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


While social distancing was crucial to slow the COVID-19 virus, it also contributed to social isolation and emotional strain. This pilot study evaluated the impact of stand-alone psychoeducational group sessions designed to build social connectedness and space for people to learn about mental health during the pandemic. The study examined if offering the stand-alone group sessions increased uptake of and receptivity to additional mental health services. People had access to free, online group psychoeducational sessions offered by a digital mental health platform company. Sessions were offered to (1) employees who had mental health benefits offered through their employer, and to (2) members of the general public. Session formats included discussions, didactic lectures, and workshops, were facilitated by a mental health provider, and used live video conference technology. Topics included race and identity, stress management, coping with political events, relationship issues, and self-compassion. First-time session registrations were tracked from June 2020 to July 2021 on 6723 participants . Among the employee subsample, 49.5% attended a group session as their first use of any available service on the platform; 52.5% of these employees sought additional services after their first session. In anonymous post-session surveys of employees and members of the general public, 86% of respondents endorsed knowledge increases, 79.5% reported improved understanding of their mental health, 80.3% endorsed gaining actionable steps to improve mental health, 76.5% said that they would consider group sessions in addition to therapy, and 43.5% said that they would consider group sessions instead of therapy. These results suggest that scalable, brief group psychoeducational sessions are a useful conduit to mental health care and have potential to reach people who may not otherwise access available mental health services. When the COVID-19 pandemic began, loneliness and social isolation drastically increased as a result of the physical distancing methods introduced to slow the spread of the virus . We tracked available information from all people who registered for sessions from June 2020 to July of 2021 when data were extracted for analysis. The data used in analyses were originally designed for programmatic operations and not originally intended for research purposes. Available data included email addresses from registrations, platform utilization, and post-session survey ratings  registration for online, group psychoeducational sessions within the study time frame; (b) aged 18 years or older at the time of registration; (c) access to a smartphone or computer with Wi-Fi or cellular service in order to connect to the online sessions.These study participants could participate in the online group sessions because they received mental health care benefits from their employer. Employees had access to the digital mental health platform offered by the company, and that access included the online group sessions. Employees could register for the online group sessions either through a public webpage on the website of the mental health platform company, or they could establish a personal account on the platform and register through the platform portal. Unique email addresses were used to track employee registrations through either route.In addition to sessions provided to the benefits-eligible employees, the mental health company offered online group sessions free-to-the-public as a corporate social responsibility action during the COVID-19 pandemic. A public-facing web page described the sessions and had a registration link. The only information collected for the free-to-the-public sessions was the email address used for registration. Given the constraints on the data, these session registrants were included in limited analyses described in the “Benefits-eligible employees learned about the sessions through direct communications sent from their employer and/or the platform company, primarily through direct email marketing and social media posts. Public registrants learned about the existence of the free sessions through mass media communications sent from the mental health platform .The online, group psychoeducational and support sessions (named “Circles”) are live community sessions delivered online. The sessions were initiated in response to the impact of social distancing restrictions imposed to slow the spread of the COVID-19 virus. The intent of the sessions was to create a forum for people to virtually connect with others and learn about and discuss their mental health during the COVID-19 pandemic. Sessions addressed a variety of topics such as follows: coping with uncertainty, race and identity, coping with racism, caregiving, parenting, improving sleep, improving health habits, stress management, coping with political events, relationship issues, self-compassion, building resilience, and combating negative thoughts. Session formats included live discussions, didactic lectures, and practical workshops. All sessions were facilitated by a mental health provider who was either a licensed clinician or certified professional coach in the network of providers managed by the mental health platform company. Individuals could register for an unlimited number of sessions, and all were offered at no charge to the attendee.Sessions were designed to be stand-alone, meaning that they were designed to be self-contained, and not expected to be taken in a specific sequence. People could attend as many sessions as they wanted, and could repeat sessions that were offered more than once. Topics were repeated throughout the study time frame, and the dates/times and facilitators varied throughout the year. The variety of session topics, formats, dates, and facilitators was intentional in order to provide people options that were appealing and accessible to them. Session durations ranged from 60 to 90 min. As the sessions were launched during the height of COVID-19 pandemic social restrictions , all sessions were conducted online using videoconferencing technology . The large didactic sessions could accommodate hundreds of participants and encouraged participants to ask questions through the chat feature. Workshops and discussions were intentionally smaller and could accommodate up to 50 participants; participants were encouraged to use their video cameras and audio connections to interact with the facilitator and other attendees during these sessions.The online group sessions are a component of a comprehensive digital mental health platform. The full platform of services was only available to benefits-eligible employees and not to the general public attending the free-to-the-public sessions. The platform was available to employees through an app. As described in more detail in the “Employees created an account on the platform with their email address and password of choice. The app surfaces questions that assess each individual’s clinical needs and care modality preferences to tailor care recommendations. Based on an individual’s baseline clinical severity and preference for care, recommendations are made to tailor services to meet their needs. Employees can follow the recommendations to connect to care, or can choose their own services if desired. In addition to the group sessions, options for care include the following: self-guided digital content ; one-to-one telehealth sessions  and in-app messaging with a licensed mental health specialist or certified professional coach; and the online, group psychoeducational sessions. Employees with full access to the platform could use any modalities of care and combine multiple modalities. We specifically focused on employees who utilized the online, group psychoeducational sessions as their first use of the platform.An email address was the only element of data required to register for a group session. These addresses were the primary data source for all session registrations for both the benefits-eligible employees and for the general public accessing the free-to-the-public sessions. The first time a unique email address was used to register for a group session, an initial time stamp was created for that unique user. We then tracked the total number of subsequent session registrations with the same email address during the study period, along with the topics for each session.Additional data from the mental health benefits platform were available only for the benefits-eligible employees. These participants were identified when the unique email address that was used for a group session registration was also associated with a platform account. Platform service utilization and demographic data were extracted from these accounts. To examine engagement in other services on the platform, we tracked use of all available services on the platform relative to the date of the individual’s registration for their first group session, including (1) use of trackable, self-guided digital content ; (2) engagement in one-on-one sessions with either an in-network coach; or (3) therapist; and/or (4) registration for additional group sessions. Some elements of the digital content usage were not trackable with timestamps and thus were not accessible for analyses. Demographic data extracted from employee platform accounts included gender  and age in years. No platform utilization data or demographic data were available for public registrants for the free-to-the-public sessions.After each online group session, all attendees were asked to complete a survey of their impressions of the session. The surveys were requested from both benefits-eligible employees and from the general public that attended the free-to-the-public sessions. The surveys were anonymous using a single online survey link, and data could not be linked back to the mental health platform or registration records; therefore, we were unable to sort or separate the data based on whether the respondent was a benefits-eligible employee or a member of the general public. All session attendees were asked to rate the group session using the questions outlined in Table We calculated the total count of session registrations received during the study time frame, the total number of session registrations per unique email address, and separated these counts by registrations from benefits-eligible employees or from the community at large.To examine utilization on the mental health platform for the benefits-eligible employee participants, we calculated the proportion of members for whom the group session was their first activity of any kind on the platform. Among these participants, we calculated the percent that accessed additional mental health services or resources from the platform after their first group session, and the percent that completed coaching sessions, therapy sessions, or accessed additional digital content after their first group session. As we only had demographic data on the benefits-eligible employees, we calculated the percentages of participants identifying as female or male and calculated the average age of these participants.From the post-session survey data, we calculated the total number of surveys returned, a breakdown of the most frequent methods through which people learned about the sessions, and the percentage who endorsed the range of responses to the survey questions. As the surveys were collected anonymously, responses could not be linked to the mental health platform or registration records; therefore, all responses were combined for analyses and not separated based on whether the respondent was a benefits-eligible employee, or a member of the general public.n = 3006) of these registrations came from the general public that had no access to the mental health services offered by the care platform. The other 55% of registrations (n = 3717) came from benefits-eligible employees who also had access to the full digital mental health platform and had a platform account with the identical email address that was used for group session registration. A total of 75 unique session topics were attended during the study time frame. Topics related to personal identity were most popular , with 58% of all registrants (n = 3899) signing up for those group sessions.A total of 6723 unique emails were used to register for an online group session for the first time during the study time frame. Forty-five percent  registered for only one session; 17% (n = 1143) went on to register for a second session; 16% (n = 1076) went on to register for 3 or more additional sessions. The employees who had employer-sponsored access to the mental health platform were more likely to sign up for more than one group session compared to members of the general public, X2  = 166.4, p < 0.001.Among all first-time group session participants , 67%  identified as female, 14% (n = 520) identified as male, and the remaining 41%  did not disclose gender identity. The average age of these participants was 36.9 years (range = 20 to 80 years).Among the benefits-eligible employees, 45% (n = 3717), the group session was the first activity of any kind on the platform for 49.5% (n = 1841). These individuals did not create an account or engage in any other platform care activity until they signed up for their first online, group session. The other 50.5% (n = 1876) of the benefits-eligible employees who signed up for their first group session during the study period had previously set up an account and used at least one other care option before they attended their first online, group session.Among the benefits-eligible employees who registered for their first group session during the study time frame (n = 967) went on to use at least one additional care component of the mental health platform after their first group session. Specifically, 21.9% (n = 213) went on to utilize at least one telecoaching session, 9.3% (n = 90) utilized at least one tele-session with a licensed therapist, and 90% (n = 870) used some combination of digital content  after their first group session.Among the 1841 employees who had not previously used any platform services, 52.5% .When asked how they learned about the group sessions, 31% of survey respondents said that they learned about the sessions from an email sent from their employer, 18% learned from an email sent from the mental health platform company, 17% learned from a public website of the company, 13% learned from the mental health platform mobile app, and 10% learned from a friend or co-worker.When asked about the impact of the group session, 79.5% of respondents agreed or strongly agreed that attending the session increased their understanding of mental health, 80.5% agreed or strongly agreed that the session gave them useful skills, and 80.3% agreed or strongly agreed that the session gave them actionable steps to improve their mental health. When asked about their receptivity towards using group sessions in addition to or instead of therapy, 76.5% said they were somewhat likely or very likely to use the group sessions in addition to therapy, and 43.6% were somewhat or very likely to use group sessions instead of therapy.The results of this pilot study evaluation suggest that online, group psychoeducational sessions were attractive and acceptable to many people during the height of the COVID-19 pandemic, and reached a segment of the population that had not previously accessed mental health care through other modalities . Among the benefits-eligible employees who had full access to the platform mental health resources, over 1800 had not previously utilized their available services before their first group session; the online group session was their first point of entry. The finding that 52.5% of these benefits-eligible employees went on to use other elements of care after their first group session suggests that the sessions may have helped to increase interest in or receptivity to additional care among employees who had full access to mental health services but had not previously used them. Our results are consistent with the extant literature that single online group sessions can serve as an important starting point or conduit to additional care Brown, .instead of therapy, and the majority being open to using groups in addition to therapy. Individual visits with a mental health provider are not necessarily the right solution initially or exclusively for many people’s mental health needs.Less than one-third of the benefits-eligible employees who had not previously used their available services went on to use either therapy or coaching; the majority (90%) used digital resources and/or additional group sessions. This finding suggests that for many individuals, receiving care in a one-on-one setting from a coach or therapist is not necessarily the first step in attending to one’s mental health. The post-session survey responses about attitudes and intentions for additional mental health care add further support to this argument, with almost half (43.6%) of the respondents with survey responses  stating that they would be open to using group sessions Thousands of people from the general public also sought participation in these group psychoeducational sessions. Almost half of the registrations for the online group sessions were from people in the public who did not have access to the mental health platform through their employer. These people would not have received personal email invitations to participate, which implies that they found the group sessions through website searches, word of mouth, or from seeing content on the mental health company website or through company posts on social media. We found that benefits-eligible employees were more inclined to register for additional group sessions than the registrants from the general public; this may be because employees may have been made more aware of their employer-sponsored benefits as a result of the session, and took advantage of the available services afterwards. Given the limited data collected on the public session participants, we do not know if the registrants from the general public had access to mental health services elsewhere. While it is possible, it is also highly likely that access was quite challenging, both due to resource constraints imposed by the pandemic  evidence that online, single sessions of group psychoeducation can be helpful for improving help-seeking attitudes and intentions , but we are not able to differentiate the results to examine this. The registration system for public users did not ask for any personal data beyond an email address, and limits our ability to understand more about the public participants . Ideally, we would want comparable demographic information to examine differences/similarities in who sought out the group sessions, and confirmation of whether or not public participants had access to mental health services elsewhere. There is also a chance that people who had employer-sponsored access to the platform chose to register through the public registrations with different email addresses, and thus, we may have some false-negative cases of public registrations. However, this would result in an under-reporting of cases of people accessing services through the platform; we may be undercounting the people who utilize group sessions and no other modalities, despite having coverage for additional care.These group sessions by design were quite diverse in terms of content topics, dates and times they were offered, formats, and facilitators that supported them. While this breadth of variety has its advantages, it also limits our ability to determine if there are any central, active ingredient(s) of these sessions that produce the desired effects. Additionally, while we were able to track registrations, we did not collect identifiers of session attendees; there is the likelihood that some individuals registered but did not attend. The observational nature of the real-world data also limits any implications about causality. Further studies will need more rigorous designs, control conditions, and intentional isolation of the hypothesized active ingredients to more precisely determine the cause(s) and their magnitude of effect.The post-session surveys were optional, and there is the likelihood of a positive response bias in that those who benefitted the most from the sessions were more likely to complete the optional feedback survey. Additionally, while social connectedness, stigma reduction, and reduced loneliness/isolation are viable outcomes for group psychoeducational sessions, we did not have measurements of these constructs incorporated into the sessions. We have since added a post-session survey question inquiring if the session helped build a sense of community and are working on other ways to collect outcomes of interest on participants without adding burden to their experience. While the data had their limits and challenges, the group sessions have been highly popular and continue to endure. This provides us with future opportunities to improve upon data collection and tracking of individuals to more closely understand how the group sessions have helped guide people towards other elements of mental health care.The results of this evaluation suggest that scalable, brief, online group educational sessions can be a useful conduit to draw people to mental health care and can be an important element in expanding the reach of mental health services to people who may underutilize their available mental health resources. During the height of the COVID-19 pandemic when social distancing measures were widespread, the introduction of these online, group psychoeducational sessions were met with a large volume of interest, and attendees who shared their experiences revealed encouraging signs of their impact . Online group sessions that are primarily psychoeducational and supportive in nature have exciting potential to be a point-of-entry; they can engage people who might not make full use of the mental health services available to them. Furthermore, some may choose to use them as a stand-alone or preferred option if given the choice. They can have a positive impact in improving people’s understanding about their mental health and giving them a sense of direction. They can also empower and encourage people to take further steps to access care for their mental health.