Rationale: Idiopathic and heritable pulmonary arterial hypertension (PAH) are rare but comprise a genetically heterogeneous patient group. RNA sequencing linked to the underlying genetic architecture can be used to better understand the underlying pathology by identifying key signaling pathways and stratify patients more robustly according to clinical risk.Objectives: To use a three-stage design of RNA discovery, RNA validation and model construction, and model validation to define a set of PAH-associated RNAs and a single summarizing RNA model score. To define genes most likely to be involved in disease development, we performed Mendelian randomization (MR) analysis.Methods: RNA sequencing was performed on whole-blood samples from 359 patients with idiopathic, heritable, and drug-induced PAH and 72 age- and sex-matched healthy volunteers. The score was evaluated against disease severity markers including survival analysis using all-cause mortality from diagnosis. MR used known expression quantitative trait loci and summary statistics from a PAH genome-wide association study.Measurements and Main Results: We identified 507 genes with differential RNA expression in patients with PAH compared with control subjects. A model of 25 RNAs distinguished PAH with 87% accuracy  in model validation. The RNA model score was associated with disease severity and long-term survival (P = 4.66 × 10−6) in PAH. MR detected an association between SMAD5 levels and PAH disease susceptibility .Conclusions: A whole-blood RNA signature of PAH, which includes RNAs relevant to disease pathogenesis, associates with disease severity and identifies patients with poor clinical outcomes. Genetic variants associated with lower SMAD5 expression may increase susceptibility to PAH. Blood transcriptomic profiles in pulmonary arterial hypertension have been examined in small limited studies indicating their potential utility in differentiating patients from control subjects and by clinical phenotypes.This study represents the most comprehensive analysis of whole-blood RNA profiles in pulmonary arterial hypertension and identifies and validates a signature that both differentiates patients from control subjects and stratifies patients by clinical risk and severity. Genetic analyses (Mendelian randomization) indicate potential causal roles for some genes including the TGF-β signaling molecule SMAD5.Pulmonary arterial hypertension (PAH) is associated with vasoconstriction and occlusion of distal pulmonary arteries, characterized by endothelial damage, smooth muscle and fibroblast proliferation, and inflammation. Increased pulmonary vascular resistance leads to right heart failure, with survival rates estimated at 52–75% at 5 years, even with modern-day therapy . The ratTranscriptome profiling through RNA sequencing permits a comprehensive analysis of gene expression in tissue samples. Whole-blood RNA analysis offers an alternative “liquid biopsy” to lung biopsy, which carries a high risk in PAH, and can be performed sequentially. This approach can also investigate immune mechanisms in PAH that have recently been highlighted . PreviouThe aim of this study was to characterize gene pathways associated with PAH and to assess their association with disease heterogeneity, specifically in terms of disease severity and outcomes including response to vasodilators and mortality, and genetic background. We compare gene expression in whole-blood samples from 359 patients with idiopathic, heritable, or drug-induced PAH from the UK PAH Cohort study with 72 age- and sex-matched healthy volunteers without any cardiac or respiratory disease as control subjects. Using equal distribution of samples into a three-stage design, we identified reproducible RNA expression differences by RNAseq. Two distinct computational approaches were used to estimate white blood cell (WBC) fractions and thus account for the potential effect of different cell numbers on gene transcript levels across samples. A predictive statistical model that combined gene expression differences performed well at identifying patients with PAH in a separate case–control analysis. The RNA-based score was also associated with disease severity and clinical outcomes . Enrichment of genetic variants that determine the levels of PAH RNAs was detected, implicating these RNAs in the pathogenesis of the disease.Comprehensive methods are included in the online supplement.www.ipahcohort.com). In each case, diagnosis was confirmed by right heart catheterization following established international guidelines (see online supplement for further details). Genomics data were obtained from a published PAH genome-wide association study  were recruited from expert centers across the UK as part of the PAH Cohort study , RNA validation (n = 120), and model validation (n = 119). Each of these three groups was then compared with an independent set of age- and sex-matched healthy volunteers as control subjects  were analyzed using Salmon v0.9.1 . Differential expression analysis was performed using edgeR v3.22.5 (P < 0.05) directionally consistent in the initial analyses and meeting false discovery rate (FDR) multiple test corrections  in the combined analysis were taken forward. Five hundred seven genes meeting these criteria were considered to generate a model to distinguish PAH from control subjects. Subset selection of RNAs that best distinguish PAH in combination was performed by least absolute shrinkage and selection operator (LASSO) regression analysis, using the glmnet v2.0-18 package from CRAN, with k-fold cross-validation (k = 10) selecting the largest value of lambda such that error is within 1 SE of the minimum. This produces an RNA score from a linear weighted combination of the mRNAs identified by the LASSO analysis. Receiver operating characteristic analysis was performed using the pROC v1.14.0 package from Bioconductor  and World Health Organization (WHO) functional class  and by cardiac biomarkers .david.ncifcrf.gov) and Ingenuity Pathway Analysis (IPA) using in-built FDR corrections for multiple tests.Functional annotation and enrichment of the genes associated with PAH was performed using DAVID (Mendelian randomization (MR) analysis using all independent genome-wide significant whole-blood expression quantitative trait loci (eQTL) from two published studies , 16 and P < 0.05) between PAH and control subjects with directional concordance in both discovery and validation analyses . All 507 genes were included after accounting for multiple testing . None were associated with exposure to the main PAH therapies (see online supplement for further details). These included RNAs in pathways relevant to PAH; for example, SMAD5 (mothers against decapentaplegic homolog-5), encoding a downstream mediator of signaling of BMPR2 . All real-time quantitative PCR measurements correlated significantly with RNAseq quantification .A set of differentially expressed RNAs relevant to PAH pathobiology were selected for validation of RNAseq quantification by real-time quantitative PCR, namely, n = 119 patients with PAH and n = 24 control subjects) and demonstrated an area under the curve of 0.868  . The model was then tested in an independent validation set (1–0.945) . The optWe next examined whether the RNA model was also associated with patients with the poorest outcomes, using all 359 patients. The optimum cutoff (1.910) for identifying PAH nonsurvivors with the LASSO model separated patients with PAH into low- and high-risk groups in survival analysis  and E3.To determine which of the 25 transcripts in the model were responsible for the association with survival, we tested each transcript for associations with all-cause mortality during follow-up. After FDR correction for multiple testing, three intronic long noncoding RNAs  were associated with survival in all the patients with PAH analyzed and cutoffs distinguished high- and low-risk patient subgroups .P = 0.008; P = 5.10 × 10−4; P = 8.7 × 10−5).To further characterize the RNA signature, we analyzed its association with three clinical measures of disease severity—WHO functional class, exercise capacity , and cardiac biomarkers (BNP or NT-proBNP). We found a significant difference in RNA scores between patients in different WHO functional classes  was consistently lower in PAH.Three hundred seventy-two out of the 507 top dysregulated genes from the current RNAseq study were present in the lung tissue microarray study and 161/372 (43%) were also directionally consistent. Forty-one out of 161 (25%) genes were nominally significant and 26 met FDR corrected significance (Table E9). Only one gene was found to be dysregulated in patients with PAH across all three studies: AMD1, the only gene in common between the current RNAseq study, the recent lung tissue microarray study and the gene expression meta-analysis, was part of the top IPA gene network .Of the 507 RNAs found to be differentially expressed in PAH, 435 were present in the functional annotations in DAVID. Enrichment of DNA-binding TFs (transcription factors), such as HIF1α (hypoxia-inducible factor 1α) and KLF10 (Krüppel-like factor 10), and many zinc finger–containing TFs was observed compared with a background of the genes detected (Table E10). The Ingenuity Knowledge Base mapped 505/507 transcripts. Double-stranded DNA repair, T-cell receptor, PI3K signaling in B lymphocytes, the role of JAK family kinases in IL6-type cytokine signaling and hypoxia signaling were among the top canonical pathways identified by IPA . SESN1 (Sestrin-1) and SMAD5, reached nominal significance using eQTL in both data sets  was associated with an 8.5% reduction in the risk of developing PAH . SMAD5 levels were similarly reduced in patients with PAH with and without pathogenic BMPR2 variants , supporting the observation that impaired signaling in the BMPR2 pathway is more common in PAH than rare mutations in BMPR2 suggest. The PAH RNA model score was similarly elevated in patients with PAH with and without pathogenic BMPR2 variants .The SMAD5 are more common in patients with PAH.Here we report an RNA signature that separates idiopathic and heritable PAH from healthy individuals. The signature also stratifies patients according to disease severity and risk of early death, adding plausibility to its association with PAH. Several of the discriminating mRNAs encode TFs, including SMAD5, HIF-1α and KLF10. MR analysis to integrate genomic data and identify underlying pathogenic signaling pathways revealed that genetic variants associated with lower expression levels of SMAD5 encodes an intracellular transcriptional modulator that is activated by ligand binding of BMPR2, the most common genetic risk factor in heritable PAH (MR is a powerful tool for separating cause from consequence, as an individual’s genetic status predates the development of PAH. We harnessed genetic data from a published international genome-wide association study  and infoable PAH . SMAD5 aable PAH . SMAD5 aable PAH , which mable PAH . Novel table PAH –25. WholAMD1 was the only gene consistently dysregulated in both these studies and our data set, with lower levels in PAH samples in all three studies. AMD1 encodes a key enzyme controlling the supply of decarboxylated S-adenosylmethionine for polyamine biosynthesis and is regulated by several mechanisms, including increased protein degradation in the presence of elevated polyamines and the inhibition of mRNA translation by spermidine and spermine (AMD1 expression in patients with PAH may be in part due to negative feedback from elevated polyamines. These data contrast with observations in hypoxic rodents. Amd1 expression is increased in hypoxic animals, and both AMD1+/− mice and mice treated with the AMD1 inhibitor, SAM486a, were partially protected from the development of hypoxic PH (ATP13A3 with PAH (We were able to externally validate our findings with a meta-analysis of published PAH blood transcriptome studies  and a PAspermine . We havespermine . Reducedpoxic PH . Interespoxic PH . This miwith PAH .Many of the transcripts that passed robust statistical evaluation in this study are novel. We identified three specific long noncoding RNAs that are reduced in PAH and associated with poor outcomes. These transcripts are not well studied, and their role in regulating lysosomal proton pump protein ATP6V0E2, protein tyrosine phosphatase PTP4A2, or small nuclear ribonucleoprotein-associated protein N remain to be established. Their association with survival selects these out as worthy of further investigation. In addition to suggesting biological relevance, the association of the RNA model, developed to distinguish PAH from control subjects, with survival and disease severity in patients with PAH suggests that the differences observed could be useful to identify patients who may require a more aggressive treatment strategy, such as upfront triple therapy . This stThere is considerable interest in developing a biochemical test to identify patients with PAH who respond well to calcium antagonists that would supersede the current test, namely, an acute vasodilator challenge while undergoing cardiac catheterization. In contrast to previous reports, we were unable to demonstrate the association of a peripheral transcript signature to vasoresponder status, including previously studied RNAs . A stren+ T cells in patients with PAH, consistent with previous reports (One of the limitations of sampling whole blood to derive transcripts is that samples comprise a mixed population of cells. We used established deconvolution methods to correct for potential confounding in RNA expression analysis. In support of the validity of this approach, we noted altered numbers of regulatory T cells and CD8 reports , 31, alt reports .This study does not assess the role of post-translational modifications in the pathogenesis of PAH, which could add further information to the circulating transcriptome. It is important that further mechanistic studies consider the role of the genes highlighted in this study in the tissues of primary interest, namely, the lung and heart. The study design included more patient samples than control subjects to account for the higher heterogeneity typically observed in patient populations. This heterogeneity is observed in the overlap in box plots of individual RNA levels or scores between subsets of patients with PAH and control subjects and emphasizes the importance of using any molecular markers in combination with best practice clinical assessments. We chose to use LASSO regression modeling not only as it is known to perform well in these kinds of data sets but also because it is widely applied and often easier to interpret than other methodologies. Another consideration is that the patients studied here were prevalent cases. Moreover, all the patients recruited had a clinical diagnosis of idiopathic, heritable, or drug-induced PAH. It would be of interest to sample patients with other presentations of PAH and other cardiopulmonary diseases to better understand the clinical utility of our RNA signature.In summary, we report a whole-blood RNA profile that distinguishes patients with PAH from healthy control subjects and reflects disease severity. Integration with genomic data suggests that SMAD5 is important in the development of PAH, prioritizing restoration of normal SMAD5 function as a target for therapeutic intervention.


Artemisia absinthium, Hypericum spp., Linum usitatissimum, Quercus robur). The four study regions located in Northern and Southern Eastern Europe did not present similar ethnoveterinary knowledge trajectories. Bukovinian mountain areas appeared to hold a living reservoir of ethnoveterinary knowledge, unlike the other regions. Setomaa  and Dzukija showed an erosion of ethnoveterinary knowledge with many uses reported in the past but no longer in use. The current richness of ethnoveterinary knowledge reported in Bukovina could have been developed and maintained through its peculiar geographical location in the Carpathian Mountains and fostered by the intrinsic relationship between the mountains and local pastoralists and by its unbroken continuity of management even during the Soviet era. Finally, our results show some patterns common to several countries and to the veterinary medicine promoted during the time of the Soviet Union. However, the Soviet Union and its centralized animal breeding system, resulted in a decline of ethnoveterinary knowledge as highly specialized veterinary doctors worked in almost every village. Future research should examine the complex networks of sources from where farmers derive their ethnoveterinary knowledge.Over the last century in the European context, animal production has been transformed by the dynamics of centralization and decentralization due to political and economic factors. These processes have influenced knowledge related to healing and ensuring the welfare of domestic animals. Therefore, our study aimed to document and compare current and past ethnoveterinary practices, and to identify trajectories in ethnoveterinary knowledge in study regions from both northern and southern Eastern Europe. In the summers of 2018 and 2019, we conducted 476 interviews, recording the use of 94 plant taxa, 67 of which were wild and 24 were cultivated. We documented 452 use reports, 24 of which were related to the improvement of the quality or quantity of meat and milk, while the other 428 involved ethnoveterinary practices for treating 10 domestic animal taxa. Cattle were the most mentioned target of ethnoveterinary treatments across all the study areas, representing about 70% of all use reports. Only four plant species were reported in five or more countries ( In many societies, livestock significantly contribute to human food security by providing several important food products, other valuable goods , agricultural inputs , and services . However, over the last century in the European context, animal production has been transformed by dynamics of centralization and decentralization , 2. ThesAnimal breeding involves maintaining animal health and welfare. Scholars have found that the knowledge related to healing and ensuring the welfare of domestic animals has been largely abandoned in industrialized areas of Europe , while iThe use of plants for veterinary practices is well-studied in some regions of Eastern Europe. For instance, in Ukraine several scholars have investigated this topic e.g., –13], foc, foc13)]Within this framework, our study aimed to document and compare current and past ethnoveterinary practices, and to identify trajectories in ethnoveterinary knowledge in rural borderland areas of eight countries from northern and southern Eastern Europe, namely Finland, Russia, Estonia, Lithuania, Poland, Belarus, Ukraine, and Romania. We further discuss what factors may have contributed to the persistence/erosion of ethnoveterinary knowledge in Eastern Europe.In the summers of 2018 and 2019, we conducted semi-structured interviews in four regions in eight countries that are home to nine main ethnolinguistic groups .Bukovina is a historical region of the Austro-Hungarian Empire that has been split between Romania and Ukraine since 1940. It is inhabited by several ethnic groups including Jews, Ukrainians, Poles, Romanians, and Hutsuls. Bukovina is partially occupied by the North-Eastern Carpathians which reach an altitude of 1,651 m a.s.l. We conducted our research among Hutsuls living in the Carpathian villages of the upper Suceava Valley in Romania and Putyla Rayon in Ukraine and among Romanians living in the pre-Carpathian hills of Straja (Romania) and Storozhenets and Glybotskyi district in Ukraine. Both in Ukraine and in Romania, most of the interviewees rely on family farming.Dzukija is a historical and cultural region located in the borderlands of Poland, Lithuania, and Belarus. This border area has long been a crossroads for trading routes and has been subject to a series of changes in national status. It is now mainly inhabited by Lithuanians and Poles. It is characterized by plain and hilly rural areas and is currently experiencing a remarkable rural emigration. Soils throughout the studied region are sandy and of little agricultural value. We conducted interviews among Lithuanians living in several villages of Augustów and Sejny counties of Podlaskie Voivodeship (Poland), Šalčininkai district of Vilnius County (Lithuania), and Hrodna, Voranava, and Ašmiany districts of Hrodna Region (Belarus).Karelia is a historical region currently divided between Finland and the Russia. Agricultural lands occupy only a small percentage of its territory, which is mainly covered by forests, a crucial resource for the local economy , 23. In Setomaa is a region located at the Estonian-Russian border inhabited by speakers of the Seto and Russian languages. After WWII, the largest part of the formerly united historical Setomaa was incorporated into the Russian Soviet Federative Socialist Republic and remained there even after Estonia regained independence. Since the twentieth century, rapid alterations to the environment  have led to the disappearance of some native plants  and the The research was part of a wider study, namely the ERC-funded DiGe project, aiming to understand the mechanisms of change in ethnobotanical knowledge that occur among cross-border minorities when a dominant group tries to modify this knowledge. The four regions were selected in order to give an overall comparative picture of the current and past uses of plant-based ethnoveterinary remedies in Eastern Europe , 2. We cIn Finnish Karelia, interviewees were also identified in advance through various social networks as it was not always possible to approach them directly. We consider this sample robust given the fact that saturation was reached after about 10–15 interviews per country.http://en.herbariumle.ru/)].Before each interview, prior informed consent was obtained following the Code of Ethics of the International Society of Ethnobiology . Upon coWhen possible, interviews were recorded upon the interviewee's approval and transcribed in the local language; in the few cases when recording was refused, we took notes. Later, we entered this information in English into an Excel spreadsheets organized as detailed use reports (DUR) of plant-based remedies, where each row contained the country and ethnic community of the interview, its code, the scientific name of the plant, its local name, the part used, when it was used, the mode of preparation, and its use. Botanical taxa were classified using World Flora Online (2021). The botanical families were classified according to the Angiosperm Phylogeny Website .The research protocol was approved by the Ethics Committee of Ca' Foscari University of Venice.In the four regions where this study was conducted, we recorded the use of 94 plant taxa, 67 of which were wild and 24 were cultivated, from 189 interviewees. We documented 452 use reports, 19 of which were related to the improvement of the quality or quantity of meat and milk, while the other 428 involved ethnoveterinary practices for treating 10 domestic animal taxa. Out of the 476 interviews we conducted in the four study regions, 189 reported ethnoveterinary uses. Below, we focus first on ethnoveterinary knowledge related to cattle as about 70% of the use reports concerned cattle illnesses .Cattle were the most mentioned target of ethnoveterinary treatments across all the study areas. We recorded the use of 55 plants belonging to 25 families , 2 for tArtemisia absinthium, Hypericum spp., Linum usitatissimum, Quercus robur).Only four species were reported in five or more countries (The majority (61%) of the DUR were currently in use, while 39% referred to past uses. However, excluding the Bukovinian Carpathian area (our Ukrainian and Romanian case studies), the proportion of currently used DUR drops to 12%, while the remaining 88% refers to past ethnoveterinary uses. Indeed, as illustrated in Rumex spp. (reported in four countries across three regions), Hypericum spp. , and Quercus robur . In regard to the reproductive system, the most common issues involved calving and the use of postpartum supplements. The most utilized plant for treating the reproductive system was Linum usitatissimum, mentioned in four countries and three regions. Among the listed plants some  could potentially have negative effects on animals. While no interviewee explicitly mentioned possible adverse side-effects, they were not assessed, being out of the scope of this article.The digestive and reproductive systems were the most common targets of ethnoveterinary remedies . For thePicea abies (three regions), Quercus robur (three regions), and Urtica dioica (two regions). The livestock most commonly treated with ethnoveterinary remedies were pigs (39 DURs), which were mainly mentioned in Russian Karelia and Russian Setomaa. As in cattle, the most widely treated illness was diarrhea, primarily in pigs. Also, five plant taxa  were used, especially in Dzukija, as feed supplements (13 DURs).Excluding cattle, we documented 130 DURs related to 44 plant taxa belonging to 28 botanical families that were used for treating nine animal taxa, including pigs, honeybees, sheep, turkeys, chickens, geese, horses, dogs, and cats. Only three species were found to be used in four countries: Urtica dioica, which was reported by six people in Bukovina for improving milk quality and by one interviewee in Lithuania for improving pork meat quality.In addition to ethnoveterinary remedies, we also recorded the use of 19 DURs referring to ten plant taxa for improving the quality of milk, meat, and in one case pig bristles . More thOut of the 476 interviews we conducted in the four study regions, 189 interviewees reported ethnoveterinary uses . SpecifiWe recorded different attitudes regarding the use of plants for treating animals . InterviIn the Dzukija region, older respondents reported the loss of ethnoveterinary knowledge as a reason for turning to official veterinary medicine: “There are no more people who know the right herbs and remedies. Now there are veterinarians. If someone needs something, they turn to veterinarians” (Belarusian Lithuanian woman born in 1946). Informants from both the Belarusian and Lithuanian sides of Dzukija noted that state-sanctioned veterinary medicine appeared with the advent of the Soviet state and the formation of kolkhozes [~1950]. On the Polish side, older Lithuanian interviewees mentioned that they mainly healed animals with drugs (Polish Lithuanian woman born in 1929).At the same time, although the older generation of interviewees living on the Belarusian and Lithuanian sides of the Dzukija region is characterized by a lack of trust in official medicine, the middle and younger generations prefer medicines over medicinal herbs. For example, a middle-aged female respondent noted that it is better to treat cows with drugs, not herbs. For her, using chemicals is a more reliable option (Lithuanian woman living in Belarus woman born in 1954).Artemisia absinthium), čystacieł (Chelidonium majus). And I carried these bottles to the kolkhoz” (Lithuanian woman living in Belarus woman born in 1941).Among the respondents who worked on kolkhoz between the 1950s and 1990s, some noted that they used home remedies for the kolkhoz animals: “I worked on the farm, raised calves, and none of them died. I made bottles for them at home. There were pałyn .In Russian Karelia and Setomaa, the decision to call a veterinary service (instead of treating livestock using plant-based remedies) was sometimes perceived as a matter of time, as one veterinary doctor mentioned, “You know, when I started working, there were already antibiotics […]. But even at the beginning of my work [in the late 1970s], in fact, they did not pay much attention to folk remedies. Well, maybe because this is a longer path to recovery, whereas one needs the result immediately. After all, they use more modern ones here” (Russian Seto woman born in 1939). This was also confirmed by a retired kindergarten nanny: “And as with pills, you call a doctor, because it is fast for calves, yeah, for calves. Quicker with them, because herbs are too slow” . Conversely, in Romania, some older interviewees were skeptical about official veterinary medicine: “Teas of In the Soviet Union, it was not difficult to receive veterinary consultations . However, some interviewees also reported that “there were specialists, but at home, you were mostly on our own” (Russian Karelian man born in 1950). Contrastingly, another interviewee stated, “Cattle were not treated at home, only by a veterinarian. In the old days, there were very good veterinarians. Only the veterinarian treated animals” (Russian Karelian man born in 1929). Indeed, during Soviet times there was a veterinary doctor working in every large village with kolkhoz who helped locals to treat cattle. An Estonian Seto woman born in 1938 narrated: “Animals had flax seeds after calving, but otherwise, if something was wrong, we had to call the vet.”In Finnish Karelia, an interviewee claimed that in the 1950s, “Veterinarians were not used as the nearest veterinarian was 35 km away. There was someone among the people, who was familiar with these problems, and he/she was called only if there was some illness and you were unsure of what you should do” .Our interviewees from the four regions often mentioned the deep connection they have or had with their livestock. For instance, in Russian Karelia, an Ingrian-Russian woman (born in 1954) mentioned, “We had goats, sheep, yes. well, at that time it was kind of necessary, because living was not very. how to say?–easy. Therefore, we kept them,” stressing the role livestock played in guaranteeing food security.Also, a Russian woman, born in 1966, living in Karelia reported using human food for feeding animals: “They just subsidized bread very, very much. […] My Karelian grandmother fed all her cattle with bread. It was cheaper than growing potatoes and buying grain from a sovkhoz. And my dad, having a car, every day brought her two loaves, four loaves, of bread. It was a strange idea of our household, but bread was subsidized, and the rest was not.”In Romania, a Hutsul interviewee (Romanian Hutsul woman born in 1961) clearly explained the nexus between “eating in a healthy way and livestock breeding. When asked about any possible product she could give animals to produce more milk she exclaimed, “Ah, no, I do not give them anything. Well, to be precise, sometimes a bit of bran, when animals come in at night and in the morning when milking, but otherwise, grass. Here we eat healthily.” It is worth noting how “we” includes both humans and animals, as a whole, and therefore if animals eat healthily humans will as well.Salix sp. twigs consecrated on Palm Sunday. They placed these twigs on the barn door to protect animals. Both Seto and Russian people recalled drawing a black cross with charcoal above the barn door, on baptism day, to protect animals.In the Dzukija region, however, among the villagers, there is often a reasonably practical approach: “We did not contact anyone. If an animal lives, it lives. Nothing was treated. As the old then died” (Lithuanian woman living in Belarus woman born in 1939). We recorded a profound respect for livestock. Many respondents said that, for example, a cow is a very clever animal that knows what to eat and what not to eat. At the same time, the respondents remembered many instances of using herbal remedies in various rituals related to the prevention of diseases in livestock. In particular, the tradition of blessing animals with a palm bouquet has survived to this day: “And also animals were smoked with that Easter Palm. Yes, if they got sick then they did it” (Lithuanian woman living in Lithuania born in 1938). Setos used Gymnocarpium dryopteris] […] which is good for the evil eye as well. […] Especially for animals, we used to collect it, if someone casts the evil eye, you smoke it, and it was a medicine” (Lithuanian woman living in Lithuania born in 1939).It should also be noted that a large number of our respondents' recollections concerned the use of various plants for the treatment and prevention of culture-bound illnesses in domestic animals, such as fright and the evil eye: “For fright, there is uročnikas . It was a difficult period then” . This was also confirmed by an elder Finnish Karelian woman who remembers that whipped cream from the only cow remaining was an important resource: “I remember, in the 1970s, when it was decided that we would be left with one cow. At that time, cream was skimmed from milk and whipped as whipped cream.” Another participant commented, “It was not considered a farm if there was no cow” (Russian Karelian man born in 1928). In addition, a retired Karelian seamstress (born in 1954) living in the Russia mentioned that cattle disappeared because “the mowing was very bad, and it was necessary first to give hay to the sovkhoz, and therefore my parents got rid of the cows.” This issue of the lack of hay was also mentioned by a Karelian forestry specialist: “It was torture to keep a cow. When I was in elementary school, my father had a MAZ lorry. Mowing was not allowed then, and they drove in the direction of Interposiolok to steal hay; they mowed on the side of the road and returned with it at night–either to the neighbors or to our own farm. They helped each other, dried. they dried hay, we dried hay, and that's all. so. and lived” (Russian Karelian woman born in 1948).Among other more recent changes, a Seto interviewee claimed, “And now you can list the cow in the Red Book, only a few cows remain. So, there are no animals to treat actually” (Russian Seto man born in 1943). An Estonian Seto woman, born in 1938, commented, “I had three cows, and for a short time also four. When the Estonian state started, it destroyed all the cows, no one needed milk anymore. Milk churn stands were lost.” A younger Estonian Seto man (born in 1953) explained his point of view, referring to the current availability of state jobs: “There were those “state” works , and, on top of them, people had their own animals and farmland. But have you seen that now no one raises animals like cows or pigs at all? Pigs can't be kept anymore, because that bastard plague (African swine fever) is here.” In Ukraine, an older Romanian woman explained, “I do not have a cow now, but I had one. I have had it for many years, but now I am old, weak and I really cannot keep it.”A couple of Romanian Hutsuls reflected upon our question regarding agricultural changes in the last few decades: “Agriculture has changed since our youth. It has changed a lot […]. Now they make much more hay, but the grass lasts, which in the past did not happen because everyone here had livestock, much more than now since there are no animals, because milk is paid almost nothing, and so why keep a cow if the milk… [is paid nothing]” . However, they also proudly stated, “Milk here is natural, from flowers, cows graze on flowers and also eat hay. The milk is fatty, it is good.” Finally, a Romanian teacher living in Romania claimed, “I have had livestock, but my children have left, parents have passed away, and working and caring for animals is hard, so I gave it up,” and then she continued counting the number of livestock in the village: “For instance, my grandparents had a lot of livestock, sheep and cows, and in summer they used to go to the mountain pastures, but now I think that in the entire village only five people go to the mountains. Also, the animals are few: we had about 50 animals, the same number as our neighbors, but now, in all the area, there are barely 10.”In the Dzukija region, the respondents noted that cows grazed everywhere in the village in the recent past [1980–1990s]. “And only this year locals killed the last cow” (Lithuanian woman living in Belarus woman born in 1939). Now, mostly older people keep at the most chickens, although, in comparison with the past, they had a rather large farm. Thus, a respondent from the Belarusian side of Dzukija noted that in her youth, she and her husband kept three cows, three pigs, a horse, a lot of chickens, geese, and sheep, and now she only has three chickens (Lithuanian woman living in Belarus woman born in 1946).The most evident difference in livestock keeping was in Poland, where the Lithuanian minority are larger-scale farmers. They still keep many animals and sell the milk to the state. In Communist times, farming in Poland was nationalized only to a small degree. Just a small proportion of food production was state based, while the majority remained in private hands. This also meant that private farming in Poland was highly fragmented, as the state did not encourage mergers and land acquisition.Our results showed that the four study regions located in Eastern Europe do not present similar ethnoveterinary knowledge trajectories. Bukovinian Romanians and Hutsuls appear to hold a living reservoir of ethnoveterinary knowledge, unlike the other regions as summarized in polonyna (summer pastures) or send their animals out with other shepherds. However, long-term (pacific) coexistence of Hutsuls with other ethnic groups, could have facilitated the sharing of their knowledge of animal breeding  among Romanians living in neighboring pre-Carpathian areas. Therefore, the mountainous nature of the area could be a key element in the resilience of ethnoveterinary knowledge, which has also been confirmed by the intrinsic relationship found between pastoralists and European mountains  and andpolons  and hass [e.g., . Other dSuch everyday practices in animal breeding (milking and cheese-making) are still currently present and so is the veterinary knowledge connected to them. Indeed, as observed by Warchalska-Troll and Troll , CarpathIn Karelia, agriculture has been marginalized, especially in terms of the area where hay is still cut and the number of natural pastures, due to the declining number of cattle and the intensification of dairy farming –45. We cIn Setomaa  and Dzukija, we recorded more uses, but mainly in the past. We can say that, in these territories, livestock were quite important in the recent past, but their importance has declined in the last few decades [e.g.,  for BelaThe process of knowledge erosion may be occurring as a result of different socio-economic changes. First, in both regions, the aging of the rural population has contributed to the abandonment of small-scale livestock breeding –53. SecoIn addition, some local drivers of decline of ethnoveterinary knowledge can be identified. For instance, in Russian Setomaa, one possible driver could be related to the drastic political and economic changes in the region. Indeed, during Soviet times, animal husbandry accounted for a relevant proportion of food production by ruble value, while after 1991 livestock herds declined precipitously and the number of livestock raised by households continues to decline , 58. AlsLinum usitatissimum and the wild Hypericum spp., Quercus robur, Alnus spp, and Rumex spp. were reported across several countries. As five out of the eight studied countries were part of the Soviet Union, we compared our field data against the uses recommended in three popular veterinary medicine books published in 1919 (Gurin), 1988 (Rabinovich), and 2007 (Korobov), representing the commonly used remedies in Imperial Russia, as well as during the Soviet and post-Soviet periods. Finally, we traced the possible sources of veterinary knowledge related to those plants listed in Bloshenko et al.  that were used in the cycle of fabric production from flax. Older informants could remember bringing flaxseed to the commonly accessible mills to produce flaxseed oil.We found that the seeds of athogens . These uathogens , Rabinovathogens , and Korathogens , althoug in rats  due to t in rats . Gurin , but they traced such a use back to the medieval European medicinal tradition. The use of Quercus robur was also recorded in Gurin . Although it was not explicitly stressed by our interviewees, we can see that some of our findings reflect the official medicinal and veterinary recommendations of the Soviet Union, possibly through the formation of veterinary specialists who actually treated the animals. The recommendations themselves relied on veterinary knowledge derived partially from ethnoveterinary knowledge that underwent scientific tests, so the local uses supported by the official veterinary medicine had a greater chance for survival.Future research should investigate the heterogeneous trajectories that ethnoveterinary knowledge takes under different drivers of change to pastoral systems, and the complex networks of sources from where farmers derive their ethnoveterinary knowledge. Additionally, it would be interesting to evaluate the effects of the use of plant-based ethnoveterinary remedies in accordance with current legislation in Europe.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ca' Foscari University of Venice Ethics Committee. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.GM, GV, and RS conceived the study. NK, VK, OB, RS, RK, JP, NS, and GM gathered the data. RS, AP, and GV supervised the study. GM analyzed the data with the help of the co-authors. GM drafted the first version of the manuscript which was then reviewed, edited, and approved by OB, RK, VK, NK, JP, NS, AP, GV, and RS. All authors contributed to the article and approved the submitted version.This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No. 714874).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.


Exosomes (or extracellular vesicles) are known to mediate intercellular communication and to transmit molecular signals between cells. Molecules carried by exosomes have their own molecular roles in affecting surrounding and distant environment, as well as recipient cells. Molecular components of exosomes can be used as cancer biomarkers for diagnosis and prognosis, being promising therapeutic targets for the interruption of cellular signals. Therefore, the understanding of the molecular compositions and their functional indications of exosomes has the potential to help doctors to diagnose and monitor diseases and to allow researchers to design and develop potential targeted therapies. This review aims to provide a comprehensive protein and lipid characterization of lung cancer exosomes and to explore their molecular functions and mechanisms regulating physiological and pathological processes. This organization offers informative insight for lung cancer diagnosis and treatment.Exosomes participate in cell–cell communication by transferring molecular components between cells. Previous studies have shown that exosomal molecules derived from cancer cells and liquid biopsies can serve as biomarkers for cancer diagnosis and prognosis. The exploration of the molecules transferred by lung cancer-derived exosomes can advance the understanding of exosome-mediated signaling pathways and mechanisms. However, the molecular characterization and functional indications of exosomal proteins and lipids have not been comprehensively organized. This review thoroughly collected data concerning exosomal proteins and lipids from various lung cancer samples, including cancer cell lines and cancer patients. As potential diagnostic and prognostic biomarkers, exosomal proteins and lipids are available for clinical use in lung cancer. Potential therapeutic targets are mentioned for the future development of lung cancer therapy. Molecular functions implying their possible roles in exosome-mediated signaling are also discussed. Finally, we emphasized the importance and value of lung cancer stem cell-derived exosomes in lung cancer therapy. In summary, this review presents a comprehensive description of the protein and lipid composition and function of lung cancer-derived exosomes for lung cancer diagnosis, prognosis, and treatment. Extracellular vesicles (EVs) and exosomes mediate cell–cell communication within the tumor microenvironment and regulate signaling pathways in their target cells ,2. ExosoThe diagnostic and therapeutic potential of exosomes allows for exosomes to possess multiple clinical applications . SeveralAccording to the World Health Organization (WHO), cancer is one of the leading causes of death, and lung cancer is the most common cause of cancer death, accounting for 1.8 million deaths in 2020 ,46. TherExosomes play an important role in cancer progression, and therefore the identification of crucial molecular targets involved in exosome-mediated cell signaling is expected to facilitate tumor growth and advancement . MoreoveCancer cells are abnormal cells that rapidly grow in an uncontrolled manner. These rapidly dividing and uncontrolled cells eventually form a mass of abnormal tissue and tumors. Tumors, stromal cells, and the extracellular matrix (ECM) form a tumor microenvironment that is suitable for tumor growth. Tumors are heterogeneous because they are composed of several cell types, including cancer cells and cancer stem cells (CSCs) . Cancer Cells within the tumor microenvironment can directly engage in crosstalk with each other through cell–cell contact, as well as indirectly through cell-secreted soluble factors and cell-derived vesicles . Direct Exosomes have been reported to be derived from several types of body fluids  and cell types  . ExosomeThe formation of ILVs (and subsequently MVBs) is driven by the endosomal sorting complex that is required for transport (ESCRT)-dependent and/or ESCRT-independent mechanisms . The ESCCells internalize exosomes via several routes: endocytosis, macropinocytosis, phagocytosis, and membrane fusion . The endLung cancer samples can be obtained from cancer cell lines and cancer patients. For cancer cell lines, cell culture medium (serum-free medium or exosome-depleted medium) has been used for exosome extraction. For cancer patients, liquid biopsies, such as blood, urine, and saliva, can be sampled, and plasma or serum can be further separated from blood. Cell-released exosomes can then be extracted from cell culture conditioned medium and patient biopsies. On the basis of their physicochemical and biochemical properties, exosomes can be isolated via ultracentrifugation-based platforms , size-based platforms , immunoaffinity-based platforms, polymer-based platforms, and microfluidics-based platforms . GuideliExosomes are cell-derived vesicles, which implies that the composition of the exosomes reflects the composition of the cells from which they are derived; therefore, the exosomal protein composition can vary by cell source . The incThe composition of exosomal proteins reflects the encapsulation of proteins originating from the cytosol and membranes of donor cells. A common set of proteins is carried in most exosomes, regardless of their cellular origin ,95,96. CIn addition to the common set of proteins, certain proteins can be found in cell type-specific exosomes. Omics data provide an overview of molecular profiles, which helps to characterize the molecular functions and mechanisms of the regulation of biological processes. Proteomics enables a comprehensive and systematic search for protein profiles. Furthermore, exosomal protein characterization has been performed with many body fluids and cell types. An extensive proteomic characterization of exosomes released from breast cancer cells  has led to the identification of proteins related to cancer cell growth, progression, migration, metastasis, and immune evasion . Ji et aSeveral exosomal proteins of lung cancer have also been characterized. We summarized the proteins that were identified in exosomes from lung cancer cell lines and patient biopsies, including plasma, serum, urine, saliva, bronchoalveolar lavage (BAL) fluid, pleural effusion, pericardial effusion, and tumor tissue. We not only included all of the major proteins identified in the original literatures but also highlighted some minor proteins mentioned in their original papers. Minor proteins could also be potential biomarkers for lung cancer. Moreover, multimarker combinations are valuable for classification prediction and could be options in addition to single markers for cancer diagnosis. Exosomal proteins and protein combinations are comprehensively organized to display their sample sources and potential applications for lung cancer . The sigFourteen proteins  were expressed in exosomes of the mouse lung cell line 3LL (Lewis lung carcinoma) with and without heat-stressed treatments . Heat stEGFR was found in microvesicles (MVs) from the human cancer cell line A431 (skin epidermoid carcinoma), as well as from A459 (lung carcinoma) and DLD-1  cell lines . ThroughAREG has been shown to be enriched in exosomes from two lung cancer cell lines (CRL-2868 and A549) and enriched in exosomes from a prostate cancer cell line (PC3) and a breast cancer cell line (MDA-MB-231), compared to their original cells . AREG haEighteen proteins  have been identified in exosomes from the serum samples of lung cancer patients, including adenocarcinoma (AC) and squamous cell carcinoma (SCC) . On the EV arrays, which represent a new application of protein microarrays printed by customized capturing antibodies, have been developed to capture and detect exosomes from unpurified samples, including plasma and cell culture supernatants . EV arrahttps://www.hprd.org/) (accessed on 27 December 2021), Park et al. found 264 MV proteins were derived from lung tissue, and 67 out of them were commonly identified in all three patients, including ANXA4, AQP1, RAS proteins, and CEACAM6. This study explored a pool of MV proteins and suggested that they could be diagnostic biomarkers and therapeutic targets. Several proteins were identified to be significantly enriched in exosomes from A549 cells (NSCLC cell line) and HCC827 cells (NSCLC cell line), compared to exosomes from HBE4 cells  [https://tcga-data.nci.nih.gov/) (accessed on 27 December 2021) data analysis, an OS prediction suggested that HGF is a promising biomarker for late stage cancer or poor prognosis. Further experiments showed that sEV-HGF could activate the HGF/c-Met signaling pathway to promote cancer cell metastasis. Exosomes released from A549 lung cancer cells under four treatment conditions  had different degrees of impact on target cells  [A proteomic analysis showed the potential to discover diagnostic and prognostic biomarkers for lung cancer ,124,125.ll line) . Proteinll line) . Via thell line) . Comparill line) . When lull line) . ProteinTGF-β and IL-10 were expressed in exosomes of NCI-H1688 cells (SCLC cell line) and NCI-H2228 cells (NSCLC cell line) . When coVIM, an intermediate filament protein, has been found in exosomes from PC14 (nonmetastatic) and PC14HM (highly metastatic) lung cancer cell lines . Both thThirteen proteins  were enriched in plasma-derived EVs of lung AC patients, with concordant expression in EVs of lung AC cell lines  . The comTim-3 and galectin-9 have shown significantly elevated expression in plasma-derived exosomes of NSCLC patients  and lung SCC patients (compared to lung AC patients) . Both exThe expression of LBP in serum-derived exosomes from NSCLC patients was observed to be significantly higher than that in exosomes from healthy individuals . FurtherBoth AHSG and ECM1 have been shown to have significantly higher expression in serum-derived exosomes of NSCLC patients than in those of healthy individuals and were proposed to be diagnostic biomarkers for NSCLC . The diaNineteen proteins  were more than fivefold more highly expressed in exosomes of NCI-H838 cells (NSCLC cell line) than in the cellular membrane of NCI-H838 cells . These pIntegrins α3 and β1 have been shown to be expressed in exosomes from four lung AC cell lines  and one NSCLC patient pericardial effusion sample . This stLRG1 has been detected in exosomes from urine samples of NSCLC patients without chemotherapy . The proThe protein expressions of HLA-class I, BAGE, PD-L1, and annexin-A2 have been shown to be elevated in exosomes from BAL fluids of smokers and NSCLC patients, compared to the expression in exosomes of healthy individuals . In theiPD-L1 has been found in exosomes from melanoma cells, as well as from lung  and breast (MDA-MB-231) cancer cells . MoreoveThe expression levels of seven proteins ) were found to be significantly increased in serum-derived EVs of lung cancer patients, including AC, SCC, and small cell lung cancer (SCLC) . FurtherKRAS mutation), H1299 (with the neuroblastoma RAS (NRAS) mutation), PC9 (with the EGFR mutation), H1650 (with the EGFR mutation), and H522 (with the TP53 mutation), but they were not identified in exosomes from human pulmonary alveolar epithelial cells (HPAEpiCs), thus indicating that the five proteins were lung cancer exosome-specific proteins [TUBA1C, GAPDH, KRT25, GCC2, and POTEKP were collectively identified in exosomes from five NSCLC cell lines, including A549  were detected in serum samples from NSCLC patients and healthy individuals . RNA expProteins in lung cancer-derived exosomes are diagramed to illustrate promising usage as diagnostic and prognostic biomarkers or therapeutic targets for lung cancer therapy .Exosomes perform their communication effects by transmitting their molecular cargo from donor cells to recipient cells . TherefoA well-known protein classification system known as PANTHER organized protein classes on the basis of protein functions and included proteins associated with each class ,152. In Proteins and their functions are briefly summarized according to the following classes: (1) tetraspanin family proteins , (2) exosome formation-/secretion-required proteins , (3) chaperones , (4) structural proteins , (5) cell–microenvironment interactors , (6) enzymes and enzyme modulators , (7) signaling proteins , and (8) immunoregulatory proteins .Within the tetraspanin family, CD9, CD63, and CD81 are commonly used as exosome markers. Tetraspanins, which are membrane proteins with four transmembrane domains, are known to possess multiple functional roles in several biological processes, such as cell adhesion, fusion, signaling, and trafficking . EvidencRABs, which are in the GTPase family, are known to modulate membrane trafficking, with roles in endocytosis, exocytosis, and exosome secretion ,165. AnnSeveral types of enzymes  and enzAlthough some proteins were not assigned to suitable protein classes according to the PANTHER classification system, they still have important molecular functions involved in several biological processes. For example, GRB2 is a pivotal protein in signal transduction . Upon thExosomes are released by the fusion of MVBs with the cell membrane, thus rendering exosome lipid-enriched membrane structures. Exosome release relies on not only proteins but also lipids that participate in exosome biogenesis and secretion . AlthougThe lipid bilayer membrane of exosomes contains various lipids to maintain exosome structure and to protect the protein and nucleic acid contents . ExosomaLipidomic profiles can reveal the lipid landscape of exosomes. For instance, Llorente et al. quantified 277 lipid species in the membranes of PC-3 cells (human prostate cancer cells) and in the exosomes they release, and they found that glycosphingolipids, SM, cholesterol, and PS were highly enriched in PC-3 cell-derived exosomes, which demonstrated a particular pattern of lipid sorting into these exosomes . NotablyIn a lung cancer study, Fan et al. explored the lipid profiles of blood plasma exosomes and distinguished 39 normal donors and 91 NSCLC patients (including 44 patients in early stage cancer and 47 patients in late stage cancer) . Lipid fThe membrane component SM helps to maintain membrane integrity and stability and is enriched in lipid rafts that help to regulate cellular signaling . DynamicCancer tumors grow in a complicated tumor microenvironment with multiple types of cells, such as cancer cells, CSCs, cancer-associated fibroblasts (CAFs), immune cells, and other normal cells . ConventAs CSCs play important roles in tumor recurrence and drug resistance, they may transmit crucial signaling to other cells via exosomes. EV-mediated crosstalk between CSCs and molecules in the tumor microenvironment can influence the fate of normal and tumor cells, thereby regulating solid tumor progression . HoweverSeveral exosomal miRNAs from various CSCs  have been found . A few mExosomes are cell–cell communicators within the tumor microenvironment. The molecular cargo carried by exosomes represents the content of the cells of origin and shows functional potential for cancer cell-mediated signaling. The identification of the molecular components of exosomes can help to pinpoint a cell–cell communication mechanism and may eventually lead to the identification of potential diagnostic and prognostic biomarkers for cancer detection and disease staging.This review presents current potential exosomal proteins and lipids for the development of lung cancer detection, diagnosis, treatment, prognosis, and follow-up monitoring. We organized lung cancer-derived exosomal proteins and lipids from the available literature, regardless of sample source. Exosomes were isolated and purified from various lung cancer sample sources, including lung cancer cell lines and liquid biopsies, such as blood (plasma and serum), urine, and saliva.Evidence has shown that exosomal protein and lipid cargos contain components necessary for exosome formation, structural components related to the cytoskeleton, and signaling molecules for intercellular communication and regulation. Proteins participate in several biological processes through different pathways and networks to regulate other molecules and to exert their own molecular functions. Enzymes and enzyme modulators help substrates to transform to active or inactive forms to control molecular transformation and metabolism during bioactive signaling. Structural proteins maintain cell structure and are packaged into exosomal cargo; they may also play versatile functional roles in mediating receptor dynamics and organization. Moreover, tetraspanin family proteins play multiple roles in the regulation of several biological processes, thus leading to changes in tumor cell growth, invasiveness, and metastatic capacity. Exosome formation-/secretion-required proteins participate in the process of exosome biogenesis and release and are involved in exosome internalization into target cells. Additionally, cell microenvironment interactors coordinate cell interactions and communication with the external environment to form a suitable niche for cell adhesion and migration. Signaling proteins transmit signals to target cells to modulate recipient cell molecular networks and can consequently affect their cellular behavior. Furthermore, chaperones protect proteins from misfolding and are important to cancer development and progression. In summary, several pathways are involved in transmitting signals from donor cells to recipient cells via exosomes, including the endocytic pathway; endosomal sorting- and transportation-related interaction pathways for cargo packaging and exosome release; exosome- and microenvironment-related pathways that promote cell adhesion and targeting; and pathways involved in exosome uptake that enable signal transduction and the promotion of enzymatic activity within target cells. In contrast, lipids not only play structural roles in maintaining exosomal membrane structure but also serve functional roles in exosome biogenesis and uptake, as well as signal modulation. The lipid bilayer membrane structure of exosomes functions as a protector of exosomal protein and nucleic acid contents.Although exosomes can be released from multiple types of cells within the tumor microenvironment, it is challenging to discriminate the original cell source of exosomes. The CSC subpopulation contributes to tumor progression and is an important source of exosomes. Nevertheless, to the best of our knowledge, after a comprehensive literature review, few or no studies have discussed the proteins and lipids of lung CSC-derived exosomes. The exploration of proteins and lipids, as well as miRNAs, in lung CSC-derived exosomes for lung cancer therapy is still an unmet need. Accordingly, we highlighted the importance of CSCs and CSC-derived exosomes in cancer therapy and listed feasible strategies to obtain CSCs for exosome study.Cancer cells and CSCs transmit tumor cell signals by packaging essential molecular components into exosomes that are carried to target cells and the tumor microenvironment, wherein the delivered exosome components can change cellular behavior and form an educated niche suitable for tumor growth and progression. Therefore, the characterization of the molecular components and functions of cancer cell- and CSC-derived exosomes helps to understand exosome-associated molecular mechanisms and to discover potential drug(s) for blocking cancer cell- and CSC-derived molecular signals.In summary, we elaborated on the comprehensive protein and lipid composition and function of lung cancer-derived exosomes. Exosomal molecules could be potential diagnostic and prognostic biomarkers and therapeutic targets for drug development. This review provides a molecular foundation for future studies and applications in exosome-based cancer diagnosis, prognosis, and treatment, especially for lung cancer.


Lung cancer is one of the most common malignant tumours worldwide. however, emerging immunotherapy and targeted therapies continue to show limited efficacy. In the search for new targets for lung cancer treatment, exosomes have become a major focus of research. Exosomes play an important role in the tumour microenvironment (TME) of lung cancer and affect invasion, metastasis, and treatment responses. This review describes our current understanding of the release of exosomes derived from different cells in the TME, the effects of exosomes on T/Tregs, myeloid-derived suppressor cells, tumour-associated macrophages, dendritic cells, and natural killer cells, and the role of exosomes in the endothelial–mesenchymal transition, angiogenesis, and cancer-associated fibroblasts. In particular, this review focuses on the potential clinical applications of exosomes in the lung cancer microenvironment and their prognostic and diagnostic value. Lung cancer is one of the most commonly diagnosed cancer and the leading cause of cancer deaths in both sexes combined , despiteTo find effective treatments and overcome low immunotherapy efficiency and drug resistance, increasing research has focused on the lung cancer microenvironment. The vasculature, immune and inflammatory cells, extracellular matrix (ECM), and cancer-associated fibroblasts (CAFs) are major components of the tumour microenvironment (TME), which is recognised as a target-rich landscape for the development of novel agents in lung cancer , 5. InteAmong these functional mechanisms, exosomes carrying large amounts of information and molecules play an important role in intercellular communication and are indispensable mediators of various processes in the TME . ExosomeExosomes transport proteins (cytosolic and transmembrane proteins), lipids, and nucleic acids  to target cells, thus regulating their behaviour –10. ExosExosomes form by inward budding in the plasma membrane, and are classified as early and late endosomes, known as multivesicular bodies. Numerous intraluminal vesicles form by the invagination of multivesicular body membranes. Exosome release is regulated by a number of genes and proteins. For example, miR-134 and miR-135b precisely regulate YKT6 expression in lung cancer cells, which in turn controls exosome release . FurtherExosomes can be ingested by a variety of cells in the TME and exert functions via proteins, nucleic acids, and other substances. First, the recognition of exosomes requires membrane protein interactions. CD169 recognises exosomes and mediates the immune response to exosome antigens. The adhesion of B cell-derived and DC-derived exosomes is CD169-dependent . After rTumour-associated macrophages (TAMs) have been linked to lung cancer cell initiation, progression, and metastasis in the TME . The keyDendritic cells (DCs) play an essential role in the regulation of tumour-specific immune responses. DC-based immunotherapy is unsatisfactory due to the poor immunogenicity of cancer cells and low uptake efficiency of antigens, even though DCs are the most potent antigen-presenting cells . Lung tuNatural killer (NK) cells are independent, non-specific immune cells. They can directly kill tumour cells without MHC restriction to the target . However+CD25+Foxp3+ T regulatory cells (Tregs) and functional alterations of T lymphocyte subsets in the TME are critical for the immune escape of lung cancer cells [+ naïve T lymphocytes incubated with tumour-derived exosomes from mutant KRAS+/+ NSCLC cells induce Foxp3+ Treg generation by phenotypic switching. Foxp3 regulates Treg functions [The proportion of CD4er cells . Lung caer cells . PD-L1-mer cells , 51. In er cells . The traer cells . Epidermer cells . The oncunctions . This counctions .Myeloid-derived suppressor cells (MDSCs) are divided into the following two groups: M-MDSCs, which are morphologically similar to monocytes, and PMN-MDSCs, which resemble polymorphonuclear cells . MDSCs rZEB1 mRNA is a major EMT transcription factor in mesenchymal cells in NSCLC. Oncogenic exosomes derived from mesenchymal NSCLC cells can transfer chemoresistance and mesenchymal phenotypes to recipient cells by ZEB1 mRNA in exosomes [Epithelial-mesenchymal transition (EMT) is a key mechanism for initiating lung cancer cell invasiveness and metastasis . Exosomeexosomes . These rexosomes . Exosomaexosomes . Increasexosomes . These rCDH1 encoding E-cadherin and VIM encoding Vimentin [hTERT mRNA has been detected in exosomes isolated from sera of patients with lung cancer. The transfer of exosomal telomerase from cancer cells into fibroblasts may contribute to alterations in the TME [Cancer-associated fibroblasts (CAFs) are a major cellular component of TME in most solid cancers. Lung cancer cells can transform the phenotype of fibroblasts via exosomes and related factors. Exosome-associated miR-142-3p promotes the transformation of lung fibroblast cells to CAFs via TGF-β signalling . InteracVimentin . In termVimentin , 70 are Vimentin . In addiVimentin , 73. Sim the TME .Various studies have evaluated the role of hypoxia during interactions between immune cells and EMT components. Lung cancer-derived exosomes increase under hypoxic conditions and play a critical role in angiogenesis. miRNAs in lung cancer cell-derived exosomes have important functions under hypoxic conditions . ExosomaThe premetastatic TME (pre-metastatic niche) in lung cancer is an important cause of induction of metastasis. Primary lung cancer-derived exosomal RNAs promote neutrophil recruitment by activating TLR3 in lung epithelial cells via the NF-kB and MAPK pathways . TGF-β hTable The progression of multidrug resistance is the major obstacle to maintain effectiveness of chemotherapy in lung cancer . The TMEExosomes in the TME may be novel diagnostic and therapeutic targets for NSCLC. YKT6 in lung cancer cells regulates exosome release. A clinical study has shown that in NSCLC, YKT6 in tumour samples is associated with a shorter disease-free survival and overall survival .Various lncRNAs have been identified as promising therapeutic targets. MiR-96 is a candidate serum biomarker and therapeutic target for NSCLC. Melanoma differentiation-associated gene-9 (MDA-9)/Syntenin is another therapeutic target for lung adenocarcinoma; this locus promotes cancer invasion and metastasis as a key regulator of Slug and Slug-mediated EMT . NSCLC cBCL2L1 could be a useful biomarker for poor survival in patients with lung adenocarcinoma [Exosomes are promising tools for tumour diagnosis and treatment , 8. Micrarcinoma . A largearcinoma . Exosomaarcinoma . Anotherarcinoma . High learcinoma . Exosomearcinoma , 107.Table Liquid biopsy is a diagnostic procedure that describes information about cancer-derived substances obtained from blood sample. The sample information of the liquid biopsy mainly comes from: circulating tumour cells (CTCs) of blood sample, circulating cell-free DNA (cfDNA) released into the blood from tumour cells and normal cells and cell-free RNA (cfRNA) enriched in exosomes from tumour cells . It makePD-L1 in serum exosomes can be used as a quantitative factor for tumour PD-L1 status, which may be helpful in predicting the clinical outcome of anti-PD-1 therapy in NSCLC patients . With thFunctional studies of non-coding RNAs and proteins in exosomes provide new insights into reshaping the TME. In terms of clinical application, a large number of non-coding RNAs and proteins have been found in exosomes, which are expected to become an indispensable tool for the diagnosis and prediction of lung cancer in clinic. However, there remains a lack of clinical studies with large samples to provide evidence support. It is particularly important to identify the precise components that act key roles in tumour processes. Ongoing experimental and clinical studies of exosomes may provide new ideas for improvement of TME and treatment of lung cancer.


Using this system, we demonstrated that both JEV RP9 and SA14-14-2 are able to cross the BBB without disrupting it at early times post viral addition. Furthermore, we find that almost 10 times more RP9 infectious particles than SA14-14 cross the model BBB, indicating this BBB model discriminates between the virulent RP9 and the vaccine SA14-14-2 strains of JEV. Beyond contributing to the understanding of early events in JEV neuroinvasion, we demonstrate this in vitro BBB model can be used as a system to study the viral determinants of JEV neuroinvasiveness and the molecular mechanisms by which this flavivirus crosses the BBB during early times of neuroinvasion.Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in South East Asia. It has been suggested that, as a consequence of the inflammatory process during JEV infection, there is disruption of the blood-brain barrier (BBB) tight junctions that in turn allows the virus access to the central nervous system (CNS). However, what happens at early times of JEV contact with the BBB is poorly understood. In the present work, we evaluated the ability of both a virulent and a vaccine strain of JEV  to cross an  Flaviviruses such as Japanese encephalitis virus (JEV) are arthropod-borne viruses (arbovirus) that are transmitted through the bite of an infected mosquito and may cause serious human diseases [diseases . JEV is diseases . About 3diseases , as welldiseases , 6. Althdiseases , suggestFlavivirus population is not clonal, but rather a mix of multiple viral genomic species (aka quasispecies) [JEV has a positive-sense RNA genome encoding a single polyprotein flanked by two untranslated regions (UTR) at the 5’ and 3’ ends. This polyprotein is co- and post-translationally cleaved into three structural proteins  involved in viral particle assembly and antigenicity and seven non-structural proteins  involved in genome replication, viral particle assembly and evasion of innate immunity [species) , 9.in vivo in murine and simian models. Expression levels of tight junction proteins involved in maintaining BBB functions such as occludin, claudin-5 and zonula occludens 1 (ZO-1) are significantly decreased in symptomatic JEV-infected mice, suggesting physical disruption of the BBB [JEV is a neuroinvasive and neurovirulent virus. It is associated with neuroinflammation of the central nervous system (CNS) , and dis the BBB . It seem the BBB  and that the BBB –13, whic the BBB . Vesicul the BBB , but it In contrast to virulent JEV strains such as RP9, the vaccine strain SA14-14-2 was shown to be essentially non-neuroinvasive and non-neurovirulent in weanling ICR mice, but is still highly neurovirulent in neonates. The JEV SA14-14-2 genome contains 57 nucleotide differences positioned along the genome when compared to the parental strain SA14, leading to 25 amino-acid substitutions . MutatioAlthough encephalitis incidents have occurred after vaccination with the SA14-14-2 JEV strain, no virus could be recovered from them . Whetherin vitro. hCMEC/D3 monolayers displays good restricted permeability to paracellular tracers and retains most of the transporters and receptors present on in vivo BBB [The BBB is the physical and physiological barrier between the brain and the blood compartments in vertebrates, and it is comprised of a network of different cell types including the brain microvascular endothelium along with pericytes, astrocytes, microglia and the basement membrane . Severalvivo BBB . Accordivivo BBB , 26.in vitro human BBB model consisting of hCMEC/D3 human endothelial cells cultivated on permeable supports above SK-N-SH human neuroblastoma cells to evaluate and compare the ability of both a virulent and a vaccine strain of JEV  to cross this BBB model.In the present study, we used an Cercopithecus aethiops monkey kidney Vero cells were maintained at 37°C in DMEM supplemented with 5% FBS. Aedes albopictus mosquito cells C6/36 were maintained at 28°C in Leibovitz medium (L15) supplemented with 10% FBS.Human endothelial cells hCMEC/D3 , were ma® (JEV SA14-14-2) vaccine was kindly provided by Dr. Philippe Dussart , and reconstituted with 500μL of DMEM. Two hundred and fifty μL of reconstituted vaccine were used to infect Vero cells for 7 days. Viral supernatants were collected and used to infect C6/36 cells cultivated in EndoGro medium supplemented with 2% FBS. Both JEV RP9 and SA14-14-2 viral supernatant stocks were collected 3 days after infection and the infectious titer was determined in Vero cells by focus-forming assay (see below).A molecular cDNA clone of JEV genotype 3 strain RP9 was kindly provided by Dr. Yi-Ling Lin . This plFlavivirus E protein were purchased from the ATCC , and a highly-purified antibody preparation was produced by RD Biotech . Mouse monoclonal anti-JEV NS5 antibody was kindly provided by Dr. Yoshiharu Matsura [Mouse hybridomas producing the monoclonal antibody 4G2 anti- Matsura . Horsera4/well) were seeded on 12-well Transwell® permeable inserts  in EndoGro medium supplemented with 5% FBS and placed at 37°C for 5 days. SK-N-SH cells (2.105/well) were seeded in 12-well tissue culture plates in EndoGro supplemented with 2% FBS. Permeable inserts containing hCMEC/D3 cells were then transferred in these culture plates and medium was replaced by EndoGro medium supplemented with 2% FBS. Aliquots of virus were diluted the next day in 50μL of EndoGro medium supplemented with 2% FBS, heated at 37°C and then added to the cells. Cells were incubated at 37°C until collection.hCMEC/D3 cells (5.105/well). Ten-fold dilutions of virus samples were prepared in DMEM and 200μL of each dilution was added to the cells. The plates were incubated for 1h at 37°C. Unabsorbed virus was removed and 800μL of DMEM supplemented with 0.8% carboxymethyl cellulose (CMC), 5 mM HEPES buffer, 36 mM sodium bicarbonate, and 2% FBS were added to each well, followed by incubation at 37°C for 48h for JEV RP9 or for 72h for JEV SA14-14-2. The CMC overlay was aspirated, and the cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min, followed by permeabilization with 0.1% Triton X-100 for 5 min. After permeabilization, the cells were washed with PBS and incubated for 1h at room temperature with anti-E antibody (4G2), followed by incubation with HRP-conjugated anti-mouse IgG antibody. The assays were developed with the Vector VIP peroxidase substrate kit  according to the manufacturer’s instructions. The foci were then counted in each well manually. The viral titers were expressed in focus-forming units (FFU) per milliliter.Vero cells were seeded in 24-well plates  supplemented with 10 mM of HEPES buffer, 1 mM of sodium pyruvate and 50μM of LY . The culture medium inside the Transwell® inserts was replaced with 500μL of HBSS buffer containing 50μM of LY. Cells were incubated at 37°C for 10 min. Permeable inserts were then transferred in culture well containing 1.5 mL of HBSS buffer and incubated at 37°C for 15 min. They were then transferred in culture well containing 1.5 mL of HBSS buffer and incubated at 37°C for 20 min. Concentrations of LY in the wells were determined using a fluorescent spectrophotometer . The emission at 535 nm was measured with an excitation light at 485 nm. The endothelial permeability coefficient of LY was calculated in centimeters/min (cm/min), as previously described [LY dye migration through the BBB monolayers was performed as previously described , 26. Briescribed .5) were seeded on coverslips in 24-well tissue culture plates in EndoGro medium supplemented with 5% FBS. After 5 days, cell medium was replaced with 1 mL of EndoGro medium supplemented with 2% FBS. SK-N-SH cells (105) were seeded on coverslips in 24-well tissue culture plates in DMEM supplemented with 2% FBS. Aliquots of virus were diluted in 200μL of medium and added to the cells. Plates were incubated for 1h at 37°C. Unabsorbed virus was removed and 1mL of EndoGro or DMEM supplemented with 2% FBS was added to the cells, followed by incubation at 37°C until collection.hCMEC/D3 cells . The slides were examined using a fluorescence microscope (EVOS FL Cell Imaging System).4/well) were seeded on 12-well Transwell® insert filters in EndoGro medium supplemented with 5% FBS for 5 days. SK-N-SH cells (2.105/well) were seeded in 12-well tissue culture plates in EndoGro supplemented with 2% FBS. Transwell® containing hCMEC/D3 cells were then transferred in these culture plates and medium was replaced by EndoGro medium supplemented with 2% FBS. Cells were incubated at 37°C. At 24h post-contact, total RNA of hCMEC/D3 cells were extracted using NucleoSpin RNA kit  following the manufacturer’s instructions. Two hundred ng of total RNA were used to produce cDNA using the SuperScript II Reverse Transcriptase  according to the manufacturer’s instructions. Quantitative PCR were performed on 2μL of cDNA using SYBR Green PCR Master Mix  according to the manufacturer’s instructions. The CFX96 real-time PCR system (Bio-Rad) was used to measure SYBR green fluorescence with the following program: an initial PCR activation at 95°C (10 min), 40 cycles of denaturation at 95°C (15s) and annealing-extension at 60°C (1 min). Results were analyzed using the CFX Manager Software (Bio-Rad) gene expression analysis tool. GAPDH was used as the reference gene. Primers used in gene expression studies are listed in hCMEC/D3 cells  following the manufacturer’s instructions.Total RNA from JEV BBB-crossing samples was extracted using NucleoSpin®RNA kit  according to the manufacturer’s instructions. The number of JEV RNA copies present in BBB-crossing samples was determined by RT-qPCR using TaqMan® Fast Virus 1-Step Master Mix kit  according to the manufacturer’s instructions. The forward and reverse primers (Sigma-Aldrich®) were t test, Mann-Whitney test and ANOVA test corrected with Tukey method for multiple comparisons were used to compare experimental data. GraphPad Prism 7 was used for these statistical analyses. The significance level for our data was set to 5% or less (P ≤0.05).Unpaired two-tailed in vitro model to study JEV neuroinvasion should consist of two main components: 1) a cell monolayer mimicking the BBB, and 2) a brain tissue-derived cell line permissive to JEV. Based on our previous work [in vitro BBB model can be used as a tool to study the neuroinvasiveness of JEV.A basic ous work , we chos10 less at 24 and 48 hpi respectively, Independent reports have found that neuroblastoma-derived SK-N-SH cells are susceptible to both the virulent JEV RP9 strain and the SA14-14-2 attenuated strain , 35. To In order to examine the susceptibility of our hCMEC/D3 BBB model to JEV infection, the cells were grown 6 days on coverslips to allow the BBB to form, and then inoculated with either the RP9 or SA14-14-2 JEV strain . As evidIt has been suggested that JEV infects brain tissue cells as a consequence of a preceding inflammatory process which in turn leads to disruption of the BBB and viral neuroinvasion , 37. Our10 higher when a MOI of 10 was used in comparison to a MOI of 1  to cross the BBB at early times post-addition. We have shown that both JEV RP9 and SA14-14-2 are able to cross the BBB without disrupting it at 6 hpi. Our finding corroborates in vivo studies that have demonstrated that JEV is able to get access to the CNS and establish a primary infection without the preceding need of BBB leakage [In this study, we have used an  leakage , 37.in vivo in mice and monkeys [in vitro BBB model and JEV strains with different neuroinvasive capabilities(such as the ones used in this work) would be useful to identify which cellular mechanisms might be "hijacked" by these pathogens to cross the BBB.Moreover, the fact that both JEV RP9 and SA14-14-2 strains crossed the BBB without infecting BBB endothelial cells, or disrupting the barrier, also suggests that the pathway JEV uses to cross the BBB is either a transcellular one, through the endothelial cells, or paracellular, between the endothelial cells. These observations are consistent with other studies conducted  monkeys , 16, 41.Interestingly, although our specific infectivity data suggest that JEV RP9 infectious particles crossed the BBB more efficiently than those of the vaccine strain JEV SA14-14-2, comparison of hCMEC/D3 cell transcriptomes from BBBs that were exposed for 6h to either JEV RP9, SA14-14-2 or no virus showed no significant difference in the levels of gene expression . This suggests that an immediate or early cellular response is unlikely to be responsible for the differential BBB crossing of JEV RP9 versus JEV SA-14-14-2 particles we observed. Instead, we suspect specific viral factors to be at play, for example, interaction of the viral particle with a strain-specific cellular surface receptor for viral entry. Other considerations to pursue such as full characterization of the viral particles that are able to cross the BBB including, by deep-sequencing of their RNA genomes, and an electron microscopic examination of the endothelial cells forming the BBB after contact with either virus, could help to shed significant light on this intriguing difference.Interestingly, we found that hCMEC/D3 were permissive to both RP9 and SA14-14-2 strains only when the BBB formation was not completed. BBB formation induces changes in cell conformation, which can then lead to the relocation of cell receptors between BBB cells . Differein vitro model to characterize viral determinants of JEV neuroinvasiveness as well as a tool to study the molecular mechanisms by which these pathogens cross the BBB.In conclusion, our study demonstrates that both a virulent and a vaccine strain of JEV are able to cross a BBB model without disruption at early times post viral addition. This BBB formed by human endothelial cells represents a useful discriminant


We present a systematic framework to study the threshold contributions of the differential rapidity distribution for the production of any number of colorless particles in the hadronic colliders. This has been achieved based on the universality structure of the soft enhancements associated with the real emissions, along with the factorization property of the differential cross section and the renormalization group invariance. In this formalism, we present a universal soft-collinear operator to compute the soft virtual differential cross section for a generic  The upcoming era of high energy physics will confront a huge boom of data driven by the upgraded run of the Large Hadron Collider (LHC). The High-Luminosity LHC will also come into effect in a few years of time. In order to fully exploit the increased quantity of data which will not only help to detect a rare phenomena but also improve the precision, an enhanced amount of effort by the theoretical physicists will be called for. In improving theoretical precision, the higher order quantum chromodynamics (QCD) and electroweak (EW) corrections play an important role. Among different observables, since the differential cross-section allows a wider range of comparisons with the experimental data, over the past few decades several attempts have been made to incorporate the higher order QCD and EW radiative corrections to this observable. The topic of this article is concerning the differential cross-section with respect to rapidity, in particular, we address the question of computing the higher order QCD corrections to this observable for any generic process at a hadron collider with all the final state particles as colorless.Despite its high importance, unlike the inclusive cross-section, the differential rapidity distribution and its radiative corrections are computed only for a limited number of scattering processes. The rapidity distributions in Drell-Yan and of the scalar Higgs boson were computed to next-to-next-to-leading order (NNLO) QCD in Refs. , 2 and , respect in Ref. . Shortly in Ref.  in the fn denotes the number of final state colorless particles. We show that combining the virtual matrix element that captures the process dependence, the universal soft part and mass-factorization kernel in an elegant way, we can calculate the SV differential rapidity distribution for any generic Needless to say, achieving a full QCD correction to any order is not easy and with increasing perturbative order, the complexity level increase substantially which often prevents us from achieving it. In absence of the full QCD correction, it is often desirable to find an alternative method averting the full complexity to capture the dominant contribution. In this article, we discuss such a method, called soft-gluon or soft-virtual (SV) approximation. In the soft limit, the momenta of all the real emission diagrams are assumed to be infinitesimally small which leads to an all order exponentiation of this contribution. In Refs. , 8, a foIn the literature, several results for the rapidity resummation employing different methods are available. In Ref. , followi in Ref.  of Drellmentclasspt{minima in Ref. , which i in Ref. , 8, 14 r in Ref. , the mergoal of this article is to present the formal methodology of computing threshold rapidity corrections including NSV terms for any generic process of Over the past decade, the formalism  for the  In Ref. , the obs in Ref.  by incor in Ref. , 18, 76.hilation . The thrhilation  and Drelhilation  are alsoons, see –33 for mons, see , 35, 77,The paper is organized as follows: in section n-number of colorless particles in hadron collisions within the framework of perturbative QCD. Our prescription that we will develop subsequently to capture this contribution is within the scope of QCD improved parton model where the collinear divergences factorize to all orders in strong coupling constant n-number of colorless particles, denoted as X, we represent an inclusive hadronic state. z, are respectively defined as the ratios of invariant mass square of the final state to the square of hadronic (S) and partonic  that take part in the scattering at the partonic level as W. The remaining delta functions reflects the momentum conservation and also the rapidity of final state invariant mass system as defined by  contribution. In addition, we also discuss how the current formalism can be extended to study the next-to-soft virtual (NSV) contribution. In order to define the soft limit for the rapidity distribution, we choose to work with a set of symmetric scaling variables y and z, the n-number of final state colorless particles, the partonic coefficient function does, in fact, depend on the Mandelstam variables constructed out of all the independent external momenta which is concisely denoted through W with respect to the variables W satisfiesThe scattering matrix element or equivalently the partonic differential distribution can be perturbatively expanded in powers of strong coupling constant q2,y) in  can be rIn this section, we setup a framework to compute the soft-virtual corrections to the rapidity distribution to all orders in strong coupling constant. The infrared safe SV rapidity distribution can be obtained by combining the ultraviolet (UV) renormalized virtual matrix elements with the soft gluon contribution and performing appropriate mass factorization to get rid of initial state collinear singularities.It is well-known that the combined soft and collinear divergences, conveniently denoted as infrared (IR), in virtual matrix elements factorize from the corresponding UV renormalized part to all orders in perturbation theory and thereby in dimensional regularization we can writein Refs. –38. For  in Ref.  and subs in Ref. . For sca in Ref.  which wa in Ref. . An all  in Ref. , 44. An  in Ref. . We provBefore moving further, we wish to iterate a well-known fact. For processes involving only conserved operator, such as Drell-Yan, the coupling constant renormalization is sufficient to get rid of all the UV divergences. However, for other processes, such as the Higgs boson production in heavy quark effective theory, an additional renormalization which often called the operator renormalization is needed. This is a property inherent to the operator itself.In order to get the infrared safe and finite differential rapidity distribution, we need to combine the UV renormalized virtual matrix element to the real emission contributions in the soft limit and perform mass factorization which ensures the removal of collinear singularities arising from the initial state colored particles. Therefore, the universal nature of IR divergences in virtual matrix element implies that the combined contribution from the real emission diagrams and mass-factorization kernels must exhibit the same universality. By employing the criteria of universal IR structure and imposing the finiteness property of the rapidity distribution, we develop the prescription to compute the rapidity distribution under SV approximation for any generic In this article, we extend the prescription, which was introduced for the Sudakov type process in Ref. , to the mentclasspt{minimak-th order UV renormalized matrix element of the underlying partonic level process The pure virtual contribution is captured through the form factor aa¯sv in  becomes mentclass2pt{minimN of this relation we getgg are related by simple scaling of quadratic Casimirs. This essentially signifies the universality of the real emission in the soft limit i.e. it is independent of the details of the process, it solely depends on the nature of the external partons. Needless to say, it is also quark flavour blind. The relation in  and finite (fin) parts asΓaa¯,fin, can be rmentclasspt{minimaur loops –56 in QCur loops , 42 fromur loops , 58 throur loops 26\documein Refs. –61.We present the new results of imension –56 alongimension –66. The in Refs. , 68 whicin Refs. –66. For in Refs. , 69 and in Refs. . MoreoveIn Online Resource, the explicit expressions of all the anomalous dimensions including the QCD y, for the production of n-colorless particles which also paves the way for a wider range of comparisons with the experiments. Earlier we have seen that there exists an operator, differential soft-collinear operator, which embeds the universality of all the soft enhancements associated with the soft gluon emissions in the production of n-colorless particles in the hadronic collision. The universality lies in the fact that the operator, Here in this section, we develop the resummation formalism for the differential distribution with respect to the rapidity variable }Sd,I in , for thez and rapidity variable y and then the threshold limit is taken only for z, but for rapidity variable y only delta \documentn-colorless particles in the final state.We have also observed that the coefficients t}DdI in  is exprerelation . Hence w differential soft-collinear operator for the NSV terms turns out to be process dependent. To the end, we have shown that both SV and NSV logarithms can be systematically summed up to all orders both in Having obtained the results for the SV part of coefficient functions, namely hilation  and deeprocesses . Later trocesses . We consument}as , 35. In n-colorless particles. We restrict ourselves to the diagonal coefficient functions (dCFs). For the diagonal channels, it is straightforward to show that the NSV terms in dCFs arise only from diagonal partonic sub processes and diagonal part of mass-factorization kernels. The pure virtual part of the partonic sub-processes does not contain any NSV terms and hence can be factored out from them. Hence, in the mass factorization formula, we need to keep both SV as well as NSV terms in N space. To end, we obtaindifferential soft-collinear operatork by studying the dimensionally regularised Feynman integrals that contribute partonic cross sections in fixed order perturbation theory. We know that z space result in the case of SV part [In the following, we apply the same formalism to study the NSV logarithms present in the rapidity of a state of rd order , 54. Receduce to  with theithms in , 39) an ann-cologiven in , 54 and  SV part , 16, 19  time in , that arained in  using thained in  and alsoained in . For theained in  using thwith the  for termfound in . The genve loops –73. The found in . For comz space result for coefficient functions expressed in the integral representation in  initiated processes. For the NSV, we find that a part of the soft-collinear functions is partially determined by universal anomalous dimensions and the remaining part depends on the underlying hard process. However, for a given process, the resummed results either in z or In summary, thanks to the remarkable simplification of mass factorised formula for the rapidity distribution for diagonal channels and the knowledge of logarithmic structure of differential soft-collinear distribution and the mass factorization kernels from fixed order results, we can systematically resum both SV and NSV terms in n-number of colorless particles in the hadronic collision within the realm of perturbative QCD.In today’s era reducing the theoretical uncertainties remain one of the main motivations for higher order radiative corrections. It is also particularly relevant for constraining beyond SM scenarios and validation of the SM itself. Among several observables, differential cross-sections allow a wider range of comparisons with the experiment and hence several attempts were made in the past for better theoretical understanding of the same. In this article, we restrict ourselves to the discussion of differential rapidity distribution for the production of y, for the production of n-colorless particles in the hadron collider. The infrared structure of rapidity distribution which was earlier studied in Ref. [N-th Mellin moment of the differential distribution has a relation with its inclusive counterpart in the limit goal of this current article is to present the general structure for the SV differential rapidity distribution up to Through this publication, we intend to present a systematic framework for the study of soft-plus-virtual corrections to the differential distribution with respect to the rapidity variable  in Ref.  for Suda through . The mern-colorless final states, one merely requires the form factor corresponding to the hard process under study provided the soft-collinear distribution for Sudakov type process is known. We present the analytical results for the fixed order up to In summary, in order to obtain the fixed order as well as resummed prediction for the differential rapidity distributions of a generic