A novel chimeric influenza virosome containing <Emphasis Type="Italic">Vesicular stomatitis</Emphasis> G protein as a more efficient gene delivery system

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.

     

Site-specific integration of CAR gene into Jurkat T cells with a linear close-ended AAV-based DNA vector for CAR-T engineering

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.

     

Mechanisms of genome instability in Hutchinson-Gilford progeria

Background

Hutchinson-Gilford progeria syndrome (HGPS) is a devastating premature aging disorder. It arises from a single point mutation in the LMNA gene. This mutation stimulates an aberrant splicing event and produces progerin, an isoform of the lamin A protein. Accumulation of progerin disrupts numerous physiological pathways and induces defects in nuclear architecture, gene expression, histone modification, cell cycle regulation, mitochondrial functionality, genome integrity and much more.

Objective

Among these phenotypes, genomic instability is tightly associated with physiological aging and considered a main contributor to the premature aging phenotypes. However, our understanding of the underlying molecular mechanisms of progerin-caused genome instability is far from clear.

Results and Conclusion

In this review, we summarize some of the recent findings and discuss potential mechanisms through which, progerin affects DNA damage repair and leads to genome integrity.

     

Crystal structure of <Emphasis Type="Italic">Escherichia coli</Emphasis> protein ybgI,a toroidal structure with a dinuclear metal site

Background

The protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function.

Results

The structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers. Each polypeptide chain binds two metal ions on the inside of the toroid.

Conclusion

The toroidal structure is comparable to that of some proteins that are involved in DNA metabolism. The di-nuclear metal site could imply that the specific function of this protein is as a hydrolase-oxidase enzyme.

     

Delivery of molecular cargoes in normal and cancer cell lines using non-viral delivery systems

Objective

In this study, transfection efficiency of human papillomavirus (HPV) E7 DNA and protein constructs into HEK-293T normal cell line, and A549 and TC-1 tumor cell lines was evaluated by four delivery systems including supercharge GFP, hPP10 cell penetrating peptide, TurboFect and Lipofectamine using fluorescence microscopy and flow cytometry.

Results

The results indicated that Lipofectamine 2000 and TurboFect produced more effective transfection for GFP and E7-GFP DNA constructs in HEK-293T cells compared to in A549 and TC-1 cells (p?<?0.05). In contrast, the supercharge GFP was efficient for E7 DNA and E7 protein delivery in both normal cell (~?83.94 and ~?77.01% for HEK-293T), and cancer cells (~?71.69 and ~?67.19% for TC-1, and ~?73.86 and ~?67.49% for A549), respectively. Indeed, in these cell lines, transfection efficiency by +36 GFP reached ~?60–80%. Moreover, the hPP10 produced the best transfection result for E7-GFP protein in HEK-293T cells (~?63.66%) compared to TurboFect (~?32.95%); however, the efficiency level of hPP10 was only ~?17.51 and ~?16.36% in TC-1 and A549 cells.

Conclusions

Our data suggested that the supercharge GFP is the most suitable transfection vehicle for DNA and protein delivery into TC-1 and A549 tumor cell lines compared to other carriers.

     

Towards the construction of high-quality mutagenesis libraries

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.

     

Expression and purification of classical swine fever virus E2 protein from Sf9 cells using a modified vector

Objective

To develop a simple method for efficient expression of classical swine fever virus (CSFV) E2 protein.

Results

The pFastBac HT B vector (pFastHTB-M1) was modified by adding a melittin signal peptide sequence. The E2 gene fragment without the transmembrane region was cloned into pFastHTB-M1. The modified vector has clear advantage over the original one, as evidenced by the purified recombinant E2 protein that was detected significantly by SDS-PAGE.

Conclusions

The modified vector has the potential for large-scale production and easy purification of the CSFV E2 protein or other proteins of interests.

     

Production of an antioxidative peptide from hairy basil seed waste by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objective

To circumvent the time-consuming and costly problems associated with natural product extraction, a potential antioxidative peptide selected from hairy basil waste after oil extraction was produced by recombinant DNA technology.

Results

Because the target peptide is short, the recombinant peptide containing seven repeats of the target sequence, QTFQYSRGWTN, and the DNA fragment coding this sequence was cloned into the pQE-30 Xa expression vector and transformed into Escherichia coli. After 6 h of recombinant peptide expression in E. coli, the target peptide was purified by Ni²⁺ affinity chromatography and gel extraction. The expected 15 kDa recombinant target peptide construct was verified by modified dot blot analysis. Compared with the chemically synthesized peptide, the recombinant peptide revealed significantly higher antioxidant activities (p < 0.05), as determined by DPPH and ABTS radical scavenging assays, and in vitro DNA damage induced by hydroxyl radicals.

Conclusion

This approach provides an alternative to produce an antioxidative peptide that provides a potential scaffold for the further development of antioxidative peptides for industrial applications.

     

A protocol for CABS-dock protein–peptide docking driven by side-chain contact information

Background

The characterization of protein–peptide interactions is a challenge for computational molecular docking. Protein–peptide docking tools face at least two major difficulties: (1) efficient sampling of large-scale conformational changes induced by binding and (2) selection of the best models from a large set of predicted structures. In this paper, we merge an efficient sampling technique with external information about side-chain contacts to sample and select the best possible models.

Methods

In this paper we test a new protocol that uses information about side-chain contacts in CABS-dock protein–peptide docking. As shown in our recent studies, CABS-dock enables efficient modeling of large-scale conformational changes without knowledge about the binding site. However, the resulting set of binding sites and poses is in many cases highly diverse and difficult to score.

Results

As we demonstrate here, information about a single side-chain contact can significantly improve the prediction accuracy. Importantly, the imposed constraints for side-chain contacts are quite soft. Therefore, the developed protocol does not require precise contact information and ensures large-scale peptide flexibility in the broad contact area.

Conclusions

The demonstrated protocol provides the extension of the CABS-dock method that can be practically used in the structure prediction of protein–peptide complexes guided by the knowledge of the binding interface.

     

Statistical analysis of fractionation resistance by functional category and expression

Background

The current literature establishes the importance of gene functional category and expression in promoting or suppressing duplicate gene loss after whole genome doubling in plants, a process known as fractionation. Inspired by studies that have reported gene expression to be the dominating factor in preventing duplicate gene loss, we analyzed the relative effect of functional category and expression.

Methods

We use multivariate methods to study data sets on gene retention, function and expression in rosids and asterids to estimate effects and assess their interaction.

Results

Our results suggest that the effect on duplicate gene retention fractionation by functional category and expression are independent and have no statistical interaction.

Conclusion

In plants, functional category is the more dominant factor in explaining duplicate gene loss.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

Versatility of chitosan/BmNPV bacmid DNA nanocomplex as transfection reagent of recombinant protein expression in silkworm larvae

Objective

To examine the feasibility of chitosan as an alternative transfection reagent candidate for protein expression in Bm5 cells and silkworm larvae using recombinant BmNPV bacmid DNA.

Result

Chitosan 100 and recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA, in amino group/phosphate group (N/P) ratios of 0.1–10, were used for formation of chitosan/DNA nanocomplexes. The chitosan/BmNPV bacmid DNA nanocomplexes showed higher specific activity of GFP_uv-β1,3-N-acetylglucosaminyltransferase 2 (β3GnT2) fusion protein (GGT2) expressed in silkworm larvae than DMRIE-C, a conventional silkworm transfection reagent. In particular, the composition of chitosan and BmNPV bacmid DNA nanocomplexes formed by an N/P ratio of 8 or 10, respectively, showed the highest specific activity of β3GnT2 in the silkworm larvae hemolymph. In addition, three different proteins were expressed in silkworm larvae to the same extent using chitosan as that using DMRIE-C.

Conclusion

This is the first finding that chitosan/BmNPV bacmid DNA nanocomplexes can rival the performance of commercially available transfection reagents for the expression of recombinant proteins in Bm5 cells and silkworm larvae.

     

A DNA-scaffold platform enhances a multi-enzymatic cycling reaction

Objective

We explored the co-localization of multiple enzymes on a DNA backbone via a DNA-binding protein, Gene-A* (A*-tag) to increase the efficiency of cascade enzymatic reactions.

Results

Firefly luciferase (FLuc) and pyruvate orthophosphate dikinase (PPDK) were genetically fused with A*-tag and modified with single-stranded (ss) DNA via A*-tag. The components were assembled on ssDNA by hybridization, thereby enhancing the efficiency of the cascading bioluminescent reaction producing light emission from pyrophosphate. The activity of A*-tag in each enzyme was investigated with dye-labeled DNA. Co-localization of the enzymes via hybridization was examined using a gel shift assay. The multi-enzyme complex showed significant improvement in the overall efficiency of the cascading reaction in comparison to a mixture of free enzymes.

Conclusion

A*-tag is highly convenient for ssDNA modification of versatile enzymes, and it can be used for construction of functional DNA–enzyme complexes.

     

re-TAMD: exploring interactions between H3 peptide and YEATS domain using enhanced sampling

Background

Analysis of preferred binding regions of a ligand on a protein is important for detecting cryptic binding pockets and improving the ligand selectivity.

Result

The enhanced sampling approach TAMD has been adapted to allow a ligand to unbind from its native binding site and explore the protein surface. This so-called re-TAMD procedure was then used to explore the interaction between the N terminal peptide of histone H3 and the YEATS domain. Depending on the length of the peptide, several regions of the protein surface were explored. The peptide conformations sampled during the re-TAMD correspond to peptide free diffusion around the protein surface.

Conclusions

The re-TAMD approach permitted to get information on the relative influence of different regions of the N terminal peptide of H3 on the interaction between H3 and YEATS.

     

Deep learning for DNase I hypersensitive sites identification

Background

The DNase I hypersensitive sites (DHSs) are associated with the cis-regulatory DNA elements. An efficient method of identifying DHSs can enhance the understanding on the accessibility of chromatin. Despite a multitude of resources available on line including experimental datasets and computational tools, the complex language of DHSs remains incompletely understood.

Methods

Here, we address this challenge using an approach based on a state-of-the-art machine learning method. We present a novel convolutional neural network (CNN) which combined Inception like networks with a gating mechanism for the response of multiple patterns and longterm association in DNA sequences to predict multi-scale DHSs in Arabidopsis, rice and Homo sapiens.

Results

Our method obtains 0.961 area under curve (AUC) on Arabidopsis, 0.969 AUC on rice and 0.918 AUC on Homo sapiens.

Conclusions

Our method provides an efficient and accurate way to identify multi-scale DHSs sequences by deep learning.