Understanding COVID-19 contagion among poor populations is hampered by a paucity of data, and especially so in remote rural communities with limited access to transportation, communication, and health services. We report on the first study on COVID-19 contagion across rural communities without road access. We conducted telephone surveys with over 400 riverine communities in the Peruvian Amazon in the early phase of the pandemic. During the first wave , COVID-19 spread from cities to most communities through public and private river transportation according to their remoteness. The initial spread was delayed by transportation restrictions but at the same time was driven in unintended ways by government social assistance. During the second wave , although people’s self-protective behaviors (promoted through communication access) helped to suppress the contagion, people responded to transportation restrictions and social assistance in distinct ways, leading to greater contagion among Indigenous communities than mestizo communities. As such, the spatial contagion during the early phase of the pandemic in tropical forests was shaped by river transportation and social behaviors. These novel findings have important implications for research and policies on pandemics in rural areas. In rural remote regions where people have limited access to transportation, communication, health services, and testing for COVID-19, available data are scant which hampers much-needed understanding10.Understanding COVID-19 contagion among poor populations in low- and middle-income countries and people’s reactions to the pandemic and corresponding government policies is critical to design effective policies to address post-pandemic problems and future pandemics2, or about 2.3 times the area of Costa Rica) 16.Our study examined the spread and evolution of COVID-19 during the early phase of the pandemic across rural communities without road access in the Peruvian Amazon. In this remote environment where rivers serve as roads, transportation restrictions could potentially prevent the spread of the virus by effectively isolating communities. Immediately after a national lockdown was lifted, we conducted telephone surveys with over 400 communities in four major river basins—the Amazon, Napo, Pastaza and Ucayali .Our study has three related objectives. First, we assess the spatial contagion of COVID-19 across rural communities relying solely on river transportation. Extant works on the spatial spread of disease highlight the role of human movement through air and ground transportation, especially across and within urban areas, suggesting policies for mitigation targeted towards highly connected areas29. We add to the literature by showing how these two factors shaped the spatial contagion of COVID-19 across rural communities through public and private river transportation in interrelated ways.Second, we examine both government policies restricting human movement  and local people’s self-protective behaviors  which are known to be critical in restricting person-to-person interactions to suppress contagionribereños) are descendants over many generations of Iberian and Indigenous peoples living in the region30. Concerns about the fate of Indigenous peoples, especially in Amazonia, are warranted and have been prominent in media reports and research on COVID-19, especially during early stages of the pandemic, but tend to overlook the larger mestizo population34. Our large-scale community surveys allow us to make comparisons between these two populations across communities, which is uncommon in the broad literature on Amazonia. We find that transportation restrictions and self-protective behaviors shaped the spatial contagion of COVID-19 in distinct ways for these two populations.Lastly, we compare Indigenous and mestizo communities (‘Study area’ in Methods). Mestizos  and a follow-up survey in August 2020 (during the second wave) (‘Surveys’ in Methods). Our analysis sample is a balanced panel of 435 communities  . Mortality was highest early during the first wave, especially in the cities of Iquitos and Pucallpa. A national lockdown was declared on March 16, 2020, lasting until early May, when restrictions were gradually relaxedWe focus on any incidence of COVID-19 case in the community, including suspected ones . With limited health facilities and testing for COVID-19 in the study area , information about confirmed cases is incomplete and data on the number of cases can be inaccurate. Although suspected case incidence may be also inaccurate, any case captures people’s perceived risk of infection in the community that would have underlain their behaviors. The first COVID-19 case had occurred in most communities (91%) by June, i.e., within three months, during the first wave Fig. A. We estWithout road access in the study area, local residents rely solely on river transportation for travelling. The river network distance from cities  varies markedly by community . The timing of initial spread across communities depended on their remoteness: the nonparametrically predicted probability of spread by April and May shows negative steep distance gradients , it took longer for the virus to reach Indigenous communities (until May), but by the time of the baseline survey in July the difference between these two types of communities had vanished . Public river transportation is effectively the only way for local people to travel from remote communities to cities and was available for 80% of all communities (Indigenous and mestizo) before the pandemic ; it was persistent and not prevalent in 18% and 43%, respectively, over time : (1) preventive measures mostly based on individual behaviors  before the baseline survey (mid-March-July), which captures initial adoption, and at the time of the follow-up survey, and (2) social restrictions consisting of community-level restrictions  at the time of each survey . Over time, preventive measures became weaker and social restrictions became somewhat stronger . Both preventive measures and social restrictions became relatively stronger in Indigenous communities than in mestizo communities .Almost three quarters of communities had communication access  . Radiophones are typically available in some communities beyond the reach of cell phone coverage. When we consider these three modes separately, preventive measures at the baseline were stronger in communities with radiophone, but not internet or cell phone access, although social restrictions were stronger in communities with any of these three modes Fig. A. In conThe status of public river transportation changed between the baseline and follow-up surveys: it was inactivated (the restriction which had been relaxed before the baseline was reimplemented) and activated (the restriction which had been maintained at the baseline was relaxed) among 10% and 13% of communities, respectively, and remained active and inactive among 54% and 3%, respectively . These patterns were similar between Indigenous and mestizo communities .We examine the impacts of a change in preventive measures and social restrictions (indices), and activated and inactivated public river transportation (indicator variables) on the mitigation and spread (mainly resurgence) of COVID-19 between the baseline and follow-up surveys . This first difference specification addresses the endogeneity of social behaviors by controlling for time-invariant community heterogeneity including unobserved factors that could cause omitted variable bias.Inactivated public transportation reduced the probability of spread , but did not increase mitigation Fig. B. Thus, Both preventive measures and social restrictions increased mitigation but did not reduce spread  in communities with inactive public transportation, as found for initial spread, but was not related to distance from cities or district capital or to indigeneity .Government cash assistance served as an important safety net for poor households during the pandemicAt the time of the follow-up survey, people had recently received cash assistance in 12% of communities . Cash assistance was more common (by 9%) in Indigenous communities than mestizo communities; it was also more common in communities closer to cities, but was not related to public river transportation  suggests that community leaders with an information advantage could better coordinate people’s adoption of individual behaviors which they were not familiar with . Such information advantage may not have been significant for restricting community-level social behaviors which were common before the pandemic .Poor communication infrastructure is likely to have caused important delays and gaps in information, or misunderstandings about COVID-19The impacts of preventive measures  on the mitigation of contagion were stronger with greater social restrictions (preventive measures), indicating that these two sets of self-protective individual and community behaviors were complementary to each other . The effectiveness of self-protective behaviors was differentiated according to whether public river transportation was activated, but not to whether it was inactivated, as follows. On one hand, preventive measures and social restrictions increased mitigation only in communities with no activated transportation Fig. C, indicaWe conjecture that the impacts of transportation restrictions on contagion were stronger in remote communities because people there rely more on public river transportation for market access. Indeed, both the diminishing effects of activated public transportation on mitigation and those of inactivated transportation on spread were stronger in communities far from cities . Moreover, in remote communities, activated public transportation increased spread and inactivated transportation increased mitigation. We also conjecture that self-protective behaviors were effective to suppress contagion in remote communities because preventive measures and social restrictions were stronger there Fig. A. IndeedAlthough the effectiveness of preventive measures and social restrictions for mitigation was not differentiated by indigeneity, both were effective at reducing the spread in Indigenous communities only Fig. C. This iThree limitations in our study are to be noted. First, as in any survey, we cannot rule out the possibility of measurement errors, though community telephone surveys such as those we conducted better circumvent reporting bias  than standard household telephone surveys (‘Surveys’ in Methods). Second, our data do not allow us to examine the extent to which the different patterns found between Indigenous and mestizo communities may be underlain by contrasting cultural and social norms. Our community-level survey data also preclude us from exploring household-/individual-level factors. Finally, although our analysis sample captures well the dynamics of spatial contagion, it is not necessarily regionally representative (‘Surveys’ in Methods). Concern about external validity has been a common issue with telephone surveys during the pandemic.47.Community-level analysis on case incidence is effective for capturing not only the spatial spread of disease, but also its spatial evolution  and people’s self-protective behaviors. This approach is practical for rural research during pandemics when researchers must rely on telephone surveys to collect data. This approach can be applied also to understand rural people’s vaccination behaviorsThe virus can spread from cities to rural communities through limited river transportation driven by people’s actions for livelihood and coping. Remoteness delays the speed of contagion but does not prevent the spread to rural communities altogether.21. Balancing this health gain against associated economic costs of limited market access and various costs of reduced social connection 2 is a central tradeoff in policymaking in remote rural areas under pandemics.It is possible to significantly delay and suppress contagion across rural communities by restricting their transportation access to cities. Failing to employ such restrictions across cities led to the rapid spread of COVID-19 in Brazil43, who receives information and how it is used may shape the adoption of unfamiliar individual behaviors44. This applies also to communication for promoting COVID-19 vaccination49.Rural people’s self-protective behaviors are also key in suppressing contagion. Although improving communication access can promote such behaviors and facilitate data collection51.The effectiveness of self-protective behaviors will likely depend on the combination of individual and community behaviors, and government policies such as transportation restrictions. Policy makers need to design pandemic policies considering their potential complementarities and possible unintended side effects. Developing a digital payment platform in places where it does not yet exist is essential for the effective and timely distribution of cash assistance while avoiding negative public health externalities52.In pandemics, people in different socio-cultural groups may adopt self-protective behaviors including vaccination and respond to government policies in distinct ways. In Peru, increased vulnerability of Indigenous people is a major concern as found also in BrazilOur study on rural communities without road access in the Peruvian Amazon revealed that COVID-19 contagion during the early phase of the pandemic was shaped by river transportation and social behaviors. Our findings have the following implications for research and policy related to pandemics more broadly.This study was approved by the Research Ethics Board of McGill University (#290–114) and performed in accordance with relevant guidelines and regulations. We obtained informed consent from all participants.53. Iquitos can be reached only by riverboat or by air; Pucallpa has also been connected with Lima, the capital city of Peru, by road since the 1940s. Small towns  which function as district capitals and secondary markets and many smaller communities (100–300 inhabitants) with limited access to public and health services line the main rivers and tributaries. Forest peoples (both Indigenous and mestizo) practice agriculture, fishing, hunting, timber and non-timber forest product gathering, and small livestock raising for subsistence and cash earnings, sending produce to market by boat55.The Departments of Loreto and Ucayali cover about 85% of the area of the Peruvian Amazon Fig.  consistihttps://parlap.geog.mcgill.ca)56. We selected four major river basins—the Amazon, Napo, Pastaza, and Ucayali—to capture the diversity of ecological conditions, economic activities, history, and ethnicity of its peoples 53, maps from the Instituto del Bien Común (IBC) for their census of Indigenous communities58, and Google Earth imagery, supplemented by local enquiries by the teams to identify unmapped settlements. The community survey conducted from December 2012 through March 2014 reached a total of 919 communities , which we estimate represents 92% of all communities in the study area . Each community was geo-referenced using a handheld Garmin GPS unit. The survey collected information through a focus group-based in-person interview among community leaders and elders following a structured questionnaire. We use data from this community survey to construct some variables.Our COVID-19 surveys were part of the Peruvian Amazon Rural Livelihoods and Poverty (PARLAP) project . We subsequently conducted a follow-up telephone survey in August and early September 2020 that reached 435 of the 469 communities in the baseline sample (7% attrition). The analysis sample is the panel sample of 435 communities.Excluding district capitals and communities with a health center from 919 communities covered in the community survey, the remaining 893 communities were eligible for the COVID-19 surveyOur surveys sought information from community leaders following a structured questionnaire. With the suspension of public telephone service since November 2019 and an unreliable radiophone system, we relied mostly on cell phone contact. Our field teams visited ports and markets in Iquitos and Pucallpa to find people from the target communities. Some telephone interviews were arranged through an intermediary when people from the target communities visited a town where the intermediary lived. In these ways the surveys also contacted people in communities with no telephone access.The non-randomly sampled communities in the COVID-19 survey are not representative of the PARLAP study area: communities in the Napo basin  and Indigenous communities are more (less) likely to have been sampled in the baseline survey and be found in the follow-up survey (Supplementary Table 4). The panel sample was not correlated with distance from cities, access to public river transportation, telephone access, and the availability of health facility. These results suggest that the findings from the COVID-19 sample may not be generalizable to the whole PARLAP study area. The limitation of external validity is a common problem of telephone surveys during the pandemic.https://www.poverty-action.org/recovr/research-projects) have been conducted at the household (or firm) level and a small number of projects have included community questionnaires59. Our COVID-19 survey is the first to our knowledge to employ a large-scale community telephone survey.Most surveys in other extant and on-going research projects on COVID-19’s socio-economic impacts in low- and middle-income countries listed at Innovations for Poverty Action (lanchas), small river boats (colectivos), and speed boats (rápidos)36. Large river boats offer regular passenger and cargo service between cities and towns, stopping in selected towns and communities along main rivers, many of which function as intermediary hubs between the cities and other rural communities. Small river boats are more common within a day’s travel from cities and towns for both passengers and cargo; serving as river buses, they run along both main rivers and tributaries which are not accessible by large river boats. In the last decade, powerful speed boats have begun offering regular passenger service along main rivers between cities and towns within a 10-h trip. Travel is about 3–4 times faster and more comfortable than on a large or small river boat but the cost is prohibitive for most rural people. Local private river transportation consists of small personal boats powered by a small air-cooled engine attached to a long shaft and a small propeller (peque-peques).In the region, public river transportation consists of three modes: large river boats  and at the time of the follow-up survey. Our social restriction index is a z-score constructed by taking the first principal component of 7 indicator measures: primary and secondary school closure at the time of the surveys (which takes 1 if there was no school), not playing soccer and volleyball during the previous 7 days, and no gatherings for communal work, community meetings, and church services during the previous 7 days (which takes 1 if there was no church).Yict is outcome variables, such as an indicator variable for case incidence and self-protective index (z-score), of community i in basin c at period t; Xit is a vector of predictors, including interviewer fixed effects which capture interview heterogeneity including potential reporting bias (8 interviewers); ϕc is a vector of basin fixed effects which capture basin heterogeneity (6 basins); and εic is an error term. Inference is based on robust standard errors. Supplementary Fig. 6A shows the distribution of transportation variables used as predictors and Supplementary Table 5 shows the definition and descriptive statistics of other predictors (the construction of transportation variables and some of other predictors is provided below). Full regression results are reported in Supplementary Tables 1, 2, 3, 6, and 7. The number of observations for some outcome variables is slightly smaller than 435 due to missing values.We estimate predictors of outcomes using Ordinary Least Squares (OLS) regression specifications of the formYict is case incidence of community i in basin c at period t ; Zit is a vector of self-protective behavior indices—preventive measures and social restrictions (z-score); Xit is a vector of public river transportation; and εict is an error term. Controlling for time-invariant community heterogeneity—both observed and unobserved factors—this first difference specification allows us to capture the relationships of COVID-19 case with social behaviors and transportation restrictions within communities. The first difference of binary outcomes takes −1, 0, or 1. We consider two binary measures: (1) an indicator variable which takes 1 if the difference takes −1, that is, the original measure decreases from 1 to 0 ; and (2) an indicator variable which takes 1 if the difference takes 1, that is, the original measure increases from 0 to 1 . Analogously, we consider two binary measures for public river transportation: (1) an indicator variable which takes 1 if the original measure decreases from 1 to 0 ; and (2) an indicator variable which takes 1 if the original measure increases from 0 to 1 . Inference is based on robust standard errors. The number of observations is 367 communities with non-missing values in self-protective behavior indices at the baseline and follow-up.We estimate impacts of social behaviors and transportation restrictions on COVID-19 case using first difference specifications of the formWhen we examine complementarity of preventive measures and social restrictions, we add their interaction term to Eq. , estimatAccess to public river transportation—access to at least one public river boat  before the COVID-19 pandemic.Access to large river/small river/speed boat—access to at least one large river/small public river/speed boat before the COVID-19 pandemic.Active/inactive public river transportation—at least one/no large river boat, small public river boat, or speed boat during the previous 7 days at the time of interviews .Active/inactive large river/small river/speed boats—at least one/no large river/small public river/speed boat during the previous 7 days at the time of interviews .Active/frequent local private river transportation—at least one/five small personal boats during the previous day at the time of interviews .60 from Iquitos or Pucallpa (closer one) to each community.Distance from city—River network distance60 from district capital (including one outside the study area) to each community. The analysis sample covered 21 districts.Distance from district capital—River network distance60 from the nearest market town out of 13 (including ones outside the study area) to each community.Distance from nearest market town—River network distance60 from the nearest community out of all other communities covered in the community survey from each community.Distance from nearest community—River network distanceRiver order—A positive whole number to indicate the level of branching in a river system. We consider the rivers on which Iquitos and Pucallpa (the source of virus) are situated as order 1. Specifically, we consider the Amazon, Ucayali, and Marañón rivers as order 1  published by INGEMMET ; available on-line at: https://portal.ingemmet.gob.pe/web/guest/carta-geologica-nacional-escala-1-100-000. Simplified reclassification of INGEMMET’s ‘Soil_NAME’ variable, as soils being formed during the Holocene period (Qh), Pleistocene (Qp), or earlier (Tertiary).Floodplain soil—A proxy for young alluvial soils in floodplain. The proportion of land area in a 5 km buffer centered on the community that is underlain by Holocene parent material. Based on Since most market towns are district capitals, we use either distance from district capital or distance from nearest market town as a predictor. We report results using distance from district capital; those using distance from nearest market town are similar.Supplementary Information.


Examinations form part of the assessment processes that constitute the basis for benchmarking individual educational progress, and must consequently fulfill credibility, reliability, and transparency standards in order to promote learning outcomes and ensure academic integrity. A randomly selected question examination (RSQE) is considered to be an effective solution to mitigate sharing of questions between students by addressing replicated inter-examination questions that compromise examination integrity and sequential intra- examination questions that compromise examination comprehensivity. In this study, a Monte Carlo approach was used to design six examination schemes for the purpose of generating and evaluating 600 RSQEs in order to investigate the effects of RSQE design on replicated inter-examination and sequential and intra-examination questions. Results revealed that the number of randomly selected questions from the pool and the number of sub-pools inversely affected the replication and sequencing of the examination questions. Thus, by designing the RSQE in many sub-pools, in equivalence to the number of examination questions and selecting only one question from each sub-pool, and updating the sub-pools after each examination, the passing of information can be prevented, ensuring the integrity of the examinations. Exams are part of the assessment processes that benchmark individuals’ educational progress and should be conducted in a way that promotes learning outcomes and upholds academic integrity. Ensuring academic integrity within online examinations has become a chief concern for educators. One such way of safeguarding academic integrity is by adopting methods to mitigate rampant breaches of the online examination procedures  online  as an effective solution to address question-sharing  sequential questions to be selected from the same question pool. Having sequential questions from a question pool might be a concern when the examiner follows a patterned order when creating the specific question pool. One scenario is when the first number of questions are derived from one specific lecture/lesson , followed by another set of questions from the next lesson , with this pattern building the entire online examination paper (OEP).Consequently, although active research is currently underway within the field of online examination design, no previous literature has yet focused on the effectiveness of differing RSQE designs to address the issues of replicated inter-examination questions or sequential intra-examination questions. This study aimed to fill this research vacuum using the Monte Carlo approach James, , by condAn empirical study using Monte Carlo analytical approach was implemented to investigate the impact of RSQE design on replicated inter-examination and sequential intra-examination questions.n = 600); (3) results and data analyses.The empirical study was performed in three main steps: (1) six RSQE proposals were designed; (2) 100 RSQEs were generated for each proposal, and results were recorded  were created using the platform’s ‘test tool’. This coding helped in tracking the appearance-list of questions within each generated examination. Although the platform’ test tool’ allows the generation of any question format, this study chose true/false-type questions. Since this study aimed to track replicated / sequential questions, consequently, the question format does not dictate the random-selection process. In addition, the true/false question format was the least complex and most rapid option for this study. Following the generation of the 100 coded questions, sub-pools and six RSQE designs were created, as indicated in Table As previously described, all online-learning management systems allow the generation of RSQEs under different names, and such RSQEs can be applied to any question-type, educational level, and subject. This study employed the ‘Random Block’ (Blackboard®) to generate the RSQE. Initially, 100 questions (Two Questions/Pool Ratio (QPR) were considered in designing the RSQE; 10% QPR - in which the examination consisted of 10 randomly selected questions from a pool of 100 questions - and 5% QPR, in which the examination consisted of five randomly selected questions from a pool of 100 questions. The reason for considering only two QPRs in this study was that a higher percentage would increase the probability of question repetition, and any lower percentage would be a demanding task for an instructor to create a vastly expanded question pool.As shown in Table The six RSQE examination designs were activated on the Blackboard™ platform, and each examination has had 100 attempts. An example of the generated examination is shown in Fig. sequential duplicate questions (SDQ), in which two sequential questions  coincided together in the same examination,sequential triplicate questions (STQ), in which three sequential questions  coincided together in the same examinationsequential quadratic questions (SQQ), in which four sequential questions  coincided together in the same examination.The percentages of replicated inter-examination questions (from the 100 generated examinations for each design) were determined. In addition, the percentages of sequential intra-examination questions were also determined. Sequential questions were categorized into:The ‘descriptive statistics package’ and the ‘histogram package’ of Microsoft Excel® 2016 were used to evaluate the statistics of all 600 examinations. The statistics included mean, median, mode, standard error, standard deviation, sample variance, range, minimum, maximum, kurtosis, skewness, and histogram of frequency.Minimum and maximum values were the lowest-and highest-observed repeated questions, respectively, and can discern if the repetition in questions has a comparable value or else vary significantly. Statistical criteria’ mean,’ ‘median,’ and ‘mode.’ are used to determine the distribution skewness. When mean,’ ‘median,’ and ‘mode’ are equals, the distribution is symmetric. However, when ‘mean’ and ‘median’ are greater than ‘mode,’ the distribution is positively skewed, indicating a flatter right side. Conversely, when ‘mean’ and ‘median’ are less than ‘mode,’ the distribution is negatively skewed, showing a flatter left side. The positive or negative skewing of data distribution can be demonstrated by ‘box and whisker analysis’, which is also helpful in indicating whether there are unusual observations (outliers) in the data set. The difference between ‘box and whisker analysis’ and normal distribution is that in ‘box and whisker analysis,’ the data are distributed in a box in which ‘median value locates in the box  of the data, while box’s right and left edges represents the second (lower 25%) and third quartiles (upper 75%) of the data included. The Left and right whiskers represent the lower and upper outliers.Another helpful statistical indicator is ‘kurtosis,’ which measures the impact of extreme observations / outliers on data distribution. Kurtosis indicates whether the data points scatter in peak or tails. If data points scatter in peak rather than tails, the distribution is (positive kurtosis) or (Leptokurtic) - characterized by heavy tails. However, (negative kurtosis) or (Platykurtic) is characterized by a flat peak - with dispersed data points having lighter tails  How does RSQE design impact replicated inter-examination questions?(2) How does RSQE design impact sequential intra-examination questions?the ratio of selected questions/question pool on the replicated inter-examination and sequential intra-examination questions (QPR)the number of sub-pools on the replicated inter-examination and sequential intra-examination questions.In order to answer each question, two points were considered:.In order to facilitate data analysis and discussion, the six RSQE designs were categorized into two groups, namely the 10% QPR group  and the 5% QPR group , as shown in Table 1The 600 examination trials that were generated, underwent statistical analyses according to the above two categories. Table , the minimum percentage of repeated questions increased proportionally with decreasing of sub-pools and QPP%. Similarly, sample variance and standard deviation that discern the percentage distribution of repeated questions increased proportionally with increasing sub-pools in both QPP% designs. Further to this, kurtosis showed negative values that decrease with increasing of sub-pools in 10%QPR.As shown in Table 2On the other hand, Table The analysis of replicated inter-examination questions assesses how many times each question in the pool was repeated when the design was generated 100 x fold. The higher-replicated questions reflected an increased probability of question-sharing between classmates. The descriptive statistics of the 100 questions (Q1-Q100) in the four designs of 10%QPR and the two designs of 5%QPR are indicated in Table Within the 10%QPR category, the standard deviation for 10% QPR/Design 4 (of one sub-pool) was 3.14%, and this increased proportionally with increasing of sub-pools, to 3.31% in the 10% QPR/Design 1 (of ten sub-pools). This increase in standard deviation from 2.91% to 3.31% reflects the tendency of asymmetrical frequency of replicated questions, by increasing the number of sub-pools within the RSQE. Skewness and kurtosis analyses indicated that this asymmetrical frequency of replicated questions trended towards the frequency with few-replicated questions. The increase of skewness positivity in design 1 compared with designs 2, 3, and 4 indicated that the tendency of few-replicated questions was higher than that of highly-replicated questions.The negative kurtosis in the four RSQE designs of 10% QPR indicates a flat peak with non-significant outliers (Platykurtic distribution). However, the negative value of kurtosis analysis decreased, from 10% QPR/Design 4 to 10% QPR/Design, revealing a decrease in minor outliers. The positive skewness in data distribution was confirmed by box-and-whisker analysis, as shown in Fig. Notably, although the number of sub-pools in 10%QPR did not affect the average frequency of replicated questions, it positively affected having increased numbers of questions with fewer replications, which helped to address the issue of question-sharing between classmates.), and was confirmed by box-and-whisker analysis in Fig. 6.Conversely, in the 5%QPR category, the average frequency of replicated questions was inferior to the 10%QPR category. The standard deviation of 5% QPR/Design 2 (of one sub-pool) was 2.91%, and this increased with increasing the number of sub-pools, to 2.29% in 5% QPR/Design 2 (of five sub-poos), reflecting the tendency of asymmetrical frequency of replicated questions, by increasing the number of sub-pools in the RSQE. Skewness and kurtosis analyses indicated that this asymmetrical frequency of replicated questions trended toward the frequency with few-replicated questions. The two designs were positively skewed, reflecting a higher number of questions with low replicates. A positive ‘kurtosis’ was revealed in 5% QPR/Design 2, reflecting a leptokurtic distribution, characterized by significant outliers. The histogram and probability distribution showed skewness in the data Fig. , and wasConsequently, it can be concluded that the number of questions that are randomly selected from the pool affects their replication at inter-examination level: where lower QPRs lead to less-replicated questions. Moreover, as the number of sub-pools increases, the tendency of increased questions with fewer replications is amplified.), design 2 exhibited few occurrences of SDQ and STD, with an average of 8% and 0.3%, respectively. In contrast, design 1 did not show any sequential questions.The percentage of SDQ, STQ, and SQQ in 10% and 5% QPRs categories are indicated in Table 3Hence, it can be concluded that the number of questions that were randomly selected from the pool affected their sequential questions in each examination; a reduction in QPR leads to reduced sequential questions. Moreover, as the number of sub-pools increased, the tendency of sequential questions was reduced.Ensuring academic integrity within online examinations has become a chief concern for educators who adopt different methods to address the rampant cheating in online examinations. Despite limitations, many of these methods  effectively reduce individual cheating. Notwithstanding, they fail with collective cheating, in which students share exam questions and answers with their classmates or use their collective memory to recall exam questions and pass them to other students who have not yet taken the exam. Due to the development of solid friendships, students experience a sense of ‘group loyalty to their peers, which cause students to excuse collective cheating by claiming that “sharing is caring” and that sharing and giving information is less of an ethical deviation than receiving information, making them feel less ethically detached.Hence, randomly- selected-questions examination (RSQE) can be considered as an effective solution to address question-sharing and interfere with students’ collective memory to recall exam questions. In RSQE, every student gets a differing selection of questions from the question pool - even if the examination allows multiple attempts, each attempt will probably contain a novel selection of questions. However, a large question pool is not synonymous with a reduction in replicated inter-examination questions due to the ‘Birthday Paradox’. Furthermore, online learning management systems do not track the selected questions from a pool, resulting in a proportion of all questions in the question pool might appear to many students. In contrast, other questions do not appear at all. In addition to replicated inter-examination questions, RSQE may result in sequential intra- examination questions that compromise examination comprehensivity.Therefore, this study aimed to design a practical RSQE to mitigate replicated inter-examination and sequential intra- examination questions by applying the Monte Carlo approach on produced 600 RSQE using six examination designs. This study revealed that the number of randomly selected questions from the question pool affected their replication at the inter-examination level: reduced QPR led to reductions in replicated questions. Moreover, as the number of sub-pools increased, the trend of additional questions with fewer replications increased. Furthermore, RSQE design impacted the sequential intra-examination questions - reduced QPR led to fewer sequential questions, and as the number of sub-pools increased, the trend of sequential questions decreased.The instructor is advised to follow the following RSQE design: Create sub-pools equal to the number of exam questions, in which each sub-pool has questions representing 10% of students . Then, pick one question from each sub-pool.The instructor is advised to dedicate a certain number of sub-pools to the required thinking order. For example, suppose the instructor designs his RSQE to be 10 question exam of 60% lower-order questions and 40% higher-order thinking questions. In that case, 6 sub-pools should be dedicated to lower-order thinking questions, including remembering information, demonstrating understanding, and using the acquired information. At the same time, 4 sub-pools should be dedicated to higher-order thinking questions, including analyzing, discovering, and organizing information, integrating knowledge, and making judgments.The instructor should ensure that each sub-pool has questions of the same difficulty level to the student.Although RSQE effectively interferes with collective memory, the instructor is advised to update the sub-pools following each examination period by including new questions and paraphrasing the exhausted questions to prevent passing examination information to the next batch of students whenever proctoring technologies are limited.Reflecting on these results, the instructor can design RSQE to measure both lower-order and higher-order thinking skills. The following strategy is helpful for a proper RSQE design;The RSQE model is considered as a potential solution to mitigate question-sharing between students. Hence, the proper design of RSQE addresses replicated inter-examination and sequential intra-examination questions. By conducting an empirical study through generating 600 RSQEs, this study could address two research questions: (1) How does RSQE design impact the replicated inter-examination question?; (2) How does the RSQE design impact sequential intra-examination questions?. Results revealed that the number of randomly selected questions from the question pool affected their replication at inter-examination level: reduced QPR led to reductions in replicated questions. Moreover, as the number of sub-pools increased, the trend of additional questions with fewer replications increased. Furthermore, RSQE design impacted the sequential intra-examination questions - reduced QPR led to fewer sequential questions, and as the number of sub-pools increased, the trend of sequential questions decreased.In essence, examiners are advised to design the RSQE in many sub-pools, in equivalence to the number of examination questions and selecting only one question from each sub-pool. They are also advised to consider the QPR to be in the 5–10% range. In addition, examiners are advised to consider sub-pools with questions of equivalent difficulty, update their question pools by including new questions and paraphrase the exhausted questions following every examination period, in order to prevent passing examination information to the next batch of students.


Creative arts interventions for people living with dementia have been shown to improve mood, emotions, communication, and relationships for older people living with dementia and their care partners. Previous research demonstrates that TimeSlips, a creative storytelling intervention, provides a “failure-free” environment and an opportunity for individuals to use their imagination. Because it does not involve memory, people living with dementia are encouraged to contribute and interact, thus creating an environment that focuses on dignity and strengths rather than deficits. This case study explores the student facilitators’ experiences of running TimeSlips sessions in different levels of care. The two facilitators ran continuous sessions over six months – with an individual at home, group sessions in memory care, and group sessions in assisted living. The facilitators journaled about their experiences after each session and the same picture prompts were used across the different types of sessions. Each journal entry was coded by two independent researchers using grounded theory principles. Through the coding, it was clear that the facilitators needed to use different skills to engage participants in storytelling based on their level of care. Themes that emerged include joy of connection, playfulness, and engagement. In addition, specific skills are needed to accommodate some behaviors of people living with dementia (such as aggressive behavior toward others in the group) and how to distract from those behaviors to continue with the storytelling. The benefits and challenges of each modality and gaps that may need to be addressed for student facilitators will be discussed.


The generation of conditional alleles using CRISPR technology is still challenging. Here, we introduce a Short Conditional intrON  that enables the rapid generation of conditional alleles via one-step zygote injection. In this study, a total of 13 SCON mouse lines were successfully generated by 2 different laboratories. SCON has conditional intronic functions in various vertebrate species, and its target insertion is as simple as CRISPR/Cas9-mediated gene tagging. In a procedure applicable to many different genes, the celebrated gene-editing technology known as CRISPR can insert a drug-controlled intronic ‘off switch’ into selected genes in mice via one-step zygote injections, allowing fine-tuned investigation of gene function. A research team led by Szu-Hsien Sam Wu and Bon-Kyoung Koo developed the procedure to generate intron-based conditional knock-out alleles. Here, the genetic modification is achieved using CRISPR gene-editing to insert a Short Conditional intrON called SCON (189 bp) into exons of genes. The authors successfully inserted SCON into 13 different genes. SCON is well tolerated in vivo when inserted into essential genes and fully compatible with the available recombinase lines for controlled switching off the gene when and where desired. SCON also shows potential application to other vertebrate species where zygote injection is feasible. Upon the induction of gRNA-directed double-strand breaks (DSBs), the preferred repair pathway, nonhomologous end-joining (NHEJ), often leads to random insertions or deletions (indels), where out-of-frame mutations can cause partial or complete loss of gene function. On the other hand, by using a DNA template, DSBs can also be repaired via homology-directed repair (HDR), which allows the precise knock-in of various sequences into target loci. Due to the versatility and wide applicability of the CRISPR/Cas9 system, it has been utilized in numerous cell lines and lab organisms, across all biological and biomedical research fields2. Recently, cKO alleles have also been generated via the CRISPR/Cas9-mediated insertion of loxP sites in zygotes. However, this approach has turned out to be rather challenging, even with additional refinements3.Despite the revolutionary advancement of CRISPR technology, the generation of conditional alleles has not been as easy as that of knockout or knock-in alleles. A conditional knockout (cKO) approach is often required to study essential genes such as housekeeping or developmentally required genes, as it allows the spatiotemporal control of gene knockout, thereby avoiding the early lethality associated with simple knockout. The use of cKO in rodents and eventually in nonhuman primates will therefore contribute to improved animal welfare. For many years, the Cre/loxP system has been widely utilized to construct cKO alleles by inserting two loxP recombination sites into the introns flanking essential exon(s). The generation of such “floxed” alleles in mice has traditionally involved the use of mouse embryonic stem (ES) cells, which are microinjected into blastocysts to generate chimeric mouse embryos6, but it has not been widely utilized in animal models because the cassettes were either too long5 or led to unexpected hypomorphic effects6. Notably, the simplest form currently is Degradation based on Cre-regulated-Artificial Intron (DECAI)6. Although the short size (201 bp) of the construct employed in this approach makes it desirable for gene targeting via zygote injection, it reduces target gene expression, compromising normal gene expression and animal development. Here, we present a fully optimized Short Conditional intrON (SCON) cassette that shows no hypomorphic effects in various vertebrate species and is suitable for targeting via one-step zygote microinjection.Here, we introduce the use of a universal conditional intron system for cKO approaches suitable for various animal models. Such a conditional intron approach has been attempted in the past, as it enables simple insertional mutagenesis with a fixed universal conditional intronic cassetteAll animal experiments were performed according to the guidelines of the Austrian Animal Experiments Act, with valid project licenses approved by the Austrian Federal Ministry of Education, Science and Research, and were monitored by the institutional IMBA Ethics and Biosafety department.Ctnnb1-SCON (Ctnnb1scon) cKO mice were generated via microinjection at the 2-cell stage, Cdh12-SCON (Cdh12scon) was generated via electroporation at the zygote stage, and the remaining 11 SCON lines  were generated via microinjection at the 1-cell stage. For microinjection, we prepared 25 μl of CRISPR injection mix in nuclease-free buffer , consisting of spCas9 mRNA (100 ng/μl), spCas9 protein (50 ng/μl), sgRNA (50 ng/μl), and ssODN . The mixture was spun down in a tabletop centrifuge at 13,000 × g at 4 °C for 15–20 min to prevent the clogging of the injection needles. Frozen 2-cell-stage embryos of the C57Bl/6JRj background (JANVIER LABS) were used for cytoplasmic injection. For electroporation, a mix of components was prepared in Opti-MEM  including ctRNA 100 ng/μl , SpCas9V3 100 ng/μl and ssODN 50 ng/μl (IDT technologies). Freshly obtained fertilized zygotes were added to the mix and electroporation was performed using NEPA21 (NEPAGENE) in a CUY501P1-1.5 slide, with the following settings: Poring pulse  and transfer pulse .Ctnnb1scon mice were crossed with Vil-CreERT2 mice (B6. Cg-Tg(Vil1-cre/ERT2)23Syr/J, JAX, 020282)7 and bred to obtain either HET  or HOM  mice. Tamoxifen  dissolved in corn oil  or corn oil alone was injected intraperitoneally into 8- to 12-week-old mice at a final concentration of 3 mg tamoxifen per 20 g body weight. Experiments were carried out in 2 mice of the respective genotype for each time point. Control mice received a matched volume of corn oil. On Day 3 or Day 5, mice were euthanized by cervical dislocation, and the intestines were harvested. The intestines were immediately cleaned with 1x PBS, flushed gently with 10% formalin solution , and fixed as ‘swiss rolls’ for 24 h at room temperature. The fixed intestines were washed three times with 1x PBS, with 2–3 h between each wash, before further processing.g. Then, 2–3 μl of the clear part of the solution was used for PCR with either Gotaq  or LongAmp 2X  according to the manufacturer’s instructions. To check the sequences of genomic DNA, PCR bands of the expected sizes were purified with a purification column.Toe clips or ear notches from the offspring were lysed in 30 μl of DirectPCR Lysis Reagent (Viagen) with 1 μl of proteinase-K  at 55 °C overnight. The resulting mixture was diluted with 270 μl of nuclease-free water and spun down for at least 5 min in a tabletop centrifuge at 13,000 × The eG-SCON-FP and eG-DECAI-FP cassettes were ordered from and synthesized by Genscript and subsequently cloned into the pcDNA4TO construct after digestion with BamHI  and XhoI  and ligation with T4 ligase . The vectors were recombined with Cre-expressing bacteria  to obtain the recombined forms. The correct clones were confirmed by restriction digestion with SalI  and Sanger sequencing.5. The mixture was transformed into Escherichia coli, and DNA was extracted from individual colonies and checked with restriction digestion and Sanger sequencing.eGFP cDNA containing SapI recognition sites at the selected intron insertion site was ordered from and synthesized by Genscript and cloned into the pcDNA4TO construct with BamHI and XhoI. Different SCON variant fragments containing the respective complementary ends were then inserted into eGFP with SapI  and T4 ligase during 20 cycles of 2 min at 37 °C and 5 min at 16 °C, followed by 15 min at 37 °C and 10 min at 80 °C incubationsHuman embryonic kidney (Hek) 293T cells were cultured in high-glucose DMEM containing 10% fetal bovine serum , 1% penicillin‒streptomycin , and 1% L-glutamine .8 in high-glucose DMEM  containing 10% FBS (Sigma), 1% P/S, 1% L-glut, 1% NEAA , 1% sodium pyruvate , 0.1 mM 2-mercaptoethanol , and 30 μg/ml of mouse LIF (stock concentration: 2 mg/ml).The mouse ES cell line AN3-12 was cultured as previously describedThe following cell lines were cultured with basal medium supplemented with 10% FBS and 1% P/S. The basal medium for each cell line is indicated in parentheses: C6 ), PK15 ), LLC-MK2 ), Vero ).A total of 500,000–750,000 cells were seeded into 6-well plates and allowed to attach and grow overnight. A total of 2.5 μg of DNA  and 1.5 μg of pcDNA4TO-eGFP, -eG-SCON-FP, or -eG-DECAI-FP or recombined forms of -eG-SCON-FP or -eG-DECAI-FP) was mixed with 8 μl of polyethyleneimine , followed by incubation at room temperature for at least 15 min before being added dropwise to the cells. The culture medium was exchanged 8–10 h after transfection. Thirty-six hours after transfection, the cells were examined under an EVOS M7000 microscope (Thermo Scientific) with brightfield, GFP, and Texas Red filters. At 36–48 h after transfection, cells were dissociated into single cells for flow cytometry analysis with a BD-LSRFortessa flow cytometer (BD). Data from the flow cytometry experiments were analyzed in FlowJo software (BD). The fluorescence intensity values were exported and used for plotting and statistical tests using R.Xenopus laevis eggs were collected and fertilized in vitro, as previously described9. A total of 100 pg of DNA, consisting of 50 pg of pcDNA4TO-eGFP, pcDNA4TO-eG-SCON-FP or pcDNA4TO-eG-SC-FP, and 50 pg of an mCherry-expressing plasmid, was injected into two dorso-animal blastomeres of X. laevis embryos at the 4-cell stage, and imaging was performed approximately 24 h later at the early tailbud stage. For Danio rerio experiments, 12 pg of DNA in total, consisting of 6 pg of pcDNA4TO-eGFP, pcDNA4TO-eG-SCON-FP or pcDNA4TO-eG-SC-FP, and 6 pg of mCherry-expressing plasmid, was injected into each fertilized egg/1-cell stage D. rerio embryo, and imaging was performed approximately 24 h later. X. laevis embryos were imaged using a stereomicroscope  equipped with GFP  and mCherry  filters. D. rerio embryos were imaged with a Stereo Lumar.V12 fluorescence microscope (Zeiss) equipped with GFP  and mCherry  filters.10 and embedded in 15 μl droplets of BME-R1  in a 48-well plate . Organoids were established in WENR + Nic medium consisting of advanced DMEM/F12  supplemented with penicillin/streptomycin , 10 mM HEPES , GlutaMAX , B27 , Wnt3 conditioned medium , 50 ng/ml recombinant mouse epidermal growth factor , 100 ng/ml recombinant murine Noggin , R-spondin-1 conditioned medium  and 10 mM nicotinamide . For the first week of culture, 100 μg/ml Primocin  and 10 μM ROCK inhibitor/Y-27632  were added to prevent microbial contamination and apoptosis, respectively. After the first passage, the established organoids were converted to ENR budding organoid cultures. Organoids were passaged at a ratio of 1:6 using mechanical dissociation.Crypts were isolated from the proximal part of the small intestine as reported previouslyBudding organoids at passage 3 or higher were passaged by mechanical dissociation and seeded in BME droplets. Medium containing vehicle (ethanol) or 500 nM 4-hydroxytamoxifen  was added after BME polymerization. Eight hours later, the medium was exchanged back to ENR and replenished every two days.Samples were processed using a standard tissue protocol on an Automatic Tissue Processor Donatello (Diapath). Samples were embedded in paraffin and cut into 2 μm sections on glass slides. Hematoxylin and eosin staining was carried out according to the standard protocol using a Gemini AS stainer (Thermo Scientific). For immunohistochemistry, the following antibodies were used: rabbit anti-Ki67  and rabbit anti-β-catenin . For signal detection, a two-step HRP-conjugated rabbit polymer system  was used. Stained slides were imaged with a 40x objective using the Pannoramic FLASH 250 III scanner (3DHISTECH), and images were cropped using CaseViewer software.Olfm4 and Wnt3 mRNAs, we performed an RNA in situ hybridization (ISH) assay using an RNAscope® Multiplex Fluorescent Detection Kit v2 (ACDBio 323110). The assay was performed according to the standard manual provided by the ACD company. Briefly, paraffin-embedded samples were freshly cut to a 4 μm thickness on glass slides one week prior to staining, and the slides were dried and stored only at room temperature. On the day before staining, the slides were manually deparaffinized, pretreated, and stored overnight at room temperature for further processing. Duplex staining was performed according to the manufacturer’s manual on the next day with the probes for Olfm4  and Wnt3 . Stained slides were imaged with a 40x objective using a Pannoramic FLASH 250 III scanner (3DHISTECH), and images were cropped using CaseViewer software.To identify g for 5 min. The supernatant and visible fraction of attached BME were removed. The pellet was resuspended in 4% paraformaldehyde and fixed at room temperature for 15–20 min. Fixed organoids were washed 3 times in 1x PBS at 10–15-min intervals. The organoids were blocked and permeabilized in a solution containing 5% DMSO, 0.5% Triton-X-100 , and 2% normal donkey serum  for one hour at 4 °C. The samples were stained overnight with Alexa 647-conjugated mouse anti-β-catenin  and ATTO 488-conjugated phalloidin . The samples were washed three times with 1x PBS. During the last wash, the samples were incubated with 2 μg/ml DAPI before mounting on coverslips in a solution containing 60% glycerol and 2.5 M fructose11, followed by imaging on a multiphoton SP8 confocal microscope (Leica).BME droplets containing organoids were carefully collected into 1.5 ml tubes and spun down in a tabletop centrifuge at 600 × http://www.ensembl.org/)12 for mouse (M. musculus), rat (R. norvegicus), macaque (M. mulatta), marmoset (C. jacchus), and medaka (O. latipes). To map canonical transcripts to genes, we derived ‘PRINCIPAL:1’ from the APPRIS database for mice and rats with Ensembl build 102 (http://appris-tools.org)13, and we regarded the longest transcript as a canonical transcript for other species. The quality features, including GC content, self-complementarity, and mismatch scores, of each candidate site were mapped by the same approach employed by CHOPCHOP (https://chopchop.cbu.uib.no)14. Of note, mismatch scores were calculated using Bowtie (v1.2.2)15 with the ‘-v 3 -a’ option with 23 bp target sequences. The codes will be made available from the corresponding authors upon request.To construct databases of SCON insert sites, we used genomic information on sequences, exons, coding regions, and gene types derived from Ensembl Biomart, build 102 (HBB (hemoglobin subunit beta) gene. SCON is 189 bp long, consisting of (from the 5′ to 3′ end) a splice donor, a loxP recombination site, a branch point, a second loxP recombination site, a polypyrimidine tract, and a splice acceptor. By design, it has a similar sequence architecture to the previously introduced conditional intronic system DECAI (201 bp)6, which showed a hypomorphic effect at the level of protein expression. To optimize conditional intron function, several features were implemented in SCON: (1) the length between the putative branch point and the splice acceptor was kept to 45 bp to allow efficient splicing to take place, (2) the distance between the two loxP sites was 100 bp for efficient Cre-loxP recombination, and (3) miscellaneous changes were incorporated for optimal splice donor, acceptor, and pyrimidine tract sequences. Upon recombination, SCON is reduced to 55 bp, in which all three reading frames contain translational stop codons within the remaining loxP sequence, such that loss of gene function occurs via premature translational termination  of the human ion Fig. .Fig. 1SC6. Thus, our SCON showed a reliable conditional intronic function with no discernable hypomorphic effect.First, we validated the functionality of SCON in an eGFP overexpression construct. We cotransfected HEK293T cells with an mCherry cassette (serving as a transfection control) and a cassette containing either intact eGFP, eG-SCON-FP, or already Cre-recombined eG-SCON-FP (eG-SC-FP) Fig.  and asseNext, we tested whether SCON could be efficiently recombined in mammalian cells. We cloned the eG-SCON-FP construct into a piggyBac transposon backbone and generated clonal mouse ES cell lines with constitutive eGFP expression containing SCON. Cotransfecting a Cre-expressing plasmid with an mCherry cassette into these eG-SCON-FP-expressing ES cells resulted in efficient reduction of eGFP levels within the mCherry+ population, whereas the mock control (transfected only with the mCherry cassette) maintained high levels of eGFP Fig. . Taken tTo better understand the functionality of different components within the SCON cassette, we designed a series of “A-stretch variants” in which 6–10 nt within SCON was converted to adenine Fig. . Among t16, the endeavors of the International Knockout Mouse Consortium17, and, recently, the advent of CRISPR technology18. However, in many other vertebrate species, such as zebrafish, frog, rat, pig, cattle, and nonhuman primate models, the application of the cKO approach has been limited, mainly due to the lack of reliable germline-competent ES cells. We therefore sought to test whether SCON may be a suitable cKO strategy for non-murine species. To this end, we used cell lines from different species, including C6 (Rattus norvegicus), PK15 (Sus scrofa), LLC-MK2 (Macca mulatta), and Vero (Cercopithecus aethiops) cells, and transfected them with eGFP, eG-SCON-FP or eG-DECAI-FP overexpression constructs and the corresponding Cre-recombined forms. In line with the results in HEK293T and mouse ES cells, the SCON intron did not cause any discernable hypomorphic effects in any of the cell lines tested  and zebrafish (Danio rerio) by injecting eGFP, eG-SCON-FP, and Cre-recombined plasmids into 4-cell-stage embryos or fertilized eggs, respectively. We examined the embryos 24 h postinjection. and observed that eGFP and eG-SCON-FP expressed readily detectable eGFP fluorescence, whereas the embryos that received the recombined form did not  showed precise heterozygous integration, with the other allele remaining intact   Fig. . From thon) Fig. , and thetes Fig. . Sox2 mites Fig. , and HETtes Fig. . In the tes Fig. , which aCtnnb1scon allele, we utilized Villin-CreERT2 (Vil-CreERT2) for intestinal epithelium-specific Cre recombination. We first isolated crypts from the duodenum of HOM  and wild-type (Ctnnb1+/+) mice to establish adult stem cell-based intestinal organoids. Then, budding organoid cultures  were transiently treated with 4-OH-tamoxifen for 8 h. Both 4-OH-tamoxifen-treated and untreated wild-type organoids as well as untreated HOM organoids continued to grow normally  and HET  mice and harvested the intestines on Day 3 and Day 5. Control samples showed a normal crypt-villus axis, detectable membrane-bound β-catenin, and a Ki67+ proliferative zone at the bottom of the crypts, where stem and progenitor cells are located  for the analysis of Olfm4 and Wnt3, which are a marker of intestinal stem cells and a marker of a molecule secreted by Paneth cells, respectively. Both Olfm4 and Wnt3 are expressed in uninduced crypts of both HET -(A/T/G)-G-(A/T/G/C))21 splice junction consensus sequences; and (3) exons must be longer than 120 bp in length, where both 5′ and 3′ split exons must be at least 60 bp   Fig. . We alsoge) Fig. . This isIn summary, our SCON-mediated cKO approach transforms the complicated process of generating conditional alleles into the simple, CRISPR-mediated knock-in of a 189 bp intronic sequence through one-step zygote injection. The complete generation of an experimental line with SCON requires 1–2 rounds of breeding. Microinjection or electroporation of targeting components  was first performed in (i) zygotes carrying inducible CreER alleles or (ii) wild-type zygotes Fig. . Founder22. The SCONing approach for generating cKO model organisms would therefore be desirable for studying functions and diseases associated with single-exon genes. Alternative splicing plays an important role in development and can be heterogeneously regulated depending on the cell population23, as revealed by advanced single-cell RNA sequencing technologies24. Being able to conditionally deplete specific isoform(s) would be desirable for further understanding complex context-dependent gene regulation. Targeting the alternatively spliced exon(s) with SCON would, in theory, produce an inactive truncated isoform without compromising the original transcript level  genes Fig. , genes wvel Fig. . Finallyion Fig. .This novel strategy gives rise to the exciting possibility of generating conditional knockouts in other vertebrate models ranging from fish to nonhuman primates. A key to our success is the creation of a neutral artificial intron ready for Cre-mediated inactivation. In contrast to DECAI, which shows undesirable hypomorphic effects that likely limit its utility in cKO animal models, SCON is well tolerated and is ideal for the rapid generation of cKO animals. Moreover, loxP sequences can be replaced by other recombination sites  for the rapid generation of FRT- or other recombinase-based conditional alleles that are not yet widely utilized. Finally, the dispensable region between the two loxP sites serves as a harboring space for the addition of other genetic elements. We expect that the SCON strategy will become the foundation for a new cKO approach in biomedical and industrial research that is well-suited for addressing animal welfare concerns.Supplementary materialsDataset 1Dataset 2Dataset 3Dataset 4Dataset 5Dataset 6Dataset 7Dataset 8Dataset 9Dataset 10


Scanning electron microscopy revealed the uniform size distribution of AgNPs on the graphene surface with minimal to no aggregation. Four main sizes of AgNPs were obtained  via controlling the printing force, z-piezo extension, and dwell time. Energy dispersive X-ray spectroscopy analysis validated the existence of elemental Ag on the graphene surface. Subsequent chemical etching of AgNPs using nitric acid (HNO3) with the aid of sonication and mechanical agitation produced holes of uniform size distribution generating hG. The obtained ID/IG ratios ≤ 0.96 measured by Raman spectroscopy were lower than those commonly reported for GO (ID/IG > 1), indicating the removal of more defective C atoms during the etching process to produce hG while preserving the remaining C atoms in ordered or crystalline structures. Indeed, SPBCL could be utilized to fabricate uniformly distributed AgNPs of controlled sizes on graphene surfaces to ultimately produce hG of uniform hole size distribution.Oxidative chemical etching of metal nanoparticles (NPs) to produce holey graphene (hG) suffers from the presence of aggregated NPs on the graphene surface triggering heterogeneous etching rates and thereby producing irregular sized holes. To encounter such a challenge, we investigated the use of scanning probe block co-polymer lithography (SPBCL) to fabricate precisely positioned silver nanoparticles (AgNPs) on graphene surfaces with exquisite control over the NP size to prevent their aggregation and consequently produce uniformly distributed holes after oxidative chemical etching. SPBCL experiments were carried out  Scanning probe block co-polymer lithography was utilized to fabricate silver nanoparticles on graphene surfaces with spatial and dimensional control. The downstream etching process produced holey graphene with uniformly distributed holes. The latter approach operates via oxidative chemical etching of graphene using either oxidants such as H2O2,6,7 O2,9 nitric acid (HNO3),10 and potassium hydroxide (KOH),11 or catalysts including metal (Ag12–14 and Cu15) or metal oxide 19 nanoparticles (NPs). The catalytic etching process involves NP deposition on the graphene basal plane, etching reaction, and catalyst removal. The deposited NPs could catalytically oxidize C atoms to produce nano-sized holes under specific conditions such as annealing or photochemical/sonochemical treatments. Although graphene is used as a mesh to enhance NP distribution, aggregation still persists,12,20–23 which is a hammering obstacle resulting in heterogeneous etching rates24,25 and thereby producing irregular sized holes.15,26 Therefore, advanced methodologies for fabricating hG with tunable hole sizes are coveted.Among various carbon nanostructures, holey graphene (hG) has attracted considerable interest owing to its exceptional properties including excellent electrical conductivity, large specific surface area, and superior chemical/electrochemical stability surpassing its pristine graphene form.27–30 we hypothesized the use of SPBCL to fabricate hG with enhanced hole distribution. SPBCL might offer an exquisite control over the size and position of the resulting nanoholes on graphene surfaces (Inspired by the successful route of scanning probe block co-polymer lithography (SPBCL) to fabricate precisely positioned single metallic NPs with control over the particle size,surfaces .31 thermal stability (≤600 °C),32 and electrical conductivity33 are the determinant properties that could permit the use of SPBCL to fabricate uniformly distributed AgNPs on its surface. Subsequent chemical etching of patterned AgNPs is expected to create patterned nanoholes on graphene with high uniformity.Graphene hydrophobicity,2 substrates (2.5 × 2.5 cm2)) were obtained from Graphena, Inc., Spain. Graphite in powder form was purchased from Sigma-Aldrich, USA. HNO3 was bought from Panreac Química SLU, Spain. Hexamethyldisilazane (HMDS) was obtained from Alfa Aesar, USA. Poly(ethylene oxide-b-2-vinylpyridine) (PEO-b-P2VP) (Mn: PEO 2800 and P2VP 1500) was obtained from Polymer Source, Inc., Canada. Polyvinylpyrrolidone (PVP) (Mw: 1 300 000) and silver nitrate (AgNO3) were purchased from Sigma-Aldrich, USA. Milli-Q® distilled deionized water (DDW) was used throughout the course of the investigation.Dettol hand soap (DHS)  was bought from a nearby grocery store. Ethanol (EtOH) and hexane were purchased from Fisher Scientific, Ltd., UK. Graphene substrates (monolayer graphene on Si/SiO2 substrates (2 × 2 cm2) and polydimethyl siloxane (PDMS) pen arrays with a 100 μm tip-spacing were purchased from TERA-Print, LLC., USA. In a typical experiment, a Si/SiO2 substrate was vapor primed with an HMDS : hexane mixture (1 : 1 v/v) for 24 h under static vacuum to render the surface hydrophobic.34 Following a procedure developed by Noroozi et al.35 with slight modification, graphene was exfoliated out of graphite (1 mg mL−1) upon mixing with PVP (0.9 mg mL−1) and DHS (12.50 μL mL−1) in DDW (4 mL) with the aid of probe sonication in a continuous mode at 60% amplitude at various sonication times (1–4 h). To self-assemble graphene flakes into a uniform layer, graphene in PVP : DHS (1 mL) was diluted with EtOH (5 mL), sonicated for 15 min, and then injected in a timely manner into DDW (12 mL). The self-assembled layer was next carefully transferred to the HMDS-primed substrate and allowed to dry under ambient conditions. The ink was prepared via dissolving PEO-b-P2VP (5 mg mL−1) in DDW under continuous stirring till a transparent solution was obtained. Subsequently, AgNO3 (0.5 mM) was added and the pH was adjusted to 3 using HNO3. The ink was kept under stirring for 3 h in the dark prior to the electrospraying process. A PDMS pen array was plasma cleaned using a Zepto plasma cleaner  directly before ink deposition via electrospraying using a Spraybase® instrument . SPBCL experiments were carried out on a TERA-Fab M series instrument, TERA-Print, LLC., USA, operating at a temperature of 27 ± 2 °C and a relative humidity (RH) of 92 ± 2%. Patterns of 12 × 12 dots were created with a step size of either 0.5 μm or 1 μm and variable dwell times (s) and z-piezo extensions (μm) while keeping the printing force constant at 200 mN. Annealing was carried out using a tube furnace  having multiple gas ports to attach cylinders of Ar and H2.Si/SiO3 aqueous solution (2.5 M), sonicated, and shaken for predetermined periods of time, after which the substrate was allowed to dry under ambient conditions.Each printed substrate was dipped in an HNOZ (atomic) number. Higher Z atoms scatter more electrons back towards the detector than lower Z atoms and thus create higher signals. For example, the heavier Ag atoms (Z = 47) scatter more electrons than the lighter C (Z = 6) or O (Z = 8) atoms and thus appear brighter in the SEM images recorded using the BED. In contrast, the SED conveys topographic information of the sample surface as it detects secondary electrons originating from the surface or near-surface regions of the sample owing to the inelastic interactions between the primary electron beam and the sample.The samples were analysed using the following characterization techniques: UV-Vis spectroscopy (PerkinElmer spectrometer-Lambda 950), thermogravimetric analysis (TGA) , Raman spectroscopy (Thermo DXR3 Raman Microscope), and scanning electron microscopy (SEM) (JEOL SEM-JSM-IT500HR-equipped with different detectors: an energy dispersive X-ray spectroscopy (EDS) detector, secondary electron detector (SED), and backscattering detector (BED)). The EDS detector is used to measure the spectrum of generated X-rays to determine the elements present in the sample  along with tracking the amount of each element from the intensity of the characteristic X-ray signals . The BED conveys information on the sample's composition wherein the number of backscattered electrons reaching the detector is proportional to the atom's Each SEM image was processed using the SEM software (SEM Operation), wherein the ruler tool was used to quantitatively measure the sizes of all AgNPs/formed holes. A line was drawn by tapping the start point of the target NP/hole and dragging the line to the end point of the target. Once done, the size will be displayed. This process was repeated for all NPs/holes in order to calculate the average size and standard deviation. The relative area of holes to that of graphene was calculated using ImageJ software.via confining NP nucleation and growth, which are primarily affected by the hydrophobicity of the substrate, the printing force, z-piezo extension, and dwell time along with the conditions utilized during the subsequent thermal annealing step.36SPBCL is increasingly becoming a preferred route to fabricate metal NPs owing to its vast capabilities to exquisitely control the size, shape, and position of NPs on solid substrates. SPBCL-defined polymeric nanoreactors govern the size of fabricated features 2 substrates were primed with HMDS via vapor coating of an HMDS : hexane mixture to ensure substrate hydrophobicity.34 To uniformly deposit graphene flakes on HMDS-primed substrates, graphene was first exfoliated out of graphite following a procedure developed by Noroozi et al.35 with slight modification. Briefly, graphite was suspended in a PVP : DHS aqueous dispersion with the aid of probe sonication as shown in  PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C bonds along with a shoulder around 320 nm attributed to n/π* transitions of CO bonds present in graphene oxide.37 A gradual increase in absorption around 270 nm was found with increasing probe sonication time, 3 h of which exhibited a comparable absorbance to that of commercial graphene with a slight red-shifting indicating the preservation of the electronic conjugation within the graphene basal plane.38 Thereby, 3 h-probe sonication was applied hereafter to obtain exfoliated graphene flakes in the PVP : DHS aqueous dispersion. Next, a uniform layer of graphene flakes was deposited on the HMDS-primed substrate via Marangoni self-assembly described by Ye et al.39 Briefly, graphene in PVP : DHS was diluted with EtOH and sonicated for 15 min to ensure the uniformity of graphene flakes, which allowed their injection in a timely manner into DDW. Besides graphene hydrophobicity, the difference in surface tension between EtOH and DDW permitted graphene flakes to float, collide, and self-assemble to ultimately form a continuous layer of graphene in a uniform fashion. This layer was carefully transferred to the HMDS-primed substrate and allowed to dry under ambient conditions (Si/SiOnditions  in ordervia dissolving PEO-b-P2VP in DDW at a concentration of 5 mg mL−1 followed by the addition of AgNO3 (0.5 mM). The pH was adjusted to 3 using HNO3, and the ink was kept under stirring in the dark for 3 h to ensure the complete coordination between the pyridine group and Ag ions. PEO-b-P2VP is expected to form micelles in DDW with P2VP core and PEO shell. The hydrophobic core coordinates with metal precursors while the hydrophilic shell ensures ink fluidity during printing in a high humidity environment. The ink was next electrosprayed on the polymer pen array and allowed to dry prior to mounting on the instrument. The sprayed polymer pen array was incubated for 2 hours in a high humidity environment (92 ± 2%) at 27 ± 2 °C prior to the printing process. This step is necessary to enable ink hydration, spreading, and diffusion, thereby facilitating its transport to the substrate. A 12 × 12 dot matrix design was used to print the ink using 200 mN of force, 1 s of dwell time, and a step size of 500 nm. Printing and patterning parameters are summarized in In parallel to substrate preparation, the ink was prepared 2. The first annealing stage induced AgNO3 particle nucleation and coarsening as the decomposition of AgNO3 wouldn't start unless the temperature reaches ∼285 °C. The second annealing stage initiated reduction and particle growth producing AgNPs29 along with decomposition of PEO-b-P2VP, PVP, and DHS since their decomposition temperatures are 409 °C,40 436 °C, and 289 °C  under sonication for 6 h resulted in no obvious hole formation probably because the holes were too small to be seen and/or the contrast was too weak as shown in The printed substrate was thermally annealed at 150 °C for 16 h under Ar atmosphere and then at 500 °C for another 16 h under HC Fig. S1, respect2 liberation and hole generation. This is generally accompanied by ‘track-marks’ formation owing to the etching-induced motion of AgNPs.12,13 Apparently, the holes' size is 4× larger than the size of AgNPs, which can be attributed to hole merging and/or excessive oxidative etching owing to the prolonged etching time.AgNPs acted as a catalyst to oxidize the surrounding carbon atoms resulting in CO/COvia applying 200 mN of force, varied dwell times (3–6 s), varied relative z-piezo extensions (0–6 μm), and a step size of 1 μm in both X & Y directions as shown in z-piezo extensions and dwell times would influence the size of the resulting AgNPs; the  block would result in fabricating the smallest NPs whereas the  block would account for the largest NPs.After proven successful, the SPBCL approach was re-utilized to fabricate AgNPs of tunable sizes on a CVD-graphene substrate in order to lessen the number of steps and simplify the whole process. The same methodology for ink preparation, electrospraying, and humidification was followed. Yet, a 12 × 12 dot matrix design was used to print the ink dispersion z-piezo extension could increase the tip-substrate contact area and thereby increase the amount of delivered ink. Parallel to this, prolonged dwell times deliver larger amounts of ink to the substrate. Thereby, a tunable gradient of feature sizes was fabricated via varying the relative z-piezo extension and dwell time as evidenced in z-piezo extension and dwell time  with the aid of sonication for 4 h and shaking for another 14 h helped with the guided breakage of the graphene sheet into smaller structures owing to the presence of catalytic AgNP chains specifically in the grafolds  produced either hG sheets  or two-d−1, a G band at ∼1630 cm−1, and a 2D band at ∼2763 cm−1. The obtained ID/IG ratios for hG flakes (0.89) and hG sheets (0.96) were lower than those commonly reported for GO (ID/IG > 1). This plausibly stemmed from removing more defective C atoms during the etching process to produce hG while preserving the remaining C atoms in ordered or crystalline structures despite the increase in the hole diameter.41,42Despite their holey structures, both hG flakes and sheets considerably retained their two-dimensional graphitic crystallinity as evidenced in N-methyl-2-pyrrolidone (NMP) followed by HMDS43 to protect its conductivity during the printing step as well as its hydrophobicity during thermal annealing. This would further enhance the size and distribution of AgNPs on the graphene surface, if needed. The downstream etching step could be optimized to improve the size of holes via adjusting the sonication/shaking time along with the HNO3 concentration or employing oxygen plasma as an alternative etchant.The graphene surface could be primed with 3-induced etching. Indeed, SPBCL could be used to fabricate custom-made metal NP–graphene composites and/or their etched counterparts with spatial and dimensional control, offering a powerful route to nanofabrication and nanomanipulation bypassing the implementation of conventional methods. The produced hG might exhibit promising properties featuring high conductivity and hence it can be used in electronics, energy storage devices, and electro-catalytic reactions. Also, it can be potentially applied in biomedical applications either in the detection or the treatment of diseases.Graphene physiochemical properties including hydrophobicity, thermal stability, and electrical conductivity permitted the use of SPBCL to fabricate uniformly distributed AgNPs on its surface with minimal to no aggregation. Regardless of all encountered challenges, the developed SPBCL approach elicited a precise control over the size and position of the fabricated AgNPs on the graphene surface and hence producing hG of uniform hole size distribution after HNOS. A. A., E. H. A., and A. H. A. designed the research; S. A. A., R. S. F., and S. A. performed the research; S. A. A. and A. H. A. analyzed the data; and S. A. A., E. H. A., A. M. A., and A. H. A. wrote and finalized the manuscript.There are no competing interests to declare.NA-004-D1NA00769F-s001NA-004-D1NA00769F-s002NA-004-D1NA00769F-s003