Background. Even though the interpretation of natural language messages is generally conceived as the result of a conscious processing of the message content, the influence of unconscious factors is also well known. What is still insufficiently known is the way such factors work. We have tackled interpretation assuming it is a process, whose basic features are the same for the whole humankind, and employing a naturalistic approach .Methodology. Our field research involved a random sample of 102 adults. We presented them with a complete real world-like case of written communication using unabridged message texts. We collected data (participants’ written reports on their interpretations) in controlled conditions through a specially designed questionnaire (closed and opened answers); then, we treated it through qualitative and quantitative methods.Principal Findings. We gathered some evidence that, in written message interpretation, between reading and the attribution of conscious meaning, an intermediate step could exist (we named it “disassembling”) which looks like an automatic reaction to the text words/expressions. Thus, the process of interpretation would be a discontinuous sequence of three steps having different natures: the initial “decoding” step , disassembling  and the final conscious attribution of meaning. If this is true, words and expressions would firstly function like physical stimuli, before being taken into account as symbols. Such hypothesis, once confirmed, could help explaining some links between the cultural (human communication) and the biological (stimulus-reaction mechanisms as the basis for meanings) dimension of humankind. Specific works will be indicated within the manuscript. Taking linguistics apart, we make reference to  The available data does not give definite answers to the researchers’ questions; our field research intends to bring some contributions to such endeavour.Human–environment interactions entail conscious knowledge, i.e., the attribution of meanings (semantic aspect) to the incoming signals and stimuli. Interpretation, namely the operation through which the meaning is attributed, is still widely unknown. A specific difficulty is represented by natural language, although it has been studied almost since the dawn of humankind, with researches ranging from the ancient rhetoric  knowledge. Incoming information is provided through the body (perception) but the “mind”2We will not enter the disputed question of mind, its existence, its nature and its relationships with the body in general and the brain in particular. For a first level of delving further into this subject: on the one hand, the early survey of  processes inputs at symbolic level, turning them into propositional representations in the brain and understanding them in terms of concepts. The answer to the inputs (reaction) is based on such comprehension and is shaped as a command to some effectors . Knowledge is the result of a sort of computation; the mind is separated from the body and rules it. The role of the motor system is totally passive.All this considered, we can roughly outline a picture with two main scientific research lines, the first of which can be named Body-centred approaches and can be synthesized as follows. Understanding/interpretation is attained through a motor reaction of the body that instantiates understanding or, at least, co-exists with conceptual knowledge. When an external stimulus/signal is perceived, it is firstly “understood” through a motor reaction which is automatic, involuntary and based on “mental maps” that are motorial, not (or not only) propositional. Understanding is a sort of motor experience that goes along with conscious  information processing; the body is not detachable from the mind and can drive it. The role of the motor system is active and decisive for understanding.The second research line can be named 3The last three works  are ascr The range of these theories goes from the merely mechanical (and naïve) hypotheses of psychoneural isomorphism  can be traced back, at least, to XIXth Century, up to the works of  neurons  and the  neurons . Accordipouring, filling, emptying, tipping, rotating, inverting, spilling (if the liquid missed its mark) or defying/ignoring/rebelling (if the pourer was instructed not to pour) …”  and Embodied simulation (ES) . About t7per se resolving the problem of knowledge  studies can be traced back to the Thirties and the works of Alan Turing on a possible “intelligent machine”. About the origins, see ; in addition, some studies make reference to deterministic chaos .See In the end, it is worth mentioning a recent specialised research field inside psychophysics, in which researchers investigate cognition and semiosis through probabilistic models , applyinic chaos  and some10passim. A recent line of research is investigating the connections among single neurons activity and the total effects detectable through indirect techniques  return very accurate measurements, but on small brain cortex surfaces. About the ethic difficulties: those techniques are almost impossible to be used on humans, and only indirect techniques as fMRI , MEG , PET (Positron Emission Tomography) or TMS  are systematically employed. They cover wider brain cortex surfaces but with inferior accuracy; moreover, they present difficulties with regards to instrument positioning and image interpreting. For a survey of these difficulties see , chapter The second main reason is the complexity of natural language  in the same way). That means: a same word can be used to refer to different meanings and different words can be used to indicate the same meaning.), which is usually overcome through studying interpretation isolated from the interpreting organism and employing simple stimuli  to understand the process of interpretation  as it works in real conditions, and design a structural model in order to adequately represent it; (ii) to produce a first check of the formulated hypothesis. We tried a naturalistic approach; this means, first, that a phenomenon must be carefully observed and precisely described in conditions the closest to “natural” ones . Second, it means that observation and description must precede analysis, being carried out independently of it. In such approach, the role of the observers is critical, either if they are involved in or external to the phenomenon. In our research, we have employed 102 observers of the first kind (the sample) and 5 (the authors) of the second one; this way, we have collected 102 self-reports  and worked out one analytical report (our research) about interpretation.This was not a clinical trial and no experiments were conducted on the participants. In addition, no personal data was collected or in any way used in the survey, and verbal informed consent was obtained. The Ethics Committee for Scientific Research of the Association ARPA-Firenze gave its approval either to the research design or to the informed consent procedure. Further details related to method, sampling and ethical aspects can be found in the Our research plan has been based on two main assumptions: first, interpretation is a process, rather than a single operation; second, the process has the same basic  universal characteristics. The rationale of our sampling was based on such assumptions: according to our objectives, we focused on the reconstruction and understanding of the process, rather than on sample features. Thus, the sample representativeness , as well as its social feature balance, were less critical; from an extreme point of view, it could be sufficient that the sample members would belong to human species. Operatively, we gathered our random sample through selecting only Italian language native speakers, all adult; we strived to reach a reasonable balance about gender and student/worker conditions. Further details (the procedure we used to randomize the sample included) can be found in The total sample  results The main operative instruments through which we have implemented our naturalistic-like approach  answer of the Project Account in which the regularity of the Project progress is declared. The message ends with the phrase: “at the moment, the progress substantially complies with the chronogram.”A 136 words reply in which XX declares herself totally unsatisfied. Her message presents two main features: (i) some minor flaws are listed; (ii) she expresses what resembles an actual threat against YY, in the case he would not take measures .Now the conflict is on and the second phase starts: YY prepares a reply to XX’s Msg #3 . The label “H” has been used because such version is a “hard” reply; a YY’s colleague suggests him a softer version (the “S” one) in order to avoid exacerbating the conflict. YY accepts the advice, he sends the Softer Msg #4(S) to XX and the case ends with the conflict resolution (XX’s satisfaction declared in Msg #5). Full-text versions of the Hard Message #4(H), the Softer #4(S) and of Msg #5 are displayed in The questionnaire has been the instrument through which we have challenged the sample with the case; it is fully documented in Our peculiar management of the survey and, specifically, of the participants/survey conductors relationship , we investigated the relationship between the interpretation of a situation and a consequent decision to be made; such decision was the selection, between the original and the colleague suggested versions of Msg #4 (“Hard” and “Softer” versions), of the one capable to solve the case . Our thought was that the consistency between interpretation and the following decision could give us either further clues for a deeper understanding of the interpretation process or elements for checking our hypothesis.The results presented in this Section are based on data regarding the first phase of the XX–YY interaction (Messages #1–3), investigated through the first part of the questionnaire (Questions #1–2). We recall that each question submitted to the sample sent two inputs: at first, participants were requested to freely interpret some aspects of the messages; then, to account for their own interpretations indicating the “concrete elements” on which these were founded. Given that the two kinds of inputs elicit different kinds of data, we will present separate analyses.The answers to the first input of the questions show that the interpretations provided by participants are widely scattered. Such scatter can be observed for all messages and for any part of them, even if accurately selected; we have delved further into one of the cases present in our research. Through Question #2, we firstly asked participants if, comparing Message #3 with Message #1, they found the attitude of XX (the sender) towards YY (the receiver) being changed , and distributing them into homogeneous categories. Such an operation was performed by one of the authors, then discussed and shared with the others; its result consisted in the macro-categories presented in per se, mere “form” components), their relative amount can be estimated in at least 35% .In order to delve further into such matter, we named “components” the categories/sub-categories of the indicated concrete elements and we tried a quantitative analysis. Given that our focus remained on the process, rather than on the sample features, our goal was to provide a rough estimate. Such an estimate was important mainly in relative terms: in case of relative small non-content (non-information) component amounts, we would have to abandon this part of our research. But those amounts were not small. Our analysis of the 1,319 detected components is displayed in The proportion of the information content components on the total is very low; even if we sum their relative amount (12.1%) to the indications of full sentences or periods  we reach just 1/3 of the total (33%). The question was important and we carried out a further check: we carefully re-examined the filled questionnaires with reference to the information content component. We found out  that oneFollowing our observations, it seemed that every aspect of even a written message , regardless of its nature and its intrinsic semantic value, could be treated as a meaningful element of the message, with an extreme degree of scatter among the participants. This was especially surprising because we had used written messages only, bereft of added signals like non-verbal language and context stimuli that usually affect verbal communication , #1-b (15) or both the questions (15).: she premised her request of a technician inspection with the words “we would be pleased if at least once…” This simple expression, apparently trivial, short (8 words in a 67 word message) and in no way highlighted in comparison with the rest of the text, has collected 68 quotations . Then, respondents have interpreted such specific passage in at least 22 different ways, summarized in Question #1 requested evaluations related to sender-receiver positions and to the relationship between them, on the basis of Messages #1 and #2  which turns written signs into words.15In our opinion, the process should be the same even in case of oral communication (reading and turning written signs into words should just be replaced by listening to and turning spoken sounds into words). We named it “decoding” and assumed that its outcomes feed the following step (the selective focusing) whose outcomes, in turn, feed the final one .Actually, the two actions of focusing on components and interpreting them seem to have different natures. In order to clear this point, we recall an observation reported in the previous sub-section: on the one hand, respondents explain the conscious meanings they attributed through the outcomes of their individual selective focusing . On the other hand, they never explain the reasons why they exactly focused on those components: such focusing manifests “immediately and automatically,” priming the attribution of a conscious meaning. In addition, if we would assume that focusing and consciously interpreting have the same nature, our reasoning would fall into an infinite regress.In the end, we outlined the model of the fact that one of these components is present in the message; it can be considered some meta-information to which readers can automatically react (the fact that XX had (redundantly) placed it at a certain point of her message.16It is particularly interesting to note that the expression “the fact that…” is spontaneously used by several respondents in their answers. For example, in the collected questionnaires we can find expressions like the following: “the fact that the arguments are presented through a dotted list”; “the fact that XX is referring to public money.”One last question remains: if a reader reacts to a given component, even though it appears to be meaningless/contentless, we need to identify what, exactly, that reader perceives. We think we can identify it as ly react . This caThe results presented in this Section are based on data regarding the second phase of the XX–YY interaction . By cross-checking the combined predictions with the final choice . Such results led us facing the core-question related to our hypothesis: given the existence of some correlations between choice and combined predictions, which is its direction? We mean: do the interpretations (the predictions) drive the choice (cognitivism stance) or, oppositely, does the choice precede and somehow drive, or overcome, the interpretations (embodied cognition stance)?The combination of the two symbols reports the combined predictions each participant expressed about the effects of the two versions on XX: H+/S+ (both the versions solving the conflict), H+/S− , H−/S+ (the opposite), H−/S− . Dichotomously displaying “H” against “S” predictions  returns l choice  we obtaiTo delve further into such subject, we created a “coherence indicator” starting from the following premises: (i) The final Message #5 clearly indicates XX’s satisfaction; therefore, the conflict has come to its end. (ii) Now, let us figure a respondent whose answers to Questions #3 and #4 return a combined prediction H+/S− . Then, we expect that this respondent indicates the “Hard” Message #4 in his final choice. Such combination (H+/S− & “Hard” Msg #4 choice) would represent the maximum coherence level. (iii) If another respondent provides the same combined prediction but chooses the “Softer” Message #4 (combination H+/S− & “Softer” Msg #4 choice), this would represent the minimum coherence level. (iv) Given the natural variability always recorded in human samples, we expected to find also intermediate coherence levels, based on the other possible combinations (H+/S+ and H−/S−). These could also be due to the predictable scattering of interpretations about the final Message #5: someone could interpret it as something different from the sign of the conflict ending (what happened in a fistful of cases).We defined four coherence levels, increasing from L (low) to LM (low-medium), MG (medium-great) and G (great); the scale is fully represented in p < 0.001). Graphic representations render even better such asymmetry: the total sample histograms . Data is displayed in stograms  show thastograms –S11, incstograms  and “Empstograms  sub-sampNow, statistical tests and graphic representations clearly indicate the existence of a correlation between the participants’ choice and the coherence level; but what about its strength and its direction? In order to investigate the strength, we calculated the odds ratio. Our success item was the L level, our failure items were all the other coherence levels. Using data from All this contrasts our “Hypothesis 0”: the participants’ choice does not seem to come as a result of the text information conscious processing. Then, the choice should be independent of the previous interpretations, what upholds our “Hypothesis 1”. After this first conclusion, we set up a second indicator (“block preference” indicator) to further check our hypothesis. For text length reasons, we present details about such indicator, its employment, and relative analysis in 17The unique doubt expressed in the whole research is the following: 1 participant (out of 102) declares uncertainties in his final choice (between the “Hard” version of Msg #4 and the “Softer” one) writing that the final effect could be obtained with both the messages. It must be noted that, with regards to the other questions, this special participant’s answers are totally doubt-free. On these bases, we have proposed a three-step model for the interpretation process .With regards to method, our work showed that studying the interpretation of natural language messages in natural-like conditions can complement laboratory studies based on isolated words/phrases and contribute to a wider comprehension of the phenomenon. With regards to results, the picture outlined through the first part of our work can be synthesized as follows: (i) The interpretation process begins with an operation that looks like a selective and subjective picking up of (or focusing on) the most different components, rather than being a systematic, conscious scanning of the text content. Such behaviour is widely scattered: in the whole research, with regards to each specific message, it is impossible to find two identical combinations of components in participants’ answers; (ii) Readers seem to make no distinction among intrinsically meaningful or meaningless components: the meaning they attribute can derive from any “chunk” of the text or from any other text or non-text element arbitrarily chosen; (iii) While the final meaning attributed to the message is justified through the indicated components, no reason  is provided for that selection: in the participants’ answers, the focused components suddenly appear; they are presented just as “given,” and without any doubt.  process ; the cruphysical stimuli) that trigger automatic reactions off in the receivers.19Such ambivalence looks interestingly (or just curiously?) similar to what happens in certain physics phenomena like the double nature of light (waves/particles) or the uncertainty about some features of many atomic particles. In those cases, the ambivalence is solved just in the process of measuring the phenomena  It also means that the third step (conscious attribution of meaning) is fed by the outcomes of the unconscious reaction (“disassembling”), rather than by the original message; our conscious direct contact with the real world would be prevented, and we would actually attribute conscious meanings just to our automatic reactions to it. In short: through the first part of our work, we have outlined the possible structure of the message interpretation process.If our hypothesis will be confirmed, this means that words are not mere symbols; they are also stimuli ; we found out significant imbalances in the coherence between interpretation and choice. Roughly, we can label “rational” the choices that show maximum coherence with the previous interpretations of the two messages ; conversely, we can label “irrational” the choices that show minimum coherence. We found that the irrational cases are significantly ascribable to “H” version choosers rather than to “S” version choosers. In other words: the elements provided by interpretations appear insufficient to determine the choice; this means that other factors intervene. Such factors should be unconscious, otherwise they would be declared by at least some participants; in addition, they must have a different and stronger source with regards to the conscious/rational analysis of the message content, otherwise their influence on the choice would not prevail.The main question is: why, in the decision process, do these factors significantly weigh just in connection with one choice and not with the other one? Further research is needed to find the answer. Provisionally, we think there are two possible hypotheses: (i) The two sub-samples follow different paths in interpreting natural language messages ; (ii) The two sub-samples actually follow the same path  and the difference they show is linked to the differences in their automatic reaction schemes .With respect to the dispute between the stance of cognitivism and the embodied cognition hypotheses, we think our research could be situated in a third position, for two reasons. The first reason is that, while these theories share (even though they come to opposite conclusions) the concept of natural language interpretation as a unique operation, we have seen it as a discontinuous process (three steps of different nature). The second reason is that, in our model, two of the three sub-processes seem to be compatible, separately, with those two theories. We mean: the embodied concept features are akin to our second step (“disassembling”); the cognitivist hypothesis is clearly akin to our third step, see .purely motorial nature; conversely, understanding it, for example, as “pouring” or “filling,” requires the interpretation of a situation which is not limited to the act per se. In order to attribute the “pouring” meaning, one must focus on the liquid flow direction (inside to outside the bottle); for the “filling” meaning, one must focus on the glass receiving the liquid; for the “emptying” meaning, one must focus on the amount of liquid inside the bottle. The attribution of conscious meanings should be preceded by the previous, unconscious selection of specific points of view (something closely resembling our “disassembling” step).Probably, we can better exemplify this through recovering the example  “body” reactions can be traced back up to the hypotheses of the Nineteenth Century philosopher and psychologist William James. In one of his examples  can act like physical stimuli.In the Twentieth Century, we can find the Gregory Bateson’s approach to human communication conceived as a system and to the question of the receiver’s active role; he uses a strictly formal presentation see . Conceivex-post justifications of unconsciously performed behaviours.About the relevance of unconscious processes in human behaviour, some fundamental clarification is provided by The role of physical stimuli in swaying communication through natural language is confirmed by a series of recent works  in human communication through natural language: some traditional empirical knowledge would find theoretical bases  and a revision of human communication current models would be needed . Simply, the fact should be taken into account that human communication through natural language could work in a slightly different way than expected and thought up until now.The second consequence would be the analogical, rather than digital, basis of interpretation. Meaning would be established starting from the body automatic reaction in the “disassembling step,” analogically triggered through individual reaction schemes. This could lead to consider natural language expertise as a system of acquired reflexes, what would mean that human beings would “communicate through their body” in a wider and deeper sense than conceived at present . Such feature could heavily affect the possibility to reproduce human interpretation process on digital computers, regardless of their processing power and data storage capacity. The two systems could result incompatible, rather than simply different. We are not the first who propose such observation  components are indifferently taken into account as sources of meaning with respect to the content components, then the difference between form and content fades, leading to a concept of “message” as a unit made up only by components, all of them having the same importance (the same ex-ante probability of being chosen).The third consequence could derive from our observations about the taking into account of the message components by the reader, that seems to be performed like a subjective operation, quite arbitrary and unpredictable. If this will be confirmed, the concept of “content of a message” should probably be revised, given that it would result impossible to The meaning attributed to a message is the receiver’s synthetic conscious report  on the final state of his/her organism after experiencing the interaction with the message.At the end of our arguing about the attribution of meaning, it is worth briefly considering the problem of “what is meaning” (what is the meaning of “meaning”). Beyond the strictly phylosophical, abstract definitions, nowadays we can record attempts to provide operative definitions; for example, 10.7717/peerj.1361/supp-1Supplemental Information 1The Supplemental Materials file contains details, unsuitable to be presented in the manuscript, about the following subjects: Foundations of the chosen methodological approach— Technical documentation —Analysis of the collected data suitability—Added materials aimed to a further check of our hypothesis.Click here for additional data file.10.7717/peerj.1361/supp-2Supplemental Information 2This file presents our raw data and consists of 8 MS Excel Sheets (one for each question or item) containing the transcriptions of all the 102 questionnaire we collected. The information provided through this file fully corresponds to the answers handwritten by participants on the paper questionnaires. The source filled paper questionnaires are stored and conserved in our archive.Click here for additional data file.


Heterogeneity at the microscopic scale in tumour blood vessel architecture has been described, and is one source of the underlying variations in oxygen tension. We seek to determine whether histologic scale measures of the erratic distribution of blood vessels within a tumour can be used to predict differing radiation response. Using a two-dimensional hybrid cellular automaton model of tumour growth, we evaluate the effect of vessel distribution on cell survival outcomes of simulated radiation therapy. Using the standard equations for the oxygen enhancement ratio for cell survival probability under differing oxygen tensions, we calculate average radiation effect over a range of different vessel densities and organisations. We go on to quantify the vessel distribution heterogeneity and measure spatial organization using Ripley’s L function, a measure designed to detect deviations from complete spatial randomness. We find that under differing regimes of vessel density the correlation coefficient between the measure of spatial organization and radiation effect changes sign. This provides not only a useful way to understand the differences seen in radiation effect for tissues based on vessel architecture, but also an alternate explanation for the vessel normalization hypothesis.Intratumoural heterogeneity is known to contribute to poor therapeutic response. Variations in oxygen tension in particular have been correlated with changes in radiation response  L function allows discrimination between vascular architectures in different density regimes in which the standard hypothesis holds and does not hold. Further, we find that this information can be used to augment quantitative histologic analysis of tumours to aid radiation dose personalisation.In this paper we use a mathematical model, called a hybrid cellular automaton, to study the effect of different vessel distributions on radiation therapy outcomes at the cellular level. We show that the correlation between radiation outcome and spatial organization of vessels changes signs between relatively low and high vessel density. Specifically, that for relatively low vessel density, radiation efficacy is decreased when vessels are more homogeneously distributed, and the opposite is true, that radiation efficacy is improved, when vessel organisation is normalised in high densities. This result suggests an alteration to the vessel normalization hypothesis which states that normalisation of vascular beds should improve radio- and chemo-therapeutic response, but has failed to be validated in clinical studies. In this alteration, we show that Ripley’s  It is increasingly recognised that an important aspect of cancers is their heterogeneity . This heRadiation biologists have, for many years, understood the importance of cell biological and microenvironmental factors on radiation response. Current radiation therapy dose planning, however, largely neglects this information and is, instead, based on years of clinical experience using intuition and trial and error. As such, there remains limited tailoring of dose planning to an individual patient’s tumour. With the advent of modern quantitative histologic  and biolResearch in this area over the last decade  has sougin vitro to understand differences in radiation effect due to oxygenation differences have been valuable, and have established an empiric relationship called the Oxygen Enhancement Ratio (OER)  and check if this number is less than a fixed, model specific threshold given by pH or pT if the cell is a ‘healthy’ or tumour cell, respectively. If it is, then we execute cell division for that cell, placing one daughter cell in the parent cell’s location and the other daughter cell in a randomly chosen empty neighbouring lattice site . Under optimal conditions then, the duration of each cell’s cycle is described by a geometric random variable.At each time step of the cellular automaton .Cells under extreme hypoxic conditions are often found to undergo autophagy (directly translated as ‘self-eating’), a state in which they become resistant to nutrient starvation , and celα and β are radiobiologic parameters associated phenomenologically with cell kill resulting from ‘single hit’ events (α) and ‘double hit’ events (β) respectively, and n represents the number of doses of d Gy of radiation. This metric, while originally derived to fit in vitro data [To model the effect of radiation on heterogeneously oxygenated tissues, we begin with the linear-quadratic model of cell survival:tro data , has sintro data , 39.α and β in αmax and βmax are the values of α and β under fully physiologically oxygenated conditions and α(c), β(c) are, respectively, the values of α, β at oxygen concentration c. We define the OER as a function of the oxygen concentration by using the empirically established relation [α and β  below which cellular division is not possible and by the spatial constraint that the cell is quiescent if there are no empty neighbouring lattice sites (using a Moore neighbourhood). In the case of a cancer cell, we require that at least one space be empty or inhabited by a normal cell, a situation in which we assume that the cancer cell will replace the normal cell if chosen for division. When the conditions for quiescence are no longer present, the cell’s state of quiescence is reversed.In response to a lack of oxygen or other nutrients or external mechanical cues indicating overcrowding, cells enter a temporary state of quiescence during which no division occurs . We modeμHr(c) and are subject to hypoxic cell death. In addition, normal cells can die through competition with cancer cells.The invasion of cancer cells into healthy tissue has been modelled extensively , 42–44. The ability to invade into normal tissue is one of the ‘Hallmarks of Cancer’ . SeveralWhile in healthy tissue vessels are normally patent, as tumours grow the vasculature can experience significant spatio-temporal changes on several timescales. There continues to be significant theoretical work to describe angiogenesis, the relatively long timescale process of new vessel formation and pruning , 28, 49,N × N, with each lattice element identified by coordinates  where i, j ∈ {1, 2, …, N}.Having described each of the constituent parts of the model, we now describe how they are coupled to form the HCA. We consider a two-dimensional lattice of size c. The specific checks that each cell undergoes, regardless of its phenotype, are:c to cap and cp;compare μirc, die, or remain quiescent;consume oxygen at rate check number of free neighbouring lattice sites;determine proliferative behaviour: if proliferative constraints are met (space and oxygen), then consume additional oxygen for proliferation  and placAs schematised in cap, below which cells will die, and be removed from the system. As cell fate decisions are made on a much slower time scale than oxygen diffusion, there is a potential for update bias which would entail cell fate decisions made long after the conditions for these decisions were met. To reduce this, we will ascribe a probability per automaton update, pd, to this death and removal, and update the HCA more frequently than the cell cycle time. We further consider a baseline oxygen consumption required for cellular processes, rc, and a threshold for cells to be proliferatively active, cp [As we endeavour to understand the effects of a heterogeneous oxygen distribution on healthy tissues and tumours, we must consider a number of parameters that govern a cell’s behaviour in response to these environmental cues. Specifically, we consider the hypoxia threshold, ive, cp .τ = 16 h [x = 50 μm for a glioma cell [−13 mol−1 cell−1) [In order to place the model in an appropriate spatial and temporal scale, we non-dimensionalise the system. We rescale time by a typical cell cycle time, τ = 16 h , and lenoma cell . We choo cell−1) , so thatIntroducing the non-dimensional variables N0 is the number of cells in the tissue before radiation therapy and SF is the surviving fraction defined by The tumour control probability (TCP) is a measure that approximates the probability of eradicating all cells within a tumour and thereby controlling (in this case ‘curing’) it. While a number of methods, both deterministic and stochastic, have been proposed to define such a measure , the mosiΔc, (i + 1)Δc) at time tk = kΔt, for i ∈ {0, …, M}. Here we define Δc = 0.01, and MΔc = 1, reflecting the fact that the oxygen concentration is bounded by its non-dimensional value in the arterial blood, which is 1. We assume that radiation effect is instantaneous, and represent the time after radiation as tk+1. The number of cells experiencing an oxygen concentration in the interval [iΔc, (i + 1)Δc) immediately following a single radiation dose d is given bytk+1 is given byαi and βi for each interval of oxygen concentration using Eqs  by utilising Eqs . We . We Nik id+βid2).Thus the=0MNik+1.To calcuWe first model a domain seeded with only healthy cells, and regular spatial distributions of vessels of varying density. Estimates in the literature for ‘normal’ vascular density span several orders of magnitude and are reported using a number of different measures , 63, 64,−3 vessels per lattice site and 3.1 × 10−3 vessels per lattice site for the parameters modelled  of cells within the domain exist at certain oxygen concentrations, a measure which will become important later when we consider the effect of radiation. In We next explore how the oxygen concentration that the population of healthy tissue supported experiences based on changes in density and patterning of vessels in irregularly vascularized domains. While this is a situation that would not likely be seen in the healthy state, understanding the behaviour of the model in this simple situation is valuable before progressing to more complex states. To this end we simulate the resulting healthy tissue growth and maintenance in a variety of randomly generated vascular patterns of the same density . We plotcap and second, the distribution becomes more and more skewed (to the left) as the vessel density decreases (skewness ranging from 0.3820 − 0.9315).As expected, we see an increase in carrying capacity with increasing vascular density, similar to the regularly vascularized scenario, with cellularity (cells / domain size) increasing from 0.557 to 0.945. In these examples of the irregularly vascularised cases, however, we find striking differences in the distribution of cell-specific oxygen concentration , right. As a tumour initially invades into healthy tissue, the vasculature that it would encounter would be that of the healthy tissue. While we are ignoring the effects of cell crowding, vascular deformation and angiogenesis, it is of value to understand how the growth rate and the pattern of cancer spread would change given differing vascular spacing in an otherwise regular vasculature. To this end, we seed a series of regularly patterned vascularised domains with increasing regular vessel spacings with a single initiating cancer cell and observe the growth. Note that, to ensure initial tumour growth, we have placed an extra vessel at the centre of the computational domain with the initial cancer cell as in certain regular spacing patterns, the centre would be an area of necrosis and the simulation would end with no cancer cells.We plot the results of these simulations in N = 73  and vary vessel number from very low until we reach saturation of cells (in this case from 3 to 236 vessels). In each simulation, we initialize the domain full of cancer cells and then allow the tissue to experience oxygen dependent birth and death until a dynamic equilibrium is reached, defined as experiencing less than a 1% change in cell number for 50 time steps. We choose this initial condition, instead of seeding the domain with a single cell, because, in this section, we are not interested in growth kinetics, but instead focus on tumour bulk equilibrium characteristics. From this point, we then record and average 100 time steps of cell and oxygen concentration data. For each vessel number, we run 500 simulations, as detailed above, with random vessel configuration and plot the result of each set of simulations and the associated standard deviation (inset) in We next investigate in further detail the effect of vessel density on the carrying capacity and cellular-oxygen distribution. We consider a domain of size As expected, we see a monotonic approach to cellular saturation as vessel number increases. When we plot the standard deviation, we find that it is significantly higher in the region that is most physiologically relevant, and that these differences can reach as high as 10% , making To begin to understand how the patterning of the vasculature affects the cellular-oxygen distribution within our simulations, we consider two cases from the sample of our simulations for two separate vessel densities (24 and 54 vessels). We plot and compare the vascular patterns which support the maximum and minimum cellular populations from two given vascular densities . We plotcap) as well as a smaller peak around an oxygen concentration of 0.5. In the maximum cellularity examples, however, these similarities disappear. In the low vessel density, we see that the hypoxic population is maintained, but we have lost the central, well oxygenated fraction, while in the high vessel density maximum population this is reversed, and we lose the hypoxic population and the entire population is centered around normoxia. This opposite effect (losing the hypoxic fraction in one case and increasing it in the other) would drastically change radiation efficacy, suggesting that vascular organization, not just density, could play an important role in clinical radiation therapy.In both minimum population cases, there is a large peak of cells near to the hypoxic minimum  which influence our computation of total surviving cells, a measure critical for comparison across heterogeneous samples, and to understanding radiation efficacy at larger scales, like TCP. How these effects are governed by vessel organisation however, we have not yet elucidated. In this section we will investigate the effects of differing vascular patterns on radiation efficacy.As discussed in the previous section, the organisation of vessels in our model can have a significant effect on both the carrying capacity of a domain and also the resulting cellular-oxygen distribution of the cells inhabiting the domain. It is exactly these two values  into a single number by taking the mean value from 0 − 19 cell diameters (from adjacent to a distance beyond the ability to affect one another). We calculate the mean Ripley’s L function for all of the 500 simulations in each vessel number case and plot it against the associated carrying capacity  as the domains begin to reach confluence for all but the minority of vessel arrangements.In order to allow for easy correlation, we first distill Ripley’s Fig C in . We findL function and surviving cells after radiation in 6 representative families of the 500 simulations with increasing vessel numbers. We find that at low vessel densities there is a strong negative correlation between Ripley’s L function and surviving cells, but this correlation changes sign at high vessel densities, explaining the counter-intuitive change in surviving fraction after Θ = 0.01 in less effective, in contrast to the vessel normalisation hypothesis.L function, which could be easily obtained from biopsy images, may predict, together with more standard measures like vessel density, radiation therapy efficacy and the effect of vascular normalisation on this. While our model is parameterised from glioblastoma data, we anticipate these results to be more widely applicable to other cancer types.We have used an HCA model of vascular tumour growth in a planar domain to investigate the dependence of cellular oxygenation and radiotherapy efficacy on vascular density and patterning. Our results indicate that simple spatial summary statistics such as Ripley’s en face, a choice which was made consciously in our model to ease future translational validation with patient tissue samples. Further, we have ignored several mechanisms of competition in order to focus on defining summary measures of cellular-oxygenation status. These differences aside, Alarcón et al. found, as we did, that heterogeneity on the scale of oxygen concentration affects cell growth, both in overall speed of tumour growth and also in shape of resulting tissue. Further, they reported significant heterogeneity in steady state oxygen concentration across their domains which was dependent on vascular organisation, but this was never explored in terms of radiation effect. A similar finding was reported by Al-Shammari et al. in a biophysical model of healthy muscle tissue [en face oxygen sources, as in our work. We have observed similar changes in cellular-oxygen distributions at equilibrium, and indeed, it is these changes that drive our findings concerning radiation effect.Our results corroborate those of Alarcón et al. , who alse tissue , this tiWe have seen that the assumption that mean vessel density, and subsequently mean oxygenation, can be used as a surrogate for the number of surviving cells after radiation is insufficient. Further, we found that the relationship between each of our vessel pattern measures and the surviving cells after radiation exhibits a sign change in the mid vessel density range. This change of sign in correlation means that the patterns, within a given vessel density, that take the extreme values of each measure of vascular organization can represent opposite extremes of radiation response. This suggests that if one were to perturb the vasculature, for example toward a more homogeneous distribution, one could induce opposite effects on radiation response, depending on the vessel density.With Folkman’s discovery, in 1971, of a master tumour angiogenesis regulating factor , the worIt was not until 2001, when Jain suggested the ‘vascular normalisation hypothesis’ , that thWhile this advance in our thinking has provided a way to explain the counter-intuitive results of many trials, it still does not explain why these combinations of anti-angiogenic drugs with chemotherapeutics (or radiation therapy) do not help all patients. Using our simple model system, we have observed a changing correlation with spatial measures and radiation response. This suggests that in certain cases, ‘improving’ the homogeneity of vascularisation would hurt the radiation effect, whilst in other cases it would help: a heterogeneous response to ‘normalisation’ of vascular patterning. We show that for certain cases, vessel density held constant, normal vascular patterning can respond either better or worse to radiation therapy .en face and all vessels are the same in terms of size and efficiency. Validation could be achieved either through in vivo window chamber experiments, or indirectly through examination of post-radiation surgical specimens. Model development, to include the addition of angiogenesis, biologically derived vessel geometries, vessel size heterogeneity and collapse, could be used to extend these predictions to more realistic situations.To translate these conclusions to the clinic would first require biological validation of these hypotheses, to include a careful study of variation in spatial vessel patterning on different spatial scales within the same tumor, or possibly between spatially distinct biopsies. Further, there are a number of model limitations to overcome. Specifically, the assumption that all vessels are L. These two pieces of information could then be incorporated by creating a temporally and a spatially optimised radiation plan whereby the appropriate radiation dose would be delivered before VNT to the area that would suffer after normalisation, and then the final radiation could be delivered after VNT to the area that would benefit from it.If these limitations were addressed, and validation was achieved, the technology exists currently to take advantage of this new idea. Specifically, macroscopic oxygen concentrations could be inferred from DCE MRI (or other advanced imaging) to create optimised dose plans, as suggested by Malinen et al. . This inS1 Software(ZIP)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Robert Koch, who discovered the tubercle bacillus in 1882, believed that M. bovis was not a danger to humans. Theobald Smith and others established beyond doubt that, contrary to Koch’s belief, M. bovis could infect humans but was not the usual source of human infection. In 1908, French scientists Albert Calmette and Camille Guérin chose an M. bovis strain for their work on a tuberculosis vaccine. They repeatedly subcultured the isolate on a mixture of glycerol, potato, and bile for 13 years until it was sufficiently attenuated to be used as a vaccine. The bacillus Calmette-Guérin (BCG) vaccine was adopted by the League of Nations as the standard tuberculosis vaccine in 1928 and continues to be used in most developing countries.From the Latin


The upcoming DSM5 will impact clinical work and research substantially, and we here point to a few problems that may have been overlooked. These concern inconsistencies and lack of clarity, and the future “not elsewhere classified” atypical anorexia nervosa category. We propose solutions in the form of working definitions and operationalizations to facilitate clinical implementation as well as streamline research. Dear Editorhttp://www.dsm5.org (recently removed from public access unfortunately) is probably definite. We may be accused of over-detailed scrutiny, especially by clinicians who daily have to reconcile the letter of manualized criteria with fluid and unwieldy reality. However, there is one instance – that will affect clinical work and research alike – which requires the kind of nitpicking we have possibly perpetrated, namely the adaptation of diagnostic instruments to DSM-5.Although research into the effects of the DSM-5 expands rapidly, we wish to point out a few issues that are not without consequence but that may have been overlooked. We are aware of the monumental task that the DSM-5 eating disorders (ED) Work Group has faced, with the near irreconcilable concerns involved, but let’s be frank: The ED section in DSM-5 is probably not to everyone’s satisfaction. However, no major changes to the criteria will occur from now on: Production is in its final stages and the proposal on In the process of just such an undertaking, we have noted the following:or shape on self-evaluation”, while the latter reads “Self-evaluation is unduly influenced by body shape and weight” (emphasis added). We would suggest that the AN version be used for both, i.e. either the one or the other, to be equally inclusive for both disorders.1. An inconsistency between the second part of the C criterion for anorexia nervosa (AN) and the D criterion for bulimia nervosa (BN). The former reads “Undue influence of body weight 2. Does AN “restriction of energy intake” concern both food intake and uptake ? For example, can it include purging, or diet pills, thereby preventing calorie uptake? We suggest that uptake should be intended, and that purging or other ways of preventing calorie absorption can fulfill the criterion, even though food is consumed.3. Enemas are mentioned as a purging example in AN, but not in BN, and “mere” examples, as so often happens with manuals, may be reified and lead to assessment differences. We suggest that enemas, although rather rarely used, are relevant for BN also.4. Significant weight loss, as well as its time frame and velocity, are not defined in the “not elsewhere classified” Atypical AN (AAN), possibly in keeping with the Work Group reluctance to define numeric limits, as in AN weight. This can cause problems for clinical use and research. For example, can it include both a 10% weight loss over the last 3 months and a 20% loss that stabilized 9 months ago? Agreeing on a working definition would be of great value in research that wants to use the category but not specifically investigate the weight loss issue, and for clinicians who need to decide whether an ED is present.5. Continuing on AAN, the B and C criteria for AN proper (both required for AAN) need clarifications:even though at a significantly low weight. If this criterion applies only to low weight patients, it does not apply to AAN. Since fear/avoidance of weight gain is common also in patients with restricting eating pathology, but without significantly low weight, the criterion should be applicable also for AAN.a. The B criterion states that fear of weight gain or becoming fat, or persistent behavior that interferes with weight gain should be present, b. In the three-part C criterion, the last part (“persistent lack of recognition of the seriousness of the current low body weight”) obviously also hinges on a low weight and is thus irrelevant for AAN. The other two parts however , work as general ED symptoms and should be retained for AAN as an either/or criterion, although a reliable operationalization of body experience disturbance for non-underweight people may prove elusive, and this issue deserves research attention.Despite significant weight loss, the individual’s weight is within or above the normal range, and there is a) an intense fear of gaining weight or becoming fat, or persistent behavior that interferes with weight gain, and b) a disturbance in the way in which one's body weight or shape is experienced, or undue influence of body weight or shape on self-evaluation. Whether such a definition truly qualifies as an ED is an empirical question, but the lack of clarity may cloud the issue. We offer our reworded definition to help streamline operationalizations.As a consequence, the AAN description may be reworded into: As noted, this level of detail may not matter greatly in everyday clinical decisions, but formally, all use of the criteria are affected, or at least we should expect them to be, when constructing the diagnoses. We hope our comments may possibly be relevant for the final DSM-5 texts in some instance, but if not they may be helpful for clinical and research colleagues. The more we use the DSM alike, the better off our attempts at cumulative research will be.The authors have no competing interests regarding the present contribution.All authors contributed to the arguments made in the manuscript and collaborated on writing, and all authors read and approved the final manuscript.


The medical sector has joined forces with the technological field in order to produce devices that facilitate health monitoring and diabetes management. However, this reality is not yet available to everyone. In this context, for patients who have diabetes and for their health care team (HCT), collecting and reviewing daily data are extremely important actions for effective treatment adjustments and improvements.Our goal was to develop and test an affordable technological alternative for most people with diabetes who use insulin, in order to record and share data with their HCT.https://youtu.be/rtj6vJFbwQU and https://youtu.be/8E5BeZQFLtI).A Microsoft® Excel® dynamic spreadsheet, called “MellitusOne.”, was created. On its first sheet personal data are inputted and in the second are the blood glucose values, insulin administered (doses and types), food consumed and corrections. On the other two sheets automated charts and graphs are presented (daily and of the last 20 days). In the tables, blood glucoses and its percentage in each category are colored according to its value . Moreover, mean, median and standard deviation values are calculated. A friendly print layout is available to print or send the reports by e-mail to the HCT. The dynamic sheet was extensively tested by the developers and then by 8 volunteers for 7 days, who answered an evaluation. Instructions were made available on the YouTube , useful to share with the endocrinologist (2), easy to use (2), easier to take quicker decisions using it (2). Slowness on Apple computers and preference for own homemade spreadsheet was the reason why one volunteer would not continue using it. The main suggestions were: transforming it into an application (to make it more portable), increasing the fields, graphs, and automating more functions. Therefore, seven people found that “MellitusOne.” contributed to a better glycemic control.The results above, pointing towards an easy alternative to follow the glycemic control and take actions to improve it, show us that the spreadsheet “Mellitus One.” is not only useful, but also may be beneficial to this population. At the same time, systemic improvements would enhance its usability.


Plain packaging requires tobacco products to be sold in packs with a standard shape, method of opening and colour, leaving the brand name in a standard font and location. We ran a randomised controlled trial to investigate the impact of plain packaging on smoking behaviour and attitudes.In a parallel group randomised trial design, 128 daily smokers smoked cigarettes from their usual UK brand, or a plain Australian brand that was closely matched to their usual UK brand for 24 hours. Primary outcomes were number of cigarettes smoked and volume of smoke inhaled per cigarette. Secondary outcomes were self-reported ratings of motivation to quit, cigarette taste, experience of using the pack, experience of smoking, attributes of the pack, perceptions of the health warning, changes in smoking behaviour, and views on plain packaging.ps > 0.279). However, smokers using plain cigarette packs rated the experience of using the pack more negatively , rated the pack attributes more negatively , and rated the health warning as more impactful .There was no evidence that pack type had an effect on either of the primary measures  contains supplementary material, which is available to authorized users. Mandatory plain (“standardised”) packaging requires cigarettes and hand-rolling tobacco to be sold in packs with a standard pack shape, method of opening and colour, leaving only the brand name in a standard font and location. In December 2012, Australia became the first country in the world to introduce plain cigarette packaging. The United Kingdom (UK) government are currently discussing the issue.Evidence to date, predominantly comprising survey and observational studies, suggests that plain cigarette packaging increases attention to cigarette pack health warnings ,2, preveWhile this research provides important information on the impact of plain cigarette packaging on attitudes to smoking and the cigarette pack, no research has directly measured the impact of plain cigarette packaging on smoking behaviour, used genuine plain cigarette packs, or used an experimental design. In the present study, we overcame these previous shortcomings and assessed whether plain, as compared with branded cigarette packaging, resulted in changes in smoking behaviour, as well as attitudes towards smoking, when using these packs over 24 hours, in a randomised controlled trial using Australian plain cigarette packs.The study was conducted at the University of Bristol. First randomisation was on March 3, 2013, and last follow-up on December 9, 2013. The published protocol [ad libitum smoking day. The smoking day started the morning after the baseline day and participants smoked cigarettes from the cigarette pack provided to them and through a smoking topography monitor for the entire day. The day after the smoking day, participants attended a final test day, during which they returned any remaining cigarettes and the smoking topography monitor and completed questionnaires about the smoking day and further taste ratings.Participants first attended a baseline day, where they completed questionnaire measures and provided blind taste ratings of a cigarette from a branded and a plain cigarette pack. Participants were then randomly allocated to either a branded or plain cigarette pack and were given instructions for the following Ethics approval was granted by the Faculty of Science Research Ethics Committee at the University of Bristol . The study was conducted according to the revised Declaration of Helsinki 2013 and Good Clinical Practice guidelines. All participants provided fully informed written consent prior to testing.Participants were randomised to receive either a plain or branded cigarette pack of their usual brand of cigarettes to smoke for the smoking day. Smokers randomised to the branded and plain cigarette pack conditions were matched by gender, with equal numbers of males and females in each condition. The lead researcher was blind to the condition assigned to participants until the participant returned on the final test day. To perform the randomisation, the lead researcher, who enrolled participants, contacted an experimental collaborator with the participant’s preferred brand of cigarettes and the participant’s gender. The collaborator then used random number generator software, along with a pre-assigned code, to allocate the participant to the branded or plain cigarette pack condition. A pack of the assigned cigarettes was then placed into a concealed envelope labelled with the participant’s anonymised identification number.Participants were given either their usual UK branded cigarette pack of cigarettes or an Australian plain cigarette pack of cigarettes which matched their preferred UK brand, to smoke for the smoking day. Since they originated from two different countries, packs differed in the shape, size and format of the health warning; however, they were selected so that both presented a message related to the effects of prenatal tobacco exposure on offspring. Examples of the Australian plain and UK branded cigarette pack cigarettes used are shown in Figure A battery-operated, hand-held CreSS Pocket topography monitor  was used to record participants’ smoking behaviour. For each cigarette smoked, the monitor recorded the date, time, start and end of smoking, puffs per cigarette, puff volume and puff duration.To assess the taste of the cigarettes smoked at the blind ‘tasting’ on the baseline day, participants were asked “How did this cigarette taste”, and when they returned on the final test day, participants were asked “How did the cigarettes in the pack given to you yesterday taste”. To answer these questions, participants were required to report their agreement with the statements “The taste of this cigarette was strong/harsh/dry/stale/dull/dirty”, each on a seven-point scale between “Strongly disagree” to “Strongly agree”. Participants also reported on a seven-point scale between “Much better” to “Much worse” the answer to the question “Compared to my usual cigarette, the taste of this cigarette is…”.Experience of smoking’, participants were asked, “To what extent did you experience the following as you smoked the cigarettes? a) Enjoyment, b) Satisfaction, c) Acceptance”. To assess ‘Experience of using the pack’, participants were asked “To what extent did you experience the following about the cigarette pack? a) Embarrassment, b) Shame, c) Acceptance”. To assess ‘Rating of cigarette pack attributes’, that is, participants’ perceptions of the packs, participants were asked to “Rate the cigarette pack on the following attributes: a) Style, b) Fashion, c) Cheapness, d) Coolness, e) Attractiveness, f) Quality, g) Appeal”. To assess participants’ ‘Rating of the health warning’, participants were asked to “Rate the health warning on the following attributes: a) Noticing, b) Seriousness, c) Believability, d) Awareness of health risks”. To assess ‘Experience of using topography monitor’, participants were asked “To what extent did you experience the following about the monitor you were asked to smoke your cigarettes through? a) Embarrassment, b) Shame, c) Acceptance”. Participants responded to each of these questions on a five-point scale, with higher scores indicating higher agreement with each of the statements. Mean responses across the sub-questions were then calculated in order to calculate an overall response for each of the five questions. ‘Changes in behaviour’, was also assessed by asking participants “During the 24 hours when you smoked cigarettes from the pack given to you in the experiment, did you 1) Stub out a cigarette early, 2) Forgo a cigarette  Keep the pack out of sight, 4) Cover the pack, 5) Smoke less around others, 6) Think about cutting down, 7) Think about quitting in the next few weeks, 8) Thinking about quitting within a year”. Each of these statements were answered with binary (yes/no) responses. ‘Yes’ responses were summed to create an overall score of smoking behaviour. With the exception of the questions relating to the topography monitor, each of these questions were taken from previous studies assessing smoking behaviour when using cigarettes in plain cigarette packs [Attitudes to plain packs’ by answering the following three questions “Do you think plain packaging would make you smoke fewer cigarettes?”, “Do you think plain packaging would help you to quit smoking?” and “Do you think plain packaging would prevent children from starting smoking?” Each of these questions were answered on a four-point scale, with higher scores indicating higher agreement with each of the questions.At the final test day, participants also answered a series of questions regarding their experiences on the smoking test day. To assess ‘On arrival at the baseline day, participants re-read the information sheet and provided informed consent. Participants were then asked to report the number of cigarettes they had smoked the previous day, and the number of minutes since their last cigarette. Smoking status was verified using a breath Carbon Monoxide (CO) test. Participants completed baseline questionnaires assessing contemplation of quitting smoking (Contemplation Ladder) , smokingi.e., not specifically cigarette packaging) and were not informed that the cigarette pack was an integral aspect of the experiment. Participants were asked to return the pack with any remaining cigarettes on the final test day (to verify the number smoked). To ensure that all unsmoked cigarettes were returned, participants were told they would be able to take these with them at the end of the study.After the taste test, participants were provided with a topography monitor and given verbal instruction on how to operate it. They were then instructed to practice using the monitor with a cigarette in the smoking laboratory, during which participants typically smoked either one or two cigarettes of their usual brand . Participants were then presented with a sealed envelope, which contained either a plain or branded cigarette pack of their usual brand, as described in the randomisation procedure. Participants were blind to the pack type given to them until they opened the envelope, which they were instructed to do the following morning. Participants were asked to use this pack of cigarettes and the monitor for the entirety of that day. Participants were informed that the main purpose of the study was to investigate factors which influence smoking behaviour . Secondary outcomes were self-reported ratings of motivation to quit smoking (as measured by the Quitting Contemplation Ladder), the cigarette taste test, experience of smoking, experience of using the pack, rating of pack attributes, rating of the health warning, changes in behaviour, and attitudes to plain cigarette packs.i.e., equivalent to one fewer cigarette per day for a 10 a day smoker) with 80% power at an alpha level of 5%, we recruited 128 participants.The sample size for the study was calculated based on the primary outcome of average volume of smoke inhaled per cigarette. Given the inelasticity of smoking behaviour, with daily smokers relatively stable with regards to the number of cigarettes they smoke per day, we hypothesised that volume of smoke inhaled would be a more sensitive measure of tobacco exposure than number of cigarettes smoked, given that this is known to be relatively plastic . A reducLinear regression was used to evaluate the effect of cigarette packaging (branded or plain) on the primary and secondary outcome measures. These analyses were conducted with and without adjustment for age, gender, heaviness of smoking and, where appropriate, corresponding baseline measures. Whether these effects differed between males and females was investigated by including appropriate interaction terms in the models. IBM SPSS Statistics 19 was used to analyse the data.Of 396 people who completed the initial assessment, 128 met the inclusion criteria and were recruited into the study, with 64 participants assigned to branded cigarette packs and 64 to plain cigarette packs  = 3.79, Standard Error (SE) = 0.11, plain: M = 3.98, SE = 0.08, with higher scores representing more acceptance of the monitor).One participant randomised to branded cigarette packaging did not provide secondary outcome data. Participants’ baseline characteristics were similar in both groups , and inhaled more smoke per cigarette . However, in both cases the confidence intervals were wide and included the null, so that we cannot conclude that plain cigarette packs had any effect on smoking behaviour after first exposure. Interaction tests did not indicate any evidence that smoking behaviour differed for males and females (ps > 0.90).As shown in Table p = 0.001), more negative ratings of the pack attributes , and that the health warning was more impactful . There was no clear evidence that plain cigarette packaging influenced the taste of the cigarette, changed self-reported behaviour, increased motivation to quit, changed the experience of smoking, or changed attitudes to plain cigarette packs (ps > 0 15). However, the point estimates for these outcomes were all in the direction of a beneficial effect . Again, interaction tests did not indicate any evidence that these results differed for males and females (ps > 0.18). These results are shown in full in Table Smokers randomised to the plain cigarette pack condition, as compared with those randomised to the branded cigarette pack condition, reported more negative experiences of using the pack , or smokers who are attempting to quit smoking, despite research suggesting that plain packaging may be more effective among these groups ,24. We dFourth, many participants were recruited from the staff and students at the University of Bristol, and one of the channels of recruitment was word-of-mouth, increasing the likelihood of contamination. However, measures such as asking all participants at the end of the experiment not to discuss the purpose with any other potential participants were taken, although compliance with this request was not directly assessed so some contamination between trial conditions is a possibility. In addition, as the majority of the participants were recruited from student populations, it is unknown to what extent the findings are generalisable to the wider population.Fifth, the study was powered to detect a reduction in inhalation volume of 50 mL per cigarette, which is a relatively large effect in the context of smoking behaviour. Our study therefore lacked power to detect smaller but nevertheless important changes in smoking behaviour, such as number of cigarettes smoked, another of our primary outcome measures. However, the point estimates we observed suggest the possibility of a reduction in cigarettes smoked, but an increase in inhalation volume. Although we cannot draw conclusions from these data, given the wide confidence intervals associated with them, they are consistent with the known plasticity of smoking behaviour . FinallyThis is the first randomised controlled trial of the effects of using genuine plain cigarette packs on actual smoking behaviour and related attitudes towards smoking. Our study provides evidence of the effect of first exposure to plain packaging on smoking behaviour and related attitudes to smoking and quitting. This first exposure acts as a precursor to later, longer-term effects on behaviour, such as quitting smoking. Although we found that plain cigarette packaging did not directly impact on smoking behaviour among regular smokers during the short time period of this trial, we observed that using a plain cigarette pack for a 24 hour period had clear effects on reducing experience of using the pack and ratings of the pack attributes and increased the impact of the health warning. A larger trial, with participants using the packs for a longer time period, would provide more detailed evidence on the impact of plain cigarette packaging on these attitudes to smoking, and whether they result in changes in smoking behaviour over the longer term. Nevertheless, these results add to the growing evidence base which suggests that plain cigarette packaging may be an effective tobacco control measure.