GEMMs) will remain an important part of preclinical studies for the foreseeable future. This review will provide an overview of the current mouse models of intestinal cancer, from the ApcMin/+ mouse, which has been used for over 25 years, to the latest ‘state‐of‐the‐art’ organoid models. We discuss here how these models have been used to define fundamental processes involved in tumour initiation and the attempts to generate metastatic models, which is the ultimate cause of cancer mortality. Together these models will provide key insights to understand this complex disease and hopefully will lead to the discovery of new therapeutic strategies. © 2015 The Authors. Pathological Society of Great Britain and Ireland.Murine models of intestinal cancer are powerful tools to recapitulate human intestinal cancer, understand its biology and test therapies. With recent developments identifying the importance of the tumour microenvironment and the potential for immunotherapy, autochthonous genetically engineered mouse models ( APC) gene, and other mutations occurring later that drive progression, eg TP53. Recent DNA sequencing studies confirmed the common co‐existence of these mutations within individual CRC tumours. A recent theory of CRC, referred to as the ‘Big Bang’ model, describes tumour growth as an expansion populated by various heterogeneous subclones. Initial mutations in genes, such as APC and KRAS (‘public mutations’), are carried by all subclones, and subsequent ‘private’ mutations are acquired later in individual subclones In the Western world, colorectal cancer (CRC) is the second‐highest cause of cancer mortality APC, and this is often called the ‘classical’ route KRAS. KRAS is mutated in 40–50% of human CRCs, with > 75% of these mutations located in codon 12, which lock KRAS in the active GTP‐bound state In 80–90% of CRCs the initial step is proposed to be the loss of the tumour‐suppressor gene PTEN or PIK3CA. This pathway is associated with driving cell growth, metabolism and tumour progression. TGFβ pathway abrogation in CRC can occur through mutation of either TGFβ‐receptor 1 (TGFBR1) or TGFBR2. Furthermore, TGFβ pathway inactivation can occur via loss of heterozygosity (LOH) of chromosome 18q, where SMAD2 and SMAD4, two downstream mediators of TGFβ signalling, are located. Another gene deleted in colorectal cancer (DCC) is also localized to 18q and encodes a netrin receptor that controls differentiation and tumourigenesis TP53TP53 and APC mutations are often associated with increased rates of chromosomal instability (CIN) Further common mutations occur to activate the PI3 kinase signalling pathway, eg in Sequencing studies have also revealed that many other mutations occur in individual CRC tumours, although at much lower frequencies (the ‘private’ mutations described above). The importance of these is still unclear and many represent passenger mutations which might have no function APC mutations, many of these are associated with mutation of DNA mismatch repair (MMR) genes or inactivation predominantly of the mismatch repair genes MLH1 and MSH2 (Lynch syndrome) TGFBR2, ACTIVIN, BAX and MBD4. Exciting recent data suggest that these cancers may be sensitized to immune checkpoint inhibition, potentially as a result of the higher levels of mutation Of the remaining 20% of CRC tumours that do not carry Given all this information on the common mutations that occur in CRC, mouse models can be developed that are based on the genetic make‐up of tumours, generating realistic mouse models of the human disease. The successes and challenges that still need to be overcome will be the focus of this review. Due to space constraints, we have limited our review to genetic models of cancer and so do not discuss colitis‐associated cancer models within the mouse , a human autosomal dominant syndrome, in which patients develop numerous colorectal polyps APC mutation in sporadic colorectal cancer and APC being the causal gene for FAP, most of the models developed to mimic colon cancer have centred on models carrying APC mutation.ApcMin/+; Figure N‐ethyl‐N‐nitrosourea (ENU) mutagenesis. The mutagen caused a loss of function mutation in the mouse Apc gene at codon 850. During adulthood, spontaneous LOH of the other Apc allele occurs and mice develop multiple intestinal adenomas and a smaller number of colonic polyps ApcMin/+ mouse model and the human disease is that human FAP patients predominantly develop colonic lesions, whereas the mice develop more polyps in the small intestine. Furthermore, human FAP, if not treated, can progress to invasive carcinoma; this is only very rarely reported in mice, probably due to the high tumour burden in these mice and the inability to intervene surgically. The ApcMin/+ model has been utilized for a broad range of studies; foremost have been chemoprevention studies and functional testing of genes that might modify intestinal tumourigenesis. Treatment studies of established tumours have also occurred, although it should be noted that if sporadic polyps appeared in patients, these would be removed surgically with no further treatment. Chemoprevention experiments have shown marked effects with non‐steroidal anti‐inflammatory drugs (NSAIDs), such as aspirin and celecoxib [cyclooxygenase (COX) inhibitor], although the mechanism of prevention by aspirin is likely pleiotropic and the suppression of tumourigenesis by celecoxib suggests that inhibition of COX2 is important for chemoprevention The most commonly used model is the multiple intestinal neoplasia (MIN) model (referred to as MOM) loci through genetic linkage studies in mice. MOM1 is located distal to chromosome 4. Interestingly, the orthologous region on the human chromosome shows frequent LOH in CRC MOM1 region are Plag2g2a and perlecan (Hspg2) and studies have identified that disruption of Plag2g2a can slow tumourigenesis MOM1 also highlighted the importance of mouse genetic background on tumourigenesis Functional genetic studies have identified numerous genes that modulate tumour development by both acceleration and deceleration. Initial studies identified modifier of MIN  Apc knockout allele that produces no protein Apc1322T/+ shows increased levels of Lgr5 and stem cell markers within tumours, although with a slight reduction in general Wnt target gene expression, eg Axin2Apc1638N/+, which harbours a neomycin cassette in antisense orientation within exon 15, resulting in a protein truncated at codon 1638, which is unstable. These mice show few tumours (<10) and a long latency, and develop adenocarcinoma along with infiltration into the mucosa and submucosa Given the high penetrance of the Cre–Lox (Cre) technologies in the 1990s enabled researchers to delete any gene in any tissue of interest Cre transgene (under the control of an inducible tissue specific promoter) are crossed to mice bearing an inducible allele where the region that is to be deleted is flanked by LoxP recombination sites. This can be either an essential exon(s) of a gene, to produce a conditional knockout, or a Stop motif to activate an oncogene, eg Kras or Pik3, within adult tissue Cre after administration of tamoxifen Cre induction either via reducing the inducing agent  or Cre recombinase also facilitates low levels of recombination, which was hoped to overcome problems of multiple tumours per mouse The advent of Apc has revealed much about the mechanism of early tumourigenesis. Shibata et alLoxP sites flanking exon 14; Apc580S/580S; Apcfl/fl) was sufficient to drive colon adenomas. Using a highly penetrant inducible Cre  within the small intestine (and to a lesser extent the colon), we et alApc loss had a dramatic impact on intestinal homeostasis. Deletion of both copies of Apc results in a crypt progenitor phenotype, which is characterized by increased proliferation and altered migration and differentiation. Notably, this phenotype was mediated by the Wnt target gene MycApc has been achieved using a Cdx2P–CreERT2 transgenic mouse and produced a very similar phenotype to that of deletion of Apc in the small intestine Cre also overcomes the problem of small intestinal tumour burden and many different colon Cres  have all been used to delete a single copy of Apc and generate colonic adenomas Acute deletion of both copies of LGR5+ intestinal stem cells (ISCs) in the small and large intestine not only led to fundamental changes in concepts on ISCs and homeostasis but also allowed us to explore the impact of deleting Apc in the ISCs ERLgr5Cre mouse was generated and interbred with the LSL–LacZRosa26 reporter mouse. Following Cre induction, LGR5 ISCs were able to stably generate all epithelial lineages Lgr5–CreER to delete Apc within ISCs led to rapid formation of intestinal adenomas, strongly suggesting that LGR5+ ISCs might be the cells of origin for intestinal cancer Cre knock‐in approach, ISCs have been shown to act as cells of origin for cancer when Apc is deleted or a constitutive‐active β‐catenin is expressed The discovery of β‐cateninΔex3/+ and the inflammatory nuclear factor‐κB (NFκB) signalling pathway, in non‐ISCs (using the ERXbp1–Cre), led to dedifferentiation and tumour development Apc deletion with aberrant KrasG12D/+ expression results again in a NF‐κB‐dependent dedifferentiation. This observation is in accordance with the ‘top‐down’ model of CRC development, which is based on the observation that early dysplastic human CRC lesions predominantly locate to the luminal part and not to the base of the crypt Apc deletion to terminal differentiated tuft cells, using a tuft cell marker, DCKL1. Although Dckl1–Cre‐mediated loss of Apc alone did not lead to tumour formation, when Apc loss was combined with dextran sodium sulphate (DSS) treatment (to induce colitis) the mice developed tumours However, two studies have recently demonstrated that activation of Wnt signalling in differentiated cells results in dedifferentiation and adenoma formation Cre‐mediated gene inactivation is that this results in the permanent deletion of a gene, and thus it is hard to assess the sustained requirement for the initiating oncogene/tumour suppressor gene. To address the continued reliance on Apc loss and downstream Wnt signalling, two recent studies using doxycycline‐inducible systems have shown that, if APC expression is restored (through inducible shRNA) or an inducible active β‐catenin allele is turned off, there is complete reversion to a normal intestinal epithelium. This underlines the continued dependence on Wnt signalling Tp53 and KrasG12D/+A fundamental drawback of Generating mouse models of metastatic CRC has proved to be difficult. One of the key steps towards modelling metastasis is generating murine models of invasive adenocarcinoma. Cellular invasion is a complex process in which tumour cells escape from the adhesive epithelium and cross the basement membrane, invading the smooth muscle of the intestine. This is often associated with a change in cellular shape, gain of motility and loss of E‐cadherin Apc mutations alone do not produce invasive tumours, later mutations in the adenoma–carcinoma sequence have been added to make mouse models of CRC more patient‐relevant.As mentioned above, CRC progression follows a distinct order of serial mutations KRAS is one of the most frequently altered genes following APC and is also described as an early event during progression Apc with aberrant expression of mutated KrasG12V/+ resulted in a higher number of intestinal tumours with an increased invasion of tumour cells to the surrounding stroma PTEN and PI3KCA mutations in human CRC Pten and Pik3ca mutant mice have been intercrossed with mice carrying Apc mutation. These additional mutations rapidly accelerate tumourigenesis and increase tumour progression so that the mice develop adenocarcinomas Pik3ca is expressed alone within the intestine, the mice develop invasive mucinous adenocarcinoma with no intermediate benign tumour stage KrasG12D/+ or G12V/+Kras alone does not show a similar phenotype; here the mice develop both adenoma and adenocarcinoma, but at very long latencies (>500 days) Apc mutation acts as an initiator, reducing latency and increasing tumour burden. This in itself is a problem, as the mice develop multiple tumours and thus may need to be euthanized due to burden before any tumours have had the opportunity to metastasize With a mutation rate of ∼40% in human CRC, Cre‐expressing viruses targeting the colon has been performed Apc and KrasG12D/+ mutation. One caveat of this model is the need for surgery, which may explain the surprising lack of uptake by the research community of what appears to be an excellent model.To overcome the issue of excessive tumour burden in mouse models, low‐level recombination with Apc mutation in combination with inactivation of various components of TGFβ signalling  generally leads to the production of invasive adenocarcinoma, although again not metastasis Smad3 loss in the ApcMin/+ model also altered tumour location, as more tumours arose in the distal colon cis‐ApcΔ716/+Smad4+/− mice was suggested to be mediated by recruitment of immature myeloid cells (iMCs) from the bone marrow, leading to secretion of matrix metalloproteinases (MMPs) at the invasion front of intestinal tumours Loss of TGFβ signalling is a common step during CRC progression. In the mouse, TP53 is altered in 50–60% of human CRCs. Surprisingly, deletion of Tp53 in an outbred mouse background did not result in increased tumour progression in the ApcMin/+ model ApcMin/+Tp53−/− compound mice revealed a tendency to higher tumour burden and the development of invasive tumours TP53 are common, particularly TP53R175H. Expression of a single copy of the mouse version of this mutant, Tp53R172H/+, with deletion of a single Apc allele, led to invasive tumour progression in all mice The tumour‐suppressor gene Apc mutation might make it harder for tumours to become metastatic in mice. This concept arose from work on two different targets of the Wnt pathway, Ephb2/3 and Tiam1. EphrinB receptors (EphB) are direct Wnt target genes that control the architecture of the normal intestinal epithelium EPHB2, EPHB3 and EPHB4 are induced during early stages but down‐regulated during CRC progression. Ephb3−/− in the ApcMin/+ mice leads to conversion of 47% of tumours to adenocarcinoma, whilst ΔcyEphb2 deletion in the ApcMin/+ model reduces the number of tumours formed, but those tumours exhibit increased invasion Tiam1, a pro‐adhesive RAC–guanine nucleotide exchange factor (GEF), strongly suppresses tumourigenesis in the ApcMin/+ mice but resultant tumours are eventually invasive. Thus, it appears (at least in mice) that induction of the Wnt signalling programme favours benign tumour formation and thus additional mutations are required to drive further progression, which may in part overcome some of the pro‐adhesive consequences of APC loss.Collectively, it is clear that, when tested in mice, nearly all the common human mutations lead to increased tumour progression and development of adenocarcinoma, although alone these additional mutations do not provoke rapid tumourigenesis. One interesting hypothesis is that nude or SCID mice) TGFβ‐over‐expressing HT29 and KM12L4a CRC cells activated IL‐11 secretion from mouse cancer‐associated fibroblasts, causing increased metastasis Transplantation models are used to test pathways involved in invasion and metastasis that might be therapeutically targetable. These xenograft (human cell line) models result in desired characteristics, such as invasion and metastasis LGR5+ stem cells in the intestine, and following the isolation of these cells, Sato et alex vivo organoid cultures. These ‘mini‐guts’ can be grown in a three‐dimensional (3D) manner and they build tissue‐like structures LGR5+ cells with niche factors Apc in these organoids results in transformation, which is characterized by a morphological change to a more rounded spheroid shape and R‐spondin‐independent growth due to hyper‐activated Wnt signalling ERApcVillin–Crefl/fl crypts only 2 days after tamoxifen application to the mice. Additional mutation of KrasG12D/+ and Tp53R172H/+ or deletion of Ptenfl/fl confers the ability of these spheres to grow in nude mice Apc, expression of KrasG12D/+ and deletion of p53T and Smad4 (AKPS). These spheres have an invasive phenotype similar to that of human CRC With the discovery of Apc, Kras, Smad4, Tp53, PIK3CAE545K (AKSTP) mutant cells grow when engrafted under the kidney capsule of Nod–scid/IL2Rγ‐null mice. However, injection of these cells into the spleen gives rise only to micrometastases in the liver, whereas cells derived from human metastatic CRC form macrometastases. This work suggested that, in addition to the major driver mutations, further alterations are required for metastatic progression and for the outgrowth of CRC metastases in the liver Further validation of the sequential alteration of major pathways in CRC has now also been proved in organoids from normal human crypt stem cells, by using CRISPR/CAS9 technology ®, and it will be important to see how microenvironmental changes and the culture of spheres might alter the response of these drugs; we have shown that basic properties, such as the ratio of E‐cadherin:β‐catenin, are very different in the in vivo setting versus cell culture in vivo responses better than other model systems.Organoids may therefore help us decipher the consequences of the major mutations in CRC and be very useful in high‐throughput screening for new therapies and potential therapeutic stratification. Already, much progress has occurred in the screening of tumour organoids from humans APC. In this section we will describe these models.One of the major goals of utilizing mouse models of cancer is to recapitulate the human disease in order to produce models to test treatments. Thus, to predict response in this setting, we need models that metastasize and these models are still lacking. Thus far, most of our more successful models of metastasis are still of long latency and low penetrance. Also, most of these models do not carry mutation of BRAF or KRAS mutations and no APC mutations BrafLSL–V637E/+ from its endogenous promoter led to the full progression of serrated hyperplasia to adenoma and finally to metastatic carcinoma. However, latency was long and the percentage of metastasis was low with BrafLSL–V637E/+ alone (one of five mice). A possible increase in metastasis was detected when mutant Tp53R172H/+ (three of 12 mice) or fl/flp16 (three of 12 mice) were also mutated in addition to Braf mutation, but latency and penetrance were still low KrasG12V/+ and Ptenfl/fl deletion; here, 41% of mice developed metastasis, with over half developing in the liver KrasG12D/+ mice combined with deletion of Tgfbr2; here, CRC cells spread to local lymph nodes and the lung in 15% of the mice. This dysplastic progression was triggered by hyper‐activated EGFR signalling KrasG12D/+ activation and −/−Ink4a/Arf deletion; primary invasive tumours showed serrated morphology and p16‐dependent depression of senescence Apc mutation. However, in all models, high levels of Wnt signalling were observed in the adenocarcinoma and metastases that arose, suggesting that Wnt activation may progress these lesions from serrated lesions into ‘bona fide’ adenocarcinomas. The relevance of these serrated models has recently come to the fore, given that CRCs which have the poorest prognosis often have a ‘serrated’ signature In addition to the classical model of CRC progression, alternative routes to CRC have been described FBXW7 is altered in 20% of human CRCs and can control Notch receptor stability Nicd1LSL–GFP) in combination with the Min/+Apc mutation generates higher numbers of adenomas which were higher‐differentiated compared to the control Nicd1 in combination with Tp53 deletion in the mouse intestine generates adenocarcinomas that exhibit markers of EMT. Analysis of these mice revealed that 23% had lymph node infiltration and 10% showed spread of tumour cells to the liver Tp53 was deleted in IEC and mice where challenged with AOM Notch signalling is a key regulator of intestinal epithelial cell fate during normal homeostasis and contributes to tumour development Min/+Apc mouse Pirc/+Apc rat, which harbours a mutation in Apc which converts lysine → Stop at codon 1137 1311Apc mutation During recent years, other animal models of CRC have been developed in both rats and pig. Both, especially the pig, can recapitulate human physiology and pharmacology in a much better way than mice. In rats, two models of CRC were developed by administration of ENU, the same mutagen used for generating the Min/+Apc mouse and this model has been extensively used to characterize the mechanism, modifiers and potential therapeutic strategies for early‐stage intestinal tumourigenesis in situ. While we have not succeeded so far in the development of metastatic CRC models, many fundamental discoveries have been made about stem cells, homeostasis and transformation, so the community has failed very successfully! Our future aims must be to better model, understand and treat the later stages of CRC.It is 25 years since the publication of reports of the OJS and RJ wrote the manuscript.


Research investigating cognitive moderators of the intention–behaviour relationship and psychological consequences of failure to enact intentions is usually conducted in a single‐behaviour paradigm. A multiple‐behaviour paradigm is introduced which overcomes bias inherent to single‐behaviour designs and allows testing of novel hypotheses. Two exploratory studies illustrate the utility of this new paradigm by investigating the role of cognitive predictors and psychological correlates of intention–behaviour relationships.n = 126) also completed self‐reported measures of everyday cognitive failures and dysexecutive behaviours, crystallised intelligence  at baseline and Quality of Life . In Study 2, objective executive function measures  were completed by N = 30 participants.The proposed method involves measuring multiple intentions across common areas of life activity at baseline and corresponding behaviours at follow‐up. In two studies, 51 intentions and behaviours were assessed . In Study 1, participants  and task errors, rather than Stroop effect and Go/No‐Go performance were related, to intention–behaviour congruence.The total number of intentions, cognitive, and QoL measures were unrelated to the percentage of intentions enacted. Crystallised intelligence was related to successful intention implementation and problems with emotion regulation were associated with forming fewer intentions and with fewer Intention–behaviour relationships might be better understood when considering the multiple intentions and behaviours that people are engaged in at once at any one point in time. A multiple‐behaviour paradigm suggests novel hypotheses. Preliminary findings reported here require replication. Anticipated applications of the paradigm are outlined and discussed. Traditional social cognition models of behaviour such as Ajzen's  Theory oOver the last 20 years, a considerable gap between intention and behaviour has been noted  and crystallised intelligence (static cognitive ability) relate to the number of intended behaviours, and overall success and failure in intention enactment?To what extent does the number of intended behaviours, and overall success and failure in enacting them, relate to QoL and depression?The aim of this manuscript is to introduce a new research paradigm that could stimulate novel research and improve the understanding of the intention–behaviour relation, its predictors and consequences. Within this paradigm the intention–behaviour gap can be assessed for each individual across an array of behaviours. Successful enactment of intentions can be related to the total number of intentions pursued. Use of this paradigm is illustrated with two initial proof‐of‐principle studies addressing four main research questions:Forming too many intentions may over‐commit individual resources and lead to self‐regulation failure. The more intentions people form, the lower the proportion of intentions they will successfully implement.successfully enact.Individuals with higher levels of crystallised intelligence will form more intentions that they executive functioning (fewer cognitive failures) will translate a higher proportion of their intentions into behaviour.People with better People who have more failed intentions will have lower levels of well‐being and higher levels of depression.To date it is not known how the number of intentions individuals form relates to the number and proportion of successfully implemented intentions. Study 1 tests four hypotheses:Study 1 was a prospective survey. Self‐reported intentions, demographics, behaviour, crystallised intelligence, and executive function were taken at baseline via an online questionnaire during autumn term in a UK university. Participants were followed up one week later with a measure of their behaviour and QoL (including well‐being and depression).N = 126 students  provided digital informed consent via a website and completed the baseline questionnaire; n = 116 (91%) completed the follow‐up one week later.All intention items from 185 original papers included in a systematic review of the TPB were extracted  Total number of intentions out of 51), (B) Number of intentions successfully translated, (C) Number of intentions which participants failed to translate,, (B) NumMill Hill Vocabulary scale  were obtained from records linked with student ID numbers.World Health Organization QoL assessment  with measures of intelligence, executive function, and quality of life.SD = 7.93) intentions out of the 51 possible options for the next week. Out of those, an average of 13 (SD = 6.0) were successfully translated and 5.37 (SD = 3.75) failed  was significantly correlated with the number of successfully translated intentions and showed a solid null correlation with the number of failures. The relationships between the MHV questionnaire and the number of intentions and the percentage rate of successfully translated intentions were not statistically significant.Neither of the CFQ subscales showed any relationship to the measures of intention–behaviour relations. The DEX subscale for dysfunctional emotion regulation, however, showed a strong correlation with the number of intentions; people with more problems in regulating their emotions have fewer intentions. The subscale of motivational dysregulation correlated negatively with the number of successfully enacted intentions, but not with the number of total or failed intentions or the percentage success rate of translated intentions.All WHOQOL measures except for depression showed significant positive relationships with the total number of intentions and, notably, with the number of failed intentions. Three WHOQOL measures showed the highest relationship with the number of intentions successfully translated: overall QoL, enjoying life, and meaningful life. The WHOQOL depression measure did not show any significant correlations with measures of total, successful, and failed intentions, or with the percentage of intentions successfully translated.Study 1 found no trade‐off between the number of intentions people form and the rate of successful enactment of intentions. Participants who formed more intentions did not show a lower percentage of successful intention enactment. Indeed, the number of intentions was positively related to QoL and participants with more intentions reported fewer problems with the regulation of emotions. The correlation between crystallised intelligence and successfully enacted intentions suggests that an adaptive pattern of behaviour may be to focus on optimising success, rather than on preventing failure of intention implementation.While cognitive failure was unrelated to intentionality or to intention–behaviour relationships, people with problems in emotional regulation may avoid forming intentions and experience thereby both fewer failures and successes. In sum, there is no evidence that failed intentions make people miserable. Intending as such is positively related to quality of life.stop and override automatic responses is therefore of particular interest  predicts engagement in physical activity behaviour over time. Study 2 tested the relationship between objectively measured executive functioning (both cognitive inhibition and cognitive flexibility) and intra‐individual measures of intention–behaviour consistency in a small pilot study.Health behaviours are often habitual and driven by learned cue‐related automaticity . In addition to the measures of intention and behaviour, a lab‐based assessment was conducted to collect the measures of executive function:An All‐Intentions Method (AIM) similar to Study 1 was conducted with 30 university students  and omission errors (not responding to a go cue) over the two 100‐trial blocks, and on choice reaction time (ms).A e et al. . ParticiStroop test. A computerised version of the Stroop test .r = .13; p = .25) or B  or to the Stroop effect . However, the intention–behaviour congruence was related to the errors made during both cognitive tasks, r = .58; p < .001 for the Go/NoGo error and r = .35; p = .028 for Stroop error. The word fluency task correlated significantly with individual intention–behaviour congruence .The individual intention–behaviour congruence was not related to Go/NoGo speed in either block A , rather than overall performance , were strongly related to the intention–behaviour congruence of individuals. Moreover, the measure of cognitive flexibility (word fluency task) also showed a significant relationship to participants’ ability to act in line with their intentions.The two studies reported here were conducted to provide an initial proof of principle for the main ideas underlying the multiple intention approach. These studies illustrated the application of this approach and suggested novel findings. There were clear limitations in both studies which we would like to highlight. They include the self‐reported predictive measures in Study 1, the small sample size in Study 2, the use of convenience student samples in both studies, and the limited follow‐up period of only a week. Based on these limitations, the main study findings require replication. Likewise, it would be feasible to conduct more sophisticated statistical analyses over the data, but the intention of this paper is to describe and illustrate the novelty of the approach, Given the limitations listed above, a more complex statistical analytic approach was not seen as desirable.The aim of the present research was to provide a simple and transparent illustration of the use of the All‐Intentions Method (AIM) in a multiple‐behaviour paradigm. The paradigm of assessing various intentions  and various behavioural outcomes simultaneously  would allow more complex multi‐level analyses of datasets addressing more complex hypotheses. Intentions and behaviours are not independent of each other and it is possible to aggregate them empirically using factor analytical methods, potentially following taxonomies of behaviours (McEachan, Lawton, & Conner, In the present research the role of a limited number of cognitive moderators of the intention–behaviour relationship was investigated. The new paradigm offers the opportunity to test a range of personality, environmental, social cognitive, habitual, and self‐regulatory moderators of cognition–behaviour relationships more rigorously in a multiple‐behaviour context (de Bruijn, Brug, & van Lenthe, Finally, future research investigating the mechanisms of behaviour change can be enhanced in particular by evaluating interventions in a multiple‐behaviour paradigm. Fleig and colleagues demonstrated that an exercise intervention which was found to be effective in increasing physical activity and exercise habit strength also had a secondary effect on fruit and vegetable intake, and that this secondary effect was moderated by changes in exercise habit strength (Fleig, Lippke, Pomp, & Schwarzer, In conclusion, the proposed multiple‐behaviour approach provides an opportunity to examine the relationship between the number of intentions and their enactment and to test novel hypotheses about intention–behaviour relationships, their predictors, and consequences. It may contribute to the understanding of intention–behaviour relationships in addition to traditional single‐intention studies.Appendix S1: Assessment of intentionsClick here for additional data file.


During summer 2013 we installed a network of 19 GPS nodes at the ungrounded margin of Helheim Glacier in southeast Greenland together with three cameras to study iceberg calving mechanisms. The network collected data at rates up to every 7 s and was designed to be robust to loss of nodes as the glacier calved. Data collection covered 55 days, and many nodes survived in locations right at the glacier front to the time of iceberg calving. The observations included a number of significant calving events, and as a consequence the glacier retreated ~1.5 km. The data provide real‐time, high‐frequency observations in unprecedented proximity to the calving front. The glacier calved by a process of buoyancy‐force‐induced crevassing in which the ice downglacier of flexion zones rotates upward because it is out of buoyant equilibrium. Calving then occurs back to the flexion zone. This calving process provides a compelling and complete explanation for the data. Tracking of oblique camera images allows identification and characterisation of the flexion zones and their propagation downglacier. Interpretation of the GPS data and camera data in combination allows us to place constraints on the height of the basal cavity that forms beneath the rotating ice downglacier of the flexion zone before calving. The flexion zones are probably formed by the exploitation of basal crevasses, and theoretical considerations suggest that their propagation is strongly enhanced when the glacier base is deeper than buoyant equilibrium. Thus, this calving mechanism is likely to dominate whenever such geometry occurs and is of increasing importance in Greenland. GPS sensor network and oblique cameras installed at Helheim Glacier during 2013Calving by bottom‐crevassing with ice downglacier of flexion zones rotatingMechanism likely to dominate whenever glacier is out of buoyant equilibrium The Greenland Ice Sheet is losing mass at an accelerating rate [Pelto and Warren, van der Veen, Benn et al. [Krug et al. [Warren et al., Boyce et al., James et al., Nick et al., Bassis and Jacobs, van der Veen, Glacier calving was originally modeled using empirical relationships that link the calving rate to water depth [n et al. , which pg et al.  proposed2 of the Greenland Ice Sheet and terminating in Helheim Fjord, an arm of Sermilik Fjord. The glacier and its fjord systems have been intensely studied in part because of its accessibility [e.g., Nettles et al., de Juan et al., Straneo et al., Sutherland and Straneo, Jackson et al., Bevan et al., Howat et al., Luckman et al., Stearns and Hamilton, Howat et al., Stearns and Hamilton, Howat et al., Murray et al., Helheim Glacier Figure  is one oStraneo et al., Motyka et al., Sutherland and Straneo, Nick et al., O'Leary and Christoffersen, Cook et al., Warm water of subtropical origin has been observed in Sermilik and Helheim fjords at depths greater than ~250 m [Nettles et al. [Joughin et al., Nettles et al., Nettles et al., de Juan et al., Rosenau et al., s et al.  reportedJames et al., In contrast, during 2010 and 2011, observations of Helheim Glacier from daily digital elevation models derived from oblique stereo terrestrial photogrammetry were interpreted to show that calving occurred driven by buoyant flexure interpreted as resulting from the propagation of basal crevasses [Bevan et al., James et al., Calving at Helheim Glacier occurs year round, and there is a relatively weak seasonal cycle of frontal position at the glacier compared to its neighbor, Kangerdlugssuaq Glacier [Nettles et al., James et al., In this paper we report the detailed dynamics of the margin of Helheim Glacier during a series of major calving events during summer 2013 recorded using a wireless network of custom‐built GPS receive‐transmit nodes. The previous projects discussed above have reported the calving process using daily averaged flow rates with more limited higher‐rate observations [2James et al., Ekström et al., Nettles and Ekström, We designed and operated a wireless network of GPS nodes at the margin of Helheim Glacier Figure . The GPS2.1Baronti et al., Martin et al., The 2013 GPS network consisted of 19 on‐ice GPS receiver nodes  never communicated) and four logging stations installed on bedrock, one of which (NW station) was co‐located with a GPS base station. At the end of the 2013 deployment five ice nodes were collected having run through the entirety of the field deployment. Zigbee transceivers were used to transmit the GPS data from the ice nodes to the logging stations [The ice nodes were purpose built and contained a dual‐frequency GPS board attached to a geodetic‐grade antenna and two Zigbee communications boards coupled to a single wireless antenna using an RF splitter circuit. Each Zigbee board communicated separately with a logging station, providing redundancy in data transmission. Each ice node was powered by a battery and solar panel. The network nodes were emplaced by helicopter and secured with crampons (15 nodes) or crampons and stakes drilled into the ice (five nodes). Known causes of node communication loss were due to calving or because the node tipped over orienting the antennas unfavourably.Zumberge et al., Bertiger et al., Lyard et al., Bar‐Sever et al., Niell, Bevan et al., The GPS base station employed a dual frequency GPS board linked to a geodetic‐grade antenna located some 20 m from the receiver unit and installed on a permanently established, highly stable geodetic monument. The coordinates of the base station were calculated using GIPSY OASIS II software v6.2 from the NASA Jet Propulsion Laboratory (JPL), and daily static coordinates were estimated using the software in Precise Point Positioning (PPP) mode [Chen, Dach et al., Processing of the ice node GPS data was performed using the Track 1.29 module of the GAMIT‐GLOBK10.50 GPS processing software Chen, . Track u0.5. While this is a considerably looser constraint than is needed to model the ~0.3 mm/s motion of the glacier, we wanted to avoid overconstraining any rapid motion during calving events. Due to the relatively short baseline lengths, ~1–7 km, no tropospheric correction was estimated as at times the low number of visible satellites degraded the final solution if this parameter estimation was included in the solution. GPS data were then filtered by excluding from plots any data where the number of unfixed biases was greater than 2, the number of double differences was less than 4, or the height uncertainty was greater than 0.1 m. Precision of the resulting coordinates based on Track formal errors and assessment of the detrended time series outside of calving events for are ~2 cm in north, 1 cm in east, and 2.5 cm in height.On‐ice node positions were estimated for each reported GPS observation. Positions were modeled as random walk stochastic process with process noise of 0.01 m/s2.2James et al. [Cameras were installed in stereo configuration at two locations on the south side of the fjord Figure . An 18 ms et al.  details Wolf and Dewitt, x‐y image coordinates of each node using (i) its XYZ position in ground space; (ii) the camera calibration parameters, focal length, and principle point; and (iii) the exterior orientation parameters of the camera including its XYZ position in ground space and azimuth, tilt, and swing orientation angles.GPS nodes were plotted on each photograph using the collinearity condition of photogrammetry which requires that a point on the ground, the camera center, and the corresponding point on the image plane lie in a straight line [James et al. [Farneback, Tracking was undertaken on images from the sideways‐looking camera to detect motion in the vertical and horizontal image directions analogous to that reported in s et al.  but usinx and 1 pixel in y. After these errors were removed, the mean apparent displacement of the bedrock in the mountains in the background of the images  from image to image as the glacier flows. Images with different lighting or obstruction (such as fog and changes in cloud cover causing shadows on the ice surface or changes in sun angle) do not track well. We therefore tracked images over 24 h when lighting is most likely to be similar. All daylight images were tracked, and then the best result during each 24 h period was chosen. Systematic tracking errors will result from any camera movement but also from changes in atmospheric conditions and temperature‐dependent lens geometry, both of which cause changes to the path of light entering the camera. For securely mounted cameras, movement is expected to be negligible except during storm events. Since storms were not a problem during our tracking periods, we attribute the majority of systematic errors in our tracking to changes in atmospheric conditions and lens geometry. This systematic bias was assessed and removed by measuring mean displacement in a 700 × 430 pixel window on stable bedrock in the foreground of each image pair and subtracting this value from the tracking results. Of the image pairs shown in this paper, only two required corrections higher than 2 pixels in x axis of the images approximates to downglacier flow. Image movement on the y axis of the images is more difficult to interpret as while most sensitive to changes in vertical and crossglacier position of the glacier, it is also sensitive to downglacier flow if the camera is slightly oblique to this flow direction or if the glacier surface is not flat. Crossglacier flow is negligible on these images as typically glacier flow measured on each GPS node varied in direction by only 1–2° over the measurement period. Vertical changes can be dynamic or melt related. Previous measurements have shown that summer ablation rates were ~3.2 cm/d at 650 m elevation [Andersen et al., y axis image movement revealed by our tracking cannot be explained by differences in ablation.On the sideways‐looking images movement on the Two aspects of the images should be noted—both related to the relatively low viewing angle of the camera from the glacier side , DOY 211–212 (a series of three events), DOY 220 and DOY 226.3.1Table 3.2Figure Nodes 3 and 8 were installed, respectively, 2.8 and 3.6 km from the glacier front Figure . The nodFigures Node 9 was installed approximately 1.9 km from the glacier front Figure a. There Figure 3.3Figures The feature (“X”) continues to advect downglacier with strong uplift downglacier of it and strong downward movement associated with its location Figure e. At the4James et al. [Van der Veen, Joughin et al., We interpret these data within the framework of the model introduced by s et al.  for calvJames et al. [The simplest explanation for the buoyancy forces required is that the rapid flow of the glacier front (25+ m/d) pushes the ice into deeper water at a depth below buoyant equilibrium  or an increase in the rate of downward movement can also occur if a new flexion zone is formed downglacier of the node.The data allow the identification and characterization of the flexion zones and quantification of the rates of vertical displacement that result from their formation and subsequent calving. We suggest that this process of calving by buoyant flexure and the exploitation of basal crevasses will dominate whenever ice is driven below buoyant equilibrium by flow into deep water, by dynamic thinning, or by surface melting. The process is of increasing importance in Greenland as tidewater glaciers retreat back to their grounding lines. Calving by this process will tend to keep the glacier front margin close to the grounding line of the glacier.


We present the first continuous, multi-day heart rate recordings of newly weaned, wild southern elephant seals. The effects of circadian periodicity and weather conditions on heart rate are examined. Changing climate regimes may affect the energetic requirements experienced during, and thus the energetic trade-off that concludes, the post-weaning fast. Mirounga leonina) pups must strike a balance between conserving energy during their post-weaning fast and simultaneously developing diving abilities to attain nutritional independence. Little is known about environmental influences on cardiorespiratory patterns, hence energy use, throughout the 6 week fast. Continuous heart rates were recorded for free-ranging, newly weaned southern elephant seals using heart rate time–depth recorders for 5–9 days at Sub-Antarctic Macquarie Island, during October 1994 (n = 1), 1995 (n = 4) and 1996 (n = 1). Daytime observations of respiration and behaviour were made throughout. We present the first instance of synchronous heart rate traces recorded simultaneously for individual weaners. Generalized additive models revealed that a sinusoidal pattern of diurnal heart rate elevation and nocturnal depression was evident in all seals and, on at least one occasion, a conspicuous break in this pattern coincided with an extreme cold weather event. Seals in this study were capable of considerable cardiorespiratory control and regularly demonstrated bradycardia during periods of resting apnoea. Apnoeic duration ranged from 33 to 291 s (mean 134 s). Apnoeic heart rates  were on average 19.7% lower than those exhibited during periods of eupnoea . The early development of the cardiorespiratory response is characterized by arrhythmic heart and respiration rates. The strong temporal patterns observed are being driven by the opposing requirements of maximizing time spent fasting in order to develop diving capabilities and of maximizing departure mass. This pilot study has highlighted a potentially large effect of ambient weather conditions on newly weaned southern elephant seal cardiorespiratory activity. Given the increasing westerlies and more erratic and increasing storminess associated with the Southern Annular Mode predicted in the Southern Ocean, the patterns observed here warrant further investigation.Southern elephant seal ( Diving mammals exhibit unique cardiorespiratory traits to cope with the extreme physiological demands of diving .Mirounga) are archetypal arrhythmic breathers. Adult individuals are typically submerged for 80–95% of the 8–10 months they spend annually at sea and are therefore highly adapted for a life where overall time spent underwater is essentially comparable to (and throughout their lifetime often exceeds) time spent on land  to examine the relationships between heart rate and respiration (apnoeic and eupnoeic periods); (ii) to quantify temporal trends in heart rate; and (iii) to evaluate possible extrinsic influences on heart rate in weaned southern elephant seals.n = 1; 1995, n = 4; 1996, n = 1; Table The study took place over 3 years on the (sheltered) east and (exposed) west coasts of the isthmus at Macquarie Island 54°35′S, 158°55′E). Weaned southern elephant seals of similar age (3–5 weeks) were randomly selected from a group of 400–800 tagged animals in each year  was also fitted to the dorsal surface of each animal to facilitate re-location of seals after deployment.A Polar−1) in wet and dry conditions . Observations were done by a single trained observer out of view of the seal to prevent disturbance, but within sight of the seal's nostrils at all times to record changes in respiratory rates. An apnoea was defined as an expiratory pause of more than 30 s terminated by an inspiration , e the water vapour pressure  and ws the wind speed (in metres per second) at an elevation of 10 m above sea level.Data for meteorological conditions  for the study period were obtained from the Australian Bureau of Meteorology (BOM) archives at the finest available resolution, namely 3 h sampling intervals. The wind chill factor (apparent temperature) was derived using the following equation :AT=Ta+0aT is the dry bulb temperature (in degrees Celsius), rh the relative humidity (as a percentage) and exp is the exponential function.The water vapour pressure is calculated as follows:The west coast of the isthmus at Macquarie Island is more exposed to passing weather systems than the east coast. There is only one meteorological station on the island and so the data cannot differentiate the slight differences in severity in weather experienced between the coasts that would have occurred ‘on the ground’.The HR-TDR data were downloaded to a computer and transformed and analysed using custom-designed software after the recovery of each unit (Wildlife Computers Software). The first 6 h of data were removed to ensure that any effects of the anaesthetic or from disturbance due to handling were excluded from analyses. Daily solar elevation values were calculated for Macquarie Island using the elevation function in the tripEstimation package  in R stan = 4). The difference between apnoeic and eupnoeic heart rates was assessed with Student's paired t-tests of heart rates from successive whole-apnoea and whole-eupnoea respiratory state pairs for each individual. Linear regression was used to test for relationships between apnoeic heart rate and duration of apnoea for individual seals.To separate inter-individual from inter-annual effects, comparative heart rate analysis was done only for seals deployed in 1995 .Continuous heart rates were recorded for six seals for periods ranging between 6.5 and 10.7 days −1].The ‘wind-swept’ west coast of the isthmus at Macquarie Island is known to be generally more exposed to prevailing westerly weather systems than the relatively sheltered east coast  on 15 October 1995 is of particular interest Figs. . Unfortu−1 during eupnoeas (mean 83 ± 15 beats min−1) to from 40 to 114 beats min−1 (mean 67 ± 15 beats min−1) during apnoeas.Heart rates during apnoeic and eupnoeic events were significantly different for all four of the seals with enough observations to run the analysis Table . The decn = 73) ranged in duration from 33 to 336 s with a mean duration of 133.5 ± 67.8 s for five seals (Table n = 62) ranged in duration from 60 to 2169 s with a mean duration of 270.3 ± 376.9 s for four seals. A weak inverse relationship between apnoeic heart rate and apnoeic duration was evident for one seal, 95161 . As this seal had double the number of successive respiratory state paired observations as the next highest individual, it is probable that small sample sizes substantially reduced the power to detect relationships in the remaining seals. Eupnoeic heart rate was also unrelated to the duration of eupnoea in all seals (n = 5).The apnoeas observed during the study .Figure Highly variable heart rates are a fundamental physiological adaptation of southern elephant seals. Seen in this light, the synchronicity observed in this study between the heart rates of simultaneously deployed wild southern elephant seal weaners uncovers a new question that may prove central to understanding the energetics of the post-weaning fast. Given that individuals must have been performing different behaviours at least some of the time throughout the study, to what extent do environmental factors have a mediatory effect on the seals' heart rates?All weaners exhibited a daily pattern of higher diurnal heart rate and nocturnal decline Fig. . EnvironM. leonina during diving and apnoeic periods  and weather variables (3 h−1) and the likelihood of weaners responding to weather cues on a scale of minutes as opposed to hours, we can currently present only a broad interpretation of the relationship between weaners' heart rates and their ambient environment.Seals equipped with HR-TDRs in pairs showed unexpectedly high synchronicity of heart rate trends over and above the general diurnal pattern, throughout their deployment periods Figs.  and 3. PThe depression of both 1995 west-coast weaners' heart rates on a single day only is of particular interest and might reflect a response to challenging weather conditions. This day coincided with high rainfall and low ambient and apparent temperatures. The degree of synchrony between heart rate traces is visibly higher in the west-coast pair compared with the east Figs  and 2. TThe findings of the present study, i.e. the first continuous multiday heart rate recordings of wild, free-ranging southern elephant seal weaners, contextualize existing descriptions of a number of physiological capabilities of newly weaned southern elephant seals. The breath-holding ability of newly weaned pups that we observed was similar to that found in other studies; the duration of apnoea  was slightly shorter than that reported for weaned southern elephant seals of a similar age in Patagonia  dropped by an average 19.7% during apnoeic periods (mean 67 ± 15 beats min−1), comparable to that recorded in elephant seals of a similar age by Eupnoeic heart rate  stressors, such as potentially increasing interactions with expanding fisheries . AlteredM. leonina and consequent survivorship at sea. Clearly, more research into the behavioural and physiological mechanisms operating in young elephant seals is required to test many of the assumptions associated with the suggestions raised in this article. More extensive behavioural and respiratory observations, additional physiological variables, finer-scale meteorological observations, greater sample sizes, temporal crossover between deployments at different locations, the quantification of thermoneutral zone and relationship between heart and metabolic rates in this species will help in the future to illuminate these remaining questions. Nevertheless, it is evident that the cardiorespiratory patterns experienced during the post-weaning fast by southern elephant seals are not fixed but exhibit some control and may fluctuate in response to changing weather conditions.There are very few studies of continuous heart rate in free-ranging pinnipeds during terrestrial life-history phases for any demographic group. Whether the patterns described in this study, especially regarding the susceptibility of heart rate to exogenous forces, will prove to have further implications for reproductive or moulting phases later in life is presently unknown. However, any sustained change in heart rate must necessarily equate to changes in energetic costs. This would thus impact upon the energetic trade-off that dictates the length, and therefore available period for development, of the post-weaning fast in This work was supported by the Australian Research Council (grant no. A09332789) and the Antarctic Scientific Advisory Committee (grant no. 589) and was conducted under permits granted by Tasmanian National Parks and Wildlife Service and the Antarctic Animal Ethics and Ionising Radiation Committee.Supplementary DataClick here for additional data file.


A constant interval of 30 °C with the heating rate of 3 °C/min was utilized to guise the temperature below and above the glass transition temperature of PC followed by thermal gravimetric, HRTEM, FESEM and hydrophobic analysis at ambient temperature. The HRTEM images of Ph-POSS nano unit demonstrated hyperrectangular structure, while FESEM image of the developed nano composite rendered separated phase containing flocculated and overlapped stacking of POSS units in the PC matrix. The phase separation in polymer nanocomposite was further substantiated by thermodynamic interaction parameter (χ) and mixing energy (Emix) gleaned via Accelrys Materials studio. The SAXS spectra has demonstrated duplex peak at higher scattering vector region, postulated as a primary and secondary segregated POSS domain and followed by abundance of secondary peak with temperature augmentation.Inorganic/organic nanofillers have been extensively exploited to impart thermal stability to polymer nanocomposite via various strategies that can endure structural changes when exposed a wide range of thermal environment during their application. In this abstraction, we have utilized temperature assisted  The exceptional mechanical, physical, thermal and magnetic properties of inorganic/organic polymer hybrid nanocomposites have attracted great attention in recent years12348111213141516171819et al. have demonstrated the enhancement in thermal degradation and combustion behavior of PC/trisilanolphenyl POSS nanocomposite via melt blending while Hao et al. have elucidated the incorporation of phenethyl POSS in polycarbonate matrix by solvent casting technique10In order to develop thermally stable and flame retardant materials, polycarbonate (PC) has allured great interest due to its high tendency of charring during combustion1023in-situ quantification of structural orientation distribution of POSS nanoparticles in polymeric composite systems. Molecular interaction and interplay can be examined by temperature assisted in-situ SAXS analysis. In this study, we have attempted to quantitatively evaluate the molecular interaction and interplay between the intercalated POSS layers in the polycarbonate matrices via temperature assisted small angle x-ray scattering, thermal gravimetric analysis, FESEM and hydrophobic analysis at ambient temperature. We have also proposed the possible mechanism involved in structural changes under the various temperatures. In order to contemplate the phase separation due to high loading of POSS nano unit in PC matrix, the dynamic molecular simulation was accomplished for computing the dynamic thermal interaction and mixing energy at various temperatures. Further, we have speculated that the formulation of free volume in PC matrix enacted pronounced effect on molecular distribution and interplay over the hyperrectangle POSS at a wide range of temperature in PC matrix.Although, the incorporation of POSS in polycarbonate has been investigated with polycarbonate for applications ranging from heat sink to the flame retardant materials, there is little literature reporting on Polycarbonate , Octaphenyl (OP) substituted POSS and Chemicals viz. Chloroform (Sigma Aldrich Corp. USA.) were used for the preparation of samples. All reagents and solvents involved were of analytical grade and were used without any further purification. The optimized amorphous unit cell of PC has been constructed by utilizing forcefield module of Materials Studio as shown in Solvent casted PC/Ph-POSS nanocomposite films were fabricated by dissolving 20 wt % of polycarbonate in chloroform followed by incorporating 10 to 30 wt % of Ph-POSS in the prepared polymer solution. The solution was then treated by ultrasonication for 5 min and subsequently casted on a glass slide in closed chamber to avoid abrupt evaporation of solvent. The detached polymer nanocomposite was dried under the vacuum at the boiling temperature of chloroform (61 °C) for 24 hr to remove retained traces of solvent. The maximum loading of Ph-POSS in PC matrix was ascertained to be 30 wt %, beyond this concentration the formation “pin hole” was observed under the scanning electron microscopy for the film thickness of approximately 100 μm as elucidated in et al. demonstrated that the evaporation of volatile solvent caused development of entangled polymer chain by the viscosity upturn of the polymer solution followed by film disruption which rendered, unorganized and aggregated structural domainin-situ small angle X-ray pattern of pristine PC at various temperatures has been exemplified in et al. Siegmann et al. have annotated that all solution casted PC thin film retains a small amount of solvent and this residual solvent (less than 1%) is sufficient to affect molecular structure of the film31et al. demonstrated that, the presence of maxima and minima in pair correlation function was likely due to overlapping of the neighboring domainsIt has been ascertained that, the polymeric chain in a solvent cast film can preferentially align in a plane of casting substrate due to the freezing of molecular conformation into the film during the drying process26et al. to calculate linear crystallinity of cold-drawn polyethylene38Polycarbonate has been regarded as crystallizable amorphous polymer with granular structure that can be affected by thermal and mechanical treatment below glass transition temperature343536ac is long spacing obtained from Brag’s law  = one dimensional correlation function, Ce(z) is electron density distribution along the stack normal, din-situ temperature assisted SAXS spectra.The inter-lamellar distance or long spacing values for all the temperature (from 30 °C to 150 °C) obtained from Brag’s law and electron density correlation function are indexed in et al. have demonstrated that octa substituted POSS nanoparticles with various end groups  form rhombohedral . In addition, the duplex peak in SAXS spectra of nanocomposite at higher q region  has beenet al. as solvent evaporates, the molecules of the film reduce their ability to respond the increased relaxation timeAs annotated by Prest mix) has been delineated via Materials Studio that utilizes off lattice method which combined extended Flory Huggins model and molecular simulation technique. Utracki has evinced that the conversion of room temperature to high temperature impart the effect of decreasing solubility parameter43mix value obtained at ambient temperature allied to the phase separation as conferred in SAXS spectra of polymer nanocomposite previously while reduction in the value with the amelioration in temperature relinquish some extent of intercalation or mixing45In order to substantiate the phase separation in polymer nanocomposite, thermodynamic interaction parameter or solubility parameter (χ) and mixing energy .


RT) for head and neck cancer. Current treatment approaches are insufficient and can only temporarily relieve symptoms. New insights into the physiopathology of radiation‐induced xerostomia might help us in this regard. This review discusses the current knowledge of salivary gland stem cells in radiation‐induced xerostomia and their value in the prevention and treatment of this complication. Salivary gland stem cell transplantation, bone marrow‐derived cell mobilization, molecular regulation of parotid stem cells, stem cell sparing RT, and adaptive RT are promising techniques that are discussed in this study.Xerostomia is an important complication following radiotherapy ( Radiotherapy (RT) is an important treatment modality in the management of head and neck cancer (HNC). RT can be used as a single modality or in combination with surgery and/or chemotherapy (CT). One of the most frequently reported side effects following RT in patients with HNC is hyposalivation and subsequent xerostomia due to co‐irradiation of the salivary glands Approximately 40% of patients with HNC will experience xerostomia to some degree after treatment with RT Xerostomia can occur early during RT treatment. Depending on the localization of the tumor and the radiation portals, a rapid decrease of the salivary flow rate is observed during the first week of RT, after which there is a continuing gradual decrease to less than 10% of the initial flow rate. This early reaction is unforeseen because the excretory, acinar cells in the salivary glands have a slow mitotic rate; a fast response to RT is therefore not expected A review of published studies suggested that severe xerostomia can be avoided if either the mean dose to both parotid glands is less than 25 Gy or one parotid gland is spared to a mean dose of less than 20 Gy Following RT, salivary gland recovery is dependent on the radiation dose and on the number of remaining viable SCs In 2006, our research group published a literature review on radiation‐induced xerostomia in patients with head and neck cancer A systematic literature search was performed in the MEDLINE/PubMed database for articles published between January 1990 and September 2015. The objective was to trace all literature containing original data on new findings and potential treatment strategies in the field of salivary gland SCs.The literature search was performed in October 2015 using combinations of Mesh terms of “xerostomia”, “radiotherapy”, “salivary gland,” and “stem cell”. Furthermore, extensive cross‐referencing of the selected articles was performed. Case reports were eliminated from this review. Furthermore, the search was limited to English language. Gender and age were not limited.Before transplantation of SCs is possible, surgical removal of salivary gland tissue before the start of the oncologic treatment is needed. Later on, SCs need to be collected. The ultimate goal of SC transplantation is regeneration of the function of the salivary gland by differentiation of these transplanted SCs into functional salivary gland cells c‐Kit. Since 2004, several studies were performed in which submandibular and parotid gland SCs were transplanted in animal models after RT CD 49f, CD 29, CD 24, and CD 133 might be used as markers for salivary gland SCs in mice On the other hand, it is suggested that clusterin—was identified as a factor to recover from hypofunction of the salivary glands. It is believed that clusterin scavenges reactive oxygen species after irradiation Side population cells have been identified in various organs as putative SCs; their precise function, however, remains unclear. In mice salivary glands, side population cells did not seem to have SC activity Mesenchymal SCs from the bone marrow can be mobilized Selection of patients for this treatment will be very important. When the damage to the salivary gland is expected to be low, this technique using bone marrow‐derived cells could be enough to limit xerostomia after RT. When the damage is expected to be high, bone marrow‐derived cell mobilization could be insufficient, so SC transplantation may be necessary β‐catenin signaling pathway is essential for maintenance and activation of different types of SCs, including these of the salivary glands β catenin, which acts as an activator of gene transcription.Until recent, little was known about the molecular regulators of SCs in human salivary glands. Nowadays, there is growing evidence that the Wnt/β‐catenin pathway in mice during RT prevents both acute and chronic hyposalivation through inhibition of apoptosis and preservation of functional salivary stem/progenitor cells There is increasing data that activation of this pathway may also have a radioprotective effect and might decrease xerostomia numbers in irradiated patients Wnt activation is also known to promote angiogenesis and innervation during development. In this regard, it is speculated that Wnt activation can help in the regeneration of the damage to blood supply and innervation after irradiation. This can play an additional role in the prevention of hyposalivation after RT These findings are promising; however, further research is mandatory to confirm the results and to investigate whether activation of this pathway is sufficient to prevent xerostomia in humans after RT.β‐catenin pathway has been linked to carcinogenesis Regarding the introduction of this technique, a major concern that needs further clarification is that overexpression of the Wnt/Furthermore, there is emerging evidence that transient activation of the Hedgehog pathway and modulation of the GDNF pathway might play a role in preserving salivary gland SCs after RT Intensity‐modulated RT (IMRT) is the modality of choice to reduce xerostomia numbers following RT for HNC A first possible explanation for this higher‐than‐expected incidence of xerostomia is the hypothesis that radiation of the parotid gland has a “bath and shower effect”. Photon‐based IMRT gives an overall low dose to the entire parotid gland Sparing of SC‐rich regions within the parotid gland could help in preventing xerostomia following RT Dijkema et al. stated that sparing of the contralateral submandibular gland, in addition to parotid gland sparing, might result in improved patient‐reported xerostomia One of the unique aspects of RT to the head and neck region is that noticeable changes in the anatomy occur during treatment. These changes include shrinkage of the tumor, but also of the surrounding organs at risk (such as the parotid and submandibular glands) We recently closed a multicenter randomized adaptive radiotherapy trial including 100 patients with HNC. All patients received a computed tomography at 2 and 4 weeks after start of treatment, replanning was performed at these time‐points. Further follow‐up of these patients will give us more insights into whether this adaptive RT technique results in less xerostomia.To date, none of the described techniques concerning SCs to treat xerostomia are ready for clinical implementation, although most of them seem very promising in animal models.It will become clear in future trials which technique or combination of techniques will be the most beneficial, cost‐effective, and easy to implement. The emphasis, nevertheless, has to be on avoiding xerostomia as much as possible. If both the shower and bath assumption, the role of the contralateral submandibular gland and the importance of SC sparing RT are confirmed in future trials, high‐specific proton RT could be a way to counteract these problems at the same time.None declared.


AD) is still an incurable neurodegenerative condition that affects an estimated 44 million individuals worldwide and this figure is expected to increase to 135 million by the year 2050. As the research community currently reflects on previous endeavours, it is essential that we maximize the use of existing knowledge to inform future trials in the field. This article describes the development of a systematically identified data set relating to over 300 interventions tested in over 10,000 animals. The data set includes cohort‐level information for six structural outcomes and six behavioural assessments. We encourage others to use this dataset to inform the design of future animal experiments modelling AD and to promote effective translation to human health.Despite many efforts by the research community, Alzheimer's disease ( Alzheimer's disease (AD) is an incurable neurodegenerative condition that affects an estimated 44 million individuals worldwide. This figure is expected to increase to 135 million by the year 2050,Currently, symptomatic relief can be achieved for some patients using acetylcholinesterase inhibitors or memantine, but these are not suitable or effective option for all patients at all stages. There is therefore a pressing need both for interventions capable of providing greater symptomatic relief, and for disease‐modifying interventions therapies that might slow, or halt or even reverse, the progression of the condition.Substantial efforts have been made in preclinical science in order to identify candidate clinical treatments. Much focus for disease‐modifying therapy has concentrated on targets identified by the amyloid hypothesis including active and passive immunization strategies  and gamma secretase inhibitors .in vivo for two decades. The first transgenic mouse model was engineered in 1995Following the identification of familial AD and those mutated genes responsible, transgenic mouse models have been designed to recapture aspects of AD in vivo and to investigate molecular mechanisms. While transgenic mouse models have certainly advanced our understanding of AD, their utility in developing new treatments has been less certain; Zahs and AsheThe prospect of testing candidate intervention strategies in animal models capable of capturing aspects of AD provides opportunities both to demonstrate efficacy There are many plausible reasons for translational failure in AD,Here we have conducted a systematic review of transgenic mouse studies testing the efficacy of candidate drugs in transgenic models of AD. We have extracted data from these studies for meta‐analysis, the results of which will be presented elsewhere. To allow others to benefit from these data we are now making these available to the scientific community as a whole. Of the many possible uses, we consider this data set will be of most immediate use to others planning preclinical AD studies  or clinical trials of candidate drugs included in this review. Those interested in using these data to empirically guide future research can find further guidance on the analyses of datasets such as these in Vesterinen et al.Studies in animal models of AD were identified from 1995 to Jan 2009 in Pubmed, EMBASE and ISI Web of knowledge with the search terms [“targeted deletion” OR “overexpression” OR “knock out” OR “vector” OR “transgenic”] AND [“dementia” OR “tau” OR “mild cognitive impairment” OR “Alzheimer's disease”] with the search limited to animals using the filters available in those databases. The searches were taken from 1995 as this year coincides with the production of the first transgenic AD mouse model. The search was conducted in January 2009. The protocol for this systematic review was defined in advance, and was amended in Feb 2011 (hierarchy for deciding which cohort to be used as a control group) and March 2011 .Publications were included that tested interventions using any amyloid‐, tau‐ or PS‐based transgenic mouse model of AD.Genetic manipulations surplus to those required to produce symptoms of the condition were excluded as with genetic treatments if intervention took place before birth (i.e. gene knock‐outs or knock‐ins). We excluded studies where more than one treatment was administered. For the MWM, reversal task behaviour nor time in opposite or adjacent quadrants were extracted.Publications identified were assessed against a five‐point study quality checklist adapted from those previously described in the “Good Laboratory Practice Guidelines” for stroke modelling.Referencing section for web address).Our systematic search identified 427 publications testing interventions in transgenic mouse models of AD. These publications described 357 interventions and 55 transgenic models representing 11,118 animals and 838 experiments. All extracted data are available through Figshare  or overall tau levels. Likewise, we extracted information regarding neurodegeneration according to the specific variable measured.We identified six key neurobehavioral outcome measures; the training (acquisition phase) and the test (probe phase) of the Morris water maze, Radial arm water maze, fear conditioning, the Y maze and T maze.For the Morris water maze, we were able to extract a number of paradigm‐specific methodological attributes. For example, we extracted data recording the number of days training, the number of training sessions per day, the temperature of the water in the Morris water maze, the diameter of the pool and the time delay between the end of the acquisition phase and the probe “test” phase assessment. In addition, for acquisition time points we measured all data points and associated errors.http://plos.figshare.com/articles/Interventions_tested_in_preclinical_studies_using_transgenic_mouse_models_of_AD/1185428. This should be referenced through reference to this publication.Other investigators are welcome to use, with attribution, the data reposited on Figshare The authors declare that there are no conflicts of interest.


Larval sturgeon swimming capacity has never been assessed. We measured critical swimming velocity of larval green and white sturgeon, and summarized published juvenile critical swimming velocity data for all sturgeon species. Recommendations for anthropogenic water diversion facility flow management were developed from the data, emphasizing Californian green and white sturgeon conservation. Acipenser medirostris) and white sturgeon (Acipenser transmontanus) inhabit the highly altered Sacramento–San Joaquin watershed, making them logical species to examine vulnerability to entrainment by altered water flows. The risk of larval sturgeon entrainment is influenced by the ontogeny of swimming capacity and dispersal timing and their interactions with water-diversion structure operations. Therefore, the aim of this study was to describe and compare the ontogeny and allometry of larval green and white sturgeon swimming capacities until completion of metamorphosis into juveniles. Despite the faster growth rates and eventual larger size of larval white sturgeon, green sturgeon critical swimming velocities remained consistently, though modestly, greater than those of white sturgeon throughout the larval life stage. Although behavioural interactions with water-diversion structures are also important considerations, regarding swimming capacity, Sacramento–San Joaquin sturgeons are most vulnerable to entrainment in February–May, when white sturgeon early larvae are in the middle Sacramento River, and April–May, when green sturgeon early larvae are in the upper river. Green sturgeon migrating downstream to the estuary and bays in October–November are also susceptible to entrainment due to their movements combined with seasonal declines in their swimming capacity. An additional inter-species comparison of the allometric relationship between critical swimming velocities and total length with several sturgeon species found throughout the world suggests a similar ontogeny of swimming capacity with growth. Therefore, although dispersal and behaviour differ among river systems and sturgeon species, similar recommendations are applicable for managers seeking to balance water demands with restoration and conservation of sturgeons worldwide.Little is known of the swimming capacities of larval sturgeons, despite global population declines in many species due in part to fragmentation of their spawning and rearing habitats by man-made water-diversion structures. Larval green ( Green sturgeon spawn in Oregon and California and are composed of at least two genetically distinct populations  salmonid starter moist feed. For each spawn, larvae were split into two 1-m-diameter flow-through tanks supplied with 18 ± 0.5°C, aerated well water (dissolved oxygen >8.2 mg l−1) creating flows of <5 cm s−1 and reared under a natural photoperiod . White sturgeon, obtained from Sterling Caviar and spawned in April 2012 and May 2013, were offspring of the F3 descendants of wild-caught Sacramento River sturgeon. Green sturgeon were tank spawned from first-generation domestic Klamath River broodstock in November 2011 and April 2013 following the methodology outlined by Ucrit were carried out at 18.5 ± 0.5°C. Due to their small size and frequent feeding requirements  equipped with a motor and a variable frequency driver. During the acclimation period, water velocity was 1 cm s−1. After 30 min, water velocity was increased to 10 cm s−1 and then increased in increments of 5 cm s−1 every 5 or 10 min, depending on the spawn year. During each increment, fish were monitored for swimming behaviour. If fish became impinged, which was defined as contact between the downstream screen of the chamber and a third of their body for any duration or less than a third of their body for 30 s, timers and flow were stopped for 2 min. After the 2 min rest, timers were restarted, and flow was resumed at the velocity at the time of impingement. On the third impingement at the same velocity increment, or failure to recommence swimming after the 2 min break, the fish was considered fatigued  with the same-aged 2011 or 2012 spawns  using a one-way ANOVA did not differ in either species .Preliminary tests with sturgeon larvae of different sizes and ages dictated that the time interval and swimming flume size and design be modified to achieve successful swimming throughout larval development. The 2013 spawn (green and white sturgeon measured at ages 20–42 dph) were swum in the 1.5 l cylindrical swimming flume for 5 min intervals. The 2011 green sturgeon and 2012 white sturgeon spawns (measured at ages 34–60 dph) were swum in the 5 l rectangular, flat-bottomed flume , set to a 2 s time constant. For each dial setting on the flume motor, three separate measurements of flow were recorded, and the mean for each dial setting was calculated. Critical swimming velocity was calculated according to the formula:Vf is the highest velocity at which the fish swam for the entire 10 or 5 min interval; Vi is the velocity increment (5 cm s−1); Tf is the duration of time the fish swam at the highest velocity attempted; and Ti is the time increment  of that fish, and expressed as body lengths per second (BL  s−1). The small fish size relative to tunnel cross-sectional area (≤5%) precluded the requirement for solid blocking effect adjustments  with days post-hatch and in determination of the allometric exponents described in the ‘Data analysis and statistics’ section below.Manufacturer calibration of the 1.5 l swimming flume was verified using the dye technique recommended by Loligoustments . Values −1) and the wet mass (in grams), girth  and TL measured.Following completion of swimming tests, all fish were euthanized by buffered anaesthetic overdose  with days post-hatch and Ucrit (in centimetres per second) with TL.Wet mass, TL and Ucrit (in body lengths per second) and absolute Ucrit (in centimetres per second) in green and white sturgeon, we independently combined the larval green and white sturgeon Ucrit values measured in this study with other Ucrit data  and published data mined from the literature for white and green sturgeon of larger sizes  and TL was fitted to a power function, and the allometric exponents for green and white sturgeon were independently determined as the slope of the linear regression of the log of TL vs. the log of Ucrit (in body lengths per second). Although not included in calculations of allometric exponents, published Ucrit data for seven other sturgeon species from nine studies were plotted for comparison with green and white sturgeon data  and white sturgeon (2012 spawned), and 79 and 78%, respectively, for green and white sturgeon spawned in 2013.Ucrit (in centimetres per second) did not differ between larval green and white sturgeon  with days post-hatch did not differ significantly between green and white sturgeon , green sturgeon Ucrit (in centimetres per second) was consistently greater than that of white sturgeon at the same age  with TL also did not differ significantly between green and white sturgeon  and days post-hatch, the intercept of the relationship between Ucrit (in centimetres per second) and TL for green sturgeon was also significantly greater than that for white sturgeon  being consistently greater than that of white sturgeon at the same TL  vs. TL in juvenile green and white sturgeon decreased with increasing TL, and the relationship took the form of a power function  increased with TL for both species  with growth, was greater for green (−0.83) than white sturgeon (−0.42). In sturgeons <5 cm long, green sturgeon relative Ucrit (in body lengths per second) was twice that of white sturgeon. However, due to the more rapid decrease in green sturgeon relative Ucrit (in body lengths per second) with increasing length, white sturgeon relative Ucrit (in body lengths per second) began to exceed that of green sturgeon in fish that were between 12 and 20 cm TL. When relative Ucrit values (in body lengths per second) of other sturgeon species were plotted with the green and white sturgeon allometric curves, both larval green and larval white sturgeon <20 cm in length appeared to have greater Ucrit (in body lengths per second) than those of other sturgeon species of the same size  were of similar masses  and lengths  to previously reported late-larval green sturgeon masses  and lengths  increases with increasing temperature up to 15°C . We experit Figs , temperare limit .Ucrit measured in both larval green and larval white sturgeon increased with size, as expected  generally increases with increasing body size in fishes , and absted Figs B and 3B.Leuciscus leuciscus), rainbow trout (Oncorynchus mykiss) and goldfish (Carassius auratus), respectively  tends to decrease with size according to a power relationship in fishes . The expectively . For pro. In combination with the behavioural tendency of sturgeons to station hold at high water velocities , these aspects of sturgeon morphology have the potential to alter the allometric exponent for the length–swimming capacity relationship for sturgeons compared with other actinopterygians.The body and tail shape of sturgeons differs from that of the typical, faster swimming actinopterygians and more closely resembles that of chondrichthyans. Differing from the more derived actinopterygian homocercal caudal fin, the sturgeon caudal fin is heterocercal, which, for lake sturgeon, has been shown to generate 66% less thrust than for the homocercal caudal fin of trout . FurtherUcrit and length of larval sturgeons from this study and juvenile sturgeons from previous studies, differed from that of other actinopterygians. The allometric exponent for all known Ucrit (in body lengths per second) data for larval to 80-cm-long green and white sturgeon shows the allometric relationship between Ucrit (in body lengths per second) and length of white sturgeon to have an exponent of −0.42, which is more like that of sockeye salmon  began to exceed that of green sturgeon. This transition appears to be due to a shallower slope of increase in Ucrit with length of green compared with white sturgeon during the early juvenile stage. The timing of this transition is consistent with previously reported evidence of a seasonal reduction in swimming capacity of green sturgeon undergoing physiological changes associated with preparation for downstream migration to estuarine or marine waters  and the onset of green sturgeon tolerance of full-strength sea water  of Chinese sturgeon (Acipenser sinensis), pallid sturgeon , shovelnose sturgeon (Scaphirhynchus platorynchus), Amur sturgeon (Acipenser schrenckii) and shortnose sturgeon (Acipenser brevirostrum) with lengths of ∼20 cm or less lie below the green and white sturgeon allometric curves , which suggests that their foraging phase may not require a high swimming capacity. Shortnose sturgeon are amphidromous . Once dispersed to foraging sites, larval green sturgeon appear to forage nocturnally on the river bottom and hide in the rocks during the day until ∼100 dph or the autumn, when they begin another active downstream migration . At this age, green sturgeon have demonstrated tolerance of and preference for near full-strength seawater  and are therefore likely to migrate to the S-SJ Delta or San Pablo and San Francisco bays. The physiological preparation process for this migration from fresh to saltwater has been associated with reductions in Ucrit of juvenile (0.5- to 1.5-year-old) green sturgeon .The threatened Southern Distinct Population Segment of green sturgeon spawn primarily in the upper reaches of the Sacramento River from April to May , green sturgeon Ucrit predicted from the linear regression increased 1.9-fold to 54 cm s−1  and Verona, California .Ucrit values ranged from 22 to 45 cm s−1, and the linear regression equation predicted a Ucrit of 29 cm s−1 , their swimming capacity, and therefore their ability to escape diversion structure flows, increased slightly to 41–48 cm s−1, with the linear regression predicting a Ucrit of 45 cm s−1  white sturgeon , fish of this size are likely to be >1 year old. Through the winter months, neither size nor swimming capacity is expected to increase, so it is unlikely that white sturgeon spending their first winter in the Sacramento River are able to hold position in water flows >50 cm s−1.Due to their high foraging activity, 30 dph larval white sturgeon, which are located throughout the lower reaches of the Sacramento River from March to June, are likely to be vulnerable to entrainment into water-diversion structures. At 35 dph (∼5.5 cm TL), white sturgeon s−1 Fig.  and, theWe developed season-specific recommendations for flow limitations around water-diversion structures of the Sacramento River and its Delta by integrating laboratory-based findings on the ontogeny of green and white sturgeon with swimming capacity and behavioural information Table .Table 3−1. This period can be extended to April for the upper reaches, where green sturgeon tend to spawn. From February to June, populations of highly susceptible larval sturgeons with limited to no swimming ability are present within the river system. This is of particular concern in the middle to lower reaches from February until April, when young white sturgeon are expected to be drifting passively downstream from spawning sites. In the upper reaches, susceptible larval green sturgeon with limited swimming capabilities are expected to be present in April and May. By the end of May, green sturgeon larvae in the upper reaches are expected to have developed sufficient swimming ability to resist flows up to 29 cm s−1, but white sturgeon in the middle to lower reaches are not expected to develop similar swimming capacities until June. By July, although green sturgeon present throughout the upper to lower reaches are likely to be able to resist water flows >50 cm s−1, white sturgeon in the lower reaches remain limited to flows of 45 cm s−1 until September. In October, as juvenile green sturgeon develop saltwater tolerance and begin their downstream migration to the estuaries and bays, their swimming capacity drops, requiring a 2 month (October and November) 40 cm s−1 limit of flows around water-diversion structures in the middle and lower Sacramento River and the Delta and bays.Throughout the entire Sacramento River system from December to February, sturgeons present are unlikely to be overwhelmed by flows below 50 cm sUcrit (in centimetres per second) of early juvenile green and white sturgeon compared with other sturgeon species suggests that acipenserids in general require lower velocities than green and white sturgeon around diversion structures, if they are to resist entrainment volitionally. However, it is also important to consider the behavioural responses of the different sturgeons and their life stages to water-diversion structures. The complete lack of published larval Ucrit data for any acipenserid species before this study underscores the urgency for studies on this vulnerable life stage of this globally imperiled family.Similar conservation recommendations can be applied to other acipenserid species, with adjustments for dispersal behaviour, river site usage and behavioural responses to flows and diversion structures. The slightly greater absolute


IP) novelty of the pathway is important. Exploratory studies can be carried using knowledge of the patent/IP landscape for synthetic biology and metabolic engineering. In this paper, we perform an assessment of pathways as potential targets for chemical production across the full catalogue of reachable chemicals in the extended metabolic space of chassis organisms, as computed by the retrosynthesis‐based algorithm RetroPath. Our database for reactions processed by sequences in heterologous pathways was screened against the PatSeq database, a comprehensive collection of more than 150M sequences present in patent grants and applications. We also examine related patent families using Derwent Innovations. This large‐scale computational study provides useful insights into the IP landscape of synthetic biology for fine and specialty chemicals production.A goal of synthetic biology bio‐foundries is to innovate through an iterative design/build/test/learn pipeline. In assessing the value of new chemical production routes, the intellectual property ( The synet al., Escherichia coli, based on our current knowledge of enzyme sequences present in biochemical routes, with the set of sequences disclosed in patents since 1970 . Performing patent analyses in emerging technologies such as synthetic biology is challenging, as specific classifications are lacking or at best lagging in the international patent classification system. Prior studies  through patenting offers important signals as to targets that might subsequently have exploitable value in biotechnology and synthetic biology  from As anticipated, biosynthetic pathways involving low number of enzymes have higher incidence in patent documents than in the full catalogue of pathways amenable to metabolic engineering, as shown in Fig. et al., et al., et al., Enumerating all pathways that can produce a target compound is not trivial, as it requires specialized algorithms. Several techniques exist based on graph theory, elementary flux modes, minimal cut sets, as well as others  Flavonoids in patent documents also had a higher market value than their average market value, which was the opposite in the case of alkaloids. These results are helpful in informing consideration of the pricing implications of patented synthetic biology compounds compared with chemical and natural production. It should be stressed that this study was focused on compounds for which biosynthetic pathways are known and their enzyme sequences reported in databases. This means that the market values in Table Our screening for biosynthetic pathways in patent documents allowed us to gain insights into the landscape of chemicals of interest. We looked at the percentage of chemical classes whose biosynthetic pathways were identified in the sequence in pathways. We focused on some of the classes of higher interest. Table We also probed the chemical space covered by pathways found in patents in comparison with the area covered by the full reachable chemical space. The main question was to explore if there were privileged regions in the reachable space that were enriched in compounds present in patents, while others had less coverage and therefore could potentially constitute target compound classes in terms of innovation. Figure We further analysed the organizations and the targets involved in the patenting of synthetic biology for fine chemical production by linking our chemical pathways database to the Derwent Innovation Index Database. This analysis focused on patent families each of which can contain multiple patent documents (see Data S1 for more details). We find modest levels of patenting of fine chemical production pathways in the 1990s, with some tail‐off in the early 2000s. From the late 2000s, however, there was a rapid growth in the number of patent applications identified in Derwent, peaking at around 500 patents per year in recent periods Fig. S4). Based on Derwent patent classes, the vast majority of patents (more than 90%) are classed in fermentation sectors, while natural products and polymers in pharmaceuticals (around 52%), biotechnology, plant genetics, veterinary vaccines (22%) and scientific instrumentation (around 14%) are the other leading classes (Table . Based oThe leading patent assignees in our dataset comprise large commercial conglomerates active in a range of industries. No single organizations held more than 5% of our patent dataset. There is an evident specialization in terms of application areas by Derwent classes. While almost all leading firms have patents in fermentation sectors, companies with strong chemical industry capabilities such as Du Pont, BASF, Bayer as well as firms operating mainly in agri‐food such as Ajinomoto and Monsanto patented in the general category of “biotechnology, plant genetics, and veterinary vaccines.” On the other hand, a number of leading firms specialized in other applications areas; for instance, Butamax focused on fuels, Goodyear patented on monomers and condensants while natural products and polymers were targeted by Mitsui and Mitsubishi Table . UniversThese results present a novel way to develop new perspectives on the patenting landscape of synthetic biology, in this case focused on biosynthetic pathways for fine and speciality chemicals. The approach provides valuable information that can help in the design process, as it can serve to select targets of interest as well as to define criteria for enzyme selection in designated pathways in chassis organisms. Ultimately, these data may serve to develop machine‐learning approaches to predict best target compounds and enzymes according to innovation criteria. There are synthetic biology applications that have not been fully covered in this patent study, as long as they are not directly related to biosynthetic pathways. Our approach can be extended to the screening of other synthetic biology‐relevant sequences such as those deposited at public registries of biological parts, extending in that way the study to other genetic parts of synthetic biology devices and systems, such as regulatory and sensing components.None declared.Fig. S1. Number of different pathways per patent document.Fig. S2. Number of different target compounds per patent document.Fig. S3. Number of alternative pathways per compound.Fig. S4. Number of patents by application year.Data S1. Experimental procedures.Click here for additional data file.