The purpose of this study is to explore the influence of consumers’ choice deferral behavior on their intertemporal choice preference. The empirical study shows that consumers’ choice deferral behavior can significantly affect their intertemporal decision preference through the level of hopefulness. Compared with non-choice deferral behavior, choice deferral behavior can improve the level of consumers’ sense of hopefulness, which then makes them prefer larger-longer interests in intertemporal decision-making. The effect of consumers’ sense of hopefulness on their intertemporal choice preference is moderated by their perceived information integrity. When the perceived information integrity is low, the effect of hopefulness on intertemporal decision preference will be enhanced, but when the perceived information integrity is high, the effect of hopefulness on intertemporal decision preference will not be affected. In addition, the theoretical and practical significance of this study and the prospect of future research are also discussed. Due to the variety of products, the uncertainty of consumers’ preferences, and other reasons, consumers often find it difficult to make decisions, which leads them to give up making purchase decisions at present and choose to postpone making decisions later . With thIndividual choice deferral behavior and intertemporal choice preference are both hot topics in recent years, but few scholars consider the relationship between them . InterteTo sum up, based on the existing research results, this paper puts forward research hypotheses, constructs a model of the impact of consumers’ choice deferral behavior on their intertemporal choice preference, and attempts to explore the relationship between the two, which is verified by empirical analysis. Finally, by comparing this study with previous studies, the theoretical contribution, practical significance, and research defects of this paper are clarified, and the future research direction is prospected.Intertemporal choice preference is a hot topic in behavioral decision-making and related fields, which refers to the decision-making subject balancing the options of different times and different interests, and then making various judgments and choices . For theChoice deferral behavior is one of many decision-making behaviors, which means that a decision-maker does not make a choice at the time a decision is required, but postpones decisions and chooses later  — in othPerceived information integrity refers to decision-makers’ subjective perception of the integrity of information required to make decisions. When consumers make purchasing decisions, they judge according to the information they have. Many scholars divide the attributes of information into quantity and quality , and mosHopefulness specifically impacts decision-making. Previous studies on hopefulness have focused on people’s mental health, healthcare, education, and other factors, while this paper focuses on the role of hopefulness in consumer behavior. Scholars have different understandings of hopefulness, which can be roughly divided into three categories. In the first category, some scholars classified hopefulness as an emotion, believing that it is an emotion generated during a difficult situation . Second,Choice deferral behavior and intertemporal choice preference are related to the time distance. Choice deferral behavior can directly increase decision-making time, while intertemporal decision making is choosing different interests at different time points. The construal level theory includes an individual’s perception mechanism of things at different time distances. It holds that individuals explain choices at a high-level interpretation of things at a long time distance, that is, the individual experience looks at things from an abstract, holistic, essential, and core point of view; the individual has a low level of interpretation of things at a short time distance, that is, the individual experience looks at things from a specific, superficial, and situational point of view . In the H1:Consumers’ choice deferral behavior can significantly impact intertemporal choice preference. Non-choice deferral behavior can cause consumers to prefer sooner-smaller interests (SS); choice deferral behavior can cause consumers to prefer LL.Hopefulness is a kind of positive emotion, which includes two dimensions: dynamic thought  and path thought  . Choice H2:Consumers’ choice deferral behavior significantly impacts their level of hopefulness. Non-choice deferral behavior can reduce consumers’ level of hopefulness, while choice deferral behavior can improve consumers’ level of hopefulness.Emotional states can influence decision-makers’ decision-making behavior. According to the broaden-and-build theory of positive emotions, positive emotions can expand a person’s instantaneous cognition and action abilities and broaden the scope of a person’s attention, cognition, and action . IndividH3:Consumers’ level of hopefulness can significantly affect their intertemporal choice preference. Higher levels of hopefulness cause consumers to prefer LL; lower levels of hopefulness cause consumers to prefer SS.When we synthesize H1, H2, and H3, we find that choice deferral gives consumers more time to think and act, reduces their negative emotions effectively, and causes their emotions to change for the positive. To optimize decision-making results, consumers are willing to think actively as well as work to discover more solutions—i.e., they have higher dynamic thought and path thought. According to the definition of hopefulness, choice deferral does give consumers a higher level of hopefulness. Simultaneously, according to the broaden-and-build theory of positive emotions, we learn that higher levels of hopefulness can broaden consumers’ thoughts, give them a longer-term vision, and help them analyze and solve problems more rationally. Therefore, when given a choice of interests, consumers will pursue long-term interests first, and then, prefer LL in intertemporal choice making. Comparatively, non-choice deferrals leave consumers with insufficient time to think and makes them reluctant to think comprehensively in a short amount of time, which means consumers have lower levels of hopefulness. As a result, lower levels of hopefulness can cause consumers to feel negative and focus only on immediate interests, which in turn means that they are unable to fully consider a purchase, are more irrational and impulsive, and will prefer sooner-smaller interests (SS). Based on these reflections, hypothesis H4 is proposed.H4:Consumers’ level of hopefulness plays a mediating role in the relationship between choice deferral behavior and intertemporal choice preference.Although consumers’ hopefulness levels can affect their intertemporal choice preferences as well as the relationship between choice deferral behavior and intertemporal choice preference, this effect may vary with the degree of information integrity consumers understand and perceive. The integrity of perceived information is one of the important factors affecting consumer decision-making, information overload and lack of information will both affect individual value judgment of options . AccordiH5:Consumers’ perceived information integrity can moderate hopefulness’ fully mediating role between choice deferral behavior and intertemporal choice preference.Based on the literature review and our research hypotheses, a research model is proposed and shown in In this study, a questionnaire survey was conducted among the staff of a university in Guangxi, China. In the pre-experiment, 20 staff were randomly invited to conduct a questionnaire survey. A total of 20 questionnaires were distributed and 20 valid questionnaires were collected. In the formal experiment, 240 staff were randomly invited to conduct a questionnaire survey, and a total of 240 questionnaires were distributed. After deleting the questionnaires with problems such as random filling and inconsistent logic, 175 valid questionnaires were finally returned. In the formal experiment, the proportion of male and female subjects was balanced . The age of subjects was mainly from 18 to 30 years old (94%), the occupation was mainly students (84%), and the education was mainly undergraduates (58%). The purpose of the pre-experiment is to select the type of products to be used in the formal experiment through a questionnaire survey. The main question of the questionnaire is for what kind of goods  the subjects will defer their choice and the reasons. Finally, the artificial intelligence box of virtual brand is selected as the experimental material.The purpose of the formal experiment is to collect the required data by issuing questionnaires to explore the relationship between variables. Two different questionnaires were developed based on whether to delay selection. Each questionnaire covered all the variables to be measured. When sending out the questionnaire, it was first explained to the subjects that the purpose of this time is to collect data for academic research rather than for commercial purposes. In addition, the questionnaire is anonymous and does not involve personal privacy. Then, random questionnaires were sent to the subjects to fill in and answer. The first part of the questionnaire is demographic information, and the second part is the items of measurement variables. Finally, after the subjects completed the questionnaire, they expressed their thanks.In the scenario design of the questionnaire, the questionnaire will be pre-set to be filled in under the two scenarios of choice deferral behavior/non-choice deferral behavior, and the subjects will be divided into two groups accordingly for the control experiment. The non-choice deferral behavior scenario is described as follows: “if you need to decide whether to buy it now, how would you think? Please answer the following questions according to your personal feelings.” The scenario of choice deferral behavior is described as follows: “now you don’t need to decide whether to buy immediately, we will contact you in a week, and then you will make a purchase decision, during which time you will have enough time to consider.”It is a continuous variable. When measuring, the subjects are shown the product information list prepared in advance. The list is made by referring to the official attribute information of the product, but the brand of the product is not disclosed to avoid the interference of the brand. Then, the definition of perceived information integrity was given to the subjects: “the quantity and comprehensiveness of information meet the needs of your purchase decision.” Then tell the subjects: “the contents in the Table are all the product information you have obtained. Please rate the information integrity you have perceived. The larger the number, the more complete it is.” Finally, consumers were asked to score with the Likert Scale 5.It is a continuous variable, referring to the State Hope Scale developed by There are many measurement models, such as hyperbolic discount model, quasi-hyperbolic discount model, the delay function , and newSPSS was used for data analysis. Firstly, Pearson correlation analysis was used to test the relationship between variables, and then the mediating analysis and moderated mediating effect analysis were carried out twice to ensure the reliability of the experimental results. The first time is to use SPSS for traditional hierarchical regression analysis, and the second time is to use macro process of SPSS to verify the mediating effect and moderated mediating effect through the bootstrap method , 2015. TThe State Hope Scale’s Cronbach’s alpha coefficient is 0.88. The scale is divided into two dimensions—path thought and dynamic thought. The Cronbach’s alpha coefficient of path thought is 0.89, and the Cronbach’s alpha coefficient of dynamic thought is 0.70. All of the coefficient scores are greater than 0.7, showing that the scale has good reliability and good internal consistency.Since only the mature scale was used to measure the level of hopefulness in the questionnaire, the validity of the scale (six items) was analyzed. The maturity scale was adapted to make it more suitable for the research situation, and it was checked by experienced experts and scholars, which can be regarded as good content validity. By confirmatory factor analysis with Amos, the AVE = 0.56 and CR = 0.88, respectively, indicating that the scale has good structural validity. Through the above analysis, the scale has good validity.First, to ensure procedure control, the order, language expression, and layout design of the questionnaire items are revised and perfected repeatedly. Additionally, an anonymous survey is adopted, and questionnaires are distributed and collected at different times and places, which, in part, controls common method biases. Second, to ensure statistical control, Harman’s one factor test is used. Exploratory factor analysis was used to examine the four variables: choice deferral behavior, level of hopefulness, degree of perceived information integrity, and time discount rate. The results show that the KMO value is 0.52, and the significance level of Bartlett’s test is 0.000. The first common factor explains 49.96% of the total variance, which does not exceed 50%. Therefore, no serious problem exists among common method biases.Testing the relationship between choice deferral behavior, hopefulness, intertemporal choice preference and perceived information integrity.SPSS was used to conduct hierarchical regression on choice deferral behavior, level of hopefulness, intertemporal choice preference and perceived information integrity. The results are shown in p < 0.05). Therefore, compared with non-choice deferral behavior, choice deferral behavior can reduce consumers’ time discount rate and make them prefer LL. Hence, H1 is supported. Regression analysis of hopefulness and choice deferral behavior forms model 2, and the result shows that choice deferral behavior is significantly correlated with hopefulness . Thus, compared with non-choice deferral behavior, choice deferral behavior can improve consumers’ hopefulness. Therefore, H2 is supported. Regression analysis of the time discount rate and hopefulness forms model 3, and the result shows that hopefulness is significantly correlated with the time discount rate . Therefore, the higher the hopefulness, the smaller the consumers’ time discount rate, and the more consumers prefer LL. As a result, H3 is supported.The regression analysis of the time discount rate and choice deferral behavior forms model 1, the result shows that choice deferral behavior is significantly correlated with the time discount rate ; (2) the independent variable  has a significant effect on the mediation variable (hopefulness) ; (3) the independent variable  and the mediation variable (hopefulness) have a significant effect on the dependent variable (time discount rate). Hopefulness has a significant effect on the time discount rate , while choice deferral behavior has no significant effect on the time discount rate . Therefore, hopefulness is a complete mediator and H4 is supported.First, this paper uses SPSS and Using the macro process of SPSS and bootstrap method, model 4 was selected and 5000 samples were repeatedly selected to verify the mediating effect , 2015, tp < 0.005); (2) hopefulness, choice deferral behavior, and perceived information integrity regressions show that the choice deferral behavior coefficient is significant ; (3) time discount rate, choice deferral behavior, perceived information integrity and hopefulness regressions show that the coefficient of hopefulness is significant ; (4) time discount rate, choice deferral behavior, perceived information integrity, hopefulness, and the interaction between perceived information integrity and hopefulness regressions show that the interaction item coefficient is significant . The above test steps fully meet the moderated mediating effect test criteria, which shows that perceived information integrity has a significant moderating effect on hopefulness’ mediating effect. Therefore, H5 is supported.First, this paper uses SPSS to test the moderating mediating effect by referring to the methods of Secondly, in order to ensure the reliability of the conclusion, the macro process of SPSS is used to verify the moderated mediating effect again by the bootstrap method, selecting model 14 and repeatedly sampling 5000 samples , 2015. AThis paper studies the influence of consumers’ choice deferral behavior on their intertemporal choice preference, and verifies the research model through questionnaire survey and data analysis. It is found that consumers’ choice deferral behavior can have a significant impact on their intertemporal choice preference, and the level of hopefulness plays a mediating role in the relationship between them. Compared with non-choice deferral behavior, consumers are more willing to make more efforts to make decisions after a delayed choice, and they will optimize their decisions through different paths and methods. That is to say, choice deferral behavior makes consumers have a higher level of hopefulness, so they prefer LL in intertemporal decision-making. The degree of information integrity perceived by consumers will have a moderating effect on the relationship between the level of hopefulness and intertemporal decision-making. When consumers perceive that they have incomplete information, they can also perceive higher risks . BecauseFirstly, based on the construal level theory, this paper reveals the influence mechanism of choice deferral behavior on intertemporal choice preference, and deepens the research on the after-effect of choice deferral behavior. Because consumers may lose sales opportunities if they do not make a purchase decision immediately, previous scholars pay more attention to the factors that lead to consumers’ choice deferral behavior, such as different decision-making scenarios , the attSecondly, based on the broaden-and-build theory of positive emotions and the prospect theory, this study explored the mediating role of hopefulness and the moderating role of perceived information integrity. Emotion is one of the important factors that affect individuals’ choice deferral behavior and intertemporal choice preference, scholars have done a lot of research, but few scholars pay attention to hopefulness . Based oThirdly, this study explored the positive role of choice deferral behavior. Most scholars believe that consumers’ choice deferral behavior means missing sales opportunities for businesses, so it is usually regarded as a negative factor , but thiFirst, businesses can adopt different marketing methods according to consumers’ different purchase behaviors . According to the previous research, non-choice deferral behavior makes consumers prefer SS, while choice deferral behavior makes consumers prefer LL. Therefore, if consumers choose whether to buy immediately, it means that consumers pay more attention to the current interests. In marketing, businesses should focus on showing consumers the benefits of current purchase, such as being able to catch up with the trend and use the product immediately. If consumers decide to postpone the choice, or have considered it for a period of time, it means that consumers pay more attention to the long-term interests, then in marketing, businesses need to highlight the cost performance of products, after-sales protection, and other aspects.Second, businesses can use differentiated marketing methods according to the level of consumers’ sense of hopefulness. As can be seen from the previous article, hopefulness includes two aspects: dynamic path and thinking path. The higher the level of hopefulness, the more consumers prefer LL. Therefore, businesses can first communicate with consumers to know their sense of hopefulness, such as whether consumers are willing to learn more about product information and whether they will compare different products through multiple channels. When the level of consumers’ sense of hopefulness is high, it indicates that they are willing and will make decision-making efforts, then businesses can carry out marketing from more rational aspects, such as highlighting the material, composition and production technology of products. And when the level of consumers’ sense of hopefulness is low, it indicates that they are not willing and will not make too much decision-making effort, then businesses can carry out marketing from more emotional aspects, such as emphasizing the products’ packaging, spokesperson, etc.Third, businesses can carry out marketing according to the degree of information integrity perceived by consumers. The results show that when consumers perceive incomplete information, they perceive higher risk, and the level of their sense of hopefulness has a strong effect on intertemporal decision preference. In marketing, businesses need to know whether consumers think they have enough information. If consumers think it is useless to have enough information, businesses should actively provide some official, professional, and authoritative information, which can also reflect the professionalism and sincerity of businesses.First of all, this paper mainly compares the changes of consumers’ intertemporal choice preference under two different behaviors of choice deferral and non-choice deferral, and does not further study whether the length of choice delay can affect consumers’ intertemporal choice preference. In the future, we can design different delay times to carry out further research on the basis of this study. Secondly, the subjects of this study are mainly Chinese college students. In the future, this paper can be used as a research paradigm to select more diverse groups, such as groups from different countries and different income levels, to compare the impact of different types of groups’ choice deferral behavior on their intertemporal choice preferences. Finally, more factors need to be included in the research model, such as product type, consumer income, and other factors. Whether they affect the relationship between consumers’ choice deferral behavior and intertemporal choice preference also needs to be explored, so as to make the research better adapt to practical application in different scenarios.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Guangxi University of Ethics Committee. Written informed consent was not required to participate in this study in accordance with the national legislation and the institutional requirements.H-LW, C-YH, S-YZ, and BL: study conception and design. H-LW, C-YH, and BL: acquisition of data. H-LW and C-YH: analysis and interpretation of data and drafting of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Histopathological diagnosis is the current standard for the classification of brain and spine tumors. Raman spectroscopy has been reported to allow fast and easy intraoperative tissue analysis. Here, we report data on the intraoperative implementation of a stimulated Raman histology (SRH) as an innovative strategy offering intraoperative near real-time histopathological analysis. A total of 429 SRH images from 108 patients were generated and analyzed by using a Raman imaging system (Invenio Imaging Inc.). We aimed at establishing a dedicated workflow for SRH serving as an intraoperative diagnostic, research, and quality control tool in the neurosurgical operating room (OR). First experiences with this novel imaging modality were reported and analyzed suggesting process optimization regarding tissue collection, preparation, and imaging. The Raman imaging system was rapidly integrated into the surgical workflow of a large neurosurgical center. Within a few minutes of connecting the device, the first high-quality images could be acquired in a “plug-and-play” manner. We did not encounter relevant obstacles and the learning curve was steep. However, certain prerequisites regarding quality and acquisition of tissue samples, data processing and interpretation, and high throughput adaptions must be considered. Intraoperative SRH can easily be integrated into the workflow of neurosurgical tumor resection. Considering few process optimizations that can be implemented rapidly, high-quality images can be obtained near real time. Hence, we propose SRH as a complementary tool for the diagnosis of tumor entity, analysis of tumor infiltration zones, online quality and safety control and as a research tool in the neurosurgical OR. The annual incidence of primary brain tumors in the USA was 23.79 per 100,000 2013–2017) and the incidence of brain metastases is expected to be approximately four times higher, although recent and robust numbers are currently not available . Neurosu–2017 and In addition to the surgeon’s anatomical skills and experience, several modalities are commonly used in the operating theater to achieve the goal of gross total resection (GTR) in brain tumor surgery including but not limited to fluorescence-guided surgery after immediate preoperative administration of 5-aminolevulinic acid (5-ALA), which has been shown to increase the extent of resection and subsequently the progression-free survival in glioblastoma patients . HoweverClassification and prognostication of brain, skull base, or spinal tumors is still based on histopathological diagnosis, which is the gold standard. In general, conventional intraoperative histopathological diagnosis is based on the interpretation of hematoxylin–eosin (H&E) staining of fast frozen sections. Conventionally, technical assistants and an experienced neuropathologist are needed to process and analyze the tissue. Afterwards, the histopathological findings are reported to the surgical team. This procedure is time consuming and labor intensive. In contrast to oncological surgeries in other disciplines where achievement of tumor-free resection borders is the goal, intraoperative histopathological examination of tumor borders is not regularly performed during neurosurgical resection.Raman spectroscopy (RS) and in particular stimulated Raman histology (SRH) are innovative techniques that allow for a near to real-time intraoperative tissue analysis , 10. TheIn this study, we want to present our first experience with intraoperative SRH imaging > 150 resected tissue specimens of 108 neurosurgical patients. We aim at sharing aspects of process optimization and avoidance of methodical pitfalls needed to maximize the information gained by SRH and to explore the potential of intraoperative SRH as a tool for analyzation of tumor infiltration at the border zone to normal tissue, online quality and safety control, and its utility as a research tool in the operating room (OR).During a period of 6 weeks, tissue specimens of more than 100 consecutive patients with various CNS lesions and mainly brain or spinal tumors were included in this study. Written informed consent was obtained and intraoperative tissue processing and imaging with the SRH system and the respected workflow were documented and analyzed for feasibility of routine clinical implementation.The local ethics committee of the University of Freiburg approved data evaluation, imaging procedures, and experimental design (protocol 5565/15). The methods were carried out in accordance with the approved guidelines, with written informed consent obtained.An initial workflow for intraoperative SRH was based on the recent literature and adapted to our institutional framework conditions. Correct and reasonable workflow steps were perpetuated and unfavorable steps were documented, erased, and replaced by more practical steps as soon as during the next sample acquisition.3  according to the manufacturer’s requirements and as previously described by Hollon et al. . Stimula et al., ).2), and stored in a − 80 °C freezer , metastases (n = 20), and meningeomas (n = 11). However, also tissue samples of rare entities were imaged, for example, subependymoma (n = 2), epidermoid cysts (n = 2), ganglioglioma, or hemangioblastoma . In the non-neoplastic collective (n = 35), we analyzed resected tissue like nucleus pulposus or arachnoid cyst wall, which was also helpful to optimize tissue prerequisites for SRH analysis.In total, tissues from 108 consecutive neurosurgical patients were imaged during a 6-week period. A total of 73 patients underwent surgery for newly diagnosed or recurrent neoplastic diseases. The three most frequent entities were gliomas  was used for tissue acquisition in microsurgical resection. A standard biopsy forceps was used for stereotactical biopsies . Especially in diffuse infiltrating tumors, multiple samples were obtained. Therefore, micro- and macroscopically different appearing putative pathological tissue was selected. After compression of the tissue specimen, multiple 2 × 2 mm sections were selected for SRH imaging . The average number of images acquired was about 14.8 per day. High-quality virtual quasi-H&E SRH images were robustly generated intraoperatively without any complex additional tissue preparation requirements. Within a few minutes of connecting the device, the first high-quality images could be acquired in a “plug-and-play” manner. The implementation of the system in our department has been uncomplicated and intuitive. The learning curve was steep, and only during the first days of usage, two persons  were needed for tissue preparation, imaging, and for data processing. Subsequently, one neurosurgical resident was sufficient for executing these steps.Rigid samples are less suitable for SRH imaging. Tissue has to be readily compressible to avoid artifact-causing air inclusions. Another problem with stiff specimens was potential glass breakage during the process of tissue compression. We therefore did not manage to image bony structures or stiff connective tissues. For example, it was not possible to image anulus fibrosus of a herniated intervertebral disc, whereas nucleus pulposus was sufficiently soft to be loaded onto the sample carriers Table .Table 2SIn order to use SRH for visualization of bacterial infections, we faced the problem that the sample carrier is not designed to image fluids but to image intact tissue, as the system needs a reference for the autofocus with a high cell density. Therefore, we have prepared an emulsion of the centrifuged fluid — for example, CSF — of a patient with fulminant bacterial meningitis or pus from abscess drainage in a prewarmed low melting agarose gel. The emulsion was then loaded onto the sample carrier. However, to date, it was not possible to display immune cells in the emulsion or even bacteria. This may be due to the fact that high-quality imaging with the SRH system is optimized for a sampling depth of 10 µm Table .SRH images were saved as DICOM files. In order to facilitate intraoperative accessibility of the SRH images for the neurosurgeon and neuropathologist, we only recently set a pipeline to upload and store the SRH files into our clinic’s imaging system IMPAX VIII  Fig. . This opSince we acquired more than 14 images from more than 3 patients on a daily average, we had to adapt the workflow for the associated high-throughput demands. The concept of the SRH system suggests the use directly in the OR. In our experience, tissue acquisition occurred rather simultaneously in different concomitant operations. Therefore, it would be bothersome to transfer the imaging system fast enough. To optimize the workflow for our needs and adapt it to our OR infrastructure, we have placed the device at a fixed location in a research laboratory co-located at the OR floor and the samples were transferred directly after acquisition into this laboratory for further processing. The SRH images were initially presented to the surgeon via a tablet with Wi-Fi connection, which was recently complemented by connection of the device to our clinic’s imaging system IMPAX VIII   . This syA major application of SRH is obtaining diagnostic information about the entity of resected putative pathological tissue. As described above, SRH images have now been integrated as DICOM files in our clinic’s imaging system IMPAX VIII immediately after imaging. Therefore, neuropathological assessment is possible at yet unprecedented speed. Previous studies  and veryAnother potential use case of Raman-based methodologies is the detection of metabolic changes in tumor tissue, which could be a surrogate parameter for specific mutations. The relevance of molecular diagnostics in brain tumors is high, as the new 2021 WHO classification of tumors of the central nervous system places a higher priority on molecular changes than ever before . Raman sSRH imaging may also be useful as an additional tool to achieve a greater extent of resection in diffusely infiltrating brain tumors. However, one should consider that there are differences between a real H&E image and the virtual H&E-like SRH image. For example, protein-rich extracellular fibers are usually red in the conventional H&E imaging, but may appear eosinophil in the virtual H&E-like SRH image. In order to use SRH as a robust modality to determine tumor histology or tissue diagnosis at the suspected tumor border, it is inevitable to fully understand the limitations and alternative illustration of SRH in comparison to classical staining in health and disease. These are also absolute prerequisites in order to use the SRH technology as an intraoperative tool for improvement of the extent of resection. One problem is the fact that the information provided by SRH imaging of white matter is very sparse . In turThe near real-time character of tissue analysis predestines SRH imaging for quality control of intraoperative biopsies. Still, if small lesions are resected or are subject to stereotactic biopsy, it is of utmost importance to increase the diagnostic yield of the specimen. Using SRH, an additional quality control step is implemented in addition to the standard of care intraoperative neuropathological assessment by H&E staining of frozen sections without disturbance of the neurosurgical workflow. One technical limitation of SRH is that the examined tissue has to be readily and uniformly compressible. Therefore, at the moment, it is not possible to image stiff specimen, which reduces the applicability of this method. On the other hand, SRH is not only limited to the field of neurosurgery. Other applications for example in Head and Neck surgery were also reported . In geneOur study shows that use of SRH imaging in a major European neurooncological center is feasible although the measures taken for this cause are not without drawbacks. Using the imaging system for multiple ORs can lead to delays in tissue processing, if the system is already imaging. This has never exceeded more than a few minutes and did not lead to noticeable lower quality of SRH imaging, but it may lead to suboptimal tissue handling in these exceptional cases.Adaptations to the high throughput application of SRH imaging proved themselves effective. Nevertheless, some difficulties regarding the exact registration and documentation of the site of tissue acquisition remain. This issue refers to the tissue acquisition, for example, in a brain tumor resection but not for stereotactic biopsy. Of course, annotation points were ideally inserted for every sample in the MRI 3D dataset of the neuronavigation system. Yet, system inherent factors like inaccuracies of the initial patient registration or the selection of the exact extraction localization with the pointer are only minor problems. Brain shift effects on the accuracy of the neuronavigation system prevent a precise and valid documentation of the exact tissue extraction points.Our experience shows that the implementation of intraoperative SRH is indeed feasible and beneficial, and is readily and rapidly adaptable to the local conditions and workflows, if certain prerequisites are considered. SRH is not a replacement of classical intraoperative neuropathological assessment, but rather a useful complementary addition for tissue analysis in neurooncological surgeries. We firmly believe that SRH has the potential to evolve into a useful tool to guide the extent of resection in the operating room (OR). Next to the practical utility in routine usage, it is also a valuable method for future neurosurgical studies and intensifies our understanding of the OR as part of a research laboratory. Augmented by deep learning/machine learning/AI-based automated analysis and classification, we are convinced that this technology will pave the way towards label-free digital histopathology in a wide range of OR scenarios and settings.


The COVID-19 pandemic has resulted in a huge number of deaths from 2020 to 2021; however, effective antiviral drugs against SARS-CoV-2 are currently under development. Recent studies have demonstrated that green tea polyphenols, particularly EGCG, inhibit coronavirus enzymes as well as coronavirus replication in vitro. Herein, we examined the inhibitory effect of green tea polyphenols on coronavirus replication in a mouse model. We used epigallocatechin gallate (EGCG) and green tea polyphenols containing more than 60% catechin (GTP60) and human coronavirus OC43 (HCoV-OC43) as a surrogate for SARS-CoV-2. Scanning electron microscopy analysis results showed that HCoV-OC43 infection resulted in virion particle production in infected cells. EGCG and GTP60 treatment reduced coronavirus protein and virus production in the cells. Finally, EGCG- and GTP60-fed mice exhibited reduced levels of coronavirus RNA in mouse lungs. These results demonstrate that green tea polyphenol treatment is effective in decreasing the level of coronavirus in vivo. The COVID-19 pandemic has resulted in millions of deaths from 2020 to 2021 due to the high mortality of SARS-CoV-2 [Green tea has been consumed for thousands of years, and many beneficial effects of green tea have been reported ,4. RecenBecause green tea is a very popular beverage, there has been attempts to evaluate the correlation between average green tea consumption and COVID-19 morbidity/mortality, and countries with higher rates of green tea consumption showed less morbidity/mortality than those with lower green tea consumption . RecentlCoronaviruses are classified into alpha, beta, gamma, and delta coronaviruses, and only alpha and beta coronaviruses are reported to infect humans . Seven cRD and HCT8 cells were maintained in MEM  containing 10% fetal bovine serum  and 1% penicillin-streptomycin solution (Welgene). RD cells were infected with HCoV-OC43, as described previously . The HCo7 PFU/mL) through intranasal injection [2) euthanasia, and samples were collected.Male C57BL/6 mice (3-week-old) were obtained from DBL  and housed with wood chip bedding, clean-air rooms with a 12-h light–dark cycle, and a relative humidity of 50%. Mice were infected with 10 μL of HCoV-OC43 virus . After dehydration, the cells were air-dried using a vacuum desiccator. Cells were coated with platinum, and images were captured using a Carl Zeiss SEM SUPRA 40 microscope .t-test using Excel software, Excel 2016 . Statistical significance was set at p < 0.05.The results of Western blotting and quantitative RT-PCR were evaluated by a 2-tailed Student’s To examine the coronavirus infection on the surface, we infected RD and HCT8 cells with HCoV-OC43 strain. Three days after infection, we used a scanning electron microscope (SEM) to analyze the infected cells. The mock-infected control cells showed a smooth surface, and coronavirus infection resulted in coronavirus virion particles on the cell surface A–F. We aWe examined the effect of green tea polyphenols on coronavirus replication in mice and produced the coronavirus in RD and HCT8 cells. As HCoV-OC43 virus produced in HCT8 cells results in efficient infection in mice, we performed the experiments with the coronavirus produced in HCT8 cells. We examined the inhibitory effect of EGCG on coronavirus infection in HCT8 cells and found that EGCG treatment effectively decreased virus production and surface projection in HCT8 cells A. In addNext, we examined the effects of green tea polyphenols on coronavirus replication. We used green tea polyphenols containing more than 60% catechin (GTP60), and SEM analysis demonstrated that GTP60 treatment decreased coronavirus-induced virus particle production and surface projections in HCT8 cells A as wellWe also confirmed the reduction in coronavirus replication by examining coronavirus infectivity upon EGCG or GTP60 treatment. RD cells were infected with HCoV-OC43 and treated with EGCG or GTP60. After the media change, the infected cells were incubated for 72 h, and the conditioned media was used to infect uninfected cells A. CoronaAfter we showed the inhibitory effect of EGCG and green tea polyphenols on coronavirus replication in vitro, we performed a mouse experiment to examine their inhibitory effect in vivo. Mice were infected with HCoV-OC43 virus, which was produced in HCT8 cells by intranasal infection, and the coronavirus RNA was evaluated by quantitative RT-PCR. We found that coronavirus RNA was readily detected in the mouse lung after two weeks A,B. HoweMounting evidence indicates that green tea polyphenols such as EGCG inhibit coronavirus replication in vitro; however, experiments using mouse models have not yet been performed . To the In this experiment, we used two types of compounds as a source of green tea polyphenols. One was EGCG, a major green tea polyphenol, and the other was a green tea extract from lab reagent suppliers (Sigma-Aldrich). Because the composition of green tea extract can be diverse based on the extraction methods, we used a standard green tea extract. Therefore, a similar experiment can be repeated by other researchers. In addition, we used the HCoV-OC43 virus for mouse experiments as a surrogate for SARS-CoV-2. Due to strict regulations, it is difficult to perform experiments with SARS-CoV-2. However, both SARS-CoV-2 and HCoV-OC43 are beta coronaviruses, and HCoV-OC43 is a good alternative for SARS-CoV-2 . FurtherInitially, we attempted to infect mice with HCoV-OC43 virus, which was produced in human RD cells; however, successful infection was not obtained with repeated trials. For this reason, we tested several other cell lines and found that the human HCT8 cell line was suitable for the production of HCoV-OC43 virus for infecting mice. Although we detected the replication of HCoV-OC43 in mice lungs, we did not observe any mouse death due to coronavirus infection, and we did not observe weight loss due to infection (data not shown). The effect of HCoV-OC43 infection on mice was mild and we did not observe any other external differences between the uninfected group and infected group. This is probably due to the pathological differences between humans and mice, and it is the limitation of animal experiments. To validate the effect of EGCG or green tea on coronavirus, further human clinical study should be conducted.In this experiment, we evaluated the mouse intake of EGCG and GTP60 based on daily water consumption and reagent concentration . As the There are many reports supporting the efficacy of green tea against coronavirus. Green tea polyphenols, including EGCG, have been reported to inhibit several coronavirus proteins . Moreove


TP53, encoding the tumor suppressor and transcription factor p53, is the most frequently mutated gene in human cancers  at the Shanghai Synchrotron Radiation Facility (SSRF). M.L. was funded by the National Key R&D Program of China (2017YFA0506200), the National Natural Science Foundation of China , the Gaofeng Clinical Medicine Grant (828318), the Shanghai Excellent Youth Academic Leader (20XD1422700), the Shanghai Medical and Health Excellent Discipline Leader Development Plan (2018BR36), the Shanghai Collaborative Innovation Center for Translational Medicine (TM201902), the Foundation of the National Facility for Translational Medicine (Shanghai) (TMSK-2020-003), and the Newton Advanced Fellowship (NAF\R1\180216). J.-L.W., S.C., and M.L. wrote the manuscript.]


Epithelial to mesenchymal transition (EMT) is a key signature in both physiologicalprocesses  and in tumor metastasis . WhiletIn particular, it was reported that HOTAIR, a well-established predictor of metastasis indifferent solid tumors, is involved in chromatin modification, acting as a scaffold for thegeneral chromatin modifier PRC2 complex in tumorigenesis , 4; howeBuilding on this evidence, we designed an RNA-based dominant negative molecular approach tocounteract HOTAIR function in hepatocellular carcinoma (HCC) cells.RNA therapeutics represent a growing field of investigation and application. The use of RNAmolecules shows several advantages since they show a very low immunogenicity, are able topenetrate the cell/nuclear membrane, and to target the desired gene even if highlyexpressed, they are cheap and easy to synthesize, and can be chemically modified, in orderto increase their stability or to stabilize secondary structures. Moreover, concerning thedelivery of these molecules, in recent years, many strategies have been developed forincreasing the efficient delivery of RNA therapeutic molecules to specific target cells.Some of these strategies are represented by the use of nanoparticles, lipid nanoparticles,and extracellular vesicles, above all exosomes, properly engineered in order to increase thedelivery and the on-target effects. Also other approaches have been proposed, like theconjugation of these nucleic acid molecules with sugars, lipids, peptides, nucleic acidsligands in order to interact with the cell membrane or with the surface receptors . TakingOverall, the described RNA-based dominant negative approach further contributes to thetranslational applications based on the use of RNA therapeutics. Specifically, this strategyappears conceivably devoid of off-target effects in EMT and tumorigenesis and holds promisesof effectiveness when the function of lncRNAs is proved as a determinant.


Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry, as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection, while a proportion is trafficked to the cytoplasm. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of these viruses is imperative in order to effectively find ways to control them. Coronaviruses (CoVs) are an important family of positive strand RNA (+RNA) viruses with a wide host range. In humans, some strains of these viruses, such as the human coronavirus (HCoV) 229E, can cause the common cold; however, there are now three CoVs that are more pathogenic and cause higher fatality rates. Until 2019, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), which were initially isolated in China and Saudi Arabia, respectively ) and theAs obligate intracellular parasites, viruses rely on their host cells to provide not only a site for replication, but much of the cellular machinery required to produce new virus particles. All +RNA viruses induce the rearrangement of intracellular membranes to form Ros ,6,7. TheThe possibility of antiviral therapies targeting the ROs has meant an increased attraction for understanding this part of the viral life cycle in recent years. Until very recently, the structures of ROs formed by the CoV family were split into the alpha- and beta-CoVs and the gamma- and delta-CoVs. The alpha- and beta-CoVs were known to induce convoluted membranes and DMVs ,33,34,35As ROs have long been purported to provide a site for viral RNA synthesis, a key point for investigation has been in understanding exactly where the sites of viral replication are within the cell, and the involvement of ROs in the process. For SARS-CoV, it has been shown using biochemical methods in vitro that sites of viral RNA synthesis are protected by membranes, possibly within DMVs , and dsRThe gammacoronavirus, IBV is a virus of economic importance to the global poultry industry, causing a highly contagious respiratory disease of chickens and other poultry. Infection results in reduced quantity and quality of eggs and meat and has an impact on animal welfare as well. Recent work has elucidated the role of ROs in viral RNA synthesis and suggested a pathway for nascent RNA to leave the DMV via a molecular pore . However2. IBV -2-aminoethanesulfonic acid (BES), 0.21% sodium bicarbonate, 2 mM l-glutamine, 250 U/mL nystatin, 100 U/mL penicillin, and 100 U/mL streptomycin). Huh7 cells  were maintained in DMEM supplemented with 10% FBS. Human coronavirus 229E  infections were carried out in DMEM + 2% FBS. VeroE6 cells  were maintained in DMEM supplemented with 10% FBS. SARS-CoV-2  infections were carried out in DMEM + 2% FBS. Porcine LLC-PK1 cells  were maintained in DMEM  supplemented with 10% FBS  at 37 °C 5% COin BeauR ) infectiC CL-101 ) were maC CL-101 ,48), infCells seeded onto glass coverslips were infected as in For pulse chase experiments, cells were pulsed with BrU and ActD for 1 h at concentrations previously used. After this time, control cells were fixed in RNase-free pfm, while the rest were chased with 50 µM uridine (Sigma Aldrich) to out-compete the labeled BrU. Cells were incubated in uridine and ActD until 24 hpi then fixed as above and labeled as in Cells seeded onto glass coverslips were mock-infected or infected as laid out in For labeling of BrU samples, in order to prevent loss of the BrU signal, immunofluorescence (IF) labeling was promptly carried out in an RNase-free environment in the presence of RNasin at 0.133 U/mL .Cells were visualized using a Leica CLSM SP5, SP8 or Stellaris 5 microscope . Super-resolution (stimulated emission depletion (STED)) microscopy was performed using a Leica TCS SP8 STED 3X microscope with inverted stand. STED Images were deconvolved using Huygens Professional software 18.10 . Figures were assembled using Adobe Photoshop CS6.We have shown previously over the course of the IBV life cycle that nsp12, the viral RNA-dependent RNA polymerase (RdRp), does not colocalize with dsRNA . The rolIn comparison, nsp12 staining was unaffected when cells were permeabilized with digitonin , indicatKnowing that dsRNA was likely within the DMVs, we sought to understand how it associated with sites of viral RNA synthesis. The uridine analog bromouridine (BrU) was incorporated into the nascent viral RNA. DF1 cells were infected with IBV then incubated in media containing BrU for 30 min prior to fixation to provide a snapshot of RNA synthesis at that timepoint. The cellular transcription inhibitor, Actinomycin D (ActD) was used to selectively inhibit cellular transcription to allow visualization of sites of active viral RNA synthesis . StartinSnijder et al.  showed tTo trace the fate of viral RNA synthesized early in infection and find out whether viral RNA produced within membrane-bound compartments is transported to the cytoplasm, cells were successively exposed to labeled uridine in the form of bromouridine (pulse) and then to unlabeled uridine (chase). In these pulse-chase experiments, viral RNA produced between 7–8 hpi was labeled with BrU as before, followed by a chase with excess unlabeled uridine until fixation at 24 hpi. The localization of viral RNA at 8 hpi was in cytoplasmic puncta, consistent with earlier observations a, left. While the structure of ROs have been shown to be conserved across all genera of CoVs, some morphological differences between the viruses remain. Mainly, while convoluted membranes are found much more widely in alpha and beta CoV, they are found to a lesser extent in delta- and gamma-CoVs. In alpha- and beta-CoVs, the spherules were found associated with CM rather than zER and most spherules were in sealed compartments rather than remaining open to the cytosol, as is the case in most spherules in gamma-CoV infections . TherefoThe formation of ROs is conserved across all +RNA viruses, an essential step in their life cycle. These ROs have long been thought to provide a site for viral RNA synthesis and have been shown to do so for several viruses ,30,31. WPrevious studies have shown for other +RNA viruses, such as SARS-CoV, FHV, and most recently in SARS-CoV-2, that dsRNA is found associated with DMVs ,31,39,40within a membrane-bound compartment. Moreover, we have confirmed that viral RNA synthesis by diverse CoVs from each of the four coronavirus genera, including recently identified SARS-CoV-2, takes place within a membrane-bound compartment. Although we cannot exclude that CoV RNA synthesis takes place when associated with spherules, as MERS-CoV and IBV RNA synthesis has been shown to be associated with DMVs [within DMVs, and that this is conserved across the whole CoV family  the site of viral RNA synthesis was bound within a membrane, consistent with being located on the interior of DMVs ,44. AlthAs summarized in


In spite of latest advances in researching the biological roles of m6A, its function in the development and progression of bladder cancer remains unclear. In this study, we used MeRIPty -55-seq and RNA-seq methods to obtain a comprehensive transcriptome-wide m6A profiling and gene expression pattern in bladder cancer and paired normal adjacent tissues. Our findings showed that there were 2,331 hypomethylated and 3,819 hypermethylated mRNAs, 32 hypomethylated and 105 hypermethylated lncRNAs, and 15 hypomethylated and 238 hypermethylated circRNAs in bladder cancer tissues compared to adjacent normal tissues. Furthermore, m6A is most often harbored in the coding sequence (CDS), with some near the start and stop codons between two groups. Functional enrichment analysis revealed that differentially methylated mRNAs, lncRNAs, and circRNAs were mostly enriched in transcriptional misregulation in cancer and TNF signaling pathway. We also found that different m6A methylation levels of gene might regulate its expression. In summary, our results for the first time provide an m6A landscape of human bladder cancer, which expand the understanding of m6A modifications and uncover the regulation of mRNAs, lncRNAs, and circRNAs through m6A modification in bladder cancer.N Bladder transitional cell carcinoma (TCC) is the most common urothelial tumor in urology departments in China. The vast majority originated from epithelial tissue, and TCC accounts for more than 90% –3. At pr6-methyladenosine methylation modification is the most common in mRNA and non-coding RNA , and mostly located near the stop codon, 3′-UTR, and long exon of transcript  values of these transcripts, and generated a cumulative curve. The result revealed that the proportion of transcripts modified by m6A was larger than that of transcripts not modified by m6A, especially in terms of the log2FC of the transcript FPKM value between 0 and 20  was overexpressed in the late stage of clear cell renal cancer and acted as a biomarker for discriminating the early and late stages of ccRCC  and enriched in coding sequence (CDS)  genes, 15 hypomethylated and downregulated (hypo-down) genes, 76 hypermethylated and downregulated (hyper-down) genes, and 51 hypomethylated and upregulated (hypo-up) genes in bladder cancer tissues compared to adjacent normal tissues  and 10 hypomethylated genes  which might participate in tumor progression in bladder cancer. We observed the almost same m6A-level changes in these genes, confirming the validity of our MeRIP-seq results (To further confirm the results of our m6A) is the most common mRNA modification in eukaryotic cells of all higher animals , Beijing, China. This study followed the Helsinki declaration and was approved by the Institutional Ethical Review Board of PKUFH. Samples were collected immediately in the operating room after surgical removal and were stored in liquid nitrogen after rapid freezing in liquid nitrogen for the following RNA isolation.6A antibody (Manga) for 2 h at 4°C. Then, the beads were prepared and incubated with the total RNA for 2 h at 4°C. Finally, the mixture was washed and the m6A-bound RNA was purified with TE buffer. After being purified, the samples were used to construct the library by Prep Kit (Illumina) on HiSeq 3000.MeRIP-seq and RNA-seq were performed at CloudSeq Biotech, Inc.  –38 and aTotal RNA was extracted from the two groups of cells by using TRIzol Reagent (Life Technologies). The quality and quantity of total RNA were assessed by using NanoDrop ND 2000 . The RNA integrity was measured using denaturing agarose. Seq-Star T M poly(A) mRNA Isolation Kit  was used to isolate mRNA from total RNA. The GenSeq™ m6A RNA IP Kit  was used to perform m6A RNA immunoprecipitation by following the manufacturer’s instructions. The input samples without immunoprecipitation and the m6A IP samples were both used for library construction with NEBNext Ultra II Directional RNA Library Prep Kit . The library quality was evaluated with the Bioanalyzer 2100 system . Library sequencing was performed on an Illumina HiSeq instrument with 150-bp paired-end reads.www.geneontology.org) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases (www.genome.jp/kegg). Clinical survival data  were downloaded from the TCGA database (https://cancergenome.nih.gov/).Paired-end reads were harvested using the Illumina HiSeq 4000 sequencer and were quality controlled by Q30. After 3′ adaptor-trimming, low-quality reads were removed by Cutadapt software (v1.9.3). First, clean reads of all libraries were aligned to the reference genome (HG19) by Hisat2 software (v2.0.4). Methylated sites on RNAs (peaks) were identified by MACS software . IdentifTotal RNA was extracted from biological samples using TRIzol Reagent (Life Technologies) according to the manufacturer’s protocol. Denatured agarose gel electrophoresis was applied to evaluate the integrity of total RNA. Seq-Star TM poly(A) mRNA Isolation Kit  was used to purify mRNA from total RNA after confirming its quantity and quality by NanoDrop ND-2000. Then, fragmented mRNA was subjected to 50-bp single-end sequencing with a BGISEQ-500 platform. Adapter and low-quality reads were trimmed by SOAPnuke , and tri6A-IP samples were subjected to RT-qPCR with gene-specific primers.Twenty genes with differentially methylated sites according to MeRIP-seq were tested by reverse transcription (RT)-qPCR. A small number of fragmented RNA was used as the input control. The rested RNA was incubated with anti-m6A antibody-coupled beads. The m6A-containing RNAs were then immunoprecipitated and eluted from the beads. Both input control and mExperiments were performed at least three times, and representative results are shown. All statistical analyses were performed and visualized using RStudio , GSEA  23, MedCalc , and GraphPad Prism . Differences between individual groups were analyzed using the chi-squared test and Student’s t-test (two-tailed and unpaired) with triplicate or quadruplicate sets. A two-tailed p < 0.05 was considered statistically significant.All RNA sequencing data were deposited in the NCBI SRA (Sequence Read Achieve) database with the accession number of PRJNA733602.The studies involving human participants were reviewed and approved by the Institutional Ethical Review Board of PKUFH. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.AL, YG, and CC performed the experiments and data analysis. CC and QuZ prepared the diagrams and wrote the manuscript. AL and YG designed the project. QiZ and LY supervised the project and provided financial support. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Natural Science Foundation of China to QZ (Nos. 82072826 and 81872088) and a grant from Peking University Medicine Fund of Fostering Young Scholars’ Scientific & Technological Innovation to YG (No. BMU2020PYB028).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cheilinus undulatus) and bumphead parrotfish (Bolbometopon muricatum). Length distributions of fish differed among habitats at the Rowley Shoals, suggesting differences in ontogenetic shifts among species. The Cocos (Keeling) Islands typically had larger‐bodied predatory species than at the Rowley Shoals. Differences in geomorphology, lagoonal habitats, and fishing history likely contribute to the differences among remote reefs. Rowley Shoals is a rare example of a reef system demonstrating ecological stability in reef fish assemblages during a time of unprecedented degradation of coral reefs.Anthropogenic impacts at isolated and inaccessible reefs are often minimal, offering rare opportunities to observe fish assemblages in a relatively undisturbed state. The remote Rowley Shoals are regarded as one of the healthiest reef systems in the Indian Ocean with demonstrated resilience to natural disturbance, no permanent human population nearby, low visitation rates, and large protected areas where fishing prohibitions are enforced. We used baited remote underwater video systems (BRUVS) to quantify fish assemblages and the relative abundance of regionally fished species within the lagoon, on the slope and in the mesophotic habitat at the Rowley Shoals at three times spanning 14 years and compared abundances of regionally fished species and the length distributions of predatory species to other isolated reefs in the northeast Indian Ocean. Fish assemblage composition and the relative abundance of regionally fished species were remarkably stable through time. We recorded high abundances of regionally fished species relative to other isolated reefs, including globally threatened humphead Maori wrasse ( Cheilinus undulatus) and bumphead parrotfish (Bolbometopon muricatum).Fish assemblage composition and the relative abundance of regionally fished species were remarkably stable through time at the Rowley Shoals. We recorded high abundances of regionally fished species relative to other isolated reefs, including globally threatened humphead Maori wrasse ( It has been fished by Indonesian artisanal fishers since at least the 1800s and is still open to fishing under a memorandum of understanding (MOU) between the Australian and Indonesian governments  has a land area of 0.17 kmAshmore Reef  is a large lagoonal platform reef with three small uninhabited islands and two lagoons separated by a calcareous rise Collins, .2 and has no lagoon  Islands  are two oceanic atolls with 27 islands. The southern Cocos (Keeling) atoll has a shallow lagoon with two northern passages connecting it to the open ocean Figure , 2016b aChristmas Island , an uplifted limestone cap metamorphized from coral reefs, has a local population of 1843 and is open to fishing Table . Christm2.3Sardinops spp.) suspended in a plastic‐coated wire mesh bag 1.2 m in front of the cameras. Each deployment was separated by at least 250 m to minimize overlap of bait plumes and reduce the likelihood of fish swimming between samples , on the fore‐reef slope (5–14 m) and at mesophotic depths (40–75 m) at two sites at each of the three atolls  to identify and count fish. To avoid repeated counts of the same fish and for standardization among historical and interinstitutional datasets, we counted the maximum number of individuals in the field of view at one time (MaxN) to estimate relative abundance  following procedures outlined by Harvey and Shortis  Islands (the only other location with length data available) and used to assess differences in length distributions between locations.BRUVS were calibrated before deployment using the CAL software  to test for differences in the overall fish assemblage due to protection status (fished vs. unfished), year , shoal , and reef habitat . Three designs were used in the multivariate PERMANOVA tests: (a) a 4‐factor design incorporating protection status , shoal , reef habitat , and site  to compare assemblages in different reef habitats during the 2018 comprehensive BRUVS survey; (b) a 3‐factor design including year , shoal, and site  to compare assemblages along the reef slope over time; and (c) a 4‐factor design incorporating year, shoal, site , and deployment  to compare assemblages over time in the lagoon. We did not compare fish assemblages in the mesophotic reef habitat through time due to differences in image quality impacting species detection and identification  relative abundances of individual species using PRIMER  and site (nested in location) to compare locations within each reef habitat. Where multiple pairwise comparisons were conducted (Appendix α) where the false discovery rate (FDR = 0.05) controls the expected rate of type I error , we focused on univariate relative abundance of regionally fished species whose range covered all locations. All selected species are retained for human consumption in both Indonesia and Australia  Islands using kernel density estimates (KDEs) when at least 20 individuals were observed in each level, using the “ggplot2” package in R to estimate the probability density function of the length‐frequency data Wickham, . Bandwid et al.,  using th et al., .33.1F = 1.31, pPERM = 0.27), and there was no effect of fishing  = 1.07, pPERM = 0.41; Table F = 6.92, pPERM < 0.001). Lagoon assemblages were characterized by high abundances of the squaretail coral trout, Plectropomus areolatus, while the reef slope supported several abundant herbivorous  and invertivorous species . The mesophotic habitat was characterized by several carnivorous  and zooplanktivorous species   = 3.45, pPERM = 0.21 and F = 1.86, pPERM = 0.23, respectively; Table Surveys of the Rowley Shoals in 2018 recorded 14,500 fish from 327 different species. Assemblages were similar among the three shoals  = 5.49, pPERM = 0.03) and Lutjanus bohar  = 5.73, pPERM = 0.03) with lower abundances observed at Mermaid and Clerke, respectively. The endangered species C. undulatus was present across all habitats with highest abundances on the slope in both 2013 and 2018. The vulnerable species, B. muricatum, was present in mesophotic habitat in low numbers in 2004 and in higher abundance in the lagoon in 2013 and 2018. Mesopredators, Lethrinus olivaceus × microdon (longnose emperor), Aprion virescens (green jobfish), and Carcharhinus spp. (requiem sharks) were present in all reef habitats, but most abundant in the mesophotic habitat. Lutjanus bohar (red bass) were present at all reef habitats with highest abundances on the reef slope. Similarly, Plectropomus spp.  were present across all reef habitats but were most common in the lagoon and rare in the mesophotic habitat. Variola spp. (coronation trout) were most abundant in the mesophotic habitat and absent from the lagoon. Cephalopholis argus (peacock rockcod) were most abundant on the reef slope and were not recorded in mesophotic habitats.The relative abundance of selected regionally fished species was consistent between years in all of the reef habitats at the Rowley Shoals, but had marked differences in abundance among habitats Appendix . Shoals 3.3C. undulatus on the reef slope compared with all other locations  Islands. No B. muricatum were observed on the slope at the Rowley Shoals, and they were rare on the slope at Cocos (Keeling) with only five individuals observed across the 80 deployments.Lethrinus olivaceus × microdon on the reef slope compared with Cocos (Keeling) Islands  and Christmas Island   and in the mesophotic habitat than at Browse Island, although neither of these differences were statistically significant  Figure . At the L. bohar were observed on the slope at all locations, except for Christmas Island , where abundances were more than six times lower. At the Rowley Shoals, abundance of L. bohar in the lagoon was similar to Cocos (Keeling), but was greater in the mesophotic habitat than at Browse Island .Plectropomus spp. on the slope, with fewer observed at Cocos (Keeling) . Plectropomus spp. was not observed at Browse or Christmas Island.Rowley Shoals, Scott Reef, and Ashmore Reef had similar abundances of ) Figure . Rowley Variola spp. were not observed in the lagoon at any location and were absent from Scott Reef. The other locations had similar abundances observed on the slope.C. argus on the reef slope which were higher than at Ashmore Reef, Cocos (Keeling), and Christmas Island.The Rowley Shoals, Scott Reef, and Browse Island had similar abundances of Carcharhinus amblyrhynchos compared with all other locations. Grey reef sharks were predominantly on the slope, with abundance in the lagoon at Cocos (Keeling) similar to abundance at the Rowley Shoals. High abundance of C. amblyrhynchos was also found in the mesophotic habitat at the Rowley Shoals and Browse Island.Cocos (Keeling) had more than twice the abundance of 3.4L. bohar occurred at a wide range of lengths on the reef slope with a higher proportion of smaller individuals in this habitat than either the lagoon or mesophotic habitats; larger individuals were mostly found in the mesophotic habitat. Conversely, L. olivaceus × microdon were smaller (<400 mm) in the lagoon than in the slope and mesophotic habitats. Plectropomus spp. also had higher density of medium and smaller (<500 mm) fish in the lagoon, and higher density of large (>500 mm) fish on the reef slope.Larger predatory fishes were observed in the mesophotic zone with smaller individuals observed in slope and lagoon habitats Figure . Eight l3.5L. bohar (>600 mm), C. amblyrhynchos , and C. argus (>280 mm) were higher at Cocos (Keeling), whereas the proportion of large Lethrinus spp. (>350 mm) and Plectropomus spp. (>600 mm) was higher at the Rowley Shoals.Length distributions for selected predator species differed between Rowley Shoals and Cocos (Keeling) when data for all species recorded on slope and lagoon habitats were combined Figure . A highe4C. undulatus and B. muricatum  Islands were the only other location with a notable number of these species. Both species are sensitive to fishing pressure, being large‐bodied, slow‐growing, and late‐maturing  indicates that fishing pressure on remote reefs may have an influence on the abundance of these taxa. However, differences in geography and island morphology among these reef systems may also contribute to the differences in abundance across locations  Islands  is unclear and an exception to the population status of this species at a global scale  due to fishing  indicates that predatory fish at Cocos (Keeling) are typically larger than conspecifics at the Rowley Shoals. This is especially true of 5C. undulatus and B. muricatum. Lagoon habitats appear to be important for supporting these and other species, especially during the early stages of their lifecycle. However, variability in abundances and length distributions across reef habitats suggest there are varied ontogenetic shifts and habitat preferences among species, some of which include mesophotic habitats. Differences among the locations seen here are likely due to multiple factors such as geomorphology, geographical location, and historical fishing pressure. Importantly, isolation from human populations does not necessarily translate to conditions that support stable fish assemblages and abundances of regionally fished species, further highlighting the rarity of reef systems like the Rowley Shoals and the importance of developing meaningful baselines to quantify the impacts of disturbances on coral reefs.The Rowley Shoals is a rare reef system that meets the criterion set for global conservation targets ; data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); project administration (lead); visualization (lead); writing – original draft (lead); writing – review and editing . Katherine Cure: Conceptualization ; investigation ; methodology ; visualization ; writing – original draft ; writing – review and editing . Shaun Wilson: Conceptualization ; investigation ; methodology ; validation ; visualization ; writing – original draft ; writing – review and editing . Stephen J. Newman: Data curation ; investigation ; methodology ; validation ; writing – review and editing . Euan S. Harvey: Data curation ; funding acquisition ; methodology ; validation ; writing – review and editing . Mark Meekan: Data curation ; funding acquisition ; methodology ; validation ; writing – review and editing . Conrad Speed: Data curation ; methodology ; validation ; writing – review and editing . Andrew Heyward: Data curation ; funding acquisition ; methodology ; validation ; writing – review and editing . Jordan Goetze: Conceptualization ; methodology ; validation ; writing – review and editing . James Gilmour: Conceptualization ; funding acquisition ; investigation ; methodology ; project administration ; resources ; validation ; writing – review and editing .Figure S1Click here for additional data file.Figure S2Click here for additional data file.Supplementary MaterialClick here for additional data file.


To protect cells under oxidative stress, bacteria induce the expression of several genes, namely the SoxRS, OxyR and PerR regulons. Cells are able to tolerate a certain number of free radicals, but high levels of ROS result in the oxidation of several biomolecules. Strikingly, RNA is particularly susceptible to this common chemical damage. Oxidation of RNA causes the formation of strand breaks, elimination of bases or insertion of mutagenic lesions in the nucleobases. The most common modification is 8-hydroxyguanosine (8-oxo-G), an oxidized form of guanosine. The structure and function of virtually all RNA species  can be affected by RNA oxidation, leading to translational defects with harmful consequences for cell survival. However, bacteria have evolved RNA quality control pathways to eliminate oxidized RNA, involving RNA-binding proteins like the members of the MutT/Nudix family and the ribonuclease PNPase. Here we summarize the current knowledge on the bacterial stress response to RNA oxidation, namely we present the different ROS responsible for this chemical damage and describe the main strategies employed by bacteria to fight oxidative stress and control RNA damage.Bacteria have to cope with oxidative stress caused by distinct Reactive Oxygen Species (ROS), derived not only from normal aerobic metabolism but also from oxidants present in their environments. The major ROS include superoxide O Conseque stress .2− is a non-diffusible free radical formed when O2 removes one electron from a donor. It can be produced both intracellularly and exogenously  (3+ (Ogenously . Inside mes P450 . In eukan system . In phagathogens . Exogenoparaquat . Superoxd thiols . Additiose (SOD)  and act SOD)   producing the highly reactive hydroxyl radicals HO•  . There iironment . Release species . H2O2 is minutes , and its minutes . The mosreaction . In thiscals HO• .• is one of the most dangerous ROS due to a half-life in the range of nanoseconds and its extreme reactivity given the high electron potential of +2.33 V . As result of oxidation, the structure and function of these macromolecules are affected, with potentially toxic consequences that can ultimately result in cell death. Most protein oxidative modifications occur in the side chains of amino acids like oxidation of thiols and the formation of carbonyl groups affecting protein structure, conformation, and function . CysteinNucleic acids are very susceptible to chemical damage given the reactivity of the nitrogen and oxygen atoms of the nucleobases . Among tRNA accounts for 80–90% of the total nucleic acids , and it • derived from the Fenton reaction by detaching the hydrogen from the C-H bonds, resulting in several oxidation adducts, namely: 8-oxo-7,8- dihydroguanosine (8-oxo-G), 8-oxo-7,8-dihydroadenosine (8-oxo-A), 5-hydroxyuridine (5-HO-U) and 5-hydrocytosine (5-HO-C) (RNA molecules can be oxidized by HO(5-HO-C)   . Althoug(5-HO-C) . Under p(5-HO-C) . This mo(5-HO-C) . Further(5-HO-C) .When compared to DNA, the study of RNA damage by oxidation has been neglected, and this is mainly due to the assumption that RNAs tend to have a short half-life due to a rapid turnover before causing deleterious effects . This neEscherichia coli it was shown that both the 16S rRNA  and the 23S rRNA  are subject to oxidation by H2O2in vivo, although the 23S rRNA seems more prone to nucleobase oxidation  were also found to reduce oxidation in tRNAs . OxidatiG adduct . SpecifiG adduct , which cG adduct . Zhong en halves . Howevernchanged . Therefonchanged . This conchanged .E. coli by interfering with the A site of mRNA codons leading to the incorporation of a different amino acid  can be removed by base excision repair enzymes   were shown to degrade 8-oxo-dGTP and 8-oxo-GTP/GDP , which specifically binds with high affinity to 8-oxo-G-containing RNA oligonucleotides . The YB-li, CspA . It is ae stress . Howevere stress . On the e stress .Bacteria (and other organisms) are only able to tolerate a certain amount of non-harmful levels of ROS. To mitigate this threat, bacteria have evolved several mechanisms to fight oxidative stress and avoid the imbalance of ROS levels that lead to cell toxicity. From these, the presence of scavenging enzymes that consume ROS  are critical in self-defense mechanisms against oxidative stress in bacteria.E. coli has two cytoplasmic enzymes (Fe-SOD and Mn-SOD) and a periplasmic enzyme (Cu-Zn-SOD)  inhibits Mn-SOD transcription while Fe-SOD is synthesized and activated by iron; when iron levels are reduced, Fur is deactivated and there is transcription of both Mn-SOD and the sRNA RyhB, which will repress Fe-SOD mRNA , and are strongly induced when aerobic cells enter stationary phase  . High ti-Zn-SOD)  in orderSOD mRNA . In anaery phase .E. coli has three main enzymes that can degrade hydrogen peroxide: alkyl hydroperoxide reductase (Ahp), catalase G (KatG) and catalase E (KatE) . KatE is strongly expressed in stationary phase cells only, since it is induced by the RpoS system  receives electrons from the respiratory chain and transfers them directly to H2O2  . Ahp is S system . These f starved . Additio to H2O2 . Ccp inspiration . This fuTo protect cells under oxidative stress, bacteria induce the expression of several genes, namely the SoxRS, OxyR and PerR systems. The activation of these multigene transcriptional pathways is critical to the bacterial response to oxidative stress and will be next presented.2− and to redox-cycling compounds, but not to H2O2 (2+) is active and able to induce transcription of soxS  SoxS is absent, and SoxR directly activates a small sets of genes in response to redox-cycling agents , heme biosynthesis (hemH), reductant supply , repression of iron import  and others . Under regular conditions, OxyR is present in its reduced form  and a regulatory metal binding site  or to ferrous iron (Fe2+), which is preferred in most conditions  superfamily of metalloregulatory transcription factors, firstly identified in response . PerR haresponse , found presponse . PerR isresponse . Each suing site . In B. snditions . The metnditions  located nditions .2+) into ferric iron (Fe3+) at the regulatory site. Under non-stress conditions, binding of the regulatory metal stabilizes the conformation of the PerR dimer to better interact with DNA, which leads to the repression of the PerR regulon , but some participate in metal homeostasis (hemAXCDBL operon) and surfactant production (srfA)  . SimilarAerobic organisms have developed over time the ability to maintain the levels of intracellular ROS within specific limits, to prevent ROS insults that can damage distinct classes of biomolecules. However, when self-defense mechanisms fail to protect the cell, ROS levels increase and as result of this imbalance, the cell faces oxidative stress. Due to its chemistry, RNA is highly susceptible to oxidation, which leads to the modification of nucleobases  causing RNA injury with potential mutagenic consequences. Since RNA is at the center of cellular regulatory pathways, the chemical damage of RNA triggered by ROS has a wide impact in gene expression. The importance of this impact is even stressed as RNA is highly expressed, comprising most nucleic acids within a cell, with the existence of many different classes of RNAs . Virtually, all these different RNA species are vulnerable to oxidation-induced damage, affecting translation at various levels. Not only the rate of protein synthesis is slowed but also the formation of aberrant proteins is potentiated, what may affect cell fitness and even cause cell death. The consequences of RNA damage by oxidation are widespread through all domains of life. Interestingly, in humans, the oxidation of RNA is implicated in several diseases, like cancer , diabete