Thus, rational models need to be supplemented with a theory of contextual shifts. We review evidence in JDM that patterns of choices are often consistent within contexts, but unstable between contexts. We also demonstrate that for a limited, though reasonably broad, class of decision-making domains, recent theoretical models can be viewed as providing theories of contextual shifts. It is argued that one particular significant source of global inconsistency arises from a cognitive inability to represent absolute magnitudes, whether for perceptual variables, utilities, payoffs, or probabilities. This overall argument provides a fresh perspective on the scope and limits of human rationality.Rational explanation is ubiquitous in psychology and social sciences, ranging from rational analysis, expectancy-value theories, ideal observer models, mental logic to probabilistic frameworks, rational choice theory, and informal “folk psychological” explanation. However, rational explanation appears to be challenged by apparently systematic irrationality observed in psychological experiments, especially in the field of judgement and decision-making (JDM). Here, it is proposed that the experimental results require not that rational explanation should be rejected, but that rational explanation is  A paradox appears to lie at the hearts of psychological explanation. The explanation of thought and behaviour in rational terms is central to psychological theorising across a vast range of psychological domains and theories, ranging from the rational analysis of memory, categorisation or reasoning ,2 expectThis perplexing state of affairs has been a principal motivation behind empirical research on rationality for more than half a century, and a variety of theoretical directions has been proposed. One approach to reconciling “rational theory” with “irrational data” is to suggest that perhaps the data is not, on inspection quite as irrational as it may seem e.g., ,11,12)—p,12—p11,1A second perspective is to argue that it is our theories of rationality that need to be challenged and perhaps even abandoned . TheorieThis paper focuses on a “third way”: namely that rational explanation is of central importance to understanding the mind and cannot be discarded, but that such explanation is local to a specific context. From this point of view, rational explanation in psychology captures the inferential relations between the beliefs, values, and actions that are created within a context; and laboratory reasoning and decision-making “anomalies” show that beliefs, values, and actions are not stable between contexts. We will argue that relevant beliefs, values and actions are created on-the-fly in a given context . Whe. Whe20])From this perspective, then, preferences, beliefs, and actions are created piecemeal, as the context demands. Rational explanation as central to the process: it underpins the arguments that allow us to make such constructions. However, different contexts will prompt the construction of different preferences, beliefs or actions.Our argument proceeds as follows. In the next section, Rational Explanation, we review the wide variety of rational approaches to understanding psychological phenomena. We then argue, in Rationality is Local, that such explanation applies locally within contexts, rather than globally across a “context of all contexts” (if such a notion even make sense). We then illustrate this viewpoint, focusing on the study of choice. We introduce the issues in Local Rationality in Choice I: Background. Next, in Local Rationality in Choice II: Empirical Data, we stress that the empirical data indicate local rationality within a context, but not between contexts. Local Rationality in Choice III: Psychological Theories provides taxonomy of psychological models of choice, which indicates that there is a wide variety of ways in which the local nature of rationality can be captured, and, by capturing the aspects of the context, these models also provide accounts of how choices change when the context shifts. Finally, we draw out broader implications of this viewpoint in the Discussion.Rational choice theory, the dominant theoretical paradigm in many areas in the social sciences, is a framework for understanding and often formally modelling social and economic behaviour. The theory is often conveniently formulated to assume that actors have perfect information, a perfect grasp of their objectives, and the perfect ability to use that information to make decisions to further their objectives (see ). Many sIn the psychology of reasoning, the cognitive system is often seen as a logical inference machine. The paradigm example of such models is “mental logic” theories in the psychology of reasoning, which regard the syntactic proof theory for logic as the basis of the algorithms that implement logical inference in the mind e.g., ,26,27). ,27. 26,2In behavioural ecology, concepts from evolutionary biology predict how an animal should respond to risk if it is maximising its fitness. In this case, the normative approach  is unique in predicting a shift in a subject’s response to risk as a function of its resource budget . In animtheory of reasoned action, which was later superseded by the theory of planned behavior [theory of interpersonal behaviour [social-cognitive theory [expectancy-value models of attitudes, which are related to subjective expected utility in economics [In social psychology, a highly influential model of attitudes and behaviour, Fishbein and Ajzen’s  theory obehavior , the theehaviour , and thee theory , are allconomics . In modehealth belief model [protection motivation theory [A similar approach is taken in health psychology. According to the prominent ef model , people n theory  proposesdual-process models of reasoning and judgment in cognitive and social psychology still preserve rationality assumptions when behavior is understood as a function of two distinct processes or systems (or sets of systems): evolutionarily older ‘System 1’ processes described as automatic, uncontrolled, effortless, associative, fast, unconscious and affective, and more recently, characteristically human ‘System 2’ processes described as reflective, controlled, effortful, rule-based, slow, conscious and rational , ,39, dualnal . expected utility theory [Rational choice is the predominant paradigm in other social sciences such as economics, law, political science, criminology, sociology, and philosophy ,46,47,48y theory , which py theory ). The asy theory ,52. In pRational explanation may also be made more complex by allowing that agents may have imperfect cognitive abilities with which to process the available information. This dimension of “bounded rationality” ,54 is thBayesian inference ,71Bayesilocal rationality is the human tendency to make local, contextualised inferences, which could lead to global inconsistency in behaviours across contexts . This seems to reflect a basic property of human cognition that applies right across psychology, from the basic psychophysics of sound perception right through to high-level cognitive processes in judgement and decision-making. Here, we explore the basic underpinnings of this cognitive limitation and also reveal the implications for psychology, economics and public policy, where we suggest they raise important questions for the central methodologies used to measure and derive human preferences.Our key proposition is that, as a result of various cognitive, biological, and environmental limitations, locally—i.e., within a context, but not globally across contexts. It is as if we have a big library in our heads and we can only look at a few books at a time when we make up our mind on a specific issue. This view also preserves the picture of humans as creatures striving to make sense of, and hold consistent beliefs about, the world and their behaviour in it [In this article, we argue that rationality assumptions typically hold ur in it , which iur in it ,74. attribution biases [bounded rationality paradigm [The cognitive system may attempt to maintain local consistency, by following rational principles. If the information available to the ‘processor’ is limited by processing resources, time constraints, motivation, and memory accessibility  for a gfocalism—people focus too much on the event in question and not enough on the consequences of other future events, but asking people to think about other future activities reduces the bias [planning fallacy, which is explained by focusing too much on the future task and not enough on past experiences with similar tasks . Such past experiences will have arisen in perhaps very different local contexts, and actively comparing present and past experience itself provides yet a further local context. In addition, in this context, a person may attempt to resolve the unexpectedly high payment with the fact that they have previously enjoyed repetitive tasks, perhaps by concluding, say, that it is the purposeless nature of the ‘peg-turning’ task, rather than its repetitive character, which is particularly unpleasant). However, the fact that local context can be linked together, to create a fresh context of deliberation, does not diminish the fact that reasoning and rationality are local.all their beliefs, values and actions across all contexts—and that, by doing so, a person would be not merely locally, but globally rational, where global rationality is a ‘limiting case’, where all contexts are linked together. However, this ‘limiting case’ view of global rationality may not even make sense. Imagine that we could record both a person’s utterances and their unspoken thoughts . At one moment, a person says “It is raining” and at another “It is not raining”. This appears to be the starkest possible example of global inconsistency. However, it may, of course, just reflect a change in context: the person looks out of the window and disconfirms their initial conjecture; or the weather changes; or, more subtly, meaning has shifted , or the contrast might have changed . If thiWe have so far argued for the local nature of rationality in general terms. We now focus on a particularly important illustrative example: the local rationality of choice.The local rationality hypothesis makes the intriguing, and counterintuitive, prediction that people have no internal value or utility scales, and instead are only able to make relative comparisons between items, and, moreover, that even these comparisons may not be invariant by may, instead, depend on local context. Thus, in a particular context , one may prefer A to B, but no meaningful “value” or “utility” can be assigned to either option. According to many theoretical accounts, instabilities of values may be attributed either to imperfect information (agents do not know what the consequences of A and B will be), or bounded rationality . However, we consider the more radical position—that the problem with conventional rational choice explanation is not that values and attitudes are well-defined but difficult to determine, but that they are not well-defined at all. Nonetheless, we shall see that this perspective is actually surprisingly consistent with large parts of standard rational choice theory.The framework described here derives some universal implications for most descriptive models of choice, across domains and disciplines in behavioural sciences, which boils down to the core principle that decisions are relative, in a specific way, to what is embedded in the local context. A plausible account is summarised by the claim that because people lack computational and time resources to integrate all relevant information from memory or the world, they have poor notions of absolute cooperativeness, risk, or utility, and, instead, humans can only make judgments and decisions in relative terms—analogously to the presented psychophysical and cognitive theories of perception and judgment of information about magnitudes representing intensities of stimulus attributes  does not change drastically. If the context  does change, then this preference ordering may reverse, so that salad is preferred to fish. This contextual view predicts that humans are unable to represent absolute magnitudes, whether perceptual variables, utilities, payoffs, or probabilities, and instead make locally consistent judgments about these quantities based on small samples of available information. Those conclusions lead to a weaker version of economists’ notion of ordinal utility: that ordinal judgments are stable within a context, or sample, containing stimuli from the current environment as well as retrieved from memory. We review evidence that behaviour is consistent within contexts and even though it is globally unstable when behaviours across different context are considered  preferences between outcomes within a particular sampled context. We conclude that economic theory ordinalist assumptions are implausible only when single utility function is used to model choice behaviour across different contexts, in order to create a global preference ordering.This ‘contextual’ view, as described from multiple levels of explanation, has implications for certain rational choice based disciplines, such as economics where people are assumed merely to have outcomes . Thus, tred (see ). We argA over B, another in which B is chosen over A. Is this is an example of irrationality? Or, as the economist and philosopher Sen points out , there preferences over bets, concerning all possible matters, and these views would need to be consistent. It is unclear whether this viewpoint is even conceptually coherent [Moreover, a global preference order appears to require that people must have well-defined and hence consistent beliefs and preferences regarding the credibility of ideas they have never entertained , and well-defined preferences over options they have never considered . An agent with a truly global preference ordering, linking together all possible contexts, would have views (however tentative), and hence coherent . In summary, our starting assumption is that human thought is about constructing rational arguments in a given context , and theWhat mental process is used each time can often be determined by the input characteristics. Consider a choice among different gambles; if the multiplication of the rewards and probabilities is straightforward, then the trade-off between the two gambles can be reframed on the dimension of “expected value” and the predictions of rational choice theory should coincide with the empirical data. This does not negate our claim that people can use one-mental-process-at-a-time, but it rather demonstrates that the decision process is adaptive and dependent on contextual factors. In summary, contextual shifts can differentiate the valuation of the choice options across different contexts , leadinConsider, for example, how context shifts are dealt with in prospect theory ,100, proThe effect of using fragmented information in decision-making has been demonstrated in a study that examined the effect of time-pressure on the magnitude of the compromise effect . The comIn the face of existing evidence, rationality is best interpreted as a rather local, instead of global, phenomenon, i.e., rationality assumptions typically apply locally, within a context. Note, however, that according to rational choice theory and economics, the appropriate psychological assumption underlying rational explanation is that people will behave consistently within a context . The drchange blindness phenomenon that occurs when observers do not notice a change in a visual stimulus . Observers do not notice those changes usually because of lack of attention, eye movements, obstacles in the visual field, or a change of location [However, people may not respond reasonably to new or hidden changes in external variables, or when they lack experience in novel contexts . Our key argument is that rational choice theory is an unrealistic model of human behaviour because the rational models try to hook together (integrate and explain) behaviour across different contexts, in order to create a global preference ordering. This approach does not work in practice because individuals are unstable in their preferences and flip around depending on context. However, the key caveat is that if the distribution of contexts across individuals is reasonably stable, then this would create stable overall behaviour. Analogous phenomena are observed in perception as demonstrated by the well-known location ,105,106.location ,108). Colocation ,110. Thelocation . In summary, most of the time, markets may behave stably and locally behaviours can also look stable and consistent. However, as a result of the fundamental instability of mental representations , such stability is not guaranteed, as we have seen, if the context changes significantly .Rational models should not be dispensed with—but they need to be supplemented with a theory of contextual shifts. We do not outline the specifics of such a theory here, but instead try to review and summarise the common characteristics of the prominent psychological theories of choice, which are comparative in nature. Hence, we suggest to carry the seed from which such unified theory may grow in the future. The comparative approach does not assume that observable binary choices result from consulting stable (independent) values. This assumption is evident in the fact that revealed preferences work only as long as the set of choice options does not change drastically. If the context  does change, then this preference ordering may reverse. Some researchers argue that these effects are due to a human inability to represent absolute magnitudes, whether perceptual variables, utilities, payoffs, or probabilities ,89. Theslocal. The extension of these abstract representational and processing assumptions, in choice problems with three or more options should be straightforward if one assumes that any multi-alternative process can be broken into elementary binary problems. This reduction makes the decision process even more susceptible to biases since some pairs might be processed more often than others due to selective attention, which is another instance of local processing/inference. Next, we categorize well-known theories of choice into: (a) theories assuming comparative value functions; and (b) theories assuming scale-less comparisons and briefly discuss the way information is processed and context is incorporated . In contrast, theories assuming comparative value function assume that the reference point for each option is some function of all other options in the set—such relative evaluation means that a value function transforms the ‘relative advantage’ of every choice options on each dimension. componential-context model [Therefore, such modern decision theories still assume scales for utility—i.e., attributes have scale-based values that can be compared on an interval scale . One of the first models based on that idea was the xt model  of conterange-frequency theory [range-frequency theory shares similar representational and processing assumption to the componential-context model .Parducci’s seminal y theory  of contestochastic difference model [Similar assumptions  are shared also by the ce model , which amultialternative decision field theory  provides the basis for subsequent choice, as value differences  are integrated over time. MDFT incorporates all six stages. In particular, stage (v) (the subjective goodness of options) is factored in by allowing the state of an accumulator (which accumulates relative evidence and tracks the preference state of an option) to be distorted by lateral inhibition from the other competing accumulators. The magnitude of the inhibitory coupling depends on the similarity of two options in the multi-dimensional choice space with the more similar options competing stronger.Comparison based value computation, but in a dynamic fashion implemented in a sequential sampling framework, is also assumed by the er MDFT, . At eachleaky competing accumulator model  is not implemented directly in DFT but instead emerges from the dynamics of the model. Both the multialternative decision field theory and leaky competing accumulator model assign to context an important role. First, the choice process is inherently local, with attention being allocated to one dimension at each time and second by allowing the options to compete through lateral inhibition, as the decision process evolves. The descriptive adequacy of these models is unique as they are the only models so far able to account for all three preference reversals in multi-attribute choice  simultaneously under single parameter sets. The ter LCA, ) takes aperceived relative argument model of decision-making under risk [regret theory, [trade-off model of intertemporal choice in which alternative-based discounting was replaced with attribute-based trade-offs between relative interval differences and payoff differences [constructed preference approaches [Other examples of this approach applied in specific domains have been recently suggested. The der risk  was prop theory, ). The saferences . Note thferences . Indeed,proaches  assume sThis tradition is followed in neuroeconomics , where sThe most recent type of comparative theories assume that people make judgments and decisions without consulting a utility scale based on the absolute magnitudes of stimuli (including key “economic” variables such as time and probability). The common feature of this class of models is the lack of value functions operating on (transforming) input variables such as the attribute levels of choice options. Thus, according to these models, ‘direct’ comparison, rather than value, is fundamental to judgment and choice, which implies that absolute differences between attribute levels often do not matter and different processing rules may apply to compare objective values  directly . Oteuristic , the winuristics , which is theory . There tThe context dependency of such comparative choice strategies makes some of them sensitive to the environmental contingencies encountered by the decision-maker . Due to this property, decision-by-sampling, for example, accounts for typical patterns of economic behaviour like diminishing marginal utility of wealth, losses looming larger than gains, hyperbolic temporal discounting, and overestimation of small probabilities and underestimation of large probabilities. Similarly, the priority heuristic also predicts behavioural phenomena such as Allais paradox, loss aversion, and the certainty effect. These results show how scale-less comparative decision theories, which do not assume any value functions, can explain decision anomalies such as loss aversion.reason-based models, follow a similar logic to scale-less comparative models. In these models, choice emerges from the comparison of the aggregated reasons for and against each option (pros and cons). An early such theory, reason-based choice [Another class of models, mentioned here as d choice  demonstrreason-based choice is query theory [query theory, valuation questions are addressed by serially posing a set of sub-questions whose order differs depending on the response mode. Because earlier queries are weighted stronger, the response mode affects the decision outcome. For example, choosers will first consider reasons why not to enter a transaction and then reasons why to accept it while sellers will follow the opposite order.Similar in spirit to the y theory , which oFuzzy trace theory [local thinking [local and selective recall of reasons. In summary, reason-based models do not assume any value function, and instead they rely only on counting-up bits of evidence for and against and on explicit thought processes such as ‘arguments’.e theory  also posthinking  demonstrThe overall argument presented in this article provides, we hope, a fresh perspective on the scope and limits of human rationality in various domains. The aim of this article is to show how the standard focus in rational choice theory on consistency can be derived out of knowledge of basic cognitive mechanisms underlying decision-making, instead of being assumed out of convenience, intuition, and theoretical helplessness. This aspect of cognition is related to how people make inferences that lead to judgments of the magnitudes of choice options, or their attributes, such as values, payoffs, and probabilities, which are essential ingredients of every decision problem. This is a very general and pervasive feature of human cognition that may have important implications for all aspects of decision-making and related fields within psychological and social sciences. Thus, rather than starting from a normative rational theory, such as expectancy-value models of attitudes/action in social psychology  and expeTraditional theories of behaviour can be grouped into four categories according to the underlying principles or mechanisms . Hunudge people to make better decisions in more sophisticated ways than the mere provision of information, in effect by changing the choice architecture [In reality, however, truly informed choice rarely happens; for example, changing intentions (as a results of changes in beliefs and attitudes) accounts for between 3% and 28% of the variance in behaviour change, depending on whether researchers employ experimental  or correitecture .when they make decisions then they might make a good choice, but otherwise some other piece of information will determine their choice. Therefore, even ‘good information’ becomes a nudge when the temporal dimension is taken into account ; and if such dynamic cognitive systems can only sample and process limited amount of information, not all relevant information, at the moment of choice, then every choice might become a ‘nudged choice’  ‘localness’ is the key, as we claim, then the information structure of the immediate environment will turn out to be crucial . Our app.g., see ,155,156).g., see , are obvSuch conclusions do not imply that people are not, in many ways, quite stable and consistent in their beliefs, desires, attitudes and behaviours, but such stability is manifested probably partly in cases where individuals have a lot of prior evidence about their own behaviours or thoughts. Such stability can also be due to recurrent environments in everyday life, but once the context changes, values and attitudes could easily change too, as demonstrated, for example, by the famous ‘Stanford prison experiment’ and other results supporting situational attribution of behaviour rather than dispositional attribution—i.e., situation causing behaviour, rather than anything inherent in individual personalities . Are suTo conclude, rather than viewing psychological data as undermining assumptions made in rational choice theories, which is often the implicit assumption in some psychological theorising, we claim here that, under specific conditions, we can still employ the standard rational choice approach to modelling decision behaviour. However, we also argued that  choice consistency emerges only within a particular context—this is the local consistency hypothesis that people are coherent within each context and rank-correlated across contexts, even though choices appear globally unstable when integrated from different contexts. In this respect, rational choice theories are often imprecise only because they are designed to model behaviour across different contexts, in order to create a global measure of attitudes or preference ordering.


Despite exhaustive efforts to detect early-stage ovarian cancers, greater than two-thirds of patients are diagnosed at an advanced stage. Although diaphragmatic metastasis is not rare in advanced ovarian cancer patients and often precludes optimal cytoreductive surgery, little is known about the mechanisms and predictive factors of metastasis to the diaphragm. Thus, as an initial step toward investigating such factors, the present study was conducted to characterize the pathological status of ovarian cancer patients who underwent debulking surgery in combination with diaphragmatic surgery.This is a retrospective and cross-sectional study of patients who underwent debulking surgery in combination with diaphragmatic surgery at our institution between January 2005 and July 2015. Clinicopathological data were reviewed by board-certified gynecologists, pathologists, and cytopathologists. The rates of various pathological findings were investigated and compared by Fisher exact test between 2 groups: 1 group that was pathologically positive for diaphragmatic metastasis (group A) and another group that was pathologically negative for diaphragmatic metastasis (group B).P = 0.001) and metastasis to the peritoneum except for the diaphragm  were significantly increased in group A compared with group B. However, no significant differences between the 2 groups were found for rates of histological subtypes (high-grade serous or non-high-grade serous), the presence of ascites, the presence of malignant ascites, exposure of cancer cells on the ovarian surface, blood vascular invasion in the primary lesion, and lymphovascular invasion in the primary lesion.Forty-six patients were included: 41 patients pathologically positive and 5 pathologically negative for diaphragmatic metastasis. The rates of metastasis to the lymph node (95.8% vs 20%, Our study demonstrated that metastasis to the lymph node and nondiaphragmatic metastasis to the peritoneum are significantly associated with metastasis to the diaphragmatic peritoneum, indicating that these factors may be pathological predictors of diaphragmatic metastasis in patients with ovarian cancer. However, as the data available are not sufficient to demonstrate the predictive power of these factors, a further comprehensive, large-scale study should be performed. Therefore, the primary aim in ovarian cancer treatment is to achieve optimal cytoreductive surgery. However, advanced ovarian cancer patients often present with upper abdominal metastases.6 In such patients, diaphragmatic metastasis is observed in approximately 40% of cases8 and in fact precludes optimal cytoreductive surgery in up to 76% of cases.6 Nevertheless, little attention has been paid to the mechanisms of diaphragmatic metastasis in ovarian cancer or to the pathological factors predictive of this metastasis.A recent meta-analysis demonstrated that the most important prognostic factor for survival in such patients is the amount of residual tumor after surgery.9 The diaphragm's main functional role is thought to involve breathing movement.9 However, the diaphragm also plays another important role by absorbing substances from the abdominal cavity via the lymphatic drainage system.10 The diaphragm is situated in the abdomen adjacent to the liver, esophago-gastric junction, inferior vena cava, abdominal aorta, thoracic duct, spleen, adrenal gland, kidney, and pancreas. In combination with ventilatory movement, these anatomical features often make diaphragmatic inspection and operation difficult and time-consuming. Accordingly, some investigators hypothesize that diaphragmatic metastasis may be underestimated during ovarian cancer surgery.11The diaphragm is one of the widest organs; it separates the thoracic and abdominal cavities, forming a dome-like structure with a very steep slope in the back.The identification of predictive factors for diaphragmatic metastasis would enable the stratification of patients with regard to the decision of whether to dedicate effort to diaphragmatic investigation during surgery. However, no adequate data are currently available for demonstrating pathological predictors of diaphragmatic metastasis. Thus, as an initial step toward investigating such factors, the present study was conducted to characterize the pathological status of ovarian cancer patients who underwent debulking surgery in combination with diaphragmatic surgery.12 and investigated pathological findings to determine the histological subtypes of the tumors (high-grade serous or non-high-grade serous), exposure of cancer cells on the ovarian surface, metastasis to the peritoneum, and metastasis to the lymph nodes. Elastica-van Gieson staining and immunohistochemistry were performed to evaluate blood vascular or lymphovascular invasion in the primary ovarian lesion using 1 representative formalin-fixed, paraffin-embedded block per case. Immunohistochemistry was performed using antibodies against Von Willebrand factor  and D2-40 , a detection kit  and an autostainer  according to the manufacturer's instructions and a method described in the literature.13 For patients with ascites accumulation, cytology was performed to examine whether the ascites contained malignant cells. Board-certified cytopathologists evaluated cytological samples prepared with Papanicolaou, periodic acid-Schiff, Alcian-blue, and Giemsa stains in a blinded manner against histological data and deemed the samples as benign, indeterminate, suspicious for malignancy, or malignant. If the initial judgment was indeterminate or suspicious, cell block samples were prepared to determine whether the specimen was benign or malignant using the rest of the cell sediment in the ascites according to a method described in the literature.14 After evaluating the clinicopathological findings, FIGO stages were determined at the time of the debulking and diaphragmatic surgery by both gynecologists and pathologists.This cross-sectional study was approved by our institutional review board (No. 2863). The inclusion criterion was that the patients underwent primary, interval or secondary debulking surgery in combination with diaphragmatic surgery for ovarian carcinomas or carcinosarcomas at Tokyo Medical University Hospital. The exclusion criterion was the lack of available pathological samples, including diaphragmatic materials. The medical records of potentially eligible patients treated from January 2005 to July 2015 were retrospectively and consecutively retrieved from our computerized database. Board-certified gynecologists reviewed patients’ medical charts and investigated information regarding patient age, clinical history, surgical procedures, and administration of chemotherapy for ovarian cancer. Board-certified pathologists evaluated histological samples and diagnosed them according to established criteriat test and the Mann–Whitney U test were used for continuous variables, and Fisher exact test was used for categorical variables. Missing values were excluded from the statistical analyses. All statistical analyses were performed using IBM SPSS Statistics 21, and a P value of less than 0.05 (2-sided) was considered to indicate statistical significance. If possible, further detailed analysis was performed for statistically significant pathological variables.Clinicopathological findings are described by reporting the mean and median values for continuous variables and the frequencies of categorical variables. To compare the rates of various clinicopathological findings between the groups pathologically positive and negative for diaphragmatic metastasis, Student P = 0.001) and the laterality of ovarian cancer between the 2 groups (P = 0.026).Between Jan 2005 and Jul 2015, 267 patients underwent surgery for ovarian carcinomas or carcinosarcomas at our institution; among them, 46 patients fulfilled our inclusion criterion, and none were eliminated based on our exclusion criterion. The clinical characteristics of the 46 patients with ovarian cancer who underwent debulking surgery in combination with diaphragmatic surgery are summarized in Table The pathological characteristics of the 46 patients are summarized in Table P = 0.001) and nondiaphragmatic peritoneal metastasis  were significantly increased in the group pathologically positive for diaphragmatic metastasis compared with the negative group. In addition, the rates of histological subtype (high-grade serous or non-high-grade serous) , the presence of ascites , the presence of malignant ascites , the exposure of cancer cells on the ovarian surface , blood vascular invasion in the primary lesion , and lymphovascular invasion in the primary lesion  did not differ significantly between the 2 groups.The rates of metastasis to the lymph nodes     .Further detailed analysis revealed significant increases in the group that was positive for diaphragmatic metastasis for the rates of metastasis to the 326B  lymph node  lymph node was significantly associated with metastasis to the diaphragmatic peritoneum (P = 0.006). Lymphogenous metastasis appears to contradict the concept of normal lymphatic flow. However, studies have shown that lymphatic pathways exist from the para-aortic lymph node to the diaphragm,38 that lymphatic flows in the diaphragm are multidirectional in nature,40 and that the diaphragmatic lymphatic system possesses a network organized into a series of confluent vessels that can functionally adapt to regional drainage requirements by recruiting lymphatics.41 In addition, changes in lymphatic dynamics caused by primary surgery or cancer dissemination might affect the direction of peritoneal or retroperitoneal lymphatic flow, directing more flow toward the diaphragm.42 The hypothesis of the lymphogenous metastatic pathway is also supported by the concept of the retrograde or rerouting lymphatic spread of cancer cells.46 Alternatively, it is possible that cancer cells spread from the metastasized peritoneum to the diaphragm via peritoneal lymphatics with or without the intercalation of lymph nodes, as suggested in an experimental study.47The second possibility is the lymphogenous metastatic route. The rich lymphatic network equipped with lymphatic stomata distinguishes the diaphragmatic peritoneum from other peritoneal regions.49 However, such shunts are seldom demonstrated by lymphangiography and then only in cases with an abnormal increase in intralymphatic pressure resulting from obstruction of the lymph flow.49 This obstruction may be due to lymphatic metastases, previous surgery, or inflammatory or infectious diseases.49 Additional research is needed to clarify whether lympho-venous shunts contribute to metastasis to the diaphragmatic peritoneum.The last possibility is the hematogenous route. If diaphragmatic metastasis occurs via this route, a significant number of metastatic lesions should be observed in extra-abdominal organs such as the lung or brain. However, no such signs were detected by imaging studies prior to debulking surgery in our cases. Some investigators suggest that cancer spread can occur via lympho-venous shunts.2 which might explain the higher rate of bilateral ovarian cancer in our patient series compared with that based on a study from a pooled German national dataset (56% vs 26.0%).50 These phenomena could be a source of bias in our study. Second, given the relatively small number of cases, it was difficult to calculate the odds ratio and perform logistic regression analysis. Third, as cases were retrieved from only 1 institution, it may be difficult to generalize the results. However, to our knowledge, this study is the first attempt to investigate the pathological status of ovarian cancer patients with and without diaphragmatic metastasis, and we believe that the results provide important information for future studies specifically designed to investigate the mechanisms of the diaphragmatic metastasis of ovarian cancers.The present study has several limitations. First, the study was performed retrospectively using surgical cases. As organs that were suspected to have metastases by gross examination were more likely to be removed by surgeons and subjected to pathological examination, the result was a small number of cases exhibiting no metastasis to the diaphragm. Moreover, although the mean age (56.5–57.6 years vs 57.6 years) and predominance of serous carcinoma histology (71.7% vs 52.4%) of our ovarian cancer patient series were roughly equal to those based on the FIGO 26th annual report, the proportions of FIGO stages in our patient series were lower in stage I (2.1% vs 35.5%) and higher in stages III (63.0% vs 44.6%) and IV (30.4% vs 11.7%) compared with those in the report,In conclusion, the present study demonstrated that metastasis to the lymph nodes and metastasis to peritoneal sites other than the diaphragm are significantly associated with metastasis to the diaphragmatic peritoneum, indicating that these factors may be pathological predictors of metastasis to the diaphragmatic peritoneum in patients with ovarian cancer. However, further comprehensive, large-scale surveys are needed to elucidate robust predictive factors and mechanisms of ovarian cancer diaphragmatic metastasis.


P < 0.05). Furthermore, high CPA4 concentration was significantly correlated with Distant metastasis, Lymph node involvement, Stage and poor overall survival of the patients with CRC. Logistic regression analysis revealed that serum CPA4 level and Lymph node metastasis were the significant parameters for predicting CRC liver metastasis. In leave-one-out-cross-validation, these two markers resulted in sensitivity (90.0%) and specificity (93.8%) for hepatic metastasis detection. Moreover, this combination could correctly classify 49 cases of the 50 CRC patients with heterochronous liver metastasis in an independent test set. Therefore, our results suggest that CPA4 is closely associated with CRC liver metastasis, and serum CPA4 concentration combined with lymph node involvement may be used as accurate predictors of liver metastasis in colorectal cancer.Hepatic metastasis is the most critical prognostic factor for colorectal cancer (CRC), and early detection of CRC liver metastasis can significantly improve cancer patient outcomes. In this study, we examined the levels of CPA4 in CRC samples, and assessed the potential of serum CPA4 as a biomarker for predicting CRC liver metastasis. CPA4 positivity was observed in 68.4% (130/190) colorectal cancer tissues, and significantly correlated with Depth of invasion, Lymph node metastasis, Distant metastasis and Stage. In addition, high CPA4 expression was associated with poor overall survival, and was an independent prognostic marker in patients with CRC. In CRC serum samples, serum CPA4 concentrations in CRC-M1(S) patients (3717.89 ± 375.98 pg/mL) were significantly increased as compared to in CRC-M1(H) patients (3692.12 ± 261.51 pg/mL), CRC patients without liver metastasis (2480.47 ± 507.90 pg/mL) or healthy controls (2183.7 ± 621.7 pg/mL) ( Colorectal carcinoma (CRC) is one of the leading causes of cancer death worldwide and prone to liver metastasis . It is eCarboxypeptidase A4 (CPA4) is a member of the metallocarboxypeptidase family, and is aberrantly overexpressed in some types of cancer tissues . As a seIn this study, we examined CPA4 level in primary colorectal cancer tissue and serum samples, and analyzed the correlation between CPA4 levels and clinicopathological characteristics of CRC patients. We propose that serum CPA4 might be a candidate diagnostic biomarker for colorectal cancer liver metastasis.P = 0.023), lymph node metastasis (P = 0.043), distant metastasis (P = 0.045) and clinical stage (P = 0.022) (Table CPA4 positivity was observed in 68.4% 130/190) CRC cases. There was weak or no staining of CPA4 in the normal colorectal epithelium. Also, CPA4 positive staining was found in interstitial tissues especially around micro-vessels areas in CRC tissues Figure . Further30/190 CRP = 0.006) , Grade , Distant metastasis ,and lymph node metastasis  were statistically independent predictive factors of poorer prognosis for CRC cancer  by ELISA. The clinicopathological characteristics of CRC patients was shown in Table P < 0.05). Further analysis indicated that the serum levels of CPA4 were 2480.47 ± 507.90 pg/mL in colorectal cancer without liver metastasis, 3717.89 ± 375.98 pg/mL in colorectal cancer with simultaneous liver metastasis, and 3692.12 ± 261.51 pg/mL in colorectal cancer with heterogeneous liver metastasis and the differences were significant (p < 0.05) , Lymph node involvement (p = 0.000), Stage (p = 0.000), serum CEA level (p = 0.015), Serum TPS level (p = 0.004) and Serum CA199 level (p = 0.001). Kaplan-Meier survival analysis showed that increased CPA4 concentrations of 3500 pg/mL or higher in colorectal cancer serum were correlated with poor overall survival (P = 0.000) , Serum CA199 level , Stage  and Distant metastasis  were independent risk factor for reduced survival in CRC cancer patients  Figure . The cli) Figure . FurtherP < 0.05) and increased the metastatic ratio. The odds ratios of CPA4 and Lymph node metastasis were 181.44 and 30.677, respectively (Table n = 50). Moreover, of the 50 heterochronous metastatic cases, this combination could correctly classify 49 cases.Logistic regression was done to calculate the respective significance of each candidate marker and clinical features for predicting liver metastasis. Both the CPA4 level and Lymph node metastasis showed predictive significance  is one of the major causes of cancer death throughout the world . The preIt has been reported that a great number of serum biomarkers including CEA, CA199 and TPS could be used for early diagnosis and monitoring of CRC liver metastasis . A singlCarboxypeptidase A4 (CPA4) is a secreted, zinc-dependent metallocarboxypeptidase that removes the C-terminal amino acid residues of proteins . Recent In this study, we firstly evaluated the expression of CPA4 in two different commercial tissue arrays containing 100 cases of colon cancer patients and 90 cases of rectal cancer patients with follow-up record. The results indicated that high CPA4 expression was observed in 68.4% (130/190) colorectal cancer samples, and significantly correlated with depth of invasion, lymph node metastasis, distant metastasis and Stage. Importantly, we found that the positivity of CPA4 in CRC primary tissues with distant metastasis was 100%. Kaplan-Meier survival analysis revealed a correlation between higher CPA4 expression levels and shorter overall survival times. Further Cox multivariate regression model demonstrated that CPA4 level was an independent predictive factors of poorer prognosis for CRC cancer. Taken together, these observations indicated that CPA4 might play a key role in the distant metastasis.To further test the efficacy of CPA4 as a serum marker for CRC liver metastasis, we collected 180 CRC serum samples. It was divided into two groups: the training set (130 cases including 31 synchronous liver metastasis cases and 14 heterochronous liver metastasis cases) was used to build up classifiers that were able to distinguish metastatic cases from non-metastatic cases, and the test set (50 heterogeneous liver metastasis cases) for validation. Firstly, we found that serum concentrations of CPA4 in CRC patients were significantly higher than those in healthy controls. Further study demonstrated that serum CPA4 level was higher in the CRC with liver metastasis group than in CRC without liver metastasis group. And the high CPA4 level was correlated with Distant metastasis, Lymph node involvement, Stage and poor survival. By using Logistic regression analysis, we identified that the expression of CPA4 and lymph node involvement were the significant predictors for detecting liver metastasis.p < 0.05). Leave-one-out method has been employed to validate the efficacy of candidate markers in predicting cancer metastasis [ was available. The expression of CPA4 in the tissues was evaluated by immunohistochemical staining with specific antibodies. Standard Avidin-biotin complex peroxidase immunohistochemical staining was performed. Briefly, after deparaffinizationin xylene and graded alcohols, heated antigen retrieval was done in citrate buffer (10 mmol/L pH 6.0) by water-bath kettle heating for 30 min. Endogenous peroxidase was blocked in 0.3% hydrogen peroxide for 10 min. Nonspecific binding was blocked by incubation in 10% normal animal serum for 10min. Sections were incubated at 4°C for 24 h with primary antibodies including polyclonal antibody against anti-CPA4 (Sigma-Aldrich). Immunohistochemistry on the TMAs was graded semiquantitatively considering both staining intensity and percentage of positive tumor cells by two pathologists blinded to the clinicopathologic variables. Protein expression levels were scored by staining intensity and the percentage of immunoreactive cancer cells. The staining intensity was arbitrarily scored on a scale of four grades: 0 (no staining of cancer cells), 1 (moderate or strong staining) and the percentage of positive cells was scored as following: 0 (0%), 1 (1–10%), 2 (10–50%), 3 (> 50%). CPA4 staining positivity was determined using this formula: overall score = positive percentage score × intensity score. Overall score = positive percentage score × intensity score. Next, Raw scores can be converted to standard scores. Colorectal cancer tissues that registered levels 0 and 1 were defined as negative for expression, whereas samples at levels 2 or 3 were defined as positive.For ELISA study, preoperative peripheral blood samples were obtained from 180 colorectal cancer patients during 2006–2008 including 31 CRC patients with Synchronous liver metastasis when it had been detected by computed tomography (CT) scan, ultrasonography, or by surgery at the time of initial diagnosis; and 64 CRC ones with heterochronous liver metastasis when liver metastases, confirmed by CT scan or ultrasonography, occurred after resection of the primary tumor. For all the specimens, clinicopathological information  was available. At last, 180 specimens were divided into two groups: the training set (130 cases) was used to build up classifiers that were able to distinguish metastatic cases from nonmetastatic cases, and the test set (50 heterogeneous liver metastasis cases) for further validation. 130 specimens of healthy individuals were donated on a voluntary basis. The study was approved by the medical ethics committee of Cancer Hospital, CAMS. CPA4  concentrations were determined by means of an enzyme linked immunosorbent assay (ELISA) method according to the manufacturer's instructions. The range of the immunoassays for CPA4 was 156 to 10000 pg/mL. Each serum sample was run in duplicate. Briefly, 100 ml of serum (1:5 dilution) were placed into each well of the ELISA plate and incubated for 45 min at 37°C. The plates were washed four times with buffer and incubated with 100 ml of detection antibody at 37°C for 45 min. After four washes, the plates were incubated with substrate solution for 15 min at room temperature, then the reaction was stopped and the plates were read by a spectrophotometer at wavelength 450 nm. A standard curve was generated with the provided standards and used to calculate the quantity of CPA4 in each serum sample.t-test. According to an optimal cutpoint, CPA4 level was analyzed as a dichotomous variable for further evaluation. The association between the markers and clinicopathologic features was analyzed using χ2-test or two-sided t-test as appropriate. All possible combinations of these factors were used to build up classifiers that were able to distinguish metastatic cases from non-metastatic cases were examined using Leave-one-out validation model. The discriminant equation and the cut-point of the most satisfactory combination were then used to predict the probability of metastasis in an independent test. The survival rates were assessed by the Kaplan-Meier method and compared by the log-rank test. Statistical significance was set at P < 0.05 (two-tailed). Receiver operating characteristics (ROC) curves were generated to compare the predictive sensitivity and specificity, and the area under the curve (AUC). The survival rates were assessed by the Kaplan–Meier method and compared by the log-rank test. Statistical significance was set at P < 0.05 (two-tailed).The SPSS 15 software package  was used for statistical analysis. The CPA4 expression was first analyzed as continuous numeric data and the mean staining intensity between the primary tumors and corresponding adjacent normal tissues was compared with a two-sided


Short Interspersed Degenerate Retroposons of the SIDER2 subfamily, largely located within 3'UTRs of Leishmania transcripts, promote rapid turnover of mRNAs through endonucleolytic cleavage within the highly conserved second tandem 79-nt hallmark sequence (79-nt SII). Here, we used site-directed mutagenesis and in silico RNA structural studies to delineate the cis-acting requirements within 79-nt SII for cleavage and mRNA degradation. The putative cleavage site(s) and other nucleotides predicted to alter the RNA secondary structure of 79-nt SII were either deleted or mutated and their effect on mRNA turnover was monitored using a gene reporter system. We found that short deletions of 8-nt spanning the two predicted cleavage sites block degradation of SIDER2-containing transcripts, leading to mRNA accumulation. Furthermore, single or double substitutions of the dinucleotides targeted for cleavage as well as mutations altering the predicted RNA secondary structure encompassing both cleavage sites also prevent mRNA degradation, confirming that these dinucleotides are the bona fide cleavage sites. In line with these results, we show that stage-regulated SIDER2 inactivation correlates with the absence of endonucleolytic cleavage. Overall, these data demonstrate that both cleavage sites within the conserved 79-nt SII as well as RNA folding in this region are essential for SIDER2-mediated mRNA decay, and further support that SIDER2-harboring transcripts are targeted for degradation by endonucleolytic cleavage.We have reported previously that  Leishmania species cause a broad spectrum of vector-borne parasitic diseases, ranging from self-healing cutaneous to fatal visceral forms that are collectively termed as leishmaniasis. The disease has expanded to over 98 countries with more than 350 million people at risk and 2 million individuals infected annually  using random oligonucleotides and Klenow fragment DNA polymerase I (New England Biolabs). Copy number values for the LUC-3810 3ʹUTR wild type and mutated plasmids as well as for the LUC-3810ΔSI+II vector were estimated following Southern blot hybridization of total DNA extracted from all the different transfectants and digested with NdeI using a probe complementary to the first 1000 nucleotides of the LmJF.36.3810 3ʹUTR, recognizing both the plasmid and the genomic copies. The ratio between the plasmid and genomic copies based on hybridization intensity signals determines the copy number of the different plasmids in all transfectants , quickly centrifuged and lysed in 2 × Laemmli buffer. To evaluate NEO expression, the blots were first reacted with a rabbit anti-NEO antibody at 1:2000 (Millipore) and then with an anti-rabbit-HRP antibody . The mouse anti-alpha-tubulin antibody (Sigma) followed by an anti-mouse-HRP antibody  was used to normalize for protein loading variations. Blots were visualized by chemoluminescence with Pierce ECL2 Western blotting kit (Thermo Scientific). Protein levels were quantified by densitometric analyses using the ImageQuant 5.2 software.Genomic DNA and total RNA of ants see . NEO-expL. major LUC-3810 3ʹUTR and LUC-3810 SIDER2 as well as in L. infantum expressing LUC-40003ʹUTR in both life stages essentially as described previously [5’-GCACAGGCCTGCTCACTGTC-3’) described before [32P]ATP using polynucleotide kinase . Total RNA was isolated by TRIzol and quantified by NanoDrop ND-1000. Approximately 10 pmol of labeled primer and 50 μg of total RNA were used for PE reactions using the SuperScript III RT kit (Invitrogen) according to the manufacturer’s recommendations. A ØX174 DNA/HinfI dephosphorylated DNA marker (Promega) was labeled with [γ-32P] ATP and PNK (New England Biolabs) and used as a size marker. Primer extension fragments and markers were separated on 8% denaturating acrylamide gels  and visualized by autoradiography.Primer extension (PE) was used to detect extended cleavage fragments in recombinant eviously . Primer d before , complemTo predict the RNA secondary structure of the wild type LmjF.36.3810 SIDER2 79-nt signature II sequence and the different mutants, we used RNAfold , one of 5'-UCCCC-3' duplicated motif and Cl2 between a CU (or a UA) dinucleotide at ~50 nucleotides downstream (3'-end of 79-nt SII) (L. major LmjF.36.3810 (3810) SIDER2-bearing transcript. The truncated 3810 3'UTR was fused downstream of the luciferase (LUC) reporter gene as part of the pSPYNEO-alphaIR expression vector and transfected into the L. major LV39 strain. Stable transfectants were analyzed by Northern blot hybridization to assess the effect of either deletion on LUC mRNA turnover. Deletion of either cleavage site blocked degradation and resulted in 3.0- and 3.3-fold accumulation for LUC-3810ΔCl1 and LUC-3810ΔCl2 transcripts, respectively , representing the hallmark of trypanosomatid retroposons  11]. We. We11]. -nt SII)  from theectively . These vL. major LV39 and total RNA was extracted from stable transfectants and used for Northern blot hybridization to quantify the impact of the mutations and of structural changes on mRNA degradation (LUC mRNA levels in the different 79-nt SII mutants (M1 to M6-2) were calculated relative to wild type SIDER2 sequences and normalized to alpha-tubulin mRNA levels. LUC mRNA values were further normalized to the copy number of LUC-expressing plasmids due to some variations between the different transfectants  , had no effect on 'UTR RNA . Interes3-fold) . The eff-fold . Tbundance . In thisay rates .in vivo, the generated 5'- and 3'-cleavage products can be visualized by different methods [Following endonucleolytic cleavage at defined sites within the 79-nt SII  methods , includibona fide cleavage sites. They also suggest that the predicted central hairpin loop structure in 79-nt SII encompassing both cleavage sites is key to the regulated SIDER2-mediated mRNA decay process.Altogether, these results indicate that both predicted cleavage sites are essential for SIDER2-mediated decay, confirming that these dinucleotides are the LUC reporter gene was sufficient to confer rapid mRNA turnover both in L. major and L. infantum [LUC yielded two cleavage products of 103-nt and 151-nt (We have shown previously that a SIDER2 retroposon (~550 bp) when placed downstream of a infantum . Here, winfantum . We usedinfantum  Fig 5A)LUC repord 151-nt  as suggeL. infantum ortholog of LmjF.36.3810 (3810), causes a rapid turnover of the 4000 mRNA in promastigotes but in amastigotes the same transcript accumulates, suggesting that SIDER2 is inactive in this life stage [NEO) gene , conserved among all SIDER2 retroposons, is essential for regulation and that it mediates rapid mRNA turnover through endonucleolytic cleavage [in silico RNA structural studies to further delineate the cis-acting region within 79-nt SII.We have reported previously a novel mechanism of regulated mRNA decay in cleavage , 14. In bona fide cleavage sites. Although the predicted changes in the LmjF.36.3810 SIDER2 79-nt SII RNA secondary structure when mutating one or both nucleotides in each dinucleotide cleavage site were not of the same magnitude, Cl1 and Cl2 folded into a double-stranded structure and were no longer single-stranded as in the wild type. These structural changes may have also contributed to the pronounced effect in blocking mRNA degradation. In fact, several classes of RNA-binding proteins, including proteins with an RNA-recognition motif (RRM), double-stranded RNA-binding motif (ds-RBD) like the Staufen 1 (STAU1) proteins mediating mRNA decay [in silico predicted RNA structures suggest that both cleavage sites are in a single-stranded conformation, it would be important to determine the structure of the 79-nt SII RNA within SIDER2 elements using nuclear magnetic resonance spectroscopy (NMR) or related technologies. Mutations predicted to disrupt the central hairpin loop structure embedding Cl2 and Cl1 sites had the most dramatic impact on mRNA decay, resulting in the highest increase in mRNA accumulation. In addition, mutations designed to specifically disrupt base-pairing in the predicted hairpin structure at the 3'-end of 79-nt SII also affected the decay process. Altogether, these data suggest that RNA secondary structure of the conserved 79-nt SII within SIDER2, especially in the vicinity of the cleavage sites, may be crucial for an efficient mRNA decay. In Trypanosoma spp., it was recently shown that the first signature sequence of the potentially functional L1Tc/ingi clade retrotransposons, the ancestors of SIDERs, forms a Hepatitis Delta Virus (HDV)-like ribozyme [in vitro ribozyme function was not detectable in the 3′UTR region of Leishmania SIDER2-harboring mRNAs undergoing degradation [et al., manuscript in preparation).We show that single or double nucleotide substitutions within both predicted cleavage sites (Cl1 and Cl2) in the LmjF.36.3810 SIDER2 element , N-pyrimNA decay , as wellNA decay –31. Althribozyme . Indeed,radation , 33 and radation . In lineWe have reported previously that a SIDER2 element outside its 3'UTR context was able to promote rapid mRNA degradation to levels comparable with a 3'UTR bearing a SIDER2 element . Here, wL. infantum LinJ.36.4000 gene, the ortholog of LmjF.36.3810, encoding an aminomethyltransferase of the glycine cleavage complex [Leishmania switches from glycolysis to amino acid catabolism and that proteins involved in protein degradation and amino acid import are significantly upregulated in amastigotes [L. infantum amastigotes. Here, we show that stage-specific accumulation of LinJ.36.4000 mRNA in amastigotes correlates with the absence of endonucleolytic cleavage, which suggests that SIDER2 is inactive. Additional experiments using an integrated reporter system where the LinJ.36.4000 genomic copy was replaced by the neomycin phosphotransferase (NEO) gene, revealed a selective NEO mRNA degradation in promastigotes but increased accumulation in amastigotes as a result of SIDER2 inactivation. A SIDER2 element may loose its ability to promote mRNA decay because of changes in the RNA structure probably caused by an increase in temperature during promastigote to amastigote differentiation. These structural changes could alter the binding of specific trans-acting regulators to SIDER2 RNA or alternatively allow stage-regulated factors to interact with the RNA and block access of the decay complex.Our previous studies have demonstrated that SIDER2-mediated mRNA decay is regulated in a stage-specific manner. For the  complex , we show complex . Increasstigotes , 35, 36.L. major as well as in L. infantum, our findings could possibly apply to a larger number of SIDER2 elements given the high conservation of the 79-nt SII. Overall, these studies provide additional insights into the complex mechanism of SIDER2-mediated mRNA decay in Leishmania.In summary, our results demonstrate that both predicted dinucleotide cleavage sites within the conserved 79-nt SII of SIDER2 elements are essential for SIDER2-mediated mRNA decay and further support that SIDER2-harboring transcripts are targeted for degradation by endonucleolytic cleavage. Furthermore, our in silico analysis underscores the role of RNA structure, especially in the vicinity of the two cleavage sites, in the decay process. Although the studies here focused on two selected SIDER2 elements in S1 Table(DOCX)Click here for additional data file.S1 FigL. major LUC-3810 3'UTR (WT), LUC-3810 3'UTRΔSI+II ΔSI,II), LUC-3810 3'UTRM1 (M1), LUC-3810 3'UTRM2 (M2), LUC-3810 3'UTRM3 (M3), LUC-3810 3'UTRM4 (M4), LUC-3810 3'UTRM5 (M5), LUC-3810 3'UTRM6 (M6), LUC-3810 3'UTRM6-1 (M6-1) and LUC-3810 3'UTRM6-2 (M6-2), digested with NdeI and analyzed by Southern blot hybridization using a radiolabeled DNA probe complementary to first 1kb region of the 3810–3'UTR. This probe recognizes both the LmjF.36.1830 genomic locus  and the linearized episomal vector (~8 kb band). Quantification of the hybridization intensity signals was carried out using PhosphorImager. The ratio of the plasmid signal vs. the genomic signal provides an estimate of the copy number of the episomal LUC-expressing vector in each transfectant. These values are shown in the upper graph.Total DNA was extracted from (TIF)Click here for additional data file.S2 FigRNA predictions were carried using the RNAfold web server according to the Minimum Free Energy (MFE). The 79-nt signature II (SII) sequence of LmJF.36.3810 SIDER2 is surrounded by a red box. Colors in the structures indicate base-pairing probabilities according to a scale of 0 to 1 .(TIF)Click here for additional data file.


Idaeovirus was proposed. Analysis of NGS-derived paired-end reads revealed the existence of bridge reads encompassing the 3′-terminus and 5′-terminus of RNA-2 or RNA-3 of BCLCaV. The full RNA-2 or RNA-3 could be amplified using outward facing or abutting primers; also, RNA-2/RNA-3 could be detected even after three consecutive RNase R enzyme treatments, with denaturation at 95 °C preceding each digestion. Evidence was obtained indicating that there are circular forms of BCLCaV RNA-2 and RNA-3.Blackcurrant leaf chlorosis associated virus (BCLCaV) was detected recently by next-generation sequencing (NGS) and a new and distinct species in the genus  Idaeovirus [r 197 kDa); (2) a bicistronic RNA-2 that encodes a putative movement protein  and the putative coat protein  of the virus, with ORFs that overlap by a single adenine (A) nucleotide (nt), representing the third position of an opal stop codon of ORF2a and the first position of the start codon of ORF2b; (3) a subgenomic form of RNA-2 (putative RNA-3) that contains ORF2b, the putative CP coding region; and (4) a concatenated form of RNA-2 that consists of an inverted RNA-3 conjoined to the full-length RNA-2 [Idaeovirus.Blackcurrant leaf chlorosis associated virus (BCLCaV) was detected by next-generation sequencing (NGS) and analysis of its genome indicated that it is a new member of the genus aeovirus . A similaeovirus . Severalaeovirus . BCLCaV 97 kDa);  a bicistRaspberry bushy dwarf virus (RBDV) is a linear, positive sense ssRNA virus, the only species of the genus Idaeovirus recognized currently by the International Committee on Taxonomy of Viruses (ICTV) and the type member of the genus [Idaeovirus [he genus . RBDV wahe genus . The genhe genus ,7,8. In aeovirus  and sincaeovirus ,7,9; andaeovirus ,7,9. In aeovirus  showed tIdaeovirus. There are certain peculiarities associated with these viruses. Their RNA-2 intergenic noncoding regions (IG-NCR) vary in size, with approximately 117 nt for RBDV (accession NC_003740), 1 nt for PrLBaV (LT221869), and 0 nt for BCLCaV (KX838924). The third nt (A) of the opal stop codon (UGA) of BCLCaV ORF2a acts as the first nt (A) of the start codon (AUG) of ORF2b [Rubus glaucus). James and Phelan [RBDV, PrLBaV, and BCLCaV are closely related phylogenetically; they possess similar genome organizations expressing similar proteins of similar sizes and their 3′ terminal nucleotide (nt) sequences form typical stem loops and are terminated by a 3′ poly(C) tail ,2,3,7, sof ORF2b  described Phelan , but witd Phelan .Herold and Andino  suggesteRibes nigrum). Bridge reads suggestive of circularity were found in essentially two types. One type included the 3′ terminus and 5′ terminus of BCLCaV RNA-2 as contiguous sequences and the other featured the 3′ terminus and 5′ terminus of BCLCaV RNA-3. We hypothesized that this may indicate the presence of other forms of end-to-end concatenated RNA-2/RNA-3 associated with BCLCaV, or possibly circular RNA forms. RT-PCR assays were developed to confirm linkages of the 3′ terminus and 5′ termini, as indicated by the NGS-derived bridge reads. Techniques were used to determine if indeed circular RNA was present including: (a) attempts at amplification of full-length fragments using outward facing or abutting primers [Taq DNA polymerase to aid in identifying concatenation. The results of these studies are described. Evidence is presented that indicates that circular RNAs are associated with BCLCaV RNA-2 and RNA-3.In this study, BCLCaV RNA-2/RNA-3-derived “bridge reads” were observed among NGS-generated paired-end reads generated from dsRNA extracted from BCLCaV-infected blackcurrant , accession number 3124-03D1, co-infected with the potexvirus Actinidia virus X [Nicotiana benthamiana by mechanical sap-transmission, as described by James and Phelan [Ribes plants and healthy N. benthamiana plants were used as negative controls.Blackcurrant leaf chlorosis associated virus (BCLCaV) was maintained in blackcurrant plants  and from a healthy control plant were carried out as described by Tzanetakis and Martin [Double-stranded RNA (dsRNA) extractions from symptomatic d Martin . ComplemN. benthamiana and blackcurrant were carried out using QIAGEN’s RNeasy Plant Mini Kit  using the modified protocol described by Kalinowska et al. [2O.Total RNA extractions from a et al. . Total RTM reverse transcriptase and the appropriate oligonucleotide primers . This was followed by PCR performed on the cDNA template using Invitrogen Platinum®Taq DNA Polymerase High Fidelity. Amplified cDNA fragments were gel purified and extracted using a MinElute Gel Extraction Kit (QIAGEN), ligated into the pCR 2.1 TOPO vector, and cloned using the TOPO TA Cloning Kit as described by the supplier . Plasmids with virus-derived cDNA inserts were sequenced by Sanger sequencing at the Nucleic Acid Protein Service Unit, UBC, , on an Applied Biosystems 3730 DNA Analyzer .Primer design was aided by Clone Manager 9  1994-2010, Scientific and Educational Software, Cary, NC, USA). cDNA was generated from total RNA using Invitrogen’s SuperScript IIwww.clcbio.com). Alignments were generated in CLUSTAL-X 2.1 [http://mfold.rit.albany.edu [Raw data files were imported, paired, and the reads trimmed with default parameters for quality and ambiguous nucleotides. Additionally, reads were trimmed of TruSeq universal and indexed adapter sequence. Bridge reads were identified by read mapping to BCLCaV sequences with increasing stringency using CLC Genomics Workbench v7.5.1  RT-PCR with primer pair BRI-0 . For the cDNA synthesis reaction, a 10 μL pre-mix containing 2.5 nmol of each dNTP, 0.01 nmol of a gene-specific primer, and 1 μL of total RNA template was denatured at 65 °C for 5 min, then placed on ice immediately. The pre-mixes were then added to 10 μL of a cDNA synthesis mix  to give a final 20 μL reaction mix. Reverse transcription was carried out at 50 °C for 50 min. When completed, 2 μL of this product was used for PCR amplification. These reactions (25 μL) used gene-specific primers at a final concentration of 0.4 μM and MgSO4 at 1.5 mM, with other reagents used as recommended by the manufacturer. PCR was preceded by a 2 min denaturation at 94 °C, followed by a touchdown phase of 17 cycles. In this phase, each denaturation was at 94 °C for 30 s, followed by a 30 s annealing step for which the temperature was set at 1 °C lower than that of the previous cycle, starting from 72 °C. Elongation steps were at 68 °C for 1 min/kb of target. For the PCR phase, cycles were identical to that of the touchdown phase, except for the annealing temperature, which was kept at 55 °C for over 25 cycles. In cases where a second round of PCR was performed, 2 μL of the previous PCR reaction was used in a similar 25-cycle reaction with no touchdown phase, and with final primer and MgSO4 concentrations at 0.2 μM and 2 mM, respectively.In order to verify the “bridge reads”—identified in the NGS data—that connected the 3′ terminus of RNA-2/RNA-3 to the 5′ terminus of the respective RNAs, RT-PCR tests were designed using outward facing primers (with respect to the linear RNA) that would flank a hypothesized junction site. The samples used in these tests included total RNA samples extracted from BCLCaV-infected ir BRI-0 B used toir BRI-0 B. The RTTaq polymerase and Taq Extender reagents were used here at the manufacturer’s recommended concentrations . Final concentrations of other reagents were: 1.5 mM MgCl2, 0.1 mM dNTP, and 0.2 μM each of the forward and reverse primers.An effective strategy used commonly for detecting the presence of circular nucleic acid forms is amplification using abutting primers to produce full-length fragments ,39. The nad5 gene [RNase R digestion was performed using a procedure similar to that described by Suzuki et al. , but witad5 gene . The seq4-73′/5′acaaagtgctaat …3′), with the 3′ terminus of RNA-3 in uppercase and its 5′ terminus in lowercase, showing clearly the variable number (4–7) of cytosine (C) residues at the 3′ terminus. This was observed also in RACE analysis by James and Phelan [Compared to RNA-1, the number of trimmed reads and the coverage associated with RNA-2 were higher by approximately 4-fold and 10-fold, respectively. In the course of analysis and genome assembly , bridge The bridging primers successfully amplified their contiguous target regions, confirming linkage. R. nigrum infected with BCLCaV (3124-03D1), using the abutting primer pair A  infected with BCLCaV. James and Phelan [RNA-2/RNA-3 contiguous bridging (3′ terminus/5′ terminus) paired-end reads were identified among NGS sequences generated from dsRNA extracted from The bridge reads described in this study are not associated with the bridge of BCLCaV RNA-2m (inverted RNA-3 conjoined to RNA-2). In this study, the bridge reads are contiguous and contain the 3′ terminus of RNA2/RNA-3 (including the variable poly(C) tail) and the 5′ terminus of the RNA-2 or of the RNA-3. This indicates a linkage that may be the result of end-to-end concatenation of BCLCaV RNA-2 and/or RNA-3 elements or associated with circular RNA. Very sensitive touchdown RT-PCRs with primers that span linkage sites were used to determine if indeed end-to-end concatenation existed, which should manifest by cDNA products or more than one cDNA products that could be amplified and sequenced to confirm concatenation. Evidence for end-to-end concatenation was never obtained. Circular RNAs are uniquely characterized by covalent linking of the 5′ and 3′ ends . RT-PCRsEvidence was obtained that is consistent with the existence of circular RNA associated with BCLCaV RNA-2/RNA-3. In addition to confirmation of the bridge reads, outward facing or abutting primers were used to generate full-length products of both RNA-2 and RNA-3. Abutting primers will only generate the full-length sequence of a target if that target is circular ,24,25,40The multiple forms of BCLCaV RNA-2/RNA-3 is likely a contributing factor to the much higher NGS read coverage associated with RNA-2/RNA-3, compared to the RNA-1-associated reads. Interestingly, this may facilitate enhanced reliability of detection using tests such as RT-PCR that target the CP encoding region present in these various RNA-2/RNA-3 forms. This is important, as BCLCaV is associated with disease symptoms [Base-pairing of flanking and complementary sequences can serve to facilitate circularization ,30. BCLCIdaeovirus [If there is indeed any accuracy to the statement by Herold and Andino  that ciraeovirus . Furtheraeovirus , will be7G(5′)ppp(5′) N cap and a 3′ poly(A) tail—and that, in some cases, as in the case of potyviruses, a 5′ VPg might act in a manner similar to the 5′ cap. RBDV [Idaeovirus, PrLBaV [Pettit Kneller et al.  stated tap. RBDV  and the , PrLBaV ,2, lack , PrLBaV ,2,3. It , PrLBaV . Althoug, PrLBaV  providedHolmes  indicate


Transcriptomic profiling of ex vivo cultured human limbal epithelial stem cells (hLESCs) will foster better understanding of corneal physiology and novel treatment paradigms to limbal stem cell deficiency (LSCD). However, currently such profiling studies are hampered due to difficulties with producing sufficient amounts of intact mRNA for deep RNA sequencing (RNA-seq) from subpopulations sorted on the basis of co-expression of membrane and intracellular antigens by fluorescence-activated cell sorting (FACS).To address this problem, we systematically analyzed the critical steps, and found that ethanol fixation together with optimized downstream procedures provided a pipeline that yielded high quality total RNA in amounts to readily support the RNA-seq procedure, while still preserving good discrimination between the individual hLESC immunophenotypes.The average RNA integrity number (RIN) was 7.7 ± 0.4, and the average yield was 4.6 ± 1.7 pg of RNA per cell. The sequencing analysis of the isolated RNA produced high quality data with more than 70% of read pairs mapping uniformly to the reference genome and 80% of bases with a Phred score of at least 30.In this study, we developed a reliable FACS-based procedure using ethanol as a fixative that would support accurate isolation of limbal epithelial progenitor subpopulations along with RNA yield and quality sufficient to enable deep transcriptomic profiling.The online version of this article (10.1186/s12575-017-0065-2) contains supplementary material, which is available to authorized users. Corneal epithelial disease due to limbal stem cell deficiency (LSCD) is a major cause of blindness worldwide . The useTo achieve this goal, defined hLESC subpopulations need to be identified and isolated, and ultimately yield a high quality RNA. In the face of difficulties to identify an archetypal stem cell profile, the stemness in the cultured hLESCs is currently believed to be associated with the surface ATP-binding cassette sub-family B member 5 (ABCB5)  antigensThe fixation and permeabilization steps have in particular been shown to have an adverse effect on the RNA integrity –10, but There is only scanty literature dealing with transcriptomic profiling of cells sorted by FACS on the basis of simultaneous surface and intracellular immunostaining. Incidentally, most of these reports are based on the use of formaldehyde as a fixative –17 but a2 in complete Keratinocyte-SFM (Life Technologies). The medium was changed every 2–3 days. At 80–90% confluency, the cells were trypsinized using TrypLE Express at 37 °C for 10 min, filtered through strainer, washed and resuspended in sPBS. Single cell suspensions of 0.5 to 1.5 × 106 cells were aliquoted into 2.0 ml RNase-free microfuge tubes  for subsequent immunofluorescence staining. The isolation and expansion process were repeated at 3 distinct time points.The hLESCs isolation and culture protocol was optimized based on our previous report . Human cFor optimization, two fixation and permeabilization methods were compared. The first method included fixation with 4% formaldehyde  at room temperature for 15 min and subsequent permeabilization with 0.1% saponin (Sigma–Aldrich) in PBS supplemented with 1:100 Rnasin Plus Rnase inhibitor  at 4 °C for 30 min. The second method was a one-step fixation and permeabilization using 70% ethanol in PBS  at 4 °C for 10 min Fig. .Fig. 1Ef6 cells. In order to discriminate dead cells, the LIVE/DEAD Fixable Dead Cell Stain Kit (Life Technologies) was used according to the manufacturer’s instructions. Following staining, the cells were resuspended in a sort/collect buffer consisting of the washing buffer with 10-fold diluted RNasin Plus Rnase Inhibitor in a concentration to yield from 0.5 to 2 × 106 cells per ml. The cell suspensions were then filtered through the Pre-Separation Filters  into 5 ml round bottom polystyrene tubes . All samples were left on ice in the dark until FACS.The cells were first stained in suspension with direct conjugated antibody against the surface marker ABCB5 . In order to avoid RNase contamination, the instrument was thoroughly decontaminated with a cleaning agent , RNaze ZAP (Sigma-Aldrich), 70% ethanol, and milli-Q water. After decontamination, RNaseAlert Lab Test kit (Ambion) was used to ensure the complete absence of RNase.+p63+CK3+ (SP1), ABCB5+p63+CK3− (SP2), ABCB5+p63−CK3− (SP3), and ABCB5−p63+CK3− (SP4). The subpopulations were sorted into polypropylene round bottom FACS tubes  pre-filled with the sort/collect buffer. After taking an aliquot for quality control, the sorted subpopulations were pelleted, the supernatant was drained completely, and the samples were flash-frozen in liquid nitrogen. Frozen cell pellets were kept at −80 °C until RNA analysis.For phenotype analysis and gate setting, data on a minimum of 10,000 events were collected. For sorting, the Summit Software v4.3 (Beckman Coulter) was used, with the sort mode set as “purify”, and the gates set with reference to the isotype and FMO controls Fig. . To maxiFor quality control the integrity of the RNA was assayed at several steps during the staining and sorting procedure with the aid of the Aurum Total RNA kit  and the Agilent 2100 Bioanalyzer  both as prescribed by the manufacture Fig. .Homo sapiens UCSC hg19 reference genome (http://hgdownload.cse.ucsc.edu/goldenPath/hg19/bigZips/). The alignment quality was further assessed by the Qualimap v2.2  producing the read coverage and GC content profiles [After flash-freezing the transcriptomic analysis of the frozen subpopulations (SP1–4) was performed by AROS Applied Biotechnology  using the SMART-Seq v4 Ultra Low Input RNA Kit for cDNA and Low Input Library Prep Kit V2 for sequencing library preparation , and Illumina HiSeq 2000 instrument  for sequencing. The sequencing data were imported as paired-ends into CLC Genomic Workbench v9.0.1  to enable RNA-seq reports, including reads average PHRED score and mapping statistics against the annotated profiles .The optimization of the procedure for RNA-seq of the ex vivo expanded and FACS sorted hLESCs was done according to the paradigm illustrated in Fig. 7 cells were processed for each run so that at least 400 ng of total RNA would be harvested, which is the amount necessary to guarantee a high quality sequencing data.When comparing different subpopulations, a considerable variability in RNA yield per cell was found, ranging from 2.25 to 6.37 pg of RNA per cell, with an average of 4.59 ± 1.71 from all four subpopulations. Additionally, a positive correlation between the number of lysed cells and the integrity of produced RNA was observed  was higher than 30 for 80% of all base calls. For mapped reads, the average GC content was 49.25% and the GC content per sample displayed a normal distribution. After alignment, on average, 82.67% of reads were mapped in pairs to the reference genome (human genome hg19), and 86.96% of total mapped reads were mapped to exonic regions. Reads coverage profile over gene body was obtained by scaling all transcripts to 100 nt and calculating the number of reads covering each nucleotide position. The result showed uniform gene body coverage. Raw sequencing data were deposited in the Gene Expression Omnibus  under the accession number PRJNA387095 and ID 387095.The performance of the sequencing was assessed by assessing the overall read quality, the mapping statistics and read coverage, and the GC content Fig. , Table 2P < 0.05 was set to discriminate statistical significance.The data are presented as mean ± SD from three independent experiments entailing a total of 2–4 biological replicates. One-way ANOVA (LSD) of the SPSS 24.0 package  was used to test for differences between the samples, and During the last decade, there has been an impressive advancement in the tools for transcriptome analysis in the scientific community and in our laboratory, from the rather simple membrane arrays allowing simultaneous analysis of a couple of hundreds of different genes , to moreFor the sorting of heterogeneous cell populations, we rely on both extracellular and intracellular markers, and therefore need to fix and permeabilize the cells prior to staining and sorting. Formaldehyde has traditionally been used as a fixative in histology and immunochemistry due to its ability to preserve cell morphology. This is due to the formation of protein-protein and protein-nucleic acid cross-links involving methylene bridges (-CH2-) . HoweverAnother major challenge in this study was to protect the RNA against the RNase-mediated degradation. The cells in our protocol were permeabilized to reveal intracellular antigens, which allowed for an increased exposure to contaminating extracellular RNase. RNase inhibitor was therefore included in all solutions following fixation and permeabilization, and rigorous cleaning was implemented to decontaminate all surfaces coming into contact with the cells. As another measure to reduce the exposure to RNase, the buffering capacity of the solutions was boosted by adding a non-phosphate HEPES system and a gentle cell detachment solution (Accumax) was implemented with the aim to minimize the cell death. All additives used in this study were previously shown to have no adverse effect on RNA integrity . Since cSince degration of RNA will greatly affect the coverage of transcripts, it is of great importance to control quality of input material before transcriptomic profiling by assessing raw reads quality and mapping to the reference genome . Before Purification and characterization of progenitor cells in human corneal epithelium remains a challenge since the stem cells constitute only a small fraction (< 10%) of the cultured limbal epithelial cell population  and thei


Proteomic and genomic techniques have reached full maturity and are providing unforeseen details for the comprehensive understanding of disease pathologies at a fraction of previous costs. However, for kidney diseases, many gaps in such information remain to inhibit major advances in the prevention, treatment and diagnostics of these devastating diseases, which have enormous global impact. The discovery of ubiquitous extracellular vesicles (EV) in all bodily fluids is rapidly increasing the fundamental knowledge of disease mechanisms and the ways in which cells communicate with distant locations in processes of cancer spread, immunological regulation, barrier functions and general modulation of cellular activity. In this review, we describe some of the most prominent research streams and findings utilizing urinary extracellular vesicles as highly versatile and dynamic tools with their extraordinary protein and small regulatory RNA species. While being a highly promising approach, the relatively young field of EV research suffers from a lack of adherence to strict standardization and carefully scrutinized methods for obtaining fully reproducible results. With the appropriate guidelines and standardization achieved, urine is foreseen as forming a unique, robust and easy route for determining accurate and personalized disease signatures and as providing highly useful early biomarkers of the disease pathology of the kidney and beyond. Following extensive genome-wide analyses and the full maturation of integrative technologies such as RNA-sequencing (RNA-Seq) techniques to reveal the crucial regulatory functions of the “non-coding” DNA bulk of human and other species  consists in structural and functional proteins, enzymes, lipids and peptides of various lengths. Notably, DNA  and a variety of RNA species are transported within EVs. These important RNA classes have now been convincingly shown to regulate all cellular functions efficiently and include small RNAs, microRNAs and messager RNAs , with their protein, enzyme or RNA content, have been discovered over the last few years , the recent technical advances in RNA-sequencing  or CHAPS (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate) are used to prevent excessive protein complexing during EV isolation, especially in order to regulate polymerization of Tamm-Horsfall glycoprotein centrifugation, has been shown to easily miss up to 20–30 % of vesicles in the pre-purification steps  to remove, for example, bacteria, cellular debris and excessive polymers of THP before filter-dialysing the sample .Musante et al.  developeIn HFD, the hydrostatic pressure of the sample pushes it through the dialysis membrane tubing. For recovery of urinary proteins and miRNA, the HFD method is clearly superior to the ultracentrifugation-based methods and provides superior quality of protein and RNA yield , Invitrogen Total Exosome Isolation Kit  and others. These products have been tested with solutions of liposomes and exosomes and have shown not only a robust isolation capacity for EV but also the co-isolation of molecules with similar physical properties. Thus, other additional purification methods may need to be used , with distinct RNA contents and constant interaction with the host defense system. As a result, a strong, although less-well understood barrier function is achieved.The number of studies on EV, especially exosomes, has been sky-rocketing during the last few years. This is because their extraordinary potential is not limited to the dissection of disease mechanisms to identify new druggable targets but can also provide early biomarkers of unforeseen accuracy. EV, which are also abundantly present in the urine, are an unparalleled resource for individualized molecular fingerprints reflecting cellular pathophysiology at their site of origin upstream in the kidney and beyond (Miranda et al. The potential of EV, especially for early diagnostics and as novel biomarkers for kidney diseases, is exciting. With full systems biology integration, combining their proteome, RNome and metabolomics information, EV now show great promise in dissecting these thus far less-well understood disease mechanisms in an individualized way (Erdbrugger and Le Whereas the physical properties and protein and RNA content of the various EV types significantly overlap, a careful selection of methods available should provide a rational basis for an appropriate selection of each type of application (Musante et al. Notably, methods for single vesicle detection are developing rapidly, including those utilizing microfluidics and nano-sized beads. These will be highly useful, especially for linking the different vesicle types to specific contents and, thus, for revealing details of their functions. The respective compact systems and techniques including micro-nuclear magnetic resonanace (Shao et al. The massively parallel Next-Generation Sequencing as applied also to uEV analytics is a welcome extension to the repertoire of the novel robust and non-biased methods for urinary analytics (Renkema et al. An exciting extension to utilizing EV biology relates to bioengineered nanoparticles. These can serve as EV mimics and can be released into the circulation with a defined target cell “address code”. Upon attachment, the engineered protein, regulatory RNA or selected medication contents can be released as previously exemplified by the delivery of targeted chemotherapeutics (Jang et al.