HIV mobile health  interventions often incorporate interactive peer-to-peer features. The user-generated content (UGC) created by these features can offer valuable design insights by revealing what topics and life events are most salient for participants, which can serve as targets for subsequent interventions. However, unstructured, textual UGC can be difficult to analyze. Interpretive thematic analyses can preserve rich narratives and latent themes but are labor-intensive and therefore scale poorly. Natural language processing (NLP) methods scale more readily but often produce only coarse descriptive results. Recent calls to advance the field have emphasized the untapped potential of combined NLP and qualitative analyses toward advancing user attunement in next-generation mHealth.In this proof-of-concept analysis, we gain human-centered design insights by applying hybrid consecutive NLP-qualitative methods to UGC from an HIV mHealth forum.UGC was extracted from Thrive With Me, a web app intervention for men living with HIV that includes an unstructured peer-to-peer support forum. In Python, topics were modeled by latent Dirichlet allocation. Rule-based sentiment analysis scored interactions by emotional valence. Using a novel ranking standard, the experientially richest and most emotionally polarized segments of UGC were condensed and then analyzed thematically in Dedoose. Design insights were then distilled from these themes.The refined topic model detected K=3 topics: A: disease coping; B: social adversities; C: salutations and check-ins. Strong intratopic themes included HIV medication adherence, survivorship, and relationship challenges. Negative UGC often involved strong negative reactions to external media events. Positive UGC often focused on gratitude for survival, well-being, and fellow users’ support.With routinization, hybrid NLP-qualitative methods may be viable to rapidly characterize UGC in mHealth environments. Design principles point toward opportunities to align mHealth intervention features with the organically occurring uses captured in these analyses, for example, by foregrounding inspiring personal narratives and expressions of gratitude, or de-emphasizing anger-inducing media. The advent of antiretroviral therapy (ART) marked an inflection point in the global AIDS epidemic, transforming HIV into a manageable chronic condition -3. With These persistent challenges suggest that traditional clinic-based treatment programs may be inadequate for fulfilling the needs of many MSM living with HIV. Mobile health  interventions, which offer tools such as informational videos, hyperlocal service guides, and peer-support forums, have shown promise in this domain -17, inclTraditional formative methods -23, ofteThe scale of UGC data can introduce analytic challenges. The extraction of meaningful units of analysis among vast unstructured data is the foremost among those challenges . NaturalNLP techniques are typically incapable of preserving narrative, subtext, and nuance ,50. WithSpecifically, within mHealth, Petersen et al  integratRecent findings point to the relative strengths of the sequential NLP-qualitative approach toward characterizing large-scale UGC, while preserving experiential nuance -55. We aAim 1: To demonstrate the viability of a novel, sequential, NLP-qualitative approach toward characterizing UGC contributed by the end users of Thrive With MeAim 2: To examine the implications of the UGC-derived insights obtained in Aim 1 toward developing user-centered design adaptations for the next generation of HIV mHealth interventionsThrive With Me is a web app–delivered intervention that combines self-monitoring tools for ART adherence, informative multimedia covering ART adherence, and asynchronous peer-to-peer support within a pseudonymous forum with the aim of improving treatment adherence among MSM living with HIV. Its components are grounded in the Information-Motivation-Behavioral skills (IMB) model of health behavior change . An earlParticipants were eligible if they (1) were HIV seropositive, (2) identified as males, (3) had a self-reported detectable viral load or suboptimal (<90%) ART adherence in the past 30 days, (4) reported sex with another man in the past 12 months, (5) could read and write English, (6) resided in the New York City area, and (7) had access to the internet and SMS text messaging for the duration of the study . This stAll study procedures and the use of associated data for secondary analyses were approved by the ethics review boards of the University of Minnesota (#1504S69721) and Hunter College of the City University of New York (#2015-0641).Initially, our procedures relied on the NLP techniques of unsupervised TM and rule-based SA to capture the semantic attributes of UGC drawn from Thrive With Me. We then employed a novel ranking technique to condense the richest and most emotionally polarized UGC. Finally, the detailed insights included in this condensed UGC were explored using the qualitative technique of interpretive thematic analysis. A flowchart of our complete procedure is shown in Textual UGC from Thrive With Me’s peer-support forums were extracted by the web app’s developer, Radiant, as a structured .csv file using the Drupal content management system’s Entity Export CSV function. Original posts and the comments they accrued were handled uniformly (referred to as posts throughout) for the sake of analysis. Content generated by study staff during prelaunch testing was removed manually before preprocessing. With test content excised, the raw UGC corpus contained 4912 posts and 147,649 total words. To accommodate necessary differentiation in the preprocessing steps, 2 UGC corpora were created: the SA corpus and TM corpus.All subsequent data preprocessing and NLP analyses were undertaken in Python  on the Windows 10 (Microsoft Corporation) operating system.In the TM corpus, first, unigram frequencies were calculated, and any unigrams occurring fewer than 3 times were discarded. The 571-term SMART  stop list was applied, excising all unigrams, such as “the” and “of,” terms whose co-occurrences are not typically indicative of the underlying topics from the raw TM corpus ,63. CapiIn the SA corpus, all semantic elements were preserved. In social media environments such as the Thrive With Me forum, peculiarities in syntax may amplify or even invert the intended sentiment of a text  and thus represent important model inputs to retain .All steps in TM were applied to the TM corpus. We used the unsupervised LDA algorithm native to the scikit-learn (“sklearn”) Python library . LDA is Each LDA model was evaluated for coherence by the first and second authors (SJS and SSJ) aided by the pyLDAvis tool. pyLDAvis plots modeled topics in 2 dimensions represented by circles, allowing for visual inspection of intertopic distances  and topic prevalence (how much content within a corpus each topic captures). A satisfactory K is characterized visually by circles with sufficiently large radii to capture a substantive share of a corpus and negligible overlap between circles, indicating discriminant interpretability across topics . DetaileFinally, informed by Schofield and colleagues ,63 and bTopic labels were developed based on domain knowledge, visual inspection of the top 30 per-topic tokens, and their particular distribution and contextual usage within the full series of posts assigned to each topic. Labels were finalized based on consensus between the first and second authors (SJS and SSJ).All steps in SA were applied to the SA corpus using the vaderSentiment library in Python . We usedReplication scripts for VADER SA are provided in Data condensation strengthens an analytic sample by honing it to its richest, most illustrative cases . To condIn the TM corpus, we calculated a simple affinity score for each post by summing the number of topic-specific tokens that appeared within that post. In this context, affinity refers to the degree to which each post is representative of the topic to which it has been assigned . Using tIn the SA corpus, we relied on VADER-generated polarity scores for percentile ranking. Posts that fell below the 90th percentile polarity score for positive and negative valences were discarded.The SA and TM corpora were percentile-ranked independently. LDA modeling, which relies on co-occurrence of terms, favors verbose UGC, whereas VADER, reliant on purer expressions of sentiment, favors concision; hence, no UGC was duplicated in the condensed TM and condensed SA corpora. Specifically, richer and verbose UGC was emphasized in the condensed TM corpus, whereas emotive and concise UGC was emphasized in the condensed SA corpus.A 90th percentile cutoff was chosen to condense a data set such that it became compact enough to be handled by 2 human coders (SJS and CMC) for the following inductive thematic analyses.κ=0.78 was achieved Pos) posts, our condensed data set included the 488 posts assigned a polarity score >0.659 by the VADER lexicon . For the negatively valenced ([–]Neg) posts, our condensed sample included the 490 posts that were assigned a polarity score >0.196 .Details of the intravalence condensation of the strongly positive and negative posts, with illustrative examples, are shown in The condensed data set contained 1276 posts: 298 associated with the 90th percentile of the affinity of topics A, B, and C from LDA Model 2, and 978 associated with the 90th percentile of positive and negative polarity. This data set was advanced to thematic analysis. The detected intratopic and intravalence themes, their operational definitions, code co-occurrences, and illustrative excerpts are displayed in a meta-matrix in Within topic A, most themes articulated the distinct, day-to-day obligations of living with HIV. The most frequently detected themes, which reflected informative prompts provided by the Thrive With Me web app, covered ART medications. These instances were rich enough to warrant the coding of dedicated subthemes capturing detailed adherence tips, personal antiretroviral regimens, and adverse effects. Issues of long-term survival were raised, as were various personal narratives and peer-to-peer recommendations for disclosing one’s HIV serostatus to potential sexual partners. Further, a code (“Raising Awareness”) captured the many instances in which users shared details of activist events, local resources, and HIV-tailored public health messaging.Within topic B, diverse personal narratives were shared, including all articulations of the specific challenges that sexual minority MSM living with HIV may encounter as they seek social and sexual bonds with other men. These included mismatched expectations around relationship longevity and extradyadic pairings, life chaos attributed to partners’ alcohol and crystal meth use, and the roles of ex-partners. Trust, broken trust, discussions of self-confidence, and expressions of loneliness and isolation were emergent intratopic themes. The roles of support networks, including direct appeals to and provision of peer-to-peer social support among Thrive With Me users also emerged within this topic.Within topic C, the overwhelming proportion of UGC was made of brief greetings. In posts where these greetings were expanded to include personal updates and peer check-ins, 2 intratopic themes predominated; the first comprised substance use, misuse, and recovery, which included disclosures of relapse among Thrive With Me users; second, an emergent theme of personal triumph was also evident, which covered accomplishments such as new physical fitness regimens, career successes, and the attainment of treatment goals such as stable CD4 counts.Strongly positive posts were characterized by gratitude, typically in response to peer-to-peer encouragements and affirmations occurring on the forum. Strongly negative posts were richer and more thematically heterogenous. Many of these posts were reactions to linked external news media, which overwhelmingly provoked anger. These media often covered acts of homonegativity and racism. Another (–)Neg theme encompassed the political climate in the United States during the period of the Thrive With Me trial, when the 2016 presidential election was decided. The final intravalence theme concerned mental health, typically through expressions of acute or ongoing struggles with depression, stress, and insomnia.We combined common NLP techniques with traditional latent thematic analysis to classify UGC drawn from an interactive HIV mHealth environment. Through multiple iterations of LDA modeling, stable topics emerged: the day-to-day concerns of living with HIV; the social, romantic, and sexual tolls of aging with HIV as a sexual minority MSM; and routine greetings and daily affirmations. Using a 90th percentile cutoff, we condensed the UGC of which these topics were composed from a total of 4912 posts to a rich, illustrative subset of 1276 posts. By further analyzing this condensed UGC as a set of meta-themes, we identified latent discourses within them, through which experiential design insights could be mined.Our work contributes to the diverse, cross-disciplinary literature exploring sequential NLP-qualitative methods ,51-55,76To that end, the results reported here offer partial fulfillment of Aim 1. Although the sequential methods we demonstrated did successfully characterize the prevailing themes of the peer forum, the future viability of these methods will depend on their routinization. Our procedures included a number of transformations and cross-platform migrations, each of which introduces friction, which in turn disincentivizes adoption . RoutineAs for Aim 2, a range of actionable design insights surfaced from these findings to guide future iterations of Thrive With Me specifically and HIV mHealth generally. An HCD approach typically reframes these insights as “how might we” (HMW) prompts, a reframing we embrace here ,26. FirsAnother topic, with several related intratopic themes, concerned relationship difficulties. In addition to the abovementioned “partnering challenges” theme emergent within topic B, unmet relationship needs were evident throughout the “trust and betrayal” and social isolation–focused “voids in my life” themes. Thus, HMW 2 is “How might we support the interpersonal needs of seropositive MSM without imposing model drift into an ART adherence intervention?” The latent need is evident, and the deliberations of end users often touched on cross-cutting topic A and (–)Neg themes; the richest instances of intertopic cross-codings are shown among the “disclosing serostatus” (topic A), “partnering challenges” (topic B), and “substance use and misuse” (topic C) themes, illustrating the entanglement of these issues in Thrive With Me users’ lives. Dedicated informational modules might address these needs more directly, tying decision-making within this domain to specific triggers for illegal drug use or missed ART doses in a manner consistent with the IMB model in which Thrive With Me is grounded ,61.Finally, a desire to narrativize the personal triumphs of HIV survivorship is often evident across topics A and C, particularly within the “survivorship” and (in vivo) “other days I move mountains” codes. These narratives, which cover grief, coming out, and the lessons imparted by long-term survival, surface as an organically occurring form of UGC, pointing out their importance to Thrive With Me users, perhaps as validations of their personal resilience. Such strength-based, person-centered affirmations may hold the potential to constructively reauthor Thrive With Me users’ experiences of societal oppressions, while finding resonances within each other’s stories ,84. If iThese findings are subject to a range of limitations. As a proof-of-concept analysis, our methods are exploratory. Nevertheless, the abovementioned migrations and transformations built into our methods allow for the imposition of human error, while rate-limiting the rapidity with which results can be generated. In contrast, the adoption of a single alternative developer environment such as R , which permits qualitative analyses via the R qualitative data analysis package  would enmHealth interventions that fulfill the needs of multiply marginalized MSM living with HIV must accommodate a diverse array of needs and experiences. The findings of this proof-of-concept analysis suggest that combined machine- and human-optimized techniques can capture actionable insights on these needs and experiences without adding to the burdens of prospective end users. By maintaining an empathic lens and focusing on refinements in method, techniques such as those demonstrated here can contribute to future innovations in HIV mHealth.


Antimicrobial self‐medication and use have significantly increased in the COVID‐19 era—increasing antibiotic consumption and resulting in a high prevalence of antimicrobial resistance in Africa (AMR). We conducted a narrative review to investigate challenges associated with curbing AMR in a post‐COVID‐19 setting in Africa, suggesting practical measures applicable for policy‐informed implementation.A narrative review was performed to pinpoint AMR challenges and actions on the African continent. A comprehensive search was conducted in the scientific databases that include PubMed, PubMed Central and Google Scholar using predetermined search terms.The emergence of the COVID‐19 outbreak has added to the challenges of tackling AMR on the continent, which has jeopardized AMR interventions' hard‐won gains. Identified challenges have been Health systems disruption, Irrational Antimicrobial Use, Weak Antimicrobials Regulatory Ecosystem, Inefficient Population Infection Prevention, and Control Practices, Inadequate access to Health Services and data challenge on AMR surveillance.The COVID‐19 pandemic fueled AMR in Africa. There is a need for AMR control post‐COVID, such as measures for ongoing antimicrobial stewardship and good infection control practices. Further, curbing AMR requires rigorous regulatory enforcement and efficient AMR Surveillance. There should be a body to raise AMR awareness among the population. Research, Innovation and Technology could play an essential role supported by capacity building and global partnership. There was a lack of accessible quality health care services, compounded by the COVID‐19 surge and paucity of equipment such as testing material. Similarly, the protective policies devised to curb the pandemic have decreased the seeking for health care, and some already‐scheduled services have been canceled or missed.During the COVID‐19 pandemic, self‐medication has extensively increased in Africa.AMR has been one of humanity's most significant global health threats today. It is a public health problem that transcends continents and national borders and is rapidly growing, as seen with the sixfold increase in resistance rates since 2017.2We conducted a narrative review of data sources to identify AMR challenges and actions in African countries. A comprehensive search on PubMed, PubMed Central and Google Scholar was conducted using predetermined search terms. The inclusion criteria were data sources, that is, relevant articles about AMR interventions and associated successes and challenges in African countries and were published in English. References of the data sources were also reviewed to identify relevant information. Supporting Information Data were also gathered from country reports, commentaries, policy briefs and other reports. The collected articles were managed using Mendeley Reference Manager. The extracted data were discussed narratively to explore the aim of the study.33.1The distribution of substandard and counterfeit antimicrobials has a significant contribution to the spread of AMR, particularly in Africa.3.2Compounded by COVID‐19, AMR can drive more people into poverty, jeopardize global health security, and obstruct progress toward the Sustainable Development Goals and universal health care.Implementing the NAPs is very cost‐intensive, a luxury Africa cannot afford. For instance, it was estimated that the implementation of Zimbabwe's NAP would cost $44.6 million over 5 years from 2017.3.3The emergence of the COVID‐19 outbreak has elevated the stakes for AMR response. Health systems responses and public health priorities have evolved due to the pandemic, and this evolution has threatened the hard‐won gains of AMR interventions  have been described as essential for AMR in Kenyan health care settings, with poor WASH deemed to increase AMR.Furthermore, better infrastructures are critical to better IPC. The poor infrastructures can lead to a significant increase in infections and AMR. Notably, an international study investigating the quality of health care facilities in 78 Lower‐ and Middle‐Income Countries (LMICs) reported poor outcomes. Overall, 39% of health care facilities had no handwashing soap, 33% had no improved toilets, 59% lacked reliable electricity, 50% had no piped water, and 39% of health care care facilities had inadequate infectious waste disposal.3.3.5Despite the considerable improvement in global health, millions of people still lack access to quality health services, including effective antimicrobial medicines.3.3.6Managing Data is among the significant obstacles to AMR surveillance in Africa. Local stewardship is usually dependent on the availability of local antimicrobial susceptibility data. The Global Antimicrobial Resistance Surveillance System (GLASS) developed by WHO to support GAPs for AMR aims to foster standard AMR surveillance globally. While AMR reporting and surveillance have seen exponential growth globally from 2017 to 2019, reports indicate that few African countries have practical and functional surveillance systems.The deficiency of trained personnel, confined expertize and experience in the discipline, deficiency of calibration of antimicrobials susceptibility testing, inadequate sampling of the case with suspected infection and many origins of data from the drug industry, international systems, hospital and private labs, and national surveillance channels cause disconnected and sporadic data. With AMR risk being highest in Sub‐Saharan Africa,4To combat and win the fight against AMR, African countries need to drive and implement comprehensive plans and strategies. This is especially important as the COVID‐19 pandemic management has been dedicated to the majority of resources and workforce in recent times. Health care delivery is a complex adaptive process, and the AMR challenge requires adequate systemic adaptation to foster sustainable change. All stakeholders, policymakers, and health systems partners should be involved in providing a wholesome policy package to combat AMR in the complicated COVID‐19 system. Adopting a systems approach is crucial in the complex and adaptive system of people, processes, resources, and institutions. Africa needs to drive the implementation of sustainable interventions with support regarding resources, including funding, strategy, data utilization, and management and capacity merged with leadership, accountability, and transparency Table .4.1In 2015, the GAP on AMR adopted by WHO listed improving awareness through effective communication, education, and training as its first objective.4.2AMR control and management require the adaptation of functional and sustainable antimicrobial stewardship measures. Rational prescribing should be promoted to advocate for stewardship. This advocacy will include developing evidence‐based guidelines for antimicrobial prescribing in private and public facilities,Collaboration with the medical and pharmaceutical professionals to ensure practice regulation and adherence to guidelines is essential. This collaboration includes rational prescribing, prescription monitoring/surveillance, patient education, formulary approval and audit, computerized decision support, and continuing medical training. Incentivising antimicrobial stewardship in LMICs health care sectors will create room for cooperation and coordination. Dedicated funding commitment and social support packages implemented by governments and stakeholders are required to drive successful interventions. Social drivers of antimicrobial practices should be incorporated and adequately explored in the policy framework for acceptable balance between access and antimicrobial stewardship.Antibiotic stewardship policies should facilitate access to medicines and care and encourage seeking the primary health care practice. “Urban advantage” of proximity to quality health services, drugs and medical supplies for health maintenance needs to be translated in rural Africa.4.3One of the five crucial objectives of the WHO GAP is to tone up data on AMR via research and surveillance. Aligning with the GLASS developed by WHO and creating local repositories of standard AMR data, analysis and distribution inform the decision making on a national and international scale.AMR surveillance in the wake of irrational antibiotic use in the context of the COVID‐19 pandemic is essential to adapt to changes in AMR epidemiology.4.4Consistent regulatory systems that work do not just materialize; they require planning, design, and concerted implementation. At the same time, African governments need in‐depth analysis and assessment and updates on NAPs.Harmonization of law and regulations on drug manufacturing procurement, logistics and treatment practices is important. Regulatory authorities require adequate funding, strategic support, authorization and autonomy to undertake enforcement procedures, including food and veterinary systems.4.5With the COVID‐19 management, health systems triggered infection prevention policies by appropriately isolating cases and public health practices, including personal protection.Beyond the biological origins of AMR, social problems also drive perspectives on disease and infection interactions and management.The AMR plight in Africa highlights the value of creating policies for birthing IPC programs, amending access to necessary second‐line antibiotics as required, and producing novel vaccines and antibiotics. In addition, high‐quality data on communicable diseases, infectious agents, and AMR are merely available in several low‐income contexts. Preventing bacterial AMR and growing microbiological lab capability and data collection systems to better scientific interpreting of such humankind's well‐being danger should be an exceedingly great priority for worldwide health policymakers.4.6Information management and innovation are at the center of AMR response. Studies on consumption practices and their consequences and national statistics on AMR and public health responses can help shape public policy, provide predictive evidence of AMR development, and serve as an essential evaluation tool. Adequate policy implementation research on AMR is critical for designing interventions in resource‐constrained settings of Africa.Africa's technology is rapidly growing. The COVID‐19 pandemic has brought to life the critical need for the rapid development of accurate diagnostic methods, vaccines and antimicrobial treatments to improve health outcomes, especially in the vulnerable population.4.7The capacity for a responsive AMR health system requires adequate infrastructure and a competent health workforce. The COVID‐19 pandemic calls for reestablishing partnerships and training programs for AMR responses. Leveraging knowledge gained from the pandemic response makes for strengthening health systems. Continued education on patient management improves appreciation of clinical guidelines across medical competencies.4.8Global dialogues drive the synthesis of knowledge and experience that informs policy development. Harnessing advocacy and the potential of the virtual and remote space can drive the AMR mandate. Policy design, research advocacy, education advocacy, and community support and development represent several advocacy contexts. Partnerships should explore communication models that drive participation and shared community amongst decision‐makers and stakeholders. While COVID‐19 mortality in Africa has been lower, the rapid spread of the virus is a call to strengthen response capacity and create a quality standard for interventions.5AMR control post‐COVID in Africa requires measures for continued antimicrobial stewardship and maintenance of good infection control practices. Robust surveillance of antimicrobial usage and resistance development is also pivotal. Deterring AMR development in Africa will thrive with more investment in implementing NAPs for AMR and demonstrating their sustained relevance. A policy framework that effectively describes the population's behavioral compass and interactions, the social and system entities that impact antimicrobial behavior, and suitable implementation design with appropriate risk communication, assessment, and regulation, establishes a strong focus for AMR management.Deborah O. Shomuyiwa, Don E. Lucero‐Prisno, Emery Manirambona conceptualization, project administration and design. Deborah O. Shomuyiwa, Don E. Lucero‐PrisnoIII, Emery Manirambona, Mohamed H. Suleman, Rehab A. Rayan, Junjie Huang, Thaint N. Zaw, Yusuf Babatunde, Salomey A. Denkyira, Shuaibu S. Musa data collection and literature review, preparation of the original draft and visualization. Deborah O. Shomuyiwa, Don E. Lucero‐Prisno, Emery Manirambona supervision, writing, reviewing, editing and proofreading. All authors have read, and confirm that they meet ICMJE criteria for authorship.The authors declare no conflict of interest.Deborah Oluwaseun Shomuyiwa affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained.


WRAP53) is an antisense gene of TP53 with three transcriptional start sites producing three transcript variants involved in the progression of non-small cell lung cancer. However, the mechanism by which these different transcript variants regulate non-small cell lung cancer cell behaviors is to be elucidated.The WD40-encoding RNA antisense to p53 (WRAP53-1α and WRAP53-1β siRNA. The biological effects were assessed via colony formation, cell viability, apoptosis, cell cycle, wound healing and cell invasion assays, as well as immunoblotting.Two non-small cell lung cancer cell lines, A549 cells with wild-type p53 and H1975 with mutated p53, were transfected with WRAP53-1α increased the mRNA and protein levels of p53; suppressed colony formation and proliferation of A549 cells but promoted them in H1975 cells; increased the proportion of cells in the G0/G1 phase in A549 cells but decreased that in H1975 cells; and suppressed migration and invasion in A549 cells but not in H1975 cells. Conversely, knockdown of WRAP53-1β had no effect on p53 expression; promoted the growth of A549 cells but not of H1975 cells; decreased the proportion of cells in the G0/G1 phase in A549 cells but not in H1975 cells; and promoted migration and invasion in A549 cells but not in H1975 cells. Knockdown of both WRAP53-1α and WRAP53-1β promoted apoptosis in A549 cells but not in H1975 cells.Knockdown of WRAP53 transcript variants exerted different functions in non-small cell lung cancer cells and regulated non-small cell lung cancer cell behaviors depending on the p53 expression. Alternative splicing is a crucial process in gene regulation as it enables a single gene to encode different protein products. It occurs in approximately 95% of multi-exon genes in eukaryotes and is involved in various disorders including cancers , 2. The TP53) gene not only cause loss of its tumor suppressor function but can also promote an oncogenic function that promotes malignant transformation. Such mutations are found in over 50% of human cancers [TP53 is the most commonly mutated gene in non-small cell lung cancer (NSCLC) [TP53 mutation is a negative prognostic factor in advanced NSCLC and that different mutations have different prognostic values [Mutations in the tumor suppressor p53 ( (NSCLC) . Mutant c values . High exc values .TP53 5’-untranslated region flanking gene, WD40-encoding RNA antisense to p53 (WRAP53), which is located on chromosome 17p13, has three different transcriptional start sites that yield three different variants: 1α, 1β, and 1γ. The start site for the 1α variant corresponds to the first exon of p53 in a cis-antisense manner. WRAP53-1α is an antisense transcript that stabilizes TP53 [WRAP53-1β transcript is not complementary to TP53 but encodes the WD repeat-containing protein WRAP53β (WD repeat domain 79) and telomerase Cajal body protein 1, which is involved in multiple cellular processes. WRAP53β regulates the maintenance of the nuclear organelles known as Cajal bodies by recruiting motor neuron proteins, small Cajal body-specific RNAs, and telomerases [WRAP53-1γ transcript is located in the first intron of p53, though its function remains unclear [The zes TP53 . The WRAomerases . WRAP53βomerases , 13. The unclear .WRAP53 has been associated with cancer pathogenesis because WRAP53-1α regulates TP53 expression. Loss of WRAP53β impairs telomere maintenance and DNA repair, thus increasing genomic instability and the probability of carcinogenesis. It is conceivable that WRAP53-1α and WRAP53-1β could play different roles in NSCLC cells. To test this hypothesis, we examined the distinct biological function of the two WRAP53 variants in vitro in cell function assays.Moreover, the effects of WRAP53 on cancer progression are controversial. 2.NSCLC cell lines (A549 and NCI-H1975) were purchased from the Type Culture Collection of the Chinese Academy of Sciences . Both cell lines were cultured in RPMI-1640 medium  supplemented with 10% fetal bovine serum (Hyclone), 100 U/mL penicillin, and 100 mg/mL streptomycin . All cells were maintained in a humidified incubator  at 37°C and 5% COTM RealTime PCR Detection System . Glyceraldehyde-3-phosphate dehydrogenase was used as the reference for normalization. The relative fold change of mRNAs was calculated using the 2 −ΔΔCt method. The primers used for qRT-PCR are presented in Total RNA was isolated from cells using TRIzol . The RNA concentrations and A260/A280 ratios were assessed with a Nanodrop spectrophotometer . Total RNA (500 ng) was reverse-transcribed using the HiScript® Q RT SuperMix for qPCR kit (Vazyme) according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using the ChamQ TM SYBR ® qPCR Master Mix (Vazyme) on the CFX ConnectWRAP53-1α and WRAP53-1β [5 cells were seeded in each well of a 6-well plate. After growing the cells to 30–50% confluence, the cells were transfected with NC or target siRNAs.Small interfering RNAs (siRNAs) targeting RAP53-1β  and a neCells were lysed in RIPA cell lysis buffer  supplemented with phosphatase inhibitors and protease inhibitors . The lysates were then centrifuged at 13300 rpm for 15 min at 4°C. The supernatants were collected, and protein concentration was measured using a bicinchoninic acid kit (Beyotime). Equal amounts of protein were applied to a 10–12% sodium dodecyl sulphate-polyacrylamide separating gel and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% non-fat milk and then incubated with primary antibodies at 4°C overnight. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. An enhanced chemiluminescence kit (Bio-Rad) was used to detect the proteins under a gel imaging analyzer (Bio-Rad). Antibodies used for immunoblotting are listed in After transfection, tumor cells were plated in 6-well plates (500 cells/well) and cultured for 2 weeks. Cell colonies were fixed with 4% paraformaldehyde (Sangon) for 20 min and stained with 0.5% crystal violet (Beyotime) at room temperature for 30 min. After washing the wells with water, the number of colonies in each well was counted under a microscope. This assay was repeated three times and performed in triplicates.Cell counting kit-8 (CCK-8) was used to quantify cell viability. The CCK-8 method is based on the conversion of the water-soluble tetrazolium WST-8 to a water-soluble formazan dye upon reduction by dehydrogenases in the presence of an electron carrier. All the experiments were conducted according to the manufacturer’s protocol. Transfected cells were seeded into 96-well plates (3000 cells/well in 100 μL medium). After culturing for 24, 48, 72, and 96 h, the supernatant was replaced with 100 μL serum-free medium with 10 μL CCK-8  and incubated at 37°C in the dark for 1 h. The absorbance was measured at 450 nm by a microplate reader . Each group had 5 wells, and the experiment was conducted three times.WRAP53 siRNAs. The cells were harvested 48 h later, washed twice with cold phosphate-buffered saline (PBS), and stained with fluorescein isothiocyanate-conjugated annexin V  for 20 min and propidium iodide  in the dark for 5 min. The stained cells were assessed by flow cytometry  and analyzed by CytExpert 2.3 software (Beckman). Each experiment was conducted three times.Cells were seeded into 6-well plates and transfected with NC or Cells were harvested with trypsin without EDTA  48 h after transfection. After washing with cold PBS, the cells were treated with a cell cycle staining kit . Flow cytometry (Beckman) was used to detect the percentage of cells in different cell cycle phases. This assay was repeated at least three times.WRAP53 siRNAs. When the cells were confluent, they were scratched with a 10-μL pipette tip and washed with PBS gently to remove cell debris. After that, 2 mL serum-free medium was added into each well, and pictures were taken using a phase contrast microscope  at 0, 24, and 48 h. The migration rate was calculated as follows:Cells were seeded in 6-well plates and transfected with NC or Wound closure (%) = /distance of initial scratch.5 cells/well) transfected with NC or WRAP53 siRNAs were seeded in the upper chambers of transwell units pre-coated with 100 μL Matrigel . After incubation for 24 h, the filters were fixed with 4% paraformaldehyde for 20 min and stained with 0.5% crystal violet for 20 min. The cells on the upper surface of the filter were completely removed using a cotton swab. The cells that invaded through the Matrigel and reached the lower surface of the filter were counted. Experiments were independently repeated for three times.Cells . All data are presented as the mean ± standard deviation (SD). Unpaired two-tailed student’s t-test was used for comparisons between groups. A P value < 0.05 was considered statistically significant.WRAP53-1α and WRAP53-1β. There was no marked difference in WRAP53-1α mRNA levels between cells transfected with si-Control and si-WRAP53-1β, whereas there was significantly less WRAP53-1α mRNA in A549 and H1975 cells transfected with si-WRAP53-1α  associated X protein and downregulated Bcl-2 in A549 cells. However, these changes were mild in H1975 cells transfected with si-WRAP53-1α or si-WRAP53-1β (WRAP53-1α and WRAP53-1β negatively regulated apoptosis in cells with wild-type p53 (A549) but not in cells with mutant p53 (H1975).Flow cytometry was used to measure apoptosis in NSCLC cells transfected with si-WRAP53-1α or si-WRAP53-1β. Knockdown of either RAP53-1β . These rWRAP53-1α knockdown, whereas the proportion of cells in the G0/G1 phase decreased by WRAP53-1β knockdown (WRAP53-1α knockdown (P < 0.05); WRAP53-1β knockdown had no significant effect on the cell cycle  was downregulated by knockdown of WRAP53-1α and upregulated by knockdown of WRAP53-1β in A549 cells. However, in H1975 cells, the levels of CDK4 were increased by knockdown of WRAP53-1α but not affected by knockdown of WRAP53-1β ; WRAP53-1β knockdown had no significant effect ; WRAP53-1β knockdown had no significant effect  and H1975 cells (which have mutated p53). WRAP53-1α regulated both the wild-type p53 and mutant p53 expressions. Knockdown of WRAP53-1α showed anti-tumor effects in A549 cells, but it had the opposite effects on H1975 cells. WRAP53-1α regulated NSCLC cell behaviors based on p53 mutation. In contrast, WRAP53-1β did not regulate the expression of either wild-type or mutant p53 and acted as a tumor suppressor in A549 cells but without effect in H1975 cells. Our results suggest that the two WRAP53 transcript variants have distinct effects on p53 and NSCLC cells.In the present study, the two WRAP53-1α is a naturally occurring p53 antisense transcript that acts as a crucial effector in several cancers [WRAP53-1α is upregulated by anticancer drugs, and miR-4732-5p has a binding site in the 5’-untranslated region of WRAP53 [WRAP53-1α methylation is significantly associated with worse survival in NSCLC. It is worth noting that WRAP53-1α stabilizes TP53 mRNA to increase the tumor suppressor activity of wild-type p53, leading to better prognosis. However, downregulation of WRAP53-1α by promoter methylation does not affect survival in p53-mutated tumors [WRAP53-1α regulates p53 signaling. The role of WRAP53-1α had not been studied in NSCLC cells.Natural antisense transcripts are long non-coding RNAs that occur naturally and play important roles in carcinogenesis, invasion, and metastasis . The lon cancers , 16. WRAf WRAP53 –19. In ad tumors . These rWRAP53β, a WD repeat-containing protein, also acts as a tumor suppressor to regulate various cellular activities . The sigin vitro and in vivo by inducing cell cycle arrest and apoptosis. WRAP53β induces cell cycle arrest at the G0/G1 phase and regulates the expression of G0/G1-related cyclins and cyclin-dependent kinase complexes. WRAP53β knockdown was also reported to induce apoptosis via the mitochondrial pathway [WRAP53β is a potential oncoprotein whose overexpression leads to transformation and promotes cancer cell survival and whose downregulation induces massive cell death –32. Addi pathway . WRAP53β pathway . Additio pathway . Moreove pathway .WRAP53 in disease progression is evident in lung cancer, but whether different targets of WRAP53 variants exert different biological functions still requires further investigation. In the present study, we used different variant-specific siRNAs to knock down WRAP53-1α and WRAP53-1β and evaluated the functions of these variants in NSCLC cells. First, WRAP53-1α was significantly associated with p53 expression, unlike WRAP53-1β expression, which is consistent with the results of a previous study [WRAP53-1α upregulated p53 in A549 and H1975 cells, which conflicts with the results of a previous study wherein WRAP53-1α stabilized p53 [WRAP53-1α might play different roles based on the p53 mutation status in NSCLC cells.The involvement of us study . Howeverized p53 , 20. At ized p53 . TherefoWRAP53-1α had different effects on NSCLC cell behaviors. It is highly probable that the different mutation statuses of p53 led to these contradictory biological functions. The tumor suppressor p53 is a cellular gatekeeper that guards against genetic abnormality and instability via sensing multiple stress signals, including DNA damage and oncogene activation [WRAP53-1α induced upregulation of wild-type p53 and activation of cell cycle arrest and apoptosis, increased protein levels of cell cycle inhibitor p21 and pro-apoptotic Bax, and decreased the expression of pro-survival Bcl2 in A549 cells. However, in H1975 cells, which harbor a p53 mutation, the knockdown of WRAP53-1α exerted the opposite effects on cell cycle arrest and had no effect on apoptosis. The p53 has been reported to directly or indirectly regulate the expression of various epithelial mesenchymal transition markers [WRAP53-1α knockdown increased the expression of epithelial marker E-cadherin and decreased the expression of mesenchymal markers MMP9 in A549 cells but demonstrated the opposite effects in H1975 cells. These results indicated that WRAP53-1α regulated cell proliferation, survival, and epithelial mesenchymal transition depending on p53 mutation.We further demonstrated that tivation . In additivation . In A549 markers . In the WRAP53-1β acts as a tumor suppressor in A549 cells. Knockdown of WRAP53-1β induced slight apoptosis and promoted the G1/S phase transition in A549 cells but had no effect on H1975 cells. The expressions of mesenchymal markers MMP9 and Vimentin were upregulated by the knockdown of WRAP53-1β in A549 cells. Though WRAP53-1β knockdown decreased the expression of E-cadherin and slightly increased the expression of Vimentin, MMP9 was not affected by the knockdown of WRAP53-1β in H1975 cells. These findings concerning the contribution of WRAP53-1β to cancer may occur depending on the p53 mutation, which is consistent with the findings in animal models [WRAP53-1β could help initiate tumor development by impairing telomere maintenance and DNA repair, leading to genomic instability. Our data suggested that WRAP53-1β is a putative tumor suppressor during the progression and metastasis of NSCLC, whereas WRAP53-1α may have a dual function, thereby suggesting potential interactions between the two transcript variants. This study provides a foundation for further examining the mechanisms by which WRAP53-1α and WRAP53-1β exert their functions in NSCLC, but more in vivo validation is needed.Cell function assays indicated that l models . InactivWRAP53 isoforms in p53 regulation, telomerase/telomere function, and DNA damage repair are to be investigated, but whether the various molecular functions are independent of each other is still not completely clear. The cDNA microarray showed that WRAP53 directly or indirectly mediated the factors related to tumorigenesis, such as p53 signaling pathway, cell cycle and apoptosis pathway, Jak-STAT signaling pathway, PI3K-Akt signaling pathway [WRAP53-1α mainly plays biological functions by regulating p53, while WRAP53-1β plays various functions, such as regulating telomerase and DNA damage repair.The roles of  pathway . We hypoWRAP53 played different and even opposite roles in NSCLC cells. NSCLC therapeutic strategies may include specifically targeting WRAP53 variants.Different transcript variants of S1 Raw images(PDF)Click here for additional data file.


Vibrio vulnificus is a ubiquitous marine bacterium that may cause rapid and deadly infection, threatening lives of people living around natural bodies of water, especially in coastal regions. However, traditional culture-based methods are time-consuming and unable to detect Viable But Non-Culturable (VBNC) V. vulnificus cells. In this work, we isolated a batch of detection aptamers specifically binding to V. vulnificus in all culture status. With traditional whole bacteria-SELEX , flow cytometer analysis and imaging, we identify 18 candidates and validated two of them (V8 and V13) as applicable aptamers. Their truncated sequences also showed comparable performance. The dissociation constant (KD) value of V8 is shown to be as low as 11.22 ± 1.32 nM. Optimal aptamers V8 and V13 are also validated to be effective to detect different Vibrio vulnificus strains under different binding environments using flow cytometry. As for detection parameters, the LOD of the V8 from cytometry is 29.96 CFU mL−1, and the linear range is 102–5 × 105 CFU mL−1. This is the first case demonstrating that aptamers can detect the existence of VBNC bacteria as well as live bacteria. V. vulnificus in VBNC Status for the first time.With whole-bacteria SELEX, we got aptamers that can bind to  As part of normal microflora, it can be found in waters, oysters and other shellfish. It is one of the most prevailing marine pathogens. As the FDA (Food and Drug Administration) of the United States reported, there were 459 cases from 1992 to 2007 with a fatality rate of 51.7%.1,2 Most cases (85%) occur in the warm water months of May to October in the Northern Hemisphere. People typically get infected through foods. V. vulnificus is responsible for 95% of seafood-related deaths in the US. Directly being exposed to contaminated water with a pre-existing lesion or cut can also cause infections. Patients with chronic and underlying diseases, especially liver diseases, are more likely to get infected. Infection symptoms can appear soon after the oyster ingestion, and the onset time can be as soon as 4 h.3 If patients can't receive antibiotic treatments in three days, the fatality rate can be 100%.4 Thus, monitoring the presence of V. vulnificus in waters and seafood is of medical and economic importance.V. vulnificus from environmental or clinical samples typically rely on selective medium culture, which require further experiments such as PCR to identify the presumptive isolates.5–7 Although such methods are quite accurate for the identification and validation of candidate microorganisms like V. vulnificus, however, there are still two major shortcomings for using this method in V. vulnificus clinical detection:Current methods for the identification and isolation of V. vulnificus can only survive three days after infection, it's IMPOSSIBLE to use culture-based diagnosis method for such acute urge infection of V. vulnificus.Firstly, the culture-based methods usually take 3 or 4 days to get the final results, which are quite time-consuming. And considering that patients infected by V. vulnificus has been reported to be able to transformed into a specific state called Viable But Non-Culturable (VBNC) state, making it possible to escape traditional culture based detection. Although with adequate cells (108/mL or more),8 the VBNC V. vulnificus can be detected through PCR, however, in nature environment, such concentration of VBNC cells can't be reached, and a small number of cells (about 100 cells or less) is enough to cause serious diseases.9Secondly, under some extreme conditions (like low temperature in winter), V. vulnificus can be used as a supplement for traditional standard methods to not only provide significant infectious information on patients quickly and effectively, like but also overcome the two shortcomings of traditional methods we mentioned above, making it possible for infected patients to get correct treatment in time.Rapid and quick direct detection of pathogens like 10–13 and they are named as aptamers. Aptamers are very suitable to be developed into diagnostic and therapeutic tools. They can be easily modified with dyes or chemical tools and can be easily immobilized on many kinds of substrates, make it more suitable for quantitative measurements.14–16 Compare with antibody, aptamer is smaller, more stable and can tolerate a wide range of temperatures. Additionally, aptamer can be produced in vitro without torturing animals and can be rapidly synthesized with high purity and little batch-to-batch variation.Multiple subtypes of molecules have been applied for pathogen diagnosis as detectors. Among them, single-stranded nucleic acid can fold into unique and stable structures and makes it an ideal choice. Some single-stranded DNA (ssDNA) or RNA chains can specifically bind to various targets, such as metal ions, small molecules, drugs, proteins, and even whole cells,17 There are two common strategies to isolate bacteria aptamers, using specific cell surface molecules as targets, or the whole cells as targets. For the first strategy, however, choosing and purifying species-specific membrane molecules can be relatively difficult, and purified molecules may not be able to reserve their native structures when their microenvironments are changed, so their aptamers may not be able to bind to cells. Under such circumstance, the whole-bacteria SELEX approach can be applied and perfectly solves the problem. Whole-bacteria SELEX approach can efficiently screen aptamers with high affinity without tedious molecule purification process, and counter SELEX can ensure the high specificity of the aptamers. In recent years, whole-bacteria SELEX approach has been applied to isolating various bacteria aptamers such as Mycobacteria tuberculosis,18Staphylococcus aureus,19Candida albicans,20Vibrio. Alginolyticus,21Vibrio. Parahaemolyticus,22etc.Aptamers are isolated by Systematic Evolution of Ligands by EXponential enrichment (SELEX) procedures.V. vulnificus. Based on FACs (Flow Cytometry) and confocal microscopy results, we identified 2 aptamers with good performance, V8 and V13. They can bind to V. vulnificus with high binding affinity and can specifically pick V. vulnificus out of other bacteria. Such optimal aptamers can detect different V. vulnificus strains, tolerate different binding environments, and detect V. vulnificus in VBNC status as well as cells in other culture phases. As for the general performance of such aptamer-based detection method, the LOD is 29.96 CFU mL−1 and its linear range is 102–5 × 105 CFU mL−1. The total test time of this fast screen method is less than 1 hour. Based on our screening methods and analysis results we described above, there are three major innovations that can be summarized in this research field.In this study, we employed whole-bacteria SELEX approaches to screen potential ssDNA sequences that can specifically bind to V. vulnificus with the best binding affinity up to now. The KD value for Aptamer V8 is 1.22 ± 1.32 nM, while the previous studie got an aptamer with KD = 26.8 ± 5.3 nM;23Firstly, we developed ssDNA aptamers for V. vulnificus VBNC status, fulfilling the gaps in this research field.Secondly, for the first time, we provided an optional method to detect environmental at V. vulnificus have various stages. Therefore, it's quite necessary for researcher to screen and validate the candidate aptamers using bacterium from different various stages. However, previous studies on V. vulnificus do not contain related screening and analysis. Our study filled the gap and identified the effective aptamers that can be applied to V. vulnificus at different phases.Thirdly, previous studies generally applied bacteria in one fixed growth period/stage, and they didn't test whether these aptamers can bind to target bacteria in different phases. Therefore, the screened-out aptamers may not be able to identify bacteria if such bacteria entered a different stage, inducing possible false negative results. As we have mentioned above, V. vulnificus with higher detection speed and accuracy, but also validated their ability to bind to V. vulnificus in different stages, especially in VBNC status.All in all, taking advantages of whole-bacteria SELEX approaches and flow cytometry, in this study, we not only screened out two effective aptamers (V8 and V13) for further clinical and environmental detection on 2.V. vulnificus. During the first seven rounds of selection, sequences bound to V. vulnificus were collected and amplified. The recovery rates increased gradually, and we decided to introduce counter-SELEX in Round 8  were incubated with various species of bacteria, including V. vulnificus cells and aptamer, laser scanning confocal microscopy was applied to observe the V. vulnificus-aptamer complex. V. vulnificus cells were incubated with 4 aptamer sequences respectively. Then the suspension was dropped on glass slide and thin smear was made. Through laser scanning confocal microscopy, we can see clearly that these sequences did binding to the cells efficiently and Limit of Detection (LOD)for V8-flow cytometer detection method is measured and calculated according to CLSI-17A. The LOB is 7 CFU mL26 Good linear relationship between plate count result and V8-flow cytometer detection result at the range between 102 to 5 × 105 CFU mL−1 is shown on The linear range of V8-flow cytometer detection method is measured according to CLSI-EP6A.V. vulnificus with Proteinase K and trypsin to see whether the target of V8 is protein. If the fluorescence signal reduces, the target of the aptamer may be protein molecules on bacterial cell wall. However, as ESI Fig. S4V8 showed best binding affinity and its target molecule was explored. We treated 3.V. vulnificus. The accuracy and efficacy of the optimal aptamers have also been validated under different circumstance and against different strains of V. vulnificus, and the aptamers showed excellent performance. Here, in this study, we divided further discussion on our project into three parts: methodological innovations, clinical significance of new aptamers and further perspectives in this field.All in all, as we have described above, we applied modified whole-bacteria SELEX and got aptamers that can specifically bind to V. vulnificus.For methodological innovations, we have made three major innovations during the selection, validation and application of the optimal aptamers against V. vulnificus. In previous studies, lots of aptamers against bacteria were selected by SELEX and many of them were developed into splendid detection method.24,27–30 However, most of them failed to pay attention to bacteria at different stages and relied on single phase of bacteria for aptamer selection and validation. Then, some scholar started to noticed the phase differences. Ying Zou et al. developed aptamers that can bind to different stages of E. coli O157:H7.31 Soo HwanSuh et al. developed aptamers specifically targeted bacterial cells at different growth phases.32 Thus, we innovatively used V. vulnificus at different stages for positive SELEX, and used different stages of bacteria to validate the aptamers. We especially validated aptamers' binding ability to VBNC state. We identified aptamers with high affinity against different phases of bacteria, improving the accuracy and efficacy for aptamer selection.First, in this study, we emphasized on developing aptamers that can bind to different stages of V. parahaemolyticus as counter selection targets to confer the optimal aptamers potential clinical significance. Previously, the aptamer selection for V. vulnificus has been reported by Yan, et al.33 Their work was more focused on fish diseases and they used V. anguillarum for counter-SELEX, for further better distinguishing different water pollution pathogens for fish. While in this study, we chose V. parahaemolyticus with more clinical significance for the counter selection, making the optimal aptamers effective enough to be applied in the differential diagnosis between V. vulnificus. and V. parahaemolyticus. For the first time, we identified effective aptamers for V. vulnificus with potential clinical significance.Moreover, we chose V. vulnificus in VBNC status. In this work we used flow cytometer as a part of detection system. Compare to other existing proved VBNC detection methods, such as PCR,34 qPCR,35 PMA-LAMP 36,37 PMA-based qPCR,38 CRENAME + rtPCR39 , the LOD of flow cytometer is slightly higher or equivalent. There is a trade-off between “easy to operate “and” extreme sensitivity”. LOD of CRENAME + rtPCR can reach to 1.2 CFU/100 mL, but this approach takes 6 h to get the results. V8-flow cytometer detection is fast, with <1 h total test time and easier sample preparation.Third, we developed an effective, fast and labour-saving method to detect et al.33 presented an effective detection method and identified effective aptamers which we have already mentioned above. Comparing to this study, we have two advantages:For biological and clinical significance of new aptamers, as we have already analysed, the optimal aptamers we screened can be further applied and modified for clinical use. Therefore, according to related publications, we compared the performance of our optimal aptamers with previous proved detection methods. In 2018, Yan, Firstly, comparing to their optimal aptamer Vapt2 with binding affinity at 26.8 ± 5.3 nM, our optimal aptamer (V8) has a better and more stable performance with binding affinity as 11.22 ± 1.30 nM. Secondly, different from previous studies using microscope to detect, which is labour-consuming and less quantitative in practical microbe detection, we applied flow cytometry to detect the performance of aptamers, which is quite fast, convenient for sample preparation and more suitable for further clinical applications.As for future perspectives, we may still focus on the optimal aptamers we screened out in this study like aptamer V8 and V13. There are two major directions that we may contribute to in the future:V. vulnificus strains, we incubated V8 with protein-free cells. And after incubation fluorescent did not change much, indicating that the target molecule of aptamer V8 is not protein but another cell wall component. Since the expression of some outer membrane protein may alter in different culture phases, it is reasonable that our SELEX strategy yielded aptamers that can bind to component that expresses on most V. vulnificus cells. Further studies are needed to identify the exact target molecule of V8.Firstly, we will try to reveal the detailed biological mechanism for the aptamer to bind the target bacteria. In this study, we have already applied related experiments to make the preliminary trials in this field. Using optimal aptamer V8 and cultured V. vulnificus at different stages. Although based on our experiments, V8-cytometry method have already been validated to be effective and accurate enough for the identification of V. vulnificus at different stages, more modification should be made and a more efficient and sensitive biosensor or clinical diagnosis kit should be established. More experiments should be applied to make sure the stability and accuracy of our kit in clinics or for environment monitoring. Therefore, we may focus on the development of novel V. vulnificus detection methods, by applying technologies like aptamer-conjugated nanoparticles, quantum dots or colorimetric approaches to increase the sensitivity, handleability and throughput of the detections.Secondly, we will try to develop a reliable and fast detection kit using the aptamers for the detection of 4.V. vulnificus ATCC 27562, V. parahaemolyticus ATCC17802 and V. alginolyticus ATCC 17749 were obtained from American Type Culture Collection (ATCC), Georgetown, DC, USA. V. vulnificus MCCC 1A08743 and V. vulnificus MCCC 1H00047 were obtained from Marine Culture Collection of China. Vibrio bacteria were grown in brain heart infusion medium  with 3% NaCl at 37 °C. V. vulnificus in three different culture phases early exponential phase (OD600 = 0.3), late exponential phase (OD600 = 0.7), and stationary phase (OD600 > 1) were mixed at the ratio of 1 : 1 : 1 in number. The mixture was used for positive SELEX. V. parahaemolyticus was cultured and prepared the same as V. vulnificus for counter-SELEX.Staphylococcus aureus ATCC25923, Listeria monocytogenes ATCC19115, Candida albicans ATCC10231, and Pseudomonas aeruginosa ATCC27853. All these bacteria were cultured overnight under aerobic condition in brain heart infusion media  with 1% NaCl at 37 °C and 150 rpm shaking. All these bacterial strains were obtained from ATCC.The following bacteria were also used to identify the specificity of the isolated aptamers: V. vulnificus under VBNC status was obtained by refrigerating cultured cells in artificial seawater (ASW) under 4 °C for 7 or more days. The viability was tested on culture plates and growth should not be detected. The VBNC cells were subject to PI stain and showed negative results in flow cytometer.40 15 min of trypsin treatment  and 10 min proteinase K treatment  were conducted. The reaction was stopped by adding Phenylmethanesulfonyl fluoride  into the suspension.Membrane protein was removed as previously described with a little modification.The initial single strand DNA (ssDNA) library and the primers used to amplify DNA were all obtained from Sangon Biotech . This ssDNA pool consisted of a central randomized region of 25 nucleotides flanked on both sides by primer regions for amplification,N25-GCAAGATCTCCGAGATATCG-3′ (66-mer).5′-AGTATACGTATTACCTGCAGC-15 different sequences, which represents high sequence diversity.The ssDNA library contained a maximum of 10Forward primer was 5′-AGTATACGTATTACCTGCAGC-3′.Reverse primer was 5′-CGATATCTCGGAGATCTTGC-3′.A poly A-labelled reverse primer (5′-AAAAAAAAAAAAAAAAAAAA-Spacer 18-CGATATCTCGGAGATCTTGC-3′) was used together with unmodified forward primer in PCR to get the asymmetry double strand DNA and to enable further purification of ssDNA by DNA PAGE. Unmodified forward primer and reverse primer were also used for PCR amplification after the final round of the selection for cloning sequencing. GoTaqHot® Start Colorless Master Mix used in PCR was purchased from Promega Corporation. A fluorescent labelled random ssDNA pool was also used as negative binding control.19,21,22 Briefly, for the SELEX screening, specific concentrations of ssDNA pool dissolved in 100 μL binding buffer , 50 mM Tris–HCl, 100 mM NaCl, 1 mM MgCl2, 5 mM KCl) were heated to 95 °C for 10 min and cooled in an ice bath for 5 min to form the optimal structural conformation of oligonucleotides. The cell mixture containing 4 × 108V. vulnificus cells was incubated with the denatured ssDNA pool at 4 °C for 2 h with end-over-end rotation, allowing the potential aptamer sequences to bind with cells. After the binding reaction, unbound ssDNA was removed by washing three times in 1 mL of binding buffer by centrifugation at 12 000 rpm for 3 min. The amounts of unbound ssDNA were measured and recovery rates were calculated. The cell pellets with adhesive ssDNA were resuspended in water, and PCR reagents were added directly to the cell suspension to amplify the tightly bound DNA sequences on the cells . Finally, the purified ssDNA was quantified using a Qubit® 2.0 Fluorometer and used for the next selection round.th round ended, ssDNA sequences selected by whole-bacteria SELEX were amplified with unmodified primers through PCR. The purified PCR products were then cloned and sequenced by Shanghai Sangon Biological Science and Technology Company . These sequences were aligned and analyzed using the Clustal X 2.0 software.When the 13via flow cytometry.BD FACSCalibur flow cytometer and CellQuest pro software  were used to assess the binding performance of different sequences. Fluorescence intensity of incubated bacterial cells were measured 2, 5 mM KCl), preheated, and then incubated with bacterial cells. After incubating with 300 nM aptamer/random ssDNA pool for 30 min, the cells were washed once and resuspended in 300 μL binding buffer for immediate flow cytometric analysis.Candidate aptamer sequences were labelled with the fluorophore (5′-FAM). A randomized ssDNA pool with fluorescent label was used as a control for nonspecific binding in each experiment. A threshold based on fluorescence intensity was set so that the fluorescence intensity of the gated cells would be greater than those incubated with randomized ssDNA pool. Gated cells were counted and gated fluorescence intensity was quantified. Candidate sequences were dissolved in binding buffer  values by incubating different concentrations of aptamers (0–300 nM) with a fixed number of cells (108 CFU mL−1).The KD were calculated with the equation y = Bmaxx/(KD + x), using SigmaPlot 12.5 software.Binding curves were created to estimate dissociation constants  were incubated with 300 nM V8 and V13 for 30 min at 25 °C. When tested on flow cytometer, set test time as 60 s.To evaluate whether different concentrations of V. vulnificus in all culture status, 300 nM aptamers were incubated with V. vulnificus in early exponential phase, late exponential phase, stationary phase and VBNC status (108 CFU mL−1) at 25 °C. for 30 min, respectively.To measure whether V8 and V13 can bind to V. vulnificus isolated from different resources, 300 nM V8 and V13 were incubated with V. vulnificus MCCC 1A08743 and MCCC 1H00047 (108 CFU mL−1) at 25 °C for 30 min, respectively.To analyse whether V8 and V13 can bind to 8 CFU mL−1) were incubated with 300 nM V8 and V13 in human serum and oyster infusion. To minimize the influence, the sediments in liquids were removed from serum and infusion by centrifugation at 12 000 rpm for 10 min. For serum incubation, serum was diluted to 30% with binding buffer. After incubation, the samples were directly detected by flow cytometer.To analyse aptamer stability, bacterial cells  were tested. Kolmogorov-Smirnov test shows that P = 0.022 < 0.05, indicates the data does not conform to the normal distribution. According to ISO suggestion, the calculation formula should be LOB = the measured value of the [NB(p/100) + 0.5] bit, α = 5%, p = 95, that is, the measured value ranked 38th. 5 different low concentrations of samples were tested to measure LOD. 300 nM aptamers were incubated with V. vulnificus at the concentrations of 25 CFU mL−1, 50 CFU mL−1, 100 CFU mL−1, 150 CFU mL−1, 300 CFU mL−1, and the volume of each sample is 12 mL. The sample is divided into 12 equal parts and the cell number in 1 mL was measured for 12 times, then the standard deviations (SD) of these tests were calculated. The formula is LoDtent = LoB + cβ SDS, where SDS is the estimated deviation of the population standard deviation (SDS2 = (n1SD1s2 + n2SD2s2 + n3SD3s2 + ……+ nnSDns2)/(n1 + n2 + n3 ……. + nn), nn = Nn − 1), cβ = 1.645/(1–1/(4 × f)), f is the degree of the freedom, f = NS − K. K is the number of groups. f = 55, cβ = 1.6525.The LOB and LOD is measured according to CLSI-17A.26 106 CFU mL−1 of Vibrio vulnificus suspension was incubated with 300 nM V8 and serially diluted into 9 concentrations. Suspension of each concentration was divided into 6 parts, 3 parts were subjected to flow cytometry detection, and 3 parts were subjected to plate colony counting method. Samples of different concentrations of bacterial suspension should be randomly tested and recorded. The mean number of bacteria in each concentration of the bacterial suspension was fitted to the curve, and the corresponding range of the linear segment was selected as the linear range.The linear range is determined by reference to the CLSI-EP6A document.V. vulnificus cells in binding buffer for 30 min at 4 °C. The concentration of V. vulnificus is 2 × 109 CFU mL−1. BSA and salmon sperm DNA were added into the binding buffer to avoid nonspecific binding. Cells were washed twice to remove the unbound aptamers by centrifugation and resuspended in 15 μL binding buffer. Then, the suspension was dropped on poly-l-lysine-coated slides. Poly-l-lysine-coated slides can avoid cell moving during scanning process. Fluorescent images of bacteria with each aptamer were observed under a fluorescence microscope  using excitation at 488 nm, with 60× magnification.Fluorescently labelled 5′-FAM-ssDNA aptamers (300 nM) were incubated individually with 42 Set DNA sequence to be linear, folding temperature to be 25 °C, and set Ionic conditions [Na+] = 100 mM and [Mg2+] = 1 mM.Secondary structures were predicted according to M. Zuker ‘s method.To test the stability, after incubated with diluted serum and oyster infusion for 30 min, 10 μL 300 nM V8 and V13 were subject to 10% urea denaturing PAGE electrophoresis to see if there was any degradation. The electrophoresis time was 30 min. 20bp DNA Lander (Takara) was used for ssDNA length indication.5.V. vulnificus. Two of them as aptamers with higher sensitivity and specificity were further confirmed to have quite good performance even in truncated forms. What's more, the performance of V8 has been confirmed to not be affected by the different strains and culture environments and V. vulnificus even in VBNC status can still be effectively detected, revealing the potentials of them to be developed as biosensors detecting the existence of V. vulnificus clinically or environmentally. In the future, we will focus on the development of more efficient and sensitive biosensors or clinical diagnosis kits.Dependent on whole-bacteria SELEX technology, we screened out ssDNA sequences as potential aptamers binding to V. vulnificus detection aptamers and identified two effective aptamers (V8 and V13) with both clinical and environmental detection significance, promoting the development of related technologies and researches in the field of bacterial aptamers. It is the first time for aptamer to be demonstrated its ability to detect VBNC bacteria.All in all, we developed There are no conflicts to declare.RA-010-C9RA10733A-s001


Life.Hypoxia is a current research topic in biology, physiology, and medicine. The effects of hypoxia and the adaptive and maladaptive responses to the hypoxic stress are extensively explored in the domains of altitude, exercise, pulmonary and cardiovascular medicine, inflammation, immunity, cancer, and metabolic diseases. However, the molecular machinery that regulates the activity of certain genes in response to hypoxia is still not explored. This Special Issue “Cellular and Functional Response to Hypoxia” presents 14 papers dealing with all aspects of responses to hypoxia, at the molecular, cellular, and integrative levels ,3. First


Flexible high-definition white-light endoscopy is the current gold standard in screening for cancer and its precursor lesions in the gastrointestinal tract. However, miss rates are high, especially in populations at high risk for developing gastrointestinal cancer  where lesions tend to be flat and subtle. Fluorescence molecular endoscopy (FME) enables intraluminal visualization of (pre)malignant lesions based on specific biomolecular features rather than morphology by using fluorescently labeled molecular probes that bind to specific molecular targets. This strategy has the potential to serve as a valuable tool for the clinician to improve endoscopic lesion detection and real-time clinical decision-making. This narrative review presents an overview of recent advances in FME, focusing on probe development, techniques, and clinical evidence. Future perspectives will also be addressed, such as the use of FME in patient stratification for targeted therapies and potential alliances with artificial intelligence.• Fluorescence molecular endoscopy is a relatively new technology that enables safe and real-time endoscopic lesion visualization based on specific molecular features rather than on morphology, thereby adding a layer of information to endoscopy, like in PET-CT imaging.• Recently the transition from preclinical to clinical studies has been made, with promising results regarding enhancing detection of flat and subtle lesions in the colon and esophagus. However, clinical evidence needs to be strengthened by larger patient studies with stratified study designs.• In the future fluorescence molecular endoscopy could serve as a valuable tool in clinical workflows to improve detection in high-risk populations like patients with Barrett’s esophagus, Lynch syndrome, and inflammatory bowel syndrome, where flat and subtle lesions tend to be malignant up to five times more often.• Fluorescence molecular endoscopy has the potential to assess therapy responsiveness in vivo for targeted therapies, thereby playing a role in personalizing medicine.• To further reduce high miss rates due to human and technical factors, joint application of artificial intelligence and fluorescence molecular endoscopy are likely to generate added value. Every year around 3.6 million people worldwide are diagnosed with cancer of the upper or lower gastrointestinal (GI) tract, resulting in nearly 2.2 million deaths annually . Early dConsiderable effort has been dedicated to the development of new imaging techniques to overcome this problem. Improving visualization of lesions based on their molecular features rather than morphology alone might aid in the early detection of lesions that are visually occult in white light. This technique is applied in fluorescence molecular imaging, which uses fluorescently labeled probes that bind to specific molecular structures or receptors expressed by (pre)malignant lesions and are made visible with dedicated light sources and camera systems. Incorporating this technique into flexible gastrointestinal endoscopy systems resulted in fluorescence molecular endoscopy (FME). In the last decade, research in the field has transitioned from preclinical to clinical studies, with promising results. Several early phase studies support FME as a successful way to detect (pre)malignant lesions, even before notable morphological changes appear –13. CoulIn this narrative review, we will discuss the current status of FME in flexible gastrointestinal endoscopy . We review current strategies including the selection of suitable molecular probes and available techniques and describe how they can be refined. We discuss the landmark clinical evidence, its gaps, and how these should be translated to clinical use. Finally, we address potential future applications of this promising diagnostic field, such as patient stratification for targeted therapies.References for this review were identified by searching PubMed using the search terms “fluorescence,” “near-infrared fluorescence,” “optical imaging,” “molecular imaging,” “fluorescence molecular endoscopy,” “fluorescent tracer,” and “targeted fluorescent tracer.” Additionally, ClinicalTrials.gov and the Netherlands Trial Register were searched for ongoing clinical trials. References published on or before Sept 15, 2021 were considered. Articles were also identified through searches of the author’s files. Only papers published in the English language were reviewed. The final reference list was generated based on relevance to the broad scope of this Review.Before the development of targeted probes, fluorescence studies were predominantly performed with non-targeted tracers like the fluorescent probe Indocyanine Green (ICG). The mechanism of these tracers relies largely on the enhanced permeability and retention (EPR) effect, by which large-sized molecules or molecule-protein complexes accumulate in tumors due to their increased vascular permeability , 15. OthStrong fluorescence signal in the (pre)malignant target area compared to the surrounding healthy tissue increases the target-to-background or tumor-to-background ratio (TBR) and enhances visualization of the lesion. This enables taking image-guided biopsies, which will direct clinical decision-making in terms of whether resection of a lesion is required, or other therapies are needed if the agent binds to specific target tissue or lesions of interest. A target for molecular detection should therefore comply with one or more of the following relevant features: (1) it is overexpressed in dysplastic or malignant cells, (2) it is minimally expressed in benign or inflamed tissue surrounding the target area, (3) it is upregulated in tumor-associated cells or structures, or (4) it is activated by the microenvironment specifically belonging to the target area , 24. WheSelecting the appropriate molecular probe is of equal importance to target selection. Every probe has its own pharmacodynamic and pharmacokinetic profile, which affects biodistribution and tumor penetration. The half-life of probes generally correlates with their molecular size: the smaller the molecular size of the probe, the faster its distribution and accumulation in the targeted area and clearance from the body. In order of size, the most well-known available molecular probes investigated (pre)clinically are antibodies, antibody fragments, nanobodies, small molecules, and peptides. The dose-to-imaging interval needs to be well-balanced for each probe, because any circulating unbound probe may cause unwanted background fluorescence . A probeIt is relevant to consider the pharmacological and optical properties of individual targets and probes. For some probes, the previously mentioned disadvantages regarding distribution and clearance can potentially be overcome by direct topical application of the probe instead of intravenous administration , 39. TheThere are certain limitations to topical administration, as it requires spraying the entire surface to enable thorough examination. The limited size and relatively clean mucosal surface of the esophagus facilitate complete coverage; however, larger volumes of spray are needed for the larger colon. Prior to a colonoscopy, patients need to “clean” their colon thoroughly using laxatives, since fecal remnants and physiologically present mucus can impair mucosal coverage. Systemic administration, on the other hand, ensures a more even distribution of the probe throughout the tissue and allows the tracer to penetrate deeper, which may also display submucosal lesions. Furthermore, dosing is easier to standardize. Lastly, while the additional time required for topical probe administration may not be a burden to the patient, it could reduce the daily number of procedures. Thus, it has to be ensured that the benefits of topical application do not outweigh the potential advantages of systemic administration.Besides selecting the most suitable molecular probe and the most viable way to administer it, other steps need to be taken to make the target visible. We will discuss how this is performed in current FME studies, as well as gaps in techniques and promising new strategies.In order to enable real-time and safe visualization, molecular probes are conjugated to a fluorescent dye — or fluorophore — which absorbs photons emitted by an external light source. Once a photon is absorbed, the fluorophore enters a state of excitation. Eventually, the fluorophore returns to its ground state, emitting the extra energy as light at a longer wavelength, creating a fluorescent signal , 48. CurVisualizing the emitted fluorescent signal requires a dedicated NIR camera system to be incorporated in wide-field endoscopes. The standard charge-coupled device cameras are unable to translate the signal to the monitor due to their NIR filter systems. In contrast to surgical systems, the endoscopes used in GI endoscopy are flexible and long in order to be able to maneuver through the GI lumen (average length of 103–133 cm with an outer diameter of 8–12 mm). This long but narrow workspace complicates installation of the required optical hardware at the tip of the endoscope. Currently, there are no flexible NIR-imaging endoscopy systems on the market. Clinical studies are performed with modified fiber-based endoscopy systems, in which a fiber is inserted through the working channel of a conventional endoscope (mother-baby approach). This fiber conducts the excitation light to the endoluminal tissue of interest and leads the emitted signal back to a NIR camera system  enables in vivo microscopic imaging of the intraluminal tissue with subcellular resolution. Tissue can be imaged with a thousand-fold magnification and tissue architecture can be evaluated during endoscopy . Clinica“What you see is not always what you get”: as with many emerging imaging technologies, a combination of data preprocessing steps is required to correct for issues associated with data acquisition. Fluorescence intensity is influenced by multiple non-pathological variables, like absorption and scattering of light in tissue, or reflectance on the smooth surface of the mucus-covered mucosa. Altering the distance and angle of the endoscope to the tissue can significantly change the detected optical signal. The variable intensity might lead to incorrect interpretation, especially if the endoscopist is unaware of these confounding variables. Proper training in signal interpretation and imaging technique is critical, as well as standardized clinical workflows. Fluorescence quantification is a way to objectify the obtained signals. In most early FME studies, quantification was performed ex vivo with algorithms to account for differences in endoscope-tissue distance and geometry over the image field of view , 54, 55.Many probes in the NIR spectrum have been tested in preclinical settings for several purposes. In selected cases they made it through to patient studies, where they were found to be safe, feasible, and effective as well. We will illustrate the need for techniques to improve intraluminal lesion detection in the GI tract and discuss promising results of probes targeting these lesions.Most studies on FME in the upper GI tract are performed in patients with Barrett’s esophagus. Barrett’s esophagus is a condition where the squamous epithelium of the esophagus is replaced with metaplastic columnar epithelium. Within this epithelium, precancerous dysplasia may arise. Because of this, Barrett’s esophagus is one of the most important risk factors for developing esophageal adenocarcinoma . AlthougIn the lower GI tract, the majority of FME studies are performed in the screening of colorectal cancer. This is one of the most common and lethal cancers worldwide, representing more than 9% of cancer-related deaths yearly . PatientSeveral clinical trials have been conducted on FME with probes targeting (pre)malignant lesions of the GI tract. The current landmark studies are summarized in Table Nagengast and colleagues were one of the first to use FME to improve dysplasia detection over standard WLE in patients with Barrett’s esophagus. They administered the fluorescently labeled monoclonal antibody bevacizumab-800CW both topically and intravenously (2 days prior to endoscopy), which led to successful real-time visualization of dysplasia and adenocarcinoma Fig.  13]. Th. Th13]. A last noteworthy clinical trial on lesion detection in Barrett’s esophagus was recently published by Chen and colleagues. In this first-in-human study, a new technique of multimodal FME was performed, using two excitation lasers of different wavelengths (638 and 785 nm) guided through a single flexible fiber. With this multiplexed imaging tool and the topical application of two different fluorescently labeled peptides (QRH*-Cy5 specific for EGFR and KSP*-IRDye800 specific for ErbB2), 92% of the present neoplastic lesions were successfully visualized . This stBesides enhancement of lesion detection, FME could also play a role in personalizing treatment strategies. This is illustrated in a clinical study by Tjalma and colleagues, using FME and spectroscopy with bevacizumab-800CW on restaging locally advanced rectal cancer after neoadjuvant chemoradiotherapy (nCRT) . At presIn vivo molecular characterization can also be used to evaluate drug delivery to targeted tissue and potentially predict therapy response. Goetz and colleagues conducted a preclinical study performing CLE with fluorescently labeled cetuximab: an antibody targeting EGFR, which is a component of the multimodal chemotherapy regimen in metastatic colorectal carcinoma. Human colorectal carcinoma cell lines were induced in mice and CLE was implemented before and after treatment with cetuximab. High fluorescence signal before treatment was related to significantly slower tumor progression, better overall survival, and better physical condition compared to low fluorescence signal . This suThe clinical findings mentioned above include current landmark studies performed with FME. Although promising and high in quality, these are proof-of-concept studies with small sample sizes. Moreover, study designs and outcome measures differ strongly. This makes interpreting and comparing studies of a certain probe hardly possible, let alone comparing different probes for a certain indication. For this reason, currently available clinical evidence need further validation with larger study populations and stratified study designs.Another research gap is that no FME studies have been carried out in patients with active IBD. These patients have a high risk of developing colorectal carcinoma and encounter high miss rates due to the camouflaging effect of the inflamed background , 9. SeleThe recent transition from preclinical to clinical studies has shown that FME is able to visualize subtle, macroscopically invisible, or uncertain lesions in the upper and lower GI tract that are regularly missed during conventional flexible white-light endoscopy. FME might therefore be a very promising tool in GI endoscopy, addressing the high miss rates of (pre)malignant lesions in both upper and lower GI tract, and improving early detection. Moreover, endoscopic interventional options are rapidly increasing. Currently, premalignant or early-stage GI cancer can often be removed endoscopically. The combination of reliable early detection of (pre)malignant lesions and minimally invasive removal yields an interesting field of action for oncological care.Moreover, the increasing number of unique probes or drugs for different molecular targets offers a wide range of potential future applications. FME could help determine the molecular characteristics of malignant lesions or inflammation, thereby paving the way for personalized targeted therapy in gastroenterology. By using fluorescently labeled drugs as a molecular probe, drug distribution and pharmacodynamics can be visualized in vivo, allowing for the possibility of predicting drug responsiveness. As discussed, this would apply for patient stratification in IBD and oncological treatment, i.e., neoadjuvant therapy in several malignancies. It might help determine whether a patient is prone to respond to therapy or not. The ultimate goal would be to label different types of drugs with different fluorescent dyes, and visualize them in vivo with multispectral camera systems. This could help to identify the optimal treatment before administering it in a therapeutic dose, which improves patient stratification, safety, and (cost) efficiency. MDSFR/SFF spectroscopy could measure mucosal concentrations, for optimizing the dose of the selected treatment.Some obstacles need to be addressed before FME can be implemented in clinical practice. The most important one is the potentially confounding effect of the human factor: all the additional information that FME and accompanying modalities offer makes interpretation more complex and leads to inter-observer variabilities. Adequate training of endoscopists is needed to benefit from the complementary input offered by FME. However, gaining experience takes time and may be costly. Furthermore, the attention span of the endoscopist — which can be lowered by distraction or tiredness — will still be of substantial influence on detection rates. Artificial intelligence (AI), and particularly deep learning, is increasingly used for computer-aided detection (CAD) of (pre)malignancies in endoscopic images. Multiple studies have shown that AI algorithms developed for image analysis in colonoscopy can successfully recognize (pre)malignant colonic lesions, as well as grade the inflammation status in IBD patients –81. ThesA recent meta-analysis by Spadaccini and colleagues showed that CAD-assisted colonoscopy significantly improves adenoma detection rates compared to high-definition WLE and available strategies that increase mucosal visualization, such as chromoendoscopy . HoweverAnother obstacle that needs to be addressed is the extra procedure time that FME requires due to administering of the probes and switching fibers and camera systems. If FME were used in all procedures, it could reduce the total number of operations by 1/5th (assuming that 5 min is added to every 20-min procedure). Therefore, technological refinement is required to streamline procedures. With integrated NIR systems — eventually with multiple spectra for simultaneous use of multiple tracers — FME could be efficiently applied in wide-field endoscopy without the need for switching fibers through the working channel. Moreover, in certain patient populations FME might reduce procedure time because fewer biopsies have to be taken. All in all, in every particular procedure the extra time that FME requires has to be balanced against the possible (time) gain it could give. Patients who are at high risk for (pre)cancerous lesions like patients with Lynch syndrome, IBD, and Barrett’s esophagus will benefit most — healthwise, costwise, and timewise.Fluorescence molecular endoscopy is a rapidly emerging field in flexible GI endoscopy that enables the visualization of lesions by detecting molecular changes rather than morphological changes. As molecular alterations in oncogenesis can appear before lesions become visible to “the naked eye,” FME can serve as a modality for early intraluminal detection of dysplastic lesions or GI cancer. It has the potential of improving screening programs for at-risk populations, as well as playing a part in personalizing medicine. Although work must be done to refine strategies and strengthen clinical evidence, we believe that FME might have an important role in GI endoscopy in the near future. Cooperation between clinicians, pharmacists, biologists, chemists, and engineers will give rise to this promising new imaging strategy in gastrointestinal endoscopy with great impact on both diagnostics and personalized medicine.