In contrast with other kinds of neoplasia, endocrine tumours are less frequently recorded, in part because many of these go undiagnosed. As endocrine tissues usually present several subtypes of cells, differentiation between normal and tumour tissue is difficult, as complex phenotypes and often paradoxical behaviour patterns are involved. This makes a reliable prognosis and successful management of endocrine tumours difficult to achieve. Hence, this situation has led to a relatively slow improvement in establishing the pathogenesis of endocrine tumours. The present Research Topic “Architects of Endocrine-Related Tumour Growth” focuses on the nature of the heterogeneous cellular features and pathways involved in the cellular growth programs that shape tumour development and considers their implications in the evolution and treatment of endocrine tumours. The involvement of inextricable signalling networks is addressed, in which the orchestration of many interdependent pathways and cellular fates may guide, preserve, or interfere with endocrine-related tumour growth.Zhu et al. This group develop a prediction model for testicular germ cell tumour metastasis and recurrence based on an mRNAlncRNA-miRNA regulatory network constructed from differentially expressed RNA profiles among tumoral and normal tissue, and integrated with RNA-Seq data and public clinical databases. They also describe the correlation between the dysregulation of mRNA with immunomodulation, cell migration and invasion, indicating a link between immune cell infiltration and testicular germ cell tumours. Finally, they identify GRK4, PCYT2, and RGSL1A as being potential specific biomarkers that could predict relapse-free survival, tumour immunity and chemotherapeutic resistance.A current approach for signalling network determination is presented by Guang et al. explores the ultrasonic features of preoperative clinical data combined with superb microvascular and contrast-enhanced ultrasound imaging of thyroid nodules and papillary thyroid cancer (PTC). These authors perform a multi-factor analysis to determine the predictive value of central lymph node metastasis from PTC. This provides a correlation between metastatic behaviour and ultrasound characteristics such as tumour size, multifocality, microcalcification particularities, vascularization, and contact with the adjacent capsule. In addition, the study establishes a criterion for a high risk of lymph node metastasis based on the grayscale ultrasound data, thereby contributing to the development of a clinical basis for the early diagnosis, treatment and prognosis of thyroid cancer.With the aim of improving early diagnosis of tumour growth, an original article by Martin et al. explore the role of nuclear localization of the insulin-like growth factor receptor (IGF1R) in the regulation of glioblastoma cellular behaviour. These authors modify a glioblastoma cell line to either allow or block IGF1R translocation to the nucleus. This study reveals that IGF1R nuclear localization increases cell motility, as well as the metabolic activity of glucose and lipids, without having any repercussions on proliferation. Furthermore, in vivo studies using subcutaneous tumour xenografts show that nuclear-localized IGF1R provoke metabolic shifts that result in increased proliferation and accelerated tumour growth. However, it also sensitised cells to pharmacological therapy with IGF1R inhibitors. These findings support the hypothesis that although IGF1R nuclear localization in glioblastoma cells may contribute to an aggressive phenotype, it also represents a potential therapeutic possibility for patients whose tumours exhibit this intracellular localization.To further the understanding of the intricate molecular dialogs involved in endocrine-related tumour growth, Sacca and Calvo review the impact of several factors and fatty acids secreted by the periprostatic adipose tissue (PPAT) on the prostate cancer (PCa) microenvironment. In this article, they describe how PPAT acts co-ordinately with PCa development by adjusting its metabolic performance according to the stage of tumour progression. These authors report that in advanced tumours, the metabolic secretome profile is enriched in energy balance and hormone response molecular signalling, with the catabolic metabolism of polyamine, protein, and nucleotide probably required for tumour growth. Thus, these metabolic reprogramming hallmarks of PPAT secretome may serve as indicators for the early diagnosis of high-risk tumours and provide positive therapeutic effects.Tumour development requires cellular physiology and metabolic reprogramming to deal with environmental modifications and new cellular demands. Moreover, the dynamical tumour ecosystem may adjust its needs and influence cellular neighbourhoods. In this regard, Ritch and Telleira extensively assess these events for epithelial ovarian cancer (EOC). In this review, they describe the sequence of microenvironments encountered by EOC cells during transcoelomic dissemination, and the special aspects of these scenarios that can influence cellular fates and tumour progression. These authors describe EOC cell fluctuations within the different cellular ecosystems, starting at the ovaries or the fallopian tubes, whose pro-inflammatory surroundings could favour precursor lesions. In addition, the detachment process is followed by the ability to survive in the peritoneal fluid, thereby evading cell death signalling and enabling suitable cellular communication. Metastatic EOC cells require adherence, and migrate as spheroids through the peritoneum, being able to reach distant tissue and invade as primary or secondary tumours. Related to this, the involvement of the microenvironment effects over EOC chemoresistance has been consistently reported. This extensive overview of the entire process allows for a better understanding of the role of cellular and non-cellular factors during EOC transcoelomic dissemination, promoting the creation of novel therapeutic strategies to treat this pathology.Tumour progression represents the coevolution and integration of phenotypic cellular changes within the microenvironment. Sabatino et al. summarise current findings in pituitary neuroendocrine tumours by examining the range of possible cellular fates involved throughout tumorigenesis. This article emphasises the relevance and importance of integrating emerging fields in pituitary research, such as cellular senescence, autophagy, mitochondrial function, metabolic reprogramming, stem cell niches and non-apoptotic cell death during tumour progression. These authors expose the need for innovative models that are able to reflect the complex interplay that occurs during tumour pathogenesis. This review also highlights the requirement for the incorporation and systematization of newly available data involving tumour origin, behaviour and plausible clinical results, in order to improve tumour classification.For tumour development to be achievable, cells must acquire new abilities beyond unregulated proliferation. Different cellular outcomes may emerge during tumour development that can either favour or constrain cell growth. Sousa et al. review existing knowledge about autophagy in adrenal tumours. As previously reported for different tumours, autophagy in this neoplasia presents a dual role towards pro-survival or pro-death, depending on the context, and its action is interconnected with other cellular processes. Both the induction and inhibition of autophagy have revealed possibilities for treatment strategies for adrenal neoplasms, with the impact of autophagy on tumour progression appearing to depend on the degree of tumour evolution and the type of therapy employed. Adrenal autophagy might be associated with steroid synthesis and extracellular vesicle biogenesis, suggesting potential targets for tumour diagnosis and management. These authors emphasise that further research is still required to clarify the clinical implication of autophagy modulation in adrenal tumours.In conclusion, the evidence presented here shows that the amount of data accumulated over recent decades highlights the complexity of endocrine-related tumorigenic mechanisms. Indeed, the pathogenesis of multiple endocrine neoplasia is still unclear, with their management mostly depending on histopathological criteria. Thus, a proper elucidation of the plethora of diverse genetic, epigenetic, and biochemical signalling pathways, together with a better understanding of the metabolically and physiological cellular outcomes implicated in tumourigenesis, would be particularly useful, as using histological characteristics alone restricts the accuracy of the diagnosis and prognosis. We have now reached a point where it is necessary to create broader modelling strategies that allow us to consider not only a single component, but the sophisticated and multifaceted network that influences tumour progression.MES, GD-T and ALD contributed to conception and design of the editorial. MES wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This work was funded by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET PIP 2020-2023 No. 11220200102210), Secretaría de Ciencia y Tecnología, Universidad Nacional de Córdoba (SECyT-UNC 2018-2022 Nº33620180100675CB), and Agencia Nacional de Promoción Científica y Tecnológica - Ministerio de Ciencia y Tecnología (FONCYT-PICT 0950-2020) to ALD.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


As biological catalysts, enzymes are vital in controlling numerous metabolic reactions. The regulation of enzymes in living cells and the amount present are indicators of the metabolic status of cell, whether in normal condition or disease. The small-molecule fluorescent probes are of interest because of their high sensitivity and selectivity, as well as their potential for automated detection. Fluorescent probes have been useful in targeting particular enzymes of interest such as proteases and caspases. However, it is difficult to develop an ideal fluorescent probe for versatile purposes. In the future, the design and synthesis of enzyme-targeting fluorescent probes will focus more on improving the selectivity, sensitivity, penetration ability and to couple the fluorescent probes with other available imaging molecules/technologies. Over the past decade, one can identify a trend towards research in drug discovery and chemical biology applications ,2. ThereA successful fluorescence probe should be noninvasive , imaging stable , selective  and sensitive (ability to exhibit strong emission change upon interaction with a target). It should also possess a low limit of detection (LOD) and a dynamic response range. If a probe is highly sensitive towards an analyte, it will have a low LOD. The wide response range is favorable when a mild-to-intense stage of an enzyme-catalyzed reaction or a similar metabolic process needs to be detected.Fluorescent probes targeting enzymes are small molecular compounds that can react specifically with specific enzymes and emit fluorescence. These compounds can accurately and rapidly bind or react with specific regions of targeted enzymes, so as to realize the monitoring function of fluorescent probes. Fluorescent probes can be applied to almost any type of cell ,13. Fluoin vivo specificity account for the weaknesses. The other available imaging methods are bioluminescence, Positron Emission Tomography (PET), Magnetic Resonance Imaging (MRI) and Single Photon Emission Tomography (SPECT).The identified strengths of fluorescence as an imaging method are high sensitivity, low cost, high throughput, ease of performing, large number of available probes, and ability to monitor two or more probes simultaneously. Low resolution, low depth penetration, and limited The fluorescent probes targeting enzymes could be broadly categorized in to two categories, active site based probes and active group based probes. Enzyme active site-based probes react with a particular class of enzymes based on the common conserved features in their catalytic domains. On the other hand, active group-based probes contain reactive groups which target multiple and diverse enzyme types, which could belong to different classes of enzymes . Therefoin vitro and in vivo.This class of enzymes catalyze the hydrolysis of specific covalent bonds such as ester bonds and amide bonds in particular substrates. Out of the hydrolase enzymes, serine hydrolase is one of the largest and most diverse eukaryotic enzyme families. Serine hydrolases are generally grouped into two large member families: serine proteases  and metabolic serine hydrolases. According to the substrate specificity of serine proteases, some high-selective fluorescent probes was developed for biosensing and bioimaging Probe 1 is the first non-peptide fluorescent probe targeting for elastase. It has excellent LOD: 68 ng/mL. However, its emission wavelength (505 nm) limits its application, and it is only applied in vitro. Near-infrared (NIR) fluorescence analysis has advantages in the detection of biological samples because of its strong penetration and low background signal ,27zebrafButyrylcholinesterase (BChE) is widely distributed in plasma, liver, muscle and brain tissues and is associated with lipid metabolism and various human diseases such as liver injury, diabetes, Alzheimer’s disease (AD) and liver metastasis. Therefore, using fluorescent probes to study and trace BChE is a good method. Yang and his colleagues developed a fluorescent probe with high sensitivity and specificity to evaluate BChE activity and inhibitor screening for high-throughput studies in drug discovery and clinical diagnosis d 28]. T. T28]. TProteases are highly diversified enzymes in living cells and they are involved in breaking down of peptide substrates. However, aberrations of proteases lead to critical diseases including cancers, inflammation and neuro-degeneration . In addiprobe 9 was developed [The aggregation of amyloid-β (Aβ) is a symptom and possible reason for Alzheimer’s disease. β-Secretase (BACE1) is the necessary protease in production of Aβ . Therefoeveloped   are calcium/zinc-dependent proteases and have 23 members. They are regarded as important extracellular matrix (ECM)-degradation enzymes. Because the concentration of ECM-degradation proteins in tumor and cancer microenvironments are always different from normal tissue, MMPs have a large potential as biomarkers for diagnosis of cancer and design of cancer imaging agents . For exaher pair  , was discovered as a new fluorescent probe for CYP34A [probe 15, was reported [Cytochrome P450s (CYPs) are a large enzyme family which includes hundreds of members and they make a vital contribution to the metabolism of toxin, drug and endogenous substrates. Meanwhile, different subtypes of CYPs have different substrate specificity and physiological function. Thus, a series of fluorescence probes for these subtypes of CYPs were developed . Among Cr CYP34A  . Importantly, it has an ultralow response time (4 min) and LOD (0.9 nM) and was used to detect NQO1 in the mitochondria of HepG2 cells (NQO1 (NAD(P)H: quinone Oxidoreductase 1) is an important oxidoreductase biomarker. It has several biological functions including the reduction of quinone compounds, maintenance of reduction stress, control of mRNA translation and stabilization of proteins and is related to many diseases, such as cardiovascular diseases, diabetes mellitus, metabolic syndrome, aging, Alzheimer’s disease and cancer . So, lotG2 cells b.As a useful detection tool, the fluorescent probe for enzymes can be applied in wide fields, such as inhibitor screening, imaging in cell and living body, biological research and diagnosis. In addition to these familiar applications, it can be used in fluorescence-guided surgery . In cancprobe 18 is aminopeptidase N (APN), a biomarker of cancer  that were observed to have aberrant overexpression in many cancers [50 = 196 nM). At 12 h after the probe was injected in the live tumor nude mice model, the tumor was clearly labeled and demonstrated a significant difference from surrounding normal tissues. Probe 20 can be activated while two cancer biomarker enzymes (cathepsin and caspase 3) all exist [The target of f cancer . The pro cancers  (Figure ll exist  (Figure Even though numerous probes have been developed to suit various applications, it is difficult to design and develop an ideal probe for all applications . Additio


Vaping or e-cigarette use has become dramatically more popular in the United States in recent years. e-Cigarette and vaping use–associated lung injury (EVALI) cases caused an increase in hospitalizations and deaths in 2019, and many instances were later linked to unregulated products. Previous literature has leveraged social media data for surveillance of health topics. Individuals are willing to share mental health experiences and other personal stories on social media platforms where they feel a sense of community, reduced stigma, and empowerment.This study aimed to compare vaping-related content on 2 popular social media platforms  to explore the context surrounding vaping during the 2019 EVALI outbreak and to support the feasibility of using data from both social platforms to develop in-depth and intelligent vaping detection models on social media.Data were extracted from both Twitter  and Reddit  from July 2019 to September 2019 at the peak of the EVALI crisis. High-throughput computational analyses  were conducted. In addition, in-depth manual content analyses were performed and compared with computational analyses of content on both platforms (577 tweets and 613 posts).P<.001, 95% CI 0.4305-0.4475) and the number of negative posts to be higher on Twitter . These results were consistent with the clinical content analyses results indicating that negative sentiment posts were higher on Twitter  than Reddit . Furthermore, topics prevalent on both platforms by keywords and based on manual post reviews included mentions of youth, marketing or regulation, marijuana, and interest in quitting.Vaping-related posts and unique users on Twitter and Reddit increased from July 2019 to September 2019, with the average post per user increasing from 1.68 to 1.81 on Twitter and 1.19 to 1.21 on Reddit. Computational analyses found the number of positive sentiment posts to be higher on Reddit (Post content and trending topics overlapped on both Twitter and Reddit during the EVALI period in 2019. However, crucial differences in user type and content keywords were also found, including more frequent mentions of health-related keywords on Twitter and more negative health outcomes from vaping mentioned on both Reddit and Twitter. Use of both computational and clinical content analyses is critical to not only identify signals of public health trends among vaping-related social media content but also to provide context for vaping risks and behaviors. By leveraging the strengths of both Twitter and Reddit as publicly available data sources, this research may provide technical and clinical insights to inform automatic detection of social media users who are vaping and may benefit from digital intervention and proactive outreach strategies on these platforms. In the United States, vaping has become dramatically more popular in recent years, with 1 in every 20 American adults using vaping devices and >2 million middle- and high-school students in the United States using e-cigarettes in 2021 ,4. HowevReddit is a similar pseudonymous social media platform used by the public to discuss personal experiences that may be stigmatizing -27, inclBoth Twitter and Reddit are popular social media platforms, but they differ in multiple ways that impact users’ posting behaviors and post content. Twitter, with >300 million monthly active users , only alLarge-scale evaluations using computer science (CS) strategies, including those using natural language processing and machine learning for text mining, have been conducted previously on vaping content from social media -42. For Studying the EVALI public health crisis specifically could aid in the identification of content and keywords related to both acute and long-term health outcomes associated with vaping shared on social media, as such signals of vaping risk may have been amplified during this period. By leveraging the strengths of both Twitter and Reddit as publicly available data sources as well as using an interdisciplinary approach to analyze complex social media content, technical and clinical insights may be garnered to inform the future development of an automatic detection model to connect with vaping users who may benefit from digital intervention on social media platforms. However, to date, there are few studies comparing insights from both Twitter and Reddit for substance misuse within the same time frame ,47, and vaping, vape, and 60 other specifying terms . After that, we removed the special terms from the tweet text, including mentions, hashtags, links, ticks, punctuations, numbers, and over spaces. Then, we applied the word lemmatization function to convert the words to their base forms.Sentiment analysis is a common computer technique to measure the subjectivity, opinions, attitudes, and emotions in texts . SentimeIn addition to the sentiment analysis described earlier, we used chi-square tests to compare differences between the frequency of keywords in Twitter and Reddit posts during each month across the following topics: (1) sentiment, (2) emotion-related keywords, (3) health-related keywords, (4) age-related keywords, (5) marketing-related keywords, (6) product-related keywords, (7) addiction-related keywords, and (8) quitting-related keywords.Term frequency–inverse document frequency (TF-IDF) is a statistical measurement that can represent the word relevant in a corpus . The TF-During the EVALI outbreak, July, August, and September 2019 were identified as months during and just before the dramatic increase in vaping-related discussions on Twitter based on both the content and sentiment analyses outlined earlier. As such, a random sample of 200 posts per month from the Twitter and Reddit data sets described earlier were extracted for in-depth human coding toward contextual content analysis. Specifically, members of our clinical team with experience in substance use research  used inductive and deductive methods to construct a codebook based on a review of sample tweets and informed by previous literature ,59. ThreTotal frequency of each theme mentioned on both Twitter and Reddit was compared across the months of July, August, and September 2019 (sum of 3 months) to demonstrate relative weight of each topic on the respective platforms.The Washington University Institutional Review Board (202101009) reviewed the methods of data extraction and analysis for this study. Given that the data are publicly available on social media, the study was determined to be nonhuman subjects research and exempt from review.This section presents the results from the high-throughput computational analyses. In total, we collected 286,703 tweets and 12,096 posts on Reddit. The sample size differences between Twitter and Reddit were related to the amount of information included in each Reddit post and in a tweet. The word limit for each tweet is 280 characters, whereas the word limit for each Reddit post is 40,000 characters. Thus, each Reddit post included much richer information than a tweet. To analyze the data set at the word level and further content analysis, the number of extracted Reddit posts was significantly smaller than the number of tweets. CS pattern analysis of sentiment found that overall posts with positive sentiment about vaping were more common than negative posts on Reddit , and negative sentiment was dominant on Twitter  during the EVALI period . ClinicaThe results of monthly sentiment trends indicated that the percentage of posts with positive sentiment was higher than that with negative sentiment in July both on Twitter and on Reddit. In August and September, the percentage of negative posts was higher than that of the positive ones on Twitter. Moreover, there was a significant decrease in the percentage of positive sentiment from July to September on Twitter, whereas positive posts were dominant on Reddit in August and September.The chi-square tests  than positive health outcomes . Additional topic mentioned within these negative health outcomes included EVALI/hospitalization, which was more prevalent on Twitter , whereas addiction or dependence on vaping products was mentioned more often on Reddit . Those mentioning positive health outcomes related to vaping were more common on Reddit, consistent with the keyword analysis described earlier; further, clinical coding found that vaping as a means of quitting combustible smoking was more often mentioned on Reddit than on Twitter .kids was the most used word on Twitter and Reddit after August 2019. Other frequently used words on Twitter included youth, young, child, and teenager. Reddit posts more often contained words such as parent, school, and family.The top 6 words related to age groups in July, August, and September 2019 are presented in kid, adult, child, young, old, youth, parent, school, age, student, family, teenager, minor, mother, husband, wife, adolescent, father, and aunt in July, August, and September 2019 separately. The chi-square test results  and alcohol were also prevalent. On Reddit, specific keywords about product components, such as juice, cartridge, and liquid, were slightly more common. The most common words on Reddit included cigarette, product, and juice, which varied across months. The most common words on Twitter included cigarette, tobacco, and product and stayed consistent across August 2019 to September 2019. On the basis of the TF-IDF scores as shown in cig, cigarette, tobacco, product, thc, and nicotine, whereas the most important words in the Reddit posts included nicotine, cigarette, juice, and weed.Vaping product-related keywords in our data set included cigarette, tobacco, product, thc, cig, nicotine, juice, juul, cartridge, liquid, cannabis, chemical, alcohol, ecigarette, weed, cbd, flavour, and ingredient based on the data sets in July, August, and September 2019. The chi-square test results . Mentions of marijuana were also >2 times as high on Reddit as on Twitter and often included queries to other Reddit users about the safety of specific vaping products and which symptoms, if any, should warrant concern or medical care. In contrast, Twitter included more mainstream media content surrounding vaping, specifically related to the rise in EVALI cases. We also observed that Twitter feeds contained attention-grabbing negative sentiment and higher use of negative emotional expressions, including In summary, we observed numerous and meaningful distinctions in the frequencies of content topics across both social media outlets. These differences may be owing to the way individuals socially network as well as their motive for discussion on each platform. For instance, information on Twitter is known as “the” social media platform for news coverage, and it is most often used by journalists and major news providers to broadcast news and update the public in real time as important events transpire . This maIt might also be that the differences we found were owing to the distinctions between the users themselves. For instance, the demographic user base of Twitter is predominantly White adults, who have a higher degree of education and are more likely to be identified as Democrat than the general public, with 10% of users creating 80% of the tweets . In contThe findings of this paper should be considered within the context of its limitations. First, we analyzed only text-based posts or messages on these platforms. Although this provided us with data-heavy information from each social media site, it did not include the multitude of multimedia content including photos, videos, and links that are available for further analysis. Second, owing to the character limits on Twitter and the unlimited length of Reddit posts, the differences between the number of words in each post could have impacted both the sentiment and keyword analyses in this study. Third, because of the timing of our data gathering, we did not garner information related to COVID-19 and its implications on those who vape and vaping policies, leaving us unable to discern more recent implications. Fourth, our original keyword list used to extract the vaping-related data sets from Twitter and Reddit may have contained more negative health-related keywords, and this could have impacted the results with regard to sentiment and health outcomes, causing a potential selection bias in our keyword list. As this study focused on the health issue regarding vaping-related topics on Twitter and Reddit during the outbreak period, the keyword list included multiple sentiment-related words. It will bring bias to our sentiment analysis results, but these words were the key to selecting the related posts and addressing our research questions. In addition, we applied the GetOldTweets and Pushshift APIs to extract the data based on the keyword list. As the extraction mechanism of these APIs is to find the posts with the same field as one of the keywords without further filtration to matched posts, the extracted data set might include the posts from bots instead of real users, which may introduce bias to our sentiment results. We plan to apply different methods to clean the posts generated by bots in our future studies. However, the use of this data set was in line with the larger aims of this study, which were to better understand the content and sentiment surrounding vaping on Twitter and Reddit to inform the development of potential identification and outreach methods on social media to those at risk of negative health outcomes to improve public health. The fifth limitation was that we applied an existing tool VADER to analyze the sentiment of the posts, and thus, it could bias to our sentiment analysis results, which are common issues for any sentiment analysis tool owing to the complex dynamics of human expressions, emotions, and contexts. In the future, we will also consider creating a sentiment analysis model optimization with social media posts to overcome the current disadvantages of not effectively identifying sarcastic sentences.Overall, the results of this study revealed the strengths of both Twitter and Reddit as publicly available social media data sources as a public health crisis transpired and evolved. Health practitioners working with those who vape or who have interest in quitting vaping should be aware of the information and possible misinformation related to vaping and work to assess whether social media engagement on various platforms could impact continued use or be a barrier to cessation. The results shared in this manuscript could also inform social media companies and public health officials by alerting them to the marketing of vaping products on these sites and encouraging protections for communities such as those on Reddit aimed to support vaping cessation. In addition, to improve public health reach, future research could explore automatic detection mechanisms that leverage each platform’s content and type of networking identified here, especially to study the potential for identifying users that are vaping and may want information and support to quit. This could help lead to efficient and timely social media informed proactive outreach strategies to distribute health education about vaping, including strategies for vaping cessation.


The activity-based probes with a benzyl guanidine side chain showed the highest inhibitory effects in the panel. Surprisingly, some of the high-affinity probes presented a reversible inhibitory mechanism. On the other hand, probes with different side chains exhibited the expected irreversible mechanism. For the first time, we demonstrate that not only irreversible probes but also reversible probes can tightly label recombinant proteases and proteases released from human mast cells. Even under denaturing SDS-PAGE conditions, reversible slow-tight-binding probes can label proteases due to the formation of high-affinity complexes and slow dissociation rates. This unexpected finding will transform the view on the required irreversible nature of activity-based probes. The diversity of this library of activity-based probes combined with a detailed enzyme kinetic characterization will advance their applications in proteomic studies and drug discovery.Activity-based probes (ABP) are molecules that bind covalently to the active form of an enzyme family, making them an attractive tool for target and biomarker identification and drug discovery. The present study describes the synthesis and biochemical characterization of novel activity-based probes targeting trypsin-like serine proteases. We developed an extensive library of activity-based probes with “clickable” affinity tags and a diaryl phosphonate warhead. A wide diversity was achieved by including natural amino acid analogs as well as basic polar residues as side chains. A detailed enzymatic characterization was performed in a panel of trypsin-like serine proteases. Their inhibitory potencies and kinetic profile were examined, and their IC In recent years, many efforts have been carried out to study the expression and function of proteins in biological organisms. This provided new insights into the pathophysiology of different diseases and disorders. Activity-based protein profiling (ABPP) is a proteomics technique that uses activity-based probes (ABPs) to visualize and characterize enzyme activity within a complex proteome. These chemical probes are designed to react covalently with the active form of a target enzyme and allow their detection or isolation. Thus, ABPs give information about the activity level of an enzyme rather than its expression level . This fein vitro samples, and visualization in animal models in vivo. However, the uptake of the ABPs by living organisms can be problematic due to their bulkiness. Alternatively, conjugation with the reporter tag can be done in situ by click chemistry. The probe precursors are added to living cells or tissues and then undergo the reaction with a fluorophore or affinity label to yield the ABP. Subsequently, the probes can be visualized or identified by different techniques  is a well-studied reactive group. The phosphorus can react with the serine hydroxyl on the active site to form a covalent bond. Thus, it has the required characteristics to design selective and irreversible ABPs. Therefore, it was previously used to develop irreversible inhibitors and ABPs targeting serine proteases .Within the human degradome, serine proteases can be divided into three main subfamilies known as trypsin-, chymotrypsin-, and elastase-like, depending on their substrate specificity. The specificity of a protease is determined by the preferred amino acid sequence that will fit into the specificity pocket of the active site . In thisa-aminoalkyl diphenyl phosphonate ABPs targeting trypsin-like proteases were described by p-guanidino-Phe-DPP) (The first synthesized Phe-DPP) . This waPhe-DPP)   .Driven by the need to identify new biomarkers in pathologies where serine proteases are upregulated and inspired by previously reported irreversible serine protease inhibitors and ABPs, this study reports the synthesis and characterization of a library of ABPs for trypsin-like serine proteases. The idea was to move away from the ABPs mimicking the natural basic amino acids lysine and arginine and to design several different synthetic analogs to improve potency and selectivity. The ABPs described are based on a DPP warhead with a biotin or desthiobiotin tag attached by click chemistry. We performed an extensive biochemical characterization and demonstrated the ability of these ABPs to differently label trypsin-like serine proteases, both recombinant enzymes and also from degranulated mast cells. Thus, these ABPs have the potential to be used in more complex pathological samples to identify active trypsin-like proteases participating in physiological processes as well as dysregulated enzymes potentially promoting disease. Therefore, these could represent a valuable tool with translational prospects for target identification and biomarker discovery.1H and 13C NMR and mass spectrometry. 1H and 13C NMR spectra were recorded on a 400 MHz Bruker Avance III Nanobay spectrometer with Ultrashield working at 400 and 100 MHz, respectively, and analyzed by use of MestReNova or TopSpin analytical chemistry software. Chemical shifts (δ) are in parts per million (ppm), and coupling constants (J) are in hertz (Hz). The signal splitting patterns were described as s = singlet, d = doublet, t = triplet, q = quartet, p = pentuplet, dd = doublet of doublet, dt = doublet of triplet, td = triplet of doublet, tt = triplet of triplet, ddd = doublet of doublet of doublet, br = broad, and m = multiplet. The LC−MS analysis was performed on a Waters UPLC−MS system equipped with a TUV and QDa detector; the column used is an Acquity UPLC BEH C18 , and as eluent, a mixture of .1% FA in H2O, .1% FA in CH3CN, H2O, and MeCN. The wavelengths for UV detection were 254 and 214 nm.Reagents were obtained from commercial sources and were used without further purification. Characterization of all compounds was done with When necessary, flash chromatography separations were carried out using a Biotage Isolera One purification system equipped with an internal variable dual-wavelength diode array detector (200–400 nm). Silica gel columns were used for normal phase purifications, and reverse-phase purifications were done using C18 cartridges, both from Büchi or Biotage. Dry sample loading was done by self-packing sample cartridges using Celite 545. Gradients used varied for each purification.3CN to a concentration of .1 mM and then diluted x10 in a solution 50:50 of CH3CN:H2O.HRMS involved the following: the dry samples of final compounds were dissolved in CHThe synthetic procedures and analytical data for all compounds reported in this manuscript can be found in the 50 value is defined as the concentration of inhibitor required to reduce the enzyme activity to 50% after a 15 min preincubation with the enzyme at 37°C before the addition of the substrate. The IC50 values were determined using a spectrophotometric assay. All the experiments were conducted in duplicate. The readout consisted of evaluating the protease-mediated release of the chromophore para-nitroaniline (pNA) or fluorophore 7-amino-4-methyl coumarin (AMC) moieties from the respective substrates. Enzymatic activity was measured for 30 min at 37°C. All compounds were initially screened at three concentrations  to estimate the range of the IC50 value. Those which were able to reduce protease activity by at least 50% at a concentration of 10 μM were submitted to an exact IC50 determination. The final IC50 values of the most potent inhibitors were the average of two independent experimental results. A third independent experiment was performed when the standard deviation of the two independent experiments was higher than three times the IC50 value. IC50 values were obtained by fitting the data with the four-parameter logistics equation using GraphPad Prism 9.The ICm.The conditions for each protease are described as follows, the concentration of substrate used was at the corresponding Km = 22.5 μM) were used in Tris buffer  at pH 8.0 (25°C). Tryptase  and fluorogenic substrate Boc-Gln-Ala-Arg-AMC (Km = 250 μM) were used in Tris buffer  at pH 8.0 (25°C). Thrombin  and fluorogenic substrate Boc-Val-Pro-Arg-AMC (Km = 15 μM) were used in TRIS buffer  at pH 8.3 (25°C). uPA  and chromogenic substrate pyro-Glu-Gly-Arg-pNA (Km = 80 μM) were used in HEPES buffer (50 mM HEPES) at pH 8.1 (25°C). Cathepsin G  and fluorogenic substrate Suc-Ala-Ala-Pro-Phe-AMC (Km = 130 μM) were used in Tris buffer  at pH 8.0 (25°C). TLCK-treated pancreatic bovine chymotrypsin (Sigma-Aldrich) and fluorescent substrate Suc-Ala-Ala-Pro-Phe-AMC (Km = 58 μM) were used in Tris buffer  at pH 8.3 (25°C). Neutrophil Elastase  and fluorogenic substrate Suc-Ala-Ala-Pro-Val-AMC (Km = 500 μM) were used in Tris buffer  at pH 8.0 (25°C).Trypsin-3  and fluorogenic substrate tosyl-Gly-Pro-Arg-AMC  varies with the inhibitor concentration (i), as described in the following equation:0 is the activity in the absence of an inhibitor, k is the second-order rate constant of inactivation, and t is the time. The inactivation rate constant was determined from the time course of inhibition.Because the compounds described in this paper were previously described as irreversible inhibitors, the IC0>>[E]0) and with less than 10% conversion of the substrate during the entire time course. In these conditions, dA/dt decreases exponentially with time. The progress curves were fitted with the integrated rate equation with GraFit7 to yield a value for kobs, a pseudo-first-order rate constant.t is the absorbance at time t, A0 is the absorbance at time zero, vi is the initial rate and vs is the velocity at the steady-state.Kinetic assays were performed in the following manner. The inhibitor was mixed with the substrate, and the buffer solution with the enzyme was added at the time zero. The progress curves show the release of pNA or AMC as a function of time. Initially, no inhibitor is bound to the enzyme, and the tangent to the progress curve (dA/dt) is proportional to the free enzyme concentration. The free enzyme concentration decreases over time due to inhibitor binding kinetics, as described above. Progress curves were recorded in pseudo-first-order conditions ([I]app) was calculated from the slope of the linear part of the plot of kobs vs. the inhibitor concentration ([I]0). In the case of competition between the inhibitor and the substrate, kapp is smaller than the “real” second-order rate constant k discussed above because a certain fraction of the enzyme is present as an enzyme-substrate complex. kapp depends on the substrate concentration used in the experiment, as described by The apparent second-order rate constant  were labeled with 1 µM ABP in reaction buffer in a final volume of 40 µL for 30 min at 37°C. After the addition of 4X Laemmli buffer  supplemented with Bond-Breaker Tris (2-CarboxyEthyl)Phosphine (TCEP) Solution , the samples were heated at 95°C for 5 min and loaded into 4%–20% Mini-Protean TGX precast gels . After electrophoresis, the proteins were blotted onto nitrocellulose membranes using the Trans-Blot Transfer Turbo System (Bio-Rad). Membranes were incubated with NeutrAvidin-HRP , and bands were visualized with ECL Select Western Blot Detection Reagent  by chemiluminescence . The molecular weight of each band was determined with the Image Lab Software v5 (Bio-Rad).+ precursors cells were isolated from PBMCs  and grown under serum-free conditions using StemSpanTM medium (STEMCELL Technologies) supplemented with recombinant human IL-6 , human IL-3  and 3% supernatant of CHO transfectants secreting murine SCF  for 1 week. Cells were next grown in IMDM Glutamax I, sodium pyruvate, 2-mercaptoethanol, .5% BSA, Insulin-transferrin selenium , penicillin (100 U/ml), streptomycin (100 μg/ml) and 3% supernatant of CHO transfectants secreting murine SCF for 8 weeks then tested phenotypically  and functionally (β-hexosaminidase release in response to FcεRI crosslinking) before use for experiments. Only primary cell lines showing more than 95% CD117+/FcεRI+ cells were used for experiments.Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats (Etablissement Français du Sang). CD345 mast cells were distributed in 50 µl Tyrode’s Buffer and adapted to 37°C for 30 min. Mast cells were next stimulated with DNP-BSA (200 ng/ml) after sensitization with 1 μg/ml of IgE anti DNP (Sigma-Aldrich) overnight. Supernatants were harvested and stored at −80°C. (β-hexosaminidase was assayed by measuring the release of p-nitrophenol from the substrate p-nitrophenyl N-acetyl-β-D-glucosaminide).1 × 10The concentration of protein in supernatants was determined using the Pierce Protein BCA Assay Kit, according to instructions (Thermo Scientific).Tryptase-like activity in mast cell supernatants was measured with .075 mM Boc-Gln-Ala-Arg-AMC hydrochloride as substrate in 50 mM Tris 120 mM NaCl + .1 mg/ml BSA + 50 μg/ml heparin pH = 8 at 25°C (Sigma-Aldrich). Substrate cleavage was calculated by the change in fluorescence , measured over 30 min at 37 °C on a FLUOstar Omega microplate reader (BMG Labtech). Sample values were interpolated into a linear regression generated with a standard curve of AMC (Sigma-Aldrich). Data were expressed as U of tryptase-like activity per L (U defined as 1 μmol/min).g for 5 min, and the solubilized sample was loaded into 4%–20% Mini-Protean TGX precast gels . After electrophoresis, the proteins were blotted onto nitrocellulose membranes using the Trans-Blot Transfer Turbo System (Bio-Rad). Membranes were incubated with NeutrAvidin-HRP, and bands were visualized with ECL Select Western Blot Detection Reagent  by chemiluminescence . The molecular weight of each band was determined with the Image Lab Software v5 (Bio-Rad) with Dual Color Protein Precision Plus Standard  as reference. The bands corresponding to active proteases were identified by their sensitivity to the pre-incubation with protease inhibitors.Mast cell supernatants  were labeled with 1 µM individual ABPs  in 50 mM Tris, 120 mM NaCl, .1 mg/ml BSA, 50 μg/ml heparin, pH 8 at 25°C (Sigma-Aldrich) in a final volume of 500 µL. As a control, supernatants were pre-incubated for 60 min at 37°C under stirring  in 4 mM AEBSF (Sigma), and 1X complete EDTA-free inhibitor cocktail (Roche) before incubation with a mixture containing the 3 ABPs mentioned above. The pre-incubation with protease inhibitors allows the identification of active proteases since enzyme inhibition abrogates their interaction with the ABP. The reaction product was then precipitated in 15% trichloroacetic acid at 4°C overnight. The pellet was washed twice in cold acetone (−20°C) and solubilized in 45 μL of Laemmli buffer (Bio-Rad) supplemented with Bond-Breaker TCEP Solution (Thermo Scientific). Samples were then heated at 95°C for 5 min, clarified by centrifugation at 12,000 g for 5 min, and 5 µg of the solubilized sample was loaded into 4%–20% Mini-Protean TGX precast gels . After electrophoresis, the proteins were blotted onto a nitrocellulose membrane using the Trans-Blot Transfer Turbo System (Bio-Rad). The membrane was incubated with primary anti-mast cell tryptase antibody  at 1:1,000 dilution overnight at 4°C. Subsequently, the membrane was labeled with the secondary HRP-conjugated antibody  for 1 h at room temperature. Bands were visualized with ECL Select Western Blot Detection Reagent  by chemiluminescence . The molecular weight of each band was determined with the Image Lab Software v5 (Bio-Rad).Protein in the mast cell supernatants  was precipitated in 15% trichloroacetic acid at 4°C overnight. The pellet was washed twice in cold acetone (−20°C) and solubilized in 75 μL of Laemmli buffer protein supplemented with TCEP (Bio-Rad). Samples were then heated at 95°C for 5 min, clarified by centrifugation at 12,000 With the aim to achieve a range of chemical tools able to identify different trypsin-like serine proteases in complex proteomes, the first goal of this study was to obtain an extensive and diverse library of ABPs. Trypsin-like serine proteases interact favorably with basic and polar residues in the S1 pocket . PreviouAlthough diaryl phosphonate ABPs targeting serine proteases have been reported in the literature, this study aimed to develop a common and accessible synthetic strategy for a wide-ranging library. Therefore, instead of adding the reporter tag by commonly used peptidic coupling , the pegpara-position were prepared from phosphorus trihalides  oxidation to achieve the desired aldehyde  . HoweverThe synthesis of 4-aminobutanal (3) required a double Boc protection of the corresponding alcohol before a Swern oxidation . In contThe Boc protecting groups of the diarylphosphonates were removed using acid treatment with hydrogen chloride in dioxane or trifluoroacetic acid in dichloromethane (DCM).2 as a catalyst in DCM and tetrahydrofuran (THF), respectively. However, for the synthesis of benzylamine 4-acetamidophendiphenyl phosphonate (17), low yields were reported (<5%) (3(OEt)2 in acetonitrile (CH3CN). For the benzylamine alkyne probe with unsubstituted diphenyl phosphonate (16), the combination of Cu(OTf)2 in DCM worked in a similar yield range as reported (R2 = H). However, for substituted diaryl phosphonates, either Bi(OTf)3 in THF (15), BF3(OEt)2 in CH3CN , or Cu(OTf)2 in THF (20–21) were more successful. Interestingly, Bi(OTf)3 has recently been reported as one of the best-performing catalysts for the Birum-Oleksyszyn reaction in an extensive optimization of the synthesis of inhibitor UAMC-00050 ed (<5%) . For thereported . These cMC-00050 .N, N′-bis(tert-butoxycarbonyl)-1-guanylpyrazole in a mixture of 1:1 of CH3CN/DCM, reducing the previously reported reaction time from 3 days to 16 h , which shares characteristics with chymotrypsin and trypsin-like proteases, and finally, chymotrypsin (ChTryp) and neutrophil elastase (NE).50 values of the alkyne probes were used as a first screening cut-off. All the alkyne probes were initially screened at three concentrations . The probes with an IC50 higher than 10 µM were not analyzed further, whereas the rest were submitted to an exact IC50 determination. The results of this first screening are presented in 1) with the biotin and desthiobiotin probes, inhibitory potencies were similar  were derived from the kinetic progress curves. The results of the kinetic assays resemble those expected for a two-step reversible slow-binding or irreversible mechanism. Thus, the rate constant kapp accounts for the initial reversible enzyme-inhibitor complex (E-I) affinity and the formation rate of the reversible high-affinity complex (E*I) or the irreversible enzyme-inhibitor complex (EI). As kapp describes the affinity and the reactivity of the inhibitors it is the best metric to discriminate between the different inhibitors. The kapp values are shown in The apparent second-order rate constants  and CatG (IC50 = .06 ± .006 µM). On the other hand, for thrombin, the benzyl guanidine probes were only in the micromolar range.Since the alkyne probes and their corresponding biotin/desthiobiotin analogs behave similarly, we will limit the discussion on the relationship between structure and activity to the alkyne probes. The IC0 values  of the bDespite being the most potent compounds, the progress curves of the benzyl guanidines show an unexpected reversible inhibition of all trypsin-like proteases tested, except for uPA, where the inhibition is irreversible . The irrapp values of the benzyl guanidines derived from the progress curves correlate with the trends observed for the IC50 values. The benzyl guanidine probes show strong inhibition of trypsin-3, β-tryptase, and uPA, weaker for CatG and much weaker for thrombin. For trypsin-3, these probes are at least 10-fold more potent than any of the other probes. The same occurs for β-tryptase, where the kapp of the bis(4-chlorophenyl)phosphonate probe 36b (kapp = 61 × 103 ± 6860 M−1 s−1) is 10-fold higher than the second best probes, arginine 33b, and phenyl guanidine 39b. Noteworthy, for uPA, the probe 36b with kapp = 32 × 104 ± 29 × 103 M−1 s−1 is at least 100-fold more potent than the other probes with different side chains. For all reversible benzyl guanidine probes, the values of KI* are at least 10-fold lower than the corresponding K1 for trypsin-3 and β-tryptase phosphonate alkyne probe (36b) and both substituted diphenyl phosphonate desthiobiotin probes .The ktryptase . This is50 values are in the same range as for the benzyl guanidine probes (34b–36b). For instance, the IC50 for β-tryptase and CatG for the bis(4-chlorophenyl)phosphonate alkyne probe (39b) are .07 ± .004 and .09 ± .022 µM, respectively, which is in the same range as for the best-performing benzyl guanidine probe 35b. For trypsin-3 and uPA, the phenyl guanidine probes lost about 100-fold potency compared to the benzyl guanidine analogs. At the concentrations tested, there was no inhibition of thrombin.The phenyl guanidine probes (37b–39b) also demonstrated strong sub-micromolar inhibitory potencies . For β-tR2) have a significant influence on the kapp, with the bis(4-chlorophenyl)phosphonate probe 39b as the best-performing for all the proteases. This probe has kapp values around 20 × 102 M−1 s−1 for trypsin-3, β-tryptase, uPA, and CatG.However, in contrast to the benzyl guanidine probes that were only irreversible for uPA, the compounds with phenyl guanidine as a side chain demonstrate irreversible inhibition for all proteases evaluated, excluding the bis(4-chlorophenyl)phosphonate probes , which, surprisingly, act reversibly on CatG . For thi50 = .03 ± .001 µM. It is an equipotent inhibitor for trypsin-3 and has weak inhibition for β-tryptase. There is no inhibition for uPA and CatG. This is also reflected in the kapp values obtained from the progress curves with irreversible inhibition of thrombin, trypsin-3, and β-tryptase  had nanomolar inhibition for trypsin-3 but were less potent for β-tryptase, thrombin, uPA, and CatG. The ornithine probe (14b) was inactive in all the proteases tested.Among the probes mimicking the natural amino acids, the arginine side chain proved to be the more interesting for most proteases. In particular, the bis(4-chlorophenyl)phosphonate probes 33b showed high potency and irreversibility for trypsin-3, β-tryptase, and uPA. A similar kIn previous studies, the Pro-Lys mimicking probe was reported to be promising for trypsin-3 with higher inhibitory potency than the Lys probe, but it was less active on β-tryptase . In our 50 higher than 1 µM. The only exceptions are the submicromolar IC50 values of 17b and 18b for CatG and chymotrypsin, reflecting the preference of these proteases for aromatic amino acids. In addition, the optimal length of the selectivity enhancing group (R1) for this panel of trypsin-like proteases lies between 6 and 8 atoms from the α-carbon. Generally, compounds with shorter or longer R1 groups significantly have reduced inhibitory potency. For instance, the benzyl methyl guanidine alkyne probe (40b) only showed affinity for uPA with an IC50 = 6.16 ± .48 µM.In general, our results demonstrate that compounds with a terminal guanidine showed significantly higher potencies than their amine analogs. For example, the less basic aniline probes 16b–18b and 19b–21b were inactive or presented ICThe compounds were tested in chymotrypsin and neutrophil elastase to test selectivity over other protease families. All compounds were not potent for neutrophil elastase, including compound 13, designed to target elastase-like proteases. Likewise, most compounds were not potent or only slightly potent for chymotrypsin, with the exception of the above mentioned 17b and 18b.app values is the benzyl guanidine 34b, with a 5-fold selectivity for uPA, 55-fold over β-tryptase and over 2000-fold for thrombin. Noteworthy, this probe shows a reversible slow-binding inhibitory mechanism. In case that an irreversible probe would be needed, the arginine 32b combines high potency with at least 30-fold selectivity over the other tested proteases. For β-tryptase, several potent reversible probes, such as 36b were obtained, but these probes are not selective for trypsin-3 and uPA. In contrast, probes 33b and 39b are also potent for β-tryptase and show an irreversible mechanism of inhibition. The preferred probe for thrombin is the 4-piperidine-1-carboximidamide (41b), with potent irreversible inhibition and excellent selectivity over β-tryptase, uPA, and CatG. All the probes described in this study are irreversible with uPA. Furthermore, this enzyme has a clear preference for the benzyl guanidine side chain. Last, CatG does not have a clear preference between benzyl guanidine and phenyl guanidine probes. However, benzyl guanidine probes have a reversible mechanism, whereas phenyl guanidine probes are generally irreversible.Concludingly, under these assay conditions, the most potent alkyne probe for trypsin-3 based on the kI*. Assuming that there is an excess of inhibitor over enzyme, the free inhibitor concentration must be very low, lower than the KI*, in order for the complex to dissociate. Therefore, the high affinity complex E*I survives during the whole process of labeling, gel electrophoresis, and visualization.To further characterize the newly synthesized compounds and their potential to be used as ABPs, nine desthiobiotin probes were used for the labeling and detection of recombinant proteases. The arginine (45b–46b) and the proline-lysine (58b) desthiobiotin probes were used as reference compounds. Whereas the benzyl guanidine  and phenyl guanidine probes (55b–57b) were used to determine whether the mechanism of inhibition (reversible or irreversible) is an obstacle to visualize the proteases. Samples of human recombinant proteases  were incubated with the different probes before electrophoresis and detection with NeutrAvidin-HRP on a Western blot. All the probes tested can label recombinant trypsin-3. However, the phenyl guanidine side chain is the least promising for this enzyme (55b–57b). For β-tryptase, the probes with natural amino acids as a side chain  are not good ABP candidates. Both benzyl guanidine and phenyl guanidine show similar labeling intensity. Nevertheless, the probes with bis(4-chlorophenyl)phosphonate  showed a slightly better resolution for the β-tryptase bands. Last, CatG could be labeled by most of the probes, except for the arginine (45b) and phenylguanidine (55b) unsubstituted diphenyl phosphonate probes.In summary, the benzyl guanidine probes are compounds with a high affinity for trypsin-like serine proteases. Although they showed a reversible mechanism of inhibition for trypsin-3, β-tryptase, and CatG, surprisingly, these can label the recombinant proteases in a Western blot assay.The last step to characterize the newly synthesized compounds as potential ABPs was to perform an experiment on biological samples and exemplify the ability of these ABPs to label proteases within a complex proteome. Human mast cells from two donors were stimulated to degranulate with IgE/DNP. Mast cells are immune cells that, upon degranulation, release mediators and play pivotal roles in allergic and inflammatory diseases . Mast ceABPP is a powerful proteomic tool which enables researchers to detect and visualize active enzymes within a complex proteome. ABPP uses chemical probes designed to react covalently with the target enzyme. This tool is attractive in target and biomarker identification and drug discovery programs.This study focuses on the serine protease family, specifically trypsin-like serine proteases. Most ABPs previously reported targeting this family used a diphenyl phosphonate as a warhead. However, there is a lack of chemical diversity as most of the reported probes contain natural amino acid mimetics as selectivity enhancing moieties. This study aimed to develop an extensive library of ABPs targeting trypsin-like serine proteases with a wide-ranging chemical diversity.An efficient synthetic route has been implemented for probes bearing a diphenyl phosphonate warhead. A combination of different side chains targeting trypsin-like proteases and the modification of the electrophilicity of the diphenyl phosphonate warhead achieved the desired extensive library of probes. First, the alkyne probes were synthesized, and then the most promising ones were clicked to biotin and desthiobiotin as reporter tags. Furthermore, this synthetic route allows a straightforward approach to couple diverse reporter tags. Thus, alternative tags will enable other applications, such as visualization by fluorescence or PET.50 values were determined as a first measurement. However, since the probes were designed to be irreversible inhibitors, the IC50 value is not a good parameter to describe their potency. Therefore, kinetic progress curves were measured to determine the mechanism and the kinetic constants of inhibition.To determine the inhibitory potency of the synthesized probes, their ICFor trypsin-3, β-tryptase, and uPA, the benzyl guanidine probes presented the highest inhibitory activities. Surprisingly, only for uPA, the interaction was irreversible, whereas a reversible slow-binding mechanism with trypsin-3, β-tryptase, thrombin, and CatG was observed. The phenyl guanidines, on the other hand, are good irreversible probes for trypsin-3, β-tryptase, and CatG. The preferred probe for thrombin is the 4-piperidine-1-carboximidamide, with potent irreversible inhibition and excellent selectivity over β-tryptase, uPA, and CatG.Last, we demonstrated that our probes can label recombinant proteases and tryptase released from mast cell degranulation. Surprisingly, not only the irreversible ABPs but also the probes that presented a reversible slow-binding mechanism can label proteases under denaturing conditions.Concludingly, these new probes for trypsin-like proteases offer significant advantages in terms of potency over the traditional probes mimicking the natural amino acids arginine and lysine. Detailed enzyme kinetic studies learn that, surprisingly, not all diaryl phosphonates show irreversible inhibition and that the inhibitory mechanism of a specific probe can differ from one protease to another. Even more surprising is the observation that irreversible binding is not needed to maintain protease labeling under denaturing conditions but that also reversible slow-tight binding is sufficient.


Paulownia leaves, which affected the ruminal parameters and methane concentration. However, there are no scientific reports on the Paulownia leaves extract (PLE) containing phenolic compounds for their mode of action in the rumen. Phenolics constituted the main group of bioactive compounds in PLE (84.4 mg/g dry matter). PLE lowered the concentration of ammonia, modulated the VFA profile in the ruminal fluid, and decreased methane production. The PLE caused a significant reduction of in vitro dry matter degradability, reduced the number of methanogens and protozoa, and affected selected bacteria populations. PLE had a promising effect on the fatty acid profile in the ruminal fluid. Paulownia as a new dietary component or its extract as a feed additive may be used to mitigate ruminal methanogenesis, resulting in environmental protection and reducing ruminal biohydrogenation, improving milk and meat quality.Paulownia is a fast-growing tree that produces a huge mass of leaves as waste that can be used as a feed source for ruminants. The previous study showed that phenolic compounds were the most active biological substances in  Growing consumer awareness  and the Paulownia tree [Paulownia dried leaves or silage positively affected the rumen fermentation parameters by changing acetate to a propionate ratio without any adverse effect on nutrient digestion, which could be exploited in the nutrition of ruminants [Paulownia leaves. These compounds can influence the rumen fermentation processes, including methanogenesis. The study carried out by Puchalska et al. [Paulownia leaves containing phenolic compounds were perhaps responsible for reducing the methanogenesis process by directly inhibiting the population of methanogens. In the study with Paulownia leaves or silage, the confounding effects of nutrients may exist on ruminal fermentation and methanogenesis. Using a Paulownia leaves extract (PLE) containing a high concentration of plant bioactive compounds, including phenolics, may avoid the influence of nutrients on microbial fermentation. However, there are no scientific reports on the mode of action of PLE in the rumen. Thus, we hypothesized that secondary metabolites present in high concentrations in PLE could alter the rumen microorganism populations, thus, modulating ruminal fermentation and mitigating methane emission. The present study aimed to explore the influence of biologically active substances contained in the PLE on in vitro rumen fermentation, microbial populations, and the fatty acid profile in buffered rumen fluid.Different nutritional strategies are implemented in livestock production to mitigate methane emissions. Recently, many biologically active substances and tree leaves have been used as a source of nutrients and plant bioactive compounds to decrease methane production and improve ruminal fermentation ,8,9. An nia tree . Paulownuminants ,9. It haa et al.  showed t30H48O3, C30H48O4, and C30H48O5, were present in the PLE extract.The UHPLC-DAD-ESI-MS results of PLE are shown in p = 0.05) was observed with increasing doses of PLE, with the highest pH at 100 mg/kg of PLE (p < 0.001), with increasing concentrations of PLE with the lowest ammonia concentration at the highest doses. PLE inclusion also affected the total VFA concentration (p < 0.001). The inclusion of PLE linearly, quadratically, and cubically (p < 0.001) increased the molar proportion of propionic, isobutyric, and isovaleric acids. However, the molar proportion of acetic acid decreased linearly, quadratically, and cubically (p < 0.001). The acetate to propionate ratio decreased linearly, quadratically, and cubically (p < 0.001). Molar proportions of butyric and valeric acids were not affected by PLE. IVDMD was affected by the PLE in a linear, quadratic, and cubic manner (p < 0.001). Logarithmic contrasts were significant for all parameters except CH4 in mmol/g DM, CH4 in mmol/L total gas, and Isotrichidae in 103/mL . The inclusion of PLE resulted in a linear, quadratic, and cubic decrease of Ophyroscolecidae and total protozoa (p < 0.001), but Isotrichidae was not affected (p > 0.10). Due to the PLE inclusion, methanogens and total bacteria populations decreased linearly, quadratically, and cubically (p < 0.001).With increasing PLE doses, the total gas and methane production  decreased linearly, quadratically, and cubically  with increasing PLE concentrations  with increasing PLE concentrations. Logarithmic contrasts were significant for all items, except Anaerovibrio lipolytica, Megasphaera elsdenii, and Streptococcus bovis , with the exceptions of C14:0, C16:0, C18:1 cis-9, the n-6/n-3 FA ratio, the LNA/LA ratio, the sum of cis C18:1, DI, and DI RA/(VA+RA). Significant linear decreases were observed only for C18:3n-6 and the sum of other FA; however, a significant cubic effect for the sum of trans C18:1 (p ≤ 0.05) was noted with the highest concentration at 400 mg/kg of PLE.With increasing PLE doses, C18:1 trans-10, C18:2 cis-9, cis-12, C18:3 cis-9, cis-12, cis-15 (3n3), C18:2 trans-10, cis-12, the sum of UFA, the sum of MUFA, the sum of PUFA, the sum of n−6 FA, the sum of n-3 FA, the sum of n6 PUFA, the sum of n3 PUFA, PUFA/SFA ratio, the sum of C18:1, the sum of trans C18:1, the sum of LCFA, and DI C18:1 cis 9/(C18:0+C18:1 cis 9) increased in a logarithmic manner  and chicory (Cichorium intybus L.) contain verbascoside in the range of 0.5 to 42 mg/g DM. The discrepancies are noted due to the differences in the time of harvest, agronomic management, soli condition, and variety variance. The activity of verbascoside as well as its derivatives was mainly assessed for anti-inflammatory and antioxidant activity [Paulownia leaves, with the content of verbascoside at 21 and 10 mg/g DM in dried and silage forms, respectively, were carried out [Paulownia extract containing significant concentrations of phenolic compounds had been conducted. The investigation of the PLE on the ruminal in vitro parameters could lead to the possibility of using Paulownia as a new dietary component or PLE as a feed additive for potentially modulating ruminal methanogenesis  and biohydrogenation .The PLE contained mainly phenolic compounds (84.4 mg/g DM). Verbascoside (acteoside) and its derivatives were the principal compounds in this group . Other biologically active substances such as iridoids  and triterpenoids  were also present. Phenolic compounds, including derivatives of verbascoside are widespread compounds present in higher plants, especially in the families ,13. The  mg/g DM  reportedactivity ,16,17,18activity ,20. StudPaulownia leaves relative to PLE. Navarette et al. [Paulownia materials. We assumed that the interaction of verbascoside derivatives or other biologically active substances contained in PLE caused a reduction of IVDMD.The results confirmed the positive effect of PLE containing a high concentration of derivatives of verbascoside and verbascoside itself on the basic ruminal parameters. PLE reduced the ammonia concentration, modulated the VFA and FA profile, and decreased methane production, the number of methanogens, and IVDMD. In the present study, the amount of dietary verbascoside derivatives was 29.0 mg/g DM. In contrast, in a previous experiment where dried leaves were used, the highest verbascoside concentration was 21 mg/g DM, which did not affect the IVDMD . The rese et al.  tested te et al. , IVDMD dp = 0.05) were observed with the increasing dietary concentration of the extract, though the total VFA concentration decreased linearly  was observed with an increasing PLE concentration. This may result from a reduction in the number of protozoa [Butyrivibrio fibrisolvens, which was confirmed in our in vitro study (p < 0.001), may be responsible for lowering the ammonia concentration. Nevertheless, the activity of Prevotella spp. increased (p < 0.001); therefore, it seems that their activity increases when the population of cellulolytic bacteria in the ruminal fluid with a lower pH decreases since they are considered to be one of the main starch-degrading bacteria [Linear decreases in pH value  [Paulownia on the inhibition of the biohydrogenation process by limiting the activity of group A bacteria, mainly Ruminococcus albus and Butyrivibrio fibrisolvens [Megasphaera elsdenii [Olea europaea L. fruit pomace, rich in polyphenols, the content of C18:2 cis-9 cis-12 and C18:3 cis-9 cis-12 cis -15 in the ruminal fluid was higher than in the control, which was in line with our results [PLE containing phenolic compounds as the main bioactive group, including verbascoside derivatives, has a promising effect on the ruminal fluid’s fatty acid profile, which may improve the milk’s fatty acid profile. An increase in UFA, including MUFA and PUFA, was observed in the experimental group containing the highest concentration of PLE. A decrease in the concentration of C15:0 was noted, which may be associated with a reduction of the y C15:0) . A higheisolvens . It is lelsdenii . With th results .All procedures were performed following the guidelines of the National Ethical Commission for Animal Research . The study was approved by the Local Ethical Commission (permission no. 14/2019).Paulownia Clone in Vitro 112 leaves were procured from a plantation nearby Trzcianka, Greater Poland Voivodeship, Poland. Then, the leaves were chopped into smaller pieces of 1.5 cm size, frozen and freeze-dried , ground using a ZM 200 mill , and kept in the dark for further use and analysis.Fresh v/v) using an ultrasonic bath at room temperature for 30 min, and also macerated using a magnetic stirrer for 30 min at room temperature. The content was centrifuged at 4000× g for 10 min. The residue was re-extracted with 1.5 L of 30% methanol (v/v) under the same conditions as above and centrifuged at 4000× g for 10 min. Finally, the residue was re-extracted with 1.5 L of 70% methanol and centrifuged at 4000× g for 10 min. The supernatants were pooled, concentrated under reduced pressure, freeze-dried, and designated as paulownia leaves extract (PLE). The yield of the extraction was 38.02%. The plant material was extracted in 8 runs with 100 g in each run.The extract of paulownia leaves was prepared in sequential extractions. The ground plant material (100 g) was extracted with 1.5 L of 5% methanol , coupled with an Acquity TQD (Waters) mass detector on an Acquity BEH C18 column . The mobile phase was composed of mixtures of solvent A (0.1% formic acid (FA) in Milli-Q water) and solvent B (0.1% FA in acetonitrile). The constituents of the samples were identified based on their molecular mass (MS), ultraviolet (UV) spectra  of a similar extract from Paulownia Clone in Vitro 112 leaves; data not shown), and literature data. Phenolic compounds and triterpenoids concentrations were determined according to previously used procedures [The phytochemical composition of the extract from leaves of ocedures . In the ocedures  was applm/z 407), formic acid adduct ion of 7-hydroxytomentoside/aucubin (m/z 391). The iridoid content was determined on the basis of a calibration curve of catalpol , and expressed as an equivalent of catalpol.Iridoid concentrations were semi-quantified by LC-MS, using a negative ion SIM method. Two ions were monitored: the formic acid adduct ion of catalpol . The triterpenoid content was determined on the basis of a calibration curve of maslinic acid (Sigma), and expressed as an equivalent of maslinic acid.Triterpenoid concentrations were determined by LC-MS, using a negative ion SIM method. The following ions were monitored : The experiment was performed using an in vitro short-term batch culture fermentation method. The in vitro experiment consisted of a control group, the ethanol group (blank group), and six experimental groups (six doses of PLE) with three repetitions in each group , and was completed in three consecutive runs (on different days) with 24 h of incubation in each run. The experimental groups were supplemented with PLE at six doses . All six doses  were dissolved in 100 µL of ethanol and added to the substrate 2 h before the start of filling the bottle with buffered rumen fluid. A small amount of ethanol addition 2 h before the start of the experiment allowed evaporation of ethanol from the substrate.ad libitum. The rumen fluid was collected before the morning feeding from each cow. Rumen fluid was squeezed through two layers of cheesecloth into a bottle , pooled, and finally mixed in an equal ratio. Then, ruminal fluid was transported to the laboratory (in anaerobic conditions at 39 °C) within 30 min and used as a source of inoculum.Ruminal fluid from three ruminal cannulated Polish Holstein-Friesian dairy cows was collected for in vitro ruminal inocula. The average weight of the animals was 625 ± 25 kg body weight. They were fed approximately 23 kg of dry matter (DM) per day. The diet consisted of 76:24 forage to concentrate and included maize silage (388 g/kg DM), alfalfa silage (82 g/kg DM), grass silage (91 g/kg DM), beet pulp (103 g/kg DM), brewer’s grain (95 g/kg DM), extracted rapeseed meal (108 g/kg DM), a concentrate mixture (119 g/kg DM), and mineral–vitamin mixture (14 g/kg DM). Water was available v/v) ratio. The bottles were constantly purged with O2-free CO2. Incubation of each experiment was run at 39 °C under CO2 in 40 mL buffered rumen fluid added to pre-warmed 125 mL vessels where 400 mg of the substrate was. The basal substrate (the diet of cannulated cows) was supplemented with various doses of PLE. The incubation flasks were sealed with rubber stoppers and aluminum caps, placed in an incubator maintained at 39 °C for 24 h, and mixed periodically.The procedure for preparing batch culture was carried out following the method described by Cieslak et al. . Briefly2 solution and stored at −20 °C for determination of VFA and NH3. Another rumen fluid sample was strained through two layers of cheesecloth and the rumen fluid was fixed with 4% formalin (6:1 ratio) for protozoa counting. For bacteria and methanogen analysis, samples were frozen in liquid nitrogen directly after collection, and then transferred to a −80 °C freezer and kept there until analysis.The microbial fermentation was stopped after 24 h of incubation. The in vitro ruminal fluid samples were collected for analyses of pH, ammonia concentration, volatile fatty acids (VFA), protozoa, bacteria, and methanogen counts. Furthermore, individual bacteria quantification and fatty acids (FA) profile analyses in ruminal fluid samples were performed. The rumen fluid samples were stored at −20 °C for further analysis regarding FA composition. Subsequently, two samples of each group of filtered rumen fluid (3.6 mL) were preserved with 0.4 mL of 46 mM HgCl® Syringes, Hamilton Bonaduz AG, Bonaduz, Switzerland) for methane analysis. The sampled gas was injected into an SRI 310 gas chromatograph , equipped with a thermal conductivity detector (TCD) and Carboxen—1000 column  according to the procedure described by Cieslak et al. [Isotrichidae and Ophyosclecidae groups. Total methanogens and total bacterial populations were quantified by fluorescence in situ hybridization (FISH), according to Puchalska et al. [After 24 h of incubation, the gas production was measured by the displacement of the syringe piston that was connected to the incubation flask. The net gas production was calculated by subtracting gas production in blank flasks (without a substrate), from the total gas produced in the flasks consisting of the buffered rumen fluid and substrate. Gas samples (500 μL) were collected from the headspace of flasks in a gas-tight syringe .Eight selected species and one genus of rumen bacteria were quantified by real-time PCR. Metagenomic DNA from rumen fluid was extracted using a QIAamp DNA Stool mini kit , according to Yanza et al. . The spep-value is ≤0.05, and tendencies are identified when the p-value is >0.05 to ≤0.10. The GenStat v. 18 statistical software package  was used for the analyses.The normality of the distribution of all the observed traits was verified with the Shapiro–Wilk’s normality test to check whether the assumption of the residuals’ normal distribution was followed for the analysis of variance (ANOVA). The homogeneity of variance was tested using Bartlett’s test. Box’s M test was used to check multivariate normality and homogeneity of variance–covariance matrices. The arithmetic means and standard error of the mean were calculated. The data were analyzed using one-way ANOVA with the treatment as a fixed factor. Linear, quadratic, cubic, and logarithmic contrasts were tested for all variables. Orthogonal polynomials were used in the analysis of variance for the construction of contrasts among unequally spaced levels of a treatment factor. Contrast is considered significant when the Secondary metabolites present in the PLE effectively modulated ruminal fermentation and inhibited methane production. The methane production inhibition was dependent on the amount of extract used. The greatest mitigation was noted with the highest dose of PLE in the buffered ruminal fluid . This mitigation was directly related to a linear decrease of the degradability of feed (substrate). Therefore, moderate doses of PLE should be used to decrease methane production while not affecting feed degradability to a great extent. The results of the research may constitute the basis for further in vivo research on new phytoadditives in ruminants.


In addition, the trapping mechanisms were ascertained by computing the streamlines and various physical parameters. The obtained results were validated with the state-of-the-art literature reports. It was claimed that our systematic approach may constitute a basis for accurately examining the impact of heat transfer on the peristaltic transport of a complex fluid through narrow tubes, useful for diverse medical applications such as the gastric fluid flow through the small intestine during endoscopy. Numerical results are computed and discussed numerically and presented through graphs. The impacts of pertinent parameters on the aforementioned quantities are examined by plotting graphs on the basis of computational results. The results indicate that the effect of parameters is very pronounced. A suitable comparison has been made with the prior results in the literature as a limiting case of the considered problem.This paper provides a rudimentary insight into the influence of heat transfer on the transport characteristics of a second-grade dusty fluid flown in a flexible tube with walls subjected to the peristaltic motion. Both dust particles and fluid movements were modeled using the coupled differential equations. The effects of different types of parameters such as Reynolds number, Prandtl number, Grashof number, wave number, wave amplitude ratio, second grade parameter as well as nature of the heat source and sink are studies on the dust particles velocity, fluid velocity, temperature, pressure profiles of the fluid and streamline patterns of the fluid. The derived equations were solved analytically via the standard perturbation method to determine the fluid temperature, streamline pattern and velocity of the dust particles as well as fluid. The values in the increase of pressure and frictional forces were calculated numerically using DSolve of the Mathematica 11 software ( Generally, the perturbation approach is used to find the solution of such complex transport in various powers of the amplitude ratios2. It was discerned that for two-dimensional (2-D) peristaltic pumping with extremely small Reynolds numbers, the fluid motion can be assumed to be inertia-free and long wavelength in nature3. In addition, the peristaltic transport of different complex fluids in an axial-symmetric tube for certain values of the Reynolds number and wavelengths was studied wherein the asymptotic solutions were obtained concerning the small amplitudes to the mean diameter’s ratios4. This work was carried out following the earlier study on the fluid motion through 2-D channels5. Meanwhile, the long wavelength approximation for 2-D peristaltic pumping enabled in relaxing the assumption regarding the small amplitudes6. An all-inclusive overview on the peristaltic pumping was made by7.Understanding the effects of heat transfer on the transport behavior of a second-grade dusty fluid streaming through a flexible tube with walls subjected to the peristaltic motion remains challenging8. The absence of unique constitutive model to describe the overall characteristics of the non-Newtonian fluids enforced the researchers to find out alternative models. To surmount this limitation, numerous classical models were introduced to obtain the nonlinear relationships among shear stresses and strain rates10. Generally, majority of the fluids including bio-fluids, synthetic lubricants, paints, oils, petroleum, honey, and so forth were shown to reveal the non-Newtonian traits11. Also, the mechanics of peristaltic pumping of a non-Newtonian fluid for a second-order fluid through an axisymmetric conduit was performed12. Over the years, several studies involving the non-Newtonian fluids have been conducted to determine the feasibility of practical applications in the field of physiology, engineering and industries19 .The emergence of Navier Stokes relations for the rheological properties of fluids provided a further impetus to the advancement of the non-Newtonian fluid dynamics research20. Examples of these dusty fluids include human urine with stones or glucose particles suspension, unrefined petroleum, crude oils and diverse foods containing pulpy granules. In the early period21, considered human blood as a binary system and analyzed its transport properties. Later22, examined the peristaltic transport behaviors of various dusty fluids under the approximation of long wavelength and small Reynolds numbers. Meanwhile23, investigated the transport properties of a solid fluid mix streaming through an axially-symmetric channel. Furthermore, the sinusoidal wave motion-induced 2-D flow of a dusty fluid moving through a tube with infinite curvy wall was examined for Reynolds numbers above one24. The peristaltic transport of a dusty fluid was demonstrated when streamed through a porous medium25. Recently26, evaluated the effects of the wall topologies of the channels on the transport attributes of tiny particles suspended in a dusty fluid. The impact of the wall topologies on the transport features of a Walters B fluid containing fine particulates flowing through a uniform channel was inspected27. The peristaltic transport characteristics of a dusty fluid streaming through a tube was demonstrated by28.Despite many dedicated efforts the behavior of pure fluids dynamics is far from being understood. Yet again, the natural fluids often contain various impurities and contaminants such as dirt and dust particles together with unknown components. The fluids are said to be dusty when they contain various dispersed solid particles in colloidal suspension, making the dynamics of such fluids very complex and yet worthy to investigate29. The influence of heat transfer on the peristaltic motion of a dusty fluid streaming through a channel was assessed30. In addition31, examined the effects of velocity slip on the magnetohydrodynamic (MHD) peristaltic transport of fluid through a porous media in the presence of both heat and mass transfer. Meanwhile32, determined the influence of rotating medium with compliant walls on the MHD fluid’s peristaltic transport. The dependence of temperatures on the peristaltic transport characteristics of a MHD fluid’s flowing through an asymmetric channel was analyzed33. The impact of compliant walls in the presence slip at the boundaries on the peristaltic transport behaviors of a MHD fluid was analytically formulated34. The influence of velocity slip on the MHD peristaltic transport of a Casson fluid and transfer of heat through an asymmetric channel cantaining a porous media was assessed35. Over a horizontal, flat plate with a constant heat flux, natural convection of power-law fluids was investigated36. Many authors have recently investigated the MHD convection for diffrent fluids under effect of heat transfer in the articles39 and41. The peristaltic transport features of a Johnson Segalman fluid flowing through an asymmetric curved channel accompanied by mass and heat transfer were analyzed42. A simulating sloshing and evaporation in a cryogenics fuel tank was studied using a new computational fluid dynamics algorithm43. A multinode-CFD simulation of a cylindrical pressurized cryogenic storage tank was examined by44. The study of cooling high temperature traveling wave tube collectors under steady state conditions with TWT collectors thermal stress and deformation was presented by45. Heat sink optimization and thermal performance enhancement in three geometry categories on energy storage system was discussed46.It is well established that the heat transfer is significant for sundry applications especially in the field of geophysical sciences and engineering like underground energy transport in the geothermal reservoirs, thermal insulation, porous solids’ drying, improvement in the recovery of fossil fuels and oils, catalytic reactors using packed-bed, and nuclear reactor’s coolinghttps://www.wolfram.com/mathematica/new-in-11/). Therefore, the analytical solution has been obtained. The peristaltic transport behavior of the studied dusty fluid was evaluated in terms of the velocity of the dust particles and fluid, fluid temperature, friction force, pressure rise and streamline pattern. Furthermore, to ascertain the trapping process, different physical parameters and streamlines were computed and discussed by the graphical results. The obtained results were analyzed, discussed, and validated by comparing the outcomes of literature review12 and20. The peristaltic transport mechanism of the proposed dusty fluid was understood.Considering the immense fundamental and applied significance involving the impact of heat transfer on the transport behaviors of various dusty fluids streaming through a flexible tube with varying wall’s topologies subjected to the peristaltic motion, this paper considers a second-grade dusty fluid flowing in a flexible tube whose walls are induced by the peristaltic movement to understand the transport process in the presence of heat transfer. The influence of heat transfer on the peristaltic transport properties of such fluid was determined. The conventional perturbation theory was used to derive the analytical solution of the modeled coupled differential equations. The obtained results are validated with the solutions obtained by DSolve a built â€“ in function in Commerical software Mathematica 11 (N). The densities of the solid particles are constant. It is further assumed that long wavelength peristaltic waves can propagate along the tube walls. Let us assume the cylindrical coordinate system a, c, b and Consider an axial-symmetric 2-D streaming of a second-grade fluid through a tube enclosing tiny solid particles of even sizes with number density  of the second-grade fluid can be written as:12:Following28:28 and heat transfer are given by15:K, m, N, g, T, The equation of motion in the fixed frame Using Eq. , one obtp as a mathematically power series with small perturbation parameter 25.Following the perturbation procedure to introduce the physical properties ing Eqs. –34 to obnditions –38 resul25, in terms of a small perturbation parameter As aforementioned, the perturbation technique was used to get the solutions of a set of nonlinear equations of the fluid and dust particles derived from the analytical model presented in the last section. Following the method referred ining Eqs. –34) and and25, iing Eqs. –38) one one25, iThe first-order approximation of O(the Eqs. –53), . In the absence of heat transfer, we get the same solutions of stream functions for a second-grade fluid and dust particles as obtained by28. Also, if there is no solid particles and heat transfer, the solution is same in the paper12.Write few lines prelude here to make this mathematical machinery meaningful and the purpose of these steps:https://www.wolfram.com/mathematica/new-in-11/). Relevant parameters associated to the impact of heat transfer on the peristaltic transport of the fluid like w, This study aimed to determine the influence of heat transfer on the transport behaviours of a second-grade dusty fluid flown in a flexible tube with peristaltic movement of the wall. The motion of dust particles and fluid were analytically modeled diverse nonlinear equations and solved analytically using perturbation approach. Numerical solutions of these modeled equations were obtained by DSolve of the Mathematica 11 software (entclass1pt{minima28.The values of entclass1pt{minimar. In short, the temperature variation of the fluid at the inlet 15. From the observations of the results, it has been noted that parameters involved have a similar role in the temperature, since the temperature determines the average kinetic energy which is related to the motion of fluid particles.The results in Fig. uid Fig. . The temFigure Figure Figure Figure With the increase of The temperature of the dusty fluid was increased with the increase of The velocity of the dust particles and fluid was increased with the increase of The parameters The frictional forces in the fluid showed reverse trend than the pressure rise.Generally, the heat transfer-mediated peristaltic transport properties of the proposed dusty fluid were affirmed to depend considerably on various physical parameters.The achieved results were in good agreement with the recent state-of-the-art works reported in the literatures.It was asserted that the present findings may be beneficial for the advancement of fluid mechanics, biomendical sciences, and engineering.The generated new knowledge can certainly help the surgeon to control the gastric fluid flow in small intestine during endoscopy.This paper comprehensively determined the effects of heat transfer on the transport features of a second-grade dusty fluid flown in a flexible tube under peristaltic motion of the wall for the first time. Both fine dust particles and fluid motion through the cylindrical tube were modeled using the nonlinear coupled differential equations. Standard perturbation method was used to get the analytical solutions of the model equations. Based on the numerical results obtained using DSolver of the Mathematica software the following conclusions may be drawn: In future one should exploit both deterministic and stochastic numerical solver for both fluid mechanics problems of paramount significance. In future, one may implement the Lobatto IIIA scheme for the numerical treatment of many potential applications arising in the fields of Bioinformatics, astro/plasma/atomic physics, nonlinear circuit models, fluid mechanics, financial mathematics and COVID-19 virus models.


In the article titled “Comparison Between Treatment Strategies of Carotid Stenosis inPatients Undergoing Coronary Artery Bypass Grafting”, DOI 10.21470/1678-9741-2020-0425,published in the Brazilian Journal of Cardiovascular Surgery, issue 37.3, thecorresponding author’s e-mail address is incorrect.In the original version, the information is:fbassan@globo.comE-mail: The correct information is:drfernandobassan@gmail.comE-mail: