They also uncover a dynamics of modular motif embedding in domain structures that is ongoing, which transfers ‘preferential’ cooption properties of ancient loops to emerging domains. Remarkably, we find that the emergence of molecular functions induces hierarchical modularity and power law behavior in network evolution as the network of motifs and structures expand metabolic pathways and translation.The formation of protein structural domains requires that biochemical functions, defined by conserved amino acid sequence motifs, be embedded into a structural scaffold. Here we trace domain history onto a bipartite network of elementary functional loop sequences and domain structures defined at the fold superfamily level of SCOP classification. The resulting ‘elementary functionome’ network and its loop motif and structural domain graph projections create evolutionary ‘waterfalls’ describing the emergence of primordial functions. Waterfalls reveal how ancient loops are shared by domain structures in two initial waves of functional innovation that involve founder ‘ The Aleph and Other Stories.“… I saw the Aleph from every vantage point, I saw in the Aleph the earth and in the earth again the Aleph, I saw my face and viscera, I saw your face and in vertigo I wept, for my eyes had seen that secret and conjectural object whose name is usurped by men …”— Jorge Luis Borges, lex continui’ of Leibnitz) that implicitly supports evolution. We recently argued that these prior states involve the combination of dipeptides to form three-dimensional loop structures and that these non-regular structures provide the necessary flexibility to develop molecular functions and genetics234Escherichia coli, subjection to stress stochastically induces biphasic-rewiring and modularity at regular time intervals of few minutesIn order to explain the structural and functional complexities of the protein world, protein domain structure must emerge from prior structural states and must fulfill the principle of spatiotemporal continuity 101214We mapped the evolutionary age of domain structures onto a bipartite graph-theoretic representation of domains and associated loop prototypes, which we then decomposed into its dual network projections using mathematical properties of finite graphsEvolutionarily, our bipartite graph and its projections describe how prior loop forms that carried the most primordial functions rearrange to create modern repertoires of domain structures and their associated molecular functions. Hence, the bipartite network portrays the makeup and evolution of ‘elementary functionomes’ (EFs). Loop prototypes represent the oldest ancestral sequence motifs, 25–30 amino acid residues in length, which do not exist contemporarily but are represented by descendant loop sequences in modern proteomesper se and its domain and loop graph projections can only display evolutionary information if an age can be assigned to its node and link components. In other words, mapping age onto the graph representations describes how the networks grow in time (nd) of each domain structure was extracted from this timeline. Calibration points of SF domains associated with microfossil, fossil and biogeochemical evidence, biomarkers, and first-appearance of clade-specific domains  converted the timeline of relative ages into geological time scalesThe EF bipartite network  in time . We usedstep function. Here we borrow the DES rationale to study how discrete evolutionary ‘time steps’  materialize in the growing structure of the undirected and unweighted EF bimodal network and its uni-modal weighted projections  of 0.043 [134/(82 × 38)] and a node average degree (edges per node) of 2.23 (±0.232), i.e. EF groups had ~2 mutual connections on average. The network was well structured from a visual clustering point of view. The Visualization of Similarity (VOS) clustering method revealed 25 communities  with a high modularity index of 0.894, calculated as a standard modularity measure24The operation of a network system can be modeled as a discrete sequence of events  using Discrete Event Simulation (DES) tools2122jections . Events arcs, arrows symbolizing the flow of time from older to younger nodes. The loop and domain directed graphs were also disconnected and had 113 and 376 arcs, network densities of 0.080 [113/(38 × (38−1))] and 0.056 [376/(82 × (82−1))], and node average degrees of 3.45 (±0.551) and 5.08 (±0.534), implying ~3 domains or ~5 loops were shared on average, respectively. The uni-modal networks showed significant community structure. The loop network had 13 clusters with a modularity index of 0.608. The domain network had a comparable number of 15 clusters but with a higher modularity index of 0.886. The number of outward (outdegree) and inward (indegree) connecting arcs of nodes endowed domains and loops as ‘donors’ (sources) or ‘acceptors’ (sinks) of functional loops and domains, respectively. The horizontal and vertical scaling of nodes were made proportional to their weighted outdegree and indegree, respectively. This facilitated the visualization of hubs. The transition from wide to tall symbols along the flow of time revealed the source-sink origination dynamics of molecular functions (see below). This transition expresses the expected increase in probability of co-opting older loops and domains with time.The projections of the EF graph were also visualized as waterfall networks . To makep-loop-related 7, 6488 and 6739 loops. The c.37.1 domain is a Rossmanoid α/β/α-layered domain structure that is the most ancient and popular in the timeline of domain history2p-loop prototype crucially enabled the nucleotide triphosphate binding functions of the P-loop hydrolase fold with its Walker A (p-loop) sequence motif located at the elbow, usually connecting the first β-strand of the main β-hairpin loop (or loop derivatives) that binds to di- and tri-nucleotides. The loop network shows that the ‘p-loop’ wave established several massive pathways of loop recruitment involving four cysteine-rich loop prototypes, the loop 536 hub, the downstream highly connected loops 1845 and 1632, and terminal loop 2524  and nucleotidyltransferase (d.218.1) domains with α/β/α-layered and sheet structures, and more derived beta and beta-prime subunits of DNA dependent RNA polymerase (e.29.1), RNA polymerase subunit (g.41.9) and prokaryotic type I DNA topoisomerase (e.10.1) domains with β-barrel and winged helix-like structures  structure, which is flanked by a 4-strand β-sheet. The structure exposes crucial elbows between the helix-turn-helix motifs that harbor the specificity of protein-protein and protein-RNA interactions typical of these enzymes. The winged domain structure plays central roles in transcription, providing flexibility and nucleic acid clamping capacity to RNA polymerasesThe second wave originated in the ‘winged helix’ DNA binding domain (a.4.5) and its uniquely connected loop 2914. The wave appeared soon after the p-loop’ and ‘winged helix’ waves embedded the first two major gateways of enzymatic recruitment we previously identified in metabolism, the first gateway mediated by the c.37 fold and originating in the energy interconversion pathways of the purine metabolism subnetwork, and the second mediated by the a.4 fold and originating in the porphyrin and chlorophyll metabolism subnetwork and the biosynthesis of cofactors2730It is remarkable that the first two waves uncovered by the EF network and its projections involve the same primordial sandwich α/β/α-layered structures, β-barrels and helical bundle structures we identified as part of the first 54 domain families that appeared in evolutionnd = 0) and the present (nd = 1)  . The dis(nd = 1)  and 3. E(nd = 1) . Howevertimeline . Furthertimeline  and patttimeline . OverwheConnectivity patterns make explicit the evolutionary dynamics of emergence of molecular functions, falsifying some alternative hypotheses that could explain it. Historically, the creation of molecular novelty most likely involved the ligation of dipeptides and small polypeptides with limited ordered structure, followed by the formation of larger peptides harboring stable loop structures11213The loop and domain connectivity of the EF network evolved gradually from ~3 to a global average of 1.63 (±0.16) loops per domain and from 1 to a global average of 3.53 (±0.59) domains per loop . RemarkaP(k) of a node being linked to k other nodes (i.e. the fraction of nodes with k links or k-neighbors) decays as a power law, P(k) ~ k−γ, without a characteristic scale. ‘Scale-free’ networks of this kind generally have exponents γ = 2.1–2.4, driving a heavy-tailed distribution in which very few nodes have high connectivity degreesNetworks whose dynamics follow the preferential attachment principle harbor large, highly connected hubs that attract increasingly more links in a ‘rich-get-richer’ fashion. In these highly inhomogeneous networks, which are remarkably popular in biology, the probability P(X = x−α) is met, only increased in degree distributions of domain nodes of the growing EF networks. These patterns are indicative of power-law decay. Finally, the log-likelihoods of the fitted power law parameters were relatively much lower than zero for most networks of the timeline, making power law distribution less likely. We found the degree distributions to be discontinuous in all of these networks.A number of statistics failed to reject power law behavior in very ancient connections of loops and most connections of domains in the EF bipartite network . Thus, c35Log based linear regression models overlapping with power law curves show that the coefficient of power law decay γ for the domain portion of the EF network increases with time and reaches a limit of 1.8, which is somehow lower than γ reported for metabolic networksnd ~ 0.2). The coefficient of determination (R2) of ~80% (but never < 50%) supports the linear models. Remarkably, loop (0.407 ± 0.028) and domain (0.656 ± 0.026) network projections maintain a lower average γ < 1, consistent with statistics that reject power law behavior of these network projections. The unexpected result of power law transfer from loop to domain components may be indicative of a global scaling phenomenon in the biphasic emergence of biological modulesThe statistical analyses of the timeline of growing EF networks therefore reveal a surprising property of power law emergence. The early-evolved loop ‘prior form’ component of the bipartite network transfers power law behavior to the domain component as molecular functions develop in protein evolution . Log basaverage clustering coefficient (C), the ratio of triangles (graph cycles of length 3) to the connected triples in the graph, averaged over all nodes, ignoring the direction and weights of the edges3742C of its projections  of nodes that connect preferentially to each other within bounds of a community3440jections . The domC of scale-free models sharply declines with network size N as N−0.75C regressed with N as N0.0022 and N−0.006, and with age nd of the networks as nd0.17 and nd−0.15 (C of strictly power-law (Barabási) reference controls were zero46. The timeline of growing domain and loop networks revealed trends of modularity and scale-free behavior that were anticorrelated. For example, the C of the domain network shows an initial decline and then a rise in modularity . This trend matches the KS fit indegree statistic that eventually rejects power law behavior   . Since tNG) algorithm iteratively calculates edge ‘betweenness’, i.e. the maximum number of shortest paths running through an edge, while systematically removing edges with high measures of inter-community centralityNG index. NG partitioned by age (NGage) ranges from −1 to 1. Positive values indicate modular connectivity within events while negative values indicate connectivity across them. NG partitioned by VOS (NGvos) describes the cohesiveness of VOS divisions24FGC) detection algorithm provides a hierarchical perspective of agglomerative community structureNGvos and FGC indices produced similar patterns of growth of community cohesiveness and agglomerative structure with age for all growing networks. Conversely, NGage dissected divergent dynamic behaviors in the EF network and its projections. The EF network revealed an age-linked modular structure along the timeline, with an initial spike of NGage ~0.5 (nd~0) followed by a gradual decrease to ~0.25 (nd~0.37). The domain and loop projections developed instead an age-independent modular structure, with initial NGage of about −0.5 and −0.25 (nd~0), respectively, which quickly flattened towards 0 (nd~0.1)  diffusing into a pervasive (red pixelated) pattern  . In thes pattern . Evident.25 nd~0.. The dom pattern . In cont . In the pattern .p-loop’ and ‘winged helix’ domain structures. We find that the dynamics of recruitment of loop motifs by domain structures is ongoing and highly modular. The emergence of molecular functions showed properties of hierarchical modularity and emergent power law behavior. Modules in the EF network behaved as integrated communities of interacting structural parts defining classes of molecular functions. Our analyses suggest that module emergence occurred through a biphasic process of diversification1Tracing evolutionary age onto growing EF networks and their projections uncovered two clear waves of functional innovation that involved ancient loops and founder ‘nd) values from phylogenomic trees, were obtained from a previously published timeline of protein domain evolutionnd + 3.628) was used to calculate geological age in GyE-value < 1 and coverage of SFs in proteomes of more than 5%. Sequence logos and additional loop information can be found elsewhereDomain structures were defined at SF level according to SCOP version 1.75igraph package2450Networks were visualized and analyzed using PajekP(k) vs. k (i.e. probability of having k-neighbors vs. k) and log-log (i.e. log of P(k) vs. log of k) mappings, with linear regression models to derive γ of the power law and the determination coefficient (R2). γ is the negative of the slope of the log linear model. Higher γ indicates increased tendency towards preferential attachment. R2 describes the percentage of the data fitting the linear model. When both γ and R2 are high, scale free behavior should be considered strongly supported. Other power law statistics included: (i) the exponent of the fitted power law distribution (α), which assumes P(X = x) is proportional to x−α; (ii) KS fit statistic, which compares the fitted distribution with the input degree vector; and (iii) the KS p-value, with the null hypothesis of data being drawn from the power law distribution35p-values (≥0.05) suggest better fit to power law distributions. We also determined the maximum log likelihood of the fitted parameters and if the power law fit pattern was continuous. Reference networks were created using ‘Barabási’ 52 methods of R’s igraph packageScale free network behavior was studied using VOS Quality index (VQ), was generated by the Pajek layout algorithm that takes into account values (weights) of lines (edges/arcs) as similarities. Similar communities were iteratively drawn closer to each other and the quality index of the final layout with least crossings and closest clusters was given. VQ is then calculated as ∑i = 1→c, j = i + 1→c (eij − ai2), where c is the number of communities. eij is the fraction of edges with one node v in community i and the other w in community j, given as ∑vw (Avw/2 m) with 1v ϵ ci, 1w ϵ cj, where m is the sum of weights in the graph and Avw = the weighted value or 0 meaning presence or absence of edge between nodes v and w, in the adjacency matrix A of the network. Finally, ai is the fraction of weighted k neighbors that are attached to nodes in community i, i.e ki/2 m24Clustering Ratio (C-ratio) considers the ratio of the number of node clusters to the count of the connected node set; (iii) The average Clustering Coefficient (C) describes the mean of the ratio of the triangles to the connected triples for all nodes in the simplified (undirected/unweighted) network3742C is only and strictly meaningful for unipartite graphsC for domain and loop network projections; (iv) The Fast Greedy Community (FGC) hierarchical agglomeration algorithm detects community structure with linear running time O(m d logn) ~ O(n log2n), with m edges, n nodes, and d, the depth of the dendrogram describing the community structureNewman-Girvan algorithm index (NG), computed with default partitions defined by age (NGage) and VOS clustering (NGvos). NG calculates the modularity of a network with respect to some division (partition) and measures how good the division is in separating the different node types from each other, to indicate assortative (positive) or disassortative (negative) mixing across modulesNG equals 1/(2 m)∑ij(Aij−1/(2 m)kikj*∆), where m is the total weights in the graph, Aij are weighted entries in the adjacency matrix, ki, kj and ci, cj are respectively the weighted degrees and the components (numeric partitions) of nodes i and j, and finally, ∆ is 1 if x = y and 0 otherwiseNG for two additional types of membership, FGC and Walk Trap Community (WTC) detection algorithm. WTC is similar to FGC but computes communities using random walksVQ, C-ratio, C and FGC range from 0 to 1, while the NG indices range from −1 to 1. Higher indices represent strong network modularity at a particular event. Heatmaps were customized from scaled modularity matrices with elements given as (Aij−kikj/(2 m))Mnd, where Aij, ki, kj and m are as defined for NGnd is network’s modularity index at event nd. Dendrograms were calculated from squared Euclidean distance matrices indicating dissimilarities between the cluster meansWe studied modularity with six indices: (i) The How to cite this article: Aziz, M. F. et al. The early history and emergence of molecular functions and modular scale-free network behavior. Sci. Rep.6, 25058; doi: 10.1038/srep25058 (2016).


The MPTP mediated neurodegeneration in substantia nigra has been well studied, but not the status of frontal cortex. The novelty of the present study is to explore the sex difference of frontal cortex during MPTP intoxication and to investigate the role of estrogen and its receptors in presence of glial cells in a time chase experiment; to identify which pathway of NF-kappaB exist to proceed the neuroinflammation; to investigate the estrogen binding with its nuclear or cytosolic receptors and whether any direct relation exists between estrogen receptor (ER) -beta and NF-kappaB molecules p65 and RelB. The progression of neurodegeneration occurred with the association of glial cells and functional (via its nuclear and cytosolic receptors) estrogen level. Both the canonical and/or non canonical pathways of NF-kappaB exist in frontal cortex of both the sexes after MPTP treatment. The homodimeric or heterodimeric form of ER-beta binds with NF-kappaB molecules p65 and RelB differently, but the canonical or non canonical pathways of NF-kappaB molecules could not be stopped or may be promoted. The changes in the molecular and cellular pattern in frontal cortex of both sexes during MPTP intoxication depends on the estrogen function via its nuclear or cytosolic estrogen receptors. There are many environmental toxins that take part in introducing the mouse model of Parkinson’s disease (PD) with alternative pathological features. Among them, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) appears to target the dopaminergic neurons and introduce itself as the most established animal models of idiopathic PD. The investigations made with MPTP induced mouse model of PD appears to reflect many important mechanistic pathways related to the disease initiation14Identifying environmental factors that predispose to the development of idiopathic PD via oxidative stress, mitochondrial dysfunction, Lewy pathology has proved elusive681012Increasing evidence suggests that estrogens protect the nigrostriatal dopaminergic pathway affected in PD. Animal studies also show that estrogens influence the synthesis, release, and metabolism of dopamine and can modulate dopamine receptor expression and functionTherefore, two opposite phenomena are present there during the progression of the disease- neuroinflammation and neuroprotection (by estrogen). Now the question arises- in spite of presence of estrogen how neuroinflammation proceeds to degenerate neurons in PD. To find the possibilities of the phenomenon, we have chosen the major transcription factor Nuclear factor κB (NF-κB) that are expressed in a wide variety of cells and tissues, including microglia, astrocytes, and neurons during the neuroinflammation. The NF-κB functions via two pathways- classical or canonical and alternative or non-canonical. Activation of the NF-κB pathway is mediated through the activity of the IKK kinase complex, targeting to alter NF-κB has been proposed as an approach to the treatment of acute and chronic inflammatory conditions, and the use of inhibitors specific for either IKKβ or IKKγ has now been found to inhibit neurodegeneration of dopaminergic neurons in murine and primate models of PDThe present study has been designed to understand the possible crosstalk between estrogen action and NF-κB during the progression of the neuroinflammation/neurodegeneration in frontal cortex of the MPTP mediated mouse model of PD. The crosstalk has also been evaluated with respect to the status of microglial and astroglial cells and also in a time dependent manner.The results indicated that the FOX3 positive immunoreactivity and the neuronal population decreased significantly compared to the respective controls in frontal cortex of both the sexes during MPTP treatment  and decrIn the present study, GFAP immunoreactivity, was the least on day3 and day7, increased on day1 of sacrifice in MPTP treated male  but decrThe percentage of astroglial population increased on day1 and decreased on day3 and day7 of sacrifice in male, however that percentage pattern was reverse in female frontal cortex of MPTP treated groups compared to their respective control . The uncIn the MPTP treated group, the expression level of Iba1 increased significantly only in the middle phase (day3) of male  and initDuring MPTP treatment, the population size of Iba1 positive microglial cells increased significantly in all the three stages of sacrifice in MPTP treated female  but thatIn the present study, TNF-α expression level increased significantly in all the three days of sacrifice in MPTP treated group with the highest level of expression in the middle phase of sacrifice in frontal cortex of both the sexes  as well It was found that estrogen level either decreased significantly or remained unchanged in frontal cortex of both male and female mice brains compared to respective controls during MPTP treatment . Again, In the present study, in MPTP treated group, the expression level of estrogen bound nuclear estrogen receptor α (nE2ERα) did not alter on all the three days of sacrifice in male  but did Further, in the MPTP treated group, the expression level of estrogen bound nuclear estrogen receptor β (nE2ERβ) did not change significantly compared to the respective controls of male and female frontal cortex . The estIt has been found that in the MPTP treated male group of frontal cortex, the higher expression level and nuclear translocation of p52 was found in all the three days of sacrifice in frontal cortex of both the sexes [(44 ± 3.7)%* p < 0.05]  and maleThe nuclear translocation of p65 increased mostly at a later stage of female [(45 ± 3.7)%* p < 0.05] and middle stage [(29 ± 3.1)%* p < 0.05] of male after MPTP treatment .The nuclear translocation of RelB decreased [(65 ± 5.7)%* p < 0.05] on all three days of sacrifice of MPTP treated male frontal cortex compared to the controls, but in the female, nuclear translocation of RelB became same on day1 and day3 but increased on day7 [(43 ± 4.3)%* p < 0.05] compared to the respective controls during MPTP treatment . When esNIK decreased on day1 [(24 ± 2.9)%* p < 0.05], remained same on day3 and day7 of the sacrifice of MPTP treated male frontal cortex compared to the controls. In females, NIK decreased on day1 [(25 ± 2.7)%* p < 0.05] and increased on day3 and increased on day7 [(47 ± 4.1)%* p < 0.05] compared to the respective controls during MPTP treatment. The NIK expression level of estrogen supplemented MPTP treated group was found to be faint on all three days of sacrifice compared to the controls in males; however that expression level increased on day1 and day3 [(39 ± 5.2)%* p < 0.05], decreased on day7 [(53 ± 6.7)%* p < 0.05] compared to their controls in female  forming a heterodimer. However, there is no true evidence has been found whether the estrogen receptors specially the ER-β can directly interact with the NF-κB molecules like p65 (ERβp65 complex) and bind with RelB (ERβRelB) at nuclear fractionation during MPTP treatment or not. In this study, we have put forward the objective to find whether the estrogen in respect with its receptors and NF-κB crosstalk exist in frontal cortex of both the sexes during MPTP mediated PD and neuroinflammation or not.In the MPTP treated group, the expression level of ERβERα became same on day1, increased on day3 and day7 compared to the control; however ERβERα did appear increasingly on all three days of sacrifice compared to the controls in female frontal cortex of the MPTP group . When esIn the MPTP treated male group, the expression level of bounded ERβp65 became absent on all days of sacrifice, but that expression level became same on day1 and on day3 and increased on day7 compared to the controls in female frontal cortex . When esIn the MPTP treated male group, the expression level of ERβRelB became same on day1, decreased on day3 and increased on day7 compared to the control. However ERβRelB decreased on day1, increased on day3 and on day7 of sacrifice compared to the controls in female frontal cortex of the MPTP group . When esThe oxidative stress and the mitochondrial dysfunction, preceding DNA fragmentation, could be early events in the apoptotic or necrotic process induced by GSH depletion by GST activation in neuronal cells192426283240The sole presence of estrogen or the activity of estrogen via binding to its receptors in both the nuclear or cytosolic part, have implication in neuroprotection in MPTP treated mouse model of PDκB as a Target for Therapy in Parkinson’s Disease has already been revealed previouslyWhen neuroinflammation persist during the progression of the disease in different days of sacrifice, we have found that selected NF-κB molecules expression pattern changes differently in frontal cortex of both the sexes. It has been reported that neuronal loss may be caused due to increased cytokines and apoptosis-related proteins via the activation of NF-κB in reactive astrocytes of the substantia nigra after MPTP treatment in miceHowever which pathway  persists during MPTP mediated neurodegeneration, is still remaining unknown. The pattern of expression level of NF-κB molecules like p65, p52, RelB and NIK changed according to the changes in estrogen level and functionality of estrogen via its nuclear and cytosolic receptors counterpart in frontal cortex of both the sexes. There is no true evidences have been found still now that how NF-κB pathways modulate during the progression of PD in frontal cortex of both the sexes and neuroinflammation persist. In our study, we have found that during the progression of the neurodegeneration in MPTP model, both the canonical or non canonical pathway exist alternatively in male and female mouse brain frontal cortex.As previously mentioned that estrogen has anti-inflammatory property that may/may not be actionable by estrogen receptors in the present study, we also examined whether there persist any direct interaction between estrogen receptor β and NF-κB molecule p65 or RelB. There is evidence that estrogen can activate the NF-κB pathway via nongenomic pathways and increase cellular resistance to injuryThe level of neuroinflammatory TNF-α in the brain depends on the activation pattern of glial cells (both microglia and astrocytes) in both the sexes in different days of sacrifice after MPTP treatment and that level of TNF-α depends on the decreased level of estrogen and functionality of estrogen by its receptors. Nuclear and cytosolic estrogen bound ER-α and ER-β differently changed during MPTP treatment in both the sexes and that binding with its expression level may be dependant on the functional status of estrogen receptors. However, no positive correlation has been established in the present study. Both the canonical and non canonical pathways exist during the MPTP mediated progression of the disease. And there is a positive binding of ER-β exists with the NF-κB molecule p65 and RelB but that could not able to suppress the NF-κB mediated neuroinflammation during the progression of the disease mediated by MPTP.These vast and rapid changes in molecular and cellular pattern during the course of the MPTP intoxication ultimately promote neuroinflammation that leads to neurodegeneration. Both the canonical and/or non canonical pathways of NF-κB exist in frontal cortex of both the sexes after MPTP treatment that is why neuroinflammation still progress. The homodimeric or heterodimeric form of ER-β binds with NF-κB molecules p65 and RelB differently, but the canonical or non canonical pathways of NF-κB molecules could not be stopped or may be promoted and neuroinflammation proceeds. One positive correlation is that the changes in the molecular and cellular pattern in frontal cortex of both the sexes during MPTP mediated neurodegeneration depend on the functionality of estrogen with its nuclear or cytosolic estrogen receptors α and β. In addition, the present experiments reinforce further studies to unveil the physiological and pathophysiological significances of the cellular and molecular interactions in different regions of mammalian brain and their sex differences during healthy and disease conditions. Therefore the diversified variability of estrogen level and its action via both the receptors have a positive implication that male is more prone to PD risk than female.ad libitum. All animal experiments were performed following the “Principles of Laboratory Animal Care” , and following the specific Indian law on “Protection of Animals” under the supervision of authorized investigator and the methods were carried out in accordance with the approved guidelines of the Institutional Animal Ethics Committee (IAEC). The experimental protocols were also approved by the IAEC at the Department of Zoology, University of Calcutta.Swiss albino male and female mice  obtained from the National Institute of Nutrition  were housed in a departmental animal facility . Animals were provided rodent chow  and filtered water The experimental mice of both sexes (n = 5) were randomly divided into different groups comprising (A) vehicle  treated control (B) MPTP-treated: Animals received four subcutaneous injections of MPTP  at 2 hours interval in a single day and were sacrificed at 24 hours (day1), day3 and day7 after the last dose was administered (53), (C) 17-β estradiol-treated: Animals were administered 17-βestradiol ] intraperitoneally once a day for 5 consecutive days and was sacrificed on the day2, day4 and day8 after the last dose was administered, (D) Tamoxifen (Estrogen receptor antagonist)-treated: Animals received intraperitoneal injections of tamoxifen  once a day for 5 days and was sacrificed on the day2, day4 and day8 after the last dose of tamoxifen administered., (E) 17-βestradiol + MPTP treated: Animals administered 17-β estradiol (2 μg/kg b.w.) intraperitoneally once a day for 5 days prior to MPTP treatment and was sacrificed on day1, day3 and day7 after MPTP treatment (day0). (F) Tamoxifen + MPTP treated: The tamoxifen + MPTP treated set received intraperitoneal injections of tamoxifen (5 mg/kg b.w.) once a day 5 days prior to MPTP treatment day and was sacrificed on day1, day3 and day7 after MPTP administration (day0). (G) 17-βestradiol + Tamoxifen treated: Animals received Tamoxifen treatment first (as mentioned above) and after four hours, received 17β estradiol (as mentioned above) for five consecutive days and sacrificed at day2, day4 and day8 after receiving the last dose and (H) 17-βestradiol + Tamoxifen + MPTP treated: Animals received Tamoxifen treatment first (as mentioned above) and after four hours, received 17β estradiol for five consecutive days and sacrificed at day1, day3 and day7 after MPTP treatment (day0). At 24 hrs, 72 hr and day7 after the final dosing, the mice in each group were euthanized by an overdose with sodium thiopentone  and brain tissue were then harvested for analyses as described in the various assays below.For estrous cycle, we placed the tip of plastic pipette, filled with PBS or saline (~10 μL), into the vagina. Then we flushed the vagina gently three to five times with same PBS/saline solution. Then we collected final flush with a pipette tip. A volume of 10 μL of saline solution allows collecting sufficient material for observation of vaginal cytology. Then we placed the final flush containing vaginal fluid on a glass slide. Then we observed unstained material under light microscope with a 10× objective. To observe the estrus stage, we identified cornified squamous epithelial cells in clusters, no visible nucleus with a granular cytoplasm and the irregular shape. The vaginal smear was taken thrice daily sparing eight hours for each female animal for the whole treatment period up to sacrifice point.et al., 2011Immunohistochemistry was performed according to Mitra et al., 2011Total cell lysate for western blot analysis were performed according to Mitra et al., 20112, 420 mM KCl, 25% glycerol, and 0.2 mM EDTA) supplemented with 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, and 1 μM pepstatin, homogenized and left on ice for 30 min. Samples were centrifuged at 14,000 rpm for 20 min at 4 °C. The supernatant, containing nuclear proteins, was separated, used in a Bradford assay and the remainder was stored at −80 °C for further experiments.According to Arumugam et al. 2011According to Mitra et al., 2009Immunoprecipitation was done according to Kundu Dissected brain region homogenized in specialized lysis buffer, containing 25 mM HEPES, 0.1% Tween-20, 5 mM magnesium chloride, 1.3 mM EDTA, 1 mM EGTA and protease inhibitors . Anti-estrogen, anti-aromatse and anti-TNFα mouse monoclonal antibodies [Abcam plc ] used to detect the level of the respective molecules in brain lysate and in blood plasma. The Estrogen and TNF-α level in FC lysates were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocol . The OD values were read in a microplate reader at 450 nm. The concentrations of TNF-α and estrogen in the sample were calculated against the standard curve generated using recombinant TNF-α.Single cell suspension from FC of mice were incubated with anti-CD16/32 (FC-block), followed by fixation with 4% paraformaldehyde and permeabilization with 0.1% saponin. The cells were then fixed, washed twice with cell staining buffer and intracytoplasmic staining was performed using anti-FOX3 for neuronal cells, anti-Iba1for microglial cells, and anti-GFAP for astrocytic cells in separate vials and incubated for 4 hours at room temperature. Rabbit polyclonal to anti-goat secondary antibody and Alexa-flour488 conjugated anti-mouse and APC conjugated anti-mouse secondary antibodies [Abcam plc ], FITC-conjugated secondary mouse anti-goat antibody [Santa Cruz biotechnologies ] were added to the respective primary antibody containing vial and incubated for 2 hours at room temperature. The percentage of respective cell types were then analyzed in a FACS Aria III with FACS diva software (BD bioscience). Viable cells were gated by forward and side scattering. Representative bar graphs indicated as percentage of cells.Values between groups were analyzed using single way ANOVA and two way ANOVA where appropriated. All values are shown as mean ± SEM, except where otherwise indicated. Data were analyzed and when appropriate, the significance of the differences between mean values was determined using Student’s t-test. The results were considered significant at p < 0.05.How to cite this article: Mitra, S. et al. Alteration in Nuclear Factor-KappaB Pathway and Functionality of Estrogen via Receptors Promote Neuroinflammation in Frontal Cortex after 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Treatment. Sci. Rep.5, 13949; doi: 10.1038/srep13949 (2015).


Variation in clinical evidence has prevented the adoption of fecal microbiota transplantation (FMT) in patients with ulcerative colitis (UC). We aimed to conduct a systematic review and meta-analysis to determine the efficacy and safety of FMT in UC.A systematic literature search was performed in 5 electronic databases from inception through September 2015. Inclusion criteria were reports of FMT in patients with UC. Studies were excluded if they did not report clinical outcomes or included patients with infections. Clinical remission (CR) was defined as the primary outcome.Q test, P = 0.139; I2 = 33%). A subgroup analysis suggested that no difference in CR was detected between upper gastrointestinal delivery versus lower gastrointestinal delivery. Furthermore, subgroup analysis revealed that there was no difference in CR between single infusion versus multiple infusions (>1) of FMT. All studies reported mild adverse events.Eleven studies , 1 open-label case-control study, and 8 cohort studies) with a total of 133 UC patients were included in the analysis. In 11 studies , the pooled proportion of patients who achieved CR was 30.4% (95% CI 22.6–39.4%), with a low risk of heterogeneity (Cochran FMT is potentially useful in UC disease management but better-designed RCTs are still required to confirm our findings before wide adoption of FMT is suggested. Additionally, basic guidelines are needed imminently to identify the right patient population and to standardize the process of FMT. Several studies have suggested that gastrointestinal microbiota might play a role in development of this disease.3 Specifically, microbial dysbiosis has been hypothesized as a trigger in UC disease development.4Ulcerative colitis (UC) is a chronic, relapsing, and remitting disease characterized by inflammation of the colonic mucosa. UC is a subtype of inflammatory bowel disease (IBD) causing significant morbidity. Epidemiological studies have shown a significant increase in the incidence of UC across the world (about 3 million of people).6 An additional alternative therapy for the management of UC is fecal microbiota transplantation (FMT), which has been shown as an effective treatment for refractory and recurrent Clostridium difficile infection (CDI).7 The success of FMT in treating C difficile infections has raised the possibility that FMT may be beneficial in other diseases through alterations in gastrointestinal microbiota or dysbiosis.Treatments that manipulate the microbiota balance have been developed and investigated including the administration of probiotics and prebiotics, with different evidences observed for their efficacy.8 In this case, Bennet treated himself, an active and severe UC patient, using FMT and he was symptom-free for 6 months subsequently. Afterward, other reports were published, most of which were case reports or noncontrol cohort studies, with variable results. Recently, some new cohort studies13 and the first 2 randomized, double-blinded, controlled trials15 were presented, but the findings in these studies are variable which has unfortunately confused UC clinicians.The use of FMT in UC patients was first described by Bennet in 1989.16 Additionally only 4 noncontrol cohort studies of UC patients with FMT (27 cases) were performed in a subgroup analysis that showed a pooled estimate for achieving remission of 24.1% (95% CI 11.1–44.9%). The validity of that data was limited by the methodological concerns and the lack of an adequate number of studies.16 Three systematic reviews of IBD also included some case reports and noncontrol cohort studies of UC patients,19 but also contained several methodological limitations. In fact, 2 systematic reviews did not analyze the subgroup of UC,18 while 1 systematic review contained mostly case reports and FMT outcomes that were measured by treatment “success rates” and not by any other more validated measures.19Only 1 meta-analysis of IBD included both unpublished and abstract data including 1 abstract of RCT.The aim of this study was to undertake a systematic review and meta-analysis of FMT in patients with UC so as to provide clinicians with a comprehensive and clear assessment of the available evidence upon which to guide current practice and future research.22 An electronic search was conducted using PubMed, Cochrane Library, Web of Science, Wanfang Data, and China National Knowledge Infrastructure. All databases were searched from their inception through September 2015. No language limits were used. Searching was limited to publications with clinical trials . In concurrence with Colman and Rubin,16 both free text and medical subject headings of this study included the following alternatives for fecal microbiota transplant: “fecal transplant,” “fecal transfusion,” “fecal implantation,” “fecal implant,” “fecal instillation,” “fecal donor,” “fecal enema,” “fecal reconstitution,” “fecal infusion,” “fecal therapy,” “fecal bacteriotherapy, “faecal transplant,” “faecal transfusion,” “faecal implantation,” “faecal implant,” “faecal instillation,” “faecal donor,” “faecal enema,” “faecal reconstitution,” “faecal infusion,” “faecal therapy,” “faecal bacteriotherapy, “microbiota transplant,” “microbiota transfusion,” “microbiota implantation,” “microbiota implant,” “microbiota instillation,” “microbiota donor,” “microbiota enema,” “microbiota reconstitution,” “microbiota infusion,” “microbiota therapy,” “microbiota bacteriotherapy,” “microflora transplant,” “microflora transfusion,” “microflora implantation,” “microflora implant,” “microflora instillation,” “microflora donor,” “microflora enema,” “microflora reconstitution,” “microflora infusion,” “microflora therapy,” “microflora bacteriotherapy,” “feces transplant,” “feces transfusion,” “feces implantation,” “feces implant,” “feces instillation,” “feces donor,” “feces enema,” “feces reconstitution,” “feces infusion,” “feces therapy,” “feces bacteriotherapy,” “faeces transplant,” “faeces transfusion,” “faeces implantation,” “faeces implant,” “faeces instillation,” “faeces donor,” “faeces enema,” “faeces reconstitution,” “faeces infusion,” “faeces therapy,” “faeces bacteriotherapy,” “stool transplant,” “stool transfusion,” “stool implantation,” “stool implant,” “stool instillation,” “stool donor,” “stool enema,” “stool reconstitution,” “stool infusion,” “stool therapy,” “stool bacteriotherapy,” “flora transplant,” “flora transfusion,” “flora implantation,” “flora implant,” “flora instillation,” “flora donor,” “flora enema,” “flora reconstitution,” “flora infusion,” “flora therapy,” and “flora bacteriotherapy.” The results were then combined using the set operator “AND” with studies identified by varied UC descriptor terms: “ulcerative colitis,” “inflammatory bowel disease,” “colitis,” “ileitis,” “IBD,” and “UC.” We also manually searched proceedings from major international conferences, including the American College of Gastroenterology, Digestive Disease Week, Advances in IBD, United European Gastroenterology Week, Asia Pacific Digestive Week, Congresses of Gastroenterology China and Chinese Congresses of Digestive Diseases from 2010 up to and including September 2015. Additional studies were identified by manually searching the references of articles retrieved from the computerized databases and relevant review articles.We followed the MOOSE, PRISMA, and Cochrane guidelines in our study.23 or pediatric UC activity index <10.24 Studies without reported clinical endpoints were excluded. If the study included patients with infections before FMT, they were excluded.Eligibility criteria were determined a priori by the study authors. FMT was defined as administration of a suspension of donor feces (either fresh or frozen) into the gastrointestinal tract for UC treatment. UC was defined by the researchers in studies according to laboratory confirmation, endoscopic evidence and/or clinical symptoms. Efficacy of FMT was assessed by clinical remission. The primary outcome was clinical remission of UC, defined as Mayo score ≤2,The studies were imported into a bibliographic database to automatically exclude duplicates. Titles, abstracts, and articles were reviewed and assessed by 2 independent reviewers (Sun and Li) based on the eligibility criteria. Data extraction from selected publication used a standardized pretested form. A third party compared the forms of data extraction (Xu and Lu). Any disagreements were corrected by consensus. Demographic data , pre-FMT therapy, transplantation procedures , choice of donor, clinical resolution, adverse events, and duration of follow-up were retrieved. If certain data points were not reported, we contacted with the authors by and obtained the detailed missing data.25  quality assessment in keeping with the previous literature25 Table . The qua27 We used a fixed-effects model assessing the pooled estimate of clinical remission in the meta-analysis with OpenMeta[Analyst]. Meta-Analyst software  was used to construct the funnel plot.28The overall meta-analysis included the clinical remission rates obtained from 9 cohort studies and from the FMT experimental arm of 2 randomized clinical studies and 1 case control study in keeping with the previous literature.Q test (χ2) and I2 method. In the Q test a P value of >0.1 was not deemed as statistically significant, it showed that the study was not heterogeneous, and hence we used fixed-effects models; otherwise, we used random-effects models. The I2 method was used to assess the degree of heterogeneity .29 Some outcomes were not analyzed but presented in a descriptive way.Statistical heterogeneity for each meta-analysis was assessed using the Cochran 11  met the eligibility criteria. Of the 11 studies, 10 were in full text and 1 was an abstract, and all of them included UC patients for whom there were no infections before FMT. With the exception of 1 study that reported mixed patients (11 UC patients and 3 patients with Crohn disease)C difficile infections;39 9 were case reports of UC;47 2 studies with the unclear definition of UC;49 and 2 studies did not clarify the primary clinical outcome.51 We could not obtain more detailed data about the primary clinical outcome from the authors in 2 studies,51 because the corresponding authors’ email addresses were not provided with the reports.Among the 23 excluded studies, 10 studies reported UC patients with 15 Adequate sequence generation and allocation concealment were not described clearly in 1 study.15 Reviewers determined that 2 RCTs had clear description of the blinding, incomplete outcome data, free of selective reporting, and free of other bias , 1 star was from comparability , and 2 stars were from exposure . The NICE quality assessment was used to evaluate 8 cohort studies. None of the 8 studies met all the criteria . Studies included both pediatric and/or adult patients. Among the 133 patients included in the review, 27 (20.3%) were described as having moderate/severe disease, 100 (75.2%) as having mild or mild/moderate disease  received weekly FMT or placebo (water) via retention enema for 6 weeks. Researchers and patients were blinded to the treatment allocation. The primary endpoint was clinical remission, defined as Mayo Score of <3 with an endoscopic subscore of 0 at week 7. Six donors  were included in the study, with the majority of subjects receiving FMT from 2 donors (A and B). The Data Monitoring and Safety Committee (DSMB) advised that the trial should be discontinued for futility because the primary end point was unlikely to be achieved as specified in the protocol. At the conclusion of the study, the authors found that patients who received FMT achieved better CR than those receiving placebo . In the second study,15 50 UC patients  of ≥4 and ≤11 and stable medication) were treated with either donor stool or autologous FMT (infusion of their own stool) delivered via nasoduodenal tube at baseline and again 3 weeks later. Participants and trial members were blinded to the treatment allocation. Only 37 subjects completed assessment for the primary endpoint, which was also CR defined as SCCAI of ≤2 in combination with ≥1-point improvement on the combined Mayo endoscopic score at week 12. This study was also terminated at interim analysis by the DSMB because of futility. There was no difference in clinical and endoscopic remission between the 2 groups in either the intention-to-treat or per-protocol analyses.Two small double-blind, randomized (1:1), controlled trials with moderate risk of bias reported use of FMT for patients with mild-to-moderate UC.9 Fifteen patients (Mayo score ≥4 with an endoscopic Mayo score ≥1) were treated with either FMT (8 patients) or standard therapy (7 patients). Enrolled patients underwent colonoscopy and received 3 administration of FMT using 200 cc of fecal slurry from a healthy donor. The CR was the second endpoint defined as partial Mayo score ≤2 with no subscore ≥1 at 2 weeks. Overall, the authors found that the CR were not significantly different between FMT and standard therapy (3/8 [37.5%] vs. 2/7 [28.6%]).An open-label case-control study was included in the review.12 4 studies used healthy adults,53 and 1 study did not describe the ages of donors.10 Two studies used related and unrelated donors.24 The delivery method included colonic delivery (including enema administration and/or colonoscopic instillation) (n = 4),53 upper gastrointestinal delivery  (n = 2),12 and the combination of colonic and upper gastrointestinal delivery (n = 2).52 The FMT dosage and number of FMT were also variable. The FMT dosage was calculated by the volume of the FMT suspension  or the weight of stool. The number of FMT ranged from 1 to 6 times among 8 studies.All 8 studies in this category were prospective, noncontrol cohort design. Incomplete data were reported for the donors, preparation, and administration of FMT Table . Eight sP = 0.139; I2 = 33%)   with low heterogeneity between studies . Six studies used lower gastrointestinal delivery. The rate of clinical remission was 29.8% (95% CI 20.2–41.6%) with low heterogeneity between studies   versus lower gastrointestinal delivery (colonoscopy and/or enema). Two studies with the combination of upper and lower gastrointestinal delivery were excluded from this analysis.13 However if the patients were administered the second FMT due to no efficacy of the first FMT, those were defined as cases with failed a single infusion.12 The rate of CR in patients with a single infusion was 28.2% (95% CI 10.4–57.0%) with moderate heterogeneity between studies . A total of 85 subjects in 6 studies received multiple infusions (>1). The rate of CR was 28.9% (95% CI 20.1–39.7%) with low heterogeneity between studies   of FMT. When studies had mixed a single infusion and multiple infusions (>1), we were able to divide and analyze individual cases, as their raw data was available.9 and the study that was categorized as “lower quality”;10 and we performed another meta-analysis including other studies, and the CR was 30.4% (95% CI 22.3–39.9%) with moderate heterogeneity between studies . The results were similar to the results of the meta-analysis of all studies.We excluded the open-label case-control study that was an abstract52 In a clinical randomized control study,14 3 patients with FMT suffered adverse events. Two patients in the FMT group developed patchy inflammation of the colon and also rectal abscess formation, which was resolved by antibiotic therapy. One patient with worsening abdominal discomfort tested positive for C difficile toxin after the study.Most of studies showed that FMT was safe Tables  and 4. A16 Because few studies were available, Colman and Rubin performed a subgroup analysis with a small sample (including 27 UC cases with FMT in 4 noncontrol cohort studies) and the confidence interval of clinical remission was distinct.To date, this is the largest systematic review and first meta-analysis on FMT in UC patients without infections. We identified 2 RCTs, 1 open-label case-control study, and 8 noncontrol cohort studies about the efficacy and safety of FMT in UC patients. According to the 11 studies  in our review, the overall efficacy of FMT was 30.4% (95% CI 22.6–39.4%) in achieving CR, which was significantly higher than the CR of 22% (95% CI 10.4–40.8%) reported by Colman and Rubin.C difficile was significantly high at 89.% (95% CI 84.0–93.3%).25 These rates are substantially higher than the 30% to 80% CR rates typically reported in various medical therapies for CDI, although direct comparison of such different studies cannot be done with confidence.54 CDI occurs as a result of outstanding disruption of the indigenous gut microbiota by antibiotics,55 while UC is a complicated disease with a complex pathologic interplay among immunologic, genetic, environmental, and gut microbial factors. Manipulating the gut microbiota might be an important treatment approach, but not the only 1 strategy for UC. The use of probiotics is the most common approach used by researchers to manipulate microbiota in UC patients. Based on the clinical trial evidence available to date, only Escherichia coli Nissle and probiotic mix VSL#3 appear to be effective in the management of UC.56 The CR rates after using Escherichia coli Nissle57 and probiotic mix VSL#358 were 68% and 49.4%, respectively. Based on limited approaches to manipulate the microbiota in UC patients, FMT with moderate efficacy (30.4% CR rate) might be an alternative approach.It is worth noting that FMT is not nearly as effective in UC as it is in CDI. In a systematic review and meta-analysis, the efficacy of FMT in patients with 15 both of which avoid methodological flaws in previous case series and cohort studies. However, results from these 2 RCTs were contradictory because of several differences in their trial design . This result was consistent with that of a RCT in patients with 52 However, better designed studies are needed to investigate this procedural aspect.In the review, our data found that multiple infusions (>1) had been used in many studies Tables  and 4; tNonetheless, the results from subgroup analyzes are mildly heterogeneous, which considerably limits the applicability of the conclusions.10 and a study in an abstract format9 were included. Even with sensitivity analysis that provides a similar result, the current results require verification of additional well-designed RCTs and enough power. Second, publication bias was a concern. Because most included studies were noncontrol cohort studies, the authors might have had a desire to publish a series of successfully treated patients, while some clinical failures might have been excluded in the reports that may result in preferential reporting of successful cases. In addition, definitions of variables associated with FMT program were not standard among studies, and publications often did not report data on these variables   Tables  and 4.Despite these limitations, these data suggest that FMT may be an efficacious and safe alternative therapy for UC, at least when the standard therapy has failed or is unacceptable to the UC patients. The positive findings in our review need to be confirmed and supported by additional well-designed randomized, double-blinded, controlled trials with enough samples. There is an urgent need to develop guidelines to standardize the process of FMT and FMT indication.


The use of etanercept, a tumor necrosis factor (TNF) inhibitor, has revolutionized the treatment of juvenile idiopathic arthritis (JIA). TNF is a key cytokine implicated in the pathogenesis of inflammatory arthritis and etanercept, which is a soluble TNF receptor fusion protein, binds and inactivates TNF-α and lymphotoxin-A.The aim of this study was to profile serum levels of TNF-α in a large cohort of adolescent patients with JIA.Serum TNF-α was measured in samples derived from 200 adolescent and young adult patients with JIA attending the adolescent and young adult rheumatology clinic at University College London Hospital using a commercial enzyme linked immunosorbent assay (ELISA) kit (eBioscience). Samples were tested in duplicate. Median age at sampling and median disease duration were 18 years and 8 years 9 months, respectively. Male:female ratio was 1:1.2. Equal numbers of patients with polyarticular (n=64) and enthesitis related arthritis  were tested in addition to 48 with oligoarticular, 16 systemic onset, and 8 psoriatic arthritis. Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) measurements were also collected. Furthermore, an L929 cell viability bioassay was used to determine if the addition of etanercept abrogates the cytotoxic effects of TNF-α in L929 cells.Surprisingly, TNF-α serum levels were shown to be markedly elevated in patients on etanercept . TNF-α levels were also higher in patients on etanercept compared to those on other biologics  or disease modifying anti-rheumatic drugs alone , p<0.0001. In addition, ESR and CRP levels had a negative correlation with high TNF-α levels in patients on etanercept (p=0.0018 and p=0.0034 respectively). Etanercept was included at its therapeutic serum concentration (2.4ug/ml) to ensure there was no cross reactivity with the assay. Finally, we showed that the addition to TNF-α to human serum leads to cytotoxicity in a TNF-α sensitive cell line, while adding etanercept at its therapeutic concentration along with TNF-α significantly reduces cell death (p = 0.0277).in vitro experiments in which the cytotoxic effect of TNF-α was abrogated upon addition of etanercept. Our hypothesis is that etanercept prolongs the half-life of circulating TNF-α. Further studies are needed to confirm these findings and dissect the mechanisms involved. As the association between high TNF-α and etanercept treatment is so strong, we hypothesise that it may be possible to measure TNF-α levels as a surrogate marker of adherence to this drug in this cohort of patients where adherence to medication can be a significant problem. This is a hypothesis that warrants further investigation.Patients treated with etanercept have higher levels of TNF-α. As the majority of patients with elevated TNF-α on etanercept were in remission, it is likely that this circulating TNF is biologically inactive. This is supported by our None declared.


Xeroderma pigmentosum complementation group A (XPA). From the analysis of the current knowledge, in addition to protein–protein docking and secondary structure prediction results presented for the first time herein, a mechanistic framework emerges, where XPA builds the NER pre-incision complex in a modular fashion, as “beads on a string”, where the protein–protein interaction “beads”, or modules, are interconnected by disordered link regions. This architecture is ideal to avoid the expected steric hindrance constraints of the DNA expanded bubble. Finally, the role of the XPA structural disorder in binding affinity modulation and in the sequential binding of NER core factors in the pre-incision complex is also discussed.Lack of structure is often an essential functional feature of protein domains. The coordination of macromolecular assemblies in DNA repair pathways is yet another task disordered protein regions are highly implicated in. Here I review the available experimental and computational data and within this context discuss the functional role of structure and disorder in one of the essential scaffolding proteins in the nucleotide excision repair (NER) pathway, namely  NER targets lesions caused by environmental mutagens, such UV light and polycyclic aromatic hydrocarbons, or by alkylating agents, such as platinum-based chemotherapeutics The regulation of molecular function by structurally disordered protein domains is currently one of the hottest topics in structural and computational biology 2Xeroderma pigmentosum complementation group C (XPC) in complex with RAD23B, replication protein A (RPA), XPA, the transcription factor II H (TFIIH) complex, XPG, and the excision repair cross-complementation group 1 (ERCC1) in complex with XPF Xeroderma pigmentosumssDNA-binding multimodular heterotrimer composed by 3 subunits, namely RPA32, RPA14, and RPA70 ssDNA stretch carrying the lesion, a structure known as DNA bubble 2) is involved in damage recognition, it would be already located on the forming bubble, however the transition from XPA2 to the DNA-bound XPA–RPA complex is not clear as yet. The XPA–RPA interaction promotes the dissociation of the XPC–RAD23B from the damage site ss to dsDNA Y junction ssDNA to dsDNA junction on the 5′ side Although significantly different in their chemistry, all NER-targeted lesions have a common structural trait, namely a severe bend in the DNA that destabilizes the double helix 3ssDNA to dsDNA junction, or Y junction 1XPA), up to residue 239; with the XPA98–239 construct found to bind DNA Y junctions with the same affinity as the full-length protein As summarized in 498–219 shown in 98–219 solution NMR structure with PDBid 1XPA are shown in ssDNA or with a Y DNA junction, recent studies have shown that two Lys, namely K168 and K179 are essential for DNA interaction 98–219 NMR structure, and for which no structural data are available yet, have been also identified as important for DNA binding, namely K221, K222, K224, and K236. As shown in The only region of XPA structurally characterized is part of its DNA and RPA70 binding core, comprising residues 98 to 219 ssDNA and RPA70, take place around the same region within the XPA98–219 domain ssDNA and RPA70 ssDNA binding of a number of other residues located on the α helix 1 of XPA and indicated in 98–219–DNA–RPA70 complex where one strand of ssDNA channels through the two proteins, interacting with both the XPA98–219 and RPA70. This hypothesis is also in agreement with kinetic data showing that the XPA–RPA complex is 2.5 fold faster than RPA alone for binding a duplex cisplatin-damaged DNA ssDNA–RPA70 complex available in the PDB with PDBids 1XPA and 1JMC, respectively, returns as the highest scoring pose a quite interesting model for a possible three-body XPA98–219–DNA–RPA70 complex. This complex, shown in ssDNA channeling within a cavity formed between XPA98–219 and RPA70. This model predicts that two poorly structured XPA98–219 regions, one located between residues 98 and 138, which includes the Zn coordination site, and the other located between residues 179 and 183, are also involved in the interaction with RPA70 and ssDNA, leaving the helical packing motif outfacing the ssDNA–RPA70 interface. Interestingly, as shown in ssDNA for residue K168. The position of the XPA98–239 core at the 3′ side of the Y DNA junction, as shown in 98–239 core would bind both ssDNA branches at the junction, with the damaged oligonucleotide interacting with α helix 1  and the undamaged branch going through the XPA98–239–RPA70 interface, as predicted by the ClusPro docking model.Interactions with both, 5ssDNA binding region of RPA32 is located in an unstructured region of the protein, between residues 43 and 171 ssDNA is moderate − 8 M Dk), obtained by surface plasmon resonance (SPR), is in the order of 2 × 10− 8 M, showing a higher affinity of XPA for RPA, relative to ERCC1 29–46 peptide bound to RPA32 is not available, 15N-HSQC data indicate that the binding mode and the binding site of the XPA29–46–RPA32 complex is identical to the one characterized for the UNG73–88–RPA32 complex, shown in 73–88 peptide, disordered in solution, adopts a helical structure upon binding 29–46 sequence, while the disorder prediction method DisEMBL In the NER pre-incision complex XPA interacts also with the other large unit of RPA, namely RPA32 6ssDNA oligonucleotide at the 5′ side of the lesion. The nuclease activity resides entirely on the XPF module ss/dsDNA Y junction and XPA 67–80 peptide, binds specifically the ERCC1 central domain (cERCC1), comprising residues 96 to 214, with submicromolar affinity. The XPA67–80 peptide binding inhibits the interaction with the full length XPA and blocks NER progression, without affecting nuclease activity 67–80 sequence is highly conserved in all species carrying NER genes 67–80 peptides bound to cERCC1 and free in solution suggest that, while specific residues, such as Asp 70, Phe 75 and Ile 76, are involved in direct interactions with the cERCC1 binding site, other residues affect the peptide conformational propensity while free (unbound) in solution, thus its recognition by cERCC1 67–80 peptide is poorly structured 67–80 peptide 67–80 conformation The ERCC1–XPF is a heterodimeric endonuclease responsible for cleaving the damaged ssDNA-binding activity 67–80 peptide has been shown to be a competitive inhibitor of ssDNA binding by cERCC1 ssDNA can bind the cERCC1 simultaneously, with the cERCC1 in contact with 3 or 4 unpaired bases at most Dk of the full length XPA–cERCC1 complex from SPR, is 2.5 × 10− 7 M − 6 M was obtained for the Dk of the cERCC1 in complex with a 10 unpaired nucleotide-long DNA bubble The XPA binding site of cERCC1, shown in 7ssDNA interactions is in apparent contrast with the lack of structure characterizing large part of the XPA sequence and with the short length of some of the XPA interaction domains. This high degree of conformational disorder is consistent with the lack of structural information we have on the full-length XPA, when bound and especially when unbound in solution. Within the whole XPA sequence, shown in 98–239 domain, specific for binding ssDNA and RPA70. In the cases of the poorly structured XPA29–46 and XPA67–80 domains, specific to RPA32 and cERCC1, respectively, secondary structure motifs may go undetected when the protein is free in solution due to the length of experimental timescales. For example, in the case of XPA67–80, a specific hairpin motif structurally similar to the bound conformation has been found to be significantly populated at the low microsecond time scale 67–80 conformational propensity in solution can be significantly affected by the mutation of the terminal residues of the cERCC1-specific region, namely Lys 67 or Glu 78 to 80, among others, where the mutants show a much higher level of structural disorder relative to the wild type The intricacy and high specificity of the XPA–protein and XPA–ssDNA binding regions fall in a range between highly (XPA98–219) to less structured (XPA29–46 and XPA67–80), where the observation timescale defines the position within this range, the linker regions connecting the different XPA binding motifs are intrinsically disordered. The presence of these structureless regions confers to XPA the ability to function as a flexible scaffold with a “bead on a string” architecture, avoiding major steric clashes in the organization of the NER core proteins around the DNA bubble framework 67–80 region confers a lower binding affinity to the ERCC1–XPA67–80 complex relative to the XPA29–46–RPA32 complex and especially to the structured XPA98–219–ssDNA-RPA70 complex 29–46 region has an intrinsic propensity to form helices, which makes XPA29–46 less disordered than the XPA67–80 region.While the XPA–protein and XPA–via its interaction with either XPC–RAD23B ssDNA 98–239 core binds RPA70 and the ssDNA in a ternary complex that constitutes the first module (Module 1) in the pre-incision bubble. Although there are no direct structural information on the XPA98–239–ssDNA-RPA70 complex, the available data, discussed in detail in a previous sections, support a structure where the ssDNA channels through an interface formed between the RPA70 and the XPA98–239, located at the 3′ side of the lesion. A model for the Module 1 complex that fits with the available experimental evidence was generated through protein–protein docking with the online tool ClusPro ssDNA alongside the RPA70 on the damaged oligonucleotide. The N-terminal XPA29–46 stretch binds specifically a globular domain in the RPA32 C-terminus ssDNA–RPA70 and XPA–RPA32 units, constitutes a loop wide enough to reach the Y junction at the 5′ side, located approximately 30 nucleotides away, thus to bind and position the ERCC1–XPF endonuclease. Once the ERCC1–XPF endonuclease is put in place, it starts the dual incision step followed by the XPG nick at the 3′ side Based on the data available and on the sequential binding discussed above, the following role for XPA in the pre-incision complex modular architecture is proposed and represented schematically in 898–239 region, the ss/dsDNA Y junction at the 3′ side of the lesion and RPA70, likely to function as the main anchoring point for XPA on the DNA bubble 29–46 region, namely binds the C-terminal globular domain of RPA32 67–80, and the central domain of ERCC1 (cERCC1) 67–80 is located in the middle of a 50 residue-long intrinsically disordered loop, connecting Modules 1 and 2. This loop is wide and flexible enough to reach the Y DNA junction at the 5′ side of the lesion and to position the ERCC1-XPF endonuclease for the dual excision. The XPA67–80–cERCC1 interaction has the lowest binding affinity between the 3 The conformational flexibility conferred to proteins by structural disorder can provide many functional advantages over highly structured domains


COL3A1 gene mutation results in reduced collagen III but with concurrent increased myofibroblast expression. This paradox has not been examined with the use of external drugs/modalities to prevent hypertrophic scars. In this paper, we injected the rabbit ear wound model of hypertrophic scarring with two doses of a protein called nAG, which is known to reduce collagen expression and to suppress hypertrophic scarring in that animal model. The higher nAG dose was associated with significantly less collagen III expression and concurrent higher degree of myofibroblast expression. We concluded that collagen III content of the extracellular matrix may have a direct or an indirect effect on myofibroblast differentiation. However, further research is required to investigate the pathogenesis of this paradoxical phenomenon.Generally speaking, the excessive expression of myofibroblasts is associated with excessive collagen production. One exception is seen in patients and animal models of Ehlers-Danlos syndrome type IV in which the  COL3A1 gene resulting in reduced amount of collagen III in their skin and other connective tissue such as the walls of the arteries and intestine. This results in areas of atrophic skin, easy bruising, aneurysms, and spontaneous visceral rupture [+/− mice” . These mice have similar features to humans including reduced production of collagen III. Volk et al. [+/− mice and compared them to similar wounds in normal (Col III+/+) mice. Mutant mice had less collagen III but much more myofibroblast expression leading to accelerated wound contraction [In unwounded skin, fibroblasts are in a quiescent state within the extracellular matrix (ECM). Acute wounding results in changes in the microenvironment of the ECM (including the release of cytokines and growth factors) leading to the transformation of fibroblasts into myofibroblasts . Myofibr rupture , 5. Howe rupture . Biopsie rupture . In cont rupture . Mice mok et al.  producedtraction . The autnAG is a salamander-derived protein mediating limb regeneration . In a prIn the current paper, we injected rabbit ear wounds with two different doses of recombinant nAG and show that the degree of expression of myofibroblasts is inversely proportional to the degree of collagen III expression.The experiment was approved by the Ethics Committee of the Medical College of our university.Details of synthesis and preparation of mammalian recombinant nAG protein was described in our previous work , 9. MammThe hypertrophic scar model in the rabbit ear is a well-established model in the literature , 11. A 7Eight white New Zealand rabbits weighing 1.5–2 kg were used. The animals were anaesthetized with intramuscular ketamine (40 mg/kg) and xylazine (4 mg/kg). One wound was created in each ear . A 7 mm punch biopsy was used to create the wound as described above. Wounds were dressed with normal saline-soaked gauze.n = 8) were injected with 100 μL of saline. Four experimental wounds were injected with 100 nM of recombinant nAG  and the other four experimental wounds were injected with 150 nM of recombinant nAG .Wound injections were done daily starting at day 14 and ending at day 27. All control wounds . Masson's trichrome staining was done to stain collagen fibers.Histological sections were fixed with 4% paraformaldehyde, embedded in paraffin, cut in 5 μm thick sections. The sections were mounted on saline coated slides and were incubated for 20 minutes in hot air oven at 60°C. Tissue sections were deparaffinized with EZ Prep  at 75°C and were heat-pretreated in Cell Conditioning 1  using “standard cell conditioning” for antigen retrieval at 100°C, and then one drop of inhibitor solution was put for four minutes at 37°C and then tissue sections were washed. The slides were incubated 30 minutes at 37°C with diluted 1 : 200 anti-α-smooth muscle actin (α-SMA) or TGFβ1 monoclonal antibody . Then ultraview universal HRP multimer as secondary antibody was added. The immunostained proteins were visualized using a copper-enhanced DAB reaction. Immunostained sections were reviewed by Olympus BX51 light Microscope and DP72 Olympus Digital Camera (magnification 200x and 400x) .Paraffin-embedded blocks of wounded skin specimens were cut into 3 RNA was extracted from tissue samples using RNeasy Protect Mini Kit  and quantified by nanodrop. 100 ng of total RNA was reverse-transcribed by using Rotor-Gene Multiplex PCR Kit . Relative mRNA levels were calculated by using the ΔΔCt method and GAPDH as a reference gene. Primers and probes were mentioned in our previous published work . Experimt-test (for SEI) and the Mann-Whitney test (for RNA extraction data). P < 0.05 indicated a significant difference.The SEI and RNA extraction data were expressed as mean ± SD. The differences between the values of the two experimental groups (the 100 nM nAG group and the 150 nM nAG group) as well as the control group  were analyzed using the independent sample P < 0.005). Masson's trichrome staining showed thick, dense, and disorganized collagen fibers in the control wounds  less collagen I and collagen III contents than controls. When the two experimental groups were compared to each other, there was no significant difference (P > 0.05) in collagen I content, but the high dose nAG group had significantly more collagen III suppression (P < 0.05) than the low dose nAG group.Compared to controls, the relative mRNA expression of collagen I and collagen III in both experimental groups is shown in Our study showed the persistent high expression of myofibroblasts in wounds deficient in collagen. Both experimental groups had similar reductions of collagen I content. However, collagen III expression was most significantly reduced in the 150 nM nAG group and this corresponded with the highest degree of myofibroblast expression. This indicates that it is the suppression of collagen III (and not collagen I) which induced the increased expression of myofibroblasts. Further research is required to investigate if this paradoxical phenomenon (low collagen III content with high myofibroblast expression) will also be seen with other modalities used to prevent hypertrophic scars.αѴβ3 and α5β1 integrin receptors. Vitronectin binds mainly to αѴβ3 receptors, whereas fibronectin binds mainly to α5β1 receptors [ COL3A1 gene mutations will not only produce less collagen III but will also recruit the αѴβ3 instead of α5β1 integrin receptors. Hence, it is possible that the low collagen III expression in the ECM will have an effect on myofibroblasts through changes in the “tension” sensing receptors.The creation of a wound with very low collagen content and a high degree of myofibroblast expression is interesting from the scientific point of view. Myofibroblasts are known to have mature focal adhesions in their cell membrane –3. Theseeceptors  have shoβ1 is the main stimulant of myofibroblast differentiation and stimulation of excessive collagen synthesis [β1 within this paradoxical environment. Our study demonstrated that the intensity of TGFβ1 expression correlates with the intensity of myofibroblast expression. In our previous study [β1 stimulation. One possible explanation of the sequence of events in our model would be the primary suppression of collagen III by nAG. This may have caused a biofeedback mechanism through which TGFβ1 is stimulated, which will then stimulate myofibroblast differentiation. Since the nAG effect on collagen is dominant over the TGFβ1 effect on collagen, collagen III remained low despite the high TGFβ1 expression.It is well known that TGFynthesis . Our curus study  we showeFinally, our findings may have clinical relevance. For example, Dupuytren's cords are now being treated with direct injection of collagenase . CollageMyofibroblast expression in the rabbit ear model is enhanced by collagen III suppression. The pathogenesis of such a paradox needs further research but is clinically relevant.


The consumption of green leafy vegetables (GLVs) has been demonstrated to reduce the risks associated with cardiovascular and other diseases. However, no literature exists that examines the influence of traditional and novel GLVs on the liver fatty acid profile of an animal model genetically predisposed to developing hypertension. The aim of the present study was to examine the effects of diets containing 4% collard greens, purslane or sweet potato greens on the liver fatty acid profiles of four-week old male spontaneously hypertensive rats . Following four weeks consumption of the diets, liver fatty acid profiles were determined by gas–liquid chromatography of transesterified fatty acid methyl esters.SHRs consuming the control diet had greater percentages of liver saturated fatty acid and less omega-3 fatty acid percentages. SHRs consuming the diets containing vegetables had significantly greater liver concentrations of γ- linolenic, docosahexaenoic and docosahexaenoic acids, as well as lower levels of lauric, palmitic and arachidonic acids. SHRs consuming the control diet had significantly greater percentages (p < 0.05) of oleic; significantly less γ-linolenic and docosahexaenoic acids.This study demonstrates the ability of GLVs to modulate liver fatty acid composition, thus providing protection against elevations in atherogenic fatty acids, which may be involved in CVD pathogenesis. Consequently, dietary recommendations for the prevention of CVD should consider the possible cardioprotective benefits and the subsequent alterations in fatty acid profiles afforded by diets containing collard greens, purslane and sweet potato greens. Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in the US as well as developing nations . In addiPlants, particularly green leafy vegetables (GLVs), serve as a major dietary reservoir of the essential PUFAs, dietary fiber, antioxidants and other bioactive compounds that exert cardioprotective and chemopreventive biological mechanisms -9. EpideBrassica oleracea), a traditional GLV consumed mainly in the southern region of the United States, in addition to purslane (Portulaca oleracea) and sweet potato greens (Ipomoea batatas L.), novel GLVs to the typical American diet, may prove beneficial in reducing the risks associated with CVD development and progression [in vivo and in vitro[Collard greens  acid  than those consuming the control diet (1.42 ± 0.06). Liver oleic acid (C18:1n9) was significantly increased following 4 weeks consumption of the control diet (12.69 ± 1.03). Among SHRs consuming diets containing GLVs, oleic acid concentration was greater with consumption of the PL diet (7.70 ± 1.14), followed by the CG (7.40 ± 1.95) and SPG (5.57 ± 1.16) diets. A more than two-fold significant increase in eicosenoic acid (C20:1n12) was present among SHRs assigned to the PL dietary group (0.28 ± 0.00) in comparison to those assigned to the control group (0.12 ± 0.03)  and docosahexaenoic  acids versus those consuming the experimental diets containing GLVs  and novel (i.e. purslane and sweet potato greens) green leafy vegetables on the liver fatty acid profile of the spontaneously hypertensive rat. The current research findings are in agreement with epidemiological evidence, which suggests the cardioprotective and chemopreventive advantages of consuming diets containing dark green leafy vegetables. The results of this research suggest the ability of diets containing collard greens, purslane and sweet potato greens to favourable shift SHR liver fatty acid compositions in such a way that may be beneficial in reducing CVD risk in spontaneously hypertensive rats.The chief limitation of the current study was the duration of feeding of the control and experimental diets to the spontaneously hypertensive rats. As CVD is a chronic, inflammatory disease occurring over the course of the lifespan, extending the duration of feeding may offer critical insights regarding long-term consumption of novel and traditional GLVs on the pathogenesis of CVD. In addition, focusing on only one aspect of CVD risk, namely the liver fatty acid profile, fails to offer a comprehensive view of the larger spectrum involved in CVD pathogenesis. Examining the effects of diets containing collard greens, purslane and sweet potato greens on factors such as the complete lipid profile, lipid and other nutrient metabolism and other relevant indices of cardiovascular health, such as cardiac hypertrophy, inflammatory biomarkers, tissue morphology, gene expression and SHR physiology, may offer critical evidence regarding the influence of these diets on disease outcomes. Further, implications and translation of the current and future studies to human nutrition and cardiovascular outcomes needs to be explored as well.In addition, future research studies may want to consider the isolation of nutrient and bioactive compounds within these diets that may have contributed to the presence of a beneficial fatty acid profile. Accordingly, moderate to long-term feeding studies involving the consumption of these diets by SHRs, as well as the consumption of these diets by “normal” rats, not genetically predisposed to developing hypertension warrant further consideration. Therefore, confirming the relationship between shifts in liver fatty acid profile on indices of cardiovascular health or CVD disease pathogenesis, through continued research studies will add credence to the physiological relevance of the current research study.Forty-four (N = 44) male spontaneously hypertensive rats (SHRs), approximately 4 weeks of age and weighing 100 grams were obtained from Charles River laboratories . The animals were housed individually in stainless steel cages at the animal care wing of the Tuskegee University Comparative Medicine Research Center. Room temperature and relative humidity were maintained between 68 and 72°C and 55%, respectively. SHRs were maintained on a 12-hour light/12-hour dark cycle.Experimental diets Table , formulaad libitum. Following the 4-week feeding experiment, animals were fasted overnight and anaesthetized by overinhalation of carbon dioxide, followed by cardiac puncture. SHR livers were removed and stored at −80°C prior to analysis. The procedures involved in the care and use of animals in this research were approved by the Tuskegee University Institutional Animal Care and Use Committee.Animals were pair fed based on the previous day’s dietary intake of the control group and allowed to consume food and water The extraction of total liver fatty acids was conducted using the method of Folch et al. . BrieflyFatty acid methyl esters were isolated and quantified by gas chromatography using a Hewlett-Packard 6890 gas chromatograph containing a fused silica capillary column (DB-23 60.0 m × 250 μm × 0.25 μm) furnished with a flame-ionization detector. A volume of 1 μL of the sample was injected by the auto sampler at a 10:1 split ratio. Helium served as the carrier gas, with a flow rate of 2.0 mL/min. Injector and column temperatures were positioned at 250°C. The initial oven temperature was 130°C and held for 1 minute followed by a 6.5°C/min increment increases in temperature until a temperature of 170°C was achieved. The temperature was again increased at 2.75°C per minute until a temperature of 215°C was reached and maintained for 12 minutes. A final temperature of 230°C was reached and maintained for 3 minutes. Eluted FAMEs were identified based on comparisons to retention times and peaks of known FAMEs contained in an external standard ). Areas under peak curves were considered proportional to the amount of the analyte present in the chromatograph. Peak area measurements were obtained and individual FAMEs within each sample were determined as a percentage of total fatty acids present.Individual liver fatty acids are presented as mean percentage ± standard error (SE) of total fatty acids. One-way analysis of variance and Duncan’s post hoc procedures were conducted to determine if statistically significant differences existed among the different dietary groups. Statistical significance was determined at p < .05. Statistical analyses were performed using Statistical Analysis System (SAS) software .The authors declare that they have no competing interests.MJ performed the animal study, liver fatty acid profile analysis, analyzed data, and drafted the manuscript. RDP contributed to the conception and design of the study, supervised the project and edited the manuscript. NLD contributed to the design of the study and edited the manuscript. KRW supervised the fatty acid profile analysis and edited the manuscript. All authors read and approved the final manuscript.


Oxyntomodulin analogues offer a novel treatment for obesity. However during analogue screening in a rat model increased food intake was consistently observed. To further investigate this finding, a series of representative analogues (OXM14 and OXM15) and their Glu-3 equivalents (OXM14E3 and OXM15E3) were administered to rats for 7 days and food intake and bodyweight measurements taken. To investigate the role of glucagon receptor activation glutamate (Glu/E) was substituted at amino acid position 3. GLP-1 and glucagon receptor efficacy of the oxyntomodulin analogues and their Glu-3 counterparts were measured at the rat receptors in vitro. Doses of 25 n mol/kg of OXM14 and OXM15 increased food intake by up to 20%. Bodyweight was not significantly increased. Food intake was not increased with the Glu-3 peptides, indicating that a glucagon receptor mechanism may be responsible for the increase in food intake. It is a product of the proglucagon precursor peptide along with glucagon-like peptide-1 (GLP-1), glucagon and glucagon-like peptide-2 (GLP-2) and is co-secreted post prandially with GLP-1 from L-cells in the distal intestine Despite the beneficial effects of oxyntomodulin on weight and glucose homeostasis, the very short half-life of native oxyntomodulin means it is not used therapeutically As part of a proprietary drug development program, analogues of oxyntomodulin were designed with modifications in regions susceptible to degradation. The changes made were conservative, thus the analogues were not derivatized, were of similar length to the native peptide, had no cross linkages and included only natural amino acids. All analogues were based on the 29 amino acids of glucagon and contained 5–7 single substitutions between residues 12–29, with 2 or 3 additional amino acid residues at the C-terminal. During screening it was observed that when administered to rats, the numerous analogues tested did cause significant reductions in bodyweight but, unexpectedly had either no effect on food intake, or actually caused an increase. The current paper focuses on a representative sample of these oxyntomodulin analogues to further investigate the mechanism behind the increase in food intake and to assess the impact these findings might have on the design and therapeutic use of oxyntomodulin analogues.22.1(7-36NH2) and oxyntomodulin) were custom synthesized by Insight Biotechnology Limited  using solid phase peptide synthesis (SPPS) methodology and purified by reverse-phase preparative HPLC. Peptide purity was determined by reverse-phase HPLC and by Matrix-assisted laser desorption ionization mass spectroscopy (MALDIMS). All peptides had a purity of >90%.All oxyntomodulin analogues tested  and native peptides . Concentrations were applied in duplicate or triplicate per experiment and each experiment repeated a minimum of 3 times.The effects of oxyntomodulin analogues and native GLP-1, glucagon and oxyntomodulin on cyclic adenosine monophosphate (cAMP) accumulation were assessed in Chinese hamster ovary (CHO-K1) cells over-expressing the rat glucagon receptor  and Chinese hamster lung (CHL) cells over-expressing the rat GLP-1 receptor . For cAMP studies, cells were seeded at a density of 150000 cells/ml in a 48 well plate  and incubated for 24 h. Cells were serum starved for 1 h and then incubated for 30 mins at room temperature with a range of peptide concentrations diluted in serum free DMEM  in the presence of 1 mM IBMX . After incubation, medium was removed and cells lysed with 0.1 M HCl +0.5% Triton-X. cAMP levels were measured using an ELISA kit according to manufacturer’s instructions , optical density was read at 405 nm on a Biotek ELx808 . EC2.3Male Wistar rats  were maintained in individual cages under controlled temperature (21–23 °C) and light conditions  with ad‑libitum access to food  and water unless otherwise stated. All animal procedures were approved under the British Home Office Animals (Scientific Procedures) Act 1986 (Project License 70/7236).2.42 slow release formulation) was administered at 1600 h daily and food and bodyweight measured at the time of administration. A minimum of 7 animals per group was required based on previous observations. n numbers varied between experiments, see figure legends for the exact sample size of each group.Rats were weighed at 09:00 on the morning of study commencement and randomisation to treatment groups was stratified by bodyweight. A subcutaneous injection of vehicle (diluent) or peptide . cAMP EC50’s were calculated using a sigmoidal dose response curve and non-linear regression (Prism v5). EC50 data is presented as a ratio of analogue average EC50/average glucagon EC50 or GLP-1 EC50. Average EC50’s were calculated from a minimum of 3 separate experiments. In all cases P < 0.05 was considered statistically significant.All food and bodyweight data are presented as mean ± SEM. Data was normally distributed according to the D’Agostino & Pearson omnibus normality test or the Shaprio–Wilk normality test for groups where 33.1At the rat receptors, oxyntomodulin and analogues showed greater efficacy at the glucagon receptor than the GLP1 receptor . The ana3.2P < 0.001; n = 7–8; A daily dose of 25 n mol/kg OXM15 caused a significant 17% increase in cumulative food intake by the end of the 7 day treatment period compared to vehicle controls  substituted for glutamate (Glu/E). This substitution had previously been reported to significantly diminish glucagon receptor activity without affecting GLP-1 receptor activity P < 0.01), and OXM14 increased cumulative food intake by 19%  severely diminishes glucagon receptor activity without affecting the activity at the GLP-1 receptor, essentially producing a GLP-1 receptor selective analogue Previous studies using native oxyntomodulin and longer lasting dual analogues have reported a reduction in food intake after repeated peripheral administration in rodents Interestingly, the increase in food intake with low doses of oxyntomodulin analogues was not replicated in a mouse model (unpublished observations), indicating that this could be a species specific effect. A number of previous studies with oxyntomodulin analogues have been carried out using only mouse models Oxyntomodulin activates both the human glucagon receptor and GLP-1 receptor with reduced efficacy compared to the native peptides There was a trend towards an increase in food intake with low doses of both analogues up to a ‘threshold dose’, beyond which food intake was then decreased. It could be postulated that the threshold point may reflect the dose at which there is sufficient GLP1r activity to cause a reduction in food intake. There is a difference in threshold dose between OXM14 and OXM15 with a higher dose of OXM14 required to cause a reduction in food intake. It is likely this can be attributed to differences in their ratio of rat GLP-1 receptor to rat glucagon receptor activity. However, OXM14 has a greater efficacy at the rat GLP-1 receptor than OXM15 which would suggest a lower dose would be required to reduce food intake, thus highlighting the likelihood of a complex underlying interaction.Exendin-4 is a naturally occurring GLP-1 receptor selective agonist with a longer half-life. The weight loss caused by administration of this peptide in standard chow fed animals is thought to be mainly due to reduction in food intake 5We have shown that in a rat model low doses of oxyntomodulin analogues often tend to increase food intake without increasing bodyweight.. Results with GLP-1 receptor selective Glu-3 analogues suggest that a glucagon receptor mediated mechanism may be involved in the increase in food intake. It is possible that the increase in food intake may be a compensatory response to an increase in energy expenditure mediated by glucagon receptor activity. It is currently unclear what impact these findings may have on the use of oxyntomodulin analogues in a clinical setting. Nausea limits the dose of GLP-1 analogues that can be used in man. It is unlikely that nausea is a feature of the oxyntomodulin analogues studied here, given the increase in food intake; however a conditioned taste aversion study in rats would help to clarify this.