DNA Protection Research Articles

Three-Dimensional Surface-Enhanced Raman Scattering Platform with Hotspots Built by a Nano-mower for Rapid Detection of MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the greatest threats to human health due to its strong drug resistance, wide distribution range, and high infection rate. Rapid identification of MRSA strains is very important for accurate diagnosis and early treatment of MRSA infections. Here, we introduced an Exo III-assisted nanomotor mower to build 3D hotspots for rapid detection of MRSA by surface-enhanced Raman scattering (SERS). As the bacteria bound to the aptamer, two trigger chains were released from the double-stranded structure, and the nano-mowers were activated by opening a hairpin probe on gold nanoparticles (AuNPs). With the continued cleavage of Exo III and cyclic release of the trigger chain, multiple hairpin DNAs on the AuNPs were cleaved to increase the motor power. The resulting nano-mower continued slicing protective DNA from larger AuNPs, exposing the AuNPs. Without the protection of DNA, Mg2+ in the buffer induced spontaneous aggregation of the AuNPs, and a large number of hotspots were formed for SERS measurements. Under optimal conditions, MRSA can be detected within 40 min, and the concentration of MRSA showed a good linear relationship with the SERS intensity at 1342 cm-1, with a limit of detection as low as 1 CFU/mL and a wide linear range (100 to 107 CFU/mL). This strategy creates a rapid bacterial detection method that performs well on actual samples utilizing portable Raman spectroscopy instruments, with potential applications in food detection, water detection, clinical treatment, and diagnosis.

Amelioration of Radiation-Induced Cell Death in Neuro2a Cells by Neutralizing Oxidative Stress and Reducing Mitochondrial Dysfunction Using N-Acetyl-L-Tryptophan.

The deleterious effects of ionizing radiation on the central nervous system (CNS) are poorly understood. Radiation exposure during an accidental nuclear explosion, nuclear war, or radiotherapy causes severe brain damage. As a result, the current work is carried out to assess the radioprotective potential of N-acetyl-L-tryptophan (L-NAT) in neuronal cells. Radiation-induced cell death and its amelioration by L-NAT pretreatment were investigated using MTT, SRB, CFU, and comet assays. Flow cytometric and microscopic fluorescence assays were used to investigate radiation-induced oxidative stress, alteration in mitochondrial redox, Ca2+ homeostasis, depolarization of mitochondrial membrane potential, and its prevention with L-NAT pretreatment. Western blot analysis of Caspase-3, γ-H2aX, p53, ERK-1/2, and p-ERK-1/2 expression was carried out to identify the effects of L-NAT pretreatment on radiation-induced apoptosis and its regulatory proteins expression. The study demonstrated (MTT, SRB, and CFU assay) significant (~80%; p <0.001%) radioprotection in irradiated (LD50 IR dose) Neuro2a cells that were pretreated with L-NAT. In comparison to irradiated cells, L-NAT pretreatment resulted in significant (p <0.001%) DNA protection. A subsequent study revealed that L-NAT pretreatment of irradiated Neuro2a cells establishes oxidative stress by increasing antioxidant enzymes and mitochondrial redox homeostasis by inhibiting Ca2+ migration from the cytoplasm to the mitochondrial matrix and thus protects the mitochondrial membrane hyperpolarization. Caspase-3 and γ-H2aX protein expression decreased, while p-ERK1/2 and p53 expression increased in L-NAT pretreated irradiated cells compared to irradiated cells. Hence, L-NAT could be a potential radioprotective that may inhibit oxidative stress and DNA damage and maintain mitochondrial health and Ca2+ levels by activating p-ERK1/2 and p53 expression in Neuronal cells.

Thioredoxin domain-containing protein 12 (TXNDC12) in red spotted grouper (Epinephelus akaara): Molecular characteristics, disulfide reductase activities, and immune responses

Thioredoxins are small ubiquitous redox proteins that are involved in many biological processes. Proteins with thiol-disulfide bonds are essential regulators of cellular redox homeostasis and diagnostic markers for redox-dependent diseases. Here, we identified and characterized the thioredoxin domain-containing protein 12 (EaTXNDC12) gene in red spotted grouper (Epinephelus akaara), evaluated transcriptional responses, and investigated the activity of the recombinant protein using functional assays. EaTXNDC12 is a 19.22-kDa endoplasmic reticulum (ER)-resident protein with a 522-bp open reading frame and 173 amino acids, including a signal peptide. We identified a conserved active motif (66WCGAC70) and ER retention motif (170GDEL173) in the EaTXNDC12 amino acid sequence. Relative EaTXNDC12 mRNA expression was analyzed using 12 different tissues, with the highest expression seen in brain tissue, while skin tissue showed the lowest expression level. Furthermore, mRNA expression in response to immune challenges was analyzed in the head kidney, blood, and gill tissues. EaTXNDC12 was significantly modulated in response to bacterial endotoxin lipopolysaccharide (LPS), nervous necrosis virus (NNV), and polyinosinic:polycytidylic acid (poly(I:C)) challenges in all of the tested tissues. Recombinant EaTXNDC12 (rEaTXNDC12) displayed antioxidant ability in an insulin reductase assay, and a capacity for free radical inhibition in a 2,2-diphenyl-1-picryl-hydrazyl-hydrate assay. In addition, a DNA nicking assay revealed that purified rEaTXNDC12 exhibited concentration-dependent DNA protection activity, while results from 2-hydroxyethyl disulfide and L-dehydroascorbic assays indicated that rEaTXNDC12a possesses reducing ability. Furthermore, fathead minnow (FHM) cells transfected with EaTXNDC12-pcDNA demonstrated significantly upregulated cell survival against H2O2-induced apoptosis. Collectively, the results of this study strengthen our knowledge of EaTXNDC12 with respect to cellular redox hemostasis and immune regulation in Epinephelus akaara.

Phenolics and Volatile Compounds of Fennel (Foeniculum vulgare) Seeds and Their Sprouts Prevent Oxidative DNA Damage and Ameliorates CCl4-Induced Hepatotoxicity and Oxidative Stress in Rats.

Researchers recently focused on studying the nutritional and functional qualities of sprouts generated from seeds. The current study investigated the total phenolic content (TPC), total flavonoids (TF), total flavonols (TFL), antioxidant activity (AOA), specific phenolic acids, and volatile chemicals in fennel seeds (FS) and fennel seed sprouts (FSS). The oxidative DNA damage prevention activity of selected FS and FSS extracts against DNA was examined. Consequently, the antioxidative stress potential of FS and FSS extracts at 300 and 600 mg kg-1 on CCl4-induced hepatotoxicity and oxidative stress in rats weas investigated. The liver's functions and oxidative stress biomarkers in rat blood were examined. FSS exhibited rich phytochemical content such as TPC, TF, TFL, and AOA with altered phenolics and volatiles. HPLC identified nineteen compounds of phenolic acids and their derivatives in FS. Thirteen phenolics and six flavonoids were predominantly identified as Vanillic acid and Kaempferol, respectively. GC-MS analysis identified fifty and fifty-one components in FS and FSS, respectively. The predominant component was Benzene, [1-(2-propenyloxy)-3-butenyl] (trans-Anethole) (38.41%), followed by trans-Anethole (Benzene, 1-methoxy-4-(2-propenyl)) (23.65%), Fenchone (11.18%), and 1,7-Octadiene, 2-methyl-6-methylene- Cyclohexene (7.17%). Interestingly, α-Pinene, Fenchone, trans-Anethole (Benzene, 1-methoxy-4-(2-propenyl)), 4-Methoxybenzaldehyde (4-Anisaldehyde), Benzeneacetic acid, α-hydroxy-4-methoxy, and Nonacosane contents were increased. While Dillapiole, 7-Octadecenoic acid, and methyl ester were newly identified and quantified in FSS. The oxidative DNA damage prevention capability of FSS and FS extracts indicated remarkable DNA protection. Administrating FS and FSS extracts at 300 and 600 mg kg-1 ameliorated AST, ALT, and ALP, as well as GSH, CAT, MDA, and SOD, in a dose-dependent manner. The most efficient treatment of FS or FSS was using a dose of 600 mg Kg-1, which recorded an improvement rate of 20.77 and 24.17, 20.36 and 24.92, and 37.49 and 37.90% for ALT, AST, and ALP, respectively. While an improvement rate of 40.08 and 37.87%, 37.17 and 46.52%, 114.56 and 154.13%, and 66.05 and 69.69% for GSH, DMA, CAT, and SOD compared to the CCl4-group, respectively. The observed protection is associated with increased phenolics and volatiles in F. vulgare. Therefore, FS and FSS are recommended as functional foods with bioactive functionality, health-promoting properties, and desired prevention capabilities that may help prevent oxidative stress-related diseases.

Are Reactive Oxygen Species (ROS) the Main Mechanism by Which Copper Ion Treatment Degrades the DNA of Mycobacterium avium subsp. paratuberculosis Suspended in Milk?

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of paratuberculosis. This pathogen is able to survive adverse environmental conditions, including the pasteurization process. Copper, a well-studied metal, is considered an important antibacterial tool, since it has been shown to inactivate even MAP in treated milk through unknown mechanisms. The aim of the present study is to show the effect of copper ions, and reactive oxygen species (ROS) generated in response to oxidative stress, on the damage to MAP DNA when exposed to a copper ion challenge in cow's milk. Spiked milk with different MAP bacterial loads was supplemented with blocking agents. These were either the copper chelators ethylenediaminetetraacetic acid (EDTA) and batocuproin (BCS) or the ROS quenchers D-mannitol, gallic acid and quercetin. The DNA protection, MAP viability and ROS production generated after exposure to a copper challenge were then measured. In a bacterial load of 104 cells mL-1, blocking effects by both the copper chelators and all the ROS quenchers offered significant protection to MAP DNA. In a concentration of 102 cells mL-1, only D-mannitol and a mix of quenchers significantly protected the viability of the bacteria, and only at a concentration of 106 cells mL-1 was there a lower production of ROS when supplementing milk with gallic acid, quercetin and the mix of quenchers. Based on these findings, it may be concluded that MAP DNA damage can be attributed to the combined effect of the direct copper ions and ROS generated. Nevertheless, taking into account the antioxidant environment that milk provides, the direct effect of copper could play a prominent role.

Radiation Protection of Yeast Cells and DNA by Epigallocatechin Gallate and Epicatechin

Radiation Protection of Yeast Cells and DNA by Epigallocatechin Gallate and Epicatechin

Targeted co-delivery of paclitaxel and anti P-gp shRNA by low molecular weight PEI decorated with L-3,4-dihydroxyphenylalanine.

Co-delivery of small chemotherapeutic molecules and nucleic acid materials via targeted carriers has attracted great attention for treatment of resistant tumors and reducing adverse effects. In this study, a targeted carrier for co-delivery was prepared based on low-molecular weight polyethylenimine (LMW PEI). Paclitaxel (PTX) was covalently conjugated onto PEI via a succinate linker. The PEI conjugate was decorated with L-DOPA in order to target large neutral amino acid transporter-1 (LAT-1) that is over-expressed on various cancer cells. This PEI conjugate was complexed with human ABCB1 shRNA plasmid to down-regulate the expression of P-glycoprotein, as one of the major efflux pumps inducing resistance against chemotherapeutics. The formation of PEI conjugate enhanced the solubility of PTX and resulted in the condensation and protection of plasmid DNA in nanosized polyplexes. The results of targeted delivery into the cells demonstrated that PEI conjugate transferred the payloads to the cells over-expressing LAT-1 transporter, while the biological effects on the cells lacking the transporter was negligible. Also, shRNA-mediated down-regulation of P-gp led to the increase of toxic effects on the cells over-expressing P-gp. This study suggests a promising approach for co-delivery of small molecules and nucleic acid materials in a targeted manner for cancer therapy.

EPICATECHIN IMPROVES FROZEN SPERM VITALITY BY ITS ANTIOXIDANT AND CRYOPROTECTIVE ACTIONS

This study served to elucidate the effects of three selected doses of epicatechin (EPI; 25, 50 or 100 μmol/L) on the motion and oxidative profile of bovine spermatozoa subjected to the process of cryopreservation. Furthermore, we focused on the impact of EPI on the expression patterns of heat shock proteins 90 and 70 as well as the BAX and Bcl-2 proteins involved in the process of apoptosis. The experimental samples were compared with a native control as well as specimens cryopreserved in the absence of any supplement. Our dada indicates that particularly 50 and 100 μmol/L EPI assured a higher preservation of the sperm motility (p&lt;0.001) in comparison with the cryopreserved control. The best protection of the sperm DNA (p&lt;0.05), lipids (p&lt;0.05) and proteins (p&lt;0.01) were observed in the samples exposed to 100 μmol/L EPI in comparison to untreated frozen samples. The presence of higher EPI concentrations in the cryopreservation medium led to significant stabilization of the heat shock protein 90 (p&lt;0.01 in case of 50 μmol/L EPI; p&lt;0.001 with respect to 100 μmol/L EPI) as well as a higher maintenance of the BAX:Bcl-2 ratio (p&lt;0.001) when compared to the untreated frozen control. In summary, we may assume that EPI exhibits antioxidant properties which enable the molecule to prevent excessive damage to biomolecules essential for the cell survival and offer an additional layer of protection to spermatozoa exposed to low temperatures, all of which may be translated into a higher post-thaw sperm motility and activity.

Design and synthesis of multi-targeted nanoparticles for gene delivery to breast cancer tissues.

Biocompatibility of nanoparticles is the most essential factor in their use in clinical applications. In this study, hyperbranched spermine (HS), hyperbranched spermine-polyethylene glycol-folic acid (HSPF), and hyperbranched spermine-polyethylene glycol-glucose (HSPG) were synthesized for DNA protection and gene delivery to breast cancer cells. The synthesis of HSPG and HSPF was confirmed using proton nuclear magnetic resonance (H-NMR), Fourier-transform infrared spectroscopy (FTIR), and thermogravimetric analysis (TGA) spectroscopy. The HS/DNA, HSPF/DNA, HSPG/DNA, and hyperbranched spermine-polyethylene glycol-folic acid/glucose/DNA (HSPFG/DNA) nanoparticles were prepared by combining different concentrations of HS, HSPF, and HSPG with the same amount of DNA. The ability of HS, HSPF, and HSPG to interact with DNA and protect it against plasm digestion was evaluated using agarose gel. Moreover, in vivo and in vitro biocompatibility of HSPF/DNA, HSPG/DNA, and HSPFG/DNA was investigated using MTT assay and calculating weight change and survival ratio of BALB/c mice, respectively. The results of agarose gel electrophoresis showed that HS, HSPF, and HSPG have the high ability to neutralize the negative charge of DNA and protect it against plasma degradation. The results of in vivo cytotoxicity assay revealed that the HSPF/DNA, HSPG/DNA, and HSPFG/DNA nanoparticles have good biocompatibility on female BALB/c mice. In vitro and in vivo transfection assays revealed that functionalization of the surface of HS using polyethylene glycol-folic acid (HSPF) and polyethylene glycol-glucose (HSPG) significantly increases gene delivery efficiency in vitro and in vivo. These results also showed that gene transfer using both HSPF and HSPG copolymers increases gene transfer efficiency compared to when only one of them is used. The HSPFG/DNA nanoparticles have a high potential for use in therapeutic applications because of their excellent biocompatibility and high gene transfer efficiency to breast cancer tissue.

Genomic Insights into the Radiation-Resistant Capability of Sphingomonas qomolangmaensis S5-59T and Sphingomonas glaciei S8-45T, Two Novel Bacteria from the North Slope of Mount Everest.

Mount Everest provides natural advantages to finding radiation-resistant extremophiles that are functionally mechanistic and possess commercial significance. (1) Background: Two bacterial strains, designated S5-59T and S8-45T, were isolated from moraine samples collected from the north slope of Mount Everest at altitudes of 5700m and 5100m above sea level. (2) Methods: The present study investigated the polyphasic features and genomic characteristics of S5-59T and S8-45T. (3) Results: The major fatty acids and the predominant respiratory menaquinone of S5-59T and S8-45T were summed as feature 3 (comprising C16:1 ω6c and/or C16:1 ω7c) and ubiquinone-10 (Q-10). Phylogenetic analyses based on 16S rRNA sequences and average nucleotide identity values among these two strains and their reference type strains were below the species demarcation thresholds of 98.65% and 95%. Strains S5-59T and S8-45T harbored great radiation resistance. The genomic analyses showed that DNA damage repair genes, such as mutL, mutS, radA, radC, recF, recN, etc., were present in the S5-59T and S8-45T strains. Additionally, strain S5-59T possessed more genes related to DNA protection proteins. The pan-genome analysis and horizontal gene transfers revealed that strains of Sphingomonas had a consistently homologous genetic evolutionary radiation resistance. Moreover, enzymatic antioxidative proteins also served critical roles in converting ROS into harmless molecules that resulted in resistance to radiation. Further, pigments and carotenoids such as zeaxanthin and alkylresorcinols of the non-enzymatic antioxidative system were also predicted to protect them from radiation. (4) Conclusions: Type strains S5-59T (=JCM 35564T =GDMCC 1.3193T) and S8-45T (=JCM 34749T =GDMCC 1.2715T) represent two novel species of the genus Sphingomonas with the proposed name Sphingomonas qomolangmaensis sp. nov. and Sphingomonas glaciei sp. nov. The type strains, S5-59T and S8-45T, were assessed in a deeply genomic study of their radiation-resistant mechanisms and this thus resulted in a further understanding of their greater potential application for the development of anti-radiation protective drugs.

Bioprospecting roots, stem and leaves extracts of Berberis baluchistanica Ahrendt. (Berberidaceae) as a natural source of biopharmaceuticals

ABSTRACT The present study was designed to explore the biological potential of aerial parts of Berberis baluchistanica. HPLC analysis revealed the highest berberine content in leaves (19453.15 ppm) followed by roots (3225.12ppm) and stem (29.11ppm). Phenolics, flavonoids, tannins, DPPH inhibition activity and total antioxidant capacity are reported in root, leaf and stem extracts. Antihaemolytic assay displayed the highest (97%) inhibition potential of extracts against H2O2-induced haemolysis than heat-induced haemolysis (75%). The highest DNA protection activity was observed in root and leaves extracts while the antimicrobial potential was recorded highest in the leaves extract against Staphylococcus aureus (23.00 ± 1.0 mm) and Klebsiella pneumonia (19.67 ± 0.2 mm). Similarly, brine shrimp cytotoxicity potential was observed highest in root extract (LC50 – 10.04 ppm) and the allelopathic potential was confirmed by radish seed phytotoxicity assay. Overall, results suggest a remarkable nutritional and biological potential B. baluchistanica, especially areal parts can help to preserve the endemic plants by discouraging the use of roots.

In vitro inhibition of glucose gastro-intestinal enzymes and antioxidant activity of hydrolyzed collagen peptides from different species.

The growing value of industrial collagen by-products has given rise to interest in extracting them from different species of animals. Intrinsic protein structure variation of collagen sources and its hydrolysis can bring about different bioactivities. This study aimed to characterize and evaluate the differences in vitro biological potential of commercial bovine (BH), fish (FH), and porcine hydrolysates (PH) regarding their antioxidant and hypoglycemic activities. All samples showed percentages above 90% of protein content, with high levels of amino acids (glycine, proline, and hydroxyproline), responsible for the specific structure of collagen. The BH sample showed a higher degree of hydrolysis (DH) (8.7%) and a higher percentage of smaller than 2kDa peptides (74.1%). All collagens analyzed in vitro showed inhibition of pancreatic enzymes (α-amylase and α-glucosidase), with the potential to prevent diabetes mellitus. The PH sample showed higher antioxidant activities measured by ORAC (67.08 ± 4.23 μmol Trolox Eq./g) and ABTS radical scavenging (65.69 ± 3.53 μmol Trolox Eq./g) methods. For the first time, DNA protection was analyzed to hydrolyzed collagen peptides, and the FH sample showed a protective antioxidant action to supercoiled DNA both in the presence (39.51%) and in the absence (96.36%) of AAPH (reagent 2,2'-azobis(2-amidinopropane)). The results confirmed that the source of native collagen reflects on the bioactivity of hydrolyzed collagen peptides, probably due to its amino acid composition. PRACTICAL APPLICATIONS: Our data provide new application for collagen hydrolysates with hypoglycemiant and antioxidant activity. These data open discussion for future studies on the additional benefits arising from collagen peptide consumption for the prevention of aging complications or hyperglycemic conditions as observed in chronic diseases such as diabetes mellitus type II (DM 2). The confirmation of these results can open new market areas for the use of collagen with pharmacological applications or to produce new supplements. Furthermore, provides a solution for waste collagen from meat industries and adds value to the product.

A biodegradable and cofactor self-sufficient aptazyme nanoprobe for amplified imaging of low-abundance protein in living cells

Despite the progress on the analysis of proteins either in vitro or in vivo, detection and imaging of low-abundance proteins in living cells still remains challenging. Herein, a novel biodegradable and cofactor self-sufficient DNAzyme nanoprobe has been deve-loped for catalytic imaging of protein in living cells with signal amplification capacity. This DNAzyme nanoprobe is constructed by assembling a DNAzyme subunit-containing aptamer hairpin (HP), another DNAzyme subunit strand (DS), and the molecular beacon (MB) substrate strand onto pH-sensitive ZnO@polydopamine nanorods (ZnO@PDA NRs) that work as DNAzyme cofactor suppliers. Such a nanoprobe can facilitate cellular uptake of DNA molecules and protection of them from nuclease degradation as well as release of them in cells by lysosomal acid-triggered dissolution of ZnO@PDA NRs into Zn2+ as DNAzyme cofactor. Upon recognition and binding with the intracellular protein target, the stem of HP is opened, after which the opened HP hybridizes with DS and generates activated DNAzymes. Each activated DNAzyme can catalyze the cleavage of many MB substrates through true enzymatic multiple turnovers, resulting in the separation of the quenched fluorophore/quencher pair labeled in MB and the generation of significantly amplified fluorescence. Using nucleolin (NCL) as a model protein, this nanoprobe enables the analysis of NCL with a detection limit of 1.8 pM, which are at least two orders of magnitude lower than that of non-catalytic imaging probe. Moreover, it could accurately distinguish tumor cells and normal cells by live cell NCL imaging. And the experimental results are also further verified by flow cytometry assays. The developed nanoprobe can be easily extended to detect other biomolecules by the change of their corresponding aptamer sequences, thus providing a promising tool for highly sensitive imaging of low-abundance biomolecules in living cells.

Mactanamide and lariciresinol as radical scavengers and Fe2+ ion chelators – A DFT study

A DFT based kinetic study of OOH radical scavenging potency of mactanamide (MA) and lariciresinol (LA), two natural polyphenols, indicates their nearly equal potential via the proton coupled electron transfer (PCET) mechanism in lipid media. Contribution of C–H bond breaking to this potency is negligible compared to O–H bond breaking, contrary to recent claims. The predicted potency of both compounds is not sufficient to protect biological molecules from oxidative damage in lipid media. In aqueous media, the scavenging potency of MA and LA phenoxide anions via the single electron transfer (SET) mechanism is much higher and may contribute to the protection of lipids, proteins, and DNA from OOH radical damage. Also, MA and LA have the potential to chelate catalytic Fe2+ ions, thus suppressing the formation of dangerous OH radicals via Fenton-type reactions. The monoanionic species of MA and LA show stronger monodentate chelating ability with Fe2+ ion compared to its neutral form. The dianionic specie LA2− exhibited the highest chelation ability with Fe2+ ion via bidentate 1:2 coordination. However, direct radical scavenging and metal chelation could be rarely operative in vivo because MA and LA presumably achieve very low concentrations in systemic circulation.

Double-stranded DNA binding function of RAD51 in DNA protection and its regulation by BRCA2

RAD51 and the breast cancer suppressor BRCA2 have critical functions in DNA double-strand (dsDNA) break repair by homologous recombination and the protection of newly replicated DNA from nucleolytic degradation. The recombination function of RAD51 requires its binding to single-stranded DNA (ssDNA), whereas binding to dsDNA is inhibitory. Using reconstituted MRE11-, EXO1-, and DNA2-dependent nuclease reactions, we show that the protective function of RAD51 unexpectedly depends on its binding to dsDNA. The BRC4 repeat of BRCA2 abrogates RAD51 binding to dsDNA and accordingly impairs the function of RAD51 in protection. The BRCA2 C-terminal RAD51-binding segment (TR2) acts in a dominant manner to overcome the effect of BRC4. Mechanistically, TR2 stabilizes RAD51 binding to dsDNA, even in the presence of BRC4, promoting DNA protection. Our data suggest that RAD51's dsDNA-binding capacity may have evolved together with its function in replication fork protection and provide a mechanistic basis for the DNA-protection function of BRCA2.

The endophytic fungus Penicillium oxalicum isolated from Ligusticum chuanxiong Hort possesses DNA damage-protecting potential and increases stress resistance properties in Caenorhabditis elegans.

The chemical composition and antioxidant activity of extracts (POE) of Penicillium oxalate isolated from Ligusticum chuanxiong Hort have been investigated. However, the biological activity of POE is limited, and its antioxidant, stress resistance and DNA protection effects in vivo are unclear. The current study aims to explore the beneficial effects of POE on DNA damage protection in pBR322 plasmid and lymphocytes and stress resistance in Caenorhabditis elegans. The results showed that POE increased the survival rate of C. elegans under 35°C, UV and H2O2 stress, attenuated ROS and MDA accumulation, and enhanced the activity of some important enzymes (SOD, CTA, and GSH-PX). In addition, the POE-mediated stress resistance involved the upregulation of the expression of the sod-3, sod-5, gst-4, ctl-1, ctl-2, daf-16, hsp-16.1, hsp-16.2, and hsf-1 genes and acted dependently on daf-16 and hsf-1 rather than skn-1. Moreover, POE also reduced lipofuscin levels, but did not prolong the lifespan or damage the growth, reproduction and locomotion of C. elegans. Furthermore, POE showed a protective effect against DNA scission in the pBR322 plasmid and lymphocytes. These results suggested that P. oxalate extracts have significant anti-stress and DNA protection potential and could be potential drug candidates in the pharmaceutical field, thus greatly broadening the understanding of the biological effects of the endophytic fungus P. oxalate.

Phytochemical Analysis and Evaluation of Antimicrobial, Antioxidant, Anti-inflammatory and Antiangiogenic activities of Methanol extract of Urochloa ramosa

Urochloa ramosa is a plant of the wet, seasonally dry and semi-arid tropics. It is also known as Bracharia ramosa or Brown top millet. Qualitative analysis of phytochemicals in methanol extracts were performed by standard methods. Partially purified phenolics from the methanol extract of leaf sample was subjected for different biological activities. In vitro antimicrobial activity was executed on Bacillus subtilis, Staphylococcus epidermidis and Staphylococcus aureus (Gram positive) and Proteus, Salmonella and Escherichia coli (Gram negative) bacteria and Candida albicans (yeast type fungi). Minimum inhibitory concentration was calculated by Resazurin method. In-vitro antioxidant assay was conducted by DPPH radical, NO. radical, superoxide anion radical scavenging assays and OH radical scavenging activity by DNA protection assay using BHT as the reference compound. IC50 value of methanol extract for each of these assays was calculated. Albumin denaturation assay, antiproteinase activity and membrane stabilization were performed to test the anti-inflammatory potential at various concentrations of methanol extract of U.ramosa. In vivo anti-angiogenesis activity was carried out by shell less chick chorioallantoic membrane assay. In the antimicrobial activity we found that Staphylococcus aureus showed considerable susceptiblity with the MIC value of 145μg and Staphylococcus epidermidis is the most resistant with the MIC value of 378μg/ml. IC50 of 10.10µg/ml, IC50 of 17.32µg/ml and IC50 of 25.12µg/ml demonstrated antioxidant activities of methanol extract of U. ramosa by scavenging DPPH, NO. and superoxide anion radicals respectively. At 100µg/ml concentration, methanol extract significantly protected DNA against the effect of hydroxyl radicals. Protein denaturation was protected between concentrations 100-500µg/ml of methanol extract, maximum proteinase inhibitory activity was significantly found at 500µg/ml concentration and even haemolysis (heat and hypotonicity induced) was prevented considerably at 500µg/ml of methanol extract. Inhibition of proliferation of capillaries was observed in chorioallantoic membrane assay proving methanol extract’s antiangiogenic property. The leaves of Urochloa ramosa are basically enriched with natural phenolics with multitudinous biological potencies that can represent a promising class as bioactive molecules.

Recent progress in structural study of DNA and its protein and small molecule complexes through NMR spectroscopy

<p indent="0mm"><b/>As the main genetic material, DNA tends to form complexes with other molecules both in its resting state, where it is packaged as heterochromatin, and in its active state during replication, recombination, and transcription. These complexes are closely associated with DNA protection, amplification and utilization. On the other hand, due to the high variability of DNA sequence and structure, aptamers are increasingly developed to recognize other molecules. The elucidation of the complex structure formed by DNA and other molecules in these systems can facilitate the understanding of basic principles of life, and provide key information for the DNA application in genetic modification, disease treatment and biosafety analysis. NMR is one of the main methods for determining molecular structure. Its advantage is that it can obtain the information of structure, intermolecular interaction sites and kinetics under near-physiological conditions. In this paper, we focus on the complex structure of DNA and other molecules, introduce the representative work recently carried out by Chinese scholars using NMR methods, and discuss the application value and development direction of NMR method in this field.

RAD51 is a druggable target that sustains replication fork progression upon DNA replication stress.

Solving the problems that replication forks encounter when synthesizing DNA is essential to prevent genomic instability. Besides their role in DNA repair in the G2 phase, several homologous recombination proteins, specifically RAD51, have prominent roles in the S phase. Using different cellular models, RAD51 has been shown not only to be present at ongoing and arrested replication forks but also to be involved in nascent DNA protection and replication fork restart. Through pharmacological inhibition, here we study the specific role of RAD51 in the S phase. RAD51 inhibition in non-transformed cell lines did not have a significant effect on replication fork progression under non-perturbed conditions, but when the same cells were subjected to replication stress, RAD51 became necessary to maintain replication fork progression. Notably, the inhibition or depletion of RAD51 did not compromise fork integrity when subjected to hydroxyurea treatment. RAD51 inhibition also did not decrease the ability to restart, but rather compromised fork progression during reinitiation. In agreement with the presence of basal replication stress in human colorectal cancer cells, RAD51 inhibition reduced replication fork speed in these cells and increased γH2Ax foci under control conditions. These alterations could have resulted from the reduced association of DNA polymerase α to chromatin, as observed when inhibiting RAD51. It may be possible to exploit the differential dependence of non-transformed cells versus colorectal cancer cells on RAD51 activity under basal conditions to design new therapies that specifically target cancer cells.

IbTLD modulates reactive oxygen species scavenging and DNA protection to confer salinity stress tolerance in tobacco

Plants accumulate reactive oxygen species (ROS) that may damage the cells under prolonged stress conditions. Reduction of the excessive ROS production can alleviate oxidative damage and enhance the survival rates under stress. TLDc–containing protein (TLD) was reported to confer tolerance to oxidative stress, but the regulatory mechanism of TLD remains unclear. In this study, we ectopically overexpressed the Ipomoea batatas TLDc gene (IbTLD) in tobacco and characterized its functions. RNA-sequencing analysis and Gene Ontology term enrichment analysis revealed that IbTLD up-regulates auxin-responsive genes in response to oxidative stress. Under salinity stress, the IbTLD transgenic lines showed higher germination rates, chlorophyll contents, and root lengths than wild type (W38). In addition, the IbTLD transgenic lines showed higher expression of ROS scavenging genes, nudix hydrolases, ROS scavenging enzyme activity, and lesser DNA damage compared to W38 under salinity stress. Therefore, our results suggest that IbTLD activates the expression of ROS scavenging genes and confers tolerance to salinity stress in planta.