Recent Perspectives in Radiation-Mediated DNA Damage and Repair: Role of NHEJ and Alternative Pathways

Radiation is one of the causative agents for the induction of DNA damage in biological systems. There is various possibility of radiation exposure that might be natural, man-made, intentional, or non-intentional. Published literature indicates that radiation mediated cell death is primarily due to DNA damage that could be a single-strand break, double-strand breaks, base modification, DNA protein cross-links. The double-strand breaks are lethal damage due to the breakage of both strands of DNA. Mammalian cells are equipped with strong DNA repair pathways that cover all types of DNA damage. One of the predominant pathways that operate DNA repair is a non-homologous end-joining pathway (NHEJ) that has various integrated molecules that sense, detect, mediate, and repair the double-strand breaks. Even after a well-coordinated mechanism, there is a strong possibility of mutation due to the flexible nature in joining the DNA strands. There are alternatives to NHEJ pathways that can repair DNA damage. These pathways are alternative NHEJ pathways and single-strand annealing pathways that also displayed a role in DNA repair. These pathways are not studied extensively, and many reports are showing the relevance of these pathways in human diseases. The chapter will very briefly cover the radiation, DNA repair, and Alternative repair pathways in the mammalian system. The chapter will help the readers to understand the basic and applied knowledge of radiation mediated DNA damage and its repair in the context of extensively studied NHEJ pathways and unexplored alternative NHEJ pathways

Distinct and Shared Roles of β-Arrestin-1 and β-Arrestin-2 on the Regulation of C3a Receptor Signaling in Human Mast Cells

BACKGROUND: The complement component C3a induces degranulation in human mast cells via the activation of cell surface G protein coupled receptors (GPCR; C3aR). For most GPCRs, agonist-induced receptor phosphorylation leads to the recruitment of β-arrestin-1/β-arrestin-2; resulting in receptor desensitization and internalization. Activation of GPCRs also leads to ERK1/2 phosphorylation via two temporally distinct pathways; an early response that reflects G protein activation and a delayed response that is G protein independent but requires β-arrestins. The role of β-arrestins on C3aR activation/regulation in human mast cells, however, remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: We utilized lentivirus short hairpin (sh)RNA to stably knockdown the expression of β-arrestin-1 and β-arrrestin-2 in human mast cell lines, HMC-1 and LAD2 that endogenously expresses C3aR. Silencing β-arrestin-2 attenuated C3aR desensitization, blocked agonist-induced receptor internalization and rendered the cells responsive to C3a for enhanced NF-κB activity as well as chemokine generation. By contrast, silencing β-arrestin-1 had no effect on these responses but resulted in a significant decrease in C3a-induced mast cell degranulation. In shRNA control cells, C3a caused a transient ERK1/2 phosphorylation, which peaked at 5 min but disappeared by 10 min. Knockdown of β-arrestin-1, β-arrestin-2 or both enhanced the early response to C3a and rendered the cells responsive for ERK1/2 phosphorylation at later time points (10-30 min). Treatment of cells with pertussis toxin almost completely blocked both early and delayed C3a-induced ERK1/2 phosphorylation in β-arrestin1/2 knockdown cells. CONCLUSION/SIGNIFICANCE: This study demonstrates distinct roles for β-arrestins-1 and β-arrestins-2 on C3aR desensitization, internalization, degranulation, NF-κB activation and chemokine generation in human mast cells. It also shows that both β-arrestin-1 and β-arrestin-2 play a novel and shared role in inhibiting G protein-dependent ERK1/2 phosphorylation. These findings reveal a new level of complexity for C3aR regulation by β-arrestins in human mast cells

Knockdown of β-arrestin-1, but not β-arrestin-2, inhibits C3a-induced degranulation in LAD2 mast cells.

<p>(A) LAD2 cells were transduced with shRNA control, β-arrestin-1, and β-arrestin-2 lentivirus. After puromycin selection, quantitative PCR was employed to assess the β-arrestin-1 or β-arrestin-2 mRNA level and results are expressed as a ratio of β-arrestin to GAPDH mRNA levels. (B, C, D) A representative desensitization experiment in shRNA control, β-arrestin-1 and β-arrestin-2 knockdown LAD2 cells is shown. (E) shRNA control, β-arrestin-1 and -2 KD LAD2 mast cells were stimulated with different concentrations of C3a and percent degranulation (β-hexosaminidase release) was determined. Data represent the mean ± SEM from three independent experiments. Statistical significance was determined by two way ANOVA with Bonferroni's post test. * indicates p<0.05 and ** indicates p<0.001.</p

C3a-induced ERK1/2 phosphorylation is enhanced in β-arrestin-1, β-arrestin-2 and double KD cells.

<p>shRNA control or β-arrestin KD HMC-1 cells (1×10⁶/ml) were exposed to C3a (100 nM) for 1, 5 and 10, 15 and 30 min. Cell lysates were separated on SDS-PAGE and blots were probed with anti-phospho-ERK1/2 antibody followed by anti-rabbit IgG-HRP. The blots were then stripped and reprobed with anti-ERK1/2 antibody followed by anti-rabbit IgG-HRP. Immunoreactive band were visualized by SuperSignal West Femto maximum sensitivity substrate. (A) Representative immunoblots from three similar experiments are shown. (B) ERK1/2 phosphorylation was quantified using Image J as shown in the line graph. Data represent the mean ± SEM from three independent experiments. Statistical significance was determined by two way ANOVA with Bonferroni's post test. ** indicates p<0.001.</p

Determination of Antioxidants by DPPH Radical Scavenging Activity and Quantitative Phytochemical Analysis of Ficus religiosa

The use of F. religiosa might be beneficial in inflammatory illnesses and can be used for a variety of health conditions. In this article, we studied the identification of antioxidants using (DPPH) 2,2-Diphenyl-1-picrylhydrazylradical scavenging activity in Ficus religiosa, as F. religiosa is an important herbal plant, and every part of it has various medicinal properties such as antibacterial properties that can be used by the researchers in the development and design of various new drugs. The 2,2-Diphenyl-1-picrylhydrazyl (DPPH) is a popular, quick, easy, and affordable approach for the measurement of antioxidant properties that includes the use of the free radicals used for assessing the potential of substances to serve as hydrogen providers or free-radical scavengers (FRS). The technique of DPPH testing is associated with the elimination of DPPH, which would be a stabilized free radical. The free-radical DPPH interacts with an odd electron to yield a strong absorbance at 517 nm, i.e., a purple hue. An FRS antioxidant, for example, reacts to DPPH to form DPPHH, which has a lower absorbance than DPPH because of the lower amount of hydrogen. It is radical in comparison to the DPPH-H form, because it causes decolorization, or a yellow hue, as the number of electrons absorbed increases. Decolorization affects the lowering capacity significantly. As soon as the DPPH solutions are combined with the hydrogen atom source, the lower state of diphenylpicrylhydrazine is formed, shedding its violet color. To explain the processes behind the DPPH tests, as well as their applicability to Ficus religiosa (F. religiosa) in the manufacture of metal oxide nanoparticles, in particular MgO, and their influence on antioxidants, a specimen from the test was chosen for further study. According to our findings, F. religiosa has antioxidant qualities and may be useful in the treatment of disorders caused by free radicals

Model for the Regulation of C3aR signaling in human mast cells by β-arrestin-1 and β-arrestin-2.

<p>(A): β-arrestin-2 causes desensitization and internalization of C3aR but both β-arrestins promote G protein-independent signaling for degranulation via the activation of Hck and/or Ral-GDS-mediated signaling pathways <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019585#pone.0019585-Barlic1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019585#pone.0019585-Bhattacharya1" target="_blank">[18]</a>. (B) β-arrestin-2 mediates inhibition of C3a-induced NF-κB activity but both β-arrestins block C3a-induced G-protein-mediated ERK1/2 phosphorylation. Red dotted lines denote inhibition.</p

Enhanced C3a-induced ERK1/2 phosphorylation in β-arrestin KD cells mast is mediated via a G protein-dependent pathway.

<p>shRNA control and double β-arrestin KD cells were pretreated with vehicle or Pertussis toxin (PTx; 100 ng/ml, 16 hr). Cell were then washed twice in serum free medium and stimulated with C3a (100 nM) for 1, 5 and 10, 15 and 30 min and ERK1/2 phosphorylation were determined. (A) Representative immunoblots from three similar experiments are shown. (B). ERK1/2 phosphorylation was quantified using Image J as shown in the bar graph. Data represent the mean ± SEM from three independent experiments. Statistical significance was determined by two way ANOVA with Bonferroni's post test. ** indicates p<0.001.</p

Knockdown of β-arrestin-2, but not β-arrestin-1, enhanced C3a-induced NF-κB activation and chemokine CCL4 generation.

<p>shRNA control, β-arrestin-1 KD or β-arrestin-2 KD HMC-1 cells were transiently transfected with NF-κB luciferase reporter gene construct. (A) Cells were stimulated with C3a (100 nM for 6 hr) and NF-κB-dependent transcriptional activity was determined by luciferase activity assay. (B) Control or β-arrestin KD cells were stimulated with C3a (100 nM for 6 hr) and CCL4 production was determined from the supernatant by ELISA. Data shown are mean ± SEM of three experiments performed in triplicate. Statistical significance was determined by two way ANOVA with Bonferroni's post test. ** indicates p<0.001.</p

Knockdown of β-arrestin-2, but not β-arrestin-1, inhibits agonist-induced C3aR internalization.

<p>(A) shRNA control HMC-1 cells (B) β-arrestin-1 KD and (C) β-arrestin-2 KD cells were exposed to buffer (−C3a) or C3a (100 nM) for 5 min. Cells were washed with ice-cold FACS buffer, incubated with a mouse anti-C3aR antibody or an isotype control antibody followed by PE-labeled donkey anti-mouse IgG antibody and analyzed by flow cytometry. Representative histograms showing cell surface C3aR expression in (A) shRNA control, (B) β-arrestin-1 KD and (C) β-arrestin-2 KD cells are shown. (D) shRNA control, β-arrestin-1 KD and β-arrestin-2 KD cells were exposed to C3a for different time periods and receptor internalization was determined as described above. Internalization is expressed as the percentage loss of C3aR following exposure to C3a. Data represent the mean ± SEM from three experiments. Statistical significance was determined by two way ANOVA with Bonferroni's post test. * indicates p<0.05.</p