Fibrin fibers form the structural backbone of blood clots; fibrinolysis is the process in which plasmin digests fibrin fibers, effectively regulating the size and duration of a clot. To understand blood clot dissolution, the influence of clot structure and fiber properties must be separated from the effects of enzyme kinetics and perfusion rates into clots. Using an inverted optical microscope and fluorescently-labeled fibers suspended between micropatterned ridges, we have directly measured the lysis of individual fibrin fibers. We found that during lysis 64 ± 6% of fibers were transected at one point, but 29 ± 3% of fibers increase in length rather than dissolving or being transected. Thrombin and plasmin dose-response experiments showed that the elongation behavior was independent of plasmin concentration, but was instead dependent on the concentration of thrombin used during fiber polymerization, which correlated inversely with fiber diameter. Thinner fibers were more likely to lyse, while fibers greater than 200 ± 30 nm in diameter were more likely to elongate. Because lysis rates were greatly reduced in elongated fibers, we hypothesize that plasmin activity depends on fiber strain. Using polymer physics- and continuum mechanics-based mathematical models, we show that fibers polymerize in a strained state and that thicker fibers lose their prestrain more rapidly than thinner fibers during lysis, which may explain why thick fibers elongate and thin fibers lyse. These results highlight how subtle differences in the diameter and prestrain of fibers could lead to dramatically different lytic susceptibilities. During ng sites –14. Thesng sites .Understanding clot lysis requires knowledge of enzyme activation kinetics, perfusion rates of enzymes into thrombi, and the effects of structural and physical properties of individual fibrin fibers on the rates of dissolution. The kinetic properties of fibrinolysis depend on the architecture and physical properties of both blood clots  and individual fibrin fibers  –21. ThroHere, we report the progression of fibrinolysis on single fibrin fibers using fluorescent optical microscopy and scanning electron microscopy (SEM). We found that fibers were transected at one point, but the ability of plasmin to cleave fibers depended upon the inherent fiber strain and initial diameter. In particular, we found that when fibers were exposed to plasmin, thin fibers were readily cleaved (lysed), but thicker fibers actually grew in length during fibrinolysis. These results suggest that the lytic susceptibility of a fiber is directly related to the intrinsic strain on the fiber resulting from the polymerization process (its “prestrain”). Based upon the current understanding of fiber architecture and polymerization, we developed continuum mechanics and polymer physics models to determine the relationship between fiber strain and fibrinolysis. We found that as the outer shell of the fiber is lysed, the equilibrium length of the fiber increased, resulting in elongation and loss of fiber prestrain. Thicker fibers elongated at a lower percentage of fibrinolysis than thinner fibers. Our findings suggest fiber prestrain is a prerequisite for lysis: thin fibers are lysed rapidly until prestrain is lost due to fiber transection, whereas thick fibers elongate, reducing prestrain and consequently lytic rates. These data emphasize the importance of the physical properties of individual fibers in determining the fate of blood clots.2 HBS buffer to 2X the concentration needed for that sample. Plasmin was then added to the samples at an equal volume to that present, and gentle pipetting was used to thoroughly mix the plasmin with the HBS on the sample. All experiments were performed over a 30-min or 24-hr period, as indicated.Structured Surfaces (SSs) were prepared as described previously  using a For samples imaged with fluorescent microscopy, after polymerization but prior to the addition of plasmin, the polymerized fibers were fluorescently labeled by gently exchanging the buffer on the SS with a 1/10,000 dilution of 20 nm thick volume labeled carboxylic acid labeled beads  in HBS for two minutes, and then the bead suspension was gently removed and replaced with 4 mM calcium HBS. For light microscopy. samples were placed face-up on a Nikon Diaphot 200 microscope with a 100X 1.3 N.A oil objective with epi-fluorescence illumination ), and appropriate filters, and images recorded with a Cooke PCO 1600 camera and CamWare capture software .The fibrin samples used for SEM imaging were suspended over channels of PDMS and prepared in the same manner, at the same concentrations, and with the batches of protein as the samples used in fluorescence microscopy. Samples were fixed by exchanging the sample buffer with 0.1% glutaraldehyde in HBS and incubated for five minutes. Samples were washed gently with HBS, and then the buffer was exchanged with a graded series of ethanol, critical point-dried sputter coated with 6 nm of gold-palladium, and imaged with a Hitachi S4700 SEM. Single fibrin fibers with no visible branching were selected for measurement, and the thinnest diameter along the length of each of these fibers was recorded. Fibers greater than 1000 nm in diameter were not counted. These were very rare, and appeared to be bundles of smaller fibers.To study individual fiber behavior during fibrinolysis and the timescales within which plasmin can lyse individual fibers, we polymerized and suspended fibrin across micropatterned ridge-and-valley structures, as previously described . Fibrin Fibrin fibers displayed two distinct conformational changes after being treated by plasmin: complete lysis (in which fibers were cleaved transversely at a single point) or elongation   Movies. Surprisingly, at least 29 ± 3% of fibers remained intact and suspended across the ridges of the structured surfaces; these fibers exhibited an increase in length from their original length . As thesWe performed control experiments to determine whether elongated fibers were in a transient, partially lysed state. First, we added 3.3 U/mL plasmin and allowed elongation to occur for one hour. We then added an additional high dose of plasmin (6.0 U/mL) while observing the already elongated fibers; in these experiments, we did not observe transverse cleavage or additional extension of the fibers, suggesting that elongation is not a result of low plasmin activity, but rater, elongated fibers are in a state where the lytic susceptibility of the fiber is greatly reduced. Second, we tested the hypothesis that fibrinolytic rates are reduced in elongated fibers, but eventually the fibers will lyse. For this experiment, we observed several samples after 24 hours of exposure to 3.3 U/mL plasmin; fiber fragments of lysed fibers were still present and elongated fibers remained in the same state without transverse cleavage or additional lengthening. These results indicate that fibrin fibers can either be cleaved at one point , or elongate. The elongated state of the fiber is not a transitional state on the pathway to lysis, but a fundamentally different state, without inherent strain, with a greatly reduced lytic susceptibility.To determine the effect of plasmin concentration on fiber lysis time, individual fibrin fibers were polymerized on structured surfaces using a fixed thrombin concentration (1.1 U/mL) and then exposed to 0.06, 0.33, 0.6, 1.0, 3.3, 4.5, or 6.0 U/mL plasmin. Fibers were imaged for thirty minutes, and the lysis time (the time required for a fiber to fully lyse after exposure to plasmin) was recorded and plotted . Fibrin For fibers that lysed, the average time measured for single fiber lysis at various plasmin concentrations is displayed in Previous work has indicated that fibrin diameter is inversely proportional to thrombin concentration ,23,28–30Next, samples of fibrin fibers were prepared on structured surfaces for fluorescent imaging using 0.11, 1.1, 11, and 33 U/mL thrombin. Samples were then treated with 3.3 U/mL of plasmin  is less than the inter-ridge (structured surface) distance, SSL, while after lysis, the final equilibrium length of the fiber, FL, is longer than the inter-ridge distance.The observation that fibrinolytic activity was reduced both after fibers are initially cleaved and in elongated fibers that were never fully cleaved suggests the ability of plasmin to cleave fibrin is directly related to the mechanical state of the fiber. Although our prior work and other investigations show that fibers are under tension after the polymerization process ,22,31, t*ε (ε = (SS-LiL)/iL ). If this prestrain is lost before the fiber completely lyses or after the initial cleavage event, then lytic activity effectively ceases. We developed two separate mathematical models based upon the structure of the fibrin fiber to test these hypotheses. Our models provide an explanation for how polymerization produces a prestrain in the fiber that is then lost during the fibrinolytic process, which in turn, leads to fiber elongation . Our data suggest that the ability of plasmin to effectively bind and engage enzymatic activity requires a certain threshold fiber strain, Q is the swelling ratio or the ratio of the network volume (polymer and solvent) relative to the dry polymer volume, and cñ is the density of crosslinks . ThiE: domain A  and domain B   . In a taectively . In thisectively . Two othectively . Togethe*ε, is necessary for lysis). Third, fiber diameter determined fiber fate; thicker fibers were more likely to elongate, whereas thinner fibers were more likely to lyse, suggesting larger fibers fall below the threshold strain when still partially lysed, whereas thinner fibers maintain strain above ε and facilitate lysis. Together, these novel findings reveal that clot lysis is regulated not only by global mechanisms , but also by biomechanical functions on the level of individual fibers.Understanding fibrinolytic mechanisms requires a detailed understanding of lysis on both the scale of whole clots and that of individual fibers. Most previous studies have evaluated whole clot lysis, while knowledge of lytic mechanisms on the scale of individual fibers is limited. Studying the lysis of isolated fibers is complementary to previous confocal and electron microscopy studies of fiber lysis within networks ,17,20 an*ε. Both the polymer network model and the two-state elastic solid model predict that the fiber will begin in a tensed, prestrained state that will relax and expand during lysis. The continuum mechanics model illustrates how the lysis of the outer protofibrils of a fiber can lead to elongation, and shows how thicker fibers lose prestrain much more rapidly than thinner ones, but does not predict a mechanism by which the unstrained core can be lysed. The polymer physics model provides a mechanism for elongation and the timescales of lysis, but does not directly account for the diameter dependence of elongation. Future modeling could combine the continuum mechanics approach with the polymeric properties of fibrin to capture the full strain and diameter dependence of lysis. This combined model should include plasmin kinetics and realistic molecular binding sites and accurate molecular packing geometries within the fiber.Our mathematical modeling provides a theoretical framework for understanding fiber prestrain and elongation, and further experimental and modeling work should attempt to determine the exact value of Our models for fiber elongation correspond with known data regarding the molecular binding sites of plasmin and tPA on fibrin, and their exposure during polymerization. Previous studies have revealed two pairs of binding sites for tPA and plasm(ogen): one pair resides in the D region with a tPA site at γ312–324 and a plasminogen site at Aα148–160, and the second pair resides on the αC region . All are*ε in order to be laterally transected. Fiber strain can be affected by cross-linking of the αC domains and γ-chains by FXIII [One potential molecular mechanism consistent with our data as well as prior studies on tPA and plasminogen binding sites, is stretching of the αC regions. Global fiber prestrain in the as-polymerized fiber implies local molecular scale strain, which we propose is accommodated by the stretching of the αC regions of the molecule. The origins of the prestrain may be related to the formation of the αC-αC network that forms through their association during polymerization. The Flory-Rehner equation predicts an equilibrium volume reduction when crosslink density increases (in this case αC-αC associations) . Since tby FXIII ; some stby FXIII ,41. The The details of how plasmin lyses fibrin fibers have previously been debated. Our observation that fibers are cleaved transversely agrees with previous studies of fibrin network digestion ,17; thes*ε required for lysis to occur. Additionally, fibers that are cleaved have been observed to aggregate, forming thicker fibers, which may increase strain and enhance lytic rates [*ε, allowing fiber lysis rather than elongation to occur. Future work should directly measure the dependence of lysis/elongation rates on fiber strain to distinguish strain from the effects of enzyme perfusion and activation rates.Our data showing that fiber elongation reduces fiber lysis, must be reconciled with the fact that blood clots and fibrin networks do, in-fact, lyse. The strain-dependence of lysis helps to reconcile these apparently contradictory results. Because of the interconnected fiber architecture of a clot, the transection of one fiber necessarily means that the stress within the network will re-equilibrate. Consequently, fibers within a lysing network are constantly subjected to dynamic stresses and strains as fibers around them lyse. The dynamic nature of lysis likely allows thicker fibers to maintain the ic rates . It is aic rates ,44. Otheic rates . We hypoPlasma clots from patients with bleeding diathesis, such as hemophilia, contain thicker fibers and are more susceptible to lysis, while clots formed from plasmas of patients with a history of thrombosis contain thinner fibers and exhibit reduced fibrinolysis . Thus, fWe have identified a relationship between a fibrin fiber’s susceptibility to lysis and its biomechanical state. Fibers either lyse transversely or elongate depending upon their initial diameter, with thicker fibers being more likely to elongate. Fibers that are transected at one location show a dramatic decrease in the rate of further degradation. Both of these observations indicate that lysis kinetics depend strongly on the inherent tension/strain in the fiber. We find that fibers must be stretched from their equilibrium length beyond a certain threshold strain, or lytic enzymatic activity is greatly reduced. Studying lysis at the individual fiber level provides a quantitative understanding of mechanisms by which fibers are digested and the enzymatic cleavage rate, which in turn, will improve models of clot lysis. Having a complete hierarchical model of fibrinolysis could enable the next generation of thrombolytic therapeutics.S1 FilePolymer physics and continuum mechanics models of fibrin fiber elongation.(DOCX)Click here for additional data file.S1 FigA A structural diagram for the fibrin fiber, consisting of protofibrils (black) with un-crosslinked αC regions (blue), suspended across the structured surfaces (1) and in free space (2). B As the unstructured αC regions become intercross linked (red), the network contracts, but the fiber constrained at both ends can only shrink in the diameter, not length-wise. This leads to the fiber being in a pre-strained state; the fiber is stretched beyond its equilibrium length. C The number of crosslinks is reduced during fibrinolysis in fibers with sufficient prestrain to undergo binding with fibrinolytic agents. The reduction in the number of crosslinks leads to an expansion of the network and the lysed αC regions form bundles.(TIF)Click here for additional data file.S2 FigSSL) and the black dot is the free length of the fiber after 100% lysis of the shell (i.e. the core length). Note that there is no radial dependence on the free length for fibers of the constant ratio model. For this plot oAL = 18 μm, S = 1.2. For the constant core, the core thickness was 15 nm; for the constant shell, the shell thickness was 35 nm; for the constant ratio, the radius of the core was half the radius of the whole fiber.The fiber free length, normalized to the length between the structured surfaces, for thin , medium , and thick  fibers as the shell is lysed. The black line signifies the SS length ((TIF)Click here for additional data file.S1 MovieThe video was sped-up 4x; the time-stamp represents real-time.(AVI)Click here for additional data file.S2 MovieThe video was sped-up 6x; the time-stamp represents real-time.(AVI)Click here for additional data file.S3 MovieThe video was sped-up 6x; the time-stamp represents real-time.(AVI)Click here for additional data file.


The Editorial on the Research TopicEngineering synthetic metabolons: from metabolic modelling to rational design of biosynthetic devicesSynthetic Biology, because the field of Synthetic Biology is multifaceted and rapidly moving. As viewed from its underlying principle, Synthetic Biology can be defined as the “application of engineering principles to biological systems.” It reaches from pathway engineering in living systems, i.e., the introduction of functional biochemical pathways into existing organisms , and the design of vesicle-based multicompartmented biochemical reactors and protocells  to the creation of entirely synthetic cells with reproductive potential that is encoded by synthetic genes [with the work of Gibson et al. (http://syntheticbiology.org covers these two extremes in that Synthetic Biology is defined as “(A) the design and construction of new biological parts, devices, and systems and (B) the re-design of existing, natural biological systems for useful purposes.” This also means that Synthetic Biology is not limited to living systems but also comprises the combination and re-design of biological parts into artificial bioreactors. In this respect, Synthetic Biology is highly interdisciplinary, bringing together molecular biology with biophysics, material sciences, bioengineering, and computational approaches.Synthetic Biology is an emerging discipline that enjoys rapidly increasing popularity. However, there is no widely accepted definition for the term n et al. , 2010 thBasler et al.), models are crucial for the rational design of synthetic systems . Metabolic modeling has the capacity to guide the biochemical layout of engineered pathways in vivo and the design of artificial bioreactors in vitro. For instance, computational modeling can play an important role for the selection of individual enzyme isoforms for engineered metabolic pathways. Vice versa, in vitro studies in artificial bioreactors has advanced our understanding of biochemically complex processes, such as starch biosynthesis, as summarized in the featured research topic review by O’Neill and Field. On the other hand, molecular dynamics simulations help to understand complex biochemical and biophysical processes such as enzymatic catalysis, molecular interactions of biomolecules , or membrane transport . The knowledge gained by these simulations can be exploited for the structural and molecular design of synthetic systems, i.e., for the choice of artificial protein or nucleic acid scaffolds for the spatial modular organization of artificial systems .Besides the invaluable role in helping biochemists understand the metabolic complexity of living systems and the extent to which observations from existing metabolic engineering strategies match predictions from large-scale modeling , to steady-state equations, testing the effect of metabolic engineering strategy in large-scale metabolic networks . A new metabolic engineering strategy for the glucosinolate pathway that increases dihomomethionine levels without increasing the levels of leucine-derived side products was experimentally validated in Nicotiana benthamiana . Sharing of modeling results and ensuring reproducibility of model-data integration requires setting and following a set of standards, which are currently established in the context of metabolomics research .This research topic focuses on the importance of computational modeling for the rational design of synthetic systems on all these levels. Consequently, a wide variety of approaches is being covered in this issue, from pathway engineering in eukaryotic cells to molecular dynamics simulations of transport processes. The modeling approaches range from partial differential equations, allowing predictions of spatiotemporal concentration of metabolites in a biochemical microreactor .LV and ZN have conceived and written the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycobacterium bovis, the main agent of bovine Tuberculosis (bTB). France became officially bTB-free in 2001, but M. bovis is still circulating in the cattle population, with about a hundred of outbreaks per year, most located in a few geographic areas. The aim of this study was to analyse the role of cattle movements in bTB spread in France between 2005 and 2014, using social network analysis and logistic regression models. At a global scale, the trade network was studied to assess the association between several centrality measures and bTB infection though a case-control analysis. The bTB infection status was associated with a higher in-degree (odds-ratio [OR] = 2.4 [1.1–5.4]) and with a higher ingoing contact chain (OR = 2.2 [1.0–4.7]). At a more local scale, a second case-control analysis was conducted to estimate the relative importance of cattle movements and spatial neighbourhood. Only direct purchase from infected herds was shown to be associated with bTB infection (OR = 2.9 [1.7–5.2]), spatial proximity to infected herds being the predominant risk factor, with decreasing ORs when distance increases. Indeed, the population attributable fraction was 12% [5%–18%] for cattle movements and 73% [68%–78%] for spatial neighbourhood. Based on these results, networks of potential effective contacts between herds were built and analysed for the three major spoligotypes reported in France. In these networks, the links representing cattle movements were associated with higher edge betweenness than those representing the spatial proximity between infected herds. They were often links connecting distinct communities and sometimes distinct geographical areas. Therefore, although their role was quantitatively lower than the one of spatial neighbourhood, cattle movements appear to have been essential in the French bTB dynamics between 2005 and 2014.Live animal movements are a major transmission route for the spread of infectious agents such as  Mycobacterium bovis. It can affect many hosts, in particular humans and cattle . Similarly, the upper quartile of the ingoing infection chain was significantly associated with bTB status (OR: 2.2 [1.0–4.7]) , and 0.85 [0.82–0.88] for the model including the ingoing contact chain, indicating a “good” fit quality for both models .The area under the ROC curve was 0.86 [0.83–0.89] for the model including the in-degree . Again, the two adjustment variables included in the model were significantly linked to bTB infection status: increasing ORs were observed with increasing herd sizes, and dairy herds were significantly less often infected than beef herds.The bTB infection status was significantly associated with the network neighbourhood of order 0 , as well as with the spatial neighbourhood of order 0 to 3 with decreasing ORs, from 4.2 to 1.7 (Table 3) was 0.86 [0.84–0.88], indicating a “good” fit quality . No spatial aggregation of the deviance residuals was observed in the corresponding map .The area under the ROC curve , and of 73% [68%–78%] for the spatial neighbourhood (order 0 to 3).The respective roles of network and spatial neighbourhood in bTB overall spread were analysed for the 3 spoligotypes that were identified in more than 50 herds reported as infected between 2005 and 2014: SB0120 (296 herds), SB021 (71 herds) and SB0134 (67 herds). For each of these spoligotypes, a network of potential effective contacts was built using the neighbourhood exposure variables found to be significantly associated with the bTB infection status in the previous step: network neighbourhood of order 0, and spatial neighbourhood of order 0 to 3.Table 4). As expected, in each of the three networks, most of the links were spatial neighbourhood links.The SB0120 network was composed of 32 components, two of which contained more than 10 nodes: a smaller component of 99 nodes , and a larger component of 157 nodes (53% of the nodes of the SB0120 network). The SB021 network was made of 6 components, one of which included most of the nodes (66 of 71 nodes). The SB0134 network contained 10 components, two of which contained >10 nodes  with nodes coloured according to the year of bTB notification (top left), according to the community (top right–only communities of more than 10 nodes are coloured) or according to the department (bottom). As expected (because of the time constraint of five years taken into account when defining the links between nodes), infected herds appear grouped by detection year in Figs Fig 3).The Table 5). In Figs Fig 1).In both large components of the SB0120 network and in the unique large component of the SB021 network, the edge betweenness was markedly and significantly higher in the network neighbourhood links than in spatial neighbourhood links (Table 4), this difference could not be assessed.Finally, the proportion of links connecting distinct communities was calculated for both types of links. For the two large components of the SB0120 network, the proportion of network neighbourhood links connecting distinct communities (85% of network neighbourhood links for component 1 and 100% for component 2) was significantly higher than for spatial neighbourhood links  . The same significant difference was observed for the largest component of the SB021 network with 78% of the network neighbourhood links connecting distinct communities, against 31% for the spatial neighbourhood links which were between-cluster links (p = 0.006). No such difference was observed for the component 1 of the SB0134 network (p = 0.164). As the component 2 of the SB0134 network did not include any network neighbourhood links  or a higher ingoing contact chain (OR = 2.2 [1.0–4.7]) and the bTB infection status of cattle herds. Indeed, both indicators are correlated and a higher in-degree (or a higher ingoing contact chain) induces an increased probability to purchase an infected animal .2 , this would mean an increased risk of bTB infection up to roughly 12km around an infected herd. It is consistent with the hypothesis of local transmission of bTB strains (distances < 14 km) in Spain put forward by de la Cruz et al. ). On the contrary, an association between bTB infection and the presence of bTB-infected herds in the spatial neighbourhood was observed up to 3 communes away . The ORs decreased from 4.2 [2.8–6.5] for the exposure to a bTB-infected herd located in the same commune as the studied herd, to 1.7 [1.1–2.6] for the exposure to a bTB-infected herd located 3 communes away. Since the mean area of a French commune is about 16 kmz et al. . However [et al. , which c [et al. . We also [et al. . However in 2014  for Grea in 2014 .Finally, we looked at networks of potential effective contacts between bTB-infected herds, for the three major spoligotypes reported in France . Cattle movements (i.e. network neighbourhood edges) were often links connecting distinct communities and sometimes distinct geographical areas. This is consistent with the hypothesis that cattle movements would allow the disease to spread into bTB-free areas, whereas local transmission (e.g. contacts on pastures or contacts with infected wildlife) would be involved in the within-area spread. Therefore, although cattle movements seemed to quantitatively have a weaker role than spatial neighbourhood, they were essential to the overall bTB dynamics in France.et al. [et al. [et al. [et al. [et al. [et al. [et al. [The role attributed to cattle movements in bTB spread varies according to the country (or area) and the infection level. For example, in Scotland, a low-incidence area, Gates et al.  showed t [et al.  conclude [et al.  showed i [et al.  explaine [et al.  estimate [et al.  establis [et al. ,32,33,35 [et al.  observedet al. [A 5-year period of time was taken into account to compute the network indicators. Indeed, bTB transmission is low and detection of the infection difficult: an infected animal can have been infectious for several years before being detected. This obliged us to consider a long period of time to compute networks indicators. On the other hand, other disease outbreaks occurred during this period in France, like the Bluetongue epidemic in 2007–2008. This could have bias our results. However, Dutta In France, when a herd is reported as bTB-infected, epidemiological investigations are implemented in order to investigate the bTB status of all the herds it has been in contact with, e.g. herds which purchased cattle from the infected one, herds which sold cattle to the infected one, or herds located or having pastures close to the infected one. Thus, it is more likely to report bTB infection in a herd that had a direct contact with an infected herd than in an isolated herd. This could therefore impact our results, artificially increasing the number of herds reported as bTB-infected herds in the neighbourhood of case herds (and not of control herds). However, as herds from both the network neighbourhood and spatial neighbourhood are investigated during these epidemiological investigations, it is unlikely that this biased the relative importance of cattle movements and local transmission estimated in our study.The spoligotype was the only molecular marker for which typing results were available for almost all herds reported as infected between 2005 and 2014. However, this marker is considered very stable, and has sometimes a limited discriminative power  . This maBased on the results of this study, it seems that control measures implemented in case of cattle movements could limit the spread of the disease from bTB-infected areas to bTB-free areas. As an example, Defra recently proposed to introduce in England statutory post-movement bTB testing of cattle moved from a bTB high-risk area to a bTB low-risk area . Howeveret al. [On the other side, as local transmission seems to play a strong role in the spread of bTB, control measures have also to focus on this type of transmission. Local transmission consists of at least three different effective contacts: contacts with other infected animals at pasture despite the fences, contacts with infected wildlife or environment and unreported local movements. In France, it is unknown which component of local transmission is the most important and which control measures are the most effective. Hence, more studies about each of local transmission, especially on neighbouring effective contacts at pasture, are needed. Nevertheless, as showed by Brooks-Pollock et al.  for Greaversus 73% for the one associated with the exposure to bTB infection in the spatial neighbourhood. However, when analysing, for several spoligotypes, the networks of potential effective contacts between bTB-infected herds, we observed that trade links were frequently the way to connect distinct communities of bTB-infected herds. Therefore, although their role was quantitatively lower than that of spatial neighbourhood, cattle movements appeared to be essential in the French bTB dynamics between 2005 and 2014.The aim of this study was to assess the role of cattle movements in the spread of bTB in France between 2005 and 2014. Three analyses were performed. At a national scale, we showed a statistical association between several centrality measures in the cattle trade network and the bTB infection status of cattle herds. At a more local scale, we estimated the relative importance of cattle movements and local transmission in bTB spread and showed that cattle movements had a lower role compared to spatial neighbourhood : the population attributable fraction associated with the exposure to bTB infection in the network neighbourhood was of 12% S1 Figth and the 95th percentiles.Red dotted line: coefficient for the real network; black dotted lines: the 5(TIFF)Click here for additional data file.S2 Figth and the 95th percentiles.Red dotted line: coefficient for the real network; black dotted lines: the 5(TIFF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S4 Fig(TIFF)Click here for additional data file.S5 Fig(TIFF)Click here for additional data file.S6 Figcommune: the smallest administrative French subdivision.(TIFF)Click here for additional data file.S7 FigGrey: spatial neighbourhood links; black: network neighbourhood links; nodes are coloured according to (A) the bTB notification year; (B) communities of more than 10 nodes within the component; (C) the department. Node locations are identical in A, B and C.(TIFF)Click here for additional data file.S8 FigGrey: spatial neighbourhood links; black: network neighbourhood links; nodes are coloured according to (A) the bTB notification year; (B) the community (only communities of more than 10 nodes within the component are taken into account); (C) the department. Node locations are identical in A, B and C.(TIFF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


The Wisconsin Card Sorting Test (WCST) and Tridimensional Personality Questionnaire (TPQ) were administered to assess neurocognitive functioning and personality traits, respectively. Compared to healthy controls, patients with alcohol dependence showed a significant reduction in dopamine transporter availability (p < 0.001), as well as diminished performance on the WCST (p < 0.001). Dopamine transporter availability was negatively correlated with both total and perseverative WCST errors among healthy controls, but only patients with alcohol dependence showed a positive correlation between dopamine transporter availability and a harm avoidance personality profile. Thus, reductions in dopamine transporter availability may play a pathophysiological role in the development of pure alcohol dependence, given its association with neurocognitive deficits. Moreover, personality may influence the development of pure alcohol dependence; however, additional clinical subgroups should be examined to confirm this possibility.Dopamine plays an important role in the development of alcohol dependence, cognitive dysfunction, and is regulated via dopamine transporter activity. Although dopamine transporter activity is critically involved in alcohol dependence, studies observing this relationship are limited. Thus the current study examined whether dopamine transporter availability is associated with developing of alcohol dependence and cognitive dysfunction. Brain imaging with  Alcohol dependence (AD) is a heterogeneous mental disorder that is confounded by several factors . StudiesDAT has been suggested as the molecular site of action for reinforcing mechanisms related to drug addiction . AlcoholPrevious research has shown reduced prefrontal cortical dopamine transmission among patients with AD ; these aDopamine is implicated in the regulation of specific personality traits , 17, andThis study enrolled male participants only because evidence suggests that gender is a significant confounding factor in research on striatal DAT availability . The Ins99mTc-TRODAT-1 for use in the imaging protocol has previously been described in detail FP-CIT have been reported to compete with endogenous dopamine in an animal study  β–CIT SPECT to subjects with alcohol dependence found a reduction in DAT availability during withdrawal followed by a significant increase with continued abstinence. This reversible neurotoxicity was also found in a previous animal study in which alcohol-preferring monkeys showed substantial improvement in alcohol-associated neurotoxicity and striatal DAT function after a period of abstinence  β -CIT SPECT and selected an antisocial-subtype AD group from a European sample. Although these results support the idea that dopamine may play a role in regulating neuronal activity based on different personality types during AD development, specific methodological differences  likely influenced divergent results. Therefore, the relationship between specific personality types and DAT availability should be further investigated using homogenous AD samples and subgroups as well as larger sample sizes.The dopaminergic system and specific personality traits have been implicated in the development of AD , 16, 47;e et al.  used [12The present study had some notable limitations. First, a cross-sectional design was used, AD is a complex disorder, and enrolled patients with pure AD could develop other mental illnesses later in life. This might be why our findings are perhaps discordant with other similar study. Second, our subjects with AD had taken lorazepam to prevent withdrawal symptoms before SPECT imaging, and there is evidence that benzodiazepines may interfere with levels of extracellular dopamine . Third, The present findings suggest that drinking behavior may influence the stabilization of brain dopamine levels and reduced striatal DAT availability. Low DAT availability may imply low dopamine levels in the brain, which would further influence neurocognitive deficits among patients with pure AD. Therefore, a reduction in DAT availability may play a pathophysiological role in the development of AD, which is also associated with neurocognitive deficits. Moreover, patients with pure AD who are high in trait–HA may show a positive relationship between DAT availability and the development of pure AD; however, different clinical subgroups and large-scale studies are needed to confirm this possibility.S1 Table(DOCX)Click here for additional data file.


We present a case of a 26 year with history of HIV/AIDS who presented with a pleural effusion. Serial radiography, pleural fluid analysis as well as clinical symptoms revealed development of Kaposi Sarcoma related immune reconstitution inflammatory syndrome (KS-IRIS) in the setting of initiation of effective anti- retroviral therapy. R.S. is a 26 year-old with HIV/AIDS, diagnosed in 2008, with intermittent compliance with antiretroviral therapy (Darunavir/ Truvada/Ritonavir), who presented to our hospital in 2014 with complaints of headache and vision changes, and was diagnosed with syphilis. During the admission, violaceous papules were noted on his skin, and a biopsy revealed histologic findings characteristic of Kaposi Sarcoma (KS), including proliferation of spindle cells, prominent, slit-like vascular spaces, and extravasated red blood cells. His viral genotype showed mutations in the reverse transcriptase  and protease (A71T) genes, indicating resistance to lamuvidine, emtricitabine, abacavir, efavirenz and nevirapine. His regimen was switched to Dolutegravir, Ritonavir, Darunavir and Rilpivirine. He had no pulmonary complaints at that time.One month later, he was admitted with an upper extremity deep venous thrombosis and found to have compression of his right axillary vein from lymphadenopathy. He continued to have no pulmonary symptoms, but a right pleural effusion was noted on imaging. A thoracentesis showed lymphocyte predominant  with pleural/serum albumin ratio of 0.64. Microbiologic cultures were negative; flow cytometric and cytologic analyses were not done. One month after this admission, he presented with complaints of dyspnea and was found to have an enlarging right-sided pleural effusion. Of note, he reported strict medical adherence to his new ARV  regimen, reflected by a rapid decline in viral load from 72,000 virions/mL to undetectable. Examination was notable for palpable cervical adenopathy and diminished breath sounds on the right side, dullness to percussion and 1–3 cm long raised violaceous nodule on the inner aspect of left upper extremity. Thoracentesis yielded 1400 cc of serosanguinous fluid that was exudative  and lymphocyte- predominant . Microbiological, fungal and mycobacterial cultures were all negative. Cytology showed reactive mesothelial cells and flow cytometry was negative for malignancy. Following drainage, the pleural effusion rapidly re- accumulated and a thoracoscopy was performed, which showed characteristic KS lesions in the visceral pleura . Talc plOn follow-up, no recurrence of the pleural effusion was noted.Kaposi Sarcoma Herpes Virus  is a double-stranded DNA virus that is associated with several clinical entities in both immunodeficient and immunocompetent patients: Castleman’s disease, Primary Effusion Lymphoma and, as in our case, Kaposi’s Sarcoma, depending on the cell type infected by KS virus. KS often presents with dermatologic manifestations but can metastasize to involve the lungs, liver and lymph nodes. Pleural and/or pulmonary manifestations occur in about half of KS patients . PulmonaTat) promote KSHV survival and tumorigenesis within infected cells, leading to continued infection and potential malignant transformation [IRIS is a complication to initiation of ART  that is fairly uncommon in western countries . It is cormation ,9. Additormation . Our patormation . Serial ormation ). Prior ormation  have repormation . Howeverormation . Indeed,In summary, we present a case of pleuropulmonary KS with clinical deterioration following ART initiation consistent with KS-IRIS. The diagnosis in the case was made via thoracoscopy, but further insight into the pathophysiology of the natural history of KS and IRIS was gained through serial analysis of pleural fluid.


In a separate sample of healthy persons, we examined whether dopamine depletion could alter insulin sensitivity.Food addiction is a debated topic in neuroscience. Evidence suggests diabetes is related to reduced basal dopamine levels in the nucleus accumbens, similar to persons with drug addiction. It is unknown whether insulin sensitivity is related to endogenous dopamine levels in the ventral striatum of humans. We examined this using the agonist dopamine D11C]-(+)-PHNO scan, 9 of which provided a scan under dopamine depletion, allowing estimates of endogenous dopamine at dopamine D2/3 receptor. Dopamine depletion was achieved via alpha-methyl-para-tyrosine . In 25 healthy persons , fasting plasma and glucose was acquired before and after dopamine depletion.Insulin sensitivity was estimated for each subject from fasting plasma glucose and insulin using the Homeostasis Model Assessment II. Eleven healthy nonobese and nondiabetic persons  provided a baseline [2/3 receptor was positively correlated with insulin sensitivity (r(7)=.84, P=.005) and negatively correlated with insulin levels (r(7)=-.85, P=.004). Glucose levels were not correlated with endogenous dopamine at ventral striatum dopamine D2/3 receptor (r(7)=-.49, P=.18). Consistently, acute dopamine depletion in healthy persons significantly decreased insulin sensitivity (t(24)=2.82, P=.01), increased insulin levels (t(24)=-2.62, P=.01), and did not change glucose levels (t(24)=-0.93, P=.36).Endogenous dopamine at ventral striatum dopamine D2/3 receptor in the ventral striatum. Moreover, acute dopamine depletion reduces insulin sensitivity. These findings may have important implications for neuropsychiatric populations with metabolic abnormalities.In healthy individuals, diminished insulin sensitivity is related to less endogenous dopamine at dopamine D Several2/3R availability and levels of fasting neuroendocrine hormones  between a baseline PET scan and a scan under acute dopamine depletion -PHNO, the first agonist PET radiotracer for D2/3R (11C]-(+)-PHNO to estimate endogenous dopamine levels at D2/3R using an AMPT challenge (11C]-raclopride (2/3R in humans. Using [11C]-(+)-PHNO body mass index (BMI) within a nonobese range was found to be positively correlated with BPND in the VS but not the dorsal striatum -PHNO.Our group has developed [or D2/3R . The usehallenge . Collectclopride  and thusstriatum . One pot11C]-(+)-PHNO and an acute dopamine depletion paradigm, we sought to examine for the first time whether estimates of endogenous dopamine at D2/3R in the VS of healthy, nonobese humans are related to IS. We hypothesized that persons with reduced IS would have less endogenous dopamine occupying D2/3R in the VS at baseline. Healthy participants were evaluated to provide: 1) a proof of concept for the relationship between IS and brain dopamine without the presence of confounding changes that may occur in disease states; and 2) a benchmark for future comparisons in clinical populations. We also sought to determine whether reducing endogenous dopamine with AMPT could lead to changes in IS in healthy individuals. Clarifying the relationship between IS and dopamine levels in the brains of humans in vivo would represent an important first step in understanding the interplay between metabolic health, energy homeostasis, and brain reward circuits in health and disease , 5:00 pm (post 8 hours), and 9:00 pm (post 12 hours) on day 1, and 6:00 am (post 21 hours) and 10:00 am (post 25 hours) on day 2. The post AMPT PET scan was scheduled at 12 pm, 28 hours after the initial metyrosine dose. Subjects were under direct observation during AMPT administration and slept overnight in hospital-designated research beds to facilitate the AMPT dosing schedule and monitor for potential side effects. In addition, subjects were instructed to drink at least 4L of fluids during the 2-day admission to prevent formation of AMPT crystals in urine, and fluid intake was monitored to ensure compliance. In addition, to alkalinize the urine, which increases AMPT solubility, sodium bicarbonate (1.25g) was given orally at 10:00 pm on the evening before day 1 and at 7:00 am on day 1 of administration.The procedure for AMPT-induced dopamine depletion has been published elsewhere . Brieflyam. For the participants who provided PET scans (n=11), fasting blood work was collected on the day of the baseline PET scan. Twenty-five healthy participants  provided fasting blood work (9:00 am) at baseline and after receiving 5 doses of AMPT. For 13 of these subjects, it was possible to collect the blood work 24 hours apart. For the remainder of subjects, 4 provided blood work 6 to 7 days apart, 4 provided 10 to 14 days apart, and 2 provided 36 to 43 days apart. Blood for glucose measurement was collected in a 4-mL grey stoppered tube containing sodium fluoride as a preservative and potassium oxalate as an anticoagulant. Plasma was assayed for glucose on the EXL 200 Analyzer (Siemens) using an adaptation of the hexokinase-glucose-6-phosphate dehydrogenase method. Blood for insulin measurement was collected in a 6-mL red stoppered tube with no additives. Serum was analyzed on an Access 2 Analyzer (Beckman Coulter) using a paramagnetic particle, chemiluminescent immunoassay for the quantitative determination of insulin levels in human serum. The IS index for glucose disposal was estimated for each subject from fasting plasma glucose and insulin using the Homeostasis Model Assessment II (HOMA2), calculated with the University of Oxford HOMA2 calculator   . Estimatulator/) .11C]-(+)-PHNO PET scans, one under baseline conditions and another at 25 hours following AMPT-induced dopamine depletion. The radiosynthesis of [11C]-(+)-PHNO and the acquisition of PET images have been described in detail elsewhere  single-photon point source to provide attenuation correction, and the emission data were acquired in list mode. The raw data were reconstructed by filtered-back projection. For baseline [11C]-(+)-PHNO scans (n=11), the mean radioactivity dose was 9 (±1.5) mCi, with a specific activity of 1087 (±341) mCi/µmol and an injected mass of 2.2 (±0.4) µg. For the dopamine-depleted scans (n=9), the mean radioactivity dose was 9 (±1.6) mCi, with a specific activity of 1044 (±310) mCi/µmol and an injected mass of 2.1 (±0.4) µg. There was no difference in mean radioactivity dose (t(8)=0.98, P=.36), specific activity (t(8)=1.09, P=.31), or mass injected (t(8)=-0.61, P=.56) between the baseline and dopamine depletion scans (n=9). [11C]-(+)-PHNO scanning data were acquired for 90 minutes postinjection. Once scanning was complete, the data were redefined into 30 frames (1–15 of 1-minute duration and 16–30 of 5-minute duration).Participants underwent 2 [lsewhere . Briefly11C]-(+)-PHNO has been described in detail elsewhere (http://www.fil.ion.ucl.ac.uk/spm). The TACs were analyzed using the Simplified Reference Tissue Method (ND), as defined by consensus nomenclature for in vivo imaging of reversibly binding radioligands . The range wherein the basis functions were generated (K2a min - K2a max) was 0.006 to 0.6. These images were spatially normalized into MNI brain space by Nearest Neighbour interpolation with a voxel size fixed in 2×2 × 2mm3 using SPM2. Regional BPND estimates were then derived from ROIs defined in MNI space. The VS and dorsal striatum  were defined according with The region of interest (ROI)-based analysis for -(+)-PHNO for D2/3R is sensitive to dopamine levels  D2/3R BPND under depletion more accurately reflects the true number status of D2/3R; and 3) the fractional increase in D2/3R BPND after dopamine depletion  is linearly proportional to baseline dopamine concentration at D2/3R, provided the process of dopamine depletion does not change the number and affinity of D2/3R. Thus, the %ΔBPND, under appropriate assumptions, is considered a semiquantitative index of endogenous dopamine levels at D2/3R . Statistical analyses were conducted using SPSS  and GraphPad . Normality of variables was determined using the D’Agostino-Pearson test. The significance level for all testes was set at P<.05 (2-tailed).Relationships between baseline BPr(9)=.76, P=.007), and waist circumference was positively correlated with fasting levels of insulin (r(9)=.80, P=.003)  participated in the PET portion of the study; a subset of these data have been previously reported upon  Caravag. Within  P=.003) .2/3R in the VS at baseline -PHNO BPND before and after dopamine depletion). Estimated baseline dopamine occupancy of D2/3R in the VS was positively correlated with IS (r(7)=.84, P=.005) (r(6)=.86, P=.007), BMI (r(6)=.72, P=.04), waist circumference (r(6)=.75, P=.03), and plasma levels of AMPT (r(6)=.84, P=.009). Concurrently, estimated baseline dopamine occupancy of D2/3R in the VS was negatively correlated with fasting insulin levels (r(7)=-.85, P=.004) but was not correlated with fasting levels of glucose (r(7)=-.49, P=.18). Dopamine occupancy in the VS was not correlated with BMI (r(7)=.09, P=.80) or waist circumference (r(7)=-.30, P=.41).Nine of the 11 subjects provided both a baseline PET scan as well as a scan under acute AMPT-induced dopamine depletion; this provided estimates of endogenous dopamine occupying D P=.005) , a corre2/3R were driven primarily by dopamine occupancy in the right VS but not the left VS. Specifically, dopamine occupancy in the left VS was not correlated with IS (r(7)=.41, P=.28), fasting levels of insulin (r(7)=-.46, P=.22), or glucose (r(7)=-.33, P=.39), whereas dopamine occupancy in the right VS was positively correlated with IS (r(7)=.75, P=.01), negatively correlated with fasting levels of insulin (r(7)=-.73, P=.02), and not correlated with levels of glucose (r(7)=-39., P=.31).Notably, the above correlations with estimated baseline dopamine occupancy of D11C]-(+)-PHNO BPND in the right VS was negatively correlated with estimated IS (r(9)=-.65, P=.02) -PHNO BPND in the right VS (r(9)=.77, P=.006), while there was no correlation with fasting glucose levels (r(9)=.27, P=.43). Notably, [11C]-(+)-PHNO BPND in the left VS was not correlated with IS (r(9)=-.35, P=.29) or fasting levels of insulin (r(9)=.53, P=.09) and glucose (r(9)=.08, P=.81).Within the full sample of subjects (n=11), baseline -(+)-PHNO and an acute dopamine depletion paradigm, we demonstrate for the first time that IS is positively correlated with endogenous dopamine levels at D2/3R in the VS. In the absence of obesity or overt glucose dysregulation, lower endogenous dopamine levels in the VS are associated with reduced IS. This novel finding is in line with previous in vivo PET studies examining baseline D2/3R availability in the VS of obese persons . Future AMPT studies examining the relationship between endogenous dopamine in the brain and IS should attempt to employ larger sample sizes. Because of our small sample size, we refrained from exploring relationships between baseline [11C]-(+)-PHNO BPND and IS in ROIs other than the VS. In particular, future [11C]-(+)-PHNO studies using larger sample sizes should examine the relationship between IS and baseline BPND in the substantia nigra and hypothalamus: regions where 100% of the [11C]-(+)-PHNO BPND signal is due to D3R vs D2R  are related to endogenous dopamine levels and dopamine release in the VS of humans. In addition, future studies should examine whether these values alter in the face of treatment for metabolic deficits. Moreover, it is important to examine across a spectrum of glucose dysregulation in humans how dopamine concentrations and functioning in the VS relate to mood, motivation, and reward processing. Finally, our sample in the current study is small. While we did not explicitly control for multiple comparisons, it is important to note that the observed relationship between IS and estimated endogenous dopamine in the VS would survive Bonferroni correction , Alzheimer’s disease , and dep2/3R in the VS of healthy humans. Furthermore, acutely reducing endogenous dopamine in healthy persons can alter estimated IS. Taken together, these findings represent an important preliminary step in elucidating how metabolic status may interface with major mental illnesses such as schizophrenia.In conclusion, using PET and an acute dopamine depletion challenge, we have demonstrated for the first time that estimates of IS are related to levels of endogenous dopamine at DDr. Nakajima reports having received grants from Japan Society for the Promotion of Science and Inokashira Hospital Research Fund and speaker’s honoraria from GlaxoSmith Kline, Janssen Pharmaceutical, Pfizer, and Yoshitomiyakuhin within the past 3 years. Dr. Graff-Guerrerro currently receives research support from the following external funding agencies: Canadian Institutes of Health Research, the U.S. National Institute of Health, and the Mexico Instituto de Ciencia y Tecnologıa para la Capital del Conocimiento en el Distrito Federal (ICyTDF). He has also received professional services compensation from Abbott Laboratories, Gedeon-Richter Plc, and Lundbeck; grant support from Janssen; and speaker compensation from Eli Lilly. Dr. Remington has received research support, consulting fees, or speaker’s fees from the Canadian Diabetes Association, the Canadian Institutes of Health Research, Hoffman-La Roche, Laboratorios Farmacéuticos Rovi, Medicure, Neurocrine Biosciences, Novartis Canada, Research Hospital Fund–Canada Foundation for Innovation, and the Schizophrenia Society of Ontario. The other authors have no competing interests to disclose.


The consensus is that both ecological and social factors are essential dimensions of conservation research and practice. However, much of the literature on multiple disciplinary collaboration focuses on the difficulties of undertaking it. This review of the challenges of conducting multiple disciplinary collaboration offers a framework for thinking about the diversity and complexity of this endeavor. We focused on conceptual challenges, of which 5 main categories emerged: methodological challenges, value judgments, theories of knowledge, disciplinary prejudices, and interdisciplinary communication. The major problems identified in these areas have proved remarkably persistent in the literature surveyed (c.1960–2012). Reasons for these failures to learn from past experience include the pressure to produce positive outcomes and gloss over disagreements, the ephemeral nature of many such projects and resulting lack of institutional memory, and the apparent complexity and incoherence of the endeavor. We suggest that multiple disciplinary collaboration requires conceptual integration among carefully selected multiple disciplinary team members united in investigating a shared problem or question. We outline a 9-point sequence of steps for setting up a successful multiple disciplinary project. This encompasses points on recruitment, involving stakeholders, developing research questions, negotiating power dynamics and hidden values and conceptual differences, explaining and choosing appropriate methods, developing a shared language, facilitating on-going communications, and discussing data integration and project outcomes. Although numerous solutions to the challenges of multiple disciplinary research have been proposed, lessons learned are often lost when projects end or experienced individuals move on. We urge multiple disciplinary teams to capture the challenges recognized, and solutions proposed, by their researchers while projects are in process. A database of well-documented case studies would showcase theories and methods from a variety of disciplines and their interactions, enable better comparative study and evaluation, and provide a useful resource for developing future projects and training multiple disciplinary researchers. Researchers from different disciplines are encouraged to come together to form a kind of chimera, in the biological sense of a single animal composed of several different populations of genetically distinct cells. Despite the assumed virtues of multiple disciplinary collaboration however, much of the literature has focused on the problems . The social sciences study human society and behavior . The humanities study the human condition . Subjects such as history straddle the latter 2 categories.Conservation Biology editorial, Mascia et al.  most papers also described as transdisciplinary or interdisciplinary.We used SCOPUS (an abstract and citation database of peer-reviewed literature) and JSTOR  to find the most productive search terms (yielding the most results) to capture articles on multiple disciplinary research on environmental problems 1960–2010). We used the 4 SCOPUS categories which yielded the most results, and added arts and humanities, as a neglected dimension. In JSTOR we used the categories area studies, biological sciences, development studies, social sciences, and humanities. We searched for the terms 960–2010.The searches yielded nearly 800 papers from 61 journals, most from 1986 onward. The most productive SCOPUS categories were environmental science, agricultural and biological sciences, social sciences, and earth and planetary sciences. We refined the selection by choosing only papers written in English that explicitly addressed the challenges of multiple disciplinary research on problems of the environment involving natural scientists with social scientists or humanities researchers or that were the outputs of such collaborative research. We sorted these papers into categories by abstract and key words: case studies (45); secondary literature (26); position papers (68); reviews or histories (31); and tools .http://www.iccs.org.uk/research-themes/past-projects/sharpen/]). S.P. read and coded these using a grounded theory approach, open to emergent themes and theoretical categories  certain challenges to multiple disciplinary work recurred. These were divided into structural, conceptual, and practical challenges. Structural challenges—educational and sectoral institutional cultures and boundaries and how these are reflected in and entrenched by allocations of funding and policy and managerial responsibilities—are too complex and wide-ranging to include here. They arise from the widely held dualism in Western society between environmental subjects and issues which are coupled with the natural sciences and social and cultural issues which are coupled with the social sciences and humanities . Within Europe, Ressurreição et al.  found loHead et al.  attack tConservation scientists are explicit about the “normative postulates” which form “the basis of an ethic of appropriate attitudes toward other forms of life” Soulé : 730. Deconservation implies action and a stance. However, when it becomes conservation science this is problematic because for many being a scientist implies a need for detached objectivity  Although natural scientists often assume they can manage the social science work required by their conservation projects, the equivalent assumption that social scientists can do natural science research is seldom heard—in fact the opposite view prevails  from different backgrounds, is judged essential for collaborative multiple disciplinary work . Prospective participants could develop working relationships and build the conceptual framework together. An anonymous questionnaire survey could help funders and researchers assess the level to which this was achieved and give advance notice of potential problems.Based on the reviewed literature, we propose that once there is an outline  question, the following steps should be followed in setting up a successful multiple disciplinary project: (1) recruit the right people  from the right disciplines  of interdisciplinary research. These are largely anecdotal and have been partially challenged by Hicks et al. . They foMany, perhaps even most, multiple disciplinary projects are ephemeral. They are also bound up in ways not adequately addressed with the career trajectories of influential individuals and the challenges of assembling and maintaining teams of people with very different personal styles, capabilities, and career aspirations Zube . For theThese challenges and recommendations have been gleaned from a long-term review of the literature. What is surprising is the periodic return of calls for major multiple disciplinary programs to tackle social–environmental problems, accompanied by assertions of novelty and calls for new kinds of researchers and research  can be dispensed with. This way we may productively pursue chimeras actual, rather than mythical.


Phenylketonuria (PKU) was the first disorder in which severe neurocognitive dysfunction could be prevented by dietary treatment. However, despite this effect, neuropsychological outcome in PKU still remains suboptimal and the phenylalanine-restricted diet is very demanding. To improve neuropsychological outcome and relieve the dietary restrictions for PKU patients, supplementation of large neutral amino acids (LNAA) is suggested as alternative treatment strategy that might correct all brain biochemical disturbances caused by high blood phenylalanine, and thereby improve neurocognitive functioning.As a proof-of-principle, this study aimed to investigate all hypothesized biochemical treatment objectives of LNAA supplementation  in PKU mice.Pah-enu2 (PKU) mice and wild-type mice received a LNAA supplemented diet, an isonitrogenic/isocaloric high-protein control diet, or normal chow. After six weeks of dietary treatment, blood and brain amino acid and monoaminergic neurotransmitter concentrations were assessed.C57Bl/6 p<0.01), while alleviating brain deficiencies of some but not all supplemented LNAA. Moreover, LNAA supplementation in PKU mice significantly increased brain serotonin and norepinephrine concentrations from 35% to 71% and from 57% to 86% of wild-type concentrations (p<0.01), respectively, but not brain dopamine concentrations (p = 0.307).In PKU mice, the investigated LNAA supplementation regimen significantly reduced blood and brain phenylalanine concentrations by 33% and 26%, respectively, compared to normal chow (This study shows that LNAA supplementation without dietary phenylalanine restriction in PKU mice improves brain biochemistry through all three hypothesized biochemical mechanisms. Thereby, these data provide proof-of-concept for LNAA supplementation as a valuable alternative dietary treatment strategy in PKU. Based on these results, LNAA treatment should be further optimized for clinical application with regard to the composition and dose of the LNAA supplement, taking into account all three working mechanisms of LNAA treatment. Phenylketonuria  is the first disorder in which severe neurocognitive dysfunction could be prevented by dietary treatment. It is caused by a deficient activity of the hepatic enzyme phenylalanine hydroxylase , normally converting phenylalanine (Phe) to tyrosine. If left untreated, classical PKU symptomatology is almost exclusively restricted to the brain, including severe neurocognitive dysfunction, seizures, and psychiatric problems, correlating with high blood Phe concentrations [This selective brain vulnerability to high blood Phe concentrations is hypothesized to be related to the transport characteristics for Phe at the blood-brain barrier (BBB) ,3. At thThus far, blood Phe reduction has been the primary target of treatment in PKU. This can be accomplished by a severe Phe-restricted diet and, in some patients, by pharmacological treatment with tetrahydrobiopterin. However, early- and continuously treated PKU patients still show impaired executive functioning and are prone to develop anxiety and depressive symptoms ,19. MorePrevious research on LNAA treatment in PKU has primarily focused on brain and blood Phe reduction as treatment objective –28. In aPah-enu2 mice had been kindly provided by Prof. B. Thöny from the division of Clinical Chemistry and Biochemistry of the University Children’s Hospital in Zurich in Switzerland. From heterozygous (+/-) mating, wild-type , heterozygous, and PKU (-/-) mice of both sexes were obtained. After weaning at four weeks of age, genetic characterization was performed by quantitative PCR analysis on DNA extracted from ear tissue. Animals were housed individually at 21±1°C on a 12-hr light-dark cycle (7:00 am-7:00 pm), and water and AM-II food pellets  were offered ad libitum. In total, 42 WT  and 46 PKU  mice were used. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health ) homogenates in phosphate-buffered saline (pH 7.4), and for tryptophan, indole and catecholamine measurements to 2% (w:v) homogenates in acetic acid (0.08 M). Brain homogenates were sonified on ice at 11–12 W. Next, samples were centrifuged at 800 rcf for 10 min (4°C), and the supernatant/internatant was put on ice to be used for further analysis.For brain amino acid (except for tryptophan) measurements, norleucine in sulfosalicylic acid was added as an internal standard to the 20% brain homogenate . Samples were vortexed and centrifuged at 20.800 rcf for 4 min. Plasma amino acid measurements were performed according to the same method, using 50 μl plasma instead of 20% brain homogenate. Amino acid concentrations were measured with a method based on ion exchange chromatography with post column derivatization with Ninhydrin on a Biochrom 30+ analyser .For tryptophan and monoaminergic neurotransmitter measurements, an anti-oxidative solution was prepared in demineralised water (0.4 g/l ascorbic acid and 1.616 g/l ethylenediaminetetraacetic acid). For tryptophan and indole measurements, 25 μl of the anti-oxidative solution was added to 25 μl of the 2% brain homogenate. For catecholamine measurements, 40 μl of the anti-oxidative solution was added to 10 μl of the 2% brain homogenate. Plasma tryptophan measurements were performed using 25 μl plasma instead of 2% brain homogenate. Analysis of tryptophan and monoaminergic neurotransmitter concentrations was performed using liquid chromatography in combination with isotope dilution mass spectrometry, essentially as described by Van de Merbel et al. .Statistical analyses were performed using the software IBM SPSS Statistics for Windows, Version 22.0. . Data of the one animal that was euthanized preterm, were excluded from analyses. All tests were performed two-sided at a significance level of α = 0.05.post-hoc tests for PKU and WT mice separately.Analyses on brain and blood biochemistry as well as on weekly food intake (per g body weight) were performed by two-way ANOVA with genotype and diet as independent variables. In case of not normally distributed data (assessed by Shapiro-Wilk test) or unequal variances (assessed by Levene’s test), analyses were performed on log-transformed data. If the interaction between genotype and diet and/or a main effect of diet was found to be significant, data were further analyzed by one-way ANOVA and Tukey’s post-hoc analysis.The effect of dietary treatment on body weight was analyzed for WT and PKU male and female mice separately by repeated-measures ANOVA with one between factor  and one within factor  and Tukey’s To investigate whether brain Phe concentrations in PKU mice were primarily determined by blood Phe concentrations or by dietary treatment, multiple linear regression analysis was performed with blood Phe concentrations and dietary treatment as independent variables.Data are expressed as mean ± standard deviation (SD).Amino acid contents of the different diets are presented in p<0.01). Moreover, in both weeks, a significant main effect of dietary treatment (p<0.01), and a significant interaction between genotype and dietary treatment on food intake was observed . In PKU mice, food intake in both weeks was lower on high-protein diet than on normal chow (p<0.05), while being even lower on LNAA supplemented diet (p<0.01). In WT mice, food intake in both weeks did not significantly differ for any of the dietary treatments (p = 0.376 and p = 0.322). Based on the weekly food intakes and amino acid contents of the different diets, mean daily intakes of individual LNAA during the six week dietary treatment were calculated for all experimental groups as shown in the Weekly food intake (expressed as g food/g body weight/week), as shown in p<0.05) or high-protein diet (p<0.01), but did not significantly differ between dietary treatments for WT male (p = 0.485) and PKU male or PKU female mice (p = 0.397 and p = 0.343)  was lower than of WT mice , but did not significantly differ between dietary groups in PKU nor WT mice. Body weight curves during treatment were significantly lower for WT female mice on LNAA supplemented diet compared to either normal chow (= 0.343) .th day after inclusion, because of too much weight loss. Pathological examination showed hydrocephalus, which is sometimes found in C57Bl/6 (both WT and PKU) mice.During the experiment, one animal  was euthanized on the 19p<0.01), and threonine concentrations (p<0.05). In PKU compared to WT mice on normal chow, plasma Phe concentrations were increased by almost 30-fold. Plasma tyrosine, tryptophan, and threonine concentrations in PKU mice were reduced to 55%, 77%, and 79%, respectively, of concentrations in WT mice on normal chow. For other LNAA, no significant main effect of genotype was observed on plasma concentrations.Plasma concentrations of individual LNAA in PKU and WT mice on control and LNAA supplemented diets are depicted in p = 0.073 for threonine; p<0.05 for tyrosine; p<0.01 for all other LNAA). Moreover, two-way ANOVA analyses showed a significant interaction between genotype and dietary treatment on plasma Phe and methionine concentrations (p<0.01 for both). In PKU mice, plasma Phe concentrations on LNAA supplemented diet were significantly reduced to 67% of concentrations on normal chow (p<0.01), while plasma Phe concentrations on high-protein diet were significantly higher than on normal chow (p<0.01). In WT mice on LNAA, plasma Phe concentrations were reduced compared to control diets (p<0.05), but concentrations on high-protein diet did not significantly differ from those on normal chow (p = 0.929). Plasma concentrations of supplemented LNAA were higher on LNAA supplementation compared to control diets, both in PKU and WT mice, although this did not reach statistical significance for all LNAA.A significant main effect of dietary treatment was observed on plasma concentrations of all LNAA except for threonine (p<0.05), just not reaching statistical significance for lysine in PKU mice (p = 0.051). In addition, plasma serine concentrations were lower on LNAA supplementation compared to control diets in WT mice (p<0.05), but not in PKU mice (p = 0.407).Plasma concentrations of non-LNAA amino acids in PKU and WT mice on control and LNAA supplemented diets are presented in p<0.01 for all LNAA but methionine, p = 0.523 for methionine). In PKU compared to WT mice on normal chow, brain Phe concentrations were increased by 8.3-fold. Also, brain histidine concentrations in PKU mice were elevated to 139% of WT concentrations on normal chow. Brain concentrations of all other non-Phe LNAA but methionine were reduced in PKU mice on normal chow, ranging from 52% of concentrations in corresponding WT mice for tyrosine to 77% for leucine.Brain concentrations of individual LNAA in PKU and WT mice on control and LNAA supplemented diets are depicted in p<0.01 for all). Moreover, two-way ANOVA analyses showed a significant interaction between genotype and dietary treatment on brain Phe, methionine, and histidine concentrations . Brain Phe concentrations in PKU mice on LNAA supplementation were reduced to 74% of concentrations on normal chow (p<0.01), still being 6.1-fold higher when compared to WT concentrations on normal chow. Brain Phe concentrations in PKU mice on high-protein diet were higher than in PKU mice on normal chow (p<0.05). Brain tryptophan, histidine, and methionine concentrations in PKU mice on LNAA supplementation were significantly higher when compared to PKU mice on normal chow (p<0.05), resulting in concentrations of 100% and 166% of WT concentrations on normal chow for tryptophan and histidine, and an elevation by 3.6-fold for methionine. In contrast, brain threonine concentrations in PKU mice on LNAA supplementation were lower when compared to PKU mice on control diets (p<0.01).A significant main effect of dietary treatment was observed on brain Phe, tryptophan, methionine, and threonine concentrations (p = 0.053). Similar to PKU mice, brain methionine concentrations were higher, whereas brain threonine concentrations were lower on LNAA supplementation as compared to control diets .In WT mice, brain Phe concentrations on LNAA supplementation tended to be lower when compared to normal chow, although this just did not reach statistical significance (p<0.05), although this did not reach statistical significance for glycine in PKU mice on LNAA supplementation compared to high-protein diet (p = 0.064). In addition, in WT mice only, brain lysine concentrations were lower (p<0.01), whereas brain taurine concentrations were higher on LNAA supplementation compared to control diets (p<0.05).Brain concentrations of non-LNAA amino acids in PKU and WT mice on control and LNAA supplemented diets are presented in p<0.01 for both). Brain dopamine and norepinephrine concentrations in PKU mice on normal chow were reduced to 76% and 57%, respectively, of concentrations in WT mice on the same diet. In the serotonergic pathway, significant main effects of genotype were observed on brain concentrations of serotonin and its associated metabolite 5-hydroxyindoleacetic acid (5-HIAA) (p<0.01 for both). Brain serotonin and 5-HIAA concentrations in PKU mice on normal chow were decreased to 35% and 26%, respectively, of concentrations in corresponding WT mice.Brain monoaminergic neurotransmitter and associated metabolite concentrations in PKU and WT mice are depicted in p<0.01 for all but dopamine, p<0.05 for dopamine). Moreover, two-way ANOVA analyses showed a significant interaction between genotype and dietary treatment on brain norepinephrine, serotonin, and 5-HIAA , but not dopamine concentration (p = 0.766).Dietary treatment had no significant effect on brain catecholamine or serotonin concentrations in WT mice, while in PKU mice it did. A significant main effect of dietary treatment was observed on brain catecholamine, serotonin, and 5-HIAA concentrations (p<0.01), and partially restored its deficit to an average of 86% of concentrations in WT mice on normal chow. In contrast, brain dopamine concentrations did not significantly differ in PKU mice between the dietary treatments (p = 0.307). In WT mice, no significant differences were observed between any of the dietary treatments for neither dopamine (p = 0.104) nor norepinephrine (p = 0.283).In the catecholamine pathway, LNAA supplementation in PKU mice resulted in significantly higher brain norepinephrine concentrations compared to normal chow (p<0.01), partially restoring their concentrations to an average of 71% and 67%, respectively, of concentrations in WT mice on normal chow. In WT mice on LNAA supplementation, brain serotonin concentrations tended to be higher compared to normal chow (p = 0.051), and brain 5-HIAA concentrations tended to be higher compared to high-protein diet (p = 0.097), but both did not reach statistical significance.In the serotonergic pathway, in PKU mice on LNAA supplementation, both brain serotonin and 5-HIAA concentrations were significantly increased when compared to control diets  by both blood Phe concentrations  and LNAA supplemented diet . Brain non-Phe LNAA concentrations in PKU mice on normal chow, LNAA supplementation, and high-protein diet did not show a clear relationship with either their respective blood concentrations nor with blood Phe concentrations (data not shown).To investigate whether the reduction of brain Phe concentrations on LNAA supplementation in PKU mice was primarily related to an effect at the BBB or especially related to reduced blood Phe concentrations, the relationship between blood and brain Phe concentrations was assessed in PKU mice on LNAA supplementation and control diets . MultiplTo investigate whether the increase of brain serotonin and norepinephrine on LNAA supplementation in PKU mice was primarily due to (1) increased brain availability of their precursors, or to (2) enhanced conversion of their precursors, brain monoaminergic neurotransmitters were assessed in relation to their respective (amino acid) precursors .p<0.01 for all), but not norepinephrine/tyrosine concentrations (p = 0.184). Ratios of brain dopamine/tyrosine were increased, while rations of brain norepinephrine/dopamine and serotonin/tryptophan concentrations were reduced in PKU compared to WT mice on normal chow.Two-way ANOVA analyses showed a significant main effect of genotype on ratios of brain dopamine/tyrosine, norepinephrine/dopamine, and serotonin/tryptophan (p<0.01). In addition, two-way ANOVA analyses showed a significant interaction between genotype and dietary treatment on ratios of brain norepinephrine/dopamine and serotonin/tryptophan concentrations (p<0.01 for both). In PKU mice on LNAA supplementation, ratios of brain norepinephrine/dopamine were increased compared to control diets (p<0.01). Also, ratios of brain serotonin/tryptophan concentrations in PKU mice on LNAA supplementation were higher as compared to control diets (p<0.05).A significant main effect of dietary treatment was observed on ratios of brain norepinephrine/dopamine concentrations only  reducing brain Phe concentrations, 2) attenuating the brain deficiencies of some, but not all, LNAA, and 3) increasing brain serotonin and norepinephrine, but not dopamine concentrations. Before discussing these results in more detail, we will first address some methodological issues.Pah-enu2 (PKU) mice, a well-established model that resembles the genetics, biochemistry and neurobiology of classical PKU in humans. Biochemically, PKU mice show high blood and brain Phe concentrations in combination with brain non-Phe LNAA and monoaminergic neurotransmitter deficits that also characterize human PKU biochemistry. Km-values for LNAA transport at the BBB have not been determined for (PKU) mice [m-values for individual LNAA as determined in vivo in rats and in human brain capillaries showed a significant correlation [Firstly, the present experiment was performed in KU) mice . Howeverrelation , while bSecondly, the LNAA supplemented diet used was based on the study by Pietz et al. (1999) that investigated the effect of concomitant LNAA supplementation during an oral Phe challenge on brain Phe uptake and EEG activity in PKU patients . The LNAAlthough LNAA supplementation is suggested to improve brain metabolism primarily by restoring the unbalanced LNAA transport at the BBB, LNAA supplementation in PKU mice was also found to significantly reduce blood Phe concentrations to 67% of concentrations on normal chow. This is in accordance with previous studies on LNAA supplementation in PKU mice showing plasma Phe reductions to 47.0–63.5% of concentrations in untreated PKU controls ,24. LNAAIn brain, Phe concentrations on LNAA supplementation in PKU mice were significantly reduced by 26% compared to concentrations on normal chow. This is in good agreement with previous studies on LNAA supplementation in both PKU patients  and miceThis is the first time that brain non-Phe LNAA concentrations have been reported in response to LNAA supplementation in PKU. The LNAA supplementation regimen that was used, changed most brain non-Phe LNAA concentrations, restoring brain tryptophan in PKU mice to WT concentrations. Brain methionine concentrations were even significantly increased by 5.5-fold in PKU mice on LNAA supplementation compared to normal chow, corresponding with the similarly strong increases in blood. Although the cerebral and systemic effects and possible toxicity due to these strongly elevated methionine concentrations are not fully understood , at leasIt can be concluded from these results that LNAA supplementation in PKU mice indeed attenuates brain Phe concentrations and attenuates brain deficiencies of (at least some) non-Phe LNAA. At the same time, results suggest that the relationships between brain non-Phe LNAA concentrations and their respective plasma concentrations as well as plasma Phe concentrations are complex and differ for each non-Phe LNAA, given the amount of LNAA supplementation used in this study. Development of the optimal LNAA supplementation regimen that can both effectively reduce brain Phe concentrations and improve brain concentration of all non-Phe LNAA therefore clearly deserves further research.Besides its effect on brain LNAA concentrations, LNAA supplementation in PKU mice significantly increased brain serotonin from 35% to 71% of concentrations in WT mice. Also, brain norepinephrine in PKU mice on LNAA supplementation increased from 57% to 86% of concentrations in WT mice, whereas brain dopamine concentrations remained unchanged. Although brain monoaminergic neurotransmitter concentrations in response to LNAA supplementation have not been reported previously, a recent study on LNAA supplementation in PKU patients showed increased melatonin (a serotonin metabolite) concentrations in plasma and urine, which—according to the authors- could be a possible new marker for brain serotonin synthesis in PKU patients . As C57Bin vitro observation that Phe inhibits TPH more strongly than TH [Both insufficient precursor availability and impaired TH and TPH activity by inhibition of excessive brain Phe concentrations have been hypothesized to account for the brain monoaminergic neurotransmitter deficits in PKU. The present results suggest that the relative contribution of each of these mechanisms may be different for the dopaminergic and serotoninergic pathways in PKU. Regarding the brain catecholamine deficiencies, the increased ratio of brain dopamine/tyrosine and unaffected ratio of brain norepinephrine/tyrosine in PKU mice could be explained in two ways, each supporting one of the two aforementioned main theories on brain monoaminergic neurotransmitter deficiencies in PKU. Firstly, it may indicate that insufficient brain tyrosine availability rather than inhibition of TH by high Phe would be responsible for the brain catecholamine deficiencies observed in PKU. This would support the report by Fernstrom et al. (2007) concluding that increased brain Phe is not likely to impair catecholamine synthesis in PKU, whereas low brain tyrosine does . In cons than TH . To furtTo conclude, this study was the first to investigate all hypothesized biochemical treatment objectives of LNAA supplementation in PKU. Results in PKU mice showed that LNAA supplementation improves brain biochemistry in PKU by three synergistic mechanisms. Thereby, this study provides proof-of-concept for LNAA supplementation as a possible alternative treatment strategy for PKU that improves brain biochemistry by targeting the unbalanced LNAA transport across the BBB. Before clinical application should be considered, however, further optimization of LNAA treatment with regard to the LNAA being supplemented and their dose is required, taking into account all three brain biochemical treatment objectives.S1 ARRIVE Checklist(PDF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.


Mycobacterium avium ssp. paratuberculosis (MAP) is an infection of the ruminant intestine. In cows, a long subclinical phase with no or low intermittent shedding precedes the clinical phase with high shedding. It is generally considered that an adaptive cell-mediated immune response controls the infection during the subclinical phase, followed by unprotective antibodies later in life. Based on recent observations, we challenge the importance of adaptive immunity and instead suggest a role of the structural organization of infected macrophages in localized granulomatous lesions. We investigated this hypothesis by mathematical modelling. Our first model describes infection in a villus, assuming a constant lesion volume. This model shows the existence of two threshold parameters, the MAP reproduction ratio RMAP determining if a lesion can develop, and the macrophage replacement ratio RMF determining if recruitment of macrophages is sufficient for unlimited growth. We show that changes in RMF during a cow’s life – i.e. changes in the innate immune response – can cause intermittent shedding. Our second model describes infection in a granuloma, assuming a growing lesion volume. This model confirms the results of the villus model, and can explain early slow granuloma development: small granulomas grow slower because bacteria leave the granuloma quickly through the relatively large surface area. In conclusion, our models show that the long subclinical period of MAP infection can result from the structural organization of the infection in granulomatous lesions with an important role for innate rather than adaptive immunity. It thus provides a reasonable hypothesis that needs further investigation.The online version of this article (doi:10.1186/s13567-015-0202-3) contains supplementary material, which is available to authorized users. Mycobacterium avium ssp. paratuberculosis (MAP) causes infection in the ruminant intestine. Cows are most susceptible to become infected in the first half year of their lives . Thus, the granuloma model reads:For adaptation of the villus model to a model for a single granuloma, we assume that the density of cells is constant and that the volume of the lesion changes; thus, we take the dimensions of the granuloma into account. For simplicity, we assume a perfect sphere with the volume determined by the total number of uninfected and infected macrophages, B/V = bQMi/(eA) and the concentration of cytokines equal to C/V = rMi/(mA). Then, we define the following quantities:RMF = ar/bm, is the macrophage replacement ratio as in the villus model.TMAP = cQ/e, is the MAP transmission efficiency. Similar to RMAP in the villus model, TMAP can be interpreted as the number of newly infected macrophages resulting from one infected macrophage in the absence of any (other) infected macrophages, but this situation never occurs as a granuloma does not exist in the absence of infected macrophages. Thus, TMAP should be more loosely interpreted as a measure of MAP transmissibility between macrophages within the granuloma.Next, we make the same quasi-steady state assumption as in the villus model. With the concentration of bacteria equal to Thus, we arrive at the reduced and final granuloma model:TMAP and RMF:ρ = 1, the volume of the granuloma per macrophage present, in arbitrary units.d = 0.04, as in the villus model.i, the rate by which uninfected macrophages enter the granuloma by background macrophage movement. It should be smaller than d/ρ, otherwise granulomas of uninfected macrophages would spontaneously arise. As a default in simulations, we will use i = 0.038 (arbitrary choice).b = 0.04, as in the villus model.This model contains four parameters aside from The aim of the model analyses is to determine for both models what types of behaviour they can display in terms of bacterial replication and resulting bacterial load in the lesion. We seek to answer these questions: will MAP disappear, will it grow until a stable steady state, or will it grow indefinitely so that it outgrows the villus and disrupts the structure of the intestinal wall? The conditions for these types of behaviour will be determined by mathematical analysis and numerical simulations with the above parameter values. All numerical simulations were done in Mathematica 7.0 .Mu* = Mu0 and Mi* = 0; and an infected steady state withMathematical analysis . The infected steady state is stable only if RMAP > 1 and RMF < 1 (each infected macrophage attracts less than one monocyte to the villus through its produced cytokines).From Equation RMAP < 1 and RMF < 1, no infected steady state exists and MAP will disappear irrespective of the initial condition , which would reflect the development of pathology and clinical signs, it does so immediately if RMAP > 1  just outbalances inefficient infection (RMAP < 1). However, this is biologically not a very likely scenario as it would result in pathology and clinical signs relatively quickly in all cows, whereas in reality most remain subclinical for many years. However, as long as RMF < 1, the number of infected macrophages in the villus remains small and the infection is kept limited. It could be that this is the situation during the subclinical phase of the infection: MAP is present in the intestine but only locally in small quantities. If conditions change which causes RMF to increase across many villi, e.g. when leucocyte levels are higher, when cows are more susceptible to infections in the last trimester of pregnancy and the first three months of lactation [The numerical simulations show that if the infection grows indefinitely ). In both steady states, the number of uninfected macrophages is proportional to the surface area of the granuloma: Mu* = A*/TMAP. Although the model is not explicitly spatial (it does not describe where the infected and uninfected macrophages are), this relation suggests that the uninfected macrophages are situated at the surface of the granuloma, and that the thickness of this layer of cells is determined by TMAP, which is the efficiency by which they are infected.Mathematical analysis of the granuloma model shows the possible existence of three steady states: the infection-free steady state with both Vsmall* exists if conditions “are favourable” , and in relation to the bursting rate b. This steady state Vsmall* is always unstable. The infected steady state Vlarge* exists only if Vsmall* exists and if RMF < 1; this steady state is always stable.Existence and stability analysis of all steady states , the only existing and stable steady state is the infection-free steady state only . The plots show that growing granulomas linger for a long time around steady state Vsmall* , before they grow more quickly towards Vlarge*. Third, if RMF > 1, there is no steady state Vlarge* and growing granulomas will grow indefinitely and outgrow the villus  so that cell densities could change. In the granuloma model the density of macrophages was fixed, and the volume of the granuloma proportional to the number of macrophages. In the granuloma model, inflow and outflow of macrophages, bacteria, and cytokines occurred at rates proportional to the surface area of the granuloma, for which a perfect sphere was assumed.RMAP or TMAP will be important in early infection, as it determines whether an animal can become infected upon ingestion of MAP. The two models are different in predicting the fate of an early lesion when it starts with very few (e.g. one) infected macrophages. With RMAP (villus model) clearly defined as the number of newly infected macrophages produced by one infected macrophage, a lesion will always develop if RMAP > 1. TMAP (granuloma model) is less clearly defined and small granulomas are predicted always to disintegrate. However, TMAP does determine the possibility of granuloma formation if it starts with some clustered macrophages right from the start, if bacteria would enter a villus and infect a couple of macrophages that are in close vicinity.Both models predict that a threshold parameter RMAP and TMAP show that initial infection control depends on a balance between availability of macrophages, rate of macrophage infection by bacteria, number of bacteria released from an infected macrophage, and bacterial clearance rate. This is similar to the results of Gammack et al. [RMAP and TMAP depend on availability of macrophages for infection and bacterial clearance shows the importance of the actual role of macrophages. Also for MAP, it has been shown that macrophages can be more permissive or become active in phagocytosis [Both k et al. , who hadk et al. ,33. In oocytosis ,42. HoweRMF, defined as the number of uninfected macrophages attracted to the lesion by one infected macrophage, through its excreted cytokines. If RMF < 1 lesions will dissolve or go to a stable steady state, depending on RMAP or TMAP. If RMF > 1, the number of infected macrophages can grow indefinitely, even if RMAP < 1, but then there should be sufficient infected macrophages to start with. RMF combines several infection processes that appear important in keeping granulomas restrained: production and outflow of cytokines, rate of attraction of macrophages by cytokines, and bursting rate of infected macrophages. Gammack et al. [RMF varies throughout a cow’s life, e.g. in lactating cows due to endocrinological changes during pregnancy and lactation [RMF remains elevated (Figures Both models also predict the existence of a second threshold parameter, the recruitment ratio of macrophages k et al.  did not actation ,25, and  Figures  and 5. IRMF due to hormonal changes during pregnancy and around calving, shedding could follow patterns as in Figure RMF. Interestingly, this size effect in models of spheres resulting from changes in efficiency of diffusion is well known from models for early tumour growth, where the result is opposite: tumours first grow quickly but come to a halt because of lack of nutrients that have to diffuse from outside [A possible explanation for the early part of the subclinical phase, when shedding levels are so low that infection is hardly ever detected, is provided by the granuloma model. It concerns the time before the stable low shedding phase is reached, even before the possible intermittent shedding as explained above. Mechanistically, this very early dynamics can be explained by considering the three-dimensional structure of the granuloma. As the steady-state solutions indicate, the number of uninfected macrophages is proportional to the surface area of the granuloma, which means that in a small granuloma most macrophages are uninfected. They remain uninfected because the concentration of bacteria is low: there are few infected macrophages and free bacteria quickly leave the granuloma through the surface area which is large relative to the volume. This results in a long phase with an almost stable granuloma size. However, the granuloma does grow slowly and at some point, infection occurs sufficiently efficient to infect most macrophages and start rapid growth of the granuloma Figures  and 5. W outside .To extrapolate dynamics in a single lesion to excretion patterns, we assume that the dynamics in many lesions is synchronized to some extent, for which we used two arguments: synchronization because parameter values change due to systemic changes in the animal –26 and cOne aspect of observed excretion patterns is not explained by our models: the initial low-level shedding in the first couple of months after infection ,46,47. TIn conclusion, our models show that the long subclinical phase during MAP infection can result from the structural organisation of the infection in granulomatous lesions, without an important role for adaptive immunity. The early subclinical phase can be explained by the three-dimensional organisation in granulomas: small granulomas grow slower because their surface area is relatively large, which causes free bacteria to leave quickly. The later phase of intermittent shedding can be explained by changes in the efficiency of macrophage recruitment to the lesions, thus by changes in the innate immune response rather than the adaptive response. This variation in immunocompetence during a cow’s life could for instance be related to hormonal changes during pregnancy or lactation. Although our model cannot prove that adaptive immunity does not play any role, it does to provide a reasonable alternative which suggests that investigation of local factors and innate immunity at the lesion site may provide a better understanding of paratuberculosis pathogenesis.