Dimethylmyricacene: An In Vitro and In Silico Study of a Semisynthetic Non-Camptothecin Derivative Compound, Targeting Human DNA Topoisomerase 1B.

Human topoisomerase 1B regulates the topological state of supercoiled DNA enabling all fundamental cell processes. This enzyme, which is the unique molecular target of the natural anticancer compound camptothecin, acts by nicking one DNA strand and forming a transient protein-DNA covalent complex. The interaction of human topoisomerase 1B and dimethylmyricacene, a compound prepared semisynthetically from myricanol extracted from root bark, was investigated using enzymatic activity assays and molecular docking procedures.

From Antarctica to cancer research: a novel human DNA topoisomerase 1B inhibitor from Antarctic sponge .

Nature has been always a great source of possible lead compounds to develop new drugs against several diseases. Here we report the identification of a natural compound, membranoid G, derived from the Antarctic sponge displaying an inhibitory activity against human DNA topoisomerase 1B. The experiments indicate that membranoid G, when pre-incubated with the enzyme, strongly and irreversibly inhibits the relaxation of supercoiled DNA.

Cow Milk Extracellular Vesicle Effects on an In Vitro Model of Intestinal Inflammation.

Extracellular vesicles (EVs) are lipid bilayer nano-dimensional spherical structures and act mainly as signaling mediators between cells, in particular modulating immunity and inflammation. Milk-derived EVs (mEVs) can have immunomodulatory and anti-inflammatory effects, and milk is one of the most promising food sources of EVs. In this context, this study aimed to evaluate bovine mEVs anti-inflammatory and immunomodulating effects on an in vitro co-culture (Caco-2 and THP-1) model of intestinal inflammation through gene expression evaluation with RT-qPCR and cytokine release through ELISA.

Natural Compounds as Therapeutic Agents: The Case of Human Topoisomerase IB.

Natural products are widely used as source for drugs development. An interesting example is represented by natural drugs developed against human topoisomerase IB, a ubiquitous enzyme involved in many cellular processes where several topological problems occur due the formation of supercoiled DNA. Human topoisomerase IB, involved in the solution of such problems relaxing the DNA cleaving and religating a single DNA strand, represents an important target in anticancer therapy.

Swapping of The N-Terminal Domain of Human Topoisomerase 1B with the Corresponding Counterpart Strongly Impairs Enzyme Activity.

Background: DNA topoisomerases 1B are a class of ubiquitous enzyme that solves the topological problems associated with biological processes such as replication, transcription and recombination. Numerous sequence alignment of topoisomerase 1B from different species shows that the lengths of different domains as well as their amino acids sequences are quite different. In the present study a hybrid enzyme, generated by swapping the N-terminal of into the corresponding domain of the human, has been characterized.

Topoisomerase IB: a relaxing enzyme for stressed DNA.

DNA topoisomerase I enzymes relieve the torsional strain in DNA; they are essential for fundamental molecular processes such as DNA replication, transcription, recombination, and chromosome condensation; and act by cleaving and then religating DNA strands. Over the past few decades, scientists have focused on the DNA topoisomerases biological functions and established a unique role of Type I DNA topoisomerases in regulating gene expression and DNA chromosome condensation. Moreover, the human enzyme is being investigated as a target for cancer chemotherapy.

The human DNA topoisomerase I mutant Gly717Asp: Higher religation rate is not always associated with camptothecin resistance.

DNA topoisomerases are key enzyme responsible for modulating the topological state of the DNA by breaking and rejoining of DNA strand. Characterization of a Gly717Asp mutation in the human topoisomerase was performed using several catalytic assays. The mutant enzyme was shown to have comparable cleavage and fast religation rate as compared to the wild-type protein.

Real-Time Label-Free Direct Electronic Monitoring of Topoisomerase Enzyme Binding Kinetics on Graphene.

Monolayer graphene field-effect sensors operating in liquid have been widely deployed for detecting a range of analyte species often under equilibrium conditions. Here we report on the real-time detection of the binding kinetics of the essential human enzyme, topoisomerase I interacting with substrate molecules (DNA probes) that are immobilized electrochemically on to monolayer graphene strips. By monitoring the field-effect characteristics of the graphene biosensor in real-time during the enzyme-substrate interactions, we are able to decipher the surface binding constant for the cleavage reaction step of topoisomerase I activity in a label-free manner.

Mutation of Gly717Phe in human topoisomerase 1B has an effect on enzymatic function, reactivity to the camptothecin anticancer drug and on the linker domain orientation.

Human topoisomerase 1B controls the topological state of supercoiled DNA allowing the progression of fundamental cellular processes. The enzyme, which is the unique molecular target of the natural anticancer compound camptothecin, acts by cleaving one DNA strand and forming a transient protein-DNA covalent adduct. In this work the role of the Gly717 residue, located in a α-helix structure bridging the active site and the linker domain, has been investigated mutating it in Phe.

Mutations of human DNA topoisomerase I at poly(ADP-ribose) binding sites: modulation of camptothecin activity by ADP-ribose polymers.

Background: DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase I belongs to the family of poly(ADP-ribose)-binding proteins and is the target of camptothecin derived anticancer drugs. Poly(ADP-ribosyl)ation occurs at specific sites of the enzyme inhibiting the cleavage and enhancing the religation steps during the catalytic cycle.

The human topoisomerase 1B Arg634Ala mutation results in camptothecin resistance and loss of inter-domain motion correlation.

Human topoisomerase 1B, the unique target of the natural anticancer compound camptothecin, catalyzes the unwinding of supercoiled DNA by introducing transient single strand nicks and providing covalent protein-DNA adducts. The functional properties and the drug reactivity of the single Arg634Ala mutant have been investigated in comparison to the wild type enzyme. The mutant is characterized by an identical relaxation and cleavage rate but it displays resistance to camptothecin as indicated by a viability assay of the yeast cells transformed with the mutated protein.

Molecular mechanism of the camptothecin resistance of Glu710Gly topoisomerase IB mutant analyzed in vitro and in silico.

Background: DNA topoisomerases are key enzymes that modulate the topological state of DNA through the breaking and rejoining of DNA strands. Human topoisomerase IB can be inhibited by several compounds that act through different mechanisms, including clinically used drugs, such as the derivatives of the natural compound camptothecin that reversibly bind the covalent topoisomerase-DNA complex, slowing down the religation of the cleaved DNA strand, thus inducing cell death. Three enzyme mutations, which confer resistance to irinotecan in an adenocarcinoma cell line, were recently identified but the molecular mechanism of resistance was unclear.

Replacement of the human topoisomerase linker domain with the plasmodial counterpart renders the enzyme camptothecin resistant.

A human/plasmodial hybrid enzyme, generated by swapping the human topoisomerase IB linker domain with the corresponding domain of the Plasmodium falciparum enzyme, has been produced and characterized. The hybrid enzyme displays a relaxation activity comparable to the human enzyme, but it is characterized by a much faster religation rate. The hybrid enzyme is also camptothecin resistant.

A small organic compound enhances the religation reaction of human topoisomerase I and identifies crucial elements for the religation mechanism.

The different steps of the human Top1 (topoisomerase I) catalytic cycle have been analysed in the presence of a pentacyclic-diquinoid synthetic compound. The experiments indicate that it efficiently inhibits the cleavage step of the enzyme reaction, fitting well into the catalytic site. Surprisingly the compound, when incubated with the binary topoisomerase-DNA cleaved complex, helps the enzyme to remove itself from the cleaved DNA and close the DNA gap, increasing the religation rate.

Specific detection of topoisomerase I from the malaria causing P. falciparum parasite using isothermal rolling circle amplification.

We present a Rolling-Circle-Enhance-Enzyme-Activity-Detection (REEAD) system with potential use for future point-of-care diagnosis of malaria. In the developed setup, specific detection of malaria parasites in crude blood samples is facilitated by the conversion of single Plasmodium falciparum topoisomerase I (pfTopI) mediated cleavage-ligation events, happening within nanometer dimensions, to micrometer-sized products readily detectable at the single molecule level in a fluorescence microscope. In principle, REEAD requires no special equipment and the readout is adaptable to simple colorimetric detection systems.

Droplet microfluidics platform for highly sensitive and quantitative detection of malaria-causing Plasmodium parasites based on enzyme activity measurement.

We present an attractive new system for the specific and sensitive detection of the malaria-causing Plasmodium parasites. The system relies on isothermal conversion of single DNA cleavage-ligation events catalyzed specifically by the Plasmodium enzyme topoisomerase I to micrometer-sized products detectable at the single-molecule level. Combined with a droplet microfluidics lab-on-a-chip platform, this design allowed for sensitive, specific, and quantitative detection of all human-malaria-causing Plasmodium species in single drops of unprocessed blood with a detection limit of less than one parasite/μL.

Interaction between natural compounds and human topoisomerase I.

Eukaryotic topoisomerase I (Top1) is a monomeric enzyme that catalyzes the relaxation of supercoiled DNA during important processes including DNA replication, transcription, recombination and chromosome condensation. Human Top1 I is of significant medical interest since it is the unique cellular target of camptothecin (CPT), a plant alkaloid that rapidly blocks both DNA and RNA synthesis. In this review, together with CPT, we point out the interaction between human Top1 and some natural compounds, such us terpenoids, flavonoids, stilbenes and fatty acids.

The need for emergency surgical treatment in carotid-related stroke in evolution and crescendo transient ischemic attack.

Objective: The purpose of this study was to examine the safety of emergency carotid endarterectomy (CEA) in patients with carotid stenosis and unstable neurological symptoms.

Methods: This prospective, single-center study involved patients with stroke in evolution (SIE) or fluctuating stroke or crescendo transient ischemic attack (cTIA) related to a carotid stenosis ≥ 50% who underwent emergency surgery. Preoperative workup included National Institute of Health Stroke Scale (NIHSS) neurological assessment on admission, immediately before surgery and at discharge, carotid duplex scan, brain contrast-enhanced head computed tomography (CT) or magnetic resonance imaging (MRI).

Role of Flexibility in Protein-DNA-Drug Recognition: The Case of Asp677Gly-Val703Ile Topoisomerase Mutant Hypersensitive to Camptothecin.

Topoisomerases I are ubiquitous enzymes that control DNA topology within the cell. They are the unique target of the antitumor drug camptothecin that selectively recognizes the DNA-topoisomerase covalent complex and reversibly stabilizes it. The biochemical and structural-dynamical properties of the Asp677Gly-Val703Ile double mutant with enhanced CPT sensitivity have been investigated.

Urgent carotid endarterectomy to prevent recurrence and improve neurologic outcome in mild-to-moderate acute neurologic events.

Objectives: This study evaluated the safety and benefit of urgent carotid endarterectomy (CEA) in patients with carotid disease and an acute stable neurologic event.

Methods: The study involved patients with acute neurologic impairment, defined as ≥ 4 points on the National Institutes of Health Stroke Scale (NIHSS) evaluation related to a carotid stenosis ≥ 50% who underwent urgent CEA. Preoperative workup included neurologic assessment with the NIHSS on admission or immediately before surgery and at discharge, carotid duplex scanning, transcranial Doppler ultrasound imaging, and head computed tomography or magnetic resonance imaging.

Comparative study on carotid revascularization (endarterectomy vs stenting) using markers of cellular brain injury, neuropsychometric tests, and diffusion-weighted magnetic resonance imaging.

Objective: Subclinical alterations of cerebral function can occur during or after carotid revascularization and can be detected by a variety of standard tests. This comparative study assessed the relationship among serum levels for two biochemical markers of cerebral injury, postoperative diffusion-weighted magnetic resonance imaging (DW-MRI), and neuropsychometric testing in patients undergoing carotid endarterectomy (CEA) or carotid artery stenting (CAS) for high-grade asymptomatic carotid stenosis.

Methods: Forty-three consecutive asymptomatic patients underwent carotid revascularization by endarterectomy (CEA, 20) or stenting (CAS, 23).

Erybraedin C, a natural compound from the plant Bituminaria bituminosa, inhibits both the cleavage and religation activities of human topoisomerase I.

The interaction of human topoisomerase I and erybraedin C, a pterocarpan purified from the plant Bituminaria bituminosa, that was shown to have an antitumour activity, was investigated through enzymatic activity assays and molecular docking procedures. Erybraedin C is able to inhibit both the cleavage and the religation steps of the enzyme reaction. In both cases, pre-incubation of the drug with the enzyme is required to produce a complete inhibition.

Evidence of the crucial role of the linker domain on the catalytic activity of human topoisomerase I by experimental and simulative characterization of the Lys681Ala mutant.

The functional and structural-dynamical properties of the Lys681Ala mutation in the human topoisomerase IB linker domain have been investigated by catalytic assays and molecular dynamics simulation. The mutant is characterized by a comparable cleavage and a strongly reduced religation rate when compared to the wild type protein. The mutant also displays perturbed linker dynamics, as shown by analysis of the principal components of the motion, and a reduced electrostatic interaction with DNA.