Transformation of cambial tissue in vivo provides an efficient means for induced somatic sector analysis and gene testing in stems of woody plant species

2006 ◽  
Vol 33 (7) ◽  
pp. 629 ◽  
Author(s):  
Kim S. Van Beveren ◽  
Antanas V. Spokevicius ◽  
Josquin Tibbits ◽  
Qing Wang ◽  
Gerd Bossinger
Keyword(s):  
Cell Fate ◽  
Gene Function ◽  
Large Scale ◽  
Secondary Growth ◽  
Wood Formation ◽  
Forest Tree ◽  
Wound Response ◽  
Sector Analysis

Large-scale functional analysis of genes and transgenes suspected to be involved in wood development in trees is hindered by long generation times, low transformation and regeneration efficiencies and difficulties with phenotypic assessment of traits, especially those that appear late in a tree’s development. To avoid such obstacles many researchers have turned to model plants such as Arabidopsis thaliana (L.) Heynh., Zinnia elegans Jacq. and Nicotiana ssp., or have focused their attention on in vitro wood formation systems or in vivo approaches targeting primary meristems for transformation. Complementing such efforts, we report the use of Agrobacterium to introduce transgenes directly into cambial cells of glasshouse-grown trees in order to create transgenic somatic tissue sectors. These sectors are suitable for phenotypic evaluation and analysis of target gene function. In our experiments the wood formation zone containing the cambium of Eucalyptus, Populus and Pinus species of varying age was inoculated with Agrobacterium containing a CaMV 35S::GUS construct. Following an initial wound response, frequent and stable transformation was observed in the form of distinct GUS-staining patterns (sectors) in newly formed secondary tissues. Sector size and extent depended on the cell type transformed, the species and the length of time treated plants were allowed to grow (more than two years in some cases). Induced somatic sector analysis (ISSA) can now be efficiently used to study cell fate and gene function during secondary growth in stems of forest tree species.

scholarly journals Deep Imaging Analysis in VISUAL Reveals the Role of YABBY Genes in Vascular Stem Cell Fate Determination

2020 ◽  
Vol 61 (2) ◽  
pp. 255-264
Author(s):  
Alif Meem Nurani ◽  
Yasuko Ozawa ◽  
Tomoyuki Furuya ◽  
Yuki Sakamoto ◽  
Kazuo Ebine ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Fate ◽  
Spatial Information ◽  
Secondary Growth ◽  
Vascular Differentiation ◽  
Mesophyll Cells ◽  
Vascular Cell

Abstract Stem cells undergo cell division and differentiation to ensure organized tissue development. Because plant cells are immobile, plant stem cells ought to decide their cell fate prior to differentiation, to locate specialized cells in the correct position. In this study, based on a chemical screen, we isolated a novel secondary cell wall indicator BF-170, which binds to lignin and can be used to image in vitro and in situ xylem development. Use of BF-170 to observe the vascular differentiation pattern in the in vitro vascular cell induction system, VISUAL, revealed that adaxial mesophyll cells of cotyledons predominantly generate ectopic xylem cells. Moreover, phloem cells are abundantly produced on the abaxial layer, suggesting the involvement of leaf adaxial–abaxial polarity in determining vascular cell fate. Analysis of abaxial polarity mutants highlighted the role of YAB3, an abaxial cell fate regulator, in suppressing xylem and promoting phloem differentiation on the abaxial domains in VISUAL. Furthermore, YABBY family genes affected in vivo vascular development during the secondary growth. Our results denoted the possibility that such mediators of spatial information contribute to correctly determine the cell fate of vascular stem cells, to conserve the vascular pattern of land plants.

scholarly journals Epigenetic Regulation in Mesenchymal Stem Cell Aging and Differentiation and Osteoporosis

2020 ◽  
Vol 2020 ◽  
pp. 1-17 ◽  
Author(s):  
Ruoxi Wang ◽  
Yu Wang ◽  
Lisha Zhu ◽  
Yan Liu ◽  
Weiran Li
Keyword(s):  
Regenerative Medicine ◽  
Cell Fate ◽  
Large Scale ◽  
Functional Decline ◽  
Cell Aging ◽  
Epigenetic Mechanisms ◽  
Reliable Source ◽  
Regenerative Therapies

Mesenchymal stem cells (MSCs) are a reliable source for cell-based regenerative medicine owing to their multipotency and biological functions. However, aging-induced systemic homeostasis disorders in vivo and cell culture passaging in vitro induce a functional decline of MSCs, switching MSCs to a senescent status with impaired self-renewal capacity and biased differentiation tendency. MSC functional decline accounts for the pathogenesis of many diseases and, more importantly, limits the large-scale applications of MSCs in regenerative medicine. Growing evidence implies that epigenetic mechanisms are a critical regulator of the differentiation programs for cell fate and are subject to changes during aging. Thus, we here review epigenetic dysregulations that contribute to MSC aging and osteoporosis. Comprehending detailed epigenetic mechanisms could provide us with a novel horizon for dissecting MSC-related pathogenesis and further optimizing MSC-mediated regenerative therapies.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

Naphthoquinones and derivatives for chemotherapy: perspectives and limitations of their anti-trypanosomatids activities

2020 ◽  
Vol 26 ◽  
Author(s):  
Luíza Dantas-Pereira ◽  
Edézio F. Cunha-Junior ◽  
Valter V. Andrade-Neto ◽  
John F. Bower ◽  
Guilherme A. M. Jardim ◽  
...  
Keyword(s):  
Large Scale ◽  
Neglected Tropical Diseases ◽  
Folk Medicine ◽  
Leishmania Species ◽  
Parasitic Infections ◽  
Mechanistic Analysis ◽  
Leishmania Spp ◽  
Nanomolar Range

: Chagas disease, Sleeping sickness and Leishmaniasis, caused by trypanosomatids Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp., respectively, are considered neglected tropical diseases, and they especially affect impoverished populations in the developing world. The available chemotherapies are very limited and a search for alternatives is still necessary. In folk medicine, natural naphthoquinones have been employed for the treatment of a great variety of illnesses, including parasitic infections. This review is focused on the anti-trypanosomatid activity and mechanistic analysis of naphthoquinones and derivatives. Among all the series of derivatives tested in vitro, naphthoquinone-derived 1,2,3-triazoles were very active on T. cruzi infective forms in blood bank conditions, as well as in amastigotes of Leishmania spp. naphthoquinones containing a CF3 on a phenyl amine ring inhibited T. brucei proliferation in the nanomolar range, and naphthopterocarpanquinones stood out for their activity on a range of Leishmania species. Some of these compounds showed a promising selectivity index (SI) (30 to 1900), supporting further analysis in animal models. Indeed, high toxicity to the host and inactivation by blood components are crucial obstacles to be overcome to use naphthoquinones and/or their derivatives for chemotherapy. Multidisciplinary initiatives embracing medicinal chemistry, bioinformatics, biochemistry, and molecular and cellular biology need to be encouraged to allow the optimization of these compounds. Large scale automated tests are pivotal for the efficiency of the screening step, and subsequent evaluation of both the mechanism of action in vitro and pharmacokinetics in vivo are essential for the development of a novel, specific and safe derivative, minimizing adverse effects.

scholarly journals Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1288
Author(s):  
Wendy Dong ◽  
Boris Kantor
Keyword(s):  
Large Scale ◽  
Lentiviral Vector ◽  
Clinical Applications ◽  
Scale Production ◽  
Base Editing ◽  
Epigenome Editing ◽  
Vector Systems ◽  
Dividing Cells

CRISPR/Cas technology has revolutionized the fields of the genome- and epigenome-editing by supplying unparalleled control over genomic sequences and expression. Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes and (ii) sustain robust and long-term expression in a broad range of dividing and non-dividing cells in vitro and in vivo. It is thus reasonable that substantial effort has been allocated towards the development of the improved and optimized LV systems for effective and accurate gene-to-cell transfer of CRISPR/Cas tools. The main effort on that end has been put towards the improvement and optimization of the vector’s expression, development of integrase-deficient lentiviral vector (IDLV), aiming to minimize the risk of oncogenicity, toxicity, and pathogenicity, and enhancing manufacturing protocols for clinical applications required large-scale production. In this review, we will devote attention to (i) the basic biology of lentiviruses, and (ii) recent advances in the development of safer and more efficient CRISPR/Cas vector systems towards their use in preclinical and clinical applications. In addition, we will discuss in detail the recent progress in the repurposing of CRISPR/Cas systems related to base-editing and prime-editing applications.

scholarly journals Pan-ERBB kinase inhibition augments CDK4/6 inhibitor efficacy in oesophageal squamous cell carcinoma

Gut ◽  
2021 ◽  
pp. gutjnl-2020-323276
Author(s):  
Jin Zhou ◽  
Zhong Wu ◽  
Zhouwei Zhang ◽  
Louisa Goss ◽  
James McFarland ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Large Scale ◽  
Cell Cancer ◽  
Oesophageal Squamous Cell Carcinoma ◽  
Squamous Cancer ◽  
Striking Success

ObjectiveOesophageal squamous cell carcinoma (OSCC), like other squamous carcinomas, harbour highly recurrent cell cycle pathway alterations, especially hyperactivation of the CCND1/CDK4/6 axis, raising the potential for use of existing CDK4/6 inhibitors in these cancers. Although CDK4/6 inhibition has shown striking success when combined with endocrine therapy in oestrogen receptor positive breast cancer, CDK4/6 inhibitor palbociclib monotherapy has not revealed evidence of efficacy to date in OSCC clinical studies. Herein, we sought to elucidate the identification of key dependencies in OSCC as a foundation for the selection of targets whose blockade could be combined with CDK4/6 inhibition.DesignWe combined large-scale genomic dependency and pharmaceutical screening datasets with preclinical cell line models, to identified potential combination therapies in squamous cell cancer.ResultsWe identified sensitivity to inhibitors to the ERBB family of receptor kinases, results clearly extending beyond the previously described minority of tumours with EGFR amplification/dependence, specifically finding a subset of OSCCs with dual dependence on ERBB3 and ERBB2. Subsequently. we demonstrated marked efficacy of combined pan-ERBB and CDK4/6 inhibition in vitro and in vivo. Furthermore, we demonstrated that squamous lineage transcription factor KLF5 facilitated activation of ERBBs in OSCC.ConclusionThese results provide clear rationale for development of combined ERBB and CDK4/6 inhibition in these cancers and raises the potential for KLF5 expression as a candidate biomarker to guide the use of these agents. These data suggested that by combining existing Food and Drug Administration (FDA)-approved agents, we have the capacity to improve therapy for OSCC and other squamous cancer.

scholarly journals A therapeutic vascular conduit to support in vivo cell-secreted therapy

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Edward X. Han ◽  
Hong Qian ◽  
Bo Jiang ◽  
Maria Figetakis ◽  
Natalia Kosyakova ◽  
...  
Keyword(s):  
Vascular Graft ◽  
Large Scale ◽  
Biological Effects ◽  
Therapeutic Protein ◽  
Close Proximity ◽  
Delivery Platform ◽  
Stage Renal Disease ◽  
End Stage

AbstractA significant barrier to implementation of cell-based therapies is providing adequate vascularization to provide oxygen and nutrients. Here we describe an approach for cell transplantation termed the Therapeutic Vascular Conduit (TVC), which uses an acellular vessel as a scaffold for a hydrogel sheath containing cells designed to secrete a therapeutic protein. The TVC can be directly anastomosed as a vascular graft. Modeling supports the concept that the TVC allows oxygenated blood to flow in close proximity to the transplanted cells to prevent hypoxia. As a proof-of-principle study, we used erythropoietin (EPO) as a model therapeutic protein. If implanted as an arteriovenous vascular graft, such a construct could serve a dual role as an EPO delivery platform and hemodialysis access for patients with end-stage renal disease. When implanted into nude rats, TVCs containing EPO-secreting fibroblasts were able to increase serum EPO and hemoglobin levels for up to 4 weeks. However, constitutive EPO expression resulted in macrophage infiltration and luminal obstruction of the TVC, thus limiting longer-term efficacy. Follow-up in vitro studies support the hypothesis that EPO also functions to recruit macrophages. The TVC is a promising approach to cell-based therapeutic delivery that has the potential to overcome the oxygenation barrier to large-scale cellular implantation and could thus be used for a myriad of clinical disorders. However, a complete understanding of the biological effects of the selected therapeutic is absolutely essential.

scholarly journals Human Oligodendrocytes and Myelin In Vitro to Evaluate Developmental Neurotoxicity

2021 ◽  
Vol 22 (15) ◽  
pp. 7929
Author(s):  
Megan Chesnut ◽  
Thomas Hartung ◽  
Helena Hogberg ◽  
David Pamies
Keyword(s):  
Large Scale ◽  
In Vitro Models ◽  
Environmental Chemicals ◽  
Developmental Neurotoxicity ◽  
In Vivo Studies ◽  
Regulatory Guidelines ◽  
In Vitro Systems ◽  
Human Oligodendrocytes

Neurodevelopment is uniquely sensitive to toxic insults and there are concerns that environmental chemicals are contributing to widespread subclinical developmental neurotoxicity (DNT). Increased DNT evaluation is needed due to the lack of such information for most chemicals in common use, but in vivo studies recommended in regulatory guidelines are not practical for the large-scale screening of potential DNT chemicals. It is widely acknowledged that developmental neurotoxicity is a consequence of disruptions to basic processes in neurodevelopment and that testing strategies using human cell-based in vitro systems that mimic these processes could aid in prioritizing chemicals with DNT potential. Myelination is a fundamental process in neurodevelopment that should be included in a DNT testing strategy, but there are very few in vitro models of myelination. Thus, there is a need to establish an in vitro myelination assay for DNT. Here, we summarize the routes of myelin toxicity and the known models to study this particular endpoint.

scholarly journals In vitro multiplication, micromorphological studies and ex vitro rooting of Hybanthus enneaspermus (L.) F. Muell. – a rare medicinal plant

2018 ◽  
Vol 77 (1) ◽  
pp. 80-87 ◽  
Author(s):  
Mahipal S. Shekhawat ◽  
M. Manokari
Keyword(s):  
Medicinal Plant ◽  
Large Scale ◽  
Nodal Segments ◽  
Ms Medium ◽  
Ex Vitro ◽  
Culture Bottle ◽  
Hybanthus Enneaspermus ◽  
Half Strength

AbstractHybanthus enneaspermusis a rare medicinal plant. We defined a protocol for micropropagation,ex vitrorooting of cloned shoots and their acclimatization. Surface-sterilized nodal segments were cultured on Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP) and kinetin (Kin). Medium supplemented with 1.5 mg L−1BAP was found optimum for shoot induction from the explants and 6.4±0.69 shoots were regenerated from each node with 97% response. Shoots were further proliferated maximally (228±10.3 shoots per culture bottle with 7.5±0.43 cm length) on MS medium augmented with 1.0 mg L−1each of BAP and Kin within 4–5 weeks. The shoots were rootedin vitroon half strength MS medium containing 2.0 mg L−1indole-3 butyric acid (IBA). The cloned shoots were pulse-treated with 300 mg L–1 of IBA and cultured on soilrite® in a greenhouse. About 96% of the IBA-pulsed shoots rootedex vitroin soilrite®, each shoot producing 12.5±0.54 roots with 5.1±0.62 cm length. Theex vitrorooted plantlets showed a better rate of survival (92%) in a field study thanin vitrorooted plantlets (86%). A comparative foliar micromorphological study ofH. enneaspermuswas conducted to understand the micromorphological changes during plant developmental processes fromin vitrotoin vivoconditions in terms of variations in stomata, vein structures and spacing, and trichomes. This is the first report onex vitrorooting inH. enneaspermusand the protocol can be exploited for conservation and large-scale propagation of this rare and medicinally important plant.

scholarly journals New inhibitors of glucosylceramide synthase and their effect on cell fate

2014 ◽  
Vol 7 (2) ◽  
pp. 99-104 ◽  
Author(s):  
Katarína Turáková ◽  
Boris Lakatoš ◽  
Andrej Ďuriš ◽  
Daniela Moravčíková ◽  
Dušan Berkeš
Keyword(s):  
Cell Viability ◽  
Cell Fate ◽  
Calcium Transport ◽  
Large Scale ◽  
Process Development ◽  
Pharmaceutical Research ◽  
Pathological Process ◽  
Uridine Diphosphate ◽  
Induction Of Apoptosis

Abstract Glucosylceramide (GlcCer) is an essential glycosylated lipid found in organisms ranging from fungi to mammals. It is composed of a hydrophilic β-linked glucose and a hydrophobic ceramide, with a predominant content of sphingosine in mammals (d18:1). GlcCer is the precursor of a large scale of different glycosphingolipids. This cerebrozide is synthesized from uridine diphosphate-glucose and ceramide by a GlcCer synthase (UDP-glucose:ceramide glucosyltransferase; UGCG, EC 2.4.1.80). GlcCer-based sphingolipids have been identified as important mediators of a variety of cellular functions and their disequilibrium leads to pathological process development and may induce several diseases progression. Therefore, design of UGCG inhibitor represents an important topic for pharmaceutical research. In this paper, we aimed to study effects of newly synthesized derivatives of (±)-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, known UGCG inhibitor) on: i) activity of UGCG in vitro; ii) thymocytes viability; iii) calcium transport through plasma membrane of thymocytes; iv) induction of apoptosis and autophagy in thymocytes. Thymocytes were isolated from thymus of three to seven weeks old mice (ICR strain). The key factors influencing the effect of PPMP analogues were their concentration, chemical structure and incubation time. Derivatives were able to change Ca2+ transport already after 15 min of cultivation, but their effects on cell viability were manifested at least after 12 h of cultivation. Four from fifteen studied compounds affected UGCG activity after four hour lasting cultivation, - but without correlation with data relating to effects on calcium transport and/or cell viability. Most potent UGCG inhibitor was chosen and applied for induction of apoptosis and autophagy in thymocytes. This inhibitor induced typical DNA fragmentation and upregulation of LC3B protein as autophagy marker, after 2 h and 4 h cultivation, respectively.

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