The diverse lipid environment of the biological membrane can modulate the structure and function of membrane proteins. However, little is known about the role that lipids play in modulating protein–protein interactions. Here we employed native mass spectrometry (MS) to determine how individual lipid-binding events to the ammonia channel (AmtB) modulate its interaction with the regulatory protein, GlnK. The thermodynamic signature of AmtB–GlnK in the absence of lipids indicates conformational dynamics. A small number of lipids bound to AmtB is sufficient to modulate the interaction with GlnK, and lipids with different headgroups display a range of allosteric modulation. We also find that lipid chain length and stereochemistry can affect the degree of allosteric modulation, indicating an unforeseen selectivity of membrane proteins toward the chemistry of lipid tails. These results demonstrate that individual lipid-binding events can allosterically modulate the interactions of integral membrane and soluble proteins. Native mass spectrometry (MS) is a technique that preserves non-covalent interactions in the mass spectrometer. Here the authors use native MS to study integral membrane proteins, and find that lipids with different headgroups and tails can allosterically modulate protein-protein interactions in different fashions. Although some channels have been shown to be regulated by specific lipid–protein interactions, such as potassium channels by phosphoinositides5, beyond these specific examples we have very limited knowledge of how the chemically diverse lipid environment of the biological membrane modulates the structure and function of membrane protein.The regulated transport of ions and solutes across biological membranes is an essential chemical process. Regulation of ion channels is carried out by their molecular interactions with regulatory proteins, small molecules, lipids, or through post-translational modificationEscherichia coli (E. coli), an integral membrane protein that is regulated by the soluble trimeric protein GlnK7. The interaction between AmtB and GlnK is tightly controlled by cellular nitrogen status through key effector molecules, while adenosine diphosphate (ADP) alone is sufficient to form the complex10. Atomic structures have revealed that GlnK blocks ammonia transport by essentially plugging the conducting channel within each subunit primarily through the insertion of a long surface loop  was used to identify a lipid that stabilized the channel, which guided co-crystallization trails leading to the first structure of AmtB bound to lipid14. However, for the other structures no bound lipids were observed, implying that crystallography will not always be adequate in the identification and biophysical characterization of lipids involved in membrane–protein interactions, emphasizing the necessity for new techniques to investigate protein–lipid interactions.As a step toward studying membrane protein and soluble protein interactions, we selected for study the ammonium channel (AmtB) from 20. Unlike other biophysical techniques, which are often ensemble measurements, native MS can preserve non-covalent interactions in the gas-phase as well as resolve and interrogate individual ligand-binding events to protein complexes22. Advances in native MS spanning nearly a decade23 have led to the ability to study membrane protein complexes in a native-like state that can provide invaluable information on their interactions with ligands, such as nucleotides, drugs, peptides, and lipids, and subunit stoichiometry29. Recently, native MS coupled with a temperature-controlled source has been applied to monitor thermal unfolding30 as well as determine thermodynamics for protein–ligand interactions, including membrane protein–lipid interactions31. Importantly, thermodynamic parameters for soluble protein–ligand interactions determined using native MS are in agreement with those obtained using other biophysical techniques, such as isothermal titration calorimetry and surface plasmon resonance (SPR)32. Building upon these recent advances, we use native MS to reveal that individual lipid-binding events allosterically modulate integral membrane protein and soluble protein interactions.To determine how individual lipid-binding events modulate the interaction of AmtB with GlnK, we employed native MS, a powerful biophysical technique that has emerged over the past two decades to study proteins and their interactions with ligands8E4), a detergent that exhibits charge reducing properties33, and at a set temperature of 298 K using a temperature-controlled source31. The mass spectrum revealed an equilibrium of GlnK, AmtB, and AmtB–GlnK 0–3 in the mass spectrum is in agreement with those calculated using reported equilibrium dissociation constants (KD) for GlnK-binding ADP1–3 in the absence of detergent  to be 1.12 ± 0.41 μM in the presence of 50 μM ADP.We recorded a mass spectrum for the AmtB–GlnK complex in ammonium acetate buffer containing 50 μM ADP and two times the critical micelle concentration of tetraethylene glycol detergent , whereas at the highest concentration of ADP (200 μM) the equilibrium-binding constant dropped to 77.68 nM. At and above an ADP concentration of 100 μM, the KD,AG approaches an asymptote around ~80 nM . In contrast, using the nonlinear form of “van’t” Hoff equation35 yielded exceptional fits with an R2 of 0.98, indicating that heat capacity is not constant over the selected temperature range (for review see ref. 36). Using the nonlinear van't Hoff equation enabled us to determine the change in heat capacity (ΔCp) and change in enthalpy (ΔH) at a reference temperature of 298 K  was calculated directly from repeated measurements of KD,AG at 298 K, and entropy (ΔS) was back calculated using both ΔH and ΔG  and different temperatures. We then performed a ““van’t”” Hoff analysis8 K Fig. . The chakon of 5.49 × 104 M−1 s−1 and koff of 3.14 × 10–2 s−1 37. The large negative ΔCp yields an upward curvature to ΔG with lower temperature ranges having smaller KD,AG values. In addition, this ΔCp value also suggests that these macromolecules adopt a flexible conformation or a variety of interconverting conformations38. Different conformations are supported by crystallographic structures, where a conformational change in AmtB is observed when bound to GlnK that arises from rigid body movements of several transmembrane helices within each subunit  for AmtB bound to n lipids (ALn) binding to GlnK (G) was observed . For POPS and TOCDL, the coupling factors exhibited neutral allosteric modulation for all lipid-binding events recorded  to abolish a specific PG site observed in the X-ray structure, resulted in reduced gas-phase stabilization of the channel by this lipid14 and a different thermodynamic signature observed for binding PG31. Equilibrium-binding constants and coupling factors for PG lipids with different tail lengths were determined using the same procedure used for lipids with different headgroups  tails in either the cis or trans configuration. Equilibrium-binding constants and coupling factors for these lipids were determined in a similar fashion to the other lipids investigated , which contains saturated and cis-monounsaturated lipid tails (SOPE). The coupling factor for SOPE did not fall between the two DOPE lipids studied but was similar to cis-DOPE. Taken together, these results reveal an exquisite selectivity toward not only the headgroup of lipids, but also the length and stereochemistry of lipid tails.Intrigued by the effect of lipid tail length on PG, we then studied the impact of the stereochemistry of monounsaturated carbon bonds within the lipid tails of PE on the AmtB–GlnK complex. In bacteria, the stereochemistry of the monounsaturated double bond is predominantly in the ted Fig. . Interes45, allostery has emerged as an underlying theme in biological macromolecules (for review see refs. 47). Here we demonstrate for the first time that individual lipid-binding events can indeed allosterically modulate integral membrane and soluble protein interactions. Given that we have identified allostery, we propose a plausible mechanism to rationalize the allosteric effects observed  or when complexed with GlnK (AmtB*). In the case of positive allosteric modulation, binding of lipid(s) to AmtB shifts the Boltzmann distribution of the interconverting states of AmtB by preferentially stabilizing (lowering ∆G) the AmtB* state. As this state resembles that when bound to GlnK, it will exhibit much higher affinity for binding GlnK giving rise to positive allosteric modulation. In contrast, the negative allosteric modulation observed for POPA could be rationalized by preferentially stabilizing the AmtBu state, and since this state resembles that of the uncomplexed state, it will exhibit reduced affinity for GlnK giving rise to negative allostery.Originating in 1965ved Fig. : in the In summary, we have shown that the interaction between AmtB and GlnK is associated with changes in equilibria of conformers and solvation. In addition, we demonstrate that individual lipid-binding events can influence integral membrane protein and soluble protein interactions, using the AmtB–GlnK complex as a model system. Interestingly, lipids with similar tails but different headgroups allosterically modulate the AmtB–GlnK complex in positive, neutral, and negative fashions. The surprising result that variable lipid tail lengths of a PG and the stereochemistry of PE tails can alter the degree of allosteric modulation illustrates that specific lipid-binding site(s) are exquisitely selective for both lipid headgroup and tail. Although a small number of lipid tail lengths and stereochemistry were examined here, by extrapolation it is likely that other modifications of lipid tails will display different degrees of allosteric modulation for protein–protein interactions. Moreover, an underlying question remains regarding the location of the bound lipids and how they induce their allosteric effect. Addressing this question is a monumental challenge as echoed by our limited knowledge in this area. However, in our native MS studies, the lipids may be binding to specific site(s) selective for headgroup and/or lipid tails, or binding to similar non-specific site(s). In closing, this is the first report demonstrating that individual lipid-binding events can have a large dependence on the chemistry of the lipid in modulating membrane protein structure and function, including allosterically modulating protein–protein interactions.31. In brief, stGlnK was expressed from pET28b (Novagen) in Escherichia coli ArcticExpress (DE3) RIL (Agilent Technologies), and protein expression was induced with 0.1 mM 1-thio-β-D-galactopyranoside (IPTG) and grown overnight at 20 °C. Cell pellets of GlnK were resuspended in NHA buffer . The cells were lysed with 4–5 passes through a Microfluidics M-110P microfluidizer at 20,000 psi and then clarified by centrifugation . The filtered supernatant containing recombinant Strep-tag II-tagged GlnK was then applied onto a StrepTrap HP 5 mL column  and eluted with the same buffer containing 2.5 mM D-desthiobiotin. The peak fractions containing Strep-tag II-tagged GlnK were pooled, concentrated, and loaded onto a HiLoad 16/600 Superdex 75 pg column  equilibrated in GF buffer . AmtB was expressed as TEV protease cleavable N-terminal fusion to maltose-binding protein (MBP) preceded by a secretion signal peptide (pelB) and 10x His-tag from pET15b (Novagen) in Escherichia coli C41(DE3) (Lucigen). Expression of AmtB was induced with 0.5 mM IPTG and cells were grown overnight at 20 °C. Cell pellets of AmtB were resuspended and lyszed as described for stGlnK. Membranes were pelleted by centrifugation  and AmtB was extracted overnight with 5% octyl glucoside. The supernatant was filtered before loading onto a 5 mL HisTrap-HP column  equilibrated in buffer NHA-DDM . After the clarified supernatant was loaded, the column was initially washed with 40–50 mL of NHA-DDM supplemented with 1% OG followed by several column volumes of NHA-DDM until a steady baseline was reached. AmtB was eluted with a linear gradient to 100% in two column volumes of NHB-DDM . Peak fractions were pooled and injected onto a HiPrep 26/10 desalting column  equilibrated in NHA-DDM. Peak fractions were pooled and supplemented with 5 mM 2-mercaptoethanol (BME) and His-tagged TEV and incubated overnight at 4 °C. After overnight incubation, the sample was filtered and passed back over a 5 mL HisTrap HP column equilibrated in NHA-DDM. Flow-through containing the untagged AmtB was collected and concentrated using a 100 kDa MWCO concentrator. The Y51F point mutation was introduced into GlnK as this residue can be modified with uridine by uridylyl transferase GlnD and prevent the interaction with AmtB48. Unless otherwise stated, all purification steps were carried out at 4 °C. To generate the AmtB–GlnK complex, purified tagless AmtB and stGlnK were mixed at a molar ratio of 1:3 in GF buffer ) and incubated overnight. The mixture of AmtB and stGlnK was loaded onto a StrepTrap HP 5 mL column  to capture both AmtB in complex with stGlnK and stGlnK, and flow through contained non-functional AmtB. Bound protein was eluted with the same buffer containing 2.5 mM D-desthiobiotin, concentrated, and loaded onto a Superdex 200 Increase 10/300 GL column  equilibrated in GF buffer supplemented with 0.5% C8E4 instead of DDM. Peak fractions containing the AmtB–stGlnK were pooled, concentrated, flash frozen in liquid nitrogen, and stored at −80 °C.AmtB wild-type and Strep-tag II-tagged GlnK (stGlnK) was expressed and purified as previously described49, and incubated at room temperature for 30 min. Heating was avoided as it promotes aggregation of AmtB. The prepared samples were loaded on a precast 12% acrylamide gel . Included on each gel was one well with pre-stained size markers (BioRad), and at least two wells loaded with a standard mixture of purified AmtB and stGlnK at a molar ratio of 1:1. Gels were run at 60 V during stacking and at 100 V once sample was in the resolving gel at room temperature. After separation, each gel was rinsed in water for 15 min, stained with 0.025% Coomassie brilliant blue R-250 (Sigma B-0149) in 10% acetic acid using a rapid, hot-staining protocol50 followed destaining overnight in a 100 mL portion of 10% acetic acid. General procedures for quantitative densitometry followed the recommendations of Gassmann and colleagues51. The gel was placed on a Ricoh 4504 scanner, imaged in transmittance mode at a resolution of 400 dpi, and stored as a 16-bit greyscale TIFF image. For densitometry analysis, each file was opened in ImageJ52 and the original greyscale values were converted to optical density (OD) values. Individual full-width lanes were then defined, their OD profiles calculated, and the integrated stain density summed for the AmtB and GlnK bands. The amount of AmtB or GlnK protein in each lane was calculated from the stain density in the lane, with reference to the average stain density of the AmtB or GlnK band in lanes loaded with the known standard mix of AmtB and GlnK. The protein concentration for AmtB and GlnK was the average of the values determined from at least two separately Coomassie-stained gels.Soluble and membrane protein concentration in the buffer not containing ADP was determined with the DC Protein Assay kit (Bio-Rad) using bovine serum albumin as the standard. For quantification of samples containing ADP, which interferes with protein assays, aliquots of the purified AmtB–GlnK complex were thawed at room temperature, immediately mixed with 0.25 volumes of 5× Laemmli sample buffer−1 in PBS . In order to generate low-density GlnK sensor, the chip surface was pretreated by sequential injection of 40 µL of activation solution (1:1 mixture of 0.4 M EDC and 0.1 M NHS) and 40 µL of deactivation solution . The pretreated surface was activated again for 2 min followed by 1 min injection of stGlnK in buffer , and then washed with 200 mM ammonium acetate (pH 7.4) until no further decline in signal response. Using this procedure, GlnK surface with different densities ranging from 150 to 400 RU was produced. A flow cell with the same treatment but without coupled protein was used as a reference surface. The sensor chip was further stabilized with 10 injections of binding buffer  followed by 1 h of continuous flow at 30 µL min−1. Binding experiments were performed at a flow rate of 30 µL min−1 at different temperatures  using freshly buffer-exchanged AmtB in binding buffer at a concentration range of 0.05–1.4 µM. Reference and buffer corrected SPR responses were collected, and the data were analyzed using Biacore evaluation software. The kinetic parameters and KD value were determined for each temperature by fitting the sensorgram series to a Langmuir 1:1 binding model.SPR experiments were performed using a Biacore 3000 optical biosensor  with CM3 sensor chip. Immobilization of GlnK was conducted at 25 ˚C using amine coupling kit  at a flow rate of 10 µL min31. In brief, stock solutions of each phospholipid were resuspended in chloroform, aliqouted, and chloroform was removed by a steady stream on nitrogen gas. Lipid films were placed under vacuum overnight to prepare to resuspend in MS buffer supplemented with 5 mM 2-mercaptoethanol. Phospholipid concentration was determined by phosphorus analysis.A stock of ADP was prepared by dissolving the ammonium salt form of ADP in water followed by adjusting the pH with ammonium hydroxide to 7.0. Serial dilutions into MS buffer  supplemented with 5 mM BME was performed to prepare ADP at certain concentrations. Phospholipids were prepared as previously described53.A frozen aliquot of purified AmtB–GlnK complex was thawed on ice prior to buffer exchange into MS buffer supplemented with a desired concentration of ADP following established methods−1 (5.2 × 10–2 mbar). The T-wave settings for trap (300 ms−1/2.0 V), IMS (300 ms−1/20 V), and transfer (100 ms−1/10 V), source temperature (110 °C) trap collision voltage (20 V) and transfer collision voltage (160 V) and trap bias (35 V) were also optimized.Native MS was performed on a Synapt G1 HDMS instrument (Waters corporation) with a 32 k RF generator. AmtB–GlnK samples were at a final concentration of 2 µM. Instrument parameters were tuned to maximize ion intensity and simultaneously preserve the native-like state of AmtB. The instrument was set to a capillary voltage of 1.7 kV, sampling cone voltage of 100 V, extractor cone voltage of 10 V, and argon flow rate at 7 mL min54 and deconvoluted using Unidec55 software with the following settings: no smoothing, m/z range 6000–12,000, charge range 10–30, mass sampling of 5 Da, and peak FWHM of 1. The intensities of apo AmtB (A) and AmtB–GlnK (AG) and AmtB (ALn) and AmtB–GlnK (AGLn) bound to Ln lipid(s) were converted to mole fraction for a given lipid titration. Notably, the non-specific ADP adducted peaks of AmtB and AmtB–GlnK and those bound to lipid(s) were taken into account when converting to mole fraction. The interaction between ALn and GlnK (G) is dependent on the apparent equilibrium association constant :n is an integer starting at 0 in the case of apo. And for a lipid binding to A:Atotal) in the system can be represented as:n (FnAL):n (FnAGL):G was calculated as follows:L:http://www.python.org) and made use of libraries scipy56, numpy57, and matplotlib58. The binding model was globally fit to the mole fraction data collected at a given temperature through minimization of the pseudo-χ2 function:n is the total number of lipids bound and d is the total number of data points.Native MS data were processed using the software program Pulsari) for a given number of lipids bound (i) was calculated as follows:59KA is the equilibrium association constant, Ko is the equilibrium association constant at the reference temperature (To), ΔH is the standard enthalpy at To, ΔCp is the change in heat capacity at constant pressure, and R is the universal gas constant. The average and standard error of the mean was determined by fitting the above equation coded into Python to experimental data for each replicate with To equal to 298 K.In the above binding model, all equilibrium association constants are treated as a separate variable in the fitting routine. The coupling factor (αThe data that support the findings of this study are available within the article and Supplementary Information. Other data and binding model Python code are available from the corresponding author on reasonable request.Supplementary Information


To verify the technique and to demonstrate its application potential, we imaged a three-component polymer blend and a melanin granule in a human hair cross-section, and demonstrate that multivariate data analysis can be applied for extracting spatially resolved chemical information. Particularly, we demonstrate that distribution and chemical interaction between the polymer components can be mapped with a spatial resolution of about 30 nm. We foresee wide application potential of hyperspectral infrared nanoimaging for valuable chemical materials characterization and quality control in various fields ranging from materials sciences to biomedicine.Infrared nanospectroscopy enables novel possibilities for chemical and structural analysis of nanocomposites, biomaterials or optoelectronic devices. Here we introduce hyperspectral infrared nanoimaging based on Fourier transform infrared nanospectroscopy with a tunable bandwidth-limited laser continuum. We describe the technical implementations and present hyperspectral infrared near-field images of about 5,000 pixel, each one covering the spectral range from 1,000 to 1,900 cm In hyperspectral imaging a broadband spectrum is recorded at each pixel, which creates information-rich images. Here, the authors combine this concept with Fourier transform infrared nanospectroscopy to achieve 5,000-pixel, nanoscale-resolution images at wavelengths between 5 and 10 micrometres. Infrared (IR) vibrational spectroscopy2346791112131415161718s-SNOM and nano-FTIR spectroscopy are based on atomic force microscopy (AFM), where either monochromatic or broadband infrared radiation, respectively, is scattered by a metallic AFM tip. The tip acts as an antenna and concentrates the incident infrared field at the very tip apex to a nanoscale spot size (nanofocus)10121220111422232417181914161415182 or cw-quantum cascade lasers are used as infrared source. As they can be tuned only over a relatively small spectral width of about 200 cm−1, several lasers are required to cover the whole mid-infrared spectral range. On the other hand, frequencies below 900 cm−1 are barely reached with table-top cw-lasers. Further, the nonlinear and typically uncontrolled drift between sample scanner and tip, as well as the tip wear due to the numerously repeated sample scanning, is challenging the construction of accurate local broadband infrared spectra.Spatially resolved spectroscopic information can be obtained by sequential s-SNOM imaging of one and the same sample area at different infrared frequencies using a tunable monochromatic laser. From the stack of images, an infrared spectrum can be constructed at each pixel282930313233−1  amounts to about 1 min  to record nanoscale-resolved hyperspectral infrared images comprising about 5,000 nano-FTIR spectra that cover the spectral range from 1,000 to 1,900 cm−1. We verify the technique with a three-component polymer blend and a hair cross-section, and demonstrate that standard multivariate analysis of the hyperspectral images can be performed. As a first application, we demonstrate the much sought-after possibility of mapping chemical interaction between polymer components with nanoscale spatial resolution.Here we introduce and describe hyperspectral infrared nanoimaging by nano-FTIR spectroscopy with a tunable laser continuum. It is based on recording and stitching together multiple bandwidth-limited nano-FTIR spectra at each pixel of a 2D sample area, which is enabled by sample drift correction during data acquisition. Specifically, we use a tunable DFG laser continuum . Throughout this work, the demodulation order was n=3. Because tip and sample are located in one interferometer arm, Fourier transform of I(d) yields amplitude ss(ω) and phase ϕs(ω) spectra14s=ss/sref and ϕ=ϕs−ϕref, the tip is positioned on a reference area on the sample 14sref(ω) and ϕref(ω).We developed hyperspectral infrared nanoimaging  using a cm−1 see . The tipx, y) of a 2D area of the sample surface  where x and y represent two spatial dimensions and ω the frequency . For the present work, we recorded and studied nano-FTIR phase spectra ϕ(ω), as they are related to the sample's infrared absorption16Ak=ϕk at three different DFG output spectra  of the individual nano-FTIR spectra. To that end, the output power of our mid-IR laser continuum (previously reported in refs y-axis). The nano-FTIR spectra of each line are then normalized to the reference spectrum included in this line.For accurate and reliable normalization of the nano-FTIR spectra, the reference spectra need to be recorded under the same experimental conditions. Due to slight spectral fluctuations of the laser continuum and drift of the interferometer arms , the sample is repositioned, yielding a data cube in which sample drift has been compensated  during the m line scans is smaller than the spatial resolution of about 30 nm. In the experiments presented in m=2 and m=4, respectively.Depending on the number of individual spectra, the acquisition of a data cube ated see . To thisAk, we need to combine at each position  the individual phase spectra ϕk obtained with the different DFG settings. A key for achieving this task is the sample drift correction during the recording of each bandwidth-limited data cube. As outlined above, the sample drift correction ensures that the position uncertainty is less than the spatial resolution, thus ensuring that spectra of the same position  are combined.To combine the individual bandwidth-limited data cubes ϕk of six subsequent pixels on the polymer blend sample. For better comparison, and to demonstrate the reproducibility of the individual spectra, the upper panel of ϕ  need to be stabilized with a precision better than 20 nm, which, however, will require sophisticated technology development in the future. Here we tackled the problem by correcting the phase offset. To that end, we shift at each pixel  the phase spectra ϕI(ω) and ϕIII(ω) by the constant phase values ϕII(ω). The offset-corrected phase spectra are shown in ϕk by the functions Fk(ω) shown by the red, green and blue graphs in The remaining challenges and solutions for combining the individual spectra are shown in ϕ(ω). We select two frequencies ωb1 and ωb2 (marked in ϕ(ωb1) and ϕ(ωb2). A comparison of baseline-corrected spectra , an acrylic copolymer (AC), and a polystyrene latex . x, y) the multivariate spectral distance D between the point spectrum and a reference spectrum (see ‘Methods' section). The distance values are converted into colour scales and plotted as specifically coloured pixels at the positions . In the resulting distance maps, high colour intensity denotes a small distance (high similarity) with the reference spectrum (and vice versa). With nano-FTIR spectra obtained from reference samples made of pure AC and FP components  but also the C=O peak of AC at 1,740 cm−1. It can be shown . Within the cortex region we find disk-shaped areas of about 300 nm diameter, where the infrared absorption is reduced . Their size and distribution corresponds to that of melanin granules observed in electron microscopy images−1 (–C–OH phenolic stretching), 1,454 cm−1 , 1,563 cm−1 (indole N–H bending) and 1,638 cm−1 −1. Further, while both particles A and B are seen in the monochromatic image at 1,670 cm−1, only particle A exhibits a contrast at 1,580 cm−1  regular sample drift correction during data acquisition, (ii) regular recording of reference spectra during data acquisition, (iii) correcting spectral offsets caused by unavoidable interferometer drift and (iv) baseline correction of the broadband nano-FTIR spectra analogous to far-field hyperspectral infrared imaging. Using a tunable DFG laser continuum with an effective spectral bandwidth of about 350 cm−1 broad laser continuum of about 100 mW that is tunable between 500 cm−1 and 3,000 cm−1 ref. We foresee a wide application potential of hyperspectral infrared nanoimaging in various fields of science and technology, ranging from materials sciences and pharmaceutical applications to biomedical imaging, and from fundamental research to quality control. In combination with multivariate data analysis, hyperspectral infrared nanoimaging yields nanoscale-resolved chemical and compositional maps, including the detection of nanoscale localized chemical interaction. By improving the power and spectral bandwidth of emerging infrared laser sources (a recent work demonstrates a 100 cmThree water-based polymer dispersions made by emulsion polymerization were employed in this work. Tg) latex based on polystyrene to improve mechanical properties without the need of any film forming aidsThree-component polymer blend for hyperspectral infrared nanoimaging: The three polymer dispersions listed in Polymer reference samples for nano-FTIR spectroscopy: The three polymer dispersions listed in Polymer reference samples for ATR-FTIR spectroscopy: The three polymer dispersions listed in A clean human hair was cut into small pieces and embedded in epoxy resin . The sample was cured during 24 h at 60 °C. The block containing the human hair was trimmed with fresh glass knife made with Leica knifemaker and then ultramicrotomed with Leica UCT using Diatome 45° diamond knife.−1 and presents an average over 250 scans with a total acquisition time of 215 s.ATR-FTIR of the hair cross section. We used a Bruker Hyperion 2,000 microscope coupled to a Vertex 70 FTIR spectrometer equipped with an ATR module  comprising a germanium crystal with a diameter of about 100 μm at the point of contact with the sample. The spectrum (showing the absorbance) was measured with a spectral resolution of 4 cm−1 and present an average over 150 scans with a total acquisition time of 796 s. The reference spectra were recorded on air. All ATR-FTIR spectra show the absorbance.ATR-FTIR of polymers. We used a Frontier FTIR/FIR spectrometer (Perkin Elmer) equipped with a universal ATR module (1 reflection with diamond/ZnSe top plates). The spectra were measured with a spectral resolution of 4 cmx, y) coordinates. In distance imaging, a high colour intensity denotes a low inter-spectral distance, or high similarity, between the actual point spectrum and a given reference spectrum. The CytoSpec software package also allows for constructing so-called composite images that are superpositions of distinct distance maps.Inter-spectral distance mapping  was carried out by means of the CytoSpec software package . Distance images were constructed by calculating Euclidean distances between the external reference spectra and the pre-processed nano-FTIR spectra contained in the hyperspectral data sets. In this approach, distance values were converted to colour scales and plotted as a function of the spatial . Basic principles of this image segmentation technique have been published elsewhere5The data that support the findings of this study are available from the corresponding author upon reasonable request.How to cite this article: Amenabar, I. et al. Hyperspectral infrared nanoimaging of organic samples based on Fourier transform infrared nanospectroscopy. Nat. Commun.8, 14402 doi: 10.1038/ncomms14402 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Notes and Supplementary References.


The related peptide hormones cholecystokinin (CCK) and gastrin are conserved throughout vertebrate clades and implicated in energy homeostasis. CCK is generally accepted as a satiety hormone in poultry, but the role of gastrin remains poorly studied. Functional dissection of these ligands is required to characterise the molecular control of growth & satiety in the domestic chicken, for which there is an increasingly pressing mandate. There are limited descriptions of physiological distributions for the two genes in birds, and these are mostly reliant on immunohistochemistry which can prove problematic due to the shared structure of the targets. Therefore, we have defined the tissue distributions of CCK and gastrin in the chicken, focussing on the gastrointestinal tract, by using transcript-dependent techniques to improve reliability by increasing specificity. Though considerably more highly expressed in the brain, gastrointestinal CCK transcripts were dispersed throughout the small intestine and particularly around the proximal ileum. Gastrin expression was strictly limited to the gastric antrum region of the intestinal tract, albeit very highly expressed. We demonstrate that CCK mRNA expression does not respond as expected for a short-term satiety hormone, and that the short-term response of gastrin expression is paradoxical compared to its role in mammals. These results partially corroborate previous peptide distribution studies and initiate exploration of the nutrient-responsive roles of these hormones in avian energy balance. The ‘broiler-breeder paradox’ – restriction of feed intake to maintain reproductive health in broiler parent flocks – is a prominent example of welfare concern arising from intense selective breeding in chickens for meat production. This might be solved or ameliorated if hormonal response to nutrition was better understood and breeding or husbandry managed to prevent aberrant ovarian follicular development . FurtherGAST (producing preprogastrin) and CCK (producing preprocholecystokinin). Gastrin and cholecystokinin represent one set of hormones relatively well-described in mammals but neglected in birds. Both genes are conserved across vertebrate species, likely arising from a duplication event early in the vertebrate lineage  Act 1986, project licence 70/7909.2.1.1in situ hybridisation was harvested from broiler breeders reared in standard conditions with commercial food restriction to achieve the breeding company’s target growth rate . The remaining pens maintained fast for the remainder of the experiment. 2.5 ± 0.5 h of feed after reintroduction of feed or maintenance of fast (10:00–11:00), five females and seven males from each treatment were culled. All remaining birds were culled 7.5 ± 0.5 h after reintroduction of feed or maintenance of fast (15:00–16:00). Chicks of this age have an average whole digestive tract transit time of 2.65 ± 0.05 h , so all gastrointestinal regions of interest should have been in recent contact with nutrients by 2.5 h. All birds were killed by cervical dislocation and immediately dissected to harvest 40–100 mg samples of gastric antrum and proximal ileum, which were snap-frozen on dry ice. All samples were taken in a coronal plane to include all intestinal tissue strata.To characterise the responses of gastrin and CCK to short-term hunger and satiety, 50 NOVOgen brown birds were sexed by genotyping  at 2d an2.2in situ hybridisation were designed manually to conform to the following parameters: ∼55% GC content (48–62%), ∼45mer length (43–47mer) and melting temperature (Tm) as high as possible within those parameters and at least 20 °C greater than the highest predicted tertiary structure Tm predicted by OligoAnalyzer 3.1 online software (Integrated DNA Technologies). Chicken CCK and gastrin preprohormone mRNA sequences were aligned using MUSCLE  using the Direct-zol RNA Kit (Zymo Research) to manufacturer’s specifications, with in-column DNase treatment. 1μg total RNA per sample was reverse transcribed using the High Capacity Reverse Transcription Kit (Applied Biosystems) in 20 μl reactions according to manufacturer’s guidelines and the product diluted to 110 μl total volume per sample with water.2.42O were mixed for each 20 μl reaction. Thermal conditions were consistent for all assays: 50 °C; 120 s, 95 °C; 120 s, , then 95 °C; 60 s, 60 °C; 30 s, 95 °C; 15 s. Apparent reaction efficiencies were between 96and 99%, as determined by analysis of the standard dilution curve. Amplicons were bidirectionally sequenced using LightRUN Sanger sequencing (GATC Biotech) to confirm identity. LBR, NDUFA1 and YWHAZ were chosen as reference genes due to their reliability in previous avian studies (Brilliant III Ultra-fast SYBR Green qPCR Mastermix and the Mx3005p qPCR System with MxPro software (Agilent Technologies) were employed according to the manufacturers’ guidelines and as described previously . Briefly studies  and quan2.5In situ hybridisation employed reagents and protocol as described previously  or sampling times , and there was no treatment by sampling time interaction  , and lower at the later sampling time compared to the earlier sampling time across both treatments , but there was no interaction between treatment and sampling time   a. Gastri = .990) b.Fig. 4RNotably, across both the distribution assay and the feeding experiment, gastrin expression was found to be far higher than CCK expression in their sites of highest expression  in real terms .4in situ hybridisation, we have corroborated and further resolved the results of previous studies of distribution of native gastrin-cholecystokinin peptide family expression in the domestic chicken  and ventriculus or gizzard (muscular stomach). The proventriculus best resembles the mammalian monogastric stomach in form and function, and so is sometimes referred to as the ‘true stomach’ . The strad libitum-fed and feed-restricted birds (CCK is heavily implicated in the short-term satiety response in vertebrate species . The cired birds b suggested birds , might had libitum for the entire rearing period before induction to experimental treatment. If this is the case, it might be sensible to consider heightened gastrin expression as a means to maintain peptide stocks for secretion upon anticipated detection of nutrients at the gastric antrum. The idea that gastrin expression might be regulated by conditioning is mimicked in the observation that a strong diurnal pattern is apparently maintained regardless of treatment, with gastrin expression decreasing across the experimental timescale for both treatments (Gastrin expression differed significantly between treatments, with fasted individuals exhibiting greater expression compared to their fed counterparts at both sampling timepoints b. This seatments b. Attenueatments , relativad libitum feeding conditions. Further studies of the expressional response of these hormones to nutritive state should consider disparate nutrient availability for longer time periods and periprandial sampling, and might clarify similarities and differences between birds and other vertebrate clades.In conclusion, we have demonstrated tissue distribution of gastrin and cholecystokinin gene expression in chicken. CCK expression does not seem to respond to short-term satiety, contrary to some antecedent vertebrate studies. Gastrin expression did alter between fed and fasted treatments, however its expression was paradoxically lower in acute satiety and higher in acute hunger, which might be an artefact of conditioning to


Surface ocean bubble-bursting generates aerosols composed of microscopic salt-water droplets, enriched in marine organic matter. The organic fraction profoundly influences aerosols’ properties, by scattering solar radiations and nucleating water particles. Still little is known on the biochemical and microbiological composition of these organic particles. In the present study, we experimentally simulated the bursting of bubbles at the seawater surface of the North-Eastern Atlantic Ocean, analysing the organic materials and the diversity of the bacteria in the source-seawaters and in the produced aerosols. We show that, compared with seawater, the sub-micron aerosol particles were highly enriched in organic matter . Also DNA, viruses and prokaryotes were significantly enriched . The relative importance of the organic components in the aerosol did not reflect those in the seawater, suggesting their selective transfer. Molecular analyses indicate the presence of selective transfers also for bacterial genotypes, highlighting higher contribution of less abundant seawater bacterial taxa to the marine aerosol. Overall, our results open new perspectives in the study of microbial dispersal through marine aerosol and provide new insights for a better understanding of climate-regulating processes of global relevance. Consequently, changes in marine aerosol abundance and/or composition can significantly influence the global climate7. Marine aerosol is composed of diverse inorganic species, but very complex mixtures of organic compounds also contribute to its overall mass6. The quantification and characterisation of these organic materials in marine aerosol is at present a largely unexplored issue and a technical challenge, with available information still very scant11. Recent evidences suggest that marine primary aerosols produced by wind-induced bubble bursting in the ocean12 can be highly enriched in organic matter15 and microorganisms19, including prokaryotes and viruses22.Marine aerosol is of primary importance for atmospheric processes from local to global scale. It influences the radiation balance of the earth, scattering and absorbing solar radiations either directly or by cloud-condensation nuclei (CCN) and ice-nucleating particles (INPs) formation23. This material is cycled/transferred through the food web, and microbes can significantly influence the exchange of organic compounds across the air/water interface and hence the production and composition of marine aerosols27. However, to date attempts to explore the relative transfer of bacteria, viruses and different biopolymeric  organic compounds from the surface seawater to the aerosol is still scarce28.Microbes in the surface oceans are intermediary sources or sinks for inorganic and organic compounds. Surface marine waters typically display higher microbial abundances and concentrations of dissolved and particulate organic matter than in sub-surface waters (up to 3-orders of magnitude)In this study, we conducted bubble-bursting experiments simulating the formation of primary airborne marine aerosols from surface seawater samples collected in the North-Eastern Atlantic Ocean, over a period of two weeks. To provide new insights on the sea-to-air transfer of different organic particles and compounds, we adapted and optimized existing analytical tools to quantify the main biochemical classes of organic compounds , viruses and prokaryotes in marine aerosol samples. Moreover, we quantified for the first time the transfer of extracellular DNA from the ocean surface layer to the aerosol. The diversity of bacterial genotypes was investigated through molecular fingerprinting of seawater samples and of the contextual produced aerosols, to provide new insights on the potential dispersal of bacteria via aerosol and the contribution of different bacterial taxa to the generation of marine aerosols.29. Despite this, the transfer of organics and microorganisms from the sea surface to the atmosphere currently remain poorly investigated when compared to harmful compounds and viral/bacterial pathogens transported through aerosols in urban and rural areas, hospitals or workplaces32.The quantity and quality of the organic fraction of marine aerosols determine their potential to influence Earth climate, with relevance on the functioning of the ecosystems at global scale34, free from the influence of major anthropogenic sources14. The experimental production of sea-sprays, as well as investigations of natural aerosols sampled in marine and coastal areas, typically reveals the presence of large fractions of organic compounds and microorganisms of marine origin35. Our results are consistent with previous findings, indicating the presence of detectable amounts of organic matter in the experimentally produced aerosols, both in the fine (<1.2 µm) and in coarse (>1.2 µm) fractions  as well as the biopolymeric carbon  are transferred from surface seawaters to marine aerosols, along with extracellular DNA, cell fragments, intact viruses and bacteria , but their concentrations in the aerosol did not reflect the patterns observed in seawater samples  displayed a high enrichment factor up to 1005, but t47, our results indicate that viruses and prokaryotes contributed only for 0.5–6.6% of the total DNA content of the organic marine aerosol, suggesting that most of the DNA was extracellular or of eukaryotic origin. These results lead to hypothesize that processes driving the release of extracellular DNA in oceanic surface waters, such as grazing and viral lysis of eukaryotic and prokaryotic cells49, may strongly influence the quantity and composition of organic matter pools of aerosols, especially of their submicron size. However, there are no studies to date on the fate of this biological molecule in the aerosol, nor evidences if aerosolized DNA can drive cloud condensation and/or or ice nucleation, influencing albedo and climate.The DNA concentrations in both fine and coarse aerosol fractions reflected those in the seawater samples (Supplementary Table 5 viruses m−3 and 0.1–2.1 × 105 cells m−3), were consistent with abundances reported so far for natural marine aerosols (0.2–1.8 × 105 viruses m−3 and 0.1–1.2 × 105 cells m−3)28. These results further support the conclusion that our experimental setup simulated realistically the aerosol production processes occurring in the field50. Assuming an average carbon content of 0.2 fg per viral particle and 20 fg per prokaryotic cell43, we estimated that viruses and prokaryotes contributed for less than 0.1% of the biopolymeric organic carbon contained in the aerosols. This result further support the conclusion that the labile fraction of organic aerosol fractions can be enriched in cell debris, exudates, colloidal materials and other dissolved forms of organic compounds of phytoplankton origin21. Our results also indicate that 15–25% of the total aerosol viruses and 10–20% of total aerosol prokaryotes were exclusively associated to the fine aerosol fraction (<1.2 µm). This suggests that previous investigations on marine organic aerosols, based on 1-µm pore size filters20, underestimated the sea-to-air transfer of microbes.The abundance of viruses and prokaryotes in aerosol samples  were filled with 100 litres of seawater through a water supply system  mounted on a five stage Berner impactor operating at a flow rate of 80 L min−1. The concentration of aerosol particles in the tank was set to 3.1 ± 0.8 × 103 cm−3, leading to an estimated production of 2.5 ± 0.7 × 107 particles m−2 s−1 to overlap the range of previously observed in situ source fluxes64. For biochemical, microbiological and molecular analyses, quartz filters were stored at −20 °C until laboratory essay. To provide contextual evaluation of the variables measured in quartz filters, the seawater used for aerosol generation was sampled and pre-filtered onto 10 µm pore-size filters, then processed for biochemical, microbiological and molecular analyses as described below. Overall, we conducted five experiments over a period of two weeks from 19 June to 04 July 2006, and three replicates were analysed in each experiment for each investigated variable. Molecular analyses were conducted only on three of the experiments, because of lack of sufficient material available for analysis in the other two.Samples of seawater were collected 400 km off the west coast of Ireland in the North Eastern Atlantic Ocean on board R/V Celtic Explorer 69. The extracted lipids were then dried at 80 °C and determined spectrophotometrically (at wavelength of 375 nm) after carbonisation at 200 °C in concentrated sulphuric acid70.Quartz filters used for collecting aerosol during bubble bursting experiments were homogenised in sterile and virus free MilliQ water (pre-filtered onto 0.02-μm pore-size filters). The homogenates of quartz filters and the polycarbonate filters  used for concentrating seawater particles were then treated with ultrasounds  before carbohydrate, protein and lipid determinations carried out using spectrophotometric analyses. Following further tests, we verified that the sonication step initially included was not strictly necessary as it did not influence the results in our specific case, thus it can be omitted in similar analyses in the future , bovine serum albumin (from 0.5 to 10 μg ml−1), tripalmitin (from 2.5 to 25 μg ml−1), respectively. For all analyses clean filters of the different pore sizes were processed according to the procedures described above and used as blanks. The detection limits determined through the analysis of blanks added with internal standards were 2.5 μg ml−1, 1.0 μg ml−1 and 5 μg ml−1 for carbohydrates, proteins and lipids, respectively.Carbohydrate, protein, and lipid concentrations in seawater and aerosol samples were obtained by calibration curves using standard solutions of glucose (from 1 to 25 μg ml−1 for 15 minutes at room temperature)71. DNA concentrations were obtained by calibration curves using standard solutions of DNA from E. coli (1–100 ng ml−1).The analyses of DNA in aerosol and seawater samples were performed fluorometrically using SYBR Green I as fluorochrome after digestion of RNA with RNase  of the aerosol, whereas concentrations obtained from the analyses of 1.2–10 μm pore-size quartz filters were defined as belonging to the coarse aerosol fraction (>1.2 μm).Seawater samples were filtered onto Anodisc 25 mm (20 nm pore size) filters. To determine viral and prokaryotic abundance in the aerosol, each size class quartz filter was homogenised in sterile and virus free MilliQ water (pre-filtered onto 0.02 μm pore-size filters) and samples were then treated by ultrasounds  and pyrophosphate  to detach viruses and prokaryotes. Aerosol samples were then centrifuged at 800 × g for 10 minutes and the supernatant filtered onto Anodisc 25 mm (20 nm pore size) filters. All Anodisc filters were stained with 20 μl of SYBR Green I (diluted 20 fold in MilliQ water) for 15 minutes in the dark and then rinsed twice with 1 ml MilliQ water in order to eliminate fluorescence background noise. The filters were mounted on slides using an anti-fade solution  and at least 100 optical fields at 1000 × magnification were examined under epifluorescence microscopy using a Zeiss Axioskop 2 Mot microscope, equipped with a 100 W lamp.−1) to remove any possible extracellular DNA contamination71. The purified extracts of intracellular DNA were then analysed by molecular fingerprinting 72 to provide information on the bacterial diversity in seawater and in the corresponding produced aerosols.The prokaryotic cells contained in the 10-μm pre-filtered source-seawater were concentrated onto 0.05 µm pore size polycarbonate filters (Sterlitech). The corresponding aerosol samples generated during the bubble-bursting experiment were obtained by pooling all quartz filters of the different pore sizes (0.05–10 μm). The DNA contained in the prokaryotic cells collected on the filters was extracted and purified using the QIAamp DNA Micro Kit (QIAGEN). This kit was preferred based on preliminary tests of DNA extractions from quartz filters, indicating higher DNA yields than with the MoBio UltraClean Microbial DNA isolation kit . PCR reactions were performed in 50 µl volumes in a thermalcycler (Biometra) using the MasterTaq® kit (Eppendorf), which reduces the effects of PCR-inhibiting contaminants. We used 30 PCR-cycles, consisting of 94 °C for 1 minute, 55 °C for 1 minute and 72 °C for 2 minute, preceded by 3 minutes of denaturation at 94 °C and followed by a final extension of 10 minutes at 72 °C. To check for eventual contamination of the filters, consumables and PCR reagents, negative controls of MoBio extracts from blank filters  containing the PCR-reaction mixture were run during each PCR reaction. All the negative controls produced no ARISA amplicons, confirming the lack of contamination and the high confidence of the analytical approach. Positive controls containing genomic DNA of E. coli were used and PCR amplicons were checked on agarose-TBE gel (1%), containing ethidium bromide for DNA staining and visualization. Four different reactions were run for each sample and then combined to form two duplicates, subsequently utilised for independent ARISA reactions. The two resulting PCR combined products were purified using the Wizard PCR clean-up system , resuspended in 50 µl of MilliQ water supplied with the clean-up system and then quantified spectrofluorimetrically as described above. For each ARISA run, 5 ng of purified amplicons were mixed with 14 µl of internal size standard  in deionised formamide, then denatured at 94 °C for 2 minutes and immediately chilled in ice. Automated detection of ARISA fragments were carried out using ABI Prism 3100 Genetic Analyzer (Applied Biosystems). ARISA fragments were determined using Peakscanner analytical software (ABI) and the results analysed using standardization of fluorescence among samples, elimination of “shoulder” and non-replicated peaks, and cut-off criterion73. Bacterial genotype richness was expressed as the total number of peaks within each electropherogram, while the evenness  was calculated in order to assess the relative importance of each taxon within the entire assemblage.For ARISA, the purified intracellular DNA was amplified using universal bacterial primers 16S-1392F (5′-GYACACACCGCCCGT-3′) and 23S-125R (5′-GGGTTBCCCCATTCRG-3′). This allowed the amplification of the ITS1 region in the rRNA operon plus ~282 bases of the 16 S and 23 S rRNA75.Measures on surface-water and contextually produced bubble bursting aerosol were compared by means of enrichment factors, following the equation:Supplementary Information


Here, multiple sensors tracked O2 and CO2, gas pressure (ΔP) between internal silage and ambient air, pH and silage temperature (Tsi) during the ensilage of maize and ryegrass. We report the first observation that CO2 produced from microbial respiration was partially dissolved in silage water, with evidence of negative or positive ΔP depending on the changing balance between CO2 production and dissolution. The ΔP < 0 reflected an apparent respiratory quotient (RQ) < 1. Net CO2 production was much greater in anaerobic fermentation stage than in initial aerobic phase or later aerobic feed-out phase. O2 transport into silage is intimately linked to the dynamics of net CO2, ΔP, microbial activity, pH and Tsi. These results suggested that both gas diffusion (based on Fick’s law) and advective transfer (Darcy’s law) play equally important roles in governing the complex temporal progression of inward and outward gas fluxes to and from the silage interior. Even though low pH suppressed microbial activity and supported aerobic stability, the negative ΔP increased the risk of O2 entry and aerobic deterioration during feed-out phase.Silage is a critical global feedstock, but is prone to aerobic deterioration. The dominant mechanism of O It is estimated that 200 million tons of dry matter (DM) are ensiled worldwide annually2. A significant fraction of the nutritive value is lost due to biological oxidation.Silage is an important preservation technique, used world-wide to provide a nutritious and palatable animal feed months after harvest3. The fermentation and storage phases are dominated by anaerobic processes. As a result, silage is susceptible to oxidation because it contains aerobic bacteria, yeasts and molds (fungi) that are dormant under anaerobic conditions. Introduction of oxygen (O2) enables these aerobic microorganisms to be quickly activated, producing carbon dioxide (CO2) and water through aerobic respiration5 and inducing loss of dry matter and nutrients, i.e. “spoilage” of the silage7. During feed-out, when the silo is opened for feeding to livestock, the face of the silage is unavoidably exposed to the oxidizing atmosphere and aerobic losses can be substantial3.The biochemical production of silage relies on lactic acid anaerobic fermentation. The silage-making process is generally divided into four temporal phases: an initial aerobic phase, a fermentation phase, a prolonged storage phase, and a final feed-out phase2 present within the silage is affected by numerous competing factors, including silage density or porosity, moisture content, wind velocity and direction, thermal buoyancy and gradients in gas concentration, density and pressure9. Two physical mechanisms of O2 transport to the silage interior during ensilage and storage have been identified as gas diffusion, which is governed by the concentration gradient between the silage pore space and ambient air, and advective transfer, driven by differential gas pressure (ΔP = Pair − Psilage) across the bunker silo seals. The first may be modelled based on Fick’s law14, and the second based on Darcy’s law18. Despite this mathematical tractability, the dominant mechanism for transport of O2 into the silage remains in question7.The amount of O19. Despite sufficient accuracy of the gas chromatography method, two technical issues are encountered. First, sample extraction may result in additional O2 entering the silage. Second, the method is time consuming and thus unable to track the dynamics of silage gasses. Measurement of pH within the silage during its solid-state fermentation has also been difficult to achieve20.While the biochemical process of silage-making has been mostly characterized, determination of conditions within the developing silage has been challenging. For gas composition and concentration inside the silage, a common method has been to extract gas samples from the silage for later analysis using a laboratory gas chromatograph2, CO2, ΔP, pH, and internal silage temperature (Tsi) over the whole silage-making process. The major objective of this study was to reveal the previously unknown dynamic interactions of the key parameters governing silage development and quality.Despite these challenges, obtaining and characterizing these process level data will contribute to understanding and improving silage yield and quality. Here we present the first multi-sensor-based, simultaneous measurements of internal OA unique suite of sensors was placed in a test silo Fig.  such tha2, CO2, pH, ΔP and Tsi  and relationships between ΔP and CO2 in Test-A were obtained from fresh-chopped maize . The decline in pH lagged the depletion of O2 in both cases , and fresh ryegrass . When pH declined to 4.6 in maize and 5.2 in ryegrass, CO2 production and further changes in pH and ΔP were greatly reduced  and for ryegrass .The relationship between COems Fig. . Piecewisi was observed in both systems during the aerobic phase , pH was low, ΔP was elevated, and all parameters were stable. The experimental system remained anoxic.An increase in Tase Fig. . This st2, CO2, pH, ΔP and Tsi and relationships between ΔP and CO2 in Test-B were obtained with three repetitions  and total-bacteria (106 Cfu g−1) were significantly lower than in ensiled maize  in maize, while both yeasts and molds were below detection threshold (102 Cfu g−1) in ryegrass throughout the experiment (Table 8 Cfu g−1) microbial populations, whereas the fresh ryegrass had higher (108 Cfu g−1) populations than the ensiled ryegrass , was significantly below 1.0 for both ensiled materials  is much greater than that of O2 (1.38 mmol L−1)26.As with the fresh material, observations of 2 concentrations in both ensiled substrates are likely to reflect several interacting factors. First, the produced CO2 was partially dissolved in the interstitial silage water. Second, after the fermentation (anaerobic phase), the residual plant sugar in the ensiled materials was sufficiently low that the CO2 produced from microbial respiration was minimal. In addition, microbial metabolic activity was further depressed by low pH21. The greater CO2 in ensiled ryegrass than ensiled maize reflects the lower density and mass of silage, greater porosity and volume of O2 entrapped, despite the similar O2 concentrations, and less available silage water for gas dissolution. Therefore, more CO2 was produced through aerobic respiration and less CO2 was dissolved in the interstitial silage water.The relatively low COΔP and CO2 in both ensiled maize  and ensiled ryegrass . In both fresh and ensiled materials, the goodness of fit demonstrated that ΔP varied primarily with CO2, rather than with Tsi.As observed for fresh material cf. Fig. , strong ΔP < 0 was observed over a quite short period (around 5 h) following introduction of fresh plant material. The transition of ΔP from negative to positive indicated that the CO2 produced by microbial respiration rapidly exceeded the capacity for CO2 dissolution, while high pH allowed sustained metabolic activity during the early stages of ensilage. Therefore, ΔP is a potentially useful indicator not only of CO2 dynamics, but also of microbial activity. On the other hand, the solubility of CO2 in H2O is a function of numerous interacting factors, including pH, partial pressure of CO2 (Henry’s law), ambient temperature (van’t Hoff equation), interaction between internal gas pressure and temperature , sugar content in water and water content in the substrate30,. Adding further complexity, dissolution of CO2 in water is exothermic27, contributing to the observed increases in both Tsi and ΔP. Since dissolution of CO2 in water is the outcome of the chemical reaction, CO2 + H2O ↔ H2CO326, the decline of silage pH over the silage-making process resulted not only from the anaerobic fermentation of plant sugars such as glucose and fructose to lactic acid by LAB, but also from the dissolution of CO2 in water. As a result, the time courses of net CO2 production and ΔP are not readily simulated using current methods. Here we characterize the trajectory of variation in ΔP (ΔP = 0) → (ΔP < 0) → (ΔP > 0) → (ΔP < 0) throughout the silage production process. Observations of ΔP < 0 indicate apparent RQ < 1, reflecting the sequestration of CO2, and ΔP > 0 reflects the greater amount of CO2 produced during anaerobic respiration. While other gases may contribute to these effects, CO2 dominates because concentrations of H2, CH4 and N2O in silage are very low compared to N2, O2, and CO233, and H2 and N2 exhibit low solubility in silage water.In Test-A, 2 penetrates into the silage. With the two ensiled plant materials, Test-B provided an analog of these biochemical and physical processes of aerobic respiration and partial dissolution of CO2 in the interstitial silage water. Here we infer changes in the dissolved CO2 in the silage water, but carefully evaluate the role of gas phase advective transfer associated with CO2 dissolution. Our unique sensor combination allowed us to follow O2 and CO2, in situ, while tracking the variations of ΔP and Tsi simultaneously, with ΔP employed as an indicator of the net CO2 dynamics based on the ideal gas law. For this reason, Test-B was conducted under gas-tight and constant volume conditions. Most real-world silage-making processes, including the maize bunker silo and ryegrass bale system used here, rarely exhibit this level of seal34. Our experimental design revealed the substantial and contrasting levels of ΔP generated by two common plant materials at the early ensiling stage and in the ensiled state. The rigorous vacuum sealing of our test silos allowed ΔP to vary over a range of −7.5 to + 45.4 kPa for maize and of −3.1 to 19.1 kPa for ryegrass.For a field-scale bunker silo during feed-out phase, the working face is exposed to air as the silage is unloaded for feeding to livestock. This results in re-ensiling of the remaining material and unavoidable aerobic deterioration in the surface zone as O2, its partial dissolution in silage water, and resulting negative and positive pressures, depending on stage of ensilage. The ΔP > 0  phenomenon has been reported previously35. One study19 observed an increase in O2 and N2 but a decrease in CO2, measured at the end of the eight-day fermentation phase, and inferred a reversal of gas movement from efflux to influx, as a result of the reversal of ΔP from positive to negative. However, fundamental processes and causative factors were not explored. Our multi-sensor measurements provide insight into these parameters with essential interactions and dynamics, and the resulting relationships between net CO2 production, O2 consumption, and variations in ΔP, pH and Tsi.To our knowledge, we provide the first evidence of the physical and chemical interactions involved in the production of COIn situ tracking of O2 depletion, CO2 production and dissolution, pH lowering-processes and ΔP dynamics has been realized during early and late phases of silage production, using multi-sensor techniques. CO2 production was much greater from anaerobic than aerobic processes. The ΔP exhibited both positive and negative values, indicative of net values of CO2 production and dissolution. For the two common silage materials tested here, the resulting ΔP is of the order of tens of kPa. Since the negative ΔP allowed more O2 entry through the silo face, risk of aerobic deterioration in the feed-out phase could be potentially higher than anticipated. These interactions are diagnostic of the previously unknown behavior during ensilage and re-ensiling processes. These results suggest that both gas diffusive and advective transfer play equally important roles in governing the complex temporal progression of inward and outward gas fluxes to and from the silage interior. Future efforts to develop simulation of the ensilage process should incorporate both Fick’s law and Darcy’s law. The fusion of our data with improved process models will support development of improved silage management.2 as it is converted to CO2, in order to interpret the resulting ΔP and Tsi dynamics. Cross calibration of the O2 and the CO2 sensors were carried out in advance of the experiment36.Table 37, by extracting 25 g of maize or ryegrass material with 0.225 L deionized water in a glass vessel for 30 min. This provided an initial reference value for comparison with the initial value of the inserted pH sensor when encased in the porous plastic wetted with the same solution. In addition, the pH sensor was repeatedly calibrated against three buffer pH-solutions  at 20 °C prior to and after the experiment.During each test period the pH sensor tip (diameter 2 mm) was enclosed in a small piece of well-wetted porous plastic to improve hygroscopic contact with the test material. The porous plastic was rinsed with deionized water and then dried naturally. Initial pH was determined using a standard protocol for silageTwo PVC cylinders  together with a lid  were used as the experimental silos   and homogenized in a mixer for one minute. From this suspension, total bacterial counts were analyzed on plate-count agar   after aerobic incubation at 30 °C for 2 days. Lactic acid bacteria (LAB) were quantified from this suspension on de Man, Rogosa and Sharpe (MRS)-agar  . MRS agar plates were incubated anaerobically using an Anaerocult system . Yeasts and molds were detected using yeast extract glucose chloramphenicol (YGC)-agar  . These agar plates were incubated at 25 °C for 3 days.Thirty grams of silage were suspended in 270 ml of ¼-strength Ringer solution (2.25 g lZea mays) including the cobs  and fresh-cut ryegrass . The resulting biomass was finely chopped and packed to high bulk density (BD) in separate cylinders  using a hydraulic ram (360 mm diameter circular foot), in approximately six layer increments to ensure uniform density. Five holes  through the top layer of silage, aligned with the holes in the lid, were drilled using a hole-saw for the installation of O2, CO2, ΔP, pH and Tsi sensors.This was an analogue to the ensiling phase using fresh chopped whole shoots of maize  were conducted in a large insulated workshop in the summer of 2016.Test-B used previously ensiled maize with DMC of 35% and ryegrass with DMC of 43%. Ensiled maize (harvested and ensiled autumn 2015) was obtained from 20 cm behind the freshly cut silage face in a concrete bunker silo (40 × 6 × 3.5 m), located at the Frankenforst research farm . Ensiled ryegrass was obtained from a silage bale (harvested and ensiled autumn 2015), which was stored outside, wrapped in four layers of plastic film (thickness: 0.025 mm). The maize and ryegrass silages were re-ensiled in the test silos, and packed with the hydraulic ram to a high BD of 625 kg m


Importantly, we show that this isolated domain is capable of de novo synthesis of the viral genome and efficient elongation without terminal nucleotide transferase activity. Altogether, this isolated polymerase domain will be a precious tool to screen and optimize specific nucleoside and non-nucleoside inhibitors to fight against Zika infections.Mosquito- and tick-borne pathogens including Chikungunya, Dengue, Japanese encephalitis, West Nile, Yellow fever and Zika virus, represent a new economic and public health challenge. In the absence of effective vaccines and specific therapies, only supportive regimens are administrated for most of these infections. Thus, the development of a targeted therapy is mandatory to stop the rapid progression of these pathogens and preoccupant associated burdens such as Guillain-Barre syndrome, microcephaly. For this, it is essential to develop biochemical tools to help study and target key viral enzymes involved in replication such as helicase complexes, methyl-transferases and RNA-dependent RNA polymerases. Here, we show that a highly purified ZIKV polymerase domain is active  While ZIKV infections in humans are mainly asymptomatic (in 80% of cases), a smaller proportion of patients present benign febrile symptoms also called Zika fever. Guillain-Barre syndrome (GBS) is characterized by a paralysis of the peripheral nervous system and usually affects 1 to 2 out of 100,000 persons per year. Yet in countries where Zika infection is present, an unexpected increase of GBS has been observed. The prevalence of these neurological reversible damages is about 20-fold higher in Zika infected patients. Thus, Zika infection appears to be an trigger of GBS3. In addition, the increase occurrence in fetal microcephaly has been linked to the recent Zika virus outbreak as ZIKV was detected in the brain tissue of deceased foetus5. Accordingly, Zika has been declared a public health emergency of national importance in Brazil6. Because there isn’t a vaccine nor a treatment specifically developed to block the worldwide expansion of this epidemic, there is an urgent need to develop a targeted therapeutic strategy which will require a better understanding of the virus replication.Zika virus (ZIKV) is a mosquito-borne member of the genus The viral genome is a (+)-sense single-stranded RNA of approximately 10,800 nucleotides containing a single open reading frame (ORF). The Cap-dependent translation of the full-length polyprotein is followed by a maturation step leading to the generation of three structural proteins , and seven non-structural proteins . The non-structural proteins associate themselves into a replication complex within membranous structures at/or derived from the endoplasmic reticulum. Efficient replication of the viral genome relies on the presence of two regulatory elements flanking this large ORF, the untranslated regions (the 5′ UTR and 3′ UTR). Actually, the RNA-dependent RNA polymerization is catalyzed by the NS5 protein which also harbors a N-terminal methyl-transferase domain (MTase) which activity combined to NS3’s 5′RNA phosphatase activity enables the capping of the nascent genomic RNA. Because the synthesis of an intermediate negative strand and subsequently new genomic copies is obviously essential, the RNA-dependent RNA polymerase (RdRp) activity constitutes a target of choice for drug development.7 or the MTase domain of NS58. That being said, little biochemical studies has been published on these essential non-structural proteins while it is clearly needed for a better understanding of the molecular process of infection and pathogenicity9. Only recently Hercik et al. described the purification of a ZIKV full-length NS5 protein (MTase and RdRp domains)10. In this study, the full-length NS5 was capable of an uncharacterized nucleotide incorporation using a primed template enabling the authors to test nucleoside analog inhibitors. Here, we report the purification of the NS5 isolated RdRp domain and characterized its enzymatic activity. Notably, we found that the RdRp domain is active in the absence of the MTase domain and is capable of de novo synthesis that was not limited to terminal nucleotide transferase activity using both homopolymers and specific RNA templates mimicking the natural viral UTR substrate. Of note, since the original submission of this work, several new studies have been published on ZIKV RdRp purification and inhibition that corroborates our results14.As of today, research on Zika has been focusing on developing diagnostic tests, setting of working cellular models of ZIKV and bio-physic approaches on certain viral proteins such as the NS2B-NS3 helicase complex15, we used the cleavage site sequence GLVKRR/GG described between NS4 and NS5 to define the ZIKV NS5 N-terminus. Then, the 903 amino acid long protein from the SPH2015 strain16 was used to perform a 3D structure prediction using the I-TASSER bioinformatics web tool18. The model identifies 2 distinct domains connected by a short linker . The resulting 1914 nucleotides long DNA was obtained by gene synthesis and cloned in a pET-21a vector. After expression in Rosetta cells, the RdRp isolated domain was purified using a regular Nickel affinity column, followed by a heparin column and subsequently a gel filtration column. To ascertain that the observed activity can be attributed to the purified NS5 RdRp and not a potential remaining contaminant, we designed a catalytically inactive version by mutating the active site from 664GDD666 to 664GND666. Mutation was introduced in the polymerase sequence and the mutant protein purified through the same procedure that was developed for the WT enzyme. The elution profile of both enzymes after exclusion chromatography was similar and showed a unique pic  from the SPH2015 strain was optimized for bacterial expression to avoid the use of rare codons, a sequence coding for an 8xHis tag was added in 5′ and silent mutations were introduced in the body of the gene to eliminate the BamHpic Fig. . Followipic Fig. , >98%. T2+ as metal cofactor with various non-specific template-primer combinations -oligo(rU) substrate  substrate was used without primer  template. Altogether, it appeared that all three steps included in our purification procedure were required in order to obtain an active RdRp exempt from other polymerase contaminant.To further assess the gain in purity of the RdRp domain along our purification procedure, we used an aliquot of each purification steps in this ant Fig. . Using 1ant Fig. . When tevs de novo synthesis leading to the replication of a template), we performed similar assays in the presence of [α-32P]-rGTP and visualized the products on a polyacrylamide gel  strand. This sequence corresponds to the entire UTR that the RdRp is expected to recognize to synthesize the genomic (+) strand. During a time-course experiment, we observed the accumulation of a product at the expected size  strand RNA as template without any primer.Then, the ability of the ZIKV RdRp to replicate ize Fig. . Accordiin vitro data obtained in our laboratory indicated that adding portion of the genomic ORF to the UTR sequence increased the replication efficiency of the RdRp from hepatitis C virus (HCV NS5B), compared to using the UTR alone as template. To investigate the possibility that additional sequences/structures may improve the RNA synthesis by the ZIKV polymerase, we generated a new RNA template of 269 nucleotides and compared the activity of the RdRp on this substrate to that of the 153 nucleotide-long template described above  strand 5′ UTR from the negative template]. Yet, this activity was limited to the use of Mn2+ as a metal cofactor and Mg2+ dependent activity was observed only with an oligo(rC) substrate. In agreement with our results, a de novo activity was also described in a concomitant study by Xu and collaborators using a ZIKV minigenome RNA12. In addition, a primer dependent activity was also recently described using a homopolymeric template12 or a short template/primer system corresponding to the 5′ end of the genome as primer and the 3′ end of the antigenome as template11. In parallel, Hercik and collaborators described the purification of the ZIKV NS5 with both the MTase domain and RdRp domain leading to a Mg-dependent activity10. However, the substrate used was different [a poly(rU) template with an oligo(rA) primer] and bypassed the de novo initiation expected for flavivirus polymerases. Besides, the experimental setting used did not enable to distinguish if the active incorporation observed was indeed synthesis or a terminal transferase activity. Importantly, recent structural studies in the context of other flavivirus NS5 proteins have shown that the MTase domain may affect the RdRp overall activity thanks to allosteric interactions. Thus, it is tedious to rule out the impact of the metal cofactor in ZIKV NS5 polymerase activity. In past decades, a tremendous effort has been made to develop efficient and specific viral polymerase inhibitors. While first generation drugs such as AZT presented important drawbacks with serious side effects, rigorous drug development has led to the identification of more advanced and selective drugs. Our work demonstrates that the isolated ZIKV RdRp domain can be used in vitro to evaluate specific compounds and will help the repositioning of already approved drugs able to inhibit other viral polymerases. A potential lead could be molecules developed against the HCV NS5B protein such as sofosbuvir and other 2′-C-methylated nucleosides that have recently been shown to be active against ZIKV NS5 in vitro or the ZIKV replication in Vero cell20. However, in silico studies performed on the ZIKV polymerase have predicted that most HCV NS5B inhibitors could be active against ZIKV21. Yet, among 30 nucleotides analogs tested in another study using cellular models, only few molecules were able to inhibit Zika infection22. This discrepancy highlights the need to biochemically validate the activity and mechanism of action of these inhibitors on the RdRp activity. In addition, this will enable the screening of chemical libraries which will also enable the identification of new drugs that similarly to HIV-1 non-nucleoside RT inhibitors may target more specifically the ZIKV polymerase preventing deleterious side effects.Here, we describe the purification of an active ZIKV RdRP isolated domain. The enzyme is fully proficient for I recognition site that was subsequently removed and the sequence corresponding to an 8xHis tag was added in 5′ of the sequence. The corresponding DNA coding for a 72 kDa protein was obtained by gene synthesis  and inserted in the multi-cloning site of the pET-21a vector between BamHI and XhoI sites.The coding sequence of ZIKV RdRp domain used in this study corresponds to the SPH2015 strain that has been optimized for codon usage using the GENEius software (Eurofins Genomics GmbH) followed by Pro codon optimization for bacterial expression (Genbank No. KY364193). The adapted gene harbored an internal BamHRosetta cells were transformed with the pET-21a-ZIKVRdRp and bacteria are grown on LB plates containing ampicillin (100 µg/ml) and Chloramphenicol (34 µg/ml) overnight at 37 °C. Selected clones were expanded in 500 ml of liquid medium with the same antibiotics until an OD of 0.8 is reached (37 °C with agitation at 220 rpm). Protein expression was then induced by addition of IPTG at a final concentration of 2 mM and culture overnight at 18 °C and 220 rpm. After centrifugation at 2500 rpm for 5 min, bacteria pellet were resuspended with 20 ml of lysis buffer  and incubated for 10 min at room temperature. After 3 × 20 sec sonication, soluble proteins were clarified by centrifugation 30 min at 12000 rpm . The supernatant was applied onto a Nickel crude Hitrap column  equilibrated with buffer A . After washing of the column with 5 volumes of buffer A, bound proteins were eluted in one step using 250 mM imidazole. Fractions containing the RdRp domain were determined by SDS-PAGE, pooled and diluted 1.5 fold with buffer B (corresponding to buffer A without NaCl and imidazole) before loading onto a Heparin-HiTrap column  equilibrated with buffer B. Proteins elution was performed following a one-step increase in NaCl to 250 mM. Fractions containing the RdRp domain were determined by SDS-PAGE, pooled (1.2 ml) and concentrated by centrifugation on Amicon 30 kDa until a final volume of about 200 µl for optimal loading onto a Superdex 200 10/300 column (24 ml). Size exclusion was performed with a running buffer composed of 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.001% reduced Triton and 10% glycerol. Purity of the protein was evaluated by SDS-PAGE. Concentration was measured using a NanoDrop 2000 spectrophotometer (Thermoscientific).DpnI digestion for 1 hour at 37 °C (10 U/50 µl reaction). DH5α chemically competent cells were transformed with the digestion product. After plasmid extraction (standard miniprep from Macherey-Nagel following manufacturer’s instructions), the presence of the desired mutations and the integrity of the ZIKV RdRp sequence were verified by DNA sequencing (MWG Eurofins). Primers sequences for mutagenesis were ATGGCAGTCTCAGGCAATGATTGTGTGGTGAAACC TATCGATGACC and CACAATCATTGCCTGAGACTGCCATGCGTTTCAAG CGGTCCCACCC. Primers AGGAGGTGTTAGAAATGCAA and TTATCCCAGCCAGTGGATGG were used for forward and reverse gene sequencing respectively.Site-directed mutagenesis was performed on the pET21a-ZIKRdRp plasmid (7323 bp) following a standard PCR-based protocol using degenerated primers obtained from MWG Biotech. The amplification reaction was performed using a step at 98 °C for 30 seconds, followed by 20 cycles of 10 seconds at 98 °C, followed by 45 seconds at 60 °C and 1 minute per kb at 72 °C; and finally a step of 10 minutes at 72 °C. Amplification products were subjected to in vitro transcription. Primers sequences were GTTGTTACTGTTGCTGACTCAGAC along with TAATACGACTCACTATAGGGCATATTGACAATCCGGAATCCTCC or TAATACGACTCACTATAGGGCTCAAAAAGGCTAGAATTGCCAAG to produce DNA templates used to generate the 153 and 269 nucleotides long RNAs, respectively. After dsDNAs purification using phenol/chloroform extraction and precipitation, substrate RNAs were obtained by in vitro transcription using the MEGAscript kit (Ambion) according to manufacturer’s instructions. After DNase I digestion  acidic phenol/chloroform extraction and precipitation, purity and integrity of the generated RNAs were determined using capillary electrophoresis on a Bioanalyzer 2100 (Agilent).The plasmid pEx-5UTRms3UTR was constructed to contain the 5′UTR and 3′UTR from the SPH2015 strain separated by a multi-cloning site with the following sequence GGATCCAAGCTTCTCGAGGCGGCCGCTCTAG. RNA sequences corresponding to 3′-end of the (−) strand were chosen to be used as substrate in the polymerase activity assay. PCR was performed using the Phusion Hotstart Flex DNA Polymerase (Biolabs) with primers designed to introduce a T7 RNA polymerase promoter enabling the generation of substrate RNAs by 3H]-XTP (rUTP at 12.2 Ci/mmole and rGTP at 41.8 Ci/mmole) and 3 mM MnCl2. For de novo synthesis, catalysis was performed in the presence of (i) a poly(rC) template (0.4 OD) and 200 µM rGTP, (ii) 1 µM oligo(rC) (20-mer with a 3′-OH extremity or blocked with cordycepin) and 5 µCi [α32P]-GTP (3000 Ci/mmole), (iii) 1.5 pmoles of RNA templates, 0.5 mM rATP, 0.5 mM rCTP, 5 µCi [α32P]-GTP (3000 Ci/mmole) and 5 µCi [α32P]-UTP (3000 Ci/mmol). De novo reactions were performed in the presence of 3 mM MnCl2 except for oligo(rC) where MgCl2 (5 mM) was used.Reactions were performed for 2 hours at 30 °C with various amounts of purified RdRp in a reaction buffer containing 20 mM Tris-HCl pH 8.0, 1 mM DTT and 13.2 U RNasin (Promega). For primer-dependent reactions, 0.48 OD of poly(rA)-oligo(rU) or poly(rC)-oligo(rG) were incubated with 2 µCi . Products were separated on 12% polyacrylamide denaturing sequencing gels. Autoradiography was performed using an imaging plate (Fujifilm) and images were obtained with a FLA-5000 Imaging System (Fujifilm).