Natural nanoparticles are fundamental building blocks of Earth’s bio- and geosphere. Amorphous silica nanoparticles are ubiquitous in nature, but fundamental knowledge of their interaction mechanisms and role in mineral replacement reactions is limited. Here we show how silica nanoparticles replace Cretaceous calcite bivalve shells in a volume- and texture-preserving process. Electron tomography reveals that mineral replacement transfers calcite crystallographic orientations to twinned photonic crystals composed of face-centered cubic silica sphere stacks. During the face-specific replacement process, silica nanoparticles continuously nucleate, aggregate, and form a lattice of uniform spheres parallel to calcite low-energy facets. We explain the replacement process with a new model that unifies recently proposed, probably universal mechanisms of interface-coupled dissolution-precipitation and aggregation-based crystallization; both key mechanisms in geological processes and nanomaterials design and synthesis. In recent years, amorphous nanoparticles have increasingly been identified as fundamental precursors for crystal formation and growth processes in natural systems7. These processes are classified as crystallization by particle attachment (CPA) (ref.8). However, despite its ubiquitous nature and prototype character for synthetic colloidal crystals9, fundamental knowledge of silica nanoparticle formation and ordering processes in nature is limited10. This knowledge gap also includes the globally widespread interaction of calcium carbonate minerals with amorphous silica13. Our analytical results and schematic model fill this gap and show that calcite dissolution and silica precipitation are linked by a fluid-mediated replacement process that transfers atomic crystallographic information to an amorphous, mesoscale-ordered material in a natural weathering environment.In a first approximation, the Earth’s crust consists of silicate rocks. Chemical weathering reactions of these rocks lead to the 3). The twinned condition of the lamellae is visible as a periodic color variation in an A-B-A-B pattern. Within some lamellae, thin straight lines intersect with twin planes at an angle of 71.2 ± 1.5° ; thus, it evidences that the replacement reaction is intimately coupled to calcite crystallographic orientations. The color variations of the photonic crystals arise from Bragg diffraction effects from colloidal arrays, visualized directly by scanning electron microscopy (SEM), showing that the crystals consist of coherently ordered silica spheres and air-filled pores  from unsilicified Early Cretaceous Bulldog Shale - a deeply chemically weathered, kaolinitic, marine siltstone fcc. For preservation in the colloidal crystal, the calcite {104} lattice planes must be thermodynamically most stable and dissolve the slowest. Both theoretical and experimental work16 support this basic principle, which underlines the importance of anisotropic dissolution rates17 to extrapolate laboratory-based results to natural systems.To evaluate how effectively the templating process captured the crystallographic elements of the calcite precursor, we analyzed the three-dimensional (3D) silica sphere packing and orientation in twin lamellae using focused ion beam (FIB)-SEM nanotomography at a voxel size of 7 × 7 × 20 nm11 or silicification controlled by the force of crystallization12. Both concepts are inconsistent with the studied material due to (i) the preservation of crystallographic orientation and (ii) the amorphous and nanoporous nature of the replacement silica. In fact, our observations point to a coupled dissolution-precipitation mechanism along a moving reaction interface18, controlled by fluid chemistry between the calcite dissolution front, {104}calcite, and the silica sphere deposition front, (111)fcc.We combine our results into a generalized model that describes the pseudomorphic replacement of an atomic crystal by the orders of magnitude larger nanoparticle architecture, via a crystallization by particle attachment process Fig. . To date2+ and CO32− into the fluid film and creates a sharp localized pH and salinity rise19 that gradually decreases toward the bulk fluid. This local change lowers the amorphous silica solubility, induces supersaturation and precipitation21, and confines particle nucleation close to the moving calcite dissolution front. In contrast to the classical LaMer nucleation-and-growth22 and Ostwald ripening mechanisms23, particle growth in the natural precipitating system is not temporarily limited by the silica monomer concentration and does not compete with nucleation. Thus, particles grow homogeneously along a multistep aggregative pathway of increasingly large units at increasing distance from the calcite surface, in a process analogous to the formation of uniform silica nanocolloids from alkoxysilanes24 and hydrothermal brine solutions4.The highly selective nature of the replacement reaction indicates that calcite dissolution initiates silica precipitation. The dissolution reaction releases Ca20. Electron probe microanalyses show that up to 1.72 wt% of the fluid cations  planes that stack layer-by-layer parallel to the {104} lattice plane of calcite. Ultimately, the colloid undergoes a phase transition into the long-range ordered, thermodynamically favored fcc lattice9 that forms the bulk photonic crystal.The uniformity of the sphere arrays described herein requires constant physicochemical conditions in the fluid film  throughout the entire replacement process. The nanoporous sphere fcc geometry (~26% porosity) is permissive and enables mass transport and the necessary permanent fluid access to the calcite dissolution front. As soon as the calcite dissolution and silica deposition fronts advance synchronously, a steady state is established and small particles form continuously until the replacement process is completed. Once these particles move due to their Brownian motion, the competition between van der Waals attraction and electrostatic repulsion drives their interaction potential. The ions in the interfacial fluid film can neutralize the particle surface charge, compress the repulsive electrical double-layer, and promote the irreversible aggregation of marginally unstable particles25, or other replacement reactions crucial for palaeoenvironmental isotopic and redox condition studies such as chert nodule5 and pyrite (FeS2) formation26. The oriented attachment of crystalline nanoparticles8 can be incorporated into our model and explains the transfer of crystallographic information (epitaxy) across the fluid film in interface-coupled dissolution-precipitation reactions. In addition, our results suggest a potential atomic crystal templating approach for the controllable synthesis of 3D self-assembled functional materials with ordered lattices and a single domain size of several millimeters. In the light of constant progress in high-resolution analytical instrumentation27 and ever-increasing evidence of nanoparticles as fundamental building blocks in natural environments8, we propose that nanoparticle-based mineral formation is a key mechanism in fluid-mediated replacement reactions in geological processes and the synthesis of advanced functional materials, such as molecular sieves, heterogeneous catalysts, semiconductors, and photonic devices29.The model proposed herein offers a crystallization by particle attachment process that potentially operates over a wide range of replacement reactions by initially nanocolloidal amorphous and crystalline phases. These replacement reactions include, for example, silica and carbonate pseudomorphism and fossilizationFour bivalve shell samples with their respective host rock were collected in the Allan Rise precious opal field (WGS84 -29.3974°N 134.8581°E) located 52 km south of the Coober Pedy Township . The samples were extracted from bulldozer cuts approximately 20 m below the present day surface. Shell samples analyzed are white to milky with moderate play-of-color (POC).Bivalve shells and their friable porous host rock .The morphologies of silica spheres and their subparticles were investigated on surfaces of thin sections and freshly fractured material by SEM. Specimens were etched in 10 vol% HF solution for 15 s, dried, and sputter-coated with ~15 nm W. Secondary electron images were obtained with a Zeiss Supra 40 VP Ultra SEM instrument, at an acceleration voltage 5 kV and beam current of 10 nA. Subparticles were identified with the InLens detection mode. For each specimen, sphere and subparticle diameters have been determined by averaging over at least 1000 manually measured particles. The sphere diameter dispersity has been calculated as relative standard deviation. The sphere stack geometry of adjacent twin lamellae was characterized by FIB-SEM nanotomography of three pre-selected regions on thin sections. The thin sections were carbon-coated (~5 nm) to enhance electrical conductivity and mounted onto a SEM specimen stub. The sample sides were covered with silver glue to improve charge conduction between the top surface and the stub. Focused ion beam (FIB)-SEM nanotomography was conducted with a FEI Helios Nanolab 600 scanning electron microscope equipped with a focused Gallium-ion beam. To protect the sample from beam damage during FIB-milling, the surface was covered twice with a platinum layer using ion beam-induced deposition. A block face was generated by milling a regular cross section perpendicular to the twin plane of adjacent lamellae. After a cleaning cross section the area for the slice and view process was defined. Slice and view was performed using the software Auto Slice & View (FEI). Ion beam milling was conducted at 30 kV with a beam current of 93 pA. Secondary electron images were obtained at an acceleration voltage of 5 kV and beam current of 86 pA. The total number of slices ranged between 140 and 200 per sampled region. With each step, 20 nm of the material was removed and the newly produced surface was imaged with the SEM (Supplementary Video 30. We used the SIFT plugin31 for automatic alignment of the slice stacks. The aligned stacks were cropped and processed to reduce image noise using a fast Fourier transform filter. Subsequently, a manual grey level thresholding was applied to each image to segment the pore space from the data. Prior to image binarization, this image correction procedure minimized the interfering effects of scan distortions and electron charging during image acquisition. Segmented image stacks were directly volume-rendered by the Voxel function in Avizo 7.0, as shown in Supplemental Figure Secondary electron images have been processed with the open source software FijiSupplementary InformationSupplementary Video S1Supplementary Video S2


Transcript abundances of hzsA genes encoding hydrazine synthase exceeded nirS and nirK transcript abundances encoding denitrifier nitrite reductase by up to two orders of magnitude, providing further support of a predominance of anammox. Anammox bacteria, dominated by groups closely related to Cand. Brocadia fulgida, constituted up to 10.6% of the groundwater microbial community and were ubiquitously present across the two aquifer assemblages with indication of active anammox bacteria even in the presence of 103 μmol L−1 oxygen. Co-occurrence of hzsA and amoA gene transcripts encoding ammonia mono-oxygenase suggested coupling between aerobic and anaerobic ammonium oxidation under suboxic conditions. These results clearly demonstrate the relevance of anammox as a key process driving nitrogen loss from oligotrophic groundwater environments, which might further be enhanced through coupling with incomplete nitrification.Despite the high relevance of anaerobic ammonium oxidation (anammox) for nitrogen loss from marine systems, its relative importance compared to denitrification has less been studied in freshwater ecosystems, and our knowledge is especially scarce for groundwater. Surprisingly, phospholipid fatty acids (PLFA)-based studies identified zones with potentially active anammox bacteria within two superimposed pristine limestone aquifer assemblages of the Hainich Critical Zone Exploratory . We found anammox to contribute an estimated 83% to total nitrogen loss in suboxic groundwaters of these aquifer assemblages at rates of 3.5–4.7 nmol L Over the last decades, human impact on the nitrogen cycle has resulted in increasing concentrations of nitrate in groundwater, which is of growing concern on a global scale  located in Thuringia, Germany. A monitoring well transect offers access to two superimposed limestone aquifer assemblages, established in the framework of the Collaborative Research Center (CRC) AquaDiva  after steady state in the physical and chemical conditions were established. Water temperature, dissolved oxygen concentration, pH, and redox potential were measured in a flow-through cell in the field using respective probes  added prior to GC analysis and relative to a standard mixture  measured in 5 different concentrations between 2 and 40 ng/μl. This approach required a recalculation of ladderane data already presented in Schwab et al. (Ladderane phospholipid derived fatty acids (PLFA) were extracted from glass fiber filters and subsequently purified using a slightly modified method of the common PLFA extraction method, as previously described  and 15N15N (30N2) as previously described  nitrite and ammonium already present was taken into account, with the labeled fraction determined as the ratio of the concentration of 15N labeled nitrite or ammonium added to the total concentration of nitrite or ammonium, respectively. For nitrite, the total concentration was determined as the sum of the background concentration and the concentration of 15N added, and for ammonium, the total concentration was measured after addition of 15A hzsA genes encoding hydrazine synthase subunit A of anammox bacteria  in accordance with the manufacturer's protocols (Promega). Plasmids were extracted using GeneJET plasmid miniprep kit  and sequencing of cloned inserts was performed at Macrogen (The Netherlands).Genomic DNA and total RNA were extracted using the PowerSoil DNA isolation kit  and the PowerWater RNA Isolation Kit , respectively, according to the manufacturer's protocol. Processing of RNA was performed as described previously  on a Mx3000P qPCR cycler (Agilent Technologies) using Maxima SYBR Green Mastermix (Thermo Fisher Scientific). QPCR targeting bacterial 16S rRNA genes, hzsA, nirK, and nirS genes was performed for monthly samples from January 2014 to August 2015, while qPCR targeting amoA genes and transcripts of hzsA, nirK, nirS, and amoA was only performed for samples obtained in August and November 2015. In detail, the following primer combinations were used: Bac8Fmod/Bac338Rabc  for the functional genes and from 5 × 102 to 5 × 108 copies per reaction for bacterial 16S rRNA genes.Quantification of bacterial 16S rRNA genes, and nirS and nirK genes were generated from genomic DNA only (August 2014) using the same primer combinations as for cloning and qPCR. Generation of barcoded amplicons and amplicon sequencing using the Illumina MiSeq platform was performed by LGC Genomics (Berlin). The PCRs included about 5 ng of DNA extract, 15 pmol of the respective forward and reverse primer in 20 uL volume of 1 × MyTaq buffer containing 1.5 units MyTaq DNA polymerase (Bioline) and 2 μl of BioStabII PCR Enhancer (Sigma). For each sample, the forward and reverse primers had the same 10-nt barcode sequence. PCRs were carried out for 30 cycles using the following parameters: 2 min 96°C predenaturation; 96°C for 15 s, 50°C for 30 s, 70°C for 90 s. For nirS, cycling conditions were similar but annealing was at 56°C. About 20 ng amplicon DNA of each sample were pooled for up to 48 samples carrying different barcodes. The amplicon pools were purified with one volume AMPure XP beads (Agencourt) to remove primer dimer and other small mispriming products, followed by an additional purification on MinElute columns (Qiagen). About 100 ng of each purified amplicon pool DNA was used to construct Illumina libraries using the Ovation Rapid DR Multiplex System 1–96 (NuGEN). Illumina libraries were pooled and size selected by preparative Gelelectrophoresis. Sequencing was done on an Illumina MiSeq using V3 Chemistry (Illumina).16S rRNA-gene based analysis to assess the structure and taxonomic affiliation of the total bacterial community was performed for samples taken in November 2015 when anammox rate measurements were carried out. To confirm the observed community patterns, we performed additional 16S rRNA gene-targeted amplicon sequencing from previous time points to link community structure information to the quantification of functional gene transcripts (August 2015) and to characterize the total bacterial population and the population with the potential for protein biosynthesis . For phylogenetic analysis of cloned hzsA fragments, reference alignments of deduced hzsA protein sequences were generated in ARB. Sequences obtained in this study were translated to deduced protein sequences and aligned against the reference alignment. Species-level OTUs were assigned based on a 0.03 distance cut-off on protein level. Closest relatives were determined based on a BLAST search using blastx against the non-redundant protein sequence (nr) database at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and using phylogenetic tree construction in ARB. Sequences obtained in this study have been submitted to the European Nucleotide Archive (ENA) for results of MiSeq Illumina amplicon sequencing  and to Genbank for clone library-based sequencing .Because of insufficient amplification of it Hall, , and theit Hall, . After r−1) and generally low microbial biomass as approximated by ~108 bacterial 16S rRNA genes per L. The two aquifer assemblages differ strongly in oxygen availability with suboxic (<10 μmol oxygen L−1) to anoxic conditions in the upper aquifer assemblage (HTU) and oxic conditions in the lower aquifer (HTL) , or in the oxic wells .Abundances of amoA genes and transcripts of ammonia-oxidizing archaea and bacteria for seven groundwater wells in August and November 2015. amoA gene abundances pointed to similar or only slightly lower total abundances of aerobic ammonia oxidizers in suboxic to anoxic wells H52 and H53 compared to the oxic well H41 and to even higher abundances compared to oxic well H51 . For both time points, transcripts of bacterial and archaeal amoA genes were detectable in the oxic groundwater of HTL but also at H52 and H53. We calculated gene and transcript ratios of [hzsA/sum of archaeal and bacterial amoA] across sites as an indicator of a predominance of either anaerobic or aerobic ammonium oxidation in the genetic potential or transcriptional activity of the groundwater microbial communities. hzsA/amoA ratios ranged from 0.1 to 444.7 and from 1.2 to 1087.6 on the gene and transcript level, respectively  followed by Nitrospirae (1–32%), Betaproteobacteria (3–17%), Deltaproteobacteria (4–12%), and Alphaproteobacteria , and two OTUs related to the genus Azospirillum . These OTUs accounted for more than 70% of the sequence reads for wells H32, H52, and H53, while they were only represented by few sequence reads at sites H42 and H43 , Ideonella sp. (83% sequence identity), and poorly characterized nirS-type denitrifiers , with the latter accounting for 20–28% of all the nirS sequence reads detected at wells H42 and H43. Since the nirS primer set used in this study may discriminate against some denitrifying genera due to mismatches in the primer binding region .MiSeq Illumina amplicon sequencing of the less abundant 2 L−1 d−1 in the groundwater of suboxic to anoxic well H53 but also demonstrated its high relevance compared to denitrification for the removal of fixed nitrogen at an estimated contribution of 83%. Well H53 and the neighboring well H52 showed a very high similarity regarding hydrochemical properties supportive of the anammox process. Moreover, previous findings from PLFA analysis and proteomics provided strong support that active anammox is also ongoing in the groundwater of well H52, where one third of the identified proteins were associated with Brocadiales , indicative of active CO2-fixation via the acetyl-CoA-pathway and characteristic of anammox bacteria . Association with aerobic heterotrophs might provide microenvironments at reduced oxygen concentrations. Moreover, a recent study reported a contribution of anammox to in situ N2 production of up to 58% in permeable riverbeds at an oxygen concentration of 134 ± 14 μmol L−1 (Lansdown et al., Cand. Brocadia, mostly described from terrestrial habitats (Humbert et al., −1 (Oshiki et al., Beyond the identified anammox hotspot, our results have clearly demonstrated the ubiquitous presence of anammox bacteria along the two limestone aquifer assemblages of the Hainich CZE, albeit at a variation of Sulfurifustis (Kojima et al., Sulfuritalea (Kojima and Fukui, Sulfuricella (Kojima and Fukui, Sideroxydans (Emerson and Moyer, nirS transcriptional activity in the groundwater of wells H52 and H53 pointed to low in situ denitrification activity. In addition to low availability of suitable organic and presumably also inorganic electron donors, a second factor favoring anammox over denitrification was most likely the constantly low groundwater temperature around 10°C, as studies of both natural and engineered systems found a lower temperature optimum for anammox compared to denitrification (Jetten, Denitrification activity observed in this study was in the lower range of rates reported from contaminated aquifers at low labile organic carbon availability or from marine oxygen minimum zones Table  and was  Jetten, .2-fixation under conditions of strong organic carbon limitation. Future studies will address whether coupling between anammox and nitrification enhances nitrogen loss from oligotrophic groundwater environments and will elucidate potential mechanisms which allow anammox bacteria to thrive in oxic groundwater.Our results have demonstrated a strong functional resemblance between oligotrophic groundwater and marine oxygen minimum zones regarding anammox activity, its high contribution to nitrogen loss, and its potential coupling with aerobic ammonia oxidation. We provided first insight into the quantitative relevance of anammox vs. denitrification in pristine groundwater. Together with knowledge gained from previous PLFA- and proteomics-based studies, our results point to the existence of an anammox hot spot within the heterogeneous carbonate-rock aquifer system of the Hainich CZE, where anammox dominates nitrogen cycling in suboxic to anoxic groundwater zones and could also make a substantial contribution to autotrophic CO15N based assessment of anammox and denitrification rates. VS performed the analysis of [3]-ladderane and [5]-ladderane phospholipid derived fatty acids. All authors contributed to the writing of the manuscript.MH, KK, and ST designed the work. SK performed most of the molecular work, contributed to chemical analysis and field work, analyzed all the data, and wrote the first version of the manuscript. ST and KT contributed substantially to the biogeochemical, transect-oriented interpretation of the results. Analysis of sequence data was carried out by SK and MH. PG performed a large part of the RNA-based work. BT carried out the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Specific, designed histidine–rhodium interactions allow a metallopeptide to bind Lyn kinase with nanomolar affinity and to activate kinase activity. ii) conjugates that deliver both potent and selective inhibition of Src-family SH3 domains. Rhodium(ii) conjugates offer dramatic affinity enhancements due to interactions with specific and unique Lewis-basic histidine residues near the SH3 binding interface, allowing predictable, structure-guided inhibition of SH3 targets that are recalcitrant to traditional inhibitors. In one example, a simple metallopeptide binds the Lyn SH3 domain with 6 nM affinity and exhibits functional activation of Lyn kinase under biologically relevant concentrations (EC50 ∼ 200 nM).Src-family kinases (SFKs) play important roles in human biology and are key drug targets as well. However, achieving selective inhibition of individual Src-family kinases is challenging due to the high similarity within the protein family. We describe rhodium( PxxP). However, our ability to chemically perturb Src-family SH3 interactions in a selective way is limited: SH3 domain interactions are weak (Kd ∼ 1–10 μM) interactions at a shallow binding interface and are highly conserved, especially among protein families such as the Src-family kinases. The Lyn kinase is a prototypical Src-family kinase. It contains a kinase domain and two regulatory domains: an SH3 and an SH2, which are believed to be involved in both upstream and downstream interactions.2 It shares significant sequence similarity with its Src-family brethren. Lyn and Lck, for example, have 63% sequence identity, similar to other comparisons within the family.The Src-family SH3 domains are functionally important mediators of protein assembly and of signaling pathways that illustrate the problems of “undruggable” targets. SH3 domains are ubiquitous and versatile subunits, appearing ∼300 times in the human genome in proteins implicated in the proliferation of cancer and other diseases. The domains recognize short, proline-rich motifs , perhaps the most effective small molecule inhibitors to date.15 The HIV Nef protein binds tightly to the Hck SH3 domain, and exhibits half-maximal activation of Hck at 130 nM,14 though Nef also displays promiscuous activation of several other Src-family kinases.17The SH3 domain represents an attractive and daunting challenge for inhibitor development. Within the Src family and in other related kinases, the catalytic kinase domain has been the primary target of inhibitor development. However, because the Src-family proteins have a high degree of similarity, kinase inhibitors can display unacceptable off-target activity. Thus the SH3 domain is a potentially powerful new target if truly selective inhibitors can be developed. In addition, SH3-selective inhibitors would shed light on kinase biology. The relative roles that Lyn SH2 and SH3 interactions play in the plethora of upstream and downstream signaling pathways known for Src-family kinases are poorly understood, apart from limited reports.23 The use of transition metals for such Lewis-basic anchoring of ligand molecules through specific interactions with the target protein is not widely studied. We have demonstrated inhibition with dirhodium cores in designed24 and natural protein26 contexts, and others have implemented similar ideas with cobalt28 and copper,29 for example. Designing metal coordination with protein target residues contrasts with alternative approaches that use transition metals as structural elements,31 oxidative damage agents,32 or cytotoxic species.34Dirhodium conjugates have unique properties that make them particularly well suited as inhibitors of specific protein–protein interactions. Dirhodium conjugates can benefit from metal–ligand interactions with histidine or other Lewis-basic residues on the surface of the target protein near the binding interface, offering potentially dramatic affinity benefits relative to traditional noncovalent organic interactions, which are typically weak. Dirhodium complexes are especially appealing in this regard due to a differential coordination environment—containing both exchange-inert equatorial sites that allow for stable conjugation and also weakly-held axial ligands for dynamic sampling of Lewis basic residues. Dirhodium complexes also have a history of biological and medical studies that indicate compatibility with living systems.cf.37 known to bind with similar affinity across Src-family SH3 domains,38 we made metallopeptides with rhodium in different sites near the beginning of the SH3-binding peptide. Several of these showed increased affinity . Under these conditions the distribution of data points on the isotherm reduces the precision of the global fit. Since the enthalpic contributions to binding in the formation of the complexes described in this work were high, we were able to use a low protein concentration (4 μM) which ensured that our C values were well below this upper range.  Isothermal titration calorimetry (ITC) measurements revealed an enthalpy-driven binding event  with a much smaller entropic penalty for binding  at 25 °C. In broad terms, the thermodynamics—favourable enthalpy and unfavourable entropy—are consistent with previous examinations of peptide–SH3 binding . The Hck domain—which shares one of two key histidine residues  with Lyn—also exhibited significant affinity . These results indicate that both histidine residues bind to the dirhodium core, presumably by binding in a co-linear fashion to each of the two rhodium atoms. The two single-histidine mutants bind with roughly similar affinity, 3–7 fold less potent than the native Lyn, and ∼100× more potent than the parent peptide, implying that both histidines are well positioned to coordinate to the dirhodium center. That the majority of the stabilization energy comes from the first histidine coordination is consistent with the negative cooperativity generally seen for axial coordination to rhodium(ii): a second coordination in solution is roughly two orders of magnitude less favorable.43The remarkable 6 nM affinity, far stronger than that of any reported non-protein ligand for SH3 domains, led us to study the structural basis of the observed affinity. Mutation experiments prove that both His78 and His96, in the Lyn SH3 binding pocket, contribute to potent binding. The H78A and H96A mutants bind to S2E44—is rare in small molecule ligands. We conducted a computational study using a combination of molecular mechanics and quantum mechanics to faithfully describe protein folding as well as rhodium coordination. Specifically, previously determined structures of Lyn  and Rh2(OAc)4 (2(OAc)4 and the respective Rh–His bonds were frozen.46 The Glu carboxylate side chain of S2E replaced an equatorial acetate ligand in order to position the peptide near the SH3 binding groove. Two-layer ONIOM calculations were performed on Lyn–S2ERh isomers using the DFT functional B3LYP for the QM layer and the force field UFF for the MM layer. The most energetically stable isomer is predicted to arise from bis-histidine binding through both γ-nitrogen atoms.47 This binding motif also necessitates the least displacement of the native Lyn structure , demonstrates what little backbone alterations are needed to accommodate bis-histidine binding to the rhodium core. Based on the model, only small conformational changes in the Glu95–Trp99 and Ile77–Asp80 loops are required to position His78 and His96 to interact with the dirhodium tetraacetate, consistent with the small entropic penalty for binding observed in the ITC measurements.The structure of the Lyn SH3 domain strongly suggests that two histidine residues are well positioned to coordinate to a dirhodium core, with the histidine side chains approaching from different directions and coordinating separate rhodium atoms. This bidentate trans coordination mode—reminiscent of metalloproteins such as cytochrome Ch2(OAc)4  were use Fig. S13. In the  Fig. S13, the met Fig. S13. A slice Fig. S13, yellow ii) conjugation represents a general way to build potent SH3 ligands in a predictable way from structural information and simple design principles. Through a combination of sequence optimization and judicious choice of rhodium location, it is possible to alter specificity to favor other SH3 domains. Lck is another Src-family protein, with high similarity to Lyn and possessing similarly SH3-binding-peptide preferences.48 However, Lck lacks the Lyn His96 residue (Rh (Rh), the affinity for Lck increased to 79 nM as a consequence of specific interactions with the unique His70 residue  is readily tagged with an alkyne group, and the modification visualized by alkyne–azide cycloaddition on a blot membrane  binding to the Yes SH3 domain, the addition of an exogenous high-affinity domain  would be predicted to out-compete metallopeptide binding to the substrate  and thus to prevent modification. Indeed, when these reactions are dosed with Lyn SH3, a drastic drop in labelling is observed, consistent with Lyn effectively outcompeting Yes and all other cellular proteins for the metallopeptide. Lyn itself is not modified by the catalyst, consistent with a Lyn–metallopeptide binding model  to minimize the alternative autophosphorylation activation pathway (2(OAc)4) displayed no activation (blue circle). However, the S2ERh metallopeptide exhibited strong Lyn activation at 200 nM, roughly three orders of magnitude lower than the parent peptide. Complete activation response was achieved at 630 nM. The only known SH3 ligand that exhibits comparable levels of activation within the Src family is the full-length HIV Nef protein, which binds tightly to Hck kinase. Taken together, the kinase activation and lysate-based inhibition demonstrate activation of the kinase target and activity in cell-like environments, and they provide confirmation of the potency gains made possible by rhodium conjugates.We examined phosphorylation of a peptide substrate at low enzyme concentration (0.074 μM) and short reaction time ( pathway . Kinase Rh metallopeptide is the first ligand with single-digit nanomolar affinity yet reported for Lyn SH3 and is among the very few highly potent SH3 ligands yet reported. In addition, the S2ERh metallopeptide is the first reported small molecule that exhibits functional activation of Lyn kinase activity under biologically relevant concentrations. Importantly, an approach based on metallopeptides allows both structure-guided inhibitor design and selective inhibition within homologous protein families that are difficult to differentiate with traditional inhibitors. By targeting unique residues at the periphery of the binding pocket, we are able to design specificity for Lyn, and, separately, for Lck and Hck, despite the large sequence homology of Src-family SH3 domains. A nanomolar Abl ligand demonstrates that the approach can be generalized to SH3 types beyond the Src family. Rhodium-containing inhibitors thus should serve as powerful tools to probe homologous protein families.The S2E


IHC staining for CD38 and ARG2 in the Stanford cohort demonstrated that combined expression of CD38 and ARG2 was prognostic. In the Canary cohort, low CD38 protein expression by IHC was significantly associated with recurrence-free survival (RFS), seminal vesicle invasion (SVI), extra-capsular extension (ECE) in univariable analysis. In multivariable analysis, ARG2 and CD38 IHC staining results were not independently associated with RFS, overall survival, or disease-specific survival after adjusting for other factors including SVI, ECE, Gleason score, pre-operative PSA, and surgical margins.The introduction of serum Prostate Specific Antigen (PSA) testing nearly 30 years ago has been associated with a significant shift towards localized disease and decreased deaths due to prostate cancer. Recognition that PSA testing has caused over diagnosis and over treatment of prostate cancer has generated considerable controversy over its value, and has spurred efforts to identify prognostic biomarkers to distinguish patients who need treatment from those that can be observed. Recent studies show that cancer is heterogeneous and forms a hierarchy of tumor cell populations. We developed a method of identifying prostate cancer differentiation states related to androgen signaling using Boolean logic. Using gene expression data, we identified two markers, CD38 and ARG2, that group prostate cancer into three differentiation states. Cancers with CD38-, ARG2- expression patterns, corresponding to an undifferentiated state, had significantly lower 10-year recurrence-free survival compared to the most differentiated group (CD38+ARG2+). We carried out immunohistochemical (IHC) staining for these two markers in a single institution (Stanford;  Although screening and early detection of prostate cancer (PC) has been associated with a drop in prostate cancer specific mortality in the US population and in the European Randomized Study of Prostate Cancer (ERSPC), PSA testing leads to significant over-detection and over-treatment of localized disease , 2. BecaMany prognostic biomarkers have been identified by association of their expression with adverse pathological and clinical features including failure after primary therapy. One of the cardinal features of cancer is dedifferentiation, often due to activation of programs active during tissue development . PreviouNormal prostate tissue is comprised of exocrine glands embedded in the prostate stroma containing fibroblasts, variable numbers of inflammatory cells, and smooth muscle cells. Current evidence demonstrates that a subset of the basal cells proliferate and give rise to terminally differentiated luminal secretory cells, suggesting that there are stem-like cells in the basal layer –18. The Based on observations in bladder, colon and myeloid cells, we hypothesized that prostate cancer cells may have a path of differentiation in which several genes are turned on at different stages of differentiation –12. We tp < 0.01 threshold for the BooleanNet analysis). To filter this list, we assembled three independent publicly available prostate cancer datasets that were annotated for recurrence-free survival and tested whether the transcripts were correlated with recurrence after surgery [We assembled a large prostate cancer mRNA dataset  for Boolean analysis as shown in Figure  surgery –25. We u surgery . These cCD38 and ARG2 have a robust Boolean implication relationship with KLK3 and AR as shown in Figure et al. dataset, Kaplan–Meier analysis demonstrated that recurrence after surgery occurred in the expected order with CD38-ARG2- showing the highest risk of recurrence, CD38-ARG2+ intermediate risk and CD38+ARG2+ showing the lowest rates of recurrence . We observed similar results in Sboner-2010 dataset  and Gerald-2004 dataset .As expected, Boolean analysis on these three datasets confirmed the existence of three differentiation states: CD38+ARG2+, CD38-ARG2+, and CD38-ARG2-. Cases were grouped using these criteria, and 10-year recurrence-free survival was compared each of the 3 groups. In the Taylor p < 0.05). However, this association was not significantly associated with RFS after adjusting for other clinical factors in the model such as grade, age and stage.Expression of CD38 and ARG2 proteins was tested by IHC and associated with patient outcome using 2 tissue microarray sets representing independent patient datasets. Scoring strategies for CD38 and ARG2 are summarized in Figure p < 0.05, Log-rank test). In addition, negative/weak CD38 expression was significantly associated with adverse pathological features including seminal vesicle invasion  and extracapsular extension . CD38 expression did not correlate with age, pre-operative serum PSA levels, positive surgical margins (PSM) or Gleason score (GS), although there was a trend for lower expression associated with higher Gleason score (p = 0.01), as were PSM, SVI, ECE, higher GS, and higher pre-operative PSA. However, CD38 IHC was not significantly associated with RFS, OS, or DSS after adjusting for other clinical factors in the multivariate analysis  and RFS. Our data clearly demonstrate that CD38 and ARG2 identify three different differentiation states in prostate cancer. There is a robust Boolean pattern that relates the expression of CD38 and ARG2; namely, when CD38 expression levels are high, ARG2 expression levels are also high. The findings in the current study, coupled with our work in bladder and colon cancer and myeloid cell development –12, stroet al. demonstrated that luminal cells with low CD38 expression are enriched in normal prostate acini adjacent to inflammation and these cells have progenitor-like features. Isolated cells expressing low levels of CD38 display increased expression of inflammatory genes, generate significantly more organoids than high CD38 expressing cells, and can generate normal prostate glands and carcinomas in an in vivo reconstitution mouse model [There have been a few relatively small studies of CD38 in the normal and malignant prostate. A graded decrease in CD38 protein expression has been observed in 23 prostate samples comparing normal prostate glands distant from cancer with normal glands adjacent to cancer, and with prostate cancer glands . Heterogse model . Chronicse model . In addise model –12.Less is known about the role of ARG2 in prostate cancer. ARG2 expression has been reported to be relatively higher in normal and non-malignant prostatic tissues compared to prostate cancer tissues . ARG2 exOur findings confirm that Boolean analysis approaches can be used to identify markers of differentiation that have biological and clinical relevance. Specifically, we have identified CD38 as a marker of differentiation in prostate cancer and confirmed that decreased of expression of CD38 transcripts and protein by IHC is associated with aggressive prostate cancer. These findings agree with recent observations demonstrating that CD38 loss correlates with a basal/progenitor class of luminal cells. As more markers of progenitor and stem cells are identifies in the prostate, Boolean approaches could yield additional genes relevant to prostate differentiation and as clinical biomarkers of prognosis.n = 891) was created from Human U133A (GPL96), Human U133 Plus 2.0 (GPL570), and Human U133A 2.0 (GPL571) Affymetrix platforms as described in n = 78, Memorial Sloan-Kettering Cancer Center) [n = 367, Memorial Sloan-Kettering Cancer Center) [n = 281, Swedish Watchful Waiting cohort) [Publicly available prostate cancer gene expression datasets with associated clinical information were downloaded from National Center for Biotechnology Information (NCBI) Gene Expression Omnibus website (GEO) and European Bioinformatics Institute ArrayExpress –36 as de Center) , Taylor- Center) , and Sbo cohort) .The expression values of each gene were ordered from low to high and a rising step function was computed to define a threshold t by the StepMiner algorithm in each individual dataset . If the All samples used in the construction of the TMA were used only for men who signed an IRB-approved Informed Consent for use of their tissues samples. A tissue arrayer  was used to construct a prostate cancer tissue microarray (Stanford-TMA) comprising an independent set of 234 formalin-fixed, paraffin-embedded primary prostate tumor cases selected from radical prostatectomy specimens collected at Stanford University, with institutional review board approval. Duplicate 0.6 mm tumor cores represented each case, and the series was associated with a minimum clinical follow-up of 5 years and a median follow-up of 8 years.Tissue blocks and accompanying clinical data were collected at each of the participating sites  under a research protocol developed by the investigators with IRB approval at each institution. The approved protocols included sharing of de-identified data and samples and correlation of clinical data with biomarker data acquired from the TMAs. A materials transfer agreement was developed jointly and approved at each site for sharing of clinical data and tissue samples.Testing of CD38 and ARG2 as clinical biomarkers of prognosis was carried out using tissue microarrays (TMA) comprised of 4 core samples from over 1300 randomly selected participants treated for PC with RP at six institutions between 1995 and 2004 [Freshly cut 5 micron sections were obtained and immunohistochemistry was performed using a commercial antibody to CD38 ) by the Department of Pathology Immunodiagnostic Laboratory using standard optimized protocols. ARG2 expression was assessed by IHC using a commercial antibody . Cancer cores were scored on a 0–3 scale based on staining intensity where negative was 0, weak 1, moderate 2 and strong 3  were provided in frequencies and percentages (Table


Longissimus thoracis (LT) muscle of Nelore steers. These genes are involved in the overrepresented pathways Metabolism of Xenobiotics by Cytochrome P450 and Butanoate and Tryptophan Metabolism. Among the DE transcripts were several proteins related to mitochondrial function and the metabolism of lipids. Our findings indicate that observed gene expression differences are primarily related to metabolic processes underlying oxidative stress. Genes involved in the metabolism of xenobiotics and antioxidant mechanisms were primarily down-regulated, while genes responsible for lipid oxidation and ketogenesis were up-regulated in HRFI group. By using LT muscle, this study reinforces our previous findings using liver tissue and reveals new genes and likely tissue-specific regulators playing key-roles in these processes.Residual feed intake (RFI), a measure of feed efficiency (FE), is defined as the difference between the observed and the predictable feed intake considering size and growth of the animal. It is extremely important to beef production systems due to its impact on the allocation of land areas to alternative agricultural production, animal methane emissions, food demand and cost of production. Global differential gene expression analysis between high and low RFI groups  revealed 73 differentially expressed (DE) annotated genes in  Feed efficiency is an important trait to cattle production systems worldwide because of the relationship between an individual’s efficiency and income over feed costs. Different measures of feed efficiency have been proposed2345The selection of animals with higher potential genetic merits for feed efficiency is an important strategy to reduce demands for feedstuff and production costs, thus increasing industry competitiveness. Furthermore, the selection of efficient animals may reduce the land area required for production, as well as methane emissions and nitrogen excretions resulting from metabolic processes19Longissimus thoracis (LT) tissue, evidence was provided that mitochondrial respiration rate was increased in the most efficient Angus animalsGenome-wide association studies (GWAS) have widely been used as a strategy for investigating mutations underlying economically important traits in livestock. Several candidate loci and genes associated with feed efficiency traits in Nelore cattle have been identified by these GWAS7Gene expression changes in the LT muscle of Nelore steers genetically divergent for RFI were detected by RNA-seq in 20 animals comprising two groups selected for their low or high estimated additive genetic merits for RFI . RNA-seq allows the examination of genome-wide gene expression differences between contrasting treatment groups and is repeatable, highly sensitive and strongly correlated with quantitative PCR experimentsBos taurus UMD3.1 reference genome.The feed efficiency phenotypes for this Nelore population had previously been used to perform a GWAS and the summary statistics for the population have previously been reported18After mapping reads with TopHat v2.0.622CYP1A1) was the only DE gene located within a quantitative trait locus (QTL) region identified by our previous GWAS for feed efficiency-related traits (explaining 0.29% of the additive genetic variance in RFI)We identified 84 DE genes  between the HRFI (less efficient) and LRFI (more efficient) groups, with 73 of these being annotated genes . CytochrB3GALTL) gene, involved in the fucose metabolic process, which was up-regulated in the less efficient, HRFI animals. On the other hand, the lowest fold change estimates were observed for genes encoding ribosomal proteins. Because the 73 annotated genes included several that encode hypothetical proteins, only 59 genes could be used in the functional annotation analysis. The functional analysis using both the up- and down-regulated genes was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.7CYP1B1) and CYP1A1 and glutathione S-transferase pi 1 (GSTP1) genes to be DE between the feed efficiency groups. With the exception of CYP1B1, which was up-regulated in the less efficient, HRFI group, the other genes involved in this pathway were down-regulated in this group. The CYP1B1 and CYP1A1 genes are also involved in the Tryptophan Metabolism pathway. On the other hand, 3-hydroxybutyrate dehydrogenase, type 1 (BDH1) and acyl-CoA synthetase medium-chain family member 1 (ACSM1) which are involved in butanoate metabolism, were up-regulated in the less efficient, HRFI group.Among the DE genes, the highest fold change estimate was for the beta 3-glucosyltransferase , NADH:ubiquinone oxidoreductase core subunit S7 (NDUFS7), ACSM1, cellular myelocytomatosis oncogene (MYC) and CYP1A1, related to mitochondrial functions to be among the DE genes. Furthermore, genes with a recognized role in feed efficiency such as Arrestin Domain Containing 3 (ARRDC3)GSTP1The summarized Biological Processes from the Gene Ontology provided by DAVID revealed many important processes underlying feed efficiency variation in Nelore cattle, such as response to organic substance, release of cytochrome c from mitochondria and macromolecule catabolic process . The enrMYC, Activating Transcription Factor 3 (ATF3), Nuclear Receptor Subfamily 4, Group A, Member 2 (NR4A2) and Eukaryotic Translation Initiation Factor 4E-Binding Protein 1 (EIF4EBP1) were responsible for expression changes detected in this study  and elongation factor 1-alpha 1 pseudogene (LOC782776) were found to be DE in all analyses. The common genes found to be DE for both RFI and ADG were activating transcription factor 3 (ATF3), mesenteric estrogen-dependent adipogenesis (MEDAG), collagen type VI alpha 6 (COL6A6), CYP1B1, collagen type XIV alpha 1 (COL14A1), EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1), follistatin-like 1 (FSTL1), immediate early response 5 (IER5), uncharacterized LOC100335754, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), MYC, nephroblastoma overexpressed (NOV), OTU deubiquitinase 1 (OTUD1), poly (ADP-ribose) polymerase 2 (PARP2) and peptidase inhibitor 15 (PI15). DE genesMHC class II antigen (BLA-DQB), elongation factor 1-alpha, somatic form-like (LOC100300305) and uncharacterized LOC100848726 were DE for both DMI and RFI. Among the DE genes for ADG, we observed adiponectin, C1Q and collagen domain containing (ADIPOQ) and thrombospondin 1 (THBS1), related to response to carbohydrate stimulus and regulation of lipid transport, in addition to fatty acid binding protein 4, adipocyte (FABP4) which is involved in the regulation of brown fat cell differentiation , which had already been identified as regulating feed efficiency in this populationNPC2) and FBJ osteosarcoma oncogene (FOS) genes, which are related to lipid transport and response to reactive oxygen species (ROS), respectively, were up-regulated in the high-DMI group.Dry matter intake (DMI) and average daily gain (ADG) were used to decompose RFI for additional gene expression analyses. This was accomplished by regrouping the same animals used for the RFI analysis based on their phenotypes for these traits. Of the 75 DE annotated genes for ADG , 17 werentiation . These gThe productivity of beef cattle is highly influenced by the rate of conversion of feed to muscle growth resulting in greater meat yields per unit of input. Therefore, any improvement in an animal’s efficiency that does not negatively affect other profitability-related traits, such as fertility or disease resistance, will result in increased herd profitability. These traits have been shown to be heritable in Nelore cattle and possess sufficient genetic variation to be valuable for selectionCYP1A1 gene; however, several common biological mechanisms were detected, such as those related to lipid and fatty acid (FA) metabolism. In fact, QTL regions do not necessary have to harbor DE genes, since QTL can be created by mutations in genes that cause post-translational disruptions affecting the functionality of proteins. Furthermore, the majority of QTLs identified in the GWAS performed for this Nelore population lay within gene desertsMost of genes found to be DE in this study do not reside within previously identified QTL regions for RFI that were found in a GWAS study using animals from the same Nelore populationB3GALTL, which is involved in fucose metabolic process and functions as an O-glucosyltransferase, contributing to the elongation of O-fucosylglycan. Genes related to fucose metabolism have previously been identified as playing a role in feed conversion efficiencyFusK) senses fucose and controls the expression of metabolic genesThe highest fold change estimate was found for ARRDC3, up-regulated in the less efficient group, plays a role in the regulation of metabolism and obesity in humans and miceARRDC3-deficiency protects against obesity by increasing energy expenditure likely acting by increasing β-adrenergic signaling to stimulate thermogenesis in brown and white adipose tissuesARRDC3 was found to be among the 10 most responsive genes to food intake, with higher transcript levels during periods of fastingGenes that have already been reported as functioning in the regulation of feed efficiency-related traits were identified. For example, GSTP1 functioning in the metabolism of Xenobiotics by Cytochrome P450 pathway to be DE between the feed efficiency groups. These genes were primarily down-regulated in the less efficient, HRFI animals, and may indicate that the malfunction of these mechanisms may result in an excess of ROS production or in a decrease in ROS reduction capacity, which in turn can cause lipid peroxidation and oxidative stressCYP1A1 has been reported in inefficient Yorkshire pigsThe functional analysis using both the up- and down-regulated genes performed by DAVID revealed that metabolism of Xenobiotics by cytochrome P450, butanoate and tryptophan metabolism pathways influence RFI in Nelore cattle. Cytochrome P450 enzymes are responsible for hydrolysis, oxidation or reduction mechanisms of xenobiotic metabolismMitochondrial dysfunction and oxidative stress have been implicated as major processes underlying variation in feed efficiency. How mitochondrial functions are associated with feed efficiency in livestock has recently been addressed13394043NDUFS7 was found to be down-regulated in the less efficient group. The protein encoded by this gene is a subunit of one of the complexes that forms the mitochondrial respiratory chain, which functions in the transfer of electrons from NADH to the respiratory chain. Supporting our findings, differences in oxygen consumption and oxygen pulse were detected when comparing more and less efficient animals of this population. Oxygen consumption per heart beat was lower for more efficient steers, and consequently, oxygen volume and oxygen pulse were also significantly lower47Mitochondrial activity may be compromised under oxidative stress. ACSM1 and BDH1 that are involved in FA oxidation and ketogenesis are members of this pathway and were found to be up-regulated in the less feed efficient animals. Oxidative stress may reprogram lipid metabolism, attenuating lipid synthesis and increasing mitochondrial fatty FA oxidationNR4A gene family membersNR4A2 was found to be up-regulated in the less feed efficient animals.We also found the Butanoate Metabolism pathway to be enriched for DE genes in our analysis. Butanoate is a metabolite of gut flora-mediated fermentation of dietary fiber and is closely involved with energy metabolismBDH1 catalyzes the interconversion of two major ketone bodies produced during FA catabolism. Ketogenesis occurs primarily in the mitochondrial matrix of liver cells and is important because it provides energy for surrounding tissues, especially cardiac and skeletal muscle. Ketogenesis is the last step of lipid energy metabolism, a pathway that links dietary lipids and adipose triglycerides to the mitochondrial respiratory chainThe protein encoded by MYC and IFI6, both up-regulated in the less efficient group, are involved in these functions. These genes have transcriptional activation functions and may regulate genes functioning in the mitochondrion and in mitochondrial respiratory chain complex activities and macromolecule catabolic processes. MYC family is known to be a proto-oncogene that activate genes involved in ribosomal and mitochondrial biogenesis, glucose and glutamine metabolism, lipid synthesis, and cell-cycle progressionMoreover, mitochondria-generated ROS play an important role in the release of cytochrome c and other pro-apoptotic proteins, which can trigger caspase activation and apoptosisATF3, a member of the ATF/cAMP-responsive element-binding protein family of transcription factors that is known to be responsive to stress and ROSATF3 in the liver results in defects in glucose homeostasis by repressing gluconeogenesisEGR1), known to encode a zinc finger transcription factor that activates “redox sensitive” genesEGR1 interacts with several other DE genes related to oxidative stress, such as GSTP1, NR4A2 and CYP1A1, for which functions have already been discussed. Our previous genome-wide gene expression study conducted on liver tissues indicated that gene expression changes between more and less efficient animals may define conditions of oxidative stress, indicating EGR1 as a potential upstream regulator of these gene expression changesOther genes with transcriptional activities were also found to be DE between the RFI groups. These genes may explain part of the expression changes that were observed in the comparison of the efficient and inefficient steers. Transcription factors, known to be induced under conditions of oxidative stress were found to be DE and up-regulated in the less feed efficient animals. Among these, we found longissimus muscle areas, suggesting greater muscle deposition in the carcasses of efficient Nelore steers from the same population that was used in this studyMYL3, partially contributes to the difference in skeletal muscle deposition between duck breedsIt has also been demonstrated that the more efficient animals from this population possessed larger Feed efficiency is a complex trait involving many processes and organs. While skeletal muscle metabolism has a key role in resting energy expenditure, liver is extremely important in metabolizing carbohydrates, lipids, and proteins into biologically useful materials, processes that produce various compounds that generate reactive oxygen radicals and may cause oxidative stress. Skeletal muscles are densely populated with mitochondria: a main producer of ATP and FA oxidation. Mitochondrial dysfunction and FA oxidation are also implicated in oxidative stress. Among the DE transcripts were several genes related to mitochondrial function and the metabolism of lipids. Our studies indicate a role for DE genes related to mitochondrial function and lipid metabolism in liverIn summary, we detected gene expression differences in the LT muscle of Nelore steers genetically divergent for residual feed intake. Our results support the hypothesis that gene expression differences between efficient and inefficient Nelore steers are primarily related to oxidative stress. Oxidative stress may arise when cellular mechanisms that reduce cellular ROS are compromised. In response, cellular transcription networks, such as metabolism of Xenobiotics by cytochrome P450, are altered. The results of this study, together with previous reports, provide a more comprehensive picture of gene expression in the muscle of animals genetically divergent for RFI. Further studies are necessary to dissect the mechanisms behind oxidative stress-induced signaling, which may help develop strategies to improve feed efficiency in beef cattle. Studies targeting the identification of functional mutations in elements regulating these DE genes may be important for the implementation of genomic selection for feed efficiency in Nelore cattle.Experimental procedures were carried out in accordance with the relevant guidelines provided by the Institutional Animal Care and Use Committee Guidelines of the Embrapa Pecuária Sudeste – Protocol CEUA 01/2013. The Ethical Committee of the Embrapa Pecuária Sudeste  approved all experimental protocols . The steers used in the expression study (n = 20) were born and raised at Embrapa Pecuária Sudeste  as described elsewhere7The fed population of steers comprised half-sib families produced by the artificial insemination of commercial and purebred Nelore dams, derived from sires representing the main breeding lineages commercialized in Brazil. BLUP estimates of genetic merit for RFI were initially generated for 585 Nelore steersLongissimus thoracis (LT) muscle samples were collected from the ribeye steak at slaughter and immediately stored in liquid nitrogen. The samples were then kept at −80 °C until RNA extraction. Total RNA was extracted from 50–100 mg of muscle tissue using Trizol (Invitrogen®), following the manufacturer’s protocol. The total RNA concentration was measured by spectrophotometry, and quality was verified initially by the 260:280 ratio, followed by assessment of integrity by agarose gel electrophoresis. Only intact samples, with a RNA 260:280 ratio greater than 1.8, were sent for sequencing. Before sequencing, eight samples were randomly chosen to double-check RNA quality on the Agilent 2100 Bioanalyzer System. The RNA Integrity Number for all samples was higher than 7. Preparation of the mRNA samples for sequencing was performed by ESALQ Genomics Center , using the TruSeq RNA Sample Preparation Kit®  according to manufacturer’s instructions. A description of the protocol was previously provided®. Paired-end reads of 2 × 100 bp were produced.Computations were performed on the HPC resources at the University of Missouri Bioinformatics Consortium (UMBC). Low-quality reads were filtered and adapter sequences trimmed using SeqClean software. The Tuxedo suite pipeline was used to perform the analysis as previously describedTranscript assembly and quantification was performed as described elsewhere22Cuffdiff2 v2.2.1 was run to test for DE genes between the divergent RFI groups. The geometric normalization method and the pooled dispersion method were used to normalize the libraries and to model cross-replicate dispersion, respectively. Briefly, the Cuffdiff2 approach is to first model the variability in fragment count for each gene across biological replicates. The algorithm uses a beta negative binomial model to represent fragment count dispersion and to estimate count variances for each transcript, which are then used to perform a statistical test to identify significantly DE genes. A change in the expression level of a gene is measured by comparing its fragment count in each biological treatmentSequences for the newly assembled transcripts were analyzed by BLAST to public database banks including SWISS-PROT , nr (NCBI non-redundant protein sequences), KEGG (Kyoto Encyclopedia of Genes and Genomes), KOG (euKaryotic Ortholog Groups), and Pfam (a widely used protein family and structure domain database).We used the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7Accession code: The RNA-seq data sets supporting the results of this study are available in the ENA repository (EMBL-EBI), under study accession PRJEB15314.How to cite this article: Tizioto, P. C. et al. Gene expression differences in Longissimus muscle of Nelore steers genetically divergent for residual feed intake. Sci. Rep.6, 39493; doi: 10.1038/srep39493 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Acute renal failure is a severe complication of lower extremity major arterial reconstructions, which could even be fatal. Levosimendan is a dual-acting positive inotropic and vasodilatory agent, which is suspected to have protective effects against cardiac ischemia. However, there is no data available on lower limb or remote organ ischemic injuries therefore the aim of the study was to investigate the effect of levosimendan on lower limb ischemia-reperfusion injury and the corollary renal dysfunction.Male Wistar rats underwent 180 min bilateral lower limb ischemia followed by 4 or 24 hours of reperfusion. Intravenous Levosimendan was administered continuously (0.2μg/bwkg/min) throughout the whole course of ischemia and the first 3h of reperfusion. Results were compared with sham-operated and ischemia-reperfusion groups. Hemodynamic monitoring was performed by invasive arterial blood pressure measurement. Kidney and lower limb muscle microcirculation was registered by a laser Doppler flowmeter. After 4h and 24h of reperfusion, serum, urine and histological samples were collected.Systemic hemodynamic parameters and microcirculation of kidney and the lower limb significantly improved in the Levosimendan treated group. Muscle viability was significantly preserved 4 and 24 hours after reperfusion. At the same time, renal functional laboratory tests and kidney histology demonstrated significantly less expressive kidney injury in Levosimendan groups. TNF-α levels were significantly less elevated in the Levosimendan group 4 hours after reperfusion.The results claim a protective role for Levosimendan administration during major vascular surgeries to prevent renal complications. Lower limb ischemic injuries (such as acute limb ischemia or even major vascular surgery on lower limb arteries) may represent serious clinical situations where profound cell destructions lead to substantial hemodynamic disturbances, systemic inflammatory changes and consequentially, severe multi-organ injuries . Critica2+ by binding to and stabilizing the Ca2+ saturated troponin C.serum), Serum [carbamide]/[creatinine] ratio, fractional sodium excretion (FENa) ([Na+]urinary × [Creatinine]serum) ÷ ([Na+]serum × [Creatinine]urinary) × 100) and renal failure index (RFI) ([Na+]urinary × [Creatinine]urinary ÷ [Creatinine]serum) were calculated.Serum (centrifuged) and urine samples were snap-frozen in liquid nitrogen immediately after harvest and stored at -80°C. Serum creatinine, carbamide, sodium concentration, urine volume, creatinine, and sodium concentration were measured. Analysis was performed by an automated clinical chemistry analyzer . Creatinine clearance . Mouse TNF-α standard , and 200 mL of TNF-α conjugate  was added to each well in the ELISA microtiter plate, and incubated. A color reaction developed with a substrate containing equal volumes of stabilized hydrogen peroxide and tetramethyl benzidine (stopped after 20 min by addition of hydrochloric acid). Absorbance was measured at 450 nm. Each optical absorbance value was calculated as a percentage of the average optical absorbance.Data were analyzed using IBM SPSS Statistics Version 22 . All values are expressed as means ± S.D. For every group of animals the assumption of normality of each measured parameter’s distribution was tested using Kolmogorov-Smirnov statistic, then Levene’s tests for homogeneity of variances were performed. Accordingly, in cases when homogeneity of variances was assumed, one way ANOVA  tests were calculated with Scheffe’s post hoc test for between groups comparisons. When heterogenity of variances was assumed, Brown-Forsythe test was performed with Games-Howell post hoc multiple comparisons. Any differences were considered statistically significant with p values less than 0.05.rd hour of ischemia. Reperfusion resulted in a drop in MAP in both groups (IR and Levosimendan) compared to the ischemic values.No significant alterations could be found during the whole registration in the Sham operated group regarding both heart rate (HR) and mean arterial blood pressure (MAP). Blood pressure elevated significantly (p<0.001) after aortic occlusion and remained elevated throughout the period of ischemia in the IR group as compared to the Sham operated group. Levosimendan administration withheld this elevation until the 3st (p = 0.004), 2nd (p = 0.002) and 3rd (p<0.001) hours versus Sham. Reperfusion resulted in another significant (p<0.001) increase in HR then remained elevated thereafter. Levosimendan administration resulted in significantly less elevated HR compared to the IR group both during ischemia and reperfusion (st (p = 0.016), 2nd (p = 0.017), 3rd (p = 0.023), 5th (p = 0.049), 6th (p = 0.038), and 7th (p = 0.025) hours.HR was significantly elevated during ischemia in the IR group, as measured after the 1erfusion  as measuCalculated ‘shock-index’ was higher for the period of reperfusion than for ischemia in the IR group (quotient >1). This was also the case in the Levosimendan group, with no statistically significant difference between the two groups .The lowest systolic blood pressure within the first 20 min of reperfusion occurred at 6.00±2.86 min in the Levosimendan, and at 7.37±4.97 min after revascularization in the IR group, which did not differ statistically (p>0.05). This drop of blood pressure was greater in the IR group (16.61±3.61% of the last registered systolic blood pressure of the ischemic time) than in the Levosimendan group (8.56±8.22%), though because of the high standard deviation, the difference was found non-significant.After the drop of the blood pressure, the pressure curves reached the plateau phase within a similar time interval in both the Levosimendan and the IR group . Sham operation resulted in no alterations in either kidney or skeletal muscle flow throughout the experiment. Kidney microcirculation remained at the baseline level after clamping of the infrarenal aorta in both IR and Levosimendan groups. After revascularization, flux deteriorated gradually in the IR group while levosimendan administration was able to maintain renal cortical microcirculation at a significantly higher level , resembling the values measured in the Sham operated group. Muscle microcirculation dropped at the onset of aortic occlusion in both the IR and Levosimendan groups. At the onset of reperfusion, microcirculation increased to about 100% of baseline flux in the Levoismendan group, in contrast to IR group where it reached only 80% of baseline values. Levosimendan treatment caused significant amelioration in every microcirculatory parameters. . Light microscopy and HE staining was not able to detect any definitive histopathological lesions in the samples taken from the tibial anterior muscle in any groups at any measured time points.Kidney samples showed signs of acute tubular injury in the IR group with swollen, vacuolized tubular cells and precipitated hyaline-cylinders within the tubular lumina after 4 hours of reperfusion. 24 hours after aortic declamping slightly milder histological damage could be observed in the IR group compared to 4 hours of reperfusion. Levosimendan administration resulted in a lesser degree of histological injury at both examined reperfusion intervals compared to the corresponding IR group .4 hours after revascularization, a marked decrease in viability was detected in the IR group, as well as results showed no regeneration even after 24 hours of reperfusion. Levosimendan administration resulted in significantly preserved (p<0.001) viability compared to the IR group at both measured time points (4 and 24 hours), however the values of the Levosimendan group failed to reach the levels of the sham operated animals. . At the end of the fourth hour of reperfusion, kidney injury induced renal hypofunction was evident in the IR group according to markedly elevated serum creatinine levels (p<0.001), as well as creatinine clearance rates (p<0.001), and reperfusion urine volumes also showed a significant  deterioration of renal function compared to the sham operated group. Meanwhile in the 4h survival levosimendan treated group, significantly (p = 0.006) less expressive kidney injury could be observed.Serum carbamide/creatinine ratio and the fractional sodium excretion indicated a tubular type kidney failure in the IR group, whereas the values in the Levosimendan group were not characteristic for any type of kidney failure, according to regulardefinitions. Differences were significant (p<0.001). Renal failure index and fractional sodium excretion indicated persistent kidney damage at the 24th hour after reperfusion with significantly lower values in the Levosimendan group (p<0.001) .TNF-α concentrations were significantly increased in the IR group compared to the Sham-operated group (p<0.001) after 4 hours of reperfusion. Levosimendan administration was able to significantly reduce serum TNF-α levels compared to the IR group at this measurement point (p<0.001). After 24 hours of reperfusion no differences  could be observed among the three experimental groups in serum TNF-α concentrations .Prolonged lower limb ischemic conditions like acute critical limb ischemia with surgical revascularization therapy as well as major vascular surgeries of the lower limb arteries with crossclamping of the abdominal aorta might result in profound ischemia-reperfusion injury of the skeletal muscle tissues. The serious injury of the skeletal muscle involves the risk for postoperative systemic complications such as multiple organ failure. The kidn2+ overload,[ATP channels in the inner membrane of mitochondria.[Besides its documented calcium sensitizer, positive inotropic and vasodilator properties mainly used in severe low output heart failure, levosimeoverload, and openchondria., 15The aim of our present study was to investigate the impact of levosimendan in a lower limb IR model—that was previously utilized by our team—on local (muscle injuries) and systemic (kidney failure) complications.In the present study levosimendan treatment was demonstrated to have a protective effect against local IR injuries as evidenced by the skeletal muscle viability and muscle microcirculation measurements. Hematoxylin and eosin staining under light microscopy failed to reveal any detectable morphological changes in this model. For assessment of subcellular lesions and alterations in mitochondrial integrity, the viability of muscle fibers was assessed with a technique developed by our group. Lower liATP) of mitochondria[ATP activates distal signaling pathways, that involve improved defence against reactive oxygen species, protein-kinase Cε (PKCε),[2+ release, pyridine nucleotide depletion, matrix swelling and a consequential rupture of the outer membrane and release of proapoptotic proteins into the cytosol.[In preservation of mitochondrial integrity by levosimendan, a couple of mechanisms may be involved. Levosimendan is a potential activator of the ATP-sensitive potassium channels , mitochonε (PKCε),, and repε (PKCε), which al cytosol.+/Ca2+ exchanger (NCX) of the plasma membrane functions in a ‘reverse’ mode, resulting in Ca2+ influx to the cell. Overloading of calcium favors the pro-apoptotic Bcl2-associated X (BAX) protein incorporation into mitochondrial outer membrane that also leads to the opening of the mPTP.[2+ overload.[The other important element of the protection of mitochondria is the regulation of cell’s calcium homeostasis. After a prolonged episode of ischemia, the Nathe mPTP. It was soverload.ATP agonist and that the signaling pathways of ischemic postconditioning also involves the opening of the channel[A less expressive IR injury in levosimendan treated animals was further confirmed by the finding of a considerably less impaired muscle microcirculation after levosimendan administration in contrast to the severely impaired muscle microcirculation in the IR group after revascularization. This finding can be explained at least in part by the vasodilating effect of levosimendan besides the positive impact on the limb tissues  IR injury. The protective effect of levosimendan on skeletal muscle viability and microcirculation shares similarities with our previous findings of the protective effects of ischemic postconditioning in the same model of limb IR injury. Given the channel it postuA protective effect against lower limb injuries would gain clinical relevance mainly by the impact it might have on the systemic complications of such injuries as for example the evolving kidney failure. Accordingly, renal dysfunction was characterized in the present model. We found that levosimendan administration resulted in milder histopathological alterations on kidney samples, less serious renal dysfunction  at the early post-revascularization period, compared to the group of animals that received no levosimendan infusion. This nephroprotective effect was preserved even 24 hours after reperfusion. Calculated clinical functional laboratory parameters  suggested a renal (tubular injury) type impairment of renal function in the IR group. The significant difference between the levosimendan treated and non-treated animals propose that levosimendan treatment results in less expressed acute tubular injury. This effect might be due to the ameliorated skeletal muscle IR injury, which resulted in less nephrotoxicity.Our previous experimental and human investigations demonstrated a release of myoglobin from the injured myocytes after vascular exclusion of the lower limbs., 30 On oATP channels, this agent is known to evoke vasodilatation, a decrease in vascular resistance and mean arterial pressure. With detailed analysis of the systemic hemodynamic changes occurring in the present experimental model we could not find any significant effect when levosimendan is applied during a prolonged ischemia and revascularization of major lower limb arteries.Although there seems to be a direct relationship between the lower limb rhabdomyolysis and the acute kidney injury in our experiment, suggesting a direct toxic mechanism and an acute tubular necrosis type of renal failure, we proposed a prerenal injury beside the acute tubular necrosis. At revascularization of large skeletal muscles (previously exposed to hypoxia and with injured microcirculation and vasorelaxation of the larger blood vessels) serious hemodynamic changes evolve, even leading to shock, and a neuroendocrine response for circulatory redistribution, both effecting kidney circulation. On one sATP channel opening properties on vascular smooth muscle cells.[In contrast to the systemic hemodynamic parameters, infusion of levosimendan during limb ischemia and revascularization had a marked effect regarding renal microcirculation. The levosimendan treated animals presented a significantly less diminished cortical microcirculation during reperfusion as opposed by the considerable reduction of blood flow in the IR group. This circulatory impairment and the reduction of renal blood flow following skeletal muscle ischemia-reperfusion may arise from the systemic hemodynamic disturbances, the compensation of renal vasoconstriction, but also from the toxic oxidoreductive effects of myoglobin directly affecting renal microcirculation. Levosimele cells., 37 All le cells. Thereforle cells.Finally our results confirm the observations regarding the anti-inflammatory effect of levosimendan. TNF-α levels were significantly lower after 4 hours of reperfusion in the levosimendan group compared to the IR group. 24 hours after reperfusion no differences could be observed among the three experimental groups in serum TNF-α concentrations displaying the temporal characteristics of the cytokin elevation. Acute inflammatory changes and TNF-α may also contribute to acute renal failure,, 41 and ATP channels and the amelioration of the skeletal muscle ischemia-reperfusion injuries, which may lead to the prevention of the myoglobinuric acute renal failure. The nephroprotective effect of the agent is further supported by the positive inotropic and hemodynamic effects of levosimendan on the level of microcirculation and the possible anti-inflammatory effect of the drug, confirmed in this study. These positive effects combined might result in the preservation of renal function.Our study presents some new results offering new perspectives for the use of levosimendan. We demonstrated that levosimendan treatment could protect muscle cells from ischemic injury after 3 hours of ischemia. In addition to these local effects, levosimendan reduced the degree of acute tubular kidney injury. An explanation of the positive effects may be the protection of mitochondria via opening the KThe study is mostly descriptive in nature and does not provide information/evidence about cellular changes during the process, most of the explanations and conclusions drawn are speculative. With purpose of standardization, specific pathogen free, healthy male Wistar rats were used only, so it is not suitable for detection the differences between genders and the population is not comparable for patients with high risk cardiovascular diseases. Moreover the anatomical differences  makes the translation difficult, thus further, mainly big-animal studies are required to eliminate these differences.S1 FileMinimal data sets of the experimental material supporting ‘Levosimendan administration in limb ischemia: multicomponent signaling serving kidney protection’ article, including heart rate, mean arterial pressure, kidney cortex as well as muscle microcirculatory, muscle viability and labouratory measurements data, as broadly discussed individually at respective sections of the article.(XLSX)Click here for additional data file.


The results of this screen can then be compared against a reference uninsulated device to select a set of insulated promoters providing a precise level of expression. We verify this method by insulating the constitutive, inducible, and repressible promotors of a four transcriptional-unit inverter (NOT-gate) circuit, finding both that order dependence is largely eliminated by insulation and that circuit performance is also significantly improved, with a 5.8-fold mean improvement in on/off ratio.Variation in the DNA sequence upstream of bacterial promoters is known to affect the expression levels of the products they regulate, sometimes dramatically. While neutral synthetic insulator sequences have been found to buffer promoters from upstream DNA context, there are no established methods for designing effective insulator sequences with predictable effects on expression levels. We address this problem with Degenerate Insulation Screening (DIS), a novel method based on a randomized 36-nucleotide insulator library and a simple, high-throughput, flow-cytometry-based screen that randomly samples from a library of 4 This prhallenge , 9: at phallenge . Insteadhallenge , 11.The poor performance of an engineered genetic regulatory network can be due to many different factors, including interactions between the various devices making up the network, poorly understood interactions of devices with their operating environment, and physical or functional interactions between engineered components and the host genetic machinery , 12. In α-subunit (α-CTD) of the RNA polymerase (RNAP) to A/T-rich Upstream Promoter (UP) elements placed between -40 and -60 bp of the transcription start site (TSS) can increase transcription by 30-60x [α-CTD has been reported to bind to DNA sequences at least as far as 100 bp upstream of the TSS resulting in altered protein expression levels [Moreover, the effect of upstream DNA sequence can be quite strong, particularly for the minimal promoters often favored in synthetic biology, which are often only a few dozen base-pairs long. Binding of the y 30-60x , the RNAn levels , 15, andn levels .et al. showed that a synthetic insulator sequence inserted at the 5’ boundary of susceptible minimal promoters did successfully inhibit increased rate of transcription by an UP element [One approach to reducing the impact of upstream DNA sequence is to extend the 5’ end of the promoter with an “insulator” sequence, thereby increasing the separation of the active components of the promoter from the potentially variable portion of upstream sequence and thus hopefully reducing its impact. Davis  element . However36 potential insulated promoters in a high-throughput, flow-cytometry-based screen, comparing against a reference device for precise expression control. We test this method by applying it to insulate a minimal constitutive promoter , an inducible promoter  and a repressible promoter . Our experiments show that using DIS-insulated promoters largely eliminates the order dependence of component transcriptional units on the performance of a four transcriptional unit genetic inverter (NOT-gate). Regularizing promoter behavior also improves circuit performance, providing an incremental increase in on/off ratio with each additional insulated promoter to an ultimate 5.8-fold mean improvement when all promoters are insulated.To address these challenges, we have developed Degenerate Insulation Screening (DIS), a novel method for insulating promoters based on a randomized 36-nucleotide insulator library. DIS draws random samples from this library of 4Degenerate Insulation Screening (DIS) is a novel promoter insulation protocol based on amplification with degenerate primers and screening to compare against reference expression levels. This method is motivated by the fact that there are many possible mechanisms by which an upstream sequence might affect promoter expression , but any given such mechanism is likely to pertain to only a small fraction of sequences . We thus hypothesized that randomly generated sequences should have a reasonable probability of behaving as good insulators, but that it might also be necessary to screen many candidate sequences in order to find insulating sequences that closely match a reference expression level from an uninsulated device. Initial tests with a small number and hand-generated random DNA sequences produced results consistent with this hypothesis , leadingIn the remainder of this section, we first present the DIS protocol and results showing that DIS produces libraries with a wide distribution of device behaviors, supporting selection of multiple insulator options matching to a desired reference behavior. We then validate the insulation provided by DIS promoters by application to a four transcriptional-unit inverter circuit, finding that DIS-insulated promoters largely eliminate order dependence, as well as significantly improving circuit performance.Basic idea of DIS is to generate a custom set of insulators for each promoter by screening a large collection of constructs with random spacers inserted upstream of the promoter. The insulators are then those spacer constructs whose expression levels match a desired reference level of expression .E. coli and cultured to produce clonal populations of individual library elements, which may then be further split for testing multiple input conditions. All samples are then screened by flow cytometry to identify samples containing DNA spacers that empirically provide the precise desired expression level or levels ; these spacers are the desired library of insulators. Finally, the insulator library can be sequenced to identify effective spacer-promoter pairs and/or used directly in construction of larger systems . Additional details may be found in Materials and Methods and in Our implementation of this concept in the DIS protocol is shown in To test DIS, we applied it to insulate three commonly used promoters: a constitutive minimal promoter (J23100), an inducible promoter (pBAD), and a repressible promoter (pTet). For a baseline to compare against, we tested the impact of upstream sequence on these promoters against four MoClo assembly , 17 fusiThe distributions of geometric mean fluorescent expression from the DIS libraries for J23100, pBAD, and pTet are shown in To test the efficacy of DIS-generated insulators, we combined the three promoters to form an “inverter” circuit comprising four transcriptional units . In thisWe test the impact of local DNA context on circuit performance by using MoClo assembly , 17 to cEach permutation set was then cultured in triplicate overnight in both the uninduced  state and the induced  states, and the expression levels of GFP and RFP measured by flow cytometry. Finally, we compare expression levels by calculating the geometric mean expression of each sample and taking the ratio of the “on” state over the “off” state . Ideally, all permutations should show similar strong levels of GFP induction and RFP repression.As expected, the behavior of the uninsulated circuit is quite sensitive to local DNA context: Insulation with DIS-generated spacers markedly improves both the strength and regularity of performance: The in-context selection of DIS does indeed appear to be important for obtaining this effective insulation: randomly-generated spacers fail to effectively insulate from local DNA context, yielding performance nearly as bad as if there were no insulation at all . We alsoIf promoters are to be used as part of complex genetic designs, it is unavoidable that their upstream genetic context will change, since they may be placed after any number of other genetic constructs. Even small variations in the DNA sequence at the 5’ boundary of a promoter can greatly alter its behavior and cause design failures, and no method has yet been identified for reliably predicting the effect of upstream sequence on promoter behavior , 13, 16.Some potential challenges in generalization of this approach remain for future investigation. For example, we note that the degree of variation observed in a collection of random insulators differs significantly between different promoters : it is tAnother important question for future work is to determine how broad a class of DNA context effects can actually be insulated against by means of this approach. The promoters tested in this paper have mechanisms that are prototypical and representative of a large class of commonly used genetic components, so it appears likely that the method we have presented will effectively insulate against a wide range of transcriptional/translational components. At the same time, there are clearly limits to the efficacy of spacers: for example, spacers provide no mechanism to protect against side effects from nearby epigenetic modification or sequence editing.We also note that our screening method is defined in terms of a reference device to screen candidate insulators: in the absence of a defined reference that is desired as a target, our approach might instead be used for “tuning” to select a desired expression level from a given promoter or to adjust and optimize the performance of engineered biological systems. Likewise, although we have focused on bacterial promoters, there is nothing to prevent this technique from being applied to promoters in other organisms or to other components that prove to be sensitive to local genetic context.Finally, many widely used bacterial promoters are either known to or likely to suffer from the upstream genetic context issues that we have addressed in this paper. We thus expect that it will be valuable for the synthetic biology community to begin applying our method to develop and characterize insulated variants of a wide variety of bacterial promoters. Furthermore, in many cases we expect that it will be useful to prepare not just one but multiple variants of an insulated promoter, as is readily supported by our method, so that if a promoter is used multiple times in a design, different insulators can be used in each case in order to minimize undesired homology. Developing such insulated promoter libraries will enable faster, simpler, and more reliable use of those promoters in new biological engineering efforts in both academia and industry.A summary of key method information follows; additional details may be found in Supporting Information Basic parts  from the CIDAR-MoClo part collection  were assE. coli. Expression of samples from the insulated library was measured by flow cytometry and compared against a reference device.Randomized 36 nt insulated promoter libraries were generated by performing an inverse PCR reaction using divergent primers to the promoter to be insulated. A phosphate group and 18N extension was added to the 5’ end of both forward and reverse primer. Overnight digestion of PCR product with DpnI  was performed to remove traces of PCR template. PCR product was purified using a GenCatch PCR Purification Kit . PCR product was ligated in an overnight 16°C blunt end ligation using T4 DNA ligase . The purified PCR product was ligated and transformed into Spacers for constitutive promoter J23100 were selected by screening candidates from an insulated GFP expression cassette library of J23100_E (12.5X lower expression than J23100_A). Spacers for inducible and repressible promoters pBAD and pTet were obtained by screening insulated expression cassettes of pBAD_F : CGTTGTTCGCGACCCTGTTCTGAGGCAATGTTGGGCpBAD (second): TAGCACGCTTTTCTGCGTATGGTTGGTCAGCTTCTApTet: CGGAGAGAATACATAGTAAAGAAACCTCACTGTTGTThe 36 nt spacers ultimately selected to serve as insulators had the following sequences:Additional information related to these sequences may be found in Supporting Information μL of MoClo reaction mixture into 12-25 μL of Alpha Select Gold Efficiency chemically competent cells  following the manufacturer’s protocol. Cells were grown overnight at 37°C on MoClo assembly level-appropriate antibiotic selective LB agar supplemented with 80 μL of 20mg/μL 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-GAL) and 100 μL of 0.1M isopropyl-β-D-thiogalactopyranoside (IPTG)  for blue-white screening.Transformations were performed using 3Plasmids were purified using the QIAprep Spin Miniprep kit (Qiagen) following the manufacturers’ protocols. Inverters were checked for correct size by performing colony PCRs on MoClo transformation colonies using 2X Taq Mastermix  and VF (5’-tgccacctgacgtctaagaa) and VR (5’-attaccgcctttgagtgagc) primers. Sequences were analyzed using Benchling .E. coli cells (Bioline) containing the confirmed plasmid construct/clone from insulated promoter cassette library of interest. Colonies were grown in 200 μL LB broth (Sigma-Aldrich) with the appropriate antibiotic in sterile 96-well, deep well plates  in triplicate (for confirmed plasmid construct) or singly (for insulated promoter expression cassette library) for 14 hours at 37°C shaking at 300 rpm. Cells were diluted into phosphate buffered saline (BioExpress) in 96-well round bottom plates before measurement using a high-throughput sampler (HTS).All fluorescent expression devices were characterized using a BD LSRFortessa SORP flow cytometer. RFP fluorescence was measured using a solid-state Coherent Sapphire 561 nm laser at 100 mw strength with a PE-Texas Red 610/20 filter. GFP fluorescence was measured using a solid-state Coherent Sapphire 488 nm laser at 200 mw strength with a FITC 530/30 filter. LB agar  plates containing the appropriate antibiotic were inoculated with Alpha-Select Gold Efficiency E. coli cells , respectively. FITC channel measurements (for GFP) are calibrated to MEFL using SpheroTech RCP-30-5-A beads.Flow cytometry data was converted from arbitrary units to compensated MEFL  using the TASBE characterization method , 20. An S1 FileAdditional information about methods employed, including all DNA sequence information.(PDF)Click here for additional data file.S1 FigA preliminary test of the variable effect of random insulators on expression was performed with 36 nt spacer sequences designed by screening random DNA base composition resembling the intergenic bacterial DNA base composition for tandem promoter elements  and abse(PDF)Click here for additional data file.S2 FigConstitutive expression from (a) J23100 promoter, (b) pBAD inducible promoter, and (c) pTet repressible promoter, when downstream of MoClo A, E, F, and G sites. Error bars show +/- two std.dev. of mean fluorescence. The J23100 and pBAD promoters both show a significant difference in expression with different fusion sites, with more than a 10-fold difference in expression between the highest and lowest expression levels, while pTet expression did not vary significantly.(PDF)Click here for additional data file.S3 FigA preliminary test of the effect of insulator length on expression was performed with spacer sequences designed to several lengths  by screening random DNA base composition resembling the intergenic bacterial DNA base composition for tandem promoter elements  and abse(PDF)Click here for additional data file.S4 FigDistribution of expression levels for induced and uninduced expression levels from spacer screen for pBAD, corresponding with fold-change distributions reported in (PDF)Click here for additional data file.S5 FigDistribution of expression levels for induced and uninduced expression levels from spacer screen for pTet, corresponding with fold-change distributions reported in (PDF)Click here for additional data file.S6 FigComparison of GFP and RFP fold changes upon induction with L-arabinose of the 24-inverter permutation set in which each transcriptional unit is insulated with a different spacer from (PDF)Click here for additional data file.S7 FigComparison of GFP and RFP fold changes upon induction with L-arabinose of the 24-inverter permutation set in which only the J23100 promoter has been insulated with a DIS spacer. Insulation is denoted by a smaller gray box positioned between upstream MoClo fusion site and colored box denoting a transcriptional unit. Performance improves over uninsulated inverters, but is not as good as inverters with more insulators.(PDF)Click here for additional data file.S8 FigComparison of GFP and RFP fold changes upon induction with L-arabinose of the 24-inverter permutation set in which only the two pBAD promoters have been insulated, each with a different DIS spacer. Insulation is denoted by a smaller gray box positioned between upstream MoClo fusion site and colored box denoting a transcriptional unit. Performance improves over uninsulated inverters, but is not as good as inverters with more insulators.(PDF)Click here for additional data file.S9 FigComparison of GFP and RFP fold changes upon induction with L-arabinose of the 24-inverter permutation set in which both the J23100 and two pBAD promoters have been insulated with a DIS spacer. Insulation is denoted by a smaller gray box positioned between upstream MoClo fusion site and colored box denoting a transcriptional unit. Performance improves over a single insulator inverter, but is not as good as insulating all promoters, particularly for change in RFP expression levels.(PDF)Click here for additional data file.S1 DatasetAll data underlying the findings described herein.(ZIP)Click here for additional data file.