Signaling pathways play important roles in the life processes of cell growth, cell apoptosis and organism development. At present the signal transduction networks are far from complete. As an effective complement to experimental methods, computational modeling is suited to rapidly reconstruct the signaling pathways at low cost. To our knowledge, the existing computational methods seldom simultaneously exploit more than three signaling pathways into one predictive model for the discovery of novel signaling components and the cross-talk modeling between signaling pathways.In this work, we propose a multi-label multi-instance transfer learning method to simultaneously reconstruct 27 human signaling pathways and model their cross-talks. Computational results show that the proposed method demonstrates satisfactory multi-label learning performance and rational proteome-wide predictions. Some predicted signaling components or pathway targeted proteins have been validated by recent literature. The predicted signaling components are further linked to pathways using the experimentally derived PPIs (protein-protein interactions) to reconstruct the human signaling pathways. Thus the map of the cross-talks via common signaling components and common signaling PPIs is conveniently inferred to provide valuable insights into the regulatory and cooperative relationships between signaling pathways. Lastly, gene ontology enrichment analysis is conducted to gain statistical knowledge about the reconstructed human signaling pathways.Multi-label learning framework has been demonstrated effective in this work to model the phenomena that a signaling protein belongs to more than one signaling pathway. As results, novel signaling components and pathways targeted proteins are predicted to simultaneously reconstruct multiple human signaling pathways and the static map of their cross-talks for further biomedical research.The online version of this article (doi:10.1186/s12859-015-0841-4) contains supplementary material, which is available to authorized users. Signaling pathways play important roles in the life processes of cell growth, differentiation and apoptosis. The stimuli from extracellular environment and cellular matrix are sensed, amplified and transducted to nucleus via signaling pathways to yield complex biological responses  . MalfuncProtein-protein interaction (PPI) networks play fundamental roles in the study of signaling transduction, because extracellular signals are generally transmitted from membrane to nucleus via a series of PPIs and molecular modifications. Thus reconstruction of PPI networks, including experimental techniques , 3 and cComparatively machine learning methods are effective to simultaneously exploit multiple experimental data of signaling pathways without prior knowledge about the underlying biochemical mechanism –16. Recoth class contains the negative data that are randomly sampled from the proteins that do not belong to the 27 signaling pathways. The scenario that a signaling component is shared by or belongs to multiple signaling pathways is modeled under multi-label learning framework. To enrich the knowledge of the protein concerned, each protein is depicted with two instances, one instance called target instance is represented with its own gene ontology annotations, and the other instance called homolog instance is represented with the gene ontology annotations of its homologs. Besides enriching the target instance, the homolog instance is especially useful to substitute the target instance when the protein concerned is completely not annotated. Unlike traditional supervised learning, the evaluation of multi-label learning model is conducted using three performance metrics, namely exact match ratio, microaverage F-measure and macroaverage F-measure. To evaluate the reliability of the proposed model, we validate the proteome-wide predictions against recent literature as well as conduct cross validation on the training data. Then we link the predicted signaling components to signaling pathways via experimental PPIs and derive the cross-talks between the 27 human signaling pathways to provide valuable cues for further biomedical research.In this work, we propose a multi-label multi-instance transfer learning method to simultaneously reconstruct multiple human signaling pathways and model their cross-talks. In this method, the data of the known signaling components from 27 human signaling pathways are used to train a 28-class multi-label SVM (support vector machine) model, wherein the 28http://www.netpath.org/) to construct the training for the reasons: (1) NetPath manually curates 35 human cancer/immune signaling pathways, the largest repository of human cancer signaling pathways at present to our knowledge; (2) The signaling components explicitly provided by NetPath are conveniently treated as the training data. KEGG is rather small and contains a limited number of human signaling pathways. The other databases like Reactome and SignaLink are timely updated, but likewise collect very limited number of human cancer signaling pathways so as not to directly serve our purpose. We incorporate the closely related cancerogenic signaling pathways into a single model to facilitate effective knowledge sharing. To date NetPath has manually curates 35 human immune/cancer signaling pathways that contain 11 sub-types of Interleukin (IL-1 ~ IL-11). For simplicity, IL-1 ~ IL-11 are merged into one single class, thus we obtain 27 human signaling pathways as shown in Table http://www.netpath.org/) are directly used as training data and the training data are further validated against SwissProt database  denotes the component g of the target instance BTi and BHi[g] denotes the component g of the homolog instance BHi. Formula  and recall (R) are defined as follows:For label j is formally defined as follows:Since F-measure is defined as Macro-average F-measure is defined as the unweighted mean of the F-measures of all class labels:Micro-average F-measure considers the predictions from all instances and calculates the F-measure across all class labels as follows:Δ|| denotes 2-norm of vector ∆ and the hyperparameter γ controls the flexibility of kernel.Both the macro-average F-measure and the micro-average F-measure take partial matches into account. In this work, we use the target instances and the homolog instances separately to estimate the exact match ratio, the macro-average F-measure and the micro-average F-measure. The performance metrics are derived using Gaussian kernel:The proposed multi-label multi-instance transfer learning model is estimated by 10-fold cross validation to derive the exact match ratio, macro-average F-measure and micro-average F-measure. In multi-instance learning scenario, each data point is represented with multiple instances, so multiple predicted outcomes are yielded for each test data point in the test or prediction phase. The outcomes are easy to combine into one single outcome in unique-label learning scenario . But outThe computational results are given in Table others, the F-measure is unsatisfactorily about 0.5, partly because of the quality of randomly sampled data. Fortunately, the proposed model achieves sound performances on the 27 human signaling pathways, implying that the misclassifications on the class others brings little adverse effect to the 27 signaling pathways. In addition, the performance difference between the homolog instance and the target instance is fairly small  and EGFR (CTRSC = 11.5). The details of the common signaling PPIs derived from HPRD database are given in Additional file CTRSPPI as illustrated in Fig. Cross-talk modeling is instrumental to study the regulatory and cooperative relationship between signaling pathways, based on which to further reveal the pathogenesis of diseases . SignaliThe static map of cross-talks between TGF-β signaling pathway and TNF-αsignaling pathway (target instance) is illustrated in Fig. We further validate the proteome-wide predictions against recent literature and signaling pathway databases. Since the data we are concerned about are scarce and sparsely scattered among hundreds of literature, it is hard to collect sufficient evidences to validate the predictions. Nevertheless, we still find dozens of supporting evidences as shown in Table The evidences that support the proteome-wide predictions are very limited, so we resort to KEGG database  for furtSignaling pathways play significant roles in the biological processes of cell growth, cell differentiation, cell apoptosis and organism development. At present, the current signaling pathways are far from complete. Computational modeling helps to accelerate the proteome-wide reconstruction and global cross-talk mapping of human signaling pathways. The existing computational methods focus on predicting signaling components and/or deriving orthologous signaling PPIs from the topology of signal transduction networks, or describing the molecular dynamics of signaling pathways. To our knowledge, no computational methods have been reported to simultaneously take more than two signaling pathways into account and explicitly predict their cross-talks. In this work, we propose a multi-label multi-instance transfer learning method to simultaneously reconstruct 27 human signaling pathways and model their cross-talks. The known signaling components of 27 human signaling pathways are directly exploited to train a 28-class predictive model (the 28th class is the negative class) and the model is used to predict proteome-wide novel signaling components. Then the predicted signaling components are linked to the current signaling pathways via the experimental PPIs in HPRD database. Based on the predicted signaling components and the derived signaling PPIs, we can conveniently reconstruct the 27 human signaling pathways and derive their cross-talks. Computational results show that both the target instances and the homolog instances achieve satisfactory multi-label learning performance and the homolog instances also yield valuable predictions. Some of the proteome-wide predictions have been validated against recent literature and KEGG database.We conduct gene ontology enrichment analysis of the predicted signaling components to get knowledge about the biological processes that the signaling pathways are involved in. Take TGF-βsignaling pathway and TNF-αsignaling pathway (predicted by the target instances)for examples, 27.4 % predicted TGF-βsignaling components are annotated with the term GO:0006355 , 15.75 % predicted TGF-βsignaling components are annotated with the term GO:0016567 (protein ubiquitination) and 11.30 % predicted TGF-βsignaling components are annotated with the term GO:0007275 . As regards with the predicted TNF-αsignaling components, the GO enrichment for GO:0006915 (apoptotic process), GO:0006457 (protein folding) and GO:0006954 (inflammatory response) are 13.63, 4.29 and 3.98 %, respectively.Next we study the molecular functions that the predicted signaling components fulfil. As for TGF-βsignaling pathway, the GO enrichment of the terms GO:0005515 (protein binding), GO:0046872  and GO:0004842 (ubiquitin-protein ligase activity) are 38.01, 27.74 and 11.99 %, respectively. As for TNF-αsignaling pathway, the GO enrichment for GO:0005524 (ATP binding), GO:0005515 (protein binding) and GO:0046872  are 23.58, 19.30 and 11.79 %, respectively. As for the cellular compartments that the predicted signaling components reside in, a majority of the predicted TGF-βand TNF-αsignaling components are located in cytoplasm (GO:0005737), nucleus (GO:0005634) and cytosol (GO:0005829). The GO enrichment analysis of predicted TGF-βand TNF-αsignaling components is illustrated in Fig. Apart from NetPath and KEGG, the other signaling pathway databases such as Reactome  and Signhttp://www.hprd.org/) for primary research since HPRD focuses on collecting reliable protein-protein interactions of Homo sapiens. However, HPRD is not so frequently updated as Reactome. In the future work, we will combine the updated PPI databases  with computational PPI predictions to update the reconstructed signaling pathways. Fortunately, the predicted signaling components are very conveniently linked to signaling pathways via newly derived PPIs.The quality of signaling PPIs largely depends on the quality of human PPI database. Here we adopt HPRD database   and recent literature. The method and the results can be used for further model development and further biomedical research.


The first step in methanol metabolism in methylotrophic yeasts, the oxidation of methanol and higher alcohols with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX), a 600-kDa homo-octamer containing eight FAD cofactors. When these yeasts are grown with methanol as the carbon source, AOX forms large crystalline arrays in peroxisomes. We determined the structure of AOX by cryo-electron microscopy at a resolution of 3.4 Å. All residues of the 662-amino acid polypeptide as well as the FAD are well resolved. AOX shows high structural homology to other members of the GMC family of oxidoreductases, which share a conserved FAD binding domain, but have different substrate specificities. The preference of AOX for small alcohols is explained by the presence of conserved bulky aromatic residues near the active site. Compared to the other GMC enzymes, AOX contains a large number of amino acid inserts, the longest being 75 residues. These segments are found at the periphery of the monomer and make extensive inter-subunit contacts which are responsible for the very stable octamer. A short surface helix forms contacts between two octamers, explaining the tendency of AOX to form crystals in the peroxisomes. AOX is a member of the GMC (glucose–methanol–choline) oxidoreductase family 21],[21],in v,[21],in tructure , glucosetructure , 23, pyrtructure –26, celltructure , cholesttructure 29], and, andin vtructure . All thetructure  and shartructure , 32. AOXPichia pastoris alcohol oxidase at a resolution of 3.4 Å, which has allowed us to determine the structure of this protein.The recent introduction of direct electron detectors in cryo-electron microscopy (cryo-EM)  has now Pichia pastoris cells that had been grown overnight on methanol as carbon source following procedures described previously and cryo-EM images were taken on a JEOL 3200 FSC electron microscope with a K2 direct electron detector in movie mode. The movie frames were aligned before image processing to correct for beam-induced particle motion [AOX was purified from e motion .The sample is pure and monodisperse; the 600-kDa AOX particles are clearly visible on the K2 images, even at defocus values well below 1 μm . 2D clasAn initial 3D model was created from the 2D class averages, applying D4 symmetry. 3D classification in Relion with this as starting model yielded consistently a single good class, containing >85% of the particles. Although no symmetry was applied during this step, the reconstruction displayed clear D4 symmetry. This symmetry was thus applied during refinement using the gold standard method in Relion . Althougde novo into the density. Apart from the N-terminal methionine, which is apparently absent in the mature enzyme [The map at 3.4 Å resolution  shows cle enzyme , the whoe enzyme , 46 and e enzyme  , but not in the GMC family.Unlike the other GMC family proteins, which act as monomers or dimers, AOX forms an octameric complex, consisting of two tetramers stacked face to face. The dimensions are 130 x 130 x 105 Å. The complex is very tightly packed, with numerous interactions between subunits and only a small cavity in the center .The sequence alignment of the GMC family proteins  reveals The most significant oligomerisation module is the 75-residue insert 477–551, which is involved both in contacts within the tetrameric ring and in dimer contacts between rings. The first 50 residues form the sole contact between the two rings with extensive interactions . The resThe residues 524–540 form a hairpin that extends from the monomer and interacts with the neighboring subunit in the tetramer via the loops 245–251 (an AOX-specific loop between two β strands of the FAD binding domain) and 435–440 . The last ten residues of the insert form a linker back to the protein core via the helix H24 (548–561), which is one helix turn longer than the equivalent element in other GMC family proteins.The highly conserved AOX-specific loop 133–140 makes contact to the also AOX-specific C-terminal end of helix H10 on the top of the octamer near the fourfold axis. The four helices 335–346 (H14) form a bundle around the four-fold axis, tightly packed at its C-terminal end but with a distance of ~15 Å at the N-terminal end. The helix bundles of the two tetramers are separated by ~12 Å . This heAnother tetramer contact is mediated by the C-terminal extensions 637–663, which form a ring at the surface around the fourfold axis . Glu656 Finally, the residues 402–411, an AOX-specific extension of the loop between the β strands C3 and C4, make a dimer contact between the two tetramer rings. The only AOX-specific region not involved in intersubunit contacts is the insert between β-strands C2 and C3, two helices H16 and H17, located on the outside of the octamer. This region is not conserved and CHOX has just a short loop between the β-strands whereas GOX and AAOX each have a different helical structure .GOX occurs as a dimer. Although the two segments involved in dimer contacts  are consAOX forms crystalline inclusions in the peroxisomes. Crystals also form readily in vitro –15. In tin vivo. Both of these kinds of contacts are important to the survival of the organism in conditions where methanol is the only substrate because they allow extremely dense packing of the large amounts of enzyme required for methanol utilization. In addition, the structure of the crystals also appears to be adapted for maximum methanol metabolism, with subsidiary enzymes able to penetrate within the lattice.In conclusion, the quality of our map has allowed us to see key features of the AOX enzyme. The residues governing substrate recognition are readily identifiable and allow for rational protein engineering whereby the specificity of the enzyme may be altered. In addition, elements responsible for the unique oligomeric structure of AOX are recognizable, as are the regions of the protein responsible for the crystallization Pichia pastoris cells were grown overnight in medium containing 1% (w/v) methanol. Cells were harvested and broken with glass beads using a Biopec BeadBeater. The soluble material was then subjected to a two-step fractionation using ammonium sulphate. The pellet obtained after incubation with ammonium sulphate at 30% saturation was discarded and the supernatant made to 60% to precipitate the AOX. The pellet was dialysed for 2 days against 20 mM sodium phosphate and loaded onto a column of Q-Sepharose. A linear gradient up to 1M NaCl was applied and the major peak pooled and concentrated. Finally, the AOX was subjected to gel-filtration on a Superose 6 column, concentrated to 14 mg/ml and frozen in aliquots.To stimulate production of AOX, -/Å2/frame, resulting in a total exposure of 51 e-/Å2.3 μl of a 0.7 mg/ml sample was applied to freshly glow discharged Quantifoil R2/2 holey carbon grids  that had been pretreated in chloroform for 2 hrs. The grids were blotted for 11 sec in an FEI Vitrobot plunge-freezer at 10°C and 70% humidity. Data was collected on a JEOL 3200 FSC operating at 300 kV, with the in-column energy filter operated at 20 eV. Data was recorded on a Gatan K2 back thinned direct electron detector at a nominal magnification of 30,000x with a calibrated specimen pixel size of 1.14 Å. The images were recorded in a defocus range of 0.6–2.5 μm using movie mode with 30 0.2-s frames using an electron dose of 1.7 e-/Å2 were used for this procedure. D4 symmetry was applied in all refinements. The post-processing procedure implemented in RELION [http://resmap.sourceforge.net) [The 30 frames of each 6-s movie were aligned using the whole-image motion correction method described in Li et al., 2013 . The firn RELION  was appln RELION . The locrge.net) .http://www.ebi.ac.uk/Tools/msa/clustalo). For model building, crystals structures of the homologous GMC family oxidoreductases choline oxidase (pdb 3nne) [Secondary structure prediction of AOX was performed with the Jpred 3 server . Multipldb 3nne) , glucosedb 3nne)  and aryldb 3nne)  using redb 3nne)  followeddb 3nne) , 66 and Figures were made using Chimera .S1 FigCandida boidinii (Cb), Pichia angusta (formerly Hansenula polymorpha) (Pa), Kuraishia capsulata (Kc) and Pichia pastoris  (AOX); aryl-alcohol oxidase from Pleurotus eryngii (pdb 3fim) (AAOX); choline oxidase from Arthrobacter globiformis (pdb 3nne) (CHOX); glucose oxidase from Aspergillus niger  (GLOX). Residues conserved in AOX are shown in green, residues conserved in the GMC family in red. Sequence where the structure of GLOX, CHOX or AAOX differs from AOX is shown in blue. α-helices are highlighted in yellow and β-strands in grey and numbered above the sequence. Sequence not seen in the structure is shown in grey font. Domain assignments for AOX and the consensus sequence are shown below the alignment: FAD-binding domain (red); FAD covering lid (violet); extended FAD-binding domain (yellow); flavin attachment loop (sea green) and intermediate region (green); substrate-binding domain (cyan) (assignment and coloring scheme as in ref. [AOX of  in ref. ). The co(PDF)Click here for additional data file.S1 MovieThe movie shows residues 201–216 (helix H10).(MP4)Click here for additional data file.S2 MovieFAD-binding domain (red); FAD covering lid (purple); extended FAD-binding domain (yellow); flavin attachment loop (green) and intermediate region (light green); substrate-binding domain (cyan). Helix H12, which forms crystal contacts, is shown in orange. AOX-specific inserts are pink.(MP4)Click here for additional data file.S3 MovieEach of the eight monomers is shown in a different colour; the FAD cofactors are red.(MP4)Click here for additional data file.S4 MovieEach subunit is shown in a different color.(MP4)Click here for additional data file.S5 MovieColors are as in (MP4)Click here for additional data file.S6 MovieEach AOX molecule is 90° rotated relative to its neighbour. As a consequence, the octameric molecules are arranged in three perpendicular directions, each shown in a different color. This arrangement leaves large holes and channels in the crystal.(MP4)Click here for additional data file.


Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase family whose members have been implicated in tumor suppression in many cancer models. In many cancers, loss of PP2A activity has been associated with tumorigenesis and drug resistance. Loss of PP2A results in failure to turn off survival signaling cascades that drive drug resistance such as those regulated by protein kinase B. PP2A is responsible for modulating function and controlling expression of tumor suppressors such as p53 and oncogenes such as BCL2 and MYC. Thus, PP2A has diverse functions regulating cell survival. The importance of microRNAs (miRs) is emerging in cancer biology. A role for miR regulation of PP2A is not well understood; however, recent studies suggest a number of clinically significant miRs such as miR-155 and miR-19 may include PP2A targets. We have recently found that a PP2A B subunit (B55α) can regulate a number of miRs in acute myeloid leukemia cells. The identification of a miR/PP2A axis represents a novel regulatory pathway in cellular homeostasis. The ability of miRs to suppress specific PP2A targets and for PP2A to control such miRs can add an extra level of control in signaling that could be used as a rheostat for many signaling cascades that maintain cellular homeostasis. As such, loss of PP2A or expression of miRs relevant for PP2A function could promote tumorigenesis or at least result in drug resistance. In this review, we will cover the current state of miR regulation of PP2A with a focus on leukemia. We will also briefly discuss what is known of PP2A regulation of miR expression. The cellular signaling pathways that control cellular homeostasis are complex and involve a diversity of activators and suppressors. As signal transduction is a dynamic process, the elements involved in turning off kinases are just as important as those that activate the signaling cascade. Protein phosphatase 2A (PP2A) is thought of as a global negative regulator of signaling. In reality, PP2A is not a single enzyme but rather a family of protein phosphatases that vary in the substrates they target, in the cell types where they are expressed, in the cellular compartments where they are found, and how they are regulated –5. The dSuppression of the PP2A family to promote global activation of cellular kinases can be achieved by targeting the catalytic core. Mechanisms include post-translational modification to inactivate protein phosphatase function , invCell from Welch and colleagues on genomic screening of mutations in AML indicate that less than half of AML patients have a RTK mutation suggesting that other mechanisms are necessary to activate leukemic signaling pathways (We have some knowledge of PP2A role in CML and Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) thanks in large part to the work of the Perrotti group in their studies on BCR–ABL regulation of PP2A in these diseases –23. In Cpathways . Gallay pathways . The Odepathways –36. Theypathways . Like Gapathways , they fopathways . Our ownpathways , 38. PP2pathways , 39–41.C. elegans in 1993 when the lin-4 gene product was identified as a RNA product that was complimentary to the lin-14 gene  demonstrated that (a) PP2A activity increases with concomitant increase in A and C subunit expression in differentiating hESC, (b) introduction of catalytic C subunit into hESC or activation of PP2A using C2-ceramide was shown to induce hESC differentiation, (c) hESC self-renewal is maintained in the presence of the PP2A inhibitor okadaic acid, and (d) suppression of PP2A C subunit expression by siRNA-induced stem cell genes such as OCT-4 and Nanog . The stuWe recently identified a number of miRs that were subject to regulation by the B55α subunit that has been found to suppress AKT and PKC α survival signaling in AML cells , 38. TheThe role of miRs as regulators of PP2A and possible control of miR expression by PP2A isoforms is only just emerging. Publications covering miR control of PP2A subunits and regulators of PP2A  are just emerging. Recent studies suggest that miR control of the PP2A genes is important as results indicate that dysregulation of PP2A targeting miRs can result in induction of signaling pathways associated with the particular PP2A isoform. An example of such a finding involves activation of AKT when miR-222 (which targets the B55α subunit) is elevated . The recThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The data reveal all six types of zone center optical phonons, along with their corresponding Davydov splittings, which have been challenging to see in other TMDCs. We discover that the anti-Stokes Raman intensity of the low energy layer-breathing mode becomes more intense than the Stokes peak under certain experimental conditions, and find the effect to be tunable by excitation frequency and number of atomic layers. These observations are interpreted as a result of resonance effects arising from the C excitons in the vicinity of the Brillouin zone center in the photon-electron-phonon interaction process.Stokes and anti-Stokes Raman scattering are performed on atomic layers of hexagonal molybdenum ditelluride (MoTe The coupling between photons, electrons, and phonons is central to understanding fundamental properties of condensed matter systems. In layered transition metal dichalcogenide (TMDC) semiconductors, electron-photon coupling enables investigation of the under-screened strong Coulomb interaction in reduced dimensions222), a prototypical TMDC semiconductor, which has recently attracted a great deal of interest. Investigations of multi- and mono-layer MoTe2 transistor devices6792 features strong photoluminescence similar to other TMDCs, but it has by far the smallest optical bandgap at the K points, lying in the infrared around 1 eV11In this work we study such interaction effects in molybdenum ditelluride  characterization in −1 (1 meV ≈ 8.06 cm−1), are given in Atomic layers of hexagonal TMDCs host six prototypical zone-center optical phonons, shown in the bilayer dispersion (see 2g symmetryi/σh) are Raman active  spectra in 2 has one Raman active mode for each of the six types of zone center optical phonons, consistent with the six peaks observed in bilayer (red) spectra.The occurrence and splitting of the observed Raman peaks can be compared with group theoretical predictions: nd panel are the same breathing mode peaks as in the 1st panel; they are present under both the z(xy)z and z(xx)z configuration, though their intensity is much less than that of the breathing mode, which is only present under the z(xx)z configuration. The IC mode appears to display non-monotonic dependence on layer numbers, and develops an asymmetric shape for 4L and 5L samples. This indicates that the IC spectra in 4L and 5L samples are composed of two peaks as suggested by the theory in 2 in 1g) and odd (A2u) OC vibrations in 2L-MoTe2 have the largest Davydov splitting.For samples thicker than bilayer the Raman spectra display Davydov splitting due to interlayer interactions. We observe two B and two S modes in 4L and 5L samples; two OC modes in 3L and 4L samples; and three OC modes in 5L samples, in perfect agreement with the theoretical expectations in 2, the OC and IMC modes are the dominant Raman features, and the B and S modes become observable if low-energy stray light is cut, but the IC and OMC modes tend to evade most Raman scattering measurements141617181920212’s small optical band gap2. In capturing all six zone center modes, our Raman spectra in 224In the other four hexagonal TMDCs, In addition to enabling observation of all six branches of zone center lattice vibrations, the C excitons in the photon-electron-phonon interaction Raman process have interesting impacts on Raman intensity. In particular we have found that the resonances have dramatic effects on the Stokes and anti-Stokes Raman signatures of the low energy breathing modes. The electronic system absorbs a photon and creates an electron-hole pair;The electron-hole pair creates (Stokes) or absorbs (anti-Stokes) a phonon;The electron-hole pair recombines and emits the scattered photon.In a typical, lowest-order Raman scattering process for zone-center optical phonons, there are three steps involved as shown in n + 1) or n, respectively, where n is the phonon occupation number. As phonons are bosonic excitations with zero chemical potential, The first and the third steps are linked to electron-photon interaction while the second step is determined by electron-phonon interaction. The rate of step 2 is proportional to the phonon creation or destruction operator acting on the system squared, giving a factor of  plays an important role in determining the Raman cross section. Step 1 in 2 excited by 488 nm (2.54 eV), 514 nm (2.41 eV), and 532 nm (2.33 eV) respectively. The anti-Stokes to Stokes intensity ratio depends sensitively on the incident laser excitation: at 2.33 eV, the anti-Stokes peak has about the same intensity as the Stokes peak; at 2.41 eV and 2.54 eV, the anti-Stokes intensity is above and below the Stokes, respectively. From these observations, we introduce a resonance correction factor, R, into Equation (1), such thatThe resonance effect becomes even clearer when we systematically measure the calibrated breathing mode intensities using different laser lines. 2 with all three excitation wavelengths. In the absence of any correction, i.e. R = 1 in Equation (2), the experimental data are expected to fall within the gray band, where the two bounding dotted black lines specify the temperature uncertainty during our experiment . The data strongly imply that the observed deviations of the R factor from unity are correlated with the overall shape and evolution of the C exciton: the measured R value across different numbers of layers for these energies. In contrast the slopes for 2.54 eV excitations have significant variations, as do the resonance effects in different numbers of layers.To further establish the connection between the C exciton and the laser-wavelength/atomic-layer-number dependent 2 breathing mode is only about 3 meV, corresponding to about 0.15% of the total photon energy. It is quite remarkable that the C exciton resonance at room temperature can amplify this minute change in energy to switch the relative Stokes–anti-Stokes Raman cross-section. The reversed intensity ratio in fact indicates that more phonons are removed from, rather than created in, the crystal, potentially forming a laser-cooling channel in the system. Indeed, the observation of intense anti-Stokes in another semiconductor system (CdS nanoribbons) is accompanied with strong laser cooling effects2We finally remark that while other materials with exciton effects may also exhibit an anomalous Stokes–anti-Stoke ratio, it is quite rare to have the anti-Stokes peak to actually be more intense than the Stokes peak. Also we take note that the Stokes–anti-Stokes photon energy difference for the 5L-MoTe2 from chemical vapor transport grown crystals. Using polarization-resolved Raman scattering we have unambiguously identified all six types of zone-center optical phonons. Splitting of these phonons, where observed, has the correct theoretically predicted number of branches. We discovered that the anti-Stokes intensities deviate from their expected values, to the point of being more intense than the Stokes intensities, with the intensity ratio tunable via resonance with the C exciton. A stronger anti-Stokes peak implies that more phonons are annihilated than created in the crystal suggesting that, from the electron-photon-phonon interaction process, anti-Stokes Raman scattering could provide a cooling channel in atomically thin TMDC crystals.We have exfoliated few layer MoTe2 crystals using chemical vapor transport with chlorine as the transport agent, as illustrated in et al.5 powders are placed in a fused silica tube, 18 mm in diameter and 300 mm in length. The purity of the source materials are Mo 99.9%, Te 99.997%, and MoCl5 95% (Sigma Aldrich). Total Mo and Te are kept in a stoichiometric 2:1 ratio with sufficient MoCl5 to achieve a Cl density of 2 mg/cm3. The tube is pump-purged with ultra-high purity argon gas and sealed at low pressure prior to growth. The reaction and growth zones were kept at 830 °C and 730 °C respectively for 100 hours. During cooling the temperature profile is inverted with the growth zone being roughly 100 °C hotter than the reaction zone so that chloride contaminants preferentially precipitate in the hot zone, away from the MoTe2 crystals.We grew bulk MoTe−1. Data for 488 and 514 nm excitations were taken at 100 μW for 120 s; data for 514 nm excitations were taken at 70 μW for 120 s. The heating effects of these powers were accounted in We collect Raman data using linearly-polarized light from an Argon laser or a frequency doubled Nd:YAG solid state laser to excite the samples in a back scattering geometry. The scattered light is collected with a 100x objective lens, dispersed by a triple stage spectrometer, and detected by a liquid nitrogen cooled CCD. In the collection path, a broadband polarization rotator and a linear polarizer are used to selectively detect scattered light with polarization either parallel 2 atomic layers onto fused silica. We then shine a white laser (NKT Photonics) on the samples and collect the reflection spectrum, which is subtracted from and normalized by the substrate reflection.We obtain differential reflection data by exfoliating MoTeHow to cite this article: Goldstein, T. et al. Raman scattering and anomalous Stokes–anti-Stokes ratio in MoTe2 atomic layers. Sci. Rep.6, 28024; doi: 10.1038/srep28024 (2016).


Reprogramming cellular gene transcription sustains HTLV-1 viral persistence that ultimately leads to the development of adult T-cell leukemia/lymphoma (ATLL). We hypothesized that besides these quantitative transcriptional effects, HTLV-1 qualitatively modifies the pattern of cellular gene expression.+ CD4+ T-cells exhibit multiple alternate exon usage (AEU) events. These affect either transcriptionally modified or unmodified genes, culminate in ATLL, and unveil new functional pathways involved in cancer and cell cycle. Unsupervised hierarchical clustering of array data permitted to isolate exon expression patterns of 3977 exons that discriminate uninfected, infected, and transformed CD4+ T-cells. Furthermore, untransformed infected CD4+ clones and ATLL samples shared 486 exon modifications distributed in 320 genes, thereby indicating a role of AEUs in HTLV-1 leukemogenesis. Exposing cells to splicing modulators revealed that Sudemycin E reduces cell viability of HTLV-1 transformed cells without affecting primary control CD4+ cells and HTLV-1 negative cell lines, suggesting that the huge excess of AEU might provide news targets for treating ATLL.Exon expression analysis shows that patients’ untransformed and malignant HTLV-1Taken together, these data reveal that HTLV-1 significantly modifies the structure of cellular transcripts and unmask new putative leukemogenic pathways and possible therapeutic targets.The online version of this article (doi:10.1186/s12977-014-0119-3) contains supplementary material, which is available to authorized users. Reprogramming of gene transcription sustains HTLV-1 viral persistence and ultimately leads to the development of adult T-cell leukemia/lymphoma (ATLL) from a CD4+-infected clone in a minority of carriers after a prolonged latency : 2.71), endocytosis (FE: 4.45), antigen processing and presentation (FE: 7.23), cell adhesion molecules , and natural killer cell-mediated cytotoxicity  permitted sample gathering in three distinct clusters, including untransformed-uninfected and infected CD4218 genes−6), MAPK signaling , focal adhesion , cytokine-cytokine receptor interactions , and endocytosis  showed distinct biological changes  that discriminate between the three groups of samples presented in Figure 7 exons t+ T-cells and the HTLV-1-negative acute lymphoblastic leukemia cell line, MOLT4. Figure + T-cells was inhibited at higher drug concentrations. In contrast, a similar Sudemycin E concentration range was found to significantly decrease the viability of HTLV-1 cell lines, while displaying weak effects on primary CD4+ and MOLT4 cells. High concentration of Sudemycin E (>5 μM) affected cell viability of all tested cells, carrying or not HTLV-1 (not shown). These data suggest that, compared to both transformed and untransformed-uninfected cells, HTLV-1 tumoral cells are highly sensitive to drugs that target splicing machinery. Time-course analysis confirmed these results and further revealed that the anti-proliferative effect of Sudemycin E was persistent in MT2 and HUT-102 HTLV-1-positive cells relative to HTLV-1-negative CD4+ and MOLT4 cells  that were composed of lethally irradiated PBMCs from three distinct allogenic HTLV-1-negative donors. Clones were phenotyped by flow cytometry using antibodies against CD4 and CD8 (DakoCytomation) and isotype-matched controls on a FACScan system using CellQuest software (Becton Dickinson). HTLV-1-positive clones were assessed by PCR using long-terminal repeat region–specific primers as previously described [Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll separation of whole blood of HTLV-1 infected individuals. For T-cell limiting dilution cloning, PBMCs were seeded at 0.1 cell/well in Terasaki plates after removal of adherent cells. T-lymphocytes were cultured in RPMI 1640 containing penicillin and streptomycin, sodium pyruvate, non-essential amino acids, 2-mercaptoethanol, 10% filtered human AB serum, 100 U/mL recombinant IL-2 (Chiron Corporation), and 75 μM HTLV-1 integrase inhibitor L-731,988 . T-lymphescribed . For eachttps://fasterdb.lyon.unicancer.fr/) [t-test on the splicing index (SI) that corresponds to comparison of gene-normalized exon intensity values between two experimental conditions [Affymetrix exon array hybridization was completed by labeling 1 μg of TRIzol-purified total RNA with Affymetrix reagents according to the manufacturer’s instructions. Hybridization cocktails containing 5–5.5 μg cDNA were prepared and hybridized to Affymetrix-GeneChip Human Exon 1.0 ST arrays (Affymetrix). Affymetrix Expression Console Software was used for quality assessment, while exon array data were normalized using quintile normalization and analyzed using FasterDB annotation (cer.fr/) . Backgrocer.fr/) . Statistnditions ,42. AccoPCR was performed using the Herculase II Fusion DNA Polymerase (Agilent Technologies) in 30–35 cycles. PCR products were analyzed by agarose gel electrophoresis with ethidium bromide under UV light. Primers were designed to encompass alternative splicing events. Primer sequences are available upon request.Tax mRNA was quantified with Rotor Gene (Qiagen) as previously described [et al. [U6 (ENSG00000206625) or hprt1 (ENSG00000165704).Expression of escribed ,43. Expr [et al. . qRT-PCRCell viability was determined by MTT assay  72 h after Sudemycin (D1 or E) exposure. The MTT dye was added during the last hour of incubation. After, supernatants were removed and cells were incubated with 100 μL/well Solubilization Solution/Stop Mix for 1 h at room temperature. The OD was measured using an ELISA reader at 570 nm, with 650 nm as a reference. Experiments were completed twice, in triplicate for each cell type, with DMSO used as control.


Paenibacillus polymyxa and relevant spoilage microorganisms by high hydrostatic pressure (HHP) treatment on apricot, peach, and pear pieces in sucrose (22°Brix) solution were assessed. Accordingly, the shelf-life was determined by evaluating both the microbiological quality and the sensory characteristics  during refrigerated storage after HHP treatment. The microbiological shelf-life of apricots, peaches, and pears was prolonged in the HHP-treated products in comparison with the untreated ones. In all HHP-treated packages for apricots, peaches, and pears, all populations were below the detection limit of the method (1 log CFU/g) and no growth of microorganisms was observed until the end of storage. Overall, no differences of the L*, a*, or b* value among the untreated and the HHP-treated fruit products were observed up to the time at which the unpressurized product was characterized as spoiled. HHP treatment had no remarkable effect on the firmness of the apricots, peaches, and pears. With regard to the sensory assessment, the panelists marked better scores to HHP-treated products compared to their respective controls, according to taste and total evaluation during storage of fruit products.In the current study, the processing conditions required for the inactivation of  High hydrostatic pressure (HHP) processing is a novel nonthermal method which has revealed great potential in producing microbiologically safer products while maintaining the natural characteristics of the food products . The use Bacillus spp. form high-pressure resistant spores and they have been suggested to be used as the target organism in the development of standards for HHP treatments [ Bacillus spp. in foods [One of the principal advantages of the HHP method is the substantial increase in shelf-life and improvement of food safety due to the inactivation affected in the microbial population , 5. HHP eatments , 10. Thein foods , 12, wheDuring the last years, processed fruit products have become well known due to the increased demand of consumers for healthy diets. Fresh-cut fruits have occurred as common snacks in food services . Overall2, which may act by reducing the formed o-quinones back to o-diphenolic compounds, avoiding thus the existence of the secondary pigment formation [A number of various methods may be used to control the browning reactions in fruit products and extend their shelf-life. In this respect, the most effective process is the heat treatment, but it leads to changes in quality characteristics of the fruit products that are not friendly to the consumer. Browning can also be delayed by the elimination of oxygen and by the use of oxygen-impermeable packaging . An alteormation . Undesirormation . Paenibacillus polymyxa (a microorganism relevant to the spoilage of fresh-cut fruit pieces) by HHP were optimized for the first time. A shelf-life study of apricot, peach, and pear pieces in sucrose solution HHP treated was assessed, where color, texture, and sensory evaluation were monitored in parallel with the microbiological control during refrigerated storage at 5°C.In the present study, HHP treatment was applied to preserve fresh-cut apricot, peach, and pear pieces in sucrose solution. To our knowledge, there is limited, if any, information relevant to the potential use of HHP treatment in fresh-cut apricot, peach, or pear pieces preservation. Consequently, the process conditions toward inactivation ofFor the series of experiments, apricots (cultivar Bebekou), peaches (cultivar Andross), and pears (cultivar Williams) were bought in a local market and thoroughly washed with water and 70% ethanol. Consequently, apricots and peaches were dipped for 30 seconds in boiling water containing 2% NaOH to loosen skins, dipped quickly in cold water, and peeled. Then the fruits were cut in half with a sterile knife and the pits were removed. The pear fruits were peeled, cut in half with a sterile knife and the core was removed. Finally, all fruits were cut with a sterile knife into approximately 0.5 cm thick cube pieces. Paenibacillus polymyxa DSM 36 . The strain was revived from a stock culture stored at −80°C, by subculturing twice in 10 mL nutrient broth , incubated for 48 h at 30°C. The fresh cultures were heat shocked (80°C for 10 min) prior to inoculation on agar plates to allow uniform sporulation. Subsequently, the above suspension was spread on nutrient agar  plates containing 0,06 g/L MgSO4  και 0,25 g/L K2HPO4 (pH adjusted at 7.0) (NA-MK) and incubated for approximately 7 days at 30°C to allow time for the cells to sporulate. When at least 90% cells have sporulated , the spores were harvested by depositing 1 mL of sterile water onto each NA-MK plate and transferred into a sterile centrifugation tube. The spore suspension was centrifuged at 4000 ×g for 20 min at 4°C and the pellet was rinsed with ice-cold distilled water. The latter procedure was repeated four times. The final pellet was resuspended in a small volume of ice-cold distilled water and stored at 4°C until use.The strain used in this study was the P. polymyxa to reach a final concentration of 400 CFU/g on the fruits. A part of the fruit samples was not inoculated with P. polymyxa to serve as samples for the sensory analyses. The inoculated or not samples were subsequently treated with the following preprocessing handlings.The prepared apricot, peach, and pear cuts were inoculated with spores ofThe fruit preparation as well as the following 1st and 2nd handling was performed according to different processing procedures followed in canned food industry for each fruit type. The 3rd handling was followed to evaluate the effect of HHP processing on the cut fruits without additional thermal treatment.1st Handling. 100 g of cut-fruit pieces was dipped in hot water (T > 95°C) for 2 min, subsequently transferred to sterile polyethylene bags  and 100 mL of boiling-hot sucrose solution (22.7% sucrose/22°Brix) was added. The average final temperature of the product was 81°C.2nd Handling. 100 g of cut-fruit pieces was washed in cold water, subsequently transferred to sterile polyethylene bags  and 100 mL of boiling-hot sucrose solution (22.7% sucrose/22°Brix) was added. The average final temperature of the product was 61°C.3rd Handling. 100 g of cut-fruit pieces was washed in cold water, subsequently transferred to sterile polyethylene bags  and 100 mL of sucrose solution (T = 30°C) (22.7% sucrose/22°Brix) was added. The final temperature of the product was 25°C.2 (30 ppm) was added, while in half packages with pears ascorbic acid (0.1%) was added. Then, the pouches were heat-sealed after careful removal of air, moved directly to the HHP unit, and treated in a single run at 600 MPa for 5 min at 10°C, as described below. Additionally, samples treated according to 1st handling without any additive and HHP treatment served as control samples. The HHP-treated and control samples were subsequently stored at 5°C. Each experiment was replicated three times (three different batches of each fruit).The handlings 1, 2, and 3 were applied to apricots and peaches, whereas handlings 1 and 3 were applied to pears. In half packages with apricots and peaches, SnClHHP inactivation experiments were conducted in triplicate at pressure of 600 MPa and temperature 10°C for processing time of 5 minutes. The high pressure unit  comprised of a pressure intensifier and a multivessel system consisting of a central vessel of 250 mL capacity, with a maximum operating pressure and temperature of 1000 MPa and 90°C. The pressure transmitting fluid used was polyglycol ISO viscosity class VG 15 . Process temperature in the vessel was achieved by liquid circulation in the outer jacket controlled by a heating cooling system , 19. TheSamples were analyzed throughout storage at regular time intervals during storage of apricot , peach , and pear . Fruit pieces were weighed aseptically, added to sterile 1/4 strength Ringer's solution, and homogenized in a stomacher  for 60 s at room temperature. Decimal dilutions in 1/4 strength Ringer's solution were prepared and duplicate 1 or 0.1 mL aliquots of appropriate dilutions was pour- or spread-plated on the following media: (i) plate count agar  for total viable counts, incubated at 30°C for 48–72 h; (ii) de Man-Rogosa-Sharp (MRS) medium  for lactic acid bacteria (LAB), adjusted in pH 5.7, overlaid with the same medium, and incubated at 30°C for 48–72 h; (iii) tryptone dextrose extract agar  containing 0.1% activated carbon; (iv) rose Bengal chloramphenicol agar base , for yeasts/molds incubated at 25°C for 72 h. In all growth media incubation time was extended by 1-2 days to allow recovery of lethally/sublethally injured or stressed by HHP treatment cells. plus texture analyser equipped with a Kramer shear cell  with the following test parameters: load cell = 500 kg, test speed = 5 mm/s, Krammer shear cell . The fruit cuts of a total weight of ca. 20 g were placed on the Kramer cell. The procedure was followed at least 4 times for each case. The speed setting was 200 mm/sec, whereas the penetration force was measured in N. The values were recorded and firmness was calculated dividing the maximum shear compression force by the total weight of fruits and expressed as N/g of product.The firmness of the fruit pieces was determined using a TA.HDX = 78.66, Y = 83.31, and Z = 88.40). The recorded values X, Y, and Z were converted to CIE L*, a*, and b* color values. The L* value indicates the visual lightness or the luminance on a scale of 0 to 100 . Positive a* values indicate red direction; negative a* value is the green direction. Positive b* values are the yellow direction, and negative b* values are the blue direction. At each sampling time, 5 random measurements of the fruits of each different treatment were performed from duplicate samples, from 3 different batches of each fruit.Color change was measured using a Minolta Chroma Meter fitted with a CR-300 measuring head . The apparatus was calibrated with a standard white tile  of the noninoculated products were assessed from a panel of eight members (staff from the laboratory). The same trained persons were used in each evaluation, and all were blinded to which sample was being tested. The sensory evaluation was carried out in artificial light and the temperature of packaged product was similar to ambient temperature. Assessment was designed to identify spoilage conditions exclusively. A persistent dull appearance, or unusual color or appearance, was considered unacceptable. Taste was scored on a three-point hedonic scale where 0 = good, 1 = acceptable, and 2 = totally unacceptable. Scores above 1 rendered the product spoiled and indicated the end of the product's shelf-life. Total evaluation was scored on a ten-point hedonic scale where 10 = very good, 5 = acceptable, and 1 = totally unacceptable. Scores below 5 rendered the product spoiled and indicated the end of the product's shelf-life. P. polymyxa was monitored during refrigerated storage of the apricot, peach, and pear pieces in sucrose solution after HHP treatment at 600 MPa for 10 min. In all HHP-treated packages for apricots  of apricot samples during storage in all treatments, with changes found to be more intense in control samples  with changes being observed earlier in 3rd handling samples without the addition of SnCl2 . It was observed that, in control samples, the firmness decreased during storage. On the other hand, the application of HHP had as a result a slight decrease in firmness values (3rd handling), with no further reduction observed during storage  of the products were assessed from a panel of eight members. The shelf-life of the products treated with the different handlings in comparison with the control is presented in Tables r Tables . In addi P. polymyxa—a microorganism relevant to the spoilage of fresh-cut fruit pieces—were studied. In addition, a shelf-life study of HHP-treated apricot, peach, and pear pieces in sucrose solution was evaluated, in which color, texture, and sensory evaluation were assessed in parallel to the microbiological quality during refrigerated storage at 5°C.In the current study, the potential of HHP treatment to preserve fresh-cut apricot, peach, and pear pieces in sucrose solution was assessed. Therefore, the processing conditions toward inactivation ofaw [ P. polymyxa spores after the application of HHP treatment is in accordance with previous reports that a minimum pressure level of 600 MPa is needed for the inactivation of bacteria and molds spores [In the present work, the syrup in which the apricot, peach, or pear pieces were dipped into did not only provide an acidic environment, but also a high concentration of sucrose. Several authors have observed the protective effect of such solutions on the inactivation of bacteria and yeasts, despite the low aw , 20–22. aw , 23, 24.aw –28, whilaw , 30. Furs spores –33.2 or ascorbic acid in the HHP-treated samples had no effect on the color parameters within the same treatment. This indicates that either the HHP application gives the maximum effect that can be given or the applied SnCl2 or ascorbic acid concentration as indicated from the industry should be elevated to meet the needs of the new processing method. However, the different handlings seemed to have an effect on the color, especially in the browning of apricot , indicating the nessecary thermal treament of fruits cuts, irrespective of the HHP application. It has been reported that, for natural peach puree and peach puree containing ascorbic acid or cysteine and treated with HHP (517 MPa/5 min), an increased color maintenance of HHP-processed purees was observed [Generally, the presence of SnCl apricot . It was observed . In anotobserved , the autThe changes in the firmness values of fruit cuts were different according to the fruit type. The application of the HHP alone (3rd handling) had no effect in the firmness of peach and apricot cuts but reduced the firmness of pears. Moreover, the previous heat treatments of the samples (1st and 2nd handling) did not initially affect the firmness of apricots but reduced the firmness of pears and peaches. However, in most of the cases, the HHP application resulted in higher firmness values of the samples during storage, in comparison with the control samples. It has been reported that when the fruits are stored in pouches with syrup, as in the current study, texture degradation may occur due to solubilization and depolymerization of water-soluble pectin sodium carbonate-soluble pectin . The pec2 or ascorbic acid, while the corresponding taste scores were found to be similar (Tables The scores of the total sensory evaluation were higher during storage of HHP-treated samples of all fruits with the SnClr Tables . In addiThe HHP treatment was applied to enhance the microbiological quality and extend the shelf-life of apricot, peach, and pear pieces in sucrose solution. Treatment at 600 MPa for 5 min with the combination of thermal processing of the fruits can remarkably extend the shelf-life of these fresh products, with minor changes on their color and firmness. However, additional research is needed in order to optimize the handling prior to HHP, aiming to better quality and sensory attributes of such products.


K-means clustering algorithm. Change in woody cover was mapped using Boolean addition Geographic Information System overlay analysis.The coexistence of woody vegetation and grass is a key characteristic of savanna ecological balance. Gains in woody vegetation at the expense of grass can lead to changes in grazer and browser carrying capacities on the savannas. This study examined long-term change in woody cover at four study sites representative of the geology and rainfall in the Kruger National Park, South Africa. Scanned 1940/1942, 1968, and 1977 high spatial resolution (0.44–1.35 m) panchromatic aerial photographs were used, supplemented by 5 and 10 m resolution 1998 and 2012 panchromatic and red band grey scale digital SPOT images. The imagery datasets of the respective study sites were georeferenced to the UTM projection. Woody cover on the imagery was enhanced using texture analysis, and mapped by unsupervised classification of the texture images using the The results indicated 29 and 40 % reductions in woody cover for the southern granites and southern basalts sites, respectively. The northern granites and northern basalts sites, on the other hand, had gains in woody cover over the analysis period. The location context-specific factors of fire frequency and elephant density, and not rainfall fluctuations, explained most of the change in woody cover.The results point to the need for location context-specific management of fire and elephant concentrations. The changes in woody cover are likely to have effects on the grazer and browser carrying capacities of the savannas in the Kruger National Park. Savannas are characterised by a coexistence of woody vegetation and grass  and relatively larger study sites.The study sites were the four research supersites in the Kruger National Park  for each woody individual  in the plot was determined by spreading measuring tape either above (for shrubs) or below the canopy. Treating the canopy as circular, the d value then gave a radius, r (i.e. r = 1/2d), and the area covered by the canopy was then computed as: Area = πr2. The individual canopy area values were then summed to derive the total area under woody canopies, and the woody fraction of cover derived as a percentage of the 1 ha area of the plot. The coordinates of the corners and centre of the plot were then taken, using a Garmin eTrex hand-held Global Positioning System (GPS) that had accuracy of 3 m. These coordinates were then used in locating the sample plots on images of the respective study sites.Field quantification of woody cover was undertaken in 2013 and 2014 at the four study sites, in order to derive data on woody cover for use in image interpretation. Sample plots measuring 100 m × 100 m (1 ha) were used during the field quantification. Rather than visual estimation of woody cover as has been employed by some studies, more reliable quantification was sought. Therefore, a method for estimating the total area covered by woody canopies in the sample plots was devised. The canopy diameter  images at the South African National Space Agency (SANSA). Aerial photographs generally have high spatial resolution. Therefore, SPOT images were judged to be more ideal for use in conjunction with the photographs than Landsat images that date back to the 1970s, based on the spatial resolution criterion. However, SPOT images are only available since the launch of the first SPOT satellite on 21 February 1986. Therefore, SPOT images of the late 1980s were selected, and then images from the late 1990s so as to approximate the decade inter-image date sequence of the 1968 and 1977 aerial photographs. However, SPOT images of the late 1980s (acquired from the SPOT 1 satellite), though listed on the SANSA catalogue, could not be supplied by SANSA due to technical difficulties. The final set of imagery was SPOT images acquired close to (or in the same season immediately after) the respective dates of the woody cover quantification fieldwork in 2013 and 2014 at the respective sites.For the period after 1977 imagery of the sites was selected from the catalogue of SPOT  were ideal, because the historical aerial photographs were panchromatic. The aerial photographs had April, June and August dates in the respective years. As a consequence, the selection of SPOT images sought images that were as close as possible to the photo acquisition months, within the constraints of image availability on the SANSA catalogue.The combined list of the imagery that was used is shown in Table For some of the study sites, the aerial photographs listed by Smit et al.  did not The aerial photographs were scanned at the NGI, using an Epson Expression 10,000 XL scanner with quite high resolution (1200 dots per inch). Since the scanning was performed at the NGI technical issues at this stage, such as the optimisation of tonal variation, could not directly be controlled. In some similar work (e.g. Hudak and Wessman The output pixel (grain) size on scanned aerial photographs can be computed using the formula in Eq. Based on Eq. ® software, and georeferenced to the UTM projection using ground control points (GCPs) that were well spread in the image scenes. The GCPs mainly consisted of road and power line junctions. During georeferencing low rectification error (expressed as Root Mean Square (RMS) error) was aimed at. Due to the large number of aerial photograph and satellite image scenes, the individual RMS error values were too many to individually report but all were less than one pixel. On the older photographs the search for GCPs was rather challenging due to the relatively fewer roads and power lines that existed in the KNP then. This limited the number of GCPs to 7–9 on the old aerial photographs, in particular the 1942 photographs of the Mooiplaas site. For this reason the 1942 aerial photographs of the north-eastern sector of the Mooiplaas site could not be georeferenced and were, therefore, not used. After the georeferencing, shapefiles of the study sites were used in subsetting the images to yield the images depicting the study sites on the respective dates shown in Fig. The aerial photographs and SPOT images were imported into ERDAS Imagine 2014Overlapping sections of the aerial photographs were carefully trimmed out in order to minimise image parallax, using image subsetting procedures within ERDAS. In order to optimise the contrast between the light and dark tones, contrast on each trimmed image was enhanced by spreading the digital number (DN) values on the full grey level scale range using a linear stretch. After separately mapping the woody cover on the respective aerial photographs from a given analysis year and study site, using image classification, the photographs were joined into thematic layer mosaics. All the images were carefully examined for presence of burn scars whose dark tone could have introduced error in mapping woody cover. Consequently the 1940 photographs of the Nhlowa site were excluded from the analysis due to extensive burn scars.The SPOT images that were used were acquired by the different sensors  on board SPOT satellites 2, 4 and 5 Table . There aPhoto texture has commonly been used in analyses of woody vegetation on aerial photographs and satellite images . The texture window should be smaller than the smallest feature being analysed, and this is optimised by high spatial resolution  of image, xcλ = DN value for spectral band λ of a window’s centre pixel, n = window number of pixels (=3).Texture analysis was employed in delineating the woody cover in this study, using the texture analysis function in ERDAS. This function enhances texture by computing new DN values based on variance, skewness, kurtosis or Mean Euclidean Distance using DN values in an Therefore, features with high reflectance (such as bare soil) had high MED values  and low reflectors (such as woody vegetation) had low values  on the resulting MED enhanced image. The contrasts were used in classifying woody vegetation on the resulting texture images.K-means algorithm in ERDAS. As a way of reducing error the automated clustering was preferred to setting thresholds on the texture images, which has been employed in some studies (e.g. Hudak and Wessman K-means clustering requires prior knowledge of the number (K) of clusters, unlike the ISODATA algorithm , comparing the classifications of the randomly sampled sites with their field (reference) classes Foody . For theChange in woody vegetation cover was then detected by GIS overlay analysis in ERDAS, using Boolean analysis. Boolean analysis employs arithmetic cell to cell comparisons of co-registered raster data sets. In order to accomplish this, the spatial resolution of all the extracted woody cover thematic layers was reduced to 10 m, the pixel size of the lowest resolution images in the data set. Numeric codes were then assigned to the woody cover thematic layers per date for the respective study sites. The codes 3, 5, 7, 11, 17 were used for 1940/42, 1968, 1977, 1998, 2012 woody cover, respectively. These codes gave unique resulting codes using Boolean addition for the GIS overlay analysis, compared to the coding sequence of 1, 2, 3, 4, 5. For example, 8 on the thematic layer resulting from the Boolean addition overlay meant woody cover in 1940/42 (coded as 3) and 1968 (coded as 5) only and none thereafter, 10 meant woody cover in 1940/42 (coded as 3) and 1977 (coded as 7) only, etc. The overlay analysis used intersects of the respective study areas, i.e. only the sections of the respective study sites that had image coverage on all the respective image dates in Fig. t = 3.68, P = 0.008), and between the Stevenson-Hamilton (southern granites) and Nhlowa  sites  in the woody cover Table ; Fig. 3.Scattered cloud affected the amount of woody cover that was mapped on the 2012  may only be taken as indicative of the direction of change, with the actual values being subject to classification error. In Fig. Combretum species, were observed during the fieldwork in May 2014. From the image analysis the indicative gain in woody cover between 1942 and 2012 was 39 % at this northern granites site.At the Ngwenyeni site, the stable woody cover was mainly in valleys and their vicinity Fig. a. Away fThe imagery intersect section of the Mooiplaas site Fig. b had a mThe Stevenson-Hamilton site’s imagery intersect section had stable woody cover in nearly all topographic positions, but underwent a general trend of loss in woody cover between 1940 and 2012. The loss in woody cover after 1977 at this southern granites site was nearly uniformly scattered in all sections of the site  had different directions of change in woody cover Table , soil tyP > 0.05) Pearson’s correlation coefficient (r) values between the woody cover and the pre-image 10-year rainfall . For the Ngwenyeni site the relationship (r) between the rainfall and woody cover was negative; the woody cover increased despite the reducing rainfall. The southern sites had losses in woody vegetation despite their higher annual rainfall  for the study sites as indicated by Smit et al.  were 4.02, which explains its loss of woody cover. The near-even spread of the woody cover losses at the Nhlowa site is consistent with elephant herbivory and destruction by fire. At the opposite end the Ngwenyeni site had the longest mean fire return interval (9.39 years), which explains its gain in woody cover. The gain in woody cover at the Mooiplaas site since 1942, despite relatively high fire frequency, can be attributed to the site’s low elephant density of 0.07 elephants/km2. Like the Ngwenyeni, the Stevenson-Hamilton site had low fire frequency  and would, therefore, also be expected to have gained woody cover in the analysis period. The loss of woody cover at this site, therefore, appears to have resulted from destruction of woody plants by elephants. The scattered nature of the sites with woody cover losses since 1977 throughout the site  change in woody cover on the savannas of the Kruger National Park using study sites in northern and southern parts of the park. A combination of panchromatic aerial photographs and black and white SPOT images was used for the analysis. The results showed gains in woody cover at the northern granites and northern basalts sites, but losses at the southern granites and southern basalts sites. The combination of fire frequency and elephant densities accounts for most of the change in woody cover. This relative significance of fire and elephant damage in causing change in woody cover in the Kruger National Park, compared to other factors like rainfall, is in accordance with established theory of the pivotal influence of the two agents of change in savanna woody cover.


Single-cell network profiling (SCNP) is a multiparametric flow cytometry-based approach that simultaneously measures evoked signaling in multiple cell subsets. Previously, using the SCNP approach, age-associated immune signaling responses were identified in a cohort of 60 healthy donors.In the current study, a high-dimensional analysis of intracellular signaling was performed by measuring 24 signaling nodes in 7 distinct immune cell subsets within PBMCs in an independent cohort of 174 healthy donors .Associations between age and 9 immune signaling responses identified in the previously published 60 donor cohort were confirmed in the current study. Furthermore, within the current study cohort, 48 additional immune signaling responses differed significantly between young and elderly donors. These associations spanned all profiled modulators and immune cell subsets.These results demonstrate that SCNP, a systems-based approach, can capture the complexity of the cellular mechanisms underlying immunological aging. Further, the confirmation of age associations in an independent donor cohort supports the use of SCNP as a tool for identifying reproducible predictive biomarkers in areas such as vaccine response and response to cancer immunotherapies. Due to an age-related decline in the function of the immune system, the elderly are more susceptible to infectious diseases and less likely to mount a sufficient response to vaccination ,2. HowevSCNP is a multiparametric flow-cytometry based analysis that can simultaneously measure both phenotypic surface markers and intracellular signaling proteins in response to extracellular modulation in multiple cell subtypes within heterogeneous populations, including PBMCs . Thus, SIn the current study, SCNP was applied to perform a multi-dimensional analysis of intracellular immune signaling in an independent cohort of healthy donors consisting of 144 elderly donors and 30 young donors. Overall, 168 immune signaling responses  were measured in PBMCs from the 174 healthy donors. Based on analyses of the previously published 60 donor cohort, 11 age-associated changes in cell signaling responses in CD45RA+ T cell subsets were pre-specified for testing in this current, independent study cohort and 9 of these age associations were verified. The confirmation of age associations in an independent donor cohort supports the use of the SCNP technology as a tool for identifying reproducible predictive biomarkers in areas such as vaccine response and response to cancer immunotherapies.In a broader exploratory analysis of age-related immune signaling changes performed in the current donor cohort, 57 immune signaling responses  were significantly associated with age. The age-associated signaling responses spanned all modulators  and all subsets , CD45RA+ Th cells, CD45RA- Th cells, CD45RA+ cytotoxic T cells, and CD45RA- cytotoxic T cells) demonstrating the importance of a holistic approach to describing immunosenescence.The current study was registered with ClinicalTrials.gov NCT01119703) and approved by an independent Canadian institutional review board. Participants were 174 healthy residents of Quebec who gave written informed consent prior to collection of blood samples. They were aged either ≥ 65 or 25–40 yrs (Table 119703 anBlood was collected into sodium heparin Vacutainer® tubes (Becton-Dickinson) from which PBMCs were isolated and cooled within 8 h of collection. Briefly, heparinized blood was diluted 1:1 with RPMI-1640 medium and overlaid on ficoll/hypaque for centrifugation – all at room temperature. Resultant PBMC were washed twice with RPMI, resuspended in ice-cold 10% DMSO/90% heat-inactivated FBS at a concentration of 10 million cells per ml, and frozen slowly using a -1°C/min device to -80°C. Cells were placed into liquid nitrogen temperatures within 72 h of freezing and transported in a liquid nitrogen dry shipper. All samples had viabilities >70% based on cleaved poly (ADP-ribose) polymerase negativity.The PBMC samples from the 60 healthy donors used for training purposes have been described previously . BrieflyThe SCNP assay was performed as described previously . ModulatThe term “signaling node” refers to a specific protein readout in the presence of a specific modulator. For example, the response to IFN-α stimulation can be measured using p-Stat5 as a readout. This signaling node is designated “IFN-α → p-Stat5”. Each signaling node is measured in each of the 7 distinct cell subpopulations simultaneously. The cell subpopulation is noted following the node e.g. “IFN-α → p-Stat5 | CD45RA- Th cells”. The raw instrument median fluorescence intensities (MFIs) were converted to calibrated intensity metrics, Equivalent Number of Reference Fluorophores (ERFs), by using rainbow calibration particles on each 96-well plate . The “FoThe “Fold” metric was calculated as follows:Fold: log2 [ERF(Modulated)/ERF(Unmodulated)]Overall, 24 signaling nodes Table . In the p value corresponding to the hypothesis that the slope is zero was used as the significance test (with a p < 0.05 cutoff). Immune signaling responses with significant associations with age in the 60 donor training set were pre-specified for confirmation in the independent set of 174 healthy donors in the current study (test) cohort. To control the type I error rate, pre-specified age associations were ranked (based on the strength of the association with age in the training cohort) and tested using a Gatekeeper strategy [p < 0.05 for the two-sided Wilcoxon rank-sum test statistic. The Wilcoxon rank-sum test was used for analyzing age associations in the current study (test) cohort because this cohort contained distinct young and elderly donor subgroups .To discount variation in cell viability as a causative factor for signaling differences between young and elderly donor subgroups, differences between the proportions of viable cells were analyzed. No significant association between cell viability and age was observed (The master set of 60 donors from the published study  was utilUsing a Gatekeeper strategy (see Methods), 9 of the 11 pre-specified age-associated signaling responses were verified in the independent cohort of 174 donors (test set) in the current study. Figure Within the CD45RA+ Th cell compartment, the verified associations included age-associated differences in IFN-α-induced p-Stat5 and in IL-2-induced p-Stat5 responses. Notably, CD45RA+ Th cells displayed higher IL-2-induced p-Stat5 in elderly donors than in young donors. Because IL-2 production is reported to decrease with age , the heiAge-associated alterations in immune signaling responses were further explored within the current study cohort by identifying all responses among the broad panel of immunomodulators and immune cell subsets profiled Figure , Table 2Of the 93 responsive signaling nodes, 57 showed significant age-associated differences with the majority (51 nodes) displaying lower responsiveness in elderly donors than in younger donors  p values < 0.05, 47 have adjusted p values < 0.05. The 10 responses that lose significance with the adjusted p values include the 9 responses in Figure p < 0.05 and 1 response with p = 0.007 (IL-27 → p-Stat3 in CD45RA-cytotoxic T cells).In the exploratory analysis described here, because the focus was on hypothesis generation, the less stringent approach of utilizing While a small number of the age-associated responses identified in the current study were not profiled in the previously published healthy donor cohort Table , the majFor example, an age-associated alteration in TCR signaling (a pathway which was not profiled in the prior study, see Table The age-associated decline in intracellular signaling responses in monocytes following modulation with the TLR7/8 ligand R848 (Figure Future work will focus on expanding the immune cell subsets interrogated for age-associated signaling alterations. The inclusion of phenotypic markers allowing for the delineation of additional cell subsets, such as dendritic cells, will allow for a greater understanding of age-related alterations in innate immunity . SubsequIn summary, SCNP provides quantitative, high dimensional measurements of the human immune cell signaling network pertinent to unraveling the complex mechanisms underlying the age-related decline in the function of the human immune system. The confirmation of age associations in two independent donor cohorts supports the utility of SCNP as a tool for identifying associations between immune signaling responses and clinical outcomes such as response to classical prophylactic vaccines, therapeutic cancer vaccines, or alternative immunotherapies.SCNP: Single-cell network profiling.DML., BL, JP, GF, EE, SP, MA, DS, AC, and REH are employees of Nodality Inc. AS, SL, RR, CRB, and LNC are employees of Merck Inc. The other authors declare no financial conflicts of interest.DML, REH, AC, CRB, and LNC conceived the study. AS and MA coordinated clinical sample acquisition and sample handling. DML. performed the experiments. DML, BL, and EE analyzed the experiments. JP, GF, SP, DS, and RR participated in the review of the study analysis. DML. drafted the manuscript. All authors read and approved the final manuscript.


While kidney injury is associated with re-expression of numerous Wnt ligands and receptors, molecular mechanisms which underlie regulation of distinct Wnt signaling pathways and ensuing biological consequences remain incompletely understood. Primary cilia are increasingly being recognized as cellular ‘antennae’ which sense and transduce signals from the microenvironment, particularly through Wnt signaling. Here, we explored the role of cilia as modulators of canonical and non-canonical Wnt signaling activities involving tubular epithelial cells in the injured kidney.We demonstrate that in the mouse model of unilateral ureter obstruction, progression of kidney injury correlates with increased expression of numerous Wnt ligands, and that increased expression of Wnt ligands corresponded with over-activation of canonical Wnt signaling. In contrast, non-canonical Wnt signaling dropped significantly during the course of kidney injury despite gradually increased expression of typical non-canonical and intermediate Wnt signaling ligands. We further demonstrate that in cultured tubular epithelial cells, cilia modulate balance between canonical and non-canonical signaling responses upon exposure to Wnt ligands.We provide evidence that in the context of renal injury, primary cilia act as molecular switches between canonical and non-canonical Wnt signaling activity, possibly determining between regenerative and pro-fibrotic effects of Wnt re-expression in the injured kidney.The online version of this article (doi:10.1186/s13069-015-0024-y) contains supplementary material, which is available to authorized users. In principle, the kidney possesses a unique capacity to repair itself, even upon severe acute injury -3. In clIn this regard, Wnt signaling is being increasingly recognized as important mediator of both renal repair and progressive renal fibrosis. Wnt proteins  in general are a family of secreted proteins, which act as ligands to activate distinct Wnt signaling pathways ,11. TherWnt signaling in general plays multiple essential roles in organogenesis, carcinogenesis, and tissue homeostasis . While iWhile the role of Wnt signaling in the injured kidney is complex, majority of studies on canonical Wnt signaling demonstrate a detrimental role. A recent study implicated activity of β-catenin signaling - the prototypical read-out of canonical Wnt signaling - within tubular epithelial cells as a predictor of tubulointerstitial fibrosis and poor prognosis of kidney allografts . MajoritIn this regard, primary cilia have emerged as principal modulators of Wnt signaling . PrimaryWe observed that in the murine model of obstructive nephropathy irreversible tubulointerstitial fibrosis (7 days after ureter obstruction), co-incides with structural damage of primary cilia, overactivation of canonical Wnt signaling, and drop of non-canonical Wnt signaling activity. We provide causal evidence that loss of intact cilia contributes to the switch from non-canonical to canonical Wnt signaling in response to Wnt ligands by tubular epithelial cells, possibly contributing to the switch from reversible to irreversible kidney injury.To enable comparison of our study with previous reports regarding involvement of Wnt signaling and kidney disease, we first analyzed the dynamics of mRNA expression of Wnt ligand genes known to be expressed in adult kidneys in our mouse model of unilateral ureter obstruction (UUO) in C57/BL6 mice. Complete UUO initiates a rapid sequence of events in the obstructed kidney, leading within 24 h to reduce renal blood flow and glomerular filtration rate . This isWe next performed immunostaining experiments using antibodies to β-catenin, the principal mediator of canonical Wnt signaling . WhiTo further substantiate our findings of unequivocal increase of Wnt ligand expression and Wnt signaling activity, we next analyzed the expression of several Wnt target genes as indicators of canonical and non-canonical Wnt signaling activity ,32. CorrXenopus and zebrafish, it has been speculated that cilia can function as molecular switches between canonical and non-canonical signaling pathways . W. W38]. WHere, we report two novel findings which add new aspects and possible explanation to the complexity of Wnt signaling in the context of kidney disease. While our study is in line with previous reports of increased canonical Wnt signaling during renal fibrogenesis (an intuitive finding in view of robust overexpression of multiple Wnt ligand genes), we report for the first time a distinct pattern of non-canonical Wnt signaling activity, as expression of non-canonical Wnt target genes drops during disease progression (despite increased expression of Wnt ligand genes). Second, we provide evidence for the first time that primary cilia  play a causal role in discerning between canonical and non-canonical Wnt signaling pathways, likely impacting the net-effect of Wnt activity in the injured kidney.While several previous studies agree that kidney injury is associated with increased expression of Wnt ligands , their iWith regard to conflicting interpretations of the sum effect of Wnt signaling on kidney fate, it may be also important to note that loss of cilia appears to be model specific and that we did not observe cilia stunting or β-catenin immune-labeling in other murine models of kidney disease (data not shown). Similarly, impact of genetic background (in our case C57BL/6 mice were used) remains unclear.We are aware of the fact that the strongest clinical evidence for a detrimental role of canonical Wnt signaling stems from a study which analyzed utility of tubular β-catenin immunostaining as predictor of chronic allograft nephropathy . Of noteper se is considered to contribute causally to manifestation of autosomal dominant polycystic kidney disease [Because cilia play a central role in pathogenesis of hereditary cystic kidney diseases (because most mutations causing hereditary cystic kidney diseases affect genes which encode for cilia proteins), it is tempting to compare our observations with existing polycystic kidney disease (PKD) literature. In this regard, increased Wnt signaling  disease . Cystoge disease . In thisWe are aware that our study focuses entirely on the impact of cilia and Wnt signaling in tubular epithelial cells. If such mechanism is relevant for other cell types such as fibroblasts remains to be seen.Primary cilia favor non-canonical Wnt signaling responses in tubular epithelial cells, while loss of intact cilia augments canonical Wnt signaling in response to Wnt ligands. As β-catenin superactivation has been identified as biomarker for negative outcome of chronic kidney disease and kidney injury is associated with increased expression of most Wnt ligands, impairment of primary cilia likely contributes causally to renal fibrogenesis by serving as molecular switch from non-canonical to canonical Wnt signaling responses.CD57BL/6 mice were anesthetized with isoflurane inhalation. Analgesia was performed by subcutaneous Buprenorphine injection. The ureter was separated from the surrounding tissues, and two ligatures were placed about 5 mm apart in upper two thirds of the ureter of the left kidney to obtain reliable obstruction. Mice were sacrificed at indicated time points after ureter ligation [Eight- to twelve-week-old ligation . All stu2O2) in methanol for 30 min. Samples were blocked with 2.5% horse serum . After the blocking step, sections were incubated with primary antibody for overnight at 4°C in a humidified chamber, and the biotinylated secondary antibody  was applied for 30 min at room temperature. The reaction products were visualized using 3,3′-diaminobenzidinetetrahydrochloride (DAB)  and slides were counterstaining with hematoxylin. Mouse monoclonal β-catenin antibody  and rabbit monoclonal non-phospho (Active) β-catenin antibody  were used as primary antibodies.Fomalin-fixed, paraffin-embedded kidney sections from mice and human biopsy samples were deparaffinized in xylene and dehydrated through graded alcohols. Antigen retrieval was undertaken in a steam cooker for 40 min in Target retrieval solution . Endogenous peroxidase was blocked with 3% hydrogen peroxide . Cells were seeded in chambered slides and fixed in ice-cold methanol for 10 min. Slides were then washed in phosphate-buffered saline (PBS) three times, and they were incubated with blocking solution (3% BSA in PBS) for 30 min. For immunofluorescence staining of mouse tissues, formalin-fixed, paraffin-embedded sections were used. Slides were deparaffinized and dehydrated. Mouse monoclonal acetylated α-tubulin antibody  and non-phospho β-catenin antibody  was used as primary antibodies. Samples were then stained with Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Fluor 568-conjugated anti-rabbit IgG antibody  and mounted with VECTASHIELD Mounting Medium  with DAPI .Cells were viewed by using the Axiovert-200 inverted light and epifluorescence microscope  or using the confocal LSM 780 microscope . Images of cilia were captured from randomly chosen high-power fields (40× objective), when the full extent of cilia could be visualized in a single plane of focus. Cilia were scored as either possessing a full length primary cilia (>2 μm) or possessing a stunted primary cilia (<2 μm). Cilia from ten proximal and ten distal tubules were measured for each mouse.2. For immunostaining of cilia in culture, cells are grown as a monolayer on chambered slides  and for RNA isolation in six-well plates . To generate ciliated and non-ciliated tubular epithelial cells, we seeded HK2 cells at different densities  and starved cells for 48 h without serum to induce cell cycle exit and ciliogenesis as described previously [HK2 human proximal tubular epithelial cells were cultured in DMEM  supplemented with 100 g/mL penicillin, 100 g/mL streptomycin, and 10% fetal bovine serum  at 37°C in 5% COeviously . Upon seTissue and cells were dissolved in TRIZOL , and tissue were shredded using TissueLyser LT . RNA was isolated by using PureLink RNA Mini Kit  according to the manufacture’s protocol.t-test or a one-way ANOVA was used. Oligonucleotide sequences are shown in Additional file For SYBR-based real-time PCR, cDNA synthesis was performed by using DNase I  treatment and SuperScript II Reverse Transcriptase  according to the manufacturer’s protocol. Reverse-transcripted cDNA was added to the reaction mixture containing the primer pair and diluted with Fast SYBR Green Master Mix . PCR reactions were performed in a 96-well reaction plate using the StepOne Real-Time System  and were done in triplicates. All qRT-PCR data for RNA expression analysis were calculated using the ΔΔCT method. For statistical evaluation, The use of parts of kidney biopsies for research purposes was approved by the Ethics Committee of the University Medical Center Göttingen, and written consent was obtained from all subjects before kidney biopsy. Detailed clinical patient data are presented in Additional file


A complex interplay of intracellular signaling networks orchestrates normal cell growth and survival, including translation, transcription, proliferation, and cell cycle progression. Dysregulation of such signals occurs commonly in many malignancies, thereby giving the cancer cell a survival advantage, but also providing possible targets for therapeutic intervention. Activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway contributes to the proliferative advantage of malignant cells and may confer resistance to chemotherapy in various hematologic malignancies. The initial mTOR inhibitor, sirolimus , was first discovered in 1975 in the soil of Easter Island. Sirolimus was originally developed as an anti-fungal agent given its macrolide properties, but was approved by the Food and Drug Administration (FDA) in 1999 as an immunosuppressive agent for renal transplantation patients once its T cell suppression characteristics were recognized. Shortly thereafter, recognition of sirolimus’s ability to inhibit cellular proliferation and cell cycle progression brought sirolimus to the forefront as a possible inhibitor of mTOR. In the subsequent decade, the functional roles of the mTOR protein have been more fully elucidated, and this protein is now known to be a key regulator in a highly complex signaling pathway that controls cell growth, proliferation, metabolism, and apoptosis. This article discusses the dysregulation of PI3K/mTOR signaling in hematologic malignancies, including acute and chronic leukemias, lymphomas, and lymphoproliferative disorders. The current repertoire of PI3K/mTOR pathway inhibitors in development and clinical trials to date are described with emphasis upon pediatric hematologic malignancies (Figure  Growth factors, such as insulin-like growth factor (IGF), interleukin 7 (IL-7), and fms-like tyrosine kinase 3 ligand (FLT3-L), bind their tyrosine kinase receptors on the cell surface membrane and induce intracellular PI3K/mTOR pathway signaling. Activation of PI3K recruits cellular protein kinases that in turn activate downstream kinases, including the serine/threonine kinase Akt. Phosphorylation of Akt at its threonine 308 residue inactivates the tuberous sclerosis complexes 1 and 2 (TSC1 and TSC2), permitting activation of the mTOR complex 1 (mTORC1) and subsequent phosphorylation of proteins 70S6K1, S6, and the eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1). This activation of mTORC1 results in increased translation and protein synthesis. A second complex of mTOR, known as mTORC2, has been more recently described and appears to act as a feedback loop via Akt. Activation of mTORC2 via phosphorylated 70S6K1 induces phosphorylation of Akt at its serine 473 residue, thereby inhibiting mTORC1 activation and resulting in G1 cell cycle arrest and apoptosis via 4EBP1. The tumor suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome 10) provides additional negative regulation of the PI3K/mTOR pathway via inhibition of Akt and phosphatidylinositol 3,4,5)-triphosphate  –3, 8, 9.t models , 11. Test models . In vivocontrols .in vitro cytotoxicity in six of seven ALL cell lines and in vivo efficacy in one of three ALL xenograft models in comparison to single-agent treatment  B-ALL  B-ALL , 17–20.t activates the PI3K/mTOR pathway directly in both chronic myeloid leukemia (CML) and Philadelphia chromosome-positive (Ph+) ALL (The BCR-ABL1 fusion protein resulting from Ph+) ALL . AlthougPh+) ALL . BCR-ABLPh+) ALL .BCR-ABL1-rearranged ALL and is associated with various genetic alterations known or predicted to activate oncogenic cytokine receptor signaling (CRLF2). The CRLF2 protein heterodimerizes with the IL-7 receptor alpha chain to form the thymic stromal lymphopoietin receptor (TSLPR) complex. Initial studies using (non-CRLF2-rearranged) murine B-ALL models identified activation of PI3K/mTOR signaling in these leukemias, which was modulated by IL-7 and TSLP and could be abrogated in vitro and in vivo by sirolimus treatment  subtype of B-ALL, which has a kinase-activated gene expression profile similar to that of ignaling –23. Adulreatment , 25. Subngements . Aberran pathway . The effsurvival . Other Pt models . The theNOTCH1 mutations , leading to decreased transcription or modification of PTEN  of pediatric T-ALL cases assessed. Correlation of mutation status with clinical outcomes data revealed that T-ALL patients with complete deletions of PTEN were far more likely to experience induction failure  than those with mutated PTEN  and likely results from a variety of mechanisms, including alterations in cellular growth factors and mutations in FLT3, c-kit, and Ras pathway proteins –34. Varie models –37. Use e models , 38. Proe models . As afore models . Such coe models , 41.JAK2 (Janus kinase 2) that result in signaling hyperactivation involving signal transduction and activator of transcription factors (STATs), but constitutive PI3K/mTOR activation has also been implicated in MPNs. Everolimus was shown to inhibit cell proliferation of JAK2 V617F mutant cell lines, and the dual mTORC1/2 inhibitor PP242 even more potently induced apoptosis (in vitro and in vivo (Many myeloproliferative neoplasms (MPNs) harbor point mutations in  in vivo .in vivo and in vitro  cases, but is less well-defined in pediatric DLBCL , 50. PreALK (anaplastic lymphoma kinase) mutations, and targeting with MTIs in preclinical models has shown some activity , a disorder of lymphocyte survival due to defective apoptosis hallmarked biologically by double negative T-cells . mTOR haAn institutional Phase 1 trial tested the safety of oral sirolimus monotherapy in nine pediatric patients with relapsed ALL. No dose limiting toxicity (DLT) was noted, and three ALL patients met the criteria for stable disease . Based u2, de-escalated to 7.5 mg/m2) with an intensive four drug re-induction backbone per the UKALL R3 relapse protocol (NCT01403415)   Phase 1 trial combined 3-weekly IV temsirolimus (10 mg/m1403415) , 61. Due1658007) .Sirolimus treatment has also been studied in a limited institutional series in children with ALPS. These patients have traditionally been treated with immunosuppression such as steroids and solid organ transplant rejection/prophylaxis medications. Five out of five patients with steroid-refractory ALPS had complete responses (CRs) with resolution of lymphadenopathy with single-agent . An ongoA Phase 1/2 study of oral everolimus in adult patients with relapsed hematologic malignancies was recently reported . No DLTsde novo AML showed improved median overall survival when compared to cytarabine alone  pan-class I PI3K inhibitors, (b) isoform-selective PI3K inhibitors, (c) Akt inhibitors, (d) MTIs/rapalogs, (e) dual PI3K/mTOR inhibitors, and (f) TORC1/TORC2 inhibitors  Figure , 72, 73.mTOR inhibitors are generally well tolerated as single-agents with common side effects such as hyperglycemia, hyperlipidemia, hepatic impairment, infection, fatigue, and mucositis . These tSingle-agents MTIs have shown some activity in slowing malignant cell proliferation, but are unlikely to succeed as monotherapy, as mTOR inhibition alone is often cytostatic and/or leads to compensatory upregulation of alternate signaling pathways. Identification of mTORC2 helped elucidate the path of compensatory aberrant phosphorylation noted in early MTI trials. Dual TORC1/TORC2 inhibitors have shown some improved cytotoxicity in preclinical leukemia models, but have not yet been evaluated clinically in patients with hematologic malignancies , 71. AktCompensatory signaling and feedback loops appear to differ among leukemia and lymphoma subtypes. While preclinical results have informed the design of clinical trials to test inhibitors of the PI3K/mTOR pathway in adults and children with hematologic malignancies, significant progress remains to be made , 38. ToxThe identification of sirolimus in the soil of Easter Island has led to better understanding of its critical protein target. In the past decade, a more comprehensive description of this key signaling network required for cellular proliferation and survival has evolved. The consistent demonstration of oncogenic abnormalities in the PI3K/mTOR signaling pathway and in cross-talk pathways demonstrates a clear rationale for development of signal transduction inhibitor-based approaches for children with hematologic malignancies. Studies are ongoing to identify the most optimal inhibitor(s) for each disease subtype. Potential synergy with other targeted inhibitors and/or with conventional chemotherapy may provide additional therapeutic options to optimize therapeutic efficacy and to minimize toxicity.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.