CLAVATA3 transcription at lower and higher concentrations, respectively. We use computational modeling and experimental analysis to investigate the properties of the cis-regulatory module. We find that intrinsically each cis-element can only activate CLAVATA3 at a higher WUSCHEL concentration. However, together, they repress CLAVATA3 at higher WUSCHEL and activate only at lower WUSCHEL, showing that the concentration-dependent interactions among cis-elements regulate both activation and repression. Biochemical experiments show that two adjacent functional cis-elements bind WUSCHEL with higher affinity and dimerize at relatively lower levels. Moreover, increasing the distance between cis-elements prolongs WUSCHEL monomer binding window, resulting in higher CLAVATA3 activation. Our work showing a constellation of optimally spaced cis-elements of defined affinities determining activation and repression thresholds in regulating CLAVATA3 transcription provides a previously unknown mechanism of cofactor-independent regulation of transcription factor binding in mediating gene expression specificity.Gene expression specificity of homeobox transcription factors has remained paradoxical. WUSCHEL activates and represses  Optimally spaced cis-elements of defined affinities regulate concentration-dependent transcriptional switching in stem cells. Drosophila melanogaster , WUSCHEL (WUS) is a homeodomain TF expressed in the rib meristem (RM) (CLAVATA3 (CLV3)  . CLV3 enr region . The incess CLV3 . The bioCLV3 CRM functions as a unit. The complexity of the CLV3 CRM regulation involving five cis-elements and the bidirectional relationship between CLV3 and WUS can be challenging to untangle experimentally. The current experimental limitations cannot provide a direct real-time view of the actual WUS/CLV3 molecular dynamics under precisely defined conditions. A multiscale computational model capable of simulating the binding and unbinding dynamics of WUS to all five cis-elements and CLV3 transcription at the tissue level can be helpful in providing mechanistic insights into the WUS concentration–dependent functioning of the CLV3 CRM.Understanding how the concentration-dependent transcriptional switch is established requires understanding how the CLV3 CRM, the number of possible configurations becomes large and it is not practical to use the thermodynamic approach. Instead, the stochastic simulation algorithm, i.e., Gillespie algorithm where the dynamics of WUS binding and unbinding to the cis-elements can be modeled as a series of probabilistic events occurring at random time steps determined, is ideal to explicitly model CLV3 transcription.Different approaches have been developed for studying TF binding dynamics. The thermodynamic models are usually based on the occupancy of the promoter by the TFs, the statistical weights of possible configurations, and the free energy  recruitment in the transcription process. Subsequently the single-cell stochastic model was applied to multiple cells represented by unit spheres to develop a cell-based three-dimensional (3D) model representing the SAM. The 3D model was applied to further test the mechanisms identified in the single-cell model in generating the spatial patterns of CLV3 expression.We developed a stochastic model to simulate the WUS binding to the CRM in a single cell. The single-cell model was applied to simulate WUS binding cis-elements under different concentrations and compared the simulation output with the experimental data on tissue-level expression patterns of different cis-element mutants of CLV3 largely to a similar extent when acting alone. Beyond residence time limit, the aged WUS monomers fail to activate transcription and they are replaced with newly synthesized WUS monomers to sustain CLV3 activation. Our experimental observations showing a correlation between higher WUS turnover and increased CLV3 activation support such a mechanism. When multiple cis-elements are involved, we found that the cooperative binding of WUS monomers and dimers is required to achieve correct CLV3 activation patterns. The model simulations also suggested a nonhomogeneous cooperativity among cis-elements that depends on the intervening distance between cis-elements. The model prediction on distance-dependent cooperativity was tested in experiments by increasing the intervening distance between cis-elements, which revealed an increase in CLV3 activation. The corresponding biochemical experiments revealed that an increase in intervening distance between cis-elements increased their affinity to WUS monomers but did not alter the concentration at which WUS monomers switch to form stable dimers/higher molecular weight complexes. These results show the importance of optimal spacing between cis-elements in determining the concentration range over which an appropriate number of WUS monomers and dimers populate on cis-elements in setting up the activation-repression thresholds. The 3D model that incorporates multiple cis-elements of different affinities that are spaced optimally allowed independent manipulation of the monomer and dimer cooperativity. Our simulations revealed that monomer cooperativity was critical for expression of CLV3 at the lower WUS concentration, while the dimer cooperativity was critical for repression at the higher WUS concentration. Moreover, a balance of the monomer and dimer cooperativity levels was critical to achieve the wild-type CLV3 expression at a WUS concentration range observed in experiments.Using a WUS gradient consistent with the experimental data, both computational models suggested a role for residence time limit (see Results for details) of WUS monomer binding to the individual cis-elements of different affinities, which have been shown to activate CLV3 expression in outer cell layers of the CZ and up-regulation in the inner cell layers of the RM, suggesting interaction among cis-elements  behavior. We generated a library of five mutant CLV3 reporters; each contained only one functional cis-element referred to as 970i, 997i, 1007i, 950i, and 1060i. The reporter expression analysis revealed a marked down-regulation of CLV3 expression in outer cell layers of CZ, including the higher-affinity cis-element 970i  phenotypes when compared to the wild-type CLV3 promoter . However, cis-element affinity is important in the context of other functioning cis-elements in the CRM, as exemplified by the repression of 970M4. In summary, the affinity-dependent collective WUS binding to all five cis-elements is required for balancing activation and repression of transcription in regulating the spatial expression and levels of CLV3.The subtle changes observed upon mutating individual lower-affinity cis-elements ruled out a simple additive interaction in regulating ent 970i  and H. Trs of CZ  and D. Tpromoter  and L. Fpromoter  and K anpromoter  and L coCLV3 CRM in a single cell, together with the RNA Pol II recruitment and CLV3 mRNA synthesis .To investigate the mechanisms of interaction among five cis-elements, we developed a stochastic modeling framework to simulate the WUS binding to the ynthesis . The modOur previous analysis revealed that each cis-element binds WUS at different concentrations as monomers first and then switches to forming dimers at increasing concentrations  footprint of RNA polymerase and mRNA elongation rate, which is estimated to be 1.2 kb/min , they were able to similarly activate CLV3 only in inner cell layers of RM where the WUS concentration is higher  in yeast  and 1060i (lowest affinity) cis-elements, we chose the residence time limit to be 10 for all simulations involving multiple cis-elements discussed in the following sections ::H2b-mYFP is initially expressed in the deeper cell layers, and the expression levels are below that of the wild-type promoter (pCLV3(DM) in the CZ weakly but failed to expand into the PZ (n = 8) when compared to the wild-type promoter, which revealed strong activation and radial expansion (pCLV3(QM)::H2b-mYFP was expressed only in the deeper layers  xpansion . The mutr layers . After 2r layers . TogetheCLV3 for single cis-elements and multiple cis-elements suggested an interaction among cis-elements within the CRM  for the low WUS concentration, compared to the highest affinity, i.e., 970 cis-element (970M)   to C. Hot (970M) , which wCLV3 at lower WUS. Therefore, distance-dependent monomer cooperativity between cis-elements was considered. The monomer and dimer cooperativity were considered separately since one is responsible for activation and the other one is responsible for repression. Considering that the affinity plays a role when multiple cis-elements interact, the residence time limit associated with single cis-element was disabled. The additional WUS monomer cooperativity along with the dimer cooperativity between all cis-elements was able to generate expected wild-type and mutant (970M and 950M) cis-element behaviors at all WUS concentrations . Similar kind of cooperativity was studied in thermodynamic models earlier . This sutrations , showingCLV3 expression in outer cell layers of CZ and up-regulate expression in the inner layers of RM ::H2B-mYFP. Increasing the distance between neighboring cis-elements led to increased CLV3 expression in all cell layers, and an increase in the deeper layers was much higher than in the outer cell layers of CZ . The full-length WUS protein could bind the oligo (970--997) at lower WUS . To test the influence of spacing between cis-elements without reducing the number or affinity, we duplicated the sequence between neighboring cis-elements. The increased distance might reduce the interaction of WUS bound to neighboring cis-elements without affecting the intrinsic binding to each independent cis-elements. Therefore, we duplicated the intervening sequence between 970-997 and 997-1007 (double space around 997) rs of CZ  to C. Thower WUS  and E. HCLV3 expression (fig. S6). However, the single-cell model can only provide average expression behavior at given WUS concentrations, without considering the tissue spatial organization and the stochasticity associated with individual cells within layers of the SAM under a broader range of WUS concentrations. Therefore, we expanded our scope of study by developing a 3D multicellular model to capture the tissue-level spatial dynamics.The single-cell model provided insights into the WUS binding dynamics with individual cis-elements, Pol II recruitment, and minimum cooperativity mechanisms required for CLV3 expression pattern throughout the tissue by simulating the stochastic single-cell model in individual cells simultaneously at different WUS concentrations. The 3D model was constructed on the basis of the framework used in our previous work , three cis-elements (DM), and single cis-element . The behaviors of several cis-element mutants are shown in CLV3 expression in wild type was generally higher than in other mutants, similar to the experimental data shown in CLV3 activation was highest in the L1 layer and lowest in the inner layers of RM. 970M showed a higher expression in the inner layers of RM than in the outer L1 layer. Of particular interest was 970M4, in which the affinity was strengthened over the default 970 affinity, expressed at a lower level in all cell layers. When simulating the mutants with a single functional cis-element in the CRM, e.g., 950i, 970i, 997i, 1007i, and 1060i, the CLV3 expression was detected in only the inner layers of RM. Other than the minor difference in the magnitude, all single cis-element mutants expressed only in the inner cell layers  had a relatively minimal effect on CLV3 activation  regardless of their WUS binding affinity . These results show that WUS dimerizes in cells that accumulate higher levels of WUS, supporting the correlation observed in biochemical analysis. However, the observed dimerization in BiFC assays does not distinguish between DNA-bound WUS complexes and unbound complexes. Moreover, it likely represents WUS complexes with cis-elements of many target genes  assays in plants expressing split enhanced green fluorescent protein (eGFP)–WUS constructs expressed from the native  in SAMs . A highe in SAMs . This suCLV3 expression. To better understand the role of cooperativity in the robust regulation of CLV3 expression quantitatively, we imposed different levels of cooperativity between monomers or dimers for both wild-type and mutant conditions. A complete removal of cooperativity led to a higher CLV3 expression in the inner cell layers of RM and a lower expression in outer cell layers of CZ under all conditions  leads to down-regulation of CLV3 expression, which could be due to a higher cooperativity among cis-elements leading to the repression. To test this hypothesis, we removed cooperativity from 970M4, which led to an increase in CLV3 expression, and the pattern of expression resembled that of wild type   could be attributed to lower dimer cooperativity leading to derepression. Together, these results show that cooperativity plays a critical role in regulating CLV3 expression when all five cis-elements are functional.As shown above, removing the overall cooperativity that includes both the monomer and dimer cooperativity leads to the internalization of (DS-997)  and C. RCLV3. WUS has been shown to activate and repress CLV3 at lower and higher levels, respectively. Our work reveals that the relative affinities of each element, the number of cis-elements, and intervening distance contribute to the collective effect. Moreover, the collective activity of the CRM arises not only because of the individual affinity but also because of cooperative binding of multiple neighboring cis-elements to WUS. WUS was previously shown to form a mixture of monomers, dimers, and oligomers in solution over a wide concentration range . Perhaps this is due to the effects of altered CLV3 signaling on the expression and nuclear accumulation of WUS establishing a new gradient. In the future, coupling the 3D stochastic model of CLV3 transcription developed here with the CLV3 signaling model of the regulation of WUS transcription and the WUS protein dynamics should allow assessment of the influence of different properties of the CLV3 CRM, including the number of cis-elements in regulating the robustness of the WUS gradient.The computational model developed in this study allows us to recreate and, in a sense, verify the plausibility of our mechanistic explanations of experimental results. It was possible to quantify properties that are very difficult to obtain through experimental means such as the residence time of WUS on cis-elements to calibrate the model and visualization of concentration-dependent ratios of WUS monomer and dimer/higher-order complexes on the Plants were grown under continuous light as described earlier in (Description of two computational models developed in this study is provided in detail in the Supplementary Materials. Parameters used in the stochastic single-cell model can be found in tables S1 and S2. Parameters used in the 3D cell-based model can be found in tables S1 and S3.N, and P values are included within each dataset.The source data associated with all experiments are presented in the additional data files. In addition, the means,


Vaccine coverage for 18-month-old children in Canada is often below the recommended level, which may be partially because of parental forgetfulness. SMS text message reminders have been shown to potentially improve childhood immunization uptake but have not been widely used in Alberta, Canada. In addition, it has been noted that language barriers may impede immunization service delivery but continue to remain unaddressed in many existing reminder and recall systems.This study aimed to assess the effectiveness and acceptability of using SMS text messages containing a link to web-based immunization information in different languages to remind parents of their child’s 18-month immunization appointment.The Childhood Immunization Reminder Project was a pilot intervention at 2 public health centers, one each in Lethbridge and Edmonton, Alberta, Canada. Two SMS text message reminders were sent to parents: a booking reminder 3 months before their child turned 18 months old and an appointment reminder 3 days before their scheduled appointment. Booking reminders included a link to the study website hosting immunization information in 9 languages. To evaluate intervention effectiveness, we compared the absolute attendance no-show rates before the intervention and after the intervention. The acceptability of the intervention was evaluated through web-based surveys completed by parents and public health center staff. Google Analytics was used to determine how often web-based immunization information was accessed, from where, and in which languages.Following the intervention, the health center in Edmonton had a reduction of 6.4% (95% CI 3%-9.8%) in appointment no-shows, with no change at the Lethbridge Health Center . The acceptability surveys were completed by 222 parents (response rate: 23.9%) and 22 staff members. Almost all (>95%) respondents indicated that the reminders were helpful and provided useful suggestions for improvement. All surveyed parents  found it helpful to read web-based immunization information in their language of choice. Google Analytics data showed that immunization information was most often read in English , Punjabi , Arabic , Spanish , Italian , Chinese , French , Tagalog , and Vietnamese .The study’s findings support the use of SMS text message reminders as a convenient and acceptable method to minimize parental forgetfulness and potentially reduce appointment no-shows. The diverse languages accessed in web-based immunization information suggest the need to provide appropriate translated immunization information. Further research is needed to evaluate the impact of SMS text message reminders on childhood immunization coverage in different settings. Immunization is a safe and cost-effective intervention that substantially reduces childhood morbidity and mortality . RoutineHaemophilus influenzae type b (DTaP-IPV-Hib) in all provinces or territories, measles-mumps-rubella-varicella (MMRV) in 7 provinces or territories, and pneumococcal conjugate 13-valent (Pneu C13) and hepatitis B in one province or territory each [It is a well-recognized phenomenon that the uptake of infant vaccines exhibits a precipitous drop at the 18-month vaccine doses -7. The vory each .The literature has shown that parental forgetfulness of immunization appointments is a key barrier to 18-month vaccine uptake ,11. FactPrevious research has shown that SMS text message reminders improve childhood immunization uptake ,15, partGiven that the effectiveness of public health interventions is context specific , assessiAlberta is a western Canadian province with approximately 4.4 million residents. The province is divided into 5 zones for the administration of health services by Alberta Health Services (AHS). Routine preschool immunization is delivered exclusively by nurses at public health centers (PHCs).The Childhood Immunization Reminder Project (ChIRP) was a pilot intervention aimed at improving attendance at 18-month immunization appointments by sending SMS text message reminders to parents. ChIRP was implemented at two PHCs: (1) Mill Woods PHC in the Edmonton health zone, which serves an ethnically diverse population in a high-density urban city with a total population of 1.1 million , and (2)Using an automated system (iceAlert), 2 SMS text message reminders were sent to each parent participating in the study. Messages were sent to the primary phone number on file provided by the parents during registration. Most were cell phone numbers, but voice message reminders were sent instead of SMS text messages if the phone number provided was a landline number. The first message was sent 3 months before the child turned 18 months  to remind parents to book the 18-month appointment or to reschedule an existing appointment if needed. The reminders were sent monthly between May 2019 and May 2020 to all parents of children who turned 15 months old in that month and had a postal code within the service area of Mill Woods or Lethbridge PHCs. This reminder included a link to the study website hosting immunization information in 9 languages .The second message was a reminder of the date and time of the booked 18-month appointment, which was sent 3 days before the scheduled visit. This reminder was initiated by AHS in December 2018, shortly before the start of ChIRP, and was, therefore, incorporated into the evaluation. This was an opt-out system . To evaluate the effectiveness of the intervention, we compared absolute no-show rates before (December 2017 to December 2018) and after (August 2019 to August 2020) the intervention in both PHCs using routinely collected administrative data from AHS. No-shows were defined as children who had missed their scheduled appointments, including those who rescheduled missed appointments. We identified all children with 18-month immunization appointments at the PHCs by using provincial patient identification numbers. Children were excluded from the analysis if their appointment was canceled or outside the preintervention or postintervention periods, they had not yet had their appointment, or were aged >24 months. No-show rates were determined by dividing the total number of no-shows by the total number of 18-month immunization appointments at each PHC. The no-show rates at the intervention sites were compared with PHCs with similar client demographics in the same health zone  using a 2-sample proportion test. The analysis was performed using R   and StatParents and PHC staff evaluated the acceptability of the reminder intervention. The PHC staff recruited parents for the web-based survey evaluation during their child’s 12-month immunization appointment, starting in March (Lethbridge) or May 2019 (Edmonton) and continuing until February 2020. Interested parents received an information sheet and consent form (available in 9 languages) that collected their name, child’s name and date of birth, and mobile phone number. There were no eligibility restrictions in terms of age, gender, or other sociodemographic characteristics other than needing a mobile phone to receive the evaluation survey link. In consultation with PHC managers, a parallel survey was sent to all staff who worked at either participating PHC during the intervention period, including nurses and clerical staff. Informed consent was obtained from all the participants.Participating parents were sent a text message containing a link to a web-based survey when their child was 19 months old, after the intended 18-month immunization appointment. Parent surveys were completed between September 2019 and October 2020. The PHC staff were sent the staff survey link via email at the end of the intervention period in December 2020 or January 2021. Survey data were collected and managed using REDCap  tools hosted and supported by the Women and Children’s Health Research Institute at the University of Alberta .Sociodemographic information collected from parents included residential location, whether they were born in Canada, language or languages read, education level, and annual household income. The PHC staff provided the PHC’s location and their job position. Parents were asked about the helpfulness of the reminders, when the reminders should be sent, what actions they took because of the reminders, and whether their child had attended the 18-month immunization appointment. The PHC staff evaluated the helpfulness and impact of the reminders, when and how many reminders should be sent, and which other routine vaccine programs  should be considered for reminders.Both parents and PHC staff evaluated the helpfulness and impact of the web-based immunization information included in the reminder. Google Analytics  was usedThis study was approved by the Health Research Ethics Board of the University of Alberta (study ID: Pro00085642).Throughout the intervention period (May 2019 to May 2020), a total of 3307 booking reminders were successfully sent, including 2885 SMS text messages and 422 voice notifications. A small number of reminders (n=133) were not delivered. Data on the number of 3-day appointment reminders sent were not available because these reminders were sent zone-wide by AHS.After removing those who had not yet had an appointment or had incomplete data, the Mill Woods PHC had 638 appointments for 18-month immunizations during the preintervention period. Of the 638 appointments, 116 (18.2%) were either no-show or initially no-show and then rebooked. During the postintervention period, there were 1508 appointments for 18-month immunizations, with 178 (11.8%) no-shows. Data from the Northeast Edmonton PHC are shown for comparison . BetweenAt Lethbridge PHC, there were 1657 appointments for 18-month immunization during the preintervention period, 186 (11.22%) of which were no-shows. During the postintervention period, there were 1653 appointments and 198 (11.97%) no-shows. Data from the Medicine Hat PHC are shown for comparison. There were no significant differences between preintervention and postintervention no-show rates in the other intervention (Lethbridge) and control site (Medicine Hat).A total of 929 parents consented to participate in the evaluation survey , whereas 107 declined to participate . Of those who consented, 222 completed the parent survey  and 10 declined the survey after receiving it , with a response rate of 23.9% (222/929). Of those who responded, 93.7% (208/222) reported attending the 18-month visit, whereas 6.3% (14/222) reported missing it. A total of 22 PHC staff members completed the staff survey . The number who received the invitation was not available, as the PHC managers were responsible for forwarding the invitation to all staff who worked during the intervention period.As seen in In total, 51.4% (114/222) of the parents surveyed reported receiving the 15-month booking reminder . Of thesAs shown in As shown in In total, 79.3% (176/222) of the surveyed parents reported receiving the 3-day appointment reminder , and 6.8Among the PHC staff, most  reported that the 3-day reminder was helpful . Over haApproximately half  of the parents who received the 15-month reminder reported reading the web-based immunization information . Most paAs shown in According to Google Analytics, ChIRP web-based immunization information pages received 207 unique visits during the intervention period. Immunization information was most often read in English , followed by Punjabi , Arabic , Spanish , Italian , Chinese , French , Tagalog , and Vietnamese .This pilot intervention aimed to assess the effectiveness and acceptability of SMS text message reminders for preschool immunization appointments. Consistent with previous literature ,27, our There was a decline in absolute no-show rates at Mill Woods PHC, which corresponds with other studies ,29 reporOverall, parents reported high acceptability of the 15-month and 3-day message reminders, with almost all stating that they were helpful. Literature has shown that parents often prefer SMS text message reminders over mail or email because of the convenience and timeliness ,30,31. JMost of the surveyed PHC staff stated that SMS text message reminders were helpful, indicating provider support for the intervention. This reflects the readiness to engage parents in positive discussions about childhood immunizations and encourage them to subscribe to reminder services . In addiParents and PHC staff agreed that the best time to send appointment reminders was 3 days before the appointment but had different preferences for the booking (15-month) reminder, with staff preferring the reminder to be sent earlier. This difference in preferences is likely because of staff needing to schedule in advance to accommodate many immunization appointments, whereas parents may not be thinking about the 18-month appointment until their child is 17 months old or may forget the appointment if the reminder is sent too early. Interestingly, the staff at Mill Woods PHC preferred the reminder to be sent earlier than the Lethbridge staff. This may reflect differences in the size of the 2 PHCs; Mill Woods serves a larger urban area and thus requires parents to book in advance, whereas Lethbridge serves a smaller urban population within a rural zone and may accommodate appointments on shorter notice.The web-based immunization information in different languages was also positively received by parents. According to the Google Analytics data, many participants accessed the information in other languages. There is increasing awareness that language barriers impede immunization service delivery, but they continue to remain unaddressed in many existing reminder and recall systems . Our stuNotably, the Google Analytics data showed a different picture of website activity than the parent survey. Specifically, Google Analytics showed more visits to the website (n=207) compared with the number of survey participants who reported accessing the information (n=51). As the link to the website was sent to all parents receiving the intervention, it is possible that nonsurvey participants accessed the information. In addition, Google Analytics showed more diversity in the languages accessed on the website  compared with the parent survey. This may reflect the fact that English-speaking participants may have been more likely to complete the survey than participants whose first language was not English. The diverse languages accessed by parents suggest the need to provide appropriately translated immunization information.Using SMS text message reminders for immunization appointments may be a convenient and cost-effective way of reducing appointment no-shows. The acceptability of the intervention by parents and PHC staff means that there is potential for SMS text message reminders to be implemented for other immunization programs, particularly 2-month and 12-month immunizations, as well as in other provinces. Future research should consider the use of experimental studies to evaluate the impact of SMS text message reminders on immunization coverage following widespread implementation.To maximize the effectiveness of an SMS text message reminder system, it is important to make it appealing to both parents and PHC staff. For example, parents preferred later booking reminders than staff; therefore, perhaps sending both early and later reminders might be a useful compromise.A strength of this study is the diverse perspectives obtained from both parents and PHC staff at 2 different sites in Alberta: one large urban site and one small urban site in a rural area. We were also able to assess the change in no-show rates at both clinics, using data over an extended time , and in comparison with a control site for each. The limitations of this study include a low parent survey response rate , which may be because of the 7-month gap between recruitment and when links to the evaluation survey were sent out. It is possible that parents who responded to the survey differed from nonrespondents. For example, as the survey was conducted in English, non-English speakers were likely to be underrepresented. In addition, a lower proportion of survey respondents did not attend their child’s 18-month immunization appointment compared with the calculated no-show rates at the 2 clinics, which means that our survey likely underrepresented no-shows. However, it is encouraging that the survey respondents had diverse sociodemographic characteristics, such as income. The number of surveyed PHC staff was also low, which may be explained by the increased strain on health care providers during the COVID-19 pandemic . In addiThis study found that parents and staff at the 2 PHCs were highly accepting of the SMS text message reminder system implemented to address the drop in coverage for 18-month immunizations. The intervention reduced the number of missed appointments at the urban intervention site. Findings support the use of SMS text message reminders as a convenient and acceptable method to minimize parental forgetfulness and potentially reduce appointment no-shows.


To analyze if recommendations given before and during the COVID-19 pandemic are still a valid option for patients suffering of ocular allergy (OA).During the last year many doctors and patients requested suggestions for the treatment of OA patients in COVID-19 time. Most of them were given by phone calls and emails following the recommendations given by Scientific Societies.Considering the current multiple problems related to the COVID-19 pandemic, OA has not been considered a priority, even though patients need treatments. Topical antiallergic drugs are still the first option to treat all kind of OA, with the addition of topical corticosteroids in the severe forms of vernal and atopic keratoconjunctivitis (VKC and AKC) even in patients at risk of COVID-19. Topical immunomodulation is still recommended in severe forms of VKC and AKC unless the patient is infected. The number of patients treated with these drugs in our Center was similar than previous years. The risk to have a VKC patient affected by COVID-19 is similar to the general pediatric population but with a lower OR. In 2021, still in COVID-time, the management of OA should follow the previous recommendations with an update due to the risk of infection. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) knows as COVID-19 pandemic posed significant challenges to medical practice and clinical care. The virus is transmittable from asymptomatic, presymptomatic, and symptomatic positive patients, by contact, droplets of saliva, nasal and oral discharge and fomites. Furthermore, the airborne transmission increases the risk of infection for healthcare workers, who routinely visits patients at close distance . All these procedures became difficult during COVID-19 pandemic.The term OA refers to a collection of ocular surface disorders that affect the eyelid, conjunctiva and cornea. Diagnosis and management of OA is a common practice for allergists, pediatricians, rhinologists, ophthalmologists and general practitioners. They are used to manage the most common IgE-mediated forms of OA, intermittent/seasonal and persistent/perennial allergic conjunctivitis (SAC and PAC) , whereas14A literature review was performed in PubMed, using the following Keywords: Allergic Conjunctivitis or Ocular Allergy [AND] COVID19, SARS-CoV-2, coronavirus pandemic. A review of European and International guidelines on the management of allergic patients during the COVID-19 pandemic has been performed. Manual search of the reference lists of selected studies was done and relevant studies identified. No time or language limitations were established. Papers were selected according to the information provided on the title and abstract. Each topic was reviewed and analyzed by all Authors.▪▪]. Considering the numerous problems caused by the COVID-19 pandemic, including the health system crisis and limited access to consultants, it was reasonable to verify whether these recommendations were still applicable. Scientific Societies have expressed recommendations concerning the assessment of allergic conditions during the COVID-19 pandemic as results of experts’ panel opinion, however, without focusing on OA. The recommendations for the management of allergy and asthma have been updated following the COVID-19 pandemic, again without mentioning OA. A recent position paper provides, in nine sections, relevant aspects for the care of allergic patients to maintain high standards in the daily clinical care of patients ensuring necessary safety [▪▪]. The use of telemedicine is considered a useful tool for providing medical advice when physical presence is impossible or limited. However, limitations of this technology are the lack of objective data from physical examination, data security and protection. Although preventive measures for allergic symptoms during the COVID-19 pandemic are similar in most of the allergic diseases, different recommendations may be provided for patients with suspected SARS-CoV-2 infection or diagnosed COVID-19 disease versus noninfected individuals or patients who have recovered from COVID-19 infection. After recovery, standard allergy care should be resumed with prior interdisciplinary consultation, before any further diagnostic or therapeutic procedure [▪▪]. Clinical trials should be continued maintaining all the anti-COVID 19 safety measures [▪▪]. Another expert panel consensus document from the USA and Canada offered a prioritization rational to help guide decision-making in COVID-19 when to limit patient access during the COVID-19 pandemic .Independently from the phenotype of OA, treatment should follow a stepwise approach based on clinical sign and symptoms severity, using topical antihistamines, mast cell stabilizers, or dual-action drugs as the first choice of treatment with the addition of brief courses of topical corticosteroids in the severe forms or long-term use of topical immunomodulators ▪▪,16▪▪.Topical corticosteroids and immunomodulators have the potential to increase local susceptibility, persistence and reactivation of viral infection. Although the presence of SARS-CoV-2 in tears has rarely been detected in infected individuals ,22, conj▪▪].Socio-psychological mechanisms play an important role in the development, exacerbation, and perception of symptoms in allergic patients. The general population is highly sensitive to people exhibiting ocular and respiratory symptoms during the COVID-19 pandemic. This increases the risk of stigmatization of patients with allergies increasing further patients’ psychosocial stress. Thus, optimal medical and psychological care for patients with allergies, including OA, during the COVID-19 pandemic is essential ▪▪.The impact of quarantine on the quality of life, daily activities, psychological health and the recurrence of allergic conjunctivitis were moderate . Males w▪▪]. The panel divided OA patients into different groups defined by different situations: a) not at risk of SARS-Cov-2 infection; b) at risk of SARS-Cov-2 infection; c) current SARS-Cov-2 infection; d) previous SARS-Cov-2 infection. Treatment recommendations were evaluated to confirm agreement with previously published recommendations [▪▪]. The panel opinion confirmed that the treatment recommendations for SAC are still valid despite COVID-19-related problems. Eye rubbing is not recommended in any type of OA, especially in time of COVID-19 and it is essential to prevent this behavior. Topical antiallergic eyedrops were confirmed to be the first choice of treatment  were avoided. The majority of consultancies were operated by phone or E-Mail. Digital health solutions, especially the use of telemedicine, have been previously proposed as a useful tool to provide medical advice remotely . Picture17https://www.azero.veneto.it), knowing the prevalence of VKC in our area, the co-morbidity of asthma and VKC in our area (14.6%) and the COVID-19 prevalence in asthma population . Particular caution is still recommended for the use of topical corticosteroids and systemic immunomodulators since no definitive consensus has been obtained, particularly for patients with current or previous COVID-19 infection.The recent recommendations of for the management of OA ▪ are sti28None.None.There are no conflicts of interest.


The success of vaccination campaigns against COVID-19 infection isvital for moving from a COVID-19 pandemic to an endemicscenario. We aimed to unravel the influence of the riskperception of epidemics along with individual and contextualfactors on adherence to COVID-19 vaccination campaigns in Italyand Sweden.We compared the results of two nationwide surveys carried out inAugust 2021 across four domains of epidemic risk perception:perceived likelihood, perceived impact on the individual andperceived individual and authority knowledge. The roles ofindividual and contextual determinants were also explored.The survey included 2144 participants in Sweden (52.3% women) and2010 in Italy (52.6% women). In both countries, we found thattrust in authorities was one of the main drivers of thisprocess, with two-fold increased odds of being vaccinated. Beinghighly educated and having a higher relative income wereassociated with a higher adherence to the vaccination campaign, whereas a right and centre-right compared with a leftand centre-left political orientation was negatively related tovaccination adherence .Increasing trust in authorities, along with an equal globaldistribution of vaccine doses, can contribute to acceleratingvaccination campaigns around the world and, in turn, to movetowards an endemic scenario. The COVID-19 pandemic has led to unprecedented medical, economic and socialconsequences. Although a wide range of potential treatments has been testedfor COVID-19, efficacy and access remain low . AtpresVaccination programmes are now facing a crucial aspect: people’s willingness tobe vaccinated. Recent surveys have shown that a proportion of the populationin many countries is unwilling to be vaccinated ,8. VacciAt odds with anti-vaccination movements, a low willingness to be vaccinatedagainst COVID-19 infection reflects more complex concerns . IndividGlobally, adherence to vaccination campaigns can contribute to achieve the goalof reducing the risk of COVID-19 outbreaks. However, the world will movefrom a pandemic to an endemic phase only if high-income countries support anequal global distribution of vaccine doses . DespiteUnderstanding the underlying  determinants of vaccineadherence is important in achieving high rates of vaccination againstCOVID-19 globally. Although individual determinants have been widely studied,8 and arA population-based anonymous survey of public risk perception was carried outin Italy and Sweden between 13 and 23 August 2021. The samples wererepresentative of the Swedish and Italian population for sex and age andwere derived from two existing national survey panels  in each country established by the marketing research companyKantar Sifo. In brief, the sample was derived as a random sample amongpanellists and weights  were applied to ensurethat the results were representative of the population. The geographicaldistribution of the sample was representative of the geographicaldistribution of the population by region in both countries, withover-representation of the capital region populations, with a 1/9 samplingratio in Italy and a 4/6 sampling ratio in Sweden. The overall participationrate was 27.5%. The survey explored the public risk perception for nineglobal threats: epidemics, droughts, flooding, wildfires, air pollution,earthquakes, economic crisis, domestic violence and terror attacks.This analytical sample included 2144 participants in Sweden (52.3% women) and2010 in Italy (52.6% women). We considered the public epidemics perceptionof four domains – likelihood, impact on the individual, and individual andauthority knowledge – using a Likert-type scale (from a minimum of 1 to amaximum of 5). Perceived authority knowledge was considered as a proxy ofthe trust in authorities. Each item was standardized for the risk perceptionof the other threats (e.g. perceived epidemics likelihood/mean perceivedlikelihood for the other threats). Vaccination acceptance was defined asself-reported vaccination against COVID-19 with at least one dose.Information on direct experience of an epidemic  and socioeconomic factors, such as employment(yes/no), relative income, university education (yes/no), politicalorientation  were consideredas possible individual-level determinants of vaccination acceptance.The prevalence of vaccination against COVID-19 with at least one dose at theregional level was retrieved from the Swedish and Italian National Institutefor Health database at the time of the survey . CumulatDescriptive statistics were used to summarize the main characteristics of thestudy sample. The associations between domains of epidemics risk perceptionand vaccination acceptance were assessed using logistic regression models.The results were expressed in terms of odds ratios (ORs) and corresponding95% confidence intervals (CIs). The role of country as an effect modifierwas examined with formal interactions. Bivariable and multivariable logisticregression models were used to examine the association betweenindividual-level factors – such as epidemic experience, employment, relativeincome, education and political orientation – as possible determinants ofvaccination acceptance (independent variables). Adjustment for age wasapplied to all models because a vaccine against COVID-19 was offered to allage groups for adults only from June 2021.We ran an ecological analysis to examine if prevalence of vaccination againstCOVID-19 with at least one dose varied according to the extent to which anarea was affected by the COVID-19 pandemic using linear regression modelsstratified by country.Some limitations should be kept in mind. Although national samples should beconsidered representative of the general populations in terms of age and sex, anoverSupplementary Table 1, available online).The sample included 2144 individuals in Sweden (52.3% women) and 2010 inItaly (52.6% women). The prevalence of vaccination against COVID-19infection with at least one dose was 93.5% in Sweden and 85.3% inItaly. The proportion of individuals with employment and universityeducation was higher in Sweden than in Italy . Overall, the perception of authorities’, but notindividuals’, knowledge was associated with a two-fold increased oddsof being vaccinated. In Sweden, there was a positive associationbetween perceived likelihood of epidemics and adherence to thevaccination programme, whereas in Italy the impact of the epidemicswas positively related to COVID-19 vaccination.Supplementary Table 3, available online, for themagnitude of ORs). High relative income and university educationcompared with low income and low level of education gave greater oddsof being vaccinated . Political orientation also influenced vaccination coverage,with right- and centre-right-oriented individuals less adherent to thevaccination campaign than centre-left- and left-oriented individuals. Of note, experience of epidemics was associated with atwo-fold higher likelihood of being vaccinated only in Sweden .n Sweden .A similWe analysed national surveillance data to evaluate the role of contextualfactors. Advancing knowledge about the influence of epidemics risk perception, alongwith individual and contextual factors on vaccination adherence, isfundamental for moving from a pandemic to an endemic global scenario. Here,we show that trust in authority (measured as authority knowledge) andpolitical orientation were among the main drivers of vaccination adherence,in particular in a context as Italy, characterized by a robust authorityresponse to the pandemic. With respect to epidemic risk perception, we foundthat trust in authority drives vaccination adherence more than the risk ofdisease. Perception of trust in authority was strongly associated withadherence to vaccination campaigns in both countries, whereas a relationwith the perceived likelihood of being affected was only observed in theSwedish context. This might be explained by two concurrent phenomena. Summermonths are characterized by a low incidence of COVID-19 infection andplausibly by a lower risk perception of likelihood of the epidemic . Along wWe also found that those who were more educated and had a higher income weremore adherent to the COVID-19 vaccination campaign. Our study shows thatindividuals with centre-right/right political orientations had a lowercoverage for COVID-19 vaccination than individuals with a centre-leftpolitical orientation. This can be plausibly explained by the politicalorientation of the current Swedish and Italian governments – that is,centre-left in Sweden and a national unity government with the right-wing asthe main party in opposition in Italy. A diverging political orientationfrom the national government may plausibly influence the perception of trustin authority. This may be exacerbated by several months of imposed andrecommended restrictive measures , along wBy comparing the results between countries, this study also contributes tounderstanding the role of contextual factors. A lower vaccination rate inthe Swedish regions with a higher cumulative incidence of COVID-19 may beexplained by doctors’ recommendation of waiting few months from the date ofinfection before being vaccinated for individuals with a history of COVID-19infection .Along wA larger population at the regional level and a different immigration patternprevented a comparison of the Swedish and Italian contexts. Although therewas no clear association between cumulative incidence of COVID-19 infectionand vaccination coverage in the Italian context, four of the five Italianregions with the lowest vaccination coverage are autonomous . Along with a self-directed organization of the vaccinationcampaign, the population of the autonomous regions may mistrust centralgovernment as a result of historical reasons.Despite massive efforts to produce and administer vaccines, a proportion of thepopulation is not willing to be vaccinated, even in countries with availablevaccines. We found that trust in authority was one of the main drivers ofthis process. Whether and how governments and public health institutesdirect efforts to improve trust in public authorities is therefore pivotalto increasing COVID-19 vaccination coverage and, in turn, moving from apandemic to a global endemic scenario.Click here for additional data file.Supplemental material, sj-docx-1-sjp-10.1177_14034948221099410 forCOVID-19 vaccine hesitancy in Sweden and Italy: The role of trust inauthorities by Elena Raffetti, Elena Mondino and Giuliano DiBaldassarre in Scandinavian Journal of Public HealthClick here for additional data file.Supplemental material, sj-docx-2-sjp-10.1177_14034948221099410 forCOVID-19 vaccine hesitancy in Sweden and Italy: The role of trust inauthorities by Elena Raffetti, Elena Mondino and Giuliano DiBaldassarre in Scandinavian Journal of Public Health


Modern fiber-reinforced polymer (FRP)-reinforced concrete structures are excepted to achieve superior mechanical performances and long service lives, even in harsh service environments. Hybrid FRP material could potentially meet this goal with its relatively high strength-to-cost ratio. This paper presents an experimental study on the compressive behavior of concrete cylinders confined by a novel hybrid fiber-reinforced polymer (HFRP) spiral. Nine types, forming a total of 27 confined or non-confined concrete cylinders, were subjected to an axial compressive-loading test. Concrete cylinders confined either with different spiral types or different spiral spacings were comparatively studied in the experiment. The results showed that the compressive failure modes and the stress–strain relationships of the HFRP-spiral-confined cylinders were similar to those of basalt-fiber-reinforced polymer (BFRP)-spiral-confined cylinders. The actual fracture strain of the HFRP spiral (tested as a single rod) was larger than that of the corresponding carbon-fiber-reinforced polymer (CFRP) bar, indicating the advantageous composite effect of the HFRP spiral. The maximum strain of the HFRP spiral reached over 70% of its ultimate strain in the cylinders compared to the BFRP spiral, which only reached 50%. Most of the existing models overestimated the ultimate stress and strain of the HFRP-spiral-confined cylinders. Wu’s model was proved to be the most accurate model, yet proper modification was required for predicting the peak strain of the HFRP-confined cylinders. Fiber-reinforced polymer (FRP) composites have been widely used in concrete structures due to their superior anticorrosion properties and high strength-to-weight ratios . ApplyinIn early applications, due to their relatively high cost FRP composites were usually introduced in retrofitting technologies for repairing or strengthening concrete structures. FRP sheets and strips were commonly used to wrap concrete columns to achieve a strong constraint effect for the columns and to enhance their compressive performances ,4. With In terms of fiber type , there are roughly two types of fibers, which either pursue maximum strength or stiffness (as carbon fiber ) but carFrom an operational level, an active confinement method  is a potOther than operational innovation and material innovation for FRP composites, many studies have focused on their design models in concrete structures. For instance, numerous compressive stress–strain models have been proposed for concrete columns confined by common FRP products, such as FRP sheets or FRP tubes composed of carbon fiber or glass fiber . HoweverThis paper presents an experimental study on the compressive behavior of concrete cylinders confined with a novel hybrid FRP (HFRP) spiral. The manufacturing method and mechanical properties of the HFRP spiral are first provided. Nine types, forming a total of 27 confined concrete cylinders, are subjected to an axial compressive-loading test. The effects of the FRP spiral type and spacing on the compressive behavior of the confined concrete cylinders are thoroughly analyzed and discussed. Several representative existing compressive stress–strain models of FRP-confined concrete are collected and evaluated with the test results. Modifications are suggested for a certain existing model to improve its accuracy in predicting the compressive performances of confined cylinders.s refers to the spiral spacing; fρ refers to the transverse reinforcement ratio; cpf and cpε refer to the compressive peak stress and strain of a cylinder with strain-softening characteristics, respectively; and ccf and ccε refer to the compressive ultimate stress and strain of a cylinder with strain-hardening characteristics, respectively.There were three series of cylinders with different reinforcements designed in the test: (1) plain concrete cylinders (pc); (2) basalt-fiber-reinforced polymer (BFRP) spiral a reinforFor cylinder type in cuf and cuε refer to the compressive ultimate stress and strain of a cylinder with strain-softening characteristics, respectively. These characteristics were mainly controlled by the confinement strength and stiffness, which could vary with spiral spacing and elastic modulus, as designed in the current test. The thickness of the concrete cover of the reinforced cylinders (cd) was 15 mm. To fix the position of the HFRP (or BFRP) spirals in cylinders, three epoxy rods  were used as longitudinal reinforcement, as shown in The reinforcement details of the hf and bf cylinders are shown in D) measurements of the cylinders were 300 mm and 150 mm, respectively [The height and diameter , their strength and modulus values  were 29 GPa (standard deviation = 1.7) and 44 MPa (standard deviation = 2.6), respectively. These were the average values tested according to ASTM C469-14 [The concrete cylinders were cured in a natural environment for 28 days before the test. The elastic modulus and compressive strength values of the concrete  (stroke = 25 mm) evenly distributed at 90° around the cylinders were used to capture the loading displacements, as shown in The spiral strain was monitored with two strain gauges (SGs) (gauge length = 2 mm) pasted on the outward surfaces of the spirals and evenly distributed at 180° on the mid-height section of the cylinders, as shown in (1)hf-2 cylinder typeTake hf-2-1 as an example, the failure process of hf-2-1 is presented in (2)hf-4 and hf-6 cylinder typesThe hf-4- and hf-6-type cylinders revealed similar failure processes. Taking hf-4-3 as an example, as shown in (3)hf-8 cylinder typeThe failure process of the hf-8 type cylinder was similar to the hf-4 (or hf-6) cylinder, as shown in The effectiveness of the HFRP confinement could be evaluated by analyzing the strain development mechanisms in concrete cylinders. For well-confined cylinders (such as the hf-2-3 cylinder), the spiral strain developed slowly with the axial strain during the initial loading process due to the relatively small transverse expansion of the concrete cylinder. Once the axial strain reached 0.002, the spiral strain quickly increased with the development of the axial strain. After the axial strain reached 0.004, despite the axial stress stopping due to continuous damage of the concrete, the spiral strain could keep rising  with the axial strain, as shown in In contrast, insufficiently confined cylinders (such as hf-8-2) showed a distinguished development mode of spiral strain, as shown in Due to the elastic properties of FRP materials, their failure in concrete structures is usually characterized by fiber fracture when the ultimate strain is reached. Since the HFRP spirals were composited with basalt fiber and carbon fiber, which have different fracture strains  in different concrete cylinders, as well as the corresponding axial stresses  and axial strains  of the cylinders, are provided in Compared to the mechanical properties of the HFRP spiral (tested as a single rod), its performance in concrete cylinders was more crucial for the evaluation and design of the confined concrete. The recorded maximum spiral strains Existing modelscc′f and cc′ε are the predicted ultimate stress and strain of a confined cylinder with strain-hardening characteristics, respectively; cu′f and cu′ε are the predicted ultimate stress and strain of a confined cylinder with strain-softening characteristics, respectively; co′ε is the ultimate strain of unconfined concrete (=0.0038); lf is the confinement strength as defined in Equation (1); and lE is the confinement stiffness as defined in Equation (2).Five typical existing models were collected in this paper, as shown in ff and 0.5 ff for the HFRP spiral and BFRP spiral, respectively) were applied for the value of ff in Equation (1).Since the actual ultimate strengths of the HFRP spirals and BFRP spirals in the cylinders were obtained through the tests in this paper, the actual ultimate strengths of the spirals in the cylinders  [iTheo. and iExpe. are the theoretical and experimental results of cylinder i, respectively; and n is the total number of cylinders (=3). Considering that the ultimate stress and strain of the spiral-confined cylinders usually happened after concrete cover failure, the experimental results for ultimate stress (or peak stress) were calculated with the ultimate load (or peak load) of a cylinder divided by its core area confined by the spiral (corA), as defined in Equation (4).corD is the diameter of the confined core area of the cylinder, which could be calculated with Equation (5).The average absolute error (Err.) , as defi(2)Evaluation for cylinders with strain-hardening characteristicsFor the cylinders revealing strain-hardening characteristics , their ultimate stress and strain were predicted with all of the collected models. The results are listed in Most of the existing models overestimated the ultimate stress and strain of the FRP-spiral-confined cylinders, as shown in Among the collected models, Wu’s model  showed t(3)Evaluation for cylinders with strain-softening characteristicscp′f) and peak stress (cp′ε) (as defined in Equations (6) and (7) [For cylinders revealing strain-softening characteristics , Wu’s model was evaluated with the test results since it was the only model that provided a prediction method for this type of cylinder (with a strain-softening characteristic). In addition, the predicted peak stress ( and (7) , respectThe peak and ultimate stresses predicted by Wu’s model fit relatively well with the test results. The maximum peak stress error equaled 0.07, which indicates that the peak stress of the FRP-spiral-confined cylinders (with strain-softening characteristics) could be precisely predicted. The ultimate stress error ranged from 0.07 to 0.21 in , relativCompared to the accuracy of the stress prediction, the accuracy of Wu’s model showed a significant decline when predicting the peak and ultimate strains of cylinders with strain-softening characteristics. The error ranged from 0.37 to 1.04 for predicting peak strain, which indicated a significant overestimation. Note that the predicted peak strains of the cylinders were close to their corresponding ultimate strains, as shown in (1)The compressive failure mode and mechanism of the HFRP-spiral-confined cylinders were similar to those of BFRP-spiral-confined cylinders, indicating that the design method of HFRP-spiral-confined concrete could be similar to that of common FRP-spiral-confined cylinders.(2)The ultimate strain of the HFRP spiral (tested as a single rod) was larger than that of the corresponding CFRP bar. This proved that an advantageous composite effect could be obtained with the HFRP spiral. The maximum strain of the HFRP spirals in concrete cylinders reached over 70% of the ultimate strain values, relatively larger than that of the BFRP spirals, indicating that an ideal in-service strength could be obtained with the HFRP spiral.(3)Most of the existing models overestimated the ultimate stress and strain of the FRP-spiral-confined cylinders. Wu’s model, derived from the ultimate Poisson’s ratio based on the strain compatibility principle, gave the most accurate prediction, both for cylinders with strain-hardening and for those with strain-softening characteristics.An experimental study on the compressive behavior of concrete cylinders confined by novel HFRP spirals was provided in this paper. The compressive behaviors of HFRP-spiral-confined concrete cylinders were comparatively analyzed and discussed. The following conclusions could be drawn:


Cutaneous melanoma (CM) is the most lethal form of skin cancer if it becomes metastatic, where treatment options and survival chances decrease dramatically. Immunotherapy treatments based on the immunologic checkpoint inhibitors programmed death cell protein 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) constituted a main breakthrough in the treatment of metastatic CM, particularly for the achievement of long-term benefits. Even though it is a very promising therapy, resistance to primary immune checkpoint blockade (ICB) arises in about 70% of CM patients treated with a CTLA-4 inhibitor, and 40–65% of CM patients administered with a PD-1-targeting treatment. Some long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) are implicated in triggering pro- and anti-tumorigenic responses to various cancer treatments. The relationship between lncRNAs, circRNAs and ICB immunotherapy has not been explored in cutaneous metastatic melanoma (CMM). The aim of this pilot study is to evaluate the potential role of circRNA and lncRNA expression variability as pre-treatment predictor of the clinical response to immunotherapy in CMM patients. RNA-seq from 12 formalin-fixed paraffin-embedded (FFPE) samples from the metastatic biopsies of CMM patients treated with nivolumab was used to identify response-associated transcripts. Our findings indicate that specific lncRNAs and circRNAs, probably acting as competitive endogenous RNAs (ceRNAs), are involved in the regulatory networks of the immune response against metastatic melanoma that these patients have under treatment with nivolumab. Moreover, we established a risk score that yields predictions of the overall survival (OS) and progression-free survival (PFS) of CMM patients with high accuracy. This proof-of-principle work provides a possible insight into the function of ceRNAs, contributing to efforts to decipher the complex molecular mechanisms of ICB cancer treatment response. BRAF, NRAS, TP53, PTEN, among others, as well as the relevant pathways involved in its carcinogenesis, such as the MAPK and PI3K/AKT pathways, and the cell-cycle control and telomerase programs. All of them are affected by pathologic somatic mutations in protein-coding genes [Melanocytes are pigment-producing cells in the skin that are derived from the neural crest during embryonic development . Epidermng genes .BRAF mutation [Interestingly, many of these mutations arise early in the clinical process: for example, over 80% of benign nevi already have a mutation . In moremutation ,11. Detemutation . Even thmutation ,14. Sevemutation . Based omutation .Long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) can act as competitive endogenous RNAs (ceRNAs)  and leadA total of 16 metastatic melanoma patients . RNA was extracted with the RNeasy FFPE kit  according to the manufacturer’s instructions.RNA-Seq libraries were prepared using TruSeq Stranded Total RNA Gold  and indexed by IDT for Illumina TruSeq RNA UD Indexes . The libraries concentration was determined by the Qubit dsDNA BR kit , and the size distribution was examined by the Agilent Bioanalyzer . Paired-end reads (75bp × 2) were acquired from the Illumina NextSeq 550 platform  according to the corresponding protocol.CDR1-AS, the most frequent circRNA, was verified by qRT-PCR using a predesigned TaqMan probe in all samples (Hs05016408_s1)   .A quality control of Fastq data from paired-end reads was performed with FastQC. Fastq files were trimmed with a cutoff of Q30. We evaluated five different pipelines to identify and quantify circRNAs reads. CIRI  CIRCExplp-value < 0.1 and an absolute value of log2 (fold change) > 1.5. For the DE analysis of both circRNAs and lncRNAs, the total linear mapped read counts were used for size factor estimation.The DESeq2 pipeline with total mapped reads were used to perform the differential expression (DE) of high-confidence circRNAs and lncRNAs. The DE analysis was based on negative binomial generalized linear models, and the threshold values were set to an adjusted We compared our differentially expressed of circRNAs to those reported in prior research using the circRNA disease databases circ2disease  circad  and circWe constructed a network composed by the circRNAs, lncRNAs and messenger RNAs (mRNAs) that were overexpressed or downregulated in relation to the response to ICB in our cohort, together with the microRNAs that interact with them. The tool Analysis of Common Targets for circRNAs (ACT) , which eFinally, we calculated the strength of the linear association between the ceRNAs  and the differentially expressed mRNAs of the network by the Pearson correlation.www.ingenuity.com/)). Upstream regulator analysis (URA), downstream effects analysis (DEA), mechanistic networks (MN) and causal network analysis (I) prediction algorithms were used to obtain functional annotations and regulatory network analysis [The differentially expressed mRNA genes targeted by predicted miRNAs were analyzed using the Ingenuity Pathways Analysis (IPA) software version 01-20-04  depletion from the FFPE tissue of clinical CMM tumors to find aberrant expressions of these ceRNAs between responders and non-responders to the PD-1 blockade. The raw sequences were processed with five different circRNA pipelines to increase the analysis specificity and sensitivity. Only circRNAs that were found in at least three of the five pipelines were selected for further analyses . Both differentially expressed circRNAs and differentially expressed lncRNAs between responders and non-responders were used to build an ICB-response ceRNA network a. Overalall five a. The diall five b. Howeveall five c. Remarkall five .hsa-ALDH1L2_0014, hsa-CD38_0001, hsa-CD74_0005, hsa-CDR1_0001 and hsa-CPM_0002.To analyze the expression patterns of lncRNAs and circRNAs in relation to response to immunotherapy, we identified the expression profile of dysregulated circRNAs and lncRNAs in eight responders versus four non-responders using transcriptome analysis. In the volcano map, we depict the differentially expressed circRNAs a and lnchsaI-SLIT2, hsa-RP11, hsa-IFI30, hsa-HLA-DRB1 and hsa-CDR1. Interestingly, hsa-CDR1 showed one of the higher ratios and was the most relevant circRNA in term of number of counts. It was transcribed with a total of 2211 counts, distributed in 1644 vs. 567 counts between responders and non-responders, respectively.To better understand the relation between linear and circular expression seeking other possible differences between responders and non-responders, we determined the circular–linear ratio of the differentially expressed circRNAs c. The inp-value < 0.1, from which 58 were found to be downregulated and 54 were found to be upregulated. Differentially expressed lncRNAs were annotated with LncAtlas [Regarding the lncRNAs, 112 were differentially expressed with a fold change of 1.5 and an adjusted LncAtlas .Finally, we were able to group responders and non-responders by their expression of circRNAs and lncRNAs. Hierarchical clustering analysis showed responders discrimination among responders and non-responders for both types of RNAs d,e.Let-7e-5p, miR-1285-3p, miR-6757-3p, miR-877-3p and miR-3689d. The second additional component comprised 154 differentially expressed mRNAs among the responders and non-responders that showed interactions with miRNAs predicted to bind with the differentially expressed ceRNAs. Furthermore, the statistical correlation between the differentially expressed mRNAs regulated by these miRNAs, and the differentially expressed ceRNAs, showed that all correlations were direct, reinforcing the notion of the ceRNAs’ inhibitory role on miRNAs’ action  and 23 circRNAs (21 upregulated and 2 downregulated). Two additional layers complemented the network. One of them consisted of 537 target miRNAs from the miRBase with strongly predicted binding sites to our differentially expressed circRNAs and lncRNAs. Of them, the ones showing the highest prediction values were ’ action . We alsoIn order to gain more quantitative and qualitative insight into the mechanism of the putative regulation of ICB response by ceRNAs, we characterized the biofunctions and diseases associated with the differentially expressed mRNAs that interacted with the differentially expressed ceRNAs, as well as the downstream and upstream modulators. Influence network analysis shows that the DE of the interactome key molecules led to activity changes, mostly in immunological processes. Activated molecules included TNF, IRF, IL27, TLR9, EIB3, TGM2 and IFNG, and inhibited ones included MAPK1 and IL1RN a. ConsisNext, by using the IPA upstream regulator analysis tool applied on the ceRNA network, we can predict upstream molecules and provide a mechanistic network that could explain the observed changes in gene expression. Interestingly, we observed the following activated transcription regulators: SOX11, IRF1, NLRC5 and SMRACA4. NEUROG1 was found uniquely inhibited. Regarding cytokines, IL1RN and IL13 were inhibited and IL27, IFLN1, TNF, IFNG and EBI3  were preFurthermore, to characterize the specific role of ceRNAs in the regulation of the mechanism of resistance to ICB, we sought to identify the pathways aberration related specifically to response genes that are correlated with such regulators. For this, we generated Z-scores of IPA canonical pathways of all differentially expressed mRNAs (DEmRNA) vs. the DEmRNA correlated with ceRNAs (DEmRNA-ceRNA) and vs. the differentially expressed mRNAs not associated with ceRNA (DEmRNA-noceRNA) . It is vMoreover, the gene ontologies and pathways were determined with IPA. The statistical overrepresentation test was used to find the enriched GO terms and pathways by matching our gene list with the human genome. The most relevant biological processes were as follows: leukopoiesis, lymphopoiesis, cell development and lymphocyte homeostasis . p-value of 0.00018. The low-risk patients’ median OS was of 28.49 months , while patients considered high risk had a median OS of 1.84 months   with a log-rank 0.92—NR) a. Concer 0.00018 b. The meIn an attempt to unmask new players in the control of ICB resistance mechanisms, we characterized the association of RNA species of recent annotation with the response to ICB in metastatic CM patients. Therefore, we performed transcriptome analysis of bulk metastatic melanoma tissue to generate information from both tumor and immune cells from a metastatic niche. Our hypothesis was that circRNAs and lncRNAs could regulate the response to ICBs by exerting a sponge function that inhibited specific miRNAs as ceRNAs. In order to test this, we integrated miRNAs that were targets of differentially expressed ceRNAs and targeted of differentially expressed mRNAs from our dataset. The resulting selection of differentially expressed mRNAs targeted by those miRNAs was subjected to a correlation test with ceRNAs. Consistent with our hypothesis, the expression correlations were always direct, suggesting that the increased expression of ceRNAs was aligned with the increased expression of the mRNA, putatively through the inhibition of the corresponding miRNA. Since miRNAs’ expression information is absent from our dataset, our conclusions cannot include the direct association with the miRNAs. In line with several previous reports that have highlighted the importance of immune function in the process of melanoma metastasis and ICB response ,53,54,55On the other hand, the identification of the pattern of pathway aberration that was exclusive to the ceRNAs-associated genes unmasked a modulatory role of the ceRNAs for a subset of specific resistance pathways. This finding implies that ceRNAs can either refine or oppose the effects on drug response processes. This is particularly important for two pathways that are intrinsically related to the ICB response: the natural-killer cell signaling pathway and PD-1–PD-L1 cancer immunotherapy. The dysregulation of the ceRNAs in responders is related to the inhibition of the natural-killer cells, while it ameliorates the inhibition of the PD-1/PD-L1 axis.TNFSF8, TNFRSF9, TNFRSF17, TNFRSF13B, TNFRSF12A and TNFRSF11B) may affect the CTL activity and the lymphocyte infiltration. This supports the relevance of TNF in response to ICB in CMM and contributes to the support of the concept of combining therapies based on anti-TNF and anti-PD-1 in CMM.With all these observations, it can be envisaged that the major disruption of immunological anti-tumor pathways related to ceRNAs and observed in responders denote the crucial role that the tumor immune microenvironment (TIME) plays in the treatment response to ICB. Some lines of evidence indicate that tumor cells can modify the TIME by recruiting immunosuppressive cells, and both circRNAs and lncRNAs are tools that tumor cells can use via extravesicular particles to obtain a favorable TIME, consequently leading to treatment failure. Interestingly, previous reports, regarding both hepatocellular carcinoma and pancreatic cancer, associated specific circRNAs with the response to targeted therapy and linked them to natural killer cell dysregulation . With IPCDR1 circRNA stands out as a potentially implementable biomarker of response. Previous studies have highlighted the relevance of CDR1 in cancer, particularly in the metastatic melanoma process. The main biological process identified to date is sponging miRNA-7, which is well established as a cancer progression marker. Recently, Hanniford D. et al. 2020 described a more complex regulation of this region via the epigenetic silencing of the lncRNA LINC00632 [CDR1 is one of the most relevant differentially expressed circRNAs in terms of abundancy and differential expression in non-responders. Moreover, we observed that the responders tend to have a more homogeneous CDR1 expression, indicating that CDR1 seems to be dysregulated in most of the non-responders. Further studies are needed to validate CDR1 for predicting and monitoring treatment response.With regards to the specific ceRNAs identified in this study, INC00632 . In our Finally, this work has found another utility as a generator of a prognosis prediction model in the context of responses to ICB in metastatic CM. A prognostic risk score has been created for the signature of ceRNAs and used to stratify patients at a high and low risk regarding OS and PFS. The application of this score can be used to predict these outcomes. To our knowledge, this the first time that a differentially expressed ceRNA’s signature could be associated with a prognosis of any ICB treatment.This study is a proof-of-principle work which develops a highly reliable bioinformatic pipeline to identify circRNAs and lncRNAs, as well as expression survival scores. Despite the small sample size and the lack of available validation datasets due to the novelty of the approach, our results and conclusions are compatible with the ceRNA hypothesis and generate a unique response signature that warrant further validation in independent cohorts.All in all, this work provides a novel insight into the modulators of ICB resistance and implies the existence of new players to be considered as prognosis biomarkers and targets to counteract resistance in ICB-treated cutaneous melanoma.In the present study, we characterized the ceRNAs in metastatic melanoma patients treated with ICB to explore the biological role of the ceRNA network on the responses. Our exploratory analysis revealed that ceRNAs can modulate specific ICB resistance processes; therefore, they need to be considered in the complex regulatory scenario of the TIME interactions. Finally, the definition of a Risk score based on the ceRNA expression signature constitutes a potentially useful tool for predicting prognoses in the context of ICB treatment in metastatic CM.


The role of villous atrophy in apoptosis, a distinctive feature of celiac disease, is a matter of controversy. The aim of this study was to determine the apoptosis rate through immunohistochemical staining for M30 and M65 in celiac disease cases. Analytical cross-sectional study in a tertiary-level center.H-scores were compared. Several laboratory parameters were recorded concomitantly, and at the one-year follow-up for celiac disease and potential celiac disease patients. Duodenal biopsies from 28 treatment-naive patients with celiac disease, 16 patients with potential celiac disease, 10 patients with a gluten-free diet and 8 controls were subjected to immunohistochemical staining for the end-apoptotic marker M30 and the total cell death marker M65. H-score for M30 expression between the celiac disease, potential celiac disease and gluten-free diet groups (P = 0.009). There was no significant difference in H-score for M65 expression. There was a positive correlation between the H-score for M30 expression and the anti-tissue transglutaminase immunoglobulin A (anti-tTgIgA) and anti-tissue transglutaminase immunoglobulin G (anti-tTgIgG) levels ; and between the H-score for M65 expression and the anti-tTgIgA and anti-tTgIgG levels . There was no difference between celiac disease and potential celiac disease patients regarding the laboratory parameters selected. There was a significant difference in  The rates of apoptosis and nutritional deficiencies in patients with potential celiac disease were similar to those in patients with celiac disease. It results from increased enterocyte destruction and deficient epithelial cell regeneration. Several studies have mentioned that apoptosis has a role in villous atrophy.M30 is a caspase-cleaved keratin 18 (CK-18) cytoskeletal protein that is known to be a marker for the end of apoptosis. M65 comprises both cleaved and uncleaved CK-18 and is used as a marker for total cell death in situations of both necrosis and apoptosis.In this study, we aimed to investigate the apoptosis rate as a marker of disease severity through immunohistochemical staining for M30 and M65 in the duodenal mucosa of patients with potential celiac disease and patients with a gluten-free diet, in comparison with patients with celiac disease; and to investigate whether there might be any difference between the groups in terms of nutritional deficiency.This was an analytical cross-sectional study and data were collected from the laboratory and pathology archives of Keçiören Training and Research Hospital. Newly diagnosed celiac disease patients, patients with a gluten-free diet and patients with potential celiac disease who came for consultations at our gastroenterology outpatient clinic between 2010 and 2017 were included the study and compared regarding apoptosis rate in the gut. Allpatients were followed up for at least one year. Some selected laboratory parameters  were compared at diagnosis and after one year of follow-up among newly diagnosed celiac disease patients and patients with potential celiac disease.This study was approved by an internal review board on June 9, 2017, under the approval number 43278876-929-441-3137, and informed consent was obtained from all patients.The sample size was calculated using 90% power and 1% margin of error, as described in the study by Shalimar etal., which used 3 patients for villus evaluation and 28 patients for crypt evaluation.The diagnosis of celiac disease was made according to the presence of clinical symptoms with positive results for anti-tTgIgA or anti-tTgIgG and presence of villous atrophy (Marsh 3). All of these patients had previously been receiving a gluten-free diet since receiving their diagnoses. The diagnosis of potential celiac disease was made according to the presence of antibody seropositivity and Marsh 0, 1 or 2 criteria in pathological examinations. Thestudy also included 10 previously diagnosed celiac disease patients who had been using a gluten-free diet for 2-8 years. Sixof these patients had duodenitis and four of them had a normal appearance in endoscopic examinations. Three of these patients had borderline positive antibody serological findings and Marsh0 appearance in pathological examinations. Patients with refractory celiac disease, coexistent systemic diseases, human immunodeﬁciency virus seropositivity or positive stool tests for parasitic infections were excluded.Eight patients with dyspepsia who underwent esophagogastroduodenoscopy were recruited as controls. The individuals included in this control group were firstly matched for age and gender. They all presented negative results from endoscopic examinations. Multiple duodenal biopsy samples were obtained from the bulbus and the second part of the duodenum. All the controls were negative for anti-tTgIgA and anti-tTgIgG. Their laboratory parameters were normal and their duodenal biopsy specimens showed normal morphology. They were diagnosed as having functional dyspepsia and it was confirmed that they were not using a gluten-free diet.Demographic data, serological (serum anti-tTgIgA and anti-tTgIgG levels), biochemical and hematological parameters and clinical and pathological data were obtained from the file records. Serum anti-tTgIgA and anti-tTgIgG levels were measured by means of the enzyme-linked immunosorbent assay (ELISA) . Anti-tTgIgA or anti-tTgIgG antibody levels > 18 U/ml were accepted as positive and levels between 12 and 18 U/ml were accepted as borderline. Upper gastrointestinal endoscopic views of the duodenal folds were recorded .Mucosal biopsies were processed in an automated tissue processor and were set into paraffinized tissue blocks. Hematoxylin and eosin (H&E)-stained slides were prepared from the biopsy samples. Biopsy specimens were considered to be sufficient if at least 3-4 crypts were seen, arranged on the muscularis mucosae. All the duodenal biopsy specimens were evaluated by the same pathologist, who was experienced in celiac disease and was unaware of the patients’ clinical data. The modified Marsh grading system described by Oberhuber was used to grade the mucosal changes for both the end of apoptosis (M30) and total cell death, i.e. apoptosis and necrosis (M65).Poly-l-lysine coated slides were prepared from 4-5 µm sections from the paraffin-embedded blocks and were processed for immunohistochemical (IHC) evaluation. All of these slides were subjected to IHC staining for: 1) the end-apoptosis marker protein M30 (CytoDEATH antibody) for detection of caspase-cleaved cytokeratin 18 neo-epitope M30 ; and 2) the end-apoptosis and necrosis marker protein M65 for detection of uncleaved cytokeratin 18 . A standard overnight IHC staining protocol was applied and the universal secondary antibody  was used. 3,3-diaminobenzidine and hydrogen peroxide were used for immunostaining.H-scores for villi were calculated by multiplying together the stain distribution grade and intensity grade in a single biopsy that contained both villi and crypt epithelium. All the biopsies were examined by one experienced pathologist.The IHC-stained sections were described in terms of the distribution and intensity of the staining of the villi in the mucosal biopsies. The intensity of marker staining was graded as follows: grade 1, pale stain expression; grade 2, moderate stain expression; or grade 3, robust stain expression. The distribution of marker staining was graded as follows: grade 0, from no staining to <10% mucosal area positivity; grade 1, 11%-25% area positivity; grade 2, 26%-60% area positivity; or grade 3, ≥ 61% area positivity. TheStatistical analyses were performed using the computer software Statistical Package for the Social Sciences (SPSS), version 22.0 . Normally distributed continuous variables were expressed as the mean and standard deviation, while non-normally distributed variables were expressed as the median and interquartile range (IQR). Comparisons were made using the Mann-Whitney U test or Kruskal-Wallis test if the distribution of the variables was not normal; and the t test or one-way ANOVA were used if the distribution of the variables was normal. Categorical data were presented as proportions, and the chi-square test or Fisher’s exact test was used to compare proportions in different groups. P-values < 0.05 were taken to be statistically significant.Mucosal biopsies were obtained from 28 patients with celiac disease, 16 patients with potential celiac disease and 10 patients with a gluten-free diet; and from eight patients with dyspepsia who formed a control group for the study. The demographic data and data from some selected laboratory parameters among the patients  are summarized in H-score for M30 expression between the celiac disease, potential celiac disease and gluten-free diet groups (P = 0.009). However, there was no significant difference specifically between the celiac disease and potential celiac disease groups (P = 0.25). The statistical difference found in the overall group comparison was due to differences between the celiac disease and gluten-free diet groups (P = 0.001) and between the potential celiac disease and gluten-free diet groups (P < 0.001). Although there was a difference in H-score for M65 expression, this was not statistically significant (P = 0.053). There was only a significant difference between the potential celiac disease and gluten-free diet group (P=0.04)   and 2.The mean serum levels of ferritin, iron, vitamin B12, 25-hydroxy-vitamin D3, folate and calcium, and the interquartile range (IQR) levels of anti-transglutaminase antibodies for IgA and IgG, were recorded at the time when the mucosal biopsy samples were taken. There were statistically significant differences in these parameters except for the serum vitamin B12 levels between the celiac disease, potential celiac disease and gluten-free diet groups . IncompaH-score for M30 expression and the anti-tTg antibody levels for IgA and IgG . Again, there was a positive correlation between the H-score for M65 expression and the anti-tTg antibody levels for IgA and IgG  (H-scores for M30 and M65 expression and the levels of some laboratory parameters.There was a positive correlation between the ctively)  and4. ThIn the present study, we observed higher expression of the markers for both the end of apoptosis (M30) and total cell death, i.e. apoptosis and necrosis (M65), in patients with celiac disease and potential celiac disease in comparison with controls and patients with a gluten-free diet. Moreover, we did not find any significant difference in the severity of apoptosis and necrosis between patients with celiac disease and those with potential celiac disease.H-scores for M30 and M65 expression and the serum anti-tTg antibody levels.There was a significant difference in nutrient deficiency levels between the patients and the control group. Comparison between the patient groups did not show any significant difference between patients with celiac disease and those with potential celiac disease regarding nutritional deficiencies. In addition, we found a positive correlation between the ,Similar mucosal injuries and nutritional deficiencies were detected objectively in the patients with celiac disease and potential celiac disease in this study. The degree of micronutrient deficiencies did not correlate with the degree of apoptosis. This result was compatible with the findings of Deora etal., who showed that the degree of micronutrient deficiencies in children with celiac disease did not correlate with the degree of villous atrophy or with the serum titers of anti-tTgIgA antibodies.It seems that potential celiac disease patients with high antibody levels (anti-tTg) present dynamic changes at the cellular level beyond what is seen through examination of the intestinal mucosa using optical microscopy. This situation may be the reason why nutritional deficiencies do not correlate with the degree of villous atrophy. Gluten-induced immune-mediated changes can be evaluated by means of apoptotic markers, even without the villous atrophy that is characteristic of celiac disease and the requirement to start using a gluten-free diet. Presence of these changes can also be used as a supportive diagnostic marker. Patients presenting such changes may need to start using a gluten-free diet.Several previous studies have emphasized the importance of a gluten-free diet for achieving clinical and histological improvements in patients with mild enteropathy. Our findings revealed the importance of a gluten-free diet through showing that there was no difference between celiac disease and potential celiac disease patients and that there was a significant difference between patients with potential celiac disease and patients with a gluten-free diet, in terms of some selected laboratory parameters. Interestingly,the hemoglobin  and vitamin B12 levels  of the potential celiac disease patients were significantly lower than those of the celiac disease patients at the one-year follow-up. This may have been due to the large amount of time that was spent on challenge testing and genetic testing to ensure that an accurate diagnosis of celiac disease was made before beginning a gluten-free diet. This may also be another reason why clinicians do not emphasize the importance of a gluten-free diet to patients with potential celiac disease as much as they emphasize it to patients with celiac disease.The most important limitation of our study was the retrospective format. Another limitation of our study was that, although there were patients with a gluten-free diet, there was no evaluation of apoptosis in mucosal biopsy specimens from the same patients after follow-up with a gluten-free diet to evaluate the changes in apoptosis and necrosis. There were no previous studies validating the use of M30 and M65 in patients with celiac disease or intestinal disease that followed a course leading to villous atrophy. Multicenter prospective studies involving large numbers of patients with celiac disease and diseases with villous atrophy other than celiac disease are needed.The rates of apoptosis and nutritional deficiencies in patients with potential celiac disease were similar to those in patients with celiac disease. Moreover, the apoptosis rate correlated with the anti-tTg levels. The apoptosis markers M30 and M65 may be candidate markers for celiac disease, especially for clinical follow-ups on celiac disease patients presenting histological remission.