Rev3l gene encoding the polymerase catalytic subunit causes a high frequency of chromosomal breaks, followed by lethality in mouse embryos and in primary cells. Yet it is not known whether the DNA polymerase activity of pol ζ is specifically essential, as the large REV3L protein also serves as a multiprotein scaffold for translesion DNA synthesis via multiple conserved structural domains. We report that Rev3l cDNA rescues the genomic instability and DNA damage sensitivity of Rev3l-null immortalized mouse fibroblast cell lines. A cDNA harboring mutations of conserved catalytic aspartate residues in the polymerase domain of REV3L could not rescue these phenotypes. To investigate the role of REV3L DNA polymerase activity in vivo, a Rev3l knock-in mouse was constructed with this polymerase-inactivating alteration. No homozygous mutant mice were produced, with lethality occurring during embryogenesis. Primary fibroblasts from mutant embryos showed growth defects, elevated DNA double-strand breaks and cisplatin sensitivity similar to Rev3l-null fibroblasts. We tested whether the severe Rev3l-/- phenotypes could be rescued by deletion of DNA polymerase η, as has been reported with chicken DT40 cells. However, Rev3l-/-Polh-/- mice were inviable, and derived primary fibroblasts were as sensitive to DNA damage as Rev3l-/-Polh+/+ fibroblasts. Therefore, the functions of REV3L in maintaining cell viability, embryonic viability and genomic stability are directly dependent on its polymerase activity, and cannot be ameliorated by an additional deletion of pol η. These results validate and encourage the approach of targeting the DNA polymerase activity of pol ζ to sensitize tumors to DNA damaging agents.DNA polymerase ζ (pol ζ) is exceptionally important for maintaining genome stability. Inactivation of the  in vivo knock-in mice, our work shows that pol ζ–mediated maintenance of genomic stability in the presence of DNA damage is absolutely dependent on its DNA polymerase activity. Others have demonstrated in chicken cells that co-deletion of pol ζ and pol η rescues the pol ζ-dependent phenotypes, but our work in mice and in mouse cell culture does not support that conclusion. These results demonstrate the physiological importance of pol ζ polymerase activity, and show that employing small-molecule inhibitors of the polymerase reaction is a valid strategy for sensitizing tumor cells to chemotherapeutic agents.Translesion synthesis allows DNA replication to occur in the presence of damaged DNA. This process is mediated by low-fidelity DNA polymerases (such as pol ζ or pol η) that maintain genomic stability. The action of these polymerases is crucial to limit cancer. In mice, complete deletion of DNA pol ζ leads to embryonic lethality, and conditional deletion enhances tumorigenesis. Pol ζ is a large protein with many domains that interact with other essential proteins and maintain the structural integrity of pol ζ. It is not known if the polymerase activity of pol ζ mediates its essential activities. Using a cell culture complementation system and  Rev3l in mice leads to death during embryogenesis  is critical for the tolerance of many types of DNA replication blocks, by playing a central role in translesion DNA synthesis (TLS). Primary replicative DNA polymerases (pol δ or pol ε) are stalled when they encounter many types of template DNA adducts or DNA sequences forming stable secondary structures. Such stalled replication forks are prone to formation of a dangerous DNA double-strand break. The process of TLS helps avoid catastrophes by using a lower fidelity DNA polymerase (such as pol ζ or pol η), to incorporate nucleotides across from a lesion. TLS may occur either in S phase during primary DNA replication or in G2 phase during post-replication DNA synthesis. In yeast and in mammalian cells, pol ζ is important for this process, but it leads to endogenous and DNA damage-induced point mutations because of errors introduced during TLS –5. Elimiiewed in ). Primarcumulate , 8. Circsitivity . Mice coll cells , 11. Thell cells .MAD2L2). REV7 is necessary for pol ζ activity in vitro and serves an important function as a bridge protein for interaction with the REV1 protein [KIAA2022 gene , with multiple functional domains. The DNA polymerase domain occupies only the last third of the protein . The str protein –15. REV1 protein –18. REV7 protein –21. An N protein . At the  protein , an Fe-S protein –26. More protein . Another022 gene .Rev3l deletion [Polh). As in mammalian cells, chicken DT40 cells with a disruption of pol ζ exhibit growth defects, chromosomal aberrations and DNA damage sensitivity [Polh and Rev3l corrects all of these phenotypes in DT40. The suggested interpretation was that pol η and pol ζ always work together in bypass of DNA damage, and that a toxic intermediate is formed by pol η that cannot be resolved in the absence of pol ζ. It is clearly important to determine, in mammalian cells, whether the genome instability caused by pol ζ disruption is dependent on pol η.A provocative hypothesis has been put forward to explain the severe genotoxic effects of deletion . It was sitivity . RemarkaRev3l-deficient mouse embryonic fibroblasts (MEFs) by expression of full-length human wild-type REV3L, and show that DNA polymerase-defective mutant REV3L cDNA is unable to complement cell survival or increased levels of DNA breaks. Using a Rev3l polymerase-dead knock-in mouse model, we show that specific disruption of the polymerase activity prevents the completion of embryogenesis. Finally, we tested whether pol ζ defects can be rescued by ablation of pol η function.Here we describe experiments with knockout cells and a specific knock-in mouse model to test whether the catalytic activity of pol ζ is responsible for the phenotypes observed in pol ζ knockout mutants. We describe complementation of Rev3l deletion in mouse cell lines is associated with an elevated baseline level of DNA breaks and an increased sensitivity to DNA damaging agents such as cisplatin and UV radiation [Rev3l. A pOZ expression vector harboring an IL2R selectable marker , as well as REV3L with a dual point mutation  in residues essential for divalent metal binding in conserved DNA polymerase motif I. Equivalent changes in all other tested DNA polymerases inactivate Mg2+ coordination in the active site, and eliminate enzymatic activity [We expressed both wild-type activity , 30.Rev3l-proficient and deficient cells expressing an empty vector (EV), as well as deficient cells expressing WT and ASM REV3L cDNA. Rev3l-deficient cells experienced S-phase associated delay and mitotic failure, leading to a population doubling time that was longer than Rev3l-proficient populations [REV3L re-expression in the deficient cell lines significantly decreased their doubling time to a level similar to Rev3l-/+ cells, whereas expression of the polymerase-inactive mutant had no effect  was not decreased in Rev3l-deficient cells , in a pure C57BL/6J background. All steps of genomic engineering were extensively monitored by Southern blotting analysis  allele of Rev3l is functional, but becomes a knockout allele  after action of the Cre recombinase. We expressed Cre recombinase in the cells to yield Rev3lM/Δ MEFs. The mice also harbored the mT/mG transgene to monitor Cre activity. This mT/mG transgene constitutively expresses red fluorescent protein (RFP). When Cre is active, the RFP gene is removed and green fluorescent protein (GFP) is expressed [Due to the early embryonic lethality in xpressed . This alRev3lM/Δ MEFs with Rev3lM/+ MEFs . We measured cell growth, cisplatin sensitivity and DNA double-strand breaks in GFP-positive cells. ASM MEFs had a growth defect compared to wild-type allele-containing MEFs   . A Rev3lin vitro activity. In addition, the central region contains a conserved positively charged domain [A major objective of this study was to determine whether the catalytic activity of pol ζ is responsible for the severe consequences observed in pol ζ mutant mouse cells. These include hypersensitivity to DNA damaging agents, a greatly increased generation of double-strand breaks in unchallenged cells, a slower growth rate, and a required role for pol ζ in embryonic viability. The impetus for this question is the existence of numerous other functional domains within the catalytic subunit of REV3L. These include a conserved N-terminal domain, two REV7 binding domains , 19, andd domain  that likERCC2/XPD gene. Complete disruption of XPD is incompatible with viability [There is ample precedent for such a situation. One example is the mammalian iability . Howeveriability . This isiability , 39. Anoiability . Recentliability .Rev3l DNA polymerase active site. No viable homozygous mice were produced, and the corresponding embryos died early in embryogenesis, as with a complete knockout allele. To investigate cell-autonomous consequences of the specific polymerase alteration, we derived primary MEFs that carried one null Rev3l allele, and one active site mutant allele. The growth defects, DNA break formation and cisplatin sensitivity of these cells were similar to cells harboring two null alleles [Here, we analyzed the consequence of a homozygous mutation of the  alleles . These rRev3l causes chromosomal instability in cells. This complicates studies of the consequences of Rev3l deficiency, as genomic alterations may accumulate during each cell cycle and lead to new phenotypes. A rigorous way to determine which phenotypes are directly caused by Rev3l loss is to complement the cells by expression of Rev3l cDNA. Here we utilized a complementation system for REV3L in mammalian cells, allowing definitive testing of whether phenotypes seen in Rev3l-deleted cells are due to Rev3l-deletion [Loss of deletion , 42. Ourdeletion .Rev3l-deficient phenotypes using human BL2 cell lines, reported to carry disruptions of REV3L [REV3L mutants. In contrast to the marked phenotypes found with Rev3l-deficient MEFs, the BL2 lines exhibited no statistically significant differences in cell doubling times, micronuclei formation or double-strand break formation as assessed by 53BP1 foci per cell. A modest sensitivity of 332 and 504 cells to UVC radiation and cisplatin was not rescued by complementation with REV3L. The limited sensitivity of 332 and 504 cells to a variety of DNA damaging agents has been noted [REV3L-deficient cells [REV3L-defective cell system. It is possible that the modest increased sensitivity of the BL2 subclones to UV radiation and cisplatin [REV3L-deficient, and are not well-suited for studies of REV3L function.We also attempted complementation of of REV3L . It is nen noted –45. Otheen noted . In a stnt cells , it was isplatin  might beisplatin –49. Our Rev3l-deficient DT40 cells are similar to Rev3l-deficient mouse cells, including elevated levels of spontaneous DNA breaks and sensitivity to DNA damaging agents. Intriguingly, it was reported that deletion of polh (pol η) could rescue the severe phenotypes of Rev3l-deficient DT40 cells [Rev3l –deficient cells are a consequence of a polh-dependent toxic intermediate. To test this model in mammalian cells, we investigated whether Rev3l-/-Polh-/- mice could be generated. We found that embryonic lethality of this double mutant was complete and similar in timing to Rev3l-/-Polh+/+ mice. Moreover, Rev3l-/ΔPolh-/- MEFs showed levels of DNA breaks and cisplatin sensitivity analogous to that seen with Rev3l deletion in the presence of pol η. The Rev3l-/loxPolh-/- MEFs were more sensitive to cisplatin than the Rev3l-/loxPolh+/+ MEFs, consistent with the cisplatin sensitivity of human polh-defective cells [Rev3l deletion cannot be rescued in murine cells by concurrent deletion of pol η. This is consistent with results found in the yeast S. cerevisiae, where a Rev3 Rad30 (pol ζ pol η) mutant is more sensitive to ultraviolet radiation than a single Rev3 mutant [TP53, and no poli gene has been found in the chicken genome. but it seems unlikely that either gene is relevant in this context. Previously reported Tp53-/-Rev3l-/- MEFs are also pol i deficient , and show major genome instability and DNA damage sensitivity [Polh poli double mutant mice are apparently normal with no deficits in development. A poli defect does not exacerbate the UV radiation sensitivity of polh-defective mouse cells, indicating that pol ι does not have a significant backup function protecting against lethality in the absence of pol η [The DT40 chicken cell line has been widely used to examine the consequences of DNA repair defects, because it is amenable to genetic manipulation by homologous recombination. Some characteristics of 40 cells . This leve cells . Notablyve cells . In summ3 mutant , 51. Altof pol η .Rev3l knock-in polymerase mutant mouse are relevant to development of REV3L as a target for chemotherapy. Suppression of REV3L sensitizes cancer cells to cisplatin in mouse model systems, and can limit chemo-resistance [Our results with the sistance , 53 becasistance –5. TheseRev3l-deficient TAg-immortalized MEFs were derived as in Lange et al [mT/mG+/-Rev3l-/lox or mT/mG+/-Rev3l+/lox, where “lox” represents a functional allele flanked by loxP sites. The mT/mG transgene constitutively expresses RFP, until Cre recombinase activity removes the RFP and allows expression of GFP [Rev3l . The cells were subcloned and selected for GFP positivity and for complete deletion of the floxed Rev3l allele. They were grown as in Lange et al [2. The primary MEFs were also derived and cultured in 2% O2 as in Lange et al [mT/mG+/-Rev3lM/lox or mT/mG+/-Rev3l+/lox, as well as from Rev3l-/loxPolh-/- or Rev3l-/loxPolh+/+ embryos. The loxP-flanked allele of the Rev3l gene was deleted using AdCre adfection, and the deletion efficiency was measured as described [ge et al . Brieflyn of GFP . The strn of GFP . All celge et al , in an age et al . They weescribed . For allThe BL2 parental cell line and subclones 332 and 504  were kinREV3L full-length cDNA was acquired in the pUC19M1 vector from Zhigang Wang [REV3L cDNA: a C-terminal Flag tag was added and the 5’-UTR was eliminated and replaced with an optimized mammalian Kozak sequence. This cDNA was cloned into the pTSIGN vector, which contains an EF1α promoter and an internal ribosomal entry site (IRES) fused to a neomycin-eGFP reporter. The active site mutation (residues D2781A/D2783A in human REV3L) was introduced into this pTSIGN-REV3L vector using PCR primers containing the REV3L mutations, and then the mutated PCR fragment was ligated into the REV3L-vector, replacing the wild-type sequence. The full-length human REV3L gene was PCR amplified from the pTSIGN-REV3L and pTSIGN-REV3L-ASM vectors and was cloned into the pETDuet-1 vector (Novagen). The REV3L gene was removed from the REV3L-pETDuet-1 vectors using XhoI/NotI digestion, and the resulting fragments were inserted into the pOZN vector . All vectors were completely sequenced to verify the integrity of the REV3L gene and the plasmid backbone. Full-length Flag-HA tagged REV3L can be expressed from this cDNA [The human ang Wang . The folted gene ). For thREV3L expression construct using the following PCR primers: NFwd: 5’ TAC ACA GTC CTG CTG ACC AC 3’, NRev: 5’ GAG GTA AGG AAA GAT GCC ATG TAG 3’, CFwd: 5’ ACC TAA CTC AGC ATG GCA TCT G 3’, CRev: 5’ CGG AAT TGA TCC GCT AGA G 3’ .The pOZN-REV3L or pCDH-REV3L vectors were transfected into HEK-293T cells using lipofectamine 2000 (Life Technologies), together with the retroviral packaging vectors psPAX2  and pMD2.G . 48 hr later, the media (containing pOZ or pCDH lentivirus) was collected. It was filtered, and polybrene was added to 4 μg/mL. This media was added to plates of immortalized MEFs (pOZ) or flasks of BL2 cells (pCDH). 48 hr later, the cells began selection for puromycin expression , or for IL2R expression (pOZ). The latter required incubation of the infected cells with IL2R-antibody conjugated magnetic beads followed by washing of the beads  from the exogenously expressed REV3L. We used primers and a probe that crossed the FLAG tag on the exogenous gene: FlagFwd: 5’–GTCTTTGTTTCGTTTTCTGTTCTG C– 3’; FlagRev: 5’–GCTTGTCATCGTCGTCCTTG– 3’; Probe: FAM-MGB-5’–GCT GTG ACC GGC GCC TAC TCT AG– 3’. Gene expression (with mouse or human GAPDH as an expression control) was measured on an Applied Biosystems 7900HT Fast Real-Time PCR System.Expression of the recombinant human REV3L was confirmed using a human-specific Taqman assay (Life Technologies) at the exon 14–15 boundary: Ex14Fwd: 5’ CAC CTG GCC TTA GCC CAT TAT 3’, Ex15Rev: 5’ CTC TTC TAA GAG TGT CAG TAT TAC TTC CTT TC 3’ Probe: FAM-MGB-5’ CAA CAG AAC CAA AAA CA 3’. In order to compare the recombinant expression to that of endogenous mouse All animal work in this study was done according to The University of Texas, MD Anderson Cancer Center Institutional Animal Care and Use Committee guidelines, and approved by the MD Anderson Animal Care and Use Committee (IACUC).Rev3l targeting vector was constructed from C57BL/6 mouse strain genomic DNA with a long (5.7 kb) homology arm upstream of exon 27, and a short (1.5 kb) homology arm downstream of exon 27. A D2773A/D2775A Rev3l mutant exon 27 and a Neo cassette (selection marker flanked by FRT sites for use in Flp-mediated excision) were inserted into intron 26. The targeting vector also incorporated a diphtheria toxin negative selection cassette. The action of Cre recombinase switched the allele to express mutant REV3L via the use of a combination of specifically oriented loxP and lox511 sites flanking the exons, as in The targeting vector construction and the FlEx strategy  . Positive selection started 48 hr after electroporation in medium containing 200 μg/ml of G418 . Rev3l resistant clones were isolated and amplified in 96-well plates. Duplicates of 96-well plates were made. The set of plates containing ES cell clones amplified on gelatin were genotyped by both PCR and Southern blot analysis.Linearized targeting vector was transfected into C57BL/6 ES cells  according to genOway's electroporation procedures : 5’-ATGCTCCAGACTGCCTTGGGAAAAG-3'Reverse: 5'-CTGGGGTGCTACTGTTCTTGTTAGAGTGC-3'loxP/lox511 sites):A second PCR was designed to confirm the integration of the FlEx cassette , which resulted in the inducible allele gene architecture. These mice were then crossed with CMV-Cre mice to remove the endogenous exon 27 and allow expression of the mutant exon 27 cassette, resulting in the constitutive ASM allele (Rev3lD2773A/D2775A mice) . Mice were bred to C57BL/6 mice expressing the Flp recombinase to remove the Neo cassette (5A mice) .Rev3lD2773A/D2775A-flox allele:The following genotyping primers were used to genotype the inducible Forward: 5’- GCCAAAGAGACATGCAGTGAGAAGAGTACC-3'Reverse: 5'- TGAGTGGGCTTGCAGAAGTCAGCA-3'Rev3lD2773A/D2775A-flox allele, 3353-bp for the Rev3lD2773A/D2775A-Neo allele. Animals were then validated by Southern blot analysis using a 3’ external probe: the wild-type allele gives rise to a 15 kb signal while the Rev3lD2773A/D2775A-flox allele gives rise to a 10.4 kb signal, and the Rev3lD2773A/D2775A-Neo allele is expected to give a signal at 11.5 kb.No amplification is expected for the wild-type allele, 1680-bp for the Rev3lD2773A/D2775A mice:The following genotyping primers were used to genotype the induced Forward: 5’- GCCAAAGAGACATGCAGTGAGAAGAGTACC-3'Reverse: 5'- TGAGTGGGCTTGCAGAAGTCAGCA-3'Rev3lD2773A/D2775A-flox allele, 1131 bp for the Rev3lD2773A/D2775A allele.No amplification is expected for the wild-type allele, 1680-bp for the Rev3lD2773A/D2775A allele gives rise to a 9.5 kb signal, and the Rev3lD2773A/D2775A-Neo allele is expected to give a signal at 11.5 kb .The presence of the mutant allele in the mice can be confirmed by PCR using the following primers: ASMWTFwd: 5’ TTG GGG CAT TGG TTT ACA GGT GGG 3’ and ASMWTRev: 5’ GCT GCT GAT ACT ACT ACT ACC ACC ACC ACT ACC 3’. Using these primers, the wild-type allele produces a 236 bp product, and the mutant allele a 345 bp product . Heterozygous mutant mice (which were maintained as pure C57BL/6 mice) were crossed in an attempt to observed homozygous mutant progeny. When this was unsuccessful, embryos were isolated at days 8.5–12.5 dpc. No viable embryos that were homozygous mutant were identified. To produce Rev3l and DNA polymerase η, we obtained mice from P. Gearhart with the genotype Rev3l-/loxPolh-/- . After crossing Rev3l-/loxPolh-/- mice, the resulting progeny were genotyped as in Lange et al [Rev3l-/-, Polh-/- embryos were identified. To study the phenotypes of Rev3l and pol η deficient cell lines, we crossed Rev3l-/lox, Polh-/- mice and isolated MEFs with this genotype.To investigate the phenotypes of animals and cell lines that were knockout for both t, as in ). These t, as in  we genotge et al , and Sarge et al . EmbryosRev3l-deleting primary MEFs, 1 day after deleting the Rev3l floxed allele with AdCre, the cells were plated into white 96-well plates . On day 3, cisplatin at various concentrations was added, and the cells were incubated for 5 days. Then ATP content was measured by luminescence, as above.To test sensitivity to chemical DNA damaging agents, the immortalized MEFs or BL2 cells were plated into white 96-well plates . The following day, various concentrations of cisplatin (Sigma) or bleomycin (Sigma) were added to the wells, and the cells were incubated for 48 hr. Then the cells were lysed, a reagent was added that emits light in the presence of ATP , and luminescence was measured using a plate reader (Biotek Synergy II). The luminescence measurement was normalized to undamaged control. To test cisplatin sensitivity in 5 cells were pelleted and resuspended in 300 μL of phosphate-buffered saline. Three 100 μL drops were placed into the middle of a plastic dish and 10 μL aliquots from each were plated into 100 μL of growth media in a white 96-well plate after 0, 2.5, 5, 7.5, 10, 15 or 20 J/m2 UVC radiation at a fluence of 0.4 J/m2 s-1. 48 hr after irradiation, ATP content was measured as above.To test sensitivity of immortalized MEFs or BL2 cells to UVC radiation, 3 x 10et al [Rev3l floxed allele using AdCre. 48 hr later they were fixed and stained for DAPI, 53BP1 and γ-H2AX as above. Photographs of the immunofluorescence were taken on the Leica microscope, and cells containing double-strand breaks were scored as those with 3 or more 53BP1 + γ-H2AX foci.To measure the formation of DNA double-strand breaks, immortalized MEFs were plated in an 8-well chamber slide. The following day they were fixed and stained for DAPI, 53BP1 and γ-H2AX, as in Lange et al  with a t-/Δ and Rev3L+/Δ cell lines for sister chromatid exchanges (SCEs) was as described [2PO4 pH 6.8). The chromosome spreads were viewed at 600X magnification under oil. Thirty to thirty-five chromosome spreads were counted for each genotype, and both total chromosome number and number of SCEs was assessed.Assessment of the Rev3Lescribed . BrdU . Statistical analysis of cell survival after cisplatin or UVC treatment, or of cell growth, was done using linear regression analysis, and the lines were compared based on equality of slope and intercepts.S1 FigS. cerevisiae Rev3, owing largely to a central domain of 1500 amino acids. A Blast search using the central domain detects similarity to the KIAA2022 gene on the human X chromosome, as we noted previously [REV3L), encoding 1386 amino acids. A Clustal X alignment, using default parameters, was generated by aligning this region of the indicated REV3L orthologs with the 1458 residue human KIAA2022 exon 3-encoded region from the same species. Most homology between the KIAA2022 and REV3L gene products is confined to a region of about 250 amino acids, with the three regions of highest similarity shown here. The third region includes a proposed nuclear localization signal [REV3L gene. This is an example of the frequent gene traffic between the mammalian X chromosome and autosomes observed during evolution [Mammalian REV3L is about twice as long as eviously . The KIAeviously . KIAA202eviously . Most ofn signal . Part ofvolution .(TIFF)Click here for additional data file.S2 FigREV3L in these cell lines using a pCDH vector system with puromycin as a selectable marker  Expression of recombinant Flag-tagged empty vector in BL2 WT cells or recombinant Flag-tagged REV3L in 332 cells. The 332 and 504 cells still expressed detectable levels of endogenous REV3L cDNA, but expression of exogenous recombinant REV3L could be achieved above the endogenous level. Expression of exogenous REV3L cDNA was confirmed using primers specific to that cDNA. (B) Doubling time (in hr) of BL2 WT, REV3L-deficient 332 and 504 cells, and 332 cells expressing exogenous REV3L. Expression of wild type REV3L cDNA did not alter the doubling time of these cells. (C) Survival of the same cell lines as in (B) as measured by ATP concentration 48 hr after addition of cisplatin. The 332 and 504 cell lines were not more sensitive to cisplatin than the parental cells. (D) Survival of the cell lines as measured by ATP concentration 48 hr after ultraviolet C (UVC) radiation. Both lines appeared moderately UVC radiation-sensitive, but REV3L re-expression did not rescue this sensitivity. (E) Quantification of percent of BL2 cell nuclei with associated micronuclei. (F) Quantification of cells with fewer than 3, 3 to 5, 6 to 10 or greater than ten 53BP1 foci (as measured using CellProfiler). There was no significant elevation in the spontaneous incidence of DNA double-strand breaks in 332 or 504 cells (compare Two reported human REV3L knockout lines designated 332 and 504 were derived from the Burkitt lymphoma cell line BL2 . We expr vectors ). We having pCDH . (A) Exp compare . Data re(TIFF)Click here for additional data file.S3 Figet al., who made the 332 and 504 cell lines, designed a targeting strategy with the intention of removing the exon encoding the initiating ATG codon for REV3L, and a downstream exon [REV3L transcript variant 1. The bottom part of the figure shows the location of the primer pair given in [neo marker intended to disrupt exon 5. The primer 3'-KO-zeta-2 for this analysis is located 100 kb away from exons 5, a distance not compatible with PCR genotyping. We note also that both primers used to test absence of Rev3l mRNA by Gueranger et al. are located in the deleted region, and therefore no PCR product would be generated in cells even if REV3L mRNA were present.The major transcript variants of human REV3L are shown. Transcript variant 1 (NM_002912) encodes DNA polymerase ζ catalytic subunit isoform a, with 33 exons translating to 3130 aa. Transcript variant 2 (NM_001286431) encodes DNA polymerase ζ catalytic subunit isoform b, with 35 exons translating to 3052 aa. The two additional exons in transcript variant 2 are indicated (yellow). A short upstream ORF is also present in transcript variant 2. Gueranger (TIFF)Click here for additional data file.


Brain arteriovenous malformation (AVM) is an important cause of hemorrhagic stroke. The etiology is largely unknown and the therapeutics are controversial. A review of AVM-associated animal models may be helpful in order to understand the up-to-date knowledge and promote further research about the disease. We searched PubMed till December 31, 2014, with the term “arteriovenous malformation,” limiting results to animals and English language. Publications that described creations of AVM animal models or investigated AVM-related mechanisms and treatments using these models were reviewed. More than 100 articles fulfilling our inclusion criteria were identified, and from them eight different types of the original models were summarized. The backgrounds and procedures of these models, their applications, and research findings were demonstrated. Animal models are useful in studying the pathogenesis of AVM formation, growth, and rupture, as well as in developing and testing new treatments. Creations of preferable models are expected. Brain arteriovenous malformations (AVMs) are vascular anomalies where arteries and veins are directly connected through a complex, tangled web of abnormal vessels instead of a normal capillary network. There is usually high flow through the feeding arteries, nidus, and draining veins. AVMs represent a high risk for hemorrhagic stroke, leading to significant neurological morbidity and mortality in relatively young adults . How theThe management in the case of sudden bleeding is focused on restoration of vital function and prevention of recurrent hemorrhage, usually with some combination of surgical resection, embolization, and stereotactic radiotherapy. But all of these treatments pose a risk of serious complications, and the optimal treatment needs to be evaluated . For nonAs considered to be embryonic origin and postnatal development, AVMs are highly dynamic rather than static , 5. AngiAnimal models are warranted to meet the needs mentioned above. Up to now, several experimental animal models have been developed in studying the AVM-related hemodynamics, pathogenesis, and treatments. Hence, a review was made about the background, the procedure, and the application of these models, and their advantages and disadvantages were briefly analyzed.The aim of the review was to encourage creating more advantageous AVM models and promote further studies of the disorder.We searched PubMed till December 31, 2014, using the term “arteriovenous malformation,” limiting results to animals and English language.Two investigators read the titles and abstracts of the publications to find out the possibly relevant ones that described creations of AVM animal models or investigated AVM-related mechanisms and treatments using these models. The articles describing the creation of the dural arteriovenous fistula models or AVM lesions in other organs were excluded. Full texts of the selected articles were obtained, and those fulfilling our inclusion criteria were identified and finally summarized.The emphases of the review were on the background, the procedure, and the application of each particular model. The chosen animals, the advantage, and the disadvantage of each model were also briefly discussed.From the result of total 911 publications found according to the search term, we picked up more than 100 articles, by the inclusion criteria of either describing the creation of original or modified animal models or adopting these models to make experimental researches.The animal models in the study of AVMs were diverse in accordance with research purpose, ranging from those based on the changes of the cerebrovascular circulation to those based on gene manipulation techniques. Eight different types of the original models were summarized and their highlights were shown in Tables 2 reactivity in the closed fistula was preserved. This model was probably not enough to clarify the mechanisms of the NPPB phenomenon [To explain a phenomenon that brain tissue surrounding the AVM lesion is subject to swelling and hemorrhage immediately following surgical excision of the lesion, Spetzler et al. firstly suggested that the chronic ischemic brain tissue near high flow AVMs might experience a loss of vascular autoregulatory capacity, the theory of normal perfusion pressure breakthrough (NPPB), by using carotid-jugular fistula (CJF) model in cats . This moenomenon –38.2 reactivity of cerebral vessels remained intact throughout the experiment. The research group recommended the avoidance of intraoperative hyperventilation and postoperative hypertension for the removal of AVM lesions. By using this model, a research group tested the hypothesis that intracerebral, extracellular norepinephrine could be the key factor influencing CBF levels [Therefore, a modified CJF model in rats was introduced by Morgan and colleagues. They made an end-to-end anastomosis of both rostral ends of the CCA and the EJV  on the right side and ligated the caudal ends of both vessels and the ipsilateral external carotid artery (ECA), creating a functional arteriovenous fistula between the circle of Willis and the right lateral sinus . After aF levels , and anoF levels .Besides, “occlusive hyperemia” was also suggested to be related to the brain edema and hemorrhage following the large AVM resection. High blood flow and mass effect of AVM lesions might cause obstruction of the venous outflow and stagnation of arterial inflow in their adjacent parenchyma, with subsequent worsening of the existing hypoperfusion and ischemia in these tissues. Bederson et al. evaluated this presumption in a rat CJF model by a proximal CCA to distal EJV anastomosis with contralateral EJV occlusion . The fisRats were mostly chosen as the model animal probably because they are economic and accessible in spite of their anatomical differences related to humans. CJF models were also tried in monkeys; however, they were hard to handle, expensive to create, and also with intricate ethical concerns .2 reactivity decreased significantly at shunt opening. The regional hemodynamic changes in this animal model simulated a real condition of brain tissues surrounding human AVMs. However, this was an acute model and the procedure was a bit complicated.Carotid-jugular fistulae resulted in the hemodynamic changes in whole brain or predominantly the hemisphere in the fistula side, but not in the regional parenchyma. A dog model with local cerebral hypoperfusion was tried using an intracranial arteriovenous fistula . The dogBoth extracranial and intracranial arteriovenous fistula models lacked a real AVM nidus, these models were focusing on the hemodynamic and pathophysiological changes of AVM adjacent parenchyma, but not the AVM lesion itself.The carotid rete mirabile (RM) of the swine is a special vascular structure with a tangle of microarteries and arterioles situated at the termination of each ascending pharyngeal artery (APA) as it perforates the cranial base. The two sides of the RM, which are connected with each other across the midline, are also supplied by other small collateral arteries and effuse to form internal carotid arteries ipsilaterally . At the To address this shortfall, Chaloupka et al. produced a high flow arteriovenous shunt in the swine RM by inserting a needle through the orbit to create communications between the rete and the surrounding cavernous sinus . SuperseMassoud et al. developed a distinguished swine AVM model with induced high blood flow across both retia, by surgical formation of a side-to-side arteriovenous fistula between the CCA and the EJV with the ligation of the CCA proximal to the fistula on the right side . The angBased on Massoud's model, modified swine AVM models were introduced. They posed a higher pressure gradient closer to values found in human AVMs, thereby reducing the rate of spontaneous thrombosis in the rete , 60.Besides, in the pig, the natural structure of carotid RM is also seen in the other artiodactyl animals such as the sheep, goat, ox, and cat, but not in the dog, rabbit, and rat. Whether the swine RM models can be duplicated in the other animals was unknown, except for a feasibility study in the sheep . The vasIn 2004, Yassari et al. described a rat model with the sham AVM nidus simply by ligating the left EJV at the confluence of the subclavian vein and making an end-to-side anastomosis of the EJV to the CCA . These rFurther analysis in this model demonstrated that the nidus vessels underwent morphological changes from normal veins to those similar to immature vessels in human AVMs, including heterogeneously thickened walls, splitting of the elastic lamina, and thickened endothelial layers . AnotherThe activation of vascular cells in the nidus made it a unique model for studying the occlusive effect of radiosurgery on AVM vessels, because little was known about the molecular mechanisms of radiation mediated vascular obliteration. One study using the model showed that the expression of endothelial adhesion molecules in the nidus cells changed after radiosurgery . Other sBoth the AVM lesions with simulated niduses using the RM and the venous plexus did not actually locate in the cerebral parenchyma. Pietilä et al. developed a novel model with an induced AVM lesion in the dog brain . A vascuThere were some highlight features of this model resembling the appearance of AVMs in human, including thickening and fibrosis of the draining venous wall, new formation of vessels, and vascular proliferation, surrounding brain tissues with signs of ischemia and hemorrhage. Although an exquisite surgical technology was required for producing the animal model, it might help discovering the pathological mechanisms involved in AVM development.Currently, radiosurgery was a kind of less invasive treatment for AVMs. It took a therapeutic effect by obliterating the AVM nidus, with a low obliterating rate and a latency period up to 2 years. Further understanding of the mechanism of radiosurgery might be helpful to develop advanced pharmacological therapies to improve the occlusive effects based on conventional radiosurgery.For this purpose, the xenograft arteriovenous fistula model was created, as a segment of main arteries from transgenic mice was interposed between the caudal end of the CCA and the rostral end of the EJV in immune-deficient nude rats . The impIn this model, the arteriovenous fistula with radiation pretreatment reproduced distinct radiation arteriopathy as observed in resected human AVM specimens pretreated with radiosurgery. If radiation pretreatment would result in a specific molecular change in the fistula graft, or if the fistula graft from different transgenic mice would have a different response to radiation, this model probably yielded clues to the vascular targeting therapy and the gene therapy. One study had detected that some robust but modified radiation responses occurred in Endoglin and eNOS knockout transgenic arteriovenous fistulae .The model was technically feasible and the overall angiographic patency rate was about 50%. However, there was a time limitation of 4 months for allowing transplanted tissues to retain their phenotypes due to the rejection reaction.The surgically resected human AVM lesions were valuable specimens for the histopathological study. When the specimens were transplanted into the corneal micropocket of the rats, they kept alive and growing. The angiogenic activity of the implanted tissues could be repeatedly measured according to a standard of neovascularization assessed by microvessel counts and VEGF expression .Based on the model, the implanted AVM tissues showed the highest angiogenesis compared to other cerebrovascular disorders, cavernous malformation, and venous angioma, indicating that the AVM niduses were more likely to be active and progressive. The implanted AVM tissues previously treated with embolization exhibited the highest angiogenic activity, followed by untreated and gamma knife treated AVM tissues; this might explain why AVM recurrence after intravascular embolization was more common. Moreover, this rat cornea model containing human AVM tissues could be used for evaluating molecular mechanisms of the neovascularization process over time .Eng) and Activin receptor-like kinase 1 (Alk1), respectively, cause HHT type 1 and type 2. It is logical that animal models containing spontaneous or induced AVM lesions could be generated by regulating the genes.Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder characterized by recurrent nosebleeds, mucocutaneous telangiectases, and AVM formations in the brain and other visceral organs . Heteroz Alk1 by its splice-site blocking morpholino caused a spectrum of morphologic and functional defects as AVM lesions in zebrafish embryos [ Eng or Alk1 genes died in embryonic period due to defects in vessel and heart developments [ Eng+/− and Alk1+/− haploinsufficient mice could be successfully generated. These mice develop vascular lesions in various organs, but spontaneous lesions in the brain were modest in Eng+/− mice and minimal in Alk1+/− mice [ Eng+/− or Alk1+/− adult mice [ Eng+/− mice than in Alk1+/− mice. Interestingly, increased cerebral perfusion by intraventricular infusion of hydralazine or nicardipine after VEGF delivery promoted capillary dysplasia in Alk1+/− mice. These studies demonstrated that VEGF delivery into the brain of wide type mice led to increased microvessel counts but not microvascular dysplasia, and saline injection did not cause significant microvascular changes even in the haploinsufficient mice, approving that the development and progression of AVM lesions in adult brains were possible, when hereditary variation was combined with endogenous or exogenous growth factor delivery. Although sharing the somewhat alike phenotype, the induced local microvascular dysplasias were not enough to stand for direct models of the disease. However, they might be useful in identifying the possible factors which took a role in the pathogenesis of AVMs.Knockdown of embryos . The tralopments , 21, 69.+/− mice , 22. A rult mice –25. The  Alk1 alleles in adult mice by tamoxifen-inducible Cre resulted in AV fistula formations and spontaneous hemorrhage mostly in the lung, gastrointestinal track, and uterus, but not remarkably in the brain, although de novo vascular malformation lesions developed upon induction of skin wounding in these mice [ Eng deletion mice, in which vessel abnormalities mimicking human AVM nidus were induced in the brain with the presence of angiogenic stimulation such as mechanical injury or VEGF delivery [2LoxP/2LoxP and Eng2LoxP/2LoxP mice [2LoxP/2LoxP mice due to more effective gene deletion. In fact, regional deletion of Eng caused more severe cerebrovascular malformation per copy than Alk1 with VEGF stimulation. These models were promising for evaluating the pathogenic mechanisms of AVMs and for discovering potential medical therapies to slow AVM growth and stabilize the rupture-prone abnormal vasculature.The conditional knockout technique with Cre/LoxP recombination system made it possible to delete target genes at the planned time or in the expected cells, because the Cre enzyme expression could be precisely controlled. Conditional deletion of bothese mice . The simdelivery . MeanwhioxP mice , 29. The Alk1 alleles in restricted endothelial cells (ECs) caused severe and fatal visceral arteriovenous malformations [ Alk1 specifically in ECs in adult mice resulted in AVM formations in the intestine, lung, and around ear-tag wounds, as well as in the brain area previously injected with vectors expressing EVGF [ Alk1 deletion [ Alk1 in pericytes alone was not sufficient to initiate AVM development in adult mice. Similarly, endothelial specific deletion of Eng led to endothelial proliferation and AVM formations in neonatal retina and local venomegaly in the adult skin induced by mechanical and VEGF stimulation [ Alk1 in the embryo by SM22-Cre, which was expressed in smooth muscle cells, ECs, and some other cell types in different organs [Eng2LoxP/2LoxP; with SM22-Cre expressed mice, in which AVMs were found in the central nervous system and intestine, more than half of the mice died from internal hemorrhage before 6 weeks of age [Antenatal deletion of bothrmations . Conditiing EVGF . Model mdeletion , indicatmulation . Owing tt organs . Most ofs of age . These d Notch4 in the endothelium by the tetracycline-regulatory system elicited cerebral arteriovenous shunting in mice, and gene repression reversed the AVM progression [ Notch1 led to AVM formations in the neonatal mouse brain, and activation of Notch1 in adult mouse caused AVM formations in other organs, but not in the brain [Other genes involved in angiogenesis would also be manipulated to create AVM models. Taking essential roles in vascular development and remolding, Notch signaling pathway was upregulated in human AVMs and might be an important molecular regulator of AVM pathogenesis . Both Nogression . Furthergression . Similarhe brain , 74. Mgp−/− mice, but not in Mgp+/− mice at 4 weeks of age. Mgp is a bone morphogenetic protein (BMP) inhibitor. Increased BMP activity due to the deficiency of Mgp induced expression of Alk1 and subsequently enhanced expression of Notch ligands Jagged 1 and Jagged 2, which abnormally increased Notch activity. As expected, reduced Jagged expression in the Mgp−/− mice by crossing them with Jagged deficient mice normalized endothelial differentiation and prevented AVM formations [ Rbpj, a mediator of Notch signaling, in postnatal day one resulted in features of AVMs in the mouse brain, including abnormal AV shunting and tortuous vessels. Deletion of the Rbpj gene in adult mice did not cause brain AVMs [The lack of matrix Gla protein (Mgp) also caused AVMs in mice. Cerebral enlarged vessels and direct connections between arteries and veins were detected in thermations . Moreoveain AVMs .Cerebrovascular abnormalities, AVM formations, and hemorrhage occurred spontaneously in some cases where relevant genes were directly or conditionally deleted at the antenatal or postnatal stages, although in most cases, the model mice either displayed minimal vascular lesions or obvious vascular lesions out of the brain. The spontaneous cerebral AVM lesions partially simulated the natural clinical course of the disease, but the lesions lacked uniformity and reproducibility in size and location. Focal angiogenic stimulation based on gene deficiency helped to create adult onset models of induced AVM lesions in the brain. These models containing comparable AVM lesions might be more suitable for mechanism and therapeutic studies. In spite of posing disadvantages such as complicated procedures, high expanding, and being time consuming, the models by gene manipulation were unique for investigating the AVM pathogenesis and testing new therapies.As shown in Tables An ideal AVM model, which completely shared the same anatomic, physiologic, biological, and clinical features as human AVM disease, was lacking. Even the transgenic mice model carried out with spontaneous but systematic vascular malformation lesions could not fully represent the sporadic cases mostly seen in clinic. In spite of limitations, these various models provided assistance to answer particular questions in the study of AVMs.β (IL-1β) with single nucleotide polymorphisms (SNPs), but the limited results were inconsistent [The origin of AVM is still a mystery. It was generally believed that the vascular disorder was initiated during embryonic development. However, evidences from animal models demonstrated that postnatal formations of AVMs were possible, due to the two causal factors of angiogenic stimulation and gene deficiency. With genome-wide association study, investigators attempted to identify mutant genes associated with AVM susceptibility in sporadic AVM patients. The possible involved genes included Alk1, Eng, interleukin-6 (IL-6), and interleukin-1nsistent , 76.The mechanisms that underlie AVM growth and progression remain poorly understood. Abnormally high blood flow and shear forces in nidal vessels activated molecular pathways in smooth muscle cells and ECs. Hypoperfusion and hypoxia in the nidal and surrounding tissues stimulated angiogenesis and inflammatory reactions. Both of them lead to vascular proliferation and remodeling . These hα (TNF-α), MMP-9, and other genes in AVM specimens appeared to influence clinical course of AVM rupture [Intracranial hemorrhage is the most severe and most common clinical presentation of AVM patients. Risk factors associated with AVM rupture include certain genetic mutations, intranidal aneurysms, exclusive or restricted venous drainage, deep or infratentorial location, and history of previous hemorrhage –81. SNPs rupture . However rupture . Further Alk1 knockout zebrafish through modulating the BMP signaling pathway [2LoxP/2LoxP mice model with focal AVMs by virus-mediated Cre and VEGF, the induced angiogenesis and vascular dysplasia were attenuated by administration of VEGF antagonist bevacizumab [Among the conventional treatments, microsurgical resection is currently recommended for Spetzler-Martin Grades I and II AVMs. For high-grade AVMs, combined treatments are often used lacking a standard procedure. Given that the majority of high-grade lesions cannot be treated without relatively high morbidity and mortality, new biological therapies and gene therapies are under development aiming toward vascular remodeling. A study showed that losartan, an angiotensin II receptor antagonist, attenuated abnormal blood vessel morphology in the pathway . In the acizumab , which lacizumab . MoreoveWe hope this review would provide the basic of currently available AVM models. The diverse techniques and methods displayed here might shed light on the creation of preferable AVM models in the future, overall promoting further studies of the disease.


This paper presents a high payload watermarking scheme for High Efficiency Video Coding (HEVC). HEVC is an emerging video compression standard that provides better compression performance as compared to its predecessor, i.e. H.264/AVC. Considering that HEVC may will be used in a variety of applications in the future, the proposed algorithm has a high potential of utilization in applications involving broadcast and hiding of metadata. The watermark is embedded into the Quantized Transform Coefficients (QTCs) during the encoding process. Later, during the decoding process, the embedded message can be detected and extracted completely. The experimental results show that the proposed algorithm does not significantly affect the video quality, nor does it escalate the bitrate. High Efficiency Video Coding (HEVC) is a relatively new video compression standard, developed by the Joint Collaborative Team on Video Coding (JCT-VC), from ITU-T VCEG and ISO/IEC MPEG Digital watermarking of HEVC encoded videos may be a difficult task, because the codec eliminates most of the redundancy that the watermarking process may exploit. Casual embedding of a watermark, thus, may escalate the final video file size or otherwise affect the quality of the video; a carefully conceived embedding strategy is thus needed. Keeping these in view, we intend to propose an HEVC watermarking scheme that would have negligible effect on both the video quality and the final file size. Our strategy is to embed a watermark, during the encoding process, that can be completely extractable during the decoding process. Normally, a watermark may either be embedded in the spatial domain or the frequency domain. With spatial domain video watermarking, the hidden data may be lost during the quantization step of the underlying video codec. One solution to this problem is to embed the watermark in such a way that it survives the quantization loss. But this may come at the cost of lower imperceptibility. A better solution is to go for the frequency domain and better embed the watermark after the quantization step, i.e. in the quantized transform coefficients (QTCs). In our approach, we adopt this later approach and embed the watermark message in the selected non-zero QTCs of all the frames of the video.The rest of the paper is organized as follows. For a better comprehension of this article, the first part of Section outlines a brief overview of the state of the art HEVC standard. The second part of the same section provides a brief literature review regarding the watermarking techniques proposed for various video coding standards, in vogue. The proposed watermarking algorithm is outlined in Section 0.2.2, wherein both the embedding and detection processes are described. Section 0.2.2 analyses the experimental results, followed by the concluding remarks in Section 0.2.2.Like its recent predecessors, HEVC is also a hybrid video compression standard based on the Intra/Inter Prediction and a 2D transform. It is an effort to improve upon the existing tools used in H.264. Besides, many new coding tools have been introduced in the HEVC; the most important change being its frame partitioning. HEVC introduces the three new concepts of Coding Unit (CU), Prediction Unit (PU) and Transform Unit (TU).The coding pipeline splits each frame into what are called Coding Tree Units (CTU). A CTU has one Coding Tree Block (CTB) covering a A CTB can be partitioned into smaller blocks using a Quad Tree structure. A given CU is a part of CTB and can be divided recursively into 4 CUs and each has an associated division into Prediction Unit (PU) and Transform Unit(TU).PU is created when a prediction method is chosen. The information of the prediction method (Intra/Inter and the related data) is contained in the PU.The Prediction Block can be split using a sampling scheme that may range 64×64 to 4×4 samples.For the intra-prediction in HEVC, 33 angular directions are used.The PU can be sub divided into 2 rectangular or 4 square partitions, in the inter-prediction. For motion compensation, the PU division may be unidirectional or bi-directional.HEVC uses transform coding of the prediction residual in the similar way as its predecessor H.264/AVC. The residual block is split into smaller square transform blocks (TBs).The transform is an approximation of DCT its block sizes can be 32×32, 16×16, 8×8 and 4×4.HEVC also has mode dependent alternative transform. An alternative integer transform derived from discrete sine transform (DST) is applied on each TB of size 4×4. The DST is only applied on luma transform blocks.Transform coefficients in the encoder side are quantized to limit the number of bits. At the start, the quantization level is defined by a quantization parameter (QP) value that controls the uniform-reconstruction quantization (URQ) scheme. To further decrease the bitrate, the QTCs are entropy coded.Only one entropy coding is specified in the HEVC, i.e. the context adaptive binary arithmetic coding (CABAC). The CABAC is used to encode the first coefficients (levels), Golomb-Rice coding to code the next and Exponential-Golomb coding is employed for encoding the last levels.The degradation of frames, which is caused by compression, is restored by applying three kinds of filters namely the sample adaptive offset (SAO), de-blocking and adaptive loop filters (ALF).A new video parameter set (VPS) is also introduced in HEVC.To increase the parallel processing capability, HEVC introduces three new features other than the slices, such as tiles, wavefront parallel processing (WPP) and dependent slices.It is pertinent to note that H.264 concepts, like the high level syntax and the Network Abstraction layer (NAL), are being retained in HEVC.While being still in its rudimentary phase, efforts regarding the watermarking of HEVC videos are scarce. The literature is, however, replete with algorithms regarding the watermarking of videos based on H.264 and other coding standards. With H.264, the watermarks are normally embedded into the DCT coefficient from the I- and P-frames Zhang et al. The MPEG-2/4 based Watermarking methods, from the literature, also rely on the DCT coefficients In MV based watermarking schemes, the watermark is embedded either directly in the video bitstream Beside the DCT based and MV-based strategies, there are approaches, like The proposed algorithm targets mainly the imperceptibility of the cover and it can be employed in applications where robustness is of secondary importance, e.g. broadcasting and hiding some sort of metadata. For embedding, we are relying on the LSB modification of the QTCs from the HEVC coding pipeline. The watermark is embedded in the coefficients whose values agree to a certain predefined threshold. The value of the threshold is selected on the basis of the size of the watermark, in bits. To avoid any significant escalation in the compression efficiency, only the non-zero QTCs are being considered for embedding - otherwise, many zero magnitude coefficient may become non-zero in the embedding process, thereby affecting the zero runlengths.The message is embedded should be completely extractable on the decoder side.bM) in each QTC. The selection criteria for QTC is based on a threshold value of 1; if the absolute value of QTC is superior to this threshold then a watermark bit is embedded in its LSB, as demonstrated by the algorithm illustrated in M) composed of bits bM. The watermarked QTCs are denoted by The proposed algorithm modifies the LSB of the selected QTCs and embeds one of the watermark bit  measure has been used to analyze the quality of watermarked video with respect to original video which is given by:Where mean square error (MSE) is a measure used to quantify the difference between the initial video frame I and the distorted video frame When it comes to payload, it will be higher for lower QP values, for obvious reasons. The frame size escalation is high at lower QP values, as illustrated in Generally, the PSNR decreases with any increase in the QP value, for both original and watermarked videos. For the video in hand the decreasing function is a straight line. Both the watermarked and un-watermarked video behave the same but, at low QP values, their PSNRs are significantly different from each other; still the watermarked video quality is good.The bitrate escalation is not that significant, as it is already an exponential function of the QP value and the watermark is embedded in the LSBs. The escalation at low QP values is more marked, however.The payload decreases exponentially with respect to an increase in QP value. The lower the QP value, higher will be the payload.We proposed a high payload watermarking algorithm for the emerging video coding standard HEVC. For the sake of imperceptibility, the watermark is embedded into the LSBs of selected non-zero coefficients from the QTC domain. The results show that the proposed scheme has the advantages of imperceptibility, bitrate conservation and high payload. These advantages are, however, highly sensitive to the QP value. The escalations are, however, somewhat marked only when the QP value is low. In future, the robustness of the method needs to be improved and a spread spectrum strategy would be explored for embedding.


The content and quality of medical information available on video sharing websites such as YouTube is not known. We analyzed the source and quality of medical information about Ebola hemorrhagic fever (EHF) disseminated on YouTube and the video characteristics that influence viewer behavior. An inquiry for the search term ‘Ebola’ was made on YouTube. The first 100 results were arranged in decreasing order of “relevance” using the default YouTube algorithm. Videos 1–50 and 51–100 were allocated to a high relevance (HR), and a low relevance (LR) video group, respectively. Multivariable logistic regression models were used to assess the predictors of a video being included in the HR vs. LR groups. Fourteen videos were excluded because they were parodies, songs or stand-up comedies (n = 11), not in English (n = 2) or a remaining part of a previous video (n = 1). Two scales, the video information and quality and index and the medical information and content index (MICI) assessed the overall quality, and the medical content of the videos, respectively. There were no videos from hospitals or academic medical centers. Videos in the HR group had a higher median number of views , more ‘likes’ , channel subscriptions , and ‘shares’ . Multivariable logistic regression showed that only the ‘clinical symptoms’ component of the MICI scale was associated with a higher likelihood of a video being included in the HR vs. LR group.. YouTube videos presenting clinical symptoms of infectious diseases during epidemics are more likely to be included in the HR group and influence viewers behavior. Filoviridae family is one of the most virulent pathogens in humans  disseminated on YouTube and the video characteristics that influence viewers’ behavior.http://www.youtube.com) was accessed on December 9th, 2014 from Cleveland, OH, USA. A new account was made for the purpose of the study. An inquiry for the search term “Ebola” was requested, and the results were sorted in decreasing order of “relevance” using the default YouTube algorithm. Out of the approximately 1.35 million results, we selected the first 100 videos and saved them in a playlist because search results on YouTube can change from day-to-day. Uniform resource locators (URLs) of the top 100 videos were also saved separately for backup.The online video hosting resource YouTube  and the medical information and content index (MICI) scale.The VIQI scale assesses the overall quality of the video, and addresses each component of the Global Quality Scale (GQS) separately  to 5  to assess the following components of the medical information included in the videos: prevalence, transmission, clinical symptoms, screening/testing, and treatment/outcomes of the Ebola virus infection.Data was collected and managed using REDCap electronic data capture tools hosted at Cleveland Clinic. REDCap (Research Electronic Data Capture) is a secure, web-based application designed to support data capture for research studies, providing: (1) an intuitive interface for validated data entry; (2) audit trails for tracking data manipulation and export procedures; (3) automated export procedures for seamless data downloads to common statistical packages; and (4) procedures for importing data from external sources.For continuous variables, means with standard deviations or medians with interquartile ranges were calculated, as appropriate. For hypotheses testing, we calculated differences in means using the unpaired Student T test and the test of medians for non-parametric data. Variables were compared between the HR and LR groups. Multivariable logistic regression analysis was conducted to investigate the predictive power of each video characteristic on the YouTube video relevance.Stata Statistical Software: Release 13. College Station, TX: StataCorp LP.). A p value <0.05 was considered significant.The data analysis was done using stata 13.1 , not in English language (N = 2) or a remaining part of a previous video (N = 1). The kappa coefficient of agreement between the two physicians that initially reviewed the videos was 0.82. Forty-two (84.0 %) and 44 (88.0 %) videos were included in the HR and LR group, respectively. Fifty videos (58.13 %) originated from IU/W and 36 (41.87 %) from GA/NA sources.The videos characteristics are summarized in Table Videos in the HR group scored significantly higher in the ‘flow’  and ‘precision’  components of the VIQI score but there was no difference in the total VIQI score between the HR and LR groups. The MICI score was higher in the HR group when compared to the LR group .Of the seven video characteristics included in the backward multivariable logistic regression analysis, only the ‘clinical symptoms’ score Table  was assoOur study provides a detailed analysis of the YouTube videos as a source of medical information. As the use of electronic multimedia infiltrates every aspect of medicine and healthcare, it is essential to study the accuracy and quality of the information delivered. During the 2014 EHF epidemic, Internet was considered to be the best tool available to health experts to predict a disease outbreak . HoweverIn our study, the comparison of the HR and LR groups confirms the validity of the YouTube algorithm in ranking videos during a search inquiry. Interestingly, no significant differences were observed in the total VIQI score between the two groups, probably because current technology enables any person to produce a high quality video. However, the HR group fared better in comparison of the ‘flow’ and ‘precision’ components of the VIQI score. This is likely because flow and appropriate title assignment lead to a higher watch time, which is already a YouTube search result criteria.Medical information and content index scores were higher in the HR vs. the LR group. Specifically, videos that discuss clinical symptoms in most details have the greatest impact on video inclusion into the HR group, showing that people utilize resources such as YouTube to better understand clinical symptoms of a disease rather than prevalence, outcomes and other attributes of the disease, and suggesting that video producers need to assign more video time on the discussion of clinical symptoms.The concerning observation that our study brings forth is the fact that amongst the top 100 videos on EHF there were none from academic medical centers or hospitals. Studies on chronic disease information delivery via YouTube have shown that university channels provided about 21 % of the data and were considered sources of useful information , we were able to minimize the possibility that large panic-driven changes in view times and other variables affected the results. Also, there is a finite possibility that the 100 videos that were studied do not completely represent the available information on EHF.Between September 2014 and the first week of January 2015, a total of 60,308 people tested positive for Influenza (CDC In the era of cost-conscious care, universities and academic centers should consider dissemination of reliable information through the social media and video sharing websites, especially in countries with good penetration of the Internet and social media. Channeling healthcare funds towards such efforts could potentially reduce unnecessary medical visits and, as a consequence, hospital admissions for diseases mistaken to be part of ongoing epidemics.


We describe a case with three focal atrial tachycardias (ATs) and focal atrial fibrillation (AF) originating from the interatrial septum (IAS) near the atrioventricular node (AVN). Contrast-enhanced computed tomography demonstrated the association of fat deposition within the anterior IAS near the AVN with successful ablation sites of these ATs and AF. This is the first report that the intramural fat deposition in the IAS could be associated with the formation of AT and AF re-entry circuits originating near the AVN. Atrial tachycardia (AT) originating near the atrioventricular node (AVN) has been shown to originate from calcium channel-dependent tissue located close to the AVN . HoweverA 69-year-old man was referred to our hospital with palpitations. His electrocardiogram showed a regular AT with narrow QRS AT-1, Fig. , Fig. 1aThis case presented both focal ATs and focal AF originating from the IAS near the AVN. Contrast-enhanced CT demonstrated a heterogeneous fat pad in the interatrial septum surrounded by focal triggers for these atrial arrhythmias. The induction of these ATs with programmed stimulation and behavior during entrainment mapping was consistent with micro re-entry as the tachycardia mechanism. These ATs may have shared a central common pathway, as suggested by the proximity of their successful ablation sites across the IAS near the AVN Fig. . In addiThis is the first study to report that the intramural fat deposition in the IAS could be associated with the formation of AT and AF re-entry circuits originating near the AVN. Further study will be required to investigate the association of fat depositions in the IAS with atrial tachyarrhythmias originating near the AVN.


Recently, the fundamental and nanoscale understanding of complex phenomena in materials research and the life sciences, witnessed considerable progress. However, elucidating the underlying mechanisms, governed by entangled degrees of freedom such as lattice, spin, orbit, and charge for solids or conformation, electric potentials, and ligands for proteins, has remained challenging. Techniques that allow for distinguishing between different contributions to these processes are hence urgently required. In this paper we demonstrate the application of scattering-type scanning near-field optical microscopy (s-SNOM) as a novel type of nano-probe for tracking transient states of matter. We introduce a sideband-demodulation technique that allows for probing exclusively the stimuli-induced change of near-field optical properties. We exemplify this development by inspecting the decay of an electron-hole plasma generated in SiGe thin films through near-infrared laser pulses. Our approach can universally be applied to optically track ultrafast/-slow processes over the whole spectral range from UV to THz frequencies. Understanding the complexity of solid-state materials such as high-temperature superconductors, functional proteins and two-dimensional materials has become a subject of intense research in the last 20 years. Nevertheless, these systems still present challenges for standard spectroscopic or imaging analysis since they exhibit an entanglement of different degrees of freedom on the mesoscopic to atomistic length scale125789101112Techniques that provide both a high spatial and temporal resolution while simultaneously being sensitive to different transient states are rare. One method that has recently been reported is time-resolved X-ray scattering and diffraction (XAS) using ultra-short X-ray pulses as delivered e.g. by X-ray free-electron lasers1418192022The approach that we follow in this paper allows for probing the optical response of a transient state of matter on the nanometer length-scale providing a pico- to femtosecond time resolution via recording the local changes of the near-field optical properties. We use a custom-made s-SNOM1126276Our s-SNOM setup is based on a non-contact atomic force microscope (AFM) as described in detail in the methods section. The typical lateral optical resolution of our setup measures ~10 nm. In order to probe the transient state behavior, we employ an optical pump-probe scheme see  as descr2 laser or a free-electron laser . Both of them are tuned to a λ = 10.6 μm wavelength. The probe beam is directed towards the s-SNOM via a geometrical beam splitter and focused onto the s-SNOM probe using a parabolic mirror, leading to a probe-focus diameter of approximately 100 μm. The same parabolic mirror is used to collect the backscattered light that is measured with a standard MCT detector.The probe lasers consist of either a tunable continuous-wave (cw) COp = 1 kHz and a pulse width of τp = 20 ps. The Nd:YAG beam is coupled into the beam path in parallel to the probe beam and focused to the probe-sample system with a focus size of 1 mm. In order to avoid stray light from the pump beam to influence the measurement a germanium filter is mounted in front of the detector.For the experiments discussed in this paper, we complemented our setup with a Nd:YAG pump laser at λ = 1064 nm with a repetition rate of fGenerally, s-SNOM constitutes a non-invasive and non-destructive scanning probe method that allows for the examination of local optical constants with a wavelength-independent lateral resolution on the order of 10 nm. For scattering-type near-field optics, the nanoscopic s-SNOM probe transforms the local near-field information of the sample into a propagating wave that reaches the detector in the far field. In order to differentiate the subtle near-field information from the typically much larger far-field scattering, we apply the method of higher-harmonic demodulation that utilizes the strongly non-linear dependence of the evanescent near field303111342Ω) reflects the time-averaged near-field response of the sample carrying information on the local optical properties of the sample region probed by the AFM tip apex.We analyze here the near-field signal with a two-fold demodulation approach. On the one hand, we extract the near fields by filtering the scattered signal with a lock-in amplifier (LIA) at the second harmonic (2Ω) frequency of the fundamental cantilever oscillation Ω312Ω), which then contains the desired transient photo-induced near-field component of our sample. Details of the lock-in amplifier settings used for this technique are given in the methods section.In a second step, we enhance our method by a novel two lock-in demodulation technique that measures the photo-induced sideband of the transient near-field signal at the corresponding harmonic see . Here, t2 laser or the pulsed FEL.In order to demonstrate the sideband-demodulated near-field technique as described above, we study the photo-induced near-field signature of transiently excited SiGe thin films (for details see methods section). The fundamental idea of this experiment is to probe the pump-induced electron plasma, that lives in the range of μs, with one of our two probe beams, namely either a cw COThe SiGe samples show a cross-hatched micro-structure383940412Ω in s-SNOM is sensitive to these local optical properties of the SiGe sample, and hence to any stress-induced variation of the optical constants in the sample2Ω must increase for all sample stripes. Moreover, since the photoexcitation of the different stripes is unequal, the recorded near-field amplitudes increase by a factor of 1.1 for stripes #2 and #4, to 2.8 for stripes #1, #3, and #4, which results in a change of the near-field contrast of up to 28%  = −5 to −1), which is not met over the full pump cycle but for an ultra-short time period only.The lifetime of the electron-hole plasma in the SiGe films is a few μs. Compared to the repetition rate of the pump laser of fp = 1 kHz, this corresponds to a duty cycle of ~10In terms of responsivity to changes in the sample properties, the sideband technique yields a solely pump-related contrast and therefore clearly exceeds the abilities of basic near-field microscopy. The main advantages of the method as presented here are the sensitivity to ultra-short transient changes rather than its time-averaged contribution, as well as the non-existence of any ground-state background signal in the sideband channel; hence, the signal-to-noise ratio reaches extremely high values even for room-temperature applications as the one detailed in this article.2 is in good agreement to macroscopic experiments quoting 1–15 mJ/cm2454638A new approach for probing transient states of matter on the nanometer length scale has been demonstrated in this work. The presented novel sideband demodulation technique allows one to separate, for the first time, transient modulated signals from the integral near-field signal. Proof-of-principle experiments were performed on SiGe thin-film samples upon optical excitation, establishing a resolution beyond the diffraction limit. The required fluence for the generation of the electron-hole plasma in semiconductors of about 1 mJ/cmThe demonstrated concept enables scanning optical near-field microscopy to be applied to transient nanoscopy with an unprecedented sensitivity. In contrast to recently performed pilot experiments of time-resolved scanning near-field infrared microscopy222349t ≈ 150 kHz and A = 50 nm, respectively. The tip apex with a ~10 nm tip radius interacts locally with the sample, allowing for a resolution beyond the optical diffraction limit. The tip scatters the local near-field signal into propagating waves that are measured in the far field. Here, we detect the backscattered light with a liquid-nitrogen-cooled MCT detector (Judson Technologies J15D12). A silicon wafer having an evaporated gold film of 100 nm is used as a geometrical beam splitter in our optical setup allowing us to monitor the incident probe-laser power Pprobe while recording the scattered near fields. All signals are normalized to Pprobe ≈ 25 mW and were demodulated at the second harmonic of the cantilever oscillation 2Ω for quantifying and differentiating the near field from any far-field signals. We define the optical contrast/visibility to V = (A–B)/(A+B) with V ranging between −1 to +1 . The AFM was operated with a RHK SPM 100 + PLL Pro 2 AFM controller for data acquisition.Our s-SNOM is based on a home-built non-contact AFM with optical accessibility to the AFM tip. The tip-sample junction is illuminated under an angle of 65° with respect to the sample normal. We use commercially available platinum-iridium-coated silicon tips from Nanosensors as the scattering probes in s-SNOM, operated in non-contact mode at a frequency and amplitude of fParameters used for the sideband-demodulation technique are a bandwidth of 18 dB and an integration time of 300 μs of the first LIA , offering enough band pass for the higher harmonic and sidebands. The second LIA (type: Stanford Research Systems SR830) triggered at the photoexcitation frequency of 1 kHz is used with an integration time of 300 ms and a filter of 24 dB. Here, the filter needs to be sufficiently sharp in order to separate the sideband from the 2Ω peak, while the integration time was adjusted such to allow for fast acquisition at a reasonable signal-to-noise ratio.1 = 12.74g = 1.07 eVe = 500–1000 cm2/Vsh ≈ 800 cm2/Vs for electrons and holesThe SiGe thin films consist of an 80 nm thick, homogeneous distribution of a SiGe mixed phase containing 77% silicon and 23% germanium which was grown epitaxially with MBE on a Si (100) substrate. In the unstrained state, optical properties are determined by the composition of the two elements. Here, typical values for optical parameters are a permittivity at λ = 10.6 μm of εHow to cite this article: Kuschewski, F. et al. Optical nanoscopy of transient states in condensed matter. Sci. Rep.5, 12582; doi: 10.1038/srep12582 (2015).


Background and aims: Over the last two decades, research into video game addiction has grown increasingly. The present research aimed to examine the relationship between video game addiction, self-control, and academic achievement of normal and ADHD high school students. Based on previous research it was hypothesized that (i) there would be a relationship between video game addiction, self-control and academic achievement (ii) video game addiction, self-control and academic achievement would differ between male and female students, and (iii) the relationship between video game addiction, self-control and academic achievement would differ between normal students and ADHD students. Methods: The research population comprised first grade high school students of Khomeini-Shahr . From this population, a sample group of 339 students participated in the study. The survey included the Game Addiction Scale (Results: Among Iranian students, the relationship between video game addiction, self-control, and academic achievement differed between male and female students. However, the relationship between video game addiction, self-control, academic achievement, and type of student was not statistically significant. Conclusions: Although the results cannot demonstrate a causal relationship between video game use, video game addiction, and academic achievement, they suggest that high involvement in playing video games leaves less time for engaging in academic work.on Scale , the Selon Scale  and the on Scale . In addi In all educational systems around the world, the level of students' academic achievement serves as one of the success indicators of their educational activities. Many different factors are involved in academic achievement such as personality and contextual factors. Self-control is considered one of these personality traits. Compared to those who have low self-control, those with high self-control are more successful during performance of their tasks. Also, they are more able to separate their leisure time activities from the other kinds, take better advantage of their study time, choose more suitable courses and classes, and control activities and entertainments which may be harmful to their educational development. Previous studies evidently indicate that the self-control may enhance the academic achievement. Another activity that can be included as a contextual factor relevant to students' academic achievement is video game addiction. According to a variety of studies, playing video games can affect the academic achievement of children and adolescents . NowadayWhile playing such games, players may forget about everything and get immersed in the game. Video game playing also has the capacity to stop players engaging in other activities . Additionally, excessive video gamers are less interested in school. Since too much playing reduces the required time for doing homework, it can consequently negatively affect the individual’s academic achievement . Some reGiven that the video game addiction and the associated issues have been the subject matter of increasing research in clinical, counseling and educational domains, the present research study aimed to examine the relationship between video game addiction, self-control, and academic achievement of both normal and ADHD high school students. Based on previous research it was hypothesized that (i) there would be a relationship between video game addiction, self-control and academic achievement, (ii) the relationship between video game addiction, self-control and academic achievement would differ between male and female students, and (iii) the relationship between video game addiction, self-control and academic achievement would differ between normal students and ADHD students.The research population comprised first grade high school students of Khomeini-Shahr . Of this population, a representative group of 339 students participated in the study. Two-stage cluster sampling was used. When data are obtained by two-stage cluster sampling, serious problems may arise if conventional methods that ignore the intracluster correlations are used. Because of this, intracluster correlation was estimated. Eighteen schools were randomly selected from 234 schools in this city. Following this, one class from each school was randomly selected. Thirteen students' questionnaires were omitted from analysis because they were not properly completed leaving a final sample of 326 high school students. Among the students, 146 (49.1%) were females, and 166 (50.9%) were males.– The data were collected via a questionnaire. In addition to questions relating to basic demographic information, students' Grade Point Average (GPA) for two terms was used as a measure of their academic achievement. The questionnaire also included the Game Addiction Scale , the Self-Control Scale  and the ADHD Diagnostic checklist . In this study, all scales were translated into Persian and back-translated into English by two independent official translators. Comparison of the original version and the back-translated into English version showed that there were only minor changes between the two forms of each scale. Cronbach’s alpha was then used to assess the internal consistency of the instruments using SPSS software. These coefficients are reported below.Computer and Video Game Addiction Scale : This questionnaire measures seven underlying addiction criteria including salience, tolerance, moodmodification, relapse, withdrawal, conflict and problems. The resulting Cronbach’s alpha coefficients in this sample respectively 0.93, 0.93, 0.69, 0.98, 0.91, 0.88 and 0.99, respectively.– Self-Control Scale : This questionnaire measures five factors . Both the reliability and validity of this questionnaire have been reported to be 0.89.– Diagnostic Checklist and Self-report Scale : This scale assesses six hyperactivity criteria as listed in the Diagnostic and Statistical Manual of Mental Disorders : (i) often fails to give close attention to details or makes careless mistakes in schoolwork, work, or other activities, often has difficulty sustaining attention in tasks or play activities, (ii) often does not seem to listen when spoken to directly, (iii) often does not follow through on instructions and fails to finish schoolwork, chores, or duties in the workplace , (iv) often has difficulty organizing tasks and activities, often avoids, dislikes, or is reluctant to engage in tasks that require sustained mental effort (such as schoolwork or homework), (v) often loses things necessary for tasks or activities at school or at home , and (vi) often easily distracted by extraneous stimuli, if often forgetful in daily activities. The internal consistency of this scale ranges from 0.63 to 0.72 based on Cronbach’s alpha and its test-retest reliability ranges from 0.58 to 0.77 based on Pearson correlation coefficient.– n = 339) selected through subsequent sampling in two stages (outlined above). The study was conducted using a paper-and-pencil method. After agreeing to participate, all participants completed the Computer and Video Game Addiction Scale, Self-control Scale, and Diagnostic Checklist and Self-report Scale. Finally, participants completed the demographics items and students' Grade Point Average (GPA) for two terms as the measure of academic achievement. Verbal instructions indicated that there were no correct answers on any of the scales and that all responses were confidential.In the current study, Iranian students were the target population. Participants were practicing students  approved the study. All subjects were informed about the study and all provided informed consent. Parental consent was also sought for those younger than 18 years of age.R2 = 0.09). In the next step, video game addiction was entered into the model, and the R2 increased to 0.154 . The contribution of video game addiction was 6.3%. Therefore, each single unit increase in self-control causes an increase of 0.278 units in student academic achievement, and that a single unit increase in video game addiction causes a decrease of 0.252 units in the student academic achievement. As expected, self-control therefore has a positive effect on academic achievement whereas video game addiction has a negative effect.The first hypothesis was that there would a relationship between video game addiction, self-control, and academic achievement. This was examined using a regression analysis. Overall, there was a significant relationship between video game addiction, self-control, and academic achievement. As shown in R2 increased to 0.263 . Meanwhile the gender contribution rate was almost 10.9% and was statistically significant. Furthermore, the Beta value of this variable was big enough (0.372) to be considered statistically significant. Thus, it can be concluded that the relationship between video game addiction, self-control, and academic achievement differs between male and female students.The relationship between gender differences and video game addiction, self-control, and academic achievement was examined through the multiple regression analysis . This is summarized in R2 increased to 0.156 . Meanwhile the type of student contribution rate is almost 0.2% which was not statistically significant.The relationship between video game addiction, self-control, academic achievement, and type of student  was also examined through multiple regression analysis . There was a significant effect for type of student see . Again, The findings of this study showed that there was a significantly negative relationship between video game addiction and students' academic achievement, while the relationship between self-control and the academic achievement of these students was significantly positive . The results are therefore similar to those of The findings of this study showed that gender had a significant effect on video game addiction, self-control, and academic achievement . This confirms much of the previous research in the area showing that boys spend more of their leisure time playing video games when compared to girls .There are many likely explanations as to why boys play video games more than girls. First, most video games are designed by males for other males, and even when the games feature strong female characters, they may be highly sexualized and alienate more females than attract them. Second, the socializing procedure is different between males and females. Women are more successful in preventing the emergence of their aggressive behaviors in presence of others, so they may feel more nervous while playing fighting games and are more attracted to more gentle and fantasy games. Furthermore, researchers have used According to the findings of this study, the relationship between self-control, video game addiction and academic achievement is significantly different between normal and ADHD students. The present research results supported the findings of Frölich et al. (2009), and Limitations and Future research: There are several limitations to the current study that should be noted. First, the sample size is fairly modest with 326 students. This sample size was smaller than would be desired. Therefore, generalization of its usefulness is limited. Second, because all students included in the analysis were from Iran only, there is no evidence that the findings can be generalized to the population of students in other country. Third, the study employed high school students as participants, and thus, the results of this study may not be generalizable to college students or students older than the age of 18 years . Fourth the cross-sectional design used in this study means that conclusions about cause and effect or sequence of events cannot be made. Finally, the results from this study also raise more general measurement concerns that should be addressed. The questionnaires that used in current study are self-report measures. Previous research suggests that for psychological constructs, self-report measures may not necessarily reflect what a person actually does. It is likely that scores from self-reports of behaviors would be reasonably valid; however, self-reports of behavior may show less consistency with other techniques.All psychology research is influenced by the participants’ characteristics and developmental stages. Future research should study a more diverse sample of ages, education level, gender, religion, and people from other cultures. Future Studies should use adequate and larger group of students. Research should encompass multiple techniques obtaining data from the same participant .