Search Results (26)

Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and is fatal if left untreated in over 95% of cases. Leishmaniasis is one of the neglected tropical diseases that tend to thrive in developing regions of the world where inadequate access to healthcare makes it difficult for some people to even receive a diagnosis. This study examined the usefulness of oral swabs as specimens for VL diagnosis, by detecting Leishmania donovani DNA in oral swabs from both VL patients and L. donovani-infected mice. Eighty oral swab (OS) and blood buffy coat (BC) samples were collected from suspected VL cases in Bangladesh. These samples were evaluated using Leishmania kinetoplast minicircle DNA (kDNA) in real-time PCR, and the results showed that 62.5% (50/80) and 67.5% (54/80) of the cases tested positive for the BC specimen and OS, respectively. The OS positivity was statistically comparable to the BC. L. donovani DNA was also detected in an oral swab of all infected BALB/c mice by conventional PCR targeting the large subunit ribosomal RNA gene (LSUrRNA), while it was negative in uninfected mice. This study highlights the potential of practical methods for the molecular diagnosis of VL using oral swabs as a non-invasive, simple, and accurate approach. Full article

Kinetoplastids display a single, large mitochondrion per cell, with their mitochondrial DNA referred to as the kinetoplast. This kinetoplast is a network of concatenated circular molecules comprising a maxicircle (20–64 kb) and up to thousands of minicircles varying in size depending on the species (0.5–10 kb). In Trypanosoma cruzi, maxicircles contain typical mitochondrial genes found in other eukaryotes. They consist of coding and divergent/variable regions, complicating their assembly due to repetitive elements. However, next-generation sequencing (NGS) methods have resolved these issues, enabling the complete sequencing of maxicircles from different strains. Furthermore, several insertions and deletions in the maxicircle sequences have been identified among strains, affecting specific genes. Unique to kinetoplastids, minicircles play a crucial role in a particular U-insertion/deletion RNA editing system by encoding guide RNAs (gRNAs). These gRNAs are essential for editing and maturing maxicircle mRNAs. In Trypanosoma cruzi, although only a few studies have utilized NGS methods to date, the structure of these molecules suggests a classification into four main groups of minicircles. This classification is based on their size and the number of highly conserved regions (mHCRs) and hypervariable regions (mHVRs). Full article

Leishmania infantum is the primary cause of visceral and cutaneous leishmaniasis in the European Mediterranean region. Subspecies-level characterization of L. infantum aids epidemiological studies by offering insights into the evolution and geographical distribution of the parasite and reservoir identity. In this study, conducted in north-east Spain, 26 DNA samples of L. infantum were analyzed, comprising 21 from 10 humans and 5 from 5 dogs. Minicircle kinetoplast DNA (kDNA) polymerase chain reaction assays using primers MC1 and MC2, followed by sequencing, were employed to assess intraspecific genetic variability. Single-nucleotide polymorphism (SNP) analysis detected seven genotypes (G1, G2, G12*–G15*, and G17*), with five being reported for the first time (*). The most prevalent was the newly described G13 (54%), while the other currently identified genotypes were predominantly found in single samples. The in silico restriction fragment length polymorphism (RFLP) method revealed five genotypes (B, F, N, P, and W), one of them previously unreported (W). Genotype B was the most prevalent (85%), comprising three SNP genotypes (G1, G2, and G13), whereas the other RFLP genotypes were associated with single SNP genotypes. These kDNA genotyping methods revealed significant intraspecific genetic diversity in L. infantum, demonstrating their suitability for fingerprinting and strain monitoring. Full article

Peridinin-containing dinoflagellate plastomes are predominantly encoded in nuclear genomes, with less than 20 essential chloroplast proteins carried on “minicircles”. Each minicircle generally carries one gene and a short non-coding region (NCR) with a median length of approximately 400–1000 bp. We report here differential nuclease sensitivity and two-dimensional southern blot patterns, suggesting that dsDNA minicircles are in fact the minor forms, with substantial DNA:RNA hybrids (DRHs). Additionally, we observed large molecular weight intermediates, cell-lysate-dependent NCR secondary structures, multiple bidirectional predicted ssDNA structures, and different southern blot patterns when probed with different NCR fragments. In silico analysis suggested the existence of substantial secondary structures with inverted repeats (IR) and palindrome structures within the initial ~650 bp of the NCR sequences, in accordance with conversion event(s) outcomes with PCR. Based on these findings, we propose a new transcription-templating-translation model, which is associated with cross-hopping shift intermediates. Since dinoflagellate chloroplasts are cytosolic and lack nuclear envelope breakdown, the dynamic DRH minicircle transport could have contributed to the spatial-temporal dynamics required for photosystem repair. This represents a paradigm shift from the previous understanding of “minicircle DNAs” to a “working plastome”, which will have significant implications for its molecular functionality and evolution. Full article

Extrachromosomal circular DNA (eccDNA) is a special class of circular DNA in eukaryotes. Recent studies have suggested that eccDNA is the product of genomic instability and has important biological functions to regulate many downstream biological processes. While NGS (Next-Generation Sequencing)-based eccDNA sequencing has led to the identification of many eccDNAs in both healthy and diseased tissues, the specific biological functions of individual eccDNAs have yet to be clearly elucidated. Synthesizing eccDNAs longer than 1 kb with specific sequences remains a major challenge in the field, which has hindered our ability to fully understand their functions. Current methods for synthesizing eccDNAs primarily rely on chemical oligo synthesis, ligation, or the use of a specific gene editing and recombination systems. Therefore, these methods are often limited by the length of eccDNAs and are complex, expensive, as well as time-consuming. In this study, we introduce a novel method named QuickLAMA (Ligase-Assisted Minicircle Accumulation) for rapidly synthesizing eccDNAs up to 2.6 kb using a simple PCR and ligation approach. To validate the efficacy of our method, we synthesized three eccDNAs of varying lengths from cancer tissue and PC3 cells and confirmed successful circularization through sequencing and restriction enzyme digestion. Additional analyses have demonstrated that this method is highly efficient, cost-effective, and time-efficient, with good reproducibility. Using the method, a well-trained molecular biologist can synthesize and purify multiple eccDNAs within a single day, and it can be easily standardized and processed in a high-throughput manner, indicating the potential of the method to produce a wide range of desired eccDNAs and promote the translation of eccDNA research into clinical applications. Full article

The role of dogs as reservoir hosts for Toscana virus (TOSV) remains undetermined. This study investigated TOSV and Leishmania infantum infections in one healthy and three infected dogs with Leishmania (A, B, C) following natural exposition to sandfly bites in a focus of zoonotic visceral leishmaniasis (ZVL) located in Northern Tunisia from June to October 2020. At the end of the exposition period, infected and healthy dogs were examined for TOSV and L. infantum infections by xenodiagnosis using a colony of Phlebotomus perniciosus. Pools of freshly engorged P. perniciosus at days 0 and those at days 7 post-feeding were screened for TOSV and L. infantum by nested PCR in the polymerase gene and kinetoplast minicircle DNA, respectively. In the exposure site, P. pernicious is the most abundant sandfly species. The infection rates of sandflies with TOSV and L. infantum were 0.10 and 0.05%, respectively. Leishmania infantum DNA and TOSV RNA were detected in P. perniciosus females fed on dog B and C, respectively. The isolation of TOSV in Vero cells was achieved from two pools containing P. perniciosus fed on dog C. No pathogens were detected in P. perniciosus females fed on dog A and on control dog. We report for the first time the reservoir competence of dog with ZVL in the transmission of TOSV to sandfly vectors in natural settings, in addition to its role as a main reservoir host of L. infantum. Full article

We evaluated four DNA vaccine candidates for their ability to produce virus-like particles (VLPs) and elicit a protective immune response against Foot-and-mouth disease virus (FMDV) in cattle. Two traditional DNA plasmids and two DNA minicircle constructs were evaluated. Both the pTarget O1P1-3C plasmid and O1P1-3C minicircle encoded a wild-type FMDV 3C protease to process the P1-2A polypeptide, whereas the O1P1-HIV-3C^T minicircle used an HIV-1 ribosomal frameshift to down-regulate expression of a mutant 3C protease. A modified pTarget plasmid with a reduced backbone size, mpTarget O1P1-3C^LT, used a 3C protease containing two mutations reported to enhance expression. All constructs produced mature FMDV P1 cleavage products in transfected cells, as seen by western blot analysis. Three constructs, O1P1-3C minicircles, pTarget O1P1-3C, and mpTarget O1P1-3C^LT plasmids, produced intracellular VLP crystalline arrays detected by electron microscopy. Despite VLP formation in vitro, none of the DNA vaccine candidates elicited protection from clinical disease when administered independently. Administration of pTarget O1P1-3C plasmid enhanced neutralizing antibody titers when used as a priming dose prior to administration of a conditionally licensed adenovirus-vectored FMD vaccine. Further work is needed to develop these DNA plasmid-based constructs into standalone FMD vaccines in cattle. Full article

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. Full article

Non-viral gene therapy has the potential to overcome several shortcomings in viral vector-based therapeutics. Methods of in vivo plasmid delivery have developed over recent years to increase the efficiency of non-viral gene transfer, yet further improvements still need to be made to improve their translational capacity. Gene therapy advances for inherited retinal disease have been particularly prominent over the recent decade but overcoming physical and physiological barriers present in the eye remains a key obstacle in the field of non-viral ocular drug delivery. Minicircles are circular double-stranded DNA vectors that contain expression cassettes devoid of bacterial DNA, thereby limiting the risks of innate immune responses induced by such elements. To date, they have not been extensively used in pre-clinical studies yet remain a viable vector option for the treatment of inherited retinal disease. Here, we explore the potential of minicircle DNA delivery to the neural retina as a gene therapy approach. We consider the advantages of minicircles as gene therapy vectors as well as review the challenges involved in optimising their delivery to the neural retina. Full article

DNA carries more than the list of biochemical ingredients that drive the basic functions of living systems. The sequence of base pairs includes a multitude of structural and energetic signals, which determine the degree to which the long, threadlike molecule moves and how it responds to proteins and other molecules that control its processing and govern its packaging. The chemical composition of base pairs directs the spatial disposition and fluctuations of successive residues. The observed arrangements of these moieties in high-resolution protein–DNA crystal structures provide one of the best available estimates of the natural, sequence-dependent structure and deformability of the double-helical molecule. Here, we update the set of knowledge-based elastic potentials designed to describe the observed equilibrium structures and configurational fluctuations of the ten unique base-pair steps. The large number of currently available structures makes it possible to characterize the configurational preferences of the DNA base-pair steps within the context of their immediate neighbors, i.e., tetrameric context. Use of these knowledge-based potentials shows promise in accounting for known effects of sequence in long chain molecules, e.g., the degree of curvature reported in classic gel mobility studies and the recently reported sequence-dependent responses of supercoiled minicircles to nuclease cleavage. Full article

Circulating levels of soluble ACE2 are increased by diabetes. Although this increase is associated with the presence and severity of cardiovascular disease, the specific role of soluble ACE2 in atherogenesis is unclear. Previous studies suggested that, like circulating ACE, soluble ACE2 plays a limited role in vascular homeostasis. To challenge this hypothesis, we aimed to selectively increase circulating ACE2 and measure its effects on angiotensin II dependent atherogenesis. Firstly, in Ace2/ApoE DKO mice, restoration of circulating ACE2 with recombinant murine soluble (rmACE2_19-613; 1 mg/kg/alternate day IP) reduced plaque accumulation in the aortic arch, suggesting that the phenotype may be driven as much by loss of soluble ACE2 as the reduction in local ACE2. Secondly, in diabetic ApoE KO mice, where activation of the renin angiotensin system drives accelerated atherosclerosis, rmACE2_19-613 also reduced plaque accumulation in the aorta after 6 weeks. Thirdly, to ensure consistent long-term delivery of soluble ACE2, an intramuscular injection was used to deliver a DNA minicircle encoding ACE2_19-613. This strategy efficiently increased circulating soluble ACE2 and reduced atherogenesis and albuminuria in diabetic ApoE KO mice followed for 10 weeks. We propose that soluble ACE2 has independent vasculoprotective effects. Future strategies that increase soluble ACE2 may reduce accelerated atherosclerosis in diabetes and other states in which the renin angiotensin system is upregulated. Full article

Nucleic acid vaccines have been proven to be a revolutionary technology to induce an efficient, safe and rapid response against pandemics, like the coronavirus disease (COVID-19). Minicircle DNA (mcDNA) is an innovative vector more stable than messenger RNA and more efficient in cell transfection and transgene expression than conventional plasmid DNA. This work describes the construction of a parental plasmid (PP) vector encoding the receptor-binding domain (RBD) of the S protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the use of the Design of Experiments (DoE) to optimize PP recombination into mcDNA vector in an orbital shaker. First, the results revealed that host cells should be grown at 42 °C and the Terrific Broth (TB) medium should be replaced by Luria Broth (LB) medium containing 0.01% L-arabinose for the induction step. The antibiotic concentration, the induction time, and the induction temperature were used as DoE inputs to maximize the % of recombined mcDNA. The quadratic model was statistically significant (p-value < 0.05) and presented a non-significant lack of fit (p-value > 0.05) with a suitable coefficient of determination. The optimal point was validated using 1 h of induction, at 30 °C, without the presence of antibiotics, obtaining 93.87% of recombined mcDNA. Based on these conditions, the production of mcDNA was then maximized in a mini-bioreactor platform. The most favorable condition obtained in the bioreactor was obtained by applying 60% pO₂ in the fermentation step during 5 h and 30% pO₂ in the induction step, with 0.01% L-arabinose throughout 5 h. The yield of mcDNA-RBD was increased to a concentration of 1.15 g/L, when compared to the orbital shaker studies (16.48 mg/L). These data revealed that the bioreactor application strongly incremented the host biomass yield and simultaneously improved the recombination levels of PP into mcDNA. Altogether, these results contributed to improving mcDNA-RBD biosynthesis to make the scale-up of mcDNA manufacture simpler, cost-effective, and attractive for the biotechnology industry. Full article

Among non-viral vectors, cationic polymers, such as poly(propylene imine) (PPI), play a prominent role in nucleic acid delivery. However, limitations of polycationic polymer-based DNA delivery systems are (i) insufficient target specificity, (ii) unsatisfactory transgene expression, and (iii) undesired transfer of therapeutic DNA into non-target cells. We developed single-chain antibody fragment (scFv)-directed hybrid polyplexes for targeted gene therapy of prostate stem cell antigen (PSCA)-positive tumors. Besides mono-biotinylated PSCA-specific single-chain antibodies (scFv(AM1-P-BAP)) conjugated to neutravidin, the hybrid polyplexes comprise β-cyclodextrin-modified PPI as well as biotin/maltose-modified PPI as carriers for minicircle DNAs encoding for Sleeping Beauty transposase and a transposon encoding the gene of interest. The PSCA-specific hybrid polyplexes efficiently delivered a GFP gene in PSCA-positive tumor cells, whereas control hybrid polyplexes showed low gene transfer efficiency. In an experimental gene therapy approach, targeted transposition of a codon-optimized p53 into p53-deficient HCT116^{p53−/−/PSCA} cells demonstrated decreased clonogenic survival when compared to mock controls. Noteworthily, p53 transposition in PTEN-deficient H4^PSCA glioma cells caused nearly complete loss of clonogenic survival. These results demonstrate the feasibility of combining tumor-targeting hybrid polyplexes and Sleeping Beauty gene transposition, which, due to the modular design, can be extended to other target genes and tumor entities. Full article

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare autosomal inherited skin disorder caused by mutations in the COL7A1 gene that encodes type VII collagen (C7). The development of an efficient gene replacement strategy for RDEB is mainly hindered by the lack of vectors able to encapsulate and transfect the large cDNA size of this gene. To address this problem, our group has opted to use polymeric-based non-viral delivery systems and minicircle DNA. With this approach, safety is improved by avoiding the usage of viruses, the absence of bacterial backbone, and the replacement of the control viral cytomegalovirus (CMV) promoter of the gene with human promoters. All the promoters showed impressive C7 expression in RDEB skin cells, with eukaryotic translation elongation factor 1 α (EF1α) promoter producing higher C7 expression levels than CMV following minicircle induction, and COL7A1 tissue-specific promoter (C7P) generating C7 levels similar to normal human epidermal keratinocytes. The improved system developed here has a high potential for use as a non-viral topical treatment to restore C7 in RDEB patients efficiently and safely, and to be adapted to other genetic conditions. Full article