scholarly journals RNA-guided piggyBac transposition in human cells

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Brian E Hew ◽  
Ryuei Sato ◽  
Damiano Mauro ◽  
Ilko Stoytchev ◽  
Jesse B Owens
Keyword(s):  
Specific Binding ◽  
Target Sequence ◽  
Safe Harbor ◽  
Gene Insertion ◽  
Guide Rnas ◽  
Clonal Cell Lines ◽  
First Time ◽  
Specific Sequences ◽  
Single Insertion

Abstract Safer and more efficient methods for directing therapeutic genes to specific sequences could increase the repertoire of treatable conditions. Many current approaches act passively, first initiating a double-stranded break, then relying on host repair to uptake donor DNA. Alternatively, we delivered an actively integrating transposase to the target sequence to initiate gene insertion. We fused the hyperactive piggyBac transposase to the highly specific, catalytically dead SpCas9-HF1 (dCas9) and designed guide RNAs (gRNAs) to the CCR5 safe harbor sequence. We introduced mutations to the native DNA-binding domain of piggyBac to reduce non-specific binding of the transposase and cause the fusion protein to favor binding by dCas9. This strategy enabled us, for the first time, to direct transposition to the genome using RNA. We showed that increasing the number of gRNAs improved targeting efficiency. Interestingly, over half of the recovered insertions were found at a single TTAA hotspot. We also found that the fusion increased the error rate at the genome-transposon junction. We isolated clonal cell lines containing a single insertion at CCR5 and demonstrated long-term expression from this locus. These vectors expand the utility of the piggyBac system for applications in targeted gene addition for biomedical research and gene therapy.

Somatic Embryogenesis and In vitro Plant Regeneration in Vigna trilobata (L.) Verd. - A Potential Pasture Legume

1970 ◽  
Vol 19 (1) ◽  
pp. 89-99
Author(s):  
K. Choudhary ◽  
M. Singh ◽  
M. S. Rathore ◽  
N. S. Shekhawat
Keyword(s):  
Somatic Embryogenesis ◽  
Growth Regulators ◽  
Somatic Embryos ◽  
Basal Medium ◽  
Long Term Study ◽  
Continuous Application ◽  
First Time ◽  
Pasture Legume

This long term study demonstrates for the first time that it is possible to propagate embryogenic Vigna trilobata and to subsequently initiate the differentiation of embryos into complete plantlets. Initiation of callus was possible on 2,4-D. Somatic embryos differentiated on modified MS basal nutrient medium with 1.0 mg/l  of 2,4-D and 0.5 mg/l  of Kn. Sustained cell division resulted in globular and heart shape stages of somatic embryos. Transfer of embryos on to a fresh modified MS basal medium with 0.5 mg/l of Kn and 0.5 mg/l of GA3 helped them to attain maturation and germination. However, the propagation of cells, as well as the differentiation of embryos, were inhibited by a continuous application of these growth regulators. For this reason, a long period on medium lacking these growth regulators was necessary before the differentiation of embryos occurred again. The consequences for improving the propagation of embryogenic cultures in Vigna species are discussed. Key words: Pasture  legume, Vigna trilobata, Globular, Heart shape, somatic embryogenesis D.O.I. 10.3329/ptcb.v19i1.4990 Plant Tissue Cult. & Biotech. 19(1): 89-99, 2009 (June)

An Evaluation of Provincial Macroeconomic Performance in Vietnam

2017 ◽  
pp. 34-47
Author(s):  
Hoi Le Quoc ◽  
Nam Pham Xuan ◽  
Tuan Nguyen Anh
Keyword(s):  
Development Strategy ◽  
Institutional Transformation ◽  
Economic Indicators ◽  
Macroeconomic Performance ◽  
Economic Development Strategy ◽  
Socio Economic Development ◽  
The Cost ◽  
Development Objectives ◽  
First Time

The study was targeted at developing a methodology for constructing a macroeconomic performance index at a provincial level for the first time in Vietnam based on 4 groups of measurements: (i) Economic indicators; (ii) oriented economic indicators; (iii) socio-economic indicators; and (iv) economic - social – institutional indicators. Applying the methodology to the 2011 - 2015 empirical data of all provinces in Vietnam, the research shows that the socio-economic development strategy implemented by those provinces did not provide balanced outcomes between growth and social objectives, sustainability and inclusiveness. Many provinces focused on economic growth at the cost of structural change, equality and institutional transformation. In contrast, many provinces were successful in improving equality but not growth. Those facts threaten the long-term development objectives of the provinces.

NEW VARIETIES OF CURRANTS AND GOOSEBERRY BREEDING PLANTS

1970 ◽  
pp. 41-46
Author(s):  
O. D. Golyaeva ◽  
O. V. Kurashev ◽  
S. D. Knyazev ◽  
А. Yu. Bakhotskaya
Keyword(s):  
Powdery Mildew ◽  
Genetic Basis ◽  
Black Currant ◽  
Leaf Spots ◽  
Agricultural Sciences ◽  
Berry Crops ◽  
New Varieties ◽  
First Time ◽  
Red Currant

The main goal of the scientific institution was and remains to improve the assortment of fruit and berry crops for the development of domestic horticulture. Black currant breeding at VNIISPK was started by A.F Tamarova and continued by the doctor of agricultural Sciences T.P.Ogoltsova and doctor of agricultural Sciences S.D. Knyazev. A long-term breeding program has been developed. The main goals of the program are to create black currant cultivars with continuous resistance to diseases, first of all powdery mildew, as wells resistance to pests, i.e. bud mite. As a result of the long-term work, over 40 black currant cultivars have been developed, 14 of them are zoned. Red currant breeding was led by the candidate of agricultural Sciences L.V. Bayanova; since 2001 the work has been continued by the candidate of agricultural Sciences O.D. Golyaeva. ‘Heinemanns Rote Spӓtlese’, the descendant of R. multiflorum Kit., was involved in the red currant breeding for the first time in Russia. On its genetic basis, a series of late maturing cultivars with long and dense racemes was created. At the Institute, in total 21cultivars of red currants have been developed, 13 of them are zoned. At present, red currant cultivars make up 25.5% of the zoned assortment in Russia. The first research on gooseberries was stated by V.P. Semakin and A.F Tamarova; since 1992 the systematic gooseberry breeding has been carried out by the candidate of agricultural Sciences O.V. Kurashev. On the basis of Grossularia robusta, we have created gooseberry forms that are resistant to powdery mildew and leaf spots. These forms are highly productive, weakly thorned, having bush habit suitable for mechanized harvest. The result of breeding activities was the transfer of 6 gooseberry cultivars to State agricultural testing: ‘Solnechny Zaychik’, ‘Nekrasovsky’, ‘Yupiter’, ‘Zemlianichny’, ‘Moryachok’ and ‘Discovery’.

ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

2020 ◽  
Author(s):  
Y.V. Mikhaylova ◽  
◽  
M.A. Tyumentseva ◽  
A.A. Shelenkov ◽  
Y.G. Yanushevich ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Correct Choice ◽  
Target Sequence ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Chemokine Receptor Ccr5 ◽  
Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

scholarly journals Structure-Properties Correlation of Cross-Linked Penicillin G Acylase Crystals

Crystals ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 451
Author(s):  
Marta Kubiak ◽  
Janine Mayer ◽  
Ingo Kampen ◽  
Carsten Schilde ◽  
Rebekka Biedendieck
Keyword(s):  
Mechanical Stability ◽  
Structural Characteristics ◽  
Penicillin G ◽  
Bacillus Species ◽  
Diffusion Limitations ◽  
Small Water ◽  
Long Term Stability ◽  
Atomic Force ◽  
First Time

In biocatalytic processes, the use of free enzymes is often limited due to the lack of long-term stability and reusability. To counteract this, enzymes can be crystallized and then immobilized, generating cross-linked enzyme crystals (CLECs). As mechanical stability and activity of CLECs are crucial, different penicillin G acylases (PGAs) from Gram-positive organisms have proven to be promising candidates for industrial production of new semisynthetic antibiotics, which can be crystallized and cross-linked to characterize the resulting CLECs regarding their mechanical and catalytic properties. The greatest hardness and Young’s modulus determined by indentation with an atomic force microscope were observed for CLECs of Bacillus species FJAT-PGA CLECs (26 MPa/1450 MPa), followed by BmPGA (Priestia megaterium PGA, 23 MPa/1170 MPa) and BtPGA CLECs (Bacillus thermotolerans PGA, 11 MPa/614 MPa). In addition, FJAT- and BtPGA CLECs showed up to 20-fold higher volumetric activities compared to BmPGA CLECs. Correlation to structural characteristics indicated that a high solvent content and low number of cross-linking residues might lead to reduced stability. Furthermore, activity seems to be restricted by small water channels due to severe diffusion limitations. To the best of our knowledge, we show for the first time in this study that the entire process chain for the characterization of diverse industrially relevant enzymes can be performed at the microliter scale to discover the most important relationships and limitations.

scholarly journals Comparable Long-Term Rabies Immunity in Foxes after IntraMuscular and Oral Application Using a Third-Generation Oral Rabies Virus Vaccine

Vaccines ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 49
Author(s):  
Verena te Kamp ◽  
Virginia Friedrichs ◽  
Conrad M. Freuling ◽  
Ad Vos ◽  
Madlin Potratz ◽  
...  
Keyword(s):  
Immune Response ◽  
Rabies Virus ◽  
Specific Binding ◽  
Lethal Dose ◽  
Genetically Engineered ◽  
Routes Of Administration ◽  
Safety Studies ◽  
Oral Rabies Vaccine ◽  
Cellular Immune

The live genetically-engineered oral rabies virus (RABV) variant SPBN GASGAS induces long-lasting immunity in foxes and protection against challenge with an otherwise lethal dose of RABV field strains both after experimental oral and parenteral routes of administration. Induction of RABV-specific binding antibodies and immunoglobulin isotypes (IgM, total IgG, IgG1, IgG2) were comparable in orally and parenterally vaccinated foxes. Differences were only observed in the induction of virus-neutralizing (VNA) titers, which were significantly higher in the parenterally vaccinated group. The dynamics of rabies-specific antibodies pre- and post-challenge (365 days post vaccination) suggest the predominance of type-1 immunity protection of SPBN GASGAS. Independent of the route of administration, in the absence of IgG1 the immune response to SPBN GAGAS was mainly IgG2 driven. Interestingly, vaccination with SPBN GASGAS does not cause significant differences in inducible IFN-γ production in vaccinated animals, indicating a relatively weak cellular immune response during challenge. Notably, the parenteral application of SPBN GASGAS did not induce any adverse side effects in foxes, thus supporting safety studies of this oral rabies vaccine in various species.

scholarly journals Exposure to Δ9-tetrahydrocannabinol during rat pregnancy leads to impaired cardiac dysfunction in postnatal life

2021 ◽  
Author(s):  
Kendrick Lee ◽  
Steven R. Laviolette ◽  
Daniel B. Hardy
Keyword(s):  
Body Weight ◽  
Cardiac Function ◽  
Cardiac Dysfunction ◽  
Cardiac Remodelling ◽  
Long Term Effects ◽  
Catch Up ◽  
First Time ◽  
Impaired Cardiac Function ◽  
In Pregnancy

Abstract Background Cannabis use in pregnancy leads to fetal growth restriction (FGR), but the long-term effects on cardiac function in the offspring are unknown, despite the fact that fetal growth deficits are associated with an increased risk of developing postnatal cardiovascular disease. We hypothesize that maternal exposure to Δ9-tetrahydrocannabinol (Δ9-THC) during pregnancy will impair fetal development, leading to cardiac dysfunction in the offspring. Methods Pregnant Wistar rats were randomly selected and administered 3 mg/kg of Δ9-THC or saline as a vehicle daily via intraperitoneal injection from gestational days 6 to 22, followed by echocardiogram analysis of cardiac function on offspring at postnatal days 1 and 21. Heart tissue was harvested from the offspring at 3 weeks for molecular analysis of cardiac remodelling. Results Exposure to Δ9-THC during pregnancy led to FGR with a significant decrease in heart-to-body weight ratios at birth. By 3 weeks, pups exhibited catch-up growth associated with significantly greater left ventricle anterior wall thickness with a decrease in cardiac output. Moreover, these Δ9-THC-exposed offsprings exhibited increased expression of collagen I and III, decreased matrix metallopeptidase-2 expression, and increased inactivation of glycogen synthase kinase-3β, all associated with cardiac remodelling. Conclusions Collectively, these data suggest that Δ9-THC-exposed FGR offspring undergo postnatal catch-up growth concomitant with cardiac remodelling and impaired cardiac function early in life. Impact To date, the long-term effects of perinatal Δ9-THC (the main psychoactive component) exposure on the cardiac function in the offspring remain unknown. We demonstrated, for the first time, that exposure to Δ9-THC alone during rat pregnancy results in significantly smaller hearts relative to body weight. These Δ9-THC-exposed offsprings exhibited postnatal catch-up growth concomitant with cardiac remodelling and impaired cardiac function. Given the increased popularity of cannabis use in pregnancy along with rising Δ9-THC concentrations, this study, for the first time, identifies the risk of perinatal Δ9-THC exposure on early postnatal cardiovascular health.

Empowering and Directive Leadership: The Cost of Changing Styles

2021 ◽  
pp. 227853372110083
Author(s):  
Smita Mukherjee ◽  
Zubin R. Mulla
Keyword(s):  
Laboratory Experiment ◽  
Leadership Styles ◽  
Leadership Style ◽  
Affective Commitment ◽  
Team Outcomes ◽  
History Of ◽  
Satisfaction With The Leader ◽  
The Cost ◽  
First Time

We examine the cost of leaders changing between empowering and directive leadership styles on team outcomes. In a laboratory experiment, we collected data from 240 participants in 80 teams. Confederates enacted different leadership styles and led teams of participants in performing a series of tasks. When leaders changed their style from directive to empowering, teams took time to respond in terms of higher satisfaction with leader and affective commitment. However, when leaders changed their style from empowering to directive, the deterioration of satisfaction with leader and reduction in affective commitment were immediate. Moreover, teams of leaders who had been consistently directive showed higher affective commitment as compared to teams of leaders who had a history of being empowering but later shifted to being directive. First time managers can get inputs on how they should enact their leadership style and be aware that switching between styles may impose long-term costs on the team’s affective commitment and satisfaction with the leader.

scholarly journals Solubility Product of Vanadinite Pb5(VO4)3Cl at 25 °C—A Comprehensive Approach to Incongruent Dissolution Modeling

Minerals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 135
Author(s):  
Justyna Topolska ◽  
Bartosz Puzio ◽  
Olaf Borkiewicz ◽  
Julia Sordyl ◽  
Maciej Manecki
Keyword(s):  
Nuclear Waste ◽  
Solubility Product ◽  
Geochemical Modeling ◽  
Incongruent Dissolution ◽  
Mineral Solubility ◽  
Actual Application ◽  
Universal Approach ◽  
Solubility Constant ◽  
First Time

Although vanadinite (Pb5(VO4)3Cl) occurs in abundance in various terrestrial geochemical systems of natural and anthropogenic origin and is seriously considered as a potential nuclear waste sequestering agent, its actual application is severely limited by a lack of understanding of its basic thermodynamic parameters. In this regard, the greatest challenge is posed by its incongruent dissolution, which is a pivotal hurdle for effective geochemical modeling. Our paper presents an universal approach for geochemical computing of systems undergoing incongruent dissolution which, along with unique, long-term experiments on vanadinites’ stability, allowed us to determine the mineral solubility constant. The dissolution experiments were carried out at pH = 3.5 for 12 years. Vanadinite has dissolved incongruently, continuously re-precipitating into chervetite (Pb2V2O7) with the two minerals remaining in mutual equilibrium until termination of the experiments. The empirically derived solubility constant Ksp,V,298 = 10–91.89 ± 0.05 of vanadinite was determined for the first time. The proposed modeling method is versatile and can be adopted to other mineral systems undergoing incongruent dissolution.

scholarly journals Dinosaurs in decline tens of millions of years before their final extinction

2016 ◽  
Vol 113 (18) ◽  
pp. 5036-5040 ◽  
Author(s):  
Manabu Sakamoto ◽  
Michael J. Benton ◽  
Chris Venditti
Keyword(s):  
Mass Extinction ◽  
Marked Reduction ◽  
Evolutionary Dynamics ◽  
General Pattern ◽  
Extinction Rate ◽  
Catastrophic Event ◽  
Extinction Event ◽  
Mass Extinction Event ◽  
First Time

Whether dinosaurs were in a long-term decline or whether they were reigning strong right up to their final disappearance at the Cretaceous–Paleogene (K-Pg) mass extinction event 66 Mya has been debated for decades with no clear resolution. The dispute has continued unresolved because of a lack of statistical rigor and appropriate evolutionary framework. Here, for the first time to our knowledge, we apply a Bayesian phylogenetic approach to model the evolutionary dynamics of speciation and extinction through time in Mesozoic dinosaurs, properly taking account of previously ignored statistical violations. We find overwhelming support for a long-term decline across all dinosaurs and within all three dinosaurian subclades (Ornithischia, Sauropodomorpha, and Theropoda), where speciation rate slowed down through time and was ultimately exceeded by extinction rate tens of millions of years before the K-Pg boundary. The only exceptions to this general pattern are the morphologically specialized herbivores, the Hadrosauriformes and Ceratopsidae, which show rapid species proliferations throughout the Late Cretaceous instead. Our results highlight that, despite some heterogeneity in speciation dynamics, dinosaurs showed a marked reduction in their ability to replace extinct species with new ones, making them vulnerable to extinction and unable to respond quickly to and recover from the final catastrophic event.

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