共查询到20条相似文献,搜索用时 31 毫秒
1.
Objectives
To test the applicability of Cpf1 from Francisella novivida in genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae.Results
An easy-to-use vector toolkit, containing a CEN6/ARS4 plasmid expressing Cpf1 from Francisella novivida (FnCpf1) and a 2 μ plasmid for crRNA or crRNA array expressing, was constructed for Cpf1-assisted genomic integration in S. cerevisiae. Our results showed that FnCpf1 allowed for targeted singleplex, doubleplex, and tripleplex genomic integration of in vivo assembled DNA parts with efficiencies of 95, 52, and 43%, respectively.Conclusions
CRISPR-Cpf1 system allows for efficient genomic integration of in vivo assembled DNA parts in S. cerevisiae, and thus provides an alternative CRISPR-Cas method for metabolic pathway engineering in addition to CRISPR-Cas9 system previously reported for yeast.2.
Objectives
To develop a site-specific integration strategy for CAR-T engineering by using a non-viral vector dependent on adeno-associated viral (AAV) genome, which tends to be integrated into AAVS1 site with the help of its Rep proteins.Results
AAV-dependent vectors were produced in Sf9 cells. Structural analyses revealed the vector as covalently close-ended, linear duplex molecules, which was termed “CELiD” DNA. A plasmid CMV-Rep was constructed to express the integrases Rep78 and Rep68. Jurkat cells were co-electroporated with “CELiD” DNA and plasmid CMV-Rep in order to specifically integrate CAR gene into AAVS1 site. We examined 71 stably transfected Jurkat clones by nested PCR, sequencing and southern blotting, of which 30 clones bore CAR gene within AAVS1 site. The site-specific integration efficiency was nearly 42.2 %.Conclusions
The AAV-dependent vector preferentially integrated CAR into AAVS1 site, which could be further used in human T cell modification and enhance the security of CAR-T therapy.3.
4.
Background
Non-viral vectors for gene transfer are less immunogenic than viral vectors but also less efficient. Significant effort has focused on enhancing non-viral gene transfer efficiency by increasing nuclear import of plasmid DNA, particularly by coupling nuclear localization peptidic sequences to plasmid DNA.Results
We have coupled a 62-aminoacid peptide derived from hSRP1α importin beta binding domain, called the IBB peptide to plasmid DNA by using the heterobifunctional linker N-(4-azido-2,3,5,6 tetrafluorobenzyl)-6-maleimidyl hexanamide (TFPAM-6). When covalently coupled to plasmid DNA, IBB peptide did not increase the efficiency of cationic lipid mediated transfection. The IBB peptide was still able to interact with its nuclear import receptor, importin β, but non-specifically. However, we observed a 20-fold increase in reporter gene expression with plasmid DNA / IBB peptide complexes under conditions of inefficient transfection. In which case, IBB was associated with plasmid DNA through self assembling ionic interaction.Conclusions
The improvement of transfection activity was not due to an improved nuclear import of DNA, but rather by the modification of physicochemical properties of IBB peptide / plasmid complexes. IBB peptide increased lipoplex size and these larger complexes were more efficient for gene transfer.5.
Yahya Mohammadzadeh Narges Rasouli Mohammad Hasan Samiee Aref Nasim Sadat Seyed Tabib Asghar Abdoli Peyvand Biglari Maryam Saleh Mansoureh Tabatabaeian Masoumeh Tavassoti Kheiri Abbas Jamali 《Biotechnology letters》2016,38(8):1321-1329
Objectives
To enhance the efficiency of influenza virosome-mediated gene delivery by engineering this virosome.Results
A novel chimeric influenza virosome was constructed containing the glycoprotein of Vesicular stomatitis virus (VSV-G), along with its own hemagglutinin protein. To optimize the transfection efficiency of both chimeric and influenza cationic virosomes, HEK cells were transfected with plasmid DNA and virosomes and the transfection efficiency was assessed by FACS analysis. The chimeric virosome was significantly more efficient in mediating transfection for all amounts of DNA and virosomes compared to the influenza virosome.Conclusions
Chimeric influenza virosome, including VSV-G, is superior to the conventional influenza virosome for gene delivery.6.
Heng Li Jing Li Ruinan Jin Wei Chen Chaoning Liang Jieyuan Wu Jian-Ming Jin Shuang-Yan Tang 《Biotechnology letters》2018,40(7):1101-1107
Objectives
To improve the quality of mutagenesis libraries in directed evolution strategy.Results
In the process of library transformation, transformants which have been shown to take up more than one plasmid might constitute more than 20% of the constructed library, thereby extensively impairing the quality of the library. We propose a practical transformation method to prevent the occurrence of multiple-plasmid transformants while maintaining high transformation efficiency. A visual library model containing plasmids expressing different fluorescent proteins was used. Multiple-plasmid transformants can be reduced through optimizing plasmid DNA amount used for transformation based on the positive correlation between the occurrence frequency of multiple-plasmid transformants and the logarithmic ratio of plasmid molecules to competent cells.Conclusions
This method provides a simple solution for a seemingly common but often neglected problem, and should be valuable for improving the quality of mutagenesis libraries to enhance the efficiency of directed evolution strategies.7.
Renato de Souza Pinto Lemgruber Kaspar Valgepea Mark P. Hodson Ryan Tappel Sean D. Simpson Michael Köpke Lars K. Nielsen Esteban Marcellin 《Metabolomics : Official journal of the Metabolomic Society》2018,14(3):35
Introduction
Quantification of tetrahydrofolates (THFs), important metabolites in the Wood–Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen.Objective
To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors.Methods
Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC–MS/MS was used for quantification.Results
Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP.Conclusion
Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards.8.
Objective
Concatenation of two NdeI–XhoI gene fragments via an oligonucleotide linker on a plasmid vector with an SfiI site was performed to evaluate success rates in construction of polycistronic genes expressible in Escherichia coli.Results
A series of plasmids with an SfiI site between the selection marker and the replication origin were constructed. The three wheat eEF1B subunit genes inserted between the NdeI and XhoI sites of pET-22b were transferred to the SfiI-containing plasmid with a spectinomycin-resistance gene. Then, the marker gene in the resultant plasmids was substituted with the ampicillin-resistance gene. These plasmids were used for concatenation of two different genes via a linker oligonucleotide containing a ribosome-binding site. During these operations, 42 clones were picked up out of which 41 had the intended product plasmid.Conclusion
This method, named as the SfiNX method, is useful for trial-and-error based testing of different combinations of fusion and co-expression partners for optimization of recombinant protein production.9.
Xue-Ting Chen Jun-Bin Ji Yong-Chuang Liu Bin Ye Chao-Yang Zhou Xin Yan 《Biotechnology letters》2016,38(12):2109-2117
Objectives
To induce natural genetic competence in Bacillus amyloliquefaciens isolates through overexpression of the master regulator, ComK, from B. subtilis (ComK Bsu ).Results
Plasmid pUBXC carrying the xylose-inducible comK expression cassette was constructed using plasmid pUB110 as a backbone. Plasmid pUBXC could be transferred from B. subtilis to B. amyloliquefaciens through plasmid pLS20-mediated biparental conjugation. After being induced by xylose, four B. amyloliquefaciens strains harbouring plasmid pUBXC developed genetic competence. Under optimal conditions, the transformation efficiencies of plasmid DNA ranged from 129 ± 20.6 to 1.7 ± 0.1 × 105 cfu (colony-forming units) per μg DNA, and the transformation efficiencies of PCR-assembled deletion constructs ranged from 3.2 ± 0.76 to 3.5 ± 0.42 × 104 cfu per μg DNA in the four tested strains.Conclusion
Artificial induction of genetic competence through overexpressing ComK Bsu in B. amyloliquefaciens completed the tasks of replicative plasmid delivery and gene knockout via direct transformation of PCR-generated deletion cassettes.10.
Andrelisse Arruda Viviane Castelo Branco Reis Vinícius Daniel Ferreira Batista Bruno Sahim Daher Luiza Cesca Piva Janice Lisboa De Marco Lidia Maria Pepe de Moraes Fernando Araripe Gonçalves Torres 《Biotechnology letters》2016,38(3):509-517
Objectives
To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.Results
P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.Conclusions
A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.11.
Background
Data integration is a crucial task in the biomedical domain and integrating data sources is one approach to integrating data. Data elements (DEs) in particular play an important role in data integration. We combine schema- and instance-based approaches to mapping DEs to terminological resources in order to facilitate data sources integration.Methods
We extracted DEs from eleven disparate biomedical sources. We compared these DEs to concepts and/or terms in biomedical controlled vocabularies and to reference DEs. We also exploited DE values to disambiguate underspecified DEs and to identify additional mappings.Results
82.5% of the 474 DEs studied are mapped to entries of a terminological resource and 74.7% of the whole set can be associated with reference DEs. Only 6.6% of the DEs had values that could be semantically typed.Conclusion
Our study suggests that the integration of biomedical sources can be achieved automatically with limited precision and largely facilitated by mapping DEs to terminological resources.12.
Kenechukwu K. Iloh Chidiebere DI. Osuorah Ikenna K. Ndu Isaac N. Asinobi Ijeoma N. Obumneme-Anyim Chijioke E. Ezeudu Ukoha M. Oluchi Onyinye U. Anyanwu Uchenna Ekwochi Christian C. Ogoke Adaeze C. Ayuk Herbert U. Obu 《International breastfeeding journal》2018,13(1):47
Background
Due to the health and economic benefits of breast milk, the World Health Organization (WHO) recommends that for infants who cannot receive breast milk from their own mothers, the next preferred option is donated breast milk. This recommendation is however rarely practiced in most developing countries where donor milk is not widely accepted.Methods
This cross-sectional multi-center study enrolled mothers attending antenatal or pediatric clinics in six tertiary institution in south-east Nigeria using purposive and convenient sampling method. Data collection was done using pretested questionnaires. The study aimed to assess the knowledge, acceptability and willingness to donate breast milk and/or use donated breast milk for their infants It also explored factors that determine this behavior.Results
A total of 1235 mothers participated; 39% (480/1225) have heard about the concept of donor milk, while only 10% (79/759) and 7% (81/1179), respectively, had adequate knowledge of the concept and policy on donor milk. Sixty percent indicated willingness to use donor milk or donate breast milk if need arises. Respondents with lower age (p?=?0.049) and with higher occupational status (p?=?0.001) were more likely to have adequate knowledge of donor breast milk, while respondents with lower educational attainment (p?=?0.002) and those who are non-Christians (p?=?0.004) were more likely to request financial inducement for donating their breast milk. Adequate knowledge of the concept of donor milk (p?=?0.001), preference of donor milk to infant formula (p?=?0.001) and requirement of financial remuneration (p?=?0.001) were the only significant predictors of willingness to donate and/or receive donated breast milk.Conclusion
The knowledge of the concept of donor breast milk and awareness of policies regulating its practice in Nigeria is low, but the prospect of its acceptability is high among mothers surveyed in south-east Nigeria. Targeted public education by relevant government agencies in collaboration with clinicians, community and religious leaders about the concept of donor breast milk to families may help increase the acceptance and practice of donating breast milk and/or use of donated breast milk among mothers in the region.13.
14.
Background
Human T-cell leukemia virus type 1 (HTLV-1) infection is associated with adult T-cell leukemia/lymphoma (ATLL), a lymphoproliferative malignancy with a dismal prognosis and limited therapeutic options. Recent evidence shows that HTLV-1-transformed cells present defects in both DNA replication and DNA repair, suggesting that these cells might be particularly sensitive to treatment with a small helicase inhibitor. Because the “Werner syndrome ATP-dependent helicase” encoded by the WRN gene plays important roles in both cellular proliferation and DNA repair, we hypothesized that inhibition of WRN activity could be used as a new strategy to target ATLL cells.Methods
Our analysis demonstrates an apoptotic effect induced by the WRN helicase inhibitor in HTLV-1-transformed cells in vitro and ATL-derived cell lines. Inhibition of cellular proliferation and induction of apoptosis were demonstrated with cell cycle analysis, XTT proliferation assay, clonogenic assay, annexin V staining, and measurement of mitochondrial transmembrane potential.Results
Targeted inhibition of the WRN helicase induced cell cycle arrest and apoptosis in HTLV-1-transformed leukemia cells. Treatment with NSC 19630 (WRN inhibitor) induces S-phase cell cycle arrest, disruption of the mitochondrial membrane potential, and decreased expression of anti-apoptotic factor Bcl-2. These events were associated with activation of caspase-3-dependent apoptosis in ATL cells. We identified some ATL cells, ATL-55T and LMY1, less sensitive to NSC 19630 but sensitive to another WRN inhibitor, NSC 617145.Conclusions
WRN is essential for survival of ATL cells. Our studies suggest that targeting the WRN helicase with small inhibitors is a novel promising strategy to target HTLV-1-transformed ATL cells.15.
16.
Wesley W. Ingwersen Ezra Kahn Joyce Cooper 《The International Journal of Life Cycle Assessment》2018,23(11):2266-2270
Introduction
New platforms are emerging that enable more data providers to publish life cycle inventory data.Background
Providing datasets that are not complete LCA models results in fragments that are difficult for practitioners to integrate and use for LCA modeling. Additionally, when proxies are used to provide a technosphere input to a process that was not originally intended by the process authors, in most LCA software, this requires modifying the original process.Results
The use of a bridge process, which is a process created to link two existing processes, is proposed as a solution.Discussion
Benefits to bridge processes include increasing model transparency, facilitating dataset sharing and integration without compromising original dataset integrity and independence, providing a structure with which to make the data quality associated with process linkages explicit, and increasing model flexibility in the case that multiple bridges are provided. A drawback is that they add additional processes to existing LCA models which will increase their size.Conclusions
Bridge processes can be an enabler in allowing users to integrate new datasets without modifying them to link to background databases or other processes they have available. They may not be the ideal long-term solution but provide a solution that works within the existing LCA data model.17.
Yuxuan Liu Meiru Zhang Tianshi Wang Xunxun Shi Jie Li Lu Jia Hui Tang Liping Zhang 《Biotechnology letters》2016,38(3):417-423
Objectives
Two genes encoding two acetyl-CoA synthetase (ACS) isoenzymes have been identified in the marine yeast Rhodosporidium diobovatum MCCC 2A00023.Results
ACS1 encoded a polypeptide with a sequence of 578 amino acid residues, a predicted molecular weight of 63.73 kDa, and pI of 8.14, while the ACS2 encoded a polypeptide containing 676 amino acid residues with a deduced molecular mass of 75.61 kDa and a pI of 5.95. Biological activity of Acs1p and Acs2p was confirmed by heterologous expression in Escherichia coli. A 1.5-kb DNA fragment of the ACS1 gene and a 2.7-kb DNA fragment of the ACS2 gene were deleted using the RNA guide CRISPR-Cas9 system. The strain lacking ACS1 was unable to grow on acetate and ethanol media, while the ACS2 deletant was unable to grow on glucose medium. ACS1-ACS2 double mutants of R. diobovatum were non-viable.Conclusions
ACS isoenzymes are essential to the yeast metabolism, and other sources of ACSs cannot compensate for the lack of ACSs encoded by the two genes.18.
Anja Gerster Claas Wodarczyk Britta Reichenbächer Janet Köhler Andreas Schulze Felix Krause Dethardt Müller 《Biotechnology letters》2016,38(12):2043-2049
Objectives
To establish a high-throughput method for determination of antibodies intra- and extracellular light chain (LC) to heavy chain (HC) polypeptide ratio as screening parameter during cell line development.Results
Chinese Hamster Ovary (CHO) TurboCell pools containing different designed vectors supposed to result in different LC:HC polypeptide ratios were generated by targeted integration. Cell culture supernatants and cell lysates of a fed batch experiment were purified by combined Protein A and anti-kappa affinity batch purification in 96-well format. Capture of all antibodies and their fragments allowed the determination of the intra- and extracellular LC:HC peptide ratios by reduced SDS capillary electrophoresis. Results demonstrate that the method is suitable to show the significant impact of the vector design on the intra- and extracellular LC:HC polypeptide ratios.Conclusion
Determination of LC:HC polypeptide ratios can give important information in vector design optimization leading to CHO cell lines with optimized antibody assembly and preferred product quality.19.
Penelope Reimers Natalie Shenker Gillian Weaver Anna Coutsoudis 《International breastfeeding journal》2018,13(1):43
Background
Donor human milk is the World Health Organization’s recommendation for infant feeding when the mother’s own breast milk is unavailable. Breast milk has been shown to reduce morbidity and mortality and in low birthweight infants, donor milk reduces the incidence of necrotising enterocolitis, late onset sepsis and improves outcomes. There is a paucity of literature documenting outcomes of using donor human milk in older children who need additional support for a variety of health issues.Case presentation
A series of seven case studies is presented of orphaned and abandoned children, many of whom were either HIV exposed or positive. All children were fed with pasteurised donor human milk at a transition home and their progress reported.Conclusions
Although detailed medical records were not always available, the case studies provide anecdotal evidence of the protective effects of donor human milk against failure to thrive, diarrhoea, atopic dermatitis, and opportunistic infections.20.
Sunandini Sharma Kritika Karri Ishwor Thapa Dhundy Bastola Dario Ghersi 《BMC medical genomics》2016,9(2):46