DNA Transport Research Articles

Influence of Metabolic, Transporter, and Pathogenic Genes on Pharmacogenetics and DNA Methylation in Neurological Disorders.

Pharmacogenetics and DNA methylation influence therapeutic outcomes and provide insights into potential therapeutic targets for brain-related disorders. To understand the effect of genetic polymorphisms on drug response and disease risk, we analyzed the relationship between global DNA methylation, drug-metabolizing enzymes, transport genes, and pathogenic gene phenotypes in serum samples from two groups of patients: Group A, which showed increased 5-methylcytosine (5mC) levels during clinical follow-up, and Group B, which exhibited no discernible change in 5mC levels. We identified specific SNPs in several metabolizing genes, including CYP1A2, CYP2C9, CYP4F2, GSTP1, and NAT2, that were associated with differential drug responses. Specific SNPs in CYP had a significant impact on enzyme activity, leading to changes in phenotypic distribution between the two patient groups. Group B, which contained a lower frequency of normal metabolizers and a higher frequency of ultra-rapid metabolizers compared to patients in Group A, did not show an improvement in 5mC levels during follow-up. Furthermore, there were significant differences in phenotype distribution between patient Groups A and B for several SNPs associated with transporter genes (ABCB1, ABCC2, SLC2A9, SLC39A8, and SLCO1B1) and pathogenic genes (APOE, NBEA, and PTGS2). These findings appear to suggest that the interplay between pharmacogenomics and DNA methylation has important implications for improving treatment outcomes in patients with brain-related disorders.

Begomovirus-Host Interactions: Viral Proteins Orchestrating Intra and Intercellular Transport of Viral DNA While Suppressing Host Defense Mechanisms.

Begomoviruses, which belong to the Geminiviridae family, are intracellular parasites transmitted by whiteflies to dicotyledonous plants thatsignificantly damage agronomically relevant crops. These nucleus-replicating DNA viruses move intracellularly from the nucleus to the cytoplasm and then, like other plant viruses, cause disease by spreading systemically throughout the plant. The transport proteins of begomoviruses play a crucial role in recruiting host components for the movement of viral DNA within and between cells, while exhibiting functions that suppress the host's immune defense. Pioneering studies on species of the Begomovirus genus have identified specific viral transport proteins involved in intracellular transport, cell-to-cell movement, and systemic spread. Recent research has primarily focused on viral movement proteins and their interactions with the cellular host transport machinery, which has significantly expanded understanding on viral infection pathways. This review focuses on three components within this context: (i) the role of viral transport proteins, specifically movement proteins (MPs) and nuclear shuttle proteins (NSPs), (ii) their ability to recruit host factors for intra- and intercellular viral movement, and (iii) the suppression of antiviral immunity, with a particular emphasis on bipartite begomoviral movement proteins.

A Fluorophore-Labeled Lysine Dendrimer with an Oxo-Anion-Binding Motif for Tracking Gene Transfection.

A transfection vector based on a peptide dendrimer (1) has been developed and its abilities for DNA binding and transport have been investigated. By attaching a fluorophore to the vector system (1*), several steps in the transfection process could be monitored directly. As DLS and AFM studies showed, the labeled vector 1* condensed DNA into tightly packed aggregates able to enter eukaryotic cells. Co-localization experiments revealed that the ligand/plasmid complex is taken up by the endosomal pathway followed by an endosomal escape or lysosomal degradation. Afterwards, the plasmid DNA seems to enter the nucleus due to a breakdown of the nuclear envelope during mitosis, as only cells that have recently undergone mitosis showed H2B-GFP expression.

Transcriptome analysis and identification of leaf, tuberous root and fibrous root tissue-specific high temperature stress-responsive genes in sweet potato

Sweet Potato is an important food crop, and its production is affected by environmental stresses, including high temperature. The gene expression patterns and molecular responses in different tissues of sweet potato under high temperature stress were studied using microarray data sets. Analysis revealed that modulation in the expression of key genes and pathways associated with various proteins including enzymes under high temperature stress in leaf, fibrous root and storage root tissues. Tissue-specific responses, with both common and unique cellular responses were observed among the tissues. Pathway analysis revealed the differential regulation of genes involved in DNA replication, metabolism, transport, signaling, and stress response during high temperature stress. Six genes viz., DnaJ-domain protein (IpDnaJ), nuclear protein (IpELF5), heat shock protein 90.1 (IpHsp90.1), ABC transporter (IpABC) hydrolase (IpNUDX1) and alternative oxidase 1a (IpAO1a), were up-regulated in the leaf, fibrous root and tuberous root tissues. These six genes might play an important role in imparting high temperature stress tolerance in the leaf, fibrous root and tuberous root tissues of sweet potato. The information generated provides valuable insights on leaf, tuberous root and fibrous root tissue-specific high temperature stress-responsive genes in sweet potato. These datasets will be helpful in selecting candidate genes and pathways for further functional and genomic analyses, facilitating the genetic improvement of sweet potato with enhanced stress tolerance.

DNA Transport through the Dynamic Type IV Secretion System.

The versatile type IV secretion system (T4SS) nanomachine plays a pivotal role in bacterial pathogenesis and the propagation of antibiotic resistance determinants throughout microbial populations. In addition to paradigmatic DNA conjugation machineries, diverse T4SSs enable the delivery of multifarious effector proteins to target prokaryotic and eukaryotic cells, mediate DNA export and uptake from the extracellular milieu, and in rare examples, facilitate transkingdom DNA translocation. Recent advances have identified new mechanisms underlying unilateral nucleic acid transport through the T4SS apparatus, highlighting both functional plasticity and evolutionary adaptations that enable novel capabilities. In this review, we describe the molecular mechanisms underscoring DNA translocation through diverse T4SS machineries, emphasizing the architectural features that implement DNA exchange across the bacterial membrane and license transverse DNA release across kingdom boundaries. We further detail how recent studies have addressed outstanding questions surrounding the mechanisms by which nanomachine architectures and substrate recruitment strategies contribute to T4SS functional diversity.

Systems biology of competency in Vibrio natriegens is revealed by applying novel data analytics to the transcriptome

Vibrio natriegens regulates natural competence through the TfoX and QstR transcription factors, which are involved in external DNA capture and transport. However, the extensive genetic and transcriptional regulatory basis for competency remains unknown. We used a machine-learning approach to decompose Vibrio natriegens's transcriptome into 45 groups of independently modulated sets of genes (iModulons). Our findings show that competency is associated with the repression of two housekeeping iModulons (iron metabolism and translation) and the activation of six iModulons; including TfoX and QstR, a novel iModulon of unknown function, and three housekeeping iModulons (representing motility, polycations, and reactive oxygen species [ROS] responses). Phenotypic screening of 83 gene deletion strains demonstrates that loss of iModulon function reduces or eliminates competency. This database-iModulon-discovery cycle unveils the transcriptomic basis for competency and its relationship to housekeeping functions. These results provide the genetic basis for systems biology of competency in this organism.

Modelling environmental DNA transport in rivers reveals highly resolved spatio-temporal biodiversity patterns

The ever-increasing threats to riverine ecosystems call for novel approaches for highly resolved biodiversity assessments across taxonomic groups and spatio-temporal scales. Recent advances in the joint use of environmental DNA (eDNA) data and eDNA transport models in rivers (e.g., eDITH) allow uncovering the full structure of riverine biodiversity, hence elucidating ecosystem processes and supporting conservation measures. We applied eDITH to a metabarcoding dataset covering three taxonomic groups (fish, invertebrates, bacteria) and three seasons for a catchment sampled for eDNA at 73 sites. We upscaled eDNA-based biodiversity predictions to approximately 1900 reaches, and assessed α- and β-diversity patterns across seasons and taxonomic groups over the whole network. Genus richness predicted by eDITH was generally higher than values from direct eDNA analysis. Both predicted α- and β-diversity varied depending on season and taxonomic group. Predicted fish α-diversity increased downstream in all seasons, while invertebrate and bacteria α-diversity either decreased downstream or were unrelated to network position. Spatial β-diversity mostly decreased downstream, especially for bacteria. The eDITH model yielded a more refined assessment of freshwater biodiversity as compared to raw eDNA data, both in terms of spatial coverage, diversity patterns and effect of covariates, thus providing a more complete picture of freshwater biodiversity.

Thermally Switchable Nanogate Based on Polymer Phase Transition.

Mimicking and extending the gating properties of biological pores is of paramount interest for the fabrication of membranes that could be used in filtration or drug processing. Here, we build a selective and switchable nanopore for macromolecular cargo transport. Our approach exploits polymer graftings within artificial nanopores to control the translocation of biomolecules. To measure transport at the scale of individual biomolecules, we use fluorescence microscopy with a zero-mode waveguide set up. We show that grafting polymers that exhibit a lower critical solution temperature creates a toggle switch between an open and closed state of the nanopore depending on the temperature. We demonstrate tight control over the transport of DNA and viral capsids with a sharp transition (∼1 °C) and present a simple physical model that predicts key features of this transition. Our approach provides the potential for controllable and responsive nanopores in a range of applications.

Morphology around Nanopores Fabricated by Laser-Assisted Dielectric Breakdown and Its Impact on Ion and DNA Transport and Sensing

Laser-assisted controlled dielectric breakdown (LaCBD) has emerged as an alternative to conventional CBD-based nanopore fabrication due to its localization capability, facilitated by the photothermal-induced thinning down in the hot spot. Here, we reported the potential impact of the laser on forming debris around the nanopore region in LaCBD. The debris was clearly observable by scanning electron microscopy (SEM) and photoluminescence (PL) spectroscopy. We found that debris formation is a unique phenomenon in LaCBD that is not observable in the conventional CBD approach. We also found that the LaCBD-induced debris is more evident when the laser power and voltage stress are higher. Moreover, the debris is asymmetrically distributed on the top and bottom sides of the membrane. We also found unexpected rectified ionic and molecular transport in those LaCBD nanopores with debris. Based on these observations, we developed and validated a model describing the debris formation kinetics in LaCBD by considering the generation, diffusion, drift, and gravity in viscous mediums. These findings indicate that while laser aids in nanopore localization, precautions should be taken due to the potential formation of debris and rectification of molecular transport. This study provides valuable insights into the kinetics of LaCBD and the characteristics of the LaCBD nanopore.

Base Pair Stacking Modulates Solid-State Charge Transport in DNA Hairpins.

Particular interest has been focused on modulation of solid-state charge transport (CT) in DNA. Nevertheless, it remains challenging to do so in a sensitive and predictive manner due to the lack of a definite relationship between DNA base pair stacking and DNA CT. The challenges can be mainly attributed to the ill-defined systems, which may lead to ambiguous and even contradictory conclusions. Here, we use DNA hairpins to construct the well-defined self-assembled monolayers. We reveal nearly positive-linear correlations between DNA conformation and CT in the DNA hairpins regulated with metal ion chelation and DNA sequence. The correlations have been confirmed by the solid-state current-voltage characteristics and circular dichroism in solution. The enhanced CT via metal ion chelated DNA hairpins is mainly from the improved DNA energy coupling to electrodes, not the almost unchanged energy barrier when Hg ion-induced DNA conformational switches toward the canonical B-form.

Dynamic concatenation leads to anomalous transport of ring DNA in concentrated solutions.

Circular DNA polymers have been widely studied due to their biological relevance as well as the unique anomalous transport properties they exhibit in concentrated solutions and blends with linear DNA. In cells, topoisomerase II relaxes torsional strain in circular DNA by cutting and then reconnecting one of the strands of the double-stranded polymer, enabling replication, transcription, and repair. When topoisomerase II acts on nicked circular DNA at high concentration, the relaxed rings can concatenate, forming Olympic ring structures reminiscent of kinetoplasts. The rate of topoisomerase activity as well as the concentration and size of the DNA can tune the nonequilibrium dynamics and conformations of the concatemers. Here, we visualize the fluorescent-labeled DNA rings comprising the concatemers and use particle tracking and differential dynamic microscopy to characterize the dynamics and time-varying size and shape of the actively linking and unlinking molecules. Beyond the insight our work provides to biological processes mediated by active restructuring of DNA, the complex fluids we engineer and study may have applications in the design of biocompatible active materials for cellular repair, toxin filtration and drug delivery.

The proteomic landscape shows oncologic relevance in cystitis glandularis.

The relationship between cystitis glandularis (CG) and bladder malignancy remains unclear. We identified the oncologic significance of CG at the molecular level using liquid chromatography-tandem mass spectrometry-based proteomic analysis of 10 CG, 12 urothelial carcinoma (UC), and nine normal urothelium (NU) specimens. Differentially expressed proteins (DEPs) were identified based on an analysis of variance false discovery rate < 0.05, and their functional enrichment was analyzed using a network model, Gene Set Enrichment Analysis, and Gene Ontology annotation. We identified 9,890 proteins across all samples and 1,139 DEPs among the three entities. A substantial number of DEPs overlapped in CG/NU, distinct from UC. Interestingly, we found that a subset of DEP clusters (n = 53, 5%) was differentially expressed in NU but similarly between CG and UC. This "UC-like signature" was enriched for reactive oxygen species (ROS) and energy metabolism, growth and DNA repair, transport, motility, epithelial-mesenchymal transition, and cell survival. Using the top 10 shortlisted DEPs, including SOD2, PRKCD, CYCS, and HCLS1, we identified functional elements related to ROS metabolism, development, and transport using network analysis. The abundance of these four molecules in UC/CG than in NU was consistent with the oncologic functions in CG. Using a proteomic approach, we identified a predominantly non-neoplastic landscape of CG, which was closer to NU than to UC. We also confirmed a small subset of common DEPs in UC and CG, suggesting that altered ROS metabolism might imply potential cancerous risks in CG.

Peptide-Based Nanoparticles for αvβ3 Integrin-Targeted DNA Delivery to Cancer and Uterine Leiomyoma Cells

Uterine leiomyoma is the most common benign tumor of the reproductive system. Current therapeutic options do not simultaneously meet the requirements of long-term efficiency and fertility preservation. Suicide gene delivery can be proposed as a novel approach to uterine leiomyoma therapy. Non-viral vehicles are an attractive approach to DNA delivery for gene therapy of both malignant and benign tumors. Peptide-based vectors are among the most promising candidates for the development of artificial viruses, being able to efficiently cross barriers of DNA transport to cells. Here we described nanoparticles composed of cysteine-crosslinked polymer and histidine-arginine-rich peptide modified with iRGD moiety and characterized them as vehicles for plasmid DNA delivery to pancreatic cancer PANC-1 cells and the uterine leiomyoma cell model. Several variants of nanoparticles were formulated with different targeting ligand content. The physicochemical properties that were studied included DNA binding and protection, interaction with polyanions and reducing agents, size, structure and zeta-potential of the peptide-based nanoparticles. Cytotoxicity, cell uptake and gene transfection efficiency were assessed in PANC-1 cells with GFP and LacZ-encoding plasmids. The specificity of gene transfection via αvβ3 integrin binding was proved in competitive transfection. The therapeutic potential was evaluated in a uterine leiomyoma cell model using the suicide gene therapy approach. The optimal formulation was found to be at the polyplex with the highest iRGD moiety content being able to transfect cells more efficiently than control PEI. Suicide gene therapy using the best formulation resulted in a significant decrease of uterine leiomyoma cells after ganciclovir treatment. It can be concluded that the application of iRGD-modified peptide-based nanoparticles has a high potential for cellular delivery of DNA therapeutics in favor of uterine leiomyoma gene therapy.

PH-Responsive Polyelectrolyte Coatings that Enable Catheter-Mediated Transfer of DNA to the Arterial Wall in Short and Clinically Relevant Inflation Times.

We report the design and characterization of pH-responsive polymer coatings that enable catheter balloon-mediated transfer of DNA to arterial tissue in short, clinically relevant inflation times. Our approach exploits the pH-dependent ionization of poly(acrylic acid) (PAA) to promote disassembly and release of plasmid DNA from polyelectrolyte multilayers. We characterized the contact transfer of multilayers composed of PAA, plasmid DNA, and linear poly(ethyleneimine) (LPEI) identified as promising in prior studies on the delivery of DNA to arterial tissue. In contrast to thinner films evaluated previously, we found thicker coatings composed of 32 repeating (LPEI/PAA/LPEI/DNA)x tetralayers to swell substantially in physiologically relevant media (in PBS; pH = 7.4). In some cases, these coatings also disintegrated or delaminated rapidly from their underlying substrates, suggesting the potential for enhanced balloon-mediated transfer. We developed a technically straightforward agarose gel-based hole-insertion model to characterize factors (inflation time, lumen size, etc.) that influence contact transfer of DNA when film-coated balloons are inflated into contact with soft surfaces. Those studies and the results of in vivo experiments using small animal (rat) and large animal (pig) models of peripheral arterial injury revealed catheters coated with these materials to promote robust contact transfer of DNA to soft hydrogel surfaces and the luminal surfaces of arterial tissue using inflation times as short as 30 s. These short inflation times are relevant in the context of clinical vascular interventions in peripheral arteries. Additional studies demonstrated that contact transfer of DNA using these short times can promote subsequent dissemination and transport of DNA to the medial tissue layer, suggesting the potential for use in therapeutically relevant applications of balloon-mediated gene transfer.

In silico and experimental validation of a new modified arginine-rich cell penetrating peptide for plasmid DNA delivery

Cell-penetrating peptides (CPPs) attracted great attention because of the capability to deliver various types of cargo molecules across into the cells. In this study, we presented a new arginine rich CPP, named MR, for efficient transporting plasmid DNA. We used a combined bioinformatic-based approach to improve the speed and accuracy of CPP evaluation. MR protein properties, structural models, interaction with DNA, as well as cell localization and membrane interaction were evaluated through multiple servers. Importantly, analysis using different algorithms showed the high CPP prediction confidence of MR. Experimental results also revealed the capacity of this gene delivery system in vitro for efficient plasmid DNA transfection. Additionally, in vitro mechanistically studies together with bioinformatic investigation suggested that MR peptide may internalize into the cell through endocytosis pathways. Moreover, in silico safety analysis such as immunogenicity, allergenicity, toxicity, and hemolysis activity as well as MTT assay also confirmed the safety of MR peptide. This study illustrated that MR peptide could be presented as remarkable potential gene delivery system for promising transport of plasmid DNA towards the therapeutic applications.

Radical Reactions Enabled By Porphyrinoids

“ Look deep into nature , and then you will understand everything better.” Albert Einstein Porphyrinoids, also known as the pigments of life, are a class of naturally occurring organic dyes. They play key roles in crucial processes that support life - oxygen transport (hem), electron transport (cytochrome c), photosynthesis (chlorophyll a), and synthesis of DNA (vitamin B12). Vitamin B12 - a co-factor in many catalytic processes. Following nature, we have been exploiting the potential of this compound in catalysis.Vitamin B12 - catalysis has been successfully translated into the laboratory and used in a small collection of reactions.[1-2] The advantage of using vitamin B12 as a catalyst lays in the complete stability of the central cobalt ion and by the definition it is nontoxic. It has also been well documented that the reaction mechanism usually follows a radical pathway, bringing a new dimension to this already interesting field.[2] Along this line, we have developed new vitamin B12-catalyzed reactions involving reduction of Co(III) to Co(I) or Co(II) and subsequent reactions with electrophiles or radicals. Vitamin B12 derivative unusually catalyzes a new olefinic sp2 C-H alkylation reaction with diazo reagents as a carbene source,[3], acylation of activated olefins, alkylation of strained molecules.[4-6] These key findings emphasize the unique feature of vitamin B12 as a catalyst to achieve something unachievable with other methodologies or to find a greener approach.

Ionic Liquid Decelerates Single-Stranded DNA Transport through Molybdenum Disulfide Nanopores.

Nanopores in two-dimensional (2D) materials have emerged to offer in principle necessary spatial resolution for high-throughput DNA sequencing. However, their fidelity is severely limited by the fast DNA translocation. A recent experiment indicates that introducing ionic liquids could slow down DNA translocation in a MoS2 nanopore. However, the corresponding in-depth molecular mechanism underlying the experimental findings is not fully understood, which is crucial for the future improvement of rational DNA translocation control. Here, we computationally investigate and then experimentally identify the effect of BmimCl ionic liquid on the retardation of ssDNA translocation through a single-layer MoS2 nanopore. Our all-atom molecular dynamics simulations demonstrate that the strong interaction between Bmim+ and ssDNA offers a considerable dragging force to decelerate the electrophoretic motion of ssDNA in the BmimCl solution. Moreover, we show that Bmim+ ions exhibit preferential binding on the sulfur edges of the nanopore. These Bmim+ in the pore region can not only act as a steric blockage but also form π-π stackings with nucleobases, which provide a further restriction on the ssDNA motion. Therefore, our molecular dynamics simulation investigations deepen the understanding of the critical role of ionic liquid in DNA translocation through a nanopore from a molecular landscape, which may benefit practical implementations of ionic liquids in nanopore sequencing.

Genetic variability in minor capsid protein (L2 gene) of human papillomavirus type 16 among Indian women.

Human papillomavirus type 16 (HPV-16) is the predominant genotype worldwide associated with invasive cervical cancer and hence remains as the focus for diagnostic development and vaccine research. L2, the minor capsid protein forms the packaging unit for the HPV genome along with the L1 protein and is primarily associated with transport of genomic DNA to the nucleus. Unlike L1, L2 is known to elicit cross-neutralizing antibodies and thus becomes a suitable candidate for pan-HPV prophylactic vaccine development. In the present study, a total of 148 cervical HPV-16 isolates from Indian women were analyzed by PCR-directed sequencing, phylogenetic analysis and in silico immunoinformatics tools to determine the L2 variations that may impact the immune response and oncogenesis. Ninety-one SNPs translating to 35 non-synonymous amino acid substitutions were observed, of these 16 substitutions are reported in the Indian isolates for the first time. T245A, L266F, S378V and S384A substitutions were significantly associated with high-grade cervical neoplastic status. Multiple substitutions were observed in samples from high-grade cervical neoplastic status as compared to those from normal cervical status (p = 0.027), specifically from the D3 sub-lineage. It was observed that substitution T85A was part of both, B and T cell epitopes recognized by MHC-I molecules; T245A was common to B and T cell epitopes recognized by MHC-II molecules and S122P/A was common to the region recognized by both MHC-I and MHC-II molecules. These findings reporting L2 protein substitutions have implications on cervical oncogenesis and design of next-generation L2-based HPV vaccines.

Adenovirus entry: Stability, uncoating, and nuclear import.

Adenoviruses (AdVs) are widespread in vertebrates. They infect the respiratory and gastrointestinal tracts, the eyes, heart, liver, and kidney, and are lethal to immunosuppressed people. Mastadenoviruses infecting mammals comprise several hundred different types, and many specifically infect humans. Human adenoviruses are the most widely used vectors in clinical applications, including cancer treatment and COVID-19 vaccination. AdV vectors are physically and genetically stable and generally safe in humans. The particles have an icosahedral coat and a nucleoprotein core with a DNA genome. We describe the concept of AdV cell entry and highlight recent advances in cytoplasmic transport, uncoating, and nuclear import of the viral DNA. We highlight a recently discovered "linchpin" function of the virion protein V ensuring cytoplasmic particle stability, which is relaxed at the nuclear pore complex by cues from the E3 ubiquitin ligase Mind bomb 1 (MIB1) and the proteasome triggering disruption. Capsid disruption by kinesin motor proteins and microtubules exposes the linchpin and renders protein V a target for MIB1 ubiquitination, which dissociates V from viral DNA and enhances DNA nuclear import. These advances uncover mechanisms controlling capsid stability and premature uncoating and provide insight into nuclear transport of nucleic acids.

ComFC mediates transport and handling of single-stranded DNA during natural transformation

The ComFC protein is essential for natural transformation, a process that plays a major role in the spread of antibiotic resistance genes and virulence factors across bacteria. However, its role remains largely unknown. Here, we show that Helicobacter pylori ComFC is involved in DNA transport through the cell membrane, and is required for the handling of the single-stranded DNA once it is delivered into the cytoplasm. The crystal structure of ComFC includes a zinc-finger motif and a putative phosphoribosyl transferase domain, both necessary for the protein’s in vivo activity. Furthermore, we show that ComFC is a membrane-associated protein with affinity for single-stranded DNA. Our results suggest that ComFC provides the link between the transport of the transforming DNA into the cytoplasm and its handling by the recombination machinery.