pUC19 Plasmid Research Articles

The Effects of Self-cleavage Intein-ELK16 Tag in the Transcript Steric Hindrance of IFN.

Intervening proteins (Inteins) are identified as protein domains in a precursor protein structure. Inteins can excise itself from precursor protein and join the remaining portions which result in forming an active protein. In this study, the transcript expression level of recombinant human Interferon beta (rhIFNβ) connected to the self-cleavage Intein-ELK16 (LELELKLKLELELKLK) tag was measured by real-time PCR in HEK293T cell line. First, the sequence of Mycobacterium tuberculosis RecA (Mtu recA) was obtained from the InBase database to do appropriate changes including adding the restriction sites, kozak sequence, signal peptide and ELK16 sequence by SnapGene software. The RNA secondary structure were also examined using the online RNA Fold 2.2 web server. Next, the construct was inserted into pUC19 plasmid. The sequence of rhIFNβ was also cloned into pBudCE4.1 vector. In the next step, the rhIFNβ was ligated into the construct (self-cleavage tag of ELK16) using T4 DNA ligase and the recombinant construct was transfected into HEK293T cell line. Finally, expression of the cassette was evaluated by real-time PCR. The analysis of secondary RNA structure indicates a minimum free energy of MEF - 261.10kcal/mol. Our results indicate that IFNβ was upregulated (37.8-fold, p < 0.0001) in cells which transfected by rhIFNβ-ELK16 compared to the mock and un-transfected conditions. Altogether, our results show that the presence of mini self-cleavage Intein-ELK16 tag along with the rhIFNβ had no interference in transcription of rhIFNβ in the HEK293T cell line.

The apoptotic and free radical–scavenging abilities of the protein hydrolysate obtained from chicken feather meal

This study examined the antioxidant capabilities of peptides derived from chicken feather meal (CFM) protein hydrolysates which were produced using 3 different microbial proteases (Neutrase, Alcalase, and flavourzyme) and tested at varying concentrations, namely 1, 2, and 5% by weight. The highest levels of 2,2-diphenyl-1-picrylhydrazl (DPPH) and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical scavenging activities were presented by CFM hydrolysate derived using 5 wt% Neutrase and digested for 4 h. Fractionation of this particular hydrolysate was then performed by applying 10, 5, 3, and 0.65 kDa molecular weight cutoff membranes. It was then determined that the molecular weight (MW) < 0.65 kDa fraction achieved the greatest level of free radical scavenging activity in the context of DPPH and ABTS. The MW < 0.65 kDa fraction then underwent additional fractionation using reverse-phase high-performance liquid chromatography to derive 3 main fractions designated as F1, F2, and F3. All of these fractions presented a high level of activity in DPPH radical scavenging, although no significant ABTS scavenging was observed. Quadrupole time-of-flight tandem mass spectrometry was used in determining the peptide contents of the fractions as Phe-Asp-Asp-Arg-Gly-Arg-X for F1 (FDDRGRX, 875 Da), Val-Thr-Leu-Ala-Val-Thr-Lys-His for F2 (VTLAVTKH, 868 Da), and Val-Ser-Glu-Ile-X-Ser-Ile-Pro-Ile-Ser for F3 (VSEIXSIPIS, 1,055 Da). Moreover, the F2 fraction was shown to be capable of preventing DNA damage induced by hydroxyl radicals, as indicated in tests using the plasmids pKS, pUC19, and pBR322 via the Fenton reaction. This outcome was demonstrated through in vitro antiproliferative activity in human cell lines based on SW620 colon cancer, using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. The F2 fraction at 0.5 wt.% was also shown to be capable of inducing weak early apoptosis, which could be measured by using the Fluorescein isothiocyanate Annexin V Apoptosis Detection Kit with Propidium Iodide Solution. Furthermore, an increase in caspase-3 and caspase-8 activity was observed in SW620 cells following exposure for 24 h and 48 h.

Ditopic binuclear copper(II) complexes for DNA cleavage

Herein we present the synthesis of two ligands containing two di(2-picolyl)amine (DPA) units linked by either a 1,1′-(pyridine-2,6-diyl)bis(3-ethylurea) (L1) or a 1,1′-(1,3-phenylene)bis(3-ethylurea) (L2) spacer. The corresponding binuclear CuII and ZnII complexes were prepared and isolated. The X-ray structures of the L1 ligand and the [Cu2L1Cl2]2+ complex evidence an unusual cis/trans conformation of one of the urea groups stabilized by an intramolecular hydrogen bond with the nitrogen atom of the pyridyl spacer. The CuII complexes form rather strong ternary complexes with phosphorylated anions. The [Cu2L1]4+ complex presents a rather high affinity for pyrophosphate (logK11 = 8.19 at pH 7, 25 °C), while [Cu2L2]4+ stands out because of its strong binding to AMP2− (logK11 = 9.3 at pH 7, 25 °C). The interaction of the CuII complexes with deoxyribonucleic acid from calf thymus (ct-DNA) was monitored using circular dichroism (CD) and luminescence spectroscopies. These studies revealed a quite strong interaction of the complexes with ct-DNA (Kb = (6.4 ± 0.7) × 103 for [Cu2L1]4+ and Kb = (6.3 ± 1.0) × 103 for [Cu2L2]4+). Competition experiments carried out in the presence of methyl green and BAPPA (N1,N3-Bis(4-amidinophenyl)propane-1,3-diamine) as major and minor groove competitors, respectively, confirm that the interaction of both complexes with DNA takes place through the minor groove, in agreement with docking studies. The [Cu2L2]4+ complex is quite efficient in promoting the cleavage of the double-stranded pUC19 plasmid DNA, by favoring the conversion of the supercoiled form to the nicked form following a hydrolytic mechanism.

Dinuclear μ‐Phenoxo and μ‐Hydroxo Bridged Copper Complexes Exhibiting Oxidation of Phenols and Isoelectronic Compounds: Cytotoxicity and Evidences for Cellular Apoptosis

AbstractThe compartmental pentadentate ligands HLR (where R=Me, OMe, tBu) have been utilized for the synthesis of dinuclear hydroxo and phenolato bridged copper complexes viz. [Cu2(LMe)(μ‐OPh)(μ‐OH](ClO4)2]CH3CN; (1), [Cu2(LOMe)(μ‐OPh)(μ‐OH)(μ‐OH2)](ClO4)2.CH3CN; (2) and [Cu2(LtBu)(μ‐OPh)(μ‐OH)(OH2)](ClO4)2; (3). All the three complexes were characterized by various spectroscopic techniques. Tyrosinase activity was studied by insitu reaction of Cu(I) and ligands exploiting 2,4‐di‐tert‐butyl phenol as substrate. The phenolic substrate (2,4‐DTBP) underwent radical mediated C−C coupling reaction with no insertion of molecular oxygen in contrast to the oxidative aromatic hydroxylation reaction. The catalytic activities of complexes 1–3 on diphenols and related substrate molecules were investigated and all three complexes efficiently oxidize 3,5‐di‐tert‐butylcatechol. Treatment of copper complexes on o‐aminophenol and o‐phenylene diamine resulted in the oxidative coupling of substrate molecules, leading to the formation of phenoxazinone and diaminophenazine respectively. Furthermore, oxidative properties of reported copper complexes were studied in relevance to their ability in inducing apoptotic cell death. Remarkably, all three copper complexes (1, 2 and 3) exhibited excellent cytotoxic activity towards MCF‐7 cell lines (human breast cancer cell lines) with IC50 values of 1.6 μM, 2.0 μM and 0.65 μM respectively and carried out self activated DNA cleavage of plasmid pUC18 DNA.

Triclosan at environmental concentrations can enhance the spread of extracellular antibiotic resistance genes through transformation

The dissemination of antibiotic resistance mediated by horizontal transfer of antibiotic resistance genes (ARGs) is exacerbating the global antibiotic crisis. Currently, little is known about whether non-antibiotic, anti-microbial (NAAM) chemicals are associated with the dissemination of ARGs in the environment. In this study, we aimed to evaluate whether a ubiquitous NAAM chemical, triclosan (TCS), is able to promote the transformation of plasmid-borne antibiotic resistance genes (ARGs). By using the plasmid pUC19 carrying ampicillin resistance genes as the extracellular ARG and a model microorganism Escherichia coli DH5ɑ as the recipient, we found that TCS at environmentally detected concentrations (0.2 μg/L to 20 μg/L) significantly enhanced the transformation of plasmid-borne ARGs into E. coli DH5ɑ for up to 1.4-fold. The combination of phenotypic experiments, genome-wide RNA sequencing and proteomic analyses revealed that TCS exposure stimulated the reactive oxygen species (ROS) production for 1.3- to 1.5-fold, induced bacterial membrane damage and up-regulated the translation of outer membrane porin. Moreover, general secretion system Sec (1.4-fold), twin arginine translocation system Tat (1.2-fold) and type IV pilus secretion systems (2.5-fold) were enhanced by TCS, which might contribute to the DNA searching/capture by pilus. Together, TCS might increase the transformation frequency of ARGs into E. coli DH5ɑ by ROS over-production, damaging cell membrane barrier, mediating the pilus capture of plasmid and the translocation of plasmid via cell membrane channels. This study reports that TCS could accelerate the transformation of extracellular ARGs to competent bacteria at environmentally relevant concentrations. The findings advance our understanding of the fate of ARGs in ecosystems and call for risk assessments of NAAM chemicals on disseminating antibiotic resistance.

An endophytic Penicillium oxalicum isolated from Citrus limon possesses antioxidant and genoprotective potential.

This study aimed at isolating endophytic fungi from Citrus limon (L.) possessing antioxidative and genoprotective potential. Endophytic fungi were screened for antioxidant activity using 2,2-diphenyl,1-picryl hydrazyl radical scavenging assayand maximum activity (79·70%) was exhibited by culture MP1 identified to be Penicillium oxalicum on the basis of morphological and molecular characteristics. The ethyl acetate extract of MP1 was subjected to silica column chromatography followed by LH 20 column chromatography for purification of active metabolites. The partially purified active fraction of P. oxalicum MP1 possessed good antioxidant activity as detected using various assays. It also exhibited a strong DNA damage protection potential on pUC19 plasmid DNA treated with Fenton reagent. On exposure to active fraction of MP1 significant reduction (P<0·05) in nuclear deformities (like nuclear buds, micronuclei, nuclear ridges and binucleated cells) was observed in human lymphocytes pretreated with a toxic concentration of H2 O2 . In vivo genoprotectivity studies were conducted in fresh water fish Channa punctatus pretreated with a damaging compound 4-nonyl phenol. The active fraction of P. oxalicum MP1 caused a reduction of 94·7 and 66·60% in micronuclei and aberrant cell formation, respectively. A significant reduction (P<0·05) in tail length and tail DNA parameters was also observed in comet assay. The endophytic P. oxalicum isolated in this study has the potential to produce metabolites possessing antioxidant and genoprotective activities. The isolated culture can be exploited in the field of therapeutics by virtue of its in vitro and in vivo genoprotective potential.

In vitro and in silico analysis of the effect of fluconazole, an antifungal drug, on DNA

Fluconazole is an important antifungal drug used worldwide for the treatment of peritonitis and cryptococcal meningitis, urinary tract infections, esophageal tract infections and vaginal candidiasis. In this research, pUC19 plasmid DNA was treated with different concentrations of fluconazole in the presence of ascorbic acid, H2O2, iron, iron plus H2O2, copper and copper plus ascorbic acid, followed by agarose gel electrophoresis. Fluconazole?DNA interactions were investigated by UV?Vis spectrophotometeric titration and in silico methods. Even in the presence of an oxidative agent or a reducing agent, at higher concentrations of fluconazole, double stranded DNA was not broken more than usually found in human plasma. Fluconazole concentrations ? 88 ?M could protect 46 ?M of DNA against hydroxyl radicals produced in the reaction between 1.5 mM of FeSO4 and 6 mM of H2O2 while drug concentrations ? 44 ?M could not provide the protection. In addition, the drug could not protect DNA against ROS originating from the reaction between copper and ascorbic acid. The binding constant of fluconazole?DNA in UV-Vis spectrophotometry analysis and docking analysis was estimated to be 1.087?103 and 6.22?105 M-1, respectively.

DNA Interaction, in vitro Antibacterial and Cytotoxic Activities of Ru(III) Heterochelates

Ruthenium(III) complexes [Ru(bphtpy)(PPh3)Cl3] (bphfpy = diphenylfuranylpyridine derivatives) were synthesized and characterized by LCMS, IR spectroscopy, elemental analysis and magnetic measurements. All the complexes were screened for their antibacterial activity in terms of minimum inhibitory concentration against two Gram-positive and three Gram-negative bacterial species. DNA binding study by absorption titration and viscosity measurement shows that complexes bind in an intercalating mode, which is also confirmed by molecular docking. All the complexes were also screened for the DNA nuclease property of pUC19 plasmid DNA. The cytotoxicity study of the synthesized complexes was performed to elucidate the LC50 values to find out the toxicity profile of the complexes.

Anticancer activity and DNA interaction of ruthenium acetate clusters bearing azanaphthalene ancillary ligands

Six triruthenium complexes (1–6) ([Ru3O(CH3COO)6(L)3]PF6, L = quinazoline (qui) (complex 1), 5-nitroisoquinoline (5-nitroiq) (complex 2), 5-bromoisoquinoline (5-briq) (complex 3), isoquinoline (iq) (complex 4), 5-aminoisoquinoline (5-amiq) (complex 5), and 5,6,7,8-tetrahydroisoquinoline (thiq) (complex 6)) have been studied regarding their anti-cancer activity (B16F10 and A549 cells) and their interactions with fish sperm DNA (fs-DNA). The crystallographic data of complex 4 show the typical triangular geometry, where the three isoquinoline ligands display different degrees of co-planarity with the [Ru3O] unit. In the range of 2 to 200 μM, these complexes make B16F10 cells less viable. The overall cytotoxicity order is complex 4 > complex 6 > complex 2 > complex 5 > complex 1. The two first complexes have IC50 of 10 and 50 μM, respectively, being less toxic to L929 non-tumoral cells. The complexes can displace ethidium bromide (EB) from DNA, but the intercalation constant values (Kapp ~105 M−1) are low due to the smaller size of the azanaphtalene ligands as compared to classic intercalating molecules. Complex 5 provides the highest constants in both the EB displacement (Kapp = 12.5 × 105 M−1) and direct spectrophotometric titration with fs-DNA (Kb = 6.3 × 104 M−1) assays, possibly because its amino group can assist interaction with DNA through hydrogen bonds. Hypochromism of complexes IC band was observed (T = 310 K), suggesting hydrophobic contributions. Low values of Kapp and circular dichroism spectra for DNA-compound 6 mixtures, suggest semi-intercalative and major groove binding interaction modes. Compared to cisplatin, incubation of complexes 1–6 with plasmid pUC19 shows no DNA cleavage. Results for the interaction with DNA do not correlate with cytotoxicity, which suggests that DNA is not the pharmacological target of this class of compounds.

RADIATION-INDUCED PLASMID DNA DAMAGE: EFFECT OF CONCENTRATION AND LENGTH.

Plasmid DNA is commonly used as a simpler substitute for a cell in studies of early effects of ionizing radiation because it allows to determine yields of primary DNA lesions. Experimental studies often employ plasmids of different lengths, in different concentrations in the aqueous solution. Influence of these parameters on the heavy-ion induced yields of primary DNA damage has been studied, using plasmids pUC19 (2686bp), pBR322 (4361bp) and pKLAC2 (9107bp) in 10 and 50ng/μl concentration. Results demonstrate the impact of plasmid length, while no significant difference was observed between the two concentrations. The uncertainty of the results is discussed.

Enhanced Gene Delivery in Bacterial and Mammalian Cells Using PEGylated Calcium Doped Magnetic Nanograin.

BackgroundBeyond viral carriers which have been widely used in gene delivery, non-viral carriers can further improve the delivery process. However, the high cytotoxicity and low efficiency impedes the clinical application of non-viral systems. Therefore, in this work, we fabricated polyethylene glycol (PEG) coated, calcium doped magnetic nanograin (PEG/Ca(II)/Fe3O4) as a genome expression enhancer.MethodsMonodisperse magnetic nanograins (MNGs) with tunable size were synthesized by a solvothermal method. The citrate anions on the spherical surface of MNGs capture Ca2+ ions by an ion exchange process, which was followed by surface capping with PEG. The synthesized PEG/Ca(II)/Fe3O4 was characterized using Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS) spectra, vibrating sample magnetometer (VSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). MTT test was utilized to assess the toxicity of PEG/Ca(II)/Fe3O4. Real time qPCR was applied for quantification of gene expression.ResultsDLS spectra and TEM images confirmed a thin layer of PEG on the nanocarrier surface. Shifting the zeta potential in the biological pH window from −23.9 mV (for Fe3O4) to ≈ +11 mV (for PEG/Ca(II)/Fe3O4) confirms the MNGs surface protonation. Cytotoxicity results show that cell viability and proliferation were not hindered in a wide range of nanocarrier concentrations and different incubation times.ConclusionPEGylated calcium doped magnetic nanograin enhanced PUC19 plasmid expression into E. Coli and GFP protein expression in HEK-293 T cells compared to control. A polymerase chain reaction of the NeoR test shows that the transformed plasmids are of high quality.

Development of a real-time loop-mediated isothermal amplification (LAMP) assay and visual LAMP assay for detection of African swine fever virus (ASFV)

African swine fever (ASF) is a fatal disease caused by a virus in domestic pigs. In this study, a real-time loop-mediated isothermal amplification (LAMP) assay and visual LAMP assay were developed for the detection of African swine fever virus (ASFV). LAMP primers targeting the p10 gene of ASFV were designed, the LAMP reaction system was optimized with plasmid pUC57 containing the p10 gene sequence, and the specificities of the real-time LAMP and the visual assays were tested with the DNA or cDNA of pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV) and ASFV, as well as the plasmid pUC57 containing the p10 gene sequence. The detection limits were determined using a serial dilution of plasmid pUC57 containing the p10 gene sequence. Our results showed that the LAMP assays could accurately and specifically detect ASFV with a detection limit of 30 copies per μl−1 of pUC57 containing p10 gene sequence. In addition, the LAMP assays were further evaluated using various genotypes of ASFV strains. Furthermore, the LAMP assays are comparable with the well-established real-time PCR assay. This study provides promising solutions for facilitating preliminary and cost-effective surveillance for prevention and control of ASFV.

Functional screening of a soil metagenome for DNA endonucleases by acquired resistance to bacteriophage infection.

Endonucleases play a crucial role as reagents in laboratory research and diagnostics. Here, metagenomics was used to functionally screen a fosmid library for endonucleases. A fosmid library was constructed using metagenomic DNA isolated from soil sampled from the unique environment of the Kogelberg Nature Reserve in the Western Cape of South Africa. The principle of acquired immunity against phage infection was used to develop a plate-based screening technique for the isolation of restriction endonucleases from the library. Using next-generation sequencing and bioinformatics tools, sequence data were generated and analysed, revealing 113 novel open reading frames (ORFs) encoding putative endonuclease genes and ORFs of unknown identity and function. One endonuclease designated Endo52 was selected from the putative endonuclease ORFs and was recombinantly produced in Escherichia coli Rosetta™ (DE3) pLysS. Endo52 was purified by immobilised metal affinity chromatography and yielded 0.437g per litre of cultivation volume. Its enzyme activity was monitored by cleaving lambda DNA and pUC19 plasmid as substrates, and it demonstrated non-specific endonuclease activity. In addition to endonuclease-like genes, the screen identified several unknown genes. These could present new phage resistance mechanisms and are an opportunity for future investigations.

Screening of Phytochemical Compounds and Assessment of Antiurolithiatic Activity of Pisonia Alba Leaves Extracts against DNA Damage

This work investigate the Phytochemical constituents of Pisoniaalba (PA) leaves using standard protocols and to determine its DNA damage inhibition and Antiurolithiatic capability. Dried leaves of Pisoniaalba were used to screen and quantify the phyto constituents in different organic solvents extracts that are analysed by GCMS, FTIR and HPTLC. In the presence of PUC18 plasmid DNA, the DNA damage inhibition assay was performed by photolysing H2O2 with UV radiation and agarose gel electrophoresis with irradiated DNA. The invivo antiurolithic assay was performed by nucleation method. Pisoniaalbain's phytochemical analysis reveals the existence of secondary metabolite components including glycosides, resins, phenols, terpenoids, flavonoids, tannins, steroids, and alkaloids, etc.. ELPA express the maximum quantity of phenols, flavonoids, alkaloids and terpenoids which were 30.44 ±0.65 mg TAE/g extract, 28.51 ± 1.19 mg RE/g extract, 28.08 ± 0.08 mg of AE/g of extract, 29.94 ± 0.32 mg RE/g extract respectively. The GC-MS assessment findings confirmed the presence of nine ELPA phyto compounds accompanied by eleven ALPA phytoc ompounds. It was also observed that Pisoniaalba leaves prevents DNA damage in UV and H2O2 treated plasmid DNA. The invitro Antiurolithiatic research indicates that ELPA was more efficient in blocking calcium oxalate nucleation. From the obtained results it was proven that the Pisonia alba leaves has potential secondary metabolites with therapeutic role mainly against urolithiasis. Both invitro and in vivo analysis demonstrates that ELPA significantly reduces crystal formation that are causative of renal stones. These evidences suggest that Pisoniaalbacan be further investigated for the prevention and treatment of urolithiais

Oxidative DNA Cleavage, Formation of μ-1,1-Hydroperoxo Species, and Cytotoxicity of Dicopper(II) Complex Supported by a p-Cresol-Derived Amide-Tether Ligand.

Metal complexes to promote oxidative DNA cleavage by H2O2 are desirable as anticancer drugs. A dicopper(II) complex of known p-cresol-derived methylene-tether ligand Hbcc [Cu2(bcc)]3+ did not promote DNA cleavage by H2O2. Here, we synthesized a new p-cresol-derived amide-tether one, 2,6-bis(1,4,7,10-tetrazacyclododecyl-1-carboxyamide)-p-cresol (Hbcamide). A dicopper(II) complex of the new ligand [Cu2(μ-OH)(bcamide)]2+ was structurally characterized. This complex promoted the oxidative cleavage of supercoiled plasmid pUC19 DNA (Form I) with H2O2 at pH 6.0-8.2 to give Forms II and III. The reaction was largely accelerated in a high pH region. A μ-1,1-hydroperoxo species was formed as the active species and spectroscopically identified. The amide-tether complex is more effective in cytotoxicity against HeLa cells than the methylene-tether one.

Development of high-throughput genotyping method of all 18 HR HPV based on the MALDI-TOF MS platform and compared with the Roche Cobas 4800 HPV assay using clinical specimens

BackgroundTo develop a new 18 high-risk human papillomavirus (HR HPV) detection and genotyping assay, which is important to evaluate the risk degree of HR HPV for causing cancers.MethodsAll 18 HR HPV and β-globin relative DNA fragments were synthesized and cloned to a plasmid pUC57 to obtain their recombinant plasmids. Based on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform, each of the 18 HR HPV genotypes were investigated using their constructed recombinant plasmids. The new 18 HR HPV genotyping assay was tested using 356 clinical specimens and the results were compared to ones detected by the Roche Cobas 4800 HPV assay (Cobas). The discrepant results between two assays were resolved by sequencing and genotyping methods.ResultsThe new 18 HR HPV MALDI-TOF MS genotyping assay was developed using HPV recombination plasmids. The sensitivity was 103 to 102 copies/reaction for the all 18 HR HPV. This new developed HR HPV genotyping test was used to detect the clinical specimens. When the results on clinical samples detected by the new MALDI-TOF MS HPV test were compared with ones detected by the Roche Cobas 4800 HPV assay in terms of 14 HR HPV, the concordance was 80.1% (kappa coefficient, 0.60; 95% confidence interval [CI], 0.52–0.69). The discrepant results were resolved by sequencing and genotyping and suggests that the developed HR HPV assay is more sensitive and specific.ConclusionsThe new developed 18 HR HPV detection method based on MALDI-TOF MS platform is a high-throughput assay for the all 18 HR HPV genotypes and a powerful complement to current detection methods.

Effects of CO2 on the transformation of antibiotic resistance genes via increasing cell membrane channels

The increase of CO2 concentration in the atmosphere, water and soil environment can lead to the changes in microbial activities. However, the transformation of antibiotic resistance genes has not been investigated in the presence of higher levels of CO2. This study demonstrated that CO2 facilitated the transformation of pUC19 plasmid, carrying ampicillin resistance genes, into Escherichia coli. Mechanism studies revealed that the type Ⅱ secretion system, type Ⅳ pilus and some other secretion systems were enhanced by CO2, leading to DNA capture by pilus, larger cell pore sizes and more cell membrane channels. CO2 also increased reactive oxygen species production, leading to SOS response and cell membrane damage. Besides, changes in intracellular Fe2+ and Mg2+ concentrations induced by CO2 caused greater damage to the cell membrane and enhanced secretion systems, respectively. Overall, increased CO2 provided more cell membrane channels for plasmid uptake and led to higher transformation efficiencies. The potential risk of a natural factor on the transformation of ARGs was first studied in this study, which helps us understand the fate of ARGs in ecosystems. As the carbon emission will continue to grow and enhance the enrichment of CO2 in water and soil, the findings revealed a more severe public health issue under the background of carbon emission and CO2 leakage.

Capsular Polysaccharide Biosynthesis from Recombinant E. coli and Chondroitin Sülfate Production

Chondroitin sulfate (CS) is an important biomedical product. CS is the basic structural component of the mammalian extracellular matrix and is widely used in many applications in the fields of medicine, veterinary medicine, pharmaceuticals and cosmetics. For CS production, mainly animal sources are used. However, in today's conditions, due to various risks and artificial synthesis, there has been an increase in alternative sources of production methods for CS, instead of using animal resources. In this study as a powerful alternative microbial production of CS has been targeted. By using recombinant E. coli strains to integrate VHb /vgb+ and kfo+ systems, the aim was to obtain high purity CS from reliable biotechnological processes. Plasmid pUC8:15 bearing the vgb gene region, and plasmid pETM6-PACF carrying the kfoA, kfoC and kfoF genes responsible for chondroitin synthesis, were transferred to E. coli bacteria. Microbial CS was obtained by adding sulfate groups to chondroitin acquired after the treatments. The results were confirmed by HPLC and NMR analyses. The product, compared to its counterparts, was found to be an effective drug, potentially with a low molecular weight value.

Pyrazole-based azo-metal(II) complexes as potential bioactive agents: synthesis, characterization, antimicrobial, anti-tuberculosis, and DNA interaction studies

In the present investigation, the bioactive azo-dye ligand 1,5-dimethyl-4-[(3-methyl-5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-4-yl)diazenyl]-2-phenyl-1,2-dihydro-3H-pyrazol-3-one (L) and its transition metal complexes have been synthesized and characterized by various physical and spectroscopic techniques to elucidate their geometrical structures. The molar conductivity measurements confirmed the non-electrolytic nature of the complexes. EPR spectroscopy indicated that the metal complexes are monomeric in nature and exhibited octahedral geometry. The redox behavior of the copper complex was studied by the cyclic voltammetric technique in DMF solution and the complex showed well-established redox behavior at a scan rate of 0.05 V s−1. The antimicrobial activity of the ligand and its metal complexes were screened against Escherichia coli, Bacillus subtilis, Aspergillus niger, and Candida albicans, and the results indicated increased activity after coordination of the ligand to the metal ions. The metal complexes exhibited enhanced antitubercular activity after chelation against M. tuberculosis. The DNA-binding experiments showed that the ligand and its metal complexes showed effective binding properties through intercalative mode against CT-DNA. All the synthesized molecules showed partial cleavage of supercoiled plasmid pUC18 DNA.

Expression and Purification of Vaccinia Virus DNA Topoisomerase IB Produced in the Silkworm-Baculovirus Expression System.

Type IB DNA topoisomerases are enzymes to change the topological state of DNA molecules and are essential in studying replication, transcription, and recombination of nucleic acids in vitro. DNA topoisomerase IB from Vaccinia virus (vTopIB) is a 32kDa, type I eukaryotic topoisomerase, which relaxed positively and negatively supercoiled DNAs without Mg2+ and ATP. Although vTopIB has been effectively produced in E. coli expression system, no studies remain available to explore an alternative platform to express recombinant vTopIB (rvTopIB) in a higher eukaryote, where the one can expect post-translational modifications that affect the activity of rvTopIB. Here in this study, rvTopIB with N-terminal tags was constructed and expressed in a silkworm-baculovirus expression vector system (silkworm-BEVS). We developed a simple two consecutive chromatography purification to obtain highly pure rvTopIB. The final yield of rvTopIB obtained from a baculovirus-infected silkworm larva was 83.25μg. We also evaluated the activity and function of rvTopIB by the DNA relaxation activity assays using a negatively supercoiled pUC19 plasmid DNA as a substrate. With carefully assessing optimized conditions for the reaction buffer, we found that divalent ions, Mg2+, Mn2+, Ca2+, as well as ATP stimulate the DNA relaxation activity by rvTopIB. The functional and active form of rvTopIB, together with the yields of the protein we obtained, suggests that silkworm-BEVS would be a potential alternative platform to produce eukaryotic topoisomerases on an industrial scale.