Escherichia coli, and one most complex and intriguing membrane transporters known to date. Analysis of wildtype AcrB and four functionally-inactive variants reveals an unprecedented mechanism that involves two remote alternating-access conformational cycles within each protomer, namely one for protons in the transmembrane region and another for drugs in the periplasmic domain, 50 Å apart. Each of these cycles entails two distinct types of collective motions of two structural repeats, coupled by flanking α-helices that project from the membrane. Moreover, we rationalize how the cross-talk among protomers across the trimerization interface might lead to a more kinetically efficient efflux system.Membrane transporters of the RND superfamily confer multidrug resistance to pathogenic bacteria, and are essential for cholesterol metabolism and embryonic development in humans. We use high-resolution X-ray crystallography and computational methods to delineate the mechanism of the homotrimeric RND-type proton/drug antiporter AcrB, the active component of the major efflux system AcrAB-TolC in DOI:http://dx.doi.org/10.7554/eLife.03145.001 The interior of living cells is separated from their external environment by an enveloping membrane that serves as a protective barrier. To regulate the chemical composition of their interior, cells are equipped with specialized proteins in their membranes that move substances in and out of cells. Membrane proteins that expel molecules from the inside to the outside of the cell are called efflux pumps.Escherichia coli bacteria, an efflux pump known as AcrB is part of a system that removes toxic substances from the bacterial cell—such as the antibiotics used to treat bacterial infections. AcrB and other closely related efflux pumps in pathogenic bacteria are often polyspecific transporters—they can transport a large number of different toxic molecules. These efflux pump systems are also more active in bacteria that have been targeted by antibiotics, and therefore they help bacteria to evolve resistance to multiple drugs. The emergence of bacterial multi-drug resistance is a global threat to human health; to combat this phenomenon, it is essential to understand its molecular basis.In E. coli, works via an ‘alternating-access cycle’; that is, the shape of the periplasmic domain changes between three different forms in such a way that antibiotic molecules are first captured and subsequently squeezed through the protein towards the outside of the cell. However, the mechanism of the transmembrane domain—which is embedded in the innermost membrane of the bacterium and is the source of energy for the transport process—was not understood.Each AcrB protein has three main parts or domains. The periplasmic domain, which is located between the two membranes that surround Here, Eicher et al. use X-ray crystallography to examine the three-dimensional structures of the AcrB efflux pump—and several inactive variants—in high detail. Combining these results with computer simulations reveals the mechanism used by the transmembrane domain to take up protons from the exterior and transport them into the cell. Proton transport also proceeds according to an alternating-access mechanism—and, although the transmembrane and periplasmic domains are far apart, their movements are tightly linked. Thus, because proton uptake releases energy, the transmembrane domain effectively powers the periplasmic domain to expel drugs and other molecules. Eicher et al. note that a similar mechanism has not been seen before in other efflux pumps or transporter proteins.Understanding how AcrB works opens up new avenues that could be exploited to develop new drugs against multidrug resistant bacteria. Furthermore, Eicher et al. suggest that efflux pumps in humans closely related to AcrB might function in a similar way—including those required for regulation of cellular cholesterol, and for the correct development of embryos.DOI:http://dx.doi.org/10.7554/eLife.03145.002 Escherichia coli, powered by the electrochemical proton gradient across the inner membrane of this Gram-negative bacterium (The AcrA-AcrB-TolC complex is a secondary active antiport system in acterium . Its bioacterium .E. coli), export of lipids  and pigment export . In humans, the RND transporters NPC1 and Patched play key roles in the regulation of cholesterol metabolism and in the Hedgehog signaling pathway of embryonic cells, respectively  family , which aectively .The first known structure of AcrB, solved by X-ray crystallography at a resolution of 3.5 Å, revealed a homotrimeric assembly in which all protomers adopt the same conformation . In eachThe intricate and unusual topology of AcrB and its trimerization state were confirmed by subsequent higher-resolution X-ray structures obtained from crystals grown in different space groups, several of which include bound substrates . These sThe transport mechanism of the TM domain of AcrB, in contrast, remains largely undefined. The asymmetric trimer structures indicate that this domain also cycles between three distinct conformations, and point to several ionizable residues in its core  that might function as a proton-relay network. Nevertheless, no clear interpretation of existing data has been formulated yet in terms of an alternating-access proton transport mechanism, which is a necessary condition for the transporter to harness the energy stored in the transmembrane electrochemical gradient. How this proton-motive-force is mechanically transduced to the porter domain, in order to power uphill drug efflux, also remains to be determined.Here, we gain insights into these crucial mechanistic questions from a series of new crystal structures of AcrB, systematically analyzed with molecular simulations and other computational methods. More specifically, we examine the TM domain of the transporter in an improved atomic structure of wildtype AcrB , at 1.9 Despite the unique topological complexity of AcrB, a systematic analysis of its structure permits to rationalize its conformational cycle in terms of collective motions of distinct structural units. In the porter domain, as noted previously, each protomer consists of two structural repeats, referred to as PN1/PC2, and PN2/PC1 . Each reGiven the modular nature of the porter domain, we reasoned that the TM domain might also consist of structural repeats, as is observed in other secondary transporters. To identify these, we quantified the degree of similarity of combinative structural superpositions of fragments of the transmembrane 12-helix bundle in the L, T and O states . This anAnalysis of R1, for example, shows that the structure of this five-helix bundle is largely preserved in the L, T and O conformations; the RMSD among these states ranges from 0.8 to 1.1 Å . These dStructural overlays and interpolations of the L, T and O conformations, based on the topological decomposition just described, reveal the nature of the relative repeat motions within the TM domain during the transport cycle , Video 6mexB gene is expressed in two halves, split at the N-terminal end of Iα  crystal structure of AcrB . In these calculations all lysine and arginine side-chains were assumed to be ionized, except for K940 and R971, which were explicitly analyzed. Ka of D407 (in the O conformation), D408(O), E346(T) and D924(T), and a weak downward shift in K940(O), thus confirming that these side-chains are most likely protonated in those protomer states. As controls we also analyzed E693(T) and K603(O), which are located on the solvent-exposed surface of the periplasmic PC2 subdomain; as expected, the calculated pKa shifts for these side-chains are small or negligible  environment within the model protein SNase are down-shifted only by ∼2 units (The proposed changes in the protonation state of D407 and D408 in the transitions from T to O (protonation) and O to L (deprotonation) are consistent with the ‘disengagement’ and ‘reengagement’, respectively, of the structural repeats R1 and R2, as the ionic interactions with K940 would be weakened by protonation of the aspartate side-chains. The finding that K940 remains ionized in the O state might be less intuitive. However, analysis of non-redundant high-resolution structures in the Protein Data Bank reveals thousands of instances in which a lysine side-chain is part of an electrostatic interaction network that does not include any negatively-charged contacts . Moreove∼2 units .As mentioned above, experimental evidence demonstrates that K940 and R971 are indispensable for the proton-driven mechanism of AcrB . Our resTo further assess the proposed proton-relay mechanism and its influence on the conformation of the TM domain, we solved the atomic structures of four AcrB variants, namely K940A, R971A, D407N and D408N , all of In all cases, the crystal structures represent an asymmetric configuration of the AcrB trimer. As in the wildtype transporter, this asymmetry originates in the porter domain due to drug binding , compounNone of the substitutions studied caused significant changes in the internal structure of the R1 and R2 repeats, compared to the wildtype L, T and O states . The relElectrostatic calculations for the R971A and D407N crystal structures indicate that, like K940A and D408N, these variants lack the ability to bind and release two protons . In R971The effect of the D407N substitution is subtler than that of R971A, but also underscores the relationship between the local configuration of the proton-relay network and the organization of the transmembrane repeats. The D407N structure shows that although the substituted asparagine can accept a hydrogen-bond from K940 in the L and T states, its orientation is geometrically inconsistent with the second hydrogen-bond with T978 seen in the wildtype . In the As mentioned above, the crystallization of these variant structures was facilitated by DARPin ligands, which recognize the porter domain of AcrB in its asymmetric form . BecauseProton translocation across membrane proteins necessarily requires a pathway of water molecules to facilitate access from the surrounding solution, and to mediate proton hopping between protonatable side-chains. In the case of proton-driven secondary transporters, these pathways must also be dependent on the conformational state of the protein, that is, they must be consistent with an alternating-access mechanism, so as to preclude leakage and ensure strict coupling of substrate and proton transport. To examine whether the conformational cycle of AcrB indeed implies changes in proton accessibility, we carried out an all-atom molecular dynamics simulation of the complete trimer in a lipid membrane, and analyzed the formation and persistence of hydration channels across the TM domain. The results, summarized in A striking feature of these putative access pathways for protons is that they are, for the most part, highly dynamic and permit individual water molecules to frequently exchange between the bulk solution and the close vicinity of the proton-binding site . For theAs mentioned, in the T state we also observed a significant number of water molecules exchanging between the proton-binding site and the cytoplasm. However, the trajectories followed by these water molecules invariably enter or exit the TM domain in close proximity to R971 (within 10 Å) . This poThat the hydration of the TM domain is conformationally dependent is entirely consistent with the electron densities from ordered water molecules in the X-ray structures of wildtype and variant AcrB. As shown in In sum, our data indicate that throughout the conformational cycle of AcrB the proton-binding sites within the TM domain become alternately accessible to the periplasm or the cytoplasm. As mentioned, this is a necessary condition in order for the transporter to harness the proton-motive-force. Interestingly, two distinct types of relative motion of repeats R1 and R2 result in alternating access, namely a rocking motion during the L to T transition, with no protons bound, and a shear motion in the T to O transition, in the protonated form. This finding indicates that other transporters may also follow significantly different conformational pathways in the substrate-bound and apo segments of their functional cycle, contrary to what is often assumed. To our knowledge, however, AcrB is the only secondary-transporter for which two distinct structural transitions resulting in alternating access have been described to date.As described above, AcrB consists of well-defined structural elements in both its periplasmic and membrane domains, whose relative arrangement changes to a significant degree throughout the conformational cycle of the transporter. In the periplasmic domain, these rearrangements reflect substrate loading from the periplasm, the re-shaping of the hydrophobic binding site, and its release into the TolC lumen. In the membrane domain, water pathways across the protein become reconfigured to facilitate access from either side of the membrane, and a network of ionic and H-bonding interactions is re-organized as a result of protonation and deprotonation events.Taken together, these observations reveal an unprecedented antiport strategy that involves the remote coupling of two alternating-access transport mechanisms, that is, one for drugs and another for protons, within each of the AcrB protomers, which we summarize in In the T to O transition, D407, D408 and K940 break away from each other , followi1Fo-ATP synthase, which also utilizes the energy harnessed from the transmembrane electrochemical proton gradient to release synthesized ATP molecules from their binding sites in the α/β subunits  for two reasons: first, both proton- and substrate-binding sites are concurrently open to the periplasm in the T state, while in the O state they become open to the cytoplasm and the TolC lumen, respectively; second, both porter and membrane domains are able to re-open to the periplasm after releasing their cargo, that is, in their apo state  indicates that this kind of kinetic optimization is what emerges from the conformational coupling among protomers. A quantification of the steric clashes between protomers in these hypothetical models indicates that OO and LO interfaces  are highly incompatible, and that OT is also suboptimal. Thus, most of the combinations that include one or more O states in the trimer would be energetically unfeasible . Figure New high-resolution crystal structures of wildtype and AcrB variants, systematically analyzed through diverse computational methods, have enabled us to formulate a novel, clear-cut theory of the mechanism of proton-coupled drug-efflux in this very complex transport system. On the basis of this theory, which is consistent with existing biochemical, structural and functional data, additional and more precise experiments and computations may now be designed to further establish the proposed molecular mechanism of transport and energy transduction. It will also be of interest to extrapolate these findings to other homologues in the RND superfamily. Such investigations could potentially aid the development of novel pharmacological approaches, such as conformational inhibitors, against Gram-negative multidrug resistant pathogens.E. coli C43 (DE3) (His (−1 (Kan50).43 (DE3)  harborinE3) (His  were useE3) (His  were useE3)  was applied. For all AcrB variants except R971A, minocycline was added prior to crystallization at a concentration of 2 mM . All crystals were grown by the hanging drop vapor diffusion method at 0.05 M ADA buffer pH 6.5, 7–9% polyethylene glycol (PEG) 4000, 6–10% glycerol, 0.2 M (NH4)SO4. Cryprotection was achieved by transfer of the crystals to the same buffer containing 30% glycerol and flash freezing into liquid N2.Overexpression and membrane preparation of wildtype and mutant AcrB was carried out as described previously . DARPin 12121 crystals were collected at beamline X06SA (wavelength 0.8–1.0 Å) of the Swiss Light Source . Data reduction was done with the XDS package  method. To assess the most likely protonation state of the ionizable side-chains in AcrB, we carried out a series of Metropolis Monte-Carlo simulations of the global protonation equilibrium of the protein at fixed pH. In a first set of calculations, we analyzed in each protomer separately, including all ionizable side-chains in the calculation. All lysine and arginine side-chains were consistently predicted to be protonated at the pH and dielectric constant values used in these calculations (see below). Thus, in subsequent simulations, in which the complete trimer was considered, all lysine and arginine side-chains were fixed in their ionized state, except for K940 and R971, which were explicitly re-analyzed along with all aspartate, histidine and glutamate residues .Each Monte Carlo simulation was initiated with a random configuration of protonation states, and consists of five cycles of temperature annealing and a subsequent equilibration. In the annealing phases, the temperature was raised to 400 K and then gradually reduced to 300 K over 100,000 simulation steps. The equilibration phases, in which the temperature was kept at 300 K, lasted 500,000 steps. The protonation probability of each group was determined by averaging the ionisation states sampled during the equilibration phase of the final cycle of the simulation. This procedure was repeated 10 times with independent starting configurations, resulting in a global average; the typical statistical error was <2%.To evaluate the energy changes associated with the Monte Carlo moves we used a continuum electrostatic model, that is, the crystal structure of the AcrB trimer is surrounded by a high-dielectric medium representing water, except for a low-dielectric rectangular slab surrounding the transmembrane domain, which mimics the lipid membrane. Specifically, we used the linearized Poisson-Boltzmann equation solver implemented in CHARMM c34a2  to set uKa values were: aspartate, 4.0; glutamate, 4.4, histidine, 6.3; lysine, 10.4; and arginine, 12.0. Residues on the periplasmic side of the protein were assumed to exchange protons with a solution at pHout = 5.5, while those on the cytoplasmic side exchanged with a solution at pHin = 7.5; interpolated pH values in this range were used for residues across the membrane. Calculations with an alternative value of pHout = pHin = 7.5 led to analogous conclusions (not shown).The reference aqueous-phase p+ counter ions when necessary to neutralize the total charge of the system.All molecular dynamics simulations were carried out at 298 K and 1 atm. Three independent all-atom systems were studied, namely the complete asymmetric AcrB trimer and truncated constructs of the T and O protomers comprising the transmembrane and porter domains only . In all http://danger.med.unc.edu/hermans/dowser/dowser.htm). To optimize the protein-solvent and protein-lipid interfaces in the model, and to thermalize the system, a series of short simulations (10 ns) were initially carried out with gradually weaker positional restraints applied to the protein and the crystallographic and DOWSER-added water molecules (excluding hydrogen atoms). To enable the protein to tumble, the equilibration phase was extended (5 ns) after replacing the positional restraints with collective conformational restraints (RMSD), applied first to the whole protein, and subsequently to each of three transmembrane domains separately. In the last state of the equilibration (10 ns), only the configuration of the proton-relay network was preserved via inter-residue distance restraints . Finally, a fully unrestrained simulation was carried out for 250 ns.This system was employed to assess the formation and dynamics of water channels within the transmembrane domain. A minocycline molecule was bound to the porter domain in the T protomer. D407 and D408 were deprotonated in the L and T protomers, and protonated in the O protomer; K940 and R971 were protonated in all protomers. The simulation was initiated with the high-resolution (1.9 Å) crystal structure of wildtype AcrB (PDB entry 4DX5), preserving crystallographic water molecules. Additional water molecules within the protein structure were placed with DOWSER  method; the substantial computational cost of this methodology is what motivated the truncation of the protein. The preparation and equilibration of these reduced simulation systems was analogous to the procedure followed for the trimer. In the final production phase (90 ns), however, only the transmembrane domain was fully unrestrained. The conformation of the porter domain was preserved using a harmonic restraint on its RMSD relative to the crystal structure, to preclude any distortions resulting from the artefactual truncation of the funnel domain. Importantly, the force constant of this harmonic restraint was selected so that the structural fluctuations of the porter domain in the truncated-protomer system are comparable to those observed in the unrestrained simulations of the complete trimer (not shown). Thus, the dynamics of the transmembrane domain during the free-energy calculations is unlikely to be significantly affected by the truncation. For both T and O protomers, snapshots after 90 ns of simulation were extracted and used to initiate the FEP calculations. Here, the protonation state of a given side-chain is exchanged alchemically, using a step-wise protocol controlled by a parameter λ that reflects the weight of either protonation state in the potential energy function of the system. All FEP calculations were carried out in the forward and backward directions, with 38 intermediate λ steps (33 for D408 in the O state). The simulation time per λ step was 1 ns; the initial 100 ps of each simulation were considered as equilibration, and so free-energy increments were obtained from the final 900 ps.2 per lipid). Electrostatic interactions were calculated using the Particle-Mesh-Ewald algorithm, with a real-space cut-off of 12 Å. A shifted Lennard-Jones potential, also cut-off at 12 Å, was used to compute van-der-Waals interactions. The CHARMM27/CMAP force field for proteins and lipids , Asn (Oδ), Glu (Oε), Gln (Oε), Ser (Oγ) or Thr (Oγ) side-chains.Alternative structures of the porter domain were modeled in all possible permutations of the L, T and O conformations, via rigid-body rotations of the PC1/PN2 and PN1/PC2 sub-domains around the trimer axis. All side-chains in all models  were then re-built using SCWRL4 . The sol review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a Reviewing editor, and 2 reviewers, both of whom (Ben Luisi and Shimon Schuldiner) have agreed to reveal their identity.Thank you for sending your work entitled “Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB” for consideration at The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.Both reviewers are in agreement that your work should be accepted pending minor changes.Reviewer #1 minor comments:1) The movies are wonderful, but it would be easier to follow them if they were annotated with some labels.2) In the Introduction the authors refer to the membrane distal portion of the periplasmic domain as the 'TolC-dockign domain'. Is this naming convention too suggestive in light of the report of the structure of the pump assembly see  Nature?3) 4) Abstract, change to read “...one of the most...”Reviewer #2 minor comments:1) What do the authors mean by saying that the ...TM domain had not previously described…?2) 3) 4) Regarding the suggested role of R971 in blocking proton leakage: is there any indication that R971A expression is toxic to the cell?5) Are the mutants D407N and D408N fully inactive? Could it be that they are moving only one proton at a time and they can then generate smaller gradients? Is it possible to look at AcrB-dependent passive loading of ethidium in whole cells? Is the assay that follows hydrolysis of cephalosporins sensitive enough for this purpose?6) The model presented here has some very clear predictions that could be challenged experimentally or supported by existing data. Is there any evidence that supports a stoichiometry of 2H+/substrate? As far as I know AcrB transports positively and negatively charged substrates as well as neutral ones. The driving force for them with this stoichiometry would be very different at different pHs. I am aware of the problem of making proteoliposomes that transport substrate but they may be able to make PLs and follow substrate-induced proton transport. Reviewer #1 minor comments:1) The movies are wonderful, but it would be easier to follow them if they were annotated with some labels.The revised movies now include labels that identify each of the conformational transitions, as well as each of the structural repeats in the periplasmic and transmembrane domains, and the coupling helicesTM2 and TM8.2) In the Introduction the authors refer to the membrane distal portion of the periplasmic domain as the 'TolC-dockign domain'. Is this naming convention too suggestive in light of the report of the structure of the pump assembly (seeNature)?The term 'TolC-docking domain' was inspired by the studies of Murakami et al. . In addition, Tamura et al.  detected cross-linking between these two components of the tripartite system, and thus inferred a direct interaction. This conclusion is at odds with the recent EM studies by Du et al. , which indicate no direct contact between TolC and AcrB. At the time of the submission of our article, the results of Du et al. had not been made public yet. A change in nomenclature seems appropriate now. Therefore, we refer to this region of the protein as the ‘funnel domain’, as it features the first section of the exit pore extended by AcrA and TolC.3)is a new interpretation of old data. The density looks a little poor in the L state: are the side chains disordered?2) than in the T and O states (between 31 and 60 A2). Nevertheless, the assignment of the side-chain conformations is unambiguous in all cases.In actuality the data shown in 4) Abstract, change to read “...one of the most...”This has been changed accordingly.Reviewer #2 minor comments:1) What do the authors mean by saying that the ...TM domain had not previously described…?Please see our answer to question #3 by Reviewer 1. We changed this sentence to “whose TM domain had not been previously analyzed”.2): there is an isolated residue in the cytoplasmic side that changes its protonation probability during the cycle. This should be labeled.The residue in question (H505) is labeled in the revised figure.3)legend. They refer to figure dashed lines and solid lines in, should beThis has been changed accordingly.4) Regarding the suggested role of R971 in blocking proton leakage: is there any indication that R971A expression is toxic to the cell?E. coli C43(DE3) cells for purification and crystallization purposes. We observed no effect on growth compared to cells producing AcrB wildtype. This variant was produced as well in E. coli BW25113ΔacrB carrying pET24acrBHis_R971A , to determine MIC values. Under conditions where no drug was added to the growth medium, these cells grew to OD600 values comparable to those cells expressing wildtype AcrB or other variants. There are a number of ways to interpret these results, but we believe the most plausible explanation is that in the absence of drug, AcrB adopts the symmetric LLL state observed in X-ray structures that lack a bound substrate. The absence of a T protomer in this ‘resting state’ of the trimer would explain the lack of H+ leakage.We overproduced the R971A variant in 5) Are the mutants D407N and D408N fully inactive? Could it be that they are moving only one proton at a time and they can then generate smaller gradients? Is it possible to look at AcrB-dependent passive loading of ethidium in whole cells? Is the assay that follows hydrolysis of cephalosporins sensitive enough for this purpose?E. coli cells.In our experience and within the limitations of the MIC value determination, D407N and D408N clones exhibit identical susceptibility towards all drugs tested. We routinely use passive loading and active efflux experiments with fluorescent dyes to test the efflux activity of AcrB variants and take either D407N or D408N clones as negative controls. Thus far, we have not observed any activity in these assays. The assay based on hydrolysis of cephalosporins  might be indeed more suitable to detect residual activity, since acrAB knock out strains produce only 1% of the efflux activity compared to wildtype. However, this assay requires a special set-up of the spectrophotometer, currently not available in our lab, to reduce scattering caused by the measurements with whole 6) The model presented here has some very clear predictions that could be challenged experimentally or supported by existing data. Is there any evidence that supports a stoichiometry of 2H+/substrate? As far as I know AcrB transports positively and negatively charged substrates as well as neutral ones. The driving force for them with this stoichiometry would be very different at different pHs. I am aware of the problem of making proteoliposomes that transport substrate but they may be able to make PLs and follow substrate-induced proton transport.+ is the magnitude of the substrate gradient sustained by the action of the AcrAB-TolC efflux pump. Comparing MIC data from cells lacking AcrB with those with active AcrB, it appears that the pump is in some cases able to sustain gradients larger than 1:1000, e.g. for the substrate oxacillin . The energetic cost required to sustain such gradients would exceed the energy gain associated with the transport of a single proton across a membrane potential of ∼-180 mV. These observations, in combination with the structural information we obtained for the three different states  and the two alternating-access pathways deduced by the MD simulation studies, strongly indicate a H+:drug stoichiometry of 2:1. Clearly, direct experimental evidence from reconstituted AcrAB-TolC in proteoliposomes would settle this question, but this remains an experimental challenge, due to the complexity of this two-membrane spanning tripartite system.In the current hypothesis, the tripartite system sequesters drugs from the periplasm/membrane boundary and expels them over the outer membrane. That is, drugs are not transported from the cytoplasm across the plasma membrane, but only across the outer membrane. For protons, however, AcrB mediates classical transport from the periplasm to the cytoplasm, to harness the electrochemical gradient and energize the conformational changes necessary for active efflux of drugs. Consequently, the charge of the transported drugs is not a relevant factor i.e. the overall process of transport will be electrogenic for every drug molecule transported from the periplasm to the outside, regardless of its charge. The thermodynamic quantity that depends on whether each transport cycle is coupled to one or two H


The formation of such domain walls is interpreted in the light of polarization discontinuity and clamping effects at phase boundaries between the nesting domains. Phase-field simulation confirms that the coexistence of both phases as encountered near the morphotropic phase boundary promotes the polarization to rotate in a continuous manner. Our results provide a further insight into the complex domain configuration in ferroelectrics, and establish a foundation towards exploring chiral domain walls in ferroelectrics.In contrast to the flexible rotation of magnetization direction in ferromagnets, the spontaneous polarization in ferroelectric materials is highly confined along the symmetry-allowed directions. Accordingly, chirality at ferroelectric domain walls was treated only at the theoretical level and its real appearance is still a mystery. Here we report a Néel-like domain wall imaged by atom-resolved transmission electron microscopy in Ti-rich ferroelectric PbO3 crystals, providing insight in exploring chiral domain walls in ferroelectrics. Oxide ferroelectrics, with coupling of polarization, strain, heat and electric field, are widely utilized in electronic devices such as multilayer capacitors, piezoelectric transducers, pyroelectric detectors and non-volatile high-density memories12346710113 (refs 3 (ref. 19201922242526x1−Tix)O3 (PZT), (1−x)PbMg1/3Nb2/3O3−xPbTiO3 and BiFeO3, where the polarization easily rotates within a crystallographic plane, offers new possibilities in the search of new types of domain walls.As known, magnetic 180° domain walls are typically of Bloch type or Néel type133 refs , 16, 17 CS) imaging (NCSI) technique in an aberration-corrected TEMCS imaging technique, the NCSI technique yields an especially high image contrastExperimentally, direct imaging of light oxygen together with heavy cations using transmission electron microscopy (TEM) serves as the decisive basis to resolve chirality at the ferroelectric domain walls. This requirement is fulfilled by the negative spherical-aberration  single crystals, where the monoclinic order coexists with the tetragonal order at the nanometre scale. The formation mechanism of the domain wall is interpreted in the light of polarization discontinuity and clamping effect at the monoclinic-tetragonal PBs.Here, by means of direct imaging of electric dipoles using the NCSI technique combined with the ultrahigh-precision measurements, we report a Néel-like rotation of polarization at monoclinic domain walls in ferroelectric PZT  is oriented along the [001]T direction in the tetragonal phase (space group P4mm), but in the monoclinic phase, it is allowed to rotate flexibly within the (110)T planex=0.54, 0.60) crystals. x=0.60 PZT specimen oriented along the [100]T direction. The observed nanometre-scale ferroelectric domains are in agreement with earlier experimental results on the second-order nature of the ferroelectric phase transition in this composition rangeP·g >0 relation (P is a component of PS and g is a scatting vector)c/a≈1.043. However, splitting of the reflection spots, which is thoroughly different from that of 90° domainsc axis relating to the left- and right-side reflection spots is ∼0.415 and 0.411 nm, respectively. The good agreement of the measured results with the data from high-resolution synchrotron X-ray diffractionT and the monoclinic  along the [001] direction. In contrast, in the monoclinic domains, M-I and M-II, the oxygen columns shift to right-up and left-up, respectively, evidently deviating from the [001] direction. A small part of M-III domain is also detected in the right part of the image area. The displacement features of oxygen columns in the M-I, T-I and M-II domains can be directly identified from the magnified images shown in cM/2, in which the displacements of oxygen columns undergo a sharp transition, is observed at the right side of the region. Measurements of the relative displacements of oxygen columns on the left-side wall segment, extending the areas upwards and downwards from the central plane, reveal a continuous change of the oxygen displacements, that is, continuous rotation of the displacements from leftward (slightly up) in M-II through upward at central plane to rightward (slightly up) in M-I. The displacement feature of oxygen atoms forms a structure similar to the Néel wall in ferromagnetsA particularly interesting feature of the atomic structure is seen in the region including the central plane of the wall between domains M-I and M-II, as denoted by a white dotted line in c axis in the M-I and M-II domains is 0.415 and 0.410 nm, respectively, which decreases in a stepwise manner across the wall centre. On the contrary, the a axis in the M-I domain is ∼0.40 nm, slightly smaller than that in the M-II domain. As a result, the c/a ratio in the M-I domain is 1.038 in average, it reaches a value of 1.048 near the central plane of the wall and extends into the M-II domain by 2 unit cells. This ratio decreases to ∼1.024 in the rest part of the M-II domain as shown in c/a ratios in the M-I and M-II domains are not necessarily different from each other. It is therefore reasonable to speculate that, to a certain degree, their tetragonality values rely on conditions of the surrounding domains.δxO2-Zr/Ti) undergo a smooth reduction from ∼12 pm in the M-I domain to zero at the central plane of the wall  phase only, in coexistence with either the rhombohedral phases333841T plane. The formation of the Néel-like wall configuration can be attributed to effects of polarization discontinuity at the PBs and local clamping, that is, to the local electrostatic and elastic conditions in the PZT crystals with coexistence of the two intertwining phases at the nanometre scale.So far, structural investigations of the PZT solid solutions at ambient pressure have revealed the monoclinic  crystal (PS=64 μC cm−2) is in a good agreement with the macroscopic electric hysteresis loop measurement  phase-field simulation on a thin (110)It is well established that sufficiently high content of the larger Zr cations in the tetragonal PZT crystal near the morphotropic PB flattens the potential profile of the system. As a result, the monoclinic states become local minima and the presence of stable monoclinic phases are expected. Although the lower-energy phase in an idealized phase-field model would always sweep-out the higher-energy phase, even if the energy difference is very small, the realistically inhomogeneous Zr distribution, as corroborated by our phase-field simulation, enables the multiphase coexistence in the PZT crystals . Facilit3 and BiFeO3. In contrast to these complex domain patterns, for example, the flux-closure structure consisting of consecutive 180°–90°–180° domains, the polarization transitions at these non-180° walls are sharp and the 180° walls are all Ising-like. However, the Néel-like domain walls observed in this work possesses the genuine chiral feature.In recent years, the 180° stripe domains4248In conclusion, our results provide direct experimental evidence for the existence of Néel-like domain walls in ferroelectric materials. The findings highlight that in the low-symmetry ferroelectric phases, which widely exist in solid solutions272614Lead zirconate titanate crystals were grown by a top-seeded solution growth technique. Optical microscopic examination showed a good optical quality of the crystals without inclusions of visible defects. The good quality of the crystals is supported also by the square-shaped saturated ferroelectric hysteresis loop and the high remnant polarization . The lamCS corrector for the objective lensThe TEM investigations were performed on an FEI Titan 80–300 microscope with a 2 times smaller those than in ref. 3 (110)T lamella of PZT crystal. The central 40% of the lamella has x=0.5, the rest x=0.6 T surfaces have constant potential and all remaining surfaces have zero free charge. Initial conditions were set in the proximity of the expected stationary solution. The model is numerically solved by the finite element method with a time-dependent solver in COMSOL 4.3a  until stationary solution at each strain was reached.The phase-field simulation results O3 single crystals. Nat. Commun. 7:12385 doi: 10.1038/ncomms12385 (2016).Supplementary Figures 1-5, Supplementary Notes 1-4 and Supplementary References


C. intestinalis. The presence of a functional collagen receptor integrin αI domain supports the origin of orthologues of the human integrins with αI domains prior to the earliest diverging extant vertebrates, a domain that has been conserved and diversified throughout the vertebrate lineage.Half of the 18 human integrins α subunits have an inserted αI domain yet none have been observed in species that have diverged prior to the appearance of the urochordates (ascidians). The urochordate integrin αI domains are not human orthologues but paralogues, but orthologues of human αI domains extend throughout later-diverging vertebrates and are observed in the bony fish with duplicate isoforms. Here, we report evidence for orthologues of human integrins with αI domains in the agnathostomes (jawless vertebrates) and later diverging species. Sequence comparisons, phylogenetic analyses and molecular modeling show that one nearly full-length sequence from lamprey and two additional fragments include the entire integrin αI domain region, have the hallmarks of collagen-binding integrin αI domains, and we show that the corresponding recombinant proteins recognize the collagen GFOGER motifs in a metal dependent manner, unlike the α1I domain of the ascidian  Integrins are multi-domain cell-surface receptors that fulfill numerous function roles at the level of cell-cell communication and interactions between cells and proteins of the extracellular matrix  and Petromyzon marinus (sea lamprey) and from cartilaginous fish .Orthologues of the human integrin α subunits with I domains are found in species extending from the bony fish (Osteichthyes) through to the mammals P. marinus, E. burgeri, Callorhinchus milii, , Raja erinacea (little skate) and Squalus acanthias (dogfish shark). Although our intuition is that orthologues of human αI domains should be found in cartilaginous fish, our searches of the chimaera, skate and shark assemblies only yielded three short fragments. Two sequence fragments from C. milii were very similar to portions of the human integrin I domains α1  and α2 , beginning by matching the αI domain αC helix; a third fragment from C. milii matched repeat 5 of the β-propeller domain of human α2 . Now, with the publication of the genome sequence of C. miliiWe have been regularly searching genomic sequencing data for integrins sequences in order to clarify the origins of different features, especially integrin α subunits, individual domains and αI domains in particular. We conducted searches Searches E. burgeri genome Additionally, one short 133-residue fragment (Ebu_f) of a possible αI domain from the hagfish Xenopus laevis), four bony fish species , four sequences from the elephant shark C. milii and sequences from the tunicates H. roretzi and C. intestinalis. The sets of trees differ in having (a) 69 sequences including the near full-length lamprey Pma_f3 sequence; (b) 72 partial sequences that include the maximum common regions – 406 to 409 residues shared by the 3 lamprey sequences Pma_f1-3; and (c) 73 αI domain regions of approximately 200 residues that include the hagfish sequence fragment Ebu_f and Pma_f1-3. The 701-residue sequence fragment of α1 from C. intestinalis is included in the αI domain and common regions trees.Here, we have constructed three separate sets of phylogenetic trees from sequence alignments and based on three different tree reconstruction methods. The sequences include representatives from 15 chordate species containing the αI domain .doc. In Phylogenetic trees were inferred from sequence pairwise distances  ends prior to the αC helical region . A searcKey residues involved in αI domain recognition of the collagen-like GFOGER and GLOGEN tripeptides were identified from known representative three-dimensional structures of complexes using Surf2 , and then we examined the similarities and differences among equivalent residues in the human set of integrin αI domains and the residues present in the agnathostome sequence fragments .doc.2+ at the MIDAS site and binds glutamic acid of ligands, although Co2+ used in the crystallization is present in the α2I domain and binds E11 from one chain of the GFOGER tripeptide ligand in the complex structure  largely determines collagen subtype preferences C of the peptide and this residue is also present in α1I, α10I and α11I.Structural models were built for the lamprey sequences and a comparison of the key features of the X-ray structure of the α2I-GFOGER complex  and the r Pma_f3 , the seq219MER221 with 219DLT221 in the α2I domain cannot be correct as the large arginine side chain in the Pma_f1 sequence cannot substitute for threonine  but it may be that the adjacent E220 can substitute for threonine; it remains to be tested.Pma_f2, like Pma_f1, is identical in sequence at 9 of 16 ligand-interacting positions seen for the α2I domain. One key position in α2I, T221, functions to chelate the metal ion at MIDAS and the equivalent residue in the Pma_f1 sequence is uncertain and there is no threonine residue nearby. In E. coli strain BL21 tuner. The expressed proteins were sufficiently pure for kinetic experiments to be carried out. A minor amount of GST was observed in each protein preparation and in Pma_f3 preparations a small amount of processed fusion protein was occasionally observed  were tested for binding to rat collagen I. Recombinant Pma_f3 αI domain shows significantly lower binding levels at a high αI concentration (400 nM) , possiblIn order to determine the binding affinity of recombinant Pma αI domains we tested their binding to rat collagen I at various αI domain concentrations and estimated the affinity as described previously 2+, to the ligand atom NH2 of arginine R5001 is 14.3 Å and the two “walls” of the α subunit, 8.8-9.7 Å between atoms near the aspartate where R5001 binds, restricts the ligand to be an extended chain), the early integrins were limited to the recognition of exposed loop regions of ligands that could occupy the restricted binding cleft and having restricted options for motif specificity. This integrin organization usefully served for the recognition of proteins from the extracellular matrix and cell surfaces with exposed loops but would have been unable to accommodate other, more bulky ligands.The basic integrin heterodimeric structure arose early , probablThis original organization of the integrin heterodimer is found across the span of metazoan species and is the sole integrin type identified in species diverging prior to the tunicates . Thus thThe insertion of the αI domain into an α subunit occurred approximately 550 MYA, after the deuterostomes first appeared and after the chordate line was established. The αI domain is observed in integrin α subunits from the tunicates but not in the lancelet , which is congruent with the lancelet now being acknowledged on the basis of genome comparison studies The αI domain has a highly solvent-exposed ligand binding surface capable of recognizing larger ligands and surfaces, thus the integrin binding site would no longer be limited to external loops that could access the fairly narrow cleft between the β-propeller and βI-like domains. With the αI domain, ligands bind to the metal at MIDAS via a glutamate residue instead of aspartate found in ligands targeting MIDAS of the βI-like domain. The αI domain allows unfettered access to the binding site facilitating recognition of ICAM immunoglobulin-fold domain surfaces and collagen triple helices bundled into large structures could be more easily accessed and recognized. The more exposed binding site also means that the interaction of the αI domain with ligands involves more residues, upwards of 15 residues in collagen-like peptide and ICAM immunoglobulin fold recognition. As a consequence of the relocation of the binding site, a C-terminal glutamate residue of the αI domain acts as an intrinsic ligand binding to MIDAS of the βI-like domain, participating in the dynamic conformational mechanisms associated with the function of integrins with αI domains.H. roretzi α1I functions in a primitive form of complement recognition and Tulla et al. C. intestinalis α1I domain can bind human recombinant collagen IX that is both metal and MIDAS independent. Orthologues of the human collagen receptor αI domains always have the αC helix and this is found in all three reported lamprey sequences, one of which is a fairly complete α subunit, lacking only the first two repeats of the β-propeller domain. The expressed lamprey αI domains bind mammalian collagens, as shown here but binding, in contrast to C. intestinalis α1I, is metal and MIDAS dependent as is the case for mammalian collagen binding with the human αI domains; thus the mechanism of mammalian collagen binding in the tunicate is clearly different from that shared by lamprey and humans. Furthermore, this study shows that the determinants for collagen recognition by integrins with αI domains was established early on in chordate evolution and persists throughout the vertebrates.Here, we show that the first appearance of features characteristic of the collagen receptor integrins, and possibly immune system integrins, are found in the agnathastomes, whereas the integrins with αI domains of the earlier diverging tunicates cluster together and have clearly not specialized into the types observed in humans . This isC. milii. Both αE and αL are present in bony fish but the presence of other bony fish integrins within the leukocyte clade show that the αM/αD/αX specialization had not yet occurred  in order to identify potential candidate sequences for this study. Various ongoing and completed genome projects at the Ensembl webpage (http://www.ensembl.org/index.html) were also searched  in order to finalize and create a dataset for our analysis . The program displays coordinates for each sequence and their locations such that the variance among the data is a maximum, and projections for various numbers of dimensions are possible. The three most informative dimensions, as a pseudo-PDB coordinate file, were visualized using Bodil http://accelrys.com/products/discovery-studio/). Furthermore, energy minimization was included by using the Charmm force field Protein structures were obtained from the Protein Data Bank Bodil E. coli BL21 tuner  was used for protein production, which was performed as earlier Lamprey nucleic acid sequences for the predicted αI domain regions were synthesized by Eurofins MWG Operon (Germany) for Pma_f1, Pma_f2, and Pma_f3 and the genes were transferred into the pGEX-2T vector for expression. The expression strain Pma_f1SGFNVSESYAPTLQKCGSYMDIVFVLDGSNSIYPWSDVQNFLVKTLQSFHIGPDQTQDDVCLPGANVVVVFKLSDTPLYERWGVSLVVLWRRWGMERGNDLNVYPSRSEAFSPERGARPDAQKVMIVVTDGESHDKYLLPEVIDQCERDGITRYAIAVLRSYSSNADDVARLINEVRSIASHPVERHFFNVTSEATLIDIVGTLGERIFSLEGTRPma_f2ADFQVTSTLTPAAQRCGLFMDIVIVLDGSNSIYPWQEVQNFVINIVKKFHIGPGQSRNGGGSTRFGVRTIHWHLGIARWACEGVQDVENIYSRPFVRTASALCQSLQVVRSEAFSPLFGAREGASKVMIVVTDGESHDSEDLTEAIAACERDNITRYAIAVLGYYKRKNIDPSNFISELKAISSEPEEKHFINVADEAALNDIVGTLGERIFSLEGTVPma_f3PNFQQLGSPFAPTMTGCRSFLDIVLVLDGSNSIWPWPSVLDFLSSILETFSIGPGQTQVGIMQYGETVSNEMNLNQFTNKAQLKIAASKIPQRGGKVTNTAMGIEAARFFFFENGGRAEASKVMIVVTDGESSDAYKLPGVIKDCNDDGITRFGIAVLDYYISSNMNVEKLQAEIRSIASTPTEKYYFDVKSTGALVDITKALGERIYSLEGTSFor both the short and long versions of Pma_f1 αI and Pma_f3 αI we did not see any differences in their binding properties (not shown), however the short version of Pma_f2 αI was not expressible.The following collagens were used in experiments: rat tail collagen I , bovine collagen II , mouse collagen IV , and recombinant human collagen IX . The GFOGER tripeptide was synthesized by Auspep . The triple-helical nature of the peptide has been checked with CD-spectroscopy.Binding studies were performed as earlier File S1Table S1: Sequences utilized in the phylogenetic analysis. Table S2. Residues in the α1I domain structure within 4.2 Å (non-hydrogen atoms) of the bound GLOGEN tripeptide Click here for additional data file.


Coliforms were not detected in the bread. Bacteria isolated were species of Bacillus, Staphylococcus, and Micrococcus while fungi isolates were species of Aspergillus, Penicillium, Rhizopus, and Mucor. The mean sensory scores  were evaluated. The color of the bread baked from WF/IPF2  blend was preferred to WF  while WF/SPF1  and WF/IPF1  aroma were preferred to WF. However, the bread baked from WF, WF/IPF2 , and WF/SPF2  was more acceptable than other blends. The use of hydrated potato flour in bread making is advantageous due to increased nutritional value, higher bread yield, and reduced rate of staling.Dehydrated uncooked potato (Irish and sweet) flour was blended by weight with commercial wheat flour at 0 to 10% levels of substitution to make bread. Comparative study of the microbial and nutritional qualities of the bread was undertaken. The total aerobic bacterial counts ranged from 3.0 × 10 Bread is universally accepted as a very convenient form of food that is important to all populations. Its origin dates back to the Neolithic era and is still one of the most consumed and acceptable staple food products in all parts of the world. It is a good source of nutrients, such as macronutrients  and micronutrients  that are essential for human health .In Nigeria, bread has become the second most widely consumed nonindigenous food product after rice. It is consumed extensively in most homes, restaurants, and hotels. It has been hitherto produced from wheat as a major raw material . In NigeAlthough there is now a substantial amount of composite bread, such bread still requires at least 70% wheat flour to be able to rise because wheat contains gluten , 5. The Potato is a food crop with potential for partial replacement of wheat in bread making. Uncooked potato flour prepared by low-cost solar dehydration technology has a long shelf life and high nutritional quality, which could be valuable in cereal-based human diets, including bread . Sweet pThe aim of this study was to replace part of the wheat flour in bread by potato flour in order to increase the fibre and other nutrients. The microbiological qualities of the bread were also assessed.Ipomea batatas) and Irish potato (Solanum tuberosum) and wheat flour were purchased from a local market in Offa, Kwara State, Nigeria, and the Central Market, Minna, Niger State, Nigeria, respectively, in polythene bags, and transported to the laboratory for processing.The dry starchy yellow-fleshed cultivar of sweet potato (The potatoes were washed under running tap water (to free them of adhering soil particles), air dried, and stored at 12°C before use. The potato flours were prepared from solar-dried slices according to the method outlined in , 11.Eight blend formulations  were bakTotal mesophilic  and fungi counts (yeast and mould counts) were carried out on the bread samples to determine the microbial load of the samples as described by American Public Health Association . Bread sThe bacterial isolates were characterized using Gram reaction and biochemical tests and were identified by comparing their characteristics with those of known taxa as outlined in Bergey's Manual of Systematic Bacteriology . The funMoisture content of the bread was determined using the procedure described by AOAC .Protein was determined using the micro-Kjeldahl method AOAC . The conThis was carried out using the standard method of AOAC .Determination of the crude fibre and the ash content in the bread samples was carried out using the standard methods described by AOAC .Carbohydrate was determined using estimation by difference AOAC . The cruSensory evaluation was preformed 24 hours after baking to evaluate loaf color, crust, aroma, crumb texture, taste, and overall acceptability of the bread sample. A panel of ten judges (using a questionnaire) of regular bread consumers using the Hedonic scale product was set up. The panel was set up in three sets (to obtain three replicates) and the sensory scores were analyzed statistically.The bread samples were stored under ambient temperature (26°C–33°C) and observed for 10 days. Visual observations for mould growth were carried out on the samples stored.P < 0.05) was used for the study.Statistical analyses were performed using the SPSS (version 20). Difference in proximate composition and sensory scores was detected using one-way analysis of variance (ANOVA). A significance level of (5 cfu/g to 1.0 × 106 cfu/g with the highest being recorded for bread made from 100% sweet potato flour (SPF) while the lowest counts (3.0 × 105 cfu/g) were obtained in bread made from 100% Irish potato flour (IPF). Bacteria were not detected in bread baked from 100% wheat flour (WF); see WF/SPF1, WF/SPF2, and WF/IPF2 in 1 cfu/g to 1.2 × 103 cfu/g with the highest counts recorded for bread baked from WF/SPF2 while the lowest counts (8.0 × 101 cfu/g) were observed in bread baked from SPF/IPF  was obtained in SPF/IPF. The respective values obtained in WF, SPF, IPF, and WF/IPF bread were 15.15%, 15.31%, 14.945, and 12.63%  was obtained in IPF bread, while the lowest ash content (1.50%) was obtained in SP/IPF bread. The carbohydrate content was higher in WF/SPF1 (87.08%) than in the rest of the flour blends, but it was, however, the least (70.10%) in SPF/IPF bread (P < 0.05).The highest moisture content of 16.0% was obtained in WF/IPFd 12.63% . The higPF bread . The staP < 0.05) among the wheat/potato blends with the exception of the WF and WF/SPF2 for color, as well as WF/SPF2 and WF/IPF2 for aroma. The scores also indicated that bread baked from WF was more acceptable than that from other blends. However, this was closely followed by bread baked from WF/IPF2 and WF/SPF2 blends.The mean sensory scores for each quality attribute evaluated  of the bread samples prepared from the wheat/potato blends are presented in 2, and WF/IPF2 lasted for 6 days while IPF and WF/IPF1 lasted for 7 days before spoilage occurred. It was also observed that SPF, SPF/IPF, and WF/SPF1 lasted for 8 days before spoilage set in  could be due to the abundance of moisture and nutrient in the potato bread, which provide for a favorable condition for growth. The bacteria isolated from the bread samples included species of heating . K. Tala heating  reported Aspergillus, Penicillium, Rhizopus, and Mucor. These organisms could have been introduced at the different stages of bread production. This finding is in line with the study of Daniyan and Nwokwu [The fungi counts were higher in bread made from SPF/IPF (50 : 50%) than all other samples probably because of raw materials, processing, handling, and storage. The fungi isolated include species ofd Nwokwu  who idenThe moisture content of food goes a long way in suggesting the shelf life of the product. The moisture content of bread made from wheat/Irish potato flour  was higher than that of the other flour blends. This may be due to the processing methods the samples were exposed to. The values of the other blended samples fall within the acceptable moisture limit for dry products (15%). Adeleke and Odedeji  obtainedIncrease in the level of potato flour resulted in decrease in the protein content from 12.25% in 100% wheat flour bread (WF) to 3.75% in bread made from 50 : 50% sweet potato/Irish potato flour (SPF/IPF). This may have been due to the low protein content of the potato flour which must have diluted the protein content of the wheat flour, thus reducing the protein level of the mixed flour. Combination of WF and IPF (95 : 5%) resulted in an increase in the protein content of the bread from 4.3% to 14.00%. This is similar to the earlier findings where protein content of snacks reduced with supplementation with starch based products  for breaBlending of wheat flour with 10% potato flour resulted in an increase in the fat content. This could be due to the fact that Irish potato contains about 1–4% fat which could have been responsible for the slight increase in the fat content. The value of ash in bread made from wheat flour blended with potato flour was low compared to the ash content in bread made from wheat flour. The value of crude fibre ranged from 0.90% to 2.01%. The crude fibre was above the 1.5% maximum allowable fibre content of bread flour as stated by Omole  but fellThe carbohydrate content of bread increased with addition of potato flour to the wheat flour. This may be due to higher carbohydrate content in potato than in wheat. Cereals store starch as a source of energy and are low in protein, fat, and ash. Madukwe et al.  recorded2 (wheat/sweet potato flour 95 : 5%), and WF/IPF2 . Bread made from SPF/IPF (50 : 50% sweet potato/Irish potato flour), IPF (100% Irish potato flour), and SPF (100% sweet potato flour) was highly rejected by the panelists. This shows that using either sweet or Irish potato flour alone to bake bread will not be accepted, except if it is blended with wheat flour. Bread made from potatoes alone has unpleasant color and is not usually soft.The mean sensory scores of quality attributes of the products indicated that, generally, panelists expressed preference for three bread samples out of the eight presented. The bread samples were those made from WF (100% wheat flour), WF/SPFThe results of the storability of bread showed that bread made from 100% wheat flour, 10% sweet potato blended bread, and bread made from 50 : 50% Irish/sweet potato could not stay longer than six days due to the presence of fungi, while other samples lasted 7-8 days. This shows that the use of 5% dehydrated potato flour in bread making reduces the rate of staling as a result of the ability to retain moisture in them.The incorporation of potato flour to wheat flour improved the nutritional value of the bread. Bacteria and molds common in the environment contaminated the bread and led to its spoilage after 6–8 days; however, sensory evaluation indicated that consumer acceptable bread could be substituted with wheat flour and dehydrated uncooked potato flour in commercial bread making without sacrificing consumer acceptability. With the use of potato flour in bread making, the cost of bread production could be less.


Plasmodium parasites: SPECT1, which is essential for host cell traversal and hepatocyte invasion Fertilization, the culminating event in sexual reproduction, occurs when haploid sperm and egg recognize each other and fuse to form a diploid zygote. In mammals this process critically depends on the interaction between Izumo1, a protein exposed on the equatorial segment of acrosome-reacted sperm, and the egg plasma-membrane-anchored receptor Juno  et al. report the crystal structure of mouse Izumo1 and show that this shares similarities with Plasmodium proteins involved in host cell invasion.Interaction between sperm Izumo1 and egg Juno is essential for triggering the fusion of mammalian gametes, but the sequences of these proteins do not resemble known membrane fusogens. Here, Nishimura  Juno and Izumo are structurally unrelated: Juno adopts a modified folate receptor family fold 5B5K). The structure shows that the Izumo domain consists of a four-helix bundle with a flexible α-helical hook/β-sheet insertion, followed by a β-hairpin that connects the bundle to a seven-stranded Ig-like domain (1HNF) — a molecule mediating the adhesion of lymphocytes to antigen-presenting cells — with a root mean square deviation (RMSD) of 2.1 Å over 86 residues.To investigate how Izumo1 might trigger gamete fusion, we expressed the entire ectodomain in mammalian cells and determined a crystal structure to 2.5 Å resolution (e domain A,B. These domain A,B. ThisThe helical character of the carboxy-terminal half of the Izumo domain, which corresponds to helices α3 and α4 in our structure A,B, was Plasmodium protein that is essential for host cell traversal by the infective sporozoite form of the parasite (Dali Z-score 6.9, RMSD 3.3 Å over 98 residues; Plasmodium protein that mediates sporozoite gliding and host cell invasion Strikingly, structural homology searches revealed a high confidence match between the four-helix bundle of Izumo1 and that of SPECT1, a secreted Plasmodium cell traversal and invasion proteins with Izumo1? Our findings suggest that the four-helix bundle of the Izumo domain and SPECT1 may orchestrate analogous fusion-related molecular recognition events at the sperm–egg and parasite–host cell interfaces. Izumo1 is not considered a fusogen per se because it cannot induce membrane fusion when heterologously expressed in commonly used cell lines What might be the functional implications of the structural similarities to Plasmodium parasites and are similarly sequestered within specialized intracellular secretory organelles called micronemes Both SPECT1 and TRAP are expressed by the sporozoite stage of The structure of Izumo1 provides new mechanistic insights into a fascinating basic biological process and, also considering the protein’s recent implication in human immunoinfertility, presents new opportunities in the rational design of new fertility treatments and contraceptives.


Background: Herpes simplex virus DNA synthesis requires seven interacting virus proteins.Results: Mapping revealed an N-terminal domain of UL52 primase interacting with UL5 helicase; a UL52 middle domain binds UL8 and the UL29 single strand DNA-binding protein.Conclusion: Protein-protein interactions in the helicase-primase were affected by an antiviral compound and resistance mutations.Significance: This work provides understanding of antiviral inhibition of replisome function. Herpes simplex virus 1 (HSV-1) UL5/8/52 helicase-primase complex is required for DNA unwinding at the replication fork and synthesis of primers during virus replication, and it has become a promising novel target for antiviral therapy. Using molecular cloning, we have identified three separate domains of UL52. Co-immunoprecipitation experiments in extracts from cells transiently expressing HA-tagged UL5, FLAG-UL8, and enhanced GFP-tagged UL52 domains revealed that the N-terminal domain of UL52 primase binds UL5 helicase and the middle domain interacts with the UL8 accessory protein. In addition, an interaction between the single strand DNA-binding protein ICP8 and the UL52 middle domain was observed. The complex between UL5 and UL52 was stabilized by the antiviral compound BAY 54-6322, and mutations providing resistance to the drug obliterate this effect. Our results also suggest a mechanism for accommodating conformational strain resulting from movement of UL5 and UL52 in opposite directions on the lagging strand template, and they identify molecular complexes that can be further examined by structural biology techniques to resolve the mechanism of primer synthesis during herpesvirus replication. Finally, they help to explain the mechanism of action of a novel class of antiviral compounds currently being evaluated in clinical trials. Herpes simplex virus type 1 (HSV-1) is one of eight different herpesviruses infecting humans. An initial infection is accompanied by establishment of latency in neuronal cells from which the virus can be reactivated. The ability to establish latent infections is shared by all herpesviruses, and as a consequence, the prevalence of herpesvirus infections is very high in the adult population. Primary infections by HSV-1 and other herpesviruses can cause serious disease, and reactivation of latent virus may occur during treatments requiring efficient immune suppression. HSV-1 infections in immunocompetent individuals can be efficiently controlled by acyclovir, a deoxyguanosine analog, which after phosphorylation by a viral thymidine kinase acts as an efficient chain terminator capturing the viral DNA polymerase in a salt-stable complex 14. Immunoul9 gene binds and unwinds double-stranded DNA at the origins of replication together with the single strand DNA-binding protein ICP8 or UL29 protein , middle (M), and C-terminal (C) domains. The UL8 protein interacts exclusively with the M domain of UL52, whereas the UL5 protein interacts strongly with the N-domain and possibly with the C-domain. UL5 also seems to enhance the interaction between full-length UL52 and UL8. Resistance mutations within the N-domain severely impaired complex formation between UL5 and UL52. On the other hand, the antiviral compound BAY 54-6322 greatly enhanced the UL5-UL52 interaction, but this effect could be efficiently counteracted by the resistance mutations in the UL5 protein and the C-domain of UL52. Finally, we note that co-expression of the UL52 M-domain, UL8, and ICP8 leads to co-localization of these proteins into prominent intranuclear foci suggesting direct interactions between ICP8, UL8, and UL52. These studies provide the first detailed interaction map for the helicase-primase complex, and they also suggest a number of dynamic structural changes within the complex with mechanistic implications for drug action and development.BHKul5 gene in the PE5 vector  as described previously . Cells wBHK monolayers were transfected with the indicated plasmids using Lipofectamine and Plus reagents (Invitrogen). At 20 h post-transfection, the cells were lysed, and the cleared extract was subjected to immunoprecipitation and subsequent Western blot analyses as described previously .Expression levels were monitored by using 1/20th of the amount used for immunoprecipitation and labeled as “input” in the figures. When indicated, the helicase-primase inhibitor BAY 54-6322  was added to the cell culture media immediately after the transfection mixture and to the cell lysate used for immunoprecipitation. EZView red anti-FLAG M2 , EZView red anti-HA , and anti-GFP mAb-agarose (MBL) affinity beads were used for FLAG, HA, and GFP pulldown experiments, as described by the manufacturer. For ICP8 immunoprecipitation assays, a rabbit polyclonal antiserum to HSV-1 ICP8 was added to the cell lysate and incubated overnight at 4 °C followed by an additional 2 h of incubation with immobilized protein A/G resin (Thermo Scientific) and several washes, as recommended by the manufacturer. For detection of eGFP-tagged proteins, FLAG-UL8, HA-UL5, ICP8, and actin in Western blots, anti-GFP , anti-FLAG , anti-HA , and anti-HSV-1 ICP8 , a rabbit polyclonal antiserum to HSV-1 ICP8 and anti-actin  antibodies was used. Proteins were detected and quantified by chemiluminescence using Bio-Rad Clarity Western ECL substrate and Bio-Rad ChemiDoc XRS imager. Restore Plus Western blot Stripping Buffer (Thermo Scientific) was used for membrane stripping.BHK cells were seeded on ibiTreated 8-well μ-slides (Ibidi) and transfected with the indicated plasmids. After 16 h of incubation at 37 °C, the cells were fixed with cold methanol and incubated with either anti-HSV-1 ICP8 , anti-HA (H9658), or anti-FLAG (F3165) followed by incubation with Alexa Fluor 568-labeled secondary antibody . Finally, the cell nucleus was stained with DAPI (Invitrogen). Confocal images were acquired using a Carl Zeiss LSM 700 Axio Observer. Z1 inverted the confocal laser-scanning microscope with either a plan-apochromat 20×/0.8 objective or with a plan-apochromat 63×/1.4 oil objective. Merging of images and profile plot measurements were done with the program ImageJ. Images were further processed for publication with Adobe Illustrator CS6.a). In contrast, the N-domain was found in lower amounts in the extract, and neither the N-domain nor the C-domain was precipitated together with FLAG-UL8. We also looked at the influence of HA-UL5 on complex formation . We found that HA-UL5 was co-immunoprecipitated with FLAG-UL8 only when full-length eGFP-UL52 was present but not with the smaller eGFP-tagged M-domain. Interestingly, addition of HA-UL5 resulted in a reproducible 2-fold increase in the amount of full-length eGFP-UL52 in the precipitate suggesting that the presence of UL5 made it more accessible to FLAG-UL8. In this experiment, we also observed a shorter form of GFP-UL52, which probably is a result of proteolysis enhanced by the presence of HA-UL5 . This finding will be further discussed below.To identify molecular interactions within the helicase-primase complex, we divided the UL52 protein into distinct domains by molecular cloning . Our staFig. 2b). This observation probably reflects decreased solubility and/or stability of the protein in the absence of the appropriate interaction partner. Further truncation of the M-domain resulted in fragments unable to bind UL8, and such fragments were also expressed less efficiently suggesting that they were unstable . Only very small amounts of the M- and C-domains were found in the immunoprecipitate. Therefore, we conclude that the N-domain is responsible for the formation of a stable UL5-UL52 complex. We also found lower amounts of HA-UL5 in the extract when HA-UL5 was expressed together with the M-domain indicating reduced stability in the absence of its interaction partner. Interestingly, higher amounts of HA-UL5 were found when co-expressed with the C-domain indicating, possibly, a stabilizing effect on UL5 (a).To investigate the interactions between UL5 and UL52, we made use of the N-, M-, and C-domains as defined above. Using HA-UL5 expressed together with eGFP-UL52 and eGFP-tagged N-, M-, and C-domains, we observed that HA-UL5 efficiently co-immunoprecipitated the full-length UL52 and even more efficiently the UL52 N-domain a. Only vt on UL5 a.The UL5-UL52 interaction could be further defined by the observation that the eGFP-tagged deletion mutant UL52 (Δ367–1058) did not bind HA-UL5 . Thus, ab). We found again efficient co-immunoprecipitation of the N-domain and full-length UL52. Interestingly, the small amounts of M- and C-domains precipitated in the absence of UL8 were significantly reduced in its presence (a and b). We also found that FLAG-UL8 was co-immunoprecipitated with HA-UL5 only when full-length eGFP-UL52 was present but not in the presence of the eGFP-tagged N-domain. Finally, HA-UL5 appeared to be unstable in the presence of only the M- and C-domains. As described in the preceding paragraph, we also performed a co-immunoprecipitation experiment using anti-GFP beads . We made an attempt to map the putative cleavage site by expressing full-length eGFP-tagged UL52 in the presence or absence of HA-UL5. We used as size markers UL52 (Δ367–1058) and the eGFP-tagged N-domain and observed that the proteolytic fragment was, approximately, the same size as the eGFP-tagged N-domain (c). The result suggests the existence of a region in UL52 that is sensitive to endogenous proteases and is located at the border between the N- and M-domains. Exposure of this sequence appears to be enhanced in the presence of UL5 suggesting that it serves as a hinge region for conformational changes in UL52 induced by UL5.We noted again the appearance of a short fragment of eGFP-UL52 in these experiments a. We madN-domain c. The reTo conclude, HA-UL5 interacts strongly with the N-domain of UL52 and in particular with amino acids 367–421. The C-domain, on the other hand, does not form a stable complex with UL5 but may enhance the solubility of that protein.We noted that mutations in UL52 causing resistance to HSV-1 helicase-primase inhibitors lie within the N- and C-domains. More specifically, mutations in the N-domain (S364G and R367H) combined with mutations in UL5 (G352V and M355I) confer a 3000-fold resistance to ASP2151 , and thea). The mutations G352V and M351I in UL5 had no effects; mutations A899T in UL52 and K356T in UL5, on the other hand, moderately increased the amounts of eGFP-UL52 brought down by the anti-HA beads.We first looked at the effects of mutations on the interactions between UL52, UL8, and UL5 in the absence of helicase-primase inhibitor. We found that the mutations S364G and R367H in UL52, alone or in combination with G352V and M351I in UL5, reduced the amount of GFP-UL52 in an HA-UL5 pulldown experiment a. The mub and c). We observed a significant and dose-dependent increase in the efficiency by which eGFP-UL52 was co-immunoprecipitated with HA-UL5 in the presence of the drug (d). At very high concentrations of the drug, the total amount of soluble UL5, UL52, and UL8 in the extract decreased, but the efficiency of eGFP-UL52 co-immunoprecipitation still increased. In contrast, using the mutant versions, K356T in UL5 and A899T in UL52, we observed the opposite effect (d).Effects of BAY 54-6322, which is structurally closely related to BAY 57-1293 , on the the drug d. At vere effect d.e). We found that BAY 54-6322 inhibited DNA replication with an IC50 of 2 μm, whereas inhibition of replication in the experiment using mutant proteins required more than 10-fold higher concentrations of the drug.To verify the physiological effects of the drug and the drug resistance mutations, we examined transient HSV-1 oriS-dependent DNA replication in cells using expression plasmids and a plasmid containing HSV-1 oriS e. We foub and 5e). We then examined whether the individual domains of UL52 all together could also support transient DNA replication, but we were unable to detect oriS-dependent DNA replication in this instance (data not shown). However, we observed, under these conditions, prominent intranuclear foci containing eGFP-tagged UL52 domains and ICP8, indicating co-localization of replication proteins (a). The results point toward the existence of complex(es) between the UL52 M-domain, UL8, and ICP8. We then performed co-immunoprecipitation experiments using anti-GFP, anti-ICP8, and anti-FLAG antibodies, respectively, in cell extracts containing eGFP-tagged UL52 M-domain, ICP8, and FLAG-tagged UL8 (b). Our results revealed that both FLAG-UL8 and ICP8 were co-immunoprecipitated with the eGFP-UL52 M-domain. Conversely, in a co-immunoprecipitate experiment using anti-ICP8, we noted that the UL52 M-domain as well as FLAG-UL8 were brought down together with ICP8; however, anti-FLAG antibodies pulled down FLAG-UL8 and the UL52 M-domain but very little ICP8 (b). Our results indicate that the UL52 M-domain can form separate complexes with UL8 and ICP8. To confirm the existence of a binary complex between the UL52 M-domain and ICP8, we performed co-immunoprecipitation experiments for ICP8 and the UL52 M-domain in the presence or absence of UL8 (c). Here, we found that anti-GFP antibodies co-immunoprecipitated the eGFP-tagged UL52 M-domain and ICP8 in the presence as well as absence of UL8. We thus conclude that the UL52 M-domain is capable of forming separate binary complexes with ICP8 and UL8.As shown above, eGFP-UL52, HA-UL5, and FLAG-UL8 together with expression plasmids encoding UL9, UL29, UL30, and UL42 can fully support transient oriS-dependent DNA replication in transfected cells b and 5e.proteins . We thenproteins a. The regged UL8 b. Our retle ICP8 b. Our ree of UL8 c. Here, a). In addition, another binary complex between the UL52 M-domain and ICP8 also exists (b and c). The formation of prominent intranuclear foci, which requires simultaneous expression of the UL52 M-domain, UL8 and ICP8, suggests that a ternary complex also might exist (a). The existence of a ternary complex is also supported by the observation that UL5-UL8-UL52 can bind ICP8 in a surface plasmon resonance experiment, whereas UL5-UL52 subcomplex cannot . It is therefore a possibility that a second UL5-binding site may involve neighboring structures from the UL52 M- and C-domains.The role of the C-domain is less clear. Mutations in the zinc-binding motif render the UL5-UL52 subcomplex enzymatically inactive with no helicase and primase activities as well as severely reduced single strand DNA ATPase activity . FurtherC-domain . This inC-domain a. It is Taken together, our results facilitate future investigations of the structure and function of helicase-primase subcomplexes and thereby provide new insights into the mechanisms by which helicase-primase inhibitors interfere with herpesvirus replication. It is to be expected that such information should also assist continued development of antiviral compounds.Finally, the herpesvirus replisome is highly conserved in evolution, but it can be assembled by different mechanism as shown by the different initiation mechanisms as observed for alpha-, beta-, and gammaherpesviruses. As recently suggested, the helicase-primase is derived from cellular ancestors to Pif1 helicase, Primpol, and a family B DNA polymerase, and it may have retained some of the functional properties once performed in cells . Further


STAT3 and Sod1 results in greater than 70% reduction of mRNA and protein. The ASOs promote aberrant exon skipping and generation of premature termination codon (PTC)-containing mRNAs. We inhibit the nonsense-mediated mRNA decay (NMD) pathway and show that the PTC-containing mRNAs are recognized by the UPF1 ATPase, cleaved by the SMG6 endonuclease and degraded by the XRN1 cytoplasmic exonuclease. NMD surveillance, however, does not entirely explain the mechanism of decreased STAT3 expression. In addition to exon skipping, ASO treatment causes intron retention and reduction of chromatin-associated STAT3 mRNA. The application of steric blocking ASOs to promote RNA degradation allows one to explore more nucleotide modifications than tolerated by RNase H or RISC-dependent ASOs, with the goal of improving ASO drug properties.Antisense oligonucleotides (ASOs) are synthetic oligonucleotides that alter expression of disease-associated transcripts via Watson–Crick hybridization. ASOs that function through RNase H or the RNA-induced silencing complex (RISC) result in enzymatic degradation of target RNA. ASOs designed to sterically block access of proteins to the RNA modulate mRNA metabolism but do not typically cause degradation. Here, we rationally design steric blocking ASOs to promote mRNA reduction and characterize the terminating mechanism. Transfection of ASOs complementary to constitutive exons in  Antisense oligonucleotides (ASOs) are powerful and extremely versatile therapeutic agents utilized in a growing number of applications including RNA reduction, translation arrest, miRNA inhibition, splicing modulation and polyadenylation site selection . The mosO-methoxyethyl, MOE) to decrease nucleolytic degradation and increase binding affinity  and 2′-sugar modifications  disrupts the open reading frame. As a general rule, when a PTC is located greater than 55 nucleotides upstream of the last exon–exon junction, the spliced PTC-containing mRNA is a likely substrate for NMD . In brieO-methyl ASOs  or H2O was added to the culture medium 20 h after ASO transfection and remained on the cells for 4 h. Cells were then washed and lysates prepared for analysis. See Supplementary Tables S1 and S2 for ASO and siRNA sequences, respectively. Silencer Select Negative Control No.1 siRNA (Life Technologies) was used as the negative control siRNA in all experiments.HeLa (CCL-2) and bEnd.3 (CRL-2299) cells were maintained according to American Type Culture Collection (ATCC) standards. For siRNA transfection, cells were plated and 24 h later transfected with 10 nM individual siRNA (Thermo Scientific) using Lipofectamine RNAiMAX (Life Technologies) for 48 h according to the manufacturer's instructions. ASO transfections were performed 24 h after cell plating or 48 h after siRNA transfection. Fifty nanomolars ASO was transfected using 3 μg/ml Cytofectin transfection reagent for 24 h. For translation inhibition experiments, emetine . For standard reverse transcriptase-polymerase chain reaction (RT-PCR), 400 ng RNA was reverse transcribed using random hexamers and SuperScript II Reverse Transcriptase (Life Technologies) followed by PCR with Platinum Taq PCR Polymerase (Life Technologies) according to the manufacturer's instructions. PCR product was resolved on 5% non-denaturing polyacrylamide gel and stained in ethidium bromide prior to imaging on the Kodak Gel Logic Imaging System. The percentage of exon skipping was calculated as [exon exclusion band/ (exon inclusion band + exon exclusion band)] x 100 with a correction factor for the amount of ethidium bromide per base pair of DNA. See Supplementary Table S3 for primer sequences.For quantitative RT-PCR, approximately 10 ng RNA was added to EXPRESS One-Step SuperScript qRT-PCR Universal (Life Technologies) with custom designed or predesigned Taqman primer and probe sets (see Supplementary Table S4 for sequences). All quantification was performed by the relative standard curve method and normalized to Ribogreen.Total cellular protein was prepared by cell lysis in Radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) containing ethylenediaminetetraacetic acid-free cOmplete Protease Inhibitor Cocktail Tablet (Roche). Cells were further lysed by passage through a needle five times, then cellular debris pelleted by refrigerated centrifugation. Protein concentration of the supernatant was determined by DC protein assay (Bio-Rad). 10–40 μg protein lysate was separated on a precast 4–20% Bis-Tris gel (Life Technologies) and transferred by iBlot (Life Technologies). See Supplementary Table S5 for antibody information.To identify endonuclease cleavage sites, 5′ rapid amplification of cDNA ends (5′ RACE) was performed using the First Choice RLM-RACE kit (Life Technologies) with minor modifications. Briefly, the 5′ RACE adapter was ligated to the free 5′ phosphate in 2 μg of RNA using T4 ligase. Reverse transcription was performed using SuperScript II followed by outer PCR for 35 cycles at 60°C annealing temperature. If necessary, a nested inner PCR was performed for an additional 12 cycles. 5′ RACE products were visualized on 2% agarose gels stained in ethidium bromide. To determine cleavage sites, bands were gel isolated, amplified by TOPO TA Cloning (Life Technologies), and sequenced (Retrogen). See Supplementary Table S3 for 5′ RACE primer sequences.STAT3 intron 21, nucleus; RN7SL1, cytoplasm) and protein .Cellular fractionation was performed as described in . Purity 6 HeLa cells were transfected with 50 nM ASO using 3 μg/ml Cytofectin transfection reagent for 24 h. RNA immunoprecipitation was performed using the Magna RIPTM RNA-Binding Protein Immunoprecipitation Kit (EMD Millipore) according to the manufacturer's instructions with 3 μg ChIPAb+ RNA Pol II monoclonal antibody (EMD Millipore) or 3 μg mouse IgG. Following RNA purification, residual DNA was removed by digestion with amplification grade DNase I (Life Technologies). Successful RNA polymerase II (RNAPII) immunoprecipitation was confirmed by western blot. RNA immunoprecipitation efficiency and specificity was assessed by calculating the percent input and fold enrichment for GAPDH (an RNAPII target) and RN7SL1 (an RNAPIII target) (data not shown).5 × 10For central nervous system administration, a single ASO dose of 500 μg in phosphate buffered saline (PBS) was delivered by intracerebroventricular injection to 8-week-old C57BL/6 female mice (JAX). Four weeks post-treatment, whole brain and thoracic spinal cord tissue was lysed using FastPrep Lysing Matrix Tubes  in RLT buffer (Qiagen) containing 1% beta-mercaptoethanol and RNA was isolated using the RNeasy kit (Qiagen). No body weight change or inflammation (Aif1 qRT-PCR) was observed.For systemic administration, 6-week-old BALB/c male mice (JAX) were dosed twice per week for 3 weeks with 50 mg/kg ASO in PBS. Liver was harvested 48 h after the last dose. Tissue was lysed using FastPrep Lysing Matrix Tubes  in guanidine isothiocyanate (Life Technologies) containing 8% beta-mercaptoethanol and RNA was isolated using the RNeasy kit (Qiagen). No changes in body weight, organ weight or plasma markers were observed.STAT3 exon 6 and Sod1 exon 2 and exon 3 including the 5′ and 3′ splice sites to identify ASOs that redirected splicing. Human cervical epithelial cells (HeLa) and mouse brain endothelial cells (bEnd.3) were used for all human STAT3 and mouse Sod1 experiments, respectively. Cells were transfected with 50 nM uniform 2′-MOE ASOs and 24 h post-transfection exon skipping was analyzed by RT-PCR. In each case, the uniform 2′-MOE ASOs with the strongest effect on splicing had binding sites located centrally in the exon  and Sod1 exon 2 (UNI2) and exon 3 (UNI3) caused decreased expression of the full-length transcript and appearance of a lower molecular weight band, indicative of exon skipping  targeting STAT3 (GAP24) or Sod1 (GAP1) that function through RNase H to reduce the target RNA  showed decreased expression of full-length HNRNPH1 and increased exon 4 skipping thereby enhancing the amount of PTC-containing mRNA present in the cell .Unlike the targeting of constitutive exons in cing-NMD . HeLa ceSTAT3, Sod1 and HNRNPH1 mRNA reduction by real-time RT-PCR (qRT-PCR). Similar to RNase H-dependent 2′-MOE gapmer ASOs, treatment with uniform 2′-MOE ASOs resulted in robust, dose-dependent RNA knockdown with maximum reductions of 70–85% compared to untransfected control cells. Correspondingly, dose-dependent reductions in protein levels were also observed . Uniform 2′-O-methyl ASOs were consistently the least effective, and in general the 2′,4′-constrained ethyl mixmers yielded mRNA reductions similar to uniform 2′-MOE ASOs. Sequence-matched uniform 2′-MOE and 2′-MOE gapmer ASOs targeting two different sites on STAT3 were nearly equipotent . For Sod1 and HNRNPH1, the 2′-MOE gapmers had IC50 values 1.5–23 times higher than sequence-matched uniform 2′-MOE ASOs .To determine if additional ASO chemical modifications supported RNA reduction, we designed sequence-matched ASOs to the active uniform 2′-MOE ASO binding sites and tested their ability to decrease the target mRNA. ASOs of multiple chemistries including uniform 2′-While 2′-MOE gapmer ASOs are known to cause RNA reduction through an RNase H enzymatic cleavage event, the molecular pathway by which these steric blocking ASOs decrease mRNA expression has not been previously characterized. We hypothesize that by redirecting splicing to generate PTC-containing mRNAs, the ASOs may invoke the NMD pathway to degrade the misprocessed transcripts. Because the PTCs are located more than 55 nucleotides upstream of the last exon–exon junction in each transcript, the messages are predicted NMD substrates but this can only be verified experimentally.STAT3 pre-mRNA and mRNA expression by qRT-PCR. Uniform 2′-MOE ASO treatment resulted in dose-dependent reduction of STAT3 mRNA upstream and downstream of the ASO binding site. In contrast, no reductions were observed in pre-mRNA levels including the last intron–exon junction in the transcript  served as a positive control for pre-mRNA knockdown by RNase H and showed dose-dependent reductions in all regions of the transcript. This data suggest that uniform 2′-MOE ASOs act to decrease the expression of STAT3 after splicing has occurred, consistent with an NMD process.To determine if uniform 2′-MOE ASOs reduced the level of pre-mRNA or mRNA, we treated cells with UNI6a and UNI6b and analyzed STAT3, Sod1 and HNRNPH1 PTC-containing transcripts 3- to 9-fold , but a different result was observed when the expression level was analyzed using STAT3 qRT-PCR primers downstream of the ASO binding site (STAT3 exon 8 and exon 22/23)  and nucleus (XRN2). In each case, the siRNAs promoted greater than 60% reduction in mRNA and protein Figure . NMD inhr Figure . XRN1 in) Figure . In thess Figure . The mos% Figure  and stab% Figure  comparedSTAT3 cleavage by performing 5′ RACE on RNA from cells transfected with UNI6a. We were unable to detect 5′ RACE products in cells treated with ASO alone, suggesting either the transcript is not cleaved or the cleaved transcript is rapidly degraded by cellular nucleases .The same detailed molecular experiments were also performed using uniform 2′-MOE ASOs targeting STAT3 RNA expression by about 40% compared to untransfected control cells. STAT3 may be degraded by a cellular surveillance pathway prior to nuclear export, so we analyzed STAT3 RNA expression in three cellular fractions—chromatin, nucleoplasm and cytoplasm. The purity of each fraction was carefully assessed by RNA and protein distribution. STAT3 intron expression was enriched 15-fold and the cytoplasmic RNA RN7SL1 was enriched 50-fold in the chromatin and cytoplasm, respectively  and exon 17 (UNI17a and UNI17b) and first confirmed they acted through splicing redirected-NMD in vitro. Treatment of bEnd.3 cells with UNI6 and UNI17 ASOs resulted in skipping of exon 6 and exon 17, respectively, and decreased Stat3 expression . Both exon 6 and exon 17 skipped transcripts contain PTCs. RT-PCR on RNA isolated from UNI17b treated cells revealed an additional lower molecular weight band that was confirmed by sequencing to be partial exon 17 skipping with utilization of a cryptic 5′ splice site. Translation inhibition by emetine treatment greatly stabilized the PTC-containing transcripts and significantly impaired the ability of the uniform 2′-MOE ASOs to knock down Stat3 . These data show that uniform 2′-MOE ASOs cause reduction of mouse Stat3 through a similar terminating mechanism as human STAT3. For in vivo experiments, uniform 2′-MOE ASOs (UNI17a and UNI17b) and a 2′-MOE gapmer ASO (GAP19) targeting mouse Stat3 were administered systemically by subcutaneous injection. Uniform 2′-MOE ASOs resulted in weak exon 17 skipping and a significant 30–35% decrease in Stat3 expression in the liver, but were much less efficacious than the 2′-MOE gapmer ASO  and in vivo delivery of ASOs targeting independent sites in Sod1 (Figure We also sought to compare the potencies of ASOs that act through NMD-dependent and RNase H-dependent mechanisms. When comparing the ability of the ASOs to cause target reduction, the uniform 2′-MOE ASOs are typically less potent than 2′-MOE gapmer ASOs. In a few cases, the uniform 2′-MOE ASOs were equipotent to the 2′-MOE gapmer ASOs, including 1 Figure . HoweverSTAT3 mRNA expression prior to cytoplasmic decay by the NMD pathway. Reduced STAT3 in the chromatin fraction was independently confirmed by a significant reduction in RNAPII-bound STAT3 mRNA. Loss of chromatin-associated STAT3 mRNA cannot be explained by impaired transcription elongation downstream of the targeted exon because chromatin-associated, or RNAPII-bound, pre-mRNA levels were not reduced by uniform 2′-MOE ASO treatment. These data suggest that STAT3 mRNA is degraded after RNAPII has reached the 3′ end of the gene, while still associated with RNAPII on chromatin. Previously it was reported that intron retention does not affect RNAPII elongation kinetics (STAT3 intron retention and/or exon skipping results in impaired RNA release from the transcription site and subsequent nuclear decay by the exosome and Xrn2.We observed that ASO treatment, unexpectedly, also reduced chromatin-associated kinetics , but impkinetics . Retentikinetics . Xrn2 iskinetics . Our datThe finding that steric blocking ASOs can reduce the level of mRNA has been previously reported –15. HoweSteric blocking ASOs are amenable to a wide range of chemical modifications because there is no need to maintain the structural and chemical requirements for RNase H or AGO2 activity. Future experiments should focus on leveraging the additional chemical space to further improve pharmacodynamic and pharmacokinetic properties, particularly for increasing distribution to tissues of interest, of steric blocking ASOs.Supplementary Data are available at NAR online, including Supplementary Figures S1–S5 and Supplementary Tables S1–S6.