Supercoiled DNA Research Articles

DNA supercoiling and regulation of intrinsic β-lactamase in pathogenic Escherichia coli.

This review addresses the involvement of DNA supercoiling in the development of virulence and antibiotic profiles for uropathogenic Escherichia coli and the emergence of new pathotypes such as strain ST131 (serotype O25:H4). The mechanism suggests a role for topoisomerase enzymes and associated mutations in altering the chromosomal supercoiling state and introducing the required DNA twists for expression of intrinsic β-lactamase by ampC and certain virulence factors. In Escherichia coli, constitutive hyperexpression of intrinsic ampC is associated with specific mutations in the promoter and attenuator regions. However, many reports have documented the involvement of slow growth interventions in the expression of intrinsic resistance determinants. There is evidence that a stationary phase transcriptional switch protein, "BolA," is involved in the expression of the intrinsic ampC gene under starvation conditions. The process involves changes in the activity of the enzyme "gyrase," which leads to a change in the chromosomal DNA topology. Consequently, the DNA is relaxed, and the expression of the bolA gene is upregulated. The evolution of the extraintestinal pathogenic E. coli strain ST131 has demonstrated successful adaptability to various stress conditions and conferred compensatory mutations that endowed the microbe with resistance to fluoroquinolones and β-lactams. The results of this study provided new insights into the evidence for the influence of DNA topology in the expression of virulence genes and various determinants of antibiotic resistance (e.g., the intrinsic ampC gene) in Escherichia coli pathotypes.

Macromolecular crowding potently stimulates DNA supercoiling activity of Mycobacterium tuberculosis DNA gyrase

Macromolecular crowding, manifested by high concentrations of proteins and nucleic acids in living cells, significantly influences biological processes such as enzymatic reactions. Studying these reactions in vitro, using agents such as polyethylene glycols (PEGs) and polyvinyl alcohols (PVAs) to mimic intracellular crowding conditions, is essential due to the notable differences from enzyme behaviors observed in diluted aqueous solutions. In this article, we studied Mycobacterium tuberculosis (Mtb) DNA gyrase under macromolecular crowding conditions by incorporating PEGs and PVAs into the DNA supercoiling reactions. We discovered that high concentrations of potassium glutamate, glycine betaine, PEGs, and PVA substantially stimulated the DNA supercoiling activity of Mtb DNA gyrase. Steady-state kinetic studies showed that glycine betaine and PEG400 significantly reduced the KM of Mtb DNA gyrase, and simultaneously increased the Vmax or kcat of Mtb DNA gyrase for ATP and the plasmid DNA molecule. Molecular dynamics simulation studies demonstrated that PEG molecules kept the ATP lid of GyrB in a closed or semi-closed conformation, which prevented ATP molecules from leaving the ATP-binding pocket of GyrB. The stimulation of the DNA supercoiling activity of Mtb DNA gyrase by these molecular crowding agents likely results from a decrease in water activity and an increase in excluded volume.

DNA supercoiling in bacteria: state of play and challenges from a viewpoint of physics based modeling.

DNA supercoiling is central to many fundamental processes of living organisms. Its average level along the chromosome and over time reflects the dynamic equilibrium of opposite activities of topoisomerases, which are required to relax mechanical stresses that are inevitably produced during DNA replication and gene transcription. Supercoiling affects all scales of the spatio-temporal organization of bacterial DNA, from the base pair to the large scale chromosome conformation. Highlighted in vitro and in vivo in the 1960s and 1970s, respectively, the first physical models were proposed concomitantly in order to predict the deformation properties of the double helix. About fifteen years later, polymer physics models demonstrated on larger scales the plectonemic nature and the tree-like organization of supercoiled DNA. Since then, many works have tried to establish a better understanding of the multiple structuring and physiological properties of bacterial DNA in thermodynamic equilibrium and far from equilibrium. The purpose of this essay is to address upcoming challenges by thoroughly exploring the relevance, predictive capacity, and limitations of current physical models, with a specific focus on structural properties beyond the scale of the double helix. We discuss more particularly the problem of DNA conformations, the interplay between DNA supercoiling with gene transcription and DNA replication, its role on nucleoid formation and, finally, the problem of scaling up models. Our primary objective is to foster increased collaboration between physicists and biologists. To achieve this, we have reduced the respective jargon to a minimum and we provide some explanatory background material for the two communities.

Guava processing waste: Biological activity profile of a natural and sustainable source of phenolic antioxidants

Guava (Psidium guajava L.) is a widely-consumed tropical fruit that can be industrially processed and generate several food products. However, about 30% of the total volume of processed guava is lost as processing waste, a homogenous fraction containing peels, seeds, and residual pulp. The valorization of the discard fraction is necessary in upcycling due to its richness of bioactive components. Therefore, the phenolic composition and biological activity of guava's processing waste and its pulp counterpart were investigated. Guava's pulp (22.32 mg gallic acid equivalents (GAE)/g) and waste (21.99 mg GAE/g) contained similar levels of total phenolic content (TPC), with the majority of phenolics occurring in the free and esterified forms. Besides, a strong positive correlation was observed between TPC and the antioxidant activity of extracts. Through HPLC-UV-MS-TOF analysis, 27 phenolic compounds were identified across the pulp (25) and waste (18) portions, with a predominance of gallic, vanillic, and ellagic acids and their derivatives. While free and esterified phenolic fractions showed higher efficiency as α-glucosidase inhibitors (84.94–98.85%), pancreatic lipase was better inhibited by the insoluble-bound phenolics in the extracts (41.55–43.31%). Guava waste's insoluble-bound phenolics strongly protected supercoiled DNA against hydroxyl radicals (80.09%). Oxidation protection by guava phenolics was also observed towards human LDL-cholesterol (10.82–22.98%), an indication of their potential cardioprotective effect. As such, guava processing waste demonstrates nutraceutical potential and should be considered as a value-added ingredient in functional foods and other related applications.

Chromatinization modulates topoisomerase II processivity

Type IIA topoisomerases are essential DNA processing enzymes that must robustly and reliably relax DNA torsional stress. While cellular processes constantly create varying torsional stress, how this variation impacts type IIA topoisomerase function remains obscure. Using multiple single-molecule approaches, we examined the torsional dependence of eukaryotic topoisomerase II (topo II) activity on naked DNA and chromatin. We observed that topo II is ~50-fold more processive on buckled DNA than previously estimated. We further discovered that topo II relaxes supercoiled DNA prior to plectoneme formation, but with processivity reduced by ~100-fold. This relaxation decreases with diminishing torsion, consistent with topo II capturing transient DNA loops. Topo II retains high processivity on buckled chromatin (~10,000 turns) and becomes highly processive even on chromatin under low torsional stress (~1000 turns), consistent with chromatin’s predisposition to readily form DNA crossings. This work establishes that chromatin is a major stimulant of topo II function.

AChE/BuChE inhibitory, DNA nuclease and cytotoxic properties of axially 5-[6-(benzyloxy)-2H-1,3-benzoxazin-3(4H)-yl]pentanoxy and 5-[6-(hexyloxy)-2H-1,3-benzoxazin-3(4H)-yl]pentanoxy substituted silicon phthalocyanines

In this work, axially di-5-[6-(benzyloxy)-2H-1,3-benzoxazin-3(4H)-yl]pentanoxy and 5-[6-(hexyloxy)-2H-1,3-benzoxazin-3(4H)-yl]pentanoxy substituted silicon phthalocyanines were synthesized and characterized for the first time. Then, AChE/BuChE inhibitory, supercoiled plasmid pR322 DNA nuclease and cytotoxic properties of the A1-C5-Si and A2-C5-Si were investigated using different methods. The results showed that A1-C5-Si and A2-C5-Si inhibited AChE and BuChE in a concentration-dependent manner whereas galantamine showed higher inhibitory effects that of the compounds. DNA nuclease experiments showed that both compounds did not damage to plasmid DNA when exposed for 30, 60 and 90 min. Both compounds exhibited a concentration-dependent cytotoxicity against SH-SY5Y cells for 24 and 48 h. The cell viabilities were 63.14±12.63% and 54.95±10.38% in the presence of A1-C5-Si at 50 and 100 µM for 24 h. Moreover, the cell viabilities were found to be 53.07±5.83% and 52.33±3.68% for A1-C5-Si and A2-C5-Si at 100 µM, respectively for 48 h.

"Cholinergic side Effect-Free anticancer drugs: paving the way for safer and more effective cancer treatment"

Cancer is a leading cause of mortality worldwide, and various anticancer medications have been developed that target different biological pathways involved in cancer growth and progression. Topoisomerase 1 (Top1) is an essential enzyme involved in unwinding supercoiled DNA, and it serves as a key target for several anti-cancer drugs. Irinotecan (FDA approved drug), a semi-synthetic camptothecin derivative, is an effective Top1 toxin that eliminates human cancer cells. Cancer patients suffer from the cholinergic syndrome caused by irinotecan and other Top1 inhibitors. Irinotecan-treated patients have developed cholinergic syndrome due to acetylcholinesterase (AChE) enzyme inhibition. It appears that irinotecan or its metabolites directly interact with AChE and inhibit its role of converting acetylcholine to choline, leading to an accumulation of acetylcholine and subsequent symptoms of the cholinergic syndrome. The phytochemicals present in Phyllanthus emblica, commonly referred to as amla, have been studied to determine their therapeutic effects. As an alternative treatment for cancer, this study explores the potential of phytochemicals found in amla to target and inhibit the Top1 protein. Additionally, the study aims to identify a non-inhibitor for AChE. Molecular docking studies assessed phytochemical binding affinities to Top1 and AChE enzymes, and ADME analyses were performed to assess their drug-likeness properties. Subsequently, molecular dynamic simulation was employed to assess the stability of these compounds. The results suggest that new anticancer medications that do not inhibit AChE or fresh Top1 inhibitors that use the camptothecin scaffold may alleviate some of the irinotecan’s side effects. Communicated by Ramaswamy H. Sarma

Transcriptional repression by a secondary DNA binding surface of DNA topoisomerase I safeguards against hypertranscription

Regulation of global transcription output is important for normal development and disease, but little is known about the mechanisms involved. DNA topoisomerase I (TOP1) is an enzyme well-known for its role in relieving DNA supercoils for enabling transcription. Here, we report a non-enzymatic function of TOP1 that downregulates RNA synthesis. This function is dependent on specific DNA-interacting residues located on a conserved protein surface. A loss-of-function knock-in mutation on this surface, R548Q, is sufficient to cause hypertranscription and alter differentiation outcomes in mouse embryonic stem cells (mESCs). Hypertranscription in mESCs is accompanied by reduced TOP1 chromatin binding and change in genomic supercoiling. Notably, the mutation does not impact TOP1 enzymatic activity; rather, it diminishes TOP1-DNA binding and formation of compact protein-DNA structures. Thus, TOP1 exhibits opposing influences on transcription through distinct activities which are likely to be coordinated. This highlights TOP1 as a safeguard of appropriate total transcription levels in cells.

Synthetic lethal mutants in Escherichia coli define pathways necessary for survival with RNase H deficiency.

Ribonucleotides frequently contaminate DNA and, if not removed, cause genomic instability. Consequently, all organisms are equipped with RNase H enzymes to remove RNA-DNA hybrids (RDHs). Escherichia coli lacking RNase HI (rnhA) and RNase HII (rnhB) enzymes, the ∆rnhA ∆rnhB double mutant, accumulates RDHs in its DNA. These RDHs can convert into RNA-containing DNA lesions (R-lesions) of unclear nature that compromise genomic stability. The ∆rnhAB double mutant has severe phenotypes, like growth inhibition, replication stress, sensitivity to ultraviolet radiation, SOS induction, increased chromosomal fragmentation, and defects in nucleoid organization. In this study, we found that RNase HI deficiency also alters wild-type levels of DNA supercoiling. Despite these severe chromosomal complications, ∆rnhAB double mutant survives, suggesting that dedicated pathways operate to avoid or repair R-lesions. To identify these pathways, we systematically searched for mutants synthetic lethal (colethal) with the rnhAB defect using an unbiased color screen and a candidate gene approach. We identified both novel and previously reported rnhAB-colethal and -coinhibited mutants, characterized them, and sorted them into avoidance or repair pathways. These mutants operate in various parts of nucleic acid metabolism, including replication fork progression, R-loop prevention and removal, nucleoid organization, tRNA modification, recombinational repair, and chromosome-dimer resolution, demonstrating the pleiotropic nature of RNase H deficiency. IMPORTANCE Ribonucleotides (rNs) are structurally very similar to deoxyribonucleotides. Consequently, rN contamination of DNA is common and pervasive across all domains of life. Failure to remove rNs from DNA has severe consequences, and all organisms are equipped with RNase H enzymes to remove RNA-DNA hybrids. RNase H deficiency leads to complications in bacteria, yeast, and mouse, and diseases like progressive external ophthalmoplegia (mitochondrial defects in RNASEH1) and Aicardi-Goutières syndrome (defects in RNASEH2) in humans. Escherichia coli ∆rnhAB mutant, deficient in RNases H, has severe chromosomal complications. Despite substantial problems, nearly half of the mutant population survives. We have identified novel and previously confirmed pathways in various parts of nucleic acid metabolism that ensure survival with RNase H deficiency.

The Impact of Substitutions at Positions 1 and 8 of Fluoroquinolones on the Activity Against Mutant DNA Gyrases of Salmonella Typhimurium.

Although many drug-resistant nontyphoidal Salmonella (NTS) infections are reported globally, their treatment is challenging owing to the ineffectiveness of the currently available antimicrobial drugs against resistant bacteria. It is therefore essential to discover novel antimicrobial drugs for the management of these infections. In this study, we report high inhibitory activities of the novel fluoroquinolones (FQs; WQ-3810 and WQ-3334) with substitutions at positions R-1 by 6-amino-3,5-difluoropyridine-2-yl and R-8 by methyl group or bromine, respectively, against wild-type and mutant DNA gyrases of Salmonella Typhimurium. The inhibitory activities of these FQs were assessed against seven amino acid substitutions in DNA gyrases conferring FQ resistance to S. Typhimurium, including high-level resistant mutants, Ser83Ile and Ser83Phe-Asp87Asn by in vitro DNA supercoiling assay. Drug concentrations of WQ compounds with 6-amino-3,5-difluoropyridine-2-yl that suppressed DNA supercoiling by 50% (IC50) were found to be ∼150-fold lower than ciprofloxacin against DNA gyrase with double amino acid substitutions. Our findings highlight the importance of the chemical structure of an FQ drug on its antimicrobial activity. Particularly, the presence of 6-amino-3,5-difluoropyridine-2-yl at R-1 and either methyl group or bromine at R-8 of WQ-3810 and WQ-3334, respectively, was associated with improved antimicrobial activity. Therefore, WQ-3810 and WQ-3334 are promising candidates for use in the treatment of patients infected by FQ-resistant Salmonella spp.

Negative DNA supercoiling induces genome-wide Cas9 off-target activity

CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology invivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate invivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology invitro and invivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.

To Break or Not to Break: The Role of TOP2B in Transcription.

Transcription and its regulation pose challenges related to DNA torsion and supercoiling of the DNA template. RNA polymerase tracking the helical groove of the DNA introduces positive helical torsion and supercoiling upstream and negative torsion and supercoiling behind its direction of travel. This can inhibit transcriptional elongation and other processes essential to transcription. In addition, chromatin remodeling associated with gene activation can generate or be hindered by excess DNA torsional stress in gene regulatory regions. These topological challenges are solved by DNA topoisomerases via a strand-passage reaction which involves transiently breaking and re-joining of one (type I topoisomerases) or both (type II topoisomerases) strands of the phosphodiester backbone. This review will focus on one of the two mammalian type II DNA topoisomerase enzymes, DNA topoisomerase II beta (TOP2B), that have been implicated in correct execution of developmental transcriptional programs and in signal-induced transcription, including transcriptional activation by nuclear hormone ligands. Surprisingly, several lines of evidence indicate that TOP2B-mediated protein-free DNA double-strand breaks are involved in signal-induced transcription. We discuss the possible significance and origins of these DSBs along with a network of protein interaction data supporting a variety of roles for TOP2B in transcriptional regulation.

A Molecular Approach to Characterize the Effects of Fluence, Fluence Rate and LET on Radiation Damage

Effects of fluence, fluence rate and LET on radiation damage are not resolved using traditional methods of measuring energy deposited by ionization events. The deficiency led to the use of empirical RBE factors in the clinical applications of particle therapy. The use of ionization dosimetry is similarly challenged when applied to development of radiation treatment at ultrahigh (FLASH) dose rate. This study reports a molecular method using plasmid DNA as a more comprehensive model for radiation dosimetry than ionization measurements. Aqueous solutions of purified supercoiled plasmid DNA, pUC19 (2686 bp), were prepared at different scavenging conditions and injected into 5x5x1 mm3 wells as detector elements. Irradiated samples were analyzed using base excision repair enzymes (Nth and Fpg) and gel electrophoresis to measure yields for DNA single and double strand breaks (SSB and DSB), and clustered lesions. The low LET characteristics of conventional radiation treatment was modeled using orthovoltage 150 kVp x-rays to deliver 2-110 Gy at 90 and 0.5 Gy/s. Higher LET irradiations in the range of 2 - 14 keV/μm were facilitated by measurements in the pristine Bragg peak region using synchrotron-produced 142.2 MeV protons to deliver dose at 2 - 160 Gy at 600 and 1 Gy/s. The DNA wells were inserted into a solid water equivalent phantom for proton irradiation. Only 4 wells could be positioned in the short Bragg peak region in water (∼ 2 cm). To alleviate the uncertainties due to rapidly varying dose and LET distributions, we innovated the use of a 3% water density (i.e., Styrofoam) medium to extend the Bragg peak region from 2 cm to 20 cm, enabling the placement of 20 well containers. Quantity and quality of molecular damage in the plasmid DNA model varies with fluence, fluence rate and LET of radiation. At high fluence (> 30 Gy) of low-LET radiations, the yields of DNA SSB and non-DSB clustered lesions depend on the fluence rate. These yields decrease by two times between ultrahigh and conventional dose rate irradiation. At a given fluence and fluence rate, the yields for the formation of DNA DSB and non-DSB clustered lesions increase linearly with LET. The low-density phantom allows significant (∼ 10 folds) increase in the number of sampling points and more accurate sample positioning at specific LET compared to water-equivalent phantom. Monte-Carlo track structure simulation of yields for different DNA lesions is being developed to model the molecular damage. In parallel, approaches to improve the sensitivity of the measurements to dose are being investigated. Our results suggest that a molecular-based approach can be used to differentiate the effects of fluence, fluence rate, and LET on radiation damage. The approach demonstrates the potential to improve on the modeling of radiation effects in biological systems than using measured ionization energy. Correlation of the molecular changes to biological outcome for in vitro and in vivo systems are under investigation.

Characterization of Rheum palmatum mitochondrial genome and comparative analysis among Caryophyllales species

Objective: This work aimed to report the first complete mitochondrial genome (mitogenome) of Rheum palmatum, summarize the features of Caryophyllales mitogenomes, and to reveal the potential of utilizing the mitogenomes of R. palmatum and other Caryophyllales species for inferring phylogenetic relationships and species identification. Methods: Both Illumina short reads and PacBio HiFi reads were utilized to obtain a complete mitogenome of R. palmatum. A variety of bioinformatics tools were employed to characterize the R. palmatum mitogenome, compare the reported mitogenomes in Caryophyllales and conduct phylogenetic analysis. Results: The mitogenome of R. palmatum was assembled into a single master circle of 302,993 bp, encoding 35 known protein-coding genes, 18 transfer RNA genes, and three ribosome RNA genes. A total of 249 long repeats and 49 simple sequence repeats were identified in this mitogenome. The sizes of mitogenomes in Caryophyllales varied from 253 kb to 11.3 Mb. Among them, 23 mitogenomes were circular molecules, one was linear, and one consisted of relaxed circles, linear molecules, and supercoiled DNA. Out of the total mitogenomes, 11 were single-chromosome structure, whereas the remaining 14 were multi-chromosomal organizations. The phylogenetic analysis is consistent with both the Engler system (1964) and the Angiosperm Phylogeny Group III system. Conclusions: We obtained the first mitogenome of R. palmatum, which consists of a master circle. Mitogenomes in Caryophyllales have variable genome sizes and structures even within the same species. Circular molecules are still the dominant pattern in Caryophyllales. Single-chromosome mitogenomes account for nearly a half of all the mitogenomes in Caryophyllales, in contrast to previous studies. It is feasible to utilize mitochondrial genomes for inferring phylogenetic relationships and conducting species identification.

Impact of R-loops on oncogene-induced replication stress in cancer cells.

Replication stress is an alteration in the progression of replication forks caused by a variety of events of endogenous or exogenous origin. In precancerous lesions, this stress is exacerbated by the deregulation of oncogenic pathways, which notably disrupts the coordination between replication and transcription, and leads to genetic instability and cancer development. It is now well established that transcription can interfere with genome replication in different ways, such as head-on collisions between polymerases, accumulation of positive DNA supercoils or formation of R-loops. These structures form during transcription when nascent RNA reanneals with DNA behind the RNA polymerase, forming a stable DNA:RNA hybrid. In this review, we discuss how these different cotranscriptional processes disrupt the progression of replication forks and how they contribute to genetic instability in cancer cells.

Phytochemical analysis and antibacterial activity of traditional plants for the inhibition of DNA gyrase

Introduction and Aim: DNA gyrase is a class of Type II Topoisomerases that plays an important role in bacterial viability. It is found in all bacteria and is involved in replication, repair, recombination, and DNA transcription. Negative supercoiling of bacterial DNA by DNA gyr B is essential in replication which further influences all the metabolic activities. Staphylococcus aureus (ATCC 25923) is one of the pathogens that can modify its genome easily under multidrug resistance. In this study, the activity of medicinal compounds to inhibit DNA gyrB is explored. Plant species Solanum nigrum, Vitex negundo, and Euphorbia hirta were studied for the potential plant-based molecules. The compounds alkaloids, glycosides, flavonoids, and terpenoids were considered to have high-potential targets. The study focuses on DNA gyrase as a target and shows insights into future drug development. The research focuses on the discovery of novel plant-based therapeutic compounds to target DNA gyrase B activity. Methods and Materials: Phytochemical screening was performed to study the medication options that could inhibit DNA gyrB. Phytochemicals were determined using GC-MS. Results: Utilizing GC MS and FT-IR analysis, the phytochemical constituents of Solanum nigrum, Vitex negundo, and Euphorbia hirta were discovered. It will be simpler to do a follow-up study on discovering bioactive compounds and evaluating their effectiveness in inhibiting DNA gyrB with the help of this preliminary data from the analytical procedures. Conclusion: There are countless applications for the phytochemicals that medicinal plants produce. Staphylococcus aureus will be stopped by DNA gyrB inhibition. The study employs DNA gyrase as its target and provides information on potential therapeutic targets. The goal of the study is to identify innovative plant-based medicinal molecules that specifically target DNA gyrase B activity.

Synthesis, anticancer, antibacterial, antifungal, DNA interactions, ADMET, molecular docking, and antioxidant evaluation of novel Schiff base and their Co(II), Ni(II) and Cu(II) complexes

Here, three bivalent transition metal complexes of [ML2] kind (where M = Co(II), Ni(II), Cu(II); HL = 2-(E)-(5-cyclohexyl-2-methoxyphenylimino)methyl)-5-methylphenol), and various analytical techniques such as elemental analysis, mass, IR, NMR, UV–visible, TGA, magnetic moments, and ESR were employed for characterization. From the analytical data it is found that the Ni (II) and Cu (II) complexes adopted a square planar geometry, while the Co (II) complex adopted an octahedral geometry. Molecular docking investigations were carried out of Schiff base ligand (HL), the results illustrated that the ligand exhibited significant affinities for a few proteins. Monometallic metal compounds interaction with CT-DNA was studied by fluorescence measurements, viscosity and UV–Vis investigations, which is illustrated that all the three metal compounds strongly intercalated to CT-DNA. The synthesized metal compounds (1a-1c) with supercoiled pBR322 DNA was used in cleavage experiments. Based on findings, DNA cleavage by the Cu(II) complexes was more effective than that of the Co(II) and Ni(II) compounds. All synthesised compounds toxic effects on cancer cell lines A-549 and MCF-7 were evaluated by MTT assay. The obtained results indicated that Cu(II) complex is more effective towards tumour cells than other complexes. In addition to the evaluation of all synthesised complexes for antimicrobial properties, it was observed that all of the metal complexes showed higher efficacy against microorganisms compared to the free ligand. Finally, drug likeness and ADMET studies was performed on Schiff base ligand (HL). These studies indicated that the ligand possessed high potency and showed potential as a drug agent for pathogens causing deformities.

Combination of bioaffinity ultrafiltration-UFLC-ESI-Q/TOF-MS/MS, in silico docking and multiple complex networks to explore antitumor mechanism of topoisomerase I inhibitors from Artemisiae Scopariae Herba

BackgroundArtemisiae Scopariae Herba (ASH) has been widely used as plant medicine in East Asia with remarkable antitumor activity. However, the underlying mechanisms have not been fully elucidated.MethodsThis study aimed to construct a multi-disciplinary approach to screen topoisomerase I (topo I) inhibitors from ASH extract, and explore the antitumor mechanisms. Bioaffinity ultrafiltration-UFLC-ESI-Q/TOF-MS/MS was used to identify chemical constitution of ASH extract as well as the topo I inhibitors, and in silico docking coupled with multiple complex networks was applied to interpret the molecular mechanisms.ResultsCrude ASH extract exhibited toxicogenetic and antiproliferative activities on A549 cells. A series of 34 ingredients were identified from the extract, and 6 compounds were screened as potential topo I inhibitors. Docking results showed that the formation of hydrogen bond and π-π stacking contributed most to their binding with topo I. Interrelationships among the 6 compounds, related targets and pathways were analyzed by multiple complex networks model. These networks displayed power-law degree distribution and small-world property. Statistical analysis indicated that isorhamnetin and quercetin were main active ingredients, and that chemical carcinogenesis-reactive oxygen species was the critical pathway. Electrophoretic results showed a therapeutic effect of ASH extract on the conversion of supercoiled DNA to relaxed forms, as well as potential synergistic effect of isorhamnetin and quercetin.ConclusionsThe results improved current understanding of Artemisiae Scopariae Herba on the treatment of tumor. Moreover, the combination of multi-disciplinary methods provided a new strategy for the study of bioactive constituents in medicinal plants.

Open Questions about the Roles of DnaA, Related Proteins, and Hyperstructure Dynamics in the Cell Cycle.

The DnaA protein has long been considered to play the key role in the initiation of chromosome replication in modern bacteria. Many questions about this role, however, remain unanswered. Here, we raise these questions within a framework based on the dynamics of hyperstructures, alias large assemblies of molecules and macromolecules that perform a function. In these dynamics, hyperstructures can (1) emit and receive signals or (2) fuse and separate from one another. We ask whether the DnaA-based initiation hyperstructure acts as a logic gate receiving information from the membrane, the chromosome, and metabolism to trigger replication; we try to phrase some of these questions in terms of DNA supercoiling, strand opening, glycolytic enzymes, SeqA, ribonucleotide reductase, the macromolecular synthesis operon, post-translational modifications, and metabolic pools. Finally, we ask whether, underpinning the regulation of the cell cycle, there is a physico-chemical clock inherited from the first protocells, and whether this clock emits a single signal that triggers both chromosome replication and cell division.

Computational evaluation of phytochemicals targeting DNA topoisomerase I in Leishmania donovani: molecular docking and molecular dynamics simulation studies

DNA topoisomerase I (Topo I) is a ubiquitous enzyme that plays a crucial role in resolving the topological constraints of supercoiled DNA during various cellular activities, including repair, replication, recombination, transcription, and chromatin remodeling. Multiple studies have confirmed the essential role of Topo I in nucleic acid metabolism of Leishmania donovani, the kinetoplastid parasite responsible for visceral leishmaniasis or kala-azar. Inhibition of this enzyme has shown promise as a strategy for therapy against visceral leishmaniasis. However, current treatment options suffer from limitations related to effectiveness, cost, and side effects. To address these challenges, computational methods have been employed in this study to investigate the inhibition of Leishmania donovani DNA topoisomerase I (LdTopo I) by phytochemicals derived from Indian medicinal plants known for their anti-leishmanial activity. A library of phytochemicals and known inhibitors was assembled, and virtual screening based on docking binding affinities was conducted to identify potent phytochemical inhibitors. To assess the drug-likeness of the docked phytochemicals, their physicochemical properties were predicted. Additionally, molecular dynamics (MD) simulations were performed on the docked complexes for a duration of 100 ns to evaluate their stability, intermolecular interactions, and dynamic behavior. Among all the docked phytochemicals, three compounds, namely CID23266147 (withanolide N), CID5488537 (fagopyrine), and CID100947536 (isozeylanone), exhibited the highest inhibitory potential against LdTopo I. These findings hold promise for the development of novel inhibitors targeting LdTopo I, which could potentially lead to improved therapies for visceral leishmaniasis. Communicated by Ramaswamy H. Sarma