Nicked DNA Research Articles

Gd-EOB-DTPA MRI Imaging and Apoptosis Research in Orthotopic Rats Model of Hepatocellular Carcinoma after Stereotactic Radiotherapy

To investigate the characteristics of Gd-EOB-DTPA MRI imaging tumor cell apoptosis and the correlation between them in orthotopic rats model of hepatocellular carcinoma after stereotactic radiotherapy. Forty healthy male Sprague-Dawley rats were randomly divided into model group (n=40) and control group (n=20). When the tumor diameter was ≥1.0cm, the rats in the experimental group underwent single stereotactic radiotherapy with a dose of 15Gy. 40 rats after radiotherapy were randomly divided into 4 groups, 10 in each group. The control group was randomly divided into 4 groups of 5 each, and the above group was added. MR rats were routinely scanned and DWI scans were performed on the 1st, 7th, 14th, and 21st day after radiotherapy. The region of interest (ROI) ADC value and signal intensity (SI) of the tumor and normal life after radiotherapy were measured, and the ratio of ADC to SI (tumor/liver) was calculated. After the end of the scan, the animals were sacrificed, and the liver cell tumor specimens were subjected to DNA nick end labeling (TUNEL) to determine the apoptosis of the tumor cells, and the apoptotic index (apoptotic cells/tumor cells) was calculated. Two independent sample t-tests were used to compare the apoptotic index and ADC ratio between the radiotherapy group and the control group. Comparison of apoptotic index and ADC ratio between the radiotherapy groups was analyzed by one-way analysis of variance; the correlation between the two was performed using the Pearson test. The ADC and SI ratios were statistically significant at 7 days, 14 days and 21 days after radiotherapy (P<0.01=, there was no significant difference at 1 day after radiotherapy (P>0.05). 1 day, 7 days, 14 days, The apoptotic index of the 21-day radiotherapy group was significantly different from that of the control group (P<0.05=. The ADC and SI ratios were positively correlated with the apoptotic index (AI) (P<0.05). The imaging characteristics of Gd-EOB-DTPA MRI can reflect the dynamic changes of apoptosis at different time points after stereotactic radiotherapy in rat liver cancer.

Chromatin-bound oxidized α-synuclein causes strand breaks in neuronal genomes in in vitro models of Parkinson's Disease

Alpha-synuclein (α-Syn) overexpression and misfolding/aggregation in degenerating dopaminergic neurons have long been implicated in Parkinson's disease (PD). The neurotoxicity of α-Syn is enhanced by iron (Fe) and other pro-oxidant metals, leading to generation of reactive oxygen species in PD brain. Although α-Syn is predominantly localized in presynaptic nerve terminals, a small fraction exists in neuronal nuclei. However, the functional and/or pathological role of nuclear α-Syn is unclear. Following up on our earlier report that α-Syn directly binds DNA in vitro, here we confirm the nuclear localization and chromatin association of α-Syn in neurons using proximity ligation and chromatin immunoprecipitation analysis. Moderate (∼2-fold) increase in α-Syn expression in neural lineage progenitor cells (NPC) derived from induced pluripotent human stem cells (iPSCs) or differentiated SHSY-5Y cells caused DNA strand breaks in the nuclear genome, which was further enhanced synergistically by Fe salts. Furthermore, α-Syn required nuclear localization for inducing genome damage as revealed by the effect of nucleus versus cytosol-specific mutants. Enhanced DNA damage by oxidized and misfolded/oligomeric α-Syn suggests that DNA nicking activity is mediated by the chemical nuclease activity of an oxidized peptide segment in the misfolded α-Syn. Consistent with this finding, a marked increase in Fe-dependent DNA breaks was observed in NPCs from a PD patient-derived iPSC line harboring triplication of the SNCA gene. Finally, α-Syn combined with Fe significantly promoted neuronal cell death. Together, these findings provide a novel molecular insight into the direct role of α-Syn in inducing neuronal genome damage, which could possibly contribute to neurodegeneration in PD.

The Aggregation Behavior and Antioxidative Activity of Amphiphilic Surfactants Based on Quinuclidin‐3‐ol

AbstractThe self‐aggregation and thermodynamic properties of three cationic quaternary ammonium surfactants were investigated. The physicochemical properties of compounds containing quinuclidin‐3‐ol with even number of carbon atoms (10, 12, and 14) in the hydrophobic tail were measured by conductivity, dynamic light scattering (DLS), and Zeta‐potential measurements. DLS and Zeta‐potential measurements show a similar size distribution for all surfactants with excellent uniformity, and Zeta‐potential increases significantly with increase in the size of hydrocarbon tail. The critical micelle concentration (CMC) and the degree of micelle ionization (β) were determined using conductivity measurements. The CMC values of surfactants were found to be between 3.4 and 23.8 × 10−3 M. The standard Gibbs free energy () was derived from conductivity measurements and suggests that surfactants containing longer chains spontaneously form micelles. The antioxidative properties of these cationic surfactants were evaluated using the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) assays. Among the tested samples, N‐tetradecyl‐3‐hydroxyquinuclidinium bromide (QOH‐C14) exhibited the highest antioxidative potential (388.30 nmol (TE) equivalents mL−1), which was further investigated by the DNA nicking assay.

Xanthine Oxidase Inhibitors Screening, Antioxidation, and DNA Protection Properties of Geranium wilfordii Maxim

Geranium wilfordii Maxim is a commonly used traditional medicine and as decoction pieces in drinks for treating acute and chronic rheumatalgia. Earlier research showed it could inhibit xanthine oxidase and had a strong antioxidative activity. In this study, four compounds namely, corilagin, geraniin, ellagic acid, and myricetrin were screened and identified as xanthine oxidase inhibitors and antioxidants using ultrafiltration combined liquid chromatography—mass spectrometry (LC—MS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) spiking combined LC—MS methods. Ellagic acid showed the greatest inhibition with IC50 of 0.018 ± 0.001 mM better than that of allopurinol. The kinetic parameters exhibited the mode of xanthine oxidase inhibitions by corilagin, geraniin, and myricetrin were the competitive types, whereas the ellagic acid was found as an uncompetitive inhibitor. In vitro DNA nicking assay showed these four compounds protected DNA from reactive oxygen species. Correlation analysis and partial least squares analysis showed there was a moderate correlation between ellagic acid and DPPH˙ scavenging activity. Our findings revealed four compounds in G. wilfordii Maxim contributed satisfied radicals scavenging, xanthine oxidase inhibition, and DNA damage protective activities. More comprehensive research on pharmaceutical applications and phytochemical profile of G. wilfordii Maxim could be further studied.

Plasmid DNA nicking- a Novel Activity of Soybean Trypsin Inhibitor and Bovine Aprotinin

Protease inhibitors, such as trypsin inhibitor, serum alpha-1 antitrypsin, or liver aprotinin, are a class of proteins that competitively bind and block the catalytic activity of proteolytic enzymes with wide ranging biological functions. A significant number of protease inhibitors have also been shown to possess antimicrobial activity, presumed to contribute in defense against pathogenic microorganisms as plants with higher levels of protease inhibitors tend to exhibit increased resistance towards pathogens. Two proposed mechanisms for the antimicrobial activity are combating microbial proteases that play roles in disease development and disruption of microbial cell wall & membrane necessary for survival. Here we show for the first time a novel activity of soybean trypsin inhibitor and bovine aprotinin that they nick supercoiled, circular plasmid DNA. A number of experiments conducted to demonstrate the observed DNA nicking activity is inherent, rather than a co-purified, contaminating nuclease. The nicking of the plasmid results in markedly reduced efficiencies in transformation of E. coli and transfection of HEK293T cells. Thus, this work reveals yet a new mechanism for the antimicrobial activity by protease inhibitors.

Dx.doi.org/10.18081/2333-5106/016-7/253-264

DNA topoisomerases are ubiquitous, enzymes needed to control topological problems encountered during DNA replication, transcription, recombination and maintenance of genomic stability. They proved to be valuable targets for cancer chemotherapy. The seeds alkaloids extract of Rue plant (Peganum harmala L. Zygophyllaceae) were fractioned by high performance liquid chromatography as a suitable technique, HPLC waters company, gradient, Nova-Pak, C18*, 4µm, 3.9x150mm, mobile phase ACN-water pH10.7. Thirty-four separate solution depending on the retention time of eluents. The aim of this study is to investigate the trace compounds relatively and used as inhibitors for the topoisomerase II enzyme, the study the poison and suppressor model inhibitions and the synergy and antagonism activity of fractions. The visicinone, harmine, visicine and harmaline, were (2.86, 0.75, 13.14, and 16.62 mg/100mg of plant powder. The harmine ˃vicicinone˃ harmaline˃ vicicine as enzyme inhibitors. The unknown trace compounds gave the best inhibition relatively, the alkaloid fractions gave higher inhibitory units of fraction effective (FE). Results of seeds alkaloid fractions showed that the FE in state of fraction mixture was 36% inhibitory units, while summation of FE of each fraction alone gave higher level than their mixture, where sum of FE of them was 1229% inhibitory units. The suppressor inhibition model according to highest percent of the remain supercoiled DNA plasmid than production of linear and open nicked circular with high value in F8 (84%), the poisons inhibition model activity according to the highest percent of linear and nicked open circular DNA plasmid with high level in F31 (88%). There are a trace compounds within some fraction but that the effectiveness of inhibitory relatively higher than known compounds (harmine, vicisinone, harmaline, and vicisine), this can be purified and used as somewhat better inhibitors. There is antagonism activity among seed alkaloids fractions. The all fractions showed a poison and a suppressor model inhibition action in varies percentage, the suppressor inhibition appear in high value in F8 (84%), while the poisons inhibition model gave high level in F31 (88%).

A Universal Assay for Making DNA, RNA, and RNA-DNA Hybrid Configurations for Single-Molecule Manipulation in Two or Three Steps without Ligation.

Despite having a great variety of topologies, most DNA, RNA, and RNA-DNA hybrid (RDH) configurations for single-molecule manipulation are composed of several single-stranded (ss) DNA and ssRNA strands, with functional labels at the two ends for surface tethering. On this basis, we developed a simple, robust, and universal amplification-annealing (AA) assay for making all these configurations in two or three steps without inefficient digestion and ligation reactions. As examples, we made ssDNA, short ssDNA with double-stranded (ds) DNA handles, dsDNA with ssDNA handles, replication-fork shaped DNA/RDH/RNA, DNA holiday junction, three-site multiple-labeled and nicked DNA, torsion-constrained RNA/RDH, and short ssRNA with RDH handles. In addition to single-molecule manipulation techniques including optical tweezers, magnetic tweezers, and atomic force microscopy, these configurations can be applied in other surface-tethering techniques as well.

Study of In vitro antioxidant and DNA damage protection activity of a novel luteolin derivative isolated from Terminalia chebula

ABSTRACTThe present study aimed to investigate the efficacy of flavonoids isolated from the fruit of Terminalia chebula against the hydrogen peroxide-induced DNA damage. Five fractions were screened and were compared for their in vitro antioxidant activity by DPPH method and DNA protection activity by DNA nicking assay. All the extracts showed excellent antioxidant activity with IC50 values ranging from 11.23 ± 0.18 to 36.70 ± 0.12 µg/ml. The band intensity of supercoiled, circular and linear DNA was measured in DNA nicking assay and it is very clear that the addition of extract significantly inhibited the formation of circular and linear forms of DNA. Methanol–Water 1:1 fraction which showed the highest activity in antioxidant and DNA protection was subjected to structural characterization using different spectroscopic techniques. The structure of the compound was identified as a derivative of luteolin. The result presented in this work strongly supports the hypothesis that DNA protection activities were positively associated with the antioxidant property.

Development of coupling controlled polymerizations by adapter-ligation in mate-pair sequencing for detection of various genomic variants in one single assay.

The diversity of disease presentations warrants one single assay for detection and delineation of various genomic disorders. Herein, we describe a gel-free and biotin-capture-free mate-pair method through coupling Controlled Polymerizations by Adapter-Ligation (CP-AL). We first demonstrated the feasibility and ease-of-use in monitoring DNA nick translation and primer extension by limiting the nucleotide input. By coupling these two controlled polymerizations by a reported non-conventional adapter-ligation reaction 3′ branch ligation, we evidenced that CP-AL significantly increased DNA circularization efficiency (by 4-fold) and was applicable for different sequencing methods but at a faction of current cost. Its advantages were further demonstrated by fully elimination of small-insert-contaminated (by 39.3-fold) with a ∼50% increment of physical coverage, and producing uniform genome/exome coverage and the lowest chimeric rate. It achieved single-nucleotide variants detection with sensitivity and specificity up to 97.3 and 99.7%, respectively, compared with data from small-insert libraries. In addition, this method can provide a comprehensive delineation of structural rearrangements, evidenced by a potential diagnosis in a patient with oligo-atheno-terato-spermia. Moreover, it enables accurate mutation identification by integration of genomic variants from different aberration types. Overall, it provides a potential single-integrated solution for detecting various genomic variants, facilitating a genetic diagnosis in human diseases.

Characterization of a relaxase belonging to the MOBT family, a widespread family in Firmicutes mediating the transfer of ICEs

BackgroundConjugative spread of antibiotic resistance and virulence genes in bacteria constitutes an important threat to public health. Beyond the well-known conjugative plasmids, recent genome analyses have shown that integrative and conjugative elements (ICEs) are the most widespread conjugative elements, even if their transfer mechanism has been little studied until now. The initiator of conjugation is the relaxase, a protein catalyzing a site-specific nick on the origin of transfer (oriT) of the ICE. Besides canonical relaxases, recent studies revealed non-canonical ones, such as relaxases of the MOBT family that are related to rolling-circle replication proteins of the Rep_trans family. MOBT relaxases are encoded by ICEs of the ICESt3/ICEBs1/Tn916 superfamily, a superfamily widespread in Firmicutes, and frequently conferring antibiotic resistance.ResultsHere, we present the first biochemical and structural characterization of a MOBT relaxase: the RelSt3 relaxase encoded by ICESt3 from Streptococcus thermophilus. We identified the oriT region of ICESt3 and demonstrated that RelSt3 is required for its conjugative transfer. The purified RelSt3 protein is a stable dimer that provides a Mn2+-dependent single-stranded endonuclease activity. Sequence comparisons of MOBT relaxases led to the identification of MOBT conserved motifs. These motifs, together with the construction of a 3D model of the relaxase domain of RelSt3, allowed us to determine conserved residues of the RelSt3 active site. The involvement of these residues in DNA nicking activity was demonstrated by targeted mutagenesis.ConclusionsAll together, this work argues in favor of MOBT being a full family of non-canonical relaxases. The biochemical and structural characterization of a MOBT member provides new insights on the molecular mechanism of conjugative transfer mediated by ICEs in Gram-positive bacteria. This could be a first step towards conceiving rational strategies to control gene transfer in these bacteria.

DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA.

Persistent hepatitis B virus (HBV) infection relies on the establishment and maintenance of covalently closed circular (ccc) DNA, a 3.2 kb episome that serves as a viral transcription template, in the nucleus of an infected hepatocyte. Although evidence suggests that cccDNA is the repair product of nucleocapsid associated relaxed circular (rc) DNA, the cellular DNA polymerases involving in repairing the discontinuity in both strands of rcDNA as well as the underlying mechanism remain to be fully understood. Taking a chemical genetics approach, we found that DNA polymerase alpha (Pol α) is essential for cccDNA intracellular amplification, a genome recycling pathway that maintains a stable cccDNA pool in infected hepatocytes. Specifically, inhibition of Pol α by small molecule inhibitors aphidicolin or CD437 as well as silencing of Pol α expression by siRNA led to suppression of cccDNA amplification in human hepatoma cells. CRISPR-Cas9 knock-in of a CD437-resistant mutation into Pol α genes completely abolished the effect of CD437 on cccDNA formation, indicating that CD437 directly targets Pol α to disrupt cccDNA biosynthesis. Mechanistically, Pol α is recruited to HBV rcDNA and required for the generation of minus strand covalently closed circular rcDNA, suggesting that Pol α is involved in the repair of the minus strand DNA nick in cccDNA synthesis. Our study thus reveals that the distinct host DNA polymerases are hijacked by HBV to support the biosynthesis of cccDNA from intracellular amplification pathway compared to that from de novo viral infection, which requires Pol κ and Pol λ.

Expression, purification and biochemical characterization of Listeria monocytogenes single stranded DNA binding protein 1

Single-stranded DNA binding proteins play an important role in DNA metabolic processes including replication, recombination, and repair. Here, we report the identification and biochemical characterization of the SSB1 protein from the foodborne pathogen Listeria monocytogenes. The L. monocytogenes SSB1 share 33% identity and 50.5% similarity with the prototype E. coli SSB protein. The electrophoretic mobility shift assay revealed that the purified L. monocytogenes SSB1 protein binds to single stranded DNA, including the M13 circular single stranded DNA and oligonucleotide, with high affinity. The plasmid based strand transfer activity showed that, in the absence of the SSB protein, L. monocytogenes RecA fails to catalyze the reaction whereas, the E. coli RecA protein has shown nicked DNA formation. Interestingly the addition of SSB1 protein stimulates both L. monocytogenes and E. coli RecA strand transfer activities however, it is sensitive to the order of addition of SSB1 protein. L. monocytogenes RecA fails to catalyze the reaction when SSB1 is added prior to RecA; nevertheless, it readily catalyzes the reaction when added after the RecA filament formation. These results suggest that the interaction among of gene product between RecA and SSB1 is required to promote optimum strand exchange activities. Altogether, these studies provide the first functional characterization of the L. monocytogenes SSB1 protein and give insights into DNA repair and recombination processes in the gram-positive foodborne pathogen L. monocytogenes.

Hookah and Vaping: Safe Nicotine Delivery Systems?

Hookah and vaping have recently been popularized as alternative to smoking tobacco. Hookah heats tobacco with a very hot piece of charcoal and then the air is pulled through the hot tobacco, this smoke is then pulled through water before it reaches the user's mouth and lungs. Vaping uses electricity to vaporize a propylene glycol/glycerine matrix that contains nicotine and flavoring agents. This vapor is then pulled directly into the mouth and lungs. The mainstream vapors from both hookah and vaping were collected on Cambridge filters, the filters were soaked in water and then the aqueous solutions were dried via vacuum centrifugation. After weighing the dried mass, the samples were reconstituted with water and then analyzed by UV spectroscopy; the Hookah samples had absorbance at 275 nm. Our original hypothesis was that the water soluble tar components would be trapped in the water that the hookah smoke is pulled through. Thus, there should have been no absorbance at 275nm. The vaping samples did not absorb at 275; since vaping does not burn or heat tobacco, we did not expect to find the same absorbance as the cigarette tar radical. Gel electrophoresis was used to analyze the nicking of plasmid (pUC18) DNA. Aqueous solutions of Hookah and vaping solutions were tested in our DNA nicking assay. Briefly, we use supercoiled pUC18 DNA, this DNA unwinds after a single nick. The supercoiled and nicked (open circular) DNA migrate as two distinct bands in the agarose gel. The density of each band is then quantitated to determine the amount of DNA nicking. The amount of nicking by both vaping and hookah solutions is orders of magnitude less that the nicking we have detected with cigarette smoke. However, we have detected aldehydes in both vaping and hookah solutions. Aldehydes are known to be toxic, primarily by their facile reaction with primary amines and thus their ability to wreck havoc with protein structure. We have implemented an assay to detect aldehydes in both hookah and vaping solutions. The reagent, Purpald, 4‐Amino‐3‐hydrazino‐5‐mercapto‐1,2,4‐triazole readily reacts with aldehydes and then the oxidized product of the reaction produces purple solutions. We then analyzed the purple solutions by UV/VIS. Based on the UV spectra, we have detected acetaldehydes in both vaping and hookah solutions.Support or Funding InformationR. Dunham is supported by the CSU, Stanislaus McNair Scholars program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel Sorghum bicolor (L.) seed polysaccharide structure, hemolytic and antioxidant activities, and laser burn wound healing effect.

This work was undertaken to determine the structural characteristics of a novel polysaccharide, Sorghum Water-Soluble Polysaccharide (SWSP), extracted from Sorghum (Sorghum bicolor (L.)) seeds and to assess its in vitro hemolytic and antioxidant activities as well as its in vivo wound healing ability to treat burns induced by fractional CO2 laser. Results obtained by 13C, 1H and HSQC Nuclear magnetic resonance (NMR) indicated that SWSP showed the characteristic bands of polysaccharides. Scanning electron microscopy of SWSP showed a rough surface with numerous cavities. SWSP exhibited strong antioxidant activities determined through three different assays: ferrous chelating activity, reducing power and DNA nicking. Also, no hemolytic activity was detected towards human erythrocytes. On the other hand, the application of SWSP hydrogel on the burn site in a rat model significantly improved wound appearance and accelerated the wound closure after 8 days of induction. Histological assessment of biopsies also demonstrated complete re-epithelialized wounds with a full epidermal regeneration. Thus, SWSP hydrogel which was efficient on wound closure might be useful as a wound healing agent in modern medicine.

Mechanism of coralyne-mediated DNA photo-nicking process

Previously, we reported that coralyne and UVA combination sensitized a wide range of human carcinoma cells regardless of their p53 status. The coralyne induced photosensitization of cancer cells may be clinically attractive, as mutation in the p53 gene is prevalent in many types of tumors. Coralyne mediated photosensitization of cancer cells is attributable to its ability to cause extensive DNA single strand breaks (SSB). However, the precise mechanism of coralyne induced DNA photo-damage is not yet known. The present study was aimed to understand the hitherto unknown mechanism of the coralyne-induced DNA photo-cleavage process. To this end, we compared the DNA photo-nicking properties of berberine, jatrorrhizine and coralyne, and deciphered involvement of the photochemical processes in the photo-nuclease action of coralyne using absorption and electron spin resonance spectroscopy, high performance liquid chromatography and mass spectroscopy (MS) techniques in conjunction with relevant in vitro studies with plasmid DNA. In association with UVA, coralyne, but not berberine and jatrorrhizine induced significant nicking of plasmid DNA via an O2-independent photo-chemical process. The Job's plot of our spectrophotometric data suggested that one coralyne molecule remains intercalated with two DNA base pairs (i. e., 1:2) and starts forming aggregates beyond this molar ratio. The DNA photo-nicking by the combination of coralyne and UVA (designated as CUVA) was primarily caused by the coralyne aggregates without any significant contribution from the DNA-intercalated coralyne monomer.

Ultrasensitive Microfluidic Paper-Based Electrochemical Biosensor Based on Molecularly Imprinted Film and Boronate Affinity Sandwich Assay for Glycoprotein Detection.

In this work, we proposed a strategy that combined molecularly imprinted polymers (MIPs) and hybridization chain reaction into microfluidic paper-based analytical devices for ultrasensitive detection of target glycoprotein ovalbumin (OVA). During the fabrication, Au nanorods with a large surface area and superior conductibility were grown on paper cellulosic fiber as a matrix to introduce a boronate affinity sandwich assay. The composite of MIPs including 4-mercaptophenylboronic acid (MPBA) was able to capture target glycoprotein OVA. SiO2@Au nanocomposites labeled MPBA and cerium dioxide (CeO2)-modified nicked DNA double-strand polymers (SiO2@Au/dsDNA/CeO2) as a signal tag were captured into the surface of the electrode in the presence of OVA. An electrochemical signal was generated by using nanoceria as redox-active catalytic amplifiers in the presence of 1-naphthol in electrochemical assays. As a result, the electrochemical assay was fabricated and could be applied in the detection of OVA in the wide linear range of 1 pg/mL to 1000 ng/mL with a relatively low detection limit of 0.87 pg/mL (S/N = 3). The results indicated that the proposed platform possessed potential applications in clinical diagnosis and other related fields.

Temporal and metabolic overlap between lipid accumulation and programmed cell death due to nitrogen starvation in the unicellular chlorophyte Chlamydomonas reinhardtii

SummaryLipid accumulation due to nitrogen depletion has been studied extensively in Chlamydomonas reinhardtii and the metabolic changes that lead to triacylglycerol biosynthesis have been of particular interest to researchers in the biodiesel industry. The induction of programmed cell death (PCD) in response to nitrogen starvation has also been documented in related chlorophytes. Here, we examined the temporal and metabolic overlap of lipid accumulation and PCD in response to nitrogen starvation in the important model organism C. reinhardtii. Nitrogen starvation induced physiological stress, measured by the progressive decline in chlorophyll a fluorescence, reduced photosynthetic efficiency and decreased growth. In keeping with previous reports, cells accumulated lipids reaching a peak after 2–3 days. At the same time, DNA nicking and caspase‐like protease activity was observed in a proportion of cells, and ultrastructural observations confirmed that death was via PCD. Our results demonstrate that DNA nicking and caspase‐like activity are observed during PCD in C. reinhardtii in response to nitrogen starvation, and that death occurs at the same time as lipid biosynthesis. Microalgal lipid production due to nitrogen depletion in C. reinhardtii is limited by the decrease in culture growth and knowing that the loss of culture density is, at least in part, due to PCD is important for the biotechnology industry.

Evaluation of bioactive compounds from Jasminum polyanthum and its medicinal properties

Jasmine plant species are widely grown in Asia and used for religious offering, is known to have wide range of bioactive compounds and its properties. Most of the jasmine species have been evaluated its bioactive properties and found positive results. This work consists of evaluating bioactive properties of jasmine species, Jasminum polyanthum and finding its potential medicinal uses. The plant leaves and flowers were powdered and added water to prepare extract. Both leaves and flower extract was evaluated for phytochemical compounds, anti oxidant, anti diabetic, anti inflammatory, anti microbial, anti cancer and DNA nicking assay. The leaf and flower extract contained most of the phytochemical compounds which leads to presence of various bioactive properties. Leaf possesses good DPPH activity, while flower possess high phenol content and FRAP activity. Leaf possesses good anti diabetic activity, while flower possess good anti inflammatory activity. Flower extract consists of higher antibacterial activity than leaf, while in antifungal it is vice versa. &#x0D; Keywords: Anti-diabetic, Anti-inflammatory, Antioxidant activity, MTT Assay, DNA Nicking study

DNA polymerase θ (POLQ) is important for repair of DNA double-strand breaks caused by fork collapse

DNA polymerase θ (POLQ) plays an important role in alternative nonhomologous end joining or microhomology-mediated end joining (alt-NHEJ/MMEJ). Here, we show that POLQ is not only required for MMEJ to repair DNA double-strand breaks (DSBs) generated by endonucleases such as I-SceI or Cas9, but is also needed for repair of DSBs derived from DNA nicks generated by Cas9 nickase. Consistently, we found that POLQ deficiency leads to sensitivity to topoisomerase inhibitors that cause DNA single-strand break (SSB) accumulation at replication forks and to ATR inhibitors that induce replication fork collapse. These studies support the function of POLQ in coping with replication stress and repairing DSBs upon fork collapse. POLQ overexpression is present in many cancer types and is associated with poor prognosis, including breast cancer regardless of BRCA1 status. We provide proof-of-concept evidence to support a novel cancer treatment strategy that combines POLQ inhibition with administration of topoisomerase or ATR inhibitors, which induces replication stress and fork collapse. Given the prevalence of POLQ overexpression in tumors, such strategy may have a significant impact on developing targeted cancer treatment.

Enzyme-free hybridization chain reaction-based signal amplification strategy for the sensitive detection of Staphylococcus aureus.

In this study, based on hybridization chain reaction (HCR) amplification and graphene oxide (GO), we developed a facile enzyme-free signal amplification strategy for sensitive detection of Staphylococcus aureus (S. aureus). Two hairpin probes (HP1 and HP2) labeled by fluorophore 6-carboxyfluorescein (FAM) are designed. The HP1 and HP2 can not only trigger to the HCR but also form a long nicked double strand DNA (dsDNA) with the target (16S rRNA). In the absence of target (16 s RNA), the free FAM-labeled HP1 and HP2 are adsorbed by the GO via π-π stacking, the fluorescence signal is quenched. In the presence of target (16 s RNA), the HCR is triggered and dsDNA complexes are generated. As a result, the fluorescence signal can be strongly amplified by the synergistic effect of FAM and the dsDNA dye SYBR Green I. Based on this mechanism, a fluorescence method is designed for the detection of 16S rRNA of S. aureus. Under the optimal conditions, it has low detection limit (50 pM) and a linear response in a concentration range of 50 pM to 100 nM for 16S rRNA. Furthermore, this method has also been successfully applied to the detection of S. aureus in milk sample with the detection limit of 4 × 102 CFU·mL-1.