Vector System Research Articles

About systems of vectors and subspaces in finite dimensional space recovering vectorsignal

The subject of this paper are the systems of vectors and subspaces in finite dimensional spaces admitting the recovery of an unknown vector-signal by modules of measurements. We analyze the relationship between the properties of recovery by modules of measurements and recovery by norms of projections and the properties of alternative completeness in Euclidean and unitary spaces. The theorem on ranks of one linear operator is considered, the result of which in some cases can be regarded as another criterion for the possibility to restore a vector-signal. As a result of this work, the equivalence of the alternative completeness property and the statement of the rank theorem for Euclidean space is proved. It is shown that the rank theorem in the real case can be extended to the systems of subspaces.
 The questions about the minimum number of vectors admissible for reconstruction by modules of measurements are considered. The results available at the moment are presented, which are summarized in the form of a table for spaces of dimension less than 10. Also the known results to the question of the minimum number of subspaces admitting reconstruction by the norms of projections are briefly given.

Adeno-associated virus vector system controlling capsid expression improves viral quantity and quality

Adeno-associated virus (AAV) vectors are promising tools for gene therapy. The current AAV vector system produces an abundance of empty capsids that are eliminated before clinical use, leading to increased costs for gene therapy. In the present study, we established an AAV production system that regulates the timing of capsid expression using a tetracycline-dependent promoter. Tetracycline-regulating capsid expression increased viral yield and reduced empty capsids in various serotypes without altering AAV vector infectivity invitro and invivo. The replicase expression pattern change observed in the developed AAV vector system improved viral quantity and quality, whereas timing control of capsid expression reduced empty capsids. These findings provide a new perspective on the development of AAV vector production systems in gene therapy.

Characterization of a cyanobacterial rep protein with broad-host range and its utilization for expression vectors.

Owing to their photosynthetic capabilities, cyanobacteria are regarded as ecologically friendly hosts for production of biomaterials. However, compared to other bacteria, tools for genetic engineering, especially expression vector systems, are limited. In this study, we characterized a Rep protein, exhibiting replication activity in multiple cyanobacteria and established an expression vector using this protein. Our comprehensive screening using a genomic library of Synechocystis sp. PCC 6803 revealed that a certain region encoding a Rep-related protein (here named Cyanobacterial Rep protein A2: CyRepA2) exhibits high autonomous replication activity in a heterologous host cyanobacterium, Synechococcus elongatus PCC 7942. A reporter assay using GFP showed that the expression vector pYS carrying CyRepA2 can be maintained in not only S. 6803 and S. 7942, but also Synechococcus sp. PCC 7002 and Anabaena sp. PCC 7120. In S. 7942, GFP expression in the pYS-based system was tightly regulated by IPTG, achieving 10-fold higher levels than in the chromosome-based system. Furthermore, pYS could be used together with the conventional vector pEX, which was constructed from an endogenous plasmid in S. 7942. The combination of pYS with other vectors is useful for genetic engineering, such as modifying metabolic pathways, and is expected to improve the performance of cyanobacteria as bioproduction chassis.

Method for obtaining recombinant antibodies produced by a cell line transduced with recombinant adenoviruses

Objectives. To develop a technology for obtaining recombinant antibodies in a suspension culture of human HEK293 cells using transduction with recombinant adenovirus serotype 5 (rAd5) carrying genes expressing heavy and light chains of antibodies on the example of two broadspectrum anti-influenza antibodies 27F3 and CR9114.Methods. Ad5-27F3-H, Ad5-CR9114-H, and Ad5-27F3-L recombinant adenoviruses carrying the 27F3 antibody heavy chain gene, CR9114 antibody heavy chain gene, and 27F3 light chain gene, respectively, were generated using the AdEasy™ Adenoviral vector system. To accumulate preparative amounts of recombinant r27F3 and rCR9114 antibodies, the HEK293 suspension cell line was transduced with recombinant adenoviruses carrying genes for heavy and light chains of antibodies. The cells were cultured in a wave-type bioreactor. Chromatography was used to purify recombinant antibodies from the culture medium. After analyzing the molecular weights of purified antibodies using protein electrophoresis, their ability to interact with influenza A and B viruses was analyzed using the Western blot technique, while their ability to neutralize influenza A and B viruses was evaluated using the virus neutralization assay.Results. A method for the accumulation and purification of recombinant r27F3 and CR9114 antibodies from the culture medium of a suspension culture of human cells following transduction with its recombinant adenoviruses carrying the genes for heavy and light chains of these antibodies was developed. The ability of the r27F3 antibody to interact with and neutralize influenza A viruses of group 1 (except influenza A virus subtype H2) and group 2 was shown. The ability of the rCR9114 antibody to interact with influenza A viruses of group 1 and influenza B viruses, as well as to neutralize influenza A viruses of group 1, was demonstrated.Conclusions. A technology for obtaining recombinant antibodies in a suspension culture of HEK293 cells using transduction with recombinant adenoviruses carrying genes expressing heavy and light chains of antibodies was developed along with a confirmation of their specificity.

Practical use of tobravirus-based vector to produce SARS-CoV-2 antigens in plants

A plant-based heterologous expression system is an attractive option for recombinant protein production because it is based on a eukaryotic system of high feasibility, and low biological risks. Frequently, binary vector systems are used for transient gene-expression in plants. However, plant virus vector-based systems offer advantages for higher protein yields due to their self-replicating machinery. In the present study, we show an efficient protocol using a plant virus vector based on a tobravirus, pepper ringspot virus, that was employed for transient expression of severe acute respiratory syndrome coronavirus 2 partial gene fragments of the spike (named S1-N) and the nucleocapsid (named N) proteins in Nicotiana benthamiana plants. Purified proteins yield of 40–60 µg/g of fresh leaves were obtained. Both proteins, S1-N and N, showed high and specific reactivities against convalescent patients’ sera by the enzyme-linked immunosorbent assay format. The advantages and critical points in using this plant virus vector are discussed.

Dissecting insect cell heterogeneity during influenza VLP production using single-cell transcriptomics.

The insect cell-baculovirus expression vector system (IC-BEVS) has been widely used to produce recombinant protein at high titers, including complex virus-like particles (VPLs). However, cell-to-cell variability upon infection is yet one of the least understood phenomena in virology, and little is known about its impact on production of therapeutic proteins. This study aimed at dissecting insect cell population heterogeneity during production of influenza VLPs in IC-BEVS using single-cell RNA-seq (scRNA-seq). High Five cell population was shown to be heterogeneous even before infection, with cell cycle being one of the factors contributing for this variation. In addition, infected insect cells were clustered according to the timing and level of baculovirus genes expression, with each cluster reporting similar influenza VLPs transgenes (i.e., hemagglutinin and M1) transcript counts. Trajectory analysis enabled to track infection progression throughout pseudotime. Specific pathways such as translation machinery, protein folding, sorting and degradation, endocytosis and energy metabolism were identified as being those which vary the most during insect cell infection and production of Influenza VLPs. Overall, this study lays the ground for the application of scRNA-seq in IC-BEVS processes to isolate relevant biological mechanisms during recombinant protein expression towards its further optimization.

Geophysical study of the Earthʼs crust (Northern regions of Russia and the Arctic sea shelf)

Research subject. The results of the geological and geophysical study of the Earth’s crust on the territory of two regions are presented: the northwestern part of the East European Platform and the adjacent water area, as well as the waters of the East Siberian, Chukchi Seas and the Russian eastern sector of the Arctic Ocean, including the Lomonosov ridge and the Mendeleev–Alpha rise. The research is focused on mapping the Moho, the thickness and type of the Earth's crust necessary to substantiate the outer boundary of the continental shelf.Materials and methods. The authors’ original materials obtained during the processing of gravimetry and magnetometry data in the VECTOR system were used. Based on the relationship between gravity anomalies and density variations of rocks, the shape, size of the studied objects, and their deep mutual position are characterized.Results. The constructed schemes and profiles of the deep structure of the Earth's crust, obtained using the VECTOR method, were tested using the European Moho global seismic model. The positive forms of the Moho section and the raised parts of the shelf surface correspond to the positive anomalies of the transformed gravitational field. The spread of the structure of the field of continental anomalies in the Pevek–Anadyr area to the marine part of the territory under consideration can indirectly confirm the belonging of the Lomonosov Cr., the Mendeleev and Alpha ridges to the continental type of crust.Conclusion. Global, i.e., related to the territory of the globe, three-dimensional models of Moho allow us to expand and deepen the amount of knowledge about the features of the shelf structure. Geopotential fields in the VECTOR system display the tectonics of the studied environment systematically and comprehensively compared to is representation according to the scheme of profile seismic studies.

Effects of Different Gene Editing Modes of CRISPR/Cas9 on Soybean Fatty Acid Anabolic Metabolism Based on GmFAD2 Family

Δ12-fatty acid dehydrogenase (FAD2) is the essential enzyme responsible for catalyzing the formation of linoleic acid from oleic acid. CRISPR/Cas9 gene editing technology has been an essential tool for molecular breeding in soybeans. To evaluate the most suitable type of gene editing in soybean fatty acid synthesis metabolism, this study selected five crucial enzyme genes of the soybean FAD2 gene family-GmFAD2-1A, GmFAD2-1B, GmFAD2-2A, GmFAD2-2B, and GmFAD2-2C-and created a CRISPR/Cas9-mediated single gene editing vector system. The results of Sanger sequencing showed that 72 transformed plants positive for T1 generation were obtained using Agrobacterium-mediated transformation, of which 43 were correctly edited plants, with the highest editing efficiency of 88% for GmFAD2-2A. The phenotypic analysis revealed that the oleic acid content of the progeny of GmFAD2-1A gene-edited plants had a higher increase of 91.49% when compared to the control JN18, and the rest of the gene-edited plants in order were GmFAD2-2A, GmFAD2-1B, GmFAD2-2C, and GmFAD2-2B. The analysis of gene editing type has indicated that base deletions greater than 2bp were the predominant editing type in all editing events. This study provides ideas for the optimization of CRISPR/Cas9 gene editing technology and the development of new tools for precise base editing in the future.

Novel rAAV vector mediated intrathecal HGF delivery has an impact on neuroimmune modulation in the ALS motor cortex with TDP-43 pathology.

Recombinant adeno-associated virus (rAAV)-based gene therapies offeran immense opportunity for rare diseases, such as amyotrophic lateral sclerosis (ALS), which is defined by the loss of the upper and the lower motor neurons. Here, we describe generation, characterization, and utilization of a novel vector system, which enables expression of the active form of hepatocyte growth factor (HGF) under EF-1α promoter with bovine growth hormone (bGH) poly(A) sequence and is effective with intrathecal injections. HGF's role in promoting motor neuron survival had been vastly reported. Therefore, we investigated whether intrathecal delivery of HGF would have an impact on one of the most common pathologies of ALS: the TDP-43 pathology. Increased astrogliosis, microgliosis and progressive upper motor neuron loss are important consequences of ALS in the motor cortex with TDP-43 pathology. We find that cortex can be modulated via intrathecal injection, and that expression of HGF reduces astrogliosis, microgliosis in the motor cortex, and help restore ongoing UMN degeneration. Our findings not only introduce a novel viral vector for the treatment of ALS, but also demonstrate modulation of motor cortex by intrathecal viral delivery, and that HGF treatment is effective in reducing astrogliosis and microgliosis in the motor cortex of ALS with TDP-43 pathology.

Metabolic engineering of Escherichia coli for the production of an antifouling agent zosteric acid

Zosteric acid (ZA) is a Zostera species-derived, sulfated phenolic acid compound with antifouling activity and has gained much attention due to its nontoxic and biodegradable characteristics. However, the yield of Zostera species available for ZA extraction is limited by natural factors, such as season, latitude, light, and temperature. Here we report the development of metabolically engineered Escherichia coli strains capable of producing ZA from glucose and glycerol. First, intracellular availability of the sulfur donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) was enhanced by knocking out the cysH gene responsible for PAPS consumption and overexpressing the genes required for PAPS biosynthesis. Co-overexpression of the genes encoding tyrosine ammonia-lyase, sulfotransferase 1A1, ATP sulfurylase, and adenosine 5′-phosphosulfate kinase constructed ZA producing strain with enhanced PAPS supply. Second, the feedback-resistant forms of aroG and tyrA genes (encoding 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase and chorismate mutase, respectively) were overexpressed to relieve the feedback regulation of L-tyrosine biosynthesis. Third, the pykA gene involved in phosphoenolpyruvate-consuming reaction, the regulator gene tyrR, the competing pathway gene pheA, and the ptsHIcrr genes essential for the PEP:carbohydrate phosphotransferase system were deleted. Moreover, all genes involved in the shikimate pathway and the talA, tktA, and tktB genes in the pentose phosphate pathway were examined for ZA production. The PTS-independent glucose uptake system, the expression vector system, and the carbon source were also optimized. As a result, the best-performing strain successfully produced 1.52 g L−1 ZA and 1.30 g L−1p-hydroxycinnamic acid from glucose and glycerol in a 700 mL fed-batch bioreactor.

First Report of Tomato Yellow Leaf Curl Virus Infecting Pepper and Tomato in Oklahoma.

Tomato yellow leaf curl virus (TYLCV), (genus Begomovirus and family Geminiviridae) is one of the devastating viruses infecting tomatoes (Solanum Lycopersicon) and could have a significant impact on tomato production worldwide (Moriones and Navas-Castillo, 2000; Rybicki, 2015; Perez-Padilla et al. 2019). TYLCV was first reported in the United States (US) in 1997 in Florida (Polston et al. 1999) and since then it has been reported in several other states but not in Oklahoma (de Sa et al. 2008). Both pepper (Capsicum spp.) and tomato are grown in >50 counties of Oklahoma for fresh produce in the local market, and for processing industries. During a survey in 2021, pepper (bell pepper cv SV3964) and tomato (cv grand marshall) plants grown in a commercial field in Bixby, Oklahoma, showed typical virus-like symptoms including yellowing, leaf curling, cupping, twisting, and mottling (Fig. 1). Estimated disease incidence was 10% in both crops. In addition, whiteflies (Bemisia tabaci) were also observed on these plants. Twenty symptomatic samples from pepper (n=16) and tomato (n=4) plants, and four asymptomatic from pepper (n=2) and tomato (n=2) plants were collected and brought to the Plant Virology Lab at the University of Tulsa for further analysis Total RNA was extracted from all samples using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, LOT: SLCG7913). RNA from two symptomatic samples, one each from pepper and tomato (named BX6 and BX11, respectively) were subjected to high-throughput sequencing (HTS) on the NextSeq 500/550 high-output kit v2.5 (Illumina, USA) at the genomic facility, Oklahoma State University (Stillwater, OK, USA). A total of 22,385,404 (average length 73.6 bp) and 19,012,605 (average length 73.7 bp) trim pair-end for both samples (BX6 and BX11) were assembled using CLC Genomics Workbench (v12.0.3) (Qiagen, Inc) and subjected to BLASTn analysis. The three contigs: 393 bp (coverage 363.24X), 670 bp (coverage 489.92X), and 990 bp (coverage 112.96X) for BX6 isolate, and four contigs: 393 bp (coverage 341.64X), 668 bp (coverage 457.23X), 814 bp (coverage 107.86X), and 990 bp (coverage 93.71X), for BX11 isolate, showed from 96 to 99% nucleotide (nt) identities with movement protein, capsid protein, C3, V2, C4, AC1, AC2, AC3 protein genes of the TYLCV isolates from Australia, China, Iran, Mexico, South Korea, and the US. The HTS data did not reveal any other viral sequence in these two samples. To further confirm the presence of TYLCV in these samples, three overlapping TYLCV-specific primer pairs (Supplementary Table 1) were designed based on the alignment of complete genome sequences of TYLCV isolates present in the Genbank. Total DNA was extracted from fresh leaf tissues of BX6 and BX11 samples using the Plant DNA Kit (Omega BIO-TEK,) and used in the PCR assay. The expected PCR products (814 bp, 1,085 bp and 1,068 bp) were generated from both samples providing further evidence of TYLCV infection. To obtain the complete genome sequence, these PCR amplified DNA fragments of both isolates were gel extracted and cloned using the pGEM-T® Easy Vector system. Three independent clones of each fragment were sequenced and analyzed by Sanger sequencing. The resulting consensus sequences were used in a BLASTn search against the GenBank and had the highest identities (99-99.2%) with TYLCV sequences. The overlapping consensus nt sequence of the BX6 isolate was 2,782 nt (Accession no ON321843), whereas that of BX11 was 2,777 nt (Accession no ON785706) showed 99.2% and 99.0% nt identities with the corresponding sequence of TYLCV isolates (KX347141 and KX347142) respectively from Australia. The nt identity between BX6 and BX11 was 99.7%. Further screening by PCR of the remaining 18 symptomatic field samples (pepper, n=15 and tomatoes, n=3) revealed TYLCV infection in two pepper and all three tomato samples consistent with the greater susceptibility of tomato. None of the asymptomatic pepper and tomato samples were positive for TYLCV. DNA from both BX6 and BX11 samples was also used in the rolling circle amplification (RCA) assay (TempliPhi Amplification Kit, Cytiva) and the presence of circular TYLCV DNA was detected. Our results demonstrated the presence of TYLCV infection in these symptomatic pepper and tomato plants. This is the first report of TYLCV infecting pepper or tomato in open fields in Oklahoma. Further surveys are needed to determine the incidence of TYLCV and the presence of its vector in other counties throughout Oklahoma.

MERCEDES-BENZ FRAMES IN $$\mathbb {R}^d$$ AS A DIRECT SUM OF A PAIR OF ORTHOGONAL TIGHT FRAMES

The well-known Mercedes-Benz frames in $$\mathbb {R}^d$$ are known to exist and they have been constructed too. They are presented here as an external direct sum of a pair of orthogonal frames. A necessary and sufficient condition for a unit norm tight frame to be a Mercedes-Benz frame in $$\mathbb {R}^d$$ is provided in terms of a vector from the null space of its synthesis operator. Also, a necessary and sufficient condition for a unit norm system of vectors to be a Mercedes-Benz frame is provided in terms of the frame potential and a vector from the null space of the synthesis operator.

Novel p22 and p30 dual-proteins combination based indirect ELISA for detecting antibodies against African swine fever virus.

African swine fever virus (ASFV) infection is one of the most complex and fatal hemorrhagic viral diseases, causing a devastating loss to the swine industry. Since no effective vaccine is available, prevention and control of ASFV heavily depends on early diagnostic detection. In this study, a novel indirect ELISA was established for detecting antibodies against ASFV using dual-proteins, p22 and p30. Recombinants p22 and p30 were expressed and purified from E.coli vector system by recombined plasmids pET-KP177R and pET-CP204L. p22 and p30 were mixed as antigens for developing the indirect ELISA. Through optimizing coating concentrations of p30 and p22, coating ratio (p30: p22 = 1:3), and serum dilution (as 1:600), the established ELISA performed higher specificity, sensitivity, and repeatability against ASFV-positive serum. Furthermore, 184 clinical serum samples from suspected diseased pigs were verified the established ELISA in clinical diagnosis. The results showed that compared with two commercial ELISA kits, the established ELISA possessed higher sensitivity and almost uniform coincidence rate. The novel indirect ELISA based on dual-proteins p30 and p22 performed a valuable role in diagnostic detection of ASFV, providing a broad insight into serological diagnostic methods of ASFV.

Generation of integration-free Down syndrome and isogenic euploid human induced pluripotent stem cells

A pair of Down syndrome (DS) human iPSCs (hiPSCs) and isogenic euploid hiPSCs generated by using an integration-free Sendai viral vector system showed trisomy 21 (47; XY) and typical (46; XY) karyotype respectively. Pluripotency of both hiPSC lines was confirmed by pluripotency marker expression and three germ layer differentiation potentials.

Cationic lipid membranes condense double-strand DNA through ion bridging.

Cationic lipid (CL)-DNA complexes have attracted much attention due to their potential application in somatic gene therapy, such as nonviral transfection vectors. The rational design of efficient synthetic vectors requires a systematic understanding of the underlying mechanisms governing CL-DNA assembly. Experimental studies show that DNA double strands form assemblies in divalent cations when confined between CL lipid systems. The physical mechanism of membrane-induced condensation is not well understood. Herein, we present a computational study of a simplified vector system, quasi-random genomic DNA chains intercalated into DOPC/DOTAP membrane bilayers. Using all-atom molecular dynamics simulations and free energy calculations, we observe attraction between dsDNA pairs when the ionic strength of the solution and mole fraction of DOPC mimic experimental conditions. Strikingly, the same salt conditions favoring condensation in CL lead to repulsion without the lipid. We investigated the role of lipids and confined cations to understand the differences in DNA-DNA interactions. Simulations suggest that the head groups of the lipids in contact with the DNA backbone reduce the DNA charges. The cationic DOTAP and zwitterionic DOPC head groups form direct contact with the DNA backbone. In addition, the DOPC phosphate head groups are glued to the DNA backbone by magnesium cations. The already reduced opposite surface of DNA double strands are brought together with dynamically bridging magnesium cations confined between the two helices. Our study offers physical insight into the mechanism of CL-DNA assembly and may help to facilitate the development of liposome transfer vectors for gene delivery.

Genome-wide nonessential gene identification of Autographa californica multiple nucleopolyhedrovirus

The Baculovirus Expression Vector System (BEVS) is an insect cell-based heterologous protein expression system that possesses powerful potential in the development of protein drugs and vaccines. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most widely-used vector in BEVS with 151 open reading frames (ORFs) containing essential and nonessential genes. Deletion of nonessential genes has many advantages including increased foreign gene insertion. In this study, the λ red recombination system was used to knock out genes in a modified AcMNPV that carried an enhanced yellow fluorescent protein (eYFP) at the Ac126-Ac127 locus. Eighty genes were almost completely deleted respectively and 69 gene knockout AcMNPVs (KOVs) were obtained to evaluate their infection efficiency. After infecting Spodoptera frugiperda 9 (Sf9) cells, 51 KOVs including 62 genes showed similar infectivity as wide type (WT) and hence were defined as nonessential genes. However, 18 KOVs produced fewer infectious virions, indicating that these genes were influential in the production of progeny viruses. Combining our research with previous studies, a desired minimal AcMNPV genome containing 86 ORFs and all of the homologous regions (hrs) was brought up, facilitating genetic modification of baculovirus vectors and improvement of recombinant protein expression in the future.

Preventative effects of the albumin nano tissue-type plasminogen activator gene plasmid ultrasound microbubble carrier system on thrombosis after cardiac valve replacement

Mechanical valve replacement is a new method for treating rheumatic heart valve disease. However, patients require long-term anticoagulation therapy after surgery, thus reducing their use of anticoagulants, making the prevention of postoperative thrombosis of great research interest. We examined the potential preventative effects of the albumin nano tissue plasminogen activator (tPA) gene plasmid ultrasound microbubble carrier system on thrombosis. The average particle size of the nano tPA gene plasmid was 131.8 nm, the surface zeta potential was 30.65±40.34 mV, and the encapsulation efficiency was 76.56%, all meeting experimental requirements. One month after gene integration, the tPA content of group B had increased beyond pre-integration levels and above post-integration levels of group A. After valve replacement operations, the venous blood D-Dimer (D-D) content of group B increased significantly, whereas the venous blood D-D content of group A decreased. In addition, at 1, 2, and 4 weeks after operation, the venous blood D-D content of group B was greater than that of group A. These results suggest that the prepared albumin nano tPA gene plasmid ultrasound microbubble vector system can prevent thrombosis after heart valve replacement.

The Basic Experimental Setup for the Vacuum-assisted, Agrobacterium-mediated Transient Expression in Tomato

Several methods are available for use, to deliver the gene of interest into plants. Among these, Agrobacterium-mediated transformation utilizes binary vector systems to achieve the stable transformation of plants. Alas, this process is labor-intensive and time-consuming as several months are needed to obtain a true transgenic plant. Transient gene expression (e.g., vacuum-infiltration) systems were offered as an alternative over stable transformation, specifically to overcome time-related drawbacks. However, this method requires expensive equipment such as vacuum chambers. In this study, we report a vacuum-infiltration protocol for the transient expression of a reporter gene, modified green fluorescent protein (mGFP), in tomato seedling. With a basic experimental setup (including a custom-built growth chamber) with affordable equipment, we showed that the entire leaf can be infiltrated by applying a mere 200 millibar vacuum for 6 minutes, and thus the transient expression can be achieved in tomato plant, evidenced by PCR-based detection of the T-DNA, detection of mGFP both analytically with SDS-page analysis, and visually by the images acquired by fluorescence microscopy. Furthermore, different Agrobacterium tumefaciens strains were tested for their transformation efficiency, and we showed that LBA4404 was the most effective strain to use in the vacuum-assisted transient expression.

Role of Pfs47 in the dispersal of ancestral Plasmodium falciparum malaria through adaptation to different anopheline vectors

Plasmodium falciparum malaria originated when Plasmodium praefalciparum, a gorilla malaria parasite transmitted by African sylvan anopheline mosquitoes, adapted to humans. Pfs47, a protein on the parasite surface mediates P. falciparum evasion of the mosquito immune system by interacting with a midgut receptor and is critical for Plasmodium adaptation to different anopheline species. Genetic analysis of 4,971 Pfs47 gene sequences from different continents revealed that Asia and Papua New Guinea harbor Pfs47 haplotypes more similar to its ortholog in P. praefalciparum at sites that determine vector compatibility, suggesting that ancestral P. falciparum readily adapted to Asian vectors. Consistent with this observation, Pfs47-receptor gene sequences from African sylvan malaria vectors, such as Anopheles moucheti and An. marshallii, were found to share greater similarity with those of Asian vectors than those of vectors of the African An. gambiae complex. Furthermore, experimental infections provide direct evidence that transformed P. falciparum parasites carrying Pfs47 orthologs of P. praefalciparum or P. reichenowi were more effective at evading the immune system of the Asian malaria vector An. dirus than An. gambiae. We propose that high compatibility of ancestral P. falciparum Pfs47 with the receptors of Asian vectors facilitated the early dispersal of human malaria to the Asian continent, without having to first adapt to sub-Saharan vectors of the An. gambiae complex.

Non-Covalent Linkage of Helper Functions to Dumbbell-Shaped DNA Vectors for Targeted Delivery.

Covalently closed dumbbell-shaped DNA delivery vectors comprising the double-stranded gene(s) of interest and single-stranded hairpin loops on both ends represent a safe, stable and efficacious alternative to viral and other non-viral DNA-based vector systems. As opposed to plasmids and DNA minicircles, dumbbells can be conjugated via the loops with helper functions for targeted delivery or imaging. Here, we investigated the non-covalent linkage of tri-antennary N-acetylgalactosamine (GalNAc3) or a homodimer of a CD137/4-1BB-binding aptamer (aptCD137-2) to extended dumbbell vector loops via complementary oligonucleotides for targeted delivery into hepatocytes or nasopharyngeal cancer cells. Enlarging the dumbbell loop size from 4 to 71 nucleotides for conjugation did not impair gene expression. GalNAc3 and aptCD137-2 residues were successfully attached to the extended dumbbell loop via complementary oligonucleotides. DNA and RNA oligonucleotide-based dumbbell-GalNAc3 conjugates were taken up from the cell culture medium by hepatoblastoma-derived human tissue culture cells (HepG2) with comparable efficiency. RNA oligonucleotide-linked conjugates triggered slightly higher levels of gene expression, presumably due to the RNaseH-mediated linker cleavage, the release of the dumbbell from the GalNAc3 residue and more efficient nuclear targeting of the unconjugated dumbbell DNA. The RNaseH-triggered RNA linker cleavage was confirmed in vitro. Finally, we featured dumbbell vectors expressing liver cancer cell-specific RNA trans-splicing-based suicide RNAs with GalNAc3 residues. Dumbbells conjugated with two GalNAc3 residues triggered significant levels of cell death when added to the cell culture medium. Dumbbell vector conjugates can be explored for targeted delivery and gene therapeutic applications.