R2=0.92). Coupled dynamics acts to improve the characteristic Niño-like spatial structure but not necessarily the variance. Perturbations of the equatorial Atlantic trade winds (∼±1.53 m s−1) can drive changes in surface latent heat flux (∼±14.35 W m−2) and thus in surface temperature consistent with a first-order autoregressive process. By challenging the dynamical paradigm of equatorial Atlantic variability, our findings suggest that the current theories on its modelling and predictability must be revised.Prevailing theories on the equatorial Atlantic Niño are based on the dynamical interaction between atmosphere and ocean. However, dynamical coupled ocean-atmosphere models poorly simulate and predict equatorial Atlantic climate variability. Here we use multi-model numerical experiments to show that thermodynamic feedbacks excited by stochastic atmospheric perturbations can generate Atlantic Niño s.d. of ∼0.28±0.07 K, explaining ∼68±23% of the observed interannual variability. Thus, in state-of-the-art coupled models, Atlantic Niño variability strongly depends on the thermodynamic component ( The nature of the El Niño-like variability in the Atlantic Ocean and its limited predictability remain unresolved. Here, via multi-model numerical experiments, the authors show that much of the variability can be explained by the interaction of stochastic atmospheric fluctuations with the ocean mixed layer. The equatorial Atlantic Ocean exhibits sporadic interannual fluctuations in zonal sea surface temperature (SST) gradients associated with westerly wind perturbations12316782101112u}' over the western equatorial Atlantic can drive ocean currents and equatorial baroclinic waves that redistribute upper ocean heat content, thereby modulating SST change in the eastern basin. Studies motivated by the Bjerknes theory show that the peak phase of the Atlantic Niño in June-July-August is preceded by equatorial westerly wind perturbations ({u}'>0)  and of the same sign as observations for only six of the 12 models. As expected, models of overly strong {u}'|{SST}' coupling  tend to overestimate equatorial Atlantic SST variability represented by the s.d. (σ{SST}') of the Atlantic Niño index. However, it is rather surprising that some models (CSIRO-Mk3.0 and MRI-CGCM2.3.2) with weak {u}'|{SST}' coupling exhibit realistic σ{SST}'. Indeed, even models with weak (ECHAM5/MPI-OM) and statistically significant (GISS-ER) but incorrect {u}'|{SST}' correlation signs, can generate realistic σ{SST}' of the Atlantic Niño.The relationship between {ll-CGCMs . The {u}u}'|{SST}' relationship. Indeed, the multi-model variance of equatorial Atlantic σ{SST}' explained by the {u}'|{SST}' coupling is only ∼37%, suggesting that processes other than the Bjerknes feedback may be key to equatorial Atlantic variability.Furthermore, some numerical models  that simulate the correct structure of seasonality — usually1In reality, SST variability is caused by a combination of dynamical  and thermodynamic  feedbacks on the ocean mixed layer. Indeed, recent studies have questioned the essence of ocean dynamics in generating the equatorial Pacific El Niño, concluding that thermodynamic feedbacks may be more important than previously thought212223σ=∼0.28±0.07 K; for the full-CGCMs, σ=∼0.47±0.09 K). σ{SST}' of the Atlantic Niño of the full-CGCMs  is linearly dependent on the σ{SST}' of the slab-CGCMs with no interactive ocean dynamics. The multi-model correlation coefficient is 0.96, corresponding to 92% of the explained variance. In comparison, the equatorial Pacific variability represented by the Niño-3 index exhibits more scatter with an explained variance of only 3%. This implies that while dynamical coupling in the equatorial Atlantic acts to amplify the σ{SST}', coupled dynamics in the Pacific tends to modify the SST variability.The monthly Atlantic Niño index of the 12 slab-CGCMs exhibits strong variability , coupling to ocean dynamics leads to unrealistically strong amplitudes of the Atlantic Niño σ{SST}'—given that the mixed layer depths are not much different from the observed. The ensemble-mean of the full-CGCMs reproduces the equatorial Atlantic SST temporal variability as may be expected from the consistency of the modelled mean mixed layer depths with observations.The full-CGCM ratios were also plotted on the same axes of To determine the comparative fidelity of the full-CGCM and slab-CGCM models in reproducing the observed spatial structure of the Atlantic Niño, we extracted the associated SST variability over the equatorial Atlantic Ocean  as the leading empirical orthogonal function . This anσ{SST}' and {u}'|{SST}' coupling  are slab-CGCMs and only two (INM-CM3.0 and MIROC3.2_medres) are fully coupled . These tcoupling .σ{SST}' despite weak or incorrect {u}'|{SST}' correlation , fail to reproduce the observed spatial structure of the equatorial Atlantic SST variability. These results suggest that while thermodynamic feedbacks can generate strong σ{SST}', the characteristic spatial structure of the Atlantic Niño could arise from coupled dynamics. Indeed, in most models , coupling to ocean dynamics actually improves the spatial correlation with observations, but not the amplitudes of the SST variability. The implication is that, overall, the fully coupled models are no better than their thermodynamic counterparts in simulating the spatial structure of the Atlantic Niño.On the other hand, those full-CGCMs with realistic Previous studies suggest that the Atlantic Niño exhibits interannual periodicity see ref. , as pred30u}'|{SST}'—suggesting that Bjerknes feedbacks operate in the model—in the equatorial Atlantic Ocean.The above analysis clearly shows that thermodynamic processes are alone able to reproduce aspects of Atlantic Niño variability. Nevertheless, there remains a wide spread of model performance that is further exacerbated by coupling to a dynamical ocean model, as may be expected from larger mean biases of the full-CGCMs. Our aim is not to fully account for inter-model differences, but instead to investigate the physical processes involved. In this respect, we further analyse GFDL-CM2.0 ref.  as its tSST}' forms off southeastern tropical Atlantic Ocean; that is, the Benguela Niño sector at t-6 leading to the peak phase of the Atlantic Niño. In subsequent months, the maximum {SST}' propagates north-westwards, amplifies to cover the equatorial Atlantic Ocean and then begins to decay. In the absence of ocean dynamics, the ocean mixed layer warming and cooling rates  are governed entirely by net surface heat flux anomaly ({QNET}') driven by stochastic atmospheric perturbations. Thus, at t–6, the {QNET}' in the Benguela Niño area is associated with weakening of the trade winds and westerly wind perturbations ({u}'>0) in the equatorial region. The following months are marked by a progressive northward intensification of the westerlies and {QNET}' that drives the {SST}'.As shown in QNET}' dampens rather than drive the {SST}' in the region, although the explained variance is relatively low37384171836QNET}' and {SST}' actually undergoes a phase change during the life cycle of the Atlantic Niño . The evolution of the {∂T/∂t}' over the equatorial Atlantic Ocean during a typical Atlantic Niño year is closely reproduced by the {QNET}' such that the residual term is quite small (T/∂t}' is largely controlled by shortwave radiation ({QSW}'=+15.83 W m−2) and by forcing from negative latent heat flux ({QLH}'=−14.35 W m−2) peaking at t–2 during the development phase of the Atlantic Niño. The {QLW}' and {QSH}' appear to play comparatively minor roles. Superimposed on the climatological-mean state, negative {QLH}' represents a suppression of evaporation due to a weakening of the trade winds, giving rise to net surface heating. Thus, the {QLH}' and {QNET}' tend to evolve together  is comparable with the sum of the advection and {QNET}' terms (2.0 e−7K s−1). On the other hand, the peak contribution of the zonal {u∂T/∂x}' and meridional {v∂T/∂y}' advection components in driving the ocean mixed layer temperature is delayed until t=0, suggesting that the Atlantic Niño event may be essentially triggered by the {QNET}' before coupled dynamics sets in , which is constructed using the observed SST. The Atlantic Niño may therefore, in reality, result largely from perturbations of the mean seasonal cycle55The tropical Atlantic Ocean has experienced profound climate shifts4849515253515253R2=0.04). The mean state biases have often been attributed to the atmospheric component of the coupled models; leading hypothesis implicates weakening of the trade winds51The mean state biases can impact equatorial Atlantic variability, as has been discussed in some recent studies54Aspects of the thermodynamically driven equatorial Atlantic variability discussed in this study—including its evolution and spatial characteristics—appear similar to the results reported for the Pacific El Niño2122234If thermodynamic feedbacks constitute the main source of Atlantic Niño variability as described in this study, then potential dynamical predictability may actually be lower than currently thought. Thus, our results call for a re-thinking of the procedures for improving the modelling and predictions of equatorial Atlantic climate that may necessarily be thermodynamically motivated5960The National Oceanic Atmospheric Administration Extended Reconstructed SST version 3b available for 2- × 2- longitude–latitude gridsTwelve numerical models that contributed skin temperature—a proxy for SST—to the ‘slab ocean control experiment' of the CMIP3 and have integration lengths of ≥30 years were selected for analysis. The slab-CGCM integration lengths (in years) studied are as follows: CCSM3 (51), CGCM3.1_T47 (30), CGCM3.1_T63 (30), CSIRO-Mk3.0 (60), ECHAM5/MPI-OM (100), GFDL-CM2.0 (50), GFDL-CM2.1(100), GISS-ER (120), INM-CM3.0 (60), MIROC3.2_medres(60), MRI-CGCM2.3.2 (150) and UKMO-HadGEM1 (70). Description of these models and experimental designs are available from the CMIP website (http://www-pcmdi.llnl.gov/ipcc/about_ipcc.php).For each model, the final years of  integration of the fully coupled configuration (full-CGCM) corresponding to the available length of the slab-CGCM were studied. As in the slab control experiment, greenhouse forcing was held constant in the pre-industrial control experiment and this allows us to explore the intrinsic variability, as the tropical Atlantic Ocean has exhibited marked warming trends4849QNET) based on the following thermodynamic equation:In this modelling set-up, the time evolution of the SST is determined by the net heat flux , respectively, and where T is the SST. QSW, QLW, QLH and QSH denote net downward shortwave radiation at the ocean surface, net upward longwave radiation, surface latent and sensible heat flux, respectively. Note that QSW is positive downward, while the other fluxes are positive upwards. Qflux is prescribed monthly climatological-mean ocean heat flux, which contributes to the annual cycle but does not drive SST variability. R is a residual term that denotes the sum of boundary flux errors from averages over the Atlantic Niño region and high frequency variability not resolved by the monthly time series analysed. Thus, the variability of ∂T/∂t is governed by the terms on the right-hand side of the equation enclosed in square bracket , which denotes the QNET.where QNET terms, coupling to interactive ocean dynamics denotes the introduction of three-dimensional ocean water advection and thus the exclusion of the restoration term (Qflux). The temperature tendency is determined as follows:Here in addition to the u and v are the horizontal ocean current velocities; and where the first two terms on the right-hand side represent zonal and meridional advection terms. R is a residual term that represents the sum of boundary flux errors from averages over the Atlantic Niño region, vertical temperature advection [w∂T/∂z] and unresolved physical processes . In the fully coupled configuration, the mixed layer depth (h) is defined as the depth at which ocean temperatures are 0.5 K lower than those at the surface; thus, h changes in space and time. Following the approach of ref. where ·}' via the least squares method and domain-average was then computed for the Atlantic Niño region.Once the heat budget terms were calculated, each was linearly de-trended, and the climatological-mean annual cycle removed to create an anomaly field {Observed data sets on the climatological-mean ocean mixed layer depth estimated using the temperature criterion described above and thermocline (represented by the depth of the 20 °C isotherm) were taken from the World Ocean Atlas 1994 (WOA94) ref. , availabThe Atlantic Niño index was computed as the domain averaged SST anomaly over the equatorial Atlantic Ocean . The Niño-3 index, determined as the domain-average at 5-N–5-S, 90-W–150-W, was used to represent eastern equatorial Pacific El Niño.P values using Student's t-test, except for the Atlantic Niño spectrum in which significance was estimated as the (lower and upper) 95% confidence bounds of the theoretical AR(1) spectrum using the lag-1 autocorrelation function.We determined the statistical significance levels based on the two-tailed How to cite this article: Nnamchi, H. C. et al. Thermodynamic controls of the Atlantic Niño. Nat. Commun. 6:8895 doi: 10.1038/ncomms9895 (2015).Supplementary Figures 1-5.


Anacardium occidentale, and fruit bodies of Merrulius tremellosus, and their effects on the electric eel acetylcholinesterase activity and conformation were studied. The observed effect distinctly depended on the chemical structure of the phenolic lipids that were available for interaction with the enzyme. All of the tested compounds reduced the activity of acetylcholinesterase. The degree of inhibition varied, showing a correlation with changes in the conformation of the enzyme tested by the intrinsic fluorescence of the Trp residues of the protein.Phenolic lipids were isolated from rye grains, cashew nutshell liquid (CNSL) from  The phenolic lipids are a heterogeneous group of natural lipids, including simple phenols, polyphenols and their derivatives. A certain sub-population of single-ring phenolic lipids are specifically found in certain plants and these contain a catechol, resorcinol or hydroquinone moiety alk(en)ylated by a multicarbon chain . PhenoliMerulius tremellosus seems to be one of the most biologically active phenolic lipids [Anacardic acids, cardols, cardanols and methylcardols a are thec lipids ,11.Acetylcholine (ACh) is a universal cell molecule found in all plants and animals and it plays a role in neuronal and non-neuronal signaling . It is aAcetylcholinesterase (AChE) is a conservative enzyme encoded by a single gene. It is a product of alternative splicing and therefore, can be bound to the membrane via a glycosyl-phosphatidylinositol anchor positioned in the exoplasmic leaflet of the membrane (erythrocyte AChE) or PRIMA peptide (synaptic AChE) or soluble in water . Each form has identical catalytic properties regardless of the place of appearance . The welElectric eel AChE (>1000 U/mg protein), acetylthiocholine iodide and DTNB -benzoic acid) were purchased from Sigma-Aldrich, Poznan, Poland. AChE solution was received at a concentration of 1.5 mg/mL and was diluted 1500 times (for the estimation of AChE activity) or 75 times (for Trp fluorescence tests).Homologues of the phenolic lipids, resorcinolic lipids and merulinic acid were isolated chromatographically according to procedures described previously . The remaining chemicals were of the best available purity and obtained from CHEMPUR .2CO3  were added to 3 mL of 0.1 M Na2HPO4/NaH2PO4 (pH 8.0). Microliter amounts (less than 50 μL) of the investigated compounds (concentration range from 8 to 94 μM) were then added. After 5 min of pre-incubation, acetylcholine iodide was added to a final concentration of 0.476 μM.AChE activity was assayed using the method described by Ellman . BrieflyThe progress of acetylthiocholine iodide hydrolysis was recorded spectrophotometrically with a Shimadzu UV-2401 PC , UV–VIS Recording Spectrophotometer at 37 °C. Changes in the absorbance of the samples at 412 nm were recorded continuously for 10 min. The rate of the reaction (V) was calculated as follows:The enzyme activity modulated by the studied compounds was calculated as a percentage of the control reaction. The influence of the solvent and lipids was considered in the control and in blank samples.2HPO4/NaH2PO4 (pH 8.0) and 25 μL of AChE with continuous stirring, and maintained at 24 °C. After 10 min of equilibration, Trp fluorescence spectra were measured with a Varian Cary Eclipse spectrofluorometer . The tryptophan residues were excited at 295 nm and emission spectra were recorded from 300 to 400 nm. Spectra were corrected for the contribution of light scattering in the presence of the tested lipids.Alterations in the microenvironment of the Trp of the peptide upon interactions with the tested compounds were monitored as described previously . Small aMerulius tremellosus  and resorcinolic lipids (cardol and methylcardol) from cashew nutshell liquid (CNSL) a, alkylrmellosus c were te50 (the inhibitor concentration causing 50% inhibition) of similar values. The IC50 values of rye bran lipids are ten times higher  and AR 19:0  causes a decrease in the fluorescence intensity of Trp residues ,29. The 0. By contrast, the lipids with a polar part of the carboxyl group (anacardic and merulinic acids) cause a decrease in the value of F/F0. The presence of methyl residues at the 1,3-dihydroxybenzene ring (methylcardol) does not alter the conformation of the protein.The tested compounds that do not have a polar charged part of the molecule  cause an increase in the value of F/F0 becomes more significant with the increase in the number of carbon atoms in the side chain of the lipid molecule. This means that the mechanism of interactions of phenolic lipids with AChE is more complex, with the enzyme probably affecting both polar and non-polar parts of the lipid molecules.The results suggest that in the case of molecules with a side chain of the same length (lipids from CNSL), the effect depends on the chemical structure of the polar parts of the molecule. On the other hand, in the case of ARs from rye bran, which have the same polar head, the effect is dependent on the length of the side chain. The change in the value of F/F0 after addition of phenolic lipids to the reaction medium. Lipids that cause a change in the conformation of the enzyme also inhibit its activity. The exceptions are methylcardol and AR 15:0, which do not cause changes in the fluorescence of Trp residues even though they inhibit the activity of the enzyme in the ranges of 50%–70% and 20%, respectively. Earlier studies have indicated several major domains within the protein: a catalytic active site composed of two sub-sites, the aromatic gorge in which the catalytic active site lies, and a peripheral anionic site, distinct from the catalytic active site [There is a correlation between the observed changes in the value of AChE inhibition and value of F/Five site . The active site . The perive site . No shifive site .E. electricus as a model and indicated that the phenolic lipids may also interact directly with the protein part of this enzyme. Our results suggest that the effect of phenolic lipids on GPI-anchored erythrocyte AChE is probably more complicated and dependent on a direct interaction of lipid molecules with the protein part of the enzyme and on changes in the physicochemical properties of the phospholipid bilayer.It has been suggested that phenolic lipids alter the activity of GPI-anchored erythrocyte AChE by changing the physicochemical properties of the phospholipid bilayer. Our experiments use AChE from


The interaction between IgE and allergen is a key event at the initiation of an allergic response, and its characteristics have substantial effects on the clinical manifestation. Despite this, the molecular details of the interaction between human IgE and the major birch allergen Bet v 1, one of the most potent tree allergens, still remain poorly investigated.To isolate Bet v 1-specific human monoclonal IgE and characterize their interaction with the allergen.Recombinant human IgE were isolated from a combinatorial antibody fragment library and their interaction with Bet v 1 assessed using various immunological assays. The structure of one such IgE in the single-chain fragment variable format was determined using X-ray crystallography.We present four novel Bet v 1-specific IgE, for one of which we solve the structure, all with their genetic origin in the IGHV5 germline gene, and demonstrate that they target two non-overlapping epitopes on the surface of Bet v 1, thereby fulfilling the basic criteria for FcεRI cross-linkage. We further define these epitopes and for one epitope pinpoint single amino acid residues important for the interaction with human IgE. This provides a potential explanation, at the molecular level, for the differences in recognition of isoforms of Bet v 1 and other allergens in the PR-10 protein family displayed by IgE targeting this epitope. Finally, we present the first high-resolution structure of a human allergen-specific IgE fragment in the single-chain fragment variable (scFv) format.We here display the usefulness of allergen-specific human monoclonal IgE as a tool in studies of the crucial molecular interaction taking place at the initiation of an allergic response. Such studies may aid us in development of better diagnostic tools and guide us in the development of new therapeutic compounds. A type I hypersensitivity response is initiated when allergen-specific IgE bound to high-affinity IgE receptors (FcεRI) on the surface of effector cells, such as mast cells and basophils, are cross-linked by allergen. This triggers a cascade of events eventually giving rise to the release of biologically active compounds that exert their effects both locally and systemically Over the years, a range of studies have characterized the genetic composition of human IgE repertoires, in terms of V(D)J germline gene usage, mutational status and evidence of antigenic selection One intriguing outcome of several of the past IgE-repertoire studies is a significant difference in the utilization of different immunoglobulin germline gene subgroups to produce the heavy chain variable (VH) domains of IgE in certain tissue as compared to IgE and other isotypes encoded by peripheral blood lymphocytes. More specifically, an overrepresentation of IgE-encoding transcripts derived from the immunoglobulin heavy variable (IGHV) 5 germline gene subgroup has been observed in such tissue samples The second partner in the initial interaction triggering an allergic response, the allergen, adds further complexity to the task of understanding the molecular basis of the early events in such a response. Not only do most allergens exist as numerous different isoforms with varying potencies to provoke allergic reactions http://scop.berkeley.edu/) entry d.129.3.1] http://pfam.sanger.ac.uk/family/PF00407). This allergen exists in at least 18 different isoallergens and isoforms, as defined by IUIS The major birch pollen allergen, Bet v 1, is one of the most potent, and therefore also the most studied, tree allergens in Europe We here approach these issues and present a molecular characterization of Bet v 1-specific human antibody fragments derived from the IgE repertoire of allergic individuals. These antibody fragments have a genetic origin in IGHV5, the germline gene subgroup sometimes implicated as overrepresented in IgE repertoires Recombinant Bet v 1.0101, Bet v 1.0102 (previously known as Bet v 1.0401), Mal d 1.0108, Aln g 1.0101 were obtained from Biomay , while Bet v 1.0112 (previously known as Bet v 1.2801) and recombinant variants Bet v 1.2744  and Bet v 1.2595  A combinatorial single-chain fragment variable (scFv) library, based on transcripts encoding the VH domain of IgE origin derived from nasal tissue of two allergic donors Escherichia coli .To allow for the production of soluble scFv, not displayed on phage, genes encoding isolated scFv were transferred in a single cloning step into a production vector NcoI and NotI, into a vector carrying a sequence encoding the second, third and fourth constant domains of human IgE E. coli .To enable the production of scFv-Fcε fusion proteins, an antibody format previously employed and evaluated in other studies 600 = 0.9 was reached. At this point, protein production was induced by addition of isopropyl β-D-1-thiogalactopyranoside to a final concentration of 1 mm. After 16 h of production at 30°C, the cells were harvested by centrifugation at 7500 × g for 12 min and treated with lysozyme . Soluble proteins were purified using affinity chromatography with Ni-NTA agarose columns .After transfection, cells carrying the vector encoding scFv were grown in 2xYT-media (supplemented with 100 μg/mL carbenicillin) until ODm L-glutamine and 10% HyClone fetal bovine serum  in 5% CO2 at 37°C. At 90% confluency, the cells were transfected with vectors encoding the scFv-Fcε fusion proteins using Lipofectamine 2000 (Invitrogen). Supernatants containing scFv-Fcε fusion proteins were collected after 72 h and sterilized by filtration (0.45 μm) prior to analysis.HEK293 cells were grown in minimum essential medium (Invitrogen) supplemented with 2 mThe ability of the isolated Bet v 1-specific IgE-derived clones, either displayed on phage, as soluble scFv or as scFv-Fcε fusion proteins to bind to recombinant allergens was determined using ELISA. Allergens and BSA (negative control) were diluted to 5 μg/mL in PBS and coated in microtiterplates (Corning). Blocking was performed with 1% w/v BSA, 0.05% Tween-20 in PBS. Bound phages were detected with horseradish peroxidase (HRP)-conjugated anti-M13 mAb , scFv with an HRP-labelled anti-FLAG M2 mAb (Sigma-Aldrich) and scFv-Fcε fusion proteins with an HRP-labelled anti-IgE antiserum  using 1-Step Ultra TMB – ELISA Substrate  as chromogen. Absorbance was measured at 450 nm.The analysis of reactivity of scFv-Fcε fusion proteins to biotinylated peptides was performed essentially in the same manner, with the exception that the microtiterplates were precoated with 1 μg/mL streptavidin in PBS prior to the addition of the biotinylated peptides diluted to 1 μg/mL in PBS. After 1 h of incubation at 37°C, unbound peptides were washed away and diluted supernatants containing scFv-Fcε fusion proteins were added. Detection was performed as described above.m HEPES pH 7.4, 150 nm NaCl, 3 mm EDTA, 0.005% v/v surfactant P20). Blocking assays were performed by injection of the blocking scFv (200 μg/mL) for 300 s followed by injection of the secondary scFv (50 μg/mL) for 120 s. As a reference, HBS-EP running buffer was injected and followed by the secondary scFv as above.Recombinant Bet v 1.0101 was coupled (2200 RU) to a CM5 sensor chip  using a standard amine coupling protocol. Samples were diluted in HBS-EP running buffer . The genetic origin of sequences encoding the heavy and light chain variable (VL) domains, and mutational status of the transcripts was determined using the IMGT/V-QUEST web tool  m MES pH 6.5 and 25% (w/v) PEG 4000. The drops contained 200 nL of protein, at a concentration of 1.4 mg/mL, and 70 nL of reservoir, and were kept at 18°C. Crystals typically started to appear after a few days and were cryoprotected in 0.1 m HEPES pH 7.5, 25% (w/v) PEG 4000 and 18% (w/v) ethylene glycol before flash cooling in liquid nitrogen.Crystals were grown using the sitting drop vapour diffusion method in MRC 96-well plates. The reservoir comprised 50 μL of 0.1 xia2 package Coot4Ser)3 linker, VL domain, Flag-tag and His-tag, is numbered sequentially.Data were collected at beamline I02 at the Diamond Light Source . Data were integrated with XDS To enable characterization of molecular interactions between antibody fragments with origin in the human IgE repertoire and the major birch pollen Bet v 1, we performed phage display selections on the three Bet v 1 isoforms Bet v 1.0101, Bet v 1.0102 and Bet v 1.0112 from an antibody fragment library created from atopic donors. The library was created from transcripts encoding the VH domain of IgE isolated from nasal tissue of two allergic subjects known to have an IgE repertoire overrepresented by antibodies derived from the IGHV5 gene subgroup Genetic analysis of VH-encoding sequences of the four Bet v 1-specific antibody fragments reveals that they all originate in the IGHV5 germline subgroup Table, a subgrDifferent Bet v 1-isoforms have shown varying ability to initiate allergic responses As the allergic response is initiated by cross-linkage of high-affinity IgE receptors (FcεRI) on effector cells, such as mast cells or basophils, IgE targeting of at least two non-overlapping epitopes is required, unless the allergen is oligomeric. We therefore investigated, using Biacore blocking assays, whether or not the isolated clones fulfilled this criterion. Indeed, clones B14 and B13 (as scFv) were each shown to target two distinctly separated epitopes in this assay . Bearing in mind the sequence similarities between clones B14 and M0418, and between clones B10 and B13, it is likely that members of each pair target the same, or very similar, epitopes. It thus seems as if these four IgE-derived clones, all with origin in IGHV5, do recognize two independent epitopes on the allergen and consequently meet one criterion needed for initiation of an allergic cascade.To map the epitopes of the available scFv-CHε2-4 fusion proteins, their ability to bind to a set of peptides covering the amino acid sequence of Bet v 1.0101 was assessed by ELISA . Clone M0418 showed strong and B14 showed weak reactivity to a peptide covering residues I56 to D69, while B13 bound weakly to a peptide covering residues G26 to S39. B10 failed to bind any of the tested peptides, but, as mentioned above, should likely bind the same epitope as B13. These binding specificities were confirmed by testing the ability of the identified peptides in soluble form to inhibit binding to immobilized Bet v 1 . In agreement with this, M0418 did not recognize Mal d 1.0108 or Aln g 1.0101, as determined by ELISA , while that from molecule A was completely modelled. In molecule A, CDRH3 extends ˜8Å above the surface of the scFv  preserves the interaction with Tyr112.1 and Tyr113 on one side, although the side chain for Tyr108 is disordered, and the position of backbone atoms for residues 112.2 and 112.1 is shifted by ˜2Å. On the other side, CDRH3 is not flanked by a symmetry-related molecule, and residues 110-112.3 are disordered.Thus, in the absence of stabilizing contacts, CDRH3 is mobile. Two glycine residues immediately precede the CDRH3 loop, at positions 108 and 109, and presumably contribute to its flexibility.Despite the proven usefulness of human monoclonal antibodies in studies of biomedically important antibody responses, for instance in viral and bacterial infection as well as in autoimmunity, researchers within the field of allergic disease have only recently started to make use of the huge potential contained in human monoclonal IgE. We have in two recent studies deconvoluted the information content of two sets of human monoclonal IgE-derived scFv specific for the two major grass pollen allergens Phl p 1 Since the structure of Bet v 1 was solved almost two decades ago The identification of novel and more precise characterization of such biologically relevant epitopes made possible by usage of antibody fragments, derived from human IgE repertoires, provides useful information that could aid in rational design of new hypoallergenic versions of Bet v 1 to be used as safer alternatives to the allergen extracts currently used in specific immunotherapy. The epitopes identified by us and previously by Hecker and colleagues Although adjacent to one another on the surface of Bet v 1, the two identified epitopes could allow for simultaneous binding of two scFv targeting their respective epitopes , thereby fulfilling the basic criteria of FcεRI cross-linkage. The optimal separation distance for such FcεRI cross-linkage, as determined by degranulation assays, is suggested to be 44-51Å The presence of skewed IgE repertoires, with overrepresentation of VH-encoding transcripts with origin in only certain germline genes or gene subgroups, has been suggested in several previous studies in vivo through the random pairing of VH and VL. The fact that several similar scFv with quite different VL were isolated in this study argues in favour of the presence of specificity determining VH domains that have originally been selected in vivo.The four IgE-derived antibody fragments included in this study were, as all other allergen-specific human monoclonal IgE available to date, selected from combinatorial antibody libraries Access to human monoclonal IgE allows for extensive characterization of such antibodies at a much more detailed resolution than that afforded by studies of polyclonal serum IgE preparations. As human and mouse antibody responses, and human IgE and IgG responses have been shown to differ in many respects Even in the absence of allergen-IgE scFv structures, the availability of human IgE-derived antibody fragments allows for very detailed analyses of human IgE–allergen interactions at a level not easily obtained with polyclonal antibodies. In this study, we have defined two human IgE epitopes on Bet v 1 and we have been able to define their very different cross-reactive potential in terms of recognition of Bet v 1 isoforms and other proteins of the PR-10 family. Importantly, it has also been possible to connect genetic studies of IgE-repertoire composition and its involvement in responses against an important allergen family. We foresee that human monoclonal IgE as they become more readily available will continue to prove their extensive value in defining allergy-causing immune responses and in providing new solutions (e.g. new hypoallergenic variants as described in the past In summary, we have in this study isolated four novel Bet v 1-specific antibody fragment with origin in human IgE repertoires and defined two peptides, each containing the epitope of one of the two clonally related IgE pairs. We have identified certain allergen residues that are crucial for the interaction with one of these human IgE-derived antibody fragments, providing an explanation, at a molecular level, for the inability of this allergen-specific IgE fragment to recognize certain allergen isoforms and to cross-react with many members of the PR-10 protein family. We also show that IgE repertoires restricted in their genetic composition, in this case to the IGHV5-51 germline gene, a major member of a gene subgroup reported


Key questions remain regarding the processes governing gliogenesis following central nervous system injury that are critical to understanding both beneficial brain repair mechanisms and any long-term detrimental effects, including increased risk of seizures. We have used cortical injury produced by intracranial electrodes (ICEs) to study the time-course and localization of gliosis and gliogenesis in surgically resected human brain tissue. Seventeen cases with ICE injuries of 4–301 days age were selected. Double-labelled immunolabelling using a proliferative cell marker (MCM2), markers of fate-specific transcriptional factors , a microglial marker (IBA1) and glial markers  was quantified in three regions: zone 1 (immediate vicinity: 0–350 μm), zone 2 (350–700 μm) and zone 3 (remote ≥2000 μm) in relation to the ICE injury site. Microglial/macrophage cell densities peaked at 28–30 days post-injury (dpi) with a significant decline in proliferating microglia with dpi in all zones. Nestin-expressing cells (NECs) were concentrated in zones 1 and 2, showed the highest regenerative capacity (MCM2 and PAX6 co-expression) and were intimately associated with capillaries within the organizing injury cavity. There was a significant decline in nestin/MCM2 co-expressing cells with dpi in zones 1 and 2. Nestin-positive fibres remained in the chronic scar, and NECs with neuronal morphology were noted in older injuries. GFAP-expressing glia were more evenly distributed between zones, with no significant decline in density or proliferative capacity with dpi. Colocalization between nestin and GFAP in zone 1 glial cells decreased with increasing dpi. In conclusion, NECs at acute injury sites are a proliferative, transient cell population with capacity for maturation into astrocytes with possible neuronal differentiation observed in older injuries. This study conforms with the code of ethics of the World Medical Association (Declaration of Helsinki). All patients had undergone resective surgery at the National Hospital for Neurology and Neurosurgery for the treatment of drug-resistant epilepsy. The cases selected had additionally undergone previous invasive intracranial monitoring  at an interval prior to resective surgery and had a focal injury site [henceforth referred to as intracranial electrode (ICE) injuries] identified in the tissue sections. The clinical details of the cases, and the intervals in days from electrode/grid insertion, their removal and tissue resection, are presented in Table2/SC/0669Sections were cut from formalin-fixed paraffin-embedded tissue blocks at 5 μm and haematoxylin and eosin (H&E) staining was performed to confirm the location of the ICE lesion. For double-labelled immunohistochemistry, sections were dewaxed, immersed in 3% hydrogen peroxide and de-ionized water for 15 min, microwaved at 800 W for 15 min, cooled and washed in phosphate buffer solution. The first set of primary antibodies, the proliferative cell marker , progenitor cell markers  and paired box gene 6  were applied overnight at 4 °C, and Dako Envision DAB  was used as the chromogen to visualize nuclear labelling. Sections were double-labelled with monoclonal or polyclonal antibodies against glial markers , or the microglial marker , for 1 h at room temperature, and cytoplasmic immunolabelling was visualized with the Vector ImmPress VIP system . This generated seven sections per case: IBA1/MCM2, GFAP/MCM2, nestin/MCM2, GFAP/PAX6, GFAP/SOX2, nestin/PAX6 and nestin/SOX2.For triple-labelled immunofluorescence, sections were incubated overnight in solutions consisting of monoclonals to combinations of MCM2, PAX6, GFAP and immature and mature neuronal cell markers, namely beta-III-tubulin , neurofilament , Doublecortin , NeuN  and polyclonal anti-nestin primary antibodies. In negative controls, one or both primary antibodies were omitted. Species-specific fluorophore-conjugated antibodies  were applied for 3 h at room temperature. Sections were coverslipped using Vectashield DAPI mounting media (Vector Labs). Images were visualized and acquired using a Zeiss LSM700 confocal microscope .2 between cases . For zone 3, an area of 0.82 mm2 was sampled in each case [equivalent to 36 (×40 objective) high-power fields], which gave reproducible data on repeat counts. Mean cell densities per unit area for each marker, as well as the percentage of double-labelled cells in each zone for each marker were calculated. Cell counts in a proportion of cases were repeated to confirm reproducibility in the counting data.Immunolabelled sections were quantified using an image analysis program  and Zeiss Axioskop microscope. Regions of interest were drawn at ×2.5 magnification in each case, using the H&E section as a reference Fig.. Zone 1 image pro plus version 6.3  for the quantification of the degree of colocalization. The image analysis program uses the Pearson coefficient as a measure of the degree of colocalization, whereby 1 indicates perfect overlap of signal, −1 indicates perfect negative correction and 0 indicates no correlation at all  using triple-labelled immunofluorescence. The ICE injury sites on nestin and GFAP co-labelled sections were tiled at ×20 objective on the Zeiss Axio Imager Z2 motorized fluorescence microscope. Zone 1 was drawn as described above for immunohistochemistry, and analysed with spss for windows , and graphpad prism software (version 6.1) was used for the graphical representation of the data. Statistical methods used included non-linear regression analysis for the variation of cell densities with age of ICE injury , non-parametric tests for comparison of values between zones, and multivariate analysis to investigate the influence of the underlying pathology and location of ICE injury (grey matter vs. white matter), with corrected exact P values shown.Statistical analysis was carried out using P = 0.02). We also did not observe any significant difference in glial cell densities in the four patients with clinical complications of local wound or surface infections (treated with antibiotics) following ICE insertion, compared with those with an uncomplicated post–operative course (TableSeventeen cases were included in the study with lesions ranging in age from 4 to 301 days post ICE injury (dpi) (TableP < 0.0001) .IBA1 staining highlighted microglial activation and macrophage influx in the immediate vicinity of the ICE injury Fig.A–D. In a001) Fig.E. They w001) Fig.D, with aet al., P < 0.001; Fig.P < 0.0001) and in zone 1 than in zone 3 (P < 0.0001). A significant decline in proliferative capacity over time was also observed in zone 2 as well as in zone 3, remote from the ICE injury site  as well as in zone 2 (P = 0.001) and zone 3  over this time series of ICE injuries highlighted a population primarily associated with the immediate vicinity of the injury site. ‘Strap-like’ or bipolar elongated cells were present in the earliest lesions (from 4 dpi) around the injury site Fig.A. From 8P < 0.0001) in ICE injuries of all age, but there was no significant difference in densities between zones 1 and 2  and 2 (P = 0.024) . More than twice the fraction of NECs were also MCM2-expressing compared with GFAP cells in zone 1 in acute ICE lesions . There were no significant differences in the percentage of SOX2-positive glia (NECs or GFAP-positive cells) between zones  but not for nestin-SOX2  has recently been established  as a baseline level on which these acute injuries are superimposed. Also, some electrodes may be positioned in more electrically active zones than others, which could also influence relative levels of gliosis. In addition, in some patients the electrodes are surgically removed at an interval prior to the tissue resection; it is possible that this procedure could further irritate the injury site. There was no observable influence of post-operative complications/infections in our small series on glial cell densities. The ICE injuries are small, limiting extensive histological studies in single cases. The injuries are also more unpredictably located in either grey or white matter and in normal vs. pathological tissue compared with controlled experimental conditions. We did not, however, identify significant differences in astrocyte numbers between pathology types or white vs. grey matter injuries [as also noted in one experimental study (Wanner


In the spring of 2014, abundant sea ice that drifted out of the Gulf of St. Lawrence caused extensive disturbance in rocky intertidal habitats on the northern Atlantic coast of mainland Nova Scotia, Canada. To monitor recovery of intertidal communities, we surveyed two wave-exposed locations in the early summer of 2014. Barnacle recruitment and the abundance of predatory dogwhelks were low at one location  but more than 20 times higher at the other location (Whitehead). Satellite data indicated that the abundance of coastal phytoplankton (the main food source for barnacle larvae) was consistently higher at Whitehead just before the barnacle recruitment season, when barnacle larvae were in the water column. These observations suggest bottom-up forcing of intertidal communities. The underlying mechanisms and their intensity along the NW Atlantic coast could be investigated through studies done at local and regional scales. In particular, abundant sea ice forms every winter on the large Gulf of St. Lawrence (Canadian Ice Service). Between late winter and early spring, fragments of sea ice drift out of the gulf through the Cabot Strait (between Nova Scotia and Newfoundland) towards the open ocean. Such drift ice then travels south following the open Atlantic coast of Nova Scotia (Canadian Ice Service).The NW Atlantic coast displays cold-temperate intertidal environments. In Nova Scotia (Canada) in winter, ice does not form on the sea surface on the open Atlantic coast. However, sea ice readily forms in relatively enclosed water bodies such as gulfs Scotia , reachinCanadian Ice Service). In the early spring of 2014, large amounts of sea ice drifted out of the Gulf of St. Lawrence and, during the first half of April, reached up to 92 km of the northern open coast of mainland Nova Scotia. Just before the arrival of the ice, seaweeds and invertebrates were abundant in many rocky intertidal communities4, as drift ice had not reached that coast for the previous 3–4 years (Canadian Ice Service). However, after the ice scoured intertidal habitats for days , intertidal biomass losses were high. For example, in wave-exposed habitats where algal canopies and sessile invertebrates (barnacles and mussels) were abundant before the arrival of the ice, only bare rock was visible after ice scour4.The open Atlantic coast of mainland Nova Scotia  is reach4: Whitehead  and Tor Bay Provincial Park  in 8 quadrats (10 cm × 10 cm) that we had randomly established along 30-m transect lines at the mid-to-high intertidal zone in late April. Because of the intense ice scour in early April, macroscopic organisms were absent at this zone in late April, so the substrate was then fully available for barnacle recruitment .To evaluate recolonization patterns, in the summer of 2014 we surveyed two wave-exposed locations that had been heavily scoured by ice in early AprilSemibalanus balanoides is the only species of intertidal barnacle on this coast1. Every year, recruits ofS. balanoides accumulate in intertidal habitats on this coast during May and June8. Our measurements  at Whitehead  occurring on 23 June 2014 in 8 quadrats (10 cm × 10 cm) that had been randomly established along 30-m transect lines at the mid-to-high intertidal zone in late April (just before the beginning of barnacle settlement) at a wave-exposed site in Whitehead and in Tor Bay Provincial Park.Number of barnacle recruits . Nearshore chlorophyll-a concentration indicates coastal phytoplankton abundance, and phytoplankton is the main food source for barnacle nauplius larvae9. ForS. balanoides from the Atlantic coast of Nova Scotia, nauplius larvae occur in coastal waters for 5–6 weeks before metamorphosis to cyprids and then intertidal settlement10, which starts in early May on our studied coast8. Thus, it is possible that the higher food supply for larvae at Whitehead than at Tor Bay Provincial Park may have ultimately contributed to determining the higher barnacle recruitment at Whitehead. A positive relationship between nearshore phytoplankton abundance and intertidal barnacle recruitment was previously documented for NW Atlantic intertidal systems at a regional scale7.The greater density of barnacle recruits at Whitehead than at Tor Bay Provincial Park was related to a higher nearshore chlorophyll-Nucella lapillus;Nucella lapillus is the main predator of barnacles on the studied coast1, so presumably a higher barnacle recruitment could locally increase dogwhelk abundance. On 15 July 2014, at each of the two studied locations we measured during low tide the density ofN. lapillus in 30 quadrats (50 cm × 50 cm) randomly established at the mid-to-high intertidal zone  at Whitehead  than at Tor Bay Provincial Park . Barnacle recruitment in June 2014 may not fully explain dogwhelk density in July 2014, as dogwhelks had not undergone their 2014 recruitment season as yet (mainly in late summer11). However, visits to both studied locations in 2012 and 2013 revealed a similar difference in barnacle recruitment between both locations , supporting the notion that dogwhelk abundance may be driven by barnacles on this coast. Interestingly, in 2014, barnacle recruits and dogwhelks were more abundant at Whitehead than at Tor Bay Provincial Park by a similar ratio (27.6 times higher for barnacles and 20.8 times higher for dogwhelks), further suggesting a possible dependency of dogwhelk abundance on barnacle recruitment.To see whether barnacle recruitment could influence higher trophic levels, we measured the abundance of dogwhelks  found at low tide on 15 July 2014 in 30 quadrats (50 cm × 50 cm) established randomly along the same 30-m transect lines used to measure barnacle recruitment in June at the mid-to-high intertidal zone at a wave-exposed site in Whitehead and in Tor Bay Provincial Park.Number of dogwhelks , the differences in food supply between these sites are strong enough to propose further studies analysing the role of bottom-up forcing in this system. Accordingly, the conclusions of the study are sensible and justified on the basis of the results. As a follow-up approach, the authors may consider first, to expand the study to a larger set of sites in order to confirm the pattern, and second, to construct competing hypotheses (in addition to the “bottom-up hypothesis”) that would be tested by means of manipulative experiments replicated at those sites. This article is a necessary benchmark from which further hypothesis-driven research should be conductedI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This study is an interesting first approach to understanding the factors that affect the ability to recruit sessile benthic organisms after disturbances in an intertidal community. Petzold and Scrosati address the issue concerning the role of larval supply and its relation to phytoplankton abundance, which leads directly to considering that a bottom up mechanism may be acting in these communities. Furthermore, the idea that began the work is clearly stated and the theoretical context is appropriate, as well as the statistical treatment of the data. Although a more deep and permanent approach to the problem requires the carrying out of further studies, perhaps with the incorporation of field experiments, this initial statement is correct and I recommend its indexing.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Rhus chinensis (Mill.) gallnut (RCG)  extract as a potent (IC50 < 1 µg/mL) inhibitor of human LDH-A (hLDH-A). In this study, through bioactivity guided fractionation of the crude extract, the data demonstrate that penta-1,2,3,4,6-O-galloyl-β-D-glucose (PGG) was a primary constituent responsible for hLDH-A inhibition, present at ~9.95 ± 0.34% dry weight. Theoretical molecular docking studies of hLDH-A indicate that PGG acts through competitive binding at the NADH cofactor site, effects confirmed by functional enzyme studies where the IC50 = 27.32 nM was reversed with increasing concentration of NADH. Moreover, we confirm protein expression of hLDH-A in MDA-231 human breast carcinoma cells and show that PGG was toxic (LC50 = 94.18 µM), parallel to attenuated lactic acid production (IC50 = 97.81 µM). In a 72-hour cell proliferation assay, PGG was found to be a potent cytostatic agent with ability to halt cell division (IC50 = 1.2 µM) relative to paclitaxel (IC50 < 100 nM). In summary, these findings demonstrate that PGG is a potent hLDH-A inhibitor with significant capacity to halt proliferation of human breast cancer cells.A characteristic feature of aggressive malignancy is the overexpression of lactic acid dehydrogenase- (LDH-) A, concomitant to pericellular accumulation of lactate. In a recent high-throughput screening, we identified  Cancer is the second leading cause of death worldwide. While major advances have been made in design of chemotherapy agents, radiation, and surgical procedures, mortality rates remain high. Terminal cancer requires innovative therapeutic approaches, which are currently limited. Traditional use of herbs to treat diverse human illnesses dates back to thousands of years, but in today's society the use of natural product remedies is defined amongst others under the classification of “complementary and alternative medicines” (CAMs) –3. The fRhus chinensis (Mill.) belongs to the genus Rhus and the family Anacardiaceae. Rhus chinensis gall (RCG) is the abnormal growth of Rhus chinensis leaves caused by Chinese aphid Schlechtendalia chinensis  and every 2–5 days, the media were replaced and cells subcultured. For experiments, plating media consisted of DMEM, 1% FBS (24-hour toxicity studies), or 5% FBS (72-hour proliferation studies) + penicillin/streptomycin (100 U/0.1 mg/mL), 25 mM glucose, 2 mM sodium pyruvate, and 3 mM L-glutamine.MDA-MB-231 (ATCC HTB-26) human breast cancer cells were obtained from ATCC . MDA-MB-231 cells were brought up in ATCC-formulated Leibovitz's L-15 medium [Catalog number 30-2008], supplemented with 10% FBS and penicillin/streptomycin (100 U/0.1 mg/mL). After confluence, the cells were subcultured and grown in DMEM containing phenol red, 10% FBS, 4 mM L-glutamine, 20 Viable cell count was quantified using resazurin (Alamar Blue) indicator dye . A worki hLDH-A assay using recombinant full length human LDHA (amino acids 1–332) with N terminal His tag, 352 amino acids with tag, and MW 38.8 kDa, enzyme commission (EC) number 1.1.1.27 (BRENDA | IUBMB) , was used, where we previously confirmed the identity of the enzyme using matrix assisted laser desorption ionisation (MALDI) mass spectrometry and analysis by Mascot ID [ hLDH-A enzyme  and β-nicotinamide adenine dinucleotide, reduced form solution (β-NADH) , and a prereading at 340 nm was established to eliminate background. The reaction was started with a solution containing the substrate pyruvate  and a reading was taken intermittently over 60 minutes at 340 nm using a 96-well microplate reader .A continuousascot ID . Briefly hLDH-A in MDA-MB-231 cells was determined using recombinant hLDH-A as the standard. Cells were washed and centrifuged and the supernatant was discarded using ice-cold sterile PBS at 4°C. The pellet was resuspended and homogenized/sonicated in RIPA lysis buffer containing protease inhibitors. Samples were placed on ice for 30 min and centrifuged at 10,000 ×g for 10 minutes at 4°C. The supernatant was added at 1 : 1 of Laemmli sample buffer (Biorad number 161-0737) + fresh β-ME and boiled for 5 minutes. Approximately 50 μg of protein was loaded/lane and separated using 5%–15% SDS-PAGE gels, running buffer, 25 mM Tris, 192 mM glycine, and pH 8.3 (Biorad #161-0734) and applying 200 constant V constant ~35 min. The proteins were transferred to polyvinylidene fluoride membranes (100 V for 30–60 minutes) in ice-cold transfer buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol. The membranes were placed in a blocking buffer consisting of 5% bovine serum albumin fraction V (BSA) w/v in TBS + 0.05% Tween-20, pH 7.4. The membranes were washed and placed in 1° rabbit anti-human LDH-A antibody (1 : 500) containing 1% BSA in TTBS at RT for 2 Hr. The membranes were washed in TTBS and incubated in 2° goat anti-rabbit IgG (Fc specific) peroxidase conjugate (1 : 4000) in 2% nonfat dried milk in PBS for 1.5 Hr at RT. After a final wash, peroxidase was detected with Sigma FAST DAB  with a metal enhancer cobalt chloride. Images were scanned using an Epson Stylus CX-8400. Intensity analysis was performed using ImageJ software provided from the National Institutes of Health [The presence of2O2 using a base lactate reagent containing lactate oxidase (400 U/L) and horseradish peroxidase 2400 U/L . The reagent was added to a chromogen and samples were incubated for 10 minutes at 37°C. Lactate was quantified at 490 nm on a UV microplate spectrophotometer .Cellular production of lactic acid was determined in 96-well plates using a colorimetric enzymatic assay . Briefly, lactate was quantified by conversion to pyruvate and HThe X-ray crystal structure of human LDH (M form), predominantly found in human muscle, was downloaded from the RCSB protein data bank (PDB: 1I10). Chain A was extracted and selected for the docking studies. The chain A of the protein was then refined using the structure preparation tool of the biopolymer module offered by Sybyl-X 1.3 suite . In thisThe 3D conformer of the compound in present study, PGG structure, was download from the PubChem database and subjected to minimization following the gradient termination of the conjugate gradient method for 5000 iterations using Tripos force field incorporating Gasteiger-Huckel charges with the nonbonding cut-off set at 8.0 and dielectric constant set at 1.0. Thus optimized compound PGG was docked in the catalytic active site of the chain A of the LDH protein complex (PDB: 1I10) using high precision Surlex-Dock GeomX program as implemented in Sybyl software by incremental construction approach of building the structure in the active site so as to favor the binding affinity. In this process, initially, protomol was generated using the bound ligand 1,4-dihydronicotinamide adenine dinucleotide (NAI), allowing us to map the active site for docking the test ligand PGG. During the entire docking process, ring flexibility of the ligand was considered. Predock and postdock minimizations were performed and a self-scoring term was included for optimum results.50s determined by regression analysis using Origin Software . Significance of difference between multiple groups was assessed using a one-way ANOVA, followed by Bonferroni's multiple comparison test or Student's t-test.Statistical analysis was performed using Graph Pad Prism  with IC Rhus chinensis gallnut ethanol extract with inhibitory activity on human recombinant hLDH-A. As previously reported, this was one of the most potent natural products elucidated with an IC50 < 1 μg/mL [ hLDH-A inhibition, bioactivity guided fractionation ( hLDH-A activity was inhibited by the ethyl acetate fraction (167–215)  and 3 (0.15 g) showed similar activity and then were identified by 1H-NMR and 13C-NMR to be the same and then later combined. The molecule is identified as PGG  which is a solid and an off-white substance. The melting point was determined on a Mel-Temp II apparatus (Laboratory Devices) and found to be 337°C. MW was 940.67 Cith HPLC  where an hLDH-A, molecular docking studies were conducted and the data show predicted competitive binding of PGG  obtained were 5.86 and 12.61, respectively, for PGG and the bound ligand NIA, respectively. These effects were also confirmed by functional studies  lysate was evaluated for the baseline expression of hLDH-A by Western blot —a component in RCG—as a newly identified hLDH-A inhibitor with potential application as a CAM for use in cancer treatment. Our work supports/builds on the findings of others in describing therapeutic value of this molecule as an anticancer agent, now having been established in the suppression of prostate cancer metastasis [The data in this study confirm penta-1,2,3,4,6-tastasis , angiogetastasis –36, and tastasis , 38 at ttastasis , corresptastasis .At the same time, the RGC extract as a whole is equally capable of inhibiting angiogenesis, inducing apoptosis in cancer cells  and as a hLDH-A which is a therapeutic target in particular given its role in tumor initiation, progression, and metastasis [The data in this study also confirm the ability of PGG to inhibittastasis , 47 and tastasis , or breatastasis . Interes hLDH-A protein expression in tumor cells, and subsequent over production of lactic acid contributes to development of radiation and chemotherapy resistance [2) was described almost a century ago by Otto Warburg, having been termed “the Warburg effect.” While many scientists theorize that this aberration is a function of altered metabolism, studies in our laboratory exploring this phenomenon using current technologies such as whole-genomic, proteomic MALDI-TOF-MS and metabolite analysis show that the Warburg effect has a functional role not in metabolism, but in regulating acidic pericellular pH (pHe). For some unknown reason, cancer cells inherently thrive in slightly acidic pH, are extremely vulnerable to necrosis as the pH shifts toward alkaline, and will sustain a steady state pH through metabolic inversion or fluctuating dominance between glycolytic-rate substrate level phosphorylation (SLP) and mitochondrial (mt) oxidative phosphorylation to control lactic acid production [ hLDH-A would comprise a vehicle to maintain acidic pH where tumor cells thrive, but the question after much work still remains as to why tumor cells require ample concentrations of acid.It is believe that elevatedsistance , 28. Tumoduction . If thisβL , a component of mTOR , PI3K/Akt signaling, and its downstream eIF4E tumor promoting target [Acidity alone, can trigger aggressive forms of malignancy, augment metastases, and initiate chemoresistance all of which correlate to low survival rates , 52. Datg target . These cg target . hLDH-A remains elusive, it is known that hLDH-A knockdown, or lowering the functional capacity of hLDH-A, can lead to suppressed tumor growth and metastasis indicating this enzyme as a novel targeted cancer therapy strategy. In this study, we provide data to support therapeutic efficacy of PGG, a component of RGC extract, to inhibit human LDH-A and halt proliferation of a human tumor cell line MDA-MB-231, which highly expresses the hLDH-A protein.While the exact role of hLDH-A at the NADH cofactor docking site. Its effect in tumor cells baring high expression of hLDH-A resulted in blocking proliferation. Future research will be required to determine in detail cause-effect relationship between hLDH-A inhibition and biochemical metabolic or process related effects in tumor cells.Pentagalloylglucose, PGG, a component of RGC, shows a remarkable competitive inhibitory activity against


The house dust mite (HDM) allergen Der p 18 belongs to the glycoside hydrolase family 18 chitinases. The relevance of Der p 18 for house dust mite allergic patients has only been partly investigated.To perform a detailed characterization of Der p 18 on a molecular, structural and immunological level.E. coli, purified to homogeneity, tested for chitin-binding activity and its secondary structure was analyzed by circular dichroism. Der p 18-specific IgG antibodies were produced in rabbits to localize the allergen in mites using immunogold electron microscopy and to search for cross-reactive allergens in other allergen sources . IgE reactivity of rDer p 18 was tested with sera from clinically well characterized HDM-allergic patients (n = 98) and its allergenic activity was analyzed in basophil activation experiments.Der p 18 was expressed in D. farinae but not with proteins from the other tested allergen sources. The allergen was mainly localized in the peritrophic matrix of the HDM gut and to a lower extent in fecal pellets. Der p 18 reacted with IgE from 10% of mite allergic patients from Austria and showed allergenic activity when tested for basophil activation in Der p 18-sensitized patients.Recombinant Der p 18 was expressed and purified as a folded, biologically active protein. It shows weak chitin-binding activity and partial cross-reactivity with Der f 18 from Der p 18 is a rather genus-specific minor allergen with weak chitin-binding activity but exhibits allergenic activity and therefore should be included in diagnostic test panels for HDM allergy. Dermatophagoides pteronyssinus and D. farinae represent the most important allergen sources for allergic patients  at a given wavelength. The secondary structure estimation program CDSSTR (reference data set 7) on the DichroWeb server  and incubated with rabbit anti-rDer p 2 antibodies, anti-rDer p 18 antibodies or normal rabbit antibodies, diluted 1: 5 000 or 1: 10 000 in buffer B o/n at 4°C. After washing, the nitrocellulose membranes were incubated with 125I-labeled donkey anti-rabbit antibodies  and bound antibodies were detected by autoradiography  [For immunoblotting, equal amounts of the extracts were separated by 12.5% SDS-PAGE and blotted onto nitrocellulose membranes . The membranes were blocked two times for 5 min and once for 30 min in buffer B [40 mM NaHGermany) .D. pteronyssinus, D. farinae extracts, 20 μg of Der p 18, Der p 15, or, for control purposes, with 20 μg of BSA, o/n at 4°C. The preincubated serum samples were then exposed to nitrocellulose-bound D. pteronyssinus or D. farinae extract o/n at 4°C and bound IgG Abs were detected as described above.For inhibition experiments, rabbit anti-Der p 18 antibodies or normal rabbit antibodies (1: 50 000 dilution) were pre-incubated with 200 μg of Der p 18-specific IgG antibodies were purified from the serum of a Der p 18-immunized rabbit using a protein G column . IgG antibodies purified from the pre-immune serum were used as control. The purified Ig fractions were used to detect Der p 18 in the mite body and feces by immunogold electron microscopy .D. pteronyssinus extract  and D. pteronyssinus-specific IgE levels above 0.7 kU/L   . The freD. pteronyssinus-specific IgE suffered from HDM allergy. Sera from these HDM-allergic individuals (sera #92–100) and from a non-allergic individual were tested for IgE reactivitiy to rDer p 2, rDer p 18 and Der p 23 in a non-denaturing dot blot assay as described [In addition, sera and fresh blood samples were obtained from HDM-allergic adult patients (sera #92–100) for dot blot, western blot inhibitions and basophil activation testing, who according to a case history indicative for HDM allergy, positive SPT and escribed . For theescribed .D. pteronyssinus extract was incubated with sera from two mite allergic patients with IgE reactivity to Der p 18 which had been diluted 1:10 in buffer B and were pre-incubated with 10 μg/ml rDer p 18 or BSA o/n at 4°C. The pre-incubated serum samples were then exposed to the nitrocellulose-strips o/n at 4°C and bound IgE antibodies were detected with 125I-labeled anti-human IgE antibodies  and visualized by autoradiography (Kodak XOMAT film).For IgE inhibition experiments, nitrocellulose-blotted Residual serum samples #1–91 which had been obtained in the course of routine allergy diagnosis when patients were enrolled for a HDM immunotherapy study  (ClinicaD. farinae (88%) as also noted earlier [B. tropicalis (I. scapulis (tick), Der p 18 contains a C-terminal putative chitin-binding domain , which is often found in peritrophic matrix proteins of insects and animal chitinases [A comparison of the amino acid sequence of Der p 18 (AAY84563.1) with sequences deposited in the NCBI database using the Basic Local Alignment Search Tool (BLAST) indicates that Der p 18 belongs to the GH 18  chitinase-like superfamily and shows a high sequence homology with Der f 18 from  earlier . In addiopicalis , Table 1opicalis , Table 1opicalis . The mulopicalis . Regionsitinases ,35,36. DThe alignment of the predicted secondary structure elements of Der p 18 with the secondary structures found in the three-dimensional structures of the templates   revealed8-TIM barrel- of the proposed chitinase core domain and the C-terminal putative chitin-binding domain with ROSETTA DOCK [Attempts to find the most probable orientation upon energy minimization calculations between the (α/β)TTA DOCK  did not E. coli and purified from the inclusion body fraction. After solubilization, around 7 mg of protein/liter bacterial culture was purified by Ni-affinity chromatography. The purified and refolded protein migrated at a molecular weight of 51 kDa in SDS-PAGE   . Preadso extract , indicat extract . Consist extract , Table 1t 51 kDa . Our resin situ localization of Der p 18 in HDMs using immunogold electron microscopy. Next we investigated the We then studied the prevalence of IgE binding to 11 HDM allergens including rDer p 18 with sera from 91 clinically well characterized HDM-allergic patients by ImmunoCAP ISAC technology . The freIn the Der p 18 positive sera (n = 9), the levels of Der p 18-specific IgE antibodies ranged from 0.42 ISU to 13.96 ISU (median: 3.45 ISU), compared to a median IgE binding of 17.20 ISU for the major allergen, Der p 2 (range: 0.32–170.07 ISU). The IgE levels to Der p 18 were much higher compared to that of the other spotted high-molecular-weight allergens Der p 11, Der p 14 and Der p 15  . The freWhen sera from two Der p 18-positive patients .The allergenic activity of rDer p 18 was analysed using basophils from four HDM-allergic individuals, who had shown IgE reactivity to Der p 18  (data not shown). We have found that certain HDM allergens  which mainly or exclusively occur in mite bodies but not in feces are more often recognized by HDM-allergic patients with atopic dermatitis. In fact, the IgE recognition rates of Der p 18 were comparable in HDM-allergic patients with only respiratory symptoms and in patients with respiratory symptoms and atopic dermatitis.The assessment of the IgE binding frequency was performed with a representative number of sera obtained from clinically well characterized HDM-allergic patients who had shown respiratory symptoms attributable to HDM exposure by controlled 8H13O5N)n, is a long chain polymeric polysaccharide comprised of N-acetyl-β-D-glucosamine (GlcNAc) residues. It is present in fungal cell walls, the shells and radulae of mollusks and the exoskeleton of crustacean and insects [It has been reported that Der p 18 is a chitinase-like allergen . Chitin  insects . Chitina insects ,39. Chit insects ,41,42 an insects .8-TIM barrel fold structure in the catalytic region [D. farinae extract but inhibition experiments indicated some cross-reactivity between Der p 18 and Der f 18.In fact, several chitinase-like allergens have been described, in particular in the context of the latex-fruit syndrome ,45. Theyc region  and oftec region . We haveSo far the allergenic activity of Der p 18 has not been studied. We compared rDer p 18 with rDer p 2 for their potential to induce basophil activation in allergic patients and found that rDer p 18 induces basophil activation in an IgE-dependent manner, albeit to a lower extent than Der p 2. This finding together with the fact that we identified a HDM-allergic patient with respiratory and skin symptoms to HDM who was only sensitized to Der p 18 but not to other known HDM allergens, indicates that Der p 18 despite being a minor allergen in terms of IgE recognition frequency has allergenic activity and therefore should be included in diagnostic test panels for HDM allergy.S1 FigMarked in orange are the chitinase core domain including the conserved cysteines of the catalytic region (C1-C4) and the putative catalytic domains . The putative chitin-binding domain (CBD) is highlighted in blue and the region rich in serine, threonine and proline in yellow. Amino acids identical to those of Der p 18 are indicated by dots; dashes represent gaps.(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.


Atopic asthma occurs in a significant percentage of patients in different age groups and the main sensitizing allergens are dust mites. The aim of this study was to assess the levels of total serum IgE, specific IgE to Dermatophagoides pteronyssinus (Dp) and Blomia tropicalis (Bt) in patients with asthma as well as the influence of gender, age and severity of disease in the total serum IgE levels and in the sensitization to mites.We conducted a retrospective, cross-sectional study with patients with asthma who were over 6 years of age and had regular follow-up. Data were collected from the Laboratory of Immunology records and database. The total serum IgE and specific IgE measurements were performed by ImmunoCAP.Of the 79 patients analyzed, aging between 6 and 81 , 65% were female. IgE levels were elevated in 64% of patients, 54% had specific IgE sensitization to Dp and 52% to Bt. IgE levels were elevated in 73% of the children, 57% of the adolescents, 81% of the adults and 27% of the elderly. Statistical analysis was significant in the comparison between children and adults versus the elderly, p = 0.01 and p = 0.0001, respectively. The frequency of specific IgE sensitization was similar in both sexes and in the different age brackets (p> 0.05). The severity of asthma had no influence in the frequency of specific IgE sensitization to Dp and Bt (p> 0.05).We observed that the majority of asthma patients showed high IgE levels and half the patients had specific IgE sensitization to Dp and Bt. Our data indicate a lower frequency of high IgE levels in the elderly, pointing to a lower sensitivity of this method in the research of atopy in elderly asthmatics. There was no correlation between the levels of total serum IgE and specific IgE sensitization with asthma severity.


Bacterial spores can remain dormant for decades, yet harbor the exceptional capacity to rapidly resume metabolic activity and recommence life. Although germinants and their corresponding receptors have been known for more than 30 years, the molecular events underlying this remarkable cellular transition from dormancy to full metabolic activity are only partially defined.Bacillus subtilis as a model organism, we performed phosphoproteomic analysis to define the Ser/Thr/Tyr phosphoproteome of a reviving spore. The phosphoproteome was found to chiefly comprise newly identified phosphorylation sites located within proteins involved in basic biological functions, such as transcription, translation, carbon metabolism, and spore-specific determinants. Quantitative comparison of dormant and germinating spore phosphoproteomes revealed phosphorylation dynamics, indicating that phospho-modifications could modulate protein activity during this cellular transition. Furthermore, by mutating select phosphorylation sites located within proteins representative of key biological processes, we established a functional connection between phosphorylation and the progression of spore revival.Here, we examined whether protein phospho-modifications occur during germination, the first step of exiting dormancy, thereby facilitating spore revival. Utilizing Herein, we provide, for the first time, a phosphoproteomic view of a germinating bacterial spore. We further show that the spore phosphoproteome is dynamic and present evidence that phosphorylation events play an integral role in facilitating spore revival.The online version of this article (doi:10.1186/s12915-015-0184-7) contains supplementary material, which is available to authorized users. Bacillus species can carry out a complex developmental process called sporulation, resulting in the formation of a highly durable spore, the most resilient cell type known. Consequently, spore forming bacteria, including dangerous pathogens, such as Clostridium difficile (C. difficile) and Bacillus anthracis (B. anthracis), are highly resistant to antibacterial treatments and difficult to eradicate [In response to nutrient deprivation, bacteria of various radicate –4. Bacteradicate .The robustness of the spore is facilitated by its unique structure and chemical composition. The spore core, containing DNA, RNA, and protein components, exhibits distinctive physiological conditions such as low pH 6.0 ± 0.3) and reduced water content, alongside a high concentration of pyridine-2,6-dicarboxylic acid  , 6. The .0 ± 0.3 +, K+) and DPA, spore rehydration, cortex hydrolysis, and coat disassembly; we have recently shown that the completion of this process requires active translation [During dormancy, the spore remains responsive to its environment and commences revival upon sensing various stimuli, such as DPA, nutrients , or muropeptides , 14. Thenslation . During nslation . Previounslation , 17. Durnslation , 19. HerStreptomyces coelicolor [B. subtilis by activating the eukaryotic-like membrane serine/threonine kinase PrkC. In turn, PrkC phosphorylates the essential translational initiation component elongation factor G (EF-G). Yet, the consequences of this phosphorylation event and its effect on germination are not fully understood [Nutrient binding to germinant receptors leads to rapid recommencement of metabolism, transcription, and translation , 20–22; elicolor . Furtherderstood .Bacillus subtilis (B. subtilis) spore Ser/Thr/Tyr phosphoproteome, and provided evidence that widespread dynamic changes indeed take place upon induction of germination. The uncovered phosphoproteome of the germinating spore contained proteins belonging to central biological processes, including translation, transcription, carbon metabolism, and spore-specific constituents. Subsequently, by altering phosphorylation sites in key factors facilitating these processes, we revealed the crucial effect phosphorylation plays in driving the exit from dormancy.Here, we examined if and to which extent protein phosphorylation occurs at the onset of spore revival. By utilizing high resolution mass spectrometry (MS)-based phosphoproteomics, we defined the reviving B. subtilis spores were purified and induced to germinate in buffer supplemented solely with the defined germinants L-Ala and AGFK  [To explore whether spore revival involves phosphorylation events in spore proteins, we sought to characterize the phosphoproteome of a germinating spore. To this end, tassium) . We reastassium) , 26. TheB. subtilis cells  using a target-decoy strategy, as described previously , 28. Theis cells , 33, 34.B. subtilis proteins and quantified their dynamics during vegetative growth [To further investigate the difference between the vegetative and spore phosphoproteomes, we compared the spore germination results with our recent study, in which we detected 177 phosphorylation sites on 140 e growth . Of 140 Next, we assessed whether germination induces multiple changes in protein phosphorylation state. To explore this possibility, differences in phosphorylation levels between the dormant stage (t = 0) and the consecutive 10 and 30 minute time points were determined by label free quantitation (LFQ) utilizing the results from one of the three biological replicates, which was measured in technical triplicates and resulted in the most comprehensive data set (see P = 0.05) of ratios of LFQ intensities at the protein level . We cannot exclude the possibility that mutating phospho-site amino acids might affect protein function for reasons other than phosphorylation state. Therefore, the consequence of the generated phosphorylation mutants was compared to that of a corresponding null mutant when possible. Of note, additional proteins were initially examined but excluded from further analysis, as deletion and/or phospho-site-specific mutations of their encoding genes had no obvious effect on revival , was not affected, while ∆sspA spores exhibited an extended ripening period , which was longer than that of the ∆sspA ∆sspB mutant spores  and the N-terminus , we next investigated the consequences of mutating this position in WT and SspA null backgrounds. The resulting phenotypes were similar to those observed for SspA-S47A/D, namely altered UV sensitivity and extended ripening period . Within the ribosome, RpsJ is located near the aminoacyl-tRNA site of the 30S subunit and interacts with 16S rRNA . Our ana32D Fig. .Fig. 5Ph600 measurements showed a clear extension of the ripening period by approximately 80 minutes for the strain harboring the rpsJ-S32D allele  and elongation factor-TU , which catalyze the translocation of the tRNA and mRNA during polypeptide elongation. Amino acid substitutions at these sites did not affect spore production or spore revival kinetics  that is part of CCR regulation but is not involved in PTS activity [crh. As can be seen in Fig. crh deletion resulted in a more pronounced HPr mutant phenotype, with profound extension of the ripening period (700 minutes) for spores harboring HPr-S46A. Furthermore, no completion of revival was observed for spores harboring HPr-S46D at the course of 1000 minutes after revival induction. Exit from the ripening period was eventually detected after approximately 1400 minutes (data not shown). These observations emphasize the importance of CCR in the revival process. Consistent with these findings, the absence of the ATP-dependent kinase/phosphatase HprK, required for phosphorylation of both HPr and Crh [HPr-S46A ∆crh or ∆hprK mutants did not display any growth phenotypes  position investigated in this study is among the most conserved sites, showing dephosphorylation trends at the course of germination. Mutating this position resulted in profound phenotypes, such as high UV sensitivity of dormant and germinating spores and extension of the ripening period accompanied with prolonged persistence over the chromosome, all consistent with higher affinity to DNA [The phosphoproteome of the reviving  revival , 62. Uniy to DNA , 43. In y to DNA .During the transition into a growing cell, the spore has to rapidly reactivate core vegetative systems. Consistently, we have recently shown that translation initiates already during germination . Our phoCarbon utilization is among the systems that need to be activated most immediately upon revival. We established a central role in spore revival for the phosphorylation state of the master regulator of carbon metabolism, HPr. HPr-S46 phosphorylation affects cell metabolism at multiple levels exerting its effect through CCR, inducer exclusion, as well as phosphorylation of transcription regulators and several metabolic enzymes . We provB. subtilis life cycle. The findings that the phospho-site mutants designed and investigated here mainly affect the extent of the ripening period, suggest that this phase has unique spore-specific features. The identification of phosphorylation dynamics as critical for this developmental transition could serve as a platform to design phosphorylation inhibitors to combat spore forming pathogens.Some of the phenotypes associated with mutations in phosphorylation sites investigated in this study could be attributed to general changes in protein properties. Nevertheless, some of the observed phenotypes were exclusive to either exit from dormancy or vegetative growth, suggesting that the constructed mutations indeed recapitulated the phosphorylation state of the examined proteins. Furthermore, this observation reinforces the view that spore revival is a developmental process regulated by unique molecular factors alongside constituents common to other stages in the B. subtilis as a model organism to comprehensively study the Ser/Thr/Tyr phosphoproteome of a germinating bacterial spore. We present first evidence that widespread dynamic changes occur upon induction of germination. Furthermore, by mutating select phosphorylation sites within proteins playing part in key biological processes , a functional link between phosphorylation and the progression of spore revival was revealed.In this research, we utilized B. subtilis strains are listed in Additional file 2PO4, 9.5 mM (NH4)2SO4, 5 mM L-lactic acid, 8 mM L-glutamic acid, 1× MT mix, 20 mM D-glucose). Spore formation was assessed by heat resistant colony forming units. Cells were induced to form mature spores by incubating them in CDSM medium at 37 °C for 36 h. The spore culture was diluted and 100 μL of the desired dilutions were plated in duplicates on LB plates. The remaining dilutions were further incubated for 30 min at 65 °C, and the same diluted samples were plated again in duplicates on LB plates. All plates were incubated for 24 h at 37 °C and colony forming units (CFU) were determined. Sporulation efficiency was evaluated by comparing the number of CFU before and after heat treatment. Spores were purified using a three-step Histodenz (Sigma) gradient. Briefly, a 10-mL 36 h CDSM culture was washed in distilled water and resuspended in 1 mL of 20 % Histodenz solution for 30 min on ice. Spores were then placed on top of a two-step gradient made up from 2 mL 40 % Histodenz on top of 6 mL 50 % Histodenz. After centrifugation , the pellet containing >99 % free spores as evaluated by phase-contrast microscopy was washed three times with distilled water to remove residual Histodenz.Sporulation of various strains was induced by exhaustion in liquid CDSM medium  , AGFK  and L-ala (10 mM) [g for 30 min to remove cell wall remnants and other insoluble matter. The protein suspension was precipitated using acetone. The resulting protein extracts were resuspended in denaturation buffer (6 M urea/2 M thiourea in 10 mMTris). The amounts of protein present from all three time points were determined through utilizing the Bradford assay . Approximately 6 mg of protein extract from each time point were digested into peptides as previously described [18 stage tips [2 chromatography as previously described [2 beads with a diameter of 10 μm  were incubated with 2,5-dihydrobenzoic acid in 80 % acetonitrile at a concentration of 30 mg/mL. Approximately 5 mg of TiO2 beads were added to each sample and incubated for 20 min, while being slightly agitated (at RT). This procedure was repeated consecutively for a total of five times. For each round of TiO2 enrichment, beads underwent a washing step with 1 mL of 30 % acetonitrile/ 3 % trifluoroacetic acid and 80 % acetonitrile/0.1 % trifluoroacetic acid for a period of 10 min. Enriched phosphopeptides were eluted from the TiO2 beads using 100 μL of 40 % ammonium hydroxide solution in 60 % acetonitrile, pH 10.5. The five elutions from each phosphopeptide enrichment step were then pooled together in order to yield one sample per experimental condition . The sample volume was reduced in a vacuum centrifuge at RT to 6 μL followed by nano-LC-MS/MS analysis. Three phosphoproteome measurements were performed; one was purely qualitative, one involved 15 N labeling of bacterial culture [For phosphoproteomic analysis, purified spores were diluted to an OD (10 mM) , and gerescribed  with sliage tips , followeescribed  with add culture , and one6 charges, followed by fragmentation via collision induced dissociation of the 10 most intense precursor ions in the linear ion trap analyzer (LTQ) at a target value of 5× 103 charges. Dynamic exclusion of precursor ions was applied for 90 sec. Additionally, the lock-mass option was enabled for internal calibration [Nano-LC-MS/MS was performed on an EasyLCnano-HPLC  coupled to the LTQ Orbitrap XL mass spectrometer. Peptides were loaded onto a 15-cm nano-HPLC column  in-house packed with reverse-phase 3 μm C18 spheres . Peptides were loaded onto the column with 0.5 % acetic acid at a flow rate of 500 nL/min. A linear, segmented 130 min gradient consisting of 5–33 % of Solvent B (80 % acetonitrile in 0.5 % acetic acid) was applied at a constant flow rate of 200 nL/min. Separated peptides were ionized through electrospray ionization using the electrospray ion source (Proxeon Biosystems). The OrbitrapXL was operated in the data-dependent positive ion mode utilizing the following acquisition cycle: one survey scan in the Orbitrapmass analyzer was acquired at the range of m/z 300–2,000 Thompson (Th) at a resolution of 60,000 (defined at m/z = 400) with a target value of 1 × 10ibration . For MS B. subtilis 168 obtained from Uniprot (taxonomy ID 1423), containing a total of 4,195 B. subtilis protein entries and 262 contaminant proteins. Briefly, trypsin was fixed as the protease with a maximum allowance of two missed cleavages. Variable modifications were set for methionine oxidation, N-terminal acetylation, and phosphorylation on serine, threonine, and tyrosine residues. Cysteine carbamidomethylation was set as a fixed modification. A mass tolerance was set to 6 ppm and 0.5 Daltons for MS and MS/MS scans, respectively. A false discovery rate of 1 % was applied at the peptide, protein, and phosphorylated site level. Finally, the LFQ algorithm was set to infer quantitative information. Phosphorylation sites with a localization probability of ≥0.75 were considered to be localized. MS/MS spectra of phosphorylated peptides were filtered through manual validation in order to ensure high data quality. Phosphorylation site intensities were normalized with their respective protein intensities in order to determine if a bias was present based on the changing protein abundance. After this normalization step, phosphorylation sites were considered as significantly changing, based on the intensity-weighed statistical test known as “Significance B” (P = 0.05). Finally, protein GO annotation and clustering analysis was done using DAVID and Subtiwiki [In terms of both processing and analyzing the MS data, all acquired MS RAW files were processed together using the MaxQuant software suite version 1.2.2.9 . Databasubtiwiki , 74. The600 of 0.5 in S7 medium 2SO4, 20 mM L-glutamic acid, 1× MT mix, 2 % D-glucose) supplemented with 10 mM L-Ala [600 was followed by Wallac Victor 2 multiwell fluorometer (Perkin Elmer) at 37 °C set with constant shaking .For spore revival experiments, purified spores were heat activated , diluted to an ODmM L-Ala , and div600 of 0.05 in S7 medium supplemented with L-Ala (10 mM) divided into a 96-well plate at a final volume of 0.2 mL culture per well, and an OD600 was followed in a Wallac Victor 2 multiwell fluorimeter at 37 °C set with constant shaking .For growth curve analysis, overnight cultures were diluted to ODgfp gene, was subtracted from the net average fluorescence intensity. Samples were photographed using AxioObserver Z1 (Zeiss), equipped with CoolSnap HQII camera . System control and image processing were performed using MetaMorph 7.7 software (Molecular Devices).Light microscopy was carried out as described previously . Briefly600 of 0.5 and different samples were exposed to increasing doses of UV (254 nm) irradiation using UVC 500 Ultraviolet Crosslinker (Amersham). The irradiated spore suspensions were serially diluted and plated on LB. After an overnight incubation at 37 °C, CFU/mL culture was determined. The survival rate was calculated as percentage of the CFU/mL obtained without UV exposure.Purified spores were diluted to an OD600 of 0.5) were incubated for 15 min at 65 °C. Then, spores were germinated at 37 °C in medium containing L-ala (10 mM) and MOPS . At various times, samples were, irradiated with UV (254 nm) using UVC 500 Ultraviolet Crosslinker . The irradiated spores were serially diluted and plated on LB. After an overnight incubation at 37 °C CFU/mL culture was determined. The survival rate was calculated percentage of the CFU/mL obtained at t = 0 minutes prior to UV exposure.Purified spores . For Immunoblot analysis of HA tag fusion proteins, membranes were blocked for 1 h at room temperature . Blots were then incubated for 1 h at room temperature with polyclonal rabbit anti-HA antibodies . Next, membranes were incubated for 1 h at RT with peroxidase conjugated goat anti-rabbit secondary antibody . EZ-ECL kit  was used for final detection. Band intensities were quantified by comparing the total intensity of identical-sized regions using MetaMorph 7.7 software (Molecular Devices) on the immunoblot image.Additional Methods including strain construction, are described in  and are freely available for download and analysis under dataset PXD002559.MS/MS raw data files have been uploaded to the ProteomeXchange Consortium via the PRIDE partner repository (


NUT (NUTM1) to the bromodomain protein family member 4 (BRD4) and thereby creating a fusion oncogene that disrupts cellular differentiation and drives the disease. In this study, we report the case of an adolescent NMC patient presenting with severe facial pain, proptosis and visual impairment due to a mass arising from the ethmoid sinus that invaded the right orbit and frontal lobe. Treatment involved radical resection, including exenteration of the affected eye with the view to consolidate treatment with radiation therapy; however, the patient experienced rapid tumor progression and passed away 79 days post resection. Molecular analysis of the tumor tissue identified a novel in-frame BRD4-NUT transcript, with BRD4 exon 15 fused to the last 124 nucleotides of NUT exon 2 (BRD4-NUT ex15:ex2Δnt1–585). The partial deletion of NUT exon 2 was attributed to a mid-exonic genomic breakpoint and the subsequent activation of a cryptic splice site further downstream within the exon. Inhibition of the canonical 3′ acceptor splice site of NUT intron 1 in cell lines expressing the most common NMC fusion transcripts  induced alternative splicing from the same cryptic splice site as identified in the patient. Detection of low levels of an in-frame BRD4-NUT ex11:ex2Δnt1–585 transcript in PER-403 confirmed endogenous splicing from this alternative exon 2 splice site. Although further studies are necessary to assess the clinical relevance of the increasing number of variant fusions described in NMC, the findings presented in this case identify alternative splicing as a mechanism that contributes to this pathogenic complexity.NUT midline carcinoma (NMC) is a fatal cancer that arises in various tissues along the upper midline of the body. The defining molecular feature of NMC is a chromosomal translocation that joins (in the majority of cases) the nuclear testis gene  Additionally, the cribiform plate was destroyed and there was involvement in the right frontal lobe . There wNUT gene , confirmNUT gene . Access BRD4-NUT fusion  of NUT exon 2 fused to BRD4 exon 15  8–332) while the previously described p300-binding domain (aa 346–593) remains intact.13 The fusion protein should thus retain its ability to sequester the histone acetyltransferase p300 - a central activity of the fusion protein that results in foci of chromatin hyperacetylation and impaired transcription of genes driving differentiation.14 Indeed, immunohistochemistry of the tumor demonstrated the punctate nuclear staining of the fusion protein that is characteristic for NMC  rearrangement consistent with the presence of a T fusion . RNA anaPER-624; . SubsequNUT exon 2 within the fusion transcript indicated the potential activation of a cryptic splice site within this exon. In silico analysis of NUT exon 2 and 100 bp of the flanking introns using the online tool ESEfinder 3.0  predicted 11 potential splice sites, including the canonical NUT intron 1 acceptor splice site, and an internal cryptic splice site that corresponds with the position of the fusion transcript breakpoint in the described NMC case  would result in premature truncation of the transcript.The partial deletion of NMC case . To testeakpoint . Hence, tic site . Even thNUT exon 2 splicing in NMC, we generated antisense oligomers (AOs) to block predicted splice sites .17 In both cases, only AO no. 1, targeting the canonical NUT intron 1/exon 2 acceptor site, induced alternative splicing , thus confirming the activation of the same cryptic 3′ acceptor splice site observed in the index case. Although different BRD4 exons are involved, the resulting fusions in both cell lines remain in frame. We did not observe any changes in phenotype in either cell line correlated with this splicing switch, in terms of growth rate or differentiation status (Ki67 or cytokeratin staining), consistent with the clinical observation that the NUT ex2Δnt1–585 fusion variant remains highly oncogenic (i.e. the patient succumbed to disease 98 days after diagnosis). Furthermore, low endogenous levels of BRD4-NUT ex11:ex2Δnt1–585 fusion transcript were detected in PER-403 , to angiogenic (VEGF-A), hyperenergetic , immune modulative  and prometastatic .18 The expression of multiple fusion isoforms within NMC could therefore have important implications for tumor phenotype as well as the design of targeted therapies.To further examine the role of ce sites  and exonce sites . We analce sites  in two csplicing . Sanger BRD4 exon 11 to the start of NUT exon 2. Only recently, two additional BRD4-NUT isoforms have been described (BRD4-NUT ex14:ex2 and ex15:ex2), which could potentially indicate the existence of clinically relevant NMC subtypes.19 In this study of an adolescent NMC patient presenting with an undifferentiated sinonasal tumor, we identify a fourth BRD4-NUT fusion variant (BRD4-NUT ex15:ex2Δnt1–585). This is the first described case with a partial deletion of NUT exon 2, which disrupts a proline-rich protein domain but without ameliorating oncogenicity. NMC is an extremely aggressive disease that is refractory to conventional treatments, yet significant preclinical and clinical responses have been reported for BET inhibitors and histone deacetylase inhibitors, making them promising candidates for NMC therapy.21 As a result of these findings, phase I clinical trials have been opened that investigate the efficacy of different BET inhibitors  and a dual phosphoinositide 3 kinase/histone deacetylase inhibitor (CUDC-907) in NMC and other advanced cancers. It will be some time before the results of these trials are known; however, there is evidence that molecular factors such as the dependency of the tumor on MYC signaling24 are likely to affect the efficacy of BET inhibitors in different cancer settings. Indeed, we have previously reported that the cytotoxicity of the BET inhibitor JQ1 may differ substantially between NMC subtypes,25 although it should be noted that observation was limited to a small number of cell lines and did not examine the effects of this drug class on cellular differentiation. The relationship between the efficacy of BET inhibitors and the genetic features of NMC , its molecular features , or its cell of origin, has not yet been described, hence there is a continued need for a better understanding of the cellular processes altered by NUT fusion proteins to develop optimal treatment strategies for NMC patients.For more than 20 years, the predominant oncogenic variant in NMC was thought to involve the fusion of