There are >200 different types of human papilloma virus (HPV) of which >51 infect genital epithelium, with ~14 of these classed as high-risk being more commonly associated with cervical cancer. During development of the disease, high-risk types have an increased tendency to develop a truncated non-replicative life cycle, whereas low-risk, non-cancer-associated HPV types are either asymptomatic or cause benign lesions completing their full replicative life cycle. HPVs can also be present as non-replicative so-called “latent” infections and they can also show superinfection exclusion, where cells with pre-existing infections with one type cannot be infected with a different HPV type. Thus, the HPV repertoire and replication status present in an individual can form a complex dynamic meta-community which changes with respect to both time and exposure to different HPV types. In light of these considerations, it is not clear how current prophylactic HPV vaccines will affect this system and the potential for iatrogenic outcomes is discussed in light of recent outcome data. Although published work overwhelmingly supports the efficacy of the current prophylactic HPV vaccines, there has been a great deal of publicity and numerous studies carried out on potential vaccine-related adverse events ,2,3,4. TAlthough there are ~200 distinct types of HPV, there is substantial evidence that many others are yet to be identified ,7 as exeOf these, ~14 are classed as high risk and are associated with cervical cancer although some are more carcinogenic than others ,9,10. FoThe unique life cycle of HPV is intimately associated with how the virus manages to evade the immune system. At its simplest, the virus hides by not penetrating below the basement membrane where the majority of immune effector cells reside. Furthermore, it does not produce secreted proteins, inflammation, viremia or cell death ,15. TherHowever, during the development of HPV-related high-grade cervical neoplasia, the production of mature virions is reduced since the virus downregulates its full replicative life cycle and expression of the L1 protein is also reduced , Gardasil targets L1 from HPV16/18/6/11  and Gardasil 9 targets L1 from 16/18/31/33/45/52/58/6/11  and their prophylactic use is currently advocated for women prior to infection with HPV. They all induce HPV type-specific L1 antibodies which provide humoral immunity against HPV infections in HPV-negative women by blocking the virus from interacting with its cellular receptors and many studies have demonstrated their effectiveness at protecting against CIN lesions caused by infection with vaccine-covered HPV types. However, for >15 years, there has been considerable debate on the ability of Cervarix and Gardasil to protect both HPV-positive and -negative women from HPV-associated cervical cancer. For example, it is well known that HPV-negative women vaccinated with either the bivalent or quadrivalent vaccines can still develop CIN lesions positive for HPV types not covered by these products although it is anticipated that the more recently introduced Gardasil 9 should improve this ,30. FurtIn women who are HPV positive at the time of vaccination, additional concerns are based on the mode of action of the L1 targeted vaccines. Since expression of the L1 protein is downregulated as a consequence of the truncated HPV life cycle that is a common feature of high-grade type 3 cervical intraepithelial neoplasia (CIN3)  19], it, it19], As previously discussed, many women are known to acquire cervical infections with multiple HPV types of which only some are associated with cervical cancer  6,9,10],9,106,9,As previously mentioned, it is known that HPV can exist at very low copy numbers, where the virus is present as a non-productive so-called latent infection  which haThese observations illustrate there is a distinct lack of knowledge concerning potential interactions within HPV meta-communities which may be present at various stages of HPV infection and how these may contribute to either progression or regression of disease . In thisTo date, long-term efficacy studies with Gardasil 9 are not available so only Cervarix and Gardasil will be considered further. Prior to, and immediately following approval, reports on the efficacy of Gardasil were produced by the Food and Drug Administration  and the Analysis of the combined results from protocols p007, 013 and 15 showed that Gardasil was 98–100% effective at preventing HPV6/11/16/18-related CIN2/3 in PPE and MITT-2 women who were sexually naive and HPV negative with normal smears at baseline. However, this dropped to 36.3% in the MITT-3 cohort which represents a real-world population of 16–26-year-old women who were positive for either vaccine or non-vaccine HPV types and also included women who had an abnormal pap test at baseline . NotablThe Future 1 p013 study was also analysed for Gardasil efficacy against all grades of CIN related to any HPV type in the baseline HPV-negative RMITT-2 population which includes women who were either sexually naïve or experienced. This demonstrated 31% efficacy where 6.4% (92/1429) of vaccinated and 9.1% (132/1441) of placebo-treated women developed CIN lesions due to any HPV type including types 16/18 . Thus, A combined analysis of both Future 1 and 2 trials (p013 and p015) was also carried out for CIN2/3 lesions related to any HPV type including types 16/18. This showed a vaccination efficacy of 46% in RMITT-2 and 27% in MITT-2 women against CIN2/3 . SignifExtent of vaccine efficacy in HPV-positive women.Extent of HPV cross-type protection.Extent of HPV type replacement and the potential role played by latent infections.The previously discussed FDA and EMA reports on Gardasil were made between 2006 and 2008 which both indicate that the vaccine provides virtually 100% protection against all grades of CIN related to HPV infection with vaccine-covered types in HPV PCR-negative women and many additional studies have since confirmed this observation. However, important questions still remain such as:Attempts to address these issues have relied on monitoring pre- and post-vaccination levels of both vaccine-covered and non-vaccine-covered HPV types in various populations. In this regard, a meta-analysis of 9 studies involving 13,886 girls and women ≤19 years of age and 23,340 women 20–24 years of age was carried out by Mesher et al. in 2016 . The resThe issue of HPV type replacement is still a subject of significant controversy and a study by Gray et al. evaluated pre- and post-vaccination HPV type prevalence in Finnish women . It was Furthermore, it was shown that a substantial proportion of this increase was due to increased prevalence of additional HPV types covered by Gardasil 9. However, there was also an overall increase of ~12% in the prevalence of CIN2 in presumably catch-up vaccinated women aged between 30 and 35 years which was attributed to changes in screening practices between 2008 and 2016.A similar study was carried out in New Zealand which analysed changes in HPV types associated with CIN2 between 2013 and 2016 in 392 women under the age of 25 . New ZeaAlthough HPV16/18 lesions decreased substantially, there was a 33% increase in lesions associated with other high-risk types and it was noted there was a significant increase in the number of women diagnosed with high-grade cytology over the same period . IntereMore recently, Innes at al evaluated the effect of vaccination on the incidence of abnormal cervical cytology and histology in 104,313 New Zealand women aged 20–24 years between 2010 and 2015 . CompareDrolet et al. carried a systematic review and meta-analysis on the population level impact and herd effects related to HPV vaccination derived from 65 articles covering 60 million individuals over a maximum period of 8 years in order to compare pre- and post-vaccination relative risk of HPV-related end points . This stUnfortunately, there are currently no studies with sufficient follow up and suitable efficacy end points to definitively answer this question although it is known that the bivalent vaccine provides 91% efficacy at protecting women older than 25 which lasts for 7 years . FurtherVaccinate prior to onset of sexual activity and begin assessment of end points at age of usual cervical screening once sexually active.Make all clinical study reports, including anonymised individual patient data, publicly available.Separate trials to assess benefits in women already exposed to HPV without restrictions based on risk factors.Analyse data by country and study site.Ensure the testing interval is in line with usual cervical screening protocols.Continue follow up for a minimum of 20 years from time of sexual debut.Power trials for primary composite outcomes CIN3/AIS/cervical cancer due to oncogenic HPV types.Define secondary outcome of persistent HPV16/18 infection at a minimum of 12 months.Use standardised methods for HPV testing.Undertake a saline placebo-controlled efficacy trial of Gardasil 9 in previously unvaccinated populations as it is difficult to draw conclusions on efficacy and risk of harms based on comparing Gardasil 9 to Gardasil.It is very clear that the previously discussed trial results raise many questions and do not provide conclusive evidence that any of the prophylactic vaccines will actually prevent cervical cancer. Indeed a very recent review by Rees et al. critically assessed the available published data in relation to this goal and drew the same conclusion . These aAlthough implementation of these suggestions would obviously clarify some of the issues raised, it is very clear that such trials would also benefit from improved HPV testing in relation to vaccine efficacy. For example, analysis of the prevalence of all 51 genital HPV types  in womenSelection of the most appropriate outcome is critical for assessing vaccine efficacy and CIN3 was judged to be preferable to either HPV status or low-grade disease since these have much higher rates of spontaneous regression . HoweverIn the UK the overall incidence of cervical cancer decreased by ~35% from 1990 to 2005 and vaccination was started in 2008.The overall incidence of cervical cancer remained at a constant level until 2017 A. HoweveAustralia started Gardasil vaccination in 2007, achieving a coverage of >80%. The age-stratified five-year average cervical cancer incidence in New Zealand from 1987 to 2016 is shown in Sweden introduced cervical screening in the mid-1960s and by 2011 the incidence of cervical cancer had declined 3 fold. Vaccination started in Sweden in 2008 firstly with Cervarix and then Gardasil from 2012 with a coverage of 10% in the period 2008–2010 rising to >80% in 2018. The conclusion from this study was that the increase may be due to insufficient detection of precursor lesions prior to 2009 and the possibility that vaccination may have played a role was dismissed since <0.5% of women in the study cohorts were vaccinated. Indeed a more recent study of vaccination and the risk of cervical cancer in Sweden concluded that vaccinated women aged 30 years or less had a significantly lower risk than unvaccinated women . HoweverNorway started single cohort vaccination of 12-year-old girls with Gardasil in 2009 achieving a coverage of 89% but, unlike most other nations, did not start catch-up vaccinations until 2016 . It is nIt is significant that the UK, Australia, New Zealand, Sweden and Norway all have vaccination coverage rates of 50–86%, whereas France started vaccination in 2007 and only achieved coverage of 19% in 16-year-old girls. Indeed France has the lowest coverage in Western Europe which begs the question, how does the incidence of cervical cancer in France compare to nations with higher vaccine coverage over the same period? In 2007, the age-standardised incidence of cervical cancer was ~10 per 100,000 women, whereas in 2018 it had declined to 6.7 . Thus, uUse of high-sensitivity methods of HPV analysis in order to detect latent infections.Determination of the impact of vaccination over time on the prevalence of all 51 HPV types known to infect genital mucosa in all age groups and combine this with vaccination status, age at vaccination, vaccine type and sexual history.Use of this to discriminate between direct and indirect (herd) effects of vaccination on the incidence of different HPV types in unvaccinated women.Carry out these analyses on normal, CIN1, 2 and 3 and cervical cancers.Do not assume that unvaccinated women residing in a vaccinated population will experience only beneficial herd effects since increased transmission of non-vaccine-covered high-risk HPV types combined with decreased transmission of low-risk types has been shown to occur .Conclusions on vaccine efficacy should not be based on comparing vaccinated and unvaccinated women over time at the same geographical location since herd-effect transmission may confound this.Ideally, the incidence and disease association of different HPV types should be evaluated in any population before and after implementing vaccination.Clearly the preceding observations do not prove any causal relationship between the current prophylactic vaccines and iatrogenic outcomes with respect to cervical cancer. However, it is also very clear that there is a distinct lack of knowledge concerning the complex biology of mucosal HPV communities and the purpose of this review was to draw attention to how this could be affected by L1 targeting prophylactic vaccines. With regard to assessing vaccine efficacy, the recommendations of Rees et al.  are cleaWhilst it is very clear that the current HPV vaccines have substantial efficacy against disease related to vaccine-covered HPV types, there are many questions left unanswered concerning the full spectrum of their effects on HPV biology. As discussed, HPVs of the genital mucosa can represent a dynamic meta-community which changes over time with respect to many factors such as age, promiscuity and type of contraception used which means it is difficult to predict outcomes. In this regard, it is notable that the French PAPCLEAR trial  has someChanges in sexual practice.Changes in screening practice.Problems with assay sensitivity.Influx of migrant communities.Finally, as discussed, the recent increase in the incidence of cervical cancer in several countries with high vaccine coverage does not in any way establish cause and effect and there have been a variety of explanations put forward to explain this, such as:Indeed it has been speculated that this may be due to relaxed screening intervals which were adopted by some countries in order to cover the cost of vaccination and HPV testing. However, it is very unlikely that these same factors will be present in all the countries cited and it is very clear it will be of crucial importance to analysed the incidence of HPV types, CINs 1–3 and cervical cancers in women of all age groups in countries with high and low vaccine coverage in the next decade.


Actinidia spp.) are nutrient-dense fruit with a number of reported health benefits that include lowering glycaemic response, improving cardiovascular and inflammatory biomarkers, and enhancing gut comfort and laxation. This study investigated the effect of consuming three whole Zespri® SunGold kiwifruit (Actinidia chinensis var. chinensis ‘Zesy002’) with or without skin on cytokine production and immune and gut health in healthy people and those with IBS-C symptoms. This study enrolled thirty-eight participants in a 16 week randomized cross-over study . Participants were randomized to consume either three kiwifruit without eating the skin or three kiwifruit including the skin for 4 weeks each, with a 4 week washout in between each intervention. There was a significant decrease in the pro-inflammatory cytokine, TNF-α, for both the healthy and the IBS-C participants when they consumed whole kiwifruit and skin, and also for the healthy participants when they ate whole kiwifruit without the skin (p < 0.001). The kiwifruit interventions increased bowel frequency and significantly reduced the gastrointestinal symptom rating scale constipation and Birmingham IBS pain scores for both participant groups. We have demonstrated that consuming the skin of SunGold kiwifruit might have beneficial effects on gastrointestinal health that are not produced by consuming the flesh alone.Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that results in constipation (IBS-C) or diarrhoea with abdominal pain, flatulence, nausea and bloating. Kiwifruit ( Kiwifruit are recognised as a natural food that enhances laxation . LaxatioThe proposed mechanisms include disturbed neural function along the gut–brain axis, abnormalities of motility, visceral sensation, and alterations in gut fermentation. More recently, important roles for low-grade inflammation and immunological changes have also become evident, including mucosal immune activation with the infiltration of mast cells and T cells into the intestinal mucosa and systemic immune activation leading to cytokine imbalance. This is based largely on the observation that there is a higher prevalence of IBS in individuals with prior exposure to an acute enteric infection than in individuals who have not been exposed and a higher prevalence of IBS-like symptoms in patients who have irritable bowel disease in remission ,5,6,7.Studies have suggested that cytokines may act as potential aetiological factors in IBS. There are increased plasma or serum concentrations of the cytokines TNF-α, IL-6, IL-8 and lower concentrations of IL-10 in individuals with IBS as opposed to healthy controls ,8,9. ThiActinidia chinensis var. deliciosa ‘Hayward’) [® SunGold kiwifruit (Actinidia chinensis var. chinensis ‘Zesy002’) [Recent research has investigated the role of kiwifruit in gut health in both healthy individuals and those suffering from IBS complicated with constipation. Green kiwifruit (ayward’)  and goldesy002’)  were shoesy002’) ,16.In the present study, our primary hypothesis was that consuming whole fruit (including the skin) of SunGold kiwifruit would reduce the production and release into the blood stream of the inflammation biomarker C-reactive protein and the cytokines Il-6, IL-10 and TNF-α in individuals with IBS-C, and that this would be different when kiwifruit flesh alone was consumed. Our secondary hypothesis was that whole SunGold kiwifruit (flesh + skin) would reduce the gastrointestinal symptom scores  associated with IBS and this would be greater than for eating only the kiwifruit flesh.This study was conducted according to guidelines laid down in the Declaration of Helsinki and was approved by the New Zealand Human Disability and Ethics Committee (18STH63). The trial was registered with the Australia New Zealand Clinical Trials Registry (ACTRN: 12618000340235p) and all participants gave written informed consent. The design of this clinical study was a randomized cross-over trial of 16 weeks duration. The interventions were SunGold kiwifruit with and without skin. This kiwifruit has smoother and thinner skin, and so may be more acceptable to consumers than that of other kiwifruit varieties.The primary outcome for this study was a decrease in the concentration of pro-inflammatory cytokines  and an increase in the anti-inflammatory cytokine (IL-10) compared with the baseline period (baseline). The secondary outcomes were improvements in the number of complete spontaneous bowel movements (CSBMs), complete bowel motions (CBMs), spontaneous bowel motions (SBMs), bowel movement frequency (BM), number of strained bowel motions and stool form as well as gut symptoms and comfort using the GSRS and Birmingham IBS score compared with the baseline. A CSBM is when the bowel is emptied completely—that is, the person feels like no more stool needs to come out and the stool came out without having to take laxatives or use manual manoeuvres. A CBM is when the bowel is emptied completely—that is, the person feels like no more stool needs to come out. A SBM is when the person did not have to use laxatives or manual manoeuvres to get the stool out. A BM is the number of times the person passed a stool per week. A strained BM is whether the person had to push really hard to get the stool out. The Bristol Stool Scale (BSS) rates the stool from 1  to 7 .2. Fasting blood glucose concentration was required to be below 6.0 mmol/L.Forty adults were recruited to this study: 20 participants classified as having IBS-C (according to Rome III criteria) and 20 healthy participants. They were recruited through newspaper and radio advertisements, local district health boards and tertiary institution newsletters, existing participant databases and community advertisements. Participants were aged between 18 and 65 years and had a BMI of ≤35 kg/mHealthy participants had normal bowel habits and waist circumference . IBS-C participants were required to meet the criteria for IBS-C as described by the Rome III criteria . The IBSParticipants continued their habitual diet and lifestyle but excluded kiwifruit, high-fibre supplements and laxatives (except the prescribed rescue laxative if needed) for at least 2 weeks before starting this study and during the trial period. Following this 2 week washout, participants were randomized to one of the treatment groups: group 1 consumed three whole (skin and flesh) SunGold kiwifruit daily for 4 weeks and group 2 consumed three SunGold kiwifruit (skins removed) daily for 4 weeks. Participants were allowed to consume the fruit in any way they wished. Baseline measurements recorded for each participant were fasting blood glucose, C-reactive protein, kidney and liver function, acid/base balance, anthropometry , and a dietary eating habits questionnaire.After the 4 week intervention period, participants entered a 4 week washout phase, in which no kiwifruit were consumed. The participants then swapped to the opposite treatment for 4 weeks before the final washout phase of 2 weeks, in which no intervention was consumed. At the beginning and end of each phase, participants were asked to complete two validated questionnaires: the gastrointestinal symptom rating scale , which rated their gastrointestinal comfort, and the Birmingham IBS score . They prDuring the baseline and washout phases of this trial, patients suffering from constipation were able to use their regular treatments to relieve symptoms up until 1 week (7 days) before any intervention phase. Any treatments used in this period were required to be reported in the daily bowel habit diary. In the week (7 days) immediately before intervention treatments, participants were to refrain from the use of any form of treatment to prevent or relieve constipation other than use of the Bisacodyl suppositories, named as the rescue treatment for this trial. In circumstances where functionally constipated participants had no bowel movements (BM) for 3 days, or up to a maximum of 2 BM per week, they were instructed to use Bisacodyl suppositories (5 g) as the rescue laxative. The use of all rescue treatments was recorded in the daily diaries.The undiluted plasma samples were assayed with Human Inflammation Panel mix and match subpanels from BioLegend, San Diego, CA following the manufacturer’s instructions for analytes TNF-α, IL-6 and IL-10. Samples were analysed on a BD FACSVerse and data were processed using LEGENDplex™ Data Analysis Software (version 8).p = 0.05 one-tailed test, it will have approximately 70% power for a p = 0.05 two-tailed test. With n = 19, it appears that this study would have approximately 79% power for a p = 0.05 two-tailed test.A power calculation was undertaken to determine the number of participants that would be required to measure a statistically significant difference. To detect changes in the level of pro-inflammatory cytokines with 80% power and 5% significance and an increase in CSBMs of 1.5 per week in the treatment group compared with the control group with 90% power and 5% significance, 16 participants for each group were required to complete the trial. To account for an expected 25% dropout, 20 subjects were recruited for each of the two subject populations, or a total of 40 subjects. On examination of the protocol and power calculations, if this study was designed with 80% power for a The cytokine data were analysed using analysis of variance (ANOVA) with the factors group  and treatment  and person as a block term. Data for IL-6 and IL-10 were skewed; the variance was stabilised by adding 0.1 and then log transforming the data. The mean number of CSBMs, total bowel movements, CBMs, SBMs, bowel movements with straining and Bristol Stool Score were calculated for the 2 weeks of the lead-in period (baseline) and the last week of the two treatment periods. This number was multiplied by seven to give the measurement per week and the data were analysed using ANOVA, as described above for the cytokine data. The Birmingham IBS score was completed at recruitment and at visits after 2, 4, 6, 8, 10, 12, 14 and 16 weeks. This self-completed questionnaire consists of 14 questions based on the frequency of IBS-related symptoms using a 6-point Likert scale ranging from 1 = all of the time to 6 = none of the time. Following Roalfe et al. , the queForty-six individuals were screened and, of these, 40 subjects met the inclusion criteria for this study. Thirty-eight participants completed this study. Two participants withdrew from this study: one withdrawal occurred shortly after the first lead-in period due to health issues that were unrelated to the study products or interventions. One participant withdrew due for personal reasons .There were no significant differences in the baseline blood biochemistry as analysed by the Chem-20 panel and results were in reference ranges (data not shown). Self-reported compliance with consumption of the study products was 87% and the products were well tolerated, with minor side effects such as flatulence being the only adverse events relating to the study product. The demographics of the participants are shown in p < 0.001) when participants from both the healthy and IBS-C groups ate the kiwifruit and skin. While there was a significant treatment effect (p < 0.001), there was no group or treatment x group effect. For IL-6, there were no significant differences for treatment, group or treatment x group. Mean concentrations of IL-6 were variable and numerically higher in the blood for both treatments in the healthy group and were lower in the IBS-C group. For IL-10, there were no significant treatment or treatment x group effects but there was a significant difference between the healthy and IBS-C participants. Mean concentrations of IL-10 were higher and more variable in the healthy participants. In the IBS-C group, IL-10 was also significantly lower than baseline following the kiwifruit flesh-only intervention (p = 0.003). Further, C-reactive protein (CRP) was measured in the blood of the participants at baseline and at the end of each intervention phase (data not shown). There were no significant effects of either the kiwifruit flesh or kiwifruit flesh and skin treatments on the level of CRP observed in either participant group.The primary measurement of the trial was change in the concentrations of pro-inflammatory cytokines  and an anti-inflammatory cytokine (IL-10) in the blood compared with the baseline. Cytokines were measured at the end of each treatment period and in the washout period and are presented in Secondary outcomes for this study were changes in bowel movement frequency per week as assessed through the measurement of the number of complete spontaneous bowel movements (CSBMs), total number of bowel movements, complete bowel movements (CBMs), spontaneous bowel movements (SBMs), and the number of strained bowel movements. Additionally, changes in the form and consistency of bowel movements were measured through the daily bowel diaries and comparisons with the Bristol Stool Scale. p = 0.004) and no treatment x group effects. CSBMs increased in both participant groups after consuming the SunGold kiwifruit and kiwifruit and skin treatments. This was statistically significant for the IBS-C participants on the kiwifruit and skin treatment compared with baseline (p < 0.001). Similarly, the total bowel movements per week, CBMs, and SBMs were numerically higher for all the participants consuming the kiwifruit and skin treatment. A significant difference from baseline (p < 0.001) was observed in CBMs for IBS-C participants eating kiwifruit and skin (6.7 vs. 9.3). Strained bowel movements decreased for both participant groups eating both treatments . Bristol Stool Scale was significantly higher than baseline for the healthy group for both kiwifruit treatments. The proportion of bowel movements using laxatives are presented here but these data were not analysed, as 92% of the participants did not use laxatives. There were no significant interactions of treatment x group for the bowel frequency data.There were significant treatment effects for the frequencies of CSBMs, total bowel movements, CBMs, SBMs, strained bowel movements as well as the Bristol Stool Scale. There was a significant effect of participant group for the strained bowel movements frequency (p = 0.004). As expected, the baseline scores for the five domains were significantly higher at baseline for the IBS-C participants than the healthy participants. There was a significant treatment x group effect for the constipation domain (p = 0.018).Participants rated their degree of gastrointestinal discomfort using the gastrointestinal symptom rating scale (GSRS) comprising fifteen questions relating to gastrointestinal discomfort rated on a 1–7 scale (no discomfort to very severe discomfort). Following Svelund et al. , the GSRp = 0.030). The IBS-C participants had significantly higher scores for constipation and diarrhoea than the healthy participants at baseline. For the constipation domain, these scores remained significantly higher than the healthy participants after consuming both kiwifruit treatments. There was no significant treatment x group effect on these scores.The Birmingham IBS gastrointestinal symptom scores are shown in Participants were asked to complete a dietary eating habits questionnaire before the start of this study to assess background habits that may relate to health. The parameters measured related to general eating behaviours and practices. Individuals were asked to comment on their current body weight and weight control methods. Most participants (92%) reported that they have a good appetite and consume a varied diet (76%) at regular meal times . Only 53p = 0.0055). There were no significant differences in any of the dietary macronutrient or micronutrient intakes of the IBS-C participants for any treatment.Participant dietary intakes were assessed at the beginning and end of each intervention period. Analysing the dietary intake data without the kiwifruit included in the intake values , we founp = 0.0055). There were no group effects on dietary intake, but there were significant treatment × group effects for SFAs and magnesium. Compared with the baseline data, the healthy group participants had higher sugar intakes during the kiwifruit flesh treatment and higher vitamin C intakes during both kiwifruit interventions. The IBS-C participants consumed more available CHO, sugar and vitamin C during the kiwifruit flesh treatment, and higher intakes of sugar and vitamin C during the kiwifruit and skin treatment. The healthy participants consumed more SFAs during the kiwifruit and skins intervention than that measured at baseline.Analysing the dietary intake data including the kiwifruit , we foun® SunGold kiwifruit with and without skin. This was measured in a population of 19 healthy individuals and a population of 19 individuals with IBS-C. We found that the consumption of kiwifruit reduced the concentrations of the pro-inflammatory cytokine, TNF-α, but had no effect on IL-6 and IL-10 concentrations. We also observed that the healthy participants had higher concentrations of IL-10 than the IBS-C participants and that overall inflammation was not affected by kiwifruit consumption as measured by C-reactive protein.The primary outcome of the trial was to examine changes in the concentrations of pro-inflammatory cytokines  and anti-inflammatory cytokine (IL-10) in the blood following consumption of ZespriTNF-α is a pro-inflammatory cell signalling protein (cytokine) that plays a key role in the regulation of immune cells . TNF-α cComponents of gold kiwifruit, such as vitamin C, vitamin B9-folate, vitamin A, lutein and zeaxanthin, have also been implicated in supporting immune function  largely TNF-α and IL6 were not significantly different between the healthy and IBS-C participants at baseline. These are not well-established biomarkers and there are no published references that the authors are aware of that show a reference range for these cytokines in healthy individuals. The literature will state however that IL-6 and TNF-α are often significantly higher in people with a range of metabolic disorders. The baseline screening process of participants was limited to a Chem 20 panel and therefore may not have accounted for all underlying inflammatory conditions that could have caused variation leading to some confounding. This is a limitation of this study and more detailed screening of individuals at enrollment might explain this.The secondary outcomes of this study investigated the potential of consuming SunGold kiwifruit to relieve gastrointestinal discomfort and improve bowel motion frequency in individuals with IBS-C in comparison to healthy individuals. Complete spontaneous bowel movements (CSBMs) increased by more than 1 per week in the healthy participants when the skin was also consumed, and increased by more than 1 per week when the IBS-C participants consumed the kiwifruit flesh only and by more than 2 per week when consuming the flesh and the skin. An increase of greater than one complete spontaneous bowel motion per week is considered a clinically significant marker of improved bowel function in individuals symptomatic of constipation . In addiNormal bowel movement frequency and consistency are highly variable among individuals and populations due to several different factors including dietary habit, gut transit time and fibre intake. What is considered normal bowel movement ranges from three stools per day to three per week and some level of straining and incomplete evacuation is usual . In thisThe increase in CSBM and CBM frequencies in the IBS-C group with kiwifruit flesh and skin provides a dietary means to improve their daily well-being. On the basis of prior research , the effThe dietary intake data show that the participants in this study generally had a dietary fibre intake lower than the recommended daily intake, which is set at 25 g/day for women and 30 g/day for men . BaselinGastrointestinal discomfort arising from constipation manifests in several different symptoms, which can considerably affect an individual’s health . Using tDietary intake was also assessed at the end of each intervention period to monitor any changes to dietary intake over the course of this study. Data were analysed both with and without the contribution made by the trial product. There was no significant effect on total energy intake during the course of this study for either set of data. Overall, the participants reported lower total energy intakes with an average daily intake of approximately 7500 kJ total energy as opposed to the adult recommendations of 8000 kJ for women and 10,000 kJ for males . IncludiThe palatability and tolerability of the study products were rated as high by participants and no adverse effects relating to either of the study products were reported. Compliance was high, with 87% of participants reporting that they took the interventions as requested. This may be because kiwifruit is already widely used by constipated individuals in the general population and is anecdotally considered to be one of the most preferred interventions. As such, many of the participants were already aware that they could tolerate SunGold kiwifruit, which was highly appealing to the participants and well liked A further limitation of this study is that consumption of the skin of kiwifruit is not appealing to all individuals and compliance with this aspect may have been difficult to adhere to, although we did not have any reports of this in the trial.® SunGold kiwifruit daily in healthy individuals and individuals with IBS-C and aimed to show that consuming whole fruit (with and without the skin) of SunGold kiwifruit would affect the production of the inflammatory marker C-reactive protein and cytokines IL-6, IL-10 and TNF-α in healthy individuals and those with IBS-C. We found the consumption of kiwifruit reduced the concentrations of the pro-inflammatory cytokine, TNF-α, but had no effect on IL-6, IL-10 or C-reactive protein concentrations in the blood. Clinically significant changes were observed in the healthy and IBS-C participants of greater than 1 CSBM per week and was enhanced in the IBS-C study population, in which CSBMs increased to greater than 2 per week with the kiwifruit flesh and skin. Symptoms of gastrointestinal discomfort were also improved in the IBS-C participants, with a decrease in constipation.This randomized, controlled study examined the consumption of three ZespriThis study is the first to assess the impact of consuming the skin with the flesh for SunGold kiwifruit. We have demonstrated that consuming the skin of kiwifruit might have beneficial effects on gastrointestinal health that are not produced by consuming the flesh alone. Further studies in this area are required to expand on these findings using the same and other populations. This will ascertain whether there are similar effects in other populations and to investigate the mechanisms of action that drive these effects.


C. elegans morphogenesis, apical constriction in the retracting pharynx drives involution of the adjacent neuroectoderm. HMR-1/cadherin mediates this process via inter-tissue attachment, as well as cohesion within the neuroectoderm. Our results demonstrate that HMR-1 is capable of mediating embryo-wide reorganization driven by a centrally located force generator, and indicate a non-canonical use of cadherin on the basal side of an epithelium that may apply to vertebrate neurulation. Additionally, we highlight shared morphology and gene expression in tissues driving involution, which suggests that neuroectoderm involution in C. elegans is potentially homologous with vertebrate neurulation and thus may help elucidate the evolutionary origin of the brain.The internalization of the central nervous system, termed neurulation in vertebrates, is a critical step in embryogenesis. Open questions remain regarding how force propels coordinated tissue movement during the process, and little is known as to how internalization happens in invertebrates. We show that in  Xenopus this occurs simultaneously to lateral intercalation between the epithelial superficial cell layer and the deep cell layer  . In tetrll layer . In zebrll layer . More anll layer , supportXenopus , and the nerve ring ultimately encircles it. Signaling from the pharynx during morphogenesis regulates the anterior-posterior placement of the nerve ring . The bilhmr-1 . The pharyngeal cells have formed a two-sheet structure in the center of the head after invaginating during gastrulation. Head neurons envelop the pharynx at this stage  and a ubiquitous nuclear marker (histone::mCherry) to track the cell lineage  fail to do so at the completion of pharynx retraction   . These rhmr-1 phenotypes, we asked if HMR-1 is localized in such a way that it could mediate this attachment (xnIs96) which shows comparable localization patterns to the endogenous HMR-1 is strongly enriched at the interface between the basal pharyngeal surface and neuroectoderm during involution in a supracellular patch  less displacement measured across three embryos, while pharynx displacement is not significantly changed  in the organizer/node region along with the notochord.  occurs between their basal sides . Upon clWhile neurons originated in radially symmetric animal phyla such as Ctenophora and Cnidaria, they began to assemble into complex centralized systems such as nerve cords and brains only in Bilateria. The traditional view holds that central nervous systems evolved multiple times, but this idea has been challenged recently. Multiple studies have suggested that the brain only evolved once in the ancestral bilaterian (the ‘urbilaterian theory’ of brain evolution). These arguments are based on similarities in anatomy and gene expression between clades .C. elegans to refer to various aspects of neural morphogenesis such as the closure of the ventral neuroblasts (C. elegans and vertebrates could imply that the process in C. elegans is homologous to bonafide neurulation (and can be referred to as such). In return, it would further strengthen the current arguments for the urbilaterian brain theory  by providing shared functional cell biological aspects in morphogenesis, as well as shared essential gene expression in tissues involved in coordinated morphogenesis beyond the nervous system itself. Since these aspects have been well characterized in chordates, which are deuterostomes, the strongest evidence would come from a protostome, which C. elegans represents.The term neurulation has been used loosely in roblasts  or the groblasts . Howeverpha-4/foxa2+ precursor cells in the organizer region, along with another structure called the dorsal endoderm , BV308  III; xnIs96 [hmr-1p::hmr-1::GFP::unc-54 3'UTR + unc-119(+)]), BV727  I; zuEx2), DCR4318 , BV745 I; zuEx2).detailed . N2 Bris2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, per liter H2O), transferred to a second drop to dilute extra OP50 bacteria, and cut open to release embryos. Depending on the goal of the imaging experiment, one of three next steps would be taken. When embryos were to have their lineage analyzed using AceTree software, 4–10 embryos at 2–4 cell stage were transferred to a small drop (~1.5 µl) of M9 media mixed with 20 µm polystyrene beads on a 24 × 50 mm coverslip in and sealed with vaseline under an 18 × 18 mm smaller coverslip. When embryos were intended only for visual phenotyping, the same protocol was used, but up to 40 embryos were imaged at once. Lastly, when embryos were to be viewed through an anterior-posterior orientation, embryos were added to a larger (~4 µl) drop of M9 without beads, a thin layer of vaseline was deposited above and below the drop, and the 18 × 18 mm coverslip was added on top and further sealed with an extra layer of vaseline to allow uncompressed imaging.Preparation of embryos for live imaging was done as previously described . Gravid Images were acquired on either a spinning-disk confocal microscope comprising a Zeiss Axio Observer Z1 frame with an Olympus UPLSAPO 60XS objective, a Yokogawa CSU-X1 spinning-disk unit, and two Hamamatsu C9100-13 EM-CCD cameras, or an instant structured illumination microscope (Visitech iSIM) using an Olympus IX73 body, an Olympus UPLSAPO40XS objective and a Hamamatsu Flash 4.0v2 sCMOS camera. Z stacks composing 30 slices of 1 micron each were used for imaging of compressed embryos, and 36 slices of 2 microns each for uncompressed embryos. Embryos were imaged every 75 s (lineaging) or between 2–5 min (non-lineaging), sufficient time was allowed to enable visualization of terminal phenotypes for easier selection of mutants. Imaging exposure time was 150 ms per slice, with 568 nm laser exposure every timepoint and 488 nm laser exposure every 5 min regardless of imaging frequency.cnd1p::RFP marker in Fiji, identified as the edge of the ventro-lateral cnd1+ chunk closest to the midline. In cnd1+ ventro-lateral neurons begin moving to the midline; second timepoints were chosen based on when these neurons were the closest toward each other around the midline.Visual analysis of embryos was done using Fiji software . In FiguIn cnd-1 marker expression in the ventro-lateral portion of the embryo. Cnd-1 labels the majority of non-interface ventro-lateral involuting neurons. ROIs for each membrane segment were drawn while blinded to HMR-1 expression. These boundaries represent the membranes between cnd-1+ follower cells with a variety of terminal fates including, in order to create a representative sample of involuting neurons. This was repeated for six embryos. None of these regions overlap with the ventral interface hmr-1 patch, as cnd-1 does not label any interface neurons. Also, only boundaries between two cnd-1+ cells were selected.In cnd-1 expression and HMR-1 expression levels were measured in six embryos. As further control to show HMR-1 is elevated on the membrane boundary, adjacent cytoplasmic regions were selected and quantified for each measured edge.To demonstrate that HMR-1is genuinely enriched on the internal membranes of the cohesive neuron mass we contrast these measurements with background HMR-1 levels in the membrane of neuronal tissue that is not part of the cohesive mass of followers. The amphids sensory neurons are adjacent to the cohesive mass, but do not move w@Sandhiyaith them, and appear to have lower HMR-1 expression. Again five internal membrane regions within the amphid group were drawn based on cnd-1+ neuron involution (~50% of distance toward midline from starting location), 5 Z slices were chosen around the ROI for both amphid and involuting neurons separately, and a MAX projection was created in order to do a 2D quantification across multiple Z planes.Timepoints were chosen halfway through involution. This was according to pharyngeal retraction distance  and Statistical tests used were as follows: For automated tracking and rendering of cell movement paths, StarryNite software was used to segment RFP tagged nuclei  and AceTCell Motion: Nuclear positions for each desired cell were extracted over the selected time window. When cell divisions occurred during this window desired cells and their parents were considered equivalent and their positions concatenated. Cell position over time was smoothed using the MATLAB smoothdata function which computes a moving window average at an automatically selected scale see .Correlation Analysis: Positions were differenced and correlation of 3d directional velocity over all timepoints was computed for all pairs of cells see .A screen was performed for neurons that maintain close contact with the pharynx over time. Based on nuclear positions a Voronoi diagram approximation of cell–cell contacts was computed. The total of neuron-pharynx contacts over time was computed from this model, using only pharyngeal neighbors from the first timepoint. A threshold of 75% indicating a neuron with near constant contact with at least one pharynx cell was established and used to determine a set of leader neurons. The SMDD neurons, the most highly cohesive left/right neuron pair in the ventral patch of neurons, were selected for use in further computational analysis.C. elegans embryo via an adjustable 3D rendering .Thank you for submitting your article "Cadherin Preserves Cohesion Across Involuting Tissues During The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.As you will see below, each of the reviewers was very positive both about the importance of the problem and the overall quality of your data – but they all point out a few issues that you can address with some textual revisions . We are looking forward to seeing a properly revised version of the paper.Reviewer #1:Elucidating conserved molecular mechanisms that contribute to coordinated tissue movement during embryogenesis, ultimately resulting in the formation of centralized nervous system across phyla, is of major interest in developmental neuroscience. Details of how this happens in invertebrates are lacking. Prior studies suggest that various families of cell adhesion molecules such as cadherins are broadly expressed in multiple tissue types during this point in embryogenesis, suggesting that they could contribute to nervous system centralization.C. elegans nervous system is internalized via coordinated movement of the retracting pharynx and the neuroectoderm. They show that inter and intra-tissue cohesion is mediated by hmr-1, a homolog of vertebrate classical cadherin. HMR-1 transiently localizes at the interface between the pharynx and neurons, as well as between neurons, at the time of involution. The authors show that animals with a loss of function mutation in hmr-1 have reduced and disorganized involution of neurons although pharynx retraction is unaffected. The present study proposes that nervous system involution in C. elegans is analogous to neurulation in vertebrates based on conserved cellular events and shared gene expression, favoring the hypothesis that the centralization of the nervous system/origin of the brain, likely occurred once in evolution.In this manuscript, the authors use 3D time-lapse imaging and cell lineage tracing to show that the C. elegans nervous system and explicates novel and exciting details to establish the similarity of C. elegans nervous system involution to mammalian neurulation. The paper is suitable for publication in eLife after addressing the points below to strengthen the support for their conclusions.This manuscript adequately shows the role of hmr-1 in internalization of the Essential revisions:not cause the lethality associated with the canonical zu289 allele. Do these alleles fail to show any of the phenotypes shown for the zu248 allele? This is not a pure bookkeeping question, but it relates specifically to the notion, discussed by the authors, that it is the longer isoform that more resembles the N-Cadherin. Meaning, their molecular homology argument may be undermined and/or confirmed by examing an "N-Cadherin" (i.e. hmr-1b)-specific allele(s).1) It is not clear what kind of an allele the authors use. There does not appear to be any molecular information about the zu248 and this needs to be remedied. Is it a nonsense/putative null? Does it affect both splice forms or just one? The authors have to provide this information and if they do not have it, they need to sequence the allele. Along those lines, I'm curious to hear why the authors did not analyze existing mutant alleles (available at the CGC) that are specific to the longer isoform, gk3258  and a missense mutation from the million mutant project. Both alleles appear to 2) The expression/localization analysis of hmr-1 is a little unsatisfactory. The authors state that they used an hmr-1b protein fusion to the "hmr-1" promoter. However, it's explicitly known that with its two different isforms, the hmr-1 locus has TWO promoters. Which was used? Using either the 5' region of the hmr-1a or the hmr-1b isoform alone, is prone to yield an incomplete expression pattern. What can the authors say about that?Reviewer #2:C. elegans. Using automatic tracking they find that neuronal cell bodies undergo coordinated involution. This is likely to be driven by force generation in an internal organ (pharynx) that undergoes apical constriction. Expression studies and genetic loss of function tests support a role for the cadherin HMR-1 in this process, linking the pharynx shape change with neural and epidermal movements. The authors speculate that this process is homologous to neurulation in vertebrates.Barnes et al., investigate morphogenetic events underlying the arrangement of the nervous system in eLife readership. The main caveat would be whether the analysis has been taken to the level that would generally be expected in this journal. The main set of data that seem lacking are tissue specific tests of the requirement of HMR-1. Those experiments would perhaps be challenging but would take the story beyond a very detailed description of a hmr-1 mutant phenotype . The authors’ claims in the Abstract to have found roles for 'localized HMR-1' would be strengthened if the requirement for localized HMR-1 had been directly tested. If such experiments are not possible, the claims should be toned down to state that.This is a well written and technically very convincing set of findings. The description of neuron involution and the role of cadherin is novel and will be of interest to Reviewer #3:Summary:C. elegans embryo using a combination of lineage analysis, microscopy, and analysis of mutants. The authors present evidence that HMR-1/cadherin is required for the integrated morphogenesis of tissues derived from multiple germ layers. The authors advance our understanding of how HMR-1/cadherin facilitates involution of neurons in the anterior by identifying a supercellular accumulation of HMR-1 at the interface between neurons and the pharyngeal primordium.This manuscript investigates involution of anterior neuroblasts in the The imaging is high quality, the use of WormGuides and other existing lineage tracing data is creative, the use of "tadpole" tracings is visually effective, and the basic story seems clear. Two main issues should be addressed to improve the manuscript, along with some minor issues. If these issues can be addressed, then this is a valuable contribution.Essential revisions:1) Statistical analysis of quantification of HMR-1 accumulation: Figure 3C seems to depict a single embryo. In the absence of aggregate data, it is really impossible to assess these data. Similarly, Figure 4B is suggestive, but in the absence of quantification, it is hard to evaluate whether HMR-1 really does accumulate on a consistent basis between anterior neuroblasts in a statistically significant manner.2) Homology discussion: The discussion of homology is deeply problematic. This study cannot distinguish between "deep homology" involving convergent evolution using conserved molecular cassettes, versus "real" homology via shared common ancestors. The idea that there was an ancestral bilaterian possessing a single tissue attached to the neuroectoderm that gradually evolved independently in the deuterostome and protostome lineages is interesting but speculative. In Figure 5, the authors try to compare worms to several chordates. If a pan-metazoan conservation of process is being proposed, however, then it would be valuable to show another protostome (annelid? arthropod?). I recommend serious overhaul of this section, including trimming it to remove excessive speculation. As you will see below, each of the reviewers was very positive both about the importance of the problem and the overall quality of your data – but they all point out a few issues that you can address with some textual revisions . We are looking forward to seeing a properly revised version of the paper.We thank the reviewers for their thoughtful and constructive feedback, especially Dr. Chisholm for making the review process more transparent. As detailed below, we have addressed the suggestions with both textual revisions and additional data analysis. In terms of textual revisions, we revised the introduction on vertebrate neurulation to better reflect the nuances as well as the discussion on the evolutionary implication. In terms of additional data analysis, we added statistics in Figure 3 and Figure 4 as suggested. We also highlighted the major changes in the manuscript.Reviewer #1:Essential revisions:1) It is not clear what kind of an allele the authors use. There does not appear to be any molecular information about the zu248 and this needs to be remedied. Is it a nonsense/putative null? Does it affect both splice forms or just one? The authors have to provide this information and if they do not have it, they need to sequence the allele. Along those lines, I'm curious to hear why the authors did not analyze existing mutant alleles (available at the CGC) that are specific to the longer isoform, gk3258  and a missense mutation from the million mutant project. Both alleles appear to not cause the lethality associated with the canonical zu289 allele. Do these alleles fail to show any of the phenotypes shown for the zu248 allele? This is not a pure bookkeeping question, but it relates specifically to the notion, discussed by the authors, that it is the longer isoform that more resembles the N-Cadherin. Meaning, their molecular homology argument may be undermined and/or confirmed by examing an "N-Cadherin" (i.e. hmr-1b)-specific allele(s).zu248 is a loss of function allele that was reported together with zu389 in the paper that defined the hmr-1 gene in C. elegans . zu389 contains a nonsense mutation and is the most widely used. However, while using JJ1079 [hmr-1(zu389)/lin-11(n566) unc-75(e950) I], we lost the mutation twice during strain maintenance (after two separate requests from CGC). Worrying about unknown complications in the genetic background, we switched to using JJ1142 . The molecular lesion of zu248 has not been defined, but according to Costa et al., zu248 has comparable phenotypes as zu389.We thank the reviewer for pointing out the additional alleles. We also agree that sorting out the requirement of the long and short isoforms is a meaningful step towards a better understanding of the molecular mechanisms, together with some suggestions below. However, we feel that these deserve a systematic set of experiments of their own rights and therefore better suited for the next step. We recognize that it is premature to discuss the N- vs E-Cadherin without these experiments and removed it from the Discussion section.2) The expression/localization analysis of hmr-1 is a little unsatisfactory. The authors state that they used an hmr-1b protein fusion to the "hmr-1" promoter. However, it's explicitly known that with its two different isforms, the hmr-1 locus has TWO promoters. Which was used? Using either the 5' region of the hmr-1a or the hmr-1b isoform alone, is prone to yield an incomplete expression pattern. What can the authors say about that?xnIs96 [hmr-1p::hmr-1::GFP::unc-54 3'UTR] reported in . To quote Achilleos et al., “hmr-1::gfp was constructed by replacing the hmr-1 genomic sequence in plasmid pW02-21  with hmr-1 cDNA containing introns 2-4, inserting an ApaI site before the stop codon, and cloning gfp plus the unc-54 3' UTR into this site. HMR-1GFP produced from the hmr-1::gfp transgene xnIs96 is localized similarly to endogenous HMR-1 as detected by immunostaining, and rescues the strict embryonic lethality of hmr-1(zu389) mutants [1064 of 1485 (72%) were viable].” Our reference to hmr-1b in the text is a typo and we apologize for the oversight.The reporter we used is Reviewer #2:This is a well written and technically very convincing set of findings. The description of neuron involution and the role of cadherin is novel and will be of interest to eLife readership. The main caveat would be whether the analysis has been taken to the level that would generally be expected in this journal. The main set of data that seem lacking are tissue specific tests of the requirement of HMR-1. Those experiments would perhaps be challenging but would take the story beyond a very detailed description of a hmr-1 mutant phenotype . The authors’ claims in the Abstract to have found roles for 'localized HMR-1' would be strengthened if the requirement for localized HMR-1 had been directly tested. If such experiments are not possible, the claims should be toned down to state that.We agree that tissue specific experiments would be the ultimate test, but as mentioned above, we do not have good promoters to target the interface neurons. We revised the Abstract as suggested to tone down the statement.Reviewer #3:Essential revisions:1) Statistical analysis of quantification of HMR-1 accumulation: Figure 3C seems to depict a single embryo. In the absence of aggregate data, it is really impossible to assess these data. Similarly, Figure 4B is suggestive, but in the absence of quantification, it is hard to evaluate whether HMR-1 really does accumulate on a consistent basis between anterior neuroblasts in a statistically significant manner.For Figure 3C, we quantified additional embryos (n=5) and revised the panel. For Figure 4B, we made quantitative measurements (6 embryos and 5 neurons/embryo) along with controls, and added a new panel .2) Homology discussion: The discussion of homology is deeply problematic. This study cannot distinguish between "deep homology" involving convergent evolution using conserved molecular cassettes, versus "real" homology via shared common ancestors. The idea that there was an ancestral bilaterian possessing a single tissue attached to the neuroectoderm that gradually evolved independently in the deuterostome and protostome lineages is interesting but speculative. In Figure 5, the authors try to compare worms to several chordates. If a pan-metazoan conservation of process is being proposed, however, then it would be valuable to show another protostome (annelid? arthropod?). I recommend serious overhaul of this section, including trimming it to remove excessive speculation.We made significant revision on this part of the manuscript as suggested. In the Results section, we reduced the section so that it only describes the observed similarities (the facts). In the Discussion section, we turned the speculations into what could be examined in both deuterostomes and protostomes to test homology vs convergent evolution.