Coli DNA Research Articles

Isolation and purification of DNA double-strand break repair intermediates for understanding complex molecular mechanisms.

Branched DNA molecules are key intermediates in the molecular pathways of DNA replication, repair and recombination. Understanding their structural details, therefore, helps to envisage the mechanisms underlying these processes. While the configurations of DNA molecules can be effectively analysed in bulk using gel electrophoresis techniques, direct visualization provides a complementary single-molecule approach to investigating branched DNA structures. However, for microscopic examination, the sample needs to be free from impurities that could obscure the molecules of interest, and free from the bulk of unwanted non-specific DNA molecules that would otherwise dominate the field of view. Additionally, in the case of recombination intermediates, the length of the DNA molecules becomes an important factor to consider since the structures can be spread over a large distance on the chromosome in vivo. As a result, apart from sample purity, efficient isolation of large-sized DNA fragments without damaging their branched structures is crucial for further analysis. These factors are illustrated by the example of DNA double-strand break repair in the bacterium E. coli. In E. coli recombination intermediates may be spread over a distance of 40 kb which constitutes less than 1% of the 4.6 Mb genome. This study reveals ways to overcome some of the technical challenges that are associated with the isolation and purification of large and complex branched DNA structures using E. coli DNA double-strand break repair intermediates. High-molecular weight and branched DNA molecules do not run into agarose gels subjected to electrophoresis. However, they can be extracted from the wells of the gels if they are agarose embedded, by using β-agarase digestion, filtration, and concentration. Furthermore, a second round of gel electrophoresis followed by purification is recommended to enhance the purity of the specific DNA samples. These preliminary findings may prove to be pioneering for various single-molecule analyses of large and complex DNA molecules of DNA replication, repair and recombination.

B-351 Genotoxic Producing Escherichia coli Protects and Promotes Breast Adenocarcinoma

Abstract Background Recent studies have linked certain colibactin producing subphylas of E. coli to colorectal cancer. The bacterium synthesizes colibactin via the pks genomic island containing a series of clbA to clbS genes. The genotoxin causes inter-strand DNA cross-links, which lead to double strand breaks and promotion of colorectal adenocarcinoma. The published studies have prioritized the colorectal regions of the body where E. coli is commonly found, but none have identified the same genotoxic relationship with other types of adenocarcinoma. In this study, we evaluate DNA damage and cellular changes of mammary breast tissue based on high and low levels of colibactin producing E. coli exposure. The goal is to evaluate if colibactin can affect cells in the mammary breast tissue promoting breast adenocarcinoma growth. Methods Two cell lines were acquired from ATCC: primary mammary epithelial cells (PCS-600-010) and MCF-7 breast adenocarcinoma cells (HTB-22). Both cell lines were split into six culture flasks and maintained until ∼80% confluence was reached using the ATCC cell culture protocols. Once all the flasks had reached the target confluence, they were divided into exposure groups of high and low concentrations of colibactin producing E. coli, a group of non-colibactin producing E. coli, a group exposed only to phosphate buffered saline, and a control group with no exposure. Cellular morphology was observed using light microscopy before, after exposure, and every 5 minutes up to 15 minutes. E. coli and colibactin DNA concentrations were quantified using quantitative polymerase chain reaction. Genomic DNA was extracted from each group of cells. The DNA from each group was whole genome sequenced and analyzed for genomic variation. Results The normal cells exposed to E. coli showed immediate lysing and continued to lyse after a 5 minute incubation; however, the adenocarcinoma positive cells showed no lysing up to 30 minutes incubation. Sequencing results show genomic variation between the groups and specific break points associated with the E. coli exposed groups. Conclusions Our results show that the presence of E. coli with mammary breast tissue can destroy normal tissue, promote lysis resistance, and allow adenocarcinoma cells to continue to grow. The colibactin producing E. coli does cause double stranded breaks and can lead to cell death. This new information points out a critical need to monitor the microbiome and infection levels of people at risk or in early stages of cancer to prevent adenocarcinoma growth and destruction of normal tissue. This study indicates that colibactin producing E. coli may play a larger role in tissue damage and promotion of other types of cancer. More research is needed to elucidate the molecular pathway and mechanisms of colibactin, which will lead to new diagnostics or therapeutics that monitor E. coli levels or inhibit colibactin’s toxic effects.

Antibacterial activity of secondary metabolites from the roots of Solanum dasyphyllum: A combined in vitro and in silico study

Solanum dasyphyllum is widely utilized for folk medicine in Ethiopia. The chromatographic separation of root extract of S. dasyphyllum led to the isolation of six compounds: tremulacin (1), scopoletin (2), stigmasterol (3), β-sitosterol (4), palmitic acid (5) and 2,3-dihydroxypropyl-9Z,12Z-octadecadienoate (6). The structures of the compounds were determined using NMR (1D and 2D), MS and comparison with literature data. The extract and isolated compounds were in vitro assayed against six bacterial strains and displaying activity against the tested strains. The extract exhibited stronger activity against E. coli, K. pneumoniae, and P. aeruginosa, with MIC values of 0.195±0.0, 0.391±0.0, and 0.195±0.0 mg/mL, respectively. The isolated compounds also showed good antibacterial activity with the highest activity was recorded for compound 2, with MIC values of 0.313±0.0 mg/mL against K. pneumoniae, while for the ciprofloxacin is 0.195±0.0 mg/mL against the same strain. In silico molecular docking analyses were performed for compounds 1, 2 and 3 against E. coli DNA gyrase B, P. aeruginosa LasR protein, and K. pneumoniae using AutoDock Vina and showed better binding activity. The in vitro antibacterial results and in silico analysis of the compounds revealed the potential of these compounds to be lead compound for antibacterial drug discovery. KEY WORDS: Solanum dasyphyllum, Chromatographic separation, Antibacterial activity, Molecular docking Bull. Chem. Soc. Ethiop. 2024, 38(6), 1843-1860. DOI: https://dx.doi.org/10.4314/bcse.v38i6.26

Synthesis and computational studies of new pyridine, pyrazole, pyran, and pyranopyrimidine-based derivatives of potential antimicrobial activity as DNA gyrase and topoisomerase IV inhibitors

In this study, new derivatives based on pyridine, pyrazole, pyran, and pyranopyrimidine scaffolds 5–14 were designed and synthesized. The newly synthesized compounds were characterized using microanalytical and spectral data. Using amoxicillin and amphotericin B, two common antibacterial and antifungal medications, respectively, the antimicrobial activity of the new compounds was assessed against a variety of strains of both Gram-positive and Gram-negative bacteria as well as fungal infections. The most promising activity was achieved by pyridine-based derivatives 5, 7a, and 7b, which exhibited MIC values ranging from 0.95 to 18.04 µM against the examined bacterial strains and ranging from 4.65 to 33.16 µM against the examined fungal strains. The later derivatives were also evaluated as E. coli DNA gyrase B/topoisomerase IV inhibitors in comparison with novobiocin as a standard drug. Although the assessed candidates 5, 7a, and 7b exhibited a promising suppression impact against E. coli DNA gyrase B (IC50 = 3.11 ± 0.14, 3.85 ± 0.20, and 4.05 ± 0.10, respectively, IC50 novobiocin= 3.64 ± 0.10 µM), they showed detectable less suppression impact against Topoisomerase IV than that of the reference drug with IC50 values of 19.72 ± 1.00, 79.33 ± 0.25, 85.14 ± 0.10, and 13.42 ± 0.05 µM, respectively. Additionally, the safety profiles of 5, 7a, and 7b against WI38 cells were significantly superior to that of novobiocin (IC50 = 138 ± 0.33, 170 ± 0.40, 163.3 ± 0.17, and 86.2 ± 0.03 µM, respectively).Lastly, molecular docking was used to examine the most active derivatives 5, 7a, and 7b's binding capacity to the target DNA gyrase B/topoisomerase IV. The molecules exhibited various H-interactions with the amino acid residues Asp73, Arg76, and Val71 in E. coli DNA gyrase, where compound 5 exhibited H-bonding with the Asp1069 side chain of E. coli Topoisomerase IV. Additional in silico ADMET studies were conducted to represent their oral bioavailability and drug-likeness characteristics.

Human Immunodeficiency Viral Reverse Transcriptases: Analyses of the Active Sites of the Polymerase Domain and Drug-Resistant Mutants of the Reverse Transcriptases

It is well-known that human immunodeficiency viruses (HIVs) cause the chronic, potentially life-threatening condition known as human Acquired Immuno-Deficiency Syndrome (AIDS). As the lifecycle of HIVs heavily depends on the crucial enzyme, the reverse transcriptase (RT), it has been used as a potential therapeutic target to treat and control the spread of AIDS. The active site amino acids of the polymerase domain of the RTs and their anti-HIV drug-binding sites are analyzed. The catalytic region of HIV RTs and the E. coli DNA polymerase I showed very similar active site amino acids, suggesting that HIV RTs would have possibly evolved from the bacterial DNA polymerase. The catalytic proton abstractor is identified as a K and the nucleotide selection amino acid as an N. However, the regular template-binding pair –YG- is slightly modified to a –YXG- in HIV RTs. Three completely conserved Ds in HIV RTs are involved in binding to the catalytic Mg2+. The sensitive and resistant strains of HIV-1 for the HIV antiretroviral drug, azidothymidine (AZT), a nucleoside analogue of thymidine, show only a few non-isofunctional amino acid replacements and are located mainly in the palm and thumb subdomains of the RT polymerase, whereas the rest of the polymerase catalytic core and metal-binding sites are completely conserved in AZT-sensitive and -resistant HIV-1 strains. In the drug-resistant mutants of non-nucleoside RT inhibitors of HIV-2, the crucial mutations are located mainly near the -MDD- motif of the catalytic metal-binding region. Even though a large number of amino acid replacements are seen between the RTs of HIV-1 and HIV-2, the polymerase active sites are completely conserved in both. The HIV-1 and HIV-2 catalytic and metal-binding sites are completely conserved in simian immunodeficiency virus (SIV) as well. The absence of DEDD-superfamily of proofreading exonuclease domain in the HIV RTs, might cause the virus to evolve rapidly in patients. A possible mechanism of action for the HIV RTs is also proposed.

Environmental and genetic influence on the rate and spectrum of spontaneous mutations in Escherichia coli.

Spontaneous mutations are the ultimate source of novel genetic variation on which evolution operates. Although mutation rate is often discussed as a single parameter in evolution, it comprises multiple distinct types of changes at the level of DNA. Moreover, the rates of these distinct changes can be independently influenced by genomic background and environmental conditions. Using fluctuation tests, we characterized the spectrum of spontaneous mutations in Escherichia coli grown in low and high glucose environments. These conditions are known to affect the rate of spontaneous mutation in wild-type MG1655, but not in a ΔluxS deletant strain - a gene with roles in both quorum sensing and the recycling of methylation products used in E. coli's DNA repair process. We find an increase in AT>GC transitions in the low glucose environment, suggesting that processes relating to the production or repair of this mutation could drive the response of overall mutation rate to glucose concentration. Interestingly, this increase in AT>GC transitions is maintained by the glucose non-responsive ΔluxS deletant. Instead, an elevated rate of GC>TA transversions, more common in a high glucose environment, leads to a net non-responsiveness of overall mutation rate for this strain. Our results show how relatively subtle changes, such as the concentration of a carbon substrate or loss of a regulatory gene, can substantially influence the amount and nature of genetic variation available to selection.

Spectroscopic characterization, DFT calculations, in vitro pharmacological potentials, and molecular docking studies of N, N, O-Schiff base and its trivalent metal complexes

In this study, trivalent metal complexes of the category: [M(L)(H2O)nCly] obtained from the interaction of metal3+ ion salts with organic N, N, O-Schiff base (HL) (where: HL = 4-{(Z)-((2-{(E)-((2-hydroxyphenyl)methylidene)amino}ethyl)imino)methyl}-2-methoxyphenol; n, y = 1 or 2 and M = Ti(III), Fe(III), Ru(III), Cr(III) and Al(III)) were synthesized and characterized viz molar conductance, FT-IR, and UV–Vis spectroscopies, elemental analyses, thermal analyses (TGA and DTA), and UV–Vis spectroscopy, theoretical calculations. A distorted octahedral structure around the metal ions was proposed based on the obtained experimental and calculated data. Thermal examination of the complexes signposts the step-by-step disintegration to give the final decomposition product as metal oxides. Moreover, DFT calculations were executed utilizing the B3LYP/LANL2DZ theory level, which revealed that the synthesized metal (III) complexes were more stable than the free ligand (HL). The value of ΔE for HL is 4.60 eV while the related values for the complexes of Cr(III) (C1), Ru(III) (C2), Fe(III) (C3), Al(III) (C4), and Ti(III) (C5) are respectively 2.59, 3.68, 3.15, 1.64, and 2.75 eV. Scavenging abilities of DPPH and ABTS radicals by the test compounds revealed promising antioxidant behavior. It was observed that the compounds are proficient DPPH radical scavengers in a dose-dependent configuration. Ru(III); IC50 = 1.69 ± 2.68 µM for DPPH and Ti(III); IC50 = 8.70 ± 2.78 µM for ABTS performed best. Similarly, the complexes demonstrated higher antimicrobial activities compared to HL against the designated strains, while ciprofloxacin acted as a standard antibiotic. Furthermore, the ligand and its most effective complexes C2 and C5 were docked against the targets S. aureus DNA gyrase (2XCT), S. pneumoniae DNA gyrase (5BOD), and E. coli DNA gyrase (5L3J). The binding sites were evaluated and the docking results showed that the studied molecules bind to the targets through classical O—H…O and/or N—H…O hydrogen bonds, as well as via hydrophobic contacts.

Disposable impedance sensors based on novel hybrid MoS2 nanosheets and microparticles to detect Escherichia Coli DNA.

The rapid and accurate detection of pathogenic bacteria is essential for food safety and public health. Conventional detection techniques, such as nucleic acid sequence-based amplification and polymerase chain reaction, are time-consuming and require specialized equipment and trained personnel. Here, we present quick, disposable impedance sensors based on the novel hybrid MoS2 nanomaterial for detecting Escherichia coli DNA. Our results indicate that the proposed sensors operate linearly between 10- 20 and 10-15 M concentrations, achieving an impressive detection limit of 10-20 M with the highest sensitivity observed at a 0.325 nM probe concentration sensor. Furthermore, the electrochemical impedance spectroscopy biosensors exhibited potential selectivity for Escherichia coli DNA over Bacillus subtilis and Vibrio proteolyticus DNA sequences. The findings offer a promising avenue for efficient and precise DNA detection, with potential implications for broader biotechnology and medical diagnostics applications.

Synthesis of CaO-Fe and CaO-Ag nanocomposites using raw and hydrolyzed waste chicken eggshell and investigation of their antimicrobial, biofilm inhibition activity, DNA cleavage ability, and antioxidant activity properties

AbstractIn this study, CaO-Fe and CaO-Ag nanocomposites were synthesized and various biological properties were characterized. E. coli cell viability, antimicrobial, antioxidant, antibiofilm, and DNA cleavage properties were examined. All nanocomposites, namely raw CaO-Ag (R-CaO-Ag), hydrolyzed CaO-Ag (H-CaO-Ag), raw CaO-Fe (R-CaO-Fe), and hydrolyzed CaO-Fe (H-CaO-Fe), were found to have good antioxidant, antimicrobial, and antibiofilm properties. They showed antioxidant activity of 83.33%, 70.60%, 74.73%, and 72.78%, respectively, at 200 mg/L nanocomposites. When DNA cleavage properties of R-CaO-Ag, H-CaO-Ag, R-CaO-Fe, and H-CaO-Fe were evaluated at different concentrations, single-strand break was observed for all samples. It was shown that R-CaO-Ag was more effective against S. aureus and C. tropicalis, and H-CaO-Ag was more effective against E. hirae. It was found that the antimicrobial activities of R-CaO-Ag and H-CaO-Ag were higher compared to R-CaO-Fe and H-CaO-Fe. The microbial cell viability of nanocomposites was examined at three different concentrations. Even at the lowest concentration (125 mg/L), high values of E. coli inhibition were found as 98.65%, 100%, 90.24%, and 88.63%, respectively. Also, it was observed that all nanocomposites exhibited excellent biofilm inhibition activities. The antibiofilm abilities of one Gr (+) and one Gr (−) microorganism at three different concentrations were investigated. Biofilm inhibition percentages of R-CaO-Ag, H-CaO-Ag, R-CaO-Fe, and H-CaO-Fe were found as 65.83%, 86.5%, 89.67%, and 93.62% for S. aureus at 500 mg/L, respectively, while it was 50.06%, 90.68%, 71.69%, and 92.36% for P. aeruginosa, respectively, at 500 mg/L.

Bioengineered DNA-decorated UiO-66-based nanocarriers for combined administration of doxorubicin and sorafenib: Hepatocellular carcinoma treatment and chemotherapy

Natural biomaterials were used to decorate UiO-66-NH2 to develop a platform for co-drug delivery of doxorubicin and sorafenib as a cost-effective and easy way to use a drug delivery system (DDS). To properly characterize the modified MOFs, standard analytical methods were used. Then doxorubicin and sorafenib were loaded into and onto the porosity of the MOF, UiO-66-NH2. The surface morphological analysis, FESEM, and TEM images revealed the hexagonal structure of the UiO-66-NH2, demonstrating the successful synthesis of these green MOFs. Internalization and loading of doxorubicin on the MCF-7 cell line was investigated with two-dimensional fluorescence microscopy, which shows that drug internalizations on the cancerous cells were successful and promising. The drug payload of 49.5% and 31.2% for doxorubicin and sorafenib were attained, respectively. Study on the MCF-7 HT-29 and HEK-293 cell lines exhibited excellent cell viability when subjected to both low and high concentrations of 0.1 µg.mL−1 and 50 µg.mL−1, respectively. After 24 h of treatment, the relative cell viability of 85.1%, 88.1%, and 76.8% was achieved for the HT-29, HEK-293, and MCF-7 cell lines, respectively. In addition, after 48 h of treatment, the relative cell viability of 82.5%, 83.5%, and 68.5% was recorded, respectively. However, the percentage of cell viability was dramatically reduced by coating the nanocarrier with Ginkgo biloba leaf extract and E. Coli DNA (62.6%, 40.9%, and 45.1% for 24 h treatment and 62.1%, 38.5%, and 38.9% for 48 h treatment in HEK-293, HT-29, and MCF-7 cell lines, respectively).

Telomere maintenance in African trypanosomes

Telomere maintenance is essential for genome integrity and chromosome stability in eukaryotic cells harboring linear chromosomes, as telomere forms a specialized structure to mask the natural chromosome ends from DNA damage repair machineries and to prevent nucleolytic degradation of the telomeric DNA. In Trypanosoma brucei and several other microbial pathogens, virulence genes involved in antigenic variation, a key pathogenesis mechanism essential for host immune evasion and long-term infections, are located at subtelomeres, and expression and switching of these major surface antigens are regulated by telomere proteins and the telomere structure. Therefore, understanding telomere maintenance mechanisms and how these pathogens achieve a balance between stability and plasticity at telomere/subtelomere will help develop better means to eradicate human diseases caused by these pathogens. Telomere replication faces several challenges, and the “end replication problem” is a key obstacle that can cause progressive telomere shortening in proliferating cells. To overcome this challenge, most eukaryotes use telomerase to extend the G-rich telomere strand. In addition, a number of telomere proteins use sophisticated mechanisms to coordinate the telomerase-mediated de novo telomere G-strand synthesis and the telomere C-strand fill-in, which has been extensively studied in mammalian cells. However, we recently discovered that trypanosomes lack many telomere proteins identified in its mammalian host that are critical for telomere end processing. Rather, T. brucei uses a unique DNA polymerase, PolIE that belongs to the DNA polymerase A family (E. coli DNA PolI family), to coordinate the telomere G- and C-strand syntheses. In this review, I will first briefly summarize current understanding of telomere end processing in mammals. Subsequently, I will describe PolIE-mediated coordination of telomere G- and C-strand synthesis in T. brucei and implication of this recent discovery.

Low-grade endotoxemia is associated with cardiovascular events in community-acquired pneumonia

Community-acquired pneumonia (CAP) is associated with low-grade endotoxemia but its relationship with cardiovascular events (CVE) has not been investigated. We evaluated the incidence of CVE including myocardial infarction, stroke, and cardiovascular death in 523 adult patients hospitalized for CAP. Serum lipopolysaccharide (LPS) and zonulin, a marker of gut permeability, were analyzed in the cohort, that was followed-up during hospitalization and up to 43 months thereafter. During the hospital-stay, 55 patients experienced CVE with a progressive increase from the lowest (0.6%) to highest LPS tertile (23.6%, p<0.001). Logistic regression analyses showed that higher LPS tertile was independently associated with CVE; LPS significantly correlated with age, hs-CRP and zonulin. In a sub-group of 23 CAP patients, blood E. coli DNA was higher in patients compared to 24 controls and correlated with LPS. During the long-term follow-up, 102 new CVE were registered; the highest tertile of LPS levels was associated with incident CVE; Cox regression analysis showed that LPS tertiles, age, history of CHD, and diabetes independently predicted CVE. In CAP low-grade endotoxemia is associated to short- and long-term risk of CVE. Further study is necessary to assess if lowering LPS by non-absorbable antibiotics may result in improved outcomes.

NAD+ mediated photoelectrochemical biosensor for histone deacetylase Sirt1 detection based on CuO-BiVO4-AgNCs heterojunction and hybridization chain reaction amplification

BackgroundHistone deacetylate Sirt1 has been involved in many important biological processes and is closely related to the occurrence and development of many diseases. Therefore, the accurate detection of Sirt1 is of great significance for the diagnosis and treatment of diseases caused by Sirt1 and the development of related drugs. ResultsIn this work, a photoelectrochemical biosensor was developed for Sirt1 detection based on the NAD + mediated Sirt1 recognition and E. Coli DNA ligase activity. CuO-BiVO4p-n heterojunction was employed as the photoactive material, rolling circle amplification (RCA), hybridization chain reaction (HCR) and AgNCs were used as triple signal amplifications. As a bifunctional cofactor, NAD+ played a crucial role for Sirt1 detection, where the peptide deacetylation catalyzed by Sirt1 consumed NAD+, and the decreased amount of NAD + inhibited the activity of E. Coli DNA ligase, leading to the failure on RCA reaction, and improving the HCR reaction. Finally, AgNCs were generated using C-rich DNA as carrier. The surface plasmon effect of AgNCs and its heterojunction with CuO and BiVO4 accelerated the transfer rate of photogenerated carriers and improved the photocurrent signal. When the detection range was 0.001–200 nM, the detection limit of the biosensor was 0.76 pM (S/N = 3). SignificanceThe applicability of the method was evaluated by studying the effects of known inhibitors nicotinamide and environmental pollutant halogenated carbazole on Sirt1 enzyme activity. The results showed that this method can be used as a new platform for screening Sirt1 enzyme inhibitors, and also provided a new biomarker for evaluating the ecotoxicological effects of environmental pollutants.

In vitro biological activities of novel Ni(II)-2-chloroquinoline derivative complex: Synthesis, characterization and computational studies

Quinoline derivative nickel complexes have received considerable attention in biomedical research. Herein, novel nickel(II) complex [Ni(II)(HL)2].2H2O was synthesized from newly synthesised ligand 2-((2-hydroxyethyl)amino)quinoline-3-carbaldehyde (H2L) through bidentate fashion of metal–ligand interaction. The complex was characterized using spectroscopic and physicochemical methods. The formation constant (KNi(II) = 2.36 × 107) of the complex was calculated spectroscopically and thermodynamic parameters (ΔG, ΔH and ΔS) were determined from it. In vitro antibacterial activity analysis of the newly synthesized complex exhibited mean inhibition zones ranging from medium to high (16.55 ± 1.03 mm, 16.69 ± 0.63 and 20.94 ± 2.01 mm) at 600 μg/mL against E. coli, S. aureus and P. aeruginosa, respectively. The complex also showed enhanced antioxidant activities compared with the corresponding free ligand. The energy band gap (1.183 eV) was calculated from the HOMO and LUMO of the most stable structure of the synthesized complex. The DFT calculated results and molecular docking analysis results showed good agreement with the experimental results. The complex showed pronounced biological activities than the free ligand. The in silico binding modes of the complex against E. Coli DNA Gyrase and S. aureus Dihydrofolate reductase (PDB: 2w9h) were found to be in excellent agreement with the in vitro biological studies.

Benzimidazole and piperidine containing novel 1,2,3-triazole hybrids as anti-infective agents: Design, synthesis, in silico and in vitro antimicrobial efficacy.

Cu alkyne-azide cycloaddition was used to easily synthesize a library of novel heterocycles containing benzimidazole and piperidine based 1,2,3-triazole(7a-7l) derivatives. The synthesized analogs were characterized by various spectroscopic techniques like FTIR, 1 H nuclear magnetic resonance (NMR), 13 C NMR, and mass spectrometry. All these novel bioactive compounds (7a-7l) were evaluated for in vitro antibacterial and antifungal efficacy. Compound 7k exhibited appreciable potent activity against Escherichia coli strain. Compounds 7a, 7b, 7f, and 7i showed excellent potent activity against all bacterial strains. Compound 7b, 7c, 7d, and 7g derivatives showed excellent effects when tested in vitro for antifungal activity against various fungal strains. Additionally, a molecular docking investigation revealed that compound 7k has the ability to bind to the active site of the E. coli DNA gyrase subunit protein and form hydrogen bonds with significant amino acid residues Asp73 and Asp49 in the active sites. In a 100 ns molecular dynamics simulation, the E. coli DNA gyrase protein's steady capacity to bind compound 7k was shown by the low measured root mean square deviation, which was an indication of the complex's conformational stability.

Quality comparison of a state-of-the-art preparation of a recombinant L-asparaginase derived from Escherichia coli with an alternative asparaginase product.

L-asparaginase (ASNase) is a protein that is essential for the treatment of acute lymphoblastic leukemia (ALL). The main types of ASNase that are clinically used involve native and pegylated Escherichia coli (E. coli)-derived ASNase as well as Erwinia chrysanthemi-derived ASNase. Additionally, a new recombinant E. coli-derived ASNase formulation has received EMA market approval in 2016. In recent years, pegylated ASNase has been preferentially used in high-income countries, which decreased the demand for non-pegylated ASNase. Nevertheless, due to the high cost of pegylated ASNase, non-pegylated ASNase is still widely used in ALL treatment in low- and middle-income countries. As a consequence, the production of ASNase products from low- and middle-income countries increased in order to satisfy the demand worldwide. However, concerns over the quality and efficacy of these products were raised due to less stringent regulatory requirements. In the present study, we compared a recombinant E. coli-derived ASNase marketed in Europe (Spectrila®) with an E. coli-derived ASNase preparation from India (Onconase) marketed in Eastern European countries. To assess the quality attributes of both ASNases, an in-depth characterization was conducted. Enzymatic activity testing revealed a nominal enzymatic activity of almost 100% for Spectrila®, whereas the enzymatic activity for Onconase was only 70%. Spectrila® also showed excellent purity as analyzed by reversed-phase high-pressure liquid chromatography, size exclusion chromatography and capillary zone electrophoresis. Furthermore, levels of process-related impurities were very low for Spectrila®. In comparison, the E. coli DNA content in the Onconase samples was almost 12-fold higher and the content of host cell protein was more than 300-fold higher in the Onconase samples. Our results reveal that Spectrila® met all of the testing parameters, stood out for its excellent quality and, thus, represents a safe treatment option in ALL. These findings are particularly important for low- and middle-income countries, where access to ASNase formulations is limited.

Chrysin based pyrimidine-piperazine hybrids: design, synthesis, in vitro antimicrobial and in silico E. coli topoisomerase II DNA gyrase efficacy.

Ten chrysin-based pyrimidine-piperazine hybrids have been evaluated in vitro for antimicrobial activity against eleven bacterial and two fungal strains. All compounds 5a-j exhibited moderate to good inhibition, with MIC values ranging from 6.25 to 250µg/ml. At 6.25µg/ml and 12.5µg/ml MIC values, respectively, compounds 5b and 5h demonstrated the most promising potency against E. coli, outperforming ampicillin, chloramphenicol, and ciprofloxacin. None of the substances had the same level of action as norfloxacin. 5a, 5d, 5g, 5h, and 5i have exhibited superior antifungal efficacy than Griseofulvin against C. albicans with 250µg/ml MIC. All the compounds were also individually docked into the E. coli DNA gyrase ATP binding site (PDB ID: 1KZN) and CYP51 inhibitor (PDB ID: 5V5Z). The most active compound, 5h and 5g displayed a Glide docking score of - 5.97kcal/mol and - 10.99kcal/mol against DNA gyrase and 14α-demethylase enzyme CYP51 respectively. Potent compounds 5b, 5h, and 5g may be used to design new, innovative antimicrobial agents, according to in vitro, ADMET, and in silico biological efficacy analyses.

Identification of Polymerase and Proofreading Exonuclease Domains in the DNA Polymerases IA, IB and Nuclear-Encoded RNA Polymerase of the Plant Chloroplasts

Chloroplast plays a crucial role in all photosynthetic plants and converts the light energy to chemical energy. It is a semi-autonomous organelle and is mostly controlled by its own genome and partly by the nuclear imports. To replicate its own genome, it uses two DNA polymerases, viz. polymerases IA and IB. DNA polymerase IA showed 72.45% identity to polymerase IB, but only 35.35% identity to the E. coli DNA polymerase I. Multiple sequence alignment (MSA) analysis have shown that the DNA polymerases IA and IB and the E. coli DNA polymerase I possess almost identical active sites for polymerization and proofreading (PR) functions, suggesting their possible common evolutionary origin. The nuclear-encoded RNA polymerase (NEP) is imported from the nucleus and involves in the transcription of all the four subunits of the chloroplast RNA polymerase. The polymerase catalytic core of the DNA polymerases IA, IB and the NEP are remarkably conserved and is in close agreement with other DNA/RNA polymerases reported already, and possess a typical template-binding pair (-YG-), a basic catalytic amino acid (K) to initiate catalysis and a basic nucleotide selection amino acid R at -4 from K. The DNA polymerases IA and IB are very similar to prokaryotic DNA polymerases, except in possessing a zinc-binding motif (ZBM) in them, like the eukaryotic replicases. Interestingly, the PR exonucleases of all three polymerases belong to the DEDD-superfamily of exonucleases. The DNA polymerases IA and IB belong to the DEDD(Y)-subfamily, whereas the NEP belongs to the DEDD(H)-subfamily.

A Comprehensive Mechanistic Antibacterial and Antibiofilm Study of Potential Bioactive ((BpA)2bp)Cu/Zn Complexes via Bactericidal Mechanisms against Escherichia coli.

Bacterial resistance to antibiotics and host defense systems is primarily due to bacterial biofilm formation in antibiotic therapy. In the present study, two complexes, bis (biphenyl acetate) bipyridine Cu (II) (1) and bis (biphenyl acetate) bipyridine Zn (II) (2), were tested for their ability to prevent biofilm formation. The minimum inhibitory concentration and minimum bactericidal concentration of complexes 1 and 2 were 46.87 ± 1.822 and 93.75 ± 1.345 and 47.87 ± 1.345 and 94.85 ± 1.466 μg/mL, respectively. The significant activity of both complexes was attributed to the damage caused at the membrane level and was confirmed using an imaging technique. The biofilm inhibitory potential levels of complexes 1 and 2 were 95% and 71%, respectively, while the biofilm eradication potential levels were 95% and 35%, respectively, for both complexes. Both the complexes showed good interactions with the E. coli DNA. Thus, complexes 1 and 2 are good antibiofilm agents that exert their bactericidal actions possibly by disrupting the bacterial membrane and interacting with the bacterial DNA, which can act as a powerful agent to restrain the development of bacterial biofilm on therapeutic implants.

Rapid profiling of DNA replication dynamics using mass spectrometry-based analysis of nascent DNA.

The primary method for probing DNA replication dynamics is DNA fiber analysis, which utilizes thymidine analog incorporation into nascent DNA, followed by immunofluorescent microscopy of DNA fibers. Besides being time-consuming and prone to experimenter bias, it is not suitable for studying DNA replication dynamics in mitochondria or bacteria, nor is it adaptable for higher-throughput analysis. Here, we present mass spectrometry-based analysis of nascent DNA (MS-BAND) as a rapid, unbiased, quantitative alternative to DNA fiber analysis. In this method, incorporation of thymidine analogs is quantified from DNA using triple quadrupole tandem mass spectrometry. MS-BAND accurately detects DNA replication alterations in both the nucleus and mitochondria of human cells, as well as bacteria. The high-throughput capability of MS-BAND captured replication alterations in an E. coli DNA damage-inducing gene library. Therefore, MS-BAND may serve as an alternative to the DNA fiber technique, with potential for high-throughput analysis of replication dynamics in diverse model systems.