While awaiting the COVID-19 vaccines, researchers have been actively exploring the effectiveness of existing vaccines against the new virus, among which the BCG vaccine  receives the most attention. While many reports suggest a potential role for BCG immunization in ameliorating SARS-CoV-2 infection, these findings remain controversial. With country-level COVID-19 outbreak data from Johns Hopkins University Coronavirus Resource Center, and BCG program data from World Atlas of BCG Policies and Practices and WHO/UNICE, we estimated a dynamic model to investigate the effect of BCG vaccination across time during the pandemic. Our results reconcile these varying reports regarding protection by BCG against COVID-19 in a variety of clinical scenarios and model specifications. We observe a notable protective effect of the BCG vaccine during the early stage of the pandemic. However, we do not see any strong evidence for protection during the later stages. We also see that a higher proportion of vaccinated young population may confer some level of communal protection against the virus in the early pandemic period, even when the proportion of vaccination in the older population is low. Our results highlight that while BCG may offer some protection against COVID-19, we should be cautious in interpreting the estimated effectiveness as it may vary over time and depend on the age structure of the vaccinated population. COVID-19 has a substantially higher morbidity rate and mortality rate for people 60 years and above3 and for people with chronic conditions including pulmonary disease, hypertension, diabetes mellitus, and cardiovascular disease4. Treatment options are limited although there has been recent progress in both therapeutic options and vaccine development recently7.The first confirmed case of COVID-19 (Coronavirus Disease 2019) was reported on January 2, 2020 in Wuhan, a metropolitan area in central China, and the causative agent of this outbreak was identified as a new strain of coronavirus, designated SARS-CoV-2. As of November 11, 2020, there are more than 50 million confirmed cases, 1.2 M deaths, and the numbers continue to climb. COVID-19, the disease caused by SARS-CoV-19, is highly infectious, with an R0 estimate between 0.4 and 4.68. The bacille Calmette-Guérin (BCG) vaccine was introduced in the 1930s and used to prevent tuberculosis (TB), which is one of the most prevalent diseases in developing countries10. In the 1970s, WHO launched massive immunization programs in developing countries, leading to substantial increase in BCG coverage among 1-year-olds: BCG coverage rate increased from 9% to 80% in African countries, and from 20% to 90% in European countries between 1980 and 201911. Though BCG vaccine was first developed as a prevention for tuberculosis, recent studies have found that BCG vaccine has nonspecific benefits in training the innate immunity through bestowing a type of immunological memory on innate cells14. This “trained innate immunity” has been shown to be protective against viral infections16.As targeted COVID-19 vaccines are being developed, researchers have been actively exploring the protective effects of existing vaccines, among which the BCG vaccine  has received much attention following observation of a coincident relationship between severity of COVID-19 outbreaks and the level of BCG vaccination in one country20. While the negative correlation between BCG vaccination and mortality and morbidity rates is observed23, studies like Hamiel et al.17 and Lindestam Arlehamn et al.20 reported no significant protection of BCG vaccination. Escobar et al.19 compared the COVID-19 death records in the early stages of the pandemic  from countries with a national BCG vaccination program to countries without, and observed significant BCG protective effect on COVID-19 mortality. Yet other studies found no significant correlation between COVID-19 mortality and BCG vaccination. For example, Lindestam Arlehamn et al.20 failed to validate the protectiveness of BCG using updated data from August 2020, and Patella et al.24 do not support significant differences in the COVID-19 incidence between “BCG vaccinated” and “BCG unvaccinated” physicians in Italy. This study aims to explain the conflicting findings from existing studies by examining the impact of BCG vaccination during different stages of pandemic.The protective effect of BCG vaccination against COVID-19 is under debate19 in that the protective effect of BCG vaccine, at least to some extent, existed during the early stage of the pandemic. However, we do not see strong evidence for protectiveness during the later stages, which is broadly in line with Lindestam Arlehamn et al.20. Our second explanation is that the age structure of the BCG “vaccinated” population matters. We see that a higher proportion of vaccinated young population helps to protect the society against the virus, while the proportion of vaccinated old population seems to show limited effects.We construct a dynamic model to investigate the effect of BCG vaccination, allowing its protection to vary across time during the pandemic. Our results reconcile the existing different opinions towards the protection of BCG against COVID-19 robustly in a variety of samples and model specifications. Our first explanation for the discrepancy between the controversial findings is that protectiveness of BCG varies over time during the pandemic. We observe a notable reduction in COVID-19 mortality in countries with a national BCG vaccination program, compared to countries without such a program during the second and the third month since the first death confirmed. This echoes Escobar et al.25. The dataset includes the daily numbers of confirmed COVID-19 cases and the numbers of COVID-19 deaths reported by 188 countries between January 22, 2020 and October 15, 2020. Since the timing of the outbreak differs from country to country, we construct a dataset that tracks each country from the day before the first confirmed death and up to 210 days after the “onset.”We obtain country-level COVID-19 outbreak data from Johns Hopkins University Coronavirus Resource Center26 provides country-level beginning and ending years for a national BCG vaccination program; we reviewed literature and governmental websites to fill in missing information whenever possible. Second, WHO/UNICEF provides Estimates of National Immunization Coverage (WUENIC) monitoring the coverage of fourteen vaccines during 1990–2018 (latest update July 1 201911). See Supplementary Table Two types of data are considered. First, World Atlas of BCG Policies and Practices27, which contain publicly available information about government responses coded into eight indicators for containment and closure, one indicator for health system (“public info campaigns”), two economic indicators (“economic support” and “debt/contract relief for households) and three indicators for health measures . Note that one country’s public health system does matter in controlling the pandemic and may also influence the implementation of the BCG vaccination program, we control for the government response index in our model to capture the time-varying confounders in the public health system. An alternative index available in Hale et al.27 is the policy stringency index. We prefer the government response index to the policy stringency index because the latter does not take health measures such as testing policy and facial coverings into account. We present our main results using the government response index and also use the policy stringency index which counts eight containment policy indicators and the indicator for health system for robustness check.Government response data are obtained from Hale et al.A country is excluded from our analysis if it has 0 confirmed cumulative death as of the last day in our analysis October 15, 2020) and has missing value in the government response index data. See Supplementary Tables , 2020 anThree regression models were employed to estimate the impact of BCG vaccination while controlling for possible confounding factors. To mitigate confounding effects, we incorporate the government response index to capture the governmental efforts, country fixed effect which helps to control for time-invariant country characteristics, and day fixed effect (since the first confirmed death) to absorb the potential confounders along with the timing of outbreak.19, which compares the COVID-19 mortality data at specific times of each country epidemic since the first death. Adjusting time according to the first day of death allows us to “align” the timelines of epidemics across countries and examine the mortality data across countries that are in the same pandemic stage. Our three models estimate the impact of BCG vaccinations per day (starting from day 31) with reference to the first month after the first death (day 0 to day 30).Model 1 estimates how current and past national BCG vaccinations affect the number of cumulative deaths per day at country level:In the 1970s, WHO launched massive immunization programs, including the BCG vaccination, in developing countries. The BCG vaccination was administered on newborns and young children. As a result, people under 50 years old in countries that have undergone the massive immunization program have a much higher vaccination rate than those 50 years and older. Model 2 estimates the impact of BCG coverage in younger and older populations in the country.Model 3 estimates the impact of BCG vaccination as an average coverage between 1990 and 2018. As shown in Supplementary Fig. In comparing across countries, we define day 1 for each country as the day the first confirmed death is reported and day 0 for the last day prior to the death reported. This is similar to the approach in Escobar et al.All three models include fixed effect terms by country  to check the robustness of our results. First, a country is excluded if its population is less than 1 millionWe summarize our findings as follows. See Table We first present the results of Model 1 that summarize the average effect of BCG vaccination program on the number of total deaths. Our dataset consists of summaries from 150 countries after filter for sample size and quality (see “Methods” section). Among these, 122 countries currently have BCG vaccination for the population, 20 countries who used to have BCG but do not currently have a national vaccination program, and 8 countries had never mandated BCG vaccination. Figure We introduce Model 2 to further examine the effect of protection by past vaccination. A surprising finding is that BCG shows a stronger protection during days 31–60 since the first death was reported relative to days 0–30 if a country has higher coverage for the population under 50 years old. As shown in Fig. In Table Model 3 analyzes how BCG coverage as a proportion protects against COVID-19. We find borderline significant effects of BCG coverage as shown in Fig. In all models, we find that the effect of BCG is stronger on the number of deaths during the early stage of the pandemic. Furthermore, we explore the robustness of our results by re-estimating all the three models with an alternative time-varying policy stringency covariate and different estimation samples. First, we replace the government response index with the policy stringency index. Second, we re-run the models in samples with different specifications. The results are all shown in Supplementary Tables After observing the dynamics in BCG effects, we further estimate Eq.  to shed entclass1pt{minimaMycobacterium tuberculosis32. While the primary mechanism of action of BCG, a mycobacterium, was originally thought to be via cross reactivity, it has also been observed that BCG has a broader effect33, that of “priming”, or what has recently been called “training” of the innate immune response19. This process stimulates the non-specific innate immune pathways to target other infections and even solid tumors in some cases34. Immunologists have long known about so called adjuvant effects of many bacterial species, including BCG, and have taken broad advantage of this phenomenon to enhance innate immune responses in a variety of settings including inclusion in autologous tumor vaccines35. However, the recent observations that this immune modulating effect can be long lasting, via trained immunity, provides an interesting mechanism that may explain some of the observations described here and may form a basis for potential COVID-19 protection in those individuals vaccinated with BCG, even years ago36. This observation motivates recent studies on the protectiveness of BCG vaccination against COVID-19. Yet the evidence is not conclusive.From the earliest history of BCG vaccination as a prevention for tuberculosis, it has been observed that BCG elicits a variety of effects that impact the immune system beyond conferring specific immunity to Our results reconcile the existing different opinions towards the protection of BCG against COVID-19. We show that the protectiveness of BCG vaccination against COVID-19 is strongest during the very early stage of the pandemic, while as the government begins to implement social distancing policy, and other actions, the effect of BCG vaccination gradually disappears in the later stages. Although the old vaccines may protect from the virus, the self-protection actions as well as compliance to social distancing policies are still the most protective measures in absence of an effective COVID-19 vaccine.19, variability of test rates28, country population size21, income (socioeconomic wellbeing and level of development) of countries8, levels of social isolation policy37 and most importantly, the timing of the outbreak in each country38. We draw on models involving country and day fixed effect to account for country heterogeneities.Our study differs from previous studies in several aspects. A major difference is that previous studies do not fully address unobserved confounding factors, which may lead to a false sense of protection of BCG, even after carefully filtering out seemingly inhomogeneous countries based on human development indexSecondly, analyses using mortality data at one specific time point during the pandemic render an aggregate effect in a specific time frame, while it is possible that the protection effect, if exists, varies across different stages of the pandemic. Our time-varying estimates of BCG protectiveness provide sufficient insights into dynamic interactions between BCG vaccination and public health policies. As the results showed, the protection effect is stronger at the beginning of the pandemic, suggesting that, without sufficient government interventions or public awareness, the BCG vaccine yields a stronger protection.39. Several factors, such as medical infrastructure, testing capacity, number of asymptomatic cases and criteria for testing could lead to the undertesting and underreporting of cases41. Therefore, mortality may serve as a better indicator for COVID-19 spread41, when high-quality cases data are not available. Future studies may incorporate corrected death or cases based on advanced projection models43.Our analysis focuses on COVID-19 mortality, since outcomes like reported cases are not as reliable as reported mortality. As suggested in literature, the number of confirmed cases could suffer more from underreporting41, testing ability and medical resource availability may evolve during the pandemic thus affecting our estimations. Government response index may partially capture the trajectory of medical resource changes, but a better measure would be desired.Although the country fixed effect in our models can net out any country-level time-invariant factors such as medical infrastructure and healthcare access at the initial stage of the pandemicAnother limitation of our analysis is that we did not take into account factors that will affect the rate of disease transmission, such as population density and traffic flow. As we observed, the severity of COVID-19 varies across urban and rural areas in a country. Since we rely on BCG program variations at country level, the population density variation within a country will be averaged out, thus possibly diluting the effect of BCG vaccine. If we can distinguish between samples in urban and rural areas and analyze them separately, then we might be able to investigate whether the effects of BCG vaccine vary across urban and rural areas. Comparing metropolitans in different countries  may shed light on the protective power of BCG against COVID-19 in an environment with a high volume of social activities and vice versa.We recognize only clinical trials can conclusively confirm the protective effect of BCG vaccination. However, the current empirical evidence indeed sheds light on the benefit of a comprehensive vaccination program. It is intriguing that a targeted vaccination such as BCG may train our immune system to be protective against other diseases. Our study shows the protective effect is most significant for individuals 50 years or younger, but less so for those older than 50. This provides new research directions for the perplexing phenomenon of many adverse outcomes in the younger low-risk population. When the next inevitable outbreak occurs, we may not have an old vaccination to find new life in that pandemic, but the lesson we learn says it is the integrated health system that shields us from the danger.Supplementary Figures.Supplementary Information 1.Supplementary Information 2.


The purpose of this randomized controlled study was to evaluate the clinical efficacy of a novel oral spray containing resveratrol (RV) in controlling bacterial biofilm and gingival inflammation in early childhood. RV, a natural polyphenol, known for its anti-inflammatory and anti-infective activities, was included in a nanovector of 2-hydroxypropyl-beta-cyclodextrins (HPβCD) to improve its bioavailability. A total of 64 children between two and five years of age with plaque-induced gingivitis were randomly included in two equal groups. Both groups were enrolled in a mechanical plaque control program for a period of four weeks, while the test group was also instructed to use the RV-HPβCD mouthwash (in spray formulation) once daily, after toothbrushing. All children underwent three oral hygiene motivation sessions, 14 days apart, during which the full-mouth presence of bacterial plaque, gingival inflammation, dental stain and salivary pH were recorded. At two-week appointment, they also received professional plaque removal. The use of RV-based oral spray significantly reduced the amount of dental plaque and the percentage of bleeding sites and improved salivary pH compared to the control group at both two- and four-week examinations. Based on these promising results, the local delivery of RV-HPβCD via oral spray could enhance the control of dental biofilm in early childhood, when antiseptic mouthwashes are not recommended. Despite the great commitment of parents, oral hygiene in early childhood has proved to be inadequate in many cases, increasing the risk of dental decay in primary dentition . A recenAlong with caries, bacterial biofilm is an essential etiological factor in gingival inflammation . AlthougIn the last decades, many natural compounds have been identified as molecules characterized by anti-inflammatory/antibacterial activity without local or systemic toxicity on mammalian tissues. Among them trans-resveratrol , a non-flavonoid polyphenol belonging to the class of stilbenes, is one of the most studied and promising compounds . It is fUnfortunately, clinical applicability of RV is limited by its chemical-physical properties, mainly related to its low solubility in water and extensive systemic metabolism leading to a rapid inactivation. Indeed, these aspects affect negatively its bioavailability and efficacy . RecentlThe aim of this randomized controlled clinical study was to evaluate the efficacy and acceptance of a novel RV-HPβCD complexation mouthrinse in oral spray formulation in controlling bacterial biofilm and gingival inflammation in early childhood.The children included in this randomized, two parallel arms clinical study were consecutively selected among those referred to the Section of Pediatric Dentistry, C.I.R. Dental School, Department of Surgical Sciences, University of Turin  from September 2019 to February 2020. The study was approved by the Institutional Ethics Committee of the “AOU Città della Salute e della Scienza”, Turin, Italy  and was conducted according to the Helsinki Declaration. Parents/guardians of each recruited child signed informed consent.Children of both genders were screened for enrolment according to the following criteria: (i) diagnosis of plaque-induced gingivitis with full-mouth bleeding score (FMBS) > 10% ; (ii) agw/v, purchased from Fagron, Bologna, Italy) and HPβCD  were mixed with deionized water in a clean and sterile recipient by means of a dissolver operated to shake at low speed without swirling for 5 h.RV-HPβCD complexation has already been described ,19. The w/v. The choice to use the strawberry aroma was based on the results obtained in a preliminary study where a sample of children preferred it over the flavors tested (data not shown) and on the findings from previous published reports [Then, a strawberry liquid aroma  was added in an amount (weight/volume) of 0.1%, together with deionized water to reach 100%  reports ,23. The Children were distributed randomly in two balanced groups using a computer-generated table by an operator not involved into the trial. The test group underwent toothbrushing and daily supervised application of the RV-HPβCD oral spray and the control group only toothbrushing. To conceal assignment, forms with the treatment modality were put into sealed and opaque envelope and provided to a dental hygienist.A sample size of 27 children per group was calculated to detect a minimum difference of 10% in FMPS between the groups with an alpha set at 5% and a power of 80% based on a previous study that found FMPS change of 32.0 ± 16.0% after mechanical plaque control . An addiA single specialist in pediatric dentistry assessed the oral status of the participants. All parents/guardians and all children aged 4 to 5 years of both groups received instruction by an experienced dental hygienist to perform the roll technique toothbrushing twice a day with a soft bristle-small head toothbrush. Oral hygiene procedures in children aged 2 to 3 years were performed by the parents, while those aged 4 to 5 years carried out the procedures by themselves, under parental supervision . The manAt baseline, 2-week and 4-week examinations clinical parameters were recorded by a blinded and experienced examiner using a 1-mm marked manual periodontal probe . Plaque accumulation and gingival inflammation were assessed dichotomously  at 6 sites per tooth and expressed as percentage of tooth surfaces harboring plaque (FMPS) and bleeding on gentle probing (FMBS) . The preAt the same assessment time points, unstimulated whole saliva samples were collected under standardized conditions  for the quantification of salivary pH using a chair side kit with a measuring range from 5.0 to 7.8 .At the end of the study, parents of test children were asked to complete a questionnaire on the acceptability of the oral spray. Furthermore, children of the test group rated their taste perception using a 3-point scale  presented with emoticons to improve their comprehension.Data were entered into a database and analyzed using statistical software . All children had coded identification so as they could not be identified. Primary outcome variable was change in FMPS from baseline to 4-week examination. Secondary variables were changes in the other clinical parameters and in salivary pH.t test or the Mann-Whitney U-test for quantitative variables. The Bonferroni correction was applied for multiple comparisons.Continuous data were examined for normality with the Kolmogorov-Smirnov and Shapiro-Wilk tests. To test the effect of treatment and time on quantitative variables within each experimental group the repeated measures analysis of variance or the Friedman test were used as appropriate, followed by post hoc tests to explore differences between time points. The statistical significance of the differences between the groups was evaluated using the Chi Square test for qualitative variables and unpaired p was <0.05.A secondary analysis was performed to explore the impact of the toothbrushing behavior  on the outcomes using the analysis of variance or the Kruskal-Wallis test for between group comparison, and the repeated measures analysis of variance or the Friedman test for intragroup analysis. Differences were considered statistically significant when As shown in The distributions of gender and age for each group are presented in The comparative analysis between test and control groups over the study period in relation to clinical parameters is shown in p < 0.001). After both 2 and 4 weeks of daily use, the RV-HPβCD group had significantly less plaque than the control group with reduction of 40–50%. A similar effect was observed for FMBS values which decreased more in the test group at both assessment time points (p < 0.001).Both treatments were associated with statistically significant reduction in the percentage of sites with plaque and in the degree of gingival inflammation with a decrease from baseline to 4-week follow-up , irrespective of toothbrushing behavior. No differences were observed in the control group between 2–3- and 4–5-year-old children. When considering clinical changes within each group, statistically significant improvement in FMPS was observed between baseline and 2 weeks and between 2 and 4 weeks in all groups. Conversely, in the test group, the oral spray application was more effective in reducing FMBS in the 4–5-year-old children between the 2- and 4-week examinations than in children aged 2 to 3 years. The same trend was observed in the control group but between baseline and 2 weeks.Clinical data were stratified based on the age of the enrolled children to explore whether there was any association between the use of the RV-HPβCD spray and the toothbrushing behavior . From thp = 0.004), and then remained nearly unchanged. In contrast, the combined mechanical and chemical plaque removal was effective in progressively increasing pH value, which reached the neutrality between baseline and 2 weeks (p = 0.010) and further slightly increased from the 2- to 4-week examination (p = 0.031).As far as salivary parameters were concerned , pH valuThe observed changes were not dependent on the age of the children: Higher pH increase was obtained in the test group as compared to the control group irrespective of the toothbrushing habits at both 2 and 4 weeks .No adverse reactions affected the hard and/or soft tissues during the 4-week study period. The 2- and 4-week intraoral examinations found no tissue changes and no teeth discoloration using the RV-HPβCD spray.All parents reported no difficulty in using the spray and all the children tolerated the agent well. The taste of the spray was well accepted by all of them, regardless of the age group. Based on the emoticon scale, all children rated the spray as good (smile face).Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans [Streptococcus mutans [Plaque-induced gingivitis and dental decay are a challenge problem in preschool children who often show inadequate quality of plaque control. In case of poor oral hygiene, primary teeth are more prone to develop carious lesions than permanent teeth due to difference in enamel structure , with pecomitans ,30 and Ss mutans .Here, we investigated the effect of RV nanoencapsulated with HPβCD (to improve its efficacy) on the existing plaque during the first two experimental weeks and on the rate of de novo biofilm accumulation on plaque-free teeth during the last two weeks of the study. The RV-HPβCD oral spray had a statistically and clinically significant effect on the amount of plaque after two and four weeks of daily use, once per day, and twice as effective as toothbrushing alone. FMPS decreased by 38.0% in the test group compared to 19.1% in the control group at two-week examination and FMPS amounted to 9.8% versus 25.8%, two weeks after the polishing session. The trend of FMBS paralleled that observed for FMPS values. This could be due to the remarkable pleiotropic activities  of RV in the oral cavity . It effiThe current findings further support the utility of chemical plaque control in addition to daily oral hygiene measures in childhood. Unfortunately, it is difficult to find products suitable for pediatric patients. Chlorhexidine (CHX) is considered as the “gold standard” of oral antiseptics for its antimicrobial activity, low toxicity and high substantivity . NeverthTherefore, a mouthwash containing a natural compound in a spray formulation seems to be a good choice: Oral spray formulation has been recommended for physically and mentally challenged patients and institutionalized elders, but it could represent an interesting possibility for healthy preschool children, too .It is meaningful that a significant clinical improvement was also observed after use of daily toothbrushing only, and this underlines the importance of oral health education either for children or parents in adopting appropriate brushing technique and frequency. It is evident that at least a temporary change in oral hygiene habits occurred, but repeated motivation sessions are needed to maintain improved gingival conditions over a long time period ,36. As rWhen data were stratified on the age of the enrolled children, the efficacy of the spray was found not dependent on the toothbrushing behavior. However, among children under three years old, the spray was effective in controlling gingival inflammation after four weeks of daily use. In the control group FMPS values were higher among children aged 2–3 years than in the 4–5 years group at both two-week and four-week examinations even if the difference did not reach statistical significance, probably due to the smaller number of children under three years of age.Streptococcus mutans [Concerning changes in salivary pH, the test group experienced a more marked tendency to neutral values than that observed in the control group. Consistent with the abovementioned findings, there was no significant difference in this parameter within each subgroup based on child’s age and toothbrushing behavior. Although the literature reveals controversial results for the associations between salivary parameters and dental decay, the research reports a positive correlation between pH value, prevalence of caries and salivary counts for s mutans ,40,41, es mutans . Therefos mutans .In order to monitor tooth staining and taste alteration as well as potential adverse effects on oral tissues as a result of RV daily use, intraoral examinations were performed during every visit. No patient experienced areas of staining on the anterior teeth after 4 weeks of using the spray and/or local effects. Parents reported that their children did not show resistance to the use of the product, due to the spray formulation so they did not have to discontinue its application, but they were willing to use it for longer periods.Taste is regarded as the main feature determining acceptability of a liquid medicine in pediatric populations . We usedStreptococcus mutans. It is also important to consider that RV application can interfere with the ability of Streptococcus mutans to produce an acidic environment inhibiting its cariogenic virulence [A limitation of this study is not having used a placebo spray in the control group. The bias introduced by the placebo effect in clinical trials has been previously documented ; so it iirulence .Oral sprays may have potential as an alternative delivery method in early childhood and natural molecules may be more appropriate to be used in these products than chemical compounds. An oral spray application with RV-HPβCD once a day resulted in marked decreases in dental plaque level and gingival inflammation, but also in neutral pH value, and this may be considered a promising new approach to improve oral health in preschool children. Long-term controlled studies on the effectiveness of RV oral spray are needed and encouraged.


Xenopus laevis larvae. We found that while Smoothened (Smo) activity is essential for proper spinal cord and skeletal muscle regeneration, transcriptional activity of the canonical Hh effector Gli is repressed immediately following amputation, and inhibition of Gli1/2 expression or transcriptional activity has minimal effects on regeneration. In contrast, we demonstrate that protein kinase A is necessary for regeneration of both muscle and spinal cord, in concert with and independent of Smo, respectively, and that its downstream effector CREB is activated in spinal cord following amputation in a Smo-dependent manner. Our findings indicate that non-canonical mechanisms of Hh signaling are necessary for spinal cord and muscle regeneration.Inducing regeneration in injured spinal cord represents one of modern medicine’s greatest challenges. Research from a variety of model organisms indicates that Hedgehog (Hh) signaling may be a useful target to drive regeneration. However, the mechanisms of Hh signaling-mediated tissue regeneration remain unclear. Here, we examined Hh signaling during post-amputation tail regeneration in  Injury in low regenerative capacity tissues such as spinal cord represents a persistent challenge in modern medicine, with millions of cases of spinal cord-related disability worldwide . There a2+-Ampk axis  and before metamorphosis (stages 49–54), with a refractory period during stages 47–48, during which regeneration is blocked , spinal cord , and skeletal muscle  in the regenerate and vicinity , and constitutive expression of near-infrared fluorescent protein as a normalizing factor for reporter expression  and the amputation plane (X), we find that relative to 0 hpa , by 4 hpThese data indicate that amputation rapidly induces widespread inhibition of canonical Gli-dependent signaling.X. laevis larvae  which binds to and disables the matched Gli2 morpholino (Gli2-MO), allowing UV-dependent uncaging of Gli2-MO at later developmental periods . ExposurThese findings indicate that canonical Hh signaling is, for the most part, not necessary for spinal cord and muscle regeneration, and suggest that Smo-dependent regeneration recruits a non-canonical signaling pathway.2+ activity is associated with both muscle regeneration  and injected (350–400 units) with human chorionic gonadotropin to induce egg laying. Eggs were squeezed into 1× Marc’s Modified Ringer solution , fertilized with minced testis, and allowed to develop at a controlled temperature in 10% MMR. Mixed sex specimens were used at either 12–18 h (Nieuwkoop-Faber [NF] stage 11–16 embryos) or 60 h (NF stage 39–40 larvae) post fertilization.2O; Calbiochem 566660), 10 μM GANT61 , and/or 10 μM KT5720 . For multi-day treatments, solutions were replaced daily.NF stage 39–40 larvae were anesthetized with 0.02% tricaine methanesulfonate  with or without the specified drugs until non-responsive. Larvae were then amputated under a dissection stereoscope using a scalpel blade at approximately 1/4 of the length from the tail tip, where the tail begins to taper. The anesthetic was then washed out and the amputated larvae were incubated at 21–23°C in 10% MMR with vehicle or 10 μM cyclopamine , 20 μM vismodegib , 1 μM SAG  or the furthest posterior 12/101+ muscle cells (muscle outgrowth). Total muscle regeneration was defined as the volume of muscle cells in the regenerate posterior to the amputation plane, not including dorsal/ventral outgrowth of muscle from the intact chevrons in SAG treatment conditions. Sox2 and P-H3 counts were performed respectively on spinal cord and total regenerate posterior to the amputation plane. All these measurements were done using the image analysis software Imaris.4) overnight at 4°C. Samples were bleached overnight in H2O2/Dent’s fixative, then permeabilized with 0.5% Triton X100 in 1× PBS (PBT) and blocked in 0.5% PBT + 2% BSA. Primary and secondary antibody incubations were performed in 0.1% PBT overnight at 4°C. Samples were washed in 0.5% PBT, mounted in 90% glycerol in 1× PBS, and imaged within 1–4 days. Antibodies were obtained and used as follows: 1:300 Sox2 , 1:100 12/101 , and 1:400 phospho-Serine10-histone-H3 . All donkey secondary antibodies were used at 1:1500–1:2000 from Thermo Fisher: anti-Goat-Alexa-647 , anti-Goat-Alexa-594 , anti-Mouse-Alexa-488 , anti-Rabbit-Alexa-594 , and anti-Rabbit-Alexa-647 . Phospho-CREB (P-CREB) immunostaining required conditions as follows: samples were fixed as above for 3 h at 4°C with gentle agitation, then washed in 0.1% PBT, dehydrated in methanol, and kept at −20°C overnight in methanol. Samples were then bleached at room temperature for 3 hr, rehydrated, permeabilized as above, blocked in 10% BSA + 1.5% normal donkey serum, and placed in primary antibody solution: 1:400 Sox2, 1:100 12/101, and 1:1500 P-CREB  for 4–5 days at 4°C, then treated as above for secondary antibody incubation, washes, and mounting.Larvae were anesthetized in 0.02% TMS in 10% MMR, then fixed in 3.7% formaldehyde in 1× MEMFA saline  at 4°C and for 2 h at room temperature with AlexaFluor conjugated secondary antibodies (1:300) in 1% BSA, 0.1% Tween in PBS. Immunostained samples were mounted and imaged in an epifluorescence microscope.Larvae were fixed in 4% PFA for 1 h at room temperature. Samples were then processed for paraffin embedding and sectioned transversely in 10-μm-thick sections. Slides were processed for immunostaining by incubating overnight with the primary antibodies Sox2 (1:300) and 5E1  and matched Gli2-PhotoMO (5′-ATGGAGCATTACPTGCGTTCT-3′) were ordered from Gene Tools. During and after injection, all steps were performed in the dark, using only >580 nm light for illumination. Embryos were injected at 4-cell stage with 4 nl of solution containing a total of 2 pmol each Gli2-MO and PhotoMO blocker with Cascade Blue-Dextran tracer and grown at 20–22°C until 32 hpf. Larvae were then split into two separate Petri dishes in 10% MMR, and either kept in the dark (inactive control) or exposed to 30 min of 365 nm UV transillumination, low-output setting on a MaestroGen 240 V UV transilluminator (active MO). Larvae were then left at 20–22°C until 28 h post-UV, then amputated, and kept at 22–23°C until anesthesia, screening for tracer and fixation at 72 hpa.Morpholino antisense oligonucleotides targeted to block Gli2 translation . All cells were filtered for minimum 3× background Sox2 or P-CREB mean signal intensity, and background subtracted for mean Sox2 or P-CREB intensity. P-CREB+ cell counts are presented as the ratio of numbers of P-CREB+ cells per total number of Sox2+ nuclei for each 100 μm bin along the spinal cord posterior and anterior to the amputation plane for controls , 10 μM cyclopamine or 1 μM SAG, then fixed in 4% PFA for 1 h at room temperature. Samples were then processed for paraffin embedding and sectioned longitudinally in 10-μm-thick sections. Slides were processed for immunostaining by incubating overnight with the primary antibodies Sox2 (1:300), P-CREB (1:800) at 4°C and for 2 h at room temperature with AlexaFluor conjugated secondary antibodies (1:300) in 1% BSA, 0.1% Tween in PBS. Immunostained samples were mounted and imaged in an epifluorescence microscope. Number of P-CREB and Sox2 immunopositive cells was quantified by thresholding the signal to twice the background and normalized per 100 μm extension of the spinal cord, determined by the length of Sox2-labeled tissue. Data was collected considering as 0 μm the tip of the tail in 4-hpa larvae.Live larvae anesthetized with 0.02% TMS in 10% MMR were imaged under a Nikon swept-field confocal microscope using 488 nm (EGFP) and 647 nm (iRFP670) lasers. Fixed samples were imaged on Nikon-A1 or C2 point laser-scanning confocal microscopes using 488 nm (Alexa488), 561 nm (Alexa594), and 640 nm (Alexa647) lasers or using an Olympus epifluorescence microscope for immunostained sections.2, protease inhibitors cocktail  on ice for 30 min and centrifuged for 10 min at 1000 g. Nuclear pellets were resuspended in 2× protein loading buffer  glycerol, 0.005% Bromophenol Blue, 5% β-mercaptoethanol) and boiled for 5 min. Samples were run in 10% SDS-PAGE and transferred to PVDF membrane. PVDF membrane was probed with anti-Gli2 goat polyclonal , 1:800 in 5% BSA at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody  and visualized by Western Lightning Plus-ECL, Enhanced Chemiluminescence Substrate . PVDF membranes were stripped in 0.2 M glycine HCl buffer, pH 2.5, 0.05% Tween for 20 min and re-probed with 1:500 anti-LaminII/III  for nucleus-specific loading control in 5% BSA.Nuclear fraction was obtained from stage 39–40 Gli2MO + PhotoMO larvae (three larvae for each group) to assess endogenous expression of Gli2. Briefly, larvae injected with Gli2MO + PhotoMO +/– UV (as described above) were frozen in liquid nitrogen, stored at −80°C, then homogenized in 25 mM HEPES pH 7.4, 50 mM NaCl, 2 mM EGTA, 5 mM MgClAB_0049-200) for whole-cell loading control in 5% BSA, followed by incubation with HRP-conjugated rabbit anti-goat secondary antibody .For assessment of Hh ligand expression, whole-cell lysates were obtained from 500-μm-long stump/regenerate from 0 and 24 hpa larvae (35/group) by freezing samples in liquid nitrogen, then solubilizing in 1× Laemmli buffer and centrifuging for 5 min at 17,900 g. Supernatant containing 2.5% β-mercaptoethanol was boiled for 4 min. Samples were run in 4–20% gradient gel SDS-PAGE and transferred to PVDF membrane, which was probed with anti-Hh antibody 5E1 (DSBH), 1:50 in 5% BSA at 4°C overnight, followed by incubation with HRP-conjugated goat anti-mouse secondary antibody  and visualized by ECL2. PVDF membranes were stripped in 0.2 M glycine HCl buffer, pH 2.5, 0.05% Tween for 20 min and re-probed with 1:1000 anti-GAPDH .All data were analyzed with Prism software (GraphPad). Data were first analyzed for normality, followed by parametric  or non-parametric tests . When normally distributed and SDs were equal among groups, unpaired two-tail t-test or ordinary one-way ANOVA followed by Tukey’s multiple comparisons test was used, when two or more groups were compared, respectively. When normally distributed and SDs were not equal among groups, Welch’s t-test or Brown–Forsythe and Welch ANOVA followed by Dunnett’s T3 multiple comparisons test was used, when two or more groups were compared, respectively. When data were not normally distributed, Mann–Whitney test or Kruskal–Wallis followed by Dunn’s multiple comparisons test was used, when two or more groups were compared, respectively. Graphed values are presented as a normalized percent of the experiment-matched control treatment, pooled across experiments. Error bars for the pooled control groups coming from different experiments in  In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Xenopus to spinal cord and muscle, after tail amputation at larva stages. The study confirms previous reports and further adds the notion that this function is mediated by a non-canonical branch of the pathway involving PKA and CREB underlies this process.This study shows that Hh signaling contributes to the regeneration of the Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Marianne Bronner as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Henry Roehl (Reviewer #2).Thank you for submitting your article "Non-canonical Hedgehog signaling regulates spinal cord and muscle regeneration" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at Summary:Xenopus to spinal cord and muscle, after tail amputation at larva stages. The manuscript suggests that a non-canonical branch of the pathway involving PKA and CREB underlies this process.Hh signalling contributes to the generation of a number of tissues. The present manuscript investigates how this signalling contributes to the regeneration of the The manuscript is well written and easy to follow. However the finding that distinguishes this manuscript from previous related reports -namely the involvement of non-canonical signalling in Hh-mediated regeneration- is poorly supported by the current data and needs to be strengthened by further experimentation.Essential revisions:1. The authors need to provide the rationale for using st 39-40 larvae instead older stages, commonly accepted as regenerative stages (i.e. st 49 to 54). This should be included in the method/results, further discussing the potential advantages/ disadvantages. More information on the timing of full regeneration at used stages should also be provided.2. The authors should address the question of whether the activation of the non canonical pathway is ligand dependent or independent, among other by analysing whether HH ligands are in the initial 24 hr of regeneration.3. The chemical inhibitors used in the study are not fully specific and therefore additional approaches should be used to validate the findings. One possibility is to use Photo-MO for Smoothened and CREB. Additional methods to support the involvement of PKA are also needed.4. A direct link between pCREB and non-canonical HH signalling is needed. This could be provided by testing pCREB reduced after Smoothened inhibition during regeneration.Reviewer #1:Xenopus larvae is mediated by non-canonical Hh signaling through the activity of PKA. This conclusion is based on a series of pharmacological treatments and a careful analysis of a number of quantified parameters.This manuscript shows that spinal cord and muscle regeneration in the The study adds to the idea that Hh signaling is important for tissue regeneration, with the additional interesting idea that the process is mediated, at least at early stages, by non-canonical signaling. The enthusiasm for these results is however undermined by a series of concerns:Xenopus laevis . Why have the authors used larvae at st 39-40? Can these earlier stages entail a contribution from developmental related aspects? How long does it take to reach full regeneration in these larvae? This information is missing and needs to be provided for non-specialists. This is important also because Figure 2 shows that Gli transcriptional activity begins to recover at 28 hr. When does it get back to normal levels? Is it before than 72 hrs ? Can Gli activity account for the differences observed between KT5720 and KT5720+ cyclop treatments?According to the literature, st 49 to 54 are considered as the regenerative stages in On the same line: does treatment with GANT61 beginning 48hr (or so) after amputation has any effect on regeneration parameters?The experiments reported in Figure 6 do not really prove that P-CREB mediates regeneration. Can the authors attempt to down-modulate its activity?Reviewer #2:Xenopus laevis. The important finding is that a non-canonical branch of the Hedgehog pathway involving Protein Kinase A and CREB is acting early during Xenopus larval tail regeneration. The paper is well-written and the findings are very interesting, but further work is needed to warrant publication in eLife. We feel that these experiments fall within the authors expertise and could be accomplished within a reasonable timeframe.In this manuscript the authors examine how Hh signalling is involved in spinal cord and muscle regeneration in the 1. Using the same regeneration model, Taniguchi et al. (2014) showed that all HH ligands become expressed in the notochord about 24 hours after injury. The analysis presented here is focused before this time. Is the hypothesis that non-canonical HH signalling is ligand independent? If so the Authors should confirm that HH ligands are not expressed during the period of their analysis (i.e. between 0 and 24 hours after injury).2. The paper relies very heavily on chemical inhibitors which may not be entirely specific . The authors should consider using Photo-MO for Smothened to confirm some key results. This would be especially convincing when compared to the Gli MO (which did not have a strong effect).3. KT5720 is used as an inhibitor of PKA. There are reports of potential off target effects including inhibiting MAPK . MAPK can act upstream of CREB too and is known to be activated immediately after wounding . Could another method be used to strengthen the PKA hypothesis?4. The Authors use pCREB as a read-out for non-canonical HH signalling in Figure 6. They need to show that pCREB is reduced by Smoothened inhibition during regeneration. What is the significance of its presence in skin after wounding, is that also dependent upon non-canonical HH signalling?Reviewer #3:Xenopus tadpoles. The downstream effectors of this non-canonical Hh signaling involves PKA and CREB activation. The finding provides an explanation of the mechanisms of Hh signaling mediated tissue regeneration. Thus, it is of some interest for regenerative biology.This manuscript demonstrated the role of non-canonical Hh signaling in spinal cord and muscle regeneration in I have two concerns about this study.Xenopus tadpole stages used for regeneration analysis, the tail buds of stage 39-40 embryos are not typically considered as differentiated tail, and the cells in the tail buds are precursor cells, rather than differentiated tissue cells. This is particularly true for the cells in the spinal cord. As has been well established, there is ontogenetic decline in the regeneration capacity of Xenopus tadpoles. Thus the stages of tadpoles used for tail amputation is an important concern when performing regeneration analysis.First, for tadpole tail amputations, the authors used NF stages 39-40 embryos. While there is no consensus in the community on the Second, for Gli activity reporting, the authors injected EGFP:iRFP670 plasmid in the early embryos. This is not ideal, as transgenic tadpoles can be easily generated now.Xenopus laevis larvae" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Marianne Bronner as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Henry Roehl (Reviewer #2).Thank you for submitting your article "Non-canonical Hedgehog signaling regulates spinal cord and muscle regeneration in The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.eLife.The manuscript is much improved but there are a couple of key issues that still need to be carefully addressed and included in the manuscript to assure its publication in Essential revisions:1. The first relates to the question of whether Hh ligand expression is activated upon amputation at stage 40. Staining of Hh ligands needs to be analyzed and included in the manuscript. This is an important support for the model presented in Figure 9.2. Graphs should be better presented. Error bars for controls are not treated consistently.3. The method section related to how regeneration is measured in Figure 1—figure supplement should be expanded and include an explanation on what is the difference between the data presentation of Figure 1 and figure supplement 1.Reviewer #1:The manuscript has been revised addressing the comments raise during the first round of review. The manuscript will add new information of the role of Hh signalling in regenerationReviewer #2:The responses to the reviewers' comments are on the whole satisfactory, except around the question of whether there is evidence that Hedgehog ligand expression is activated by excision at stage 40.The authors state that "Taniguchi et al. reported an upregulation in shh expression upon amputation in the regenerating tail, but they do not argue that Hh is not expressed prior to amputation or during the first 24 hours, nor do their findings support this position." This is not accurate. Figure 1A of Taniguchi et al. analyses expression of shh and bhh over time. At 0hrs post amputation expression of shh is barely detectable, and bhh is not detected. At 24hrs post amputation there is no expression of either ligand. Starting at 48 hours post amputation, shh is induced strongly and bhh to a lesser degree. This expression is stated as being exclusively in the notochord. This has led to the model that Hedgehog signalling becomes slowly activated peaking four to five days after amputation. The authors should familiarise themselves with the literature on the role of Hedgehog signalling during tail regeneration. The comparison with other published analysis of tail regeneration is important even if it is at a different stage.The authors show a staining of Hedgehog ligands in their rebuttal but do not present analysis of this data and have not added the data to their manuscript. It appears to show that Hedgehog ligands are strongly upregulated within 4 hours after amputation, and that this activation is primarily in the neural tube (not notochord?). This finding would be consistent with the author's model that Hedgehog signalling is activated by wounding and stimulates muscle and neural tube regeneration . Importantly this data would indicate that Hedgehog expression becomes active much earlier than has been previously described for stage 49. This data should be properly analysed and presented in the manuscript. It would provide important support for their model. This is not a labour intensive request.Reviewer #3:Gli2-mo. The revision adequately addressed my concerns.The revised manuscript added new data from additional experiment, explained the reasoning of using stage 39-40 tadpoles for tail amputation experiment, and replaced the western blot images showing the efficiency of However, I find it confusing in understanding the graph presentations, especially with the new data. Error bars for controls are not treated consistently. For example, in Figure 1D-I, there are no error bars shown for the control groups, but in Figure 1—figure supplement 1 B-G, there are error bars shown for the control. Yet, the means for the controls in Figure 1—figure supplement 1 B-G are all 100. Could you please explain a bit more in the method section, how the regeneration is measured in Figure 1—figure supplement? What is the difference between the data presentation of Figure 1 and figure supplement 1? Essential revisions:1. The authors need to provide the rationale for using st 39-40 larvae instead older stages, commonly accepted as regenerative stages (i.e. st 49 to 54). This should be included in the method/results, further discussing the potential advantages/ disadvantages. More information on the timing of full regeneration at used stages should also be provided.Xenopus larvae as a model for tail regeneration results in a total of 63 original research articles, with 37 in which larvae of stages 39-42 are used and 26 in which stages older than 48 are used. Hence, the earlier stages that we used in this study are well accepted in the field as a good model for tail regeneration in this system. The reasoning behind this is that unlike the tip of the tail that is still developing and growing, the tissues of the stump that become exposed after amputation correspond to mature larval tissues, thus the replenishing of these tissues represents a regenerative process and not the mere developmental progression of tissue growth.A review of the literature on studies using Nevertheless, this is an important point that we are thankful the reviewers brought to our attention so that we can better introduce the readers to this model system. We revised the Results section accordingly .2. The authors should address the question of whether the activation of the non canonical pathway is ligand dependent or independent, among other by analysing whether HH ligands are in the initial 24 hr of regeneration.in situ hybridization assays . From the 4 hedgehog genes identified in Xenopus laevis, Shh, expressed in notochord and/or floor plate, and banded hedgehog (bhh), expressed in somites , are likely the potential ligands of Patched that activate Smo signaling during regeneration at these larval stages. Moreover, immunostaining of transverse sections of non-amputated and 4 hpa-amputated larvae shows immunolabeling for Hh  in somites of larvae at these stages and in spinal cord of regions proximal to the amputation plane .3. The chemical inhibitors used in the study are not fully specific and therefore additional approaches should be used to validate the findings. One possibility is to use Photo-MO for Smoothened and CREB. Additional methods to support the involvement of PKA are also needed.Gli2-photo-morpholino. In addition, we also report the decrease in Gli activity upon amputation. We employed multiple approaches to demonstrate the main scope of this study.Cyclopamine and SAG have been extensively validated and utilized as both highly specific and efficient antagonist and agonist respectively of Smoothened, Hh signaling major effector. The main focus of this study is to demonstrate the necessity for non-canonical Hh, Smoothened-dependent, Gli-independent signaling for muscle and spinal cord regeneration. Hence, we complemented the pharmacological loss and gain of Smoothened function approaches with both pharmacological and genetic loss of Gli function by treating amputated larvae with GANT61, Gli transcriptional activity inhibitor, and by microinjecting Nevertheless, we performed additional experiments that further support the conclusion of Smoothened-dependent regeneration of muscle and spinal cord by incubating amputated larvae with another highly specific Smoothened inhibitor, vismodegib, that binds to a different domain than cyclopamine, and obtained similar effects on regeneration of muscle and spinal cord as with cyclopamine. These new data are included as a new supplement to Figure 1 in the revised manuscript  and described in the Results section .vivo-morpholino, that, unlike the regular morpholinos, permeate into cells, thus, do not need to be injected in the fertilized embryo. However, high variability in regeneration metrics observed in pilot experiments and difficulty in successfully staining for Smo in whole mounts led to inconsistent results and uncertainty of the efficiency of the intended manipulation.We chose pharmacological means to manipulate signaling because this study requires temporal control so that the inhibition of the signaling is restricted to the regeneration period. In addition, Gene Tools, the company that develops morpholinos, has discontinued the production of photo-morpholinos. We have attempted to utilize Smo We agree with the reviewers that further investigation is needed to identify the non-canonical Smo-dependent signaling involved in the regeneration of muscle and spinal cord. However, we consider this belongs to a follow up study and believe the findings presented in this manuscript contribute with significant and relevant understanding on Hh-dependent tissue regeneration.4. A direct link between pCREB and non-canonical HH signalling is needed. This could be provided by testing pCREB reduced after Smoothened inhibition during regeneration.We have performed additional experiments to directly address the link between the non-canonical HH signaling and pCREB during regeneration by incubating amputated larvae in the presence or absence of SAG or cyclopamine followed by immunostaining of samples for pCREB. Results show that cyclopamine or SAG incubation for the first 4 hpa respectively decreases or increases the number of pCREB+ nuclei in the spinal cord proximal to the tip of the amputated tail. These results suggest that HH signaling modulates CREB activity in the regenerating spinal cord. Moreover, we find that 24-h incubation with cyclopamine or SAG decreases or increases respectively the number of pCREB immunopositive nuclei in the regenerating spinal cord. We revised the manuscript to include these new sets of data .Xenopus larvae is mediated by non-canonical Hh signaling through the activity of PKA. This conclusion is based on a series of pharmacological treatments and a careful analysis of a number of quantified parameters.Reviewer #1:This manuscript shows that spinal cord and muscle regeneration in the The study adds to the idea that Hh signaling is important for tissue regeneration, with the additional interesting idea that the process is mediated, at least at early stages, by non-canonical signaling. The enthusiasm for these results is however undermined by a series of concerns:Xenopus laevis . Why have the authors used larvae at st 39-40? Can these earlier stages entail a contribution from developmental related aspects? How long does it take to reach full regeneration in these larvae? This information is missing and needs to be provided for non-specialists.According to the literature, st 49 to 54 are considered as the regenerative stages in Xenopus larvae as a model for tail regeneration results in a total of 63 original research articles, with 37 in which larvae of stages 39-42 are used and 26 in which stages older than 48 are used. Hence, the earlier stages that we used in this study are well accepted in the field as a good model for tail regeneration in this system. The reasoning behind this is that unlike the tip of the tail that is still developing and growing, the tissues of the stump that become exposed after amputation correspond to mature larval tissues, thus the replenishing of these tissues represents a regenerative process and not the mere developmental progression of tissue growth.A review of the literature on studies using Nevertheless, this is an important point that we are thankful the reviewers brought to our attention so that we can better introduce the readers to this model system. We revised the Results section accordingly .Studies have shown that full regeneration is achieved after 7-10 days post tail amputation in these larval stages. Nonetheless, the current study is focused on the first stages of regeneration and the signaling mechanisms responding to the acute injury. Hence, the latest end point for the assays presented is 3 days post amputation, when regeneration of muscle and spinal cord are appreciable and measurements of extent of regeneration are reproducible, to allow comparison across controls and experimental groups.This is important also because Figure 2 shows that Gli transcriptional activity begins to recover at 28 hr. When does it get back to normal levels? Is it before than 72 hrs ? Can Gli activity account for the differences observed between KT5720 and KT5720+ cyclop treatments?Gli2-MO incubated for the full 72 h post amputation are almost inactive upon the process of spinal cord and muscle regeneration. Hence, we conclude that the canonical, Gli-dependent Hh signaling does not contribute significantly to this process during the first 3 days of regeneration.Because cyclopamine effect in 24-h only group is already significant, this suggests that Smo activity during these first 24 h is important. In contrast, GANT61 and Gli2-MO does not result in any appreciable effect on regeneration of muscle or spinal cord. Instead, we think that other non-canonical Smo-dependent signaling, independent of PKA is triggered; and conversely some PKA-dependent regeneration may operate independently from Smo pathway.We do not think that Gli activity explains the differences in regeneration between inhibiting PKA alone or along with Smo inhibition because inhibiting Gli directly either pharmacologically with GANT61 or genetically by activating On the same line: does treatment with GANT61 beginning 48hr (or so) after amputation has any effect on regeneration parameters?Treatments for 72 h were done so that the drugs were replenished in the saline that larvae were incubated in every day. Hence, the 72-h treatment implemented for GANT61 consisted on replenishing the solution with GANT61 each day of the 3-day period. Any effect of inhibiting Gli1/2 transcriptional activity during the last day of regeneration (48-72 h) should have been observed. We interpret the lack of significant effect on almost every parameter of regeneration as a lack of participation/necessity of Hh Gli-dependent canonical pathway in the early stages (first 3 days) of the regenerative process.We do not expect a different result from starting incubation with GANT61 at 48 hpa, compared to 0 hpa.The experiments reported in Figure 6 do not really prove that P-CREB mediates regeneration. Can the authors attempt to down-modulate its activity?We believe that inhibiting PKA inhibits CREB activation, as we and many others have demonstrated in previous studies (Belgacem and Borodinsky 2015). Thus, the effect of PKA inhibitor on spinal cord and muscle regeneration may be revealing a role for P-CREB on the regeneration of these tissues.We agree with the reviewers that assessing more specifically the role of P-CREB will be an interesting future direction for this investigation. However, we believe it belongs to a follow-up study beyond the scope of the present manuscript.Reviewer #2:Xenopus laevis. The important finding is that a non-canonical branch of the Hedgehog pathway involving Protein Kinase A and CREB is acting early during Xenopus larval tail regeneration. The paper is well-written and the findings are very interesting, but further work is needed to warrant publication in eLife. We feel that these experiments fall within the authors expertise and could be accomplished within a reasonable timeframe.In this manuscript the authors examine how Hh signalling is involved in spinal cord and muscle regeneration in the 1. Using the same regeneration model, Taniguchi et al. (2014) showed that all HH ligands become expressed in the notochord about 24 hours after injury. The analysis presented here is focused before this time. Is the hypothesis that non-canonical HH signalling is ligand independent? If so the Authors should confirm that HH ligands are not expressed during the period of their analysis (i.e. between 0 and 24 hours after injury).Taniguchi et al. (2014) used stage 49 tadpoles (12 day-old) instead of stage 40 larvae (2.75 day-old) as in this study. Hence it is difficult to compare. Nevertheless, Taniguchi et al. reported an upregulation in shh expression upon amputation in the regenerating tail, but they do not argue that Hh is not expressed prior to amputation or during the first 24 hours, nor do their findings support this position.in situ hybridization assays . From the 4 hedgehog genes identified in Xenopus laevis, Shh, expressed in notochord and/or floor plate, and banded hedgehog (bhh), expressed in somites , are likely the potential ligands of Patched that activate Smo signaling during regeneration at these larval stages. Moreover, immunostaining of transverse sections of non-amputated and 4 hpa-amputated larvae shows immunolabeling for Hh  in somites of larvae at these stages and in spinal cord of regions proximal to the amputation plane .2. The paper relies very heavily on chemical inhibitors which may not be entirely specific . The authors should consider using Photo-MO for Smothened to confirm some key results. This would be especially convincing when compared to the Gli MO (which did not have a strong effect).Every approach has its pros and cons. The pharmacological approach offers a higher temporal resolution of the down or upregulation of a signaling pathway, which is particularly important in this case when we want to impose the loss or gain of function of a certain pathway strictly during injury and regeneration.Gli2-photo-morpholino. In addition, we also report the decrease in Gli activity upon amputation. We employed multiple approaches to demonstrate the main scope of this study.We agree with the reviewer that showing the same outcome with multiple approaches strengthens the relevance and significance of the finding. Nevertheless, Cyclopamine and SAG have been extensively validated and utilized as both highly specific and efficient antagonist and agonist respectively of Smoothened, Hh signaling major effector. The main focus of this study is to demonstrate the necessity for non-canonical Hh, Smoothened-dependent, Gli-independent signaling for muscle and spinal cord regeneration. Hence, we complemented the pharmacological loss and gain of Smoothened function approaches with both pharmacological and genetic loss of Gli function by treating amputated larvae with GANT61, Gli transcriptional activity inhibitor, and by microinjecting We also performed additional experiments that further support the conclusion of Smoothened-dependent regeneration of muscle and spinal cord by incubating amputated larvae with another highly specific Smoothened inhibitor, vismodegib, that binds to a different domain than cyclopamine, and obtained similar effects on regeneration of muscle and spinal cord as with cyclopamine. These new data are included as a new supplement to Figure 1 in the revised manuscript  and described in the Results section .vivo-morpholino, that, unlike the regular morpholinos, permeate into cells, and thus do not need to be injected. However, high variability in regeneration metrics observed in pilot experiments and difficulty in successfully staining for Smo in whole mounts led to inconsistent results and uncertainty of the efficiency of the intended manipulation.In addition, Gene Tools, the company that develops morpholinos, has discontinued the production of photo-morpholinos. We have attempted to utilize Smo 3. KT5720 is used as an inhibitor of PKA. There are reports of potential off target effects including inhibiting MAPK . MAPK can act upstream of CREB too and is known to be activated immediately after wounding . Could another method be used to strengthen the PKA hypothesis?We agree with the reviewer and appreciate the reviewer’s suggestion of strengthening the results from pharmacologically inhibiting PKA. However, the main focus of this manuscript is to demonstrate that a non-canonical, Hh signaling is necessary for spinal and muscle regeneration, while the canonical Gli-dependent signaling is dispensable and suppressed during the early stages of tissue regeneration.We believe we convincingly demonstrate that and provide further evidence of potential partners in the Hh non-canonical signaling without being exhaustive or definitive about the identity of these signaling mechanisms.Although we used in previous studies overexpression of dominant negative and constitutively active forms of PKA, by microinjecting these constructs in 2-cell stage embryos , for the current study focused on later larval stages and restricted to the regenerative process after tail amputation, this approach is not adequate because it interferes with embryonic development.We present the inhibition of PKA as a potential candidate pathway, but we agree with the reviewer that other pathways may be affected by the PKA inhibitor and participating in the regeneration of muscle and spinal cord. Further investigation is needed and will be pursued in a follow up study. We have revised the manuscript to indicate that the drug may affect other kinases that may participate in this process, that are also known to activate CREB .Nonetheless, we now provide further evidence in the revised manuscript of Smo-dependent activation of P-CREB in the spinal cord proximal to the site of injury , strengthening the link between Smo and the non-canonical signaling, similar to what we have identified in the embryonic spinal cord .4. The Authors use pCREB as a read-out for non-canonical HH signalling in Figure 6. They need to show that pCREB is reduced by Smoothened inhibition during regeneration. What is the significance of its presence in skin after wounding, is that also dependent upon non-canonical HH signalling?We have performed additional experiments to directly address the link between the non-canonical HH signaling and pCREB during regeneration by incubating amputated larvae in the presence or absence of SAG or cyclopamine followed by immunostaining of samples for pCREB. Results show that cyclopamine or SAG incubation for the first 4 hpa respectively decreases or increases the number of pCREB+ nuclei in the spinal cord proximal to the tip of the amputated tail. These results suggest that HH signaling modulates CREB activity in the regenerating spinal cord. Moreover, we find that 24-h incubation with cyclopamine or SAG decreases or increases respectively the number of pCREB immunopositive nuclei in the regenerating spinal cord. We revised the manuscript to include these new sets of data .Reviewer #3:Xenopus tadpoles. The downstream effectors of this non-canonical Hh signaling involves PKA and CREB activation. The finding provides an explanation of the mechanisms of Hh signaling mediated tissue regeneration. Thus, it is of some interest for regenerative biology.This manuscript demonstrated the role of non-canonical Hh signaling in spinal cord and muscle regeneration in I have two concerns about this study.Xenopus tadpole stages used for regeneration analysis, the tail buds of stage 39-40 embryos are not typically considered as differentiated tail, and the cells in the tail buds are precursor cells, rather than differentiated tissue cells. This is particularly true for the cells in the spinal cord. As has been well established, there is ontogenetic decline in the regeneration capacity of Xenopus tadpoles. Thus the stages of tadpoles used for tail amputation is an important concern when performing regeneration analysis.First, for tadpole tail amputations, the authors used NF stages 39-40 embryos. While there is no consensus in the community on the Xenopus larvae as a model for tail regeneration results in a total of 63 original research articles, with 37 in which larvae of stages 39-42 are used and 26 in which stages older than 48 are used. Hence, the earlier stages that we used in this study are well accepted in the field as a good model for tail regeneration in this system. The reasoning behind this is that unlike the tip of the tail that is still developing and growing, the tissues of the stump that become exposed after amputation correspond to mature larval tissues, thus the replenishing of these tissues represents a regenerative process and not the mere developmental progression of tissue growth.A review of the literature on studies using Nevertheless, this is an important point that we are thankful the reviewers brought to our attention so that we can better introduce the readers to this model system. We revised the Results section accordingly .Second, for Gli activity reporting, the authors injected EGFP:iRFP670 plasmid in the early embryos. This is not ideal, as transgenic tadpoles can be easily generated now.We appreciate the reviewer’s suggestion. However, we consider that the mosaic expression of the reporter actually plays to our advantage in allowing us to better assess in sparsely labeled cells Gli activity through live imaging. If reporter expression is extended to all cells as in transgenic larvae, the ability to distinguish between individual cells would have been difficult, if not impossible. Additionally, transgenesis is not an established methodology in the lab and currently we do not have the capability of breeding transgenic animals. Moreover, although transient F0 transgenic larvae would be doable in the lab, the efficiency of transgenesis for big constructs is low and the reporter we developed for this study is 6,378 bp, on the large side of constructs.The manuscript is much improved but there are a couple of key issues that still need to be carefully addressed and included in the manuscript to assure its publication in eLife.Essential revisions:1. The first relates to the question of whether Hh ligand expression is activated upon amputation at stage 40. Staining of Hh ligands needs to be analyzed and included in the manuscript. This is an important support for the model presented in Figure 9.To assess Hh ligand protein expression upon amputation, we performed Western blot assays from 500 mm-long regions of the tail stump at 0 and 24 h after amputation. We probed membranes with whole-cell lysates from these samples with the Hh 5E1 antibody (DSHB). Results show that Hh ligands are detected before amputation and during early regeneration. Moreover, we find an increase in Hh protein levels in regenerating tail after 24 h post amputation compared to 0 h. We included these additional results in a new Figure 1- supplement 1, along with an image of 4 hours post amputation larvae stained with the 5E1 antibody to illustrate the spatial distribution of Hh ligands during the first hours post amputation.We attempted to do a quantitative analysis of 5E1 immunostaining in tissue sections of non-amputated compared to amputated larvae, but we were not satisfied with the reliability of this approach. Due to the pattern of the staining, which mostly localizes to cell membrane, we were not able to count cells or threshold the signal in a consistent manner across samples. Nevertheless, data on the expression of Hh ligand transcripts in the same model system at the same developmental and post amputation times that we used for our study have been recently published using single-cell RNA seq . We browsed the data shared (https://marionilab.cruk.cam.ac.uk/XenopusRegeneration/) by the authors and found that Hh ligands are indeed expressed in stage 40 larvae. Transcripts for shh are detected in posterior notochord and floor plate in pre-amputated and in 1 through 3 days post amputation of the regenerating tail, with an increase in levels of shh transcript as early as 1 day post amputation, which is the earliest time point presented by the authors. Transcripts for shh are also expressed in neural tissue, including spinal cord progenitors and subtypes of spinal cord neurons, while dhh is detected in neural tissue and floor plate.It should be noted that the pattern of immunohistochemistry that we included in our prior responses to reviewers and in the new Figure 1- supplement 1 may detect not only cells/tissues that express the Hh ligands but also target cells that receive it. Indeed, Aztekin and colleagues reported expression of ptch1 and ptch2 genes in the myotome, neural tissue and floor plate.2. Graphs should be better presented. Error bars for controls are not treated consistently.We have revised graphs in Figures 1, 3, 4, 5 and 7D to keep a consistent presentation of error bars for control groups.3. The method section related to how regeneration is measured in Figure 1—figure supplement should be expanded and include an explanation on what is the difference between the data presentation of Figure 1 and figure supplement 1.We have revised the presentation of the data so that is consistent across figures and figure supplements and revised the text in the Methods section  to explain how control groups and error bars were treated for each set of data.Reviewer #2:The responses to the reviewers' comments are on the whole satisfactory, except around the question of whether there is evidence that Hedgehog ligand expression is activated by excision at stage 40.The authors state that "Taniguchi et al. reported an upregulation in shh expression upon amputation in the regenerating tail, but they do not argue that Hh is not expressed prior to amputation or during the first 24 hours, nor do their findings support this position." This is not accurate. Figure 1A of Taniguchi et al. analyses expression of shh and bhh over time. At 0hrs post amputation expression of shh is barely detectable, and bhh is not detected. At 24hrs post amputation there is no expression of either ligand. Starting at 48 hours post amputation, shh is induced strongly and bhh to a lesser degree. This expression is stated as being exclusively in the notochord. This has led to the model that Hedgehog signalling becomes slowly activated peaking four to five days after amputation. The authors should familiarise themselves with the literature on the role of Hedgehog signalling during tail regeneration. The comparison with other published analysis of tail regeneration is important even if it is at a different stage.X. laevis larvae at the same developmental and post amputation times that we used for our study, and found that Hh ligands are indeed expressed in stage 40 larvae. Transcripts for shh are detected in posterior notochord and floor plate in pre-amputated and in 1 through 3 days post amputation of the regenerating tail, with an increase in levels of shh transcript as early as 1 day post amputation, which is the earliest time point presented by the authors. Transcripts for shh are also expressed in the neural tissue, including spinal cord progenitors and subtypes of spinal cord neurons, while dhh is detected in neural tissue and floor plate.We have revised the text to include more recent studies that have addressed the expression of Hh ligands during regeneration of the tail at the developmental stages used in this study . We browsed the single-cell RNA seq data shared (https://marionilab.cruk.cam.ac.uk/XenopusRegeneration/) by Aztekin et al. (Science 2019), given that they used Comparing patterns of expression in the regenerating tail between stage 40 and stage 49 larvae is problematic, and it is not that surprising that there are differences between the findings of Taniguchi et al. in stage 49 larvae and Aztekin et al. in stage 40 animals. It is also important to consider the methodological approaches used to assess expression, which exhibit different sensitivities and spatiotemporal resolution of expression levels. In addition, these studies report on Hh ligand transcript expression; whether transcripts correlate directly with protein levels is doubtful. For instance, The authors show a staining of Hedgehog ligands in their rebuttal but do not present analysis of this data and have not added the data to their manuscript. It appears to show that Hedgehog ligands are strongly upregulated within 4 hours after amputation, and that this activation is primarily in the neural tube (not notochord?). This finding would be consistent with the author's model that Hedgehog signalling is activated by wounding and stimulates muscle and neural tube regeneration . Importantly this data would indicate that Hedgehog expression becomes active much earlier than has been previously described for stage 49. This data should be properly analysed and presented in the manuscript. It would provide important support for their model. This is not a labour intensive request.To assess Hh ligand protein expression upon amputation, we performed Western blot assays from 500 mm-long regions of the tail stump at 0 and 24 h after amputation. We probed membranes with whole-cell lysates from these samples with the Hh 5E1 antibody (DSHB). Results show that Hh ligands are detected before amputation and during early regeneration. Moreover, we find an increase in Hh protein levels in regenerating tail after 24 h post amputation compared to 0 h.We included these additional results in new Figure 1- supplement 1, along with an image of 4 hours post amputation larvae stained with the 5E1 antibody to illustrate the spatial distribution of Hh ligands during the first hours post amputation.We attempted to do a quantitative analysis of 5E1 immunostaining in tissue sections of non-amputated compared to amputated larvae, but we were not satisfied with the reliability of this approach. Due to the pattern of the staining, which mostly localizes to cell membrane, we were not able to count cells or threshold the signal in a consistent manner across samples. Nevertheless, data on the expression of Hh ligand transcripts in the same model system at the same developmental and post amputation times that we used for our study have been recently published using single-cell RNA seq . We browsed the data shared (https://marionilab.cruk.cam.ac.uk/XenopusRegeneration/) by the authors and found that Hh ligands are indeed expressed in stage 40 larvae. Transcripts for shh are detected in posterior notochord and floor plate in pre-amputated and in 1 through 3 days post amputation of the regenerating tail, with an increase in level of shh transcript as early as 1 day post amputation, which is the earliest time point presented by the authors. Transcripts for shh are also expressed in neural tissue, including spinal cord progenitors and subtypes of spinal cord neurons, while dhh is detected in neural tissue and floor plate.It should be noted that the pattern of immunohistochemistry that we included in our prior responses to reviewers and in the new Figure 1-supplement 1 may detect not only cells/tissues that express the Hh ligands but also target cells that receive it. Indeed, Aztekin and colleagues reported expression of ptch1 and ptch2 genes in the myotome, neural tissue and floor plate.Reviewer #3:The revised manuscript added new data from additional experiment, explained the reasoning of using stage 39-40 tadpoles for tail amputation experiment, and replaced the western blot images showing the efficiency of Gli2-mo. The revision adequately addressed my concerns.However, I find it confusing in understanding the graph presentations, especially with the new data. Error bars for controls are not treated consistently. For example, in Figure 1D-I, there are no error bars shown for the control groups, but in Figure 1—figure supplement 1 B-G, there are error bars shown for the control. Yet, the means for the controls in Figure 1—figure supplement 1 B-G are all 100. Could you please explain a bit more in the method section, how the regeneration is measured in Figure 1—figure supplement? What is the difference between the data presentation of Figure 1 and figure supplement 1?Error bars in control groups were used in Figure 1 supplement 1  because the control conditions were perfectly cohort-matched with treatment conditions, such that the error bars for controls would apply exactly to the treatments depicted; i.e. all of the data that went into said bar graphs came from the same experimental cohorts. In contrast, the control bars in Figure 1 and others did not have error bars because some independent experimental sets were combined for presentation: i.e. the cohort-matched controls for each treatment bar were not the same samples. For example, some cyclopamine treatments did not have matched SAG treatments in their cohort, and hence the controls for the SAG and cyclopamine bars were not the same samples. All metrics presented were pooled for analysis as a percent of the average, cohort-paired control values for each parameter in question.In the interest of consistency in the presentation of the data and to report on the variability inherent to control groups in all figures, we have added error bars to the controls corresponding to the largest standard error values in the control groups of the different independent experiments that were included in the given bar graph.Hence, we revised graphs in Figures 1, 3, 4—figure supplement 1, 5 and 7 to keep a consistent presentation of error bars for control groups.We revised the text in the Methods section  to explain how control groups and error bars were treated for each set of data presented.


Research has shown that SARS-CoV-2 can survive on personal protective equipment, such as commonly used surgical masks. Methods are needed to inactivate virus on contaminated material. We show here that embedding viral-disinfecting compounds during the manufacturing of surgical masks inactivates a high dose (up to 1 × 10 These data suggest that PPE can be protected against viral contamination, which has implications for increased safety and life span of common PPE materials.SARS-CoV-2 has caused a devastating pandemic that is still ongoing. Public health measures were undertaken to slow the spread of the virus, including a heavy focus on social distancing and the use of personal protective equipment (PPE) to decrease rates of infection. The most common form of PPE used by the general public has been masks, including common disposable surgical masks. However, it has been well-documented that infectious SARS-CoV-2 can live on surfaces and masks for 1–28 days, depending on the material tested , raising2 at 3450 KPa. This produced a microscopic, invisible polymer film with the embedded disinfectants or natural products bonded to the outside of the mask. The heat and pressure sterilized the masks, and they were subsequently wrapped in sterile aluminum foil. Masks were cut in small pieces to provide 2–3 samples for each chemical treatment, with two samples in the single-experiment screens in 5 plaque forming units (pfu) of live SARS-CoV2 virus  in a BSL-3 laboratory for the indicated times. The virus stocks were amplified in Vero E6 cells in DMEM/5% FCS and supernatants were harvested and quantitated. Mask pieces were inoculated with 12.5 µL of virus. The inoculated fabric was incubated for the indicated times, and the virus was extracted by incubating mask pieces with 300 µL of cell culture medium for 30 min. The eluted virus was added at a 1:10 dilution to Dey-Engley Neutralizing Broth  for 15 min at room temperature, and then the live virus was quantitated using a plaque assay on Vero E6 cells as we have previously published [All surgical masks tested  had a spunbond polypropylene (PP) outer fabric, an electrostatically charged meltblown PP middle filtration layer, and an inner spunbond PP fabric that contacts the face. A mixture of disinfectant chemicals were embedded into a polymer film  applied to the outside (spunbond PP layer) of the surgical mask. Several different disinfectant chemistries were studied and evaluated: (1) phenolics (2-phenylphenol and thymol), (2) quaternary ammonium compounds of different compositions and water solubility, and (3) the natural products peppermint oil and citric acid. Monomer chemistry was selected to dissolve the disinfectant chemicals and was coated onto only the outside spunbond layer of the masks by gravure coating at 2–3 g per square meter. The coated film was polymerized by heating to 100 °C in a pressure vessel with N5 pfu of live, authentic SARS-CoV-2 after 2 h  used to anchor the compounds and combinations of a water-soluble quaternary ammonium compound (labeled 3310), a water insoluble quaternary ammonium compound (labeled 1297), and the disinfectant 2-phenylphenol (with or without 10% PEG 200) completely inactivated masks inoculated with 1 × 10fter 2 h A. Masks In a second screen, masks treated with additional disinfectant chemicals were inoculated with virus for 2 h B. In thiBased on these initial results, we conducted similar experiments with shorter time points. We found complete inactivation of SARS-CoV-2 with masks infused with a combination of 1297, 3% thymol, and citric acid after 20 or 60 min, and, in some experiments, in as few as 5 min, showing remarkable rapid potency A–C. CompThese data show rapid and complete inactivation of high titers of live, authentic SARS-CoV-2 by masks infused with quaternary ammonium salts. The advantage of such an approach is the elimination of infectious virus on the surface of a mask, increasing its protective capabilities and minimizing accidental spread to the wearer or to shared PPE. An interesting inadvertent finding of these experiments were that there was recoverable live virus in untreated masks at the 24-h time points (data not shown), confirming our earlier studies that this virus can survive on masks used as PPE . These dQuaternary ammonium salts are known to inactivate pathogens and are routinely used to disinfect surfaces. Their mechanism of action against enveloped viruses is thought to be disruption of the viral lipid envelope . There iTo address the concern of potential toxicity to the wearer, we embedded the quaternary ammonium compounds into the polymer film that is bonded to the outside of the spunbond polypropylene fabric of the 3-layer mask. Our polymerization technology, which we also use for polymeric, water repellency finishing of apparel, consists of a hydrocarbon-based monomer with an organic peroxide polymerization initiator. Because we apply the coating only to the outside of the mask, the resultant polymeric film anchors and concentrates the disinfectant chemicals in a film applied to the outside of the mask. This polymerization anchors the chemicals in place, preventing flaking, loss, or inhalation of particles. In addition, this approach minimizes any contact risk of the quaternary ammonium salts with the skin. It is important to note that the middle and inner PP mask layers are not modified by our process. The PP layer that contacts the skin has no chemical treatment. Untreated PP is strongly hydrophobic, meaning that water drops will not wick into untreated PP. Similarly, viral-containing droplets may have a long lifetime on PP because this protective mechanism. The quaternary compounds used are hydrophilic and, thus, cause the polymer film to be hydrophilic. As such, water droplets will rapidly wick into the polymer film. Any virus particles will be similarly absorbed with the water and will be brought into contact with the entrapped quaternary ammonium compounds. This helps kill the virus by maximizing contact of the virus into contact with the disinfectants.This study was aimed at demonstrating an anti-viral treatment on disposable masks, in support of an FDA certification process. As such, the re-use and longevity of the treatment was not studied. However, preliminary studies by our group have shown that compounds embedded into the 265 L polymer persist through multiple laundry cycles.Together, these data suggest that compounds placed on PPE can be used to inactivate SARS-CoV-2 in a rapid and safe manner. Quaternary ammonium salts are used to inactivate a wide range of viruses, bacteria and fungi ,10,11,12A patent application is pending on this technology (GSS).