and subsequent recovery of three Eucalyptus grandis clonal hybrids. The aim was to investigate the degree to which stomatal conductance (gs) and stomatal density differ between the clonal hybrids across seasons and in response to water stress.

Mass spectrometric characterization of elements and molecules in cell cultures and tissues

International Nuclear Information System (INIS)

Arlinghaus, H.F.; Kriegeskotte, C.; Fartmann, M.; Wittig, A.; Sauerwein, W.; Lipinsky, D.

2006-01-01

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and laser post-ionization secondary neutral mass spectrometry (laser-SNMS) have been used to image and quantify targeted compounds, intrinsic elements and molecules with subcellular resolution in single cells of both cell cultures and tissues. Special preparation procedures for analyzing cell cultures and tissue materials were developed. Cancer cells type MeWo, incubated with boronated compounds, were sandwiched between two substrates, cryofixed, freeze-fractured and freeze-dried. Also, after injection with boronated compounds, different types of mouse tissues were extracted, prepared on a special specimen carrier and plunged with high velocity into LN 2 -cooled propane for cryofixation. After trimming, these tissue blocks were freeze-dried. The measurements of the K/Na ratio demonstrated that for both cell cultures and tissue materials the special preparation techniques used were appropriate for preserving the chemical and structural integrity of the living cell. The boron images show inter- and intracellular boron signals with different intensities. Molecular images show distinct features partly correlated with the cell structure. A comparison between laser-SNMS and ToF-SIMS showed that especially laser-SNMS is particularly well-suited for identifying specific cell structures and imaging ultratrace element concentrations in tissues

Effects of Clonal Reproduction on Evolutionary Lag and Evolutionary Rescue.

Science.gov (United States)

Orive, Maria E; Barfield, Michael; Fernandez, Carlos; Holt, Robert D

2017-10-01

Evolutionary lag-the difference between mean and optimal phenotype in the current environment-is of keen interest in light of rapid environmental change. Many ecologically important organisms have life histories that include stage structure and both sexual and clonal reproduction, yet how stage structure and clonality interplay to govern a population's rate of evolution and evolutionary lag is unknown. Effects of clonal reproduction on mean phenotype partition into two portions: one that is phenotype dependent, and another that is genotype dependent. This partitioning is governed by the association between the nonadditive genetic plus random environmental component of phenotype of clonal offspring and their parents. While clonality slows phenotypic evolution toward an optimum, it can dramatically increase population survival after a sudden step change in optimal phenotype. Increased adult survival slows phenotypic evolution but facilitates population survival after a step change; this positive effect can, however, be lost given survival-fecundity trade-offs. Simulations indicate that the benefits of increased clonality under environmental change greatly depend on the nature of that change: increasing population persistence under a step change while decreasing population persistence under a continuous linear change requiring de novo variation. The impact of clonality on the probability of persistence for species in a changing world is thus inexorably linked to the temporal texture of the change they experience.

Identification of Stevioside Using Tissue Culture-Derived Stevia (Stevia rebaudiana) Leaves

Science.gov (United States)

Karim, Md. Ziaul; Uesugi, Daisuke; Nakayama, Noriyuki; Hossain, M. Monzur; Ishihara, Kohji; Hamada, Hiroki

2015-01-01

Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for identification of stevioside from tissue culture-derived stevia leaf. Stevioside in the sample was identified using HPLC by measuring the retention time. The percentage of stevioside content in the leaf samples was found to be 9.6%. This identification method can be used for commercial production and industrialization of stevia through in vitro culture across the world. PMID:28008268

Multiplex polymerase chain reaction on FTA cards vs. flow cytometry for B-lymphocyte clonality.

Science.gov (United States)

Dictor, Michael; Skogvall, Ingela; Warenholt, Janina; Rambech, Eva

2007-01-01

Two-colour flow cytometry was compared with multiplex PCR with capillary electrophoresis for clonality determination in specific categories of B-cell lymphoma. FTA cards were evaluated for preserving DNA from node imprints and expediting molecular analysis. A single-tube multiplex PCR targeted IGH and lymphoma-specific translocations in DNA extracted from 180 frozen lymphoid tissues and DNA bound to FTA cards from 192 fresh tissues and 137 aspirates. PCR results were compared with flow cytometry in the extracted and aspirated samples. Overall, single-tube multiplex PCR sensitivity was equivalent in the sample groups (intergroup range 79%-91%). False negatives were associated with tumour origin in the follicle centre. Multiplex PCR and flow cytometry were equally sensitive and together detected 98% of B-cell lymphomas. Additional two-tube targeting of IGK suggested an overall molecular sensitivity >90%. False positive (pseudoclonal) single-tube multiplex PCR was associated with necrosis and sparse lymphocytes. Multiplex PCR using template DNA bound to an FTA card effectively detects B-lymphocyte clonality, obviates DNA extraction and refrigeration, and can be used without diminished sensitivity in fine needle aspirates or node imprints as a replacement for or complement to flow cytometry at any point in the diagnostic work-up.

Banana Musa tissue culture plants enhanced by endophytic fungi

African Journals Online (AJOL)

Mo

Merging biotechnology with biological control: Banana Musa tissue culture plants enhanced by endophytic .... While working in the laminar flow cabinet, sterile filter papers were placed in ..... University of Bonn, Bonn, Germany. Niere, B., 2001.

Research progress in plant mutation by combining ion beam irradiations and tissue culture

International Nuclear Information System (INIS)

Zhou Linbin; Li Wenjian; Qu Ying; Li Ping

2007-01-01

About a new mutation breeding method which combines plant tissue culture technique with heavy ion beam irradiations were discussed in this paper with the principles, operation steps, molecular mechanisms, etc. The mutation method developed a few advantages coming from plant tissue culture, which can produce offspring by asexual ways. Meanwhile, using this method, the study of biological effects of high energy particles with different linear energy transfer values on plant tissues or cells can be explored and optimized in theory or practice. (authors)

HYPOLIPIDEMIC EFFECT OF ARGLABIN IN HEPATOMA TISSUE CULTURE

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A. V. Ratkin

2015-01-01

Full Text Available Objective. Investigation of hypolipidemic effect of sesquiterpene γ-lactone Arglabin in hepatoma tissue culture (HTC.Materials and methods. In this study we’ve evaluated the effect of sesquiterpene γ-lactone Arglabin and gemfibrozil (reference drug on the lipid content in the hepatoma tissue culture (HTC which were incubated with a fat emulsion “Lipofundin” by fluorescent method with vital dye Nile Red. The cell viability was investigated using the MTT-test and staining by Trypan blue.Results. Cultivation of cell cultures of rat’s hepatoma cell line HTC with Arglabin and gemfibrozil in concentrations from 10 to 50 μmol and from 0.25 to 0.5 mmol, respectively, had no cytotoxic effect. HTC cell viability did not change compared with the corresponding rate in the control culture. Experimental hyperlipidemia in hepatoma culture was induced by the addition in the incubation medium of fat emulsion “Lipofundin” in a final concentration of 0.05 %. The fluorescence intensity of Nile Red in the cells was increased 4-fold (p < 0.05, which indicates a significant accumulation of lipids in the cytosol of cells. In these steady-state Arglabin and gemfibrozil at concentrations 75–100 μM and 0.25–1.0 mM, respectively, reduced the content of lipid in cells. Conclusion. In the model of hyperlipidemia induced by lipofundin, sesquiterpene γ-lactone Arglabin prevents the accumulation of lipids in the HTC cell line, as evidenced by a decrease in Nile Red fluorescence. However hypolipidemic effect of Arglabin is associated with cytotoxic effects, which is typical for anticancer drugs.

Human colon tissue in organ culture: calcium and multi-mineral-induced mucosal differentiation.

Science.gov (United States)

Dame, Michael K; Veerapaneni, Indiradevi; Bhagavathula, Narasimharao; Naik, Madhav; Varani, James

2011-01-01

We have recently shown that a multi-mineral extract from the marine red algae, Lithothamnion calcareum, suppresses colon polyp formation and inflammation in mice. In the present study, we used intact human colon tissue in organ culture to compare responses initiated by Ca(2+) supplementation versus the multi-mineral extract. Normal human colon tissue was treated for 2 d in culture with various concentrations of calcium or the mineral-rich extract. The tissue was then prepared for histology/immunohistochemistry, and the culture supernatants were assayed for levels of type I procollagen and type I collagen. At higher Ca(2+) concentrations or with the mineral-rich extract, proliferation of epithelial cells at the base and walls of the mucosal crypts was suppressed, as visualized by reduced Ki67 staining. E-cadherin, a marker of differentiation, was more strongly expressed at the upper third of the crypt and at the luminal surface. Treatment with Ca(2+) or with the multi-mineral extract influenced collagen turnover, with decreased procollagen and increased type I collagen. These data suggest that calcium or mineral-rich extract has the capacity to (1) promote differentiation in human colon tissue in organ culture and (2) modulate stromal function as assessed by increased levels of type I collagen. Taken together, these data suggest that human colon tissue in organ culture (supporting in vivo finding in mice) will provide a valuable model for the preclinical assessment of agents that regulate growth and differentiation in the colonic mucosa.

Effect of environmental stress on clonal structure of Eucypris virens (Crustacea, Ostracoda)

OpenAIRE

Martins M. J. F.; Vandekerkhove J.; Adolfsson S.; Rossetti G.; Namiotko T.; Jokela J.

2010-01-01

Environmental stress imposes strong natural selection on clonal populations, promoting evolutionary change in clonal structure. Environmental stress may also lead to reduction in population size, which together with clonal selection may reduce genotypic diversity of the local populations. We examined how clonal structure in wild-collected samples of two parthenogenetic populations of the freshwater ostracod Eucypris virens responded to hypersalinity and starvation, and the combination of the ...

[Comparative study on alkaloids of tissue-culture seedling and wild plant of Dendrobium huoshanense ].

Science.gov (United States)

Chen, Nai-dong; Gao, Feng; Lin, Xin; Jin, Hui

2014-06-01

To compare the composition and content of alkaloid of Dendrobium huoshanense tissue-culture seedling and wild plant. A comparative evaluation on the quality was carried out by HPLC and TLC methods including the composition and the content of alkaloids. Remarkable variation existed in the two kinds of Dendrobium huoshanense. For the tissue-culture plant, only two alkaloids were checked out by both HPLC and TLC while four alkaloids were observed in the wild plant. The alkaloid content of tissue-culture seedling and wild plant was(0. 29 ± 0. 11)%o and(0. 43 ± 0. 15) %o,respectively. Distinguished difference is observed in both composition and content of alkaloids from the annual shoots of different provenances of Dendrobium huoshanense. It suggested that the quality of tissue-culture seedling of Dendrobium huoshanense might be inconsistent with the wild plant. Furthermore, the established alkaloids-knock-out HPLC method would provide a new research tool on quality control of Chinese medicinal materials which contain unknown alkaloids.

Plasmid-dependent attachment of Agrobacterium tumefaciens to plant tissue culture cells.

Science.gov (United States)

Matthysse, A G; Wyman, P M; Holmes, K V

1978-11-01

Kinetic, microscopic, and biochemical studies show that virulent Ti (tumor inducing)-plasmid-containing strains of Agrobacterium attach to normal tobacco and carrot tissue culture cells. Kinetic studies showed that virulent strains of A. tumefaciens attach to the plant tissue culture cells in increasing numbers during the first 1 to 2 h of incubation of the bacteria with the plant cells. Five Ti-plasmid-containing virulent Agrobacterium strains showed greater attachment to tobacco cells than did five avirulent strains. Light and scanning electron microscopic observations confirmed that virulent strains showed little attachment. Bacterial attachment was blocked by prior incubation of the plant cells with lipopolysaccharide extracted from A. tumefaciens, but not from A. radiobacter, suggesting that bacterial lipopolysaccharide is one of the components involved in the attachment process. At least one other bacterial product may be required for attachment in tissue culture because the virulent A. tumefaciens NT1, which lacks the Ti plasmid, does not itself attach to tobacco cells, but its lipopolysaccharide does inhibit the attachment of virulent strains.

Influence of postmortem time on the outcome of blood cultures among cadaveric tissue donors.

Science.gov (United States)

Saegeman, V; Verhaegen, J; Lismont, D; Verduyckt, B; De Rijdt, T; Ectors, N

2009-02-01

Tissue banks provide tissues of human cadaver donors for transplantation. The maximal time limit for tissue retrieval has been set at 24 h postmortem. This study aimed at evaluating the evidence for this limit from a microbiological point of view. The delay of growth in postmortem blood cultures, the identification of the species isolated and clinical/environmental factors were investigated among 100 potential tissue donors. No significant difference was found in the rate of donors with grown blood cultures within (25/65=38%) compared with after (24/65=37%) 24 h of death. Coagulase-negative staphylococci and gastro-intestinal microorganisms were isolated within and after 24 h of death. Two factors--antimicrobial therapy and "delay before body cooling"--were significantly inversely related with donors' blood culture results. From a microbiological point of view, there is no evidence for avoiding tissue retrieval among donors after 24 h of death.

A method for establishing human primary gastric epithelial cell culture from fresh surgical gastric tissues.

Science.gov (United States)

Aziz, Faisal; Yang, Xuesong; Wen, Qingping; Yan, Qiu

2015-08-01

At present, biopsy specimens, cancer cell lines and tissues obtained by gastric surgery are used in the study and analysis of gastric cancer, including the molecular mechanisms and proteomics. However, fibroblasts and other tissue components may interfere with these techniques. Therefore, the present study aimed to develop a procedure for the isolation of viable human gastric epithelial cells from gastric surgical tissues. A method was developed to culture human gastric epithelial cells using fresh, surgically excised tissues and was evaluated using immunocytochemistry, periodic acid-Schiff (PAS) staining and cell viability assays. Low cell growth was observed surrounding the gastric tissue on the seventh day of tissue explant culture. Cell growth subsequently increased, and at 12 days post-explant a high number of pure epithelial cells were detected. The gastric cancer cells exhibited rapid growth with a doubling time of 13-52 h, as compared to normal cells, which had a doubling time of 20-53 h. Immunocytochemical analyses of primary gastric cells revealed positive staining for cytokeratin 18 and 19, which indicated that the culture was comprised of pure epithelial cells and contained no fibroblasts. Furthermore, PAS staining demonstrated that the cultured gastric cells produced neutral mucin. Granulin and carbohydrate antigen 724 staining confirmed the purity of gastric cancer and normal cells in culture. This method of cell culture indicated that the gastric cells in primary culture consisted of mucin-secreting gastric epithelial cells, which may be useful for the study of gastric infection with Helicobacter pylori and gastric cancer.

Identification of Stevioside Using Tissue Culture-Derived Stevia () Leaves

OpenAIRE

Ziaul Karim Md.; Daisuke Uesugi; Noriyuki Nakayama; M. Monzur Hossain; Kohji Ishihara; Hiroki Hamada

2015-01-01

Stevioside is a natural sweetener from Stevia leaf, which is 300 times sweeter than sugar. It helps to reduce blood sugar levels dramatically and thus can be of benefit to diabetic people. Tissue culture is a very potential modern technology that can be used in large-scale disease-free stevia production throughout the year. We successfully produced stevia plant through in vitro culture for identification of stevioside in this experiment. The present study describes a potential method for iden...

Migration assay on primary culture isolated from patient's primary breast cancer tissue

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ED Yuliana

2014-12-01

Full Text Available Background: Migration is an essential component of breast cancer metastasis, which studyhas been concentrated on culture of established breast cancer cell lines that do not accuratelyrepresent the sophistication and heterogeneity of patient's breast cancer. An attempt toperform migration assay using Boyden Chamber Assay (BCA on primary culture originatingfrom patient's breast cancer tissue was developed to accommodate upcoming study of breastcancer migration in lndonesian patients.Methods: Pathologically proven primary breast cancer tissue samples were obtained fromCiptomangunkusumo Hospital during core (n=4 and incisional (n=3 biopsies of stage llAup to stage lllA breast cancer patients. Following biopsy, the breast cancer tissue samplesunderwent processings to isolate the cancer cells. These cancer cells were -then resuspendedwithin Dulbecco's modified Eagle's medium (DMEM ahd cultured in 12-well plate. The growthof primary culture were observed and compared between the core biopsy and the incisionalbiopsy specimens. Optimization of BCA method was later performed to investigate themigration of the breast cancer primary culture towards different experirnental conditions, whichwere control, Fetal Bovine Serum (FBS, and Stromal Derived Factor-l (SDF-1. Two differentnumber of breast cancer cells were tested for the optimization of the BCA, which were 1 x 105and3x105cells.Results: None of the culture performed on core biopsy specimens grew, while one out ofthree incisional biopsy specimens grew until confluence. The one primary culture that grewwas later assesed using BCA to assess its migration index towards different experimentalconditions. Using 1 x 10s breast cancer cells in the BCA , the result of the absorbance level ofmigrated cells showed that the migration towards SDF-1 (0.529 nearly doubled the migrationtowards controlmedium (0.239 and FBS (0.209. Meanwhile, the absorbance levelwas simiiarbetween the control medium (1.050, FBS (1 .103

Usefulness of fibroblast culture for testing of cattle tissues polluted with heavy metals

International Nuclear Information System (INIS)

Weglarz, L.; Drozdz, M.Wa.; Wardas, M.; Kula, B.; Pawlaczyk-Szpilowa, M.

1990-01-01

Cattle tissues (liver, kidney, brain, and lung) that had been polluted with heavy metals were tested for their ability to alter fibroblast culture growth, cellular protein and DNA content, and fibroblast DNA synthesis. At 72 hr of incubation a significant increase in cellular DNA and [14C]thymidine incorporation was noted in the primary cultures as well as in the subcultures compared to controls. Fibroblast cultures also displayed growth inhibition and reduction in protein content. The measurement of basic biochemical parameters of the fibroblast culture may represent a sensitive means of assessing rapidly the activity of heavy metals deposited in the tissues of cattle as a result of their grazing on polluted soil

Project on production of mutants by irradiation of in vitro cultured tissues of coconut and banana and their mass propagation by the tissue culture technique

International Nuclear Information System (INIS)

Guzman, E.V. de

1975-01-01

Fruit pulp tissue, ovary segments with or without ovules and sections from shoot tips of banana were used for studies on growth stimulating or morphogenetic effects of irradiation. Irradiation at 0.1-1.0 kR tended to induce faster callus growth in the otherwise slow-growing cultures. The physical condition and composition of the culture media especially with respect to growth regulators were studied, as were techniques to overcome discoloration of explants, the best choice of plant tissue for explant, and radiation effects on growth and morphogenesis. Due to the difficulty of callus induction with coconut, only the effects of irradiation on embryos cultured in vitro were studied. They were irradiated at various stages of development, i.e. during the early and final stage of liquid culture, and several days after transfer to a solid medium. Adverse effects of irradiation became evident only during the subsequent growth in solid, during the latter stage of which morphological changes were observed. Whereas irradiation of the liquid as well as solid media up to 50 kR had no adverse effect; survival and development became adversely affected at a dose of 1 kR

Isolation, culture, characterization, and osteogenic differentiation of canine endometrial mesenchymal stem cell

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A. K. Sahoo

2017-12-01

Full Text Available Aim: In this study, the canine endometrium tissue is characterized for its stem cell properties such as adherence to tissue culture plate (plasticity, short population doubling time, serial clonal passaging, long-term culturing properties, stem cell marker expression, and multilineage differentiation potential. Materials and Methods: The present work describes a novel isolation protocol for obtaining mesenchymal stem cells from the uterine endometrium and is compared with cells derived from umbilical cord matrix as a positive control. These cells are clonogenic, can undergo several population doublings in vitro, and can be differentiated to the osteocytes in mature mesenchymal tissues when grown in osteogenic differentiation media as detected by Alizarin Red-S staining. Results: It is reported for the first time that the cells derived from the canine endometrium (e-multipotent stem cells [MSCs] were able to differentiate into a heterologous cell type: Osteocytes, thus demonstrating the presence of MSCs. Thus, the endometrium may be told as a potential source of MSCs which can be used for various therapeutic purposes. Conclusion: The endometrium can be used as a potential source of MSCs, which can be used for various therapeutic purposes.

Substrate specific hydrolysis of aromatic and aromatic-aliphatic esters in orchid tissue cultures

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Agnieszka Mironowicz

2014-01-01

Full Text Available We found that tissue cultures of higher plants were able, similarly as microorganisms, to transform low-molecular-weight chemical compounds. In tissue cultures of orchids (Cymbidium 'Saint Pierre' and Dendrobium phalaenopsis acetates of phenols and aromatic-aliphatic alcohols were hydrolyzed, whereas methyl esters of aromatic and aromatic-aliphatic acids did not undergo this reaction. Acetates of racemic aromatic-aliphatic alcohols were hydrolyzed with distinct enantiospecificity.

Comparative drug susceptibility study of five clonal strains of Trichomonas vaginalis in vitro

Institute of Scientific and Technical Information of China (English)

Hemantkumar Somabhai Chaudhari; Prati Pal Singh

2011-01-01

Objective: To produce comparative data on a group of Trichomonas vaginalis clonal strains with varied drug responses using identical methods and materials. Methods: Five clonal strains of Trichomonas vaginalis were isolated from reference strain using agar plate technique. The variability of growth kinetic and susceptibility of clonal strain to metronidazole, tinidazole, satranidazole and nitazoxanide were observed in 96 well microtitre plate. Results: Among these clonal strains there was a good correlation between rates of growth with the relative susceptibility of the strains to drugs in vitro. Regarding metronidazole, tinidazole and satranidazole susceptibility, different degrees of susceptibility were determined. However, no difference in nitazoxanide susceptibility was found between the clonal strain tested and a reference strain.Conclusions: This is the first description of biological variability in clonal stock of Trichomonas vaginalis. Different degrees of drug susceptibility were determined among clonal strains tested. Further studies will be necessary to ascertain the importance of this variability in clinical infection.

Self-Condensation Culture Enables Vascularization of Tissue Fragments for Efficient Therapeutic Transplantation

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Yoshinobu Takahashi

2018-05-01

Full Text Available Summary: Clinical transplantation of tissue fragments, including islets, faces a critical challenge because of a lack of effective strategies that ensure efficient engraftment through the timely integration of vascular networks. We recently developed a complex organoid engineering method by “self-condensation” culture based on mesenchymal cell-dependent contraction, thereby enabling dissociated heterotypic lineages including endothelial cells to self-organize in a spatiotemporal manner. Here, we report the successful adaptation of this method for generating complex tissues from diverse tissue fragments derived from various organs, including pancreatic islets. The self-condensation of human and mouse islets with endothelial cells not only promoted functionalization in culture but also massively improved post-transplant engraftment. Therapeutically, fulminant diabetic mice were more efficiently treated by a vascularized islet transplant compared with the conventional approach. Given the general limitations of post-transplant vascularization associated with 3D tissue-based therapy, our approach offers a promising means of enhancing efficacy in the context of therapeutic tissue transplantation. : Takahashi et al. report on generating vascularized islet tissue from humans and mice. After transplantation, vascularized islets significantly improve survival of diabetic mice, demonstrating the quick normalization of blood glucose compared with conventional islet transplantation. Keywords: tissue engineering, tissue-based therapy, vascularization, islet transplantation, organoid

Monitoring Dynamic Interactions between Breast Cancer Cells and Human Bone Tissue in a Co-Culture Model

Science.gov (United States)

Contag, Christopher H.; Lie, Wen-Rong; Bammer, Marie C.; Hardy, Jonathan W.; Schmidt, Tobi L.; Maloney, William J.; King, Bonnie L.

2015-01-01

Purpose Bone is a preferential site of breast cancer metastasis and models are needed to study this process at the level of the microenvironment. We have used bioluminescence imaging (BLI) and multiplex biomarker immunoassays to monitor dynamic breast cancer cell behaviors in co-culture with human bone tissue. Procedures Femur tissue fragments harvested from hip replacement surgeries were co-cultured with luciferase-positive MDA-MB-231-fLuc cells. BLI was performed to quantify breast cell division and track migration relative to bone tissue. Breast cell colonization of bone tissues was assessed with immunohistochemistry. Biomarkers in co-culture supernatants were profiled with MILLIPLEX® immunoassays. Results BLI demonstrated increased MDA-MB-231-fLuc proliferation (pbones, and revealed breast cell migration toward bone. Immunohistochemistry illustrated MDA-MB-231-fLuc colonization of bone, and MILLIPLEX® profiles of culture supernatants suggested breast/bone crosstalk. Conclusions Breast cell behaviors that facilitate metastasis occur reproducibly in human bone tissue co-cultures and can be monitored and quantified using BLI and multiplex immunoassays. PMID:24008275

Air exposure induced characteristics of dry eye in conjunctival tissue culture.

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Hui Lin

Full Text Available There are several animal models illustrating dry eye pathophysiology. Current study would like to establish an ex vivo tissue culture model for characterizing dry eye. Human conjunctival explants were cultured under airlift or submerged conditions for up to 2 weeks, and only airlifted conjunctival cultures underwent increased epithelial stratification. Starting on day 4, the suprabasal cells displayed decreased K19 expression whereas K10 keratin became evident in airlift group. Pax6 nuclear expression attenuated already at 2 days, while its perinuclear and cytoplasmic expression gradually increased. MUC5AC and MUC19 expression dramatically decreased whereas the full thickness MUC4 and MUC16 expression pattern disappeared soon after initiating the airlift condition. Real time PCR showed K16, K10 and MUC16 gene up-regulated while K19, MUC5AC, MUC19 and MUC4 down-regulated on day 8 and day 14. On day 2 was the appearance of apoptotic epithelial and stromal cells appeared. The Wnt signaling pathway was transiently activated from day 2 to day 10. The inflammatory mediators IL-1β, TNF-α, and MMP-9 were detected in the conditioned media after 6 to 8 days. In conclusion, airlifted conjunctival tissue cultures demonstrated Wnt signaling pathway activation, coupled with squamous metaplasia, mucin pattern alteration, apoptosis and upregulation of proinflammatory cytokine expression. These changes mimic the pathohistological alterations described in dry eye. This correspondence suggests that insight into the pathophysiology of dry eye may be aided through the use of airlifted conjunctival tissue cultures.

Adjusting to global change through clonal growth and epigenetic variation

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Richard S Dodd

2016-07-01

Full Text Available The earth is experiencing major changes in global and regional climates and changes are predicted to accelerate in the future. Many species will be under considerable pressure to evolve, to migrate, or be faced with extinction. Clonal plants would appear to be at a particular disadvantage due to their limited mobility and limited capacity for adaptation. However, they have outlived previous environmental shifts and clonal species have persisted for millenia. Clonal spread offers unique ecological advantages, such as resource sharing, risk sharing, and economies of scale among ramets within genotypes. We suggest that ecological attributes of clonal plants, in tandem with variation in gene regulation through epigenetic mechanisms that facilitate and optimize phenotype variation in response to environmental change may permit them to be well suited to projected conditions.

Pathogen and biological contamination management in plant tissue culture: phytopathogens, vitro pathogens, and vitro pests.

Science.gov (United States)

Cassells, Alan C

2012-01-01

The ability to establish and grow plant cell, organ, and tissue cultures has been widely exploited for basic and applied research, and for the commercial production of plants (micro-propagation). Regardless of whether the application is for research or commerce, it is essential that the cultures be established in vitro free of biological contamination and be maintained as aseptic cultures during manipulation, growth, and storage. The risks from microbial contamination are spurious experimental results due to the effects of latent contaminants or losses of valuable experimental or commercial cultures. Much of the emphasis in culture contamination management historically focussed on the elimination of phytopathogens and the maintenance of cultures free from laboratory contamination by environmental bacteria, fungi (collectively referred to as "vitro pathogens", i.e. pathogens or environmental micro-organisms which cause culture losses), and micro-arthropods ("vitro pests"). Microbial contamination of plant tissue cultures is due to the high nutrient availability in the almost universally used Murashige and Skoog (Physiol Plant 15:473-497, 1962) basal medium or variants of it. In recent years, it has been shown that many plants, especially perennials, are at least locally endophytically colonized intercellularly by bacteria. The latter, and intracellular pathogenic bacteria and viruses/viroids, may pass latently into culture and be spread horizontally and vertically in cultures. Growth of some potentially cultivable endophytes may be suppressed by the high salt and sugar content of the Murashige and Skoog basal medium and suboptimal temperatures for their growth in plant tissue growth rooms. The management of contamination in tissue culture involves three stages: disease screening (syn. disease indexing) of the stock plants with disease and endophyte elimination where detected; establishment and pathogen and contaminant screening of established initial cultures

Probable clonal origin of acute myeloblastic leukemia following radiation and chemotherapy of colon cancer

International Nuclear Information System (INIS)

Wiggans, R.G.; Jacobson, R.J.; Fialkow, P.J.; Woolley, P.V. III; Macdonald, J.S.; Schein, P.S.

1978-01-01

A 64-yr-old female developed acute myeloblastic leukemia following radiation and drug therapy for colon carcinoma. The patient was heterozygous for glucose-6-phosphate dehydrogenase [G-6-PD) and displayed types A and B isoenzymes in nonhematopoietic tissue. In contrast, only type B G-6-PD was observed in peripheral blood white cells. In addition, a karyotypic abnormality was found in peripheral blood and marrow cells but not in skin fibroblasts. The data are consistent with a clonal origin of this leukemia

Invasive clonal plant species have a greater root-foraging plasticity than non-invasive ones.

Science.gov (United States)

Keser, Lidewij H; Dawson, Wayne; Song, Yao-Bin; Yu, Fei-Hai; Fischer, Markus; Dong, Ming; van Kleunen, Mark

2014-03-01

Clonality is frequently positively correlated with plant invasiveness, but which aspects of clonality make some clonal species more invasive than others is not known. Due to their spreading growth form, clonal plants are likely to experience spatial heterogeneity in nutrient availability. Plasticity in allocation of biomass to clonal growth organs and roots may allow these plants to forage for high-nutrient patches. We investigated whether this foraging response is stronger in species that have become invasive than in species that have not. We used six confamilial pairs of native European clonal plant species differing in invasion success in the USA. We grew all species in large pots under homogeneous or heterogeneous nutrient conditions in a greenhouse, and compared their nutrient-foraging response and performance. Neither invasive nor non-invasive species showed significant foraging responses to heterogeneity in clonal growth organ biomass or in aboveground biomass of clonal offspring. Invasive species had, however, a greater positive foraging response in terms of root and belowground biomass than non-invasive species. Invasive species also produced more total biomass. Our results suggest that the ability for strong root foraging is among the characteristics promoting invasiveness in clonal plants.

Variable effects of dexamethasone on protein synthesis in clonal rat osteosarcoma cells

International Nuclear Information System (INIS)

Hodge, B.O.; Kream, B.E.

1988-01-01

We examined the effects of dexamethasone on protein synthesis in clonal rat osteoblastic osteosarcoma (ROS) cell lines by measuring the incorporation of [ 3 H]proline into collagenase-digestible and noncollagen protein in the cell layer and medium of the cultures. In ROS 17/2 and subclone C12 of ROS 17/2.8, dexamethasone decreased collagen synthesis with no change in DNA content of the cultures. In ROS 17/2.8 and its subclone G2, dexamethasone stimulated collagen and noncollagen protein synthesis, with a concomitant decrease in the DNA content of the cells. These data indicate that ROS cell lines are phenotypically heterogeneous and suggest that in normal bone there may be distinct subpopulations of osteoblasts with varying phenotypic traits with respect to the regulation of protein synthesis

To clone or not to clone? Induced pluripotent stem cells can be generated in bulk culture.

Science.gov (United States)

Willmann, Charlotte A; Hemeda, Hatim; Pieper, Lisa A; Lenz, Michael; Qin, Jie; Joussen, Sylvia; Sontag, Stephanie; Wanek, Paul; Denecke, Bernd; Schüler, Herdit M; Zenke, Martin; Wagner, Wolfgang

2013-01-01

Induced pluripotent stem cells (iPSCs) are usually clonally derived. The selection of fully reprogrammed cells generally involves picking of individual colonies with morphology similar to embryonic stem cells (ESCs). Given that fully reprogrammed cells are highly proliferative and escape from cellular senescence, it is conceivable that they outgrow non-pluripotent and partially reprogrammed cells during culture expansion without the need of clonal selection. In this study, we have reprogrammed human dermal fibroblasts (HDFs) with episomal plasmid vectors. Colony frequency was higher and size was larger when using murine embryonic fibroblasts (MEFs) as stromal support instead of HDFs or human mesenchymal stromal cells (MSCs). We have then compared iPSCs which were either clonally derived by manual selection of a single colony, or derived from bulk-cultures of all initial colonies. After few passages their morphology, expression of pluripotency markers, and gene expression profiles did not reveal any significant differences. Furthermore, clonally-derived and bulk-cultured iPSCs revealed similar in vitro differentiation potential towards the three germ layers. Therefore, manual selection of individual colonies does not appear to be necessary for the generation of iPSCs - this is of relevance for standardization and automation of cell culture procedures.

Selection of seed lots of Pinus taeda L. for tissue culture

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Diego Pascoal Golle

2014-06-01

Full Text Available The aim of this work was to identify the fungi genera associated with three Pinus taeda L. seed lots and to assess the sanitary and physiological quality of these lots for use as selection criteria for tissue culture and evaluate the in vitro establishment of explants from seminal origin in different nutritive media. It was possible to discriminate the lots on the sanitary and physiological quality, as well as to establish in vitro plants of Pinus taeda from cotyledonary nodes obtained from aseptic seed germination of a selected lot by the sanitary and physiological quality higher. The nutritive media MS, ½ MS and WPM were equally suitable for this purpose. For the sanitary analysis the fungal genera Fusarium, Penicillium and Trichoderma were those of the highest sensitivity. For the physiological evaluation were important the variables: abnormal seedlings, strong normal seedlings; length, fresh and dry weight of strong normal seedlings. The analyzes were favorable to choose lots of seeds for in vitro culture and all culture media were adequate for the establishment of this species in tissue culture.

Transcriptomic comparisons between cultured human adipose tissue-derived pericytes and mesenchymal stromal cells

Directory of Open Access Journals (Sweden)

Lindolfo da Silva Meirelles

2016-03-01

Full Text Available Mesenchymal stromal cells (MSCs, sometimes called mesenchymal stem cells, are cultured cells able to give rise to mature mesenchymal cells such as adipocytes, osteoblasts, and chondrocytes, and to secrete a wide range of trophic and immunomodulatory molecules. Evidence indicates that pericytes, cells that surround and maintain physical connections with endothelial cells in blood vessels, can give rise to MSCs (da Silva Meirelles et al., 2008 [1]; Caplan and Correa, 2011 [2]. We have compared the transcriptomes of highly purified, human adipose tissue pericytes subjected to culture-expansion in pericyte medium or MSC medium, with that of human adipose tissue MSCs isolated with traditional methods to test the hypothesis that their transcriptomes are similar (da Silva Meirelles et al., 2015 [3]. Here, we provide further information and analyses of microarray data from three pericyte populations cultured in pericyte medium, three pericyte populations cultured in MSC medium, and three adipose tissue MSC populations deposited in the Gene Expression Omnibus under accession number GSE67747. Keywords: Mesenchymal stromal cells, Mesenchymal stem cells, Pericytes, Microarrays

Demographic consequences of greater clonal than sexual reproduction in Dicentra canadensis.

Science.gov (United States)

Lin, Chia-Hua; Miriti, Maria N; Goodell, Karen

2016-06-01

Clonality is a widespread life history trait in flowering plants that may be essential for population persistence, especially in environments where sexual reproduction is unpredictable. Frequent clonal reproduction, however, could hinder sexual reproduction by spatially aggregating ramets that compete with seedlings and reduce inter-genet pollination. Nevertheless, the role of clonality in relation to variable sexual reproduction in population dynamics is often overlooked. We combined population matrix models and pollination experiments to compare the demographic contributions of clonal and sexual reproduction in three Dicentra canadensis populations, one in a well-forested landscape and two in isolated forest remnants. We constructed stage-based transition matrices from 3 years of census data to evaluate annual population growth rates, λ. We used loop analysis to evaluate the relative contribution of different reproductive pathways to λ. Despite strong temporal and spatial variation in seed set, populations generally showed stable growth rates. Although we detected some pollen limitation of seed set, manipulative pollination treatments did not affect population growth rates. Clonal reproduction contributed significantly more than sexual reproduction to population growth in the forest remnants. Only at the well-forested site did sexual reproduction contribute as much as clonal reproduction to population growth. Flowering plants were more likely to transition to a smaller size class with reduced reproductive potential in the following year than similarly sized nonflowering plants, suggesting energy trade-offs between sexual and clonal reproduction at the individual level. Seed production had negligible effects on growth and tuber production of individual plants. Our results demonstrate that clonal reproduction is vital for population persistence in a system where sexual reproduction is unpredictable. The bias toward clonality may be driven by low fitness returns

Frozen and fresh ovarian tissue require different culture media to promote in vitro development of bovine preantral follicles.

Science.gov (United States)

Castro, Simone Vieira; Carvalho, Adeline Andrade; Silva, Cleidson Manoel Gomes; Santos, Francielli Weber; Campello, Cláudio Cabral; de Figueiredo, José Ricardo; Rodrigues, Ana Paula Ribeiro

2014-10-01

The aim of this study was to evaluate the efficiency of different media in the in vitro culture of bovine preantral follicles that were used either fresh or following slow freezing treatment. Frozen and fresh noncultured or cultured ovarian fragments were processed for histological, viability, and cell proliferation analyses. For cryopreservation, a solution containing 1.5 M ethylene glycol was frozen in a programmable biological freezer. After thawing, a portion of the samples was destined for frozen controls. The remainder were cultured in vitro for 5 days in three media: α-MEM, McCoy, or M199. Samples from these culture media were collected on days 1 and 5 for quantification of reactive oxygen species (ROS) and for hormonal assays. In fresh-cultured tissues, the percentage of morphologically normal follicles was significantly higher when cultured in M199 compared to that in the other media. In frozen-cultured tissues, McCoy medium was significantly superior to the other media, and was the only treatment that helped in maintaining the viability similar to fresh and frozen controls. Upon quantification of the nucleolus organizer region, we observed greater proliferation of granulosa cells in the frozen-cultured tissues with McCoy medium, and lesser proliferation in fresh-cultured tissues only with α-MEM. In frozen-cultured tissues, ROS levels were highest at day 1 and progressively reduced during culture, independent of the media used. In conclusion, under the conditions used in this study, the M199 and McCoy media are recommended for the culture of follicles derived from fresh and frozen ovarian tissues, respectively.

Clonal in vitro propagation of peat mosses (Sphagnum L.) as novel green resources for basic and applied research.

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Beike, Anna K; Spagnuolo, Valeria; Lüth, Volker; Steinhart, Feray; Ramos-Gómez, Julia; Krebs, Matthias; Adamo, Paola; Rey-Asensio, Ana Isabel; Angel Fernández, J; Giordano, Simonetta; Decker, Eva L; Reski, Ralf

As builders and major components of peatlands, Sphagnopsida (peat mosses) are very important organisms for ecosystems and world's climate. Nowadays many Sphagnum species as well as their habitats are largely protected, while their scientific and economic relevance remains considerable. Advanced methods of in vitro cultivation provide the potential to work in a sustainable way with peat mosses and address aspects of basic research as well as biotechnological and economical topics like biomonitoring or the production of renewable substrates for horticulture ( Sphagnum farming). Here, we describe the establishment of axenic in vitro cultures of the five peat moss species Sphagnum fimbriatum Wils. and Hook., Sphagnum magellanicum Brid., Sphagnum palustre L., Sphagnum rubellum Wils. and Sphagnum subnitens Russ. and Warnst. with specific focus on large-scale cultivation of S. palustre in bioreactors. Axenic, clonal cultures were established to produce high quantities of biomass under standardized laboratory conditions. For advanced production of S. palustre we tested different cultivation techniques, growth media and inocula, and analyzed the effects of tissue disruption. While cultivation on solid medium is suitable for long term storage, submerse cultivation in liquid medium yielded highest amounts of biomass. By addition of sucrose and ammonium nitrate we were able to increase the biomass by around 10- to 30-fold within 4 weeks. The morphology of in vitro-cultivated gametophores showed similar phenotypic characteristics compared to material from the field. Thus the tested culture techniques are suitable to produce S. palustre material for basic and applied research.

Introduction to special issue on the ecology of clonal plants

Czech Academy of Sciences Publication Activity Database

Gross, K. L.; Herben, T.; Klimešová, Jitka

2017-01-01

Roč. 52, 3-4 (2017), s. 265-267 ISSN 1211-9520 Institutional support: RVO:67985939 Keywords : Introduction to special issue * clonal plants * clonal meeting Subject RIV: EH - Ecology, Behaviour OBOR OECD: Ecology Impact factor: 1.017, year: 2016

Prediction for the occurrence of clonal chromosome aberrations in human blood lymphocytes

International Nuclear Information System (INIS)

Nakano, M.; Kadama, Y.; Ohtaki, K.; Itoh, M.; Awa, A.; Cologne, J.; Nakamura, N.

2003-01-01

Full text: Identical chromosome aberrations among multiple blood lymphocytes in a blood sample (clonal aberrations) are encountered occasionally during cytogenetic examination of radiation-exposed people. Clonal aberrations are found primarily among high-dose exposed people but no systematic surveys were ever conducted. Therefore, the underlying mechanism is unknown. Here we conducted a large-scale screening for detecting clonal aberrations using FISH followed by Q-banding. Examinations of 500 cells from each of 513 A-bomb survivors led us to detect 96 clones. The clonal cell fraction (Cf) varied from 0.6% to 20% among the 500 cells. As the number of clonal event was inversely proportional to Cf, we hypothesized that the progenitor cells vary extensively in the number of offspring that they can produce and relative number of progenitor cells decreases as the increase of treatment, while other genes such as DNA repair proteinsnumber of progenitor cells capable to form clones (Cf >=0.6%) to be 2 (1 to 3) in non-exposed individuals. The number increased to up to 7 among the high-dose exposed survivors. Further, our preliminary results for the origins of 10 clones indicated that both hematopoietic stem cells (HSCs) and mature T cells contributed to the clone formation roughly equally. Thus, the estimated number of 2 in non-exposed individuals is shared as one HSC and one mature T cells. The model could neatly explain the frequency of clones in two reports. Our model predicts that clonal aberrations are rarely found but clonal expansion of T lymphocytes occurs commonly. In fact, clonal expansions of non-aberrant cells are reported using TCR gene rearrangement patterns as a marker. We now understand the rough structure of lymphocyte pool in humans and can predict the probability of detecting a clone if the individual frequency of non-clonal translocations and the number of cells scored are given

[18S-25S rDNA variation in tissue culture of some Gentiana L. species].

Science.gov (United States)

Mel'nyk, V M; Andrieiev, I O; Spiridonova, K V; Strashniuk, N M; Kunakh, V A

2007-01-01

18S-25S rDNA of intact plants and tissue cultures of G. acaulis, G. punctata and G. lutea have been investigated by using blot-hybridization. The decrease of rDNA amount was found in the callus cultures as compared with the plants. In contrast to other species, G. lutea showed intragenome heterogeneity of rRNA genes as well as qualitative rDNA changes in tissue culture, in particular appearance of altered repeats. The relationship between the peculiarities of rRNA gene structure and their rearrangements in in vitro culture was suggested.

Macroporous Hydrogel Scaffolds for Three-Dimensional Cell Culture and Tissue Engineering.

Science.gov (United States)

Fan, Changjiang; Wang, Dong-An

2017-10-01

Hydrogels have been promising candidate scaffolds for cell delivery and tissue engineering due to their tissue-like physical properties and capability for homogeneous cell loading. However, the encapsulated cells are generally entrapped and constrained in the submicron- or nanosized gel networks, seriously limiting cell growth and tissue formation. Meanwhile, the spatially confined settlement inhibits attachment and spreading of anchorage-dependent cells, leading to their apoptosis. In recent years, macroporous hydrogels have attracted increasing attention in use as cell delivery vehicles and tissue engineering scaffolds. The introduction of macropores within gel scaffolds not only improves their permeability for better nutrient transport but also creates space/interface for cell adhesion, proliferation, and extracellular matrix deposition. Herein, we will first review the development of macroporous gel scaffolds and outline the impact of macropores on cell behaviors. In the first part, the advantages and challenges of hydrogels as three-dimensional (3D) cell culture scaffolds will be described. In the second part, the fabrication of various macroporous hydrogels will be presented. Third, the enhancement of cell activities within macroporous gel scaffolds will be discussed. Finally, several crucial factors that are envisaged to propel the improvement of macroporous gel scaffolds are proposed for 3D cell culture and tissue engineering.

Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells

International Nuclear Information System (INIS)

Rettig, W.F.; Garin-Chesa, P.; Beresford, H.R.; Oettgen, H.F.; Melamed, M.R.; Old, L.J.

1988-01-01

Normal differentiation and malignant transformation of human cells are characterized by specific changes in surface antigen phenotype. In the present study, the authors have defined six cell-surface antigens of human sarcomas and normal mesenchymal cells, by using mixed hemadsorption assays and immunochemical methods for the analysis of cultured cells and immunohistochemical staining for the analysis of normal tissues and > 200 tumor specimens. Differential patterns of F19, F24, G171, G253, S5, and Thy-1 antigen expression were found to characterize (i) subsets of cultured sarcoma cell lines, (ii) cultured fibroblasts derived from various organs, (iii) normal resting and activated mesenchymal tissues, and (iv) sarcoma and nonmesenchymal tumor tissues. These results provide a basic surface antigenic map for cultured mesenchymal cells and mesenchymal tissues and permit the classification of human sarcomas according to their antigenic phenotypes

Pre-irradiation of tissue culture flasks leads to diminished stem and progenitor cell production in long-term bone marrow cultures

International Nuclear Information System (INIS)

Rooney, P.; Wright, E.G.

1993-01-01

Empty plastic tissue culture flasks were exposed to X-irradiation doses of 0.3-10.0 Gy, prior to the establishment of long-term bone marrow cultures. During the course of a 10 week culture period, all irradiated plastic flasks exhibited a dramatic decrease in the number of both haemopoietic stem cells and myeloid progenitor cells, in the non-adherent layer, when compared with controls. This decrease was not due to a decrease in the number of non-adherent cells produced. Histological examination of non-adherent cells showed an increase in mature granulocytic cells with few blast cells. Morphologically, the adherent layers of irradiated flasks demonstrated a delay in appearance or absence of fat cell production. X-irradiation of glass tissue culture flasks had no deleterious effect. (author)

Lemongrass-Incorporated Tissue Conditioner Against Candida albicans Culture

Science.gov (United States)

Amornvit, Pokpong; Srithavaj, Theerathavaj

2014-01-01

Background: Tissue conditioner is applied popularly with dental prosthesis during wound healing process but it becomes a reservoir of oral microbiota, especially Candida species after long-term usage. Several antifungal drugs have been mixed with this material to control fungal level. In this study, lemongrass essential oil was added into COE-COMFORT tissue conditioner before being determined for anti-Candida efficacy. Materials and Methods: Lemongrass (Cymbopogon citratus) essential oil was primarily determined for antifungal activity against C. albicans American type culture collection (ATCC) 10231 and MIC (minimum inhibitory concentration) value by agar disk diffusion and broth microdilution methods, respectively. COE-COMFORT tissue conditioner was prepared as recommended by the manufacturer after a fixed volume of the oil at its MIC or higher concentrations were mixed thoroughly in its liquid part. Antifungal efficacy of the tissue conditioner with/without herb was finally analyzed. Results: Lemongrass essential oil displayed potent antifungal activity against C. albicans ATCC 10231and its MIC value was 0.06% (v/v). Dissimilarly, the tissue conditioner containing the oil at MIC level did not cease the growth of the tested fungus. Both reference and clinical isolates of C. albicans were completely inhibited after exposed to the tissue conditioner containing at least 0.25% (v/v) of the oil (approximately 4-time MIC). The tissue conditioner without herb or with nystatin was employed as negative or positive control, respectively. Conclusion: COE-COMFORT tissue conditioner supplemented with lemongrass essential oil obviously demonstrated another desirable property as in vitro anti-Candida efficacy to minimize the risk of getting Candidal infection. PMID:25177638

Effects of Apollo 12 lunar material on lipid levels of tobacco tissue and slash pine cultures

Science.gov (United States)

Weete, J. D.

1972-01-01

Investigations of the lipid components of pine tissues (Pinus elloitii) are discussed, emphasizing fatty acids and steroids. The response by slash pine tissue cultures to growth in contact with Apollo lunar soil, earth basalt, and Iowa soil is studied. Tissue cultures of tobacco grown for 12 weeks in contact with lunar material from Apollo 12 flight contained 21 to 35 percent more total pigment than control tissues. No differences were noted in the fresh or dry weight of the experimental and control samples.

Chemical evaluation of strawberry plants produced by tissue culturing of gamma irradiated seedlings

International Nuclear Information System (INIS)

Maraei, R.W.

2007-01-01

studies were conducted to evaluate the influence of gamma irradiation as a supplementary factor precedes tissue culture application on strawberry seedlings (c.v.Rosa Linda). the strawberry seedling were irradiated using 8 doses of co 60 gamma rays 50.75.100.125 ,150,250, 350 and 500 gray. tissue culture technique was applied on irradiated and unirradiated strawberry seedling. different characteristics of plantlets, plant and fruit of strawberry produced from the double treatment (irradiation followed by tissue culture) were studied as well as the early, total and exportable fruit yields. data indicated that, low radiation doses 50,75 and 100 gray increased all morphological and chemical characteristics of the plantlets, plant and fruit of strawberry, whereas radiation doses higher than 100 gray decreased them significantly. moreover 350 and gray were lethal doses. radiation dose 50 gray increased the survival percentage and the length of plantlets by 1.5% and 50% respectively more than the unirradiated treatment in all multiplication stages

Studies on the reaction in tissue culture of tomato genotypes under biotic stress

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Ewa Hanus-Fajerska

2014-01-01

Full Text Available Plant regeneration in vitro from virus-infected somatic tomato (Lycopersicon sp. tissue was performed. Regeneration experiments were started after the determination of virus presence, using enzyme-linked immunosorbent assay, in leaves used as a source of explants. Leaf explants infected with selected strains of tomato mosaic Tobamovirus or cucumber mosaic Cucumovirus respectively, were cultured on a standarised MS agar medium to induce adventitious shoots, which were afterwards excised, rooted in vitro and cultured to plants. Explants were also screened for their ability to produce callus. Diverse effects of viral infection, ranging from stimulation to inhibition of callus formation and of morphogenesis rate, were observed. The health condition of the tissue proved to affect regeneration potential of Lycopersicon esculentum, whereas wild accesions did not react in that case so distinctly. In cultivated tomato was encountered the decline in competence to reproduce shoots adventitiously in infected tissue. There was also relationship between donor plant health condition and adventitious root formation in regenerated shoots. Experiments with short-term cultures of L. esculenum reveled also that a certain number of shoots regenerated from diseased tissue can be virus-free.

Simple and high yielding method for preparing tissue specific extracellular matrix coatings for cell culture.

Science.gov (United States)

DeQuach, Jessica A; Mezzano, Valeria; Miglani, Amar; Lange, Stephan; Keller, Gordon M; Sheikh, Farah; Christman, Karen L

2010-09-27

The native extracellular matrix (ECM) consists of a highly complex, tissue-specific network of proteins and polysaccharides, which help regulate many cellular functions. Despite the complex nature of the ECM, in vitro cell-based studies traditionally assess cell behavior on single ECM component substrates, which do not adequately mimic the in vivo extracellular milieu. We present a simple approach for developing naturally derived ECM coatings for cell culture that provide important tissue-specific cues unlike traditional cell culture coatings, thereby enabling the maturation of committed C2C12 skeletal myoblast progenitors and human embryonic stem cells differentiated into cardiomyocytes. Here we show that natural muscle-specific coatings can (i) be derived from decellularized, solubilized adult porcine muscle, (ii) contain a complex mixture of ECM components including polysaccharides, (iii) adsorb onto tissue culture plastic and (iv) promote cell maturation of committed muscle progenitor and stem cells. This versatile method can create tissue-specific ECM coatings, which offer a promising platform for cell culture to more closely mimic the mature in vivo ECM microenvironment.

Culture of equine fibroblast-like synoviocytes on synthetic tissue scaffolds towards meniscal tissue engineering: a preliminary cell-seeding study

Directory of Open Access Journals (Sweden)

Jennifer J. Warnock

2014-04-01

Full Text Available Introduction. Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be an ideal source of cells for in vitro meniscal fibrocartilage formation, however, favorable in vitro culture conditions for synovium must be established in order to achieve this goal. The objective of this study was to determine cellularity, cell distribution, and extracellular matrix (ECM formation of equine fibroblast-like synoviocytes (FLS cultured on synthetic scaffolds, for potential application in synovium-based meniscal tissue engineering. Scaffolds included open-cell poly-L-lactic acid (OPLA sponges and polyglycolic acid (PGA scaffolds cultured in static and dynamic culture conditions, and PGA scaffolds coated in poly-L-lactic (PLLA in dynamic culture conditions.Materials and Methods. Equine FLS were seeded on OPLA and PGA scaffolds, and cultured in a static environment or in a rotating bioreactor for 12 days. Equine FLS were also seeded on PGA scaffolds coated in 2% or 4% PLLA and cultured in a rotating bioreactor for 14 and 21 days. Three scaffolds from each group were fixed, sectioned and stained with Masson’s Trichrome, Safranin-O, and Hematoxylin and Eosin, and cell numbers and distribution were analyzed using computer image analysis. Three PGA and OPLA scaffolds from each culture condition were also analyzed for extracellular matrix (ECM production via dimethylmethylene blue (sulfated glycosaminoglycan assay and hydroxyproline (collagen assay. PLLA coated PGA scaffolds were analyzed using double stranded DNA quantification as areflection of cellularity and confocal laser microscopy in a fluorescent cell viability assay.Results. The highest cellularity occurred in PGA constructs cultured in a rotating bioreactor, which also had a mean sulfated glycosaminoglycan content of 22.3 µg per scaffold. PGA constructs cultured in static conditions had the lowest cellularity. Cells had difficulty adhering to OPLA and the PLLA

Apollo 12 lunar material - Effects on lipid levels of tobacco tissue cultures.

Science.gov (United States)

Weete, J. D.; Walkinshaw, C. H.; Laseter, J. L.

1972-01-01

Tobacco tissue cultures grown in contact with lunar material from Apollo 12, for a 12-week period, resulted in fluctuations of both the relative and absolute concentrations of endogenous sterols and fatty acids. The experimental tissues contained higher concentrations of sterols than the controls did. The ratio of campesterol to stigmasterol was greater than 1 in control tissues, but less than 1 in the experimental tissues after 3 weeks. High relative concentrations (17.1 to 22.2 per cent) of an unidentified compound or compounds were found only in control tissues that were 3 to 9 weeks of age.

Co-culture with infrapatellar fat pad differentially stimulates proteoglycan synthesis and accumulation in cartilage and meniscus tissues.

Science.gov (United States)

Nishimuta, James F; Bendernagel, Monica F; Levenston, Marc E

2017-09-01

Although osteoarthritis is widely viewed as a disease of the whole joint, relatively few studies have focused on interactions among joint tissues in joint homeostasis and degeneration. In particular, few studies have examined the effects of the infrapatellar fat pad (IFP) on cartilaginous tissues. The aim of this study was to test the hypothesis that co-culture with healthy IFP would induce degradation of cartilage and meniscus tissues. Bovine articular cartilage, meniscus, and IFP were cultured isolated or as cartilage-fat or meniscus-fat co-cultures for up to 14 days. Conditioned media were assayed for sulfated glycosaminoglycan (sGAG) content, nitrite content, and matrix metalloproteinase (MMP) activity, and explants were assayed for sGAG and DNA contents. Co-cultures exhibited increased cumulative sGAG release and sGAG release rates for both cartilage and meniscus, and the cartilage (but not meniscus) exhibited a substantial synergistic effect of co-culture (sGAG release in co-culture was significantly greater than the summed release from isolated cartilage and fat). Fat co-culture did not significantly alter the sGAG content of either cartilage or meniscus explants, indicating that IFP co-culture stimulated net sGAG production by cartilage. Nitrite release was increased relative to isolated tissue controls in co-cultured meniscus, but not the cartilage, with no synergistic effect of co-culture. Interestingly, MMP-2 production was decreased by co-culture for both cartilage and meniscus. This study demonstrates that healthy IFP may modulate joint homeostasis by stimulating sGAG production in cartilage. Counter to our hypothesis, healthy IFP did not promote degradation of either cartilage or meniscus tissues.

Distribution of phospholipase C isozymes in various rat tissues and cultured cells

International Nuclear Information System (INIS)

Suh, P.G.; Ryu, S.H.; Choi, W.C.; Lee, K.Y.; Rhee, S.G.

1987-01-01

Monoclonal antibodies prepared against PLC-I or PLC-II enzyme did not cross-react with the other. Using a pair of antibodies which recognizes 2 different antigenic sites on the same molecule, radioimmunoassays were developed for the quantitation of PLC-I and PLC-II in homogenates of various tissues and cultured cells, prepared by homogenization in a 2 M KCl buffer. The contents of PLC enzymes were measured in 19 rat tissues, in human platelets and in 17 cultured cells. Results indicate that the concentration of PLC-I and PLC-II is very high in brain, PLC-I is localized mainly in brain and partly in seminal vesicles, PLC-II is found in most tissues and cells. PLC-I is highly localized even in brain: 5 different neuroblastoma did not contain PLC-I while 2 glioma and 1 astrocytoma contained significant amounts

On the ecological genetics of the clonal perennial Agrostis stolonifera

NARCIS (Netherlands)

Kik, Christoffel

1987-01-01

There have to date been few studies specifically addressed to the evolution of clonal organisms. The present study attempts to fill this gap and aims to analyse the distribution pattern of a clonal plant species, using the wide-spread grass Agrostis stolonifera L.(Creeping Bent) as a model species.

Glioma tissue obtained by modern ultrasonic aspiration with a simple sterile suction trap for primary cell culture and pathological evaluation.

Science.gov (United States)

Schroeteler, Juliane; Reeker, Ralf; Suero Molina, Eric; Brokinkel, Benjamin; Holling, Markus; Grauer, Oliver M; Senner, Volker; Stummer, Walter; Ewelt, Christian

2014-01-01

Ultrasonic aspiration is widely used in the resection of brain tumors. Nevertheless, tumor tissue fragments obtained by ultrasonic aspiration are usually discarded. In this study, we demonstrate that these fragments are possible sources of material for histopathological study and tissue culture and compare their microscopic features and viability in tissue culture of cavitron ultrasonic surgical aspirator tissue fragments. Brain tumor tissue collected by ultrasonic aspiration (CUSA EXcel®; Integra Radionics Inc.) in a simple sterile suction trap during resection was processed for primary cell culture. Cell viability and immunohistological markers were measured by the WST-1 test, microscopy and immunofluorescent evaluation. Six gliomas are presented to demonstrate that these tissue fragments show good preservation of histological detail and tissue viability in culture. Utilization of this material may facilitate pathological interpretation by providing a more representative sample of tumor histology as well as an adequate and sterile biosource of material for tissue culture studies.

Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade

Science.gov (United States)

McGranahan, Nicholas; Furness, Andrew J. S.; Rosenthal, Rachel; Ramskov, Sofie; Lyngaa, Rikke; Saini, Sunil Kumar; Jamal-Hanjani, Mariam; Wilson, Gareth A.; Birkbak, Nicolai J.; Hiley, Crispin T.; Watkins, Thomas B. K.; Shafi, Seema; Murugaesu, Nirupa; Mitter, Richard; Akarca, Ayse U.; Linares, Joseph; Marafioti, Teresa; Henry, Jake Y.; Van Allen, Eliezer M.; Miao, Diana; Schilling, Bastian; Schadendorf, Dirk; Garraway, Levi A.; Makarov, Vladimir; Rizvi, Naiyer A.; Snyder, Alexandra; Hellmann, Matthew D.; Merghoub, Taha; Wolchok, Jedd D.; Shukla, Sachet A.; Wu, Catherine J.; Peggs, Karl S.; Chan, Timothy A.; Hadrup, Sine R.; Quezada, Sergio A.; Swanton, Charles

2016-01-01

As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8+ tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non–small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. T cells recognizing clonal neoantigens were detectable in patients with durable clinical benefit. Cytotoxic chemotherapy–induced subclonal neoantigens, contributing to an increased mutational load, were enriched in certain poor responders. These data suggest that neoantigen heterogeneity may influence immune surveillance and support therapeutic developments targeting clonal neoantigens. PMID:26940869

How-To-Do-It: Using Cauliflower to Demonstrate Plant Tissue Culture.

Science.gov (United States)

Haldeman, Janice H.; Ellis, Jane P.

1988-01-01

Presents techniques used for disinfestation of plant material, preparation of equipment and media, and laboratory procedures for tissue culture using cauliflower. Details methods for preparing solutions and plant propagation by cloning. (CW)

Generation of Immunoglobulin diversity in human gut-associated lymphoid tissue.

Science.gov (United States)

Spencer, Jo; Barone, Francesca; Dunn-Walters, Deborah

2009-06-01

The organised gut associated lymphoid tissue (GALT) exists adjacent to an extensive and diverse luminal flora. The follicle associated epithelium and associated dendritic cells and lymphocytes form a tightly fortified gateway between the flora and the host that permits connectivity between them and chronic activation of the lymphoid compartment. As a consequence, plasma cell precursors are generated continuously, and in abundance, in GALT by clonal proliferation. Clonal proliferation alone on this scale would reduce the spectrum of B cell specificity. To compensate, GALT also houses molecular machinery that diversifies the receptor repertoire by somatic hypermutation, class switch recombination and receptor revision. These three processes of enhancing the diversity of mature B cells ensure that although clonally related plasma cells may secrete immunoglobulin side by side in the mucosa they rarely have identical antigen binding sites.

Enlargement of induced variations by combined method of chronic irradiations with callus culture in sugarcane

International Nuclear Information System (INIS)

Nagatomi, Shigeki

1993-01-01

The present study was conducted to elucidate the effects of gamma ray irradiation and callus culture upon induced variation of the regeneratives. The populations regenerated from young leaf tissue of chronic irradiated plnats grown under a gamma field receiving a total dose of 300 and 100 Gy, showed rather wider variation on quantitative characters than plants from populations of the non-irradiated. This variation extended in both negative and positive directions. Analysis of variance also revealed that variation and heritability in broad sense of most agronomic characters increased significantly among the subclones as the irradiation done rose. Principal component analysis also indicated that the subclones from the irradiated population were more variable than the non-irradiated. Such variation with higher heritability could be transmitted to the following generations by clonal propagation and utilized as genetic sources in mutation breeding. The combined method with chronic irradiation followed by tissue culture is evaluated as an effective method of widening mutation spectrum and increasing mutation frequency in regenerated plants. In addition, this method is valid to improve any crop species which can regenerate plants through callus culture. (author)

Clonal Occurrence of Salmonella Weltevreden in Cultured Shrimp in the Mekong Delta, Vietnam

Science.gov (United States)

Noor Uddin, Gazi Md.; Larsen, Marianne Halberg; Barco, Lisa; Minh Phu, Tran; Dalsgaard, Anders

2015-01-01

This study investigated the occurrence, serovar and antimicrobial resistance of Salmonella spp. in shrimp samples from intensive and extensive farms located in three different provinces in the Mekong Delta, Vietnam. Shrimp from 11 of the 48 farms all contained S. Weltevreden, except for one farm yielding S. Agona, with no difference in Salmonella occurrence between the two production systems. Pulsed field gel electrophoresis (PFGE) of S. Weltevreden showed closely related XbaI pulse types, suggesting a clonal relationship despite the farms and shrimp samples being epidemiologically unrelated. S. Weltevreden was susceptible to most antimicrobials tested, with a few strains being resistant to florfenicol, chloramphenicol, sulfamethoxazole or trimethoprim. Future studies of the ecology of S. Weltevreden should establish if this serovar may survive better and even multiply in warm-water shrimp farm environments compared to other Salmonella serovars. PMID:26222547

Clonal Occurrence of Salmonella Weltevreden in Cultured Shrimp in the Mekong Delta, Vietnam.

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Gazi Md Noor Uddin

Full Text Available This study investigated the occurrence, serovar and antimicrobial resistance of Salmonella spp. in shrimp samples from intensive and extensive farms located in three different provinces in the Mekong Delta, Vietnam. Shrimp from 11 of the 48 farms all contained S. Weltevreden, except for one farm yielding S. Agona, with no difference in Salmonella occurrence between the two production systems. Pulsed field gel electrophoresis (PFGE of S. Weltevreden showed closely related XbaI pulse types, suggesting a clonal relationship despite the farms and shrimp samples being epidemiologically unrelated. S. Weltevreden was susceptible to most antimicrobials tested, with a few strains being resistant to florfenicol, chloramphenicol, sulfamethoxazole or trimethoprim. Future studies of the ecology of S. Weltevreden should establish if this serovar may survive better and even multiply in warm-water shrimp farm environments compared to other Salmonella serovars.

Prevention of pink-pigmented methylotrophic bacteria (Methylohacterium mesophilicum) contamination of plant tissue cultures.

Science.gov (United States)

Chanprame, S; Todd, J J; Widholm, J M

1996-12-01

Pink-pigmented facultative methylotrophic bacteria (PPFMs) have been found on the surfaces of leaves of most plants tested. We found PPFMs on the leaf surfaces of all 40 plants (38 species) tested and on soybean pods by pressing onto AMS medium with methanol as the sole carbon source. The abundance ranged from 0.5 colony forming unit (cfu) /cm(2) to 69.4 cfu/cm(2) on the leaf surfaces. PPFMs were found in homogenized leaf tissues of only 4 of the species after surface disinfestation with 1.05% sodium hypochlorite and were rarely found in cultures initiated from surface disinfested Datura innoxia leaves or inside surface disinfested soybean pods. Of 20 antibiotics tested for PPFM growth inhibition, rifampicin was the most effective and of seven others which also inhibited PPFM growth, cefotaxime should be the most useful due to the expected low plant cell toxicity. These antibiotics could be used in concert with common surface sterilization procedures to prevent the introduction or to eliminate PPFM bacteria in tissue cultures. Thus, while PPFMs are present on the surfaces of most plant tissues, surface disinfestation alone can effectively remove them so that uncontaminated tissue cultures can be initiated in most cases.

Long-term culture of human liver tissue with advanced hepatic functions.

Science.gov (United States)

Ng, Soon Seng; Xiong, Anming; Nguyen, Khanh; Masek, Marilyn; No, Da Yoon; Elazar, Menashe; Shteyer, Eyal; Winters, Mark A; Voedisch, Amy; Shaw, Kate; Rashid, Sheikh Tamir; Frank, Curtis W; Cho, Nam Joon; Glenn, Jeffrey S

2017-06-02

A major challenge for studying authentic liver cell function and cell replacement therapies is that primary human hepatocytes rapidly lose their advanced function in conventional, 2-dimensional culture platforms. Here, we describe the fabrication of 3-dimensional hexagonally arrayed lobular human liver tissues inspired by the liver's natural architecture. The engineered liver tissues exhibit key features of advanced differentiation, such as human-specific cytochrome P450-mediated drug metabolism and the ability to support efficient infection with patient-derived inoculums of hepatitis C virus. The tissues permit the assessment of antiviral agents and maintain their advanced functions for over 5 months in culture. This extended functionality enabled the prediction of a fatal human-specific hepatotoxicity caused by fialuridine (FIAU), which had escaped detection by preclinical models and short-term clinical studies. The results obtained with the engineered human liver tissue in this study provide proof-of-concept determination of human-specific drug metabolism, demonstrate the ability to support infection with human hepatitis virus derived from an infected patient and subsequent antiviral drug testing against said infection, and facilitate detection of human-specific drug hepatotoxicity associated with late-onset liver failure. Looking forward, the scalability and biocompatibility of the scaffold are also ideal for future cell replacement therapeutic strategies.

Mass Spectrometry-Based Proteomics in Molecular Diagnostics: Discovery of Cancer Biomarkers Using Tissue Culture

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Debasish Paul

2013-01-01

Full Text Available Accurate diagnosis and proper monitoring of cancer patients remain a key obstacle for successful cancer treatment and prevention. Therein comes the need for biomarker discovery, which is crucial to the current oncological and other clinical practices having the potential to impact the diagnosis and prognosis. In fact, most of the biomarkers have been discovered utilizing the proteomics-based approaches. Although high-throughput mass spectrometry-based proteomic approaches like SILAC, 2D-DIGE, and iTRAQ are filling up the pitfalls of the conventional techniques, still serum proteomics importunately poses hurdle in overcoming a wide range of protein concentrations, and also the availability of patient tissue samples is a limitation for the biomarker discovery. Thus, researchers have looked for alternatives, and profiling of candidate biomarkers through tissue culture of tumor cell lines comes up as a promising option. It is a rich source of tumor cell-derived proteins, thereby, representing a wide array of potential biomarkers. Interestingly, most of the clinical biomarkers in use today (CA 125, CA 15.3, CA 19.9, and PSA were discovered through tissue culture-based system and tissue extracts. This paper tries to emphasize the tissue culture-based discovery of candidate biomarkers through various mass spectrometry-based proteomic approaches.

Mass Spectrometry-Based Proteomics in Molecular Diagnostics: Discovery of Cancer Biomarkers Using Tissue Culture

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Paul, Debasish; Kumar, Avinash; Gajbhiye, Akshada; Santra, Manas K.; Srikanth, Rapole

2013-01-01

Accurate diagnosis and proper monitoring of cancer patients remain a key obstacle for successful cancer treatment and prevention. Therein comes the need for biomarker discovery, which is crucial to the current oncological and other clinical practices having the potential to impact the diagnosis and prognosis. In fact, most of the biomarkers have been discovered utilizing the proteomics-based approaches. Although high-throughput mass spectrometry-based proteomic approaches like SILAC, 2D-DIGE, and iTRAQ are filling up the pitfalls of the conventional techniques, still serum proteomics importunately poses hurdle in overcoming a wide range of protein concentrations, and also the availability of patient tissue samples is a limitation for the biomarker discovery. Thus, researchers have looked for alternatives, and profiling of candidate biomarkers through tissue culture of tumor cell lines comes up as a promising option. It is a rich source of tumor cell-derived proteins, thereby, representing a wide array of potential biomarkers. Interestingly, most of the clinical biomarkers in use today (CA 125, CA 15.3, CA 19.9, and PSA) were discovered through tissue culture-based system and tissue extracts. This paper tries to emphasize the tissue culture-based discovery of candidate biomarkers through various mass spectrometry-based proteomic approaches. PMID:23586059

Three-dimensional spheroid culture targeting versatile tissue bioassays using a PDMS-based hanging drop array.

Science.gov (United States)

Kuo, Ching-Te; Wang, Jong-Yueh; Lin, Yu-Fen; Wo, Andrew M; Chen, Benjamin P C; Lee, Hsinyu

2017-06-29

Biomaterial-based tissue culture platforms have emerged as useful tools to mimic in vivo physiological microenvironments in experimental cell biology and clinical studies. We describe herein a three-dimensional (3D) tissue culture platform using a polydimethylsiloxane (PDMS)-based hanging drop array (PDMS-HDA) methodology. Multicellular spheroids can be achieved within 24 h and further boosted by incorporating collagen fibrils in PDMS-HDA. In addition, the spheroids generated from different human tumor cells exhibited distinct sensitivities toward drug chemotherapeutic agents and radiation as compared with two-dimensional (2D) cultures that often lack in vivo-like biological insights. We also demonstrated that multicellular spheroids may enable key hallmarks of tissue-based bioassays, including drug screening, tumor dissemination, cell co-culture, and tumor invasion. Taken together, these results offer new opportunities not only to achieve the active control of 3D multicellular spheroids on demand, but also to establish a rapid and cost-effective platform to study anti-cancer therapeutics and tumor microenvironments.

A Method to Preclude Moisture Condensation in Plated Tissue Cultures

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Alex M. Diner

1992-01-01

Excessive condensate normally accumulates in in vitro-illuminated petri dishes containing plant tissue cultures, causing avariety of problems. A dark-colored rubber net-mesh placed over the petri dishes prevented such condensation, even when charcoal-supplemented media are used under high light intensity in a growth chamber.

Cost-effective nutrient sources for tissue culture of cassava ( Manihot ...

African Journals Online (AJOL)

Application of tissue culture technology is constrained by high costs making seedlings unaffordable. The objective of this study was to evaluate the possibility of using locally available fertilizers as alternative nutrient sources for cassava micropropagation. A Low Cost Medium (LCM) whereby the conventional sources of four ...

GENETIC VARIABILITY OF CULTURED PLANT TISSUES UNDER NORMAL CONDITIONS AND UNDER STRESS

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Dolgikh Yu.I.

2012-08-01

Full Text Available The genetic variability induced by in vitro conditions known as somaclonal variation is of practical interest due to its potential uses in plant breeding but, on the other hand, if clonal propagation or transformation is main goal, it becomes an unwelcome phenomenon. Thus, it is important to know frequency, the genomic distribution, the mechanisms and factors influencing somaclonal variation. We studied variability of PCR-based DNA markers of cultured tissues and regenerated plants of maize and bread wheat. The original A188 line of maize and the somaclones obtained were tested using 38 RAPD and 10 ISSR primers. None of the A188 plants showed variation in the RAPD and ISSR spectra for any of the primers used. However, the PCR spectra obtained from the somaclones demonstrated some variations, i.e., 22 RAPD primers and 6 ISSR primers differentiated at least one somaclonal variant from the progenitor line. Six SCAR markers were developed based on several RAPD and ISSR fragments. The inheritance of these SCAR markers was verified in the selfing progeny of each somaclone in the R1–R4 generations and in the hybrids, with A188 as the parental line in the F1 and F2 generations. These markers were sequenced and bioinformatic searches were performed to understand the molecular events that may underlie the variability observed in the somaclones. All changes were found in noncoding sequences and were induced by different molecular events, such as the insertion of long terminal repeat transposon, precise miniature inverted repeat transposable element (MITE excision, microdeletion, recombination, and a change in the pool of mitochondrial DNA. In two groups of independently produced somaclones, the same features (morphological, molecular were variable, which confirms the theory of ‘hot spots’ occurring in the genome. The presence of the same molecular markers in the somaclones and in different non-somaclonal maize variants suggests that in some cases

Clonal expansion under the microscope: studying lymphocyte activation and differentiation using live-cell imaging.

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Polonsky, Michal; Chain, Benjamin; Friedman, Nir

2016-03-01

Clonal expansion of lymphocytes is a hallmark of vertebrate adaptive immunity. A small number of precursor cells that recognize a specific antigen proliferate into expanded clones, differentiate and acquire various effector and memory phenotypes, which promote effective immune responses. Recent studies establish a large degree of heterogeneity in the level of expansion and in cell state between and within expanding clones. Studying these processes in vivo, while providing insightful information on the level of heterogeneity, is challenging due to the complex microenvironment and the inability to continuously track individual cells over extended periods of time. Live cell imaging of ex vivo cultures within micro fabricated arrays provides an attractive methodology for studying clonal expansion. These experiments facilitate continuous acquisition of a large number of parameters on cell number, proliferation, death and differentiation state, with single-cell resolution on thousands of expanding clones that grow within controlled environments. Such data can reveal stochastic and instructive mechanisms that contribute to observed heterogeneity and elucidate the sequential order of differentiation events. Intercellular interactions can also be studied within these arrays by following responses of a controlled number of interacting cells, all trapped within the same microwell. Here we describe implementations of live-cell imaging within microwell arrays for studies of lymphocyte clonal expansion, portray insights already gained from these experiments and outline directions for future research. These tools, together with in vivo experiments tracking single-cell responses, will expand our understanding of adaptive immunity and the ways by which it can be manipulated.

Metabolic heterogeneity in clonal microbial populations.

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Takhaveev, Vakil; Heinemann, Matthias

2018-02-21

In the past decades, numerous instances of phenotypic diversity were observed in clonal microbial populations, particularly, on the gene expression level. Much less is, however, known about phenotypic differences that occur on the level of metabolism. This is likely explained by the fact that experimental tools probing metabolism of single cells are still at an early stage of development. Here, we review recent exciting discoveries that point out different causes for metabolic heterogeneity within clonal microbial populations. These causes range from ecological factors and cell-inherent dynamics in constant environments to molecular noise in gene expression that propagates into metabolism. Furthermore, we provide an overview of current methods to quantify the levels of metabolites and biomass components in single cells. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Clonal Clusters and Virulence Factors of Group C and G Streptococcus Causing Severe Infections, Manitoba, Canada, 2012-2014.

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Lother, Sylvain A; Demczuk, Walter; Martin, Irene; Mulvey, Michael; Dufault, Brenden; Lagacé-Wiens, Philippe; Keynan, Yoav

2017-07-01

The incidence of group C and G Streptococcus (GCGS) bacteremia, which is associated with severe disease and death, is increasing. We characterized clinical features, outcomes, and genetic determinants of GCGS bacteremia for 89 patients in Winnipeg, Manitoba, Canada, who had GCGS bacteremia during 2012-2014. Of the 89 patients, 51% had bacteremia from skin and soft tissue, 70% had severe disease features, and 20% died. Whole-genome sequencing analysis was performed on isolates derived from 89 blood samples and 33 respiratory sample controls: 5 closely related genetic lineages were identified as being more likely to cause invasive disease than non-clade isolates (83% vs. 57%, p = 0.002). Virulence factors cbp, fbp, speG, sicG, gfbA, and bca clustered clonally into these clades. A clonal distribution of virulence factors may account for severe and fatal cases of bacteremia caused by invasive GCGS.

The effect of plant growth regulators on optimization of tissue culture ...

African Journals Online (AJOL)

USER

2010-04-05

Apr 5, 2010 ... ISSN 1684–5315 © 2010 Academic Journals ... tissue culture system in Malaysian upland rice ... Scientists believe that using new cultivars which have potential ..... providing the financial support and to Firouzeh Ashjazadeh ...

The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin.

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Pellegrini, G; Ranno, R; Stracuzzi, G; Bondanza, S; Guerra, L; Zambruno, G; Micali, G; De Luca, M

1999-09-27

Cell therapy is an emerging therapeutic strategy aimed at replacing or repairing severely damaged tissues with cultured cells. Epidermal regeneration obtained with autologous cultured keratinocytes (cultured autografts) can be life-saving for patients suffering from massive full-thickness burns. However, the widespread use of cultured autografts has been hampered by poor clinical results that have been consistently reported by different burn units, even when cells were applied on properly prepared wound beds. This might arise from the depletion of epidermal stem cells (holoclones) in culture. Depletion of holoclones can occur because of (i) incorrect culture conditions, (ii) environmental damage of the exposed basal layer of cultured grafts, or (iii) use of new substrates or culture technologies not pretested for holoclone preservation. The aim of this study was to show that, if new keratinocyte culture technologies and/or "delivery systems" are proposed, a careful evaluation of epidermal stem cell preservation is essential for the clinical performance of this life-saving technology. Fibrin was chosen as a potential substrate for keratinocyte cultivation. Stem cells were monitored by clonal analysis using the culture system originally described by Rheinwald and Green as a reference. Massive full-thickness burns were treated with the composite allodermis/cultured autograft technique. We show that: (i) the relative percentage of holoclones, meroclones, and paraclones is maintained when keratinocytes are cultivated on fibrin, proving that fibrin does not induce clonal conversion and consequent loss of epidermal stem cells; (ii) the clonogenic ability, growth rate, and long-term proliferative potential are not affected by the new culture system; (iii) when fibrin-cultured autografts bearing stem cells are applied on massive full-thickness burns, the "take" of keratinocytes is high, reproducible, and permanent; and (iv) fibrin allows a significant reduction of the cost

Anaerobic Cultures from Preserved Tissues of Baby Mammoth

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Pikuta, Elena V.; Hoover, Richard B.; Fisher, Daniel

2011-01-01

Microbiological analysis of several cold-preserved tissue samples from the Siberian baby mammoth known as Lyuba revealed a number of culturable bacterial strains that were grown on anaerobic media at 4 C. Lactic acid produced by LAB (lactic acid bacteria) group, usually by members of the genera Carnobacterium and Lactosphera, appears to be a wonderful preservative that prevents other bacteria from over-dominating a system. Permafrost and lactic acid preserved the body of this one-month old baby mammoth and kept it in exceptionally good condition, resulting in this mammoth being the most complete such specimen ever recovered. The diversity of novel anaerobic isolates was expressed on morphological, physiological and phylogenetic levels. Here we discuss the specifics of the isolation of new strains, differentiation from trivial contamination, and preliminary results for the characterization of cultures.

The gene expression profile of non-cultured, highly purified human adipose tissue pericytes: Transcriptomic evidence that pericytes are stem cells in human adipose tissue

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Silva Meirelles, Lindolfo da, E-mail: lindolfomeirelles@gmail.com [Center for Cell-Based Therapy (CEPID/FAPESP), Regional Center for Hemotherapy of Ribeirão Preto, University of São Paulo, Rua Tenente Catão Roxo 2501, 14051-140 Ribeirão Preto, SP (Brazil); Laboratory for Stem Cells and Tissue Engineering, PPGBioSaúde, Lutheran University of Brazil, Av. Farroupilha 8001, 92425-900 Canoas, RS (Brazil); Deus Wagatsuma, Virgínia Mara de; Malta, Tathiane Maistro; Bonini Palma, Patrícia Viana [Center for Cell-Based Therapy (CEPID/FAPESP), Regional Center for Hemotherapy of Ribeirão Preto, University of São Paulo, Rua Tenente Catão Roxo 2501, 14051-140 Ribeirão Preto, SP (Brazil); Araújo, Amélia Goes; Panepucci, Rodrigo Alexandre [Laboratory of Large-Scale Functional Biology (LLSFBio), Regional Center for Hemotherapy of Ribeirão Preto, University of São Paulo, Rua Tenente Catão Roxo 2501, 14051-140 Ribeirão Preto, SP (Brazil); and others

2016-12-10

Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with an AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology. - Highlights: • Non-cultured adipose tissue-derived human pericytes (ncATPCs) exhibit a distinctive gene expression signature. • ncATPCs express key adipose tissue stem cell genes previously described in vivo in mice. • ncATPCs express message for anti-proliferative and antiangiogenic molecules. • Most ncATPC-specific transcripts are absent in culture-expanded pericytes or ATMSCs • Gene expression changes ncATPCs undergo as they acquire a cultured ATMSC phenotype are pointed out.

The gene expression profile of non-cultured, highly purified human adipose tissue pericytes: Transcriptomic evidence that pericytes are stem cells in human adipose tissue

International Nuclear Information System (INIS)

Silva Meirelles, Lindolfo da; Deus Wagatsuma, Virgínia Mara de; Malta, Tathiane Maistro; Bonini Palma, Patrícia Viana; Araújo, Amélia Goes; Panepucci, Rodrigo Alexandre

2016-01-01

Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with an AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology. - Highlights: • Non-cultured adipose tissue-derived human pericytes (ncATPCs) exhibit a distinctive gene expression signature. • ncATPCs express key adipose tissue stem cell genes previously described in vivo in mice. • ncATPCs express message for anti-proliferative and antiangiogenic molecules. • Most ncATPC-specific transcripts are absent in culture-expanded pericytes or ATMSCs • Gene expression changes ncATPCs undergo as they acquire a cultured ATMSC phenotype are pointed out.

Clonal integration facilitates spread of Paspalum paspaloides from terrestrial to cadmium-contaminated aquatic habitats.

Science.gov (United States)

Luo, F-L; Xing, Y-P; Wei, G-W; Li, C-Y; Yu, F-H

2017-11-01

Cadmium (Cd) is a hazardous environmental pollutant with high toxicity to plants, which has been detected in many wetlands. Clonal integration (resource translocation) between connected ramets of clonal plants can increase their tolerance to stress. We hypothesised that clonal integration facilitates spread of amphibious clonal plants from terrestrial to Cd-contaminated aquatic habitats. The spread of an amphibious grass Paspalum paspaloides was simulated by growing basal older ramets in uncontaminated soil connected (allowing integration) or not connected (preventing integration) to apical younger ramets of the same fragments in Cd-contaminated water. Cd contamination of apical ramets of P. paspaloides markedly decreased growth and photosynthetic capacity of the apical ramets without connection to the basal ramets, but did not decrease these properties with connection. Cd contamination did not affect growth of the basal ramets without connection to the apical ramets, but Cd contamination of 4 and 12 mg·l -1 significantly increased growth with connection. Consequently, clonal integration increased growth of the apical ramets, basal ramets and whole clones when the apical ramets were grown in Cd-contaminated water of 4 and 12 mg·l -1 . Cd was detected in the basal ramets with connection to the apical ramets, suggesting Cd could be translocated due to clonal integration. Clonal integration, most likely through translocation of photosynthates, can support P. paspaloides to spread from terrestrial to Cd-contaminated aquatic habitats. Amphibious clonal plants with a high ability for clonal integration are particularly useful for re-vegetation of degraded aquatic habitats caused by Cd contamination. © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.

Application of Tissue Culture and Transformation Techniques in Model Species Brachypodium distachyon.

Science.gov (United States)

Sogutmaz Ozdemir, Bahar; Budak, Hikmet

2018-01-01

Brachypodium distachyon has recently emerged as a model plant species for the grass family (Poaceae) that includes major cereal crops and forage grasses. One of the important traits of a model species is its capacity to be transformed and ease of growing both in tissue culture and in greenhouse conditions. Hence, plant transformation technology is crucial for improvements in agricultural studies, both for the study of new genes and in the production of new transgenic plant species. In this chapter, we review an efficient tissue culture and two different transformation systems for Brachypodium using most commonly preferred gene transfer techniques in plant species, microprojectile bombardment method (biolistics) and Agrobacterium-mediated transformation.In plant transformation studies, frequently used explant materials are immature embryos due to their higher transformation efficiencies and regeneration capacity. However, mature embryos are available throughout the year in contrast to immature embryos. We explain a tissue culture protocol for Brachypodium using mature embryos with the selected inbred lines from our collection. Embryogenic calluses obtained from mature embryos are used to transform Brachypodium with both plant transformation techniques that are revised according to previously studied protocols applied in the grasses, such as applying vacuum infiltration, different wounding effects, modification in inoculation and cocultivation steps or optimization of bombardment parameters.

In vitro differentiation of rat spermatogonia into round spermatids in tissue culture.

Science.gov (United States)

Reda, A; Hou, M; Winton, T R; Chapin, R E; Söder, O; Stukenborg, J-B

2016-09-01

Do the organ culture conditions, previously defined for in vitro murine male germ cell differentiation, also result in differentiation of rat spermatogonia into post-meiotic germ cells exhibiting specific markers for haploid germ cells? We demonstrated the differentiation of rat spermatogonia into post-meiotic cells in vitro, with emphasis on exhibiting, protein markers described for round spermatids. Full spermatogenesis in vitro from immature germ cells using an organ culture technique in mice was first reported 5 years ago. However, no studies reporting the differentiation of rat spermatogonia into post-meiotic germ cells exhibiting the characteristic protein expression profile or into functional sperm have been reported. Organ culture of testicular fragments of 5 days postpartum (dpp) neonatal rats was performed for up to 52 days. Evaluation of microscopic morphology, testosterone levels, mRNA and protein expression as measured by RT-qPCR and immunostaining were conducted to monitor germ cell differentiation in vitro. Potential effects of melatonin, Glutamax® medium, retinoic acid and the presence of epidydimal fat tissue on the spermatogenic process were evaluated. A minimum of three biological replicates were performed for all experiments presented in this study. One-way ANOVA, ANOVA on ranks and student's t-test were applied to perform the statistical analysis. Male germ cells, present in testicular tissue pieces grown from 5 dpp rats, exhibited positive protein expression for Acrosin and Crem (cAMP (cyclic adenosine mono phosphate) response element modulator) after 52 days of culture in vitro. Intra-testicular testosterone production could be observed after 3 days of culture, while when epididymal fat tissue was added, spontaneous contractility of cultured seminiferous tubules could be observed after 21 days. However, no supportive effect of the supplementation with any factor or the co-culturing with epididymal fat tissue on germ cell differentiation in

Spread of clonally related Escherichia coli harboring an IncA/C1 plasmid encoding IMP-8 and its recruitment into an unrelated MCR-1-containing isolate.

Science.gov (United States)

Elena, Alan; Cejas, Daniela; Magariños, Francisco; Jewtuchowicz, Virginia; Facente, Andrea; Gutkind, Gabriel; Di Conza, José; Radice, Marcela

2018-04-16

Ten IMP-8-producing Escherichia coli isolates were recovered from the surveillance cultures of a neonatal intensive care unit, of which eight were clonally related. A 168.2-kb- bla IMP-8 plasmid was fully sequenced, and it corresponded to the recently described IncA/C1-ST13. This plasmid was detected in all isolates, even in those no clonally related. One unrelated isolate was also resistant to colistin and positive for mcr-1. This marker was located in a 62.7-kb-IncI2 plasmid, which was also fully sequenced. Copyright © 2018 American Society for Microbiology.

Beating the odds: The poisson distribution of all input cells during limiting dilution grossly underestimates whether a cell line is clonally-derived or not.

Science.gov (United States)

Zhou, Yizhou; Shaw, David; Lam, Cynthia; Tsukuda, Joni; Yim, Mandy; Tang, Danming; Louie, Salina; Laird, Michael W; Snedecor, Brad; Misaghi, Shahram

2017-09-23

Establishing that a cell line was derived from a single cell progenitor and defined as clonally-derived for the production of clinical and commercial therapeutic protein drugs has been the subject of increased emphasis in cell line development (CLD). Several regulatory agencies have expressed that the prospective probability of clonality for CHO cell lines is assumed to follow the Poisson distribution based on the input cell count. The probability of obtaining monoclonal progenitors based on the Poisson distribution of all cells suggests that one round of limiting dilution may not be sufficient to assure the resulting cell lines are clonally-derived. We experimentally analyzed clonal derivatives originating from single cell cloning (SCC) via one round of limiting dilution, following our standard legacy cell line development practice. Two cell populations with stably integrated DNA spacers were mixed and subjected to SCC via limiting dilution. Cells were cultured in the presence of selection agent, screened, and ranked based on product titer. Post-SCC, the growing cell lines were screened by PCR analysis for the presence of identifying spacers. We observed that the percentage of nonclonal populations was below 9%, which is considerably lower than the determined probability based on the Poisson distribution of all cells. These results were further confirmed using fluorescence imaging of clonal derivatives originating from SCC via limiting dilution of mixed cell populations expressing GFP or RFP. Our results demonstrate that in the presence of selection agent, the Poisson distribution of all cells clearly underestimates the probability of obtaining clonally-derived cell lines. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017. © 2017 American Institute of Chemical Engineers.

Clonal diversity analysis using SNP microarray: a new prognostic tool for chronic lymphocytic leukemia.

Science.gov (United States)

Zhang, Linsheng; Znoyko, Iya; Costa, Luciano J; Conlin, Laura K; Daber, Robert D; Self, Sally E; Wolff, Daynna J

2011-12-01

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. The methods currently used for monitoring CLL and determining conditions for treatment are limited in their ability to predict disease progression, patient survival, and response to therapy. Although clonal diversity and the acquisition of new chromosomal abnormalities during the disease course (clonal evolution) have been associated with disease progression, their prognostic potential has been underappreciated because cytogenetic and fluorescence in situ hybridization (FISH) studies have a restricted ability to detect genomic abnormalities and clonal evolution. We hypothesized that whole genome analysis using high resolution single nucleotide polymorphism (SNP) microarrays would be useful to detect diversity and infer clonal evolution to offer prognostic information. In this study, we used the Infinium Omni1 BeadChip (Illumina, San Diego, CA) array for the analysis of genetic variation and percent mosaicism in 25 non-selected CLL patients to explore the prognostic value of the assessment of clonal diversity in patients with CLL. We calculated the percentage of mosaicism for each abnormality by applying a mathematical algorithm to the genotype frequency data and by manual determination using the Simulated DNA Copy Number (SiDCoN) tool, which was developed from a computer model of mosaicism. At least one genetic abnormality was identified in each case, and the SNP data was 98% concordant with FISH results. Clonal diversity, defined as the presence of two or more genetic abnormalities with differing percentages of mosaicism, was observed in 12 patients (48%), and the diversity correlated with the disease stage. Clonal diversity was present in most cases of advanced disease (Rai stages III and IV) or those with previous treatment, whereas 9 of 13 patients without detected clonal diversity were asymptomatic or clinically stable. In conclusion, SNP microarray studies with simultaneous evaluation

[Effective productions of plant secondary metabolites having antitumor activity by plant cell and tissue cultures].

Science.gov (United States)

Taniguchi, Shoko

2005-06-01

Methods for the effective production of plant secondary metabolites with antitumor activity using plant cell and tissue cultures were developed. The factors in tannin productivity were investigated using culture strains producing different types of hydrolyzable tannins, i.e., gallotannins (mixture of galloylglucoses), ellagi-, and dehydroellagitannins. Production of ellagi- and dehydroellagitannins was affected by the concentrations and ratio of nitrogen sources in the medium. The formation of oligomeric ellagitannins in shoots of Oenothera tetraptera was correlated with the differentiation of tissues. Cultured cells of Eriobotrya japonica producing ursane- and oleanane-type triterpenes with antitumor activities were also established.

Clonal proliferation of multipotent stem/progenitor cells in the neonatal and adult salivary glands

International Nuclear Information System (INIS)

Kishi, Teruki; Takao, Tukasa; Fujita, Kiyohide; Taniguchi, Hideki

2006-01-01

Salivary gland stem/progenitor cells are thought to be present in intercalated ductal cells, but the fact is unclear. In this study, we sought to clarify if stem/progenitor cells are present in submandibular glands using colony assay, which is one of the stem cell assay methods. Using a low-density culture of submandibular gland cells of neonatal rats, we developed a novel culture system that promotes single cell colony formation. Average doubling time for the colony-forming cells was 24.7 (SD = ±7.02) h, indicating high proliferative potency. When epidermal growth factor (EGF) and hepatocyte growth factor (HGF) were added to the medium, the number of clonal colonies increased greater than those cultured without growth factors (13.2 ± 4.18 vs. 4.5 ± 1.73). The RT-PCR and immunostaining demonstrated expressing acinar, ductal, and myoepithelial cell lineage markers. This study demonstrated the presence of the salivary gland stem/progenitor cells that are highly proliferative and multipotent in salivary glands

Tissue culture-induced alteration in cytosine methylation in new rice ...

African Journals Online (AJOL)

Zizania DNA introgression could induce a large number of genetic and epigenetic changes of the new rice recombinant inbred lines genome. In this present study, we employed inter-simple sequence repeat (ISSR) to further study the genetic and epigenetic changes that are induced by tissue culture. Changes induced by ...

Cells in human postmortem brain tissue slices remain alive for several weeks in culture

NARCIS (Netherlands)

Verwer, Ronald W. H.; Hermens, Wim T. J. M. C.; Dijkhuizen, PaulaA; ter Brake, Olivier; Baker, Robert E.; Salehi, Ahmad; Sluiter, Arja A.; Kok, Marloes J. M.; Muller, Linda J.; Verhaagen, Joost; Swaab, Dick F.

2002-01-01

Animal models for human neurological and psychiatric diseases only partially mimic the underlying pathogenic processes. Therefore, we investigated the potential use of cultured postmortem brain tissue from adult neurological patients and controls. The present study shows that human brain tissue

Citrus tissue culture employing vegetative explants.

Science.gov (United States)

Chaturvedi, H C; Singh, S K; Sharma, A K; Agnihotri, S

2001-11-01

Citrus being a number one fruit of the world due to its high nutritional value, huge production of fruits and fruit products, the citrus industry may be considered a major fruit industry. Though citrus orchard area in India is comparable to USA, the produce is far less, while its export is nil. Biotechnology has played an outstanding role in boosting the citrus industry, e.g., in Spain, which is now the biggest exporter of citrus fruit with the application of micrografting. Amongst the fruit trees, perhaps the maximum tissue culture research has been done in citrus during the past four decades, however, the results of practical value are meagre. The shortfalls in citrus tissue culture research and some advancements made in this direction along with bright prospects are highlighted, restricting the review to vegetative explants only. Whilst utilization of nucellar embryogenesis is limited to rootstocks, the other aspects, like, regeneration and proliferation of shoot meristems measuring 200 microm in length--a global breakthrough--of two commercially important scion species, Citrus aurantifolia and C. sinensis and an important rootstock, C. limonia, improvement of micrografting technique, cloning of the same two scion species as well as some Indian rootstock species, employing nodal stem segments of mature trees, of immense practical value have been elaborated. A rare phenomenon of shift in the morphogenetic pattern of differentiation from shoot bud differentiation to embryoid formation occurred during the long-term culture of stem callus of C. grandis. Stem callus-regenerated plants of C. aurantifolia, C. sinensis and C. grandis showed variation in their ploidy levels and a somaclonal variant of C. sinensis, which produced seedless fruits was isolated. Tailoring of rooting in microshoots to a tap root-like system by changing the inorganic salt composition of the rooting medium, resulting in 100% transplant success, and germplasm preservation through normal growth

Analysis of allelic expression patterns in clonal somatic cells by single-cell RNA-seq.

Science.gov (United States)

Reinius, Björn; Mold, Jeff E; Ramsköld, Daniel; Deng, Qiaolin; Johnsson, Per; Michaëlsson, Jakob; Frisén, Jonas; Sandberg, Rickard

2016-11-01

Cellular heterogeneity can emerge from the expression of only one parental allele. However, it has remained controversial whether, or to what degree, random monoallelic expression of autosomal genes (aRME) is mitotically inherited (clonal) or stochastic (dynamic) in somatic cells, particularly in vivo. Here we used allele-sensitive single-cell RNA-seq on clonal primary mouse fibroblasts and freshly isolated human CD8 + T cells to dissect clonal and dynamic monoallelic expression patterns. Dynamic aRME affected a considerable portion of the cells' transcriptomes, with levels dependent on the cells' transcriptional activity. Notably, clonal aRME was detected, but it was surprisingly scarce (aRME occurs transiently within individual cells, and patterns of aRME are thus primarily scattered throughout somatic cell populations rather than, as previously hypothesized, confined to patches of clonally related cells.

Advanced cell culture technology for generation of in vivo-like tissue models

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Stefan Przyborski

2017-06-01

Full Text Available Human tissues are mostly composed of different cell types, that are often highly organised in relation to each other. Often cells are arranged in distinct layers that enable signalling and cell-to-cell interactions. Here we describe the application of scaffold-based technology, that can be used to create advanced organotypic 3D models of various tissue types that more closely resemble in vivo-like conditions (Knight et al., 2011. The scaffold comprises a highly porous polystyrene material, engineered into a 200 micron thick membrane that is presented in various ways including multi-welled plates and well inserts, for use with conventional culture plasticware and medium perfusion systems. This technology has been applied to generate numerous unique types of co-culture model. For example: 1 a full thickness human skin construct comprising dermal fibroblasts and keratinocytes, raised to the air-liquid interface to induce cornification of the upper layers (Fig.1 (Hill et al., 2015; 2 a neuron-glial co-culture to enable the study of neurite outgrowth interacting with astroglial cells to model and investigate the glial scar found in spinal cord injury (Clarke et al., 2016; 3 formation of a sub-mucosa consisting of a polarised simple epithelium, layer of ECM proteins simulating the basement membrane, and underlying stromal tissues (e.g. intestinal mucosa. These organotypic models demonstrate the versatility of scaffold membranes and the creation of advanced in vivo-like tissue models. Creating a layered arrangement more closely simulates the true anatomy and organisation of cells within many tissue types. The addition of different cell types in a temporal and spatial fashion can be used to study inter-cellular relationships and create more physiologically relevant in vivo-like cell-based assays. Methods that are relatively straightforward to use and that recreate the organised structure of real tissues will become valuable research tools for use in

Canine osteosarcoma karyotypes from an original tumor, its metastasis, and tumor cells in tissue culture

International Nuclear Information System (INIS)

Taylor, N.; Shifrine, M.; Wolf, H.G.; Trommershausen-Smith, A.

1975-01-01

Radiation-induced osteosarcoma, its metastasis, and cells grown in tissue culture were karyotyped. Both hypodiploid and hyperdiploid stem lines were observed. The hypodiploid line contained 45-55 chromosomes with 10 to 15 abnormal metacentric and submetacentric chromosomes and one subtelocentric marker. The hyperdiploid line contained 90 to 105 chromosomes with 20 to 30 abnormal metacentric and submetacentric chromosomes with two subtelocentric markers. Karyotypic analysis can be used to monitor osteosarcomas maintained in tissue culture

Tissue culture of three species of Laurencia complex

Science.gov (United States)

Shen, Songdong; Wu, Xunjian; Yan, Binlun; He, Lihong

2010-05-01

To establish a micropropagation system of three Laurencia complex species ( Laurencia okamurai, Laurencia tristicha, and Chondrophycus undulatus) by tissue culture techniques, we studied the regeneration characteristics and optimal culture conditions of axenic algal fragments cultured on solid medium and in liquid medium. Regeneration structures were observed and counted regularly under a reverse microscope to investigate the regeneration process, polarity and optimal illumination, and temperature and salinity levels. The results show that in most cultures of the three species, we obtained bud regeneration on solidified medium with 0.5% agar and in liquid medium. Rhizoid-like regeneration was filamentous and developed from the lower cut surface of fragments in L. okamurai, but was discoid and developed from the apical back side of bud regeneration in L. tristicha and C. undulatus. Regeneration polarity was localized to the apical part of algal fronds in all three species, and on fragments cut from the basal part of algae buds could develop from both the upper and the lower cut surfaces. Buds could develop from both the medullary and the cortical portions in L. okamurai and C. undulatus, while in L. tristicha, buds only emerged from the cortex. The optimal culture conditions for L. okamurai were 4 500 lx, 20°C and 35 (salinity); for C. undulatus, 4 500 lx, 20°C and 30; and for L. tristicha, 4 500 lx, 25°C and 30.

Low cost options for tissue culture technology in developing countries. Proceedings of a technical meeting

Energy Technology Data Exchange (ETDEWEB)

NONE

2004-02-01

Tissue culture technology is used for the production of doubled haploids, cryopreservation, propagating new plant varieties, conserving rare and endangered plants, difficult-to-propagate plants, and to produce secondary metabolites and transgenic plants. The production of high quality planting material of crop plants and fruit trees, propagated from vegetative parts, has created new opportunities in global trading, benefited growers, farmers, and nursery owners, and improved rural employment. However, there are still major opportunities to produce and distribute high quality planting material, e.g. crops like banana, date palm, cassava, pineapple, plantain, potato, sugarcane, sweet potato, yams, ornamentals, fruit and forest trees. The main advantage of tissue culture technology lies in the production of high quality and uniform planting material that can be multiplied on a year-round basis under disease-free conditions anywhere irrespective of the season and weather. However, the technology is capital, labor and energy intensive. Although, labor is cheap in many developing countries, the resources of trained personnel and equipment are often not readily available. In addition, energy, particularly electricity, and clean water are costly. The energy requirements for tissue culture technology depend on day temperature, day-length and relative humidity, and they have to be controlled during the process of propagation. Individual plant species also differ in their growth requirements. Hence, it is necessary to have low cost options for weaning, hardening of micropropagated plants and finally growing them in the field. This publication describes options for reducing costs to establish and operate tissue culture facilities and primarily focus on plant micropropagation. It includes papers on the basics of tissue culture technology, low cost options for the design of laboratories, use of culture media and containers, energy and labor saving, integration and adoption of

Low cost options for tissue culture technology in developing countries. Proceedings of a technical meeting

International Nuclear Information System (INIS)

2004-02-01

Tissue culture technology is used for the production of doubled haploids, cryopreservation, propagating new plant varieties, conserving rare and endangered plants, difficult-to-propagate plants, and to produce secondary metabolites and transgenic plants. The production of high quality planting material of crop plants and fruit trees, propagated from vegetative parts, has created new opportunities in global trading, benefited growers, farmers, and nursery owners, and improved rural employment. However, there are still major opportunities to produce and distribute high quality planting material, e.g. crops like banana, date palm, cassava, pineapple, plantain, potato, sugarcane, sweet potato, yams, ornamentals, fruit and forest trees. The main advantage of tissue culture technology lies in the production of high quality and uniform planting material that can be multiplied on a year-round basis under disease-free conditions anywhere irrespective of the season and weather. However, the technology is capital, labor and energy intensive. Although, labor is cheap in many developing countries, the resources of trained personnel and equipment are often not readily available. In addition, energy, particularly electricity, and clean water are costly. The energy requirements for tissue culture technology depend on day temperature, day-length and relative humidity, and they have to be controlled during the process of propagation. Individual plant species also differ in their growth requirements. Hence, it is necessary to have low cost options for weaning, hardening of micropropagated plants and finally growing them in the field. This publication describes options for reducing costs to establish and operate tissue culture facilities and primarily focus on plant micropropagation. It includes papers on the basics of tissue culture technology, low cost options for the design of laboratories, use of culture media and containers, energy and labor saving, integration and adoption of

HIV genetic information and clonal growth

Science.gov (United States)

Based on an analysis of blood cells from five HIV-infected individuals, NCI researchers have identified more than 2,400 HIV DNA insertion sites. Analysis of these sites showed that there is extensive clonal expansion (growth) of HIV infected cells.

Tissue culture of osteogenic sarcoma in rats, induced by radioactive phosphorus P-32 and the effect of the anti-cancerous agents on these tumor cells under tissue culture

International Nuclear Information System (INIS)

Osaka, Shunzo

1976-01-01

Small pieces of osteogenic sarcoma, induced into albino rats of the C.F. Wistar strain by injection of radioactive phosphorus 32 P, were cultured in mixtures of Eagle's minimum essential medium and 20% calf serum. The tumor cells cultured in this way were transplanted into the subcutaneous tissue or the intraabdominal cavity to healthy albino rats. The effect of the anticancerous agents was evaluated by the decrease of nucleic acid composition in these cultured tumor cells. As anti-cancerous agents, cyclophosphamide (CPA), mitomycin C(MMC), and 5-fluorouracil(5-FU) were put into contact with the tumor cells in cultures for two hours under the following dilutions: CPA; 10 -6 , 10 -5 , 10 -4 g/ml. MMC; 2 x 10 -8 , 2 x 10 -7 , 2 x 10 -6 g/ml. 5-FU; 2 x 10 -6 , 2 x 10 -5 , 2 x 10 -4 g/ml. The results are as follows: Three of the seven osteogenic sarcomas in rats were successfully cultured, one of them through more than eighteen generations. After about five hundred thousand cultured cells had been transplanted into the subcutaneous tissues or abdominal cavities of rats, tumors grew in all of them. The histological findings of the tumors in the second generation were quite similar to those of the original tumor. The same process was repeated three times and the tumor showed histogical findings similar to those of the original ones. The capability of nucleic acid synthesis in these cells was decreased at twenty fours after CPA contact and at forty eight hours after MMC. (J.P.N.)

Whole genome characterization of non-tissue culture adapted HRSV strains in severely infected children

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Kumaria Rajni

2011-07-01

Full Text Available Abstract Background Human respiratory syncytial virus (HRSV is the most important virus causing lower respiratory infection in young children. The complete genetic characterization of RSV clinical strains is a prerequisite for understanding HRSV infection in the clinical context. Current information about the genetic structure of the HRSV genome has largely been obtained using tissue culture adapted viruses. During tissue culture adaptation genetic changes can be introduced into the virus genome, which may obscure subtle variations in the genetic structure of different RSV strains. Methods In this study we describe a novel Sanger sequencing strategy which allowed the complete genetic characterisation of 14 clinical HRSV strains. The viruses were sequenced directly in the nasal washes of severely hospitalized children, and without prior passage of the viruses in tissue culture. Results The analysis of nucleotide sequences suggested that vRNA length is a variable factor among primary strains, while the phylogenetic analysis suggests selective pressure for change. The G gene showed the greatest sequence variation (2-6.4%, while small hydrophobic protein and matrix genes were completely conserved across all clinical strains studied. A number of sequence changes in the F, L, M2-1 and M2-2 genes were observed that have not been described in laboratory isolates. The gene junction regions showed more sequence variability, and in particular the intergenic regions showed a highest level of sequence variation. Although the clinical strains grew slower than the HRSVA2 virus isolate in tissue culture, the HRSVA2 isolate and clinical strains formed similar virus structures such as virus filaments and inclusion bodies in infected cells; supporting the clinical relevance of these virus structures. Conclusion This is the first report to describe the complete genetic characterization of HRSV clinical strains that have been sequenced directly from clinical

Diagnostic utility of melanin production by fungi: Study on tissue sections and culture smears with Masson-Fontana stain

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Challa Sundaram

2014-01-01

Full Text Available Background: Dematiaceous fungi appear brown in tissue section due to melanin in their cell walls. When the brown color is not seen on routine H and E and culture is not available, differentiation of dematiaceous fungi from other fungi is difficult on morphology alone. Aims and Objective: To study if melanin production by dematiaceous fungi can help differentiate them from other types of fungi. Materials and Methods: Fifty tissue sections of various fungal infections and 13 smears from cultures of different species of fungi were stained with Masson Fontana stain to assess melanin production. The tissue sections included biopsies from 26 culture-proven fungi and 24 biopsies of filamentous fungi diagnosed on morphology alone with no culture confirmation. Results: All culture-proven dematiaceous fungi and Zygomycetes showed strong positivity in sections and culture smears. Aspergillus sp showed variable positivity and intensity. Cryptococcus neoformans showed strong positivity in tissue sections and culture smears. Tissue sections of septate filamentous fungi (9/15, Zygomycetes (4/5, and fungi with both hyphal and yeast morphology (4/4 showed positivity for melanin. The septate filamentous fungi negative for melanin were from biopsy samples of fungal sinusitis including both allergic and invasive fungal sinusitis and colonizing fungal balls. Conclusion: Melanin is produced by both dematiaceous and non-dematiaceous fungi. Masson-Fontana stain cannot reliably differentiate dematiaceous fungi from other filamentous fungi like Aspergillus sp; however, absence of melanin in the hyphae may be used to rule out dematiaceous fungi from other filamentous fungi. In the differential diagnosis of yeast fungi, Cryptococcus sp can be differentiated from Candida sp by Masson-Fontana stain in tissue sections.

Clonal Spread in Second Growth Stands of Coast Redwood, Sequoia sempervirens

Science.gov (United States)

Vladimir Douhovnikoff; Richard S. Dodd

2007-01-01

Coast redwood (Sequoia sempervirens) is one of the rare conifers to reproduce successfully through clonal spread. The importance of this mode of reproduction in stand development is largely unknown. Understanding the importance of clonal spread and the spatial structure of clones is crucial for stand management strategies that would aim to maximize...

Comparison of radiosensitivity between tumor and normal tissue in terms of cell population kinetics

International Nuclear Information System (INIS)

Sugahara, Tsutomu; Utsumi, Hiroshi

1975-01-01

Puck and Marcus in 1956 established the in vitro colony formation of mammalian cells and demonstrated a dose-survival curve of mammalian cells well fitted to the target theory. Since then almost all of the work on the radiosensitivity of malignant and normal cells has been based on the reproductive integrity of cells. However, in the author's laboratory, a recent work was done on the effect of ionizing radiation on the differentiative trait, using clonal cell cultures developed by Coon (1966) in chick embryonic cartilage cells. This work demonstrated clearly that the differentiative trait is more radiosensitive than is reproduction. Based on this finding a new compartment model is proposed for a cell renewal system which demonstrates the difference between normal and malignant tissue. (author)

Tobacco clones derived from tissue culture with supersensitivity to ozone

International Nuclear Information System (INIS)

Sun, E.J.; Kang, H.W.

2003-01-01

New tobacco clones supersensitive to ozone were obtained from tissue culture. - At least two supersensitive tobacco somaclones were obtained from tissue culture (TC) , when this approach was used to asexually propagate Bel-W3 tobacco indicator plants. These somaclones can detect as low as 30 ppb ozone for a 4-h exposure duration both within CSTR exposure chambers and in ambient air. Comparison of the injury index and their coefficient of variance showed that the TC plantlets usually have more uniform performance in response to ozone in addition to their higher sensitivity. A quick regeneration procedure was established to preserve the supersensitive germplasm immediately when it was found. The TC plantlets will flower and produce seed similar to seed-grown tobacco. The TC approach proved to be a better propagation system for valuable indicator plant species. The mechanism that causes the variation and the possible difference in their genome from seed-grown tobacco is still unknown. Further studies are needed in the future to determine if factors in the TC system may be responsible for the sensitivity difference

EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations

NARCIS (Netherlands)

Langerak, A. W.; Groenen, P. J. T. A.; Brüggemann, M.; Beldjord, K.; Bellan, C.; Bonello, L.; Boone, E.; Carter, G. I.; Catherwood, M.; Davi, F.; Delfau-Larue, M.-H.; Diss, T.; Evans, P. A. S.; Gameiro, P.; Garcia Sanz, R.; Gonzalez, D.; Grand, D.; Håkansson, A.; Hummel, M.; Liu, H.; Lombardia, L.; Macintyre, E. A.; Milner, B. J.; Montes-Moreno, S.; Schuuring, E.; Spaargaren, M.; Hodges, E.; van Dongen, J. J. M.

2012-01-01

PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to

Organ and plantlet regeneration of Menyanthes trifoliata through tissue culture

Directory of Open Access Journals (Sweden)

Urszula Adamczyk-Rogozińska

2014-01-01

Full Text Available The conditions for the regeneration of plants through organogenesis from callus tissues of Menyanthes trifoliata are described. The shoot multiplication rate was affected by basal culture media, the type and concentration of cytokinin and subculture number. The best response was obtained when caulogenic calli were cultured on the modified Schenk and Hildebrandt medium (SH-M containing indole-3-acetic acid (IAA 0,5 mg/l and 6-benzyladenine (BA 1 mg/l or zeatin (2 mg/l. Under these conditions ca 7 shoots (mostly 1 cm or more in length per culture in the 5th and 6th passages could be developed. In older cultures (after 11-12 passages there was a trend for more numerous but shorter shoot formation. All regenerated shoots could be rooted on the SH-M medium supplemented with 0.5 mg/l IAA within 6 weeks; 80% of in vitro rooted plantlets survived their transfer to soil.

Screening of clonal chromosome aberrations present in A-bomb survivors by FISH method

International Nuclear Information System (INIS)

Nakano, Mimako; Kodama, Yoshiaki; Ito, Masahiro; Otaki, Kazuo; Nakamura, Nori

1997-01-01

Significance of FISH method for detection of clonal chromosome aberration was reviewed. A clonal chromosome aberration is derived from one abnormal cell clone and gives the influence on the frequency of the aberration. As well, the size and frequency of the aberration give an important information concerning lymphocyte kinetics. FISH method is meaningful for detection of the clonal aberration. Fifteen kinds of clonal aberrations were detected in A-bomb survivors, of which 10 were specifically detected by the method, indicating that its detection rate was 2-3 time as high as the ordinary method. The results were those on the DNA probe on no.1, no.2 and no.3 chromosomes, which consisting of about 23% of the genome. (K.H.)

Rhizome elongation and seagrass clonal growth

NARCIS (Netherlands)

Marbà, N.; Duarte, C.M.

1998-01-01

A compilation of published and original data on rhizome morphometry, horizontal and vertical elongation rates and branching patterns for 27 seagrass species developing in 192 seagrass stands allowed an examination of the variability of seagrass rhizome and clonal growth programmes across and within

Drug hypersensitivity in clonal mast cell disorders

DEFF Research Database (Denmark)

Bonadonna, P; Pagani, M; Aberer, W

2015-01-01

and severity of immediate hypersensitivity reactions. Mastocytosis in adults is associated with a history of anaphylaxis in 22-49%. Fatal anaphylaxis has been described particularly following hymenoptera stings, but also occasionally after the intake of drugs such as nonsteroidal anti-inflammatory drugs......, opioids and drugs in the perioperative setting. However, data on the frequency of drug hypersensitivity in mastocytosis and vice versa are scarce and evidence for an association appears to be limited. Nevertheless, clonal MC disorders should be ruled out in cases of severe anaphylaxis: basal serum...... tryptase determination, physical examination for cutaneous mastocytosis lesions, and clinical characteristics of anaphylactic reaction might be useful for differential diagnosis. In this position paper, the ENDA group performed a literature search on immediate drug hypersensitivity reactions in clonal MC...

Clonal relatedness between lobular carcinoma in situ and synchronous malignant lesions

Science.gov (United States)

2012-01-01

Introduction Lobular carcinoma in situ (LCIS) has been accepted as a marker of risk for the development of invasive breast cancer, yet modern models of breast carcinogenesis include LCIS as a precursor of low-grade carcinomas. We provide evidence favoring a clonal origin for LCIS and synchronous estrogen receptor-positive malignant lesions of the ductal and lobular phenotype. Methods Patients with prior LCIS undergoing mastectomy were identified preoperatively from 2003 to 2008. Specimens were widely sampled, and frozen blocks were screened for LCIS and co-existing malignant lesions, and were subject to microdissection. Samples from 65 patients were hybridized to the Affymetrix SNP 6.0 array platform. Cases with both an LCIS sample and an associated ductal carcinoma in situ (DCIS) or invasive tumor sample were evaluated for patterns of somatic copy number changes to assess evidence of clonal relatedness. Results LCIS was identified in 44 of the cases, and among these a DCIS and/or invasive lesion was also identified in 21 cases. A total of 17 tumor pairs had adequate DNA/array data for analysis, including nine pairs of LCIS/invasive lobular cancer, four pairs of LCIS/DCIS, and four pairs of LCIS/invasive ductal cancer. Overall, seven pairs (41%) were judged to be clonally related; in five (29%) evidence suggested clonality but was equivocal, and five (29%) were considered independent. Clonal pairs were observed with all matched lesion types and low and high histological grades. We also show anecdotal evidence of clonality between a patient-matched triplet of LCIS, DCIS, and invasive ductal cancer. Conclusion Our results support the role of LCIS as a precursor in the development of both high-grade and low-grade ductal and lobular cancers. PMID:22776144

Treatment of chronic desquamative gingivitis using tissue-engineered human cultured gingival epithelial sheets: a case report.

Science.gov (United States)

Okuda, Kazuhiro; Momose, Manabu; Murata, Masashi; Saito, Yoshinori; lnoie, Masukazu; Shinohara, Chikara; Wolff, Larry F; Yoshie, Hiromasa

2004-04-01

Human cultured gingival epithelial sheets were used as an autologous grafting material for regenerating gingival tissue in the maxillary left and mandibular right quadrants of a patient with chronic desquamative gingivitis. Six months post-surgery in both treated areas, there were gains in keratinized gingiva and no signs of gingival inflammation compared to presurgery. In the maxillary left quadrant, preoperative histopathologic findings revealed the epithelium was separated from the connective tissue and inflammatory cells were extensive. After grafting with the gingival epithelial sheets, inflammatory cells were decreased and separation between epithelium and connective tissue was not observed. The human cultured gingival epithelial sheets fabricated using tissue engineering technology showed significant promise for gingival augmentation in periodontal therapy.

Characterization of cytoskeletal and junctional proteins expressed by cells cultured from human arachnoid granulation tissue

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Mehta Bhavya C

2005-10-01

Full Text Available Abstract Background The arachnoid granulations (AGs are projections of the arachnoid membrane into the dural venous sinuses. They function, along with the extracranial lymphatics, to circulate the cerebrospinal fluid (CSF to the systemic venous circulation. Disruption of normal CSF dynamics may result in increased intracranial pressures causing many problems including headaches and visual loss, as in idiopathic intracranial hypertension and hydrocephalus. To study the role of AGs in CSF egress, we have grown cells from human AG tissue in vitro and have characterized their expression of those cytoskeletal and junctional proteins that may function in the regulation of CSF outflow. Methods Human AG tissue was obtained at autopsy, and explanted to cell culture dishes coated with fibronectin. Typically, cells migrated from the explanted tissue after 7–10 days in vitro. Second or third passage cells were seeded onto fibronectin-coated coverslips at confluent densities and grown to confluency for 7–10 days. Arachnoidal cells were tested using immunocytochemical methods for the expression of several common cytoskeletal and junctional proteins. Second and third passage cultures were also labeled with the common endothelial markers CD-31 or VE-cadherin (CD144 and their expression was quantified using flow cytometry analysis. Results Confluent cultures of arachnoidal cells expressed the intermediate filament protein vimentin. Cytokeratin intermediate filaments were expressed variably in a subpopulation of cells. The cultures also expressed the junctional proteins connexin43, desmoplakin 1 and 2, E-cadherin, and zonula occludens-1. Flow cytometry analysis indicated that second and third passage cultures failed to express the endothelial cell markers CD31 or VE-cadherin in significant quantities, thereby showing that these cultures did not consist of endothelial cells from the venous sinus wall. Conclusion To our knowledge, this is the first report of

Implementation of Microfluidic Chip Electrophoresis for the Detection of B-cell Clonality

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Vazan M

2016-04-01

Full Text Available Introduction: A clonal population of B-cells is defined as those cells arising from the mitotic division of a single somatic cell with the same rearrangement of immunoglobulin genes. This gives rise to DNA markers for each individual lymphoid cell and its progenies and enables us to study clonality in different B-cell malignancies using multiplex polymerase chain reaction - PCR. The BIOMED-2 protocol has been implemented for clonality detection in lymphoproliferative diseases and exploits multiplex PCR reaction, subsequently analyzed by heteroduplex analysis (HDA using polyacrylamide gel electrophoresis (PAGE. With the advent of miniaturization and automation of molecular biology methods, lab-on-chip technologies were developed and replace partially the conventional approaches. We tested device for microfluidic chip, which is used for B-cells clonality analysis, using a PCR reaction for three subregions called frameworks (FR of the immunoglobulin heavy locus (IGH gene.

Solid tissue culture for cytogenetic analysis: a collaborative survey for the Association of Clinical Cytogeneticists.

Science.gov (United States)

Rodgers, C S; Creasy, M R; Fitchett, M; Maliszewska, C T; Pratt, N R; Waters, J J

1996-01-01

AIMS: To survey the diagnostic service provided by UK laboratories for the culture of solid tissue samples (excluding tumours) and in particular to examine the variation in culture success rates and the problems of maternal cell overgrowth. METHODS: Twenty seven laboratories took part in a collaborative survey during 1992. Each laboratory submitted data on up to a maximum of 60 consecutive specimens (n = 1361) over a six month period. RESULTS: Skin specimens, the largest category received (n = 520), were the most problematic (51% success rate). Culture success rates were significantly lower (43%) when skin specimens (n = 140) were transported dry to the laboratory. Success rates for skin specimens also varied, depending on the origin of the specimen, from 18% for intra-uterine deaths (IUD) (n = 94) to 85% for neonatal deaths (n = 33) and 83% for live patients (n = 54). Culture of selected extra-fetal tissues from IUD, stillbirths and following elective termination of pregnancy (TOP) gave comparable success rates to those achieved for skin samples from neonatal deaths and live births. Skewed sex ratios, female > male, were identified for products of conception (POC) (n = 298) and placental biopsy specimens (n = 97). CONCLUSIONS: By appropriate selection, transport and processing of tissues, and in particular by avoiding relying solely on skin samples from IUD, stillbirths and TOP, an increase in culture success rates for solid tissue samples submitted for cytogenetic analysis could be achieved. The high risk of maternal cell contamination from POC and placental biopsy specimens was also identified in this survey. PMID:8881913

Study of the agroindustrial alterations induced by the irradiated tissue culture in sugar cane, variety NA 56-79

International Nuclear Information System (INIS)

Figueiredo Junior, O.

1991-01-01

The use of plant tissue culture and the application of gamma radiation as mutation inducing agents, in the sugar cane plant, variety NA 5679, are studied. The variation in the contents of brix, pol, fiber, purity, extraction, phosphorus, nitrogen, reducing sugars as well as the morphological characteristics are analysed. The 'callus' obtained by the tissue culture were irradiated with 20, 40, and 60 Gy doses. The statistical analysis indicated that the method of tissue culture may, eventually, increase the contents of the technological parameters and the dosages of gamma radiation were not efficient for such purpose. (M.A.C.)

Heritability of regeneration in tissue cultures of sweet potato (Ipomoea batatas L.).

Science.gov (United States)

Templeton-Somers, K M; Collins, W W

1986-03-01

A population of open-pollinated progeny from 12 parents, and the 12 parents, was surveyed for in vitro growth and regeneration characteristics. Four different tissue culture procedures involving different media and the use of different explants to initiate the cultures were used. Petiole explants from young leaves were used as explants for initiation of callus cultures. These were evaluated for callus growth rate, friability, and callus color and texture, before transferring to each of three different regeneration media for evaluation of morphogenetic potential. Small shoot tips also were used to initiate callus cultures, which were evaluated for the same growth characteristics and transferred to growth-regulator free regeneration media. Regeneration occurred through root or shoot regeneration or through embryogenesis. Tissue culture treatment effects, as well as genotypic effects, were highly significant in determining: the types of callus produced, callus growth rates, color and texture on the two types of media used for the second and third subcultures. The family x treatment interaction was generally not statistically significant, affecting only callus color. Estimates of narrow sense heritability for callus growth rate in both the second and third subcultures were high enough (0.35 and 0.63, respectively) for the evaluation of parental lines for selection procedures. These characteristics were also the only early culture callus traits that were consistently correlated with later morphogenesis of the cultures. They were negatively correlated with root or shoot regeneration. The occurence of somatic embryogenesis was not correlated with early callus growth characteristics. Genetic and treatment effects were highly significant in the evaluation of morphogenetic potential, through root or shoot regeneration, or through embryogenesis. Regeneration of all types was of low frequency for all procedures, expressed in ≦ 11% of the cultures of the total population.

Mutational Profiling Can Establish Clonal or Independent Origin in Synchronous Bilateral Breast and Other Tumors.

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Lei Bao

Full Text Available Synchronous tumors can be independent primary tumors or a primary-metastatic (clonal pair, which may have clinical implications. Mutational profiling of tumor DNA is increasingly common in the clinic. We investigated whether mutational profiling can distinguish independent from clonal tumors in breast and other cancers, using a carefully defined test based on the Clonal Likelihood Score (CLS = 100 x # shared high confidence (HC mutations/ # total HC mutations.Statistical properties of a formal test using the CLS were investigated. A high CLS is evidence in favor of clonality; the test is implemented as a one-sided binomial test of proportions. Test parameters were empirically determined using 16,422 independent breast tumor pairs and 15 primary-metastatic tumor pairs from 10 cancer types using The Cancer Genome Atlas.We validated performance of the test with its established parameters, using five published data sets comprising 15,758 known independent tumor pairs (maximum CLS = 4.1%, minimum p-value = 0.48 and 283 known tumor clonal pairs (minimum CLS 13%, maximum p-value 0.99, supporting independence. A plausible molecular mechanism for the shift from hormone receptor positive to triple negative was identified in the clonal pair.We have developed the statistical properties of a carefully defined Clonal Likelihood Score test from mutational profiling of tumor DNA. Under identified conditions, the test appears to reliably distinguish between synchronous tumors of clonal and of independent origin in several cancer types. This approach may have scientific and clinical utility.

Plant Clonal Integration Mediates the Horizontal Redistribution of Soil Resources, Benefiting Neighboring Plants.

Science.gov (United States)

Ye, Xue-Hua; Zhang, Ya-Lin; Liu, Zhi-Lan; Gao, Shu-Qin; Song, Yao-Bin; Liu, Feng-Hong; Dong, Ming

2016-01-01

Resources such as water taken up by plants can be released into soils through hydraulic redistribution and can also be translocated by clonal integration within a plant clonal network. We hypothesized that the resources from one (donor) microsite could be translocated within a clonal network, released into different (recipient) microsites and subsequently used by neighbor plants in the recipient microsite. To test these hypotheses, we conducted two experiments in which connected and disconnected ramet pairs of Potentilla anserina were grown under both homogeneous and heterogeneous water regimes, with seedlings of Artemisia ordosica as neighbors. The isotopes [(15)N] and deuterium were used to trace the translocation of nitrogen and water, respectively, within the clonal network. The water and nitrogen taken up by P. anserina ramets in the donor microsite were translocated into the connected ramets in the recipient microsites. Most notably, portions of the translocated water and nitrogen were released into the recipient microsite and were used by the neighboring A. ordosica, which increased growth of the neighboring A. ordosica significantly. Therefore, our hypotheses were supported, and plant clonal integration mediated the horizontal hydraulic redistribution of resources, thus benefiting neighboring plants. Such a plant clonal integration-mediated resource redistribution in horizontal space may have substantial effects on the interspecific relations and composition of the community and consequently on ecosystem processes.

Plant clonal integration mediates the horizontal redistribution of soil resources, benefiting neighbouring plants

Directory of Open Access Journals (Sweden)

Xuehua eYe

2016-02-01

Full Text Available Resources such as water taken up by plants can be released into soils through hydraulic redistribution and can also be translocated by clonal integration within a plant clonal network. We hypothesized that the resources from one (donor microsite could be translocated within a clonal network, released into different (recipient microsites and subsequently used by neighbour plants in the recipient microsite. To test these hypotheses, we conducted two experiments in which connected and disconnected ramet pairs of Potentilla anserina were grown under both homogeneous and heterogeneous water regimes, with seedlings of Artemisia ordosica as neighbours. The isotopes [15N] and deuterium were used to trace the translocation of nitrogen and water, respectively, within the clonal network. The water and nitrogen taken up by P. anserina ramets in the donor microsite were translocated into the connected ramets in the recipient microsites. Most notably, portions of the translocated water and nitrogen were released into the recipient microsite and were used by the neighbouring A. ordosica, which increased growth of the neighbouring A. ordosica significantly. Therefore, our hypotheses were supported, and plant clonal integration mediated the horizontal hydraulic redistribution of resources, thus benefiting neighbouring plants. Such a plant clonal integration-mediated resource redistribution in horizontal space may have substantial effects on the interspecific relations and composition of the community and consequently on ecosystem processes.

Analysis of clonal expansions through the normal and premalignant human breast epithelium reveals the presence of luminal stem cells.

Science.gov (United States)

Cereser, Biancastella; Jansen, Marnix; Austin, Emily; Elia, George; McFarlane, Taneisha; van Deurzen, Carolien Hm; Sieuwerts, Anieta M; Daidone, Maria G; Tadrous, Paul J; Wright, Nicholas A; Jones, Louise; McDonald, Stuart Ac

2018-01-01

It is widely accepted that the cell of origin of breast cancer is the adult mammary epithelial stem cell; however, demonstrating the presence and location of tissue stem cells in the human breast has proved difficult. Furthermore, we do not know the clonal architecture of the normal and premalignant mammary epithelium or its cellular hierarchy. Here, we use deficiency in the mitochondrial enzyme cytochrome c oxidase (CCO), typically caused by somatic mutations in the mitochondrial genome, as a means to perform lineage tracing in the human mammary epithelium. PCR sequencing of laser-capture microdissected cells in combination with immunohistochemistry for markers of lineage differentiation was performed to determine the clonal nature of the mammary epithelium. We have shown that in the normal human breast, clonal expansions (defined here by areas of CCO deficiency) are typically uncommon and of limited size, but can occur at any site within the adult mammary epithelium. The presence of a stem cell population was shown by demonstrating multi-lineage differentiation within CCO-deficient areas. Interestingly, we observed infrequent CCO deficiency that was restricted to luminal cells, suggesting that niche succession, and by inference stem cell location, is located within the luminal layer. CCO-deficient areas appeared large within areas of ductal carcinoma in situ, suggesting that the rate of clonal expansion was altered in the premalignant lesion. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Optimization of an Efficient Non-Tissue Culture Transformation Method for Brassica Juncea

International Nuclear Information System (INIS)

Naeem, I.; Munir, I.; Iqbal, A.; Ullah, F.

2016-01-01

The major hurdles in successful in vitro transformation of Brassica juncea through standard tissue culture (STC) method are: culture contamination, somaclonal variations, and lack of expertise. Moreover, the current STC method is time consuming and needs continuous electricity. In the present study, the in planta transformation method through floral dip with or without vacuum infiltration was optimized for successful transformation of B. juncea. The B. juncea CV RAYA Anmol was used for transformation through Agrobacterium tumefaciens strain GV3101 harboring the binary vector plasmid pBinGlyBar4-EADcT. Based on the resistance reaction to the herbicide Basta, 20 and 40 resistant seedlings were obtained from 2000 seed germinated from the plants transformed through floral dip and vacuum infiltration methods, respectively. The PCR analyses further confirmed the presence of transgene in 3 floral dipped plants without vacuum infiltration and 17 floral dipped plants with vacuum infiltration, giving the transformation frequencies of 1.5*10/sup -3/ and 8.5*10/sup -3/, respectively. This method, which avoids tissue culture, will reduce the somaclonal variation accompanying prolonged culture of cells in a dedifferentiated state, will facilitate functional genomics and improvement of Brassica juncea with novel desirable traits while reducing time and expense. (author)

Virulence, sporulation, and elicitin production in three clonal lineages of Phytophthora ramorum

Science.gov (United States)

Phytophthora ramorum populations are clonal and consist of three lineages. Recent studies have shown that the clonal lineages may have varying degrees of aggressiveness on some host species, such as Quercus rubra. In this study, we examined virulence, sporulation and elicitin production of five P. ...

Clonal growth and plant species abundance

Czech Academy of Sciences Publication Activity Database

Herben, Tomáš; Nováková, Z.; Klimešová, Jitka

2014-01-01

Roč. 114, č. 2 (2014), s. 377-388 ISSN 0305-7364 R&D Projects: GA ČR GA526/09/0963 Institutional support: RVO:67985939 Keywords : clonal plants * frequency * plant communities of Central Europe Subject RIV: EF - Botanics Impact factor: 3.654, year: 2014

Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

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Gesiane Ribeiro

2013-12-01

Full Text Available The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.

Thymic epithelial cells. I. Expression of strong suppressive (veto) activity in mouse thymic epithelial cell cultures

DEFF Research Database (Denmark)

Claesson, Mogens Helweg; Ropke, C

1990-01-01

We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level. It is conclu......We show that thymic epithelial cells grown under serum-free conditions in a chemically defined culture medium can act as veto cells in vitro. The veto activity of thymic epithelial cells results in inactivation of specific alloreactive cytotoxic T-cell precursors at the clonal level...

Comparative investigations of T cell receptor gamma gene rearrangements in frozen and formalin-fixed paraffin wax-embedded tissues by capillary electrophoresis

DEFF Research Database (Denmark)

Christensen, M; Funder, A D; Bendix, K

2006-01-01

AIM: To compare clonal T cell receptor gamma (TCRgamma) gene rearrangements in frozen and formalin-fixed paraffin wax-embedded (FFPE) tissue, using capillary electrophoresis for use in diagnostics, as T cell lymphomas may be difficult to diagnose by conventional methods.METHODS: The DNA for PCR......% for patient specimens and the specificity 100%. The junctional region between the Vgamma and Jgamma segments was specific for each patient.CONCLUSIONS: Capillary electrophoresis of PCR products from frozen and FFPE tissue is suitable for detecting clonal TCRgamma gene rearrangements. It is important, however...

Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade

DEFF Research Database (Denmark)

McGranahan, Nicholas; Furness, Andrew J S; Rosenthal, Rachel

2016-01-01

demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8(+)tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non-small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4...

Clonal relation in a case of CLL, ALCL, and Hodgkin composite lymphoma

NARCIS (Netherlands)

van den Berg, Anke; Maggio, Ewerton; Rust, R; Kooistra, K; Diepstra, A; Poppema, S

2002-01-01

Large cell lymphomas and Hodgkin disease may develop during the course of chronic lymphocytic leukemia (CLL). In some cases the transformed cells are Epstein-Barr virus (EBV)-positive and not clonally related to the CLL cells. In other cases the transformed cells have the same clonal rearrangements

Discrimination and similarity evaluation of tissue-cultured and wild Dendrobium species using Fourier transform infrared spectroscopy

Science.gov (United States)

Chen, Nai-dong; Chen, Han; Li, Jun; Sang, Mang-mang; Ding, Shen; Yu, Hao

2015-04-01

The FTIR method was applied to evaluate the similarity of tissue-cultured and wild Dendrobium huoshanense C.Z. Tang et S.J. Cheng, Dendrobium officinale Kimura et Migo and Dendrobium moniliforme (Linn.) Sw and discriminate different Dendrobium species, especially D. huoshanense and its main goldbrick Dendrobium henanense J.L. Lu et L.X. Gao. Despite the general pattern of the IR spectra, different intensities, shapes and peak positions were found in the IR spectra of these samples, especially in the range of 1800-600 cm-1, which could be used to discriminate them. The methanol, aqueous extracting procedure and the second derivative transformation obviously enlarged the tiny spectral differences among these samples. The similarity evaluation based on the IR spectra and the second derivative IR spectrum revealed that the similarity of the methanol extracts between tissue-cultured and wild Dendrobiums might be lower than that between different Dendrobium species. The similarities of the powders and aqueous extracts between tissue-cultured and wild Dendrobiums were higher than those between different Dendrobium species. The further principal component analysis showed that the first three components explained 99.7%, 87.7% and 85.1% of data variance for powder, methanol extract and aqueous extract, respectively, demonstrating a good discrimination between samples. Our research suggested that the variations of secondary metabolites between different origins of the investigated Dendrobiums might be higher than what we had supposed. Tissue culture techniques were widely used in the conversation of rare and endangered medicinal amedica, however, our study suggested that the chemical constituents of tissue-cultured plants might be quite different from their wild correspondences.

Transgenerational plasticity in clonal plants

Czech Academy of Sciences Publication Activity Database

Latzel, Vít; Klimešová, Jitka

2010-01-01

Roč. 24, č. 6 (2010), s. 1537-1543 ISSN 0269-7653 R&D Projects: GA ČR GD206/08/H044; GA ČR GA526/09/0963; GA ČR GPP505/10/P173 Institutional research plan: CEZ:AV0Z60050516 Keywords : maternal effect * evolution * clonal plants Subject RIV: EF - Botanics Impact factor: 2.398, year: 2010

Co-cultures and cell sheet engineering as relevant tools to improve the outcome of bone tissue engineering strategies

OpenAIRE

Pirraco, Rogério

2011-01-01

Taking into consideration the complex biology of bone tissue it is quite clear that the understanding of the cellular interactions that regulate the homeostasis and regeneration of this remarkable tissue is essential for a successful Tissue Engineering strategy. The in vitro study of these cellular interactions relies on co-culture systems, a tremendously useful methodology where two or more cell types are cultured at the same time. Such strategy increases the complexity of typ...

Clonal Growth Forms in Eastern Ladakh, Western Himalayas: Classification and Habitat Preferences

Czech Academy of Sciences Publication Activity Database

Klimešová, Jitka; Doležal, Jiří; Dvorský, Miroslav; de Bello, Francesco; Klimeš, Leoš

2011-01-01

Roč. 46, 2-3 (2011), 191-217 ISSN 1211-9520 R&D Projects: GA AV ČR IAA600050802 Institutional research plan: CEZ:AV0Z60050516 Keywords : Clonal growth form * Clonal space occupancy strategies * Habitat types Subject RIV: EF - Botanics Impact factor: 1.500, year: 2011

Analytical and diagnostic performance of a qPCR assay for Ichthyophonus spp. compared to the tissue culture 'gold standard'.

Science.gov (United States)

Lowe, Vanessa C; Hershberger, Paul K; Friedman, Carolyn S

2018-06-04

Parasites of the genus Ichthyophonus infect many fish species and have a non-uniform distribution within host tissues. Due in part to this uneven distribution, the comparative sensitivity and accuracy of using molecular-based detection methods versus culture to estimate parasite prevalence is under debate. We evaluated the analytical and diagnostic performance of an existing qPCR assay in comparison to the 'gold standard' culture method using Pacific herring Clupea pallasii with known exposure history. We determined that the assay is suitable for use in this host, and diagnostic specificity was consistently high (>98%) in both heart and liver tissues. Diagnostic sensitivity could not be fully assessed due to low infection rates, but our results suggest that qPCR is not as sensitive as culture under all circumstances. Diagnostic sensitivity of qPCR relative to culture is likely affected by the amount of sample processed. The prevalence values estimated by the 2 methods were not significantly different when sample amounts were equal (heart tissue), but when the assayed sample amounts were unequal (liver tissue), the culture method detected a significantly higher prevalence of the parasite than qPCR. Further, culture of liver also detected significantly more Ichthyophonus infections than culture of heart, suggesting that the density and distribution of parasites in tissues also plays a role in assay sensitivity. This sensitivity issue would be most problematic for fish with light infections. Although qPCR does not detect the presence of a live organism, DNA-based pathogen detection methods provide the opportunity for alternate testing strategies when culture is not possible.

Clonal distribution of osteoprogenitor cells in cultured chick periostea: Functional relationship to bone formation

International Nuclear Information System (INIS)

McCulloch, C.A.; Fair, C.A.; Tenenbaum, H.C.; Limeback, H.; Homareau, R.

1990-01-01

Folded explants of periosteum from embryonic chick calvaria form bone-like tissue when grown in the presence of ascorbic acid, organic phosphate, and dexamethasone. All osteoblast-like cells in these cultures arise de novo by differentiation of osteoprogenitor cells present in the periosteum. To study the spatial and functional relationships between bone formation and osteoprogenitor cells, cultures were continuously labeled with [3H]thymidine for periods of 1-5 days. Radioautographs of serial 2-microns plastic sections stained for alkaline phosphatase (AP) showed maximal labeling of 30% of fibroblastic (AP-negative) cells by 3 days while osteogenic cells (AP-positive) exhibited over 95% labeling by 5 days. No differential shifts in labeling indices, grain count histograms of fibroblastic and osteogenic cells or numbers of AP-positive cells were observed, indicating no significant recruitment of cells from the fibroblastic to the osteogenic compartment. Despite the continuous presence of [3H]thymidine, less than 35% of both osteoblasts and osteocytes were labeled at 5 days, indicating that only one-third of the osteoprogenitor cells had cycled prior to differentiation. Spatial clustering of [3H]thymidine-labeled cells was measured by computer-assisted morphometry and application of the Poisson distribution to assess contagion. Cluster size and number of labeled cells per cluster did not vary between 1-3 days, but the number of clusters increased 20-fold between Day 1 and Day 3. Three-dimensional reconstruction from serial sections showed that clusters formed long, tubular arrays of osteogenic cells up to eight cells in length and located within 2-3 cell layers from the bone surface. Selective killing of S-phase cells with two pulse labels of high specific activity [3H]thymidine at 1 and 2 days of culture completely blocked bone formation

Functional specialization of ramets in a clonal plant network functional specialization of ramets in a clonal plant network

NARCIS (Netherlands)

Ikegami, M.

2004-01-01

Functional specialization of ramets and co-operation between interconnected ramets in heterogeneous environments are studied in this thesis. Since clonal plants can spread horizontally by vegetative growth, a genet has the potential to grow across a heterogeneous environment. Therefore, ramets can

Rotating three-dimensional dynamic culture of adult human bone marrow-derived cells for tissue engineering of hyaline cartilage.

Science.gov (United States)

Sakai, Shinsuke; Mishima, Hajime; Ishii, Tomoo; Akaogi, Hiroshi; Yoshioka, Tomokazu; Ohyabu, Yoshimi; Chang, Fei; Ochiai, Naoyuki; Uemura, Toshimasa

2009-04-01

The method of constructing cartilage tissue from bone marrow-derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow-derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per microg DNA of the tissues were 10.01 +/- 3.49 microg/microg DNA in the case of the RWV bioreactor and 6.27 +/- 3.41 microg/microg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three-dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow-derived cells.

Human serum-derived protein removes the need for coating in defined human pluripotent stem cell culture

Science.gov (United States)

Pijuan-Galitó, Sara; Tamm, Christoffer; Schuster, Jens; Sobol, Maria; Forsberg, Lars; Merry, Catherine L. R.; Annerén, Cecilia

2016-01-01

Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications. PMID:27405751

Clonal diversity and estimation of relative clone age: application to agrobiodiversity of yam (Dioscorea rotundata).

Science.gov (United States)

Scarcelli, Nora; Couderc, Marie; Baco, Mohamed N; Egah, Janvier; Vigouroux, Yves

2013-11-13

Clonal propagation is a particular reproductive system found in both the plant and animal kingdoms, from human parasites to clonally propagated crops. Clonal diversity provides information about plant and animal evolutionary history, i.e. how clones spread, or the age of a particular clone. In plants, this could provide valuable information about agrobiodiversity dynamics and more broadly about the evolutionary history of a particular crop. We studied the evolutionary history of yam, Dioscorea rotundata. In Africa, Yam is cultivated by tuber clonal propagation. We used 12 microsatellite markers to identify intra-clonal diversity in yam varieties. We then used this diversity to assess the relative ages of clones. Using simulations, we assessed how Approximate Bayesian Computation could use clonal diversity to estimate the age of a clone depending on the size of the sample, the number of independent samples and the number of markers. We then applied this approach to our particular dataset and showed that the relative ages of varieties could be estimated, and that each variety could be ranked by age. We give a first estimation of clone age in an approximate Bayesian framework. However the precise estimation of clone age depends on the precision of the mutation rate. We provide useful information on agrobiodiversity dynamics and suggest recurrent creation of varietal diversity in a clonally propagated crop.

Functional enhancement of chitosan and nanoparticles in cell culture, tissue engineering, and pharmaceutical applications

Directory of Open Access Journals (Sweden)

Wenjuan eGao

2012-08-01

Full Text Available Abstract: As a biomaterial, chitosan has been widely used in tissue engineering, wound healing, drug delivery, and other biomedical applications. It can be formulated in a variety of forms, such as powder, film, sphere, gel and fiber. These features make chitosan an almost ideal biomaterial in cell culture applications, and cell cultures arguably constitute the most practical way to evaluate biocompatibility and biotoxicity. The advantages of cell cultures are that they can be performed under totally controlled environments, allow high throughput functional screening, and are less costly, as compared to other assessment methods. Chitosan can also be modified into multilayer composite by combining with other polymers and moieties to alter the properties of chitosan for particular biomedical applications. This review briefly depicts and discusses applications of chitosan and nanoparticles in cell culture, in particular, the effects of chitosan and nanoparticles on cell adhesion, cell survival, and the underlying molecular mechanisms: both stimulatory and inhibitory influences are discussed. Our aim is to update the current status of how nanoparticles can be utilized to modify the properties of chitosan to advance the art of tissue engineering by using cell cultures.

A Protocol for Rapid, Measurable Plant Tissue Culture Using Stem Disc Meristem Micropropagation of Garlic ("Allium Sativum L.")

Science.gov (United States)

Peat, Gerry; Jones, Meriel

2012-01-01

Plant tissue culture is becoming an important technique for the mass propagation of plants. Problems with existing techniques, such as slow growth and contamination, have restricted the practical work in plant tissue culture carried out in schools. The new protocol using garlic meristematic stem discs explained in this article addresses many of…

Tissue culture-induced genetic and epigenetic alterations in rice pure-lines, F1 hybrids and polyploids.

Science.gov (United States)

Wang, Xiaoran; Wu, Rui; Lin, Xiuyun; Bai, Yan; Song, Congdi; Yu, Xiaoming; Xu, Chunming; Zhao, Na; Dong, Yuzhu; Liu, Bao

2013-05-05

Genetic and epigenetic alterations can be invoked by plant tissue culture, which may result in heritable changes in phenotypes, a phenomenon collectively termed somaclonal variation. Although extensive studies have been conducted on the molecular nature and spectrum of tissue culture-induced genomic alterations, the issue of whether and to what extent distinct plant genotypes, e.g., pure-lines, hybrids and polyploids, may respond differentially to the tissue culture condition remains poorly understood. We investigated tissue culture-induced genetic and epigenetic alterations in a set of rice genotypes including two pure-lines (different subspecies), a pair of reciprocal F1 hybrids parented by the two pure-lines, and a pair of reciprocal tetraploids resulted from the hybrids. Using two molecular markers, amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP), both genetic and DNA methylation alterations were detected in calli and regenerants from all six genotypes, but genetic alteration is more prominent than epigenetic alteration. While significant genotypic difference was observed in frequencies of both types of alterations, only genetic alteration showed distinctive features among the three types of genomes, with one hybrid (N/9) being exceptionally labile. Surprisingly, difference in genetic alteration frequencies between the pair of reciprocal F1 hybrids is much greater than that between the two pure-line subspecies. Difference also exists in the pair of reciprocal tetraploids, but is to a less extent than that between the hybrids. The steady-state transcript abundance of genes involved in DNA repair and DNA methylation was significantly altered in both calli and regenerants, and some of which were correlated with the genetic and/or epigenetic alterations. Our results, based on molecular marker analysis of ca. 1,000 genomic loci, document that genetic alteration is the major cause of somaclonal variation in rice

Unforeseen clonal evolution of tumor cell population in recurrent and metastatic dermatofibrosarcoma protuberans.

Directory of Open Access Journals (Sweden)

Ensel Oh

Full Text Available Dermatofibrosarcoma protuberans (DFSP is a very rare soft tissue sarcoma, generally of low-grade malignancy. DFSP is locally aggressive with a high recurrence rate, but metastasis occurs rarely. To investigate the mechanism of metastasis in DFSP, we analyzed the whole exome sequencing data of serial tumor samples obtained from a patient who had a 10-year history of recurrent and metastatic DFSP. Tracking various genomic alterations, namely somatic mutations, copy number variations, and chromosomal rearrangements, we observed a dramatic change in tumor cell population during the occurrence of metastasis in this DFSP case. The new subclone that emerged in metastatic DFSP harbored a completely different set of somatic mutations and new focal amplifications, which had not been observed in the primary clone before metastasis. The COL1A1-PDGFB fusion, characteristic of DFSP, was found in all of the serial samples. Moreover, the break position on the fusion gene was identical in all samples. Based on these observations, we suggest a clonal evolution model to explain the mechanism underlying metastasis in DFSP and identified several candidate target genes responsible for metastatic DFSP by utilizing The Cancer Genome Atlas database. This is the first study to observe clonal evolution in metastatic DFSP and provide insight for a possible therapeutic strategy for imatinib-resistant or metastatic DFSP.

Development of human nervous tissue upon differentiation of embryonic stem cells in three-dimensional culture.

Science.gov (United States)

Preynat-Seauve, Olivier; Suter, David M; Tirefort, Diderik; Turchi, Laurent; Virolle, Thierry; Chneiweiss, Herve; Foti, Michelangelo; Lobrinus, Johannes-Alexander; Stoppini, Luc; Feki, Anis; Dubois-Dauphin, Michel; Krause, Karl Heinz

2009-03-01

Researches on neural differentiation using embryonic stem cells (ESC) require analysis of neurogenesis in conditions mimicking physiological cellular interactions as closely as possible. In this study, we report an air-liquid interface-based culture of human ESC. This culture system allows three-dimensional cell expansion and neural differentiation in the absence of added growth factors. Over a 3-month period, a macroscopically visible, compact tissue developed. Histological coloration revealed a dense neural-like neural tissue including immature tubular structures. Electron microscopy, immunochemistry, and electrophysiological recordings demonstrated a dense network of neurons, astrocytes, and oligodendrocytes able to propagate signals. Within this tissue, tubular structures were niches of cells resembling germinal layers of human fetal brain. Indeed, the tissue contained abundant proliferating cells expressing markers of neural progenitors. Finally, the capacity to generate neural tissues on air-liquid interface differed for different ESC lines, confirming variations of their neurogenic potential. In conclusion, this study demonstrates in vitro engineering of a human neural-like tissue with an organization that bears resemblance to early developing brain. As opposed to previously described methods, this differentiation (a) allows three-dimensional organization, (b) yields dense interconnected neural tissue with structurally and functionally distinct areas, and (c) is spontaneously guided by endogenous developmental cues.

Dynamic Culturing of Cartilage Tissue: The Significance of Hydrostatic Pressure

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Pereira, Ana L.; Duarte, Ana R.C.; Frias, Ana M.; Pedro, Adriano J.; Oliveira, João T.; Sousa, Rui A.; Reis, Rui L.

2012-01-01

Human articular cartilage functions under a wide range of mechanical loads in synovial joints, where hydrostatic pressure (HP) is the prevalent actuating force. We hypothesized that the formation of engineered cartilage can be augmented by applying such physiologic stimuli to chondrogenic cells or stem cells, cultured in hydrogels, using custom-designed HP bioreactors. To test this hypothesis, we investigated the effects of distinct HP regimens on cartilage formation in vitro by either human nasal chondrocytes (HNCs) or human adipose stem cells (hASCs) encapsulated in gellan gum (GG) hydrogels. To this end, we varied the frequency of low HP, by applying pulsatile hydrostatic pressure or a steady hydrostatic pressure load to HNC-GG constructs over a period of 3 weeks, and evaluated their effects on cartilage tissue-engineering outcomes. HNCs (10×106 cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 3 weeks: (1) 0.4 MPa Pulsatile HP; (2) 0.4 MPa Steady HP; and (3) Static. Subsequently, we applied the pulsatile regimen to hASC-GG constructs and varied the amplitude of loading, by generating both low (0.4 MPa) and physiologic (5 MPa) HP levels. hASCs (10×106 cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 4 weeks: (1) 0.4 MPa Pulsatile HP; (2) 5 MPa Pulsatile HP; and (3) Static. In the HNC study, the best tissue development was achieved by the pulsatile HP regimen, whereas in the hASC study, greater chondrogenic differentiation and matrix deposition were obtained for physiologic loading, as evidenced by gene expression of aggrecan, collagen type II, and sox-9; metachromatic staining of cartilage extracellular matrix; and immunolocalization of collagens. We thus propose that both HNCs and hASCs detect and respond to physical forces, thus resembling joint loading, by enhancing cartilage tissue development in a frequency- and

Clonal forestry, heterosis and advanced-generation breeding

Energy Technology Data Exchange (ETDEWEB)

Tuskan, G.A.

1997-08-01

This report discusses the clonal planting stock offers many advantages to the forest products industry. Advanced-generation breeding strategies should be designed to maximize within-family variance and at the same time allow the capture of heterosis. Certainly there may be a conflict in the choice of breeding strategy based on the trait of interest. It may be that the majority of the traits express heterosis due to overdominance. Alternatively, disease resistance is expressed as the lack of a specific metabolite or infection court then the homozygous recessive genotype may be the most desirable. Nonetheless, as the forest products industry begins to utilize the economic advantages of clonal forestry, breeding strategies will have to be optimized for these commercial plant materials. Here, molecular markers can be used to characterize the nature of heterosis and therefore define the appropriate breeding strategy.

Big Bang Tumor Growth and Clonal Evolution.

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Sun, Ruping; Hu, Zheng; Curtis, Christina

2018-05-01

The advent and application of next-generation sequencing (NGS) technologies to tumor genomes has reinvigorated efforts to understand clonal evolution. Although tumor progression has traditionally been viewed as a gradual stepwise process, recent studies suggest that evolutionary rates in tumors can be variable with periods of punctuated mutational bursts and relative stasis. For example, Big Bang dynamics have been reported, wherein after transformation, growth occurs in the absence of stringent selection, consistent with effectively neutral evolution. Although first noted in colorectal tumors, effective neutrality may be relatively common. Additionally, punctuated evolution resulting from mutational bursts and cataclysmic genomic alterations have been described. In this review, we contrast these findings with the conventional gradualist view of clonal evolution and describe potential clinical and therapeutic implications of different evolutionary modes and tempos. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Clonal Streptococcus equi subsp. zooepidemicus post breeding endometritis in thoroughbred broodmares

DEFF Research Database (Denmark)

Christoffersen, Mette; Söderlind, Maja; Rydemann Rudefalk, Sofia

Streptococcus equi subsp. zooepidemicus is one of the most commonly isolated pathogens from the uterus of mares with infectious endometritis. Its ability to cause chronic latent infection by residing deep within the endometrial tissue has previously been described. The aim of the study was to inv......Streptococcus equi subsp. zooepidemicus is one of the most commonly isolated pathogens from the uterus of mares with infectious endometritis. Its ability to cause chronic latent infection by residing deep within the endometrial tissue has previously been described. The aim of the study...... was to investigate whether clonal or genetically distinct S. zooepidemicus strains isolated from mares with endometritis were associated with mare risk factors and the outcome of natural cover. Uterine swabs were obtained from mares with intrauterine fluid after natural cover (n=31) at thoroughbred stud farms...... in Australia. Fifty two percent of the mares (n=16) were diagnosed with infectious endometritis, and S.zooepidemicus was isolated in 81% (n=13) of these mares. Up to four S. zooepidemicus isolates were selected from each mare with growth of S. zooepidemicus and isolates from an additional five mares were...

High-throughput monitoring of integration site clonality in preclinical and clinical gene therapy studies

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Frank A Giordano

Full Text Available Gene transfer to hematopoietic stem cells with integrating vectors not only allows sustained correction of monogenic diseases but also tracking of individual clones in vivo. Quantitative real-time PCR (qPCR has been shown to be an accurate method to quantify individual stem cell clones, yet due to frequently limited amounts of target material (especially in clinical studies, it is not useful for large-scale analyses. To explore whether vector integration site (IS recovery techniques may be suitable to describe clonal contributions if combined with next-generation sequencing techniques, we designed artificial ISs of different sizes which were mixed to simulate defined clonal situations in clinical settings. We subjected all mixes to either linear amplification–mediated PCR (LAM-PCR or nonrestrictive LAM-PCR (nrLAM-PCR, both combined with 454 sequencing. We showed that nrLAM-PCR/454-detected clonality allows estimating qPCR-detected clonality in vitro. We then followed the kinetics of two clones detected in a patient enrolled in a clinical gene therapy trial using both, nrLAM-PCR/454 and qPCR and also saw nrLAM-PCR/454 to correlate to qPCR-measured clonal contributions. The method presented here displays a feasible high-throughput strategy to monitor clonality in clinical gene therapy trials is at hand.

Biomaterials and Culture Technologies for Regenerative Therapy of Liver Tissue.

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Perez, Roman A; Jung, Cho-Rok; Kim, Hae-Won

2017-01-01

Regenerative approach has emerged to substitute the current extracorporeal technologies for the treatment of diseased and damaged liver tissue. This is based on the use of biomaterials that modulate the responses of hepatic cells through the unique matrix properties tuned to recapitulate regenerative functions. Cells in liver preserve their phenotype or differentiate through the interactions with extracellular matrix molecules. Therefore, the intrinsic properties of the engineered biomaterials, such as stiffness and surface topography, need to be tailored to induce appropriate cellular functions. The matrix physical stimuli can be combined with biochemical cues, such as immobilized functional groups or the delivered actions of signaling molecules. Furthermore, the external modulation of cells, through cocultures with nonparenchymal cells (e.g., endothelial cells) that can signal bioactive molecules, is another promising avenue to regenerate liver tissue. This review disseminates the recent approaches of regenerating liver tissue, with a focus on the development of biomaterials and the related culture technologies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of reference genes for quantitative real-time PCR in oil palm elite planting materials propagated by tissue culture.

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Pek-Lan Chan

Full Text Available BACKGROUND: The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR. With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. RESULTS: In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569 outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN. PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. CONCLUSIONS: Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection

Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture

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Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

2014-01-01

Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate

Expression of lumican in hidroacanthoma simplex and clonal-type seborrheic keratosis as a potent differential diagnostic marker.

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Takayama, Ryoko; Ansai, Shin-Ichi; Ishiwata, Toshiyuki; Yamamoto, Tetsushi; Matsuda, Yoko; Naito, Zenya; Kawana, Seiji

2014-08-01

Lumican, a member of the small leucine-rich proteoglycan family, regulates the assembly and diameter of collagen fibers in the extracellular matrix of various tissues. The lumican expression correlates with pathological conditions and the growth and metastasis of various malignancies. In cutaneous neoplasms, the lumican expression is lower in advanced-stage malignant melanomas that invade the dermis than in early-stage melanomas. Furthermore, we have recently reported that the expression pattern of lumican is different from that of actinic keratosis and the Bowen disease. Lumican is positive in the poroid cells of intraepidermal sweat ducts; therefore, we examined the expression patterns of lumican in acanthotic-type seborrheic keratosis and Pinkus-type poroma followed by clonal-type seborrheic keratosis and hidroacanthoma simplex. The neoplastic cells of acanthotic-type seborrheic keratosis exhibited positive immunostaining in only 1 of 31 cases (3.23%), whereas the poroid cells of Pinkus-type poroma exhibited positive immunoreactivity in 26 of 28 patients (92.8%). In the hidroacanthoma simplex cases, lumican was expressed in poroid cells forming intraepidermal nests in 22 of 28 patients (78.6%), whereas the neoplastic cells in most cases of clonal-type seborrheic keratosis were negative for lumican. In some seborrheic keratosis cases that were positive for lumican in neoplastic cells, lumican was observed in squamoid cells but not in basaloid cells. Therefore, it is necessary to evaluate the immunoreactivity of lumican in seborrheic keratosis and in basaloid cells. These findings suggest that lumican is a potent differential diagnostic marker that distinguishes hidroacanthoma simplex from clonal-type seborrheic keratosis.

Clonal growth strategy, diversity and structure: A spatiotemporal response to sedimentation in tropical Cyperus papyrus swamps.

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Geremew, Addisie; Stiers, Iris; Sierens, Tim; Kefalew, Alemayehu; Triest, Ludwig

2018-01-01

Land degradation and soil erosion in the upper catchments of tropical lakes fringed by papyrus vegetation can result in a sediment load gradient from land to lakeward. Understanding the dynamics of clonal modules (ramets and genets) and growth strategies of plants on such a gradient in both space and time is critical for exploring a species adaptation and processes regulating population structure and differentiation. We assessed the spatial and temporal dynamics in clonal growth, diversity, and structure of an emergent macrophyte, Cyperus papyrus (papyrus), in response to two contrasting sedimentation regimes by combining morphological traits and genotype data using 20 microsatellite markers. A total of 636 ramets from six permanent plots (18 x 30 m) in three Ethiopian papyrus swamps, each with discrete sedimentation regimes (high vs. low) were sampled for two years. We found that ramets under the high sedimentation regime (HSR) were significantly clumped and denser than the sparse and spreading ramets under the low sedimentation regime (LSR). The HSR resulted in significantly different ramets with short culm height and girth diameter as compared to the LSR. These results indicated that C. papyrus ameliorates the effect of sedimentation by shifting clonal growth strategy from guerrilla (in LSR) to phalanx (in HSR). Clonal richness, size, dominance, and clonal subrange differed significantly between sediment regimes and studied time periods. Each swamp under HSR revealed a significantly high clonal richness (R = 0.80) as compared to the LSR (R = 0.48). Such discrepancy in clonal richness reflected the occurrence of initial and repeated seedling recruitment strategies as a response to different sedimentation regimes. Overall, our spatial and short-term temporal observations highlighted that HSR enhances clonal richness and decreases clonal subrange owing to repeated seedling recruitment and genets turnover.

Exploring plant tissue culture in Withania somnifera (L.) Dunal: in vitro propagation and secondary metabolite production.

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Shasmita; Rai, Manoj K; Naik, Soumendra K

2017-12-26

Withania somnifera (L.) Dunal (family: Solanaceae), commonly known as "Indian Ginseng", is a medicinally and industrially important plant of the Indian subcontinent and other warmer parts of the world. The plant has multi-use medicinal potential and has been listed among 36 important cultivated medicinal plants of India that are in high demand for trade due to its pharmaceutical uses. The medicinal importance of this plant is mainly due to the presence of different types of steroidal lactones- withanolides in the roots and leaves. Owing to low seed viability and poor germination, the conventional propagation of W. somnifera falls short to cater its commercial demands particularly for secondary metabolite production. Therefore, there is a great need to develop different biotechnological approaches through tissue and organ culture for seasonal independent production of plants in large scale which will provide sufficient raw materials of uniform quality for pharmaceutical purposes. During past years, a number of in vitro plant regeneration protocols via organogenesis and somatic embryogenesis and in vitro conservation through synthetic seed based encapsulation technology have been developed for W. somnifera. Several attempts have also been made to standardize the protocol of secondary metabolite production via tissue/organ cultures, cell suspension cultures, and Agrobacterium rhizogenes-mediated transformed hairy root cultures. Employment of plant tissue culture based techniques would provide means for rapid propagation and conservation of this plant species and also provide scope for enhanced production of different bioactive secondary metabolites. The present review provides a comprehensive report on research activities conducted in the area of tissue culture and secondary metabolite production in W. somnifera during the past years. It also discusses the unexplored areas which might be taken into consideration for future research so that the medicinal properties and

Current status and future prospects for cultured limbal tissue transplants in Australia and New Zealand.

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Harkin, Damien G; Apel, Andrew J; Di Girolamo, Nick; Watson, Stephanie; Brown, Karl; Daniell, Mark D; McGhee, J Jane; McGhee, Charles N J

2013-04-01

Cultured limbal tissue transplants have become widely used over the last decade as a treatment for limbal stem cell deficiency (LSCD). While the number of patients afflicted with LSCD in Australia and New Zealand is considered to be relatively low, the impact of this disease on quality of life is so severe that the potential efficacy of cultured transplants has necessitated investigation. We presently review the basic biology and experimental strategies associated with the use of cultured limbal tissue transplants in Australia and New Zealand. In doing so, we aim to encourage informed discussion on the issues required to advance the use of cultured limbal transplants in Australia and New Zealand. Moreover, we propose that a collaborative network could be established to maintain access to the technology in conjunction with a number of other existing and emerging treatments for eye diseases. © 2012 The Authors. Clinical and Experimental Ophthalmology © 2012 Royal Australian and New Zealand College of Ophthalmologists.

COMPARISON OF CULTURE OF SYNOVIAL FLUID, PERIPROSTHETIC TISSUE AND PROSTHESIS SONICATE FOR THE DIAGNOSIS OF KNEE PROSTHESIS INFECTION

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Andrej Trampuž

2003-03-01

Full Text Available Background. Synovial fluid and periprosthetic tissue specimens are the standard specimens cultured for the diagnosis of prosthetic joint infection (PJI. We hypothesize that ultrasonication of the explanted prosthesis may improve diagnosis of PJI by dislodging biofilm bacteria from the prosthesis surface and improve the sensitivity and specificity of diagnosis of PJI.Methods. Included were patients undergoing knee prosthesis exchange for septic or biomechanical failure and have not received antimicrobial therapy in the last 2 weeks prior specimen collection. Cultures of synovial fluid and periprosthetic tissue specimens were performed per the usual clinical practice. Additionally, explanted joint components were sonicated for 5 minutes at frequency 40 kHz in sterile Ringer’s solution; aliquots of 0.5 ml sonicate were plated onto five aerobic and five anaerobic blood agar plates, and incubated at 37 °C and examined for the next seven days. The number and identity of each colony morphology was recorded.Results. 35 patients undergoing knee replacement have been studied (24 for aseptic biomechanical failure and 11 for suspected PJI. In patients with PJI, coagulase-negative staphylococci (7 cases, Corynebacterium spp. (2 cases, Staphylococcus aureus (1 case, and viridans group streptococcus (1 case were recovered. Culture sensitivity and specificity were for synovial fluid 88% and 100%, for periprosthetic tissue 83% and 81%, and for explant sonicate 91% and 100%, respectively. In sonicate cultures higher numbers of microorganisms than in periprosthetic tissue cultures were consistently detected.Conclusions. Using synovial fluid, periprosthetic tissue, and explant sonicate cultures, 12%, 17% and 9% of PJI were missed, respectively. Explant sonicate cultures were the most sensitive with respect to the diagnosis of PJI, indicating that explant ultrasonication may improve bacterial recovery. In sonicate cultures, infecting organisms were detected in

Metabolic aspects of growth in HU-treated crown-gall tissue cultures. I. Nicotiana tabacum

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Aldona Rennert

2015-01-01

Full Text Available An influence of hydroxyurea (HU on the growth, DNA and RNA contents and protein synthesis in the tobacco tumour tissue culture was studied in comparison with a homologous callus tissue. In conformity with expectations considerable decrease of DNA level in both tissues is a primary effect of HU activity. This results in the growth inhibition and in the secondary metabolic effects; these effects depend not only on the concentration of inhibitor but also on the age of tissue. In spite of some common features the character of these changes shows a distinct differentiation depending on the tissue type. TMs points to specific modifications of the biochemical regulation of growth in a tumour.

Tissue culture-induced genetic and epigenetic variation in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927) regenerants.

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Machczyńska, Joanna; Zimny, Janusz; Bednarek, Piotr Tomasz

2015-10-01

Plant regeneration via in vitro culture can induce genetic and epigenetic variation; however, the extent of such changes in triticale is not yet understood. In the present study, metAFLP, a variation of methylation-sensitive amplified fragment length polymorphism analysis, was used to investigate tissue culture-induced variation in triticale regenerants derived from four distinct genotypes using androgenesis and somatic embryogenesis. The metAFLP technique enabled identification of both sequence and DNA methylation pattern changes in a single experiment. Moreover, it was possible to quantify subtle effects such as sequence variation, demethylation, and de novo methylation, which affected 19, 5.5, 4.5% of sites, respectively. Comparison of variation in different genotypes and with different in vitro regeneration approaches demonstrated that both the culture technique and genetic background of donor plants affected tissue culture-induced variation. The results showed that the metAFLP approach could be used for quantification of tissue culture-induced variation and provided direct evidence that in vitro plant regeneration could cause genetic and epigenetic variation.

Generation of Functional Thyroid Tissue Using 3D-Based Culture of Embryonic Stem Cells.

Science.gov (United States)

Antonica, Francesco; Kasprzyk, Dominika Figini; Schiavo, Andrea Alex; Romitti, Mírian; Costagliola, Sabine

2017-01-01

During the last decade three-dimensional (3D) cultures of pluripotent stem cells have been intensively used to understand morphogenesis and molecular signaling important for the embryonic development of many tissues. In addition, pluripotent stem cells have been shown to be a valid tool for the in vitro modeling of several congenital or chronic human diseases, opening new possibilities to study their physiopathology without using animal models. Even more interestingly, 3D culture has proved to be a powerful and versatile tool to successfully generate functional tissues ex vivo. Using similar approaches, we here describe a protocol for the generation of functional thyroid tissue using mouse embryonic stem cells and give all the details and references for its characterization and analysis both in vitro and in vivo. This model is a valid approach to study the expression and the function of genes involved in the correct morphogenesis of thyroid gland, to elucidate the mechanisms of production and secretion of thyroid hormones and to test anti-thyroid drugs.

Metabolomics reveals the heterogeneous secretome of two entomopathogenic fungi to ex vivo cultured insect tissues.

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Charissa de Bekker

Full Text Available Fungal entomopathogens rely on cellular heterogeneity during the different stages of insect host infection. Their pathogenicity is exhibited through the secretion of secondary metabolites, which implies that the infection life history of this group of environmentally important fungi can be revealed using metabolomics. Here metabolomic analysis in combination with ex vivo insect tissue culturing shows that two generalist isolates of the genus Metarhizium and Beauveria, commonly used as biological pesticides, employ significantly different arrays of secondary metabolites during infectious and saprophytic growth. It also reveals that both fungi exhibit tissue specific strategies by a distinguishable metabolite secretion on the insect tissues tested in this study. In addition to showing the important heterogeneous nature of these two entomopathogens, this study also resulted in the discovery of several novel destruxins and beauverolides that have not been described before, most likely because previous surveys did not use insect tissues as a culturing system. While Beauveria secreted these cyclic depsipeptides when encountering live insect tissues, Metarhizium employed them primarily on dead tissue. This implies that, while these fungi employ comparable strategies when it comes to entomopathogenesis, there are most certainly significant differences at the molecular level that deserve to be studied.

Clonal fidelity of chrysanthemum regenerated from long term cultures

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Jevremović Slađana

2006-01-01

Full Text Available Morphological characteristics of flowers of long term regenerated chrysanthemum, cv. "White Spider", after ten years of micropropagation are investigated. Shoot cultures are established and maintained more than ten years by stem segment culture on MS medium supplemented with BAP and NAA (1.0, 0.1 mgL-1, respectively. Rooting of shoots (100 % has done on MS medium without hormones and it was very successful after ten years, as well as, after two or eight years of micropropagation. Acclimation of rooted chrysanthemum plantlets at greenhouse conditions was excellent and after appropriate photoperiod "in vitro" plants flowered 90.3 % and have the same flower color, shape and size as mother plants. Flower color changes of "in vitro" plants are observed during another flowering cycle one year after acclimatization. Observed variations of chrysanthemum flowers could be attributed to epigenetic factors.

Likelihood-Based Inference of B Cell Clonal Families.

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Duncan K Ralph

2016-10-01

Full Text Available The human immune system depends on a highly diverse collection of antibody-making B cells. B cell receptor sequence diversity is generated by a random recombination process called "rearrangement" forming progenitor B cells, then a Darwinian process of lineage diversification and selection called "affinity maturation." The resulting receptors can be sequenced in high throughput for research and diagnostics. Such a collection of sequences contains a mixture of various lineages, each of which may be quite numerous, or may consist of only a single member. As a step to understanding the process and result of this diversification, one may wish to reconstruct lineage membership, i.e. to cluster sampled sequences according to which came from the same rearrangement events. We call this clustering problem "clonal family inference." In this paper we describe and validate a likelihood-based framework for clonal family inference based on a multi-hidden Markov Model (multi-HMM framework for B cell receptor sequences. We describe an agglomerative algorithm to find a maximum likelihood clustering, two approximate algorithms with various trade-offs of speed versus accuracy, and a third, fast algorithm for finding specific lineages. We show that under simulation these algorithms greatly improve upon existing clonal family inference methods, and that they also give significantly different clusters than previous methods when applied to two real data sets.

Nanotechnology, Cell Culture and Tissue Engineering

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Kazutoshi Haraguchi

2011-01-01

Full Text Available We have fabricated new types of polymer hydrogels and polymer nanocomposites, i.e., nanocomposite gels (NC gels and soft, polymer nanocomposites (M-NCs: solid, with novel organic/inorganic network structures. Both NC gels and M-NCs were synthesized by in-situ free-radical polymerization in the presence of exfoliated clay platelets in aqueous systems and were obtained in various forms such as film, sheet, tube, coating, etc. and sizes with a wide range of clay contents. Here, disk-like inorganic clay nanoparticles act as multi-functional crosslinkers to form new types of network systems. Both NC gels and M-NCs have extraordinary optical and mechanical properties including ultra-high reversible extensibility, as well as a number of new characteristics relating to optical anisotropy, polymer/clay morphology, biocompatibility, stimuli-sensitive surfaces, micro-patterning, etc. For examples, the biological testing of medical devices, comprised of a sensitization test, an irritation test, an intracutaneous test and an in vitro cytotoxicity test,was carried out for NC gels and M-NCs. The safety of NC gels and M-NCs was confirmed in all tests. Also, the interaction of living tissue with NC gel was investigated in vivo by implantation in live goats; neither inflammation nor concrescence occurred around the NC gels. Furthermore, it was found that both N-NC gels consisting of poly(N-isopropylacrylamide(PNIPA/clay network and M-NCs consisting of poly(2-methoxyethyacrylate(PMEA/clay network show characteristic cell culture and subsequent cell detachment on their surfaces, although it was almost impossible to culture cells on conventional, chemically-crosslinked PNIPA hydrogels and chemically crossslinked PMEA, regardless of their crosslinker concentration. Various kinds of cells, such ashumanhepatoma cells (HepG2, normal human dermal fibroblast (NHDF, and human umbilical vein endothelial cells (HUVEC, could be cultured to be confluent on the surfaces of N

Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

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Sunita Nayak

Full Text Available The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-β, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1β and nitric oxide production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

Quality improvement in Vignoles through clonal selection

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Our goal is to select an improved, loose-clustered clone of Vignoles that will contribute to an integrated approach to disease control. Clonal selection has historically proven useful in reducing cluster compactness through a variety of mechanisms, including decreased berry size, lengthening of the ...

Micropropagation and maintenance of phytoplasmas in tissue culture.

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Bertaccini, Assunta; Paltrinieri, Samanta; Martini, Marta; Tedeschi, Mara; Contaldo, Nicoletta

2013-01-01

Maintenance of phytoplasma strains in tissue culture is achievable for all strains transmitted to periwinkle (Catharanthus roseus), and also for other naturally infected plant host species. Shoots of 1-3 cm length are grown in a solid medium containing Murashige and Skoog (MS) micro- and macroelements and 0.12 mg/L benzylaminopurine. The continued presence of phytoplasmas in infected shoots of periwinkle that have been maintained in micropropagation for up to 20 years can be shown by diagnostic methods such as nested PCR tests using the 16S rDNA gene (see Chapters 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,and 26 for phytoplasma diagnostic methods).

Study on rapid propagation of Zanhuang Chinese jujube by tissue culture

International Nuclear Information System (INIS)

Li Yun; Wang Yu; Tian Yanting

2002-01-01

Zanhuang jujube is a very precious and rare variety of Chinese jujube. Its development was restricted by the under-developed propagate technique in history. The rapid propagation by tissue culture was studied and the optimum media were screened out. Through studying the condition of initial, proliferating, acclimatizing and rooting culture, 4 media, MS +6-BA 0.5 mg/L+IBA 0.1 mg/L, MS+6-BA 1.5 mg/L+IBA 0.1-0.2 mg/L, MS+KT 0.5 mg/L+NAA 0.2 mg/L and 1/2 MS+IBA 0.6 mg/L+NAA 0.2-0.3 mg/L were selected respectively

Plant cell tissue culture: A potential source of chemicals

Energy Technology Data Exchange (ETDEWEB)

Scott, C.D.; Dougall, D.K.

1987-08-01

Higher plants produce many industrially important products. Among these are drugs and medicinal chemicals, essential oils and flavors, vegetable oils and fats, fine and specialty chemicals, and even some commodity chemicals. Although, currently, whole-plant extraction is the primary means of harvesting these materials, the advent of plant cell tissue culture could be a much more effective method of producing many types of phytochemicals. The use of immobilized plant cells in an advanced bioreactor configuration with excretion of the product into the reactor medium may represent the most straightforward way of commercializing such techniques for lower-value chemicals. Important research and development opportunities in this area include screening for plant cultures for nonmedical, lower-value chemicals; understanding and controlling plant cell physiology and biochemistry; optimizing effective immobilization methods; developing more efficient bioreactor concepts; and perfecting product extraction and purification techniques. 62 refs., 2 figs.

Isolation of Lysosomes from Mammalian Tissues and Cultured Cells.

Science.gov (United States)

Aguado, Carmen; Pérez-Jiménez, Eva; Lahuerta, Marcos; Knecht, Erwin

2016-01-01

Lysosomes participate within the cells in the degradation of organelles, macromolecules, and a wide variety of substrates. In any study on specific roles of lysosomes, both under physiological and pathological conditions, it is advisable to include methods that allow their reproducible and reliable isolation. However, purification of lysosomes is a difficult task, particularly in the case of cultured cells. This is mainly because of the heterogeneity of these organelles, along with their low number and high fragility. Also, isolation methods, while disrupting plasma membranes, have to preserve the integrity of lysosomes, as the breakdown of their membranes releases enzymes that could damage all cell organelles, including themselves. The protocols described below have been routinely used in our laboratory for the specific isolation of lysosomes from rat liver, NIH/3T3, and other cultured cells, but can be adapted to other mammalian tissues or cell lines.

Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

DEFF Research Database (Denmark)

Meyer, Morten; Widmer, H R; Wagner, B

1998-01-01

of grafted dopaminergic neurons and to correlate that with the behavioral effects. Additional cultures and acutely prepared explants were also fixed and stored for histological investigation in order to estimate the loss of dopaminergic neurons in culture and after transplantation. Similar behavioral...... numbers of TH-immunoreactive (TH-ir) neurons in grafts of cultured tissue (775 +/- 98, mean +/- SEM) and grafts of fresh, dissociated cell suspension (806 +/- 105, mean +/- SEM). Cell counts in fresh explants, 7-day-old cultures, and grafted cultures revealed a 68.2% loss of TH-ir cells 7 days after......Ventral mesencephalon (VM) of fetal rat and human origin grown as free-floating roller-tube (FFRT) cultures can survive subsequent grafting to the adult rat striatum. To further explore the functional efficacy of such grafts, embryonic day 13 ventral mesencephalic tissue was grafted either after 7...

[Chronologic analysis of clonal evolution in acquired aplastic anemia and sMDS].

Science.gov (United States)

Yoshizato, Tetsuichi

2016-04-01

Acquired aplastic anemia (AA) is a prototype of idiopathic bone marrow failure, which is caused by immune-mediated destruction of hematopoietic progenitors but is also characterized by frequent evolution to clonal myeloid disorders, such as myelodysplastic syndromes or acute myeloid leukemia. However, the chronological behavior of the clonality and its link to myelodysplastic syndrome or acute myeloid leukemia has not been fully explored. To define the clonality and its chronological behavior in AA, we performed targeted sequencing (N=439) in cases with AA. Somatic mutations were detected in 1/3 of our cases. Mutations were most frequently found in DNMT3A, followed by BCOR, PIGA and ASXL1. The prevalence of mutations increased with age. The clone sizes in DNMT3A and ASXL1 were prone to increase, whereas those of BCOR and PIGA were more likely to decrease or remain stable. Mutations in PIGA, BCOR and BCORL1 correlated with a better response to immunosuppressive therapy and more favorable survival. On the other hand, other mutations were associated with worse outcomes. The chronological dynamics of clonality showed marked variability and were not necessarily associated with prognosis.

[Lymphocytic Clonal Expansion in Adult Patients with Epstein-Barr Virus-Associated Lymphoproliferative Disease].

Science.gov (United States)

Zhong, Feng-Luan; Zhang, Hong-Yu; Zhang, Qian; Feng, Jia; Zhang, Wen-Li; Xu, Lei; Xu, Hai-Chan; Wen, Juan-Juan; Meng, Qing-Xiang

2017-12-01

To explore the lymphocytic clonal expansion in adult patients with Epstein-Barr virus-associated lymphoproliferative diseases (EBV+LPD), and to investigate the experimental methods for EBV+LPD cells so as to provide a more objective measure for the diagnosis, classification and prognosis in the early stage of this disease. Peripheral blood samples from 5 patients with EBV+LPD, 4 patients with adult infectious mononucleosis(IM) as negative control and 3 patients with acute NK-cell leukemia(ANKL) as positive control were collected. Prior to immunochemotherapy, viral loads and clonality were analysed by flow cytometry (FCM), T cell receptor gene rearrangement (TCR) was detected by real-time polymerase chain reaction (RT-PCR), and diversity of EB virus terminal repeat (EBV-TR) was detected by Southern blot. FCM showed only 1 case with clonal TCRVβ in 5 patients with EBV+LPD, TCR clonal expansion could be detected both in patients with IM(4 of 4) and 4 patients with EBV+LPD(4 of 5), Out of patients with EBV+LPD, 1 patient displayed a monoclonal band and 2 patients showed oligoclonal bands when detecting EBV-TR by southen blot. Detecting the diversity of EBV-TR by Southern blot may be the most objective way to reflex clonal transformation of EBV+LPD, which is of great benefit to the diagnosis, classification and prognosis in the early stage of this disease.

PCR-based clonality assessment in patients with lymphocytic ...

Indian Academy of Sciences (India)

study was to assess the clinical benefits of clonality testing with previously evaluated .... with the samples, negative controls containing no DNA were ..... 2003 Standardization and quality con- ... In Molecular cloning, a laboratory manual (ed.

Cryptic changes in the genetic structure of a highly clonal coral population and the relationship with ecological performance

Science.gov (United States)

Williams, Dana E.; Miller, M. W.; Baums, I. B.

2014-09-01

Elkhorn coral , Acropora palmata, relies heavily on clonal propagation and often displays low genotypic (clonal) diversity. Populations in the Florida Keys experienced rapid declines in tissue cover between 2004 and 2006, largely due to hurricanes and disease, but remained stable from 2006 to 2010. All elkhorn colonies in 150 m2 permanent study plots were genotyped in 2006 ( n = 15 plots) and 2010 ( n = 24 plots), and plots sampled in both years were examined for changes in allelic and genotypic diversity during this period of stable ecological abundance. Overall, genetic diversity of Florida plots was low and declined further over the 4-yr period; seven of the 36 original genets and two of 67 alleles (among five microsatellite loci) were lost completely from the sampled population, and an additional 15 alleles were lost from individual reefs. In 2010, Florida plots (~19 colonies) contained an average of 2.2 ± 1.38 (mean ± SD) genets with a significant negative correlation between colony abundance and genotypic diversity. When scaled to total tissue abundance, genotypic diversity is even lower, with 43 % of genets below the size of sexual maturity. We examined the hypothesized positive relationship of local genotypic diversity with ecological performance measures. In Florida plots ( n = 15), genotypic diversity was not significantly correlated with tissue loss associated with chronic predation, nor with acute disease and storm-fragmentation events, though this relationship may be obscured by the low range of observed diversity and potential confounding with abundance. When more diverse plots in Curaçao ( n = 9) were examined, genotypic diversity was not significantly correlated with resistance during an acute storm disturbance or rate of recovery following disturbance. Cryptic loss of genetic diversity occurred in the apparently stable Florida population and confirms that stable or even increasing abundance does not necessarily indicate genetic stability.

Review of vascularised bone tissue-engineering strategies with a focus on co-culture systems.

Science.gov (United States)

Liu, Yuchun; Chan, Jerry K Y; Teoh, Swee-Hin

2015-02-01

Poor angiogenesis within tissue-engineered grafts has been identified as a main challenge limiting the clinical introduction of bone tissue-engineering (BTE) approaches for the repair of large bone defects. Thick BTE grafts often exhibit poor cellular viability particularly at the core, leading to graft failure and lack of integration with host tissues. Various BTE approaches have been explored for improving vascularisation in tissue-engineered constructs and are briefly discussed in this review. Recent investigations relating to co-culture systems of endothelial and osteoblast-like cells have shown evidence of BTE efficacy in increasing vascularization in thick constructs. This review provides an overview of key concepts related to bone formation and then focuses on the current state of engineered vascularized co-culture systems using bone repair as a model. It will also address key questions regarding the generation of clinically relevant vascularized bone constructs as well as potential directions and considerations for research with the objective of pursuing engineered co-culture systems in other disciplines of vascularized regenerative medicine. The final objective is to generate serious and functional long-lasting vessels for sustainable angiogenesis that will enable enhanced cellular survival within thick voluminous bone grafts, thereby aiding in bone formation and remodelling in the long term. However, more evidence about the quality of blood vessels formed and its associated functional improvement in bone formation as well as a mechanistic understanding of their interactions are necessary for designing better therapeutic strategies for translation to clinical settings. Copyright © 2012 John Wiley & Sons, Ltd.

Antiandrogenic actions of medroxyprogesterone acetate on epithelial cells within normal human breast tissues cultured ex vivo.

Science.gov (United States)

Ochnik, Aleksandra M; Moore, Nicole L; Jankovic-Karasoulos, Tanja; Bianco-Miotto, Tina; Ryan, Natalie K; Thomas, Mervyn R; Birrell, Stephen N; Butler, Lisa M; Tilley, Wayne D; Hickey, Theresa E

2014-01-01

Medroxyprogesterone acetate (MPA), a component of combined estrogen-progestin therapy (EPT), has been associated with increased breast cancer risk in EPT users. MPA can bind to the androgen receptor (AR), and AR signaling inhibits cell growth in breast tissues. Therefore, the aim of this study was to investigate the potential of MPA to disrupt AR signaling in an ex vivo culture model of normal human breast tissue. Histologically normal breast tissues from women undergoing breast surgical operation were cultured in the presence or in the absence of the native AR ligand 5α-dihydrotestosterone (DHT), MPA, or the AR antagonist bicalutamide. Ki67, bromodeoxyuridine, B-cell CLL/lymphoma 2 (BCL2), AR, estrogen receptor α, and progesterone receptor were detected by immunohistochemistry. DHT inhibited the proliferation of breast epithelial cells in an AR-dependent manner within tissues from postmenopausal women, and MPA significantly antagonized this androgenic effect. These hormonal responses were not commonly observed in cultured tissues from premenopausal women. In tissues from postmenopausal women, DHT either induced or repressed BCL2 expression, and the antiandrogenic effect of MPA on BCL2 was variable. MPA significantly opposed the positive effect of DHT on AR stabilization, but these hormones had no significant effect on estrogen receptor α or progesterone receptor levels. In a subset of postmenopausal women, MPA exerts an antiandrogenic effect on breast epithelial cells that is associated with increased proliferation and destabilization of AR protein. This activity may contribute mechanistically to the increased risk of breast cancer in women taking MPA-containing EPT.

Participation of cob tissue in the transport of medium components into maize kernels cultured in vitro

International Nuclear Information System (INIS)

Felker, F.C.

1990-01-01

Maize (Zea mays L.) kernels cultured in vitro while still attached to cob pieces have been used as a model system to study the physiology of kernel development. In this study, the role of the cob tissue in uptake of medium components into kernels was examined. Cob tissue was essential for in vitro kernel growth, and better growth occurred with larger cob/kernel ratios. A symplastically transported fluorescent dye readily permeated the endosperm when supplied in the medium, while an apoplastic dye did not. Slicing the cob tissue to disrupt vascular connections, but not apoplastic continuity, greatly reduced [ 14 C]sucrose uptake into kernels. [ 14 C]Sucrose uptake by cob and kernel tissue was reduced 31% and 68%, respectively, by 5 mM PCMBS. L-[ 14 C]glucose was absorbed much more slowly than D-[ 14 C]glucose. These and other results indicate that phloem loading of sugars occurs in the cob tissue. Passage of medium components through the symplast cob tissue may be a prerequisite for uptake into the kernel. Simple diffusion from the medium to the kernels is unlikely. Therefore, the ability of substances to be transported into cob tissue cells should be considered in formulating culture medium

Comparison of Stevia plants grown from seeds, cuttings and stem-tip cultures for growth and sweet diterpene glucosides.

Science.gov (United States)

Tamura, Y; Nakamura, S; Fukui, H; Tabata, M

1984-10-01

The growth and sweet diterpene glucosides of Stevia plants propagated by stem-tip cultures were compared with those of the control plants propagated by seeds. There was no significant difference between the two groups both in growth and in chemical composition. As for the contents of sweet diterpene glucosides, however, the clonal plants showed significantly smaller variations than the sexually propagated plants; they were almost as homogeneous as the plants propagated by cuttings. These results suggest that the clonal propagation by stem-tip culture is an effective method of obtaining a population of uniform plants for the production of sweet diterpene glucosides.

Integration Site and Clonal Expansion in Human Chronic Retroviral Infection and Gene Therapy

Science.gov (United States)

Niederer, Heather A.; Bangham, Charles R. M.

2014-01-01

Retroviral vectors have been successfully used therapeutically to restore expression of genes in a range of single-gene diseases, including several primary immunodeficiency disorders. Although clinical trials have shown remarkable results, there have also been a number of severe adverse events involving malignant outgrowth of a transformed clonal population. This clonal expansion is influenced by the integration site profile of the viral integrase, the transgene expressed, and the effect of the viral promoters on the neighbouring host genome. Infection with the pathogenic human retrovirus HTLV-1 also causes clonal expansion of cells containing an integrated HTLV-1 provirus. Although the majority of HTLV-1-infected people remain asymptomatic, up to 5% develop an aggressive T cell malignancy. In this review we discuss recent findings on the role of the genomic integration site in determining the clonality and the potential for malignant transformation of cells carrying integrated HTLV-1 or gene therapy vectors, and how these results have contributed to the understanding of HTLV-1 pathogenesis and to improvements in gene therapy vector safety. PMID:25365582

Variation in primary and culture-expanded cells derived from connective tissue progenitors in human bone marrow space, bone trabecular surface and adipose tissue.

Science.gov (United States)

Qadan, Maha A; Piuzzi, Nicolas S; Boehm, Cynthia; Bova, Wesley; Moos, Malcolm; Midura, Ronald J; Hascall, Vincent C; Malcuit, Christopher; Muschler, George F

2018-03-01

Connective tissue progenitors (CTPs) embody the heterogeneous stem and progenitor cell populations present in native tissue. CTPs are essential to the formation and remodeling of connective tissue and represent key targets for tissue-engineering and cell-based therapies. To better understand and characterize CTPs, we aimed to compare the (i) concentration and prevalence, (ii) early in vitro biological behavior and (iii) expression of surface-markers and transcription factors among cells derived from marrow space (MS), trabecular surface (TS), and adipose tissues (AT). Cancellous-bone and subcutaneous-adipose tissues were collected from 8 patients. Cells were isolated and cultured. Colony formation was assayed using Colonyze software based on ASTM standards. Cell concentration ([Cell]), CTP concentration ([CTP]) and CTP prevalence (P CTP ) were determined. Attributes of culture-expanded cells were compared based on (i) effective proliferation rate and (ii) expression of surface-markers CD73, CD90, CD105, SSEA-4, SSEA-3, SSEA-1/CD15, Cripto-1, E-Cadherin/CD324, Ep-CAM/CD326, CD146, hyaluronan and transcription factors Oct3/4, Sox-2 and Nanog using flow cytometry. Mean [Cell], [CTP] and P CTP were significantly different between MS and TS samples (P = 0.03, P = 0.008 and P= 0.0003), respectively. AT-derived cells generated the highest mean total cell yield at day 6 of culture-4-fold greater than TS and more than 40-fold greater than MS per million cells plated. TS colonies grew with higher mean density than MS colonies (290 ± 11 versus 150 ± 11 cell per mm 2 ; P = 0.0002). Expression of classical-mesenchymal stromal cell (MSC) markers was consistently recorded (>95%) from all tissue sources, whereas all the other markers were highly variable. The prevalence and biological potential of CTPs are different between patients and tissue sources and lack variation in classical MSC markers. Other markers are more likely to discriminate differences

Clonal diversity and fine-scale genetic structure in a high andean treeline population

Czech Academy of Sciences Publication Activity Database

Peng, Y.; Macek, P.; Macková, Jana; Romoleroux, K.; Hensen, I.

2015-01-01

Roč. 47, č. 1 (2015), s. 59-65 ISSN 0006-3606 Grant - others:GA AV ČR(CZ) IAA601110702; GA MŠk(CZ) LM2010009 Program:IA Institutional support: RVO:60077344 Keywords : AFLP * clonal diversity * clonal propagation * fine-scale genetic structure * Polylepis reticulata * treeline Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.944, year: 2015

Variation of functional clonal traits along elevation in two fern species

International Nuclear Information System (INIS)

Song, Y.B.; Chen, L.Y.; Xiong, W.

2015-01-01

Phenotypical plasticity is generally considered among adaptive strategies by which plants can cope with environmental variation in space and time. Although much is known about plasticity in seed plants in terms of functional clonal traits while little is known about ferns. Variation of functional clonal traits of two ferns Dicranopteris dichotoma and Diplopterygium glaucum among plots differing in elevation in a subtropical evergreen broad-leaved forest in southern China was investigated. Along with elevation increasing the two fern species showed similar variation pattern of functional clonal traits: stable spacer length, increasing specific spacer length and decreasing spacer weight per ramet and specific spacer weight. The two ferns species had similar variation pattern of ramet performance traits but different variation pattern of ramet population properties. These results suggest an evolutionary trade-off between functions of foraging for and storing of resources in the two ferns, with a functional preference for the foraging in response to environmental change. (author)

Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

International Nuclear Information System (INIS)

Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M.

1990-01-01

The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures

Cryopreservation of testicular tissue before long-term testicular cell culture does not alter in vitro cell dynamics

NARCIS (Netherlands)

Baert, Yoni; Braye, Aude; Struijk, Robin B.; van Pelt, Ans M. M.; Goossens, Ellen

2015-01-01

To assess whether testicular cell dynamics are altered during long-term culture after testicular tissue cryopreservation. Experimental basic science study. Reproductive biology laboratory. Testicular tissue with normal spermatogenesis was obtained from six donors. None. Detection and comparison of

Lipid-mediated glial cell line-derived neurotrophic factor gene transfer to cultured porcine ventral mesencephalic tissue

DEFF Research Database (Denmark)

Bauer, Matthias; Meyer, Morten; Brevig, Thomas

2002-01-01

Transplantation of dopaminergic ventral mesencephalic (VM) tissue into the basal ganglia of patients with Parkinson's disease (PD) shows at best moderate symptomatic relief in some of the treated cases. Experimental animal studies and clinical trials with allogenic and xenogenic pig-derived VM...... tissue grafts to PD patients indicate that one reason for the poor outcome of neural transplantation is the low survival and differentiation of grafted dopaminergic neurons. To improve dopaminergic cell survival through a gene-therapeutic approach we have established and report here results of lipid-mediated...... numbers of tyrosine hydroxylase-positive neurons in the cultured VM tissue. We conclude that lipid-mediated gene transfer employed on embryonic pig VM explant cultures is a safe and effective method to improve survival of dopaminergic neurons and may become a valuable tool to improve allo...

Altered Loyalties of Neuronal Markers in Cultured Slices of Resected Human Brain Tissue

NARCIS (Netherlands)

Verwer, Ronald W. H.; Sluiter, Arja A.; Balesar, Rawien A.; Baayen, Johannes C.; Speijer, Dave; Idema, Sander; Swaab, Dick F.

2016-01-01

Organotypic cultures from normal neocortical tissue obtained at epilepsy surgery show a severe injury response. This response involves both neuronal degeneration and the proliferation of reactive cells. A salient feature of the reactive cells is the co-expression of microglial and astrocytic

Diverse cellular architecture of atherosclerotic plaque derives from clonal expansion of a few medial SMCs

DEFF Research Database (Denmark)

Jacobsen, Kevin; Lund, Marie Bek; Shim, Jeong

2017-01-01

Fibrous cap smooth muscle cells (SMCs) protect atherosclerotic lesions from rupturing and causing thrombosis, while other plaque SMCs may have detrimental roles in plaque development. To gain insight into recruitment of different plaque SMCs, we mapped their clonal architecture in aggregation...... in the cap and heterogeneous ACTA2– SMCs in the plaque interior, including chondrocyte-like cells and cells with intracellular lipid and crystalline material. Fibrous cap SMCs were invariably arranged in endothelium-aligned clonal sheets, confirming results in the aggregation chimeras. Analysis of the clonal...

Emergence of clonal hematopoiesis in the majority of patients with acquired aplastic anemia.

Science.gov (United States)

Babushok, Daria V; Perdigones, Nieves; Perin, Juan C; Olson, Timothy S; Ye, Wenda; Roth, Jacquelyn J; Lind, Curt; Cattier, Carine; Li, Yimei; Hartung, Helge; Paessler, Michele E; Frank, Dale M; Xie, Hongbo M; Cross, Shanna; Cockroft, Joshua D; Podsakoff, Gregory M; Monos, Dimitrios; Biegel, Jaclyn A; Mason, Philip J; Bessler, Monica

2015-04-01

Acquired aplastic anemia (aAA) is a nonmalignant disease caused by autoimmune destruction of early hematopoietic cells. Clonal hematopoiesis is a late complication, seen in 20-25% of older patients. We hypothesized that clonal hematopoiesis in aAA is a more general phenomenon, which can arise early in disease, even in younger patients. To evaluate clonal hematopoiesis in aAA, we used comparative whole exome sequencing of paired bone marrow and skin samples in 22 patients. We found somatic mutations in 16 patients (72.7%) with a median disease duration of 1 year; of these, 12 (66.7%) were patients with pediatric-onset aAA. Fifty-eight mutations in 51 unique genes were found primarily in pathways of immunity and transcriptional regulation. Most frequently mutated was PIGA, with seven mutations. Only two mutations were in genes recurrently mutated in myelodysplastic syndrome. Two patients had oligoclonal loss of the HLA alleles, linking immune escape to clone emergence. Two patients had activating mutations in key signaling pathways (STAT5B (p.N642H) and CAMK2G (p.T306M)). Our results suggest that clonal hematopoiesis in aAA is common, with two mechanisms emerging-immune escape and increased proliferation. Our findings expand conceptual understanding of this nonneoplastic blood disorder. Future prospective studies of clonal hematopoiesis in aAA will be critical for understanding outcomes and for designing personalized treatment strategies. Copyright © 2015 Elsevier Inc. All rights reserved.

Clonality, virulence and antimicrobial resistance of enteroaggregative Escherichia coli from Mirzapur, Bangladesh.

Science.gov (United States)

Chattaway, Marie Anne; Day, Michaela; Mtwale, Julia; White, Emma; Rogers, James; Day, Martin; Powell, David; Ahmad, Marwa; Harris, Ross; Talukder, Kaisar Ali; Wain, John; Jenkins, Claire; Cravioto, Alejandro

2017-10-01

This study investigates the virulence and antimicrobial resistance in association with common clonal complexes (CCs) of enteroaggregative Escherichia coli (EAEC) isolated from Bangladesh. The aim was to determine whether specific CCs were more likely to be associated with putative virulence genes and/or antimicrobial resistance. The presence of 15 virulence genes (by PCR) and susceptibility to 18 antibiotics were determined for 151 EAEC isolated from cases and controls during an intestinal infectious disease study carried out between 2007-2011 in the rural setting of Mirzapur, Bangladesh (Kotloff KL, Blackwelder WC, Nasrin D, Nataro JP, Farag TH et al.Clin Infect Dis 2012;55:S232-S245). These data were then analysed in the context of previously determined serotypes and clonal complexes defined by multi-locus sequence typing. Overall there was no association between the presence of virulence or antimicrobial resistance genes in isolates of EAEC from cases versus controls. However, when stratified by clonal complex (CC) one CC associated with cases harboured more virulence factors (CC40) and one CC harboured more resistance genes (CC38) than the average. There was no direct link between the virulence gene content and antibiotic resistance. Strains within a single CC had variable virulence and resistance gene content indicating independent and multiple gene acquisitions over time. In Bangladesh, there are multiple clonal complexes of EAEC harbouring a variety of virulence and resistance genes. The emergence of two of the most successful clones appeared to be linked to either increased virulence (CC40) or antimicrobial resistance (CC38), but increased resistance and virulence were not found in the same clonal complexes.

Dynamic culture induces a cell type-dependent response impacting on the thickness of engineered connective tissues.

Science.gov (United States)

Fortier, Guillaume Marceau; Gauvin, Robert; Proulx, Maryse; Vallée, Maud; Fradette, Julie

2013-04-01

Mesenchymal cells are central to connective tissue homeostasis and are widely used for tissue-engineering applications. Dermal fibroblasts and adipose-derived stromal cells (ASCs) allow successful tissue reconstruction by the self-assembly approach of tissue engineering. This method leads to the production of multilayered tissues, devoid of exogenous biomaterials, that can be used as stromal compartments for skin or vesical reconstruction. These tissues are formed by combining cell sheets, generated through cell stimulation with ascorbic acid, which favours the cell-derived production/organization of matrix components. Since media motion can impact on cell behaviour, we investigated the effect of dynamic culture on mesenchymal cells during tissue reconstruction, using the self-assembly method. Tissues produced using ASCs in the presence of a wave-like movement were nearly twice thicker than under standard conditions, while no difference was observed for tissues produced from dermal fibroblasts. The increased matrix deposition was not correlated with an increased proliferation of ASCs, or by higher transcript levels of fibronectin or collagens I and III. A 30% increase of type V collagen mRNA was observed. Interestingly, tissues engineered from dermal fibroblasts featured a four-fold higher level of MMP-1 transcripts under dynamic conditions. Mechanical properties were similar for tissues reconstructed using dynamic or static conditions. Finally, cell sheets produced using ASCs under dynamic conditions could readily be manipulated, resulting in a 2 week reduction of the production time (from 5 to 3 weeks). Our results describe a distinctive property of ASCs' response to media motion, indicating that their culture under dynamic conditions leads to optimized tissue engineering. Copyright © 2011 John Wiley & Sons, Ltd.

Organotypic culture of human bone marrow adipose tissue.

Science.gov (United States)

Uchihashi, Kazuyoshi; Aoki, Shigehisa; Shigematsu, Masamori; Kamochi, Noriyuki; Sonoda, Emiko; Soejima, Hidenobu; Fukudome, Kenji; Sugihara, Hajime; Hotokebuchi, Takao; Toda, Shuji

2010-04-01

The precise role of bone marrow adipose tissue (BMAT) in the marrow remains unknown. The purpose of the present study was therefore to describe a novel method for studying BMAT using 3-D collagen gel culture of BMAT fragments, immunohistochemistry, ELISA and real-time reverse transcription-polymerase chain reaction. Mature adipocytes and CD45+ leukocytes were retained for >3 weeks. Bone marrow stromal cells (BMSC) including a small number of lipid-laden preadipocytes and CD44+/CD105+ mesenchymal stem cell (MSC)-like cells, developed from BMAT. Dexamethasone (10 micromol/L), but not insulin (20 mU/mL), significantly increased the number of preadipocytes. Dexamethasone and insulin also promoted leptin production and gene expression in BMAT. Adiponectin production by BMAT was BMAT, in which adiponectin protein secretion is normally very low, and that BMAT may exhibit a different phenotype from that of the visceral and subcutaneous adipose tissues. BMAT-osteoblast interactions were also examined, and it was found that osteoblasts inhibited the development of BMSC and reduced leptin production, while BMAT inhibited the growth and differentiation of osteoblasts. The present novel method proved to be useful for the study of BMAT biology.

Analysis of the clonal repertoire of gene-corrected cells in gene therapy.

Science.gov (United States)

Paruzynski, Anna; Glimm, Hanno; Schmidt, Manfred; Kalle, Christof von

2012-01-01

Gene therapy-based clinical phase I/II studies using integrating retroviral vectors could successfully treat different monogenetic inherited diseases. However, with increased efficiency of this therapy, severe side effects occurred in various gene therapy trials. In all cases, integration of the vector close to or within a proto-oncogene contributed substantially to the development of the malignancies. Thus, the in-depth analysis of integration site patterns is of high importance to uncover potential clonal outgrowth and to assess the safety of gene transfer vectors and gene therapy protocols. The standard and nonrestrictive linear amplification-mediated PCR (nrLAM-PCR) in combination with high-throughput sequencing exhibits technologies that allow to comprehensively analyze the clonal repertoire of gene-corrected cells and to assess the safety of the used vector system at an early stage on the molecular level. It enables clarifying the biological consequences of the vector system on the fate of the transduced cell. Furthermore, the downstream performance of real-time PCR allows a quantitative estimation of the clonality of individual cells and their clonal progeny. Here, we present a guideline that should allow researchers to perform comprehensive integration site analysis in preclinical and clinical studies. Copyright © 2012 Elsevier Inc. All rights reserved.

Distribution of clonal growth forms in wetlands

Czech Academy of Sciences Publication Activity Database

Sosnová, Monika; van Diggelen, R.; Klimešová, Jitka

2010-01-01

Roč. 92, č. 1 (2010), s. 33-39 ISSN 0304-3770 R&D Projects: GA ČR GD206/08/H044 Institutional research plan: CEZ:AV0Z60050516 Keywords : clonal growth organ * the Netherlands * wetland plant community Subject RIV: EF - Botanics Impact factor: 2.087, year: 2010

Potato transformation and potato cyst nematode infection on potato plantlets in tissue culture

Science.gov (United States)

These two protocols describe the methods for generating transgenic potato plants and for evaluating potato cyst nematode (Globodera rostochiensis and G. pallida) infection on potato plantlets in tissue culture. These methods are useful tools that can be used in the study of the interactions between ...

Clonal evolution following chemotherapy-induced stem cell depletion in cats heterozygous for glucose-6-phosphate dehydrogenase

International Nuclear Information System (INIS)

Abkowitz, J.L.; Ott, R.M.; Holly, R.D.; Adamson, J.W.

1988-01-01

The number of hematopoietic stem cells necessary to support normal hematopoiesis is not known but may be small. If so, the depletion or damage of such cells could result in apparent clonal dominance. To test this hypothesis, dimethylbusulfan [2 to 4 mg/kg intravenously (IV) x 3] was given to cats heterozygous for the X-linked enzyme glucose-6-phosphate dehydrogenase (G-6-PD). These cats were the daughters of domestic X Geoffroy parents. After the initial drug-induced cytopenias (2 to 4 weeks), peripheral blood counts and the numbers of marrow progenitors detected in culture remained normal, although the percentages of erythroid burst-forming cells (BFU-E) and granulocyte/macrophage colony-forming cells (CFU-GM) in DNA synthesis increased, as determined by the tritiated thymidine suicide technique. In three of six cats treated, a dominance of Geoffroy-type G-6-PD emerged among the progenitor cells, granulocytes, and RBCs. These skewed ratios of domestic to Geoffroy-type G-6-PD have persisted greater than 3 years. No changes in cell cycle kinetics or G-6-PD phenotypes were noted in similar studies in six control cats. These data suggest that clonal evolution may reflect the depletion or damage of normal stem cells and not only the preferential growth and dominance of neoplastic cells

Formation of proteoglycan and collagen-rich scaffold-free stiff cartilaginous tissue using two-step culture methods with combinations of growth factors.

Science.gov (United States)

Miyazaki, Tatsuya; Miyauchi, Satoshi; Matsuzaka, Satoshi; Yamagishi, Chie; Kobayashi, Kohei

2010-05-01

Tissue-engineered cartilage may be expected to serve as an alternative to autologous chondrocyte transplantation treatment. Several methods for producing cartilaginous tissue have been reported. In this study, we describe the production of scaffold-free stiff cartilaginous tissue of pig and human, using allogeneic serum and growth factors. The tissue was formed in a mold using chondrocytes recovered from alginate bead culture and maintained in a medium with transforming growth factor-beta and several other additives. In the case of porcine tissue, the tear strength of the tissue and the contents of proteoglycan (PG) and collagen per unit of DNA increased dose-dependently with transforming growth factor-beta. The length of culture was significantly and positively correlated with thickness, tear strength, and PG and collagen contents. Tear strength showed positive high correlations with both PG and collagen contents. A positive correlation was also seen between PG content and collagen content. Similar results were obtained with human cartilaginous tissue formed from chondrocytes expanded in monolayer culture. Further, an in vivo pilot study using pig articular cartilage defect model demonstrated that the cartilaginous tissue was well integrated with surrounding tissue at 13 weeks after the implantation. In conclusion, we successfully produced implantable scaffold-free stiff cartilaginous tissue, which characterized high PG and collagen contents.

Implementing oxygen control in chip-based cell and tissue culture systems.

Science.gov (United States)

Oomen, Pieter E; Skolimowski, Maciej D; Verpoorte, Elisabeth

2016-09-21

Oxygen is essential in the energy metabolism of cells, as well as being an important regulatory parameter influencing cell differentiation and function. Interest in precise oxygen control for in vitro cultures of tissues and cells continues to grow, especially with the emergence of the organ-on-a-chip and the desire to emulate in vivo conditions. This was recently discussed in this journal in a Critical Review by Brennan et al. (Lab Chip (2014). DOI: ). Microfluidics can be used to introduce flow to facilitate nutrient supply to and waste removal from in vitro culture systems. Well-defined oxygen gradients can also be established. However, cells can quickly alter the oxygen balance in their vicinity. In this Tutorial Review, we expand on the Brennan paper to focus on the implementation of oxygen analysis in these systems to achieve continuous monitoring. Both electrochemical and optical approaches for the integration of oxygen monitoring in microfluidic tissue and cell culture systems will be discussed. Differences in oxygen requirements from one organ to the next are a challenging problem, as oxygen delivery is limited by its uptake into medium. Hence, we discuss the factors determining oxygen concentrations in solutions and consider the possible use of artificial oxygen carriers to increase dissolved oxygen concentrations. The selection of device material for applications requiring precise oxygen control is discussed in detail, focusing on oxygen permeability. Lastly, a variety of devices is presented, showing the diversity of approaches that can be employed to control and monitor oxygen concentrations in in vitro experiments.

Age-related mutations associated with clonal hematopoietic expansion and malignancies.

Science.gov (United States)

Xie, Mingchao; Lu, Charles; Wang, Jiayin; McLellan, Michael D; Johnson, Kimberly J; Wendl, Michael C; McMichael, Joshua F; Schmidt, Heather K; Yellapantula, Venkata; Miller, Christopher A; Ozenberger, Bradley A; Welch, John S; Link, Daniel C; Walter, Matthew J; Mardis, Elaine R; Dipersio, John F; Chen, Feng; Wilson, Richard K; Ley, Timothy J; Ding, Li

2014-12-01

Several genetic alterations characteristic of leukemia and lymphoma have been detected in the blood of individuals without apparent hematological malignancies. The Cancer Genome Atlas (TCGA) provides a unique resource for comprehensive discovery of mutations and genes in blood that may contribute to the clonal expansion of hematopoietic stem/progenitor cells. Here, we analyzed blood-derived sequence data from 2,728 individuals from TCGA and discovered 77 blood-specific mutations in cancer-associated genes, the majority being associated with advanced age. Remarkably, 83% of these mutations were from 19 leukemia and/or lymphoma-associated genes, and nine were recurrently mutated (DNMT3A, TET2, JAK2, ASXL1, TP53, GNAS, PPM1D, BCORL1 and SF3B1). We identified 14 additional mutations in a very small fraction of blood cells, possibly representing the earliest stages of clonal expansion in hematopoietic stem cells. Comparison of these findings to mutations in hematological malignancies identified several recurrently mutated genes that may be disease initiators. Our analyses show that the blood cells of more than 2% of individuals (5-6% of people older than 70 years) contain mutations that may represent premalignant events that cause clonal hematopoietic expansion.

Effect of plant growth regulators on in vitro shoot multiplication of ...

African Journals Online (AJOL)

STORAGESEVER

2009-11-16

Nov 16, 2009 ... to other regions and continents which include the Middle. East, China, the Mediterranean region and America. (Ladizinsky, 1999). Besides ... makes the plant tissue culture techniques more valuable for the clonal propagation ...

Morphological, biochemical and genetic influence of mutagen treatments on medicinal plant tissue cultures

International Nuclear Information System (INIS)

Onisei, T.; Toth, E.; Tesio, B.; Floria, F.

1994-01-01

Gamma rays and/or alkylant agents have been applied on callus tissue, young regenerants and cell suspension in order to establish their effect on morphogenesis, regeneration ability and biosynthetic potential. Growth dynamics, morpho-anatomic variables, secondary metabolite production, cell cytogenetics, enzyme specific activities, isoperoxidase and isoesterase patterns were analyzed in relation to the morphogenetic response of Atropa belladonna, Datura innoxia, Lavandula angustifolia, Chamomilla recutita, Digitalis lanata and Vinca minor tissue cultures. The effects of gamma-ray doses varied from one species to another; 10 to 20 Gy were generally able to stimulate growth and plant regeneration (via organogenesis and somatic embryogenesis), while 10 to 50 Gy enhanced secondary metabolite biosynthesis both in callus and cell suspension culture. Semnificative increase of secondary metabolite production was obtained when treatments with EMS (0.1-0.2%) have been applied to young regenerants. Many differences in biological features and biochemical behaviour were registered 20 days and one year, respectively, after treatment. (author)

Novel R tools for analysis of genome-wide population genetic data with emphasis on clonality

Science.gov (United States)

To gain a detailed understanding of how plant microbes evolve and adapt to hosts, pesticides, and other factors, knowledge of the population dynamics and evolutionary history of populations is crucial. Plant pathogen populations are often clonal or partially clonal which requires different analytica...

Toxicity and oxidative stress of canine mesenchymal stromal cells from adipose tissue in different culture passages

Directory of Open Access Journals (Sweden)

Arícia Gomes Sprada

2015-12-01

Full Text Available Abstract: Stem cells in regenerative therapy have received attention from researchers in recent decades. The culture of these cells allows studies about their behavior and metabolism. Thus, cell culture is the basis for cell therapy and tissue engineering researches. A major concern regarding the use of cultivated stem cell in human or veterinary clinical routine is the risk of carcinogenesis. Cellular activities require a balanced redox state. However, when there is an imbalance in this state, oxidative stress occurs. Oxidative stress contributes to cytotoxicity, which may result in cell death or genomic alterations, favoring the development of cancer cells. The aim of this study was to determine whether there are differences in the behavior of cultured mesenchymal stem cells from canine adipose tissue according to its site of collection (omentum and subcutaneous evaluating the rate of proliferation, viability, level of oxidative stress and cytotoxicity over six passages. For this experiment, two samples of adipose tissue from subcutaneous and omentum where taken from a female dog corpse, 13 years old, Pitbull. The results showed greater levels of oxidative stress in the first and last passages of both groups, favoring cytotoxicity and cell death.

The release of bystander factor(s) from tissue explant cultures of rainbow trout (Onchorhynchus mykiss) after exposure to gamma radiation.

Science.gov (United States)

O'Dowd, Colm; Mothersill, Carmel E; Cairns, Michael T; Austin, Brian; McClean, Brendan; Lyng, Fiona M; Murphy, James E J

2006-10-01

The bystander response has been documented in cell lines and cell cultures derived from aquatic species over the past several years. However, little work has been undertaken to identify a similar bystander response in tissue explant cultures from fish. In this study, indirect effects of ionizing gamma radiation on tissue explant cultures of fish were investigated. Tissue explants in culture were exposed to 0.5 Gy and 5 Gy gamma radiation from a 60Co teletherapy unit. A bystander response in Epithelioma papulosum cyprini (EPC) cells exposed to gamma-irradiated tissue conditioned medium from rainbow trout explants was investigated, and the effects on cell survival were quantified by the clonogenic survival assay. Dichlorofluorescein and rhodamine 123 fluorescent dyes were used to identify alterations in reactive oxygen species (ROS) and mitochondrial membrane potential (MMP), respectively. Results indicate a different response for the three tissue types investigated. Clonogenic assay results vary from a decrease in cell survival (gill) to no effect (skin) to a stimulatory effect (spleen). Results from fluorescence assays of ROS and MMP show similarities to clonogenic assay results. This study identifies a useful model for further studies relating to the bystander effect in aquatic organisms in vivo and ex vivo.

Usefullness of IGH/TCR PCR studies in lymphoproliferative disorders with inconclusive clonality by flow cytometry.

Science.gov (United States)

Ribera, Jordi; Zamora, Lurdes; Juncà, Jordi; Rodríguez, Inés; Marcé, Silvia; Cabezón, Marta; Millá, Fuensanta

2013-07-25

In up to 5-15% of studies of lymphoproliferative disorders (LPD) flow cytometry (FCM) or immunomorphologic methods cannot discriminate malignant from reactive processes. The aim of this work was to determine the usefulness of PCR for solving these diagnostic uncertainties. We analyzed IGH and TCRγ genes by PCR in 106 samples with inconclusive FCM results. A clonal result was registered in 36/106 studies, with a LPD being confirmed in 27 (75%) of these cases. Specifically, 9/9 IGH clonal and 16/25 TCRγ clonal results were finally diagnosed with LPD. Additionally, 2 clonal TCRγ samples with suspicion of undefined LPD were finally diagnosed with T LPD. Although polyclonal results were obtained in 47 of the cases studied (38 IGH and 9 TCRγ), hematologic neoplasms were diagnosed in 4/38 IGH polyclonal and in 1/9 TCRγ polyclonal studies. There were also 14 PCR polyclonal results (4 IGH, 10 TCRγ), albeit non-conclusive. Of these, 2/4 were eventually diagnosed with B-cell lymphoma and 3/10 with T-cell LPD. In 8 IGH samples the results of PCR techniques were non-informative but in 3/8 cases a B lymphoma was finally confirmed. We concluded that PCR is a useful technique to identify LPD when FCM is inconclusive. A PCR clonal B result is indicative of malignancy but IGH polyclonal and non-conclusive results do not exclude lymphoid neoplasms. Interpretation of T-cell clonality should be based on all the available clinical and analytical data. © 2013 Clinical Cytometry Society. Copyright © 2013 Clinical Cytometry Society.

Three-Dimensional Culture Model of Skeletal Muscle Tissue with Atrophy Induced by Dexamethasone.

Science.gov (United States)

Shimizu, Kazunori; Genma, Riho; Gotou, Yuuki; Nagasaka, Sumire; Honda, Hiroyuki

2017-06-15

Drug screening systems for muscle atrophy based on the contractile force of cultured skeletal muscle tissues are required for the development of preventive or therapeutic drugs for atrophy. This study aims to develop a muscle atrophy model by inducing atrophy in normal muscle tissues constructed on microdevices capable of measuring the contractile force and to verify if this model is suitable for drug screening using the contractile force as an index. Tissue engineered skeletal muscles containing striated myotubes were prepared on the microdevices for the study. The addition of 100 µM dexamethasone (Dex), which is used as a muscle atrophy inducer, for 24 h reduced the contractile force significantly. An increase in the expression of Atrogin-1 and MuRF-1 in the tissues treated with Dex was established. A decrease in the number of striated myotubes was also observed in the tissues treated with Dex. Treatment with 8 ng/mL Insulin-like Growth Factor (IGF-I) for 24 h significantly increased the contractile force of the Dex-induced atrophic tissues. The same treatment, though, had no impact on the force of the normal tissues. Thus, it is envisaged that the atrophic skeletal muscle tissues induced by Dex can be used for drug screening against atrophy.

Clonal architectures and driver mutations in metastatic melanomas.

Directory of Open Access Journals (Sweden)

Li Ding

Full Text Available To reveal the clonal architecture of melanoma and associated driver mutations, whole genome sequencing (WGS and targeted extension sequencing were used to characterize 124 melanoma cases. Significantly mutated gene analysis using 13 WGS cases and 15 additional paired extension cases identified known melanoma genes such as BRAF, NRAS, and CDKN2A, as well as a novel gene EPHA3, previously implicated in other cancer types. Extension studies using tumors from another 96 patients discovered a large number of truncation mutations in tumor suppressors (TP53 and RB1, protein phosphatases (e.g., PTEN, PTPRB, PTPRD, and PTPRT, as well as chromatin remodeling genes (e.g., ASXL3, MLL2, and ARID2. Deep sequencing of mutations revealed subclones in the majority of metastatic tumors from 13 WGS cases. Validated mutations from 12 out of 13 WGS patients exhibited a predominant UV signature characterized by a high frequency of C->T transitions occurring at the 3' base of dipyrimidine sequences while one patient (MEL9 with a hypermutator phenotype lacked this signature. Strikingly, a subclonal mutation signature analysis revealed that the founding clone in MEL9 exhibited UV signature but the secondary clone did not, suggesting different mutational mechanisms for two clonal populations from the same tumor. Further analysis of four metastases from different geographic locations in 2 melanoma cases revealed phylogenetic relationships and highlighted the genetic alterations responsible for differential drug resistance among metastatic tumors. Our study suggests that clonal evaluation is crucial for understanding tumor etiology and drug resistance in melanoma.

Unexpected heterogeneity derived from Cas9 ribonucleoprotein-introduced clonal cells at the HPRT1 locus.

Science.gov (United States)

Sakuma, Tetsushi; Mochida, Keiji; Nakade, Shota; Ezure, Toru; Minagawa, Sachi; Yamamoto, Takashi

2018-04-01

Single-cell cloning is an essential technique for establishing genome-edited cell clones mediated by programmable nucleases such as CRISPR-Cas9. However, residual genome-editing activity after single-cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR-Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single-cell cloning of Cas9 RNP-transfected cells affected the heterogeneity of the resultant clones. We carried out transfection of Cas9 RNPs targeting several loci in the HPRT1 gene in HCT116 cells, followed by single-cell cloning at 24, 48, 72 hr and 1 week post-transfection. After approximately 3 weeks of incubation, the clonal cells were collected and genotyped by high-resolution microchip electrophoresis and Sanger sequencing. Unexpectedly, long-term incubation before single-cell cloning resulted in highly heterogeneous clones. We used a lipofection method for transfection, and the media containing transfectable RNPs were not removed before single-cell cloning. Therefore, the active Cas9 RNPs were considered to be continuously incorporated into cells during the precloning incubation. Our findings provide a warning that lipofection of Cas9 RNPs may cause continuous introduction of gene mutations depending on the experimental procedures. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices.

OpenAIRE

Christensen, G D; Simpson, W A; Younger, J J; Baddour, L M; Barrett, F F; Melton, D M; Beachey, E H

1985-01-01

The adherence of coagulase-negative staphylococci to smooth surfaces was assayed by measuring the optical densities of stained bacterial films adherent to the floors of plastic tissue culture plates. The optical densities correlated with the weight of the adherent bacterial film (r = 0.906; P less than 0.01). The measurements also agreed with visual assessments of bacterial adherence to culture tubes, microtiter plates, and tissue culture plates. Selected clinical strains were passed through ...

[Effects of endophytic fungi from Dendrobium officinale on host growth and components metabolism of tissue culture seedlings].

Science.gov (United States)

Zhu, Bo; Liu, Jing-Jing; Si, Jin-Ping; Qin, Lu-Ping; Han, Ting; Zhao, Li; Wu, Ling-Shang

2016-05-01

The paper aims to study the effects of endophytic fungi from D. officinale cultivated on living trees on growth and components metabolism of tissue culture seedlings. Morphological characteristics and agronomic characters of tissue culture seedlings infected and uninfected by endophytic fungus were observed and measured. Polysaccharides and alcohol-soluble extracts contents were determined by phenol-sulfuric acid method and hot-dipmethod, respectively. Monosacchride composition of polysaccharides and alcohol-soluble extracts components were analyzed by pre-column derivatives HPLC and HPLC method, respectively. It showed that effects of turning to purple of stem nodes could be changed by endophytic fungus. Besides, the endophytic fungus could affect the contents and constitutions of polysaccharides and alcohol-soluble extracts. The strains tested, expect DO34, could promote growth and polysaccharides content of tissue culture seedlings. The strains tested, expect DO12, could promote the accumulation of mannose. Furthermore, DO18, DO19 and DO120 could increase alcohol-soluble extracts. On the basis, four superior strains were selected for mechanism research between endophytic fungus and their hosts and microbiology engineering. Copyright© by the Chinese Pharmaceutical Association.

Primary microglia isolation from mixed glial cell cultures of neonatal rat brain tissue.

Science.gov (United States)

Tamashiro, Tami T; Dalgard, Clifton Lee; Byrnes, Kimberly R

2012-08-15

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted. Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study. The principle and protocol of this methodology have been described in the literature. Additionally, alternate methodologies to isolate primary microglia are well described. Homogenized brain tissue may be separated

Metabolic aspects of growth in HU-treated crown-gall tissue cultures. II. Helianthus annuus

Directory of Open Access Journals (Sweden)

Aldona Rennert

2015-01-01

Full Text Available The dynamics of growth and changes in nucleic acid and protein contents in sunflower calluses and tumours cultured in hydroxyurea (HU containing media were examined. HU-induced changes in healthy tissues ran in parallel always in the same direction, in tumourous ones however an uncoupling between DNA synthesis and tissue growth on one hand and RNA and protein synthesis on the other took place. A detailed analysis of the results allows to suppose that the specific activity of HU on tumourous tissue could be an index of: 1 quantitative disturbances in its genes function (2 degree of the lass of sensitivity to the factors of regulation.

Productivity gains by fertilisation in Eucalyptus urophylla clonal ...

African Journals Online (AJOL)

Productivity gains by fertilisation in Eucalyptus urophylla clonal plantations across gradients in site and stand conditions. ... The control plot may typically be a permanent plot of an inventory network, providing representative information for a company's decisionmaking. The paired twin-plot receives intensive management ...

Detection of clonal B cells in microdissected reactive lymphoproliferations: possible diagnostic pitfalls in PCR analysis of immunoglobulin heavy chain gene rearrangement

DEFF Research Database (Denmark)

Zhou, X.G.; Sandvej, K.; Gregersen, Niels

1999-01-01

Aims-To evaluate the specificity of standard and fluorescence based (GENESCAN) polymerase chain reaction (PCR) immunoglobulin heavy chain (IgH) gene rearrangement analysis in complete and microdissected paraffin wax embedded sections from lymphoid proliferations. Methods-PCR IgH gene rearrangement...... because of preferential priming or detection of local B cell clones. Data from clonal analysis of small, microdissected or lymphocyte poor samples must be evaluated critically. It is recommended that analyses should be run in parallel on at least two tissue specimens. Only reproducible bands present...

Uses of in vitro propagation for crop improvement

International Nuclear Information System (INIS)

Lazarte, J.E.

1984-01-01

For crop improvement the uses of tissue culture are multiple; tissue culture alone is important in clonal propagation and for developing new genotypes. Together with other techniques such as the ones used in mutation breeding, tissue culture will increase the chance for mutations to occur by increasing the number of meristems per area of treatment. With this combination of techniques there is the possibility of developing better crops in a shorter period, thus complementing the work done by nature and by traditional breeding methodology. This is important since world population is expected to double by the year 2000, which is only 18 years away. Tissue culture is a general term used to describe any method of aseptic culture, i.e. meristem, callus, cell, shoot tip, anther culture, etc

Limpeza clonal de mudas de videira infectadas por Xanthomonas campestris pv. viticola Clonal cleaning of grapevine plants infected by Xanthomonas campestris pv. viticola

Directory of Open Access Journals (Sweden)

Adriano Márcio Freire Silva

2013-03-01

Full Text Available O cancro bacteriano da videira é causado por Xanthomonas campestris pv. viticola (Xcv. Visando à limpeza clonal de mudas de 'Red Globe', foram estudados: tamanho ideal de ápices e gemas axilares para cultivo em meio de Galzy modificado (MGM; efeito da termoterapia (38ºC/30 dias; e ação de antibióticos na eliminação de Xcv em videiras infectadas. Os percentuais de contaminação por Xcv e de regeneração foram analisados, e as plantas obtidas foram indexadas em meio ágar nutritivo-dextrose-extrato de levedura-ampicilina (NYDAM, seguindo-se teste de patogenicidade. O cultivo de explantes com 3 mm possibilitou a obtenção de plantas livres da bactéria, com regeneração 14,3 vezes maior que explantes com 1 mm. A termoterapia de mudas infectadas, associada ao cultivo in vitro, não eliminou o patógeno. O cultivo de explantes com 10 mm, durante 40 dias em MGM + cefotaxima (300 mg L-1, proporcionou limpeza clonal das mudas. A indexação de plantas de videira regeneradas in vitro, quanto à infecção por Xcv utilizando NYDAM, seguida de teste de patogenicidade, é uma alternativa econômica e eficiente para produção de mudas de alta qualidade fitossanitária.Bacterial canker is caused by Xanthomonas campestris pv. viticola (Xcv. In order to eliminate Xcv from 'Red Globe' plants it was studied: optimal size of meristem tips and axillary buds for cultivation in modified Galzy's medium (MGM; effects of thermotherapy (38ºC/30 days; and action of antibiotics in the elimination of Xcv in infected grapevines. The percentages of contamination by Xcv and regeneration were analyzed and plants obtained were indexed using the semi-selective culture medium nutrient agar-dextrose-yeast extract-ampicilin (NYDAM followed by a pathogenicity test. The cultivation of 3 mm explants permitted to obtain plants free of bacteria with regeneration 14.3 times higher than 1 mm explants. The thermotherapy of infected plants associated to the in vitro culture

Identification of QTLs Associated with Callogenesis and Embryogenesis in Oil Palm Using Genetic Linkage Maps Improved with SSR Markers

Science.gov (United States)

Ting, Ngoot-Chin; Jansen, Johannes; Nagappan, Jayanthi; Ishak, Zamzuri; Chin, Cheuk-Weng; Tan, Soon-Guan; Cheah, Suan-Choo; Singh, Rajinder

2013-01-01

Clonal reproduction of oil palm by means of tissue culture is a very inefficient process. Tissue culturability is known to be genotype dependent with some genotypes being more amenable to tissue culture than others. In this study, genetic linkage maps enriched with simple sequence repeat (SSR) markers were developed for dura (ENL48) and pisifera (ML161), the two fruit forms of oil palm, Elaeis guineensis. The SSR markers were mapped onto earlier reported parental maps based on amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers. The new linkage map of ENL48 contains 148 markers (33 AFLPs, 38 RFLPs and 77 SSRs) in 23 linkage groups (LGs), covering a total map length of 798.0 cM. The ML161 map contains 240 markers (50 AFLPs, 71 RFLPs and 119 SSRs) in 24 LGs covering a total of 1,328.1 cM. Using the improved maps, two quantitative trait loci (QTLs) associated with tissue culturability were identified each for callusing rate and embryogenesis rate. A QTL for callogenesis was identified in LGD4b of ENL48 and explained 17.5% of the phenotypic variation. For embryogenesis rate, a QTL was detected on LGP16b in ML161 and explained 20.1% of the variation. This study is the first attempt to identify QTL associated with tissue culture amenity in oil palm which is an important step towards understanding the molecular processes underlying clonal regeneration of oil palm. PMID:23382832

Growth and stem form quality of clonal Pinus taeda following fertilization in the Virginia Piedmont

Science.gov (United States)

Jeremy P. Stovall; Colleen A. Carlson; John R. Seiler; Thomas R. Fox

2013-01-01

Clonal forestry offers the opportunity to increase yields, enhance uniformity, and improve wood characteristics. Intensive silvicultural practices, including fertilization, will be required to capture the full growth potential of clonal plantations. However, variation in nutrient use efficiency that exists among clones could affect growth responses. Our research...

Assessment of three types of spaceflight hardware for tissue culture studies: Comparison of skeletal tissue growth and differentiation

Energy Technology Data Exchange (ETDEWEB)

Klement, B.J. [Space Medicine and Life Sciences Research Center Department of Anatomy Morehouse School of Medicine 720 Westview Dr. SW Atlanta, Georgia30310-1495 (United States); Spooner, B.S. [NASA Specialized Center of Research and Training Division of Biology Ackert Hall Kansas State University Manhattan, Kansas66506 (United States)

1997-01-01

Three different types of spaceflight hardware, the BioProcessing Module (BPM), the Materials Dispersion Apparatus (MDA), and the Fluid Processing Apparatus (FPA), were assessed for their ability to support pre-metatarsal growth and differentiation in experiments conducted on five space shuttle flights. BPM-cultured pre-metatarsal tissue showed no difference in flight and ground control lengths. Flight and ground controls cultured in the MDA grew 135 {mu}m and 141 {mu}m, respectively, in an 11 day experiment. Only five control rods and three flight rods mineralized. In another MDA experiment, pre-metatarsals were cultured at 4{degree}C (277K) or 20{degree}C (293K) for the 16 day mission, then cultured an additional 16 days in laboratory dishes at 37{degree}C (310K). The 20{degree}C (293K) cultures died post-flight. The 4{degree}C (277K) flight pre-metatarsals grew 417 {mu}m more than the 4{degree}C (277K) ground controls post-flight. In 5 and 6 day experiments done in FPAs, flight rods grew longer than ground control rods. In a 14 day experiment, ground control and flight rods also expanded in length, but there was no difference between them. The pre-metatarsals cultured in the FPAs did not mineralize, or terminally differentiate. These experiments demonstrate, that while supporting pre-metatarsal growth in length, the three types of hardware are not suitable to support routine differentiation. {copyright} {ital 1997 American Institute of Physics.}

Products of cells from gliomas: VIII. Multiple-well immunoperoxidase assay of immunoreactivity of primary hybridoma supernatants with human glioma and brain tissue and cultured glioma cells.

Science.gov (United States)

McKeever, P E; Wahl, R L; Shakui, P; Jackson, G A; Letica, L H; Liebert, M; Taren, J A; Beierwaltes, W H; Hoff, J T

1990-06-01

To test the feasibility of primary screening of hybridoma supernatants against human glioma tissue, over 5000 combinations of hybridoma supernatants with glioma tissue, cultured glioma cells, and normal central neural tissue were screened with a new multiple-well (M-well) screening system. This is an immunoperoxidase assay system with visual endpoints for screening 20-30 hybridoma supernatants per single microscope slide. There were extensive differences between specificities to tissue and to cultured glioma cells when both were screened with M-wells and when cultured cells were screened with standard semi-automated fluorescence. Primary M-well screening with glioma tissue detected seven hybridoma supernatants that specifically identified parenchymal cells of glioma tissue and that were not detected with cultured cells. Immunoreactivities of individual supernatants for vascular components (nine supernatants), necrosis (five supernatants), and nuclei (three supernatants) were detected. Other supernatants bound multiple sites on glioma tissue and/or subpopulations of neurons and glia of normal tissue. The results show that primary screening with glioma tissue detects a number of different specificities of hybridoma supernatants to gliomas not detected by conventional screening with cultured cells. These are potentially applicable to diagnosis and therapy.

Clinical significance of T-cell clonality in mycosis fungoides and other cutaneous T-cell lymphomas

NARCIS (Netherlands)

Muche, Joachim Marcus

2010-01-01

The purpose of this thesis was to obtain more insight into T-cell clonality in blood of mycosis fungoides (MF) patients. Investigation of the frequency of blood T-cell clonality clearly indicated early dissemination of neoplastic T-cells into skin and blood as a sign of physiological recirculation.

Population Genomic Analysis of 1,777 Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Isolates, Houston, Texas: Unexpected Abundance of Clonal Group 307.

Science.gov (United States)

Long, S Wesley; Olsen, Randall J; Eagar, Todd N; Beres, Stephen B; Zhao, Picheng; Davis, James J; Brettin, Thomas; Xia, Fangfang; Musser, James M

2017-05-16

Klebsiella pneumoniae is a major human pathogen responsible for high morbidity and mortality rates. The emergence and spread of strains resistant to multiple antimicrobial agents and documented large nosocomial outbreaks are especially concerning. To develop new therapeutic strategies for K. pneumoniae , it is imperative to understand the population genomic structure of strains causing human infections. To address this knowledge gap, we sequenced the genomes of 1,777 extended-spectrum beta-lactamase-producing K. pneumoniae strains cultured from patients in the 2,000-bed Houston Methodist Hospital system between September 2011 and May 2015, representing a comprehensive, population-based strain sample. Strains of largely uncharacterized clonal group 307 (CG307) caused more infections than those of well-studied epidemic CG258. Strains varied markedly in gene content and had an extensive array of small and very large plasmids, often containing antimicrobial resistance genes. Some patients with multiple strains cultured over time were infected with genetically distinct clones. We identified 15 strains expressing the New Delhi metallo-beta-lactamase 1 (NDM-1) enzyme that confers broad resistance to nearly all beta-lactam antibiotics. Transcriptome sequencing analysis of 10 phylogenetically diverse strains showed that the global transcriptome of each strain was unique and highly variable. Experimental mouse infection provided new information about immunological parameters of host-pathogen interaction. We exploited the large data set to develop whole-genome sequence-based classifiers that accurately predict clinical antimicrobial resistance for 12 of the 16 antibiotics tested. We conclude that analysis of large, comprehensive, population-based strain samples can assist understanding of the molecular diversity of these organisms and contribute to enhanced translational research. IMPORTANCE Klebsiella pneumoniae causes human infections that are increasingly difficult to

Polymerase chain reaction-based clonality testing in tissue samples with reactive lymphoproliferations : usefulness and pitfalls. A report of the BIOMED-2 Concerted Action BMH4-CT98-3936

NARCIS (Netherlands)

Langerak, A. W.; Molina, T. J.; Lavender, F. L.; Pearson, D.; Flohr, T.; Sambade, C.; Schuuring, E.; Al Saati, T.; van Dongen, J. J. M.; Van Krieken, J. H. J. M.

Lymphoproliferations are generally diagnosed via histomorphology and immunohistochemistry. Although mostly conclusive, occasionally the differential diagnosis between reactive lesions and malignant lymphomas is difficult. In such cases molecular clonality studies of immunoglobulin (Ig)/T-cell

Polymerase chain reaction-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls. A report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

NARCIS (Netherlands)

Langerak, A.W.; Molina, T.J.; Lavender, F.L.; Pearson, D.; Flohr, T.; Sambade, C.; Schuuring, E.; Saati, T. Al; Dongen, J.J.M. van; Krieken, J.H.J.M. van

2007-01-01

Lymphoproliferations are generally diagnosed via histomorphology and immunohistochemistry. Although mostly conclusive, occasionally the differential diagnosis between reactive lesions and malignant lymphomas is difficult. In such cases molecular clonality studies of immunoglobulin (Ig)/T-cell

Primary chondrocytes enhance cartilage tissue formation upon co-culture with expanded chondrocytes, dermal fibroblasts, 3T3 feeder cells and embryonic stem cells

NARCIS (Netherlands)

Hendriks, J.A.A.; Miclea, Razvan L.; Schotel, Roka; de Bruijn, Ewart; Moroni, Lorenzo; Karperien, Hermanus Bernardus Johannes; Riesle, J.U.; van Blitterswijk, Clemens

2010-01-01

Co-culture models have been increasingly used in tissue engineering applications to understand cell–cell interactions and consequently improve regenerative medicine strategies. Aiming at further elucidating cartilage tissue formation, we co-cultured bovine primary chondrocytes (BPCs) with human

[Extraction and analysis of chemical components of essential oil in Thymus vulgaris of tissue culture].

Science.gov (United States)

Li, Xiao-Dong; Yang, Li; Xu, Shi-Qian; Li, Jian-Guo; Cheng, Zhi-Hui; Dang, Jian-Zhang

2011-10-01

To extract the essential oils from the Seedlings, the Aseptic Seedlings and the Tissue Culture Seedlings of Thymus vulgaris and analyze their chemical components and the relative contents. The essential oils were extracted by steam distillation, the chemical components and the relative contents were identified and analyzed by gas chromatography-mass spectrometry (GC/MS) and peak area normalization method. The main chemical components of essential oil in these three samples had no significant difference, they all contained the main components of essential oil in Thymus vulgaris: Thymol, Carvacrol, o-Cymene, gamma-Terpinene, Caryophyllene et al. and only had a slight difference in the relative content. This study provides important theoretical foundation and data reference for further study on production of essential oil in thyme by tissue culture technology.

Comparison of tumour age response to radiation for cells derived from tissue culture or solid tumours

International Nuclear Information System (INIS)

Keng, P.C.; Siemann, D.W.; Rochester Univ., NY; Rochester Univ., NY; Wheeler, K.T.

1984-01-01

Direct comparison of the cell age response of 9L and KHT tumour cells derived either from tissue culture or solid tumours was achieved. Cells from dissociated KHT and 9L tumours (the latter implanted either subcutaneously or intracerebrally) and cells from tissue culture were separated into homogenous sized populations by centrifugal elutriation. In both tumour models these homogeneous sized populations correspond to populations enriched at different stages of the cell cycle. The survival of these elutriated cell populations was measured after a single dose of Cs-137 gamma rays. For cells isolated from 9L solid tumours, there was little variation in radiosensitivity throughout the cell cycle; however, a very small but significant increase in resistance was found in late G 1 cells. This lack of a large variation in radiosensitivity through the cell cycle for 9L cells from solid tumours also was seen in 9L cells growing in monolayer tissue culture. When similar experiments were performed using the KHT sarcoma tumour model, the results showed that KHT cells in vitro exhibited a fairly conventional increase in radioresistance in both mid G 1 and late S. However, the cell age response of KHT cells from solid tumours was different; particularly in the late S and G 2 + M phases. (author)

Allelopathy of small everlasting (Antennaria microphylla) : Phytotoxicity to leafy spurge (Euphorbia esula) in tissue culture.

Science.gov (United States)

Hogan, M E; Manners, G D

1990-03-01

Media and media extracts from callus cultures of small everlasting (Antennaria microphylla) inhibited leafy spurge (Euphorbia esula L.) callus tissue and suspension culture growth (50 and 70% of control, respectively) and were phytotoxic in lettuce and leafy spurge root elongation bioassays (64 and 77% of control, respectively). Hydroquinone, a phytotoxic compound previously isolated from small everlasting, was also biosynthesized by callus and suspension cultures of this species. Exogenously supplied hydroquinone (0.5 mM) was toxic to leafy spurge suspension culture cells and was only partially biotransformed to its nontoxic water-soluble monoglucoside, arbutin, by these cells. This report confirms the chronic involvement of hydroquinone in the allelopathic interaction between small everlasting and leafy spurge.

Longitudinal Claudin Gene Expression Analyses in Canine Mammary Tissues and Thereof Derived Primary Cultures and Cell Lines

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Susanne C. Hammer

2016-09-01

Full Text Available Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies.

Planar cell polarity signaling coordinates oriented cell division and cell rearrangement in clonally expanding growth plate cartilage.

Science.gov (United States)

Li, Yuwei; Li, Ang; Junge, Jason; Bronner, Marianne

2017-10-10

Both oriented cell divisions and cell rearrangements are critical for proper embryogenesis and organogenesis. However, little is known about how these two cellular events are integrated. Here we examine the linkage between these processes in chick limb cartilage. By combining retroviral-based multicolor clonal analysis with live imaging, the results show that single chondrocyte precursors can generate both single-column and multi-column clones through oriented division followed by cell rearrangements. Focusing on single column formation, we show that this stereotypical tissue architecture is established by a pivot-like process between sister cells. After mediolateral cell division, N-cadherin is enriched in the post-cleavage furrow; then one cell pivots around the other, resulting in stacking into a column. Perturbation analyses demonstrate that planar cell polarity signaling enables cells to pivot in the direction of limb elongation via this N-cadherin-mediated coupling. Our work provides new insights into the mechanisms generating appropriate tissue architecture of limb skeleton.

Rapid detection of Mannheimia haemolytica in lung tissues of sheep and from bacterial culture

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Jyoti Kumar

2015-09-01

Full Text Available Aim: This study was aimed to detect Mannheimia haemolytica in lung tissues of sheep and from a bacterial culture. Introduction: M. haemolytica is one of the most important and well-established etiological agents of pneumonia in sheep and other ruminants throughout the world. Accurate diagnosis of M. haemolytica primarily relies on bacteriological examination, biochemical characteristics and, biotyping and serotyping of the isolates. In an effort to facilitate rapid M. haemolytica detection, polymerase chain reaction assay targeting Pasteurella haemolytica serotype-1 specific antigens (PHSSA, Rpt2 and 12S ribosomal RNA (rRNA genes were used to detect M. haemolytica directly from lung tissues and from bacterial culture. Materials and Methods: A total of 12 archived lung tissues from sheep that died of pneumonia on an organized farm were used. A multiplex polymerase chain reaction (mPCR based on two-amplicons targeted PHSSA and Rpt2 genes of M. haemolytica were used for identification of M. haemolytica isolates in culture from the lung samples. All the 12 lung tissue samples were tested for the presence M. haemolytica by PHSSA and Rpt2 genes based PCR and its confirmation by sequencing of the amplicons. Results: All the 12 lung tissue samples tested for the presence of PHSSA and Rpt2 genes of M. haemolytica by mPCR were found to be positive. Amplification of 12S rRNA gene fragment as internal amplification control was obtained with each mPCR reaction performed from DNA extracted directly from lung tissue samples. All the M. haemolytica were also positive for mPCR. No amplified DNA bands were observed for negative control reactions. All the three nucleotide sequences were deposited in NCBI GenBank (Accession No. KJ534629, KJ534630 and KJ534631. Sequencing of the amplified products revealed the identity of 99-100%, with published sequence of PHSSA and Rpt2 genes of M. haemolytica available in the NCBI database. Sheep specific mitochondrial 12S r

MIRU-VNTR allelic variability depends on Mycobacterium bovis clonal group identity.

Science.gov (United States)

Hauer, Amandine; Michelet, Lorraine; De Cruz, Krystel; Cochard, Thierry; Branger, Maxime; Karoui, Claudine; Henault, Sylvie; Biet, Franck; Boschiroli, María Laura

2016-11-01

The description of the population of M. bovis strains circulating in France from 1978 to 2013 has highlighted the discriminating power of the MLVA among predominant spoligotype groups. In the present study we aimed to characterize clonal groups via MLVA and to better understand the strain's population structure. MLVA was performed with eight MIRU-VNTR loci, most of them defined by the Venomyc European consortium. The discriminatory index of each MLVA loci was calculated for SB0120, SB0134, SB0121 and the "F4-family", the main spoligotype groups in France. Differences in global DI per spoligotype, but also by locus within each spoligotype, were observed, which strongly suggest the clonal complex nature of these major groups. These MLVA results were compared to those of other European countries where strain collections had been characterized (Spain, Portugal, Italy, Northern Ireland and Belgium). Overall, QUB 3232 and ETR D are respectively the most and the least discriminative loci, regardless of the strains geographical origin. However, marked DI differences are observed in the rest of the MIRU-VNTR loci, again highlighting that strain genetic variability in a country depends on the dominant existing clonal complexes. A web application for M. bovis, including spoligotyping and MIRU-VNTR typing data, was developed to allow inter-laboratory comparison of field isolates. In conclusion, combination of typing methods is required for M. bovis optimum discrimination and differentiation of groups of strains. Thus, the loci employed for MLVA in a country should be those which are the most discriminative for the clonal complexes which characterize their M. bovis population. Copyright © 2016 Elsevier B.V. All rights reserved.

Umbilical cord Wharton's jelly repeated culture system: a new device and method for obtaining abundant mesenchymal stem cells for bone tissue engineering.

Directory of Open Access Journals (Sweden)

Zhengqi Chang

Full Text Available To date, various types of cells for seeding regenerative scaffolds have been used for bone tissue engineering. Among seed cells, the mesenchymal stem cells derived from human umbilical cord Wharton's jelly (hUCMSCs represent a promising candidate and hold potential for bone tissue engineering due to the the lack of ethical controversies, accessibility, sourced by non-invasive procedures for donors, a reduced risk of contamination, osteogenic differentiation capacities, and higher immunomodulatory capacity. However, the current culture methods are somewhat complicated and inefficient and often fail to make the best use of the umbilical cord (UC tissues. Moreover, these culture processes cannot be performed on a large scale and under strict quality control. As a result, only a small quantity of cells can be harvested using the current culture methods. To solve these problems, we designed and evaluated an UC Wharton's jelly repeated culture device. Using this device, hUCMSCs were obtained from the repeated cultures and their quantities and biological characteristics were compared. We found that using our culture device, which retained all tissue blocks on the bottom of the dish, the total number of obtained cells increased 15-20 times, and the time required for the primary passage was reduced. Moreover, cells harvested from the repeated cultures exhibited no significant difference in their immunophenotype, potential for multilineage differentiation, or proliferative, osteoinductive capacities, and final osteogenesis. The application of the repeated culture frame (RCF not only made full use of the Wharton's jelly but also simplified and specified the culture process, and thus, the culture efficiency was significantly improved. In summary, abundant hUCMSCs of dependable quality can be acquired using the RCF.

Loss of heterozygosity drives clonal diversity of Phytophthora capsici in China.

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Jian Hu

Full Text Available Phytophthora capsici causes significant loss to pepper (Capsicum annum in China and our goal was to develop single nucleotide polymorphism (SNP markers for P. capsici and characterize genetic diversity nationwide. Eighteen isolates of P. capsici from locations worldwide were re-sequenced and candidate nuclear and mitochondrial SNPs identified. From 2006 to 2012, 276 isolates of P. capsici were recovered from 136 locations in 27 provinces and genotyped using 45 nuclear and 2 mitochondrial SNPs. There were two main mitochondrial haplotypes and 95 multi-locus genotypes (MLGs identified. Genetic diversity was geographically structured with a high level of genotypic diversity in the north and on Hainan Island in the south, suggesting outcrossing contributes to diversity in these areas. The remaining areas of China are dominated by four clonal lineages that share mitochondrial haplotypes, are almost exclusively the A1 or A2 mating type and appear to exhibit extensive diversity based on loss of heterozygosity (LOH. Analysis of SNPs directly from infected peppers confirmed LOH in field populations. One clonal lineage is dominant throughout much of the country. The overall implications for long-lived genetically diverse clonal lineages amidst a widely dispersed sexual population are discussed.

Effect of clonal reproduction on genetic structure in Pentaclethra macroloba (Fabaceae: Mimosoideae).

Science.gov (United States)

Gaddis, Keith D; Zukin, Helen L; Dieterich, Inca A; Braker, Elizabeth; Sork, Victoria L

2014-06-01

The existence of monodominant forests on well-drained soils in tropical regions has been widely reported. Such forests most likely result from a combination of both ecological and evolutionary factors. Under conditions of high seed and seedling mortality, vegetative reproduction could create a reproductive advantage leading to forest dominance, and profoundly affect the distribution of genetic variation in a clonal species. We investigated these effects in a low diversity forest site in Northeastern Costa Rica dominated by the species Pentaclethra macroloba, which sprouts from the root mass of fallen trees and from snapped trunks. We examined the population structure of juvenile P. macroloba growing in different soil types and across an elevational gradient. Using seven molecular markers, we genotyped 173 juvenile P. macroloba from 18 plots (six plots in seasonally inundated swamps, and 12 plots in upland non-swamp) spanning 50-300m in elevation at La Selva Biological Station and the adjacent Reserva Ecológica Bijagual in Northeastern Costa Rica. We answered two specific questions: (1) How extensive is clonal reproduction? and (2) what is the distribution of genetic diversity and structure? We found that clonal reproduction occurred exclusively within inundated swamp areas. However, there was no significant difference between genetic diversity measures in swamp and non-swamp plots, which were both generally low when compared with other tropical forest species. Genetic structure was significant across all plots (F(ST) = -0.109). However, genetic structure among swamp plots (F(ST) = 0.128) was higher than among non-swamp upland plots (F(ST) = 0.093). Additionally, spatial autocorrelation among individuals within non-swamp upland plots was significant from the 25 to 100m spatial scale, but not within swamp plots. The degree of overall genetic structure we found in P. macroloba is high for a tropical forest tree. The incidence of clonal reproduction is a contributing

Effect of clonal reproduction on genetic structure in Pentaclethra macroloba (Fabaceae: Mimosoideae

Directory of Open Access Journals (Sweden)

Keith D. Gaddis

2014-08-01

Full Text Available The existence of monodominant forests on well-drained soils in tropical regions has been widely reported. Such forests most likely result from a combination of both ecological and evolutionary factors. Under conditions of high seed and seedling mortality, vegetative reproduction could create a reproductive advantage leading to forest dominance, and profoundly affect the distribution of genetic variation in a clonal species. We investigated these effects in a low diversity forest site in Northeastern Costa Rica dominated by the species Pentaclethra macroloba, which sprouts from the root mass of fallen trees and from snapped trunks. We examined the population structure of juvenile P. macroloba growing in different soil types and across an elevational gradient. Using seven molecular markers, we genotyped 173 juvenile P. macroloba from 18 plots (six plots in seasonally inundated swamps, and 12 plots in upland non-swamp spanning 50-300m in elevation at La Selva Biological Station and the adjacent Reserva Ecológica Bijagual in Northeastern Costa Rica. We answered two specific questions: (1 How extensive is clonal reproduction? and (2 what is the distribution of genetic diversity and structure?. We found that clonal reproduction occurred exclusively within inundated swamp areas. However, there was no significant difference between genetic diversity measures in swamp and non-swamp plots, which were both generally low when compared with other tropical forest species. Genetic structure was significant across all plots (F ST=0.109. However, genetic structure among swamp plots (F ST=0.128 was higher than among non-swamp upland plots (F ST=0.093. Additionally, spatial autocorrelation among individuals within non-swamp upland plots was significant from the 25 to 100m spatial scale, but not within swamp plots. The degree of overall genetic structure we found in P. macroloba is high for a tropical forest tree. The incidence of clonal reproduction is a

Core Genome Multilocus Sequence Typing for Identification of Globally Distributed Clonal Groups and Differentiation of Outbreak Strains of Listeria monocytogenes.

Science.gov (United States)

Chen, Yi; Gonzalez-Escalona, Narjol; Hammack, Thomas S; Allard, Marc W; Strain, Errol A; Brown, Eric W

2016-10-15

Many listeriosis outbreaks are caused by a few globally distributed clonal groups, designated clonal complexes or epidemic clones, of Listeria monocytogenes, several of which have been defined by classic multilocus sequence typing (MLST) schemes targeting 6 to 8 housekeeping or virulence genes. We have developed and evaluated core genome MLST (cgMLST) schemes and applied them to isolates from multiple clonal groups, including those associated with 39 listeriosis outbreaks. The cgMLST clusters were congruent with MLST-defined clonal groups, which had various degrees of diversity at the whole-genome level. Notably, cgMLST could distinguish among outbreak strains and epidemiologically unrelated strains of the same clonal group, which could not be achieved using classic MLST schemes. The precise selection of cgMLST gene targets may not be critical for the general identification of clonal groups and outbreak strains. cgMLST analyses further identified outbreak strains, including those associated with recent outbreaks linked to contaminated French-style cheese, Hispanic-style cheese, stone fruit, caramel apple, ice cream, and packaged leafy green salad, as belonging to major clonal groups. We further developed lineage-specific cgMLST schemes, which can include accessory genes when core genomes do not possess sufficient diversity, and this provided additional resolution over species-specific cgMLST. Analyses of isolates from different common-source listeriosis outbreaks revealed various degrees of diversity, indicating that the numbers of allelic differences should always be combined with cgMLST clustering and epidemiological evidence to define a listeriosis outbreak. Classic multilocus sequence typing (MLST) schemes targeting internal fragments of 6 to 8 genes that define clonal complexes or epidemic clones have been widely employed to study L. monocytogenes biodiversity and its relation to pathogenicity potential and epidemiology. We demonstrated that core genome MLST

Jatropha tissue culture: A critical review on present scenario and future prospects

KAUST Repository

Kumar, Nitish

2012-10-26

Ever increasing fuel prices and depletion of fossil reserves have ignited worldwide search for alternative renewable energy sources. Development of biofuels as an alternative and renewable source of energy has become critical in the national efforts towards maximum self-reliance, the corner stone of our energy security strategy. Ever since it was established that Jatropha methyl esters yields biodiesel of an exceptional quality and easy adaptation to semi-arid marginal lands, there has been a surge of interest in biodiesel miracle tree. Large scale cultivation remains the single most important factor that will ultimately determine the success of Jatropha curcas as a source of biofuel. Non-availability of superior clones/varieties, shortage of cuttings, low multiplication rate, gaps in knowledge of clonal technology, higher cost of clonal plantation, etc. are the major factors that limit large-scale cultivation. Recent advances in DNA technology and genetic transformation offer a credible approach for the genetic improvement of the species. The last decade witnessed a blooming interest in the development of micropropagation and transformation techniques for this energy crop. In this review, the achievements made during the last three decades in J. curcas micropropagation are presented. © Springer Science+Business Media New York 2013. All rights are reserved.

Clonal replacement and expansion among invasive meningococcal isolates of serogroup W in France.

Science.gov (United States)

Hong, Eva; Barret, Anne-Sophie; Terrade, Aude; Denizon, Mélanie; Antona, Denise; Aouiti-Trabelsi, Myriam; Deghmane, Ala-Eddine; Parent du Châtelet, Isabelle; Levy-Bruhl, Daniel; Taha, Muhamed-Kheir

2018-02-01

Neisseria meningitidis group W (NmW) belonging to the clonal complex ST-11 (NmW/cc11) spread in Europe and in France in 2000 and declined thereafter. In France, invasive meningococcal disease (IMD) due to NmW increased again in 2012 and thereafter since 2015. Several sub-lineages of NmW/cc11 are circulating worldwide with successive epidemic waves. We aimed to describe recent epidemiological trends of NmW in France and to explore the microbiological and epidemiological characteristics associated with different NmW/cc11 sub-lineages. The epidemiology of NmW was described based on data collected through mandatory notification of IMD and strain typing data for culture-confirmed and PCR-confirmed cases for the period 2000-2016. All culture-confirmed cases due to NmW from the period 2010-2016 were characterised by whole genome sequencing (WGS). A detailed epidemiological analysis was performed for culture-confirmed cases on the basis of WGS data. During the period 2010-2016, genotyping was obtained for 148 cases including all the 132 culture-confirmed cases, among which 127 were matched with epidemiological data, and 16 PCR-confirmed cases (out of a total of 47 PCR-confirmed cases). An increase in IMD was observed in 2012 and was linked to isolates belonging to the "Anglo-French-Hajj" sub-lineage. These isolates have decreased significantly since 2013 and have been replaced by NmW/cc11 isolates related to the "South American - UK" sub-lineage which caused a marked increase in the number of cases of NmW in 2016. In this sub-lineage, the "original UK strain" was first detected in 2012 and increased thereafter, followed by the recently described "UK 2013-strain". Isolates related to the "South American-UK" sub-lineage represented 45% of all NmW cultured isolates from the whole period 2010-2016 but were the most frequent isolates in 2016, representing 76% of the total NmW typed isolates and 94% of the typed NmW/cc11 isolates. A changing pattern in the epidemiology of Nm

Proteomic analysis reveals the mechanisms of Mycena dendrobii promoting transplantation survival and growth of tissue culture seedlings of Dendrobium officinale.

Science.gov (United States)

Xu, X B; Ma, X Y; Lei, H H; Song, H M; Ying, Q C; Xu, M J; Liu, S B; Wang, H Z

2015-06-01

Dendrobium officinale is an important traditional Chinese medicinal herb. Its seedlings generally show low survival and growth when transferred from in vitro tissue culture to a greenhouse or field environment. In this study, the effect of Mycena dendrobii on the survival and growth of D. officinale tissue culture seedlings and the mechanisms involved was explored. Mycena dendrobii were applied underneath the roots of D. officinale tissue culture seedlings. The seedling survival and growth were analysed. The root proteins induced by M. dendrobii were identified using two-dimensional (2-D) electrophoresis and matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF-MS). Mycena dendrobii treatment significantly enhanced survival and growth of D. officinale seedlings. Forty-one proteins induced by M. dendrobii were identified. Among them, 10 were involved in defence and stress response, two were involved in the formation of root or mycorrhizae, and three were related to the biosynthesis of bioactive constituents. These results suggest that enhancing stress tolerance and promoting new root formation induced by M. dendrobii may improve the survival and growth of D. officinale tissue culture seedlings. This study provides a foundation for future use of M. dendrobii in the large-scale cultivation of Dendrobiums. © 2015 The Society for Applied Microbiology.

In Vivo-Like Culture Conditions in a Bioreactor Facilitate Improved Tissue Quality in Corneal Storage.

Science.gov (United States)

Schmid, Richard; Tarau, Ioana-Sandra; Rossi, Angela; Leonhardt, Stefan; Schwarz, Thomas; Schuerlein, Sebastian; Lotz, Christian; Hansmann, Jan

2018-01-01

The cornea is the most-transplanted tissue worldwide. However, the availability and quality of grafts are limited due to the current methods of corneal storage. In this study, a dynamic bioreactor system is employed to enable the control of intraocular pressure and the culture at the air-liquid interface. Thereby, in vivo-like storage conditions are achieved. Different media combinations for endothelium and epithelium are tested in standard and dynamic conditions to enhance the viability of the tissue. In contrast to culture conditions used in eye banks, the combination of the bioreactor and biochrom medium 1 allows to preserve the corneal endothelium and the epithelium. Assessment of transparency, swelling, and the trans-epithelial-electrical-resistance (TEER) strengthens the impact of the in vivo-like tissue culture. For example, compared to corneas stored under static conditions, significantly lower optical densities and significantly higher TEER values were measured (p-value <0.05). Furthermore, healing of epithelial defects is enabled in the bioreactor, characterized by re-epithelialization and initiated stromal regeneration. Based on the obtained results, an easy-to-use 3D-printed bioreactor composed of only two parts was derived to translate the technology from the laboratory to the eye banks. This optimized bioreactor facilitates noninvasive microscopic monitoring. The improved storage conditions ameliorate the quality of corneal grafts and the storage time in the eye banks to increase availability and reduce re-grafting. © 2017 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Micropropagation of important bamboos: A review | Mudoi | African ...

African Journals Online (AJOL)

... tissue culture mediated biotechnological interventions made in bamboo for large scale micropropagation, that being the need of the hour. Keywords: Bamboo, micropropagation, somatic embryogenesis, in vitro flowering, macroproliferation, field performance, clonal fidelity. African Journal of Biotechnology Vol. 12(20), pp.

Clonally Expanding Thymocytes Having Lineage Capability in Gamma-Ray-Induced Mouse Atrophic Thymus

International Nuclear Information System (INIS)

Yamamoto, Takashi; Morita, Shin-ichi; Go, Rieka; Obata, Miki; Katsuragi, Yoshinori; Fujita, Yukari; Maeda, Yoshitaka; Yokoyama, Minesuke; Aoyagi, Yutaka; Ichikawa, Hitoshi; Mishima, Yukio; Kominami, Ryo

2010-01-01

Purpose: To characterize, in the setting of γ-ray-induced atrophic thymus, probable prelymphoma cells showing clonal growth and changes in signaling, including DNA damage checkpoint. Methods and Materials: A total of 111 and 45 mouse atrophic thymuses at 40 and 80 days, respectively, after γ-irradiation were analyzed with polymerase chain reaction for D-J rearrangements at the TCRβ locus, flow cytometry for cell cycle, and Western blotting for the activation of DNA damage checkpoints. Results: Limited D-J rearrangement patterns distinct from normal thymus were detected at high frequencies (43 of 111 for 40-day thymus and 21 of 45 for 80-day thymus). Those clonally expanded thymocytes mostly consisted of CD4 + CD8 + double-positive cells, indicating the retention of lineage capability. They exhibited pausing at a late G1 phase of cell cycle progression but did not show the activation of DNA damage checkpoints such as γH2AX, Chk1/2, or p53. Of interest is that 17 of the 52 thymuses showing normal D-J rearrangement patterns at 40 days after irradiation showed allelic loss at the Bcl11b tumor suppressor locus, also indicating clonal expansion. Conclusion: The thymocytes of clonal growth detected resemble human chronic myeloid leukemia in possessing self-renewal and lineage capability, and therefore they can be a candidate of the lymphoma-initiating cells.

Picroside I and Picroside II from Tissue Cultures of Picrorhiza kurroa

Science.gov (United States)

Ganeshkumar, Yamjala; Ramarao, Ajmera; Veeresham, Ciddi

2017-01-01

Background: Picrorhiza kurroa (PK) belongs to Scrophulariaceae family and is a representative endemic, medicinal herb, widely distributed throughout the higher altitudes of alpine Himalayas from west to east, between 3000 and 4500 m above mean sea level. Objective: The objective of the present study is to assess the production of picroside I and picroside II from tissue cultures of PK. Materials and Methods: Auxiliary shoot tips of PK were incubated in Murashige and Skoog medium supplemented with indole-3-butyric acid and kinetin phytohormones. The callus produced was collected at different time intervals and was processed for extraction of picroside I and picroside II followed by thin layer chromatography and high-performance liquid chromatography HPLC analysis. Results: The maximum growth index was found to be 5.109 ± 0.159 at 16-week-old callus culture. The estimation of picroside-I and picroside-II was carried out by (HPLC) analysis; quantity of secondary metabolite found to be 16.37 ± 0.0007 mg/g for PK-I and 6.34 ± 0.0012 mg/g for PK-II. Conclusion: This is the first attempt to produce the Picroside-I and II in large amount by the tissue culture technique. It can be observed that the method of callus culture can be used in production of secondary metabolites Picroside-I and II from PK SUMMARY Picrorhiza kurroa is a high value medicinal herb due to rich source of hepatoprotective metabolites, Picroside-I and Picroside-II. The medicinal importance of P. kurroa is due to its pharmacological properties like hepatoprotective, antioxidant (particularly in liver), antiallergic and antiasthamatic, anticancer activity particularly in liver and immunomodulatory. Shoot apices which were produced a good response was inoculated on selected medium i.e., on MS medium containing 2, 4 D (mg/l) + KN (1mg/l) for induction of callus. The initiation of callus was observed after 4weeks and it was light green and fragile Maximum growth was observed with 3% w/v of sucrose

Clonal integration supports the expansion from terrestrial to aquatic environments of the amphibious stoloniferous herb Alternanthera philoxeroides.

Science.gov (United States)

Wang, N; Yu, F-H; Li, P-X; He, W-M; Liu, J; Yu, G-L; Song, Y-B; Dong, M

2009-05-01

Effects of clonal integration on land plants have been extensively studied, but little is known about the role in amphibious plants that expand from terrestrial to aquatic conditions. We simulated expansion from terrestrial to aquatic habitats in the amphibious stoloniferous alien invasive alligator weed (Alternanthera philoxeroides) by growing basal ramets of clonal fragments in soils connected (allowing integration) or disconnected (preventing integration) to the apical ramets of the same fragments submerged in water to a depth of 0, 5, 10 or 15 cm. Clonal integration significantly increased growth and clonal reproduction of the apical ramets, but decreased both of these characteristics in basal ramets. Consequently, integration did not affect the performance of whole clonal fragments. We propose that alligator weed possesses a double-edged mechanism during population expansion: apical ramets in aquatic habitats can increase growth through connected basal parts in terrestrial habitats; however, once stolon connections with apical ramets are lost by external disturbance, the basal ramets in terrestrial habitats increase stolon and ramet production for rapid spreading. This may contribute greatly to the invasiveness of alligator weed and also make it very adaptable to habitats with heavy disturbance and/or highly heterogeneous resource supply.

Molecular marker analysis to differentiate a clonal selection of ...

African Journals Online (AJOL)

Lalit Kumar

2013-04-03

Apr 3, 2013 ... Microsatellite and amplified fragment length polymorphism (AFLP) markers were used to differentiate. Manjari Naveen, a clonal selection of Centennial Seedless variety of grape. Twenty one (21) microsatellite primers could not detect variation between parent variety and its clone. AFLP analysis.

Clonal Evaluation of Prostate Cancer by ERG/SPINK1 Status to Improve Prognosis Prediction

Science.gov (United States)

2017-12-01

19 NIH Exploiting drivers of androgen receptor signaling negative prostate cancer for precision medicine Goal(s): Identify novel potential drivers...AWARD NUMBER: W81XWH-14-1-0466 TITLE: Clonal evaluation of prostate cancer by ERG/SPINK1 status to improve prognosis prediction PRINCIPAL...Sept 2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Clonal Evaluation of Prostate Cancer by ERG/SPINK1 Status to Improve Prognosis Prediction 5b

Clonal expansion of the Pseudogymnoascus destructans genotype in North America is accompanied by significant variation in phenotypic expression.

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Khankhet, Jordan; Vanderwolf, Karen J; McAlpine, Donald F; McBurney, Scott; Overy, David P; Slavic, Durda; Xu, Jianping

2014-01-01

Pseudogymnoascus destructans is the causative agent of an emerging infectious disease that threatens populations of several North American bat species. The fungal disease was first observed in 2006 and has since caused the death of nearly six million bats. The disease, commonly known as white-nose syndrome, is characterized by a cutaneous infection with P. destructans causing erosions and ulcers in the skin of nose, ears and/or wings of bats. Previous studies based on sequences from eight loci have found that isolates of P. destructans from bats in the US all belong to one multilocus genotype. Using the same multilocus sequence typing method, we found that isolates from eastern and central Canada also had the same genotype as those from the US, consistent with the clonal expansion of P. destructans into Canada. However, our PCR fingerprinting revealed that among the 112 North American isolates we analyzed, three, all from Canada, showed minor genetic variation. Furthermore, we found significant variations among isolates in mycelial growth rate; the production of mycelial exudates; and pigment production and diffusion into agar media. These phenotypic differences were influenced by culture medium and incubation temperature, indicating significant variation in environmental condition--dependent phenotypic expression among isolates of the clonal P. destructans genotype in North America.

Clonal expansion of the Pseudogymnoascus destructans genotype in North America is accompanied by significant variation in phenotypic expression.

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Jordan Khankhet

Full Text Available Pseudogymnoascus destructans is the causative agent of an emerging infectious disease that threatens populations of several North American bat species. The fungal disease was first observed in 2006 and has since caused the death of nearly six million bats. The disease, commonly known as white-nose syndrome, is characterized by a cutaneous infection with P. destructans causing erosions and ulcers in the skin of nose, ears and/or wings of bats. Previous studies based on sequences from eight loci have found that isolates of P. destructans from bats in the US all belong to one multilocus genotype. Using the same multilocus sequence typing method, we found that isolates from eastern and central Canada also had the same genotype as those from the US, consistent with the clonal expansion of P. destructans into Canada. However, our PCR fingerprinting revealed that among the 112 North American isolates we analyzed, three, all from Canada, showed minor genetic variation. Furthermore, we found significant variations among isolates in mycelial growth rate; the production of mycelial exudates; and pigment production and diffusion into agar media. These phenotypic differences were influenced by culture medium and incubation temperature, indicating significant variation in environmental condition--dependent phenotypic expression among isolates of the clonal P. destructans genotype in North America.

IN VITRO PROPAGATION OF DENDROBIUM AND PHALAENOPSIS THROUGH TISSUE CULTURE FOR CONSERVATION

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Lita Soetopo

2012-06-01

Full Text Available The studies were focused on developing an efficient and effective propagation protocol for orchid species from genera Dendrobioum and Phalaenopsis through tissue culture. The Materials used were explants from adventive shoot tip, floral stalk buds and PLBs derived from seeds. The results indicated growth and development of adventive shoot tip explants of Dendrobium: a high survival percentage for explant with green color was shown by D. racianum, followed by D. laxiflorum, D. pseudo-conantum, D. strebloceras, D. lineale, and D. veratrifolium. However, plantlets regeneration occurred only on D. pseudoconantum, and D. strebloceras. Explant regeneration from seed derived protocorm-like bodies on D. spectabile occurred 40 days after inoculation transfer and subculture. High survival percentage of explant from floral stalk shoot was shown by P. amabilis. There were several plantlets surviving in acclimatisation. Explant regeneration from seed derived from protocorm-like bodies on P. hieroglypha occurred 40 days after inoculation and subculture. It was suggested that for ex situ conservation on certain species of Dendrobium and Phalaenopsis in the category of rare germplasms, tissue culture could be applied effectively and efficiently by using explant from adventive shoot tip, floral stalk buds and seed derived protocorm-like body explant for vegetative seed multiplication.

Somatic Mutations and Clonal Hematopoiesis in Aplastic Anemia.

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Yoshizato, Tetsuichi; Dumitriu, Bogdan; Hosokawa, Kohei; Makishima, Hideki; Yoshida, Kenichi; Townsley, Danielle; Sato-Otsubo, Aiko; Sato, Yusuke; Liu, Delong; Suzuki, Hiromichi; Wu, Colin O; Shiraishi, Yuichi; Clemente, Michael J; Kataoka, Keisuke; Shiozawa, Yusuke; Okuno, Yusuke; Chiba, Kenichi; Tanaka, Hiroko; Nagata, Yasunobu; Katagiri, Takamasa; Kon, Ayana; Sanada, Masashi; Scheinberg, Phillip; Miyano, Satoru; Maciejewski, Jaroslaw P; Nakao, Shinji; Young, Neal S; Ogawa, Seishi

2015-07-02

In patients with acquired aplastic anemia, destruction of hematopoietic cells by the immune system leads to pancytopenia. Patients have a response to immunosuppressive therapy, but myelodysplastic syndromes and acute myeloid leukemia develop in about 15% of the patients, usually many months to years after the diagnosis of aplastic anemia. We performed next-generation sequencing and array-based karyotyping using 668 blood samples obtained from 439 patients with aplastic anemia. We analyzed serial samples obtained from 82 patients. Somatic mutations in myeloid cancer candidate genes were present in one third of the patients, in a limited number of genes and at low initial variant allele frequency. Clonal hematopoiesis was detected in 47% of the patients, most frequently as acquired mutations. The prevalence of the mutations increased with age, and mutations had an age-related signature. DNMT3A-mutated and ASXL1-mutated clones tended to increase in size over time; the size of BCOR- and BCORL1-mutated and PIGA-mutated clones decreased or remained stable. Mutations in PIGA and BCOR and BCORL1 correlated with a better response to immunosuppressive therapy and longer and a higher rate of overall and progression-free survival; mutations in a subgroup of genes that included DNMT3A and ASXL1 were associated with worse outcomes. However, clonal dynamics were highly variable and might not necessarily have predicted the response to therapy and long-term survival among individual patients. Clonal hematopoiesis was prevalent in aplastic anemia. Some mutations were related to clinical outcomes. A highly biased set of mutations is evidence of Darwinian selection in the failed bone marrow environment. The pattern of somatic clones in individual patients over time was variable and frequently unpredictable. (Funded by Grant-in-Aid for Scientific Research and others.).

Clonal structure and variable fertilization success in Florida Keys broadcast-spawning corals

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Miller, M. W.; Baums, I. B.; Pausch, R. E.; Bright, A. J.; Cameron, C. M.; Williams, D. E.; Moffitt, Z. J.; Woodley, C. M.

2018-03-01

Keystone reef-building corals in the Caribbean are predominantly self-incompatible broadcast spawners and a majority are threatened due to both acute adult mortality and poor recruitment. As population densities decline, concerns about fertilization limitation and effective population size in these species increase and would be further exacerbated by either high clonality or gametic incompatibility of parental genotypes. This study begins to address these concerns for two Caribbean broadcasting species by characterizing clonal structure and quantifying experimental pairwise fertilization success. Orbicella faveolata showed surprisingly high and contrasting levels of clonality between two sampled sites; Acropora palmata was previously known to be highly clonal. Individual pairwise crosses of synchronously spawning genotypes of each species were conducted by combining aliquots of gamete bundles immediately after spawning, and showed high and significant variability in fertilization success. Over half of the individual crosses of O. faveolata and about one-third of A. palmata crosses yielded ≤ 40% fertilization. Total sperm concentration was quantified in only a subset of O. faveolata crosses (range of 1-6 × 107 mL-1), but showed no correlation with fertilization success. We interpret that both parental incompatibility and individual genotypes with low-quality gametes are likely to have contributed to the variable fertilization observed with important implications for conservation. Differential fertilization success implies effective population size may be considerably smaller than hoped and population enhancement efforts need to incorporate many more parental genotypes at the patch scale to ensure successful larval production than indicated by estimates based simply on preserving levels of standing genetic diversity.

Deep Sequencing of T-cell Receptor DNA as a Biomarker of Clonally Expanded TILs in Breast Cancer after Immunotherapy.

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Page, David B; Yuan, Jianda; Redmond, David; Wen, Y Hanna; Durack, Jeremy C; Emerson, Ryan; Solomon, Stephen; Dong, Zhiwan; Wong, Phillip; Comstock, Christopher; Diab, Adi; Sung, Janice; Maybody, Majid; Morris, Elizabeth; Brogi, Edi; Morrow, Monica; Sacchini, Virgilio; Elemento, Olivier; Robins, Harlan; Patil, Sujata; Allison, James P; Wolchok, Jedd D; Hudis, Clifford; Norton, Larry; McArthur, Heather L

2016-10-01

In early-stage breast cancer, the degree of tumor-infiltrating lymphocytes (TIL) predicts response to chemotherapy and overall survival. Combination immunotherapy with immune checkpoint antibody plus tumor cryoablation can induce lymphocytic infiltrates and improve survival in mice. We used T-cell receptor (TCR) DNA sequencing to evaluate both the effect of cryoimmunotherapy in humans and the feasibility of TCR sequencing in early-stage breast cancer. In a pilot clinical trial, 18 women with early-stage breast cancer were treated preoperatively with cryoablation, single-dose anti-CTLA-4 (ipilimumab), or cryoablation + ipilimumab. TCRs within serially collected peripheral blood and tumor tissue were sequenced. In baseline tumor tissues, T-cell density as measured by TCR sequencing correlated with TIL scores obtained by hematoxylin and eosin (H&E) staining. However, tumors with little or no lymphocytes by H&E contained up to 3.6 × 10 6 TCR DNA sequences, highlighting the sensitivity of the ImmunoSEQ platform. In this dataset, ipilimumab increased intratumoral T-cell density over time, whereas cryoablation ± ipilimumab diversified and remodeled the intratumoral T-cell clonal repertoire. Compared with monotherapy, cryoablation plus ipilimumab was associated with numerically greater numbers of peripheral blood and intratumoral T-cell clones expanding robustly following therapy. In conclusion, TCR sequencing correlates with H&E lymphocyte scoring and provides additional information on clonal diversity. These findings support further study of the use of TCR sequencing as a biomarker for T-cell responses to therapy and for the study of cryoimmunotherapy in early-stage breast cancer. Cancer Immunol Res; 4(10); 835-44. ©2016 AACR. ©2016 American Association for Cancer Research.

Production of mutants by irradiation of in vitro-cultured tissues of coconut and banana and their mass propagation by the tissue culture technique

International Nuclear Information System (INIS)

Guzman, E.V. de; Rosario, A.G. del; Pagcaliwagan, P.C.

1982-01-01

Regeneration of buds/shoots as well as plantlets was induced from banana shoot tip explants cultured in highly modified Murashige and Skoog's medium supplemented with coconut water and benzyladenine. Initially shoot regeneration was sparse, but on further subculture became profuse. Gamma irradiation at low dosage (1.0 kR) was stimulating to explant growth and bud formation with the two types of explants used. With Bungulan stimulation was observed even at 2.5 kR. Several morphological aberrations were exhibited by shoots of 'irradiated' in vitro plants growing in potted soil. A highly and continuously proliferating tissue strain has been isolated from a subculture which was ultimately derived from an irradiated explant. Its continued proliferation is dependent on an external supply of coconut water and benzyladenine. In vitro-produced plants have been established under field conditions. The 'irradiated' plants are comparable with, and some seem to be better than, the unirradiated controls with respect to height, girth, sucker production and number of hands and fingers per bunch. Higher doses of irradiation are required to produce an adverse effect on growth of coconut embryos during the liquid culture than when growing in solid medium. (author)

Hormonal effect on polyphenol accumulation in Cassia tissues cultured in vitro

Energy Technology Data Exchange (ETDEWEB)

Shah, R R; Subbaiah, K V; Mehta, A R

1976-06-01

Effects of auxin and kinetin on growth and production of phenolic compounds in cultured Cassia fistula L. tissues were examined. Initiation of polyphenols was largely determined by the auxin concentration in the medium. Growth of the cells in relation to accumulation of polyphenols was studied at different auxin and kinetin concentrations. The accumulation of phenolic materials was essentially restricted to the most rapid phase of the growth cycle. Progressive changes in the pattern of peroxidase activity were followed and their relationship with polyphenol synthesis is examined.

Use of X-Chromosome Inactivation Pattern to Analyze the Clonality of 14 Female Cases of Kaposi Sarcoma.

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Yuan, Ding; XiuJuan, Wu; Yan, Zhang; JunQin, Liang; Fang, Xiang; Shirong, Yu; Xiaojing, Kang; Yanyan, Feng; Weidong, Wu; Dong, Luo; Qingli, Lu; DeZhi, Zhang; XiongMing, Pu

2015-06-16

Kaposi sarcoma (KS) has features of both neoplastic growth and hyperplastic proliferation. It is the most common tumor seen in patients with HIV infection. Whether KS is a real tumor or a benign hyperplastic disease is not known. Tissues from KS and cutaneous hemangioma lesion DNA were extracted, and then digested with methylation-sensitive restriction endonuclease HpaII. Human androgen receptor gene (HUMARA) was amplified with PCR method and the product was separated on 10% denaturing polyacrylamide gels and stained with ethylene dibromide (EB) to show the polymorphism of HUMARA. Phosphoglycerate kinase (PGK) was amplified and the product was digested by BStXI, agarose gel and EB stained to show the polymorphism of PGK. Finally, we analyzed the clonality of KS. In the 14 patients with KS, heterozygosity of the HUMARA gene was observed in 12 (85.7%) cases. Loss of heterozygosity of HUMARA gene on X-chromosome (without HpaII digestion there were 2 bands, after HpaII digestion there were just 1 of the bands), representing monoclonal origin, was present in 11 cases of Kaposi sarcoma. Heterozygosity of the PGK gene was observed in 5 (35.7%) cases, which all represent monoclonal origin. There was no significant difference according to country, stage, or HIV and HHV-8 (P>0.05). The current findings suggest that Kaposi sarcoma is a clonal neoplasm, not a reactive proliferation.

Royal Jelly Prevents Osteoporosis in Rats: Beneficial Effects in Ovariectomy Model and in Bone Tissue Culture Model

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Saburo Hidaka

2006-01-01

Full Text Available Royal jelly (RJ has been used worldwide for many years as medical products, health foods and cosmetics. Since RJ contains testosterone and has steroid hormone-type activities, we hypothesized that it may have beneficial effects on osteoporosis. We used both an ovariectomized rat model and a tissue culture model. Rats were divided into eight groups as follows: sham-operated (Sham, ovariectomized (OVX, OVX given 0.5% (w/w raw RJ, OVX given 2.0% (w/w RJ, OVX given 0.5% (w/w protease-treated RJ (pRJ, OVX given 2.0% (w/w pRJ, OVX given 17β-estradiol and OVX given its vehicle, respectively. The Ovariectomy decreased tibial bone mineral density (BMD by 24%. Administration of 17β-estradiol to OVX rats recovered the tibial BMD decrease by 100%. Administration of 2.0% (w/w RJ and 0.5–2.0% (w/w pRJ to OVX rats recovered it by 85% or more. These results indicate that both RJ and pRJ are almost as effective as 17β-estradiol in preventing the development of bone loss induced by ovariectomy in rats. In tissue culture models, both RJ and pRJ increased calcium contents in femoral-diaphyseal and femoral-metaphyseal tissue cultures obtained from normal male rats. However, in a mouse marrow culture model, they neither inhibited the parathyroid hormone (PTH-induced calcium loss nor affected the formation of osteoclast-like cells induced by PTH in mouse marrow culture system. Therefore, our results suggest that both RJ and pRJ may prevent osteoporosis by enhancing intestinal calcium absorption, but not by directly antagonizing the action of PTH.

Beauveria bassiana (Balsamo) Vuillemin as an endophyte in tissue culture banana (Musa spp.).

Science.gov (United States)

Akello, Juliet; Dubois, Thomas; Gold, Clifford S; Coyne, Daniel; Nakavuma, Jessica; Paparu, Pamela

2007-09-01

Beauveria bassiana is considered a virulent pathogen against the banana weevil Cosmopolites sordidus. However, current field application techniques for effective control against this pest remain a limitation and an alternative method for effective field application needs to be investigated. Three screenhouse experiments were conducted to determine the ability of B. bassiana to form an endophytic relationship with tissue culture banana (Musa spp.) plants and to evaluate the plants for possible harmful effects resulting from this relationship. Three Ugandan strains of B. bassiana (G41, S204 and WA) were applied by dipping the roots and rhizome in a conidial suspension, by injecting a conidial suspension into the plant rhizome and by growing the plants in sterile soil mixed with B. bassiana-colonized rice substrate. Four weeks after inoculation, plant growth parameters were determined and plant tissue colonization assessed through re-isolation of B. bassiana. All B. bassiana strains were able to colonize banana plant roots, rhizomes and pseudostem bases. Dipping plants in a conidial suspension achieved the highest colonization with no negative effect on plant growth or survival. Beauveria bassiana strain G41 was the best colonizer (up to 68%, 79% and 41% in roots, rhizome and pseudostem base, respectively) when plants were dipped. This study demonstrated that, depending on strain and inoculation method, B. bassiana can form an endophytic relationship with tissue culture banana plants, causing no harmful effects and might provide an alternative method for biological control of C. sordidus.

Clonality and Micro-Diversity of a Nationwide Spreading Genotype of Mycobacterium tuberculosis in Japan

Science.gov (United States)

Wada, Takayuki; Iwamoto, Tomotada; Tamaru, Aki; Seto, Junji; Ahiko, Tadayuki; Yamamoto, Kaori; Hase, Atushi; Maeda, Shinji; Yamamoto, Taro

2015-01-01

Mycobacterium tuberculosis transmission routes can be estimated from genotypic analysis of clinical isolates from patients. In Japan, still a middle-incidence country of TB, a unique genotype strain designated as ‘M-strain’ has been isolated nationwide recently. To ascertain the history of the wide spread of the strain, 10 clinical isolates from different areas were subjected to genome-wide analysis based on deep sequencers. Results show that all isolates possessed common mutations to those of referential strains. The greatest number of accumulated single nucleotide variants (SNVs) from the oldest coalescence was 13 nucleotides, indicating high clonality of these isolates. When an SNV common to the isolates was used as a surrogate marker of the clone, authentic clonal isolates with variation in a reliable subset of variable number of tandem repeat (VNTR) genotyping method can be selected successfully from clinical isolates populations of M. tuberculosis. When the authentic clones can also be assigned to sub-clonal groups by SNVs derived from the genomic comparison, they are classifiable into three sub-clonal groups with a bias of geographical origins. Feedback from genomic analysis of clinical isolates of M. tuberculosis to genotypic markers will be an efficient strategy for the big data in various settings for public health actions against TB. PMID:25734518

An electrochemical approach to monitor pH change in agar media during plant tissue culture.

Science.gov (United States)

Wang, Min; Ha, Yang

2007-05-15

In this work, metal oxide microelectrodes were developed to monitor pH change in agar media during plant tissue culture. An antimony wire was produced by a new approach "capillary melt method". The surface of the obtained antimony wire was oxidized in a potassium nitrate melt to fabricate an antimony oxide film for pH sensing. Characterization results show that the oxide layer grown on the wire surface consists of Sb(2)O(3) crystal phase. The sensing response, open-circuit potential, of the electrode has a good linear relationship (R(2)=1.00) with pH value of the test solution. Adding organic compounds into the test media would not affect the linear relationship, although the slope of the lines varied with different ingredients added. The antimony oxide electrodes were employed to continuously monitor pH change of agar culture media during a 2-week plant tissue culture of Dendrobium candidum. The antimony oxide electrode fabricated this way has the advantages of low cost, easy fabrication, fast response, and almost no contamination introduced into the system. It would be suitable for in situ and continuous pH measurement in many bio applications.

Identification of QTLs associated with callogenesis and embryogenesis in oil palm using genetic linkage maps improved with SSR markers.

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Ngoot-Chin Ting

Full Text Available Clonal reproduction of oil palm by means of tissue culture is a very inefficient process. Tissue culturability is known to be genotype dependent with some genotypes being more amenable to tissue culture than others. In this study, genetic linkage maps enriched with simple sequence repeat (SSR markers were developed for dura (ENL48 and pisifera (ML161, the two fruit forms of oil palm, Elaeis guineensis. The SSR markers were mapped onto earlier reported parental maps based on amplified fragment length polymorphism (AFLP and restriction fragment length polymorphism (RFLP markers. The new linkage map of ENL48 contains 148 markers (33 AFLPs, 38 RFLPs and 77 SSRs in 23 linkage groups (LGs, covering a total map length of 798.0 cM. The ML161 map contains 240 markers (50 AFLPs, 71 RFLPs and 119 SSRs in 24 LGs covering a total of 1,328.1 cM. Using the improved maps, two quantitative trait loci (QTLs associated with tissue culturability were identified each for callusing rate and embryogenesis rate. A QTL for callogenesis was identified in LGD4b of ENL48 and explained 17.5% of the phenotypic variation. For embryogenesis rate, a QTL was detected on LGP16b in ML161 and explained 20.1% of the variation. This study is the first attempt to identify QTL associated with tissue culture amenity in oil palm which is an important step towards understanding the molecular processes underlying clonal regeneration of oil palm.

GROWTH AND ROOTING SYSTEM OF ACACIA MANGIUM OBTAINED BY TISSUE CULTURE

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SUPRIYANTO

1991-01-01

Full Text Available Since 1980/1981, the government of Indonesia through the Ministry of Forestry has started to reforest logged-over, alang-alang, unproductive areas and to convert them to Forest Industry Plantation. The target is 300 000 ha per year. It means, 750 million seedlings should be provided per year (planting distance 2 m x 2 m. The tree species to be planted in forest industry plantation should have shorter life cycle (8 - 10 years, good stem-form, good rooting system, and should be fast growing. Acacia mangium has been selected as one of the important tree species for forest industry plantation due to its growth, quality of fiber wood (pulp and paper industry and rooting system (produce a lot of secondary root and nitrogen fixater (Soebardjo 1986. The reforestation of logged-over Dipterocarp forests in Malaysia with A. mangium has also been considered (Appanah and Weinland 1989. Generally, reforestation with A. mangium is done with seedlings obtained by seed germination. A. mangium produce a lot of seeds but its production is still limited by the season, while the conventional method of vegetative propagation through cuttings gave very low percentage of rooted-cuttings (1% (Umboh and Syamsul Yani 1989. The micropropagation of A. mangium through tissue culture is a promising method. The production of A. mangium plantlets through that method has been done at the Forest Genetic Laboratory, Tropical Forest Biology, SEAMEO BIOTROP (Situmorang 1988, Umboh 1988, Umboh et al. 1989, 1990. These rooted-plantlets (plantlings were first put in the green house (acclimatization before planting in the field. Field tests of some agricultural plants have been done but information on forest trees species is still lacking because the production of plantlings through tissue culture is still limited as there are still problems of their rooting. In fact, the progress of reproducing woody plants by tissue culture has been much slower than with herbaceous plants. The major

Seismomorphogenesis: a novel approach to acclimatization of tissue culture regenerated plants.

Science.gov (United States)

Sarmast, Mostafa Khoshhal; Salehi, Hassan; Khosh-Khui, Morteza

2014-12-01

Plantlets under in vitro conditions transferred to ex vivo conditions are exposed to biotic and abiotic stresses. Furthermore, in vitro regenerated plants are typically frail and sometimes difficult to handle subsequently increasing their risk to damage and disease; hence acclimatization of these plantlets is the most important step in tissue culture techniques. An experiment was conducted under in vitro conditions to study the effects of shaking duration (twice daily at 6:00 a.m. and 9:00 p.m. for 2, 4, 8, and 16 min at 250 rpm for 14 days) on Sansevieria trifasciata L. as a model plant. Results showed that shaking improved handling, total plant height, and leaf characteristics of the model plant. Forty-eight hours after 14 days of shaking treatments with increasing shaking time, leaf length decreased but proline content of leaf increased. However, 6 months after starting the experiment different results were observed. In explants that received 16 min of shaking treatment, leaf length and area and photosynthesis rate were increased compared with control plantlets. Six months after starting the experiment, control plantlets had 12.5 % mortality; however, no mortality was observed in other treated explants. The results demonstrated that shaking improved the explants' root length and number and as a simple, cost-effective, and non-chemical novel approach may be substituted for other prevalent acclimatization techniques used for tissue culture regenerated plantlets. Further studies with sensitive plants are needed to establish this hypothesis.

Screening Test of Greenhouse Seeding Exercise Matrix for Tissue Culture Seeding of Dendrobium Officinale Kimura et Migo

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Zhou Yuan

2015-01-01

Full Text Available The Dendrobium officinale Kimura et Migo has a high demand on planting matrix, while its tissue culture seeding has much more demands on planting matrix. To find out a seeding exercise matrix to enhance the survival rate of tissue culture seeding of Dendrobium officinale Kimura et Migo more efficiently, this article carries out a screening test of greenhouse seeding exercise matrix material for tissue culture seeding of Dendrobium officinale Kimura et Migo. The test adopts full random test design, mainly for screening test of five matrix materials, namely pine bark, camphor tree bark, fern root, peanut shell and longan bark. Compare the impact of prepared seeding exercise matrix on the survival rate and growth trend (including plant height, growth rate and bud growth rate. The test result shows that: The seeding exercise matrix prepared by fern root is the most efficient, and the survival rate, plant height, growth rate and bud growth rate have achieved 100%, 4.5cm, 43.67% and 54.33% respectively. The main reason may be that the seeding exercise matrix C prepared by fern root is fairly loose and has a great water permeability, which is conducive to the growth of Dendrobium officinale Kimura et Migo.

Finite element study of scaffold architecture design and culture conditions for tissue engineering.

Science.gov (United States)

Olivares, Andy L; Marsal, Elia; Planell, Josep A; Lacroix, Damien

2009-10-01

Tissue engineering scaffolds provide temporary mechanical support for tissue regeneration and transfer global mechanical load to mechanical stimuli to cells through its architecture. In this study the interactions between scaffold pore morphology, mechanical stimuli developed at the cell microscopic level, and culture conditions applied at the macroscopic scale are studied on two regular scaffold structures. Gyroid and hexagonal scaffolds of 55% and 70% porosity were modeled in a finite element analysis and were submitted to an inlet fluid flow or compressive strain. A mechanoregulation theory based on scaffold shear strain and fluid shear stress was applied for determining the influence of each structures on the mechanical stimuli on initial conditions. Results indicate that the distribution of shear stress induced by fluid perfusion is very dependent on pore distribution within the scaffold. Gyroid architectures provide a better accessibility of the fluid than hexagonal structures. Based on the mechanoregulation theory, the differentiation process in these structures was more sensitive to inlet fluid flow than axial strain of the scaffold. This study provides a computational approach to determine the mechanical stimuli at the cellular level when cells are cultured in a bioreactor and to relate mechanical stimuli with cell differentiation.

Tissue culture of black pepper (piper nigrum l.) in Pakistan

International Nuclear Information System (INIS)

Hussain, A.; Naz, S.; Nazir, H.; Shinwari, Z.K.

2011-01-01

Black pepper (Piper nigrum L.) the 'King of Spices' is a universal table condiment. It is extensively used in Pakistani cuisines and herbal medicines and imported in bulk from neighboring countries. The black pepper vine is generally cultivated by seed because other vegetative propagation methods are slow and time consuming. Therefore the tissue culture technique is considered more efficient and reliable method for rapid and mass propagation of this economically important plant. The present study was initiated to develop protocol for micro-propagation of black pepper vine. The stem, leaf and shoot tip explants from mature vine were cultured on MS medium supplemented with different concentrations of plant growth regulators (2,4-D, BA, IBA). Best callus was produced on MS medium with 1.5 mg/l BA by shoot tip explant. Shoot regeneration was excellent on MS medium with 0.5 mg/l BA. The plantlets formed were rooted best on 1.5 mg/l IBA. The rooted plants were transplanted in soil medium and acclimatized in growth room. The plants raised were test planted under the local conditions of Hattar. (author)

Dynamics of Tumor Heterogeneity Derived from Clonal Karyotypic Evolution

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Ashley M. Laughney

2015-08-01

Full Text Available Numerical chromosomal instability is a ubiquitous feature of human neoplasms. Due to experimental limitations, fundamental characteristics of karyotypic changes in cancer are poorly understood. Using an experimentally inspired stochastic model, based on the potency and chromosomal distribution of oncogenes and tumor suppressor genes, we show that cancer cells have evolved to exist within a narrow range of chromosome missegregation rates that optimizes phenotypic heterogeneity and clonal survival. Departure from this range reduces clonal fitness and limits subclonal diversity. Mapping of the aneuploid fitness landscape reveals a highly favorable, commonly observed, near-triploid state onto which evolving diploid- and tetraploid-derived populations spontaneously converge, albeit at a much lower fitness cost for the latter. Finally, by analyzing 1,368 chromosomal translocation events in five human cancers, we find that karyotypic evolution also shapes chromosomal translocation patterns by selecting for more oncogenic derivative chromosomes. Thus, chromosomal instability can generate the heterogeneity required for Darwinian tumor evolution.

Genet-specific DNA methylation probabilities detected in a spatial epigenetic analysis of a clonal plant population.

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Kiwako S Araki

Full Text Available In sessile organisms such as plants, spatial genetic structures of populations show long-lasting patterns. These structures have been analyzed across diverse taxa to understand the processes that determine the genetic makeup of organismal populations. For many sessile organisms that mainly propagate via clonal spread, epigenetic status can vary between clonal individuals in the absence of genetic changes. However, fewer previous studies have explored the epigenetic properties in comparison to the genetic properties of natural plant populations. Here, we report the simultaneous evaluation of the spatial structure of genetic and epigenetic variation in a natural population of the clonal plant Cardamine leucantha. We applied a hierarchical Bayesian model to evaluate the effects of membership of a genet (a group of individuals clonally derived from a single seed and vegetation cover on the epigenetic variation between ramets (clonal plants that are physiologically independent individuals. We sampled 332 ramets in a 20 m × 20 m study plot that contained 137 genets (identified using eight SSR markers. We detected epigenetic variation in DNA methylation at 24 methylation-sensitive amplified fragment length polymorphism (MS-AFLP loci. There were significant genet effects at all 24 MS-AFLP loci in the distribution of subepiloci. Vegetation cover had no statistically significant effect on variation in the majority of MS-AFLP loci. The spatial aggregation of epigenetic variation is therefore largely explained by the aggregation of ramets that belong to the same genets. By applying hierarchical Bayesian analyses, we successfully identified a number of genet-specific changes in epigenetic status within a natural plant population in a complex context, where genotypes and environmental factors are unevenly distributed. This finding suggests that it requires further studies on the spatial epigenetic structure of natural populations of diverse organisms

Genet-specific DNA methylation probabilities detected in a spatial epigenetic analysis of a clonal plant population.

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Araki, Kiwako S; Kubo, Takuya; Kudoh, Hiroshi

2017-01-01

In sessile organisms such as plants, spatial genetic structures of populations show long-lasting patterns. These structures have been analyzed across diverse taxa to understand the processes that determine the genetic makeup of organismal populations. For many sessile organisms that mainly propagate via clonal spread, epigenetic status can vary between clonal individuals in the absence of genetic changes. However, fewer previous studies have explored the epigenetic properties in comparison to the genetic properties of natural plant populations. Here, we report the simultaneous evaluation of the spatial structure of genetic and epigenetic variation in a natural population of the clonal plant Cardamine leucantha. We applied a hierarchical Bayesian model to evaluate the effects of membership of a genet (a group of individuals clonally derived from a single seed) and vegetation cover on the epigenetic variation between ramets (clonal plants that are physiologically independent individuals). We sampled 332 ramets in a 20 m × 20 m study plot that contained 137 genets (identified using eight SSR markers). We detected epigenetic variation in DNA methylation at 24 methylation-sensitive amplified fragment length polymorphism (MS-AFLP) loci. There were significant genet effects at all 24 MS-AFLP loci in the distribution of subepiloci. Vegetation cover had no statistically significant effect on variation in the majority of MS-AFLP loci. The spatial aggregation of epigenetic variation is therefore largely explained by the aggregation of ramets that belong to the same genets. By applying hierarchical Bayesian analyses, we successfully identified a number of genet-specific changes in epigenetic status within a natural plant population in a complex context, where genotypes and environmental factors are unevenly distributed. This finding suggests that it requires further studies on the spatial epigenetic structure of natural populations of diverse organisms, particularly for

Tissue culture media supplemented with 10% fetal calf serum contains a castrate level of testosterone.

NARCIS (Netherlands)

Sedelaar, J.P.M.; Isaacs, J.T.

2009-01-01

BACKGROUND: Human prostate cancer cells are routinely maintained in media supplemented with 10% Fetal Calf Serum (FCS) to provide androgen. In the present study, total and free testosterone levels in 10%FCS supplemented tissue culture media were determined and compared to levels in intact and

Transmission of clonal chromosomal abnormalities in human hematopoietic stem and progenitor cells surviving radiation exposure

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Kraft, Daniela, E-mail: d.kraft@gsi.de [GSI Helmholtz Center for Heavy Ion Research, Department of Biophysics, Planckstr. 1, 64291 Darmstadt (Germany); Institute for Transfusion Medicine und Immunohematology, DRK-Blutspendedienst Baden-Wuerttemberg—Hessen, Johann Wolfgang Goethe-University Hospital, Sandhofstrasse 1, 60528 Frankfurt (Germany); Ritter, Sylvia, E-mail: s.ritter@gsi.de [GSI Helmholtz Center for Heavy Ion Research, Department of Biophysics, Planckstr. 1, 64291 Darmstadt (Germany); Durante, Marco, E-mail: m.durante@gsi.de [GSI Helmholtz Center for Heavy Ion Research, Department of Biophysics, Planckstr. 1, 64291 Darmstadt (Germany); Institute for Condensed Matter Physics, Physics Department, Technical University Darmstadt, Hochschulstraße 6-8, 64289 Darmstadt (Germany); Seifried, Erhard, E-mail: e.seifried@blutspende.de [Institute for Transfusion Medicine und Immunohematology, DRK-Blutspendedienst Baden-Wuerttemberg—Hessen, Johann Wolfgang Goethe-University Hospital, Sandhofstrasse 1, 60528 Frankfurt (Germany); Fournier, Claudia, E-mail: c.fournier@gsi.de [GSI Helmholtz Center for Heavy Ion Research, Department of Biophysics, Planckstr. 1, 64291 Darmstadt (Germany); Tonn, Torsten, E-mail: t.tonn@blutspende.de [Institute for Transfusion Medicine und Immunohematology, DRK-Blutspendedienst Baden-Wuerttemberg—Hessen, Johann Wolfgang Goethe-University Hospital, Sandhofstrasse 1, 60528 Frankfurt (Germany); Technische Universität Dresden, Med. Fakultät Carl Gustav Carus, Institute for Transfusion Medicine Dresden, German Red Cross Blood Donation Service North-East, Blasewitzer Straße 68/70, 01307 Dresden (Germany)

2015-07-15

Highlights: • Radiation induced formation and transmission of chromosomal aberrations were assessed. • Cytogenetic analysis was performed in human CD34+ HSPC by mFISH. • We report transmission of stable aberrations in irradiated, clonally expanded HSPC. • Unstable aberrations in clonally expanded HSPC occur independently of irradiation. • Carbon ions and X-rays bear a similar risk for propagation of cytogenetic changes. - Abstract: In radiation-induced acute myeloid leukemia (rAML), clonal chromosomal abnormalities are often observed in bone marrow cells of patients, suggesting that their formation is crucial in the development of the disease. Since rAML is considered to originate from hematopoietic stem and progenitor cells (HSPC), we investigated the frequency and spectrum of radiation-induced chromosomal abnormalities in human CD34{sup +} cells. We then measured stable chromosomal abnormalities, a possible biomarker of leukemia risk, in clonally expanded cell populations which were grown for 14 days in a 3D-matrix (CFU-assay). We compared two radiation qualities used in radiotherapy, sparsely ionizing X-rays and densely ionizing carbon ions (29 and 60–85 keV/μm, doses between 0.5 and 4 Gy). Only a negligible number of de novo arising, unstable aberrations (≤0.05 aberrations/cell, 97% breaks) were measured in the descendants of irradiated HSPC. However, stable aberrations were detected in colonies formed by irradiated HSPC. All cells of the affected colonies exhibited one or more identical aberrations, indicating their clonal origin. The majority of the clonal rearrangements (92%) were simple exchanges such as translocations (77%) and pericentric inversions (15%), which are known to contribute to the development of rAML. Carbon ions were more efficient in inducing cell killing (maximum of ∼30–35% apoptotic cells for 2 Gy carbon ions compared to ∼25% for X-rays) and chromosomal aberrations in the first cell-cycle after exposure (∼70% and �

Fusarium growth on culture media made of tissue juice from irradiated and unirradiated potato tubers

International Nuclear Information System (INIS)

Taczanowski, M.

1994-01-01

Fusarium Sulphureum Schlecht is one of the tuber pathogens causing potato storage disease knowing as dry rot. Because irradiation can disturb the tissue defence mechanism against the pathogen, it was decided to carry out experiments on influence of the treatment on subsequent tuber tissue reaction to a maceration process. The maceration as a physical stress was a substitute for the pathogen activity. Tubers of two potato varieties were tested: Mila -a resistant variety to Fusarium and Atol - susceptible one. Tubers of both varieties were irradiated with a dose of 105 kGy. Unirradiated tubers were taken as a control. A day after irradiation the cortex tissue was macerated using an ordinary rasper and the resulted tissue pulp was strained through medical gauze to obtain crude juice. The juice was clarified by centrifugation and then added to dissolved PDA. The volume ratio of juice to PDA was 1:1. The prepared media were dispensed into Petri dishes. Small pieces of the Fusarium culture were put on the surface of the medium at the centre of each Petri dish. Subsequent growth of the fungus was assessed by measurement of culture diameters every 24 hours. Linear functions of the Fusarium growth were obtained for Mila control and Atol control. In the case of Mila, the Fusarium found more favourable conditions for its growth in the presence of juice from irradiated tubers than from the control ones. Making the same comparison for Atol, no difference was detected. (author)

Joint use of developed collagen-containing complexes and cell cultures in creating new tissue equivalents

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K. V. Kulakova

2016-01-01

Full Text Available The purpose of the study is to assess the possibility of applying the integrated module as the basis of a celltissue equivalent for treatment of wounds of skin and soft tissues. In the frame of the set task the following problems were being solved: research of the spatial structure and architectonics of the surface of the developed base collagen-containing materials and their biocompatibility with cell cultures.Materials and methods. The study of a material which is a two-layer complex film, consisting of collagen and polysaccharide components was carried out. The collagen was separated from the dermis and was then impregnated with particulate demineralized bone matrix (DCM according to the original methodology. For the purposes of the study the dehydrated material was created in the form of a film. Electron microscopic examination of surfaces was performed on scanning electron microscope JEOL JSM-IT300LV in high vacuum and at low values of probe current (< 0,1 nА. Studies to assess the viability of the cells cultivated on films of collagen material (tested for cytotoxicity and the adhesive capacity were performed in vitro using strains of diploid human fibroblasts 4–6 passage. The culture condition was visually assessed using an inverted Leica microscope DM IL (Carl Zeiss, Austria, equipped with a computerizes program of control of culture growth (Leica IM 1000.Results. The data obtained in the study of the surface structure of the developed complex module showed that it seems to be promising as a basic component of the cellular-tissue system with its large number of structural formations for fixation of the cells and a well-organized barrier layer capable of vapor - permeability. Experiments in vitro confirmed the absence of toxicity of the material being studied in relation to the culture of dermal human fibroblasts, suggesting the possibility of creation on its basis of cell-tissue complex and further experimental studies in vivo

Hypertrophy of cultured bovine aortic endothelium following irradiation

International Nuclear Information System (INIS)

Rosen, E.M.; Vinter, D.W.; Goldberg, I.D.

1989-01-01

The vascular endothelium is a vital multifunctional tissue which covers the entire luminal surface of the circulatory system. Loss of continuity of the endothelial lining normally results in cell migration and proliferation to make up for cell loss and to ensure that exposure of the thrombogenic subendothelium to platelets and clotting factors is minimized. We showed that ionizing radiation (400-3000 cGy) causes dose-dependent cell loss from confluent monolayer cultures of bovine aortic endothelium, which cannot immediately be compensated by cell proliferation. Within 24 h, the remaining attached cells undergo substantial somatic hypertrophy (evidenced by increased protein content, cell volume, and attachment area) but remain diploid. If cell loss is not excessive, monolayer continuity is restored within several days. Although reduced protein degradation may contribute, most of the protein accumulation is due to synthesis of new protein. Unlike endothelium, irradiation of smooth muscle cultures causes neither cell loss nor increased protein synthesis. Hypertrophy of irradiated endothelial cells appears to be a consequence of a proliferative stimulus (cell loss) in a population of cells which is unable to divide. It can be modulated by replating irradiated cells at different densities. We suggest that endothelial hypertrophy is an early vascular homeostatic response before clonal proliferation of surviving cells or repopulation by cells from outside of the irradiated field can compensate for cell loss

ACAM2000 clonal Vero cell culture vaccinia virus (New York City Board of Health strain)--a second-generation smallpox vaccine for biological defense.

Science.gov (United States)

Monath, Thomas P; Caldwell, Joseph R; Mundt, Wolfgang; Fusco, Joan; Johnson, Casey S; Buller, Mark; Liu, Jian; Gardner, Bridget; Downing, Greg; Blum, Paul S; Kemp, Tracy; Nichols, Richard; Weltzin, Richard

2004-10-01

The threat of smallpox as a biological weapon has spurred efforts to create stockpiles of vaccine for emergency preparedness. In lieu of preparing vaccine in animal skin (the original method), we cloned vaccinia virus (New York City Board of Health strain, Dryvax by plaque purification and amplified the clone in cell culture. The overarching goal was to produce a modern vaccine that was equivalent to the currently licensed Dryvax in its preclinical and clinical properties, and could thus reliably protect humans against smallpox. A variety of clones were evaluated, and many were unacceptably virulent in animal models. One clonal virus (ACAM1000) was selected and produced at clinical grade in MRC-5 human diploid cells. ACAM1000 was comparable to Dryvax in immunogenicity and protective activity but was less neurovirulent for mice and nonhuman primates. To meet requirements for large quantities of vaccine after the events of September 11th 2001, the ACAM1000 master virus seed was used to prepare vaccine (designated ACAM2000) at large scale in Vero cells under serum-free conditions. The genomes of ACAM1000 and ACAM2000 had identical nucleotide sequences, and the vaccines had comparable biological phenotypes. ACAM1000 and ACAM2000 were evaluated in three Phase 1 clinical trials. The vaccines produced major cutaneous reactions and evoked neutralizing antibody and cell-mediated immune responses in the vast majority of subjects and had a reactogenicity profile similar to that of Dryvax.

Cell culture density affects the proliferation activity of human adipose tissue stem cells.

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Kim, Dae Seong; Lee, Myoung Woo; Ko, Young Jong; Chun, Yong Hoon; Kim, Hyung Joon; Sung, Ki Woong; Koo, Hong Hoe; Yoo, Keon Hee

2016-01-01

In this study, we investigated the effect of cell density on the proliferation activity of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT-MSCs) over time in culture. Passage #4 (P4) and #12 (P12) AT-MSCs from two donors were plated at a density of 200 (culture condition 1, CC1) or 5000 (culture condition 2, CC2) cells cm(-2) . After 7 days of incubation, P4 and P12 AT-MSCs cultured in CC1 were thin and spindle-shaped, whereas those cultured in CC2 had extensive cell-to-cell contacts and an expanded cell volume. In addition, P4 and P12 AT-MSCs in CC1 divided more than three times, while those in CC2 divided less than once on average. Flow cytometric analysis using 5(6)-carboxyfluorescein diacetate N-succinimidyl ester dye showed that the fluorescence intensity of AT-MSCs was lower in CC1 than in CC2. Furthermore, expression of proliferation-associated genes, such as CDC45L, CDC20A and KIF20A, in P4 AT-MSCs was higher in CC1 than in CC2, and this difference was also observed in P12 AT-MSCs. These data demonstrated that cell culture density affects the proliferation activity of MSCs, suggesting that it is feasible to design a strategy to prepare suitable MSCs using specific culture conditions. Copyright © 2016 John Wiley & Sons, Ltd.

Effects of cyclic compression on the mechanical properties and calcification process of immature chick bone tissue in culture.

Science.gov (United States)

Maeda, Eijiro; Nakagaki, Masashi; Ichikawa, Katsuhisa; Nagayama, Kazuaki; Matsumoto, Takeo

2017-06-01