Our aim was to evaluate, in a second data analysis of the prospective randomized controlled trial conducted by Austrian Urogynaecology Working Group, the effect of age, BMI and parity at the time of surgery on short- and long-term outcomes of women primarily treated for SUI (stress urinary incontinence) with midurethral slings.In the original study 554 patients received randomly a retropubic (TVT) or a transobturator midurethral (TVT-O) sling procedure. 480 (87%) and 277 (50%) patients were available for a follow-up efficacy evaluation at 3 months and 5 years respectively.Higher age and BMI at surgery appear to lead to a larger probability to have a positive stress test 5 years after surgery, but not after 3 months. Older patients seem to have a worse perception of improvement 5 years after surgery as compared to younger ones, as described by the PGI-I score. Age and BMI do not affect significantly the quality of life of women surgically treated for SUI, as reflected by the results of King´s Health Questionnaire. Parity does not seem to have any effect on objective and subjective surgical outcomes.Higher age and BMI at surgery have a detrimental influence on the objective cure rate at 5 years after midurethral sling surgery; higher age also has a negative influence on subjective long-term outcomes. However, these demographic parameters do not influence significantly the quality of life of patients after anti-incontinence surgery. Parity does not show any significant influence on success rate of midurethral sling. Retropubic tension-free vaginal tape (TVT) or transobturator tape (TOT) are a standard of care for the surgical treatment of stress urinary incontinence in women . These tSeveral studies have demonstrated the feasibility and safety of midurethral slings in elderly –8 and obHowever, the literature shows significant variability in terms of influence of demographic characteristics on midurethral sling outcomes –13.The Austrian Urogynaecological Working Group conducted a randomized controlled trial (RCT) to compare objective and subjective outcomes of TVT with those of TVT-O ,14. The The current study is a secondary analysis utilizing an established database from a previously reported prospective randomized controlled noninferiority study by the Austrian Urogynaecology Working Group ,14. The For the RCT, preoperative evaluation included a demographic questionnaire , urogynaecological history, bladder diary, urodynamic studies  and cough stress test. Condition-specific quality of life (QoL) was assessed with the validated German-language version of the King’s Health Questionnaire (KHQ) , and witPostoperatively, participants were evaluated at 3 months and at 5 years: both evaluation included a clinical and urodynamic evaluation, with a cough stress test at bladder filling of 300 ml; patients were also asked to complete the same QoL questionnaires used for the preoperative evaluation.Cough stress test was performed in the supine and standing positions at bladder filling of 300 ml. Patients were considered objectively cured when this test in the supine and standing positions were negative.Secondary analysis of the RCT data, evaluated if age, BMI and parity affect the objective and subjective cure rate of SUI at 3 months and 5 years postoperatively. Objective measurement of SUI cure was considered as negative cough stress test at 3 months and at 5 years postoperatively. Subjective cure was measured at 3 months and 5 years using QoL questionnaires . King’s Health Questionnaire were assessed in 3 parts according to Hebbar et al. : Part I 2.For this analysis we assumed that TVT and TVT-O have comparable success rate, as demonstrated by our original study  and by oThe STROBE Statement guidelines for reporting observational cohort research were followed .To analyse the influence of age, BMI and parity (as continuous variable) on the probability of a positive stress test, logistic regression models (one for 3 month and one for 5 year results) were performed. To analyze the results in more detail, polynomial regression models (using the LOESS approach) were performed for the influence of age and BMI. To investigate the influence of parity in more detail, proportions of failing stress test and corresponding 95%-confidence intervals were calculated for each observed parity value. The three parts of the King’s Health Questionnaire were calculated according to Hebbar et al. . The infA total of 554 patients were included in this study between January 2005 and July 2007: 285 (51%) TVT and 269 (49%) TVT-O. Of them, 480 (87%) patients were assessed at 3 months and 277 (50%) patients at 5 years. At the time of surgery, mean age was 59.2  years, mean BMI was 28.1 , median parity was 2 (range 0–8). The intraoperative complications occurred were: 11 (1.9%) bladder perforations, 5 (0.9%) intraoperative bleeding. Reoperations due to postoperative complications were 5 (0.9%).At 3 months a positive stress test was seen in 70/480 (16%) women. At 3 months no significant influence of age, BMI and parity on a positive stress test was found . However, the polynomial regression model showed a trend for an increasing probability for surgery to fail with increasing of age. The probability of stress test failure seems to be rather constant for all BMI values. Also for parity, no discernible pattern was identified .At 5 years a positive cough stress test was seen in 56/277 (20%) women. The probability to have a positive stress test at 5 years was significantly increasing with age (p = 0.05) and with BMI (p = 0.01). This trend can also be seen using polynomial regression models . For parA significant difference in mean BMI between PGI-S classes at 3 months after surgery was observed: patients with lower PGI-S scores (those with better perception of urinary tract condition) had higher BMI values. A reverse trend, although not significant, was found after 5 years. No significant influence of age and parity on PGI-S scores at 3 months and 5 years was found .At 3 months a significant difference of BMI between the PGI-I classes was found. On average a higher BMI was found for patients with lower PGI-I scores (those with a better perception of improvement as compared with before treatment). However, at 5 years, although not significant, a reverse trend was found. A significant difference in age was found between PGI-I classes after 5 years: older patients had higher PGI-I classes (worse perception of improvement as compared with before treatment). No significant difference in parity between the PGI-S classes was found, neither 3 months nor 5 years after surgery .The descriptive statistics of the three scores of KHQ are described on Age, BMI and parity did not significantly influence any of the three KHQ scores at 3 months and 5 years  .This secondary analysis of data from a large prospective randomized trial of the surgical outcomes of tension-free vaginal tape shows that at 3 months, the patient´s age or BMI did not have a significant influence on either objective and subjective outcomes. While at 5 years older patients with higher BMI had a higher probability of a positive stress test. Parity had no significant influence on surgical outcomes of midurethral slings.The results of our study indicate that age and BMI at surgery may affect the objective long-term outcomes of SUI surgery, but not the early. This is plausible considering that the immediate mechanical effect of midurethral slings is independent of ageing of the tissue and patient´s weight. When placed correctly, a midurethral sling supports the urethra in a “hammock-like” way and shows its curative effect after 3 months. However, after 5 years, the remodelling process around the tape and the deterioration of pelvic floor support occur. During this time the overweight may affect pelvic floor structures thereby, increasing intra-abdominal pressure in older and overweight patients, resulting in the loss of the therapeutic effect of the sling. In fact, the ageing of pelvic floor tissue is due to decreased number of vascular plexuses and collagen type II/III content in the urethral submucosa and to changes in striated pelvic floor muscle.This is a risk factor for the deterioration of the continence mechanisms, and consequently, also for the efficacy of anti-incontinence surgery . FurtherControversial results were found with PGI-S and PGI-I questionnaires which indicated that patients with higher BMI defined their urinary tract condition as normal and had a better global impression of improvement 3 months after surgery than patients with lower BMI. However at 5 years a reverse trend was found. This can be explained by the immediate mechanical effect of midurethral sling at 3 months after surgery in the more overweight patients, which perceived a greater impression of improvement than in thinner women. This effect is lost over the time, particularly in the more overweight patients, since the prolonged increased intra-abdominal pressure counteracts the urethral support. This interpretation should however take in account that from 3 months to 5 years, several patients were lost to follow up. These results could therefore be also a result of selection bias.At 5 years, there is a trend showing that older and overweight women perceive a severe urinary tract condition and a diminished perception of improvement. However this was only significant for age. This shows that the long-term subjective outcomes seem to be influenced by age and BMI also.On the other hand, our results show that age, BMI and parity at the time of surgery do not significantly affect the QoL of patients after anti-incontinence surgery. This can be seen in all the KHQ´s domains: distress, anxiety, loss of self-esteem, social, cultural, marital, domestic, physical, psychological and sexual wellbeing related to urinary incontinence, are not significantly influenced by age, weight and parity at the time of surgery.There is little consensus in literature about the influence of age and weight on surgical success of patients candidates for suburethral slings. That can be explained by the use of different outcome parameters and different follow up timing utilized in the different trials.Our results, studying long-term follow-up are consistent with those reported by Anger et al , who demRegarding BMI, some authors have shown that BMI did not influence the midurethral sling success rates ,21,22 wi2 as a risk factor when discussing the suitability of the midurethral sling procedure in a patient with SUI. Some important limitations have to be taken in account when assessing this meta-analysis. First, the follow-up times are different among the different studies, ranging from 6 months [A recent review  of 2846 6 months ,28 to 866 months . TherefoOur finding that higher BMI at surgery leads to a larger probability to have a positive stress test 5 years after surgery but not 3 months after surgery are supported by the fact that all the outcome parameters were standardized, validated and uniformly applied at 3 months and 5 years postoperatively.The strength of this analysis is that it was performed in one of the largest prospective randomized trial of retropubic vs. transobturator midurethral tapes. The post hoc statistical power calculation with the present sample size is >90%, with an a error of 0.05. The study population was homogeneous and representative, including peri/post-menopausal (mean age 59± 11 years) and overweight (mean BMI 28.1± 5) women at the time of surgery. Additionally, the study had clear, robust and validated outcome measures.One limitation of the study is the fact that 50% of patients were lost to 5 years follow-up. All attempts to reach study-patients for follow-up within the scope of the ethics-committee agreement were made such as contacting by telephone, mail and e-mail. Attempts we also made by inquiring new residential address from the national registration office, arranging at least three possible follow-up appointments as well as sending postal questionnaires to women who did not attend the clinical appointment. As previously described , 58% of However, to date there have been few randomized trials of surgery for stress incontinence with long-term follow up; it has to be taken in account that it is one of the few studies reporting on 277 patients prospectively 5 year after midurethral sling procedures.Higher age and BMI at surgery have a detrimental influence on the objective and subjective long-term outcomes of midurethral tape surgery. On the other hand, our results suggest that these demographic parameters do not affect the QoL of patients after anti-incontinence surgery. As population demographics continue to evolve, specifics on age-related and weight-related outcomes of urinary incontinence interventions deserve further investigations. The results of this study add robustness and validated evidence on risk factors of treatment failure of midurethral sling procedures, giving the clinician the ability to provide correct and exhaustive information, that better modulates a patient's expectations.S1 FileData used for the statistical analysis of the study, collected in excel files.(ZIP)Click here for additional data file.


The results indicated that radiation exposure, 4 Gy of gamma rays, induced 9.6 indels and 2.5 multisite mutations in spermatogonia and 4.7 indels and 3.1 multisite mutations in mature oocytes in the autosomal regions of each F1 individual. Notably, we found two types of deletions, namely, small deletions (mainly 1~12 nucleotides) in non-repeat sequences, many of which showed microhomology at the breakpoint junction, and single-nucleotide deletions in mononucleotide repeat sequences. The results suggest that these deletions and multisite mutations could be a typical signature of mutations induced by parental irradiation in mammals.The exposure of germ cells to radiation introduces mutations in the genomes of offspring, and a previous whole-genome sequencing study indicated that the irradiation of mouse sperm induces insertions/deletions (indels) and multisite mutations (clustered single nucleotide variants and indels). However, the current knowledge on the mutation spectra is limited, and the effects of radiation exposure on germ cells at stages other than the sperm stage remain unknown. Here, we performed whole-genome sequencing experiments to investigate the exposure of spermatogonia and mature oocytes. We compared  IR induces various types of DNA damage ranging from nucleotide modifications to DNA strand breaks. Among the DNA damage induced by IR in germ cells, those that are not correctly repaired will lead to de novo mutations in the genomes of offspring2. The types and extent of mutations induced by IR in the mammalian germline have been a particularly important issue in the field of radiation biology. A substantial number of studies on human populations  have been conducted, but the heritable effects following exposure to IR have not been clearly elucidated5. Much of the decisive evidence has been obtained from studies using mouse models. In particular, screening experiments based on the visible phenotypes of the offspring of exposed parents, known as specific-locus tests, have provided important evidence for transgenerational effects. For example, the frequency of mutants increases with increases in the radiation dose 6. The exposure of maturing and mature oocytes induces an approximately two-fold higher number of mutations than the exposure of spermatogonia6. However, because specific-locus tests have been limited to specific regions, typically seven protein-coding genes, it has been difficult to evaluate the effects on mutation induction at a genome-wide level.The history of mutagenesis experiments began with exposure to ionizing radiation (IR) in 1926de novo mutations at the genome-wide level. One previous study using a WGS approach indicated that parental irradiation of 3 Gy at the sperm stage induces insertions/deletions (indels) and “multisite mutations”, clusters of single nucleotide variants (SNVs) and indels in close proximity, but not SNVs in the offspring7. However, the current knowledge on the mutation spectra is limited, and the genome-wide effects of IR on germ cells at stages other than the sperm stage have not been assessed using WGS techniques.The recent progress in high-throughput sequencing technologies enables us to perform whole-genome sequencing (WGS) and provides us with the chance to survey 9. For example, the paternal and maternal gametogenesis processes are quite different, and a large portion of the genomic DNA in sperm is tightly packed with protamines instead of histones10. Indeed, specific-locus tests in mice have suggested that the effects on mutation induction in offspring show variations among different parental exposures: male germ cells that have entered meiosis, as well as maturing and mature oocytes, show increased sensitivity to IR than spermatogonia11. These data suggest that it is important to understand the effects of IR on germ cells other than those at the sperm stage.The heritable effects of IR are thought to differ depending on the developmental stage of gametogenesis at which the exposure occurs because these stages are characterized by significant differences in the chromosome conformation and the ability to repair DNA damage12, and the exposure of mice to 0.45 Gy of radiation causes a severe loss of small primordial follicles from the ovary13. Here, we characterized and quantified the de novo mutations, mainly base substitutions and small indels, on spermatogonia and mature oocytes through WGS and consider the transgenerational effects of IR at the genome-wide level.In this study, we focused on the effects of IR on paternal germ cells at the spermatogonial stage and on maternal germ cells at the mature oocyte stage using a mouse model. The effects of IR on spermatogonia and immature oocytes are particularly important for assessing the risk of human exposure to IR. However, in this study, we investigated the effect on mature oocytes as a maternal exposure model. This stage was selected because immature mouse oocytes are sensitive to the lethal effect of IR, which makes it difficult to assess the effect on the genome14, where it takes approximately 5 weeks for cells to develop from spermatogonia to sperm8. On the other hand, it is also known that 4 Gy of IR exposures have a killing effect on some portion of mature oocyte and reduce litter size of the offspring6. But, in our experiment, we found no apparent reduction of the litter size of the F1 mice born to the mother irradiated with 4 Gy of gamma rays 15. In the mature oocyte exposure experiment . To achieve an accurate comparison of the de novo mutations between the groups, we focused on a specific autosomal genomic region , and high-quality sequences of all the nucleotides in this region were obtained from the WGS of all the mice in the spermatogonia and oocyte exposure experiments. This region corresponds to 53.9% of the entire mouse autosomal genome  and includes 70.5% of all autosomal exon regions.A total of 24 F1 mice (six F1 mice born before and six F1 mice born after irradiation in the spermatogonia and mature oocyte exposure experiments) and their parents were subjected to WGS with at least 33× total read coverage (Supplementary Data de novo SNV candidates and 79 de novo indel candidates in the EWC region (Supplementary Data de novo variant list (see Methods) because they were difficult to detect precisely. In addition, we defined any cluster of mutations (SNVs and indels) with a shared haplotype within 100 base pairs (most of the mutations occurred within 15 bp) as a multisite mutation. In this study, a multisite mutation was counted as a single mutation separate from SNVs and indels. A subsequent validation experiment using Sanger sequencing showed highly reliable results, which indicated that all the tested candidates (53 SNVs and 43 indels) were successfully confirmed as de novo variants in the F1 mice with the exception of three SNV candidates that could not be amplified by PCR.For the 24 F1 mice, we obtained a total of 336 de novo mutations in the offspring born before and after the parental exposures are shown in Table −9  per nucleotide, and no significant difference in the pre-irradiation estimates was found between the spermatogonia and oocyte exposure experiments . This rate is comparable to a previous estimate in laboratory mice 7 and somewhat lower than the rate of 5.4 × 10−9  mutations per nucleotide per generation reported for a mean parental age of 12 weeks at conception16. For indels, the spontaneous per-generation mutation rate (assuming mating at 8 weeks of age) was estimated as 3.9 × 10−10  per nucleotide, and the estimates from the paternal and maternal exposure experiments were not significantly different . Although the values cannot be easily compared due to the different detection conditions, particularly the method used to address variants in repeat sequences (Methods), the indel rate was similar to our previous estimate of 3.1 × 10−10  per nucleotide per generation 16.The numbers of de novo mutations between the F1 mouse groups born before and after irradiation. In both the spermatogonia and mature oocyte exposure experiments, the offspring born after parental irradiation exhibited higher numbers of de novo SNVs, indels, and multisite mutations compared with those found in the offspring born prior to irradiation  between offspring born from parents aged 8 weeks (before irradiation) and offspring born later (after irradiation)  and 2.3%  of the total number of de novo SNVs observed in the offspring of parents aged 8 weeks at conception would be added each week , we obtained another estimate for the effect of parental aging as an 8.8% increase in de novo SNVs per week . Because the estimates from two independent methods appeared to be close, we used a 6.8% increase  per week in the current analysis.At present, the quantitative relationship between parental aging in mice and increases in on) Fig. . Using non) Fig. . A compade novo mutations adjusted for parental aging effects is shown in Table 7.According to this estimate  in our evaluation of the aging effects of indels  and in the offspring from the exposed mother (P = 0.0095). Assuming that the parental aging effect is two-fold greater than our estimates of SNVs, the increase in indels after irradiation was still significant (P = 0.0012 for the spermatogonia exposure experiment and P = 0.011 for the mature oocyte exposure experiment). In addition, the age-adjusted number of indels in the offspring of the father with irradiated spermatogonia was somewhat greater than that in the offspring of the mother whose mature oocytes were exposed .Although the increase in the absolute number of indels is thought to be small, we estimated the parental aging effect on indels to evaluate the effects of radiation exposure. According to a human study, the ratio of indels to SNVs is nearly constant, regardless of the parental age at conceptionde novo multisite mutations was also significantly higher in the offspring of the irradiated parents. According to a human study22, multisite mutations  occurred at a very low frequency, ~0.07 de novo variants per diploid per generation, and no significant effect of parental aging at conception was observed. Therefore, we assumed that parental aging has no effects on de novo multisite mutations in mice and therefore assumed that multisite mutations are induced by parental irradiation in both the spermatogonia and mature oocyte exposure experiments (P = 0.031 for the spermatogonia exposure experiment and P = 0.0031 for the mature oocyte exposure experiment)  and 4.7-fold increase of that in the offspring of the mother with exposed mature oocytes (P = 0.014) compared with the controls (offspring born to unexposed parents), whereas no significant increase in the number of insertions in the offspring of the exposed father (P = 0.13) and mother (P = 0.19) was observed. We also noted that the number of deletions in the offspring of the exposed father was significantly higher than that in the offspring of the exposed mother . These results indicated that deletions were the main type of mutation and that the number of de novo deletions might depend on the germ cell stage at the time of exposure.To ascertain the features of radiation-associated mutations, we first examined the spectra of P = 0.00018 for single-nucleotide deletions and P < 0.00001 for non-repeat sequence deletions; mature oocyte exposure, P = 0.089 for single-nucleotide deletions and P = 0.0047 for non-repeat sequence deletions; a two-tailed test by Poisson-based simulation). In addition, both types of deletions appeared at a higher frequency in the offspring of the exposed father than in the offspring of the exposed mother .Regarding the deletions, we frequently found two types of mutations in the offspring of the irradiated parents Fig. . One mut23. We also found that the spectrum of base substitutions within a multisite mutation was different from the spectrum of common germline de novo base substitutions in mice . In contrast, C > A substitutions were increased in the multisite mutations compared with the common germline base substitutions (P = 0.0035 by Bonferroni-corrected Fisher’s exact test). C > A substitution is a well-known feature derived from reactive oxygen species, such as 8-oxoguanine24.In most of the multisite mutations, two changes occurred within 15 nucleotides Fig. . Out of ice Fig. . C > T sP = 0.41 for indels and P = 0.58 for multisite mutations by a Chi-square test). These findings indicated that IR would not induce mutations exclusively in specific functional regions. A single nucleotide deletion identified in the offspring of the exposed mother caused a frameshift of the Eps8l3 gene, whose homozygous knockouts show a craniofacial phenotype25, but caused no apparent abnormal phenotype. Among the 12 F1 mice in the spermatogonia exposure experiment, one mouse that was born to the irradiated father had a 4,967 kb deletion that we identified in our previous study using array CGH15. On the chromosome with the large deletion, only one multisite mutation was detected.We also considered the functional impact of the induced mutations. Out of the 67 indels and 20 multisite mutations observed in the offspring born to exposed parents, three indels (~4.5%) were identified in exonic regions, constituting 3.5% of the EWC region; 20 indels (~30%) and nine multisite mutations (~45%) were found in intronic regions, constituting 38% of the EWC region; and 44 indels (~66%) and 11 multisite mutations (~55%) were located in intergenic regions, constituting 58% of the EWC region. A significant difference in mutation occurrence was not observed among regions . Extrapolation of our results to regions outside of the EWC region indicated that paternal exposure (spermatogonia) to 4 Gy induced 9.6 indels, most of which were deletions, and 2.5 multisite mutations in the autosomes of each F1 mouse and that maternal exposure (mature oocytes) to 4 Gy induced 4.7 indels and 3.1 multisite mutations per F1 mouse (Table 26. The fact that the number of mutations detected in these WGS studies (~10 de novo mutations) is notably smaller than the number of such estimated damaged sites supports the contemporary view that almost all sites damaged in spermatogonia, sperm cells, and mature oocytes would be correctly repaired2. Presumably, most of the damaged sites in sperm cells and some of the damaged sites in mature oocytes would be repaired in fertilized eggs9. After such repair activity, a very small portion of the damage is inherited as a de novo mutation in the genome of the offspring.Many prior studies have indicated that 4 Gy of gamma rays leads to approximately 160 double-stranded DNA breaks, more than 500 clustered lesions, and more than 10,000 other instances of DNA damage (single-strand breaks and base damage) in the mammalian genome29. In addition, these features of deletions are consistent with the results of a previous WGS study examining secondary malignancies after radiotherapy30, which showed that small deletions exhibiting microhomology at the breakpoint junction and single nucleotide deletions in the mononucleotide repeat sequence frequently occurred in the radiation-associated tumours. These deletions might be a typical signature of mutations induced by irradiation in mammalian cells.To our knowledge, this report is the first to show the spectrum of small size indels induced genome-widely by IR in mammalian germlines. We observed two types of deletions: deletions (main range: 1~12 nucleotides) occurred in the non-repeat sequences and single-nucleotide deletions occurred in mononucleotide repeat sequences Fig. . Many of7 showing the effect of radiation exposure on mouse sperm cells, the induction of multisite mutations was observed in both the spermatogonia and mature oocyte exposure experiments. Multisite mutations are believed to result from clustered lesions caused by IR31. A recent human study indicated that de novo multisite mutations were increased in the offspring of fathers who worked with radar units32. Considered in this context, a multisite mutation is also a potential signature of mammalian germline mutations induced by IR.Consistent with a previous study7. Our results suggest that the number of de novo SNVs is associated with the parental age at conception rather than the effect of IR. Further studies will be necessary to characterize in detail the effects of IR on SNVs.In contrast to indels and multisite mutations, we found no significant increase in SNVs derived from IR in either the spermatogonia or the mature oocyte exposure experiment Table , consistP = 0.0053, a two-tailed test by Poisson-based simulation). Higher frequencies of both types of deletions (deletions in non-repeat sequences and deletions in mononucleotide repeat sites) were observed in the spermatogonia exposure experiment than in the mature oocyte exposure experiment , mainly large deletions up to a mega base scale, in the offspring15 and that the frequency of induced CNVs obtained after paternal exposures at the spermatogonia and sperm stages is similar. In addition, according to a previous study3 using a mouse specific-locus test, parental radiation exposure increases the number of mutations, which disrupts specific gene loci, in the offspring, and this effect depends on the dose. The exposure to 3-Gy radiation at the sperm stage induced twice as many mutations as irradiation at the spermatogonia stage11. The exposure of maturing or mature oocytes to 3~4-Gy radiation also induced twice as many mutations as the irradiation of spermatogonia cells3. These data suggested that the timing at which germ cells are exposed to IR can induce higher frequencies of some types of mutations. It is possible that cell competition among germ cells might impact the selective exclusion of some large-scale mutations. Future research is needed to uncover the effects of IR exposure at various germ cell stages on various types of mutations, and improvements in WGS technology, including long-read sequencing techniques, will make it easier to analyse wider ranges of mutations.In this current study, we assessed only relatively small mutations (mainly up to a few base pairs in size) due to technical limitations. Previous studies using array-CGH indicated that the exposure of mouse male germ cells to 3~4-Gy radiation induced 35. Therefore, it doesn’t mean that the mutations found in the mouse experiment are directly induced in humans. In addition, the effect of these mutations on the phenotype of the offspring remains obscure. In this study, we found no phenotypic effects in the 12 offspring mice born to the exposed parents. Previous phenotype-based studies on human populations (offspring of exposed people such as atomic bomb survivors) have not shown clear heritable effects of parental exposure to IR5. It remains controversial whether the frequency of minisatellite mutations is increased in the human offspring of irradiated parents37. In the near future, the picture of the effect of IR at the whole-genome level will become clearer and more comprehensive. The knowledge about the genome-wide effects of IR would provide new insights into the results of phenotype-based studies such as large-scale cohort studies on IR-exposed human populations.Our observations showed that both spermatogonia and mature oocyte exposure (4 Gy) would induce several small mutations in mouse offspring. It is known that there are differences in radiation sensitivity between human and mouse. For example, 1 Gy or less is the threshold of immature oocyte sensitivity to be sterile in mice, but about 5 Gy for humans15. F1 mice were derived from the cross of a C57BL/6J male  and C3H/HeN females  before and after irradiation with 4 Gy of 137Cs gamma rays  at a dose rate of 0.5 Gy/min at the NIRS. The male mouse “B8” at 8 weeks of age was mated with the female mouse “C8” at 8 weeks of age and was then exposed to IR at 10 weeks of age. After 16 weeks, the “B8” mouse, which was 26 weeks of age, was mated with the female mouse “C45” at 17 weeks of age. The second group of mice was used for the female exposure experiment, in which mature oocytes were irradiated. At 8 weeks of age, a female C57BL/6J mouse was mated with a male C3H/HeN mouse to produce F1 mice before irradiation. Once the female mouse reached 15 weeks of age, the animal was exposed to gamma rays at 4 Gy. Later that day, the irradiated female was mated with the same C3H/HeN male mouse. F1 mice obtained from this mating were used for subsequent experiments as the offspring produced after irradiation.All animal experiments were approved by the Institutional Animal Care and Use Committee of the National Institute of Radiological Sciences   and by the Experimental Animal Care Committee of the Radiation Effects Research Foundation  . They were performed in accordance with the Guidelines for Animal Experiments of the NIRS and with the Guidelines for Animal Experiments of the RERF. We used two groups of mice .High-molecular-weight genomic DNA was extracted from the spleen, liver, and kidneys through the proteinase K/phenol extraction method. Spleen DNA samples from F1 and parental mice were used for WGS, and the remaining DNA was used for confirmation of the mutation candidates. The extracted DNA solutions were used to prepare paired-end libraries according to the user manual of the Illumina sample preparation kit, either with PCR amplification (spermatogonia exposure experiment) or without PCR amplification (mature oocyte exposure experiment). The libraries were sequenced on the Illumina HiSeq 4000 (spermatogonia exposure experiment) or HiSeq X (mature oocyte exposure experiment) platform using 150 bp paired-end reads. The average total read coverage among the samples was 35 reads per nucleotide for the spermatogonia exposure experiment and 49 reads per nucleotide for the mature oocyte exposure experiment , and hard-clip and soft-clip reads were excluded from variant calling. Variants that were different from the mouse reference genome were called using GATK40 HaplotypeCaller to obtain high-mapping-quality reads of mapped autosomal data with a minimum base quality of at least 20. The variant calling of multiple samples consisting of F1 mice and their parents was performed with HaplotypeCaller. Annotations of genomic variants and predictions of their functional effects were conducted using SnpEff41.We used mapped sequence data from autosomes to call variants. To decrease the number of false variant calls, we used only high-mapping-quality reads from the mapped autosomal data, i.e., those reads with a mapping quality of 60  a variant allele frequency (VAF) less than 0.1 in both parents and (2) a VAF of at least 0.25 in F1 mice. Using these filters, we called de novo SNV and small indel candidates, and the de novo mutation candidates were reviewed by visual inspection using Integrative Genomics Viewer42. For the validation experiment, a subset of the mutation candidates  were subjected to Sanger sequencing.de novo mutations among samples from the different experiments, we defined the EWC region as the region that satisfied the following conditions: i) the lower and upper coverage bounds were set to 50% and 300%, respectively, of the peak coverage of high-mapping-quality reads mapped to autosomal data and ii) the minimum depth ratio of high-mapping-quality reads of mapped autosomal data to all reads of mapped autosomal data at each site was set to 80%. The EWC region was composed of the regions shared by all F1 mice and their parents in both the spermatogonia exposure experiment and the mature oocyte exposure experiment. The de novo mutations detected in the EWC region were highly reliable compared with the mutations detected outside of the EWC region. Most mutations in the EWC region exhibited higher variant quality scores, as demonstrated using EAGLE43 software.To clearly compare the de novo indel mutations, we used an additional filter. We divided the indel variants into two groups: indels that occurred in repeat sequences and indels that occurred in non-repeat sequences. We defined “indels that occurred in repeat sequences” as indels that increased (or decreased) the number of tandem repeat units (tandem repeat units were defined as the sites having more than one repeat unit). The called indel variants located in repeat sequences, particularly those consisting of many repeat units, exhibited a particularly high false positive rate. Therefore, we excluded the following indels located in repeat sequences: (1) mutations in mononucleotide repeat sequences having more than 7 units, (2) mutations in dinucleotide repeat sequences having more than 4 units, and (3) mutations in trinucleotide (or more) repeat sequences having more than 2 units of the repeat. To search for deletions that exhibit microhomology, we defined deletions that satisfied the following criteria as microhomologous deletions: (1) the deletion length was at least three nucleotides and (2) two nucleotides of the deleted sequence were identical to the flanking sequences of the deletion.To obtain a credible list of We defined a multisite mutation as more than one alteration (SNV or indel) occurring within 100 base pairs in the same allele. Multisite mutations were counted separately as mutations different from SNVs and indels. For analysis of the spectra of base substitution within multisite mutations, the condition with the fewest alterations was used for each multisite range. The statistical significance of the spectral difference between the base substitutions within multisite mutations and spontaneous SNVs was assessed in two ways. First, to obtain an entire indication of whether the mutation distribution differed between two groups, we applied Fisher’s exact test comparing the number of mutations and mutation types by group. Second, if there was a significant difference between two groups, we conducted Fisher’s exact test to identify which type of mutations differed. To account for multiple testing, we applied a Bonferroni correction by multiplying the obtained P values by the number of mutation categories (seven in this case).μ was calculated as μ = m/(n × G), where m is the total number of mutations, n is the number of individual mouse offspring, and G is the size of the analysed genome in base pairs . In this study, to define the mutation rates of indels and multisite mutations, we counted an occurrence of one indel or one multisite variant as one mutation. The 95% confidence intervals of the mutation rate were calculated using an exact Poisson test in R (poisson.test)44.The mutation rate de novo SNVs by comparing the numbers of mutations of paternal and maternal origin in unexposed alleles before and after exposure . We conducted 100,000 extractions of mutations according to the Poisson distribution assuming that the numbers of mutations before and after irradiation are the same. We estimated the significance of the differences by calculating the proportions of the 100,000 simulations that gave an excessive number of mutations compared with observed values. We used a two-tailed test for the comparisons of simulated with observed data. We incorporated the uncertainty of the aging effects on de novo SNVs and indel mutations in our calculations using the numbers of mutations after adjusting for the parental age effects. In the calculation of multisite mutations, we did not calculate the uncertainty of the aging effects because multisite mutations with an inter-mutational distance of less than 100 bp in humans did not show aging effects22, and we assumed that the effects were similar in mice. To calculate the 95% confidence intervals shown in Table We tested the significance of the differences in the numbers of SNVs, indels, and multisite mutations between offspring born before and after parental irradiation using a similar Poisson simulation as described in a previous reportSupplementary data.


Smoking cessation treatment programs have been widely available for patients with nicotine dependence. Despite intensive programs, the continuous abstinence rate (CAR) from weeks 9-12 is still about 50%. Recently, a smartphone app emerged as a novel tool for therapeutic interventions, including nicotine dependence. In this study, we developed “CureApp Smoking Cessation” (CASC), which consists of a smartphone app for patients and a Web-based patient management software for doctors with a mobile carbon monoxide (CO) checking device to improve the efficacy of the smoking cessation treatment.This study aims to evaluate whether the CASC app is effective for individuals with nicotine dependence in addition to standard smoking cessation programs.This will be a randomized, sham-controlled, open-label, multicenter trial. We will recruit participants with nicotine dependence, but are otherwise healthy adults. We will randomize and allocate participants 1:1 to the CASC treatment group or a control app group. Both groups will receive a 12-week standard smoking cessation program with pharmacotherapy and counseling. In addition, participants in the treatment group will have the CASC app installed on their smartphone, which will provide video tutorials, advice from an artificial intelligence nurse, a digital diary, and measure daily exhaled CO concentration. In contrast, the control group will have the control app installed on their smartphone, where all the functions that can potentially effect smoking cessation are removed. The primary outcome will be the biochemically validated CAR from weeks 9-24. The success of smoking cessation will be defined as self-reported continuous abstinence from weeks 9-24 and exhaled CO concentration ≤10 ppm both at weeks 12 and 24. The main secondary outcomes will be the CAR from weeks 9-12, weeks 9-52, and 7-day point prevalence abstinence at weeks 4, 8, 12, 24, and 52.We will recruit 580 participants with nicotine dependence from October 2017 to September 2018 or until the recruitment process is complete. The final 52-week follow-up will be completed in October 2019. We expect all trial results to be available by the end of 2019. The trial is funded by CureApp, Inc.This is the first randomized controlled trial to evaluate the efficacy of CASC. We expect that CASC, in addition to standard smoking cessation programs, has a significantly higher CAR during weeks 9-24 than the control app.University Hospital Medical Information Network Clinical Trials Registry UMIN000031589; https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000033555DERR1-10.2196/12252 Smoking is a risk factor that causes various diseases such as malignant tumors, heart disease, cerebrovascular disease, and chronic obstructive pulmonary disease . It has Smoking cessation treatment programs have been widely available for patients with nicotine dependence supported by the Japanese national insurance system . This prMedical apps for smartphones are emerging as “therapeutic” intervention tools to ameliorate diabetes mellitus ,10, deprRecently, we developed “CureApp Smoking Cessation” (CASC), which consists of the latest version of CureApp Smoking Cessation smartphone app for patients and a Web-based patient management software for primary physicians with a mobile CO checking device to improve the success of the smoking cessation treatment. The CASC provides users with accurate knowledge of nicotine dependence, clues to change their behaviors, and measurements of their own exhaled CO concentration levels at home. In addition, patients can share these data remotely with their primary physicians. Masaki et al reported that, in the previous prospective studies with participants using prototypes of CASC smartphone app without a mobile CO checker, the results showed the feasibility and usability in the phase I study  and demoTherefore, this study aims to evaluate whether the latest version of CASC in addition to a standard smoking cessation program is effective in treating individuals with nicotine dependence.. Consent forms must be dated and signed both by trial participants and researchers. When consent is obtained, the first copy of the form will be kept in each institution, while the other part will be kept by participants and will not be collected even after the trial is over. In addition, we will inform all participants that their medical care will not be affected if they refuse to enroll in this trial. Participants will be able to leave the trial whenever they want to discontinue it. All participants will receive a compensation of 10,000 yen per clinic visit .We will recruit nicotine-dependent adult participants from October 2017 to September 2018 or until the recruitment process completes; we plan to follow them until October 2019. Participants who meet all inclusion criteria will be included in the trial and those fulfilling the exclusion criteria will be removed from this trial . We willRandomization will be performed for eligible participants. They will be allocated 1:1 to both arms—the CASC treatment group and the control app group. Randomization will be performed by a computer-generated random sequence with stratification for smoking cessation medications.In addition to the 12-week standard treatment procedure for smoking cessation , we willCASC consists of a CureApp Smoking Cessation smartphone app based on a cloud system paired with a mobile CO checker for participants and a Web-based patient management software for primary care physicians. Participants in the treatment group will install the app in their smartphones and then begin taking a few minutes every day for watching a video tutorial regarding smoking cessation; in addition, participants from the treatment group will keep a digital diary of quitting smoking, chat with artificial intelligence (AI) nurse, and check their exhaled CO concentration by the mobile CO checker at least once a day. Physicians will be able to follow participants’ exhaled CO concentration data and physical conditions through a secure cloud system and review them during the clinic visits .Inclusion:Participants diagnosed with nicotine dependence (Tobacco Dependence Screener≥5 points)Participants with Brinkman index≥200Participants who wish to quit smoking immediatelyParticipants who agree to receive the smoking cessation treatment program and sign the written consent formParticipants who can use a smartphone (operating system: Android 5.0 and above or iPhone 8.0 and above)Exclusion:Participants with severe mental illnessParticipants who cannot come to their follow-up clinic visits during the 1 yearParticipants who have started taking a smoking cessation medication within 1 year before the registrationParticipants who plan to use any smoking cessation aids and participate in any kind of smoking cessation activities (not limited to smoking cessation therapy) outside the trialParticipants whose investigator or clinical trial doctor judged them to be unsuitable for participation in this trial owing to other reasonsOn the other hand, participants from the control app group will be able to perform only 6 basic functions in their app as follows: (1) show the user guide (how to use the app); (2) enter their profile and set the start date of smoking cessation; (3) display the course of 5 visits during the 12-week treatment with a summary of objectives of each visit; (4) show the date of next appointment; (5) get the contact form for technical support; and (6) display an app version, privacy policy, and other administrative information. The other functions of the CASC app will not be incorporated in the control app. The control app will not include the mobile CO checker either. Note that exhaled CO concentrations will be measured by the medical staff at each clinic visit regardless of the study groups, and these CO data at the clinic will be used in the analysis of study outcomes.The CASC, control app, and mobile CO checker delivered by CureApp Inc.  should be carefully managed to ensure they are not used outside the clinical trial at each medical institution. A prescription code will be required to activate the investigational device, and the control group app will be issued by sponsors specific to each practicing medical institution; these sponsors regularly (at least once a year) perform inventory confirming there will be no usage of the app outside the trial.The CASC smartphone app was developed at CureApp Inc. supervised by the Division of Pulmonary Medicine, Department of Medicine, Keio University School of Medicine. The app runs on both iOS and Android smartphones. Primary physicians will provide the app prescription code to participants at their outpatient clinics. Participants will download the app through their smartphones, activate the app entering the code, and enter their baseline data including age, sex, years of smoking, number of cigarettes per day, prescribed medications (nicotine patch or varenicline), and motivation and self-confidence regarding smoking cessation. These data will be securely sent to the cloud storage, and the AI of our system will assemble tailor-made smoking cessation programs suitable to each participant based on their personal information. Personal data stored on the cloud can be reviewed by primary physicians. The CASC app has the following 4 main components to maximize the therapeutic effects of pharmacological therapy and counseling provided by health care professionals: (1) keeping a smoking cessation diary (once a day); (2) lectures and educational videos helping its users quit smoking; (3) interactive counseling by chat-bot (AI nurse); and (4) daily measurement and recording of exhaled CO concentration levels at home through a mobile CO checker.Regarding a Web-based personal computer software for primary doctors, it provides a data management app from patients’ CASC mobile apps and advice for physicians to follow the national clinical guidelines appropriately.The primary outcome of this study will be the biochemically validated CAR from weeks 9-24. The success of smoking cessation has been defined as (1) self-reported continuous abstinence from weeks 9-24 and (2) exhaled CO concentration no more than 10 ppm at both weeks 12 and 24. In current smoking cessation programs, the CAR from weeks 13-24 drastically decreases, whereas our investigational device is expected to prevent this reduction by covering “treatment gaps” to promote the retention of appropriate recognition and behavioral changes. A single-arm prospective pilot study showed that a CASC app, even without a mobile CO checker, had a significantly higher CAR from weeks 9-24 than the historical cohort that had received standard smoking cessation program ,17.In addition, we will evaluate the following secondary outcomes: (1) 7-day point prevalence abstinence at weeks 4, 8, 12, 24, and 52; (2) the CAR from weeks 9-12 and from weeks 9-52; (3) changes in several scores related to smoking cessation, including the Mood and Physical Symptoms Scale (MPSS) , 12-itemAn independent monitoring staff will conduct on-site data monitoring. In addition, the staff will review trial database to confirm whether principal and subinvestigators at each clinic perform the clinical trial according to the research proposal and standard operation procedures, and data queries will be asked if necessary.th week or participant no longer intends to continue the smoking cessation program; (8) sponsor cannot continue the trial; and (9) any other cause in which researchers decide discontinuation is appropriate.Participants will be discontinued from follow-up visits for this trial when they meet either of the following criteria: (1) participants request discontinuation; (2) researchers consider discontinuation is necessary owing to severe adverse effects, for example; (3) the eligibility criteria was violated; (4) significant deviation from the protocol occurred; (5) study institution terminated trial participation because of good clinical practice violation, for example; (6) participants’ further follow-up visits are considered very difficult to accomplish; (7) continuing smoking even at the 9The primary endpoint will be the CAR from weeks 9-24. We estimated conservatively 52.8% in the CASC treatment group and 40.8% in the control group as the primary endpoint (CARs from weeks 9-24) based on the survey of smoking cessation . RegardiP values for each outcome. A statistical analysis plan detailing all statistical computations will be completed prior to the lock of the database. SAS statistical software, version 9.4 or upper version (SAS Institute Inc), will be used for all the analyses.All statistical analyses will be performed according to the intention-to-treat principle and using 2-sided at a .05 significance level. The baseline characteristics will be described by the mean and SD, median and quantiles (continuous variables), or proportion . We will examine the outcomes using the full analysis set. For the primary outcome, we will compare CARs from weeks 9-24 comparing the CASC treatment group and the control group using a logistic regression model adjusted for prescribed smoking cessation medications. In addition, secondary outcomes at each defined period will be evaluated with logistic regression analysis adjusted for appropriate covariates between the groups. We will provide descriptive statistics, odds ratios, 95% CIs, and A total of 580 participants with nicotine dependence will be recruited from October 2017 to September 2018 or until recruitment is complete. The final 52-week follow-up will be completed in October 2019. We expect that all trial results will be available by the end of 2019.This is the first randomized controlled trial to evaluate the long-term efficacy of CASC, a smoking cessation support app for participants, which also includes a Web-based software for doctors and a mobile CO checker. We expect that participants in the CASC treatment group, in addition to a 12-week standard smoking cessation program, will exhibit a significantly higher CAR from weeks 9-24 than participants in the control app group.In recent years, several medical apps for smartphones have shown better clinical outcomes compared with conventional treatment -12. ThesWe developed and used a mobile Internet of Things (IoT) device to measure an exhaled CO concentration level in this trial. A level of exhaled CO concentration is a useful biomarker for patients with nicotine dependence . It helpThere are several benefits to including smartphone apps with an IoT device to clinical settings. First, a mobile app covers treatment gaps between the outpatient clinic visits. It is beneficial to continuously contact participants and keep providing effective treatment programs, which could be one of the most important points for the program success ,13. SecoImproving nicotine dependence treatment programs with therapeutic smartphone apps and IoT devices is important in terms of preventing chronic obstructive pulmonary disease, cardio- and cerebrovascular diseases, and malignant tumors. In addition, these mobile apps and devices can contribute to preventing secondhand smoke, which kills approximately 15,000 people every year in Japan . In addi


Danio rerio) is emerging as an increasingly successful model for translational research on human neurological disorders. In this review, we appraise the high degree of neurological and behavioural resemblance of zebrafish with humans. It is highly validated as a powerful vertebrate model for investigating human neurodegenerative diseases. The neuroanatomic and neurochemical pathways of zebrafish brain exhibit a profound resemblance with the human brain. Physiological, emotional and social behavioural pattern similarities between them have also been well established. Interestingly, zebrafish models have been used successfully to simulate the pathology of Alzheimer’s disease (AD) as well as Tauopathy. Their relatively simple nervous system and the optical transparency of the embryos permit real-time neurological imaging. Here, we further elaborate on the use of recent real-time imaging techniques to obtain vital insights into the neurodegeneration that occurs in AD. Zebrafish is adeptly suitable for Ca2+ imaging, which provides a better understanding of neuronal activity and axonal dystrophy in a non-invasive manner. Three-dimensional imaging in zebrafish is a rapidly evolving technique, which allows the visualisation of the whole organism for an elaborate in vivo functional and neurophysiological analysis in disease condition. Suitability to high-throughput screening and similarity with humans makes zebrafish an excellent model for screening neurospecific compounds. Thus, the zebrafish model can be pivotal in bridging the gap from the bench to the bedside. This fish is becoming an increasingly successful model to understand AD with further scope for investigation in neurodevelopment and neurodegeneration, which promises exciting research opportunities in the future.Zebrafish ( Zebrafish can be used as a model to study various disease pathologies in Alzheimer’s disease.Laser axotomy coupled with time-lapse imaging and 3D imaging reveal interesting facts about degeneration/regeneration in Zebrafish larvae.New approaches to treat Alzheimer’s disease can be further unearthed with this model.How does zebrafish serve as a model for understanding Alzheimer’s disease?Can real-time imaging in zebrafish address the major breakthroughs in the field of Alzheimer’s research?Can zebrafish merge the gap between neurospecific drug discovery and clinical research?Danio rerio) is a prominent vertebrate model system for comprehensive analysis of the unique functions of genes along with their signalling pathways during development and neurodegeneration1. Such studies have been possible as the zebrafish possesses several distinct advantages over other vertebrate models  throughout the developmental process using non-invasive imaging techniquesOwing to the small size of the larvae, high-throughput screening of neuroactive compounds can be easily performed.10 . Very recently in an interesting study, scientists have upgraded the web tool for analysing zebrafish genes using gene ontology as the entire zebrafish genome has been sequencedThe zebrafish  and sporadic AD (SAD)23.Alzheimer’s disease (AD) is a progressive neurodegenerative disease. It is the most prevalent form of dementia in the world affecting almost 47 million people worldwide (Alzheimer’s Association)24. FAD accounts for only ~ 1–5% cases of AD, whereas the rest is attributed by SAD25. SAD is attributed mainly by a combination of environmental risk factors and genetic susceptibility. Reports suggest a functional role of Apolipoprotein E (ApoE) phenotype in the late-onset AD26, ε4 allele being the major risk factor for AD, whereas ε2 allele is protective28.FAD shows an autosomal dominant inheritance and is usually caused by mutations occurring mainly in three genes, Amyloid precursor protein (APP), Presenilin 1 (PSEN1) and Presenilin 2 (PSEN 2)29. It suggests the pivotal role of Aβ in initiating and triggering the pathology of the disease including inflammation and oxidative stress30. For decades, Aβ deposition and aggregation have been treated as the primary mechanisms underlying the disease pathology. Though Aβ hypothesis is central to the disease, scientists, however, have propounded several other hypotheses32. Among them, the earliest proposed was the cholinergic hypothesis.33 Decrease in the cholinergic transmission in AD is responsible for the abnormalities in the cognitive and functional domains of AD patients. However, it is now clear, that cholinergic dysfunction can cause cognitive impairment only through indirect mode33. Another most prevalent hypothesis is the tangles hypothesis.34 A better correlation exists between presence of tangles and status of the disease progression; however, the amyloid hypothesis holds as it has been noticed that mutations in Tau do not result in plaque deposition. Among other hypotheses, the calcium hypothesis explains how the activation of the amyloidogenic pathway regulates the calcium signalling in the neurons to affect cognition36. However, the relation between dysregulated calcium signalling, aging and cognitive decline are still not clear36. The mitochondrial hypothesis suggests that the mitochondria plays a major role for the production of reactive oxygen species and subsequent neurodegeneration37. The complete link between mitochondria and the disease is yet to be established. Hence, a better understanding of the various hypotheses in AD will definitely direct our approach for better treatment strategies of the disease.The presence of Aβ plaques in the brain led to the origin of the Aβ hypothesis38. A great degree of similarity between their neuroanatomic41 and neurochemical pathways42 are reported. The medial, dorsal and lateral pallium of the zebrafish are similar to the amygdala43, isocortex44 and hippocampus46 in other vertebrates, respectively. The zebrafish encephalon comprises of the forebrain, mid brain and hind brain, . Similar to mammalian brain, the zebrafish brain harbours the main excitatory glutamatergic and inhibitory GABAergic neurotransmitter circuits47 along with the presence of muscarine cholinergic receptors48. Besides, they possess GABA, glutamate, serotonin, dopamine, histamine, acetylcholine neurotransmitters49, enzymes of synthesis and for metabolism51. Similarity exists even at the cellular level, as the cell types astrocytes52, microglia53, oligodendrocytes54, cerebellar Purkinje cells55, myelin56 and motor neurons57 are found similar to human cells. Further studies on the neuronal patterns in the spinal cord of adult zebrafish, neural differentiation and development of spinal network establish their similarity to higher order vertebrates60.Research shows a high conservation between zebrafish and human brain organisation63, learning64, hearing65, vision66, touch69 and emotions like fear72, pain73, helplessness74, courtship75, social interactions77, anxiety78 and decision making79. Further, reports indicate similarity between expression patterns and axonal projections of hypocretin/orexin neurons of larval zebrafish and humans80. Interestingly, because of the high degree of behavioural similarity between zebrafish and humans; researchers have also employed the use of circadian rhythm in zebrafish locomotor activity to understand the mechanism, which can regulate sleep in humans83. Sleep behaviour pattern has been studied in the zebrafish and has been simulated with sleep-like state in mammals. This study shows that the rest phase in zebrafish and the behavioural manifestations of sleep in mammals exhibit considerable fundamental similarities. They further report that the zebrafish exercise homoeostatic control over rest behaviour which is regulated by the circadian rhythm, features similar to that of mammals84. Disruptions in the circadian rhythm in AD have been reported by several groups90. Although a link between circadian cycle and AD is clearly implicated, a proper mechanistic explanation connecting them is still lacking. The interactions among the proteins involved in maintaining circadian rhythm in the zebrafish is quite similar to those observed in the mammals94. Apart from this, the cholinergic neurotransmitter system modulates drug induced reward activity in the zebrafish making it compatible to study the neurobiology of addiction. This establishes the zebrafish as a robust model for studying the biology of behaviour in vertebrates95.Several anthropomorphic assays have been performed to depict similar behavioural pattern between zebrafish and humans, which implicates a conserved behavioural mechanism and circuitry paradigm of both systems. Researchers have employed the zebrafish behavioural pattern to study physiological behaviour like feeding96. Quite interestingly, another group found both APP and Aβ-deficient larvae displayed cerebrovascular defects. Interestingly, these anomalies could be reversed by treating the embryos with human Aβ1–42 peptide. However, there was no effect when they were treated with p3 (the shorter APP cleavage product)97. This finding brought to light the significant cerebrovascular growth promoting function of Aβ. Further, the embryos treated with 2.5 µM of Aβ1–40 led to abnormal vasculature development and cell death98.Several zebrafish models have been established by placing Aβ central to the disease pathology to simulate AD. A group of researchers suggest that higher levels of Aβ monomers can stimulate angiogenic sprouting in the developing zebrafish hind brain99. Cytoskeletal disruption occurred on expressing frontotemporal dementia with Parkinsonism linked to chromosome 17, a mutant form of human Tau in the neurons of the zebrafish, which resembled the neurofibrillary tangles observed in AD101. Another group expressed mutated form of the human τ protein in the zebrafish neurons which disrupted the cytoskeletal structure101. Although removal of exon2 from Enolase GFP-transgenic zebrafish and its replacement with complementary DNA that encoded for the four-repeat isoform of the human Tau resulted in eight times overexpression of Tau in the zebrafish brain compared with normal human brain. The overexpressed Tau localised to axons and resembled neurofibrillary tangles102. These reports exemplify the use of zebrafish as a model for AD.Besides the Aβ model, research has also focussed on the generation of zebrafish models with Tauopathy103. Another group injected Aβ1-42 into the hind brain ventricle of 24 h post fertilisation (hpf) zebrafish embryos. They observed significant cognitive deficits in the embryos with increased Tau phosphorylation in target residues of GSK-3β in the 5 dpf larvae104. Zebrafish embryos exposed to Trimethyltin chloride (TMT) exhibited neurobehavioural toxicity, specifically, apoptosis in the tail, modulations in photomotor response and frequency of tail flexion105. Most recently, another pharmacologic model of AD has been developed by adding okadaic acid106. Both Aβ plaques and phosphorylarion of Tau have been found to increase with increasing concentrations of okadaic acid. Learning and memory deficits have also been observed in these fish106. Zebrafish treated with Aluminium in an acid environment displayed behaviour with AD-like condition wherein their locomotor activity and learning ability got abrogated107. Furthermore, it has been reported that intraventricular injection of Aβ1–42 in the embryonic brain leads to memory loss and cognitive deficits along with increased Tau phosphorylation108. The zebrafish is therefore emerging as a powerful model for research in the field of neuropharmacology.Researchers have checked non-associative learning in the zebrafish larvae based on cognitive and behavioural responses of AD. Seven days post fertilisation (dpf) larvae were exposed to a series of acoustic stimuli, to which the larvae displayed significant reduction of startle response110. In addition, it has been shown that the Sortilin-Related Receptor (SORL1)-dependent switch can divert APP from the late endosomal pathway. This sequesters the APP into the endosomes, thus preventing the formation of Aβ112. Another group has showed that the wild type Psen1 in the zebrafish facilitates anomalous Aβ1–42 secretion similar to mutations associated with FAD113. Zebrafish embryos injected with morpholinos, which block Psen1 translation are viable but show p53-dependent apoptotic death of neurons117. A unique function of the Psen1 has come to light through studies in a mutant zebrafish lacking Psen1 activity. These mutant fish are viable and reveal the regulation of histaminergic neuronal development by Psen1 in the fish118. Unlike Psen1, loss of Psen2 expression hinders the synthesis of dorsal longitudinal ascending interneurons from the spinal cord during the development of zebrafish larvae119. In an interesting study, the scientists report the pathological role of a new truncated isoform, PS2V of Psen2 and its implication in neurodegenerative diseases117. Research has focussed on the β secretase as well, whereas Bace1 and Bace2 knockout zebrafish were generated using zinc finger nuclease-mediated genome editing. Bace1 mutants showed hypomyelination in the peripheral nervous system while Bace2 mutants showed anomalous migration of the melanocytes120. However, the Bace1/2 double knockout zebrafish did not display any enhancement in the mutant phenotypes, pointing out to the non redundant function of Bace1 and Bace2. Another Aβ toxicity model was generated by using the mitfa (nacre) gene promoter for the expression of the Aβ gene. The gene was chosen as it could produce a distinct disrupted pigmentation pattern in the larvae. However, the model did not turn out to be successful as the differential pigmentation pattern was evident in the adult fish, only at 16 months, by which time, the fish were infertile and too old for breeding121. Fluorescently labelled Tau transgenic zebrafish model of AD with a mutation Tau-P301L were also generated, which presented with the key pathological features of Tauopathy including neurofibrillary tangles, neuronal loss and cell death122. The zebrafish therefore harbour a huge resource of genetic information, which need to be manipulated to reveal the molecular details of AD.Researchers have unravelled the role of APP in FAD using the zebrafish model, whereby, APP knockdown zebrafish displayed defective convergent-extension movements with reduced body length and short curled tail. They showed that Swedish mutant APP is unable to overcome the developmental defects unlike the wild type APP123. Further, the fact that zebrafish-specific Aβ peptide is yet to be elaborated remains a drawback. More research also needs to be performed to study whether the post translational processing of APP in humans is also prevalent in the zebrafish124.There are several advantages in using zebrafish as a model system for studying AD and a few limitations in using them in translational neuroscience research. Pharmacological modifications in the fish are easily brought about by adding the desired chemicals in water. However, the quantification of chemical compound entering the fish is unpredictable, as substances can be absorbed randomly through the gills and skin of the fish owing to exposure of the whole body in the aqua medium1–42 peptide showed regeneration of neurons, specifically neural cell/progenitor cell proliferation and neurogenesis125. They studied the regenerative ability in old and young fish to understand the influence of aging and Aβ deposition on neuroregeneration. They show that in neurodegeneration induced by Aβ, microglia get activated, to prevent synaptic degeneration and promote neurogenesis. Thus they establish a potential link between neurodegeneration, neuroinflammation and neurogenesis125. This might pose a threat to the feasibility of the AD model but it definitely opens up a whole new world of research to delve into the molecular mechanisms of signalling pathways that could be active in playing a paramount role in the regeneration of neurons. This will surely pave the way for understanding the molecular programmes required for regeneration of the mammalian central nervous system.Zebrafish possesses a unique ability of regenerating neurons along their rostrocaudal brain axis throughout life unlike mammals. In an interesting observation, the researchers found that zebrafish microinjected with Aβ130. Zebrafish larvae are small transparent vertebrates, which are highly suitable to confocal microscopy when labelled with dyes131. Thus, it facilitates studies regarding the in vivo physiological signalling processes in intact organ systems132.The zebrafish has a relatively simple nervous system, which allows imaging of neurons easily. This creates the possibility to visualise specific neuronal proteins of interest and thus provide a wonderful opportunity to study the neurological processes in detail122. After a few days, the larvae developed substantial neurodegeneration displaying all pathological features including neurofibrillary tangles by 5 weeks of development. The pathological features develop much earlier in zebrafish as compared with the other available rodent models. As the zebrafish model provides the opportunity for easy manipulation and visualisation of the optically clear embryos122. Based on this, real-time imaging of microglial phagocytosis has enhanced the perception of microglia mediated neurodegeneration53. A group of scientists used laser imaging technique to measure the activity of nerve cells in zebrafish, propounding a novel association between Neurexin2 and spinal muscular atrophy; establishing neurexin2 as a potential target for the treatment of SMA133. In vivo fragmentation of mitochondria was observed by real-time imaging when exposed to apoptosis-inducing agents in a mitochondrially targeted GFP-transgenic zebrafish134. Another group studied the effect of mitochondrial oxidation on vulnerability to axonal degeneration by time-lapse confocal imaging135. Detailed real-time imaging analysis of the timing of cell division in zebrafish embryo has also been analysed by real-time imaging136. Recently developed techniques like laser axotomy coupled with time-lapse imaging have revealed the role of extrinsic cell types in degeneration and regeneration in the zebrafish larvae137. Real-time imaging using zebrafish has therefore unearthed a vast amount of information in AD, whereas the potential exists for a lot more to be brought to light.Real-time imaging using zebrafish has had a profound impact on the understanding of major physiological processes such as neurodegeneration Table . Researc138. Bulk loading approach involves the use of synthetic Ca2+ indicators to label cells within the tissue. This technique has been utilised to study the reticulospinal neurons or Mauthner cells in the larval zebrafish140. Microinjection of a chemical dye, Oregon Green BAPTA-1 results in specific labelling of neuronal cell bodies in the zebrafish brain140. Another group of researchers injected the calcium green dextran to study the Mauthner cell circuit in the living fish139. The next improvisation in this field is the use of aequorin, a bioluminescent photoprotein that emits light upon binding to Ca2+141. Aequorins have been successfully used for Ca2+ imaging in the zebrafish143. The subsequent development in this field is the use of in vivo two-photon Ca2+ imaging. Understanding of the functional topology of neural activity patterns has been possible using this technique145. Another recent development in this area is the use of genetically encoded calcium indicators such as cameleon. Their major advantage is the ability to monitor neuron activity in a non-invasive manner in the living zebrafish146. Calcium imaging has also provided insights into the cellular events that execute axonal dystrophy arising in neurodegeneration148. Thus the Ca2+ imaging technique has helped in understanding the functional role of neurons which would not have been possible by other approaches.Neuronal development of the larva and adult has been studied using the calcium imaging technique. These studies have presented us with a better understanding of the basic biological principles during development and adulthood of vertebrates149. Selective plane illumination microscopy, also known as light sheet fluorescence microscopy is another technique used in 3D imaging150. Apart from this, researchers have also used fluorescent probes to construct 3D imaging in live zebrafish151. This serves for high-throughput imaging ensuring sufficient reproducibility of observations. Vertebrate automated screening technology has been used to take multiple axial views of the zebrafish larvae. These were later reconstructed to produce 3D volumetric representation of the larvae and also its measurements, thus providing a more holistic view152. Extensive automated processing techniques have also been developed to analyse the complex images for easier comprehension153. 3D high resolution imaging is a rapidly developing technique but is accompanied by some technical issues like high expense, normalisation, artefact corrections and web-based publication of results among others. A recent discovery has addressed one such issue by coming up with a multi-usage observation chamber called UniverSlide adapted for live 3D bio imaging of the zebrafish larvae154.The zebrafish is yet again adept for one of the most recent imaging techniques, which is the three-dimensional reconstruction of images to produce a 3D volumetric representation of the specimen. It includes three-dimensional time-lapse imaging of embryos either by confocal or multiphoton laser scanning microscopy. This technique employs the use of varied signals for analysis of the image, including two-photon-excited fluorescence, second harmonic generation and third harmonic generation3D imaging in zebrafish is a most recent technique and still rapidly evolving, which allows the visualisation of the whole organism, thus leading to an elaborate understanding of the in vivo scenario in the organism. It is therefore a highly promising technique that could produce a breakthrough in the field of zebrafish imaging and thus help it to stand out as a star model for future research.155. Till date, very few studies involving neurological drug discovery have been performed in the zebrafish, most of the studies being performed outside the nervous system160. Evaluation of a number of psychoactive compounds have also been performed using zebrafish161. Although it has been found that both nicotine162 and ethanol163 affect the development of the nervous system, cocaine165, amphetamine95, morphine167, ethanol169 and nicotine have been noted to elicit reward or anxiety-related behaviour in the zebrafish. Interestingly, nicotine has been reported to have cognition enhancing effects in a learning paradigm in zebrafish170.Several approaches have been made to effectuate the discovery of potential therapeutic compounds exploiting the optical clarity of the zebrafish embryos and larvae. Owing to the small size of the embryo and larvae, drug discovery in a high-throughput scale can be achieved using this model system171. Small molecules that could relieve cell cycle arrest172 and cardiovascular problems173 and also modulate the embryonic heart rate174 by suppressing mutations have also been screened using zebrafish model system. Most importantly, research shows that the effect of these drugs is similar in humans and in zebrafish175, thus validating that zebrafish models for drug discovery have the potential to produce or identify therapeutic compounds suiting human conditions177.A group of researchers identified a small molecule called prostaglandin E2, which regulates hematopoietic stem cell homoeostasis9-tetrahydrocannabinol and heroin (diacetylmorphine) have been tested using zebrafish184. Rapid behaviour-based screening has also evolved as an inexpensive assay for identifying small neuroactive molecules185.Studies have also been performed to analyse the effect of several neuroactive compounds in the zebrafish adults, whereby addition of such compounds in the water of the fish tanks have been shown to differentially change their swimming pattern and diving behaviour. Several neuroactive compounds like piracetam, methylenedioxymethamphetamine, hallucinogenic agents like mescaline and phencyclidine, a nitroamine explosive 1,3,5-Trinitroperhydro-1,3,5-triazine, kynurenic acid, Δ188. Analysis performed using transmission electron microscopy, fluorescent markers, chromatography and tandem mass spectrometry have revealed that BBB in the zebrafish is both structurally and functionally similar to that of mammals. Its development starts by 3 dpf and continues upto 10 dpf in the zebrafish larvae189. Another group reports the presence of tight junctions in the BBB as the brain endothelial cells show immunoreactivity to Claudin-5 and Zona Occludens-1190. Recently a group of researchers suggested an orthotopic glioblastoma model of zebrafish, which could be used as an efficient assay system for visualising the BBB penetrating efficiency of anti-GBM drugs191. Given the feasibility of high-throughput screening and the advantage of similarity with mammals, it is reasonable to say that zebrafish offers an excellent opportunity for screening neurospecific compounds.The blood–brain barrier (BBB) of the zebrafish has molecular and functional similarity to the higher vertebrates193. They lead to synaptic dysfunction, disruption of neuronal connectivity and neuron death199. Interestingly, research in the zebrafish model of AD suggests that Aβ may also play a role in maintaining cerebrovascular functions97. They show that Aβ deficiency leads to reduction in cerebrovascular branching and vessel length in the developing hind brain of zebrafish embryo. In corroboration to this, another group of researchers reported that Aβ is involved in regulation of angiogenesis in the human umbilical cord vein and in the zebrafish hind brain96. This role of Aβ in the zebrafish stands in complete refutation to its functionality in the humans; wherein it causes cerebrovascular dysfunction, leading to cognitive defects201. One probable reason for this discrepancy could be the fact that Aβ in the teleosts is different from the other vertebrate Aβ including humans97. The group further explains why drugs targeting Aβ production failed in recent clinical trials as the potential function of Aβ in regulating angiogenesis could be interfering with the mechanism of action of the drug. Further research on the molecular mechanism of this functionality shall help understand the discrepancy better92. This also points out to the existence of other non-amyloid hypotheses  in AD. Several zebrafish AD models have already been established with focus on Tauopathy122 and cholinergic hypothesis202 while better models exhibiting a combination of several pathologies still remain to be studied. Elaborate studies involving real-time imaging in the zebrafish model may unravel potential roles of these hypotheses in AD pathogenesis. With these controversies, zebrafish emerges to be even more interesting as a model system for neurodegeneration which needs further research. It also holds promise as a complete model for understanding AD and providing a platform for research on areas which have not been dealt with earlier but could prove to be beneficial in the field of drug development.Aβ plaques are most widely believed to be pivotal to the pathogenesis of AD203. The gap between drug discovery based on cellular models and pre clinical assays can be efficiently bridged by research using this model system. The zebrafish presents itself as the best candidate for high-throughput pharmacological screening of drugs before validating them in rodent models. Abundant research has already been performed to elaborate on the scope of zebrafish as a model to understand AD. However, arenas like the behaviour, physiology, neuroanatomical circuitry of the fish and the link between neurodevelopment and neurodegeneration (Table In the present review, we aim to illustrate the eminent progress that zebrafish propounds in order to comprehend the pathological mechanisms of AD. It can be envisaged that zebrafish model has emerged as an interesting tool for strategic study of AD