Palindromic Sequence Research Articles

Type II and IV toxin-antitoxin systems coordinately stabilize the integrative and conjugative element of the ICESa2603 family conferring multiple drug resistance in Streptococcus suis.

Integrative and conjugative elements (ICEs) play a vital role in bacterial evolution by carrying essential genes that confer adaptive functions to the host. Despite their importance, the mechanism underlying the stable inheritance of ICEs, which is necessary for the acquisition of new traits in bacteria, remains poorly understood. Here, we identified SezAT, a type II toxin-antitoxin (TA) system, and AbiE, a type IV TA system encoded within the ICESsuHN105, coordinately promote ICE stabilization and mediate multidrug resistance in Streptococcus suis. Deletion of SezAT or AbiE did not affect the strain's antibiotic susceptibility, but their duple deletion increased susceptibility, mainly mediated by the antitoxins SezA and AbiEi. Further studies have revealed that SezA and AbiEi affect the genetic stability of ICESsuHN105 by moderating the excision and extrachromosomal copy number, consequently affecting the antibiotic resistance conferred by ICE. The DNA-binding proteins AbiEi and SezA, which bind palindromic sequences in the promoter, coordinately modulate ICE excision and extracellular copy number by binding to sequences in the origin-of-transfer (oriT) and the attL sites, respectively. Furthermore, AbiEi negatively regulates the transcription of SezAT by binding directly to its promoter, optimizing the coordinate network of SezAT and AbiE in maintaining ICESsuHN105 stability. Importantly, SezAT and AbiE are widespread and conserved in ICEs harbouring diverse drug-resistance genes, and their coordinated effects in promoting ICE stability and mediating drug resistance may be broadly applicable to other ICEs. Altogether, our study uncovers the TA system's role in maintaining the genetic stability of ICE and offers potential targets for overcoming the dissemination and evolution of drug resistance.

Polyvalent Aptamer-Functionalized NIR-II Quantum Dots for Targeted Theranostics in High PD-L1-Expressing Tumors.

Ag2S quantum dots (QDs) show superior optical properties in the NIR-II region and display significant clinical potential with favorable biocompatibility. However, inherent defects of low targeting and poor solubility necessitate practical modification methods to achieve the theranostics of Ag2S QDs. Herein, we used rolling circle amplification (RCA) techniques to obtain long single-stranded DNA containing the PD-L1 aptamer and C-rich DNA palindromic sequence. The C-rich DNA palindromic sequences can specifically chelate Ag2+ and thus serve as a template to result in biomimetic mineralization and formation of pApt-Ag2S QDs. These QDs enable specific targeting and illuminate hot tumors with high PD-L1 expression effectively, serving as excellent molecular targeted probes. In addition, due to the high NIR-II absorption of Ag2S QDs, pApt-Ag2S QDs exhibit remarkable photothermal properties. And besides, polyvalent PD-L1 aptamers can recognize PD-L1 protein and effectively block the inhibitory signal of PD-L1 on T cells, enabling efficient theranostics through the synergistic effect of photothermal therapy and immune checkpoint blocking therapy. Summary, we enhance the biological stability and antibleaching ability of Ag2S QDs using long single-stranded DNA as a template, thereby establishing a theranostic platform that specifically targets PD-L1 high-expressing inflamed tumors and demonstrates excellent performance both in vitro and in vivo.

Molecular insights into FucR transcription factor to control the metabolism of L-fucose in Bifidobacterium longum subsp. infantis

Bifidobacterium longum subsp. infantis commonly colonizes the human gut and is capable of metabolizing L-fucose, which is abundant in the gut. Multiple studies have focused on the mechanisms of L-fucose utilization by B. longum subsp. infantis, but the regulatory pathways governing the expression of these catabolic processes are still unclear. In this study, we have conducted a structural and functional analysis of L-fucose metabolism transcription factor FucR derived from B. longum subsp. infantis Bi-26. Our results indicated that FucR is a L-fucose-sensitive repressor with more α-helices, fewer β-sheets, and β-turns. Transcriptional analysis revealed that FucR displays weak negative self-regulation, which is counteracted in the presence of L-fucose. Isothermal titration calorimetry indicated that FucR has a 2:1 stoichiometry with L-fucose. The key amino acid residues for FucR binding L-fucose are Asp280 and Arg331, with mutation of Asp280 to Ala resulting in a decrease in the affinity between FucR and L-fucose with the Kd value from 2.58 to 11.68 μM, and mutation of Arg331 to Ala abolishes the binding ability of FucR towards L-fucose. FucR specifically recognized and bound to a 20-bp incomplete palindrome sequence (5’-ACCCCAATTACGAAAATTTTT-3’), and the affinity of the L-fucose-loaded FucR for the DNA fragment was lower than apo-FucR. The results provided new insights into the regulating L-fucose metabolism by B. longum subsp. infantis.

Application of prime editing system to introduce TP53 R248Q hotspot mutation in acute lymphoblastic leukemia cell line.

In childhood acute lymphoblastic leukemia (ALL), TP53 gene mutation is associated with chemoresistance in a certain population of relapsed cases. To directly verify the association of TP53 gene mutation with chemoresistance of relapsed childhood ALL cases and improve their prognosis, the development of appropriate human leukemia models having TP53 mutation in the intrinsic gene is required. Here, we sought to introduce R248Q hotspot mutation into the intrinsic TP53 gene in an ALL cell line, 697, by applying a prime editing (PE) system, which is a versatile genome editing technology. The PE2 system uses an artificial fusion of nickase Cas9 and reverse-transcriptase to directly place new genetic information into a target site through a reverse transcriptase template in the prime editing guide RNA (pegRNA). Moreover, in the advanced PE3b system, single guide RNA (sgRNA) matching the edited sequence is also introduced to improve editing efficiency. The initially obtained MDM2 inhibitor-resistant PE3b-transfected subline revealed disrupted p53 transactivation activity, reduced p53 target gene expression, and acquired resistance to chemotherapeutic agents and irradiation. Although the majority of the subline acquired the designed R248Q and adjacent silent mutations, the insertion of the palindromic sequence in the scaffold hairpin structure of pegRNA and the overlap of the original genomic DNA sequence were frequently observed. Targeted next-generation sequencing reconfirmed frequent edit errors in both PE2 and PE3b-transfected 697 cells, and it revealed frequent successful edits in HEK293T cells. These observations suggest a requirement for further modification of the PE2 and PE3b systems for accurate editing in leukemic cells.

NGS Technique for Palindromic Sequencing of DNA Through Effective PST-PCR

DNA Sequencing technologies have been in use since 1970 and has diversified to much more effective transformations till the date. Initially due to certain drawbacks like cost, time period and requirement of toxic and radioactive elements for the compilation of the process, it remained unintroduced to research setting for complex data in earlier times. One of a technique named as the Sanger technique had more practical approach for sequencing the desired data of the fragments. But the need of DNA sequencing surged after the commencement of the Human Genome Project (HGP) which was a 13 year long collaboration to sequence human genome for understanding its applicable uses.1 At the current stage, the progress moved towards Next Generation Sequencing (NGS) to sequence the fragments of DNA for a better acknowledgement but somehow this did not reported for a fair outcome for palindromic DNA while sequencing. In this rugged sequencing platforms, Roche’s 454 with emulsion PCR technique amplified the templates through beads which later went through pyrosequencing with DNA Polymerase gave the desired output but had a major drawback of high cost.2 Therefore, this study deals with effective PST – PCR technique for studying palindromic DNA in NGS and how it can be considered to fit in all advantages for the above task.

Lilium liangiae, a new species in the genus Lilium (Liliaceae) that reveals parallel evolution within morphology.

The former genus Nomocharis, which has been merged as a clade within the genus Lilium (Liliaceae), represents one of the most complicated and unclear groups included in the latter. Research on members of the Nomocharis clade has been quite limited due to the sampling difficulties caused by its selective environmental preferences. In this study, we propose a new species within this clade, Lilium liangiae, as a further bridge connecting the former genus Nomocharis with other members of the genus Lilium. We conducted morphological clustering, phylogenetic, and comparative genomics analyses of nuclear internal spacers and the newly generated complete chloroplast genome, in conjunction with previously published sequences, and performed ancestral state reconstruction to clarify the evolutionary pattern of important traits in Lilium. The clustering results of 38 morphological traits indicated that the new species is allied to Nomocharis, further increasing the morphological polymorphism in the latter. The phylogenetic results and morphological clustering both supported L. liangiae belonging to the subclade Ecristata in Nomocharis, its closest affinity being Lilium gongshanense. Inconsistencies in phylogenetic relationships were detected between nuclear and plastid datasets, possibly due to ancient hybridization and ongoing introgression. Comparative genomics revealed the conservation and similarity of their chloroplast genomes, with variations observed in the expansion and contraction of the IR regions. A/T and palindromic repeat sequences were the most abundant. Seven highly variable regions (Pi≥0.015) were identified as potential molecular markers based on the chloroplast genomes of 47 species within Lilium. Both nuclear and plastid genes exhibited very low variability within the Nomocharis clade, contrasting with their highly variable morphological appearance. The ancestral state reconstruction analysis suggests that the campanulate flower form, as in L. liangiae, arose at least three times within the genus Lilium, revealing parallel evolution in the latter. Overall, this study adds important genetic and morphological evidence for understanding the phylogenetic relationships and parallel evolution patterns of species within the genus Lilium.

Pyoluteorin regulates the biosynthesis of 2,4-DAPG through the TetR family transcription factor PhlH in Pseudomonas protegens Pf-5.

Soil and rhizosphere bacteria act as a rich source of secondary metabolites, effectively fighting against a diverse array of pathogens. Certain Pseudomonas species harbor biosynthetic gene clusters for producing both pyoluteorin and 2,4-diacetylphloroglucinol (2,4-DAPG), which are polyketides that exhibit highly similar antimicrobial spectrum against bacteria and fungi or oomycete. A complex cross talk exists between pyoluteorin and 2,4-DAPG biosynthesis, and production of 2,4-DAPG was strongly repressed by pyoluteorin, yet the underlying mechanism is still elusive. In this study, we find that the TetR family transcription factor PhlH is involved in the cross talk between pyoluteorin and 2,4-DAPG biosynthesis. PhlH binds to a palindromic sequence within the promoter of phlG (PphlG), which encodes a C-C bond hydrolase responsible for degrading 2,4-DAPG. As a signaling molecule, pyoluteorin disrupts the PhlH-PphlG complex by binding to PhlH, leading to decreased levels of 2,4-DAPG. Proteomics data suggest that pyoluteorin regulates multiple physiological processes including fatty acid biosynthesis and transportation of taurine, siderophore, and amino acids. Our work not only reveals a novel mechanism of cross talk between pyoluteorin and 2,4-DAPG biosynthesis, but also highlights pyoluteorin's role as a messenger in the complex communication network of Pseudomonas.IMPORTANCEAntibiosis serves as a crucial defense mechanism for microbes against invasive bacteria and resource competition. These bacteria typically orchestrate the production of multiple antibiotics in a coordinated fashion, wherein the synthesis of one antibiotic inhibits the generation of another. This strategic coordination allows the bacterium to focus its resources on producing the most advantageous antibiotic under specific circumstances. However, the underlying mechanisms of distinct antibiotic production in bacterial cells remain largely elusive. In this study, we reveal that the TetR family transcription factor PhlH detects the secondary metabolite pyoluteorin and mediates the cross talk between pyoluteorin and 2,4-DAPG biosynthesis in the biocontrol strain Pseudomonas protegens Pf-5. These findings hold promise for future research, potentially informing the manipulation of these systems to enhance the effectiveness of biocontrol agents.

Plastid genome data provide new insights into the dynamic evolution of the tribe Ampelopsideae (Vitaceae)

BackgroundAmpelopsideae J. Wen & Z.L. Nie is a small-sized tribe of Vitaceae Juss., including ca. 47 species from four genera showing a disjunct distribution worldwide across all the continents except Antarctica. There are numerous species from the tribe that are commonly used as medicinal plants with immune-modulating, antimicrobial, and anti-hypertensive properties. The tribe is usually recognized into three clades, i.e., Ampelopsis Michx., Nekemias Raf., and the Southern Hemisphere clade. However, the relationships of the three clades differ greatly between the nuclear and the plastid topologies. There has been limited exploration of the chloroplast phylogenetic relationships within Ampelopsideae, and studies on the chloroplast genome structure of this tribe are only available for a few individuals. In this study, we aimed to investigate the evolutionary characteristics of plastid genomes of the tribe, including their genome structure and evolutionary insights.ResultsWe sequenced, assembled, and annotated plastid genomes of 36 species from the tribe and related taxa in the family. Three main clades were recognized within Ampelopsideae, corresponding to Ampelopsis, Nekemias, and the Southern Hemisphere lineage, respectively, and all with 100% bootstrap supports. The genome sequences and content of the tribe are highly conserved. However, comparative analyses suggested that the plastomes of Nekemias demonstrate a contraction in the large single copy region and an expansion in the inverted repeat region, and possess a high number of forward and palindromic repeat sequences distinct from both Ampelopsis and the Southern Hemisphere taxa.ConclusionsOur results highlighted plastome variations in genome length, expansion or contraction of the inverted repeat region, codon usage bias, and repeat sequences, are corresponding to the three lineages of the tribe, which probably faced with different environmental selection pressures and evolutionary history. This study provides valuable insights into understanding the evolutionary patterns of plastid genomes within the Ampelopsideae of Vitaceae.

Characterization and mitigation of artifacts derived from NGS library preparation due to structure-specific sequences in the human genome

BackgroundHybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors.ResultsWe used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation “blacklist” in the BED region to reduce errors in downstream analyses.ConclusionsWe first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.

Abstract A009: Targeting Structural Variants (DNA palindromes) in plasma cell-free DNA for Liquid Biopsy-based Cancer Detection

Abstract We aim to expand the utilities of our genome-wide detection method of chromosomal structural aberrations (structural variants, SVs) for blood-based cancer detection (liquid biopsy). SVs, such as genomic amplification, are prevalent DNA abnormalities and define tumor phenotypes. With cell-free DNA (cfDNA) in plasma, a genome-wide assay for SVs seems feasible for cancer detection. However, most current tests probe a part of the genome for cancer gene mutations and differential DNA methylation. Considering liquid biopsy's promise in clinics, approaches targeting SVs must be considered and developed. The low tumor fraction in plasma cfDNA is a major obstacle to developing liquid biopsy-based tests. Targeted assays overcome low signals by ultra-deep NGS, yet deep sequencing for the entire genome for SV detection would be costly and computationally challenging. In this regard, we have an established approach for enriching SVs (DNA palindromes or inverted repeats, also called fold-back inversions) from total genomic DNA to determine their frequency and genomic locations by sequencing (genome-wide analysis of palindrome formation, GAPF-seq). DNA palindromes arise from common events of genome instability, such as illegitimate repair of DNA double-strand breaks and telomere-telomere fusions, as an early step of genomic amplification. Therefore, a technique to identify DNA palindromes could serve as a tumor DNA detection test across tumor types, including breast cancer. The simple GAPF-seq procedure confers advantages over other methods because it simultaneously amplifies the target signal and reduces background noise. GAPF-seq exploits the inherent propensity of denatured DNA palindromes to form double-stranded DNA (dsDNA) by intra-molecular annealing. Genomic DNA will be denatured and quickly renatured to enrich DNA palindromes to favor intra-molecular annealing. Single-strand-specific nuclease S1 will eliminate the remaining single-stranded, non-palindromic DNA. The dsDNA from palindromes will be amplified by the process of preparing libraries for NGS, by which DNA palindromes appear as coverage peaks in the genome. As a proof-of-concept, we examined the read coverage of nearly 3 million 1-kb bins from low coverage NGS GAPF-seq data for 39 breast paired tumor/normal DNA. We found that the chromosomal distribution of the top 1000 high-coverage bins (GAPF profiles) could effectively differentiate breast tumor DNA, including all stage I tumor DNA, from paired normal DNA (AUROC=0.9885). Automated, three-fold cross-validation with machine learning (ML) algorithms from the STREAMLINE platform confirmed the highly accurate binary classification by GAPF profiles. The same approach was applied for 15 cfDNA from breast cancer patients and effectively differentiated cancer patients' cfDNA from healthy individuals' cfDNA (AUROC=0.900). These results support the potential of GAPF-seq as a unique, SV-targeting liquid biopsy approach for breast cancer. Citation Format: Fumie Igari, Hisashi Tanaka, Michael M Murata, Mitsue Saito, Armando E Giuliano. Targeting Structural Variants (DNA palindromes) in plasma cell-free DNA for Liquid Biopsy-based Cancer Detection [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A009.

Enhancing the Features of DNA Mimic Foldamers for Structural Investigations.

DNA mimic foldamers based on aromatic oligoamide helices bearing anionic phosphonate side chains have been shown to bind to DNA-binding proteins sometimes orders of magnitude better than DNA itself. Here, we introduce new features in the DNA mimic foldamers to facilitate structural investigations of their interactions with proteins. Thirteen new foldamer sequences have been synthesized and characterized using NMR, circular dichroism, molecular modeling, and X-ray crystallography. The results show that foldamer helix handedness can be quantitatively biased by means of a single stereogenic center, that the foldamer structure can be made C2-symmetrical as in palindromic B-DNA sequences, and that associations between foldamer helices can be promoted utilizing dedicated C-terminal residues that act as sticky ends in B-DNA structures.

Elevated incidence of somatic mutations at prevalent genetic sites.

The common loci represent a distinct set of the human genome sites that harbor genetic variants found in at least 1% of the population. Small somatic mutations occur at the common loci and non-common loci, i.e. csmVariants and ncsmVariants, are presumed with similar probabilities. However, our work revealed that within the coding region, common loci constituted only 1.03% of all loci, yet they accounted for 5.14% of TCGA somatic mutations. Furthermore, the small somatic mutation incidence rate at these common loci was 2.7 times that observed in the non-common. Notably, the csmVariants exhibited an impressive recurrent rate of 36.14%, which was 2.59 times of the ncsmVariants. The C-to-T transition at the CpG sites accounted for 32.41% of the csmVariants, which was 2.93 times for the ncsmVariants. Interestingly, the aging-related mutational signature contributed to 13.87% of the csmVariants, 5.5 times that of ncsmVariants. Moreover, 35.93% of the csmVariants contexts exhibited palindromic features, outperforming ncsmVariant contexts by 1.84 times. Notably, cancer patients with higher csmVariants rates had better progression-free survival. Furthermore, cancer patients with high-frequency csmVariants enriched with mismatch repair deficiency were also associated with better progression-free survival. The accumulation of csmVariants during cancerogenesis is a complex process influenced by various factors. These include the presence of a substantial percentage of palindromic sequences at csmVariants sites, the impact of aging and DNA mismatch repair deficiency. Together, these factors contribute to the higher somatic mutation incidence rates of common loci and the overall accumulation of csmVariants in cancer development.

The 60 nm gold nanoparticles improve qPCR amplification efficiency through specific palindromic sequences (GGATCC or ACCGGT) in primers

BackgroundPolymerase chain reaction (PCR) technology and quantitative real-time PCR (qPCR) technology are widely used in clinical diagnosis and research, but amplification efficiency and sensitivity are still key problems for researchers. An increasing number of reports show that gold nanoparticles (AuNPs) can be used to improve the sensitivity and amplification efficiency of PCR. Here, we found that 60 nm gold nanoparticles with a positive charge (60 nm- Au+) can enhance the amplification efficiency of qPCR. MethodsMouse DNA was extracted by the alkaline lysis method. Primer 5.0 software was used to design primers and mutation primers, and the DNA fragments were obtained by the method of synthesizing plasmids. The qPCR was applied to amplify target gene fragments. ResultsThe amplification efficiency of qPCR was improved by about 1.828 times in the experimental group with 60 nm- Au+ compared with the control group without 60 nm- Au+. The primer pair contains a specific palindromic sequence (GGATCC or ACCGGT). And 60 nm Au+ did not enhance the amplification efficiency of qPCR when the above primer was mutated. ConclusionsThe primers contain special palindrome sequences (GGATCC or ACCGGT) with 60 nm- Au+ can enhance the amplification efficiency of qPCR. Therefore, it suggests a more in-depth understanding of the mechanism and function of gold nanoparticles and primer sequences. This study has presented some implications for gold nanoparticles application in the development of qPCR technology.

Interleaved Binary Demodulation Technique for Low-Cost and Fast-Response Visible Light Positioning Using Palindromic Sequences

Recent advancements in visible light positioning (VLP) systems have continuously improved positioning accuracy, extending the application fields in Internet-of-things and factory automation technologies. However, their multiplexing and modulation methods still limit the applicability of VLP systems. Multi-level driving and sensing techniques that use orthogonal frequencies increase the design complexity and are strictly constrained by the bandwidths of the illuminators and sensor devices. Existing binary modulations require additional amendments to the code synchronization. In addition, real-time VLP is fundamentally affected by the distorted signal after orthogonal demultiplexing. Herein, we present a low-cost expansion of VLP that employs binary demodulation in code-division multiplexing. The proposed method utilizes DC-biased binary sequences using the palindromic variant of the Hadamard matrix, which provides perfectly orthogonal sequences while maintaining robustness to signal changes. The proposed demodulation regroups the signal collections of orthogonal sequences in an interleaved manner and determines the source signals via re-modulation. Compared with existing methods, it does not require extra-synchronizing structures and naturally compensates for dynamic sensor signals solely via binary operations. Following its implementation in an FPGA using commercial LEDs and a PIN-PD, we validated that the method achieved 48 dB SNR at a source-to-sensor distance of 10 m. The experiments showed that the proposed method eliminates 45–66% of signal errors and 33–43% of positional displacement during the sensor movements at 800 mm/s. Combined with trilateration, the static positioning error is 0.0158 m on average in 1.31, 0.91, 2.36 m, whereas the standard deviation of the raw position is limited to 0.0016 m.

Application of CRISPR-Cas 9 system in cancer therapy

There are several gene editing technologies under the background of that time, such as ZFN and TALEN which are hard to use and have high cost. On the contrary, the efficiency and accuracy of CRISPR-Cas system has attracted the attention of scientists. CRISPR is a Short palindromic repeat sequence, and it is widespread in many prokaryotes. The first CRISPR was cloned in E. coli by scientists in 1987, and several other sequences were subsequently cloned. The Cas is a nuclease, then two of them cooperate to become the CRISPR-Cas system. It is not only an acquired immune defense mechanism of prokaryotes against virus, but also a tool for gene editing. As an emerging gene editing technology in recent years, CRISPR-Cas9 technology has been widely applied to the treatment of a variety of diseases. This review focused on the application of this technology in cancer. This review systematically introduced the working principle and immune mechanism of CRISPR-Cas9 and compared CRISPR-Cas9 technology with two previous generations of gene-editing technology, current application of CRISPR-Cas9 in cancer treatment, elaborating it with relevant examples, and finally pointed out the challenges of CRISPR-Cas9 in cancer treatment, looking forward to the development of this technology in the future. The purpose of the paper is to introduce and popularize CRISPR-Cas9 technology, and to provide a direction for future cancer treatment.

Enforcing Local DNA Kinks by Sequence-Selective Trisintercalating Oligopeptides of a Tricationic Porphyrin: A Polarizable Molecular Dynamics Study.

Bisacridinyl-bisarginyl porphyrin (BABAP) is a trisintercalating derivative of a tricationic porphyrin, formerly designed and synthesized in order to selectively target and photosensitize the ten-base pair palindromic sequence d(CGGGCGCCCG)2 . We resorted to the previously derived (Far et al., 2004) lowest energy-minimized (EM) structure of the BABAP complex with this sequence as a starting point. We performed polarizable molecular dynamics (MD) on this complex. It showed, over a 150 ns duration, the persistent binding of the Arg side-chain on each BABAP arm to the two G bases upstream from the central porphyrin intercalation site. We subsequently performed progressive shortenings of the connector chain linking the Arg-Gly backbone to the acridine, from n=6 methylenes to 4, followed by removal of the Gly backbone and further connector shortenings, from n=4 to n=1. These resulted into progressive deformations ('kinks') of the DNA backbone. In its most accented kinked structure, the DNA backbone was found to have a close overlap with that of DNA bound to Cre recombinase, with, at the level of one acridine intercalation site, negative roll and positive tilt values consistent with those experimentally found for this DNA at its own kinked dinucleotide sequence. Thus, in addition to their photosensitizing properties, some BABAP derivatives could induce sequence-selective, controlled DNA deformations, which are targets for cleavage by endonucleases or for repair enzymes.

Biophysical model for DNA mutations induced by retroviral genome insertion based on the probability density function of mutation distribution

Research into the biophysical properties of deoxyribonucleic acid (DNA) and the mechanisms underlying genetic mutations has undergone marked advancements. The intriguing nature of mutations resulting from retroviral DNA insertion has garnered considerable attention. Whether these mutations are random or region-specific, the distribution patterns of mutation sites have been the focus of numerous research endeavours. This mutation mechanism originates from interactions between host DNA and the pre-integration complex (PIC), comprising retroviral DNA and an integrase enzyme that facilitates its incorporation into the host DNA. Our study focused on the Zfp521 gene locus, recognised for its pronounced susceptibility to insertional mutations, particularly around unique palindromic sequences. We employed two biophysical models to predict mutation distribution within a range of 50 base pairs centred on these sequences. The first is a probabilistic collision model emphasising PIC and target DNA interactions. The second model is a DNA diffraction lattice, where the PIC behaves according to probability density. Although both models adeptly illuminated the probability distributions of target sites, the second model was more successful in predicting the PIC integration sites based on DNA biophysical properties. This highlights the pivotal role of intricate interactions between the PIC and target DNA, suggesting that mutations can be predicted in a stochastic manner.

Control of A/D type CpG-ODN aggregates to a suitable size for induction of strong immunostimulant activity

Among several types of CpG-ODNs, A/D-type CpG-ODNs have potent adjuvant activity to induce Th-1 immune responses, but exhibit a propensity to aggregate. For the clinical application of A/D-type CpG-ODNs, it is necessary to control such aggregation and obtain a comprehensive understanding of the relationship between their structure and the immune responses. This study revealed that a representative A/D-type CpG ODN, D35, adopted a single-stranded structure in water, while it assembled into aggregates in response to Na+ ions. From polyacrylamide gel electrophoresis and circular dichroism analyses, D35 adopted a homodimeric form (duplex) via palindromic sequences in low-Na+-concentration conditions (10–50 mM NaCl). After replacement of the solution with PBS, quadruplexes began to form in a manner coordinated by Na+, resulting in large aggregates. The duplexes and small aggregates prepared in 50 mM NaCl showed not only high cellular uptake but also high affinity to Toll-like receptor 9 (TLR9) proteins, leading to the production of a large amount of interferon-α for peripheral blood mononuclear cells. The much larger aggregates prepared in 100 mM NaCl were incorporated into cells at a high level, but showed a low ability to induce cytokine production. This suggests that the large aggregates have difficulty inducing TLR9 dimerization, resulting in loss of the stimulation of the cells. We thus succeeded in inducing adequate innate immunity in vitro by controlling and adjusting the formation of D35 aggregates. Therefore, the findings in this study for D35 ODNs could be a vital research foundation for in vivo applications.

A novel RofA-family transcriptional regulator, GadR, controls the development of acid resistance in Listeria monocytogenes.

The ability to survive the acidic conditions found in the stomach is crucial for the food-borne pathogen Listeria monocytogenes to gain access to the mammalian gastrointestinal tract. Little is currently known about how acid resistance is regulated in this pathogen and why this trait is highly variable between strains. Here, we used comparative genomics to identify a novel RofA-family transcriptional regulator, GadR, that controls the development of acid resistance. The RofA family of regulators was previously found only in a small group of bacterial pathogens, including streptococci, where they regulate virulence properties. We show that gadR encodes the dominant regulator of acid resistance in L. monocytogenes and that its sequence variability accounts for previously observed differences between strains in this trait. Together, these findings significantly advance our understanding of how this important pathogen copes with acid stress and suggest a potential molecular target to aid its control in the food chain.

The complete chloroplast genome of Diplodiscus trichospermus and phylogenetic position of Brownlowioideae within Malvaceae

BackgroundMalvaceae is an economically important plant family of 4,225 species in nine subfamilies. Phylogenetic relationships among the nine subfamilies have always been controversial, especially for Brownlowioideae, whose phylogenetic position remains largely unknown due to the lack of samples in previous analysis datasets. To greatly clarify the phylogenetic relationship of Malvaceae, we newly sequenced and assembled the plastome of Diplodiscus trichospermus taxonomically located in Brownlowioideae, and downloaded the allied genomes from public database to build a dataset covering all subfamily members of Malvaceae.ResultsThe annotation results showed that the plastome of Diplodiscus trichospermus has a typical quadripartite structure, comprising 112 unique genes, namely 78 protein-coding genes, 30 tRNA genes and 4 rRNA genes. The total length was 158,570 bp with 37.2% GC content. Based on the maximum likelihood method and Bayesian inference, a robust phylogenetic backbone of Malvaceae was reconstructed. The topology showed that Malvaceae was divided distinctly into two major branches which were previously recognized as Byttneriina and Malvadendrina. In the Malvadendrina clade, Malvoideae and Bombacoideae formed, as always, a close sister clade named as Malvatheca. Subfamily Helicteroideae occupied the most basal position and was followed by Sterculioideae which was sister to the alliance of Malvatheca, Brownlowioideae, Dombeyoideae, and Tilioideae. Brownlowioideae together with the clade comprising Dombeyoideae and Tilioideae formed a sister clade to Malvatheca. In addition, one specific conservation SSR and three specific palindrome sequences were observed in Brownlowioideae.ConclusionsIn this study, the phylogenetic framework of subfamilies in Malvaceae has been resolved clearly based on plastomes, which may contribute to a better understanding of the classification and plastome evolution for Malvaceae.