In this study, we showed that the equivalent CSF-1R D802V mutation does not promote such structural effects on the JMR despite of a reduction on some key H-bonds interactions controlling the JMR binding to the KD. In addition, this mutation disrupts the allosteric communication between two essential regulatory fragments of the receptors, the JMR and the A-loop. Nevertheless, the mutation-induced shift towards an active conformation observed in KIT D816V is not observed in CSF-1R D802V. The distinct impact of equivalent mutation in two homologous RTKs could be associated with the sequence difference between both receptors in the native form, particularly in the JMR region. A local mutation-induced perturbation on the A-loop structure observed in both receptors indicates the stabilization of an inactive non-inhibited form, which Imatinib cannot bind.The colony stimulating factor-1 receptor (CSF-1R) and the stem cell factor receptor KIT, type III receptor tyrosine kinases (RTKs), are important mediators of signal transduction. The normal functions of these receptors can be compromised by gain-of-function mutations associated with different physiopatological impacts. Whereas KIT D816V/H mutation is a well-characterized oncogenic event and principal cause of systemic mastocytosis, the homologous CSF-1R D802V has not been identified in human cancers. The KIT D816V oncogenic mutation triggers resistance to the RTK inhibitor Imatinib used as first line treatment against chronic myeloid leukemia and gastrointestinal tumors. CSF-1R is also sensitive to Imatinib and this sensitivity is altered by mutation D802V. Previous  KIT, whose activating mutations are hallmarks of systemic mastocytosis FLT3, whose activating mutations are frequently observed in acute myeloid leukemias (AML) CSF-1R gene have been rarely detected in human tumors Receptor tyrosine kinases (RTKs) are cell-surface transmembrane receptors that possess a tightly regulated tyrosine kinase (TK) activity within their cytoplasmic domain Fig. S1). The TK domain has a bi-lobar structure, with an ATP-binding cleft located between the N- and C-terminal lobes. The N-lobe is composed of twisted five-stranded anti-parallel β-sheet adjacent to an α-helix (Cα-helix) and the C-lobe shows predominantly α-helical structure (The type III RTKs have a common architecture that includes extracellular immunoglobin (Ig)-like domains to which polypeptide ligands bind, a single-pass transmembrane helix, an autoinhibitory juxtamembrane region (JMR) and a cytoplasmic tyrosine kinase (TK) domain including a kinase insert domain (KID) tructure Fig. 1. i.e., its phenylalanine is flipped into the ATP-binding site, thus preventing ATP and Mg2+ co-factor binding In the absence of ligand, the receptors are in dynamic equilibrium between two states: the inactive autoinhibited state that is highly dominant, and the active state Analysis of the crystallographic structures of KIT, CSF-1R and FLT3 in their inactive state i. e., ligand-independent) activation and/or resistance to the tyrosine kinase inhibitors. Particularly, mutation of an aspartic residue in the A-loop, namely D835(V/Y/H/V/E/N) in FLT3, D816(V/H/N/Y/E/I) in KIT, and D842V in PDGFRα, is a typical example of mutation that confers a proliferative signal. The KIT D816V oncogenic mutation in addition triggers resistance to the RTK inhibitor Imatinib Mutational hotspot regions in type III RTKs are mainly located in the JMR and the A-loop, although mutations have also been found in the extracellular and in the transmembrane regions CSF-1R and KIT have considerable sequence identity (68%) and their auto-inhibited states display great structural similarities (RMSD is 1.14 Å) 10-helix in the A-loop and reorganization of the DFG motif conformation is consistent with the inactive non-autoinhibited conformation observed in both receptors. The results of this study offer a plausible common mechanism, according to which the equivalent mutation may induce resistance to the tyrosine kinase inhibitors targeting the inactive autoinhibited state of type III RTKs.We have reported that KIT D816V mutation, positioned in the A-loop, induced a long-range structural reorganization of the JMR, followed by its release from the KD in the absence of extracellular ligand binding The secondary structure prediction for the JMR residues was performed using six methods based on the protein primary sequence:GOR4 Jpred SOPMA SCRATCH NetSurfP Psipred Finally, STRIDE, a knowledge-based algorithm that assigns the secondary structure from atomic coordinates based on the combined use of hydrogen bond energy and statistically derived backbone torsional angle information http://plasma-gate.weizmann.ac.il/Grace/).VMD http://www.poissonboltzmann.org/pdb2pqr/d/web-servers).Electrostatic potential surfaces were calculated on the crystal structures of CSF-1R (PDB code: 20GV) and KIT (PDB code: 1T45) using APBS at PDB2PQR web-based server  auto-inhibited form of CSF-1R cytoplasmic region (PDB id: 2OGV) In silico substitution of Asp (D) to Val (V) at position 802 was performed by MODELLER, using the WT structure as template, making them comparable starting models. Generated models of the native CSF-1R and its mutant D802V were referred to as CSF-1RWT and CSF-1RMU respectively.Set up of the systems. The setup of the systems (CSF-1RWT and CSF-1RMU) was performed using AMBER force field, parameter set 99SB − counter ions were added when necessary to neutralize the overall charge (3 for CSF-1RWT and 4 for CSF-1RMU). The minimization procedure consisted of 2 steps: steepest descent energy minimization (EM) with the solute atoms restrained; (ii) EM with all atoms free. The equilibration procedure was performed on the solvent, keeping the solute heavy atoms restrained for 500 ps at 310 K and a constant volume .Production of trajectories. Two production runs of 50 ns were carried out for both receptors, CSF-1RWT and CSF-1RMU. The temperatures of solute (protein) and solvent (water and ions) were separately coupled to the velocity rescale thermostat Analysis of the trajectories. The trajectories for each pair of molecular dynamics (MD) simulations were analyzed with tools included in the GROMACS package. When concatenating the MD simulations replicas, the first 5 ns of each replica trajectory needed to achieve relaxation were not considered. Analyses were performed on the resulting merged trajectory of 90 ns for each protein or based on the 45 ns individual replicas. We have also produced a 60 ns concatenated trajectory from the last 30 ns from each replica to be further used for IDSs calculations with MONETA reference structures are identified, (ii) the MD conformational ensemble was clustered into corresponding reference groups. Each reference structure was first picked up at random and associated with a bin of conformations distant by less than an arbitrary cutoff r. Then the merged trajectory was split in four halves  and conformations from each half were grouped based on their RMSD from each reference structure. A good convergence quality was assessed when each reference group was populated by conformations from the four halves of the trajectory at equivalent levels, meaning that every reference structure is equivalently represented in both replicas of the trajectory.Geometrical measurements. Two characteristic distances were monitored every 10 ps over the MD simulations of each model: (i) the distance d1 between the centroid (C) of the JM-B region  and the C of the remaining residues in the N-lobe ; (ii) the distance d2 between the C of the JM-S  and the C-lobe . The hydrogen (H-) bond analyses were done with the program g_hbond available in GROMACS. Time occupancy of H-bonds stabilizing the JMR and the A-loop was recorded every 100 ps of simulation for each model of CSF-1R. H-bonds (D•••H–A) where defined with a DHA angle cutoff of 120° and a D•••A distance cutoff of 3.5 Å (D and A are donor and acceptor atoms).Secondary structure prevalence. The secondary structure profile was calculated using the program do_dssp available in GROMACS. The program makes use of DSSP WT and CSF-1RMU. Free energies were evaluated using the Molecular Mechanism Generalized Born Surface Area (MMGBSA) method, implemented in AMBER 12 The free energy of JMR or its segments  binding to KD  defined asWT taken at 1 526, 49 390, 66 530 and 81 680 ps, spanning both replicas contained in the 90-ns merged trajectory, and (ii) CSF-1RMU mutant taken at 5 510, 23 530, 40 670 and 84 680 ps. The selected MD conformations were found to be the most representative of the trajectories, according to the convergence analysis. The first hydration shell (5 Å) around the MD conformations was kept to help prevent the solvent-exposed regions of the protein from collapsing during the minimization procedure 2 were applied to the starting structure and reduced by a factor of 2 every 1000 minimization steps until they fell below a threshold value of 5 kcal/mol/A2. The constraints were then removed and the system was minimized by conjugate gradient and adopted-basis Newton-Raphson steps until the RMS energy gradient fell below 10−5 kcal/mol/A2. Normal modes were computed by diagonalizing the mass-weighted Hessian matrix of the energy-minimized conformations and the 96 non-zero lowest-frequency modes were analyzed. The degree of collectivity of the JMR motions in a given mode l was calculated as iα is defined as:ix, iy and iz are the components of mode l showing the three degrees of freedom of atom i and such that 1/n indicates that only one atom is involved in the motion while a value close to 1 indicates high collectivity.Normal modes (NM) analysis was performed using the diagonalization in a mixed basis (DIMB) method i.e. the norm of the resultant displacement vector, of any fragment of the protein was calculated as:M of the m atoms belonging to the fragment –172 for JM-Switch and 181 for JM-Zipper.The resultant displacement, m orthogonal vectors 1w,…,mw with a reference subspace spanned by n orthonormal vectors 1v, …,nv and it can be quantified as follows:A Principal Component Analysis (PCA) was applied to each model to identify the main eigenvectors (3N directions) along which the majority of the collective motions are defined. The calculations were performed on the backbone atoms positions recorded every ps along the trajectories for each 45 ns simulation replica. The 100 first modes of each trajectory were extracted. The calculation was performed using the g_covar module of GROMACS package. The overlap between the first 10 modes of each trajectory was calculated using the g_anaeig module of GROMACS package. Briefly, the method consists in overlapping the subspace spanned by m and will be 1 when set v is a subspace of set w.The overlap will increase with increasing WT and CSF-1RMU were built and visualized with MONETA independent dynamic segments (IDSs) and chains of residues representing communication pathways (CPs). The representation is derived from the protein topology and the inter-residue dynamical correlations calculated on a conformational ensemble obtained by MD simulations. CPs were generated based on the communication propensities IDSs and CPs in CSF-1RWT and CSF-1RMU were determined using a protocol described in details in our previous work IDSs were identified from Local Feature Analysis (LFA) WT and CSF-1RMU, respectively. These vectors were used to apply the LFA formalism as described in cut was arbitrary chosen by the program to keep 1.0% of all LFA cross-correlations above it. The value was set to 0.035 for the WT and 0.038 for the D802V CSF-1R. Distance matrices consisting of the average of the smallest distance between each residue pairs were computed using the g_mdmat module of GROMACS package, v.4.5.6. Two residues were considered neighbors if the average smallest distance between them was lower than a given threshold dcut of 3.6 Å. Since we have observed a slightly different dynamical behavior in the two MD simulation replicas, we have computed the CPs on the individual MD simulations, considering the last 30 ns only, in order to distinguish between the communication pathways of CSF-1RWT and CSF-1RMU. One replica of each form of receptor was retained for the illustrations. The CPs definition was based on the concept of communication propensity described elsewhere CPs are grown ensuring that the adjacent residues are connected by non-covalent interactions and that every residue in the CP is connected to any other point by a shorter commute time (CT). Non-bonded interactions were recorded along the MD simulations using LIGPLOT CTs, a threshold cutCT was chosen so that highly connected residues communicate efficiently with about 10% of the total number of residues in the protein Modular network representations of CSF-1RStatistical analyses were performed with the R software 543–922) and its mutant D802V (referred to as CSF-1RWT and CSF-1RMU respectively) were generated from the crystallographic structure of the wild-type (WT) receptor in an auto-inhibited inactive state (2OGV) WT and KITMU abbreviation was used for cross-receptor comparisons.Models of the native cytoplasmic region of CSF-1R  of backbone atoms with respect to the initial frame plotted versus simulation time and showed separately for N- and C-lobes, the JMR and the A-loop regions (WT and two for CSF-1RMU) displayed comparable conformational drifts, with RMSD mean values in the range 0.12–0.30 nm indicating a tolerable stability of the simulated systems after a 5 ns relaxation interval. However, the RMSD profile of the A-loop region showed high deviation after 17 ns for one CSF-1RMU replica, with RMSD values up to ∼0.26 nm, which was not observed in the other trajectories. We observed a similar behavior for the A-loop in KIT MD simulations Molecular dynamics (MD) simulations of the generated models (two 50-ns trajectories for each form) were carried out to investigate and compare the structure and internal dynamics of the two proteins, CSF-1R regions Fig. 2A.WT and CSF-1RMU (MU (The root mean square fluctuation (RMSF) values, describing atomic fluctuations averaged over the protein residues, ranged from 0.1 to 0.4 nm, and were overall quantitatively comparable between CSF-1RCSF-1RMU Fig. 2B.WT and CSF-1RMU and shows in general a similarity between these two forms (10-helices formed by residues 798–800 (H1) and 803–805 (H2). Over the MD simulations of CSF-1RWT the structure of H1 region was mainly folded as a 310-helix while the H2 region secondary structure type alternated between 310-helix (5%), bend (20%), turn (30%) and coil (45%)  in silico studies in KITD816VD816H/N/Y mutants (our data submitted to publishing). The disappearance of the H2 310-helix changed the local H-bond network in the A-loop of CSF-1RMU (data not shown) as it was observed in KITMU.Such disappearing of the well-ordered structural element, previously observed in KITMU. The JMR structure and dynamics were strikingly similar in CSF-1RWT and CSF-1RMU. The quantitative analysis of the secondary structure pattern over the MD simulations revealed a retained secondary structure of the JMR in CSF-1RMU compared to CSF-1RWT . This secondary structure prediction matches well with the JMR structure of the native receptor (CSF-1RWT) observed by X-ray crystallography and obtained by MD simulations of CSF-1RWT and CSF-1RMU. This observation prompts to hypothesize that either the JMR structure in CSF-1R does not depend on the KD − a behavior quite different from the allosterically regulated JMR folding in KIT, − or D802 in CSF-1R and D816 in KIT do not play a similar role in the activation mechanisms.To explore the secondary structure profile of CSF-1R JMR (residues 538–580), we used six sequence-based secondary structure prediction methods and one structure knowledge-based method. Predictions indicated a relatively high probability of the polypeptide organization in well-folded structural elements, particularly β-strands in the segments 551–555 and 563–564 linked by a random coil including 4 residues, probably stabilized as a turn  (i) to clarify the mutation effects in the context of collective motions between functional CSF-1R fragments in the cytoplasmic region, (ii) to compare the impact of mutation on dynamical features of CSF-1R and KIT, and (iii) to connect motions with communications between spatially distant regulatory fragments, namely A-loop and JMR. The most relevant movements of CSF-1R fragments were identified by emphasizing the amplitudes  and directions (eigenvectors) of the protein motions dominating the residue pair covariance matrix calculated from the MD ensemble. The calculation was done for the individual MD simulation trajectories of each model and the best overlap between CSF-1RMU display eigenvalues twice as big as those of CSF-1RWT . To ensure the robustness of the method, we performed the analyses using five different random seeds for the reference structure picking up. For each form of the receptors, the fourth run containing the set of conformations that was better represented among the different replicas was chosen.Normal modes analyses (NMA) were carried out to further characterize the collective movements related to the JMR. The choice of the initial CSF-1R conformations was based on a convergence analysis performed on the merged trajectories Table S1 and Fig. S3. The computed degrees of collectivity of JMR atomic motions, JMRk, ranged from 1/n (only one atom among n is involved in the motion) to 1 (highly collective). The mean JMRk value for CSF-1RWT and CSF-1RMU of 0.44 and 0.42 respectively, indicating a low and statistically identical degree of collectivity in both proteins denoting the absence of independent motions associated with the JMR. The modes correlated to movements located at the JMR clearly indicated a great similarity between CSF-1RWT and CSF-1RMU. Altogether, the NM analysis confirmed the absence of JMR displacement from the KD in the mutated protein, evidenced by the PCA.The results of this analysis are summarized in MU. The d2 profiles of the two proteins blend into each other, demonstrating that JM-S and C-lobe retained their relative position in the mutated receptor. Secondly, we calculated the binding energy associated to the interaction between JMR and KD. The free energy of binding (ΔG) computed over the individual MD simulations by the MMGBSA method showed a tendency of JMR to display a lower affinity with the KD in CSF-1RMU than in CSF-1RWT (MUIn order to probe a possible coupling of the JMR with the kinase domain (KD), we first characterized the relative position of these two receptor's portions using two geometrical parameters, d1 and d2, describing the distance between the centroids defined on JM-B and N-lobe, and JM-S and C-lobe, respectively Fig. 5A.We also used the MD simulations data to calculate the occurrences of H-bonds involving key residues that maintain the inactive auto-inhibited form of CSF-1R MU appeared to be unfavorable to the H-bonds pattern  as a pseudo-substrate with the catalytic loop residue D778 (C-lobe) in CSF-1Rrds D778 Table 1Further, we compared the electrostatic potential surfaces of JMR and kinase domain in both receptors. The calculations were performed by the Adaptive Poisson-Boltzmann Solver (APBS) software using the crystallographic structures describing the inactive auto-inhibited state of the native receptors, CSF-1R  analyze the communication pathways in the cytoplasmic domain of the receptor, (ii) evaluate the role of residue D802 in communication pathways and finally (iii) assess the impact of the D802V mutation on the protein internal communication network.Independent Dynamic Segments (IDSs) that are formed around each seed and display concerted local atomic fluctuations and independent dynamical behavior Identification of the protein regions representing the most striking local features of the two proteins' internal dynamics was carried out using a statistical technique known as Local Feature Analysis (LFA) WT and 19 in CSF-1RMU, the number of IDSs identified by MONETA being 8 in CSF-1RWT and 9 in CSF-1RMU, respectively. The IDSs differences between the two receptors concern their feature, location, and size. To optimize the comparative analysis, the distinct IDSs were referred to as Si, where i = 1,2,…,N.The number of PCA modes retained for LFA was 17 in CSF-1RIDSs common to the two forms of receptor are located in JM-B , in JM-S , in the solvent-exposed loop that connects β2 and β3  in the N-lobe, in the pseudo-KID , in the A-loop , and in the C-terminal tail  . The three IDSs specifically observed in CSF-1RMU were localized in the N-lobe  and in the A-loop . Interestingly, the residues forming S9′ in CSF-1RMU were also found in S1, suggesting that the JM-B and the loop linking β3 and Cα-helix were associated in an entire self-reliant IDS (not shown). The other unexpected observations were the participation of D802V and Y809 in S5′ and S5, respectively.IDS in the N-lobe of CSF-1RWT and three in that of KITWTIDSs in the JMR, the A-loop, the pseudo-KID, and the G-helix were identical in the two native receptors. The impact of the equivalent mutation on the IDSs in the cytoplasmic region of the two receptors is dissimilar. In CSF-1RMU three novel IDSs, S5′, S9′ and S10′, are a consequence of increased concerted local motions of the A-loop and the loops linking β3 with Cα-helix, and β4 with β5  in CSF-1RMU, while in KITMU, only the point mutation is located in IDS.Using MONETA, we identified only one IDSs. To quantify the inter-residues communications, we computed the number of communication pathways (CPs) for each protein. In virtue of the strong influence of the dynamical behavior onto the communication pathways, the calculation of CPs was performed based on the individual MD simulations. For instance, the communication network computed over the 60 ns concatenated trajectory contains 1692 and 1626 non-redundant paths in CSF-1RWT and CSF-1RMU respectively, indicating the mutation-induced diminishing of the communication network in the receptor 802, the A-loop tyrosine Y809 and the CPs that connect JMR residues to other functional TKD segments, such as the P-loop, the Cα-helix and the C-loop, all involved in the stabilization of the inactive auto-inhibited conformation of the JMR  loop residue D778, and further, through the H-bond Y809•••D778, to the A-loop residues. The JM-S residues are involved in distinct CP networks providing connection of the JMR to the other functionally crucial fragments of the kinase domain.The differences in communication are illustrated using replica MD 2 for both CSF-1Rulations Table 1.communication pathways formed by the JM-B residues (Y546 and V548) with the P-loop (F593) and the Cα-helix (residues 628–633), the extended CPs from the JMR residues reaching the C-loop, and the Eα- Fα- and Hα-helices, constitute a developed multi-branched CP network capable to coordinate the movements of N- and C-lobes involved in CSF-1R activation mechanisms, i.e. post-translational modifications and catalytic functions. Interestingly, the CPs of each α-helix, Cα, Eα, Fα and Hα, are extended over the entire helix length, making a structurally preformed communication fiber. A considerable part of this extended CP network is completely lost in MD 2 from CSF-1RMU, i.e., no CP was observed between the JM-B and the P-loop, the Cα-, or the Hα-helices. Nevertheless, a relatively extended CP network is still observed between the JMR and the C-loop and the Eα- helix in CSF-1RMU  and S2 (JM-Switch) mainly to the molecular fragments not manifesting the concerted local atomic fluctuations (IDSs), except S5 formed by residues from the A-loop β-sheets. The links between residues belonging to IDSs and the other receptor fragments involved in CPs are held by H-bonds  loop is crucial for maintaining the allosteric regulation of the KD in KIT and its disruption in KITMU is a major contribution to its constitutive activation Our analysis showed that despite a comparable pattern of Fig. S4). Mutual CPs of the JM-B residues with the Cα-helix are extended over the entire helix length in the native protein, while few and relatively small CPs are observed in KIT.Another particularity of the CSF-1R communication pattern consists of the JMR communication with the glycine-rich P-loop and with the Cα-helix, not observed in KIT . Indeed, the P-loop and the Cα-helix in the active state of KIT are shifted respectively to their positions in the inactive autoinhibited state. The relative position of the P-loop and the Cα-helix in the active and inactive forms, which is equivalent in CSF-1R and divergent KIT, may reflect their different implication in the mechanisms regulating the activation of the two receptors. This hypothesis is coherent with the different communication pathways observed in the inactive autoinhibited state of these receptors. Nevertheless, such hypothesis requires an advanced examination of the structural features of both receptors in the active state. The crystallographic structure of CSF-1R active form  equivalent in the inactive state of both receptors; (ii) conserved over the inactive-to-active forms transition in CSF-1R; and (iii) highly dissimilar in KIT active and inactive forms  to analyze and compare the structure and molecular dynamics of the native and mutated KIT and CSF-1R, the present study demonstrates that the two homologous mutations do not have the same consequences in terms of receptor conformation and dynamics, providing a plausible explanation for the differential incidence of these mutations in oncology. The local impact of D802V mutation, which is a partial unfolding of the small 3The two mutations also disrupt the allosteric communication between two essential regulatory fragments of the receptor, the JMR and the A-loop. Nevertheless, the similarity is limited to only this effect. The mutation-induced shift towards an active conformation observed in KIT D816V is not observed in CSF-1R D802V. This differential impact on the conformational dynamics of the receptor, which might be related to differences in the primary sequence between the two wild-type receptors, particularly in the JMR region, could explain why the CSF-1R D802V does not confer a competitive advantage to the cell, thus is not retained as a driver oncogenic event. Nevertheless, it will be of interest to follow the response to new drugs, including small molecule kinase inhibitors, currently developed to target CSF-1R in cancer in which the receptor is often over-expressed, either on tumor cells or in the microenvironment Figure S1Structural organization of RTK III receptors. Receptor tyrosine kinases of type III comprise an extracellular cytokine binding region subdivided into five domains (from D1 to D5), a single transmembrane (TM) helix, a juxtamembrane region (JMR), a conserved tyrosine kinase (TK) domain containing a kinase insert domain (KID) and a carboxy-terminal tail. Specifically for CSF-1R, locations of mutation D802V and the main phosphorylation sites implicated in receptor activation are represented in the JMR and the activation (A-) loop.(TIF)Click here for additional data file.Figure S2WT.Secondary structure prediction of the JMR sequence (residues 538–580) from CSF-1R Prediction was performed using sequence-based algorithms GOR4 (TIF)Click here for additional data file.Figure S3WT (WT) and CSF-1RMU (D802V) models performed on the 90 ns concatenated trajectories.Convergence analysis of the MD simulations for CSF-1R Grouping of MD conformations was made using five independent runs calculated for each model. The populations of each group for each run are presented as histograms in the logarithmic scale denoted by different colors, black and grey from the 1st and 2nd halves of the two replica respectively. The identification numbers of each reference structure denotes the time (ns) in which it was picked from the MD trajectory. The fourth run contains reference structures that are better represented in both replicas and it was chosen for further NM calculations.(TIF)Click here for additional data file.Figure S4WT and KITMU.3D structural mapping of the inter-residues communication in KIT The average MD conformation is presented as cartoon. The proteins fragments are presented with different colors: JMR (blue), Cα-helix (violet), P-loop (yellow), C-loop (green) and A-loop (red). Communication pathways (CPs) between residues atoms  are depicted by coloured lines: CPs formed by the A-loop residues in orange; by the JMR-residues in magenta. The key residues in the communication networks are labelled (in KITWT) and depicted as bulky circles.(TIF)Click here for additional data file.Figure S5Structure of the cytoplasmic domain of CSF-1R and KIT in the native form. Superimposition of the CSF-1R and KIT crystallographic structures : (A) CSF-1R (2OGV B) CSF-1R in the inactive (2OGVC) KIT in the inactive (1T45) and active Click here for additional data file.Table S1Characteristics of convergence analysis of the native CSF-1R (WT) and its mutant (D802V) MD trajectories.(DOC)Click here for additional data file.


Primary central nervous system (CNS) melanoma is a type of rare and aggressive tumor that can easily spread to the leptomeninges, and in fact, leptomeningeal metastasis is one of the most serious complications in patients with this carcinoma. Prognosis is extremely poor if a CNS melanoma has metastasized, and there are no effective treatments. Here, we present a case of a 37-year-old woman who presented with horizontal diplopia and progressive headache. Magnetic resonance imaging findings were consistent with the diagnosis of melanoma. The results of cytological examination of cerebrospinal fluid (CSF) showed malignant cells characteristic of melanoma. No extracranial lesions were observed. All of the available evidence confirmed a diagnosis of leptomeningeal metastases from a primary CNS melanoma. The patient received aggressive treatment, which consisted of concurrent radiotherapy and weekly intra-CSF methotrexate (MTX) followed by adjuvant monthly intra-CSF MTX. Her survival time was 13 months after diagnosis. This case report suggests that the modality of concurrent radiotherapy and weekly intra-CSF MTX followed by adjuvant monthly intra-CSF MTX may be used as the mainstay of treatment for such patients. Primary central nervous system (CNS) melanoma is an uncommon disease and represents only 1% of all cases of melanoma . MelanobA 37-year-old woman was referred to our hospital because of horizontal diplopia and progressive headache for 2 weeks. She had a medical history of non-frequent epilepsy as a child. Scattered melanocytic nevi with hair were observed on her skin when she was born, without darkened color, enlargement, pruritus or ulceration. Neurological examination on admission revealed right eye abduction. No other focal neurological signs, such as abnormal reflexes, sensory deficits or neck stiffness, were noted.Cranial computed tomography (CT) scans revealed an isodense or slight hyperdense lesion  in the left temporal lobe  and erythrocytes (110 cells/mm3). Malignant cells with the characteristics of melanoma were found by cytological examination of the CSF  was hematic and cloudy, and the opening pressure was 30.4 mmHg (5 to 15 mmHg). Biochemical analysis of the CSF revealed that the glucose level was 28.8 mg/dL (41.4 to 73.8) and the protein level was 66 mg/dL (15 to 45). The cells consisted of leucocytes (15 cells/mm3) and erythrocytes (10 cells/mm3). No malignant cells were found in the CSF. Symptomatically, the patient was in complete remission. No severe treatment-related adverse events were noted, and her memory and cognitive ability had not obviously changed before and after treatment. Thereafter, it was suggested that she undergo monthly intra-CSF MTX. However, the patient herself refused the treatment on the fifth month. Headache and vomiting reappeared, but resolved following intra-CSF MTX (15 mg) monthly. Treatment was again interrupted until month 9, and recurrence was noted. She refused any examinations including brain MRI and CSF analysis. Unfortunately, re-administration of intra-CSF MTX was ineffective. The patient died from progression of the nervous system disease at 13 months after initial presentation with diplopia.The patient received aggressive treatment including concurrent whole brain radiotherapy  and intra-CSF chemotherapy. Methotrexate (MTX) 15 mg and dexamethasone 5 mg were given intrathecally once weekly from the second day of radiotherapy, for a total of 8 weeks. On the eighth administration of intra-CSF MTX, CSF examination showed that the opening pressure was 18.2 mmHg, with a colorless transparent appearance. The glucose and protein levels were 28.8 mg/dL and 18.5 mg/dL, respectively. Cellular components consisted of leucocytes  [Involved-field radiotherapy at sites of symptomatic or bulky disease is an important treatment modality for the majority of patients with leptomeningeal neoplasm. Paolo hibitors . The patactions) . Remissiactions) .Chamberlain and Kormanik carried out a phase II study of combined modality therapy for leptomeningeal metastases due to melanoma, which enrolled 16 patients . The majBased on previous studies, concurrent radiotherapy and intra-CSF chemotherapy were chosen for the present patient. Symptoms related to meningeal irritation were relieved quickly, and the patient survived for more than one year. No severe toxicity was observed. The therapeutic advantages of the present treatment modality are as follows: (1) MTX is an S-phase specific antineoplastic agent, and tumor cells in the G2 and M phases are more sensitive to radiotherapy than cells in other phases. Therefore, radiotherapy combined with intra-CSF MTX may improve the antitumor effects because of a synergistic effect on different phases of the cell cycle. (2) MTX has a sensitizing effect on radiotherapy . (3) WhoIn general, primary CNS melanoma is a rare disease associated with poor prognosis when the leptomeninges are involved. The treatment modality of concurrent radiotherapy and weekly intra-CSF MTX followed by adjuvant monthly intra-CSF MTX may be an efficacious combination. However, additional studies are needed to improve treatment and prognosis.Written informed consent was obtained from the next of kin of the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Thymus vulgaris L.), tincture and syrup, on carbon tetrachloride-induced acute liver injury in rats.Herbal supplements are widely used in the treatment of various liver disases, but some of them may also induce liver injuries. Regarding the infuence of thyme and its constituents on the liver, conflicting results have been reported in the literature. The objective of this study was to examine the influence of two commonly used pharmaceutical formulations containing thyme (Chemical composition of investigated formulations of thyme was determined by gas chromatography and mass spectrometry. Activities of enzyme markers of hepatocellular damage in serum and antioxidant enzymes in the liver homogenates were measured using the kinetic spectrophotometric methods. Liver morphology was characterized by light microscopy using routine hematoxylin and eosin staining.Thymol was found to be predominant active constituent in both tincture and syrup. Investigated thyme preparations exerted antioxidant effects in liver by preventing carbon tetrachloride-induced increase of lipid peroxidation. Furthermore, co-treatment with thyme preparations reversed the activities of oxidative stress-related enzymes xanthine oxidase, catalase, peroxidase, glutathione peroxidase and glutathione reductase, towards normal values in the liver. Hepatotoxicity induced by carbon tetrachloride was reflected by a marked elevation of AST and ALT activities, and histopathologic alterations. Co-administration of thyme tincture resulted in unexpected exacerbation of AST and ALT values in serum, while thyme syrup managed to reduce activites of aminotransferases, in comparison to carbon tetrachloride-treated animals.Despite demonstrated antioxidant activity, mediated through both direct free radical scavenging and activation of antioxidant defense mechanisms, thyme preparations could not ameliorate liver injury in rats. Molecular mechanisms of diverse effects of thyme preparations on chemical-induced hepatotoxicity should be more in-depth investigated. Herbal medicinal products are increasingly being used and many of them have shown promising potential for the treatment of various diseases. However, many of herbal medicines remain untested and their use also not monitored, which makes knowledge of their safety very limited . BesidesThe liver is highly susceptible to drug-induced toxicity due to its significant role primarily in drug metabolism. The pathophysiological mechanisms of drug-induced hepatotoxicity are still to be elucidated, but are mostly initiated by generation of hepatotoxic metabolites in the phase I of drug metabolism that often leads to the mitochondrial inhibition and accumulation of reactive oxygen species (ROS). Oxidative stress in hepatocytes finally results in apoptosis and necrosis of cells , 4. ConsThymus vulgaris L., Lamiaceae), a small subshrub native to the western Mediterranean region of Europe, is commonly used as a culinary herb, but also for different medicinal purposes. Antitussive, expectorant and spasmolytic effects are considered to be the major pharmacological properties of thyme, and therefore it is used for diseases of the upper respiratory tract, alone or in combination with other herbs [Thyme  and flavonoids  are proposed to be the polyphenolic compounds responsible for the antioxidant effects of ethanolic and aqueous extracts . It shou4)-induced liver injury in rats [Regarding the infuence of thyme and its constituents on the liver, conflicting results have been reported in the literature. Although thyme has been listed as Generally Recognized as Safe (GRAS) for use in food, it is known that long-term or high-dose administration of thyme preparations such as tinctures and essential oils that contain thymol and carvacrol in high amounts, can be hepatotoxic . Use of  in rats . The pro in rats  and esse in rats  have bee4-induced acute liver injury in rats.Therefore, the objective of our study was to examine the influence of two pharmaceutical formulations containing thyme, tincture and syrup, which are commonly used in humans, on CClThymus vulgaris L., Lamiaceae) used in this study were tincture and syrup. They were obtained from the Institute for Studies on Medicinal Plants ’Dr Josif Pančić’, Belgrade, Serbia, in 2012. A voucher specimen of the plant  was confirmed and deposited in the Herbarium of the Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad [Thymi herba) with 70 % ethanol in a 1:5 ratio of weight to volume. Thyme syrup consists of thyme tincture (15 % w/w) and potassium bromide, simple syrup, methyl and propyl p-hydroxybenzoate as preservatives, and water.Pharmaceutical formulations of thyme . All chemicals for biochemical assays were purchased from Sigma-Aldrich .The identification and quantification of thymol and carvacrol, as main chemical constituents of thyme tincture and syrup, were carried out by gas chromatography coupled with mass spectrometric detection (GC/MS) and flame ionization detection (GC/FID). GC/MS analysis was performed using an Agilent 7890A GC system equipped with 5975C VL MSD . The capillary column used in this study was DB-5MS . The temperature program was set as follows: initial temperature 50 °C held for 1 min, 5 °C per min to 100 °C, 9 °C per min to 200 °C held for 7.89 min, and the total run time was 30 min. The flow rate of helium as a carrier gas was 0.811851 ml/min. The MS system was operated in electron ionization (EI) mode with selected ion monitoring (SIM). The ion source temperature and quadrupole temperature were set at 230 °C and 150 °C, respectively. Identification of thymol and carvacrol was performed by comparison of their retention times and mass spectra with those of authentic standards and with spectra in the NIST08.L and Wiley7n.l libraries.GC/FID analysis was carried out using a Hewlett Packard 6890 chromatograph equipped with flame ionization detector, autosampler and a split/splitless injection system. The column DB-5MS  and helium as a carrier gas with flow rate of 1.9 ml/min were used in this analysis. Samples in volumes of 1 μl were injected in a split mode . The injector and detector temperatures were set at 250 °C and 300 °C, respectively, and the column temperature program was set as follows: initial temperature 60 °C held for 1 min, 10 °C per min to 265 °C held for 8.5 min, with the total run time of 30 min. The concentrations of thymol and carvacrol were determined using the calibration curves prepared with standards of thymol and carvacrol, and cumene as internal standard.50 value, which represents the concentrations of the sample required to cause 50 % inhibition of DPPH radical, was estimated by linear regression analysis from the obtained RSC values and was expressed in mg/ml.Antioxidant activity of the tincture of thyme was evaluated as free radical scavenging capacity (RSC). The ability of tincture to donate an electron and scavenge the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was investigated . The tinPhenolic content of tincture was determined using the Folin-Ciocalteu reagent . The tinad libitum. Animal care and all experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals edited by Commission of Life Sciences, National Research Council (USA). The study was approved by the Ethical Committee of the University of Novi Sad .The experiment was performed on 7–8 week old albino Wistar rats of both sexes, weighing 230–300 g, obtained from Military Medical Academy, Belgrade. The rats were housed in standard laboratory cages, under a 12 h light-dark cycle, at a constant ambient temperature (23 °C) and humidity (30–50 %), and were allowed to adapt for 2 weeks before the experiments were started. The animals were maintained on standard pellet diet  and allowed access to tap water 4-induced liver injury, the animals were randomly divided into six experimental groups, each containing six individuals, and treated as follows:p.o.Con S: Control group, saline 1 ml/kg 4: saline 1 ml/kg p.o. + a single CCl4 dose 1 ml/kg i.p.Con CClp.o.Tin: thyme tincture 0.18 ml/kg 4: thyme tincture 0.18 ml/kg p.o. + a single CCl4 dose 1 ml/kg i.p.Tin + CClp.o.Syr: thyme syrup 5.6 ml/kg 4: thyme syrup 5.6 ml/kg p.o. + a single CCl4 dose 1 ml/kg i.p.Syr + CClTo evaluate the effects of thyme formulations on CClper os gavage, previously diluted with saline. On the 7th day, 1 h after the last dose of thyme preparations or saline, animals were treated intraperitoneally with CCl4 dissolved in olive oil  [Applied daily doses of thyme tincture and syrup for rats were 0.18 ml/kg and 5.6 ml/kg, respectively. Recommended human daily doses of thyme tincture and syrup for a male of approximately 70 kg weight  were ada1 ml/kg) . After 2Total cholesterol and triglycerides levels, concentration of bilirubin, activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and the concentrations of urea and creatinine were determined in serum using commercially available kits based on the well-established spectrophotometric methods, according to the manuals supplied. All analyses were performed in triplicate for every sample on the Olympus AU 400 autoanalyzer .Oxidative status in the liver was estimated by measuring the levels of lipid peroxidation (LPx), and the activities of oxidative stress-related enzymes, including catalase (CAT), peroxidase (Px), glutathione peroxidase (GPx), glutathione reductase (GR) and xanthine oxidase (XOD) in liver homogenates, using the spectrophotometric methods.2O2 decomposition rate at 240 nm [The intensity of LPx was estimated by measuring the amount of malondialdehyde, a terminal product of lipid breakdown due to peroxidation damage, using the method of Buege and Aust, 1978 . XOD actt 240 nm , and Px t 240 nm . The actt 240 nm  and Goldt 240 nm , respect4, a small piece of hepatic tissue was removed for histological analysis. Liver tissue samples from each rat were fixed in Bouin’s solution for 24 h, then dehydrated in a graded series of isopropyl alcohol, and embedded in paraffin blocks. Five-micrometer tissue sections were cut using a rotation microtome (Leica Microsystems) in triplicate for each animal, and stained with hematoxylin and eosin for histological analysis under Olympus BX-43 light microscope  with an attached Olympus DP 73 video camera .Liver morphology was characterized by light microscopy using routine hematoxylin and eosin (H&E) staining. Twenty-four hours after administering CClp < 0.05. Data were analyzed using SPSS software, version 19 .Data are expressed as mean ± standard error of the mean (SEM). The intergroup variation between various groups was measured by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Results were considered statistically significant when Thymol and carvacrol, as the major phenolic active compounds of thyme, were identified and quantified in examined thyme formulations by GC/MS. Thymol concentrations of 175.3 μg/ml and 9.73 μg/ml were measured in tincture and syrup, respectively. Concentrations of carvacrol were much lower than those of thymol in both formulations, and they were 10.54 μg/ml and 0.55 μg/ml in tincture and syrup, respectively. GC/MS chromatograms of thymol and carvacrol standards and thyme syrup are shown in Fig. 50 value, defined as the concentration of the antioxidant required to scavenge 50 % of DPPH present in the test solution. The results demonstrated that investigated tincture had a strong radical scavenging activity with an IC50 value of 0.169 mg/ml. A comparison was made with ascorbic acid (vitamin C), well-known potent antioxidant and free radical scavenger, which exhibited IC50 value of 0.642 μg/ml. According to that, 1 mg of ascorbic acid was found to be equivalent to 263.2 mg of the thyme tincture.The antioxidant activity of thyme tincture was evaluated by the DPPH free radical scavenging test and the result was presented by ICAntioxidant activity of thyme was reported to be derived mostly from the presence of phenolic compounds, particularly thymol and carvacrol. We measured total phenolic content in order to determine the extent of contribution of these chemical compounds to free radical scavenging capacity of thyme tincture. Total phenolic content of investigated tincture was measured using the Folin-Ciocalteu method and the high amount of total phenols (4859.77 mg GAE/l) was determined.4-treated animals. Thyme tincture and syrup could partially revert those values, but they were still significantly lower than those of saline-treated group. CCl4induced a slight decrease in cholesterol levels, which was partially recovered after administration of thyme tincture. The intake of thyme preparations alone did not affect significantly the metabolic function of the liver. Impaired excretory function of the liver was observed after treatment of animals with CCl4, which is indicated by significant increase in both total and direct bilirubin levels in serum, compared to the control group. Co-administration of thyme formulations partially recovered bilirubin levels, while administration of thyme tincture and syrup alone did not change significantly these values when compared to saline-treated group.Biochemical parameters, related to hepatic and renal function, were determined in serum of rats and the results are presented in Table 4-treated group, compared to the control group. This effect was additionally exacerbated after the co-treatment with thyme tincture. On the contrary, co-administration of thyme syrup reduced the values of AST and ALT in serum, compared to CCl4-treated animals. Activities of these enzyme markers of liver injury were not significantly changed in animals treated only with thyme preparations compared to the control group.The extent of hepatocellular damage was assessed by measuring activities of AST and ALT in serum, which were increased 7- and 3-fold respectively in CCl4 has been observed, since it induced a slight decrease in urea and creatinine levels, which were not significantly changed after co-treatment with thyme preparations. The intake of thyme tincture and syrup alone did not affect significantly renal excretory function.Concentrations of urea and creatinine, as indicators of renal function, were also determined in serum. Nephrotoxic potential of CCl4-induced liver injury and to explore whether its mechanism of action is associated with modulation of hepatic oxidative status. Thus, we measured malondialdehyde (MDA) levels and activities of xanthine oxidase (XOD), catalase (CAT), peroxidase (Px), glutathione peroxidase (GPx), and glutathione reductase (GR), as biomarkers of the effects of studied thyme formulations, and the results are presented in Fig. In the present study, we aimed to evaluate the effects of thyme on CCl4 induced 5-fold increase of hepatic MDA level, when compared to the control group. Co-treatment with both thyme tincture and syrup during 7 days succeeded to decrease MDA significantly in liver tissue, compared to CCl4-treated group. The intake of thyme preparations alone induced a slight increase in MDA levels, but it was not statistically significant. The activity of pro-oxidant XOD enzyme was changed in a similar manner after treatment of animals with CCl4 and thyme preparations. XOD activity was significantly elevated in CCl4-treated group, while co-treatment with thyme tincture and syrup could significantly reduce activity of this enzyme. In animals co-treated with thyme syrup, XOD activty remained significantly higher when compared to that of saline-treated group.The single dose of CCl4 resulted also in a modification of antioxidant enzymes activities in the liver. Activities of CAT, Px, and GR were significantly reduced in CCl4-treated animals, compared to the control group, while GPx showed significantly increased activity in the liver. Co-administration of thyme preparations restored activities of CAT and Px towards normal values, and these increases were more pronounced in the group co-treated with tincture than with syrup. Thyme preparations significantly reduced the activity of GPx when applied with CCl4 in comparison to animals treated only with CCl4, and the tincture and syrup exerted these effects in a similar manner. On the other hand, the intake of thyme tincture alone resulted in significant increase of GPx, while syrup did not have that effect. Thyme tincture, when applied alone, also induced significant elevation of hepatic GR activity in comparison to both saline- and CCl4-treated animals, while that activity was not significantly changed in other studied groups.Oxidative stress induced by CClThe general liver morphological changes were examined by standard H&E staining. Histopathological examination of liver sections of control group showed normal cellular architecture with distinct hepatic cells, sinusoidal spaces and central vein. Hepatocytes were well formed with homogeneous cytoplasm and regular sized, centrally located nuclei. The sinusoidal capillaries were normal-sized without lymphocytic infiltrate. Hepatic artery, portal vein and bile ducts could be observed in portal spaces Fig. . Seven-d4-intoxicated rats, alone and in combination with thyme tincture, showed similar histopathological findings of steatosis or steatohepatitis. Most of hepatocytes contain lipid droplet that may be either macrovesicular or microvesicular . In our study, hepatotoxicity induced by CCl4 was reflected by a marked elevation of AST and ALT activities, but also by notable histopathologic alterations. Significant elevation of ALT and AST in serum after administration of CCl4 indicates a loss of the structural and functional integrity of the liver, and the release of these cytoplasmic enzymes into systemic circulation. In addition, our results reported increased level of serum bilirubin, thereby suggesting impaired excretory function of the liver. This is in agreement with the results of many earlier studies using a model of CCl4-induced liver damage. In this work, necrosis of hepatocytes was confirmed histologically.The influence of thyme preparations on CCl4-treated animals. Opposing effects of thyme tincture and syrup on the liver suggest that the dose of tincture is too high, since it is known that herbal polyphenols in high concentrations and under some circumstances such as the presence of transition metals or peroxidases, can become pro-oxidants and cause cytotoxicity and liver damage through generation of ROS [Administration of thyme preparations alone did not change significantly biochemical nor histological markers of hepatic function, when compared to saline-treated group. On the other hand, co-administration of thyme tincture resulted in unexpected exacerbation of AST and ALT values in serum, while thyme syrup managed to reduce activites of aminotransferases, in comparison to CCln of ROS . Besidesn of ROS .4 [4-induced liver injury in mice and it was suggested that it suppressed cytochrome P450 mediated metabolic activation of CCl4 [Previous studies demonstrated that oral administration of a concentrated ethanolic extract of thyme in subacute toxicity tests resulted in increased weights of liver in mice . Further4 . The res of CCl4 . Further of CCl4 .Promising results in terms of antidiabetic activity of thyme have been observed in both streptozotocin  and allo4 induced a slight decrease in urea and creatinine levels in serum of rats. In our previous studies [4 was applied in a higher dose (1 ml/kg) that exerted more pronounced nephrotoxic effects indicated by significant increase of urea and creatinine concentrations in serum. The parameters of renal function were not significantly changed after co-treatment with thyme preparations, which is in accordance with results of the study of Ozkol et al., 2013 [In addition to determination of hepatic function, the influence of thyme preparations on renal function has been also investigated in our study. CCl studies , 50, CCll., 2013 .4, since this compound is metabolized via reductive dehalogenation involving cytochrome P450, resulting in formation of ROS that cause the peroxidation of membrane lipids. Administration of CCl4 induced significant elevation of hepatic levels of MDA, a major secondary product of lipid peroxidation and indicator of cell membrane injury (Fig. 4-induced increase of MDA, suggesting their ability to preserve cellular integrity. Similar to our results, it was shown that dietary supplementation of thyme extract and essential oil during 8 weeks was able to ameliorate CCl4-induced lipid peroxidation in liver of rats [4 also increased the activity of pro-oxidant enzyme XOD that forms superoxide anion radical and hydrogen peroxide, and the intake of thyme significantly reversed these activities towards normal values.Considering the role of oxidative stress in etiopathogenesis of liver injury induced by most xenobiotics, we evaluated the influence of pharmaceutical formulations containing thyme on lipid peroxidation and endogenous antioxidant defense system in liver. We used the model of acute liver damage induced by CClury Fig. . Co-admi of rats . CCl4 al4 (Fig. 4. Similar results were obtained in our previous study [4 treatment. It is well known that CAT has an important role in the elimination of ROS derived from the redox process of xenobiotics in the liver, and that it is easily inactivated by lipid peroxides. On the other hand, under oxidative stress conditions, GPx can be upregulated as the adaptive response of the hepatocytes to oxidative damage and its activity can be increased [4, but these differences may be attributed to different treatment protocols.The activities of antioxidant enzymes were also significantly changed after administration of CCl4 Fig. . This tous study  and the us study , where tncreased . In sevencreased , 22, allin vivo models, beside models of liver damage. The essential oil and particularly aqueous tea infusion obtained from thyme exerted a protective role on copper-induced LDL oxidation [Antioxidant activity of thyme has been ascertained in several xidation . Furtherxidation . Severalxidation . Likewisxidation .in vivo, the isolated active compounds thymol and carvacrol have shown antagonistic effects. It is known that thymol may have an irritative effect that likely involves histamine and prostanoids, contrary to carvacrol with established anti-inflammatory activity [The results of our study have determined that thyme mediates its antioxidant activity through both direct free radical scavenging and activation of physiological defense mechanisms. Despite demonstrated antioxidant activity, thyme preparations unexpectedly could not ameliorate liver injury in rats. The role of oxidative stress in liver injuries is highly significant, but it should be emphasized that several other pathophysiological mechanisms of drug-induced hepatotoxicity have been shown to be involved, including inflammatory processes. Although anti-inflammatory effects of thyme have been demonstrated activity . Given tPharmaceutical formulations containing thyme may aggravate existing hepatotoxicity, probably through involvement in cellular inflammatory processes. The lack of clinical safety and toxicity data for thyme, and many other herbs that are increasingly being used, suggests the necessity of further investigations regarding their influence on hepatic function, including the evaluation of molecular mechanisms involved in order to exploit them for potential therapeutic benefits.


The hydrodynamics of the cerebrospinal fluid (CSF) is well-known to contribute to neurological disorders of the brain. However, little attention has been paid to the CSF dynamics in the lumbosacral spine. Meanwhile, the pathomechanism of neurogenic claudication, a characteristic symptom of spinal stenosis, has not been clearly elucidated. In this study, we suspected that spinal stenosis may be associated with altered CSF dynamics and compared CSF flow velocities at the lumbosacral spinal level between patients with spinal stenosis and healthy controls, at rest and at claudication.Twelve subjects  underwent phase-contrast magnetic resonance imaging (PC-MRI) to quantify CSF dynamics at the lumbosacral spinal level. Using PC-MRI, the CSF flow velocities were measured at the L2 and S1 levels. All of the subjects underwent PC-MRI at rest and after walking (to provoke neurogenic claudication in the patients).The flow rate in the sacral spine  was greatly attenuated compared to the flow in the lumbar spine  in both patients and controls. The caudal peak flow was slower in patients (-0.65 ± 0.22 cm/s) than controls (-1.07 ± 0.49 cm/s). The difference between the L2 caudal peak flow became more pronounced after walking . The sacral CSF flow after walking was barely detectable in patients  compared with controls . The severity of structural stenosis  did not correlate with the flow velocities within the participants.CSF dynamics in the lumbosacral spine were more attenuated in patients with spinal stenosis than healthy controls in a manner that was not proportionate to the structural stenoses. After walking, the CSF flow rate did not exhibit an appropriate increase in patients experiencing claudication, whereas the flow rate did increase appropriately in controls. Altered CSF dynamics may partially explain the pathophysiology of spinal stenosis.


Pythium insidiosum, is the etiologic agent of the life-threatening infectious disease called “pythiosis”. Diagnosis and treatment of pythiosis is difficult and challenging. Novel methods for early diagnosis and effective treatment are urgently needed. Recently, we reported a 74-kDa immunodominant protein of P. insidiosum, which could be a diagnostic target, vaccine candidate, and virulence factor. The protein was identified as a putative exo-1,3-ß-glucanase (Exo1). This study reports on genetic, immunological, and biochemical characteristics of Exo1. The full-length exo1 coding sequence  was cloned. Phylogenetic analysis showed that exo1 is grouped with glucanase-encoding genes of other oomycetes, and is far different from glucanase-encoding genes of fungi. exo1 was up-regulated upon exposure to body temperature, and its gene product is predicted to contain BglC and X8 domains, which are involved in carbohydrate transport, binding, and metabolism. Based on its sequence, Exo1 belongs to the Glycoside Hydrolase family 5 (GH5). Exo1, expressed in E. coli, exhibited ß-glucanase and cellulase activities. Exo1 is a major intracellular immunoreactive protein that can trigger host immune responses during infection. Since GH5 enzyme-encoding genes are not present in human genomes, Exo1 could be a useful target for drug and vaccine development against this pathogen.The oomycete organism,  Pythium insidiosum  and incubated at 37°C for 7 days.Seven strains of P. insidiosum was cultured on SD agar for 7 days at 37°C. Several small agar pieces with actively-growing mycelium were transferred to 100 ml SD broth and shaken (150 rpm) at 37°C for 7 days. Merthiolate (0.02% wt/vol) was added to kill the organism before the hyphae were filtered through a Durapore membrane filter . The resulting hyphae-free broth, containing PMSF (0.1 mg/ml) and EDTA (0.3 mg/ml), was called culture filtrate antigen (CFA). The separated hyphal mass was ground in a pre-cooled mortar in the presence of 25 ml of cold sterile distilled water containing PMSF (0.1 mg/ml) and EDTA (0.3 mg/ml). The hyphal lysate was centrifuged  at 4°C, for 10 min. The supernatant, called soluble antigen from broken hyphae (SABH), was filtered through a Durapore membrane filter . SABH and CFA were concentrated ~80 fold using an Amicon Ultra-15 centrifugal filter . The concentrated CFA and SABH were stored at -80°C until use.Antigens were prepared using the method described by Krajaejun et al . BrieflyP. insidiosum hyphae were harvested for gDNA extraction, using the conventional-extraction protocol described by Lohnoo et al  was added at 100 μl/well, and incubated at room temperature for ~5 min. The reaction was terminated by adding 100 μl/well of 0.3 N sulfuric acid. Optical density was measured using an Infinite200 Pro ELISA reader  at the wavelength of 595 nm. The cutoff value for a positive signal was calculated from the mean of the control ELISA signals plus 4 SDs, as reported by Chareonsirisuthigul et al  [The d water] . Diameteexo1-coding sequences from P. insidiosum strain P06, Pi-S, P16, P24, P29, and P17 have been submitted to the DNA Data Bank of Japan database, under accession numbers LC033486 to LC033491, respectively.All exo1 gene sequence  [5’-TCAGTCG-3’; the adenosine triphosphate (1+) is a predicted transcription start site; 5’-CGTTTTTCC-3’).Upstream  and downstream (484-bp long) regions of the partial d region . BLAST sdatabase  identifiTNYY-3’) , as indiexo1 open reading frame encodes a 742-amino-acid protein , and seexo1-coding sequences (accession number: LC033486 to LC033491) from six strains of P. insidiosum, and 35 top exo1-BLAST hit sequences from 9 oomycetes and 26 fungi [P. insidiosum and other oomycetes form a clade that is separate from the sequences of fungi  and 28c-14d-1x (1.9-fold higher), while the normalized expressions of exo1 in 28c-7d-0x was significantly decreased (0.9-fold lower) (P. insidiosum strain P06 (data not shown). These findings indicate that exo1 transcript accumulates over culture time and is regulated by temperature and carbon source availability.The d lower) . SimilarRabbit anti-Exo1 peptide serum was raised against the combination of Peptides-A, -B, and -C . Based oP. insidiosum infection, humans mount an antibody response to peptides of the Exo1 protein.Peptide-A, -B, -C, or combination of these peptides was used to coat wells of an ELISA plate, and incubated with serum samples from pythiosis patients  and healthy blood donors . With an exception of the Peptide-C , all pytE. coli expression vector pRSET-C, to make pPinsEXO1, as a HIS-tagged fusion (E. coli strain harboring pPinsEXO1 produced only trace amounts of protein (molecular weight: ~60–90 kDa) recognized by the rabbit anti-Exo1 peptide serum . Taken together, these findings indicate that the transgenically-expressed Exo1 has glycoside hydrolase activity.In order to demonstrate the expected hydrolytic activity of Exo1, we cloned the full-length coding region into an d fusion . The E. de serum , and nicde serum  to measu was cloned, expressed, and characterized. The deduced 742-amino-acid-long protein (Exo1) contains the BglC and X8 domains, suggesting a possible role in carbohydrate transport, binding, and metabolism. Results of analyzing Exo1 with the InterProScan [http://www.cazy.org/) [The full-length exo-1,3-β-glucanase gene of rProScan  softwarezy.org/) , suggestzy.org/) : EC 3.2.zy.org/) . The agaP. insidiosum) were phylogenetically grouped together and separate from the glucanase-encoding genes from fungi (P. insidiosum was more closely-related to Saprolegnia parasitica (a member of the Saprolegnian lineage), than Pythium ultimum and also Phytophthora spp  [P. insidiosum and S. parasitica are animal pathogens, while all other oomycetes included in this study are plant pathogens [As expected from their evolutionary histories , the gluom fungi ; Fig 3. lineage) . This paathogens .exo1 in response to stresses, such as elevated temperature, aging, and limited carbon source, have been investigated here. The ability to grow at human body temperature (37°C) is a crucial factor for successful pathogens, including P. insidiosum. As demonstrated by quantitative real-time PCR  up-regulated exo1 expression , and trace amounts of several lower molecular weight proteins (~20–60 kDa), in SABH (representing intracellular proteins) . All of P. insidiosum [E-value < -87; P. insidiosum transcriptome-derived protein sequences found matches to putative exo-1,3-β-glucanases from Phytophthora spp , but not by control sera CS1-3; . Additioexo1) was successfully cloned and expressed. Glucanase genes from oomycetes, including P. insidiosum, form a clade that is distantly related to the glucanase genes from fungi. Exo1 was predicted to contain the BglC and X8 domains found in proteins with a role in carbohydrate metabolism. Exo1 was characterized as a major intracellular immunoreactive protein that exhibits GH5 hydrolytic activity. exo1 is up-regulated upon exposure to body temperature and prolonged incubation, but down-regulated upon exposure to long term carbon-depleted conditions. Exo1 contains B-cell epitopes that trigger host immune responses during P. insidiosum infections in humans. Since GH5 enzyme-encoding genes are not present in the human genome, Exo1 could be a good candidate for development of drugs or vaccines against P. insidiosum.In conclusion, full-length exo-1,3-β-glucanase-encoding gene  or post-immune (Lane: Post-Immune) serum. The arrowheads indicate multiple protein bands (sizes: 60–90 kDa), only present when probed with the rabbit post-immune serum.Cell lysate proteins prepared from the (TIF)Click here for additional data file.


Endothelial progenitor cells (EPCs) are bone marrow-derived precursor cells that may repair vascular injury in atherosclerosis. Chemokine (C-C motif) receptor 5 (CCR5) promotes mobilization of EPCs. In this study, we investigated the therapeutic potential of CCR5-overexpressing EPCs on plaque stabilization in an apolipoprotein E (ApoE)−/− C57BL/6 J mice fed a high-fat diet for 24 weeks were intravenously injected with EPCs transfected with CCR5 overexpression lentivirus. The recruitment of EPCs over the atherosclerotic plaques was evaluated by immunofluorescence. The content of lipid, smooth muscle cells, monocytes/macrophages, and endothelial cells in atherosclerotic plaques was assayed by specific immunostaining. The serum levels of atherosclerosis-related inflammatory factors in ApoE−/− mice were measured by mouse atherosclerosis antibody array I.The expression of CCR5 and its cognate ligand chemokine (C-C motif) ligand 5 (CCL5) was examined in atherosclerotic aortas of humans and mice by immunohistochemistry. Splenectomized ApoE−/− mice with CCR5-overexpressing EPC treatment demonstrated a more stable plaque formation with enhanced recruitment of EPC, reduced lipid, and macrophage content in the atherosclerotic plaques. CCR5-overexpressing EPC treatment also increased the content of endothelial cells and nitric oxide production in the plaques. In addition, the serum levels of interleukin-3 (IL-3), IL-5, IL-6, IL-13, CD40, and tumor necrosis factor-alpha and the plaque contents of IL-6 and matrix metalloproteinase-9 were reduced in mice with CCR5-overexpressing EPC treatment.CCR5 and CCL5 are highly expressed in atherosclerotic plaques in both humans and mice. The ApoEThese findings suggest that CCR5 is a novel therapeutic target in EPC treatment for stabilization of atherosclerotic plaques.The online version of this article (doi:10.1186/s13287-015-0026-0) contains supplementary material, which is available to authorized users. Atherothrombotic events, such as myocardial infarction and strokes, are the most devastating clinical manifestations of atherosclerosis ,2. The lex vivo to an endothelial phenotype -RANTES/CCL5 and [E66A]-RANTES/CCL5 mutants displayed a reduced ability to induce the migration of EPCs [It has been shown that specific interactions between chemokines and their receptors mediate the chemotaxis activation of EPCs ,14,48. O of EPCs . CCL5 isIn summary, we provided evidence for improving atherosclerotic plaque stability through treatment of CCR5-overexpressing EPCs. If CCR5 has a similar function in humans, stimulation of CCR5 expression in EPCs may promote EPC recruitment, restore EC function, decrease pro-inflammatory factors, and eventually improve the stabilization of atherosclerotic plaques.


N=331) in the BioFINDER study, using cerebrospinal fluid (CSF) Aβ40 as a surrogate for amyloidogenic APP processing. We find that levels of brain Aβ fibrils (measured by 18F-flutemetamol PET) are independently associated with high CSF Aβ40 (P<0.001) and APOE ɛ4 (P<0.001). The association between CSF Aβ40 and brain Aβ is stronger in APOE ɛ4-negative than in positive people (P=0.0080). The results are similar for CSF Aβ38 and for a combination of CSF Aβ38 and CSF Aβ40. In conclusion, sporadic Aβ accumulation may be partly associated with increased amyloidogenic APP production, especially in APOE ɛ4-negative subjects. The risk for sporadic AD may consequently depend on increased Aβ production, in addition to decreased Aβ clearance.Increased APP (amyloid precursor protein) processing causes β-amyloid (Aβ) accumulation in autosomal dominant Alzheimer's disease (AD), but it is unclear if it also affects sporadic Aβ accumulation. We tested healthy controls and patients with mild cognitive symptoms ( Autosomal dominant Alzheimer's disease is thought to be caused by increased amyloidogenic APP processing. Mattson et al. show that association between brain Aβ and cerobrospinal fluid Aβ40 levels is stronger in APOE ɛ4 negative people, suggesting that increased processing may also underlie sporadic disease. APOE ɛ4 allele is the main genetic susceptibility factor for late-onset AD and sporadic Aβ pathologyAPOE ɛ4 gene product apoE4 has reduced capacity to clear Aβ peptides from the brainAPOE ɛ4 APOE ɛ4. We hypothesized that there would be independent correlations between Aβ accumulation and the predictors APOE ɛ4 and CSF Aβ40, and that increased amyloidogenic APP processing would be related to Aβ accumulation mainly in APOE ɛ4-negative subjects. We also hypothesized that CSF Aβ40 would not be associated with APOE ɛ4 . Finally, we hypothesized to see similar results when using CSF Aβ38 instead of CSF Aβ40 to estimate amyloidogenic APP processing.Brain accumulation of amyloid β (Aβ) is a hallmark of Alzheimer's disease (AD) which may precede dementia by up to two decades124 ɛ4 ref.  and ∼40–P<0.001) and APOE ɛ4 (P<0.001) and that the association between CSF Aβ40 and brain Aβ is stronger in APOE ɛ4-negative than in positive people (P=0.0080). The results are similar when using CSF Aβ38 or a combination of CSF Aβ38 and CSF Aβ40 to estimate amyloidogenic APP production. We conclude that sporadic Aβ accumulation may be partly associated with increased amyloidogenic APP production, especially in APOE ɛ4-negative subjects. Thus, the risk for sporadic AD may partly depend on increased Aβ production, in addition to decreased Aβ clearance.Our results confirmed our hypothesis. We show that 18F-flutemetamol PET levels are independently associated with high CSF Aβ40 (APOE status). In sum, APOE ɛ4 positivity was more common in SCD and MCI than in CN, CSF Aβ42 levels were lower in MCI compared with the other groups, and the frequency of PET Aβ positivity was lowest in CN and highest in MCI. CSF Aβ38 and CSF Aβ40 did not differ between the diagnostic groups. APOE ɛ4 was not associated with CSF Aβ40 or with CSF Aβ38  121, subjective cognitive decline (SCD) 102 and mild cognitive impairment (MCI) 108). Demographics and data on cognition and biomarkers are summarized in CSF Aβ38 . The lacAPOE ɛ4-positive and -negative groups. In a linear regression model with PET Aβ as the dependent variable, high levels of CSF Aβ40 , APOE ɛ4-positivity  and the interaction between APOE ɛ4 and CSF Aβ40  were all significant predictors of continuous PET Aβ. Note that since APOE ɛ4 and CSF Aβ40 were both included as predictors the main effect of CSF Aβ40 indicates the effect within APOE ɛ4-negative subjects. The significant interaction between CSF Aβ40 and APOE ɛ4 indicates that the correlation between CSF Aβ40 and brain Aβ was stronger in APOE ɛ4-negative than in positive people . The results support the hypotheses that high CSF Aβ40 and APOE ɛ4 are independent predictors of PET Aβ, and that the relationship between CSF Aβ40 and PET Aβ varies with APOE ɛ4 carrier status. As expected, CSF Aβ42 was a significant covariate , but CSF Aβ40, APOE ɛ4 and the interaction between CSF Aβ40 and APOE ɛ4 remained significant also when not adjusting for CSF Aβ42 . Age  and diagnosis  were also significant predictors of PET Aβ, but sex was not (P=0.23). White matter lesions (WML) were evaluated as a covariate, but were not significant (P=0.68) and were therefore excluded from the final model. We also evaluated plasma levels of Aβ40 as a covariate to exclude the possibility that the results depended on peripheral APP processing. Plasma Aβ40 was not a significant covariate (P=0.99) and including it in the model did not change the other estimates.−4, P<0.001) was a significant predictor in this model but APOE ɛ4 (P=0.13), and the interaction between CSF Aβ40 and APOE ɛ4 were not (P=0.73). Age and sex (P=0.13–0.14) were not significant but CSF Aβ42 (P<0.001) and diagnosis  remained significant covariates. When CSF Aβ42 was excluded from the model, CSF Aβ40 (P=0.014) and APOE ɛ4 (P<0.001) were both significant predictors of PET Aβ positivity.To further examine if clinically significant Aβ accumulation  >1.42 ref.  was assoβ=4.04 × 10−4, P<0.001), APOE ɛ4  and the interaction between APOE ɛ4 and CSF Aβ38  were all significant, and CSF Aβ42 was a significant covariate . CSF Aβ38 (P=0.017), APOE ɛ4 (P<0.001) and the interaction between APOE ɛ4 and CSF Aβ38 (P=0.015) remained significant when removing CSF Aβ42 from the model. When predicting PET Aβ positivity using logistic regression, CSF Aβ38  and APOE ɛ4  were independent predictors and there was a tendency for significant interaction between APOE ɛ4 and CSF Aβ38 (P=0.061). Again, CSF Aβ42 was a significant covariate .To corroborate our findings, we repeated the analyses using CSF Aβ38 instead of CSF Aβ40, with very similar results. When predicting continuous PET Aβ, the effects of CSF Aβ38 (−1). Again, the results were very similar. When predicting continuous PET Aβ, the effects of CSF Aβ , APOE ɛ4  and the interaction between APOE ɛ4 and CSF Aβ  were all significant predictors. CSF Aβ42 remained a significant covariate . CSF Aβ (P=0.0090), APOE ɛ4 (P<0.001) and the interaction between APOE ɛ4 and CSF Aβ (P=0.021) remained significant when removing CSF Aβ42 from the model. When predicting PET Aβ positivity using logistic regression, CSF Aβ  was a significant predictor and CSF Aβ42 remained a significant covariate .We also used a combination of CSF Aβ40 and CSF Aβ38 based on their molar amounts  compared with PET Aβ- & APOE ɛ4− subjects. The model was adjusted for WML , age , sex  and diagnostic group . When also adjusting for CSF Aβ42 as a covariate the effect of PET Aβ & APOE ɛ4 was even stronger, with higher CSF Aβ40 in the PET Aβ+ & APOE ɛ4− group compared with PET Aβ- & APOE ɛ4− (P<0.001) and PET Aβ- & APOE ɛ4+ (P<0.001) but no significant difference compared with the PET Aβ+ & APOE ɛ4+ group (P=0.45).In a linear regression model with CSF Aβ40 as the dependent variable and a four level combination of PET Aβ and APOE ɛ4 were independent predictors of brain Aβ fibril accumulation. In accordance with our hypotheses, CSF Aβ40  and APOE ɛ4 were independent predictors of PET Aβ, and the effect of CSF Aβ40 was strongest in the APOE ɛ4-negative individuals. To our knowledge, this is the first study showing that increased Aβ production are associated with increased risk for sporadic brain Aβ accumulation. These novel results provide indirect evidence that brain Aβ pathology in humans may arise from two pathways, where one involves the APOE ɛ4 allele (likely causing reduced apoE4-mediated Aβ42 clearance), and the other involves increased amyloidogenic processing of APP. This may correspond to two pathways to sporadic AD, namely reduced clearance and increased production of Aβ peptides.We tested the hypothesis that biomarker surrogates of amyloidogenic APP processing (CSF Aβ40 and Aβ38) and APOE ɛ4, since the apoE4 protein isoform has reduced capacity to clear Aβ peptides compared with other apoE isoformsAPOE ɛ4 may also contribute to increased AD risk by other mechanisms, for example, by affecting inflammation and neuronal repair15APP gene polymorphism which reduces Aβ production is associated with reduced risk of AD in the general populationThe amyloid cascade hypothesis postulates that Aβ pathology arises due to an imbalance between Aβ production and clearanceAPOE ɛ4-mediated impaired Aβ clearance. The later was demonstrated by our finding that there was no overall difference in CSF Aβ40 depending on APOE ɛ4 status  may impact Aβ clearance in APOE ɛ4-negative subjectset al.The main limitation of this paper was that we used an indirect measure of APP processing, which was estimated by CSF Aβ40. The rationale for this approach was that Aβ40 is a major Aβ isoform produced by neurons4 status . Alterat4 status . It is nAPOE ɛ4 and APP processing in the development of brain Aβ pathology in non-demented subjects. Previous studies did not find correlations between CSF Aβ40 (or Aβ38) and PET Aβ (ref. APOE ɛ4 (and/or CSF Aβ42). Adjusting for APOE ɛ4 is important since the relationship between CSF Aβ40 and PET Aβ differs between APOE ɛ4-positive and -negative individuals. Furthermore, adjusting for APOE ɛ4 and CSF Aβ42 reduces the residual errors of the models, and some of this error may contribute to the variance of CSF Aβ40. Once this error is removed the correlation between CSF Aβ40 and PET Aβ can be better estimated. Our results add novel information and point to different possible pathways to Aβ pathology in humans. In sum, our results support the idea that sporadic Aβ accumulation may be partly associated with increased amyloidogenic APP production, especially in APOE ɛ4-negative subjects. The risk for sporadic AD may consequently depend on increased Aβ production, in addition to decreased Aβ clearance. This provides novel insight into disease mechanisms in AD and may be important for development of drugs targeting Aβ metabolism in early stages of AD.Until now, there have been few attempts to examine the independent roles of  Aβ ref. . This isThe study population came from the Swedish BioFINDER study (Biomarkers For Identifying Neurodegenerative Disorders Early and Reliably). All available CN and non-demented patients with mild cognitive symptoms characterized as having SCD or MCI were included.CN subject were originally enrolled from the population-based EPIC cohort. The inclusion criteria were: age ≥60 years old, MMSE 28-30, and fluent in Swedish. Exclusion criteria were: presence of subjective cognitive impairment, significant neurologic disease , severe psychiatric disease , dementia or MCI. All CN subjects underwent a thorough clinical assessment, including neurological, psychiatric and cognitive testing all performed by a medical doctor, in addition to MRI of the brain and relevant blood tests. The cognitive battery included MMSE, ADAS-cog (items 1–3), Trail Making A & B, Symbol Digit modalities, A quick test of cognitive speed, clock drawing, cube coping, letter S fluency and animals fluency. The medical doctor made a global assessment of whether the individual was cognitively healthy based on the test results in relation to education and age. All CN subjects had a Clinical Dementia Rating scale score of 0.The SCD and MCI cases were recruited consecutively and were thoroughly assessed by physicians with special competence in dementia disorders. The inclusion criteria were: referred to a memory clinic due to possible cognitive impairment, not fulfilling the criteria for dementia, MMSE 24–30, age 60–80 years and, fluent in Swedish. The exclusion criteria were: cognitive impairment that without doubt could be explained by another condition ; severe somatic disease; and refusing lumbar puncture or neuropsychological investigation. The classification in SCD or MCI was based on a neuropsychological battery and the clinical assessment of a senior neuropsychologist. The battery included tests for verbal ability . Most analyses in this study used continuous PET Aβ but when indicated a previously defined cutoff for Aβ positivity was used , plasma samples were aliquoted into polypropylene tubes and stored at −80 °C pending biochemical analyses. Plasma Aβ40 was analysed using Simoa immunoassay .For plasma collection, blood was drawn into tubes containing EDTA as anticoagulant. After centrifugation (2000http://www.applied-statistics.de/lst.html), generating a total WML volume. Before this, manual segmentation for reference of WML was performed on FLAIR images co-registered to the native MPRAGE in four MCI patients, with the segmented volume ranging from 0.5 to 106.3 ml; the resulting optimal κ based on the Dice coefficient was 0.4 . Automated segmentation of WML was performed using the Lesion Segmentation Tool implemented in SPM8 (0.4 ref.  and was APOE ɛ4 (dichotomous), and the interaction between CSF Aβ40 and APOE ɛ4. Second, we tested the correlation between clinically significant PET Aβ accumulation and CSF Aβ40 and APOE ɛ4 in a logistic regression model with PET Aβ positivity as the dependent variable. Third, we tested a linear regression model with CSF Aβ40 as the dependent variable and a four level combination of PET Aβ and APOE ɛ4 as the independent variable . All models were adjusted for age (years), sex and diagnostic group. We also adjusted for CSF Aβ42 to test if CSF Aβ40 was associated with PET Aβ beyond CSF Aβ42, and to reduce the residual error of the model, allowing a better estimate of the correlation between CSF Aβ40 and PET Aβ. We adjusted for WML (ml) except for when WML was clearly nonsignificant, as detailed in the results section. The primary analyses were done using CSF Aβ40, but we also performed analyses using CSF Aβ38 and a combination of CSF Aβ38 and Aβ40 .We tested correlations between CSF Aβ40 and PET Aβ in different regression models. The main model was a linear regression model where the dependent variable was PET Aβ and the independent variables were CSF Aβ40, l−1 ref. . StatistHow to cite this article: Mattsson, N. et al. Increased amyloidogenic APP processing in APOE ɛ4-negative individuals with cerebral β-amyloidosis. Nat. Commun. 7:10918 doi: 10.1038/ncomms10918 (2016).


Troponin from a patient with the K280N TnT mutation showed no difference in Ca2+-sensitivity when compared with donor heart troponin and the Ca2+-sensitivity was also independent of the troponin I phosphorylation level (uncoupled). The recombinant K280N TnT mutation increased Ca2+-sensitivity 1.7-fold and was also uncoupled. The R92Q TnT mutation in troponin from transgenic mouse increased Ca2+-sensitivity and was also completely uncoupled. Five TnT mutations  studied in recombinant troponin increased Ca2+-sensitivity and were all fully uncoupled. Thus, for HCM-causing mutations in TnT, Ca2+-sensitisation together with uncoupling in vitro is the usual response and both factors may contribute to the HCM phenotype. We also found that Epigallocatechin-3-gallate (EGCG) can restore coupling to all uncoupled HCM-causing TnT mutations. In fact the combination of Ca2+-desensitisation and re-coupling due to EGCG completely reverses both the abnormalities found in troponin with a TnT HCM mutation suggesting it may have therapeutic potential.We investigated the effect of 7 Hypertrophic Cardiomyopathy (HCM)-causing mutations in troponin T (TnT) on troponin function in thin filaments reconstituted with actin and human cardiac tropomyosin. We used the quantitative  •in vitro motility assay.7 HCM-causing mutations in cardiac TnT were studied using •2+-sensitivity (range 1.5–2.7 fold).All the mutations increased myofilament Ca•2+-sensitivity by TnI phosphorylation.All mutations suppressed the modulation of Ca•Epigallocatechin-3-gallate (EGCG) restored this modulation to all mutations.•This suggests a therapeutic potential for EGCG in the treatment of HCM. A recent study has shown that 11% of the identified mutations are in the proteins of the thin filament; actin, tropomyosin, troponin I (TnI), troponin C (TnC) and troponin T (TnT) 2+-sensitivity is reduced in familial DCM (the opposite of HCM) has proven to be incorrect since changes of Ca2+-sensitivity do not correlate with the phenotype 2+-sensitivity is not modulated by TnI phosphorylation, a process we have called uncoupling, and we have proposed that this is a disease-mechanism Mutations in the same sarcomeric protein genes are also associated with familial dilated cardiomyopathy (DCM), a disease characterised by a hypo-contractile phenotype with dilation of the heart chamber and thinning of cardiac muscle 2+-sensitivity 2–3 fold and correspondingly increases the rate of Ca2+ dissociation from TnC. This is an essential component of the lusitropic response to adrenergic stimulation, consequently DCM mutations cause a blunting of the response to β1 agonists, and this leads to a reduced cardiac reserve and predisposes the heart to failure under stress TnI is one of several substrates of protein kinase A (PKA). In normal heart, on adrenergic stimulation PKA is activated and TnI phosphorylation at Ser22 and 23 is increased. Phosphorylation of TnI decreases myofibrillar CaTNNT2) are the most common HCM-causing mutations after MYH7 and MYBPC3. There have been persistent reports from in vitro studies that HCM-causing mutations in the other thin filament proteins: TnI, TnC, actin and tropomyosin uncouple changes in myofilament Ca2+-sensitivity from TnI phosphorylation level in addition to increasing absolute Ca2+-sensitivity  monoclonal antibody affinity column as described 2.2Troponin isolated from human heart samples has a high level of phosphorylation, which was reduced by treatment with shrimp alkaline phosphatase (Sigma P9088). Recombinant TnI was phosphorylated by treatment with protein kinase A (PKA) catalytic subunit  as previously described 2.32+-regulation of filament motility by the quantitative in vitro motility assay (IVMA) Thin filaments were reconstituted with 10 nM rabbit skeletal or mouse cardiac muscle α-actin , tropomyosin (40–60 nM) and troponin (60 nM) to study Ca2+ concentrations to generate Ca2+-activation curves as shown previously 50 and nH. EC50 values from replicate experiments were analysed by paired t-test since the distribution of EC50 has been shown to be normal.The fraction motile and the sliding speeds were measured over a range of Ca33.1We initially studied a TnT mutation in a unique cardiac muscle sample from a patient with HCM due to a homozygous mutation in TnT, K280N. The patient had a septal myectomy but subsequently required a heart transplant at age 26. The mutation was identified and contractility of skinned myocytes was investigated in a previous study We isolated troponin from the sample and confirmed that asparagine had exclusively replaced a lysine at amino acid 280 (K280N) by mass spectrometry . The TnI2+-regulation of unloaded movement over immobilised heavy meromyosin. When we compared thin filaments containing donor heart and TnT K280N patient heart troponin there was no difference in the Ca2+-sensitivity or maximum sliding speed  patients that generally also had an uncoupled relationship between Carylation , Table 1rylation  and obse2+-sensitivity was similar to donor heart troponin, as observed here, and did not change with TnI phosphorylation level independently of the gene that was mutated 2+-sensitivity 1.7-fold; a similar shift in Ca2+-sensitivity was obtained with 90% and 45% exchanged  and related compounds ntration B. The otrylation C. Thus E44.12+-sensitivity was not very different from donor heart controls: the two were indistinguishable in IVMA whilst TnT K280N patient skinned muscle had a 20% higher Ca2+-sensitivity in skinned fibres. The studies in myofibrils indicated that the kinetics of contraction at saturating Ca2+ were altered by the mutation, with kACT and slow kREL both being increased compared to donor heart myofibrils, similar to a previous study with an HCM mutation in myosin, however, Ca2+ dependence was not measured This sample enabled us to study troponin with an HCM related mutation in TnT in the context of the patient's heart. Contractility in the same sample has also been studied in skinned muscle fibres and in single myofibrils 2+-sensitivity 1.7-fold in the IVMA, as is common with HCM-causing mutations  and the Seventh Framework Program of the European Union ‘BIG-HEART’ (grant agreement 241577).This work was supported by grants from the The authors declare no conflict of interest.


In 2011, vaccination with a serogroup A meningococcal polysaccharide conjugate vaccine was implemented in 3 of 23 regions in Chad. Cases of meningitis declined dramatically in vaccinated areas, but an epidemic continued in the rest of Chad. In 2012, the remaining Chad population was vaccinated, and the epidemic was halted. For >100 years, countries in the meningitis belt of Africa have experienced intermittent epidemics of meningococcal meningitis, caused mainly by the serogroup A meningococcus  was undertaken in the capital N’Djamena, Mayo Kebbi Est, and Chari Baguirmi . In 2012N. meningitidis were sent to the World Health Organization (WHO) Intercountry Support Team, Ouagadougou, Burkina Faso, and to the WHO Collaborating Centre for Reference and Research on Meningococci in Oslo. Information about the laboratory methods used to isolate and characterize meningococci is provided elsewhere . The proportion of reported cases for which CSF samples were submitted from the N’Djamena regions was highest in 2010, when the main pediatric unit for N’Djamena was in the same hospital as the national reference laboratory  and in 2012, when case-based surveillance was introduced. In Moissala, the proportion of cases for which CSF samples were submitted increased from 22% (74/341) in 2012 to 119% (56/47) in 2013, the latter figure being attributed to undernotification. During weeks 1–26 of 2013, a total of 106 CSF specimens were received by the national reference laboratory; 13 yielded Streptococcus pneumoniae, 4 Haemophilus influenzae type b, 2 N. meningitidis serogroup W, and 1 (obtained from a 3-year-old child who had not received PsA-TT) serogroup A N. meningitidis. Four infections were caused by other pathogens. This finding differed markedly from those of previous years (2010−2012), when the predominant organism was N. meningitidis serogroup A, and only a few cases caused by N. meningitidis serogroup W and S. pneumoniae were also identified. The predominance of serogroup A infection in Chad during 2010–2012 was confirmed among the CSF specimens examined at the National Institute of Public Health in Oslo. All fully characterized serogroup A strains were porA 20.9, FetA F3.1, sequence type (ST) 7 (ST5 complex), and all serogroup W strains were porA 5.2, FetA F1–1, ST11 (ST11 complex). Although national reference laboratory data were not available in Chad for 2009, the predominant organism identified from CSF specimens received at the National Institute of Public Health in Oslo in 2009 was N. meningitidis serogroup W (11/14 serogrouped strains), also porA 5.2, FetA F1–1, and ST11 (ST11 complex) (Fewer CSF specimens were submitted to the national reference laboratory in N’Djamena in 2013 than in 2012 , but theN. meningitidis serogroup A epidemic in Chad was waning and that fewer cases of serogroup A meningitis would have occurred during 2013 than during 2012, even in the absence of vaccination. However, the incidence of meningitis dropped lower in 2013 than would have been expected as a result of a natural decline, and only 1 serogroup A isolate was obtained at the national reference laboratory despite improved CSF sampling. This finding provides strong additional evidence of vaccine effectiveness for preventing serogroup A meningococcal disease in Chad.We previously reported a >90% reduction in incidence of meningitis among vaccinated populations in Chad in 2011  suggestsIf the effectiveness of PsA-TT vaccination seen in Chad and Burkina Faso (