Topology optimization holds great potential to achieve the best performance for various fluid-related applications like aircraft components and microfluidic mixers. This paper reviews comprehensively the technical progress of this field over the last decade from the viewpoint of structural expression. The density-based approach has been widely adopted to design structures due to its simple concept, ease of implementation, and robustness. Different designs using such a pointwise method for systems under Stokes, laminar Navier–Stokes, turbulent, non-Newtonian, and steady-state/unsteady-state fluid flows are reviewed and discussed in depth. Both isogeometric analysis and the moving morphable components/voids methods will demonstrate their advantages regarding integration with computer-aided design. The moving morphable components/voids method also significantly reduces computing costs. From the viewpoint of boundary smoothness, we are most concerned about whether smoother boundaries can reduce objective functions such as energy dissipation even lower. Therefore, this work also concentrates on level set and spline expression methods. Furthermore, we identify isogeometric analysis and machine learning in shaping the field’s future. In addition, the review highlights the following two challenges: achieving accurate fluid model construction and the relatively limited experimental validation of fluid topology optimization outcomes. Since the seminal work on homogenization proposed by Bendsoe and Kikuchi in 1988 [One of its stunning achievements, like lightweight structures , is the The remainder of this paper is organized as follows. Topology optimization using a density-based method is a powerful approach to optimize the material distribution within a given design space to achieve the desired structural performance. The fundamental idea behind density-based methods is to represent the design domain using a mesh and to assign artificial density values based on the material occupying ratio within an element. The optimization process involves iteratively adjusting these density values to find the optimal distribution that meets specified objectives while satisfying design constraints. These objectives could include minimizing compliance, maximizing stiffness, reducing stress concentration, or achieving other performance criteria. The optimal artificial density ranging from 1 (solid) to 0 (void) allows for the elimination of inefficient materials, as shown in The density-based method is one of the most extensively utilized techniques in fluid dynamics. Its incorporation into fluid topology optimization dates back to the early 21st century, marking a pivotal development in the field. The seminal paper by Borrvall and Petersson first solved the topology optimization problems for fluid systems . The optρ is the fluid density, u indicates the fluid flow velocity, µ represents the fluid dynamic viscosity, p denotes the pressure, f symbolizes the external body forces, and α is the inverse permeability. Based on lubrication theory, this method formed a fictitious fluid/solid domain by adding a friction term αu to the equation with the inverse permeability α, shown in The governing equations commonly used in density-based fluid topology optimization problems are the laminar Navier–Stokes equations, which can be characterized as follows :(1)ρ∂u∂tTwo years later, Gersborg-Hansen et al., extending the previous work, achieved optimal design of the flow channel topology for two-dimensional constant incompressible laminar viscous flow, steady-state Navier–Stokes model, which makes the governing equation change from linear to nonlinear . Later, Kreissl, Pingen, and Maute first proposed a method to optimize unsteady flow problems, indicating that its results differ significantly from steady-state systems. They employed a stabilization technique to effectively address unsteadiness, effectively mitigating velocity oscillations within porous materials. Notably, they emphasized that while the Brinkman method effectively partitions the domain into fluid and solid regions, it only partially prevents pressure diffusion through the solid phase, potentially impacting overall predictive accuracy . ExtendiYoon developed a new finite element-based turbulence topology optimization using the Spalart–Allmaras equation . Dilgen As for non-Newtonian fluids, Pingen and Maute studied topological optimization problems for non-Newtonian fluids based on the Lattice Boltzmann method. They also presented an outlook on topological optimization of non-Newtonian fluids, including applications to biomedicine and different non-Newtonian fluid properties . Suárez Some experimental validations were based on the density method of fluid topology optimization and computational fluid dynamics. Both Lin et al. and Lim et al. studied fixed-geometry fluid diodes and conducted experimental validation of this design using 3D printing, which verified that the structure possesses the characteristics of a fluid diode after topology optimization ,30. Sá eThe moving morphable components topology optimization approach was initially introduced by Guo et al. in 2014 . SubsequResearchers focus more on fluid-related thermal systems than purely fluid structures in this method. Yu et al. first introduced the moving morphable components method to thermal-fluid problems, in which the objective is minimizing heat compliance and fluid power dissipation. Because cooling device pipes often exhibit larger curvature than bearing structure, a higher-order component is utilized, and elliptic joints are introduced to enhance the connections between high-order components. Additionally, they developed a feature size control technique to limit the minimum width of pipes based on manufacturing capacity and removed unnecessary components. They compared the results with those obtained using the density-based method, revealing differences in the optimization processes of the two optimal structures. The density-based method initially establishes primary fluid pathways. As the optimization progresses, additional thin channels emerge to link these main paths. The results obtained using the moving morphable components method, on the other hand, exhibit relatively stable evolution processes due to the initial components setup. Additionally, the feature size control technique prevents the formation of those thinner channels . Their team employed a mixed topology optimization approach based on the framework to perform thermal-fluid topology optimization problems. They treated the cooling channels with the density-based method and considered the heat sources as moving components. Also, they proposed an exchange algorithm redistributing heat intensities during optimization to reduce the impact of the initial distribution of heat sources . As for Besides the density-based approach, the level set method, as a classical method, has also been widely used for topology optimization, starting with this seminal article by Wang et al. . TopologDuan et al. first used a variational level set method to solve Navier–Stokes flow topology optimization ,45,46. IKreissl, Pingen, and Maute combined the Lattice Boltzmann method with the level set method for shape optimization of fluids . Based oSasaki et al. used the moving particle semi-implicit and level set method to optimize fluid topology. The algorithm demonstrates that the optimized structure is consistent with that obtained by the finite element method . Nguyen Additionally, blood vessels are a critical component of many biological systems, and the presence of non-Newtonian fluid behavior within these vessels adds a layer of complexity to fluid flow dynamics. Some critically crucial vascular fluid studies have already been published ,62,63,64In this approach, spline functions, which are mathematical curves that can be smoothly adjusted, are employed to define the shape and topology of the design. On the other hand, the traditional linear shape functions of the finite element method are replaced with higher-order NURBS basis functions used in the isogeometric analysis, allowing a more accurate representation of complex geometries. The fundamental idea is to use the same geometric description for analysis as used in computer-aided design models, promoting seamless data exchange between design and analysis. However, isogeometric analysis for fluid topology optimization is still rare. To date, only a few fluid-related isogeometric shape optimizations have been published. Based on the trimming isogeometric analysis , Park etMachine learning has shown great potential in various fields, which focuses on utilizing data using statistical methods to learn meaningful patterns. Deep learning has also been widely used as a subset of machine learning. The human brain’s neural structure inspires it and trains hierarchical neural networks using the data to enhance learning . BecauseGaymann and Montomoli introduced the concept of making the fluid-structure topology optimization problem gamification. They proposed a method for optimizing the fluid-structure to minimize pressure difference using deep neural networks and Monte Carlo tree search. In this optimization approach, the deep neural networks determine the winner and decide whether the game ends while predicting the pressure difference. The player’s future movements are determined through iterative Monte Carlo searches based on these outcomes, achieving the final optimized shape .Ban and Yamazaki used clustering/classification methods and exploration strategies to solve the optimization problems of discontinuities, infeasible regions, and high-dimensional optimization problems in advanced global topological optimization. Combining these methods, they used machine learning technologies to publish the black-box function for the aerodynamic topology optimization problem. By only using the objective function and constraints, the newly developed design tool can perform effective fluid topology optimization . HammondDeng et al. presented a novel approach called self-directed online learning optimization, which combines deep neural networks with finite element method calculations. Deep neural networks are employed to learn and replace the objective function regarding design variables. The deep neural networks adjust to the evolving training data, leading to improved predictions within the targeted region until convergence . This apAlthough density-based and level set methods have been well developed, their topology optimization results often need help to integrate effectively with computer-aided design systems. When dealing with 3D problems, the increased number of design variables also leads to higher computational costs. As a result, the moving morphable components/voids method was proposed. Compared to the previous two methods, the method allows for better integration of topology optimization with computer-aided design systems, resulting in a more geometrically explicit and flexible process. This approach employs the geometric information of components as design variables, and the design domain can be composed of a small number of components, thus significantly reducing the number of design variables . NeverthSince the optimization outcomes are predicted based on pixel similarity, there might be a lack of connectivity, leading to less manufacturability and lower resolution of the topology optimization results. For enhancing manufacturability, this method  can be cFluid topology optimization identifies the most advantageous fluid flow regions that enhance metrics like drag, lift coefficients, pressure drop, and heat transfer. Its core aim is to craft fluid shapes aligning with designated engineering criteria, improving the system’s effectiveness. It has been developed for two decades and has shown significant potential for future advancements and applications in various industries. This paper briefly outlines the pros and cons of recent optimization methods for fluid-structure design. For instance, density-based approaches are computationally efficient and easily shape-controllable, while level-set techniques ensure clear, well-defined fluid–solid interfaces throughout the optimization. Due to its ability to combine with computer-aided design and significantly reduce computational costs, the moving morphable components/voids method also represents an alternative direction. Additionally, integrating isogeometric analysis can enhance geometric representation and optimization accuracy, while machine learning techniques bring data-driven insights and predictive capabilities to the forefront of fluid topology optimization.Because of the diversity and complexity of fluid flow models, one of the critical challenges in fluid topology optimization is the need to model fluid flow accurately. Given that much research is now directed at dealing with 2D simple fluid topology optimization problems, more complicated fluid patterns and 3D meshing techniques must be developed to simulate fluid flow more accurately although they are computationally expensive, time-consuming, and require more specialized skills and knowledge. Existing reports using simplified models ,88,89,90Using 3D printing technology, the experimental validation methods in fluid topology optimization include flow visualization, pressure drop measurements, and computational fluid dynamics simulations ,30,31,32


Scholars have used data from in-person interviews, administrative systems, and surveys for sexual violence research. Using Twitter as a data source for examining the nature of sexual violence is a relatively new and underexplored area of study.We aimed to perform a scoping review of the current literature on using Twitter data for researching sexual violence, elaborate on the validity of the methods, and discuss the implications and limitations of existing studies.We performed a literature search in the following 6 databases: APA PsycInfo (Ovid), Scopus, PubMed, International Bibliography of Social Sciences (ProQuest), Criminal Justice Abstracts (EBSCO), and Communications Abstracts (EBSCO), in April 2022. The initial search identified 3759 articles that were imported into Covidence. Seven independent reviewers screened these articles following 2 steps: (1) title and abstract screening, and (2) full-text screening. The inclusion criteria were as follows: (1) empirical research, (2) focus on sexual violence, (3) analysis of Twitter data , and (4) text in English. Finally, we selected 121 articles that met the inclusion criteria and coded these articles.We coded and presented the 121 articles using Twitter-based data for sexual violence research. About 70%  of the articles were published in peer-reviewed journals after 2018. The reviewed articles collectively analyzed about 79.6 million tweets. The primary approaches to using Twitter as a data source were content text analysis  and sentiment analysis . Hashtags  were the most prominent metadata feature, followed by tweet time and date, retweets, replies, URLs, and geotags. More than a third of the articles  used the application programming interface to collect Twitter data. Data analyses included qualitative thematic analysis, machine learning , and quantitative analysis. Only 10.7% (13/121) of the studies discussed ethical considerations.We described the current state of using Twitter data for sexual violence research, developed a new taxonomy describing Twitter as a data source, and evaluated the methodologies. Research recommendations include the following: development of methods for data collection and analysis, in-depth discussions about ethical norms, exploration of specific aspects of sexual violence on Twitter, examination of tweets in multiple languages, and decontextualization of Twitter data. This review demonstrates the potential of using Twitter data in sexual violence research. Sexual violence is a global public health problem, threatening individuals’ health and well-being worldwide . It is dTwitter, launched in 2006, has become one of the most widely used social media platforms and an important source of public health information -8 and thResearch has also explored social reactions to sexual violence disclosures on Twitter, with the use of hashtags such as #NotOkay . There hIn the digital age, Twitter has emerged as a crucial platform for understanding sexual violence. Understanding how the Twitter data set can be harnessed is essential to contribute to our understanding of sexual violence research. Therefore, exploring the ways in which Twitter data can be effectively used in sexual violence research can provide valuable insights to practitioners and researchers to effectively use the platform’s potential for implementing violence prevention and intervention strategies.This study aims to provide a scoping review of the current literature on using Twitter data for sexual violence research. This review focuses primarily on the current state of Twitter data in sexual violence research, including (1) describing the main objectives addressed by previous studies in the field; (2) evaluating the methodology of these studies, including aspects such as study design, data collection, and data analysis strategies; (3) presenting a taxonomy describing the ways in which Twitter is used as a data source in sexual violence research; (4) presenting the various ways in which Twitter metadata features are used in this regard; and (5) discussing the implications and limitations of using Twitter data for sexual violence research. By providing a scoping overview of the existing research, this study contributes to a better understanding of the potential of Twitter data for sexual violence research and provides insights into the evidence-based practices for conducting such research.This study was guided by Arksey and O’Malley’s scoping review framework . This stA social and health sciences librarian (JL) developed a search strategy for this review. The librarian drafted an initial search strategy validated against a predetermined test set of articles and peer-reviewed by an independent librarian colleague using the Peer Review of Electronic Search Strategies (PRESS) framework . This seTo be included in the review, studies had to meet the following criteria: (1) be empirical research, (2) focus on sexual violence , (3) analyze Twitter data , and (4) be written in English. In this review, we defined sexual violence as “a sexual act that is committed or attempted by another person without freely given consent of the victim or against someone unable to consent or refuse” . AligninThe search retrieved a total of 3759 studies from the 6 selected databases. After removing duplicates (n=1063), 2695 articles were entered into Covidence for abstract screening. To ensure the accuracy of article extraction, 7 research assistants  were trained to participate in both abstract and full-text screening processes to extract relevant studies. Each study was independently reviewed by 2 research assistants using the screening software, Covidence. The principal investigator (PI) (JX) resolved all disagreements. Before the screening process began, the PI trained all research assistants on screening guidelines. Following the abstract screening, 258 studies were selected for full-text screening. Of these, 137 studies were excluded in the full-text screening stage, resulting in a final sample of 121 articles that met the inclusion criteria. A PRISMA  flowchart, shown in The team developed a charting guideline for extracting essential information from the articles included in the review. The charting guideline was organized into several main categories, including publication year, study objectives, theoretical or conceptual framework, use of Twitter data in the study, use of Twitter metadata, data collection methods, sample size, data analysis strategy, key findings, ethical considerations, and study limitations. Seven research assistants participated in the data charting process. Prior to the charting launch, all research assistants completed a charting exercise by independently reviewing the same 4 selected articles to familiarize themselves with the charting guideline and identify any areas that needed further clarification. The research team met to debrief the charting exercise and refine the charting guideline accordingly. The intercoder reliability score was excellent (Cohen κ >90%). The PI then assigned each article to 2 research assistants for independent charting. The charting results from all research assistants were compiled into an Excel sheet, and any discrepancies were resolved by the PI. The team spent 3 months completing the screening and another 3 months for data extraction.Using a scoping review design, this study aimed to provide a comprehensive knowledge synthesis that maps the current state of sexual violence studies using Twitter data and identifies gaps for future research. As per the scoping review framework , the quaIn this study, a total of 121 articles were reviewed out of 3759 studies initially identified through the search. We reviewed and described the current state of sexual violence research using Twitter data, including publication year, publication sources , research topic, research methods, data collection methods, sample size, timeframe of tweets collected, data analysis strategy, and institutional review board procedures for using Twitter data in sexual violence research. To characterize Twitter use as a data source in these articles, we developed a codebook to define the taxonomy for different metadata features that can be extracted from Twitter, including Twitter use , Twitter functions , and Twitter users . We also extracted information about the results and discussions of the assessed studies but did not report them in this article.We extracted information about the publication year, publication source , and research topics. Discussions related to institutional review boards or ethics reviews were also extracted.Journal of Interpersonal Violence, Journal of Medical Internet Research, Feminist Media Studies, Violence and Gender, and Journal of Community Psychology. The remaining journal articles were published across various disciplines. A total of 28 of 121 (23.1%) articles were published at conferences. The findings suggest a growing interest in using Twitter data for sexual violence research across different fields.Findings showed that journals were the most common publication type for the reviewed articles, with 73.6% (89/121) of the articles published in journals. The reviewed articles most favored the following journals: Regarding research topics, the reviewed articles used 511 different hashtags to reference their research focus. The top 10 most commonly used hashtags were #MeToo (n=71), #NotOkay (n=21), #WhyIStayed (n=18), #WhyILeft (n=12), #WhyIDidntReport (n=9), #YesAllWomen (n=8), #BeenRapedNeverReported (n=5), #BlackLivesMatter (n=5), and #domesticviolence (n=5). However, 451 hashtags only appeared once in the reviewed articles. The findings indicate that most articles focused on similar themes surrounding focusing events or social movements in the society.This scoping review examined the current state of using Twitter data for sexual violence research, with a focus on publication year, publication source , research objectives, and ethical considerations surrounding using Twitter data. We identified 7 main objectives after reviewing and summarizing the stated research objectives, including (1) exploring online disclosures and public opinions of sexual violence victimization ,31; (2) Out of the 121 reviewed articles, only 2 indicated that they had obtained ethics approval for using Twitter data for sexual violence research from an Institutional Review Board . Eleven We employed a predefined taxonomy codebook to categorize how Twitter data were used as a data source in the reviewed articles. The taxonomy included the following 5 categories: (1) content/text, (2) sentiment/emotions, (3) images/photos, (4) engagement , and (5) social networks . Among tIn the 121 reviewed articles, hashtags were the most common Twitter metadata feature used , followed by tweet time and date , retweets , replies , URLs , and geotags  . Among tTo determine metadata inclusion, we coded articles with 1 if they explicitly mentioned the 140-character limitation for tweets and 0 if not. Similarly, for URLs and hashtags, articles were coded as 1 if the URLs were explicitly shown as preserved in the data and 0 if the URLs were explicitly stated as cleaned from the data. The image/photo feature was coded as 1 for articles that included tweet images in the data set, regardless of their inclusion in the analysis. Few studies analyzed the demographic characteristics of Twitter users, such as gender , race , age , and occupation  .The application programming interface (API) was used to collect tweets in more than a third of the reviewed studies 51/121, 42.1%), followed by Twitter’s advanced search function  and NVivo Addition NCapture . The remaining studies used various tools such as Topsy, GetOldTweets, Meltwater, Netlytic.org, NodeXL, Radian6, Discover Text, LexisNexis, and R. However, we found that 8 articles, such as the study by More and Francis , did not.1%, follThe range of sample sizes varied greatly from 93 tweets  to 32 miAbout 21%  of the reviewed articles collected tweets within 7 days, while one-third of the articles  collected tweets over an 8- to 31-day period. About 30%  of the articles collected tweets for longer than 1 month (31 days) but less than 1 year (365 days), and 10 articles collected tweets for over 12 months.To obtain the average number of tweets collected per day, the sample size was divided by the number of days collected. Among the articles, 30 collected fewer than 100 tweets daily, 31 collected between 100 and 999 tweets daily, 26 collected between 1000 and 9999 tweets daily, 15 collected between 10,000 and 99,999 tweets daily, and 3 collected more than 100,000 tweets daily.In terms of the year of tweets collected, the highest number of tweets were posted in 2017 , with more than half of the tweets posted between 2016 and 2018 .Our review revealed 2 main aspects, namely coding strategies and analysis methods. Most studies (n=76) relied on manual coding of tweets, while 13 studies used a combination of manual coding and machine learning coding strategies. For example, Chowdhury et al  developeRegarding the analysis methods employed in the reviewed articles, we identified 6 main approaches: qualitative analysis, sentiment analysis, supervised machine learning, unsupervised machine learning, social network analysis, and quantitative analysis. These categories extend the previously discussed taxonomies of Twitter data usage and coding strategies. A combined approach of qualitative and unsupervised machine learning strategies was reported by 5 studies -48. The Regarding the software and programming languages used for data analysis, Python was found to be the most commonly used language n=19) [ 9,15,2525,56, foOver the past decade, researchers have shown an increased interest in using Twitter data to conduct sexual violence research. This scoping review included 121 articles that were published between 2006 and 2022, with an increase in the number of publications since 2013, peaking in 2019 and 2020. Almost 75%  of the reviewed articles were published in peer-reviewed journals across different disciplines. This review has a couple of main findings. First, it described the current use of Twitter data in sexual violence. Second, it presented a taxonomy outlining how Twitter data are used in this field, including analyzing content/text, sentiment/emotions, images/photos, engagement , and social networks. Third, it described and evaluated the methodology applied in the reviewed studies.This review developed a taxonomy to describe the use of Twitter data in sexual violence research, which included the following 5 categories: (1) content/text, (2) sentiment/emotions, (3) images/photos, (4) engagement , and (5) social networks. More than 90%  of the reviewed studies used Twitter text for analysis. For example, Aurrekoetxea-Casaus  examinedThe technical paper by Kumar and Aggarwal  reportedSocial network analysis, which examines the relationship and interactions between Twitter users , was empLa Manada case in Spain on April 26, 2018. They analyzed the images in the posts and found that the images broadened the layers of meaning in the public discourse on Twitter. Despite being a small category, the analysis of Twitter images and campaigns can provide valuable insights into the public’s attitudes and perceptions related to sexual violence.The smallest category of studies in our review focused on the analysis of Twitter images (n=2) or engaged people in campaigns (n=5). Hassan et al  examinedThis review represents sexual violence research that uses Twitter data, with an estimated 79.6 million tweets analyzed. The most common approach used to access Twitter data was Twitter’s API or Twitter’s advanced search function. Other platforms, such as Discover Text, provided access to the entire collected tweet data set at a cost. Twitter’s API has several advantages for academic research, including free access and the ability to retrieve up to 10 million tweets per month using advanced search operators . NotablyA previous review stated that 38 data features could be extracted for each tweet, but Twitter metadata was not fully used in the studies . In thisAdditionally, the passive nature of data analysis can only retrieve demographic information if it is available on the users’ profiles . HoweverThis review highlights a potential limitation of underutilizing demographic information about Twitter users, which could decontextualize study findings. Understanding the specific communities that contribute to public opinion on Twitter is crucial to fully grasp the social impact of sexual violence on society. However, researchers must balance this with the ethical considerations of protecting Twitter users’ privacy and anonymity, particularly in sensitive topics such as sexual violence. As a result, demographic information, such as gender, race, and location, may be intentionally removed to protect the identity of users. This suggests a research gap and an opportunity to explore ways of better contextualizing social media data while maintaining confidentiality and privacy.There are several limitations in this review that need to be acknowledged. First, we only included articles that used Twitter data  to research sexual violence or assault. Studies that used Twitter for intervention or participation recruitment purposes were excluded. Second, the search terms were limited to certain types of sexual violence, such as sexual assault, rape, sexual abuse, sexual harassment, and gender-based violence . StudiesThis scoping review demonstrates the potential of Twitter data as a valuable resource in sexual violence research. This method allows researchers to expand beyond traditional data collection methods like surveys, interviews, and administrative data for sexual violence research. However, it is important to note that gaps exist in the current literature, and a further study is needed to fully understand the potential and limitations of using Twitter as a data source for sexual violence research.This scoping review highlights the need to develop more robust methods for data collection and analysis. Fourteen studies reported that a small sample size restricted the generalizability of their findings . In addiOn the other hand, Lowenstein-Barkai  suggesteThe ethical considerations of using Twitter data in sexual violence research have been discussed in a limited number of studies. Only 10.7% (13/121) of the reviewed studies explicitly reported ethical considerations. In these articles, direct interaction with human subjects was not involved, and formal informed consent was not required. To ensure the anonymity of the participants, quotes were modified to avoid reverse identification , and traFurther examination is needed to gain a more comprehensive understanding of particular aspects of sexual violence on Twitter. One crucial area for exploration is the manifestation of intersectionality in tweets. Kachen et al  suggesteMost of the reviewed studies only coded and analyzed English language tweets, leading to concerns about potential bias. Sixteen articles noted the inherent bias in their methodology, as they only included English tweets . ResearcUsing Twitter as a data source for sexual violence research may not provide a representative sample of individuals who have experienced sexual violence. Thirty-two reviewed articles revealed that Twitter users do not represent the general population and only reflect opinions at a time, which do not accurately define the users. It is important to note that not all individuals who have experienced sexual violence use Twitter or disclose their experiences on this platform. Guidry et al  noted thExisting literature shows the potential of using Twitter-based data for sexual violence research, as evidenced by the increasing number of publications after 2018. To contribute to this field, this study reviewed and extracted 121 articles that collectively analyzed about 79.6 million tweets. We described the current state of using Twitter data for sexual violence research, developed a new taxonomy that describes Twitter as a data source, and evaluated the methodologies used. Based on our review, several research recommendations are proposed, such as development of data collection and analysis methods, in-depth discussions about ethical norms, exploration of specific aspects of sexual violence on Twitter, analysis of tweets in multiple languages, and a more inclusive representation of the findings. These recommendations may guide future studies on using Twitter data for sexual violence research.


COVID-19 vaccines in pregnancy protect both pregnant individuals and young infants from severe illness via transplacental transfer of maternally-derived IgG. However, the impact of multiple (e.g. twin) pregnancy on transplacental transfer of SARS-CoV-2 IgG is unknown. We aimed to evaluate anti-Spike (S) antibody transfer among infants from twin pregnancies compared to singleton pregnancies.th percentile) birthweight.We conducted a prospective cohort study among individuals with twin and singleton pregnancies who received at least 2 doses of an mRNA COVID-19 vaccine prior to delivery. We tested paired maternal and cord samples for anti-S IgG and used linear regression to evaluate associations between multiple or singleton pregnancy and anti-S antibody. We included as covariates timing of last vaccine dose, gestational age at delivery, number of doses prior to delivery, and small for gestational age  and 2971 BAU/mL  for twin and singleton pregnancies, respectively. Median cord anti-S IgG was 4110 BAU/mL  and 3636 BAU/mL  for twin and singleton infants, respectively . Cord:maternal IgG ratios were significantly lower in twin pregnancies compared to singleton pregnancies . After adjustment for covariates, there was no difference between maternal anti-S IgG concentrations , cord anti-S IgG concentrations  or cord:maternal IgG ratios  between twin and singleton pregnancies.Twin and singleton infants benefit similarly from maternal COVID-19 vaccine during pregnancy. Higher risk pregnancies including multiple pregnancies should be considered in health policy discussions regarding COVID-19 vaccine timing in pregnancy.Alisa B. Kachikis, MD, MSc, Merck: Grant/Research Support|Pfizer: Grant/Research Support Mindy Pike, PhD, Merck: Grant/Research Support Alexander L. Greninger, MD, PhD, Cepheid: central contracts|Hologic: central contracts|Janssen: central contracts|Novavax: central contracts|Pfizer: central contracts Janet A. Englund, MD, Ark Biopharma: Advisor/Consultant|AstraZeneca: Advisor/Consultant|AstraZeneca: Grant/Research Support|GlaxoSmithKline: Grant/Research Support|Meissa Vaccines: Advisor/Consultant|Merck: Grant/Research Support|Moderna: Advisor/Consultant|Moderna: Grant/Research Support|Pfizer: Advisor/Consultant|Pfizer: Grant/Research Support|Sanofi Pasteur: Advisor/Consultant


The relative contributions of these sites and targets may depend on the type of model being used and the timeline of the response. Shorter models emphasise the effect of reinvigoration in the absence of recruitment of new clones but studies spanning longer time periods examining T cell clones in patients demonstrate clonal replacement. Ultimately, further work is needed to determine which of the diverse effects of immune checkpoint inhibitors are the fundamental drivers of anti-tumour responses in patients.The introduction of immunotherapy, in particular immune checkpoint inhibition, has revolutionised the treatment of a range of tumours; however, only a minority of patients respond to these therapies. Understanding the mechanisms by which different immune checkpoint inhibitors work will be critical for both predicting patients who will respond and to developing rational combination therapies to extend these benefits further. The initiation and maintenance of anti-tumour T cell responses is a complicated process split between both the tumour microenvironment and the tumour draining lymph node. As understanding of this process has increased, it has become apparent that immune checkpoint inhibitors can act both within the tumour and in the draining lymph node and that they can target both already activated T cells as well as stimulating the priming of novel T cell clones. Currently, it seems likely that immune checkpoint inhibition acts both within the tumour and in the tumour draining lymph node both reinvigorating existing clones and driving further  The initial groundwork establishing the immune system as having a potential role in cancer therapy can be traced back as early as the late 1800s German physicians, when Busch and Fehleisen noted an epidemiological association between erysipelas infections and spontaneous tumour regression . Later wAs stated previously, the first ICI approved by US regulators targeted CTLA-4 which was first described as a negative regulator of T cell function . By comp+ T cell responses [+ T cells primed in the tdLN acquired expression of the activation marker PD-1 while maintaining expression of the transcription factor Tcf7 and remaining negative for effector cytokines [PEX) [PEX and that T cells which exited these niches gradually became exhausted (TEX), losing expression of Tcf7 and, ultimately, effector molecules [EX remain exhausted even once removed from the TME [PEX can reverse migrate back to the tdLN potentially seeding the tdLN-derived tumour-specific memory cells (TSM) population there [SM cells are marked by expression of PD-1 and Tcf7 [de novo priming of new T cell clones is required to drive the anti-tumour effects of ICIs as compared to reinvigoration of already primed, existing anti-tumour T cells.Despite these advances, a major hindrance to solving these challenges lies in our relatively poor understanding of the initiation and maintenance of anti-tumour T cell responses. As in other settings, anti-tumour T cell responses are initiated when conventional dendritic cells (cDC) take up tumour antigens in the context of damage associated molecular patterns, mature and migrate to the tumour draining lymph node (tdLN) where they present antigen in the context of MHC to antigen specific T cells . As in tesponses ,23 with esponses . This suesponses . There aesponses . More reytokines . These Tytokines . Indeed es [PEX) . In a violecules . This lo the TME ,30. Receon there . These Tand Tcf7 , thus reand Tcf7 . All thiREG) function [PEX population residing within the TME rapidly expand and regenerate TEX cells leading to tumour control [PEX reside in niches rich in cDC and so these effects on cDC likely impact TPEX disproportionately. The centrality of these cells in responses to ICI has been highlighted by a recent study where ICI was combined with FTY-720 to eliminate the contribution of the tdLN to the therapeutic response. In the absence of migration from the tdLN there was equivalent control of B16 tumours over a period of 6 days and this required the presence of Tcf7+ TPEX within the TME [PEX within the TME of a range of tumours in response to ICI [PEX are centrally important to responses to ICI there is a significant caveat that by using rapidly growing subcutaneous models, responses are monitored over a brief time-span and so the question remains about whether these intra-tumoural TPEX cells are sufficient to maintain anti-tumour responses over the longer time periods associated with treatment of patients. Despite this, the importance of TME resident TPEX cells in human cancer can be highlighted by the fact that the abundance of Tcf7+ CD8+ T cells within the TME of melanoma patients is associated with responsiveness to ICI [PEX niche within the TME, it has also been shown that the abundance of cDC1 also correlates with responsiveness to αPD-1 therapy in both melanoma and head and neck squamous cell carcinoma [PEX appears to be associated with more positive outcomes. The ability to deduce the site of action of ICI in these human studies is, however, limited due to the absence of matched samples from the tdLN and the reasonable hypothesis that TPEX abundance in the TME may correlate with the environment in the tdLN given that TPEX both originate from precursors within the tdLN and themselves act as a source for TSM [Given that both CTLA-4 and PD-1 are up-regulated upon T cell activation, early work centred around the roles of ICI reactivating already primed T cells predominantly within the TME . CTLA-4 function ,35 or byfunction . PD-1 onfunction ,38 recrufunction . PD-L1 ifunction . Thus PDfunction . Interesfunction  and othefunction ,43. This control . As stat the TME . This dee to ICI . While ts to ICI . Interesarcinoma . Thus, t for TSM ,31.REG specifically within the TME in a model of melanoma [REG cells led to increased activation of cDC2 which migrated to the tdLN and subsequently improved anti-tumour CD4+ T cell responses. This priming in the tdLN was critical for the therapeutic benefit as inhibition of T cell egress from the lymph node with FTY-720 was sufficient to prevent the tumour regression [SM which have migrated back from the TME to the tdLN [SM within the tdLN raises the question of whether these interventions function through reinvigoration of existing responses or, in fact, drive de novo priming.Despite these studies showing the centrality of tumour resident T cells for tumour control, there is also conflicting evidence for the importance of the tdLN in responses to ICI . Conceptmelanoma . In thisgression . In factgression . While tgression  and indegression ,31,50. Fthe tdLN . Interesthe tdLN ,53. As sthe tdLN . Even mothe tdLN . These Pthe tdLN . In factthe tdLN  highlighthe tdLN . Furtherthe tdLN . This cade novo priming of new T cell clones as opposed to reinvigoration of existing responses arises since the tdLN contains both naïve tumour-specific T cells, partially primed T cells which may arise during priming due to a separation of activation and full acquisition of effector function and TSM cells which may be seeded by T cells returning to the tdLN from the TME  were administered Nivolumab every 2 weeks and their tumours were removed 4 weeks after the initiation of treatment. Peripheral blood was analysed and T cell clones reactive against candidate neo-antigens prior to treatment were identified. Subsequently to treatment this process was repeated and several new T cell clones were found to have expanded which reacted to candidate neo-antigens; these new T cell clones were not identified in the pre-treatment samples [SM cells and were instead likely derived from naïve T cells undergoing de novo priming. This appears a reasonable conclusion, especially given previous data showing that treatment with αPD-1 improves cDC ability to prime in the tdLN in mouse models. Despite these findings, it is unclear as to how important these newly primed T cells are in generating the anti-tumour response post ICI treatment. Given the ability for tumour clones to evolve and lose expression of neo-antigens, the generation of new T cell responses post ICI treatment is an attractive prospect. However, despite these tantalising patient data it is unclear how important these new responses are in driving ICI responsiveness. A further consideration is that both the PD-1 and CTLA-4 pathways are important in the maintenance of peripheral tolerance through a variety of mechanisms in both peripheral tissues and within the lymph node [de novo priming of self antigen specific T cell clones [The difficulty of defining the contribution of  the TME . As suchEX cells . Criticathe node . This stthe node . Crucialthe node . The T c samples . This in samples . Given tmph node . As suchmph node . These il clones . This reEX or TPEX, as compared with that of tdLN-derived T cells. Furthermore questions remain about the relative contribution of reactivation of TSM as compared to de novo priming of naïve T cells. As noted, many of these questions remain due to the difficulties inherent in unpicking a complex interconnected system represented by the wider tumour macroenvironment incorporating both the TME and the tdLN; however, other questions arise from the necessity to better understand and position the mouse models being used for such studies. A pertinent example of this comes from the different conclusions reached about the impact of FTY-720 treatment on ICI outcomes. In some mouse models FTY-720 leads to no alteration in responsiveness to ICI whilst in others it completely abrogated any response. These questions are not trivial as understanding better the mechanism of action of ICI will inform better combination therapeutic approaches moving forward. As such, there is a pressing need to better characterise existing tumour models and to develop new ones which may address outstanding questions. Despite these caveats, it appears likely that in patients at least there is a contribution made to the ongoing T cell response from de novo priming during ICI therapy, however, whether that is the main driver or merely a passenger in the effective response remains to be seen.In conclusion, due to the complex nature of the anti-tumour T cell response, it is unclear the relative contribution of rejuvenation of tumour infiltrating T cells, be they TNew approaches are needed to increase the number of patients benefitting from ICI therapy and this requires greater understanding of their mechanism of action.Different model systems provide data suggesting roles for ICI both within the TME and in the tdLN.de novo priming of new T cell clones although the necessity of this for responsiveness has not been elucidated.Data from patients treated with ICI suggest clonal replacement of T cells indicating


Shwannomas are rare benign tumours especially in tracheal. A 16‐year‐old male presented with a chronic cough, and a thoracic CT scan revealed a pedunculated tumour measuring approximately 11 × 13 mm in size, located 22 mm away from the main carina. Tissue sample was obtained via rigid bronchoscopy and cryobiopsy, and the pathological analysis confirmed the diagnosis of a benign nerve sheath tumour consistent with schwannoma. The patient subsequently underwent resection of the tumour and tracheal anastomosis. Schwannomas are uncommon pulmonary tumours that typically occur in adults, with a higher incidence among females. The presenting symptoms vary depending on the size and location of the tumour. Treatment options include therapeutic bronchoscopy or surgical resection, with the choice of approach based on tumour characteristics (pedunculated or sessile), preoperative surgical risk, and risk of recurrence. The prognosis is generally favourable, with a low risk of recurrence and excellent outcomes. Schwannomas are benign tumours that originate from Schwann cells. Primary trachea tumours are uncommon. Here we report a case of primary schwannoma of the trachea in a 16 year‐old male patient. Schwannomas are benign tumours that originate from Schwann cells.Primary trachea tumours are uncommon. Among these tumours, 75% are composed of squamous cell carcinoma and adenoid cystic carcinoma. The remaining tumours have diverse histological subtypes and can be benign, malignant, or of intermediate grade.Neurogenic tumours of the tracheobronchial tumours, such as schwannomas and neurofibromas, are exceedingly rare.The purpose of this report is to present a case of primary schwannoma of the trachea.A 16‐year‐old male with an unremarkable medical history presented to the thoracic surgery clinic with a chief complaint of a non‐productive cough that had been persistent for approximately 3 months despite the use of cough suppressant medications. The patient was asymptomatic for weight loss, fever, diaphoresis, anorexia, or hemoptysis. There was no history of tobacco or substance use, drug or food allergies, familial respiratory or malignant diseases, or any recent close contact with an ill individual. The patient demonstrated stable vital signs and a normal cardiac and pulmonary examination. Sputum cultures were negative for mycobacteria on three separate occasions. Chest CT scan revealed a pedunculated polypoid mass measuring approximately 11 × 13 mm in cross‐sectional diameter, situated in the distal trachea and extending over a cranio‐caudal length of 21 mm , and malignant transformation of the tumour is extremely infrequent.Kashara et alIn the literature review, four cases of this rare disease were reported and are referred to in Table The clinical manifestations of intratracheal schwannoma depend on the size, location, and degree of obstruction caused by the tumour.Schwannoma of the intratracheal is a disease of adults and is more common in women.The location of tracheal schwannomas is frequently in the distal third of the tracheal, followed by the proximal third and middle third, respectively.Diagnosis is confirmed by bronchoscope sampling, and CT scan helps determine tumour size and extent. Other evaluations include spirometry, which demonstrates fixed obstruction of the upper airway on volume‐flow curve.PET/CT imaging reveals increased uptake in the tumour, although routine use is not recommended.In our patient, the purpose of performing CT scan was to evaluate the presence of nodules in the lung parenchyma, in addition to assessing the tracheal and the extent of the tumour.Schwannoma of the tracheal can be treated by several methods, including primary tumour excision or endoscopic treatment using laser, snare resection, argon plasma coagulation (APC), CO2 laser, electrocautery, cryotherapy, with or without endoscope and microdebrider. The choice of surgical approach should be determined by the clinical manifestations of the tumour (pedunculated versus flat), the risk of tracheal resection, and the presence or absence of extratracheal extension.In our patient, due to the low risk of surgery and incomplete tumour removal by cryobiopsy, the decision was made to perform tracheal resection.In patients with pedunculated lesions without extratracheal extension or in individuals with high surgical risk, the tumour can be removed using endoscopic techniques. However, tumour recurrence occurs in a quarter of patients.Due to the slow growth rate of these tumours, bronchoscopic follow‐up every year is recommended. In another group of patients with flat tumours, where the risk of surgery is low or there is extratracheal extension, surgical removal is preferred. Recurrence has not been reported in this group.The prognosis for patients with schwannoma is excellent, whether treated surgically or endoscopically.Hesam Amini and Alireza Samimiat gathered and interpreted the patient data regarding history and physical examination. Shahab Rafieian and Reza Ershadi wrote the manuscript. Matin Vahedi supervised and designed the project. Shahab Rafieian and Matin Vahedi perform surgery on the patient. As, Hesam Amini and Mohsen Gholinataj Jelodar contributed to the discussion of the research. All the authors read, revised and approved the final manuscript.None declared.The authors declare that appropriate written informed consent was obtained for the publication of this manuscript and accompanying images.


Influenza virus infections are common in children and lead to numerous ED visits each year. The Infectious Diseases Society of America, American Academy of Pediatrics, and the Centers for Disease Control and Prevention recommend outpatient antiviral treatment for children at increased risk of severe influenza illness  and recommend treatment within 48-hours of symptom onset. We describe antiviral prescription in children presenting to the ED with influenza from 2016-2020.We analyzed data from NVSN, a prospective seven-site acute respiratory illness surveillance study. We enrolled children presenting to the ED with confirmed influenza by research molecular testing, stratified by children at increased risk of severe influenza and with symptom onset ≤ 48-hours. We compared characteristics of children who were prescribed antivirals to those who were not. We used logistic regression to compare the odds of prescription, adjusting for age, gastrointestinal symptoms, symptom duration, underlying conditions, clinical testing before antiviral prescription, and site.Table). Odds of prescription were higher for those clinically tested for influenza and for those with underlying respiratory and cardiovascular conditions, and lower for those who were younger, presented with diarrhea, and had longer duration since symptom onset .Of the 16,822 children presenting in the ED and tested for influenza, 2,387 (14%) tested positive. Among influenza positive cases, 1,873 (79%) were defined as children at increased risk of severe influenza, and 571 (30%) were prescribed an antiviral. Additionally, there were 281 (12%) children with symptom onset ≤ 48-hours, and only 70 (25%) were prescribed an antiviral , FIDSA, FAAM, Abbott: Honoraria|Altona Diagnostics: Grant/Research Support|Baebies Inc: Advisor/Consultant|BioMerieux: Advisor/Consultant|BioMerieux: Grant/Research Support|Bio-Rad: Grant/Research Support|Cepheid: Grant/Research Support|GSK: Advisor/Consultant|Hologic: Grant/Research Support|Lab Simply: Advisor/Consultant|Luminex: Grant/Research Support Natasha B. Halasa, MD, MPH, Merck: Grant/Research Support|Quidell: Grant/Research Support|Quidell: donation of kits|Sanofi: Grant/Research Support|Sanofi: vaccine support


In most countries, men are more likely to die by suicide than women. Adherence to dominant masculine norms, such as being self-reliant, is linked to suicide in men in Western cultures. We created a suicide prevention media campaign, “Boys Do Cry,” designed to challenge the “self-reliance” norm and encourage help-seeking in men. A music video was at the core of the campaign, which was an adapted version of the “Boys Don’t Cry” song from “The Cure.” There is evidence that suicide prevention media campaigns can encourage help-seeking for mental health difficulties.We aimed to explore the reach, engagement, and themes of discussion prompted by the Boys Do Cry campaign on Twitter.We used Twitter analytics data to investigate the reach and engagement of the Boys Do Cry campaign, including analyzing the characteristics of tweets posted by the campaign’s hosts. Throughout the campaign and immediately after, we also used Twitter data derived from the Twitter Application Programming Interface to analyze the tweeting patterns of users related to the campaign. In addition, we qualitatively analyzed the content of Boys Do Cry–related tweets during the campaign period.During the campaign, Twitter users saw the tweets posted by the hosts of the campaign a total of 140,650 times and engaged with its content a total of 4477 times. The 10 highest-performing tweets by the campaign hosts involved either a video or an image. Among the 10 highest-performing tweets, the first was one that included the campaign’s core video; the second was a screenshot of the tweet posted by Robert Smith, the lead singer of The Cure, sharing the Boys Do Cry campaign’s video and tagging the campaign’s hosts. In addition, the pattern of Twitter activity for the campaign-related tweets was considerably higher during the campaign than in the immediate postcampaign period, with half of the activity occurring during the first week of the campaign when Robert Smith promoted the campaign. Some of the key topics of discussions prompted by the Boys Do Cry campaign on Twitter involved users supporting the campaign; referencing the original song, band, or lead singer; reiterating the campaign’s messages; and having emotional responses to the campaign.This study demonstrates that a brief media campaign such as Boys Do Cry can achieve good reach and engagement and can prompt discussions on Twitter about masculinity and suicide. Such discussions may lead to greater awareness about the importance of seeking help and providing support to those with mental health difficulties. However, this study suggests that longer, more intensive campaigns may be needed in order to amplify and sustain these results. Suicide is a significant public health issue worldwide, with more than 700,000 people dying by suicide every year . In 2019It is also possible that traditional masculinity, the cultural set of norms dictating how men should think and behave -14, may In collaboration with an independent film production company (Heiress Films) and an advertising company , and with guidance from several Australian suicide prevention and men’s health community organizations, we developed Boys Do Cry, a suicide prevention media campaign designed to challenge adherence to masculine norms such as self-reliance and encourage help-seeking. At the core of the campaign was a video of a diverse group of Australian men singing an adapted version of the song “Boys Don’t Cry” from the British rock band “The Cure.” The adapted lyrics relate to the importance of men talking to others and reaching out for help if they are struggling emotionally . The viThe Boys Do Cry campaign was publicly released on November 21, 2021, and the video was made available through Twitter, Facebook, Instagram, TikTok, and YouTube. In addition, a website was developed  which seThe evaluation of the Boys Do Cry campaign included a randomized controlled trial of the effects of the video on help-seeking intentions that was conducted before the campaign was launched . This stWe addressed the study’s aim through 4 research questions:What was the reach of the Boys Do Cry campaign, and how did the Twitter users engage with it?What were the highest- and lowest-performing tweets, and how did the Twitter users engage with them?What was the pattern of Twitter activity for campaign-related tweets over the course of the campaign and in the postcampaign period?What were the key themes of discussion on Twitter prompted by the Boys Do Cry campaign?To address our research questions, we used Twitter data from 2 sources: (1) Twitter analytics; and (2) Twitter application programming interface (API). To address research questions 1 and 2, we downloaded Twitter data using the Twitter analytics tool. This tool is available to all Twitter users; it tracks user metrics such as top performing tweets and mentions, the number of times a tweet is seen on Twitter, and the number of engagements . The Twitter analytics tool allows users to freely download their data.On November 21, 2022, we downloaded monthly tweet activity metrics from the campaign’s hosts (@BoysDoCry2021) for the active campaign period (during campaign), which included the 2 phases of the campaign. We then combined the monthly data files into 1 data set. These data allowed us to identify and describe the campaign’s reach and users’ engagement with the campaign. We described reach as the number of times users saw a tweet posted by @BoysDoCry2021 and engagement as the number of times users interacted with @BoysDoCry2021 tweets  . A totalTo address research questions 3 and 4, we used the Twitter API. There are several Twitter API streams; we used the Academic Research Twitter API after submitting a project proposal to Twitter. This free-of-charge data collection method is no longer available due to recent changes in Twitter’s business model .On November 30, 2022, we collected Twitter data using the R package “academictwitteR” . We searWe conducted quantitative analyses to answer research questions 1 to 3 and qualitative analysis to answer research question 4. For the analyses, we used R (version 4.2.2)   attracted more reactions, so tweets could be either a (1) video, (2) image, or (3) link. For instance, if a tweet involved an image or video and a link, we coded it as under image or video, so we checked all links to confirm if they involved any visual component. Videos were either embedded in the tweet or links to the campaign’s video (either through YouTube or through the campaign’s homepage). Tweets classified under “link” were therefore purely text-based content. We note that the 10 lowest-performing tweets included @BoysDoCry2021 replies or mentions to users without meaningful data , so we coded the next lowest-performing tweets with meaningful content in those instances.For research question 3, we used the Twitter API data to calculate the number of campaign-related tweets per week and plotted their frequencies by campaign period (during-campaign vs postcampaign). Then we calculated the average number of tweets by campaign period. For research question 4, we used a subset of the Twitter API data to conduct the qualitative analysis. This data subset only included tweets in the English language. We excluded retweets as they did not have any new text data and had the potential to bias the results. We also excluded tweets coming from the campaign’s hosts, as our primary goal was to explore the audience discussions prompted by the campaign. We followed Braun and Clarke’s  methodolThis study was approved by the Human Research Ethics Committee at the University of Melbourne (HREC No. 2022-25141-34361-3). All research was conducted in accordance with the National Health and Medical Research Council’s National Statement on Ethical Conduct in Human Research and the Declaration of Helsinki.The 247 tweets posted by @BoysDoCry2021 were seen a total of 140,650 times by Twitter users during the campaign. Users engaged a total of 4477 times with those 247 tweets. A total of 9 out of 10 of the highest-performing tweets were from phase 1, while 7 out of 10 of the lowest-performing tweets were from phase 2. All the 10 highest-performing tweets included some visual content, either a video  or an image . Likewise, the 10 highest-performing tweets involved sharing the campaign’s video or an image or screenshot promoting the campaign video. Among the 10 highest-performing tweets, the highest-performing tweet was the first tweet posted by @BoysDoCry2021, and it included the campaign’s core video embedded in the tweet.Each day in Australia, an average of 7 men take their own lives. Together we can change the stats.Introducing a new mental health campaign #BoysDoCry which aims to encourage men to get talking when they are struggling with their #MentalHealth.https://t.co/9AVe8TYKaM(with 748 engagements)The second highest-performing tweet was a screenshot of the tweet posted by Robert Smith, the lead singer of The Cure, who wrote and performed the original song, shared the campaign’s video on YouTube and tagged the campaign account . to @RobertSmith from @TheCure for sharing our adaptation of his song, and for supporting #mensmentalhealth! Huge Watch the music video now:https://t.co/HOEkCBcfKU#BoysDoCry2021 #RobertSmith #TheCure #GetTalking #MentalHealthMatters https://t.co/Hu2Y8xZkw7(with 641 engagements)The 10 lowest-performing tweets shared either a link  or an image . A total of 9 out of 10 of the 10 lowest-performing tweets involved sharing information related to masculinity, help-seeking resources, or broad resources such as articles and interviews. Examples of tweets posted by @BoysDoCry2021 with the lowest engagement are:“When society creates an impossible standard of #masculinity to maintain, nobody wins.”Interesting #newstudy published in Social Psychological and Personality Science @SPSPnews<link>#Relationships #Sociology(with 1 engagement)If you need support @LifelineAust have a 24/7 telephone counselling and crisis support line that’s free to call on 13 11 14. You can also visit our website for a list of organisations that can help on our website:https://t.co/CuKsKcOUD5(no engagements)In total, 1237 out of 1318 (93.9%) tweets were related to the Boys Do Cry campaign on Twitter in the during-campaign 19-week period (excluding campaign-generated tweets), while there were 81 out of 1318 (6.1%) campaign-related tweets in the postcampaign 19-week period. The summary statistics of weekly tweet counts by campaign period are shown in The 237 campaign-related unique tweets were a subset of the Twitter Application Programming Interface data; it only included tweets in the English language and with new text data in ; see The most common theme was “campaign support” . This theme was about showing support for the Boys Do Cry campaign by sharing its video or the link to its website or mentioning the campaign handle (@BoysDoCry2021). Examples of tweets for campaign support are:keep up the great work!@BoysDoCry2021 We are more than happy to support such an amazing campaignSuch a powerful important message… to all my male #firefighter and #firstresponder colleagues and friends. This could save your life #SuicidePrevention #FRNSW https://t.co/ldHg9YQX4xThe second most frequent theme was “reference to original song/band/lead singer” . This theme included tweets that cited the original song, band or its lead singer. Examples of tweets coded under the reference to original song/band/lead singer theme are:@RobertSmith @BoysDoCry2021 It's no surprise, but always a pleasure, to see you and The Cure supporting a strong cause @RobertSmith @username @BoysDoCry2021 Beautiful, I’ve always thought this is the emotion from where you wrote this song.“Reiterate the message” was the third most common theme . This theme was related to tweets that incorporated core messages from the campaign around men reaching out for help when struggling and challenging traditional masculine ideas. Examples of tweets for reiterate the message are:#boysdocrywhen the going gets tough, get talkingLove this <link>Sometimes old messages need replacing - this is one of those times. @BoysDoCry #MentalHealth https://t.co/WIJak25tSiThe fourth most frequent theme was “emotional response” . This theme involved tweets referencing emotional reactions in response to the campaign. Examples of tweets coded under this theme are:Our men are broken. I know a lot over 40 that I worry about their mental health going forward. Hopefully hearing from other men to talk about their inner most feelings will help someone out there!It feels awesome to have a big cry! Not a weakness but a strength https://t.co/fTNNqQwS7C@RobertSmith @BoysDoCry2021 HeartwarmingThe fifth most common theme was “mental health-related hashtags” . The mental health-related hashtags theme included tweets with hashtags such as #suicide, #mentalhealth, and #mensmentalhealth. Examples of tweets coded under this theme are:Absolutely spot on. Gents, it's OK not to be OK, and men DO cry. We lose far too many of you to suicide and we need to break the stigma NOW. #boysdocry #SuicidePrevention #MentalHealthMatters <link>I’m listening#BreakTheBias #Mentalhealth #boysdocry <link>This study is part of a larger evaluation of the Boys Do Cry campaign. In this study, we explored the campaign’s reach, engagement, and themes of discussion prompted by the Boys Do Cry campaign on Twitter. In assessing the content of conversations elicited by this campaign, we provide new insights regarding the critical elements for crafting future suicide prevention media campaigns targeting boys and men.We found that the Boys Do Cry campaign reached a large number of Twitter users, prompting them to engage with its content. The most effective aspect for eliciting engagement was the addition of visual content in the campaign’s tweets. Evidence suggests that including visual content is an important strategy in public health communication because images have the potential to increase people’s attention to key health messages, especially when there is an emotional response to them . NotablyMost public campaign-related Twitter activity occurred during the first week of the Boys Do Cry campaign, when an international celebrity—Robert Smith from The Cure—tweeted about the campaign. However, it is also likely that many campaign followers were more interested in the campaign when it was new. This may be a characteristic of this type of health promotion campaign; for example, another study monitoring suicide and depression discussions on Twitter over time (176 million tweets) found that Twitter activity faded quite rapidly following expected events, such as mental health campaigns, compared to unexpected events, such as a celebrity death by suicide .This study also examined the key discussion themes prompted by the Boys Do Cry campaign. Key among these themes was that people supported the campaign and reinforced its messages. This finding may reflect a desire to challenge masculine norms, such as the notion that boys and men should not cry and should keep their emotions in check, even in difficult times. Evidence suggests that the masculine norm of self-reliance is associated with suicidal thinking in men  and thusOur findings are in part similar to those from our Twitter evaluation of another suicide prevention media campaign focused on challenging traditional masculinity, called “Man Up” . We noteThis study had some limitations. Our search query for the Twitter API only searched for the string “boysdocry” , so it is possible that we did not collect tweets that used the term “boys do cry” with spaces. However, our pilot searches revealed that the inclusion of spaces in the query would return more false positives than we could feasibly manage manually, so we did not include spaced terms. Another limitation is the potential bias in this study sample based on the demographics of Twitter users, as there are documented differences on social media preferences by age group . This stSocial media platforms offer another avenue through which to engage with boys and men on the topic of masculinity and suicide. This study demonstrates that a brief media campaign such as Boys Do Cry can achieve good reach and engagement and can prompt discussions on Twitter around the issue, which may lead to greater awareness about the importance of seeking help and providing support. However, this study suggests that longer, more intensive campaigns may be needed in order to amplify and sustain these results.


The mice were treated with resveratrol per os for five consecutive days beginning on day 2 after tumour injection and/or by injecting cisplatin intraperitoneally (ip) at a dose of 2.5 mg/kg on days 10 and 12 and at a dose of 5 mg/kg on day 15. Immediately thereafter, the mice were exposed to systemic hyperthermia for 15 min at a temperature of 41 °C. The obtained results showed that the administration of resveratrol did not significantly contribute to the antitumour effect of cisplatin and hyperthermia, but it partially contributed to the immunomodulatory effect and to the reduction of cisplatin toxicity and to a slight increase in animal survival. This treatment schedule did not affect microvessel density, but it inhibited tumour growth and modulated macrophage polarization to the M1 phenotype. Furthermore, it abolished the resistance of tumour cells to cisplatin by modulating HDAC activity and the concentration of HSP70 and HSP90 chaperones, contributing to the increased lifespan of mice. However, the precise mechanism of the interaction between resveratrol, cisplatin, and hyperthermia needs to be investigated further.The aim of this study was to investigate the therapeutic potential of resveratrol in combination with cisplatin on the inhibition of tumour angiogenesis, growth, and macrophage polarization in mice bearing the solid form of an Ehrlich ascites tumour (EAT) that were exposed to whole-body hyperthermia treatment. In addition, we investigated whether a multimodal approach with hyperthermia and resveratrol could abolish cisplatin resistance in tumour cells through the modulation of histone deacetylase (HDAC) activity and levels of heat shock proteins (HSP70/HSP90) and contribute to the direct toxicity of cisplatin on tumour cells. The tumour was induced by injecting 1 × 10 Angiogenesis is the process of the formation of new blood vessels and is one of the main factors that promote tumour growth. Angiogenesis occurs as a response to hypoxia, which, together with a lack of essential nutrients, increases the expression of inflammatory signals and cytokines that recruit blood vessel cells to create a network of new tumour blood vessels . HypoxiaTumours with higher angiogenic potential show higher metastatic capacity and aggressiveness . MMPs plWith the aim of improving treatment options for cancer, there is a growing interest in the scientific community about the potential therapeutics originating from biogenic sources with HDAC inhibitory activities. Based on that, we hypothesised that combinations of natural compounds and chemotherapeutics could enhance the sensitivity of cancer cells, overcome resistance, increase the cytotoxicity, improve the treatment outcome, and reduce the cisplatin-induced side effects. An example of such a compound is resveratrol, a phytoestrogen and polyphenol with antioxidant, anti-inflammatory, cardioprotective, and antitumour properties. The administration of resveratrol can reverse multidrug resistance and in combination with cisplatin has an additive and/or synergistic effect in increasing the chemosensitivity of cancer cells, reducing cytotoxic effects on healthy cells and modulating the immune response in the tumour microenvironment by regulating cytokines/chemokines secretion, immune-checkpoints/MHCI (major histocompatibility complex 1), the nuclear factor-κB (NF-κB) signalling pathway, the AKT-mammalian target of rapamycin (mTOR) signalling, and the activation of receptors on natural killer (NK) cells ,12. YangHence, the aim of this study was to investigate the effects of the combined treatment with cisplatin and resveratrol on metalloproteinase activity, angiogenesis, tumour growth, and survival in mice bearing the solid form of Ehrlich’s ascites tumour. This model is a suitable model in tumour biology and is useful for studying tumour pathogenesis and the development of antitumour drugs . FurtherIn patients and animals, cisplatin causes prominent side effects that increase morbidity and impair quality of life. Currently, around 40 side effects of cisplatin have been reported , includiTreatment with cisplatin and resveratrol under hyperthermic conditions increased the lifespan by about 68% and reduced tumour growth by about 50% compared to the Control group . Increasig treatment of animals with resveratrol (50 mg/kg) or therapeutic treatment with cisplatin (2.5 or 5 mg/kg) under normal and hyperthermic conditions significantly reduced the tumour volume of mice bearing the solid Ehrlich tumour compared to controls (Control and Control + HT). Preventive Preventive treatment with resveratrol has reinforced the delay of tumour growth in mice treated with cisplatin and increased the survival of animals .p = 0.00233 and p = 0.00506, respectively).The survival rate of mice after treatment with test components in physiological and hyperthermic conditions was shown by the Kaplan–Meier method  and analp = 0.68082). In addition to the comparison with the control groups  and Res + HT vs. Res + Cis + HT (p = 0.02158)  see .Resveratrol as a natural product could partly affect the inhibition of tumour growth through epigenetic mechanisms by acting on HDACs. HDACs are enzymes involved in numerous steps of tumourigenesis, including the initiation of tumour growth, angiogenesis, metastasis, and numerous other processes of tumour progression ,11,12,17p < 0.05. However, hyperthermia further enhanced the inhibitory effect of cisplatin on HDAC activity. The HDAC inhibition values obtained were: 2.84 ± 0.04 (Cis + HT) and 2.91 ± 0.11 (Res + Cis + HT) vs. 8.84 ± 0.57 (Control), p < 0.001 for both groups  vs. 8.84 ± 0.57 (Control), h groups .Chaperones are usually classified according to their molecular weight. HSP70 and HSP90 chaperones participate in de novo folding and refolding of proteins. Thus, HSP90 participates in the posttranslational folding and stabilization of more than 200 client proteins involved in the regulation of those cell signals that promote tumour cell growth and radioresistance , while HInterestingly, levels of HSP70 a were hip ≤ 0.001), Control + HT (p ≤ 0.05), and Res (p ≤ 0.001). The second highest value was observed for the treatment with Cis + HT (compared to Control p ≤ 0.05 and Res p ≤ 0.01). According to the obtained data, the group treated with Res + Cis + HT had only a slightly lower level of HSP-70 compared to the Control and Control + HT groups, which indicates greater sensitivity of tumour cells since the inhibition of HSP70 can enhance the chemotherapy-induced tumour-killing effect. This group showed a significant decrease of HSP70 concentration compared to Cis + HT (p ≤ 0.05) and Res (p ≤ 0.001).Treatment with Res + Cis showed the highest value of HSP70 compared to Control (p ≤ 0.001), followed by the values obtained in groups Res + HT and Res + Cis + HT .HSP90 concentrations b were reHyperthermia causes numerous changes of cellular membranes, the cytoskeleton, the nucleus, DNA, RNA, proteins, and cell organelles, but also damages the microvasculature and suppresses the angiogenesis of malignant tumours. Angiogenesis is essential for tumour development and metastasis, and tumour-derived angiogenic factor(s) such as VEGF play(s) an important role in the formation of new vessels and tumour progression in vitro and in vivo.p < 0.01; p < 0.01), Res + Cis , and Res + Cis + HT (p < 0.05).From VEGF is the key factor in tumour neovascularization. It increases endothelial cell proliferation and migration, tumour growth in vivo, and remodels the perivascular matrix by increasing levels of proteinases, including matrix metalloproteinases MMP2 and MMP9 ,4,5.p < 0.01 in both groups).MMP2 concentration a was redp < 0.001 or p < 0.001) and Cis + HT groups (p < 0.001 or p < 0.01). MMP9 concentration in the group treated with Res + Cis + HT was significantly lower compared to the Res + HT and Cis + HT groups (p < 0.001 and p < 0.001). Contrary to MMP2, the concentration of MMP9 b was uprp < 0.05), Cis (p < 0.01), Cis + HT (p < 0.05), and Res + Cis + HT (p < 0.05) compared with the tumours of Control animals that showed extensive neovasculature (Tumour progression and the escape of tumour cells (metastasis) require a dynamic interaction between cancer cells and their microenvironment. The induction of tumour angiogenesis is a critical step in the early development of solid tumours. Formation of new blood vessels requires the angiogenic factor VEGF and its proteolytic release from tumour matrix by MMPs, predominantly by the MMP-9 that is delivered into the tumour microenvironment by tumour-infiltrating leukocytes. Given the reduced level of VEGF and tumour growth, we next investigated whether the number of blood vessels was also reduced within the solid form of EAT treated with resveratrol and cisplatin with or without hyperthermia. The obtained results clearly indicated that there was a significant inhibition of neovascularization in mice treated with Res  plays an important role in the regulation of interactions between the tumour and its microenvironment, thereby affecting tumour progression . The tum and Re + Cis + HT .Arg activity  in the tp < 0.01; p < 0.01), Control + HT , and Res (p < 0.05).For the same groups , the level of NO  was incrThe pathological examination of the paraffin-embedded solid EAT samples showed visible central necrosis in all specimens, the degree of which cannot be assessed . In all Hypoxia inducible factors 1 and 2 (HIF) are important transcriptional regulators of angiogenesis and energy metabolism in tumours and an important target for cancer therapy . AccumulAfter immunohistochemical staining, we observed weak cytoplasmic staining for HIF-1α in control and single-agent treated samples . AlthougImmunohistochemical staining for inducible nitric oxide synthase (iNOS) showed weak to moderate cytoplasmic staining for iNOS in control and single-agent treated samples. The strongest expression was observed in samples treated with Cis. Breast cancer was used as a positive control for iNOS with strong cytoplasmic staining . After immunohistochemical staining, we observed moderate cytoplasmic staining for HIF-1α in all samples treated with combined agents . The staThe immunohistochemical staining for iNOS also showed moderate cytoplasmic staining in all samples treated with combined agents. The strongest expression of iNOS was observed in samples treated with Cis + HT and Res + Cis + HT.p ≤ 0.001), Res + Cis (p ≤ 0.001), and Res + Cis + HT goups (p ≤ 0.001), while the treatment of animals with Res + HT was statistically different compared to Control and Res + Cis (p ≤ 0.001 for both groups). In addition, changes in the level of iNOS were noticed in the Res group compared to Control (p ≤ 0.001) and Cis + HT (p ≤ 0.01), and between Control + HT vs. Cis + HT (p ≤ 0.01). Quantitative analysis of HIF-1α showed statistically significant changes in the group treated with Cis + HT, Res + Cis, and Res + Cis + HT compared to the Control . Immunohistochemical staining intensity presented as units  of HIF-1 in tumour tissue of these groups was as follows: 10.6 ± 1.0 (Res + Cis), 20.7 ± 6.3 (Cis + HT), and 32.8 ± 3.0 (Res + Cis + HT), while the staining intensity in the tumour Control tissue was 110.5 ± 5.6 units (p ≤ 0.001), as well as between Cis + HT vs. Control + HT and Res (p ≤ 0.01). For the quantitative analysis of the intensity of immunohistochemical staining of the images represented in .6 units b. A statTumour hyperthermia is a powerful therapeutic approach and chemotherapy sensitizer. It increases the cytotoxicity of cancer cells and stimulates the immune response through the activation of immune cells. Hyperthermia modulates local pro-inflammatory responses, immune activation, cell death, and microenvironmental changes at target sites, mainly by specific heat shock proteins (HSPs) ,7,8,9,18Previous studies have shown that resveratrol can increase the antitumour efficacy of cisplatin and reverse multidrug resistance by increasing the chemosensitivity of cancer cells ,11,12. AIn this study, we investigated whether the pre-treatment with the natural antioxidant resveratrol, in combination with systemic hyperthermia and cisplatin, can enhance the effect of cisplatin on tumour cells through the modulation of HDAC activity and the HSP70 and HSP90 levels and consequently contribute to direct toxicity on tumour cells, the inhibition of angiogenesis and tumour growth, and ultimately increase the lifespan of mice.p < 0.01) compared to the Control (p < 0.001) in relation to Control , affecting the size of the tumour and the process of metastasis, which are closely related to the levels of VEGF, MMP-2, and MMP-9 ,9.Hyperthermia with Cis can inhibit VEGF production in vivo and shows the anti-angiogenic effect, which was confirmed by the reduced number of blood vessels (MVD) . Hence, p < 0.01; p < 0.01). In other treated groups, the concentration of MMP-2 was also lower compared to the Control, but without significance  (p < 0.001)  b. Additi< 0.001) b. The ob< 0.001) , which iActivation of VEGF as a pro-angiogenic factor is mediated by HIF-1α to induce angiogenesis. Hypoxic conditions also regulate HDAC activity directly or indirectly through the HDAC involvement in oxygen-regulated gene expression and hypoxia-induced angiogenesis ,19. An iCells respond to stressful conditions by activating stress response proteins, which may be important in maintaining a fine balance between cell death and survival. Stress proteins induced after chemotherapy, radiation, or hyperthermia can have a dual role. Thus, HSPs may display cytoprotective functions due to the ability to protect cells from damaging stress, acting as survival regulators through their anti-apoptotic properties . On the The reduced level of HSP90 in all groups, except for the control groups in physiological and hyperthermic conditions and resveratrol, confirms the sensitivity of tumour cells to the inhibition of HSP90 b, whose The Induction of protective antitumour immunity and its manifestations at local and distal sites result from a complex interplay between different cell types and soluble mediators such as cytokines and stress proteins. An increasing number of studies have shown that hyperthermia, as an adjuvant approach to cancer immunotherapy, correlates with the increased presence of infiltrating monocytes and macrophages in tumours, and that this tends to be associated with increased macrophage activity ,31,32,33It is known that chemotherapeutic “drugs” such as cisplatin, doxorubicin, mitomycin C, 5-fluorouracil, and camptothecin, which exert direct cytotoxic effects, also exhibit an indirect cytotoxic activity by “preparing” tumour cells for elimination by immune cells such as NK or cytotoxic T lymphocytes using a Fas or TNF-related apoptosis-inducing ligand (TRAIL)-dependent pathway . ChemothThus, platinum-based chemotherapies, including cisplatin, exert immunogenic effects through the cell death induction and the release of death-associated molecular patterns, which activate pro-inflammatory signalling pathways. It has been reported that cisplatin-induced macrophage activation stimulates the synthesis and release of NO, tumour necrosis factor (TNF)-α, interleukin (IL)-1, interferon (IFN)-γ, and IL-12, leading to M1 polarisation ,33,34. IThe activation of M1 macrophages induces the production of NO. An increased concentration of NO has a cytotoxic effect on tumour cells and promotes apoptosis. M1 macrophages increase the activity of the iNOS enzyme, which catalyses the conversion of L-arginine to NO and citrulline, while M2 macrophages increase the expression of the Arg-1 enzyme. Hence, these two enzymes are in competition for L-arginine ,37. M1 mp < 0.001) compared to the Control group  such as CRT, HSP70, and HMGB1, and via the remodelling of the tumour microenvironment by the increasing maturation of DCs and macrophages. Activated DCs and macrophages, as antigen presenting cells, reactivate the effector function of cytotoxic T cells on tumour cells, as well as the activation of NK cells by HSP70 ,32,33,34It should also be noted that resveratrol is one of the most important dietary constituents capable of chemoprevention and vasculoprotection.Aging induces numerous changes such as genomic instability, epigenetic alterations, telomere shortening, loss of proteostasis, deregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, and stem cell exhaustion. Various changes induced by microenvironmental factors and aging-dependent changes are partially related to blood vessels, which are important for the maintenance of tissue function and the transport of oxygen, food, and waste materials. During aging, the density of blood vessels and the number of pericytes decrease ,53, togeIt seems that HIF inhibition with resveratrol ultimately prevents resistance to anti-angiogenic therapy, blocks epithelial-mesenchymal transition and differentiation of myeloid suppressive cells into M2-proangiogenic TAM, contributes to the reactivation of NK and T cell activity, and prevents genetic instability in tumour endothelial cells and the selection of invasive metastatic tumour cell clones that are resistant to anti-angiogenic agents ,53,57,58In conclusion, resveratrol can enhance the antitumour effect of cisplatin by modulating HDAC activity and HSP70 and HSP90 levels, thus contributing to direct toxicity on tumour cells and tumour growth, and consequently prolonging mice life. Furthermore, it seems that hyperthermia can create a favourable tumour microenvironment for an immune chain reaction that improves the efficacy of the combined treatment with cisplatin and resveratrol. Hyperthermia enhances local tumour blood flow and increases the accumulation of cisplatin within tumour tissues and remodulates the tumour microenvironment by realizing danger signals and antigens originating from tumour cells that contribute to the reactivation of macrophage polarization into the M1 phenotype and the activation of the immune response to tumour cells. Summarizing the data, treatments of animals with Cis and HT and Res + Cis + HT have the greatest effects in antitumour activity, but apparently via the slightly different synergistic/antagonistic mechanisms. According to the obtained results, the use of resveratrol did not significantly contribute to the antitumour effect of cisplatin and hyperthermia, but it partially contributed to minor differences in animal survival, MMP2 concentration, and a significant decrease in arginase activity, which is important in HIF-NO signalling. With an increased understanding of the mechanisms underlying these processes, more accurate and effective therapies can be developed for the growing number of diseases influenced by HIF-NO signalling. It is possible that the microenvironmental conditions of the tumour, especially the availability of oxygen, affects the redox balance of Res and changes its chemotoxic activity, but also weakens the activity of cisplatin. Although the use of natural components in combination with cisplatin often gives an advantage in attenuating tumour growth, angiogenesis, chronic inflammation, and immune tolerance, thus reducing the consequences of toxicity, caution is needed when using multimodal therapy because the microenvironmental conditions of the tumour can significantly affect the final outcome of tumour therapy. However, the precise mechanism of the interaction between resveratrol, cisplatin, and HT remains unclear and needs to be investigated further. The research was conducted on inbred Balb/c mice obtained from the Department of Animal Physiology, Faculty of Science, University of Zagreb. The mice were 2–3 months old, weighing 20–25 g. A total of 120 animals were divided into 8 groups of 15 animals. They were fed standard food for laboratory animals  with constant access to water. The conditions were standard and included a daily rhythm of 12 h of light and 12 h of darkness and a temperature of 24 °C with controlled air humidity. The study was approved by the Ethics Committee  and was conducted in accordance with the ethical principles valid in the Republic Croatia  ; Law on Ehrlich’s ascites tumour (EAT) is an undifferentiated carcinoma originating from mouse mammary cells. It is hyperdiploid, highly transmissible, fast-growing, completely malignant with extremely sensitive and heterogeneous cells, and lacks tumour-specific transplantation antigens (TSTA), which makes it a valuable model for research. EAT is very similar to human tumours that are most susceptible to chemotherapy since it is an undifferentiated, angiogenesis-dependent, fast-growing tumour and as such is suitable for studying the effect of chemical and natural compounds on tumour growth, angiogenesis, inflammation, and immunomodulation.ip) in Balb/c mice by serial transplantation of cells every 7 or 9 days in the form of ascites. After washing the abdominal cavity with 5 mL of saline and gently massaging the abdominal wall, an extensive incision was performed to open the peritoneal cavity of the mouse. Using a Pasteur pipette, peritoneal fluid with tumour cells was taken and diluted with physiological solution  to the final concentration of EAT cells (1 × 106 cells in 0.5 mL/mouse). The number of living cells was determined by staining with trypan blue in a Bürker–Türk chamber. In this study, we used a solid model of tumour growth caused by the subcutaneous (sc) application of EAT cells into the right femoral region of the mouse.EAT cells were maintained intraperitoneally (po) by intragastric cannula at a dose of 50 mg/kg.The resveratrol solution was prepared by dissolving resveratrol  in water with the addition of DMSO. The solution was administered per os  was prepared immediately before use in a dose of 5 mg/kg by diluting it in water for injections  and injected in a dose of 2.5 mg/kg 6 EAT cells subcutaneously in the right femur to induce Ehrlich solid tumour formation. Before and during the experiment, the weight of the animals was individually measured, and the dose of test components was adjusted according to the mice’s daily weight. All animals were divided into two large groups , each containing 4 groups, . After the injection of tumour cells, the animals were intragastrically (ig) treated with resveratrol at a dose of 50 mg/kg for 5 consecutive days, starting 2 days after the tumour injection. The treatment with resveratrol started after the injection of the tumour, considering that resveratrol is a common flavonoid present in many plants (more than 70 plant species) and wine. Cisplatin was injected three times  where T is the reaction time in minutes, V is the volume of the sample added to the wells, 1 mM is the concentration of urea standard, 50 is the reaction volume, and 103 is the conversion factor from mM to μM.Arginase activity = [A(sample) − A(empty well)]/[A(standard) − A(water)] × [. Tissue sections 3–5 μm thick were cut from paraffin blocks, mounted on glass slides, and stained with hematoxylin and eosin for pathological examination.TMFLEX Target Retrieval Solution, High pH, K8000). The following primary antibodies were used for immunohistochemical staining: (a) Hypoxia-inducible factor 1 alpha ; (b) Inducible Nitric Oxyde Synthase ; (c) Proliferating Cell Nuclear Antibody . Primary antibodies against HIF1α and PCNA were applied on tumour tissue for 2 h at room temperature, while iNOS was applied overnight at 4 °C. After washing with wash buffer, the secondary antibody, which is part of DAKO’s EnVisionTM FLEX/HRP detection kit (DAKO K8000), was applied for 45 min at room temperature. This kit used to visualize the reaction contains peroxidase, secondary antibodies, chromogen, and wash buffer. Immunohistochemical staining was performed in automated Dako Autostainer Link 48. The sections were contrast-stained with hematoxylin. Representative photomicrographs of immunohistochemical slides were taken.Several sections were cut and placed on positively charged slides for immunohistochemical staining. Antigen unmasking was conducted by heat treatment in the PTLink module using Dako 3 in 1 Antigen retrieval buffer pH 9.0  resulting in a 24-bit colour image. Areas of cancerous tissues that show a positive immunohistochemical reaction have brown pigmentation. The median intensity of the RGB signal was read on a completely taken image using the Histogram option in the program, and the median intensity was recorded for each individual colour of the RGB spectrum. The brown colour consists mostly of the Red but also a part of the Green as well as Blue contrasting colouring, so we used the mathematical equation published by Fu et al.  to solveAccording to their publication, when the α to β ratio is two, the images produce better contrast between brown DAB-stained tissues and blue hematoxylin-stained tissues. Therefore, we also used α = 0.5 and β = 0.25. Most DAB-stained pixels have low BN values, which are lower as the brown colour becomes darker.Small blood vessel density (MVD) was determined according to Weidner’s method . The numFor an estimation of the minimum sample size required for the experiment, STATA 13.1  was used. Using a non-parametric Kruskall–Wallis rank sum test with an alpha level of 0.05, a power level of 0.8, and an effect size of 0.22, the estimated minimum sample size was 15 mice per treatment group. Statistical analyses were performed using STATISTICA 13 software  and data were presented as mean ± standard error (SE) of the mean. All data were analysed through a Kruskal–Wallis ANOVA test and further analyses of the differences between the groups were made with multiple comparisons of mean ranks for all groups. p < 0.05. Treatment-dose specific survival curves were calculated by the Kaplan–Meier method, and comparison between the survival curves was made by log-rank test (α = 5%) as described in [The data were considered significant at ribed in ,7,17.