This simple strategy holds great potentials to generate highly robust and stable SERS substrates for real applications.Silver nanostructures have been considered as promising substrates for surface-enhanced Raman scattering (SERS) with extremely high sensitivity. The applications, however, are hindered by the facts that their morphology can be easily destroyed due to the low melting points (~100 °C) and their surfaces are readily oxidized/sulfured in air, thus losing the SERS activity. It was found that wrapping Ag nanorods with an ultrathin (~1.5 nm) but dense and amorphous Al Surface-enhanced Raman scattering (SERS) has emerged into an important analytical technique in biological, clinical, chemical and environmental sensing for its superior capability to nondestructively provide plenteous structural information of analytes at extremely low amounts1234568101213Ag nanostructures especially one-dimensional (1D) structures such as nanorods, nanowires, etc., have long been considered as the most promising candidate for SERS substrates due to their high plasmonic efficiency and EM enhancement effect, as well as the lower cost versus Au1516181920212324252628292O3 films could maintain their SERS activity for a period of 34 days2 thin films by glancing angle deposition (GLAD) technique could sustain their morphology and stabilize the SERS performance at 100 °C 2933It has been reported that coating Ag nanostructures with protective metal oxide layers was able to suppress their surface reactions with air232528312O3 films that fully wrapping Ag nanorods at a temperature of 50 °C, and investigated the coating influences on the morphological stability of Ag nanorods at elevated temperatures in air, as well as their SERS sensitivity and activity at ambient conditions. It was found that an ultrathin (~1.5 nm) but dense and amorphous Al2O3 layer was thick enough to make Ag nanorods robust in morphology to a temperature of 400 °C, and passivate sufficiently their surfaces to stabilize the SERS activity in air, without largely decreasing the SERS sensitivity.In this study, we employed atomic layer deposition (ALD) technique to prepare ultrathin Al−6 M methylene blue (MB) as the probe molecule, while the nanorods heated at 50 °C were of a similar SERS sensitivity as the as-deposited ones. We thus selected 50 °C to prepare Al2O3 coatings by ALD approach.352O3 thin films of various thickness were deposited on Ag nanorods at 50 °C by changing the ALD cycle numbers to 1, 2, 3 and 5, respectively. 2O3 layers by 1, 2, 3 and 5 ALD cycles. It is seen that the coating layers are amorphous in structure and of different thickness (from <1 nm to ~4 nm), fully wrapping the nanorods. A linear relationship between the thickness of Al2O3 layers and ALD cycle numbers was observed and plotted in 33372O3 shells were analyzed by XPS. 2O3395/2 and Ag 3d3/2 doublet peaks of this coated sample and the uncoated one are both centered at ~367.8 and ~373.8 eV, respectively, see 4130Al2O3 layers on the morphological stability of Ag nanorods was investigated by thermally annealing these samples in air at temperatures of 200, 300 and 400 °C for 30 minutes. It was found that the uncoated Ag nanorods melted completely at these temperatures, evolving from irregular aggregations to spherical particles as the annealing temperature rising, see 2O3 layer was <1 nm thick) were robust in morphology at 200 °C (not shown), but melted partly at 300 and 400 °C. For the nanorods coated by 2 ALD cycles (the Al2O3 layer was ~1.5 nm thick), however, no obvious morphological change was observed after being heated at 300 and 400 °C, in comparison with the as-deposited ones. Similar results were also obtained for Ag nanorods coated by 3 and 5 ALD cycles, see 43442O3 layer of <1 nm was too thin to protect the morphology of Ag nanorods at temperatures >200 °C.The influence of Al−6 M MB as the probing molecule. 2O3 layer by 2 ALD cycles was effective to protect Ag nanorods in both morphology stiffness and SERS sensitivity up to a temperature of 400 °C in air.The SERS performance of the above Ag nanorods before/after annealing was evaluated using 5 × 102O3 coating layers on the SERS activity of Ag nanorods at room temperature in air was also investigated as a function of time. As the starting point, the Raman spectra of 5 × 10−6 M MB on as-prepared, uncoated Ag nanorods, and on as-prepared, coated Ag nanorods by 1, 2, 3 and 5 ALD cycles were measured and compared as references, see 2O3 coating layers2O3 by 2 ALD cycles remained almost constant. For a clear comparison, we normalized the intensity of the peak of MB at 1622 cm−1 obtained on various SERS substrates to that obtained on the as-prepared, uncoated Ag nanorods, and plotted in 2O3 coating. It is also noticed that after coating with an Al2O3 layer by 2 ALD cycles (~1.5 nm thick), the SERS sensitivity of Ag nanorods was still ~50% that of the as-prepared, uncoated ones. For nanorods coated with a thinner layer (<1 nm thick) by only 1 ALD cycle, their SERS sensitivity was a little higher at the beginning, but decreased ~18% after 50 days in air, i.e., their SERS stability was not as good as that of Ag nanorods coated by 2 ALD cycles or more. These suggest that a ~1.5 nm Al2O3 layer be optimal to combine both excellent SERS sensitivity and outstanding SERS stability of Ag nanorods in air.The effect of Al2O3 coating layer deposited by 1 ALD cycle (<1 nm thick) was not capable to effectively protect Ag nanorods is intriguing. It is known that pyridine has a small size and a relatively large Raman cross-section and can be adsorbed on the surface of Ag nanostructures intensively. Therefore, it can be used as a mark to check out whether the Al2O3 coating layers were compact enough to protect Ag nanorods. If there are any pinholes in the Al2O3 layer, pyridine could penetrate through pinholes and be adsorbed on the surface of Ag nanorods, thus exhibiting Raman siganls4647−2 M pyridine solution, and measured the Raman spectra. 2O3 layer of <1 nm thick (by 1 ALD cycle deposition) was too thin to avoid pinholes, and was not dense enough to protect effectively Ag nanorods, and confirm again that an Al2O3 layer of ~1.5 nm thick was an optimal choice by this approach.The reason why an Al2O3 layers, and found that an Al2O3 layer of ~1.5 nm thick was effective to preserve the morphology of Ag nanorods to a temperature of 400 °C, and stabilize their SERS sensitivity in air for at least 50 days. The present study provides a simple way to fabricate Ag nanostructures with both excellent SERS sensitivity and outstanding SERS stability in air, which could be used in real SERS-based sensors.In conclusion, we developed a low-temperature ALD approach to completely cover Ag nanorods with thin but dense and amorphous Al–6 Pa. During deposition, the incident angle of the vapor flux was set at ~86° off the surface normal of substrates, with a deposition rate of 0.75 nm/s. The deposition stopped at a thickness of 500 nm read by a quartz crystal microbalance (QCM). The detailed deposition procedure can be found elsewhere495051Slanted Ag nanorods were prepared on Si (001) substrates by oblique angle deposition (OAD) technique in an electron-beam system  with a background vacuum level on the order of 102O3 layers were coated on as-prepared Ag nanorod in an ALD reactor  at 50 °C. The Al2O3 precursors, i.e., trimethylaluminum  and water (maintained at 40 °C), were alternatively pumped into the reaction chamber using high purity N2  as the carrier and purge gas. Typically, one complete reaction cycle took ~30 s, and consisted of four steps: (1) TMA reactant exposure time, 20 ms; (2) N2 gas purging time, 10 s; (3) water vapor exposure time, 10 ms; and (4) N2 gas purging time, 20 s. The thickness of alumina was controlled by varying the number of reaction cycles.Al2O3-coated Ag nanorods were annealed in a quartz tube furnace at 200, 300 and 400 °C for 30 minutes in air.The as-prepared, uncoated and Al2O3 layers were characterized by scanning electron microscope , high-resolution transmission electron microscope  and X-ray photoelectron spectroscopy  with Mg Kα as the excitation source, respectively. The reflectance spectra were measured by a R1 angle-resolved spectroscopy system (Idea Optics Co.) in the 200–1000 nm range, using a mirror-like Al film as the reference.The morphology, structure and chemical states of Ag nanorods and the Al−6 M MB or 1 × 10−2 M pyridine aqueous solutions for 30 minutes, and dried naturally in air. The Raman spectra were obtained using a 785 nm laser as the excitation source, with its beam spot focused to ~80 μm in diameter and using an excitation power of 120 mW. The data collection time of one spectrum was set to 10 s. For every sample, the spectrum was obtained by averaging the spectra obtained from five different areas of the SERS substrate.The SERS measurements were conducted with an optical fiber micro-Raman system , using methylene blue (MB) and pyridine as the probing molecules. Before SERS measurements, the substrates  were merged into 5 × 10How to cite this article: Ma, L. et al. Silver Nanorods Wrapped with Ultrathin Al2O3 Layers Exhibiting Excellent SERS Sensitivity and Outstanding SERS Stability. Sci. Rep.5, 12890; doi: 10.1038/srep12890 (2015).


We report here the assembly of the functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme capable of DNA synthesis consists of α, β and DnaX (τ and γ), δ and δ′ components of the clamp-loader complex. The proteins were each cloned and expressed in a native form. Each component of the system was purified extensively. The minimum holoenzyme from these five purified subunits reassembled is sufficient for rapid and processive DNA synthesis. In an isolated form the α polymerase was found to be unstable at temperatures above 65°C. We were able to increase the thermostability of the pol III HE to 98°C by addition and optimization of various buffers and cosolvents. In the optimized buffer system we show that a replicative polymerase apparatus, Tth pol III HE, is capable of rapid amplification of regions of DNA up to 15,000 base pairs in PCR reactions.DNA replication in bacteria is accomplished by a multicomponent replicase, the DNA polymerase III holoenzyme (pol III HE). The three essential components of the pol III HE are the  Thermus thermophilus (Tth) is an extreme thermophile which can grow at temperatures above 75°C dTTP (20 cpm/pmol). Enzyme concentrations were the same as used in the thermostability optimization. The reaction was allowed to proceed through 20 cycles composed of 94°C/30 s, 60°C/1 min, and 70°C/2 min. Individual reactions were stopped after every other cycle to the end of the 20 cycles. The assays were carried out in duplicate, one for gel analysis and one for quantification of DNA synthesis. For gel analysis each 25 μL reaction was loaded into a well of a 0.7% agarose gel (15 × 15 cm) and electrophoresis was for 45–90 min at 100 V using a subcell GT apparatus (BioRad). Gels were stained for 5 min in TAE buffer containing 5 μg/mL ethidium bromide. The gels were destained for 15 min in TAE buffer and DNA bands visualized using a Kodak Image Station 440.Assays (25 μL) using pET Blue-2 plasmid (Novagen) as a template contained the optimized buffer system plus 200 μM each dNTP, 2 μM each primer, and 0.05 μg plasmid. Enzyme concentrations were the same as used in the thermostability optimization. PCR cycles consisted of 94°C/30 s, 55°C/1 min, and 72°C/2 min. Following cycling, the samples were incubated at 72°C for an additional 5 min. PCR reactions were analyzed using 0.7% agarose gel electrophoresis as described above. Reactions using Taq DNA polymerase  for comparison were carried out as per the manufacturer's conditions. The Tth holoenzyme and Taq polymerase reactions contained the same amount of primer and template in the same reaction volume. PCR reactions (50 μL) using Lambda DNA-HindIII Digest (New England BioLabs) as a template contained double the amount of enzyme.PCR reactions  under control of the pTAC promoter . To translationally couple the genes encoding the Tth proteins to the gene encoding the tRNA NT, most of the tRNA NT gene was removed, leaving only 13 codons of the 5′ end of the gene. A sequence containing an RBS followed by a TAA stop codon was added in the same reading frame as the gene encoding the tRNA NT. In similar constructs, genes encoding Tth α, DnaX, δ, δ′, and β were added in such a way that the start codon of each gene overlapped the second A residue of the stop codon. This coupled the Tth gene to the sequence encoding the tRNA NT 5′ by virtue of the new RBS, but in a different reading frame. All of the Tth genes were overexpressed in this system to 5–20% of total cellular protein.We have previously expressed all of the proteins in NHed forms . Howeverpromoter . In this Tth pol III HE were expressed and purified as described in “τ and γ proteins revealed minor bands migrating between the two subunits. However, western blot analysis using monoclonal antibodies against the DnaX protein indicated that the minor bands between τ and γ were degradation products of τ (data not shown). The γ form of DnaX also appeared to resolve as a doublet. Previous work indicated that even though the τ and γ forms of DnaX are encoded by the same gene, in Tth the mRNA encoding γ is distinct from that encoding τ, as a result of transcriptional slippage [γ were present. The smaller form of γ results from a −1 slippage bringing a UGA stop codon located two codons downstream into the same reading frame as the dnaX gene. The larger form of γ is a result of a −2 slippage which brings a UGA stop codon located sixteen codons downstream into the same reading frame as the dnaX gene. The dnaX gene in Tth also contains a translational frame-shift signature [γ from a single mRNA encoding both τ and γ by a translation frame-shifting mechanism. Which mechanism is occurring during overexpression of Tth DnaX in E. coli cells is not clear.Native forms of the subunits comprising theslippage . This woignature  which al2-terminal His-tagged forms of α, β, τ/γ, δ, and δ′, we previously showed that each subunit was required for processive DNA synthesis [ Tth pol III HE purified here was titrated into identical assays to determine optimal activity for downstream assays . This is likely due to the fact that δ acts to load and unload the β subunit onto the DNA [δ (greater than the DnaX complex), the β subunit is preferentially unloaded, thereby causing a decrease in the processivity of the holoenzyme. It is possible that the NH2-terminal His-tag may interfere with the optimal ability of δ to unload β.Using NHynthesis . Each ofm assays . Each suecreases . When th the DNA , 33 and α subunit has proven to be an Achilles heel for the Tth pol III HE to be active at increased temperatures. As an initial purification step, fractions containing α lost activity when heated to 65°C. To ascertain the heat stability, purified α was heated to 90°C for 2 min and 100% loss of activity was observed . The primers  improved the yield of active proteins and addition of metals was shown to stabilize the structure of misfolded proteins [ Tth pol III HE. By iteratively testing additives and combinations of additives, we were able to increase the stability of the Tth pol III HE so that full activity was retained to 98°C for at least five PCR-like cycles and for 30-cycle PCR reactions in which 94°C was used as a heat step. To determine if the activity could be retained for the denaturation steps required for PCR, the Tth pol III HE was compared with Taq polymerase initially using primer sets that yielded short PCR products. The length of the region of DNA to be amplified was increased incrementally to determine how well Tth pol III HE performed compared to Taq. In these reactions the Tth pol III HE PCR products were comparable to that of Taq polymerase but contained fewer abortive product fragments.Previous work has identified conditions that would allow increased stabilization of individual enzymes from both mesophilic and thermophilic origins. Certain cosolvents have been shown to enhance the thermostability of xylanase frompora sp.  and Thertoga sp. , alpha-ctoga sp. , and paptoga sp. . The statoga sp. , 44. Deteratures . Betaineeratures , 47 or tproteins , 49. The Tth pol III HE is able to synthesize the entire Tth genome in one binding event, we were encouraged to test whether it would be capable of long-range PCR (greater than 5000 bp). PCR reactions using λ DNA as a template were designed to determine if Tth pol III HE could produce long-range PCR products, and we showed that amplification of DNA regions up to 15,000 bp was possible. Taq polymerase produced no products in comparable reactions. The observation that Taq polymerase could not amplify DNA regions above 5000 bp highlights the importance of speed of polymerization for amplification of long regions of DNA. Taq polymerase in the presence of a secondary polymerase is able to amplify much longer regions of DNA (up to 35 kbp). To accomplish this, extension times have to be extended to as much as 26 min [Since PCR was first developed, the production or longer PCR product has been an ongoing goal and continues today –53. Sincs 26 min , 26. Thi Taq polymerase, a lack of a high level of fidelity seen with replicative polymerases [ Taq polymerase with no added proofreading function misincorporates nucleotides at a rate of 0.01 to 0.27 × 10−6 [ E. coli, pol III HE, in vitro, is approximately 700 times higher than that of Taq polymerase [ E. coli system, base selection by the polymerase accounts for up to 10,000-fold higher fidelity than does the proofreading function of epsilon and the addition of epsilon only decreases the error rate by 3- to 5-fold [This is the first time that a replicative multicomponent polymerase has been shown to have the ability to perform PCR. The advent of a single-enzyme system that is capable of amplifying long regions of DNA in a short period of time opens up exciting possibilities. Aside from the ability to do longer-range PCR the other major limitations of enzymes in use today remain to be, even though increased from that ofymerases . Taq pol7 × 10−6 , 55, whi7 × 10−6 , 56. Howlymerase . The curo 5-fold , 58. The


Diffusion characteristics of cobalt atoms were investigated using radioactive isotope method in phase-hardened Fe-18wt.%Mn-2wt.%Si alloy. The observed significant increase of diffusion coefficient of cobalt atoms under the cyclic γ-ε-γ martensitic transformations was due to the action of two independent mechanisms - an athermal one and a thermally activated one. The first one arose from the direct γ-ε and the reverse ε-γ transformations with corresponding direct and reverse lattice shears during alternating stresses and simultaneous lattice restructuring. Another mechanism arose under the diffusion annealing of the phase-hardened alloy. Phase transformations are the ones that most significantly affect the diffusion properties of interstitials and substitution atoms in metastable alloys . The extAs a result of γ-ε-γ transformations in Fe-Mn alloys, the dislocation density increases in the reverted austenitic phase by an order of magnitude . Such diOur investigations were carried out in Fe-18wt.%Mn-2wt.%Si alloy with high completeness of γ-ε transformation (more than 90% of martensitic ε-phase). This has enabled us to reach a high scale phase hardening by cyclic γ-ε-γ transformations.3 + 45%KNO3) at the temperature of 380°C. The temperature points of the investigated alloy were: Ms = 80°C, Mf = −110°C, As = 220°C, Af = 380°C.The direct, γ-ε transformation in the alloy, occurred as the result of cooling in liquid nitrogen, and the reverse, ε-γ one, took place during consequent heating in a salt bath γ and (002)ε ratio :1\documeThe use of the specified reflexes from austenite and ε-martensite areas, which were parallel in accordance with orientation correlations between FCC and HCP lattices, has enabled us to calculate the amount of ε-phase without a reduction in the measurement precision that could arise in case of a texture appearance under the thermocycling. The amount of martensitic phase was measured with the accuracy of 0.5%.60Co was deposited on one of the austenitic alloy surfaces. The thickness of the isotope was less than 0.5 microns and β-activity was (5 × 103 ± 50) pulses/min. The concentration distribution of cobalt in depth after the cyclic martensitic γ-ε-γ transformations and diffusion annealing at temperatures 250°C and 325°C was determined by removing the layers of the sample of the alloy and measuring the integral radioactivity of the rest of the sample. The thickness of each layer was determined by weighing on microanalytical scales and calculating with the formula:h0 and P0 are respectively the thickness and the weight of the initial sample, Δh and ΔP are the thickness and the weight of the removed layer.The diffusion characteristics were determined by a radioactive method. A layer with radioactive isotope tg(α) was determined from the graph:t is the diffusion annealing time, x is the penetration depth of the isotope into the sample, I is the intensity of the isotope at a certain depth. The measurement accuracy of the diffusion coefficient was 20%.To calculate the diffusion coefficient, we used the following formula (from Fick’s first law) :3\documeThe analysis of the distribution of cobalt atoms in depth has showed that, as a result of cyclic γ-ε-γ transformations without additional diffusion annealing, they penetrate into the alloy volume to certain depths. After the first cycle of transformations, the penetration depth of cobalt atoms was already 3.5 microns Figure , curve 1We could not calculate the diffusion coefficient of cobalt using diffusion profiles because we had no way to determine the time length of the cobalt diffusion penetration into the bulk of the alloy, which is obviously comparable with the time of martensitic transformation. It should be considered that the mechanism of transfer of cobalt atoms is connected with actual structural f.c.c. - h.c.p. - f.c.c. restructuring and related resulting stress field due to changes in the size and volume of crystals that turn. The increase of penetration depth under repeated cycles indicated the possibility of accumulation of internal stresses that consistently stimulated further diffusion of cobalt atoms. Obviously, this mechanism of moving substitution atoms can be considered mainly athermal. Previously, it was shown that an athermal mechanism of substitution and interstitial atom mass transfer was realized by explosive kinetics of γ-α transformation in iron alloys ,9.Under γ-ε-γ transformations, in local areas of alloy, internal tensile and compressive stresses cyclically occurred, in accordance with the character of the direct γ-ε (increasing of specific volume) and reverse ε-γ (decreasing of specific volume) transformations. Stretched regions occurred as a result of the direct γ-ε transformation at low temperatures, when the relaxation processes were inhibited and internal stresses did persist. This created the conditions for the transport of cobalt atoms into such areas. Obviously, the reverse ε-γ transformation created more weak conditions for the migration of substitution atoms under a thermal mechanism.In order to detect the thermally activated diffusion part in phase-hardened Fe-18wt.%Mn-2wt.%Si alloy, we subsequently conducted an investigation of isothermal annealing influence on the parameters of the diffusion of cobalt. After γ-ε-γ transformations, we additionally annealed the phase-hardened specimens for 2500 hours at 250°C and 325°C, thus realizing the diffusion annealing cycle for further stimulation of the diffusion of cobalt atoms. The annealing time at maximum temperature under cycling was rather small compared to the additional time of the diffusion annealing. To avoid relaxation processes in the reverted austenite, we prevented overheating above the temperature at the final point of inverse transformation. After thermal cycling and additional isothermal annealing, the amount of ε-phase in the samples was over 90%. This indicated that the diffusion parameters of the phase-hardened alloy related mainly to ε-phase.Diffusion annealing of the phase-hardened alloy led to the penetration of cobalt atoms to macroscopic distances. Thus, for example, after the first and the hundredth cycles the penetration depth was respectively 3.5 and 12 microns, whereas after following annealing at 250°C the penetration depth increased up to 80 and 133 microns. Increasing of annealing temperature to 325°C caused an additional penetration of tracer atoms into the alloy , the penetration depth of atoms of the isotope increased twice and the diffusion coefficients after the first cycle and 100 cycles were equal to 2.95 × 10The crystal structure defects formation in metastable alloys under the cyclic martensitic γ-ε-γ transformations and the resulting significant increase of the diffusion coefficient of substitution atoms at low temperatures opens up new opportunities for creating more intensive methods of chemical and thermal treatment. Due to the preliminary phase hardening, the temperature of the surface metallization of metastable iron-manganese alloys can be reduced by several hundred degrees.


Cell-surface Fcγ receptors mediate innate and adaptive immune responses. Human Fcγ receptor I (hFcγRI) binds IgGs with high affinity and is the only Fcγ receptor that can effectively capture monomeric IgGs. However, the molecular basis of hFcγRI's interaction with Fc has not been determined, limiting our understanding of this major immune receptor. Here we report the crystal structure of a complex between hFcγRI and human Fc, at 1.80 Å resolution, revealing an unique hydrophobic pocket at the surface of hFcγRI perfectly suited for residue Leu235 of Fc, which explains the high affinity of this complex. Structural, kinetic and thermodynamic data demonstrate that the binding mechanism is governed by a combination of non-covalent interactions, bridging water molecules and the dynamic features of Fc. In addition, the hinge region of hFcγRI-bound Fc adopts a straight conformation, potentially orienting the Fab moiety. These findings will stimulate the development of novel therapeutic strategies involving hFcγRI. FcγRs are cell-surface receptors for IgGs that play key roles in the humoral and cellular immune response to infection. Here, the authors present a high-resolution crystal structure of the hFcγRI-Fc complex to reveal the molecular mechanisms underlying the high specificity of this important immunological interaction. Fcγ receptors are cell-surface receptors for immunoglobulin G (IgG) that play pivotal roles in humoral and cellular protection against infection125Three different classes of human Fcγ receptors have been identified: hFcγRI (CD64), hFcγRII  and hFcγRIII . Each receptor exhibits distinctive tissue distribution, structure and binding specificity towards various IgG subclasses7891011The hFcγRI is a major immune receptor expressed on the surface of macrophages, monocytes, neutrophils, eosinophils and dendritic cells1317181920212223KD ∼10−9−10−10 M). The specific exclusion of IgG2 is thought to be related to sequence determinants within the lower hinge of the Fc moiety . The combination of structural, kinetic and thermodynamic data reveals details of the interaction between hFcγRI and Fc with unprecedented detail. We observe unexpected differences in the conformation of the lower hinge region of Fc bound to hFcγRI with respect to that bound to other Fcγ receptors. Based on our observations, we propose that the Fab region of IgG1 may adopt a distinct orientation at the cell surface, in which domain D3 of FcγRI acts as a spacer to accommodate the Fab moieties. Overall, these results shed light on an important immunological process and should enable the development of novel therapies in the fight against autoimmune diseases in which hFcγRI is involved.We determined the crystal structure of an optimized version of hFcγRI in complex with the Fc fragment of a humanized IgG1 (rituximab) at a resolution of 1.80 Å. The data collection and refinement statistics are summarized in 2 of contact interface with high shape complementarity with Fc (Sc=0.86)2). In subsite 2, the relative contribution of the lower hinge of Fc to the interaction with the receptor is modest in comparison with that in subsite 1. However, despite its smaller size, subsite 2 engages in a similar number of water-mediated and protein–protein H-bonds to that in subsite 1 . Since the rigid body displacement of D3 takes place away form the binding interface, it is unclear if this movement influences the interaction with Fc or it reflects the intrinsic mobility of the receptor. We note that the different environment experienced by the receptor in different crystal lattices may influence the relative position of D3 with respect to D1–D2. No other significant change in the secondary or tertiary structure of hFcγRI is observed, demonstrating that the mutations introduced to increase its stability , whereas the CH2 domains are much more flexible  with respect to the unbound state (RMSDunbound=2.5±1.5 Å). We note that the conformation of the CH3 domain remains constant when bound to receptors (RMSD=0.3±0.1 Å).The analysis of the crystal structure of Fc bound to four different Fcγ receptors revealed that the fluctuations of the CH2 domain are greatly reduced when bound to receptors  accounts for 25% of the interaction surface of chain A of Fc, and suggest a critical role of this residue for binding. Previous mutational studies have suggested the importance of Leu235 for the high affinity of hFcγRI2543Detailed examination of the binding interface of Fc-hFcγRI reveals two distinct features not observed in the structure of Fc with other human Fcγ receptors. First, residue Leu235 of the lower hinge of Fc chain A is buried in a deep hydrophobic pocket of the receptor. This pocket is lined by residues Trp104, Lys130, Trp127, Val132, Lys173 and Tyr176 . The burSecond, because of the high resolution and good quality of the electron density maps of our crystal structure, we identified eight well-defined water molecules bridging Fc with hFcγRI . Half ofThe affinities of Fcγ receptors for the different IgG subclasses vary by nearly 1,000-fold despite their similarity at the sequence and structural levels302) with respect to other receptors  explaining its high affinity for IgG1.Comparison of the key residues contributing to the interaction between Fc and Fcγ receptors reveals a greater number of interactions in subsite 1 of hFcγRI than in any other receptor, consistent with the important role of subsite 1 for binding . Indeed,eceptors . As a reeceptors . OverallALK to hFcγRI were evaluated by surface plasmon resonance (SPR). No significant differences were found among the three constructs assayed  and the dissociation rate constant (koff) were 2.7 × 104 M−1 s−1 and 1.1 × 10−4 s−1, respectively, resulting in a dissociation constant (KD) of 4.2 nM. These parameters are typical for high-affinity interactions. For IgG, the values of kon, koff and KD were 4.1 × 104 M−1 s−1, 1.2 × 10−4 s−1 and 2.9 nM, respectively. The similarity between these values and those found for Fc demonstrates that the influence of the two Fab moieties of IgG1 is essentially negligible.SPR is uniquely suited to determine a broad range of useful kinetic and thermodynamic parameters, as well as to examine the rate-limiting step (transition state) of the binding reaction. Kinetic parameters for the binding of Fc (or IgG1) to a surface decorated with hFcγRI were determined from the dose-response sensorgrams . For Fc,kon and KD yields useful information about the two fundamental thermodynamic states of the binding reaction coordinate: the activation (transition) state and the equilibrium state. The thermodynamic parameters of the transition state were determined from the temperature dependence of kon , activation enthalpy  and activation entropy  demonstrate the key influence of breaking non-covalent interactions at the transition state, rather than a dynamic reorganization of solvent and/or protein. Similar observations were made when examining the binding of IgG1 .The temperature dependence of e of kon 47. For FKD , a process driven by the formation of numerous non-covalent interactions , and opposed by the loss of entropy . The values obtained with IgG1 led to the same conclusions, although we note that the magnitude of the entropy/enthalpy compensation was even greater for the full-length antibody . Overall, the binding of Fc to hFcγRI displayed a greater favourable change of enthalpy than to any other receptor reported in the literatureThe equilibrium parameters (van't Hoff) were obtained from the temperature dependence of KD . The valΔH‡=+11.2 kcal mol−1) without a net change in the dynamics of the system . Moreover, the electron density of the lower hinge (residues before Pro238) cannot be modelled, indicating it is disordered in the crystal structure and probably in solution. On the contrary, the B-factors of Fc in the complex with hFcγRI are relatively uniform throughout the entire sequence. These two observations are consistent with the dynamic model inferred from the thermodynamic data. We note that no significant differences were found between the B-factor profiles of bound/unbound hFcγRI , but also crystal structures determined in two other different space groups (P6122 and C2). Although crystal-packing forces are likely to play a role in the conformation adopted by the Fc chains, these observations suggest that the Fc portion of IgG may exist as a dynamic (flexible) and asymmetric ensemble in solution, as recently reported by other groupsInterestingly, a similar pattern of flexible and asymmetric ensembles is observed in other crystal structures of Fc in the unbound form, except that of entry code 1LX6  governed by the insertion of Leu235 in a unique hydrophobic pocket of hFcγR1. In subsite 2, however, the interaction surface is relatively flat and does not involve local conformational changes. Given the similarities between the contact interface of Fc engaging subsite 2 of hFcγRI to that engaging subsite 2 in other Fcγ receptors, we propose this region is the common recognition platform for all receptors. In contrast, the residue Leu235 of Fc that interacts with subsite 1 represents the high-affinity element specific for hFcγRI.These ideas are represented schematically in Importantly, the crystal structure of the immune complex with hFcγRI reveals a unique orientation of the lower-hinge residues of Fc compared with that in other Fcγ receptors . In contIn summary, we determined the recognition mechanism between human IgG1 and the major cell-surface receptor hFcγRI from structural, kinetic and thermodynamic standpoints. This is an important step for the understanding of a key effector of the immune system that may guide therapeutic efforts to combat autoimmune diseases.Escherichia coli cells to generate a baculovirus shuttle vector (bacmid), which was subsequently transfected into Sf9 insect cells  using Lipofectamine 2000 reagent (Invitrogen). After 4 days, the primary baculovirus (P1) was used to infect fresh Sf9 insect cells . Two days later, the amplified (high-titre) baculovirus (P2) was collected from the infected Sf9 cells and used for protein expression.The protein sequence of WT and optimized receptor is shown in 6 cells per ml at 27 °C were infected by recombinant viruses (P2) at a concentration of 4% (v/v). After 4 days incubation, the cells were harvested, centrifuged at 5,800g and the supernatant applied onto a complete His-Tag purification resin (Roche) equilibrated with buffer A . Protein was eluted by increasing of concentration of imidazole stepwise  in buffer A. The fractions containing hFcγRI were pooled and dialysed against buffer B . The dialysed sample was subjected to cation exchange chromatography in an AKTA system . The protein was eluted in a gradient of NaCl between 0.1 and 1.5 M.Sf9 mimic cells incubated in Sf-900 III SF medium (Gibco) supplemented with 10% (v/v) fetal bovine serum at a density of 1.8 × 10The humanized IgG1 monoclonal antibody Rituximab was purchased from Chugai Pharma. The Fc fragment of the IgG1 monoclonal antibody was obtained by papain digestion (Pierce). Digested Fc was purified with a Protein A kit (Pierce) following the instructions of the manufacturer. The fractions containing Fc were pooled, and subjected to size exclusion chromatography using a HiLoad 26/60 Superdex 200-pg column  equilibrated with 40 mM Tris-HCl (pH 7.4) and 300 mM NaCl.−1. The initial crystallization screening was carried out using an Oryx8 protein crystallization robot (Douglas Instruments). Single crystals were obtained in a solution of 0.1 M sodium acetate, 0.1 M zinc acetate, 4% (v:v) 1,4-butanediol and 12% PEG 4,000 (pH 4.6).Purified Fc was mixed with hFcγRI (molar ratio was 1:1), followed by dialysis overnight against 0.1 M NaCl, 20 mM Tris-HCl (pH 7.4). The Fc–hFcγRI complex was subjected to size exclusion chromatography using a HiLoad 26/60 Superdex 200-pg column  equilibrated with 20 mM Tris-HCl, 100 mM NaCl (pH 7.4) to separate the unbound protein. The fractions containing the complex were pooled and concentrated to 5.0 mg ml−1 in 10 mM Tris-HCl (pH 7.4) were screened using commercial kits (Hampton Research). After a brief optimization, the best single crystals were prepared in a solution composed of 0.2 M calcium acetate, 0.1 M sodium cacodylate (pH 6.1) and 19% (w:v) PEG 8,000 at 20 °C.Suitable crystallization conditions for Fc at 2.5 mg mlSuitable crystals of the Fc–FcγRI complex or Fc were harvested, immersed in crystallization solution supplemented with 20% glycerol and frozen by submersion in liquid nitrogen. Data collection was carried out at beamline BL5A at the Photon Factory  at a wavelength of 1.000 Å and a temperature of 100 K. Diffraction images were processed with the programme MOSFLMALK, antibody at 3.5 μM was first incubated with 10 mM dithiothreitol at 37 °C for 1 h, followed by treatment with 50 mM iodoacetamide at 37 °C for 30 min. The reagents were eliminated by dialysis with PBS supplemented with 0.005% Tween-20. Alkylation was corroborated by non-reducing SDS–polyacrylamide gel electrophoresis.To prepare alkylated IgG1−1 for 1 min to load the sensor chip with Ni2+ ions. Subsequently, hFcγRI displaying a hexa-histidine at the C-terminus was immobilized on the surface chip at a density of ∼550 RU (when examined with Fc) or 220 RU (when examined with IgG). Sensorgrams corresponding to the binding of Fc or IgG1 to hFcγRI were obtained by injecting increasing concentrations of analytes at a flow rate of 30 μl min−1. Contact and dissociation time were 250 and 600 s, respectively. Regeneration was carried after completion of each sensorgram by injecting a solution of 0.35 M EDTA.The interaction between hFcγRI and Fc or IgG was analysed by SPR in a Biacore T200 instrument . Two methods were used. In the first one, proteins were dialysed against running buffer (PBS supplemented with 0.005% Tween-20). A NTA sensor chip  was used for immobilization of hFcγRI. Before each run, the NTA sensor chip was treated with 500 μM nickel chloride dissolved in running buffer at 10 μl minkon) and dissociation (koff) rate constants were calculated by a global fitting analysis assuming a Langmuir binding model and a stoichiometry of (1:1). The dissociation constant (KD) was determined from the ratio of the rate constants.Data analysis was performed with the BIAevaluation software . Association , using a second procedure. In this case, research grade CM5 sensor chip  was activated by a short treatment with N-hydroxysuccinimide and N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride as described elsewhereALK) to FcγRI were obtained by injecting increasing concentrations into the sensor chip at a flow rate of 30 μl min−1. The measurements were carried out in the same running buffer. Contact time and dissociation time were 200 and 600 s, respectively. Data analysis was performed as described in method 1.Kinetic parameters of the binding between hFcγRI and Fc, IgG or IgGΔHvH) and entropy (ΔSvH) were calculated from the slope and intercept, respectively, of the temperature dependence of the dissociation constant using the van't Hoff approximationChanges in enthalpy . Each protein was concentrated to 10 μM in PBS buffer. Data collection was performed in a VP-DSC instrument  at a scanning rate of 1.0 °C mind 110 °C .E. coli BL21 (DE3). Protein expression was induced by addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside. The cells were harvested and lysed by the sonication method. The supernatant was collected and purified using immobilized metal affinity chromatography.The affinity of IgG1 for WT and optimized hFcγRI was examined by isothermal titration calorimetry (ITC) in an VP-ITC instrument , equilibrium-binding constant (KA) and binding stoichiometry (n) were determined with ORIGIN7.Receptor and IgG1 were dialysed in PBS. The solution containing IgG1 was placed in the injection syringe at 5–8 μM. The cell contained hFcγRI (WT or mutein) at 50 μM. The volume of each injection was 10 μl except the first injection was set at 3 μl. The binding enthalpy .Supplementary Figures 1-10, Supplementary Tables 1-11, Supplementary Note 1 and Supplementary References


Globigerina bulloides collected from tow samples along a transect from the equatorial Indian ocean to the Southern Ocean (45°E and 80°E and 10°N to 53°S) were analysed and compared with the equilibrium δ18O and δ13C values of calcite calculated using the temperature and isotopic composition of the water column. The results agree within ~0.25‰ for the region between 10°N and 40°S and 75–200 m water depth which is considered to be the habitat of Globigerina bulloides. Further south (from 40°S to 55°S), however, the measured δ18O and δ13C values are higher than the expected values by ~2‰ and ~1‰ respectively. These enrichments can be attributed to either a ‘vital effect’ or a higher calcification rate. An interesting pattern of increase in the δ13C(DIC) value of the surface water with latitude is observed between 35°S and~ 60°S, with a peak at~ 42°S. This can be caused by increased organic matter production and associated removal. A simple model accounting for the increase in the δ13C(DIC) values is proposed which fits well with the observed chlorophyll abundance as a function of latitude.Oxygen and carbon isotope ratios in planktonic foraminifera  Globigerina bulloides as a paleoclimate proxy was demonstrated by comparison of results of foraminiferal shell samples recovered by net from the water column and that from sediment core top samples. This comparison assumes that core top samples represents a time after the last glacial period when the oceanic environment remained relatively warm and stable and did not suffer any major changesG. bulloides is sensitive to oceanographic change from the results of its increased abundance due to changes in the monsoonal activity during the HoloceneG. bulloides proxy has been calibrated using modern sea-floor samplesIsotopic compositions of planktonic foraminifera are commonly used in the reconstruction of past oceanographic conditions but their reliability as environmental index depends on assumption of equilibrium condition at the time of growth and absence of subsequent alteration. Investigations on tow samples123567G. bulloides species are at a temperature of 13.4 °C and a salinity of 34.8PSU14G. bulloides inhabits mainly the mixed layer214161819G. bulloides occurs over a large range of depth from 20 to 200 m in spring. Furthermore, there are recent suggestions that its calcification continues to depths even greater than 200 m17. G. bulloides secretes its shell by adding new chambers intermittently. Each new chamber has a thicker wall and a larger volume than the preceding one2216The planktonic foraminifera species are mostly confined to the photic zone but the exact depth habitat inside that zone depends on their temperature and salinity preferences. Expected isotopic composition of a species can only be calculated if we know their depth habitat. Based on observations of the tow and core top samples the optimum growth preferences of 18O and 13C into the foraminiferal test of G. bulloides. We also compare results of tow samples with the published data of core top samples collected earlier in the same region. In addition, the observed latitudinal distribution of δ13C(DIC) in surface water of the Southern Ocean obtained by our earlier studyIn this work, we aim to study the effect of changes in the water mass properties  with depth and latitudinal position on the incorporation of Based on the presence of different water masses in the Southern Ocean, characterized by temperature and salinity changes with latitude, the region has been divided in to several zones described as follows. The Tropical Indian Ocean (TIO) is characterized by a confluence of diverse water masses, each with distinct physical characteristics, nutrient conditions, and production rates extending from 20°N to 20°S. The Subtropical zone (STZ)2728et al.,(2015)et al., (2004)The frontal structure over the Indian Sector of the Southern Ocean which marks the transition between water masses has its own characteristic temperature and salinity as seen from an analysis of CTD and XCTD data. The fronts described in the present study are (1) Subtropical front (2) Sub-Antarctic front and (3) Polar front. The Subtropical front (STF) extends from 40 to 42°S30The water and tow samples were collected from various locations across these zones and analysed for isotopes as described below.ORV Sagar Kanya from the TIO and Southern Ocean region during her 199th and 200th cruises respectively have been reported earlier13C and δ18O values of contemporary living specimens of foraminifera collected by towing from the same region. These data are shown in oS), the δ13C values display wide scatter, from −2.0 to −0.5‰, up to about 40 oS. Further south, the values are mostly high and lie within a narrow range of −0.3 to +0.7‰. In contrast, the δ18O values increase systematically from the tropical zone to the Antarctic zone from a low value of −2.0 to a high value +3.0‰.Isotopic analyses of foraminifera specimens obtained from sediment samples collected on board 18O values of surface sea water is shown in oS. There is a sharp drop to −0.6 ± 0.2‰ even as one moves by a few degrees south.The latitudinal distribution of δ13C(DIC) values for the surface water samples taken from Prasanna et al., (2015)3) obtained from satellite measurement  across this latitudinal belt. There is large inter-annual variability of mean δ13C(DIC) for the years 2012 and 2013. The mean value for the TIO and STZ (20°N to 35°S) is 0.81 ± 0.14‰. An overall enrichment of 0.29‰ was noted in TZ with mean value of 1.21 ± 0.16‰. SAFZ and PFZ show a large enrichment of ~1‰ with mean values of 1.54 ± 0.08‰ and 1.51 ± 0.02‰ respectively. Further south, AAZ showed a small extent of depletion compared to the peak zones with a mean value of 1.34 ± 0.1‰  are calculated by plugging the δ13C values of surface water and temperature for that depth. For depths 50–1000 m, the δ13C(DIC) values and temperature of the water column were adapted from WOCEI9N and WOCEI8S datahttp://www-pord.ucsd.edu/whp_atlas/indian_index.html). Using these inputs, depth contours of δ13C(DIC) values are generated and shown in Expected δ13C(DIC) values in the surface waters as a function of latitude (2 = 0.64). This is quite interesting since the two parameters refer to averaging over widely different time periods. The δ13C(DIC) is probably the steady state value of the local sea water representing many years (residence time) whereas the chlorophyll-a concentration refers to a snapshot picture obtained by the passing satellite. The enrichment of 13C/12C ratio with latitude, in particular, agrees well with the increase in photosynthetic activity. Similarly, the decreases at higher latitudes beyond 55 oS are found to be similar. We think that the correlated change in δ13C(DIC) and chlorophyll-a concentration with latitude can be explained by a simple mechanism. The Southern Ocean (SO) is characterised by high productivity and in places the phytoplankton density can yield as much as 1 gC/m2/day. Over about 100 day season, that yields 100 gC/m2/year (http://public.wsu.edu/~dybdahl/lec10.html). It is well known that production of organic matter preferentially uses the lighter carbon (12C) and leaves behind the heavier (13C) in sea water. For example, available data of ocean organic matter around 50 oS indicate a δ13C(POM) value of~ −25‰41oS) would imply corresponding increment in the δ13C(DIC) values by the logic of mass balance. Thereafter, as the production decreases further south, the δ13C(DIC) values also drop but not as much as the northern flank of the peak zone. This qualitative expectation is modelled below using a simple isotopic mass balance formulation. The near-Gaussian type increase and decrease of chlorophyll concentration is indicative of phytoplankton production which in turn is related to supply of nutrients and availability of light for photosynthesis. The macro-nutrients are abundant in the SO due to yearly upwelling caused by formation and melting of sea ice. When ice forms the salt is rejected which increases the salinity of the sea water around the sea ice, causing it to become denser than surrounding water. The cold and salty brine sinks and is replaced by deeper water which is warmer and less dense and has high concentration of nutrients. This causes the ocean to overturn. This process of brine rejection causes the exchange between surface and deep water that keeps the surface of the SO supplied with nutrients. The nutrient supply is also controlled to a large extent by ocean currents (Antarctic Bottom Water circulation). The sea ice formation is higher as one moves further south and that induces more productivity (due to more brine rejection) and may partially explain the higher δ13C(DIC) values in the southern flank of the Gaussian type variation (δlatitude  show gooariation . The sup13C(DIC) values for the productive regions in the Southern Ocean (13C(DIC) values in a given location based on the production rate of organic carbon(Po), total organic carbon [OM] and the removal rate of organic carbon assumed to be equal to -λ*[OM]. In the surface ocean, phytoplankton takes up inorganic carbon (DIC) to produce organic carbon via photosynthesis. As mentioned before, this process discriminates against the heavy isotope; as a result, surface DIC becomes enriched in13C44o) and the value of λ a stationary state is obtained after certain time when the amount of [OM] in the box and the δ13C(DIC) remain constant with time. Under this condition, the equation which governs the process of organic biomass (expressed as carbon) generation is:We use a box model to simulate the δrn Ocean . The modo is the production rate of organic carbon, λ*[OM] is the rate of organic carbon removal. The organic carbon biomass thus calculated is used to calculate the δ13C (DIC) using the mass balance equation (t and 0 refer to the state at time ‘t’ and ‘0’ respectively) given below:Where [OM] is the total organic carbon biomass (DOM + POM), Pet al.et al.3 at 3 oN. The DIC concentration is taken to be 2100 μmole/kg13C(DIC) values as a function of time are calculated (oS and 60 oS). It is possible that in these locations the organic matter production has been estimated wrongly due to incorrect assumption about the Chl-a concentration. For example, this could be due to the inability of the satellite sensors to detect frequently occurring subsurface chlorophyll patches3 in the organic carbon the δ13C(DIC) changes by about 0.15‰. The prediction can be improved by simultaneous measurement of POM, DOM and DIC in the same location.For the purpose of calculation, the DOM value is taken from Tremblay lculated  and plot13C and δ18O values of both tow samples and sediment core top samples from Khare and Chaturvedi (2012) have been plotted in 13C and 1‰ for δ18O. At 50°S δ13C value matches within 0.5‰ whereas δ18O value matches within 0.1‰. At three different stations at 43°S, δ13C values match within 0.3‰ whereas δ18O values match within 0.2‰. At 10°27′N the sample was collected near a coastal station. Here the δ13C value is higher by 0.58‰ due to possible riverine input. In contrast, the nearest location of sediment core top is at 9°24′N which has a value of −1.4‰. However, in the same sample the δ18O values match within 0.1‰ since the sea water represents a very large reservoir and is less affected by river input. The close agreement of tow samples with core top samples is quite interesting since the time interval covered by a typical core top sample is on a different scale (of the order of 1000 years) compared to the contemporary tow samples (less than a year). It shows that in these locations oceanographic conditions have not changed significantly during this time scale and the core top samples have not suffered major alterations since then.δ18O and δ13C values of foraminifera are compared with the δ18O and δ13C values expected at different depths across the latitude. As shown in 18O and δ13C values of foraminifera in the region between 10°N and 40°S agree within ~0.25‰ with the expected δ18O and δ13C values at ~75–200 m. Further southward from 40°S to 55°S, the measured δ18O values are higher than the expected δ18O values by ~2‰ at surface while the measured δ13C values of foraminifers are higher by ~1‰ than the expected δ13C values at surface  observations show high primary production in the Southern Ocean. As discussed above (13C(DIC) values are higher13C enriched water due to high productivity along with a possible higher calcification rate can thus account for the observed disequilibriumThird probable reason for the deviation  from theed above , with hi2 has depleted δ13C composition since the industrial revolution due to the burning of fossil fuels2 into atmosphere the nearly half of the CO2 is taken up by the ocean. Previous studies regarding change in δ13C of the surface ocean reported that the carbon isotopic composition of the oceans have become lighter through time due to the uptake of atmospheric CO2606213C(DIC) in the surface waters. However, in 50 and 75 m there is clear decrease as reflected in the profile of δ13C(DIC) with latitude  played a major role.Globigerina bulloides retrieved from tow samples from various locations across the Southern Ocean between 10°N−60 oS were analysed for δ18O and δ13C values. The measured δ18O values, along with the earlier published values of sediment core top samples, are in good agreement with the values expected at equilibrium in the depth range of ~75–200 m upto 40°S latitude. Beyond 40°S, the δ18O values of the foraminifera show a deviation from prediction. Similarly, the δ13C values of the foraminifera agree with the expected equilibrium values based on ambient water δ13C(DIC) values upto 40°S, but further south, a deviation is observed.18O values, we conclude that the calcification depth of G. bulloides is confined to a depth of ~75–200 m till 40°S latitude. This is consistent with earlier studies. However, further south (>40 oS) an unknown disequilibrium process sets in which is responsible for a large offset between the observed and the predicted equilibrium values. Unless appropriate corrections can be made for this deviation, the G. bulloides data obtained from sediment cores cannot be considered as a reliable paleo-climatic proxy in the >40 oS region of the Southern Ocean. We suspect that this deviation may be due to genetic effect on the calcification but more work is needed to confirm this.Based on the similarities between the estimated and measured δ13C values of sea water with latitude (up to about 43°S) which is observed in the surface seawater matches well with the variation in the chlorophyll concentration obtained by satellite observations. This type of correlated distribution (13C) enriches the ambient water. The model also shows that a steady state of the carbon isotope ratio of water can be achieved in a relatively short time of ~5000 days. Beyond 50 oS, the pattern changes and one sees lower δ13C(DIC) values. The overall pattern is controlled by the nutrient supply by ocean currents (Antarctic Bottom Water) and upwelling (by brine rejection process) and availability of light.The pattern of enrichment in the ribution  can be eORV SagarNidhi garNidhi . The MOCE as see . The loc18O analyses were carried out on the 150–250-μm fraction of G. bulloides collected from the tow samples. These samples were manually separated on-board, cleaned and stored. All samples were roasted under vacuum at 375 °C for 1 hour prior to isotopic analysis. Measurements of δ18O and δ13C values in both foraminifera and sea water were carried out at Indian Institute of Science  using a Gas bench II peripheral coupled with our Thermo Fisher- MAT 253 isotope ratio mass spectrometer 18O and 0.05‰ for δ13C based on repeated analysis.In order to minimize possible size-related differences, δ18O and δ13C values against measured foraminiferal values we estimated the δ18O and δ13C of calcite precipitated in isotopic equilibrium with seawater as described in section 2.3.In order to compare equilibrium calcite δHow to cite this article: Prasanna, K. et al. Isotopic disequilibrium in Globigerina bulloides and carbon isotope response to productivity increase in Southern Ocean. Sci. Rep.6, 21533; doi: 10.1038/srep21533 (2016).


In recent years attention has focused on γH2AX as a very sensitive double strand break indicator. It has been suggested that γH2AX might be able to predict individual radiosensitivity. Our aim was to study the induction and repair of DNA double strand breaks labelled by γH2AX in a large cohort.In a prospective study lymphocytes of 136 rectal cancer (RC) patients and 59 healthy individuals were ex vivo irradiated (IR) and initial DNA damage was compared to remaining DNA damage after 2 Gy and 24 hours repair time and preexisting DNA damage in unirradiated lymphocytes. Lymphocytes were immunostained with anti-γH2AX antibodies and microscopic images with an extended depth of field were acquired. γH2AX foci counting was performed using a semi-automatic image analysis software.Distinct increased values of preexisting and remaining γH2AX foci in the group of RC patients were found compared to the healthy individuals. Additionally there are clear differences within the groups and there are outliers in about 12% of the RC patients after ex vivo IR.The γH2AX assay has the capability to identify a group of outliers which are most probably patients with increased radiosensitivity having the highest risk of suffering radiotherapy-related late sequelae. In radiation therapy there is a need to find a monitoring assay which can predict a patients’ individual radiosensitivity and the risk to develop early and late tissue reaction and to adjust the radiation dose when indicated. A pretreatment identification of cancer patients by a predictive assay of normal-tissue radiosensitivity may allow appropriate adjustment of treatment and could reduce the risk of therapy related side effects. Several different predictive assay strategies were tested, for review see .To monitor the development of DNA double strand breaks (DSB) γH2AX was established as a well-known biomarker and over the last years the γH2AX assay has come into the focus of attention as a predictive assay. H2AX is a member of the histone H2A family . The numThe efficiency of γH2AX detection as a biomarker for DNA DSBs makes this protein a good candidate as a therapeutic marker for improving the efficiency of radiation, drug and other therapies -8. DickeThe prospective study included a total of 195 individuals. Enrolled were 136 rectal cancer patients (RC) and 59 healthy individuals  were isolated by Ficoll gradient centrifugation and were cytocentrifuged  onto a specimen. The samples were fixed with methanol and acetone and afterwards washed in a phosphate-buffered saline with foetal calf serum. The slides were then incubated with a mouse anti-γH2AX antibody , washed in PBS and incubated with a secondary goat anti-mouse Alexa 488 fluorescent antibody . Afterwards lymphocytes were washed in PBS and mounted with Vectashield mounting medium .2 (magnification 630×) was captured automatically. For each of the samples at least 1000 lymphocytes were identified using image analysis software . All DAPI-stained nuclei were morphologically considered by eye and apoptotic cells were excluded. Using the image analysis software the γH2AX foci inside each nucleus were counted [Fluorescence labelled lymphocytes were visualised by a fluorescence-microscope  and image acquisition software . Digital images of five optical planes separated by a distance of 0.75 μm were recorded and combined to an extended focus image using the maximum intensity algorithm (Metasystems). An area of 2 mmData analysis and statistics were performed using SPSS 21 for Windows . For comparing the results referred to the different groups, the Kolmogorov–Smirnov test and Lilliefors test were applied for testing normality and data were fitted by a Gaussian distribution. Standard deviations of the Gaussian distributions have been used to designate an individual’s categorization. The cut-off values of 2 and 3 standard deviations are equivalent to the 95% and 99% confidence intervals. Different groups were compared using the two-sample t-test. Graphics were plotted with TechPlot for Windows 3.0.11 .γH2AX foci rates in a group of 136 RC patients were compared to 59 healthy individuals . No difference between the two groups was found in initial γH2AX rates after 0.5 Gy and 30 minutes incubation time. The residual foci after a dose of 2 Gy and 24 hours recovery time were significantly increased in the RC patients group compared to the healthy individuals (p < 0.001)  with increased preexisting numbers of γH2AX foci. We could not correlate the measured values with radiotherapy related late effects because of the short period since IR exposure of patients. It is therefore difficult to judge whether the outliers have an increased radiosensitivity and an increased risk of therapy related late effects. Nevertheless, there is an unambiguous difference between the patients group having a distinct increased number of outliers compared to the healthy individuals. We found three different outlier groups (Figure Some studies reported an association between γH2AX foci and individual radiosensitivity. Fleckenstein et al. found a correlation between a serious mucositis and a rising number of foci 24 hours after ex vivo irradiation . SimilarOn the other hand there have been some studies reporting contradictory results and showing some weakness in the γH2AX assay. One group stated that γH2AX focus measurement has limited scope as a pre-RT predictive assay in lymphoblast cell lines from RT patients; however, the assay can successfully identify DNA DSB repair-defective patient’s cell lines . WerbrouThere is much hope that the γH2AX assay has the capability to predict individual radiosensitivity ,22. The In conclusion the γH2AX assay has the capability to identify a group of outliers which are probably patients with increased radiosensitivity which have the highest risk suffering from radiotherapy-related late sequelae. Future follow-up on these patients will correlate ex vivo data with clinical outcome.


R45° – 2O/Cu(100)] by scanning tunneling microscopy (STM) and low energy electron diffraction (LEED). Our results confirm the (4√2 × 5√2)R45° superstructure of PTCDA/Cu(100) reported by A. Schmidt et al. . However, contrary to Schmidt et al., we have no indication for a dissociation of the PTCDA upon adsorption, and we propose a detailed structure model with two intact PTCDA molecules within the unit cell. Domains of high lateral order are obtained, if the deposition is performed at 400 K. For deposition at room temperature, a significant density of nucleation defects is found pointing to a strong interaction of PTCDA with Cu(100). Quite differently, after preadsorption of oxygen and formation of the (√2 × 2√2)R45° – 2O/Cu(100) superstructure on Cu(100), PTCDA forms an incommensurate monolayer with a structure that corresponds well to that of PTCDA bulk lattice planes.We have investigated the adsorption of perylene-3,4,9,10-tetracarboxylic acid dianhydride (PTCDA) on the clean and on the oxygen pre-covered Cu(100) surface [referred to as (√2 × 2√2) Interfaces between organic semiconductors and metals have gained large interest because they are present in many applications, e.g., organic field-effect transistors  or organR45° superstructure  and the [010] direction of Cu(100), respectively. This is deduced from the angle φ0 between a0 and the substrate vector as and the angle γ0 between a0 and b0  are possible. For comparison the unit cell parameters of the commensurate (4√2 × 5√2)R45° superstructure reported by Schmidt et al.  direction of (100) surface and also the dimensions of the unit cell vectors differ with respect to those of PTCDA on bare Cu(100). We note that contrary to the situation on bare Cu(100) we get a more pronounced submolecular contrast of the PTCDA for similar tunneling parameters  surface. The respective unit cells are indicated in p2gg symmetry of the PTCDA layer. Due to its incommensurability with the 2O/Cu(100) substrate, the scattering of the substrate does not contribute to the LEED spots of the PTCDA layer and hence does not have an effect on the extinction of the  and  spots. This is different from the situation discussed above for the commensurate structures of PTCDA on Cu(100) or Ag(111), where the substrate can lift the extinction rule.A close up of the ordered PTCDA structure is shown in R45° – 2O superstructure, yielding its symmetry equivalent rotational domain. However, we propose that all four symmetry equivalent PTCDA domains exist on both rotational domains of the (√2 × 2√2)R45° – 2O/Cu(100) structure, because the PTCDA superstructure overgrows the respective domain boundaries (see above).As noted, the four symmetry equivalent domains of PTCDA consist of two pairs of mirror domains. A structure model of one domain is shown in R45° superstructure of PTCDA on Cu(100) with that within the commensurate (4√2 × 4√2)R45° superstructure of PTCDA on Ag(100) [2. This value is very comparable to that for PTCDA on Ag(100) (132.9 Å2). Both values are about 10% larger than the area per molecule in the lattice planes of PTCDA bulk crystals of the α or β-modification (120 Å2) [ and the  directions.It is interesting to compare the lateral ordering within the commensurate (4√2 × 5√2) Ag(100) . Both st(120 Å2) . Interes and the  directions, i.e., the close packed rows of the substrate, because the resulting high symmetry of the adsorbate complex presumably optimizes the molecule/substrate interactions. On Cu(100), the two hypothetic commensurate structures with a quadratic unit cell, which yield this orientation of the molecules along the  and the  directions, are either too large ((5√2 × 5√2)R45° /163.8 Å2 per molecule) or too small ((4√2 × 4√2)R45° /105.1 Å2 per molecule). Other quadratic commensurate superstructures on Cu(100) with a similar size to that of PTCDA/Ag(100) are rotated with respect to the former. Such a structure is, e.g.,  with 134.5 Å2 per molecule and φ0 = 38.7°. However, under the assumption that the favorable orientation of the long molecular axes along the  and the  directions was maintained in this structure, the spaces between the anhydride groups would be too small, which is unfavorable due to the negative partial charges on these groups. We note that we actually found this superstructure as a minor phase at the beginning of our experiments due to a  contamination, e.g., carbon, which stabilized the closeness of the anhydride groups. Due to an increasing cleanliness of the sample during the following experiments this structure disappeared. In conclusion, on Cu(100), a rectangular unit cell is preferred because this cell yields orientations of the long molecular axes along the  and the  directions and simultaneously optimizes the intermolecular interaction of the negatively charged anhydride groups with the positively charged perylene cores.The bonding of the PTCDA molecule to the Cu(100) substrate is of course most important for the structure formation. It causes the preferential orientation of the molecular axes along the We suppose that UPS will reveal a chemisorptive bonding of PTCDA on Cu(100) with filling of the former LUMO, similar to the situation that was found on Ag(100) . We noteHowever, the question whether the chemisorption is dissociative or not still needs to be answered. So far, all our structural findings stand against dissociative adsorption: (a) In STM images, like m/z = 124 for a coverage of 1 ML. This observation could be due to PTCDA dissociation during the adsorption process, but it is also conceivable that the dissociation occurs at elevated temperatures during the TPD experiment itself. This scenario is known from other chemisorptive systems, like PTCDA/Ag(111), [R45° superstructure for 5 min at 550 K. Some chains are isolated and some chains are packed closer together, probably with remaining PTCDA molecules in between them (see upper right corner of Data reported in the literature does not give a firm proof of a dissociative adsorption of PTCDA on Cu(100). The XPS measurements by Schmidt et al. were carried out with PTCDA on a polycrystalline copper foil . TherefoAg(111), . Our STM2, which is hence very similar to that of the respective lattice plane in bulk PTCDA crystals of the β-modification. The area per molecule is 117.7 Å2 which has to be compared with 120.6 Å2 for the β-modification. This indicates a dominant character of the intermolecular interactions in this system. All these observations reveal that for this system the adsorbate/adsorbate interactions have more influence on the structure formation than the adsorbate/substrate interactions, which contrasts with the findings for PTCDA on bare Cu(100), where the latter interactions are dominant.The situation changes drastically for the adsorption of PTCDA on the oxygen induced reconstruction of the Cu(100) surface. The structure is incommensurate and has a unit cell of a size of 12.2 × 19.3 ÅR45° – 2O superstructure passivates the reactive Cu(100) surface effectively. A similar phenomena is known for the adsorption of PTCDA on the reactive Ni(111) surface. In that case the interaction on the free surface is so strong that the molecules do not diffuse over the surface and hence do not form an ordered structure [p(2 × 2) – O/ /Ni(111) a well ordered herringbone structure was observed [R45° – 2O/Cu(100) already at rt.Thus the (√2 × 2√2)tructure , whereasobserved . The pasR45° superstructure of PTCDA on Cu(100) proposed by Schmidt et al. [R45° – 2O/Cu(100) surface, the interfacial interaction is strongly reduced, leading to an ordered PTCDA layer with an incommensurate structure.From STM and LEED data we confirm the commensurate (4√2 × 5√2)t et al. . A strucFile 1Two possible adsorption sites of PTCDA on Cu(100).


The mystery is how a single signalling ion can lead to such diverse and specific changes in cell function. This is partly due to aspects of the Ca2 + signal itself, including its magnitude, duration, localisation and persistent or oscillatory nature. The transduction of the Ca2 + signal requires Ca2 + binding to various Ca2 + sensor proteins. The different properties of these sensors are important for differential signal processing and determine the physiological specificity of Ca2 + signalling pathways. A major factor underlying the specific roles of particular Ca2 + sensor proteins is the nature of their interaction with target proteins and how this mediates unique patterns of regulation. We review here recent progress from structural analyses and from functional analyses in model organisms that have begun to reveal the rules that underlie Ca2 + sensor protein specificity for target interaction. We discuss three case studies exemplifying different aspects of Ca2 + sensor/target interaction. This article is part of a special issue titled the 13th European Symposium on Calcium.Changes in the intracellular free calcium concentration ([Ca •2 + regulates multiple aspects of neuronal function over varying time scales.Ca•2 + signalling is determined by the properties of Ca2 + sensorsThe specificity of Ca•2 + sensors contributes to specificity.Differing properties of closely related Ca•2 + sensors determines specificity of target protein interactions.The structure of Ca•We discuss three case studies involving calmodulin, NCS proteins and CaBP proteins.•2 + sensors.The examples illustrate the structural–function relationships of Ca Changes in [Ca2 +]i can be local or global, highly transient, oscillatory or persistent. Localisation of [Ca2 +]i changes to particular neuronal compartments is important for the generation of specific neuronal responses. For example, certain stimuli strictly require nuclear rather than cytoplasmic changes in [Ca2 +]i2 +]i changes restricted to dendritic spines may play an important role in activity-dependent synaptic plasticity 2 +]i can be manifest in microseconds (neurotransmitter release), milliseconds (channel facilitation or inactivation) or over much longer time scales leading to changes in gene expression The intracellular free calcium concentration  in the same neurons are coupled through distinct signalling pathways to changes in gene expression 2 + signals are translated into changes in cellular function through various types of Ca2 + sensor proteins that in general terms detect increases in [Ca2 +]i by becoming loaded with Ca2 +, undergo a conformational change and then interact with and regulate various target proteins. The subtlety of neuronal Ca2 + signalling is underpinned by differential signal processing by these Ca2 + sensor proteins, which in turn determines the physiological specificity of Ca2 + signalling pathways.Clearly Ca2 + sensor proteins 2 +-sensor for fast (microsecond) neurotransmitter release 2 +-binding motif 2 + sensor protein calmodulin 2 +/calmodulin-dependent protein kinase II 2 + sensors expressed in neurons include the calcium-binding protein (CaBP) family Neurons express a large number of Ca2 +-signals is affected by multiple aspects of the properties of Ca2 + sensors, and we suggest that they have a key role in determining signalling specificity. Factors that influence differential Ca2 + signalling include varied expression levels of sensors between neuronal cell types 2 +-binding 2 +-sensors to signalling specificity requires analysis of both the Ca2 +-sensors and their target interactions at a structural level 2 + signalling in neurons have begun to emerge in recent years but still remain to be fully understood. In this review, we will present three case studies to illustrate current understanding of the molecular and structural basis of the contribution of the properties of Ca2 + sensors to differential neuronal signal processing.The differential processing of neuronal Ca22 + regulation of phototransduction. One other subfamily, consisting of the visinin-like protein (VILIP) 1–3, neurocalcin δ and hippocalcin has less well-defined functions. Hippocalcin, as suggested by its name, has a very restricted pattern of expression mainly in hippocampal neurons where it shows dynamic membrane association  1–3 have specialised roles in the CaCaenorhabditis elegans as a consequence of its restricted neuronal expression DrosophilaSaccharomyces cerevisiae NCS-1 (Frq1) is essential for survival as a consequence of its requirement for activation of the phosphatidylinositol-4-kinase Pik1 2 +-dependent exocytosis NCS-1 appears to be expressed in most neuronal cell types 2 + sensors particularly calmodulin in vitro binding assays, and so their biological importance is unclear. It is possible that those binding partners that are also calmodulin targets in vitroNCS-1 has many known interacting partners The other clinically important interaction is with the interleukin 1 receptor accessory protein-like 1 protein (IL1RAPL1), which appears to be specific for NCS-1 2 + sensors and more particularly other members of the NCS protein family which have their own specific targets? The NCS proteins show differences in membrane-association with some such as recoverin and hippocalcin showing transient membrane-association through their Ca2 +/myristoyl switch mechanism 2 +/myristoyl switch 2 + binding, the NCS proteins undergo a conformational change that exposes a hydrophobic groove within which one or two helices from target proteins can bind. Once Ca2 + loaded the NCS, proteins undergo relatively limited structural changes on target ligand binding unlike the more dramatic changes seen in calmodulin conformation on ligand binding (described below).One key question is, what determines the specificity of target recognition for NCS-1 over other CaKey hydrophobic amino acids within the hydrophobic groove of the NCS proteins that are conserved across the whole NCS family are directly involved in the target interactions in multiple NCS proteins . DespiteC. elegansThe structural studies on NCS protein/target interaction have been based on the use of short peptides derived from the target protein. These studies have provided a consistent picture in which conserved hydrophobic residues are crucial. Nevertheless, it is important to assess the significance of the structural data using a physiological model. This would, for example, allow a test of requirement for the key residues in the hydrophobic groove and also the requirement for the C-terminal tail for NCS-1 function. This was approached using an assay for temperature-dependent locomotion in Drosophila frequenins. In Drosophila species, a gene duplication event generated two genes encoding closely similar proteins known as Frq1  of CaBP7 was solved by solution NMR 3 receptor in the docked complex CaBP7 and CaBP8  or Ca2 +-dependent inhibition (CDI) on a millisecond timescale. The speed of this regulation is explained by the response of the channels being due to nanodomains of Ca2 + near the mouth of the channel 2 + and progress on the characterisation of the mechanisms particularly those involving calmodulin 2 + sensors has also been the subject of a recent review 2 + sensor required for CDI of L-type (CaV1.2) Ca2 + channels 2 +-free (apo) calmodulin was pre-bound to the pore-forming α1 channel subunit and that this was mediated through the so-called IQ domain  channels calmodulin is required for both CDI and CDF with the N-lobe of calmodulin involved in CDI and the C-lobe in CDF V2.1 1 subunit  neurons was used to probe the general role for interaction of Ca2 + sensors in synaptic plasticity. In this study, it was shown that mutation of the IQ (IM in Cav2.1) motif or the CBD that would prevent CDF or CDI reduced facilitation or short-term synaptic depression, respectively Many of the studies on the regulation of CDF or CDI of VGCCs by CaV1.2 V1.3 channels as a consequence of CaBP4 binding to regions other than the IQ domain One problem with the originally proposed competitive model of action for CaBPs in which they need to compete endogenous calmodulin from the IQ domain is that calmodulin is expressed in brain at considerably higher levels than CaBPs. CaBP1 has been shown, however, to interact with an additional site in the N-terminus of CaV1.4 V1.2 channels v1.3 channels Findings consistent with an important role for CaBP4 in the regulation of phototransduction have come from the study of a CaBP4 mouse knock-out and also the presence of mutations in human CaBP4 2 + sensors exert their Ca2 +-dependent effects on VGCCs has been a subject of intensive study. Key issues to be resolved include (1) how can the two lobes of calmodulin have divergent functions when both bind to the IQ domain, (2) why does the C-lobe dominate in CDI in CaV1.2 channels but the N-lobe does so in CaV2 channels, (3) how can binding of calmodulin to an intracellular region of the channel α1 subunit affect the gating properties of the channel pore and (4) how can additional Ca2 + sensors that mediate differing effects do so particularly in the presence of calmodulin tightly bound to the IQ domain 2 +-bound calmodulin with an IQ-like peptide from CaV1.2 (V2.1, CaV2.2 or CaV2.3 channels in an anti-parallel manner giving a possible explanation for the reverse role of the N- and C-lobes in CDI of the two classes of channels 1 subunit involving the Ca2 +-loaded N-lobe may be important The structural basis for how calmodulin or other Cam CaV1.2 . These iV1.2 channels. This has demonstrated that both lobes of CaBP1 are functionally important with the C-lobe anchoring to the channel subunit as does the C-lobe of calmodulin. The N-lobe and also the inter-lobe linker specific for the CaBPs are required for modification of channel function and this could contribute to the different effects on the channel of CaBP1 compared to calmodulin 1 subunit may be important, but this will require further structural characterisation. There is currently no published data on the structural bases for the interactions of other Ca2 + sensors with VGCCs.A full structural characterisation of the interaction of CaBP1 with VGCCs is not available but one analysis has used chimeras with calmodulin and specific mutants to dissect the requirements for the regulation of Ca52 + sensors which is determined by fundamental structural characteristics of the proteins. Calmodulin is the most promiscuous with many target proteins. It consists of two independent lobes joined by a flexible linker that allows calmodulin to wrap around and interact with target proteins in multiple different ways using mutually induced fit to ensure high affinity interaction One aspect that emerges from consideration of the three case studies above is the variation in level of specificity versus promiscuity of the Ca2 + binding, Ca2 + sensors undergo a conformational change that allows them to bind ligands and may then undergo further conformational changes. This in turn will allow them to regulate the function of the target proteins. Most of the available structural characterisation has provided information on the binding mechanism and specificity but so far has provided little information on how the activity of the target protein is regulated. This is particularly notable in the case of VGCCs where even different lobes of calmodulin can have different functional effects after binding to a site on the channel α1 subunit some distance from the channel pore. For VGCC regulation, we need further information on the structural basis of interactions of Ca2 + sensors outside of the IQ domain, with the rest of the C-terminus, with sites in the N-terminus of the α1 subunit and also potentially with other subunits of the holo-channel. The NCS proteins such as NCS-1 have a smaller range of targets and differ from calmodulin in having two lobes that have a very rigid inter-relationship severely reducing the conformational flexibility between the two lobes. Target binding is due to the exposure of a hydrophobic groove the size and surrounding residues of which in conjunction with a variable and more flexible C-terminal tail determine which ligands can bind. A completely un-investigated aspect of NCS-target interaction is how NCS protein binding influences target conformation and structure and how this translates to function. Again, structural insights in this regard are limited by the complexes characterised to date containing only short peptides derived from the target protein. Further progress in this field will, therefore, require characterisation of Ca2 + sensors complexes with larger, ideally full-length, target proteins in order to contribute to an understanding of the mechanistic basis of differential signal processing in response to Ca2 + signals.On Ca