Previous studies of immunoglobulin gene sequences in patients with allergic diseases using low-throughput Sanger sequencing have limited the analytic depth for characterization of IgE repertoires.(IGH) repertoires in patients with seasonal allergic rhinitis (AR) with the aim of better understanding the underlying disease mechanisms.We used a high-throughput, next-generation sequencing approach to characterize immunoglobulin heavy-chain gene IGH sequences in matched peripheral blood and nasal biopsy specimens from nonallergic healthy control subjects (n = 3) and patients with grass pollen–related AR taken in season (n = 3) or out of season (n = 4) were amplified and pyrosequenced on the 454 GS FLX+ System.IGH  sequences were analyzed. Use of immunoglobulin heavy-chain variable region gene families 1 (IGHV1) and 5 (IGHV5) was higher in IgE clonotypic repertoires compared with other antibody classes independent of atopic status. IgE repertoires measured inside the grass pollen season were more diverse and more mutated (particularly in the biopsy specimens) and had more evidence of antigen-driven selection compared with those taken outside of the pollen season or from healthy control subjects. Clonal relatedness was observed for IgE between the blood and nasal biopsy specimens. Furthermore in patients with AR, but not healthy control subjects, we found clonal relatedness between IgE and IgG classes.A total of 97,610 IGH repertoires in patients with respiratory allergic disease. We demonstrate that natural pollen exposure was associated with changes in IgE repertoires that were suggestive of ongoing germinal center reactions. Furthermore, these changes were more often apparent in nasal biopsy specimens compared with peripheral blood and in patients with AR compared with healthy control subjects.This is the first report that exploits next-generation sequencing to determine local and peripheral blood  Although Sanger sequencing has been applied to study IgE repertoires in patients with allergic disease,12The immunoglobulin repertoire in the periphery is shaped by somatic hypermutation (SHM), class-switch recombination (CSR), and affinity maturation within the germinal centers (GCs) of lymphoid tissues in response to antigens. The pivotal role of IgE in allergic inflammation is well characterized,Over recent years, sequencing technologies have evolved dramatically, advancing from the low-throughput Sanger-based methods to massively parallel and high-throughput approaches that are enabled by several next-generation sequencing (NGS) platforms.(IGH) repertories in matched peripheral blood and nasal mucosal biopsy specimens from patients with AR inside the grass pollen season (AR.IS group), patients with AR outside the pollen season (AR.OS group), and nonallergic healthy control subjects (NA group). We detected significant changes in the IgE repertoire (as well as those of other antibody classes) in the AR.IS group with evidence of enhanced affinity maturation for IgE as a result of natural exposure to seasonal grass pollen. This report demonstrated the technical feasibility and usefulness of high-throughput NGS repertoire analysis in respiratory allergic disease research.Seasonal allergic rhinitis (AR) affects a quarter of the population of westernized countries, and a large proportion of these patients are allergic to pollens. In this report a high-throughput NGS approach is introduced to characterize immunoglobulin heavy-chain gene www.jacionline.org). Samples were collected after obtaining written informed consent, as approved by the East London & The City REC Alpha (09/H0704/67).Subjects with different atopic statuses, the AR.OS group (n = 3), the AR.IS group (n = 4), and the NA group (n = 3), were recruited from the Royal Brompton Hospital London allergy clinic or through local advertisement  were taken from the inferior turbinate after achievement of local anesthesia and subsequently homogenized with a Qiagen TissueLyser . Peripheral blood lymphocytes were isolated from venous blood by using Ficoll density gradient separation . Total RNA was extracted with the RNeasy Mini Kit (Qiagen), and cDNA was synthesized by using SuperScript III RT .IGH sequences were generated by means of seminested PCR reactions (see the www.jacionline.org) with a mixture of sense primers (framework region 1/immunoglobulin heavy-chain variable region [IGHV] gene families 1-7 for respective framework 1 regions) in conjunction with antisense primers . Processed library IGH sequences were pyrosequenced on the 454 GS FLX+ System .As previously described,As previously described,Selection strength for complementarity-determining regions (CDRs) and framework regions in sampled immunoglobulin sequences was estimated by using BASELINe (see the The Phylogeny Inference Package (PHYLIP)Depending on the nature of data sets, different statistical methods were used for multiple group comparisons by using GraphPad Prism 6.0 software . QC-filtered sequences can be searched on the National Center for Biotechnology Information's Sequence Read Archive  DNA motifs and identify 35,175 clonotypic sequences to represent their clonal families (see www.jacionline.org).A total of 152,784 sequence reads were generated from 20 samples. After QC analysis, 97,610 IGHV5 genes have been associated with antibody responses to superantigens. Therefore we compared the relative abundance of immunoglobulin genes to determine whether natural pollen exposure increased particular IGH gene rearrangements. We observed that the use of IGHV gene families (A), IGHD gene families , and IGHJ gene families (see B) for IgE clonotypes did not significantly differ between allergic and nonallergic subjects. Differences between peripheral blood and biopsy specimens were very minor. However, we detected increased use of IGHV3 gene families and a trend for reduced use of IGHV4 gene families in nasal biopsy specimens from the NA and AR.OS groups but not the AR.IS group compared with their counterparts in peripheral blood.Specific antigen challenge might enrich B cells expressing particular immunoglobulin genes, changing the landscape of immunoglobulin repertoires.families , A, IGHDlies see , B for IIGH gene rearrangements between IgA and IgG repertoires allowed us to group them as one switched IgE− (IgA and IgG) population.IGHV1 and IGHV5 gene families but a reduction for IGHV2 and IGHV3 (B) compared with IgM and switched IgE− sequences. Similar patterns were observed separately in peripheral blood (see C) and nasal biopsy specimens (see D). IgE repertoires were also distinct significantly from other classes in their use of IGHD gene families (see E), IGHJ gene families (see F), and individual IGHV genes (C). Approximately 11 of 58 IGHV genes, such as IGHV5-a, differed in abundance for IgE clonotypic repertoires compared with other classes. In addition, significant changes between patient groups were observed in the use of some IGHV families  for IgM (see G and H) and switched IgE− (see I and J) repertoires.Similar patterns of nd IGHV3 , B compalood see , C and nmens see , D. IgE lies see , E, IGHJlies see , F, and HV genes , C. Appr IgM see , G and HIgE− see , I and JIGHV region are indicative of higher-affinity antigen binding.IGHV mutations was determined as the number of nucleotide differences compared with germline sequence per 10,000 bases. IgE and switched IgE− sequences had significantly more mutations than IgM sequences (A and B). Within the IgE class, there were more mutations in the AR group (C and D), with further increases observed in biopsy specimens taken in season (E and F). Regardless of antibody class, the mutation frequency was increased , and the proportion of germline-like  sequences was significantly reduced in biopsy specimens taken in season . In contrast, the opposite pattern for IgM and switched IgE− sequences was observed in peripheral blood.Increased mutation numbers in the equences , A and BAR group , C and Dn season , E and FIGHV mutations among all clonally related sequences. IgE clones taken in season had the highest degree of intraclonal variations for larger clones  and were more mutated compared with those taken out of season (see D).We further analyzed the degree of sequence variations among related mutants within each IgE clonal family as a way of assessing intraclonal diversification as a cause of clonal expansion and SHM. Thus intraclonal variation was calculated as the SD of ason see , D.A and B). IgE sequences from allergic subjects (in and out of season combined) had significantly longer CDR-H3  than the NA group (80% <15 nucleotides). This was mainly due to increased N-nucleotide numbers and IGHD lengths in allergic subjects (C). This pattern remained evident for IgE isolated from peripheral blood, as well as IgE from biopsy specimens (D). Although the CDR-H3 length for IgE did not differ in allergic subjects between samples taken in or out of season (data not shown), we observed different patterns in IgM and switched IgE− repertoires between different atopic status (see B and C).CDR-H3 is regarded as the most important motif within immunoglobulin genes because of its central position in the antigen-binding pocket. Therefore we analyzed the physiochemical properties of CDR-H3 peptides. CDR-H3 motifs of unmutated (100% germline) IgM sequences were extracted from the whole data set to represent the most diverse fraction for comparison. Among antibody classes, CDR-H3 was longest in length for IgM but shortest for IgE , A and Bsubjects , C. Thispecimens , D. Althatus see , B and CIGHV mutations within an immunoglobulin sequence often negatively correlates with its CDR-H3 length.IGHV mutations for IgE from the NA group  and AR.OS group (see B) samples. However, this correlation was not observed in the AR.IS group (C). In addition, we did not detect any significant differences of CDR-H3 peptide properties, including the aliphatic index, grand average of hydropathicity index, and theoretic isoelectric point (see www.jacionline.org), when comparing IgE sequences between the patient groups.The number of roup see , B samplIS group , C. In aIGH repertoires. When comparing IgE sequences between the AR.IS and AR.OS groups, we observed distinctive patterns of selection (A); however, nasal biopsy specimens showed stronger positive selection (B) compared with other atopic statuses.B-cell selection is a pivotal process for affinity maturation of antibodies in which positive selection can often improve antibody affinity and negative selection can contribute to the structural integrity of antibodies.election . For IgEelection , A; howeelection , B compa0D) within each population of clones, we used the generalized diversity model proposed by Hill (see the www.jacionline.org).A and B).C), whereas IgE clones from nasal biopsy specimens taken in season had the highest measure of diversity (D).The repertoire diversity of B cells is influenced by selection strength, and a repertoire with a higher degree of diversity is more likely to recognize a wider array of antigens  than a more restricted repertoire. To quantify diversity  but none in the 1,094 trees from nonallergic samples.Lineage tree analysis was used to identify sequences across different antibody classes, sample types, or both. By clustering clonally related sequences and analyzing their mutation patterns, we were able to generate 1,752 lineage trees (data not shown). Of these, IgE sequences were present in 146 lineage trees see . Trees cIGH repertoire in a small cohort of patients with AR.Previous studies of the human IgE repertoire were restricted by the use of low-throughput Sanger sequencing. To the best of our knowledge, the largest number of IgE sequences analyzed in a single report was limited to 1,366 IgE sequences isolated from the blood of 13 asthmatic children.IGH gene rearrangements, resulting in altered repertoire diversity and abundance of immunoglobulin genes. In this report we observed a more limited diversity for IgE clones compared with other antibody classes, indicating that IgE clones might be more stringently selected. Furthermore, in contrast to a previous report describing similar use of IGHV between IgM and IgE repertoires,IGHV1 and IGHV5 gene families but lower IGHV3 use independent of atopic status compared with IgM. Although our sequence data alone cannot determine the nature of antigen stimulation, the distinct repertoire profile of IgE clonotypes suggests that IgE+ B cells might be subject to selection pressures different from other antibody classes. The overabundance of the minor IGHV5 subgroup in the IgE repertoire has been suggested to associate with the pathogenesis of allergic disease,IGHV5 might be specific to IgE but not to allergy per se. Further studies are required to confirm this finding and exclude any confounding variables related to disease status or sampling efficiency.Antigen stimulation often leads to selection for or against particular + B cells in allergic patients under the influence of allergen exposure. Alternatively, increased mutations for IgE transcripts in season could be associated with enhanced sequential CSR from IgG to IgE, as previously reported in asthmatic patients.in vivo, given the unlikeliness of capturing all clonal members at the time of sampling. For example, it would be biologically implausible for IgG2/4 to switch to IgG1/3, as illustrated in our own lineage trees.Recent data from a mouse model suggest that high-affinity IgE antibodies might arise through high-affinity, hypermutated IgG intermediates that undergo sequential CSR to IgE.Hypermutation patterns have detrimental effects on the selection of B cells and, subsequently, the diversity and affinity of antibodies. A previous study showed evidence of negative selection in framework regions but did not observe any specific patterns for CDRs in IgE sequences.+ B cells undergo affinity maturation. Increasing evidence suggests that GC-like structures containing IgE+ B cells are present in both the upper and lower airways in patients with AR and allergic asthma, respectively.IGHV mutations and diversity of IgE clones isolated from the nasal mucosa. These observations were consistent with stronger positive selection that we observed in mucosal IgE in patients with AR in season. In part, these results might reflect selection of IgG clones and formation of IgG+ memory cells in the primary sensitization to allergens localized in the target organ, followed by allergen stimulation of clonal expansions and CSR to IgE. Using lineage tree analysis, we also found molecular footprints that linked IgE to other antibody classes in the nasal biopsy specimens, supporting this hypothesis. Altogether, our observations point to the possibility that the allergic respiratory mucosa supports local affinity maturation of IgE, which is consistent with the detection of activation-induced cytidine deaminase and switch circle transcripts in the target organ in patients with AR and asthma, as well as nasal polyposis.It remains unclear exactly where, if at all, IgE+ population is derived through direct CSR from IgM to IgE and has been suggested to arise through polyclonal activation of B1-like CD5+ B cells.+ populations was different between allergic and healthy subjects, directly characterizing these IgE+ populations both phenotypically and functionally might help us better understand their differing roles in allergic responses.There are important questions that remain to be answered. First, as previously reported for atopic dermatitis,Second, we observed that IgE sequences taken in season from patients with AR were more mutated but had longer CDR-H3. Increased hypermutation and shorter CDR-H3 are suggestive of specific antigen-driven responses, whereas polyreactivity has been associated with longer CDR-H3.45+ B cells and the nature of allergic response in patients with AR remains to be investigated in future studies.Third, whether the alterations observed in non-IgE repertoires are relevant to the development of IgEIGH gene rearrangements in the IgE repertoire was similar for all groups but distinct from other antibody classes independent of atopic status. Our data demonstrate seasonal and compartmental differences in clonal diversity and selection strength of IgE and provide direct evidence of increased hypermutation for IgE+ cells in patients with AR under the influence of natural pollen exposure. Our lineage tree analysis reveals intraclonal diversification of IgE clones and association of IgE repertoires between the blood and nasal mucosal compartments. Finally, clones shared between IgE and IgG classes are detected in allergic subjects but absent in nonallergic subjects, which is indicative of preferential sequential switching through IgG in allergic subjects. Taken together, these data demonstrate NGS as a powerful approach to study the immune repertoire in patients with allergic diseases.•Natural exposure to grass pollen is associated with enhanced SHM, increased diversity, and changes in selection and CSR patterns for IgE repertoires in patients with AR.•IGH rearrangements independent of atopic status.IgE repertoires linking peripheral blood and nasal mucosal compartments have a distinct pattern of •We demonstrate the technical feasibility and analytic power of NGS for the determination of immunoglobulin repertoires in patients with respiratory allergic disease.In conclusion, we observe that the pattern of


Neurons in the brain are known to operate under a careful balance of excitation and inhibition, which maintains neural microcircuits within the proper operational range. How this balance is played out at the mesoscopic level of neuronal populations is, however, less clear. In order to address this issue, here we use a coupled neural mass model to study computationally the dynamics of a network of cortical macrocolumns operating in a partially synchronized, irregular regime. The topology of the network is heterogeneous, with a few of the nodes acting as connector hubs while the rest are relatively poorly connected. Our results show that in this type of mesoscopic network excitation and inhibition spontaneously segregate, with some columns acting mainly in an excitatory manner while some others have predominantly an inhibitory effect on their neighbors. We characterize the conditions under which this segregation arises, and relate the character of the different columns with their topological role within the network. In particular, we show that the connector hubs are preferentially inhibitory, the more so the larger the node's connectivity. These results suggest a potential mesoscale organization of the excitation-inhibition balance in brain networks. One of the salient characteristics of neurons is their ability to either excite or inhibit other neurons, depending on the type of neurotransmitter they use to act upon them. In fact, a careful balance between excitation and inhibition is required for the brain to operate in a sustained state, away from both epileptic activity (which would arise if excitation dominated over inhibition) and complete quiescence (which would emerge if inhibition prevailed). While much work has been devoted to study how excitation and inhibition are organized at the level of neural microcircuits, little is known about how these two effects are structured at larger scales. Here we are interested in the scale of cortical macrocolumns, large collectives of neurons that can be described computationally by population models known as neural mass models. These models are routinely used to describe the brain rhythms observed with non-invasive techniques such as EEG. In this paper we show, using a neural mass model, that a collection of coupled cortical macrocolumns self-organizes spontaneously into a dynamical state in which excitation and inhibition are segregated at the mesoscopic level, with some cortical columns (the most connected ones) being inhibitory, and the rest being excitatory. The correct operation of the brain requires a high degree of self-organization, one of whose paradigmatic examples is a careful balance between excitation and inhibition at the neuronal level –3. Much The columnar structure of the cerebral cortex was initially described by Lorente de Nó  as a morIn order to describe the dynamics of cortical macrocolumns we use a neural mass model (NMM) originally developed by Jansen and co-workers to reproduce the evoked EEG activity elicited in neuronal populations by visual stimuli , 35, in Neural mass models have a rich bifurcation structure  that is In order to explore the organization of excitation and inhibition in brain networks, we have to make assumptions regarding the architecture of those networks. The nature of the topology of brain networks has been under intense investigation and debate in recent years –59. FuncWe focus in networks formed by 50 identical nodes, whose dynamics is represented by an extension of the model by Jansen and co-workers , 35. EacHz, corresponding to the alpha band.The activity of an isolated cortical column, corresponding to a node in our network, is represented by a neural mass model, as described in the here see , this moδsin(2πft), to the constant external input, δ and frequency f. A positive MLE corresponds to a chaotic dynamical evolution of the system. It is noticeable that many combinations of driving frequency and amplitude lead to chaos, and that a small domain characterized by negative MLE, with f ∼ 8.7 Hz and scaled driving amplitude δ/C around 0.7 Hz, appears inside the chaotic domain.Chaotic dynamics appears in this system when adding a periodic spike density, nt (MLE) , as showReg. This quantity is calculated using the second maximum of the autocorrelation function for ye(t)−yi(t) signal (see Eqs. (δ and f. The regions characterized by large positive (negative) values of the MLE in Reg in f = 8.5 Hz and scaled driving amplitude δ/C = 0.49 Hz is illustrated by the signal dynamics and its corresponding power spectrum density (PSD) in Reg will be used hereafter to quantify the regularity of the signals that characterize the network dynamics.Calculating the MLE becomes computationally costly when dealing with a large number of columns. Thus, in the rest of the paper we will use a different measure of the regularity of the dynamics, given by the parameter see Eqs. , 10, 11,δ and f, which for isolated nodes produced irregular dynamical evolutions  periodic, quasi-periodic and chaotic nodes coexist in a single network, even though all nodes receive the same driving. For other α-β combinations, the whole network exhibits either highly regular or highly irregular dynamics.The inter-columnar coupling intensities are determined by parameters . We α-β values, different degrees of synchronization appear between different pairs of nodes of the network. Cmax(τ), for all pairs of nodes i,j, ordered for increasing correlation, for scaled α/C = 0.56 and β/C = 0.26 (labeled as point 1 in Cmax(τ) in the range 0.2–1, and lie mostly above Cmax(τ) = 0.4, indicating that the activity of direct neighbors is highly correlated. For pairs of nodes that are not connected directly  and yi(t), respectively). Hence, each node  can be considered inhibitory if ⟨ye(t)−yi(t)⟩ < 0, and excitatory if ⟨ye(t)−yi(t)⟩ ≥ 0, where ⟨⋯⟩ denotes temporal average. y(t) = ye(t)−yi(t) for a pair of directly connected nodes characterized by high regularity and low correlated activity. In this case, the activity of both nodes is dominantly inhibitory. In contrast, the pair of nodes illustrated in segregation, and which is discussed in detail in the next section. As described in the α and β. For scaled α/C = 0.790 and β/C = 0.037 (point 2 in y = ⟨ye(t)−yi(t)⟩ of all nodes is positive −yi(t)⟩ , whereas large values of EIS mean that the number of nodes is evenly distributed in each side of the histogram, with a large difference of the ⟨ye(t)−yi(t)⟩ value between excitatory and inhibitory nodes. α-β parameter space studied so far. We observe that the dynamics is roughly separated in three domains. A domain characterized by no segregation, labeled as ‘N’ in the plot, corresponds to a dominantly excitatory input. An intermediate domain, labeled ‘L’, is characterized by a low level of segregation. Finally, if β is large enough and α is small enough, a regime of high segregation, labeled ‘S’, arises. Note that the transitions between the three domains are rather sharp.In order to establish the robustness of the segregation, we now compute the excitation/inhibition segregation index (EIS), as defined in the section ye(t)−yi(t)⟩, and its regularity. α-β combinations used in In order to determine the influence of the network characteristics on the dynamics of the nodes, we examined the relationship between the number of connections of each node (degree), its average activity ⟨There is also an important difference in the distribution of regularity of the nodes, depending on the segregation exhibited by the network. In the absence of segregation , the nodx(t)−y(t), which resembles the observable typically used in the Jansen and Rit model. Besides, segregation occurs when the excitatory and the inhibitory blocks of each oscillator receive a different level of external stimulus, that is, p1 ≠ p2 as well as q1 ≠ q2 (being pi ≠ qi).As a final remark, the emergence of segregation of excitation and inhibition is also present in other mesoscopic descriptions of the neuronal activity, showing the possible universality of the phenomenon. To proof so, we studied the dynamics of two coupled Wilson-Cowan oscillators , 78 see, which sx−y⟩t and plotted the x1−y1⟩t and ⟨x2−y2⟩t signals remain segregated. For the case of Wilson-Cowan oscillators segregation might be excitatory or inhibitory dominated, depending on which coupling term . These results are also in agreement with the description of chaotic dynamics at a cellular level following periodic inhibitory inputs , 81. TheOne of the main features of the networks studied here is that even when the nodes are identical and receive a common driving input, their activity can be segregated in two different modes, being predominantly excitatory or predominantly inhibitory. This segregation results from the combined effect of the heterogeneous network connectivity, the specific excitatory-inhibitory couplings, and the external excitatory input to which all network nodes are subject. However, if no external input is fed into the columns segregation also emerges, though in lower levels, as shown in small motifs of connected cortical modules . The samm0 = 10  than much simpler and regular signals. The normal functioning state of the brain, however, requires a certain degree of synchrony . A dysfumpm(t), into an average postsynaptic (excitatory or inhibitory) membrane potential yn(t). A linear convolution implements this transformation in terms of the kernelA and B are related with the maximum height of the excitatory and inhibitory postsynaptic potentials , whereas a and b represent the inverse of the membrane time constants and the dendritic delays.We study a system of coupled cortical macrocolumns described by an extension of the model developed by Jansen et al . This moe0 determines the maximum firing rate of the neural population, ν0 sets the net PSP for which a 50% firing rate is achieved, r is the steepness of the sigmoidal transformation, and m(t) corresponds to the net PSP input into the considered population (see pm(t) = λS(m(t)), where λ weights the coupling between two population contacts. This coupling constant quantifies the efficiency of the synaptic contacts. The intra-columnar connectivity constants values are defined in terms of Cp, with p = 1,...,4, as described by Jansen et al. [C = 133.5.The second dynamical transformation is the conversion of the net average membrane potential into an average spike density. This conversion is done at the somas of neurons in the population, and is described mathematically by a sigmoidal function defined as:n et al. . Here C pα(t), for the excitatory input into the pyramidal population, and pβ(t) for the inhibitory input into the pyramidal population, respectively. We chose this contact arrangement because pyramidal cells are widely regarded as the responsible for non local connectivity.In our model the different cortical columns interact with each other through both excitatory and inhibitory connections. The pyramidal-pyramidal excitatory couplings and the inhibitory interneuron-pyramidal inhibitory couplings mimic short range inputs from nearby columns. These inter-column interactions correspond to incoming pulse densities pT(t) from external sources . The excitatory pulse densities reaching a cortical column i can be written asNi corresponds to the in-degree (number of connections entering the node) of the receiver node, Nj is the out-degree (number of connections leaving the node) of the emitter nodes and K is node’s i number of neighbors. All the couplings between neighbor nodes in the network are bidirectional. The transformation described by Equations n = 0,1) describing the excitatory integration of the average density of action potentials reaching the population:n = 2) to describe the inhibitory integration of the average density of action potentials.Besides the intra-columnar inputs, the pyramidal population receives a constant incoming pulse train of density N = 50 cortical columns with a heterogeneous connectivity, constructed using a preferential attachment rule [i  are as follows:We are centered in a network formed by a set of ent rule , 89. Thei, the excitatory postsynaptic potential input into the interneuron population, the excitatory postsynaptic potential input into the pyramidal population (ye(t) in the yi(t) in the The parameter values used in this study are depicted in x (y) being the average activity of the excitatory (inhibitory) populations. a and d are the self excitation parameters for x and y units, and b and c are the couplings from the inhibitory (excitatory) unit y (x) to the excitatory (inhibitory) unit x (y), respectively  gives the proportion of excitatory (inhibitory) neurons receiving thresholded excitation per unit time. We chose as parameter values a = 16, b = 12, c = 16, d = −2 and varied the external inputs p−q to find values that allowed segregation in the coupled scenario.We have also used the Wilson-Cowan model  to show i,j = 1,2. We have explored which values of the coupling strengths Kij  allow the system to remain segregated for fixed values of external stimuli p1 = −3.5, q2 = −6.5, p2 = −1.0 and q2 = −4.0.The coupling is performed as shown in s stands for the number of realizations for different initial conditions—50 in the case studied in N is the total number of cortical columns—50 for all cases—and Maximal Lyapunov Exponent (MLE). The maximal cross-correlation for each pair of columns in the network, Cmax(τ), was computed using the maximum value of the cross-correlation function. We computed the excitatory-inhibitory segregation (EIS) index as EIS = ∣CMeAeCMiAi∣, where CMe (CMi) stands for the position of the center of mass of the excitatory—positive (inhibitory—negative) part of the activity distribution (Ae (Ai) is the corresponding area of the excitatory (inhibitory) distributions, respectively.We characterize the regularity of the signals in terms of the autocorrelation function and the Maximal Lyapunov Exponent (MLE), the synchronization in terms of the cross-correlation, and the segregation by using the average activity lgorithm  but we jribution , and Aet = 50 s. A period of 25 s was omitted to avoid transients.The integration of the model equations was performed using two methods. The Adams-Bashforth method was used for the MLE computation , with a m0 initial nodes connected randomly to which other nodes are added gradually. These new nodes are connected to m ≤ m0 existing nodes with a probability that increases with the number of links of the already connected nodes. This procedure gives rise to networks with heavily connected nodes, and thus with a power-law distribution of degrees. In Figs.m0 = 1. In α and β pair. In the Supporting Information m0 = 10 (m0 = 20 (RPs) of 0, 0.5 and 1 [RP defines a different network: ring (RP = 0), small-world (RP = 0.5) and random (RP = 1.0). We constructed 10 networks for each RP. In all cases every network was simulated using 10 sets of different initial conditions for the nodes and were constructed using the NetworkX Python package.Scale-free networks were constructed using the Barabási-Albert algorithm , which mm0 = 10  and m0 m0 = 20 , respect.5 and 1 . Each RPS1 Figm0 = 10  and the nodes display irregular dynamics. When inhibition dominates over excitation (bottom-right network) the most connected nodes (hubs) tend to become inhibitory and the nodes become more regular. (B) Same as panel (a) but for larger clustering. Segregation here is higher than in networks with lower clustering (see colorbar ranges). There is a region of maximal segregation for dominating β. Map showing the excitatory-inhibitory segregation index (EIS) for a scale-free network constructed using the Barabási-Albert algorithm with (ZIP)Click here for additional data file.S2 Figα and β values explored.  Segregation appears for complex topologies such as small-world with a rewiring probability of 0.5.  Random networks also display segregation. In panels from (C) to (F) it is shown that segregation is strongly dependent on the number of neighbors of each node (see segregation color bar ranges). Map showing the excitatory-inhibitory segregation (EIS) index for a ring shaped network (with 2 neighbors in (A) and 10 neighbors in (B)) and examples of the segregation and regularity of the networks. No segregation occurs for any (ZIP)Click here for additional data file.S3 Figix−iy for the two segregated oscillators, in an excitatory-dominated situation .(A) Coupling scheme between the two oscillators. (B) Resulting segregation map. (C) Time traces of the subtracted signal (ZIP)Click here for additional data file.S1 TextDescription of the Wilson-Cowan neural model and the emergence of segregation in two coupled Wilson-Cowan oscillators for increasing excitatory and inhibitory coupling strengths.(PDF)Click here for additional data file.


Many studies showed that purified cGKI autophosphorylates multiple sites at its N-terminus. Autophosphorylation might be involved in kinase activation, but it is unclear whether this happens also in intact cells. To study cGKI autophosphorylation in vitro and in vivo, we have generated phospho-specific antisera against major in vitro-autophosphorylation sites of the cGKI isoforms, cGKIα and cGKIβ. These antisera detected specifically and with high sensitivity phospho-cGKIα (Thr58), phospho-cGKIα (Thr84), or phospho-cGKIβ (Thr56/Ser63/Ser79). Using these antisera, we show that ATP-induced autophosphorylation of cGKI in purified preparations and cell extracts did neither require nor induce an enzyme conformation capable of substrate heterophosphorylation; it was even inhibited by pre-incubation with cGMP. Interestingly, phospho-cGKI species were not detectable in intact murine cells and tissues, both under basal conditions and after induction of cGKI catalytic activity. We conclude that N-terminal phosphorylation, although readily induced in vitro, is not required for the catalytic activity of cGKIα and cGKIβ in vivo. These results will also inform screening strategies to identify novel cGKI modulators.Although cGMP-dependent protein kinase type I (cGKI) is an important mediator of cGMP signaling and upcoming drug target, its  Cyclic guanosine monophosphate (cGMP) acts as a second messenger in various cell types and tissues of eukaryotes in vivo-use are of great interest to basic and clinical research. However, the development of such drugs has been hampered, in part, because the in vivo-biochemistry of cGKI is not well understood.The cGMP-dependent protein kinase type I  is considered a major mediator of cGMP signaling in mammals. Many studies suggest that pharmacologic regulation of cGKI might interfere with diverse patho-physiological processes prkg1 gene encodes two cGKI isoforms, cGKIα and cGKIβ. Each isozyme forms a homodimer of two ≈75 kDa subunits. cGKIα and cGKIβ have identical cGMP-binding and catalytic domains, but differ in their N-terminal regions (≈100 amino acids). This region mediates dimerization via a leucine zipper motif, regulates the affinity of the cGMP-binding pockets via allosteric mechanisms, and interacts, presumably in an isoform-specific manner, with anchoring and substrate proteins. It also contains an autoinhibitory/autophosphorylation region that might be involved in enzyme activation 4-(Lys)2-Lys-β-Ala-OH, using standard Fmoc/tBu chemistry Peptides containing known N-terminal cGKI autophosphorylation sites were selected based on the murine proteins (NP_001013855.1 for cGKIα and NP_035290.1 for cGKIβ) and synthesized with and without the corresponding phospho-amino acids. The peptides were synthesized as single peptides and as multiple antigen peptides, (peptide)Affinity purification of antiserum PS3 was performed as described previously 2O2 in 0.1 M citrate buffer (pH 4.5) were added to each well. After 20 min at 37°C, light absorbance was measured at 405 nm.ELISAs were performed as described Recombinant bovine cGKIα and cGKIβ were expressed in Sf9 insect cells and purified by affinity chromatography as described L-/L-) 2. MEFs were used for experiments between passage 20 and 39. VSMCs were isolated from aortae of 5- to 6-week-old mice and analyzed in primary culture without passaging.Wild-type and cGKI-deficient  and heated for 5 min at 95°C. Samples were stored at −20°C.Cultured cells were serum-starved (MEFs for 3 h and VSMCs for 48 h) in DMEM containing 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C and 6% COL-/L-) 2 and stored at −70°C. To evaluate the effects of various agents on cGKI phosphorylation, tissues were rapidly dissected and then incubated with the test compounds in Tyrode buffer  in the absence or presence of 100 nM calyculin A as specified in the respective figure legends. For Western blot analysis, tissue homogenates were prepared in lysis buffer B  using a FastPrep homogenizer . Samples were heated for 5 min at 95°C and stored at −20°C.Native tissues  were isolated from 4- to 12-week-old wild-type and cGKI-deficient mice . The cell suspension was subjected to sonication  followed by centrifugation at 18,000 g for 10 min at 4°C. The supernatant was collected and the protein concentration was measured with the Bradford assay.MEFs were serum-starved for 3 h in DMEM containing 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C and 6% COTo induce phosphorylation of cGKI, MEF extracts were incubated at 30°C for 15 min in a total volume of 500 µL. The reaction mix contained 50 mM Mes, 0.4 mM EGTA, 1 mM magnesium acetate, 10 mM NaCl, 10 mM dithiothreitol, and 200 µg cell extract. Phosphorylation was initiated by adding 0.1 mM ATP or 0.1 mM ATP combined with 0.1 mM cGMP. In some experiments, the reaction mixtures were pre-incubated with cGMP before ATP was added. The reactions were stopped by adding 1x SDS-PAGE loading buffer and heating for 5 min at 95°C. Samples were stored at −20°C.Proteins were separated by SDS-PAGE and blotted on polyvinylidene difluoride membranes (Millipore). Antibodies used were rabbit anti-cGKI common (DH) (1∶5000), a pan-specific (nonphospho-specific) antiserum detecting both cGKIα and cGKIβ t test.Data are expressed as mean ± SEM. Significance was determined by using Student's in vitro-autophosphorylation sites reported previously: phospho-Ser50, phospho-Thr58, phospho-Ser72, and phospho-Thr84 in cGKIα in silico by a group-based phosphorylation predicting and scoring method in silico method did also identify Thr56 of cGKIβ as a potential autophosphorylation site and, therefore, this site was also selected for antibody generation.To detect N-terminally phosphorylated cGKI species, we sought to generate phospho-specific rabbit polyclonal antisera against the major As depicted in e.g., 20 ng were loaded in s), the newly generated antisera appeared to recognize phospho-cGKI species at least with the same or even higher sensitivity in the lower ng-range. In good correlation with the ELISA data  and primary aortic vascular smooth muscle cells (VSMCs) were investigated. In general, these cells express both cGKIα and cGKIβ, but MEFs express predominantly cGKIα and VSMCs express more cGKIβ than cGKIα To study cGKI autophosphorylation upper panels), VASP phosphorylation was induced in wild-type MEFs by treatment with 8-Br-cGMP, 8-Br-cGMP and 8-Br-cAMP, or CNP for 15 min at 37°C. In cGKI-deficient MEFs, 8-Br-cGMP and CNP did not induce VASP phosphorylation, confirming that the cGMP-induced VASP phosphorylation observed in wild-type cells was indeed mediated by cGKI. Combined treatment with 8-Br-cGMP and 8-Br-cAMP still resulted in an increase of phospho-VASP in cGKI-deficient MEFs, most likely because 8-Br-cAMP had activated the cAMP-dependent protein kinase. Surprisingly, although our stimulation protocol clearly induced cGKI phosphotransferase activity in intact MEFs, our phospho-specific cGKI antisera did not detect epitopes on the Western blot that could potentially represent autophosphorylated cGKI species, both under basal and stimulated conditions, and when huge amounts of protein (50 µg) were loaded (lower panels). Very weak signals were detected by AffPS3 and PS7, and stronger bands were detected by PS6. However, it was unlikely that these signals were derived from phosphorylated cGKI. First, most of these bands did not co-migrate with purified autophosphorylated cGKI. Second, similar signals were detected under basal and cGKI-activated conditions. Third, and most importantly, these bands were also detected in cGKI-deficient MEFs. A similar experiment was performed with primary VSMCs . Next, we tested whether phospho-cGKI could be detected when the experiment was performed in the presence of the cell-permeable protein phosphatase inhibitor calyculin A, which inhibits the Ser/Thr phosphatases PP1 and PP2A in vivo is very low under basal conditions, but increases to detectable levels after stimulation of cGKI activity. To test this hypothesis, we treated acutely isolated bladder and lung tissues in the presence of calyculin A with 8-Br-cGMP, 8-Br-PET-cGMP, or DEA-NONOate, a NO-releasing compound that promotes endogenous cGMP synthesis via soluble guanylyl cyclase. Although these treatments increased the level of phospho-VASP indicating that cGKI was activated, phosphorylated cGKI species were not detectable with our antisera in the stimulated tissues  indicated that, at least in in vitro, (a) cGKIα can undergo N-terminal phosphorylation in a non-cGMP-activated state and (b) autophosphorylation alone does not activate heterophosphorylation of substrate proteins like VASP.ATP-induced phosphorylation of endogenous cGKIα and its inhibition by cGMP were also observed when cell extracts prepared from MEFs were incubated ompounds . This exompounds , lane 2 in vitro experiments with purified cGKI. According to this model  and three potential phospho-sites of cGKIβ . We cannot exclude phosphorylation of cGKI at Ser/Thr residues that were not recognized by our antibodies. Alternative in vivo phosphorylation sites of cGKI under baseline and activating conditions could be identified by a hypothesis-free approach based, for instance, on mass spectrometry. However, autophosphorylation of these alternative sites would be expected to coincide with autophosphorylation of at least some of the major sites that were detected by our antisera.In this study, phospho-specific antibodies were generated that detect autophosphorylated cGKIα and cGKIβ with high specificity and sensitivity. Autophosphorylation of cGKI could be induced in purified preparations or cell extracts by addition of ATP. However, phospho-cGKI species could not be detected in intact cells and tissues, both under basal conditions and after induction of cGKI catalytic activity. These findings challenge the physiological relevance of the current cGKI activation model that is based on is model  [6], autin vivo-phosphorylation status of cGKI. Hou et al. 32P]PPi and could not detect an increase of [32P]-cGKIα after treatment of the cells with 8-Br-cGMP. Pinske et al. in vivo as a phosphoprotein that is mainly phosphorylated at Thr516, but shows no N-terminal autophosphorylation.Our failure to demonstrate N-terminal autophosphorylation of cGKI in intact cells with phospho-specific antibodies is in agreement with previous studies that used other methods to determine the in vitro. Phosphorylation of cGKI could be induced by addition of ATP (0.1 mM) to the purified enzymes or cell extracts. Interestingly, ATP-induced autophosphorylation of cGKIα was inhibited by pre-incubation with saturating amounts of cGMP (0.1 mM). These data are in line with previous studies that reported cGKI autophosphorylation in the presence of ATP alone and its inhibition by the addition of cGMP lane 2). Taken together, our data indicate that N-terminal phosphorylation of cGKI (a) does not require, and can be even inhibited by a cGMP-activated conformation of the kinase and (b) does not increase the basal catalytic activity of the kinase toward exogenous substrates in the absence of cGMP.Our experiments with purified proteins  and 6A ain vitro but not in vivo? Considering that purified cGKI autophosporylates in the presence of 0.1 mM ATP, and that the intracellular ATP concentration is typically 1–10 mM, one would expect that autophosphorylated cGKI occurs in vivo already under basal conditions. However, we did not detect phospho-cGKI in intact cells. This suggests that the conformation and/or environment of the kinase in intact cells differ fundamentally from purified protein and broken-cell preparations, in which autophosphorylation occurred. The balance between auto- and heterophosphorylation could be influenced by the availability of physiological partner proteins of cGKI, such as anchoring and substrate proteins. Purified cGKI preparations lack these factors and cell extracts contain them in much lower concentrations than intact cells. Interestingly, cell extracts showed cGKI autophosphorylation in the absence of VASP phosphorylation (lane 2), whereas intact cells demonstrated VASP phosphorylation in the absence of autophosphorylation (in vitro conditions autophosphorylation is preferred as compared to phosphorylation of exogenous substrates. However, autophosphorylation is obviously prevented in intact cells by the interaction of cGKI with other proteins, and after cGMP activation only heterophosphorylation of substrate proteins occurs. This also implies that autophosphorylation is not involved in cGKI activation in vivo, and we propose to revise the working model of cGKI accordingly (in vitro assays with purified cGKI protein. Contrary to what would be suggested by the previous model that incorporated autophosphorylated cGKI as a relevant enzyme species, our present results strongly suggest that these assays should not be performed with autophosphorylated cGKI.Why does cGKI readily autophosphorylate rylation , lane 2,rylation , 4, 5. Tordingly . The finin vitro, autophosphorylation of cGKIα and cGKIβ does most likely not occur in vivo. Thus, the catalytic activity of cGKI in intact cells appears to be independent of N-terminal autophosphorylation. These findings also support the general notion that the in vitro- and in vivo-biochemistry of a given protein can be fundamentally different.In conclusion, this study provides important new insights into the structure-function relationship of cGKI in intact cells. Although readily induced


Although Indonesia has been rabies-infected since at least the 1880s, some islands remain rabies-free, such as Lombok. However, due to its adjacency to rabies-infected islands such as Bali and Flores, there is considerable risk of a rabies incursion. As part of a rabies risk assessment project, surveys were conducted to estimate the size of the dog population and to describe dog management practices of households belonging to different ethnic groups. A photographic-recapture method was employed and the number of unowned dogs was estimated. A total of 400 dog owning households were interviewed, 300 at an urban site and 100 at a rural site. The majority of the interviewed households belonged to the Balinese ethnic group. Owned dogs were more likely male, and non-pedigree or local breed. These households kept their dogs either fully restricted, semi-free roaming or free-roaming but full restriction was reported only at the urban site. Dog bite cases were reported to be higher at the urban site, and commonly affected children/young adults to 20 years old and males. A higher number of unowned dogs was observed at the urban site than at the rural site. Data generated within these surveys can inform rabies risk assessment models to quantify the probability of rabies being released into Lombok and resulting in the infection of the local dog population. The information gained is critical for efforts to educate dog owners about rabies, as a component of preparedness to prevent the establishment of rabies should an incursion occur. Dogs are closely associated with humans, being kept by households as companions and are often considered as part of the family, especially in developed countries ,2. In sopers. comm., 2013). Rabies eradication efforts in most infected provinces have not been successful for several reasons, but most importantly the difficulties encountered in vaccinating free-roaming dogs [Rabies was first reported in Indonesia during the 1880s , and is ing dogs . The faiing dogs . With thing dogs , the spring dogs .Consideration of the role of the dog, level of dog ownership and nature of dog management in a community appears warranted when planning for rabies control and prevention in Indonesia. Rabies was successfully eradicated from areas of Java, where the control program was aided by a high proportion of confined owned dogs compared to free-roaming dogs that allowed vaccination of a sufficient proportion of the dog population to prevent rabies transmission . CommuniLombok Island, a rabies free island in eastern Indonesia situated between rabies infected Bali Island (35km east) and Flores Island (300km west), is at risk of rabies introduction. Understanding the presence of dogs within ethnic groups living on Lombok Island, and management of their dogs, is necessary to develop strategies to prevent and to control a potential rabies incursion. However, no study in Indonesia has yet been conducted on rabies-free islands to understand the role of ethnicity. Our overall program goal was to investigate pathways for the entry of rabies to Lombok via dog movement, and assess the probability of rabies entry and exposure to the susceptible dog population on Lombok. To inform this risk assessment, a survey of dog-owning households and photographic capture methods were conducted. Our objectives were to describe dog management practices of dog-owning households belonging to different ethnic groups at an urban and a rural site on Lombok Island, and to describe dog bite occurrence and estimate dog population size at these sites.2 and is inhabited by 3,200,686 people, with fishing, agriculture and tourism the important sources of income [Lombok is one of two larger islands within West Nusa Tenggara (NTB) province, located in the eastern part of Indonesia. It has a total land area of about 4,725 kmf income . Lombok f income ,23. The The study sites purposely selected for this survey were one city and one district on Lombok, representing an urban area and a rural area, considered high risk for the entry of rabies and inhabited by more than one ethnic group. Mataram City, the selected urban area, is located near the west coast of Lombok and is the major commercial center on the island . This site was selected as it includes all ethnic communities living on Lombok Island, has a higher socio-economic status than other areas of Lombok and a high density of people and dogs. The selected rural area was West Lombok District due to its close proximity to rabies-infected Bali, the presence of several informal ports, and the presence of more than one ethnic group.Within the two sites, we purposively selected: Cakranegara District (urban) and Batu Putih village . Cakranegara District, a central business area in Mataram City, is inhabited predominantly by Balinese and Sasakese ethnic groups with a minority of other ethnic groups, and has a high density of people and dogs compared to other districts. Batu Putih village was selected because it has the informal port which is closest to Bali of all the ports in Lombok . This informal port has no quarantine post and is often visited by boats from Bali. Furthermore, this village is inhabited predominantly by Balinese and Sasakese ethnic groups and fishing is an important occupation.http://www.clive.ed.ac.uk) assuming a 95% confidence level, 5% precision and an assumed pedigree ownership proportion of 20%. For the urban site an infinite population was assumed  whilst a finite population of 150 households per ethnic group was assumed in the rural site. The target sample size per ethnic group was 235 and 94 households in the urban and rural site, respectively.The study population for this survey was dog-owning households at the urban and rural sites. These were divided into two categories according to the ethnic group: Balinese and non-Balinese. The required sample size to estimate the proportion of households owning a pedigree dog in each ethnic group was calculated  were selected using the random process described below for this study. At each of these 12 locations, the interview team consulted with the community leader  to seek information on dog-owning households in the area. Of the 12 locations, five were known to be inhabited by mainly people of the Balinese ethnic group. At each of these five locations, 50 Balinese dog-owning households were conveniently selected and interviewed. Households were approached by an interview team in conjunction with the community leader; if the dog owner or the person who took care of the dog was present, he/she was interviewed.In contrast, at the remaining seven locations, where most people were from a non-Balinese  ethnic group, the community leader did not have information on the dog-owning households residing at these locations. Thus an alternative convenience approach was implemented after consultation with a veterinarian from Mataram City, who provided addresses of approximately 80 non-Balinese dog-owning households from across the city. Of these, 32 were identified and interviewed, who also provided information on 18 additional households. This resulted in 50 non-Balinese dog-owning households located in an urban area being interviewed. As a result the non-Balinese participants also included households in Mataram City outside Cakranegara district.At the rural site, although the required sample size was 94 households for each ethnic group, only 60 Balinese dog-owning households were identified by the village leader. No information about dog ownership among the non-Balinese (Sasakese) was available and therefore the same number was targeted for this ethnic group. For the Balinese ethnic group, each household in the village was visited and an interview conducted when the occupant was at home at time of visit and if currently owning dog/s. A total of 50 interviews were completed. Contact and interviews with 50 Sasakese dog-owning households was achieved using the snowballing method with initial households identified by Balinese participants and the remainder by Sasakese participants.A structured questionnaire was designed to obtain data on number of dogs owned, dog function, dog management and dog bites from dog-owning households. The questionnaire was developed in English and translated into Bahasa Indonesian by an independent university academic, then back translated from Bahasa into English by a member of the Indonesian research team. Points of difference were identified and discussed by the two translators and clarified to determine the correct final wording in Bahasa. Minor modification of the questionnaire was performed after piloting it with ten dog-owning households at the urban and rural sites, including Balinese and non-Balinese households. The data obtained from the pilot trial were included in the data analysis. The questionnaire is available from the corresponding author upon request.The questionnaire survey was conducted from January to April 2012 by the lead author and a team of eight people from the Research Centre for Rural Development, University of Mataram, NTB Province. This team had extensive experience in conducting questionnaire surveys, including animal health surveys in NTB province and had a 1-day training to become familiar with the dog-management questionnaire.The questionnaire was administered via a face-to-face interview with the person responsible for the dog management at the household residence. The interviews were completed within 30 to 35 minutes and a rabies brochure and incentive of 20,000 IDR (about USD 2) were given to participants on completion of the interview. Ethics approval for this survey was obtained from the Ethics Committee at the University of Mataram, and participants provided written informed consent to participate in this study.http://www.animalsheltering.org/training-events/expo/expo-2013-handouts/hsi-humane-dog-management-gamboa1.pdf, accessed 20 May 2013) to achieve a random selection of Lingkungan for sampling that are well distributed across the chosen district, with each Lingkungan having a known and equal probability of selection. Due to logistic considerations, the total number of Lingkungan that could be managed by the research team was 12. Of these, seven were mainly inhabited by non-Balinese and five were mainly inhabited by Balinese. As results from the dog-owning household survey reported a low number of dog-owning households in the seven non-Balinese Lingkungan, only the five Lingkungan inhabited by the Balinese ethnic group were selected to conduct the unowned dog counting activity. At the rural site , dog counting was conducted throughout the entire site.The aim of this counting exercise was to compare the size of the dog population in the urban and in the rural study areas. For the estimation of unowned dog population size, the same sites described above were used. At Cakranegara District, 12 of 51 Lingkungan were selected using the World Society for the Protection of Animals approach  and visibility (good or not). The age, sex, body size and coat colour of each dog seen during counting were also recorded. Further the location where the dog was seen, the amount of garbage in the village  and the ‘sighting status’  were recorded.The dog counting activity was conducted by the lead author and two personnel from the Animal Health Care Centre of the Agriculture, Marine and Fisheries Office in Mataram City. It was undertaken during April 2012, toward the end of the rainy season (November to May in Lombok). A preliminary visit was made to the urban and rural sites to find the most appropriate time of the day to conduct the unowned dog counting, according to the traffic and presence of street lighting. At Cakranegara District, the most feasible time was at night from 12:00 am to 02:00 am; whilst at Batu Putih village, the most feasible time was early morning between 6:00 am and 8:00 am, when traffic was minimal and natural light sufficient for observations (the lack of street lighting at this site prohibited dog counting at night-time). The counting was carried out at each location on two consecutive days.The target dogs for the dog counting activity were unowned dogs defined as being free to roam and having no identifiable owner. As distinguishing free-roaming owned dogs from unowned dogs was challenging, dog owners (through village leaders during community gatherings) at these sites were asked to tether or collar their dogs during the dog counting activity.The photographic recapture method was used to estimate the population of unowned dogs, implementing a modification of Schnabel’s variation of the Petersen-Lincoln Index using multiple recaptures . As BeckPermission to undertake dog counting was obtained from community leaders with Cakranegara District and Batu Putih village. This field study did not involve endangered or protected species, and animal ethics committee approval was not required since no handling of dogs or activities that would change their behaviour were undertaken.th edition . Categorical data were described using frequency tables and continuous variables using mean, median and range. Standard statistical tests were used to determine differences between categories for categorical variables (chi-square) and between means for continuous variables (t-test). The estimated number of unowned dogs was calculated using a Chapman estimate, with corresponding variance , Sasakese , Javanese , Flores , Batak , Bima  and expatriate . At the rural site, there were 50 Balinese and 50 non-Balinese  households.Among the urban dog-owning households, 186 (62%) owned one dog, 81 (27%) owned two dogs, 22 (7.3%) owned three dogs and 11 (3.7%) owned between four and six dogs. Of the 100 rural dog-owning households, 57 (57%) owned one dog, 31 (31%) owned two dogs, three (3%) owned three dogs and nine (9%) owned between four and seven dogs. The mean number of dogs owned per household at the urban (mean 1.6) and rural sites (mean 1.7) was not significantly different (P = 0.938).The most common type of dog kept by all ethnic groups interviewed was non-pedigree, except for the Chinese ethnic group, in which 77.3% of households owned pedigree dogs. The percentage of households owning pedigree dogs was higher at the urban site than the rural site .Guard dog was the main function of dogs kept by both ethnic groups  at both sites: guarding the house at the urban site and guard the house and plantation at the rural site. Dogs were kept solely as pets by Balinese, Chinese, Javanese, Timorese and expatriate households at the urban site, with the proportion notably higher for Chinese households. Only one Chinese household reported keeping dogs to trade .n = 468) and 67.6% at the rural site (n = 170) , constituting 74.1% of dogs at the urban site (n = 170) , with a n = 170) . The majn = 170) .The movement of dogs living at the urban site was better controlled than at the rural site. Of the 468 dogs at the urban site, 137/468 (29.3%) were restricted at all times, whereas dogs at the rural site were either always allowed to roam outdoors or semi-free roaming (roaming freely for some hours each day), and none of the dogs were restricted at all times . The disA total of 27 households that were interviewed reported 29 dog bite cases during the last five years. One Balinese household reported 3 dog bite cases. At the urban site, 12 Balinese households reported 14 dog bite cases: the median age of the 14 people bitten was 34 (1−77) years. One non-Balinese (Timorese-Flores) household reported one dog bite case, aged 17 years old. At the rural site, 6 dog bite cases were reported by 6 Balinese households, median age 14 (5−65) years, while 8 non-Balinese households reported 8 dog bite cases, median age 37.5 (10−65) years.Of the total 29 dog bite cases, half were children and young adults . There were more dog bite cases in males  than in females  (P = 0.194). The leg was the part of the body reportedly bitten the most (68.9%), then hand/arm (17.4%), stomach (6.9%), hip (3.4%) and shoulder/neck (3.4%). The dog bite cases were frequently bitten by another person’s dog or their own dog ; only 6.9% of bite cases were bitten by a free-roaming dog. Only five people at the urban site and one person at the rural site went to hospital for treatment of bite wounds: all belonged to the Balinese ethnic group. The other cases treated their wound/s at home by cleaning the wound with or without application of iodine. Other types of treatment, such as using traditional medication, were reported at both the urban and rural sites, but were more common at the rural site. Non-treatment of the wound was higher at the rural site .During dog counting, it was assumed that all dogs present without a collar on the counting days were ownerless dogs because dog owners had been asked to confine or collar their dogs. Only one dog with a collar was viewed during that time. The majority of houses at the two sites were fenced. Only dogs present outside residences' fences were counted. Unowned dogs were seen near garbage piles, on the streets, and sleeping in front of closed shops, around unfinished buildings and in empty water drainage. The estimated number of unowned dogs at the urban site was 180 ± 14.2 SD and at the rural site 65 ± 7.6 SD .pers. comm., 2013). The preference appears to be related to guarding duties, given the majority of interviewed households keep dogs for this reason. A preference for keeping dogs for guarding purposes has also been observed in other countries such as Madagascar, Tanzania and the Philippines [Of the dog-owning households interviewed in this survey, the majority  owned only one dog, most commonly non-pedigree and more commonly male. Dogs are used to guard houses, and in rural areas to guard plantations against monkeys and feral pigs destroying valuable crops. Based on common local understanding, female puppies are likely to be abandoned soon after birth  and a variety of dog breeds sold by pet shops, traditional live animal markets, dog traders and dog breeders; on Lombok Island, pedigree dogs are not easily found. This creates the possibility of further illegal dog imports not only for household purposes, but also for trading purposes.In this survey, most (71%) owned dogs were semi-free roaming or free-roaming at the urban site, and all owned dogs at the rural site were semi-free roaming or free-roaming. A substantial number of roaming dogs is a threat for rabies transmission, should an incursion occur in Lombok, and for transmission of other zoonotic diseases. Therefore, control and management of owned dogs is needed through education of dog owners by relevant government authorities and veterinarians.This survey recorded dog bite cases at both urban and rural sites. The reported dog bite cases were common in children/young adults (up to 20 years old) and more common in males than females. This distribution has also been found in other countries in Asia, such as Bangladesh, Bhutan and India.−30 HumanThe survey conducted to estimate dog population size used the WSPA approach to select locations at the urban site. Of the 12 locations selected, only five Lingkungan that were inhabited mainly by the Balinese ethnic group were actually used. Given that dogs are more tolerated and appreciated in these communities, targeting Balinese locations provides an estimate of the likely maximum dog population size to inform rabies exposure assessment and response strategies. According to our survey, the estimated number of unowned dogs at the urban site is higher than at the rural site . It appears that the type of garbage, such as fish waste, leftover meals and other household kitchen garbage seen at the urban site provides food for the unowned dogs. A study conducted at Sao Paulo also showed the importance of garbage for free-roaming dogs .Lack of dog population control can lead to higher numbers of free-roaming dogs . ApproacFeasible methods of unowned dog population reduction on Lombok may include reducing dog access to garbage disposal areas: for example, fencing the dump area. Further, regular sterilisation of free-roaming dogs (unowned and owned dogs) will also assist population reduction. Inexpensive identification should be provided for the sterilised dogs. Education of dog owners to promote responsible dog ownership is also necessary, as many semi-free roaming and free-roaming owned dogs are seen on Lombok. Owned dogs that are allowed to roam freely will likely contribute to the unowned dog population (through breeding) and to disease transmission.Dog counting at the urban and rural sites provided initial data about the unowned dog population in urban and rural Lombok. The main limitation of this was utilisation of a simple dog population estimation method to estimate the unowned dog population. This method is not the best way to estimate the number of dogs . Other mPromoting responsible dog ownership  on Lombok should is an important component of activities to prevent the introduction and establishment of rabies on Lombok. However, this needs to consider the diversity of cultural and religious beliefs. Dog management could be included in community-based programs on rabies and thus presented in the context of rabies prevention, surveillance and early detection of a rabies incursion.The pattern of dog ownership on Lombok varies between ethnic groups. Education of dog owners about the control and management of their dogs is needed as part of a program to reduce the likelihood of a potential rabies incursion becoming established. There needs to be an emphasis on responsible dog ownership (particularly dog confinement and desexing).S1 Dataset(XLSX)Click here for additional data file.


Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that is a primary regulator of cellular proliferation through targeting of proliferative kinases, cell cycle regulators, and apoptosis inhibitors. It is through the regulation of these regulatory elements that gives PP2A tumor suppressor functions. In addition to mutations on the regulatory subunits, the phosphatase/tumor suppressing activity of PP2A is also inhibited in several cancer types due to overexpression or modification of the endogenous PP2A inhibitors such as SET/I2PP2A. This review focuses on the current literature regarding the interactions between the lipid signaling molecules, selectively sphingolipids, and the PP2A inhibitor SET for the regulation of PP2A, and the therapeutic potential of sphingolipids as PP2A activators for tumor suppression via targeting SET oncoprotein. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase, which serves as a regulator of cell death and/or cell division, functions performed by targeting kinases and other effectors of these processes including Bcl-2 and the Aurora kinases –3. In noStructurally, PP2A is a holoenzyme, which typically contains a catalytic “C” subunit, coupled with an “A” scaffolding subunit, and a regulatory “B” subunit Figure A 4). So. So4). SProtein phosphatase 2A was first identified as a tumor suppressor when it was discovered that the known carcinogen okadaic acid is a specific PP2A inhibitor –7. SimilProtein phosphatase 2A can be regulated through changes in phosphatase activity as well as changes in substrate specificity, the latter of which is primarily controlled by the regulatory B subunits. The B subunits are often tissue-specific and mediate subcellular localization of the holoenzyme. The catalytic activity is controlled by post-translational modification of the C-subunit and through endogenous inhibitory proteins. Although the specific interplay between each of these inhibitory proteins and the PP2A holoenzyme is not fully understood, they are typically found together in a complex suggesting a role for direct inhibition.One of these endogenous inhibitors is Inhibitor 1 of PP2A  . I1PP2A Cancerous Inhibitor of PP2A (CIP2A) was first described as a fusion protein detected in a leukemia patient , which iInhibitor 2 of PP2A  is an endogenous PP2A inhibitor and a well-described proto-oncoprotein. Although the specific mechanism by which SET inhibits PP2A is believed to be through direct binding to the catalytic domain of PP2A Figure , 30 and Several of the domains of the SET protein have been attributed to specific functions. An internal span close to the N-terminus (residues 36–124) has been found to be critical for PP2A inhibitory function . Convers+ primary patient samples restored PP2A activity indicating a causal role for SET in PP2A inhibition in these cells. Additionally, CML cells expressing SET knock-down had reduced BCR/ABL1 phosphorylation (activity) as well as reduced clonogenic potential. SET was also found to be overexpressed in lung tumors when compared to normal lung tissue with an accompanying increase in PP2A phosphorylation (inhibition)  leads to an enhanced mRNA stability, which produces an increase in SET protein levels . Knock-dibition) . Knock-dnd Ramos . Reactivnd Ramos .de novo through a series of steps starting with serine palmitoyltransferase (SPT), which uses acetyl-CoA and serine to form 3-keto-dihydrosphingosine. This product is desaturated by dihydrosphingosine desaturase (DES) to produce dihydrosphingosine, which is the acylated by ceramide synthase (CerS1-6) to form ceramide with the desaturation reaction by dihydroceramide desaturase. Alternative pathways of synthesis of ceramide are through the breakdown of sphingomyelin by sphingomyelinases or originating directly from sphingosine produced by the dephosphorylation of sphingosine-1-phosphate (S1P) via the salvage pathway involving CerS function  is a synthetic sphingosine (myriocin) analog currently approved for the treatment of multiple sclerosis. Originally designed as an immunosuppressant, FTY720 is given orally as a pro-drug. Once internalized within a target cell, FTY720 is mainly phosphorylated by sphingosine kinase 2 (SphK2), producing FTY720-P. Following phosphorylation, FTY720-P is exported out of the cell, where it functions in an autocrine/paracrine manner by binding to the sphingosine-1-phosphate receptor 1 (S1PR1). Following binding to S1PR (except S1PR2), the receptor is internalized and degraded, leading to immune suppression. Thus FTY720-P is a functional antagonist for S1PR1. FTY720 was found to activate PP2A by Nagahara et al. in HL-60RG and Jurkat cells . As a spWhile SET can be regulated through expression levels or phosphorylation changes, the inhibitory properties of SET are also controlled through sphingolipid signaling. While sphingolipids have been shown to bind another PP2A inhibitor, I1PP2A/ANP32A, SET is unique in that it contains a binding “pocket,” which affords high affinity and substrate selectivity for ceramide.Ceramide was described as a PP2A activator in 1993  and, alt6-ceramide to pull-down ceramide associated proteins found in A549 lung carcinoma cell extracts using avidin-conjugated columns  D-e-C6-ceramide, while its isomers Pyr-dihydro-C6-ceramide, Pyr-L-e-C6-ceramide, Pyr-D-theo-C6-ceramide, or Pyr-D-theo-C6-ceramide produced no PP2A activation. Taken together, these data indicate that ceramide functions as a PP2A activator through, at least in part, the binding to SET.In fact, SET was found to be a direct target of ceramide by Mukhopadhyay et al. by employing biotin-labeled C columns . The proin silico modeling to be significant for ceramide binding. When mutated to K209D (replacing the positively charged R-group of lysine with the negatively charged group of aspartate), the ability of ceramide to bind SET was decreased by >75%. Moreover, ceramide-mediated PP2A activation induced proteasomal degradation of c-Myc, and tumor suppression , which is responsible for the generation of C18-ceramide were decreased in lung tumor tissue compared to normal lung tissue. Interestingly, the mRNA of CerS6 and its product, C16-ceramide were both increased in lung tumor tissue  and biotin-tagged FTY720 pull-downs FTY720, but not immunosuppressive FTY720-P, was found to bind directly to SET  suggest that it too, may be a ligand for SET. y to SET . Similary to SET . These sy to SET . Howeverin situ and in vivo. Surprisingly, through the use of caspase 3/7 knockout MEFs and the caspase inhibitor Z-VAD, FTY720-induced cell death was found to be caspase-independent  reduced c-Myc levels in each of the cancer cell lines. Moreover, pretreatment with the PP2A inhibitor okadaic acid (10 nM) increased basal c-Myc levels and prevented a decrease following treatment with C6-ceramide .To increase ceramide levels, synthetic Creatment . While n+ cell lines with FTY720-induced caspase-depended apoptosis through a PP2A-dependent pathway  and dose (7.5–10 μM)-dependent manner. Particularly, these same ceramides were enriched in the mitochondrial fraction of FTY720-treated cells with C22-ceramide showing a dominant increase. Intriguingly, sphingosine-1-phosphate (S1P), a known inhibitor of PP2A activity (Recently, Chen et al. found that treating AML-ETO pathway . To unraactivity , was fouactivity .Protein phosphatase 2A is commonly deregulated in cancers, typically due to inhibition by SET. The protein level of SET can be increased or SET can be subject to post-translational modification through a variety of oncogenic pathways, each resulting in the inhibition of PP2A activity Figure . The comThe 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.


Crk is the prototypical member of a class of Src homology 2 (SH2) and Src homology 3 (SH3) domain-containing adaptor proteins that positively regulate cell motility via the activation of Rac1 and, in certain tumor types such as GBM, can promote cell invasion and metastasis by mechanisms that are not well understood. Here we demonstrate that Crk, via its phosphorylation at Tyr251, promotes invasive behavior of tumor cells, is a prominent feature in GBM, and correlating with aggressive glioma grade IV staging and overall poor survival outcomes. At the molecular level, Tyr251 phosphorylation of Crk is negatively regulated by Abi1, which competes for Crk binding to Abl and attenuates Abl transactivation. Together, these results show that Crk and Abi1 have reciprocal biological effects and act as a molecular rheostat to control Abl activation and cell invasion. Finally, these data suggest that Crk Tyr251 phosphorylation regulate invasive cell phenotypes and may serve as a biomarker for aggressive GBM. The CT10 regulator of kinase (Crk) family of adaptor proteins, CrkI and CrkII, and a related gene product CrkL, are versatile Src homology 2 (SH2) and Src homology 3 (SH3) domain-containing proteins that regulate protein-interactions downstream of tyrosine kinases . The besRecently, we described a non-canonical pathway for Crk signaling whereby Tyr221 phosphorylation, rather than turning off Crk signaling, redirects signaling from the conventional SH2->SH3(N) circuitry to an unconventional SH3(N)->pSH3(C) circuitry, mediated by iterative tyrosine phosphorylation of Crk on Tyr221 and Tyr251 or Tyr239 . Using bIn contrast to Crk, other SH3 domain-containing adaptor proteins concomitantly interact with Abl but do so with opposing effect on Abl activity. For example, Abi1, an adaptor protein identified in screens for spectrin , Eps8 1 and Abl In addition to interacting with Abl, Abi1 contributes to the composition of the WAVE protein complex that regulates actin polymerization downstream from a variety of receptors , and upsIn the present study, we show that Crk and Abi1 have reciprocal and opposing effects on Abl kinase activity, whereby Abl is affirmatively regulated by pTyr251 Crk phosphorylation, and negatively regulated by Abi1 isoform 2. We demonstrate that Crk Tyr221 and Tyr251 are routinely and iteratively tyrosine phosphorylated by EGF in EGFR-expressing glioblastoma cell lines (GBM), and at both the biochemical and cell biological level, the level of Tyr251 Crk phosphorylation strongly correlates with Abl kinase activation, as well as the invasive properties of GBM cells. Moreover, in patient-derived clinical GBM samples, Crk expression and Y251 phosphorylation is upregulated, and Abi1 levels are downregulated, and the level of Crk and phospho Crk Y251 is inversely correlated with patient survival outcomes. These data support the idea that Abi1 has tumor suppressor-like properties in GBM, and does so by unleashing the Crk non-canonical phosphorylation axis to activate Abl kinase and induce invasive phenotypes.We investigated the potential interplay between Crk Y251 phosphorylation, loss of Abi1, and Abl activation, by meta-analyzing genomic datasets obtained from annotated next generation sequencing studies in tumor samples of GBM cancers as described by CBioportal cancer genome analysis , 27 and n = 32) versus matched normal tissue samples (n = 18), and consistent with the data using cell lines, GBM samples have up-regulated protein levels of EGFR (1.7 fold), CrkpY251 (1.5 fold), Crk (1.45 fold) and decreased level of Abi1-Iso2 (0.82 fold)  Figure .p = 0.02, p = 0.029, respectively). Inversely, Abi1-Iso2 expression was downregulated in undifferentiated (G4) GBM cancer tissues as compared to lower grade G2 and G3 glioma cancer tissues  was noted by chi-square test and that Crk and EGFR expression were significantly associated with the age of glioma patients (Table p < 0.001 and p = 0.048). No significant relationship was found between EGFR, Crk, Crk pY251 and Abi1 protein expression with the gender of glioma patients . By contrast, although low expression of Abi1-Iso2 appeared to have lower overall survival, this could not reach statistical significance . H&E staining were performed on all the specimens to assess the tumor grades , were enriched when Crk and Abi1 expression was altered . In fact, when co-incubated with GST-Crk, GST-Abi1 reduced the Crk-mediated Abl transactivation in a concentration dependent manner to background levels  cells stimulated with EGF showed enhanced CrkY251 phosphorylation and AblY245 phosphorylation  MEFs reconstituted with Abi1 to rule out the possibility that other Abi1 isoforms may compensate for Abi1-Iso2 in the HS683 tumor cells. Consistent with the aforementioned n Figure . Moreoven Figure . Finallyn Figure , the ratn Figure  while thn Figure .in vitro kinase assay. Consistent with the co-transfection experiment, co-incubation of Abl PP with purified Abi1-Iso2, significantly suppresses Abl PP's constitutive kinase activity (shown by reduction in Abl pY412 phosphorylation) in a concentration-dependent manner  and Crk(−/−) MEFs, and genetically modified GBM cells, our studies show that Abi1 functionally suppresses Crk binding to Abl, functionally suppresses Crk Tyr251 phosphorylation, and functionally suppresses Crk-mediated enhancement in cell motility and invasion. Together, these data are consistent with a potential tumor suppressor role for Abi1 in GBM, and raise a paradigm by which the non-canonical Crk pY251 pathway becomes functionally operational in tumor cells that down-regulate Abi1.An important consequence of the aforementioned scenario, in which Crk and Abi1 compete for the same proline-rich motifs in Abl, implicate a net equilibrium in these adaptor proteins exist that can shift the equilibrium towards Abl activation or Abl inactivation depending on the net balance of Crk and Abi1 expression. Indeed, using meta-analysis of genomic datasets derived from human GBM, a general trend is observed towards Crk up-regulation and Abi1 down-regulation. Moreover, using a combination of Indeed, both experimental results and the accompanying database mining analysis for Crk and Abi1 suggest that Crk Tyr251 phosphorylation might be informative as a biomarker for invasive GBM. The expression studies shown here support the idea that high levels of Crk, pCrkY251, and EGFR predict aggressive behavior of GBM and patient survival outcomes. Moreover, the fact that Crk Tyr251 can also be phosphorylated by EGFR  indicateIn summary, our studies have identified a novel regulatory network by which Abi1 and Crk reciprocally interact with Abl kinase to drive the malignant phenotypes in EGFR-expressing human GBM cells Figure  and funcGBM tissues were histopathologically diagnosed at First Affiliated Hospital of Wenzhou Medical University from 2013 to 2015. Normal brain tissue samples were obtained during autopsy, after accidental death, with no clinical or pathological evidence of brain tumor or other abnormality. All GBM and normal tissues were immediately shock frozen in liquid nitrogen and stored at −80°C. The pathological grade of GBM was defined according to WHO-criteria. This study was approved by the Board and Ethical Committee of Wenzhou Medical University. Written informed consents were obtained from all patients who enrolled in this study.Glioma TMA containing a total of 43 formalin-fixed, paraffin-embedded tissue sections (4μm thickness) were constructed as previously described . IHC wasCells were lysed in 1% HNTG buffer supplemented with sodium vanadate, sodium molybdate and protease inhibitor cocktail. Western blot analyses were performed using EGFR, phospho-Y221 CrkII, previously described phospho- Crk Y251 (CST), anti-pY245 Abl, and anti-pY412 Abl , phosphotyrosine antibody PY99, GST (Santa Cruz Biotechnology Inc.), Abl Ab-3 , and Abi1 1B9 antibodies. For immunoprecipitation, cell lysates were made in 1% HNTG buffer, incubated with respective antibodies and supplemented with protein A/G agarose beads. After incubation and repeated washing with 0.1% HNTG buffer, samples were boiled and analyzed by immunoblotting.2). The reaction mixtures were kept on ice with intermittent mixing for 1hr. 0.1mM ATP was added to the individual reaction mix and incubated in at 25°C for 10min. The reactions were terminated by addition of SDS sample buffer and analyzed by immunoblotting.10nM Abl 1a was incubated with a 100-1000 fold molar excess of GST, GST-CrkII or GST-Abi1 isoform2 proteins in kinase buffer , were added in triplicates in upper chamber and change in Cell Index (CI) were measured every 5min for mentioned time periods. For invasion assays, an additional 10% matrigel plug was used in the upper chamber.Cells were serum starved overnight, washed with 1X PBS and supplemented with 100ng/ml hEGF containing medium. Using a 200uL sterilized tip, single scratches/ well were made in sterile conditions. Images were taken quickly after this step followed by imaging every 3hrs.ABI1 and CRK expression were selected based on the normalized expression of these genes relative to the remainder of the dataset.Bioinformatics meta-analysis for genomic and gene expression changes were performed on next-generation sequencing data from multiple studies made available through CBioportal for Cancer Genomics and Oncomine databases. Results were represented as on a log2 scale as Box- Whisker plots. P value of less than 0.003 was considered significant. The gene expression data of CrkII and Abi1-Iso2 were used to classify human GBM cases into high and low groups by SurvExpress analysis. Gene Set Enrichment Analysis (GSEA) was performed on the Broad Institute GenePattern Server using 75th-percentile normalized RSEM gene expression values downloaded from the NCI Data Matrix Portal. Cases of coupled or uncoupled p < 0.05.All the experiments were repeated at least 3 or more times. Unless otherwise mentioned, all measurements are shown as mean ± SEM. ANOVA and Student's t-test were performed as mentioned using GraphPad Prism software. The χ2 test was used to analyze the relationship between respective protein expressions and clinicopathological characteristics. Statistical significance was set at