pUC19 Plasmid Research Articles

Antimicrobial dynamics and DNA nicking within 1,3-propanediaminium dibromide and dichloride supramolecular frameworks: The critical role of strong hydrogen bonding and subtle molecular interactions

Amine salt compounds are attracting increased attention due to their potential bioactive properties. This study explores the synthesis and detailed characterization of two novel amine salts, 1,3-propanediaminium dibromide (13DPBr) and 1,3-propanediaminium dichloride (13DPCl), through single-crystal X-ray diffraction, Hirshfeld surface analysis, and optical absorption spectroscopy. Structurally, 13DPBr crystallizes in the orthorhombic space group Pbcn, while 13DPCl crystallizes in the monoclinic space group P2/n. Supramolecular assemblies in both compounds are driven by strong N–H···X (X = Cl, Br) hydrogen bonds, alongside weaker C–H···X interactions. The optical absorption spectra indicated band gaps of 4.32 eV for 13DPBr and 4.56 eV for 13DPCl. The biological activities of these compounds were assessed through DNA nicking assays and antimicrobial tests. The antimicrobial properties were evaluated using agar diffusion assays against pathogenic bacterial strains, including Salmonella enterica, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The Minimum Inhibitory Concentration (MIC) results demonstrated that both salts exhibited strong antibacterial activity, with 13DPBr showing higher efficacy against Pseudomonas aeruginosa. DNA nicking assays using pUC19 plasmid DNA revealed that both compounds offered significant protection against hydroxyl radical-induced DNA damage, preventing the degradation of plasmid DNA. These results highlight the potential of 13DPBr and 13DPCl as promising agents for antimicrobial and DNA protection applications.

In vitro and in silico studies of a Zn(II) complex as a potential therapeutic agent for breast cancer.

Breast cancer (BC) is one of the most life-threatening diseases of women's health worldwide. This work was conducted to assess the anti-BC potency of a new Zn(II)-based complex. The Zn(II) complex coordinated to dimethoxy-substituted bipyridine was synthesized and its molecular structure was elucidated as [Zn(2Meobpy)3](clo4)2 (2Meobpy-Zn) by single-crystal X-ray diffraction, 2Meobpy represents 4,4'-dimethoxy-2,2'-bipyridine. The cytotoxicity results indicated that 2Meobpy-Zn, unlike cisplatin, acts potently and selectively on the human breast cancer cells (MCF-7) compared to normal murine embryo cells (NIH/3T3) by IC50 value of 4.6 ± 0.5µm and selectivity index (SI) of 2.0 over 48h. 2Meobpy-Zn and cisplatin showed anti-metastatic activity as evidenced by inhibition of the colony formation and cell migration. The flow cytometric assessment of MCF-7 cells supported that 2Meobpy-Zn and cisplatin exert their cytotoxic effect through the apoptotic pathway. Moreover, 2Meobpy-Zn could induce overproduction of intracellular reactive oxygen species (ROS) in MCF-7 cells. The apoptotic mechanism in 2Meobpy-Zn-treated MCF-7 cells is probably related to the regulation of apoptosis-relevant genes expression, including BAX and BCL2. Moreover, 2Meobpy-Zn is able to cleave pUC19 plasmid DNA through the hydrolytic reaction pathway. Finally, 2Meobpy-Zn's affinity towards antiapoptosis-related proteins, as a potential apoptosis inducer, as well as breast cancer-relevant proteins, as a potential anti-BC agent, was evaluated by in silico molecular docking studies. Altogether, the results of this work strongly evidenced that 2Meobpy-Zn can be the subject of experimental validation and clinical trials to introduce this complex as a promising BC therapeutic agent.

Degradation of extracellular antibiotic resistance genes using peracetic acid (PAA) and performic acid (PFA)

Antibiotic resistant bacteria (ARB) associated infections are identified as a major threat to human health in the 21st century. Wastewater contains antibiotic resistance determinants and they are discharged to aquatic environments with treated or untreated effluents. These antibiotic resistance determinants can be taken up by environmental bacteria via horizontal gene transfer (HGT) and they can become antibiotic resistant. Among antibiotic resistance determinants, extracellular DNA containing antibiotic resistance genes can be up taken by environmental bacteria via natural transformation. Therefore, we evaluated the suitability of low-cost disinfectants peracetic acid (PAA) and performic acid (PFA) on inactivating total and functional extracellular ARGs in ultrapure water and secondary wastewater effluent matrices using pUC19 as a model ARG. Performance of PAA and PFA in inactivating extracellular ARGs was compared with the performance of free chlorine disinfection. RT-qPCR results showed that inactivation of ampR gene in pUC19 plasmid suspended in ultrapure water and wastewater secondary effluent by free chlorine was higher compared to PAA and PFA. However, the reduction of functional pUC19 by PAA and PFA were comparable to free chlorine. Transformation frequency of pUC19 treated by PAA and PFA achieved the maximum possible reduction within a shorter time compared to free chlorine treatment, highlighting the possibility of having disinfection chambers with smaller footprints in wastewater treatment plants. These results suggest the suitability of PAA and PFA for inactivation of ARGs in aquatic samples as alternative disinfectants to free chlorine.

Unveiling the role of uranium in enhancing the transformation of antibiotic resistance genes

Transformation represents one of the most important pathways for the horizontal transfer of antibiotic resistance genes (ARGs), which enables competent bacteria to acquire extracellular ARGs from the surrounding environment. Both heavy metals and irradiation have been demonstrated to influence the bacterial transformation process. However, the impact of ubiquitously occurring radioactive heavy metals on the transformation of ARGs remains largely unknown. Here, we showed that a representative radioactive nuclide, uranium (U), at environmental concentrations (0.005–5 mg/L), improved the transformation frequency of resistant plasmid pUC19 into Escherichia coli by 0.10–0.85-fold in a concentration-dependent manner. The enhanced ARGs transformation ability under U stress was demonstrated to be associated with reactive oxygen species (ROS) overproduction, membrane damage, and up-regulation of genes related to DNA uptake and recombination. Membrane permeability and ROS production were the predominant direct and indirect factors affecting transformation ability, respectively. Our findings provide valuable insight into the underlying mechanisms of the impacts of U on the ARGs transformation process and highlight concerns about the exacerbated spread of ARGs in radioactive heavy metal-contaminated ecosystems, especially in areas with nuclear activity or accidents.

Assessment of cytoprotective and genoprotective effects of Moringa oleifera and Tinospora cordifolia extracts in vitro.

Moringa oleifera and Tinospora cordifolia is extensively used as an ingredient of food and in traditional medicine for the management of a variety of diseases. The extracts of leaf of Moringa oleifera and stem of Tinospora cordifolia were assessed to examine their ability to inhibit the oxidative DNA damage (by DNA protection assay), cytoprotective and genoprotective potential (by Comet assay) in V79 cells individually and in combinations. It was found that these extracts could significantly inhibit the OH-dependent damage of pUC18 plasmid DNA. M. oleifera extract (160 and 320μg/mL) and Tinospora cordifolia extract (640, 1,280 and 2,560μg/mL) individually showed higher DNA protection activity. M. oleifera (1,280μg/mL) combined with Tinospora cordifolia (640μg/mL) showed best cytoprotective and genoprotective activities among different concentration combinations and various concentrations of individual plants in V79 cell line against hydrogen peroxide induced cytotoxicity and genotoxicity. This study demonstrates the cytoprotective and genoprotective activity of M. oleifera and Tinospora cordifolia individually or in combination.

Novel antipyrine substituted 4-thiazolidinones: Synthesis, DNA binding and topoisomerase inhibition activities, and in-silico studies

In this study, we report the synthesis and characterization of novel 4-thiazolidinones containing antipyrine (3a-e) derived from the starting compounds of Schiff bases (2a-e). The Schiff bases were synthesized using aldehydes of natural origin, such as citronellal, cuminaldehyde, 2-thiazolecarboxaldehyde, 5-methylfurfural, and syringaldehyde. Their FTIR spectra revealed the enol- tautomeric solid-state structures of compounds 3b and 3d. The pUC18 plasmid DNA binding specificity and capacity of the compounds to catalytically generate topoisomerase I activity were also examined using agarose gel electrophoresis. The compounds induced changes in DNA mobility and showed a topoisomerase I inhibitory effect at all doses. 4-Thiazolidinones had a higher affinity in DNA and topoisomerase I than the Schiff bases. DNA binding and topoisomerase I activities were most effective at low concentration (6 μM) for the compounds of Schiff bases 2a, d, and 4-thiazolidinones 3a, d. Furthermore, this study presents computational methods for addressing the molecules’ drug-like properties. 2a, d, and 3a, d Compounds were used as representative of the most bioactive among the newly synthesized molecules in in-silico studies. The web tools of ADMETlab, SwissADME, OSIRIS, and the BOILED-Egg method were used to conduct in-silico biological studies involving ADME prediction, BBB penetration, gastrointestinal absorption, and toxicity studies. The ADME profile of the query compounds gets within the range of applicability and is at an appropriate level. In-silico studies revealed that compounds 2a, d, and 3a, d are neither hERG blockers nor AMES toxic. However, compounds 2a and 3a have hepatotoxicity.

Comparison of mutation spectra induced by gamma-rays and carbon ion beams.

The ionizing radiation with high linear energy transfer (LET), such as a heavy ion beam, induces more serious biological effects than low LET ones, such as gamma- and X-rays. This indicates a difference in the DNA damage produced by low and high LET radiations and their biological effects. We have been studying the differences in DNA damage produced by gamma-rays and carbon ion beams. Therefore, we analyze mutations induced by both ionizing radiations to discuss the differences in their biological effects in this study. pUC19 plasmid DNA was irradiated by carbon ion beams in the solution containing 1M dimethyl sulfoxide to mimic a cellular condition. The irradiated DNA was cloned in competent cells of Escherichia coli. The clones harboring some mutations in the region of lacZα were selected, and the sequence alterations were analyzed. A one-deletion mutation is significant in the carbon-irradiated DNA, and the C:G↔T:A transition is minor. On the other hand, the gamma-irradiated DNA shows mainly G:C↔T:A transversion. These results suggest that carbon ion beams produce complex DNA damage, and gamma-rays are prone to single oxidative base damage, such as 8-oxoguanine. Carbon ion beams can also introduce oxidative base damage, and the damage species is 5-hydroxycytosine. This was consistent with our previous results of DNA damage caused by heavy ion beams. We confirmed the causal DNA damage by mass spectrometry for these mutations.

Influence of alkylthio and arylthio derivatives of tert-butylquinone on the induction of DNA damage in a human hepatocellular carcinoma cell line (HepG2)

The aim of this study was to investigate the effects of tert-butylquinone (TBQ) and its alkylthio and arylthio derivatives on DNA in vitro, using acellular and cellular test systems. Direct interaction with DNA was studied using the plasmid pUC19. Cytotoxic (MTS assay) and genotoxic (comet assay and γH2AX focus assays) effects, and their influence on the cell cycle were studied in the HepG2 cell line. Our results show that TBQ and its derivatives did not directly interact with DNA. The strongest cytotoxic effect on the HepG2 cells was observed for the derivative 2-tert-butyl-5,6-(ethylenedithio)-1,4-benzoquinone (IC50 64.68 and 55.64 μM at 24-h and 48-h treatment, respectively). The tested derivatives did not significantly influence the cell cycle distribution in the exposed cellular populations. However, all derivatives showed a genotoxic activity stronger than that of TBQ in the comet assay, with 2-tert-butyl-5,6-(ethylenedithio)-1,4-benzoquinone producing the strongest effect. The same derivative also induced DNA double-strand breaks in the γH2AX focus assay.

Role of cspA on the Preparation of Escherichia coliCompetent Cells by Calcium Chloride Method.

One of the fundamental techniques in genetic engineering is the creation of Escherichia colicompetent cells using the CaCl2 method. However, little is known about the mechanism of E. coli competence formation. We have previously found that the cspA gene may play an indispensable role in the preparation of E. coli DH5αcompetent cells through multiomics analysis. In the present study, the cellular localization, physicochemical properties, and function of the protein expressed by the cspA gene were analyzed. To investigate the role of the cspA gene in E. coli transformation, cspA-deficient mutant was constructed by red homologous recombination. The growth, transformation efficiency, and cell morphology of the cspA-deficient strain and E. coli were compared. It was found that there were no noticeable differences in growth and morphology between E. coli and the cspA-deficient strain cultured at 37°C, but the mutant exhibited increased transformation efficiencies compared to E. coli DH5α for plasmids pUC19, pET-32a, and p1304, with enhancements of 2.23, 2.24,and 3.46 times, respectively. It was proved that cspA gene is an important negative regulatory gene in the CaCl2 preparation of competent cells.

Photoaged nanoplastics with multienzyme-like activities significantly shape the horizontal transfer of antibiotic resistance genes

Nanoplastics (NPs), identified as emerging pollutants, pose a great risk to environment and global public health, exerting profound influences on the prevalence and dissemination of antibiotic resistance genes (ARGs). Despite evidence suggesting that nano-sized plastic particles can facilitate the horizontal gene transfer (HGT) of ARGs, it is imperative to explore strategies for inhibiting the transfer of ARGs. Currently, limited information exists regarding the characteristics of environmentally aged NPs and their impact on ARGs propagation. Herein, we investigated the impact of photo-aged NPs on the transfer of ARG-carrying plasmids into Escherichia coli (E. coli) cells. Following simulated sunlight irradiation, photo-aged nano-sized polystyrene plastics (PS NPs) exhibited multiple enzyme-like activities, including peroxidase (POD) and oxidase (OXD), leading to a burst of reactive oxygen species (ROS). At relatively low concentrations (0.1, 1 μg/mL), both pristine and aged PS NPs facilitated the transfer of pUC19 and pHSG396 plasmids within E. coli due to moderate ROS production and enhanced cell membrane permeability. Intriguingly, at relatively high concentrations (5, 10 μg/mL), aged PS NPs significantly suppressed plasmids transformation. The non-unidirectional impact of aged PS NPs involved the overproduction of ROS (•OH and •O2−) via nanozyme activity, directly degrading ARGs and damaging plasmid structure. Additionally, oxidative damage to bacteria resulted from the presence of much toxic free radicals, causing physical damage to cell membranes, reduction of the SOS response and restriction of adenosine-triphosphate (ATP) supply, ultimately leading to inactivation of recipient cells. This study unveils the intrinsic multienzyme-like activity of environmentally aged NPs, highlighting their potential to impede the transfer and dissemination of ARGs.

Ru(II) Complexes with Absorption in the Photodynamic Therapy Window: 1O2 Sensitization, DNA Binding, and Plasmid DNA Photocleavage.

Two Ru(II) complexes, [Ru(pydppn)(bim)(py)]2+ [2; pydppn = 3-(pyrid-2'-yl)-4,5,9,16-tetraaza-dibenzo[a,c]naphthacene; bim = 2,2'-bisimidazole; py = pyridine] and [Ru(pydppn)(Me4bim)(py)]2+ [3; Me4bim = 2,2'-bis(4,5-dimethylimidazole)], were synthesized and characterized, and their photophysical properties, DNA binding, and photocleavage were evaluated and compared to [Ru(pydppn)(bpy)(py)]2+ (1; bpy = 2,2'-bipyridine). Complexes 2 and 3 exhibit broad 1MLCT (metal-to-ligand charge transfer) transitions with maxima at ∼470 nm and shoulders at ∼525 and ∼600 nm that extend to ∼800 nm. These bands are red-shifted relative to those of 1, attributed to the π-donating ability of the bim and Me4bim ligands. A strong signal at 550 nm is observed in the transient absorption spectra of 1-3, previously assigned as arising from a pydppn-centered 3ππ* state, with lifetimes of ∼19 μs for 1 and 2 and ∼270 ns for 3. A number of methods were used to characterize the mode of binding of 1-3 to DNA, including absorption titrations, thermal denaturation, relative viscosity changes, and circular dichroism, all of which point to the intercalation of the pydpppn ligand between the nucleobases. The photocleavage of plasmid pUC19 DNA was observed upon the irradiation of 1-3 with visible and red light, attributed to the sensitized generation of 1O2 by the complexes. These findings indicate that the bim ligand, together with pydppn, serves to shift the absorption of Ru(II) complexes to the photodynamic therapy window, 600-900 nm, and also extend the excited state lifetimes for the efficient production of cytotoxic singlet oxygen.

Silencing of Resistance Mechanism in Vancomycin-Resistance Enterococci Using Antisense RNA of vanA Gene

The World Health Organization has included the problem of antibiotic resistance among the top 10 important health problems in the world. Treatment of infectious diseases has become more difficult due to the spread of antibiotic resistance between bacteria via transposable elements. Vancomycin-resistant enterococci (VRE) are of critical medical and public health importance due to their association with serious nosocomial infections and high risk of death. One of the most important features of VREs is that they have multiple antibiotic resistance and treatment options are reduced. Therefore, new treatment methods are needed. The vanA gene constitutes the building block of the vancomycin resistance mechanism and causes high resistance to vancomycin. In this study, it was aimed to investigate the neutralization of the vancomycin resistance mechanism by creating vanA antisense RNA (asRNA). The vanA positive VRE50 strain in our culture collection which was isolated from the clinical sample, was used to amplify the vanA gene by polymerase chain reaction (PCR). The amplified vanA amplicon was inserted inversely into the pUC19 plasmid by means of the enzyme cutting sites in the primers used. The resulting plasmid was combined with the pAT392 plasmid which can replicate in gram-positive bacteria and a fusion plasmid was created. The fusion plasmid whose orientation was confirmed, was transferred to the wild strain VRE50 by electroporation method. Minimum inhibitory concentration (MIC) values of transformed VRE (tVRE50) and wild type VRE50 strains used as control were determined by the E-Test method. The vancomycin MIC value of the wild type VRE50 strain was determined as 1024 µg/mL and that of the tVRE50 strain was 32 µg/mL and it was determined that the vancomycin resistance of the tVRE50 strain decreased with asRNA (antisense RNA). Antisense RNA technology is an important method for neutralizing the expression of genes. This study showed that neutralization of the vancomycin resistance gene may provide a lower MIC value in a vancomycin-resistant enterococcus strain and lead to increased susceptibility. This new approach provides a new method for VRE treatment by neutralizing the vancomycin resistance mechanism. The result obtained in this study needs to be supported by in vivo tests.

Development of cancer-targeted nano gold functionalized ruthenium(III)-doxorubicin complex: Synthesis, characterization and evaluation of DNA cleavage, anticancer and bioimaging activity

Metal-based drugs have attracted greater attention for cancer diseases because of biological relevance. However, some drawbacks are present in the metal complexes such as low stability and low bioavailability. Nanomaterials functionalized metal complexes can protect the stability and enhanced the bioavailability. In the present study, Nano gold functionalized ruthenium(III) complex from doxorubicin have been synthesized from ruthenium(III) complex and characterized using various spectrochemical techniques such as IR, UV, and NMR. In the metal complex, Doxorubicin (DOX) acts as a monobasic bidentate ligand and is coordinated through the carbonyl oxygen and phenolate oxygen of the anthracene ring. Based on spectral evidences, the complex [Ru(DOX)(PPh3)2Cl2] has an octahedral geometry. The Nano Au-Ru(III) DOX is analysed for their size, and morphology using XRD, SEM and HRTEM. X-ray diffraction pattern revealed that the complex has nanocrystalline in nature. The Nano Au-Ru(III) DOX is spherical shape and particle size is less than 15 nm. The DNA cleavage activity of [Ru(DOX)(PPh3)2Cl2] and Nano Au-Ru(III) DOX has been evaluated using pUC18 plasmid DNA and the results showed significant cleavage. The [Ru(DOX)(PPh3)2Cl2] and Nano Au-Ru(III) DOX have been tested against human breast cancer (MCF-7) and leukemia cancer cell lines (K-562) in vitro for its antitumor effects. The tumour activity of the Nano Au-Ru(III) DOX showed more promising activity than the [Ru(DOX)(PPh3)2Cl2]. The cell imaging of the Nano Au-Ru(III) DOX was examined against epithelial human breast cancer cell line (MDA-MB-231) using fluorescent microscopy. The fluorescence images showed high scattering light in tumour tissue at 100 µg/mL Hence, Nano Au-Ru(III) DOX can serve as a promising drug for cancer therapy and cellular imaging.

Synthesis, theoretical analysis, and biological properties of a novel tridentate Schiff base palladium (II) complex.

Schiff base complexes play a crucial role in bioinorganic chemistry. A novel curcumin/phenylalanine tridentate Schiff base ligand and its palladium (II) complex were synthesized so that they were stable in aqueous buffer. The structure of the complex was investigated using a variety of methods, including DFT, NBO analysis, FMOs, and MESP. The interaction of the complex with a plasmid (pUC19) and CT-DNA was studied. The anticancer, antibacterial, and antioxidant activities of the complex were examined. The statistical analysis of the MTT assay was compared using the 1-way ANOVA and Tukey test. Results showed that the complexes were stable in aqueous buffer, pH 8. The extrinsic fluorescence emission of the plasmid and CT-DNA was quenched while interacting with the complex. The complex had an IC50 of 72.47 µM against MCF-7 cells. The ANOVA and Tukey analysis of MTT data demonstrated a statistically significant difference between groups (P < 0.0001). The minimum inhibitory concentrations (MIC) of the complex for E. coli and S. aureus were 300 and 200µg/mL, with 96.3 and 95.2% biofilm growth inhibition at 250µg/mL, respectively. The sample concentrations contributing to 50% radical inhibition in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) test for curcumin, ligand, and palladium (II) complex were 33.62, 21.27, and 51.26 µM, respectively. The results suggest that the complex interaction with DNA is one of the potential mechanisms for eliminating cancer cells and bacteria in the planktonic and biofilm. On the other hand, while stability in an aqueous buffer at pH 8 increases, the modified curcumin antioxidant effect decreases.

Nutrient limitation and oxidative stress induce the promoter of acetate operon in Salmonella Typhimurium.

Glyoxylate shunt is an important pathway for microorganisms to survive under multiple stresses. One of its enzymes, malate synthase (encoded by aceB gene), has been widely speculated for its contribution to both the pathogenesis and virulence of various microorganisms. We have previously demonstrated that malate synthase (MS) is required for the growth of Salmonella Typhimurium (S. Typhimurium) under carbon starvation and survival under oxidative stress conditions. The aceB gene is encoded by the acetate operon in S. Typhimurium. We attempted to study the activity of acetate promoter under both the starvation and oxidative stress conditions in a heterologous system. The lac promoter of the pUC19 plasmid was substituted with the putative promoter sequence of the acetate operon of S. Typhimurium upstream to the lacZ gene and transformed the vector construct into E. coli NEBα cells. The transformed cells were subjected to the stress conditions mentioned above. We observed a fourfold increase in the β-galactosidase activity in these cells resulting from the upregulation of the lacZ gene in the stationary phase of cell growth (nutrient deprived) as compared to the mid-log phase. Following exposure of stationary phase cells to hypochlorite-induced oxidative stress, we further observed a 1.6-fold increase in β galactosidase activity. These data suggest the induction of promoter activity of the acetate operon under carbon starvation and oxidative stress conditions. Thus, these observations corroborate our previous findings regarding the upregulation of aceB expression under stressful environments.

In vitro Production and Characterization of a Recombinant Viral Protein (rVP1) of Coxsackievirus B3 (CVB3): Contribution for the Development of a Subunit Vaccine or an Immunodiagnostic Reagent

Coxsackievirus B3 is an Enterovirus implicated in diverse human pathologies, from viral myocarditis to neurological disorders. There isn’t a medicinal agent or vaccine for CVB3 in clinical use at the moment, despite the possibility that vaccination could lower the prevalence of these illnesses. This study focuses on the in vitro production and characterization of the viral protein 1 (VP1) in the objective to use it as subunit vaccine and/or immunodiagnostic reagent. VP1 is considered as the most immunogenic capsid protein of the CVB3 surface. We amplified the VP1 whole gene by RT-PCR from the extracted wild type Nancy strain RNA, then cloned it into the pUC19 plasmid expression vector, and expressed it in E. coli DH5a prokaryotic cells. The obtained recombinant proteins were then analyzed by SDS-PAGE and characterized by Bioinformatic software tools. Our results revealed that the produced recombinant amino acid VP1 (rVP1) is highly identical to the VP1 of the CVB3 wild-type strain and has very similar physicochemical properties. In addition, we demonstrated that rVP1 has the highest number of phosphorylation sites which means that rVP1 can translate the host cell signal via the phosphorylation mechanism. Moreover, The Linear B cell epitope analysis showed that the rVP1 contains many epitope regions that should be recognized by the humoral host immune response. Taken together, results demonstrate that the cloned and recombined expressed viral protein could be used to carry out any studies concerning the development a protein subunit vaccine against CVB3 infections or an immunodiagnostic reagent for detecting the virus in samples.

Proximity Estimation and Quantification of Ionizing Radiation-induced DNA Lesions in Aqueous Media using Fluorescence Spectroscopy.

Clustered DNA damage (cluster) or a multiply damaged site, which is a region with two or more lesions within one or two helical turns, has a high mutagenic potential and causes cell death. We quantified fluorophore-labeled lesions and estimated their proximity through fluorescence anisotropy measurements depending on Förster resonance energy transfer (FRET) among the fluorophores close to each other. pUC19 plasmid DNA (2,686 base pairs) dissolved in water or 0.2 M Tris-HCl buffer at a concentration of 10 μg/μL was irradiated by several ionizing radiations with varying linear energy transfers (LET, 0.2-1890 keV/μm). Electrophilic carbonyls (aldehydes and ketones) at abasic sites (APs) produced in DNA were labeled with Alexa Fluor 488 fluorescent dyes with an O-amino functional group. Regardless of the presence or absence of the buffer, AP yields (the number of APs/base pair/Gy) tended to decrease with increasing LET, and the ratio of the AP yield (in 0.2 M Tris-HCl/in water) was less than 0.1 in the LET range of 0.2-200 keV/μm. However, in a higher LET range, the ratios were greater than 0.1. At a low dose, fluorescence anisotropy decreased with increasing LET in 0.2 M Tris-HCl, whereas, in water, this LET dependence was almost insignificant. These findings suggest that 1. the damage distribution on a DNA molecule formed by indirect effects (e.g., by hydroxyl radicals) does not depend on radiation quality and 2. greater LET radiation is more likely to produce a cluster and/or to produce a cluster with shorter distances between lesions by direct effects. This FRET-based proximity estimation of DNA lesions will contribute not only to the identification of clusters and their complexity in a whole genome, but also to the study of their repair mechanism by single-molecular level fluorescence microscopy.

Bioinformatics analysis of gene bhsA and its role in Ca2+ -treated Escherichia coli.

One of the commonly employed methods in molecular biology is to utilize calcium chloride to treat Escherichia coli for the preparation of competent cells to facilitate foreign gene expression. However, the molecular mechanisms underlying Ca2+ mediation of competent cell formation and identification of the key genes involved in the process remain unclear. In previous studies, the combined analysis of transcriptomics and proteomics revealed bhsA as one of the crucial genes. The gene ontology functional annotation of bhsA identified it as a member of the YhcN family encoding an outer membrane protein that confers resistance to various stresses. The IPR0108542 domain found within the protein plays a significant role in stress response and biofilm formation in E. coli. Analysis of the STRING database showed that the proteins interacting with bhsA are primarily involved in biofilm formation and stress resistance. Using the RED homologous recombination method, a bhsA deletion strain was constructed to verify its role in E. coli genetic transformation. Although the mutant strain showed no significant differences in morphology or growth trend when compared to the wild-type strain, its transformation efficiency decreased by 1.14- and 1.64-fold with plasmids pUC19 and pET-32a. Furthermore, the 1-N-phenylnaphthylamine assay indicated a 1.71-fold reduction in cell membrane permeability in the mutant strain.

Construction and functional verification of size-reduced plasmids based on TMP resistance gene dfrB10.

Plasmid size is one of the factors affecting transfection efficacy in most of the molecular genetic research studies. One effective approach for reducing plasmid size is to replace relatively large, conventional antibiotic resistance genes with the short-size dfrB10 gene. The successful construct of a series of dfrB10-based tool plasmids and their functional validation, via comparison with original plasmids, suggest that dfrB10 is a potent drug resistance selection marker. The antibiotic trimethoprim offers convenient usage comparable to that of ampicillin or kanamycin. Additionally, fluorescence analysis has demonstrated the compatibility of TMP with protein expression in various host cells. Based on these findings, TMP-dfrB10 could be an alternative choice for future use in molecular genetic research studies that require miniature plasmids to achieve optimal results.

In vitro anticancer activity of a head-to-toe constructed heterobimetallic [CoFeL]2+ metallocylinder

ABSTRACT [M2L3]-type metallo-supramolecular architectures (MSAs) are widely explored for their biological activity, whereas data on non-symmetric, heterobimetallic [MM’L3] structures is comparatively rare. [M2L3] compounds are known to form helicates and/or mesocates. Here, we report the self-assembly of a discrete non-symmetrical, heterobimetallic MSA by combination of an amine-functionalised cobalt(III)-tris((picolinamido)ethyl)amine (PICTREN) base unit with FeCl2 and 2,2’-bipyridine-5-carboxaldehyde. NMR spectroscopy in tandem with computational studies revealed the formation of a mesocate rather than a helicate. The ability of the heteromesocate to bind to DNA was examined by gel electrophoresis with pUC19 plasmid DNA, which showed DNA binding although longer incubation periods were required than found for a known DNA-interacting helicate. The mesocate displayed promising antiproliferative activity in cancer cells, including a cisplatin-resistant cell line, with IC50 values in the 17–26 μM range. Lower biological activity of the mesocate compared to a metallocylinder reference and cisplatin may be explained by differences in the DNA interactions. This work further supports efforts to establish methods with which to construct heterobimetallic complexes, establishing the utility of the self-assembly method in [M2L3]-type assemblies using the cobalt(III)-PICTREN platform as a robust building block towards more complex assemblies of the [MM’L]-type.