This review systematically analyzes the effect of the electrolyte-to-sulfur ratios on battery energy density and the challenges for sulfur reduction reactions under lean electrolyte conditions.The strengths and limitations of different transition metal compounds are systematically presented and discussed from a fundamental perspective.Three promising strategies for sulfur hosts that act as anchors and catalysts are proposed to boost lean electrolyte Li–S battery performance. −1), and the strengths and limitations of different transition metal compounds are presented and discussed from a fundamental perspective. Subsequently, three promising strategies for sulfur hosts that act as anchors and catalysts are proposed to boost lean electrolyte Li–S battery performance. Finally, an outlook is provided to guide future research on high energy density Li–S batteries.Lithium–sulfur (Li–S) batteries have received widespread attention, and lean electrolyte Li–S batteries have attracted additional interest because of their higher energy densities. This review systematically analyzes the effect of the electrolyte-to-sulfur (E/S) ratios on battery energy density and the challenges for sulfur reduction reactions (SRR) under lean electrolyte conditions. Accordingly, we review the use of various polar transition metal sulfur hosts as corresponding solutions to facilitate SRR kinetics at low E/S ratios (< 10 µL mg Cg−1 Fig. h. The saatteries . The higIn general, transition metal nanoparticles and alloy nanoparticles coupled with carbon materials are some of the most promising electrocatalysts to realize high sulfur utilization and long cycle life under lean electrolyte conditions. The dispersion of the metal onto suitable carbon substrates allows for maximizing the surface area and the number of active sites. However, the use of these catalysts is often limited in practical applications due to their high cost, as they may involve expensive noble metals or heavy metals that are not economically feasible.Transition metal oxides (TMOs) have been used primarily to trap and block the diffusion of highly soluble LiPS at the cathode, thereby alleviating the shuttle effect of LiPS between the cathode and anode –161. A s2N2O/NC-6) possessed rapid Li+ transfer, LiPS anchoring and multiple functional sites . . 2O5−x)  battery . In 2023behavior . Guo’s tles Fig. e 165]. . 2O5−x) ode Fig. f. The GPIn conclusion, most TMOs possess high surface polarity that facilitates the reaction with polar LiPS. Furthermore, TMOs with oxygen defects provide excellent catalytic activity and stability for the conversion of LiPS. This, together with their simple synthesis makes them a promising sulfur host. However, TMOs need to be combined with carbon materials to increase their electrical conductivity and dispersion.Like TMOs, transition metal phosphides (TMPs) are also characterized by notable polarity associated with the differential electronegativity between phosphorous and transition metals. P atoms with high electronegativity can gain electrons from metal atoms and serve as bases for trapping positively charged species –176. In 2S. In addition, DFT calculations show that amorphous CoP has higher binding energy and a lower diffusion energy barrier to LiPS. Furthermore, amorphous CoP decreases the energy gap and increases the electron concentrations of adsorbed LiPS close to the Fermi level. As a result, combining the advantages of conductive rGO-CNT and the amorphous CoP, the S/rGO-CNT-CoP(A) cathode provides a discharge capacity of 1006 mAh g−1 at 0.8 mA cm−2 under a sulfur loading of 5 mg cm−2 and an E/S ratio of 7 μL mg−1.Cobalt phosphides, like typical TMPs, exhibit low overpotential for LiPS transitions, suggesting that they could enhance the sluggish redox kinetics of LiPS and improve the rate performance of Li–S cells , 189. Al−2 was realized at 0.4 mA cm−2 under lean electrolyte (E/S = 5 μL mg−1) and high sulfur loading (7.7 mg cm−2) . . 3C2Tx) atteries . The aut of LiPS . As showatteries . Figure In general, TMCs have the potential for lean electrolyte Li–S batteries due to their polarity and high electric conductivity. However, the use of TMCs as host materials is still in the early research stage due to the harsh conditions required for their synthesis. Therefore, there is still a need to develop simple synthetic approaches to prepare TMCs with tailored morphologies.Metal–organic frameworks (MOFs) are a relatively new class of crystalline porous materials consisting of metal ions or metal clusters coordinated by organic linkers –259. MOF+  showed a sustainable capacity of 1007 mAh g−1 at 0.1C under a low E/S (6 μL mg−1).In 2020, Knibbe’s group demonstrated that Lewis acid–base interactions between MOFs and LiPS are beneficial to improving the electrochemical performance of Li–S batteries . In this+ Fig. d. The au+ Fig. e–h. Comp+ Fig. g and UIO+ Fig. h, the li+ Fig. f is stil). A very slow conversion process was observed for the Li–S cells with the traditional UiO-66-V/S electrode . In addition to conventional insulating MOFs, Wang and his colleagues synthesized conductive MOFs to facilitate the conversion of LiPS . . 2S oxidatalysts . Co–N–C 4 moieties , FeN4 active sites supported on a highly wrinkled graphene (wG) matrix (FeNC/wG) induced the modification of the local structure of Fe, thereby promoting the conversion reaction kinetics of LiPS. The relative content of Fe in FeNC/wG was estimated to be 0.78 wt%. Due to the beneficial interaction between the atomically modified FeN4 active sites and the morphological engineering of graphene support, the Fe–N–C-based Li–S batteries showed excellent cycling stability with low electrolyte usage. The morphology of Li2S deposition has a significant impact on the deposition capacity of Li2S. Li’s group proposed that nitrogen-doped carbon fibers decorated with SA Cu change the Li2S deposition morphology from the traditional 2D lateral morphology to 3D spherical clusters by consecutive 3D nucleation and growth  on SA-Fe, Co, Ni, V, Cr and Mn  around the Fermi energy level (EF), thus significantly increasing the electronic conductivity of the Li2S molecule  as electrocatalysts for Li–S batteries . . 4 moiet Mn Fig. f. Howeveule Fig. g. As a ratteries . The Co ess Fig. j. The enOverall, SAs catalysts hold great potential for use in Li–S batteries due to their high atom utilization efficiency, strong catalytic activity and the ability to adjust their structure at the atomic level. Although SAs catalysts offer the above-mentioned advantages, the development of large-scale synthesis strategies which allow increased loadings is crucial for their application in lean electrolyte Li–S batteries.In addition to the previously mentioned transition metal-based systems, the applications of transition metal borides, transition metal fluorides and nickel foam in lean electrolyte Li–S batteries are also discussed in this section. Table 2) as a sulfur host . . 2@rGO c−2 and achieve a high areal capacity of over 40 mAh cm−2 at 0.2C with a low E/S ratio of 7. This work provides a new strategy for developing optimized cathodes to achieve high energy density Li–S batteries.During the past decade, the use of nickel foam as an electrode substrate in Li–S cells has been investigated. For example, Manthiram’s group proposed a new pie-like electrode consisting of an electrocatalytic nickel foam and an outer carbon shell for high-energy Li–S batteries with low-electrolyte content Fig. r 282]. . 282]. TThis review provided a systematic summary of a range of composites of carbon with transition metal-based compounds used in lean electrolyte Li–S batteries. The first topic of discussion was the fundamentals and limitations of the electrochemical conversion of LiPS with low E/S ratios. Then, the functional mechanism and characteristics of the transition metal-based carbon compounds for lean-electrolyte Li–S cell cathode components were comprehensively reviewed. Finally, several possible improvement trends for Li–S batteries in low electrolyte environments were foreseen. Specifically, we discussed the application of carbon-based sulfur hosts containing metal elemental, alloy nanoparticles, metal oxides, phosphides, chalcogenides, nitrides, carbides, borides, fluorides, as well as metal organic frameworks and single atoms of these compounds in lean electrolyte Li–S batteries. By exploiting the inherent characteristics of hybrid nanostructure geometries and developing their external properties, transition metal-based carbon compound engineering holds a lot of potential and provides a path for constructing high-performance Li–S systems with low electrolyte usage.i.Heterogeneous electrocatalysts have a limited number of specific catalytic sites; therefore, it is necessary to increase the number of redox-active sites per unit surface area to enhance catalytic activity without changing the catalyst structures. However, the understanding of how the number of active sites influences the electrochemical performance of Li–S cells is still unclear. Therefore, an in-depth study of the relationship between the catalytic site density of catalysts and electrochemical performance of Li–S batteries is required. Furthermore, the catalytic effect of catalysts can be enhanced by increasing the catalyst dosage; however, a large dosage of catalysts can greatly decrease the gravimetric energy density of Li–S cells; thus, a balance needs to be discovered between the high catalyst dosage and the high gravimetric energy density of Li–S cells.ii.As the redox reaction proceeds, “dead sulfur” can lead to catalyst deactivation; therefore, the effectiveness protection of active sites to maintain sustainable electrocatalytic effects for long-cycle Li–S batteries deserves more attention. Emphasis should be devoted to developing catalysts with improved poisoning resistance to mitigate the negative impacts of "dead sulfur" deposition and catalytic interface poisoning. Furthermore, the exploration of liquid metal catalysts with self-healing and reversible properties holds promise for enhancing the long-term stability and durability of the catalytic system.iii.2S on the electrolyte/electrode surface should be studied, as it significantly influences the transport of Li ions and electrons at the reactive interfaces. Optimized reaction interfaces help to regulate the redox conversion of LiPS, control the nucleation of Li2S and achieve 3D growth of Li2S. Exploring candidates such as stepwise and selective electrocatalysis can offer opportunities for manipulating the kinetics of Li2S nucleation versus growth. In addition, the decomposition of Li2S also deserves special mention. Li2S is hard to be completely oxidized due to the high energy barrier (Eb) of Li2S delithiation during charging. In this sense, the design of bidirectional electrocatalysts holds great prospects in accelerating the transformation from insoluble Li2S to long-chain LiPS.The precipitation process and the deposition morphology of Liiv.The balance of carbon content and electrolyte/sulfur ratio should be carefully considered. The minimization of carbon content in the cathode is very important at low E/S ratios as carbon can lead to severe electrolyte depletion due to high electrode porosity. However, recent studies have shown that the rate constant of sulfur redox in the presence of carbon is significantly increased, indicating the importance of electrical conductivity in accelerating sulfur redox reactions. Thus, the balance between carbon content and electrolyte/sulfur ratio should be studied.v.−1. The characteristics of multilayer pouch cells and transparent calculations of their components need to be explored further, as these aspects are critical for the commercial application of Li–S batteries.More research efforts should be directed toward evaluating the electrochemical performance of Li–S pouch cells, especially at high areal sulfur loadings and low E/S ratios, to achieve high energy density Li–S pouch cells exceeding 400 Wh kgWhile a lot of progress has been made in the development of lean electrolyte Li–S batteries based on these cutting-edge cathode materials, the following points have not received sufficient attention:


Fundamental understandingof the structure and assembly of nanoscalebuilding blocks is crucial for the development of novel biomaterialswith defined architectures and function. However, accessing self-consistentstructural information across multiple length scales is challenging.This limits opportunities to exploit atomic scale interactions toachieve emergent macroscale properties. In this work we present anintegrative small- and wide-angle neutron scattering approach coupledwith computational modeling to reveal the multiscale structure ofhierarchically self-assembled β hairpins in aqueous solutionacross 4 orders of magnitude in length scale from 0.1 Å to 300nm. Our results demonstrate the power of this self-consistent cross-lengthscale approach and allows us to model both the large-scale self-assemblyand small-scale hairpin hydration of the model β hairpin CLN025.Using this combination of techniques, we map the hydrophobic/hydrophiliccharacter of this model self-assembled biomolecular surface with atomicresolution. These results have important implications for the multiscaleinvestigation of aqueous peptides and proteins, for the predictionof ligand binding and molecular associations for drug design, andfor understanding the self-assembly of peptides and proteins for functionalbiomaterials. Of particular interestto organic and biochemistry is the self-assembly of organic moleculesdue to their interactions with their aqueous environment.9 To address this challenge, multiscale11 and integrative14 approaches are becoming increasingly important, ashighlighted by the 2013 Nobel prize in chemistry for coarse grainedsimulations of protein folding.15 Multiscaleapproaches are often entirely simulation based, from quantum mechanicalsimulations, through all atom molecular dynamics, to coarse grainingapproaches.8 Each method operates at a different spatiotemporalresolution and informs the next scale method, either sequentially,9 or in parallel.16 Integrativeapproaches use experimental data from techniques sensitive to nanoscalestructure, such as cryo-EM, small-angle X-ray scattering, and nuclearmagnetic resonance, to constrain simulation approaches14 and employ machine learning (ML)-acceleratedcomputational methods.17A pervasive challenge across a wide rangeof scientific disciplines,18 as neutrons are deeply penetrating, cause no radiation damage, exhibitmuch greater sensitivity to hydrogen than X-rays, and allow for deuteriumsubstitution to achieve contrast variation of different chemical environments.In the present work we apply a novel approach of combining small andwide angle neutron scattering (SANS/WANS) measurements to constrainthe integrative atomistic modeling technique empirical potential structurerefinement (EPSR), yielding an all-atom simulation refined againstexperimental data of a concentrated aqueous solution of the nanoscaleβ-hairpin “mini-protein”: CLN025.19 CLN025 is a synthetic 10 residue peptide that was chosendue to its well-defined folded structure19 and high solubility. The hairpin structure is a variant of a 10residue synthetic peptide chignolin,19 designedusing residues 41–56 of protein GB1 as a target scaffold. Itshares several key features with naturally occurring proteins, suchas it contains only naturally occurring amino acids, it is thermallystable , stable reversible foldingwith a funnel-like energy surface,20 andcomparable enthalpy of unfolding (47.7 kJ/mol). It is also of considerableinterest, as its fast folding time of ∼100 ns,22 due to a negligible global energy barrier to folding,22 means it can be used for all-atom simulationsof protein folding23 and is rapidly becominga benchmark for testing new force fields.24 Its sequence and structure are shown in A particularlypowerful experimental technique for integrativemultiscale modeling is neutron scattering,19 It has also been shown that its folding is drivenby a gain in water entropy, primarily originating from an excluded-volumeeffect.27 Its small size, well-defined structure,and fast folding time make it an ideal model system for all atom simulationsof protein folding in explicit water. Such studies have shown thatits funnel-like energy landscape is also lost if water is modeledimplicitly rather than explicitly,28 andis sensitive to the choice of water reference potential/force fieldin the molecular dynamics simulations,30 with hairpin stability having been shown to be correlatedto the average hairpin–water interaction energy.31 The hydration of the CLN025 hairpin is thereforeof crucial importance to understand its folding behavior.The structure of theCLN025 hairpin derived from X-ray crystallographyshows several water molecules hydrating polar residues of the hairpin,suggesting hairpin–water interactions maintain its fold.24 it is vital that its hydration structure isaccurately understood. Equally important is the understanding of hairpinself-assembly, given that nanoscale β hairpins have become anestablished building block for the creation of stimuli-responsivebiomaterials (hydrogels).34 A pressing challenge, therefore,is to understand hairpin hydration with atomic resolution in the contextof a self-assembled system. This necessitates a multiscale approach.Combining SANS and WANS with computational modeling addresses thischallenge. SANS is sensitive to structural correlations involvinglarge scale structures (up to ∼ hundreds of nm) and is wellestablished to study the structure of polymers, hydrogels, and biomaterials.39 In this work, it allows us to study self-assembly of the CLN025hairpin in aqueous solution. Conversely, WANS  is sensitive to structural correlations involvingsmall scale structures (on the order of ∼ angstroms) and iswell established for the study of atomic structure of systems suchas aqueous solutions,42 ionic liquids44 and crucially to thiswork, aqueous amino acids.48 In this work, it allows us to study hairpin hydrationwith atomic resolution. Multiple length scales are important for thestructure of concentrated aqueous peptides, from hydrogen bonds (∼2Å), through to the monomeric peptide structure (∼10 Å),to large-scale peptide self-assembly (up to ∼100 Å inthe case of amyloid fibrils49). A combinedSANS and WANS investigation is very important for the CLN025 hairpinas it possesses a well-defined folded structure that is defined byits hydration, and it can self-assemble to form larger length scalestructures at high concentration. Combining these techniques allowsus to map out the hydrogen bonding character of a multiscale self-assembledbiomolecular complex with atomic resolution, with important implicationsfor simulation force field design, molecular recognition prediction,and biochemical self-assembly.GivenCLN025’s popularity as a tractable model for ultrafastfolding and the opportunity it provides for all atom simulations,50 The synthesiswas performed at a 0.25 mmol scale on Rink-amide resin using 5 equivof Fmoc-protected amino acid, 5 equiv of N,N′-diisopropylcarbodiimide (DIC), and 5 equiv of2-cyano-2-(hydroxyimino)acetate (Oxyma) in N,N-dimethylformamide (DMF) with microwave heating at 90 °C.The deprotection was achieved using 20% piperidine in DMF with microwaveheating at 90 °C. The procedure was iterated until the completepolypeptide was formed, then the sample was removed, washed throughwith DMF, resuspended in DMF and reacted for 30 min with 10 equivof acetic anhydride to form the acetyl cap on the unprotected N-terminus.The hairpin was then cleaved from the resin using 10 mL of a cleavingcocktail containing 92.5% trifluoroacetic acid (TFA), 2.5% water,2.5% triisopropylsilane (TIPS), and 2.5% 3,6-dioxa-1,8-octanedithiol(DODT). The hairpin therefore differs slightly from the one investigatedby Honda et al.16 as the hairpin employedin this work has protective acetyl and amide caps at the N and C terminus,respectively. The solution was filtered to remove resin, and diethylether was added at −20 °C to precipitate the hairpin.After centrifugation for 5 min at 6000 rpm, the diethyl ether wasdecanted and the hairpin was rehydrated and freeze-dried. It was laterrehydrated and purified using preparative high pressure liquid chromatography(HPLC) coupled with mass spectrometry before being freeze-dried againfor storage. High resolution mass spectrometry data and HPLC data,presented in Supporting Information, Figures S1 and S2, confirm that the hairpin was produced with the expectedmolecular weight and to a high purity. Circular dichroism data presentedin Figure S3 demonstrates the secondarystructure of the hairpin produced in this work is identical to thatproduced by Honda et al.,19 despite theadditional capping groups.A microwave-assisted solid-phaseFmoc-based synthesis was completed in a CEM Liberty Blue peptide synthesizeras described previously.–1 cm–1. pH was measuredto be 5.5, hence, all amino acids are expected to occur as they woulddo at neutral pH. Circular dichroism data presented in Supporting Information, Note S1 and Figure S3, suggest that this method of concentrating the hairpin causes negligibleirreversible aggregation.CLN025 onlyforms a higherorder structure at a high concentration. Consequently, a ratio of1:500 CLN025:water molecules was chosen, corresponding to 111 mM or150 mg/mL. Due to the presence of two acidic residues in the hairpin(Glu5 and Asp3), it was dissolved in 250 mM NaOH to aid solubility.This was achieved by packing an Eppendorf with freeze-dried purifiedhairpin, adding solution, dissolving, and centrifuging at 5000g for1 min to pellet any aggregated hairpin. The solution was then removed,leaving any visible pelleted hairpin behind and used to dissolve morefreeze-dried hairpin packed into a second Eppendorf. This was repeateduntil the desired concentration was reached, as validated throughabsorbance spectrometry at 280 nm using an extinction coefficientof 9970 M18 This is monitored according to the wavevectorof the scattered neutrons Q, which is equal to thedifference in momentum between the initial ki and scattered kf neutron. In the case of elastic scattering, Q, is dependent on the wavelength of the neutron λand the scattering angle 2θ, as shown in Neutron scatteringrelies on theconstructive interference of a beam of neutrons diffracted throughan experimental sample to extract spatial correlations.Q isan inverse length scale metric. For example, forperiodically repeating structures the Bragg condition is satisfiedsuch that the distance between repeated layers, d, is inversely related to Q as shown in The wave vector 51 at a sample-to-detector distanceof 8 m, covering a Q range of 0.0022–0.45Å–1 (corresponding to a distance range of ∼14–3000Å), and the intermediate to wide angle instrument NIMROD,52 covering a Q range of 0.02–50.0Å–1 (corresponding to a distance range of ∼0.1–300Å). In both instances, the measured quantity is the differentialscattering cross section I(Q). NIMRODdata are conventionally reported in units of barns steradian–1 atom–1, while Zoom data are reported in unitsof cm–1. The NIMROD data is converted and rescaledto units of cm–1 and then stitched to the Zoom datawithin the software suite MANTID. For SANS analysis, I(Q) is modeled as proportional to the product ofthe form factor, F(Q), that describesthe shape of the scattering object and the structure factor, S(Q), that describes the positional correlationsbetween scattering objects. In WANS measurements, I(Q) is related by scattering theory to a sum ofthe partial atomic structure factors Sαβ that describe positional correlations between distinct atom typesα and β weighted by their relative concentrations ci and coherent scattering lengths bi, as described in Neutron scattering data was collected on the small angle instrumentZoomSαβ(Q) is then related to the associated atom–atomradial distributionfunction (RDF) by Fourier transform, as shown in –3, gαβ(r) is the RDF between atom types α and β, and r is the distance in Å. RDFs represent the local densityof a given atom type β normalized to the bulk density of βas a function of distance from a given atom type α. Therefore,in the case of liquids, features such as hydration shells occur asa series of broad peaks of diminishing intensity as the local structurearound α below r ≈ 10 Å gives wayto the disordered bulk liquid structure, at which point gαβ(r) = 1.Each Q ≈ 0.5 Å–1 as “low Q”, data around Q ≈ 1 Å–1 as “mid Q”, and data above Q ≈ 1 Å–1 as “high Q”. To allow for fitting and analysis of the midto low Q data, the data for CLN025 suspended in 250mM NaOH/100% D2O solution, from both NIMROD and Zoom, werefirst buffer subtracted using a 100% D2O scan. While ithas previously been demonstrated by Imberti et al.53 that hydroxide salts have an effect on the structure ofwater, these results were shown for OH– concentrationsin excess of 1:12 mol ratio of salt:water. These concentrations areover 18 times higher than those used in this study, suggesting thatthe structure of water is insignificantly impacted; hence, pure D2O should serve as a reasonable estimate for buffer subtraction.As stated previously, the rescaled NIMROD data were stitched withthe absolutely scaled data from the Zoom instrument to extend thelow Q range by another order of magnitude, from 0.03Å –1 to 0.003 Å –1.This was fitted with a repulsive elliptical cylinder model with theaddition of two Gaussian functions and a power law term shown in http://www.sasview.org). The additionof the Gaussian functions is to accurately model the mid-Q data, while the power function is to capture the increasing intensityprofile at the lowest Q values.Vblock are the volumefraction, neutron contrast,and volume of the CLN025 stack scattering object, respectively, PEC(Q) is an elliptical cylinder54 form factor and SER(Q) is a electrostatic repulsion structure factor.56 α and β are scaling factors to describe the amplitudeof the two Gaussian functions centered around Qip and Qis, respectively, observed in the mid-Q data.γ is the scaling factor for the power law described using exponent n observed in the lowest Q data (Q < ∼ 10–2 Å–1). The background, bkg, is a constant that models the incoherentbackground scattering signal.To aidthe following discussion, we will refer to data below 57 This data was analyzed using the Monte Carlo based structural refinementtechnique EPSR58 over the range 0.05 < Q < 30 Å–1. This technique addressesthe challenge that for a system containing J distinctatomic species, the number of distinct interatomic correlations N is equal to J(J + 1)/2,hence, deconvolution of total scattering data arising from the weightedcontribution of every distinct interatomic correlation present withinthe sample into a complete set of partial structure factors by studying N distinct isotopic variants rapidly becomes impossible.EPSR therefore provides atomistic information to produce a completeset of partial structure factors by building a simulation that issimultaneously constrained by experimental data on fewer distinctisotopic variants .58 Neutron scattering combined with EPSR has previously beenwell applied to the study of simple binary solutions, including thosecontaining biomolecule building blocks such as amino acids48 and short peptides,61 as well as a host of other aqueous systems including pure waterunder various pressures and temperatures,62 alcohols,65 salts,68 and other osmolytes.72 EPSR builds a simulated box of molecules matching the concentrationsand density of the experimental sample, with each atom characterizedby a reference potential consisting of a charge q and the Lennard–Jones (LJ) parameters σ and εusing the standard Lorentz–Berthelot mixing rules. Exchangeablehydrogens are accounted for when calculating the predicted weightsof each interatomic correlation to the total scattering data for eachisotopic variant. The simulation is then equilibrated through a MonteCarlo procedure using this reference potential. Once this is complete,an iterative, evolving empirical potential is applied, derived fromthe difference between the predicted scattering data from the simulationand the experimentally obtained scattering data. The simulation continuesuntil a satisfactory agreement exists between the simulation and thesupplied scattering data. Monte Carlo steps are allowed to continuefurther for >1000 iterations to gather statistics on interatomiccorrelations.The dynamic liquid nature of the sample requires this as atomic positionswill be constantly evolving, and a single “crystal-like”structure with well-defined atomic positions is unfeasible. Gatheringstatistics over large numbers of iterations therefore allows one todetermine average interatomic correlations and positional uncertaintiesthrough tools such as RDFs. The result is an ensemble of structuresfrom a simulated box of atoms whose predicted scattering data is consistentwith the measured scattering data. The nature of this procedure doesnot guarantee a single unique solution to the supplied scatteringdata, but it is consistent with the scattering data and based on sensiblestarting potentials and known intramolecular structures. The workflowof these procedures and quantification of quality of fit is outlinedin Supporting Information, Figure S4.The experimental WANS data fromNIMROD was first corrected for incoherent, multiple scattering, andattenuation effects using Gudrun software.+ ions (0.1 atoms/Å3). The concentrationof Na+ ions is slightly lower than that in the experimentalsample. This is a simple way to ensure charge neutrality without requiringfree hydroxide ions, as there is no capacity within EPSR for dissociation/reassociationof water into its constituent ions . The reference potential for waterwas the SPC/E model,74 the Na+ force field was taken from Mancinelli68 and the CLN025 force field from the AMBER-FB15 potential from McKiernanet al.23 The number of atom types for CLN025was reduced by grouping similar atoms of a similar charge as describedin Supporting Information, Note S2. Theraw and modified force fields can be found in Supporting Information, Tables S1 and S2.The EPSR simulation contained 6944 water molecules, 14 CLN025 molecules,and 28 Na19 and multiple hairpin self-assembly was incorporated basedon results from the SANS analysis. The CD data reported in Supporting Information, Figure S3 show that thisis a reasonable approach, as the strongly overlapping spectra suggestessentially identical secondary structure between the hairpin usedin this work and that of Honda et al. The acetyl and amide caps werethen added to the N and C termini, respectively, without further structuralminimization. This method was chosen as the minimization step employedthrough EPSR, which causes the hairpin bonds along the length of thehairpin backbone to be distorted away from the trans-conformation and, therefore, produces a physically unreasonablestructure. No bond rotations were permitted, and the CLN025 moleculetherefore remains folded during the simulation by virtue of intramolecularharmonic potentials between 1–2 neighbors (bonded atoms), 1–3neighbors (bond angles), and 1–4 neighbors .These are defined by an average distance and a width term that allowsfor natural fluctuation of bond lengths/angles and mimics thermalfluctuations.75The CLN025hairpin was modeled within EPSR by directly incorporatingits crystal structure as reported by Honda et al.2O (with 250 mM NaOH)from the combined neutron data obtained on both the NIMROD and Zoominstruments. Below Q = 0.03 Å–1 a power law with decreasing Q is observed, yieldinga fitted exponent of 3 ± 0.2, which suggests the presence oflarge (>6000 Å) dense fractal aggregates of unfolded protein.However, this power law is not observed over the entire Q range, suggesting that the volume fraction of these large aggregatesis low. CD analysis does not show the presence of any aggregates,confirming that any aggregates present, while large, must be at relativelylow concentrations compared to the remainder of the CLN025 hairpin,as detailed in Supporting Information, Note S1. The intensity profile in the region 0.03 < Q < 3 Å–1 is characterized by two overlappingpeaks at ∼1.0 Å–1, and a power law increasein the intensity ∝ Q–2.5 in the region of 0.1 < Q < 0.5 Å–1 followed by an inflection at ∼0.1 Å–1. The overlapping peaks at ∼1.0 Å–1 suggest the presence of repeated characteristic structurallength scales, while the Q–2.5 powerlaw suggests there is a nonspherical 3D scattering object, e.g., athick rod and the inflection at Q ∼ 0.1 Å–1 suggests that these objects are electrostaticallyrepulsive. This is supported by previous observations of electrostaticrepulsion between proteins in aqueous solution measured by SAXS ationic strengths <1 M76 .Supporting Information, Note S3 for further details)suggests that CLN025 hairpins assemble into a cylinder with a lengthof 60 ± 10 Å and an elliptical cross section with Rminor and Rmajor of 6.5 ± 0.6 and 12.6 ± 0.8 Å, respectively , suggesting that the plane of the β-hairpinCLN025 is aligned perpendicular to the axis of the elliptical cylinder.If the hairpin is aligned such that the self-assembled cylindricalstructure is made up of a planar stacked hairpin, then we would expectthere to be 7 hairpins per cylinder on average, as the minor equatorialdiameter of the hairpin is 9.6 Å . As the average charge of CLN025 atneutral pH is −2 e, we would expect the average overall chargeof the self-assembled cylinder to be −14 e; indeed, from thefitting . The χ2 values ofthis procedure are also reported in the SI.Fitting ectively 2b. The fSupporting Information, Note S4 and Figures S10–S15). Following the buildingof the 7-hairpin stack, two stacks were loaded into EPSR and, hence,a total of 14 hairpin monomers, into a cubic box of dimension 61.506Å separated by a horizontal distance of half the box width. Thisrepresents a necessary simplification of the modeled system, as inthe solution we expect from the 2D SANS pattern  that the stacks will berandomly oriented with respect to each other and not fixed in theirpositions. However, the primary objective of the EPSR analysis isto understand local hydration, which should not be affected by theinterstack structure at longer length scales. Each CLN025 moleculewas tethered to its starting coordinate by its center of mass to preservethe stack; however, other movements  were permitted. The hairpin stacks were then hydratedusing 6944 water molecules and 28 Na+ ions to ensure overallcharge neutrality leading to a density of 0.1 atoms/Å3. Following the procedure of EPSR, the box was allowed to equilibrateunder a reference potential and then the empirical potential. Statisticswere then gathered over >3000 iterations. The final fits betweenexperimentaldata and EPSR simulation are shown in R ∼ 0.01, as defined in Supporting Informatin, Figure S4. The fits are observed to be of high quality above Q ∼ 1 Å–1, however, the strongupturn at low Q is only observed in the fully H2O sample, as shown in Supporting Informatin, Figure S16. This suggests that treating the hairpin self-assemblyas two stacks of 7 hairpins held at fixed positions partially capturesthe low Q behavior. In the future, a larger box containingmultiple hairpin stacks that are free to diffuse may be required tofully capture the low Q behavior. It is also importantto note that the simulated scattering data produced through EPSR isonly reliable to a minimum Q of 4π/Lbox, where Lbox isthe full width of the cubic simulation box, hence, a larger box willintrinsically improve the ability of EPSR to fit the low Q behavior. However, this is outside the scope of the EPSR’scurrent capabilities. The data above Q ∼ 1Å–1 contains information on small scale structures,such as peptide–water and water–water interactions and,hence, can be reliably studied to inform hairpin hydration characteristics.The SANS datasuggest a stacked hairpin structure involving seven CLN025 monomers;however, it is incapable of providing the structure of a stack withatomic resolution. To achieve this length scale resolution, we builta stack of seven all atom hairpin monomers using a custom built moleculardocking Monte Carlo simulation  and normalized to the OH bond length (Rdonor = 0.96 Å) to yield the hydrogen bonding index Δ.The Δ therefore ranges from 0 to 1 for hydrogen bond acceptingsites where 1 is a perfect hydrogen bond acceptor and 0 is a sitethat causes the hydrating water molecule to show no tendency to orientits hydrogen atoms toward or away from the site. For hydrogen bonddonating sites rwX-O – rwX-H wasnormalized to Racceptor, as detailed in Racceptor representsthe rwX-O – rwX-H distance that would occurif the hydrating water molecule were to orient both hydrogens simultaneouslyas far away as possible from site X, to yield the value Δ. TheΔ ranges from 0 to −1 for hydrogen bond donating sites,where −1 represents a perfect hydrogen bond donor and 0 againis a site that causes the hydrating water molecule to orient its hydrogenstoward or away from the site. Δ values calculated for each atomon the hairpin stack surface with identical atoms on each of the 14hairpins are assumed to have identical Δ values. The resultsof this procedure are shown in Using the accumulated data from the >3000 iterations it is possibleto derive the hydration structure of each atom belonging to the peptidestacks. A snapshot of a single iteration for the hydration structurearound a tyrosine side chain and a glutamic acid side chain is shownin Supporting Information, Table S2  to form soft materials that retain large volumes (>90%)of water with tunable physical properties.93 (simulation size in this work was 23268 atoms),atomistic resolution of a complete hydrogel network on the order of100s of nm would be unreachable, however more focused simulationsof hydration around individual structural motifs of interest, suchas single fibers, branches, or entanglements that have been previouslyobserved in peptide hydrogels,33 couldpotentially be achieved. With the development of the Dissolve software,93 access to potential refinement techniques onlarger simulation sizes ca. 106 atoms is now possible. Coupled with the ability toemploy directed construction of simulation starting points, permittingbias toward specific structural motifs and arrangements as the startingpoint for refinements, Dissolve has the potential to open up structuralanalysis of these larger and more complex systems. This could allowfor the study of a small model hydrogel featuring several structuralmotifs to be present within the same simulation. In addition, furtherdevelopments of the coarse grained simulation scattering calculationtool MuSSIC would allow for neutron diffraction constrained computationalmodeling of yet larger and more complex systems.103We envisage that this could occur in severaldifferent ways. Asthe current limitation of EPSR is approximately 140000 atoms104 such as in the coarse-grained MARTINI,105 and PRIME models.106 The MARTINI model has been primarily applied to the study of peptideassembly,108 proteins in lipid membranes,110 and protein conformational changes,112 whereas PRIMEis mainly applied to the study of protein aggregation.114 In both instances, side chains are described using coarse-grainedbeads that explicitly describe hydrogen bonding and hydrophobicity.The information gathered through the study herein can help parametrizesuch force fields, for example the description of hydrogen bondingused in the MARTINI force field, in which coarse grained sites aredescribed by hydrogen bonding ability and polarity (1–5). The site-specific RDFs generated throughthis approach can also aid in the development of force fields throughprocesses, such as iterative Boltzmann inversion,115 inverse Monte Carlo,116 or Newtoninversion.117Finally, we highlight the importance of the resultspresentedherein to the field of biomolecular simulation. An atomistic mappingof the surface hydrophobicity of a peptide or protein is an importantconsideration for biomolecular engineering and modeling,


Nanog and Oct4 primes cells for neural commitment, whilst loss of Sox2 expression primes cells for primitive streak commitment. Furthermore, we find that Wnt signalling acts through Nanog to direct cells towards a primitive streak fate, but that transcriptionally active β-catenin is associated with both neural and primitive streak commitment. These observations confirm and extend previous suggestions that pluripotency genes influence lineage commitment and demonstrate how their dynamic expression affects the direction of lineage commitment, whilst illustrating two ways in which the Wnt signalling pathway acts on this network during cell fate assignment.Understanding how interactions between extracellular signalling pathways and transcription factor networks influence cellular decision making will be crucial for understanding mammalian embryogenesis and for generating specialised cell types in vitro. To this end, pluripotent mouse Embryonic Stem (mES) cells have proven to be a useful model system. However, understanding how transcription factors and signalling pathways affect decisions made by individual cells is confounded by the fact that measurements are generally made on groups of cells, whilst individual mES cells differentiate at different rates and towards different lineages, even in conditions that favour a particular lineage. Here we have used single-cell measurements of transcription factor expression and Wnt/β-catenin signalling activity to investigate their effects on lineage commitment decisions made by individual cells. We find that pluripotent mES cells exhibit differing degrees of heterogeneity in their expression of important regulators from pluripotency, depending on the signalling environment to which they are exposed. As mES cells differentiate, downregulation of  The mammalian embryo is derived from a small group of cells, the epiblast, which is first segregated from extraembryonic tissue just before implantation, at the blastocyst stage . This grMouse Embryonic Stem (mES) cells are clonal populations of cultured cells derived from the blastocyst of the embryo that can give rise to all of the cell types that constitute the adult organism, i.e. they are pluripotent . SeveralHere we have analyzed the controlled exit of mES cells from pluripotency under established neural differentiation conditions (N2B27) by measuring gene expression in single cells. This technique has previously been used to study mouse blastocysts , pluripoNanog, Oct4 and Sox2) restricts differentiation to specific lineages as cells exit pluripotency. Our findings lead us to suggest that during the first four days of differentiation in culture, cells undergo a transition that mimics events in vivo between the blastocyst and the postimplantation epiblast, by the end of which cells are biased towards commitment to different lineages. We also find that, during this period, ES cells display a widespread activation of canonical Wnt/β-catenin signalling which is not affiliated with specific lineages and discuss the possible implications of this observation.We find that initial differentiation of mES cells in N2B27 is not restricted to neural lineages, but that during the first four days of differentiation, individual cells express markers for a variety of lineages. Furthermore, our single cell analysis suggests that the expression of individual components of the core pluripotency network  and T/Bra   . These c markers . This deSox1  and an early decrease in the expression of Sox2 as cells exit pluripotency, followed by an increase as cells adopt neural fates. However, we also observed expression of T/Bra and Eomes, which are genes associated with Primitive Streak and endodermal fates (the sharp decline in T/Bra levels on day 1 most likely represents the failure of cells expressing this gene in BMP4+LIF conditions to re-adhere when reseeded). These profiles additionally reveal a small, but significant and reproducible, rise in the levels of Nanog and Oct4 on day 3 of differentiation, which can be observed, but is rarely remarked on, in many reports. Dynamic expression of Nanog and Oct4 probably reflects reactivation of these genes as cells pass through the epiblast state, where both are expressed , epiblast differentiation (Fgf5), neural commitment (Sox2 and Sox1) and primitive streak commitment (T/Bra) was measured to determine the differentiation state of individual cells over the course of the experiment  of the gene expression profiles of individual cells shows that in 2i conditions cells have very similar expression profiles, which gradually diverge as they differentiate . In 2i, Rex1 , indicatNanog, Oct4 and Sox2 homogeneously. However, differentiating cells express different combinations of these genes, suggesting that the sequence in which the expression of pluripotency genes is downregulated differs between differentiating cells. Interestingly, cells that retain high levels of Sox2 expression often coexpress Sox1, but very rarely express T/Bra, whilst the opposite is true of cells that retain high levels of Nanog and/or Oct4 expression. These observations suggest that retention of Sox2 expression predisposes cells towards neural commitment, whilst retention of Nanog or Oct4 expression biases cells towards primitive streak commitment.Patterns of pluripotency gene expression across the plot suggest that individual pluripotency genes influence the direction of lineage commitment . PluripoNanog and T/Bra on the one hand, and Sox2 and Sox1 on the other, in differentiating cells. The relationships between expression of Oct4 and the other genes were similar to those of Nanog, but generally much weaker. These findings confirm earlier suggestions that different pluripotency genes specifically inhibit commitment to individual lineages, and additionally demonstrate how these interactions are relieved over time in order to guide cells towards different lineages.To test these relationships more formally we looked at probabilities of coexpression after four days differentiation . This anNanog acts as a gatekeeper for pluripotency but, as we have shown here and others have observed previously, it is also associated with the emergence of a primitive streak fate  in Serum, mES cells undergo preferential neural differentiation and we have observed that they activate a reporter of Wnt signalling . This isFgf5 expression and low levels of the pluripotency genes Rex1, Nanog, Oct4, Sox2, Tcf15 and Otx2 relative to the rest of the population. These cells also express lineage associated genes, but without a bias for particular fates. Thus we observe expression of markers of primitive streak and its derivatives , as expected, and also genes associated with neural development (Sox1 and Zfp521). These results suggest that, as cells exit pluripotency in culture, they activate Wnt signalling and that this activation is not associated with specific lineages, but rather appears to be a feature of the early stages of differentiation. It is known that during self-renewal there is little expression of Wnt genes and that as cells differentiate they express many members of the Wnt family and increase expression of those expressed during self-renewal , as well as differentiation markers in a proportion of cells. In ground state pluripotency (2i conditions), these genes are expressed more homogeneously and the expression of differentiation markers is reduced, though we observe a small degree of heterogeneity, which is in agreement with previous observations  is associated with variable but generally high levels of expression of pluripotency genes  (2.E14Tg2A wild-type (129/Ola), (44Cre6)  and Wnt (44Cre6)  mES cell5 trypsinised and pelleted mES cells using the RNeasy Mini kit  according to the manufacturer's instructions, and resuspended in 30 µL distilled water. RNA was reverse transcribed as follows. RNA samples (1 µg in 38 µL nuclease free water) were combined with 2 µL Oligo-dT anchored primers  and incubated at 80°C for 2 minutes before transferring immediately to ice for 2 minutes. PCR master mix was then added to each sample: 1.5 µl dNTPs , 12 µl 5× First Strand buffer , 3 µl 0.1 M DTT , 1.5 µl RNaseOUT  and 2 µl Superscript III Reverse Transcriptase . Thermal cycling was carried out as follows: 25°C 10 minutes, 50°C 30 minutes, 70°C 15 minutes. To remove RNA from the resultant cDNA samples 1 µL RNaseH  was added and samples were incubated at 37°C for 20 minutes and then at 80°C for 20 minutes. Samples were diluted 1 in 10 before being analysed for gene expression using a Rotor-gene Q instrument. Reactions were set up using a Qiagility liquid-handling robot , 0.5 µL primer mix (50 µM each), 9 µL PCR grade water and 3 µL diluted cDNA) and analysed using the 2-step protocol described in the Quantifast manual . Fluorescence curves were quantified using the MAK3 algorithm found in the R package qpcR. Primer sequences are listed in supplementary material Table S1.RNA was isolated from ∼5×10FACS was used to distribute individual mES cells into the wells of a 96-well plate containing lysis buffer (see below). The transcriptomes of isolated cells were then amplified according to a previously published protocol (up to step 30) . The resCells were trypsinised and dissociated, spun down in a microcentrifuge, resuspended in serum-containing medium, then analysed and/or sorted on a Beckman–Coulter Cytomation MoFlo High Performance Cell Sorter.


We review the conceptual bases of XDMR and recast them in the general framework of the linear and nonlinear theories of ferromagnetic resonance (FMR). Emphasis is laid on the information content of XDMR spectra which offer a unique opportunity to disentangle the precession dynamics of spin and orbital magnetization components at given absorbing sites. For the sake of illustration, we focus on selected examples in which marked differences were found between FMR and XDMR spectra simultaneously recorded on ferrimagnetically ordered iron garnets. With pumping capabilities extended up to sub-THz frequencies, high-field XDMR should allow us to probe the precession of orbital magnetization components in paramagnetic organometallic complexes with large zero-field splitting. Even more challenging, we suggest that XDMR spectra might be recorded on selected antiferromagnetic crystals for which orbital magnetism is most often ignored in the absence of any supporting experimental evidence.X-ray Detected Magnetic Resonance (XDMR) is a novel spectroscopy in which X-ray Magnetic Circular Dichroism (XMCD) is used to probe the resonant precession of  In the early nineties, X-ray Magnetic Circular Dichroism (XMCD) measurements made this possible within the limits of validity of the magneto-optical sum-rules  in which the spin Hamiltonian  was given the following form:We are left here with an oversimplified picture in which essentially the spin part of the angular momentum is precessing. Van Vleck  went to g and μB are standard notations for the spectroscopic splitting factor and the Bohr magneton, Jjk being the isotropic exchange coupling constant between sites j and k. Whereas the first and second right-hand members of Djk and Ejk can depart a lot from the expected values for dipolar/quadrupolar couplings between spins located at the j, k sites. This is precisely where spin-orbit interaction and orbital valence effects are taken into account to yield pseudo-dipolar (S = 1/2) or pseudo-quadrupolar (S > 1/2) couplings K is the difference in the absorption cross-sections of left- and right-circularly polarized X-rays in the vicinity of the K-edge, Cp being a constant factor. Note that such a differential formulation refers to a fixed energy of the photoelectron: ΔE = ERX + E0 – EF in which ERX, E0 and EF respectively denote the energy of the X-ray photons, the binding energy and the reference Fermi level. Whereas 〈Lz 〉p is the expectation value of the orbital angular momentum operator integrated over all excited states featuring p-type symmetry, 〈ℓz 〉p defines the orbital polarization of p-projected densities of states (DOS) at energy ΔE. Taking into account the weaker electric quadrupole (E2) transitions simply results in mixing final states associated with atomic orbitals of different symmetry h = 0. The first equation yields: h = −▿ψ and the second: ▿ · μ̄ ▿ψ = 0. These are Walker’s Equations. For a plane wave such as: ψ ∝ exp[ik · r], the Landau–Lifshitz–Gilbert and Walker’s Equations can be solved simultaneously only if a dispersion relation is satisfied  =  =  = 0. In particular, this implies that the Heisenberg exchange Hamiltonian should also commute with the longitudinal (z) component of the magnetization:  = 0.In the quasi-particle language of second quantification, Akhiezer , but alsAkhiezer . Energy in waves  because in waves . Given ty Keffer , this isy Keffer ) that thM(s) probed by XDMR are directly affected by collective excitations of exchange spin waves. Even though exchange interactions should have no direct effect on orbital magnetization components M(ℓ), one should not forget that M(ℓ) and M(s) are dynamically coupled by spin-orbit interactions at a time-scale much shorter than the time-scale of precession. Let us insist, however, that it cannot be taken yet as firmly established that M(ℓ) couple to exchange spin waves.There is no doubt that the local spin magnetization components k = 0) can be annihilated with the simultaneous creation of one or more nonuniform magnons (k ≠ 0): the latter scattering process was identified as the critical step controlling transverse relaxation processes, i.e., T2 in the Bloch–Bloembergen model.Recognizing that dipolar interactions open a band of degenerate magnons is the clue to understand the relaxation mechanisms in FMR , since aStatistical thermodynamics led to a pair of important equations which proved to be very helpful in discussing the relevance of various relaxation processes:nk is the number of magnons with wavevector k in volume V, including thermal magnons. Mz; |M|. where M(ℓ) couple to spin waves; (ii) energy can be redistributed within additional degrees of freedoms involving orbital ordering (Oord) in orbitally degenerate systems. In the first case we shall speak of pseudo-spin waves or pseudo-magnons. In the second, we have to deal with true orbital ordering waves or orbitons which can be best described using a formalism that was recently reviewed by Khaliullin , [(ℓx)i2 – (ℓy)i2] and [(ℓxℓy)i + (ℓyℓx)i], in which ℓx, ℓy and ℓz are the components of a fictitious orbital angular momentum built up from the 3dxz, 3dyz and 3dxy transition metal orbitals. The superexchange Hamiltonian  is modulated by an operator  which looks like the orbital quadrupole-quadrupole interaction  between sites i and j, i.e., the higher-order terms in the Van Vleck anisotropic exchange interaction. Note that  also appears in the theory of the Jahn–Teller effect.Regarding triply degenerate states  joint spin-orbital fluctuations should significantly lower the ground state as first predicted by Cyrot and Lyon-Caen (Fe3)O12 Fe4.84O12 (Y-La-LuIG). Note that in film 2, “diamagnetic” (1S0) rare earth cations  substitute for Y3+. In Section 3.4, we shall also briefly discuss XDMR spectra recorded with a thin, polished platelet of gadolinium iron garnet (GdIG) single crystal.In Section 3, we shall reproduce XDMR spectra collected essentially on two iron garnet films that were grown by liquid phase epitaxy on oriented gadolinium gallium garnet (GGG) substrates: χ″) and dispersive (χ′) XDMR components of film 1 can be identified with the real and imaginary parts of the vector detection scheme. A small (instrumentation dependent) phase-shift (ΔΦ ≃ 2°) was added in order to let χ′pass through zero when χ″is maximum as well as |XDMR|. In this experiment, the energy of the circularly polarized X-ray photons was tuned to the maximum of the Fe K-edge XMCD spectrum (E1 = 7113.91 eV) and the film was rotated by βY ≃ 420 in order to minimize the demagnetizing field anisotropy. We checked that the XDMR peak intensity varied linearly with the square root of the pumping power up to ca. 10 mW.We like first to show that XDMR spectra of unprecedented quality can now be recorded in the transverse detection geometry (TRD) on YIG and related thin films, thanks to our superheterodyne detection scheme that relies on the powerful concepts of either bpsk or qpsk (bi- or quadrature phase shift keying) used in satellite telecommunications. A vector detection scheme also allows us to recover the phase information that is preserved in a TRD geometry. This is illustrated with locally, the orbital magnetization components M(ℓ) couple to non uniform magnetostatic spin waves through dipole-dipole interactions. Note that forward/backward MSW seem to have different phases. At this stage, one should realize that there is no chance to excite and detect standing waves resonances associated with magnetostatic modes unless there is a net transverse magnetization component interacting with both the microwave pump field and the circularly polarized X-rays: in the YIG thin film 1, this can be envisaged only for standing waves of rather low order and featuring an odd number of semiperiods  = 13 – 19°, was much larger than the apparent precession cone angle of the spin magnetization component M(s) measured at the La L2,3-edges, i.e., θ0(s) [La] = 4.7°. Even more puzzling was the large precession cone angle θ0(ℓ) [Fe] measured for film 2, especially if we compare it to the opening angle (θ0(ℓ) [Fe] = 7.2°) measured for the YIG film 1 under identical experimental conditions. In contrast, θ0(s) [La] was found to be slightly smaller than the cone angle measured at the Y L-edges in film 1  [Y] = 5.9°).Long before we started the XDMR experiments reported below, a u3+, Y3+ . Technic2 under high pumping power (630 mW). The XDMR spectrum was again recorded in the longitudinal detection geometry (kRX|| || B0). The FMR and XDMR spectra both exhibit strong foldover distortions resulting into broad lineshapes (ΔH0 ≥ 400 Oe). Most intriguing to us was the very sharp increase of the XDMR signal just before the foldover jump in the downfield scan, in a range where the FMR absorption spectrum seems to saturate. Even far from the foldover jump, the precession cone angle (θ0(ℓ) [Fe] ≃ 13°) is much too large to be realistic: this definitively ruled out the crude assumption that the length of the precessing moment (|M(ℓ)|) was invariant and convinced us to envisage the parametric excitation of nonuniform modes.We compare in |M(ℓ)| and to increase the transverse relaxation rate 1/T2. Regarding Mz(ℓ) unaffected since two uniform magnons are replaced by two degenerate magnons, so that the total number of magnons is left unchanged. This, however, does not hold true if the life-times of uniform and degenerate magnons are different. Schlömann (Bu4N)3). Recall that 4 is a building block for the chemical synthesis of heterobinuclear molecular magnets 1/2 < BC2, the magnetic system undergoes a first order transition (spin-flop) resulting in the new situation sketched in M = M1 +M2 will be pumped:When the external field BE||= 2BE − BA and BE⊥= 2BE + BA, so-that BC1 = [BABE||]1/2 and BC2 = [BABE⊥]1/2. For completeness, it should be mentioned that there exists also a soft mode (ω → 0) but the latter will be undetectable by XDMR since it cannot be excited by the external pump field 2 × (BC/BE)2, in which BC/BE does not exceed, at best, a few percents. This would bring us very close to (or below) the detection limit.Given that the precession cone angles BE is not too strong, i.e crystals with a rather low Néel temperature . Regarding AFMR, MnF2 (TN = 68 K) is a good example given that a very narrow linewidth (ΔB = 5 G) was measured at T = 4 K for the uniform precession mode (ω −) excited by a microwave pump field (Fp = 23 GHz) in a bias field B0 ≃ 8.5 T [B = 15 G) were also obtained with epitaxial films grown on MgF2 substrates [∝ ωBE. Unfortunately, MnF2 may not be the ideal candidate for Mn K-edge XDMR experiments due to the vanishing orbital moment 〈Lz》 of Mn2+ ions in a high spin (6S) state. Recall, however, that 〈ℓz〉 may still be finite in excited states even though 〈Lz〉 vanishes in the ground state. There is the further handicap that, in an octahedral crystal field with 6A1g ground term, only electric quadrupole (E2) transitions contribute to the pre-edge structures assigned to final states involving magnetic 3d orbitals.For XDMR experiments, it may be preferable to select AFM crystals in which the exchange field  ≃ 8.5 T  so that bstrates . Note th3 (TN = 39 K) looks more attractive owing to the fact that this crystal is the archetype of orbitally ordered systems [c axis. On the other hand, careful studies of the angular dependence of the EPR spectra of KCuF3 in the paramagnetic phase revealed the existence of a strong contribution of the Dzyaloshinskii–Moriya antisymmetric exchange interaction:The case of KCuF systems . In thisdjk ⊥ c [djk could be expressed in terms of transition orbital moments:with djk ⊥ c . Moriya djk ⊥ c . Then, tλ denotes the spin-orbit coupling factor, ΔEj(k) are the energy separations between the ground state orbital levels gj(k) and the relevant excited states ej(k), Jj(k) being the effective superexchange constants at sites j and k respectively. Clearly, the coupling field vanishes if the two sites transform into each other by inversion symmetry. It is well documented that a large antisymmetric exchange interaction can cause a small canting effect of the AFM-ordered magnetization vectors resulting in a weak ferromagnetism [B0 ⊥ c, KCuF3 should behave just like a weak ferromagnet satisfying the resonance condition [in which agnetism . Typicalondition :Under such conditions, one may reasonably expect a (weak) XDMR signal to be detectable at low temperature in TRD geometry.ω − could be excited at microwave frequencies, single crystals of ilmenites  could also be good candidates for XDMR: they had rather low Néel temperatures [Lz〉 at the sites of the Jahn–Teller cations [2O3 (TN = 308 K) looks less favorable even though we already produced definitive evidence of orbital magnetism in this crystal, at least in its magnetoelectric phase [2O3 is rather low (BC1 ≃ 5.9 T), it cannot be taken yet for granted that Δm⊥(±)will be large enough to yield a XDMR signal easily detectable at microwave pumping frequencies.Among transition metal oxides for which the low frequency mode eratures ,94 and s cations . With a  cations ,96, the ic phase . Althougd transition elements and for which much stronger XDMR signals could be expected at L-edges. Unfortunately, neither single crystals nor epitaxial films of K2ReBr6  or ReO2M(ℓ). There is no doubt left that locally, M(ℓ)can couple to magnetostatic spin waves through dipole-dipole interactions but there is no experimental result which would definitively prove that M(ℓ)could similarly couple to exchange spin-waves. On the other hand, we pointed out strong anomalies in the XDMR spectra which support our view that orbitons may contribute to new relaxation mechanisms. Several important questions were left aside, e.g., the question to know whether, locally, the spin and orbital magnetization components M(s) and M(ℓ)do precess in or out of phase and possibly around different effective axes. Also the whole problem of the magnetoelastic waves resulting from the modulation of the spin-orbit interactions by the lattice phonons will be discussed elsewhere. Nevertheless, in Section 4, we tried to anticipate over the emergence of XDMR as a parallel method to study orbital magnetism in paramagnetic as well as in antiferromagnetic phases.XDMR is a novel spectroscopy which is still in an early stage of development often dominated by severe instrumentation problems. In this review, we tried to convince the reader that XDMR could develop as a unique tool to study dynamical aspects of orbital magnetism including collective excitations. For the first time, direct experimental evidence was produced of the forced, elliptical precession of orbital magnetization components


We related composition of cerebral thrombi to stroke subtype and attenuation on non-contrast CT (NCCT) to gain more insight in etiopathogenesis and to validate thrombus attenuation as a new imaging biomarker for acute stroke.r).We histopathologically investigated 22 thrombi retrieved after mechanical thrombectomy in acute stroke patients. First, thrombi were classified as fresh, lytic or organized. Second, percentages of red blood cells (RBCs), platelets and fibrin and number of red, white (respectively RBCs or platelets outnumbering other components with ≥15%) or mixed thrombi were compared between large artery atherosclerosis (LAA), cardioembolism, dissection and unknown subtype. Third, correlation between attenuation and RBCs, platelets and fibrin was calculated using Pearson's correlation coefficients (Thrombi were fresh in 73% (n = 16), lytic in 18% (n = 4) and organized in 9% (n = 2). The stroke cause was LAA in eight (36%), cardioembolism in six (27%), dissection in three (14%), and unknown in five (23%) patients. LAA thrombi showed the highest percentage RBCs (median 50 (range 35–90)), followed by dissection (35 (20–40), p = 0.05), cardioembolism (35 (5–45), p = 0.013) and unknown subtype (25 (2–40), p = 0.006). No differences in platelets (p = 0.16) and fibrin (p = 0.52) between subtypes were found. LAA thrombi were classified as red or mixed (both n = 4), cardioembolisms as mixed (n = 5) or white (n = 1) and dissection as mixed (n = 3). There was a moderate positive correlation between attenuation and RBCs , and weak negative correlations with platelets  and fibrin .The majority of cerebral thrombi is fresh. There are no differences in age of thrombi between subtypes. LAA thrombi have highest percentages RBCs, cardioembolism and unknown subtype lowest. No relationship exists between subtype and platelets or fibrin percentages. We found a correlation between the RBC-component and thrombus attenuation, which improves validation of thrombus attenuation on NCCT as an imaging biomarker for stroke management. Thromboembolism is the cause of ischemic stroke in the majority of acute stroke patients The use of mechanical thrombectomy devices allows retrieval of cerebral thrombi from the intracranial vessels and subsequent histopathologic analysis. The first histopathologic studies showed that the architecture of cerebral thrombi is variable with different main components such as fibrin, red blood cells (RBC) and platelets Furthermore, the age of thrombi could also play a role in success rate of thrombolysis, which is only about 50% with current IV-rtPA therapy. Recanalisation depends on the time from stroke onset to intravenous (IV) rtPA-treatment, hence it may be more effective in fresh than old thrombi On non-contrast CT (NCCT) platelet-rich thrombi have lower attenuation than RBC-rich thrombi since the attenuation has a linear correlation with the concentration of hemoglobin Improved knowledge about thrombus age and composition and its relation with stroke subtypes and attenuation could help to gain more insight in the etiopathogenesis of stroke. The aims of this study were to relate the age and histopathologic composition of thrombi to the stroke subtype and to thrombus attenuation on NCCT.We histopathologically investigated thrombi retrieved after mechanical thrombectomy of acute stroke patients admitted to the University Medical Center Utrecht or the St. Antonius Hospital Nieuwegein, between December 2010 and April 2013. Inclusion criteria were: age ≥18 years, National Institutes of Health Stroke Scale (NIHSS) ≥2, acute stroke patients with an intracranial thrombus on CT-Angiography (CTA) eligible for thrombectomy, retrieval of thrombus material available for histopathologic analysis. Exclusion criteria were the collection of thrombus material that was unsuitable for histopathologic analyses and a poor quality NCCT that was unsuitable for thrombus density measurements.The following baseline data were collected: demographic features, cardiovascular risk factors , NIHSS on admission, location of the thrombus, and onset time to CT-imaging, to IV-rtPA treatment and thrombus retrieval.LAA was defined as imaging findings of either significant (>50%) stenosis or occlusion of a major cervical  artery or intracranial artery on CTA ipsilateral to the symptomatic hemisphere, presumed to be due to atherosclerosis according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria The medical ethics committee of the University Medical Center Utrecht approved the study. If patients had the capacity to consent, written informed consent was obtained from themselves. If this was not the case, written informed consent was obtained from their nearest relative(s).Based on clinical and imaging information IV-rtPA was administered to patients when considered eligible by a team of experienced neuroradiologists and neurologists. If IV-rtPA failed or could not be administrated due to contra-indications, the administration of intra-arterial (IA) rtPA followed by thrombectomy or thrombectomy alone was done, in all cases under systemic heparin. The heparin was given IV at a dose of 2500 IU.Informed consent was obtained prior to the procedure. Mechanical thrombectomy was performed in accordance with local guidelines. All patients had heparin administered during the percutaneous intervention. The occlusion was reached using a guiding catheter through the femoral artery. The thrombus was removed by a Merci retriever, a Trevo retriever or a Solitair stent and thrombus material was collected in a formalin equipped bottle.Specimens were embedded in paraffin and 4 consecutive 5-µm thick slices were cut. All thrombi were stained with hamatoxylin and eosin, Mallory's phosphotungstic acid-hematoxilin (identifying fibrin), glycophorin A (identifying RBCs) and CD31 (identifying platelets) immunostains.Subsequently, thrombi were histopathologic analysed by an experienced vascular pathologist (A.V.) together with two other investigators (J.N. and L.v.D.) on all 4 slices. Histological analysis was conducted in a blinded fashion without any knowledge of patient and stroke characteristics. First, all thrombi were classified as fresh (<1 day), lytic (1–5 days) or organized (>5 days), based on previously published definitions of thrombus age All imaging studies were performed using 64- or 256-slice MDCT–scanner . CT parameters of the NCCT were: 120 kVp, 300 mAs, and 3-mm reconstructed slice thickness. For the CTA 65–70 ml of contrast agent (300 mg Iopromide/mL) was injected into the antecubital vein (18-gauge needle) at a rate of 6 mL/s followed by a 40-mL saline flush at the same rate.The attenuation of the thrombus was measured on 3-mm NCCT in Hounsfield Units (HU) by two independent observers, both blinded for any data except localization of the thrombus on CTA. Within the thrombus a standardised small round region of interest was drawn three separate times and mean HU-values were used for analysis. The same measurements were performed in the corresponding contralateral vessel and by calculating the relative HU  we corrected for hematocrit. If the thrombus was situated in the basilar artery, a more proximal non-thrombosed portion of the basilar artery was used for the control measurements. If the proximal end was not open, the supraclinoid ICA was used (n = 3).Baseline characteristics were summarised as means (with standard deviation (SD)), medians (with range) or frequency counts and proportions.First, the percentages of RBCs, platelets and fibrin were compared between the subtypes of stroke by the Kruskal-Wallis test. Significant group differences were further analysed by the Mann-Whitney U test to determine individual differences.r) of 0.90–1 as very strong; 0.70–0.89 as strong; 0.40–0.69 as moderate; 0.20–0.39 as weak and 0.01–0.19 as a negligible relationship. Statistical analysis was performed using SPSS . Results were considered statistically significant if P-values were <0.05.Intraclass correlation coefficients (ICCs) were used to evaluate the interobserver variation in HU-measurements of the thrombus. We considered ICC values <0.20 as poor, 0.21–0.40 as fair, 0.41–0.60 as moderate, 0.61–0.80 as good, and 0.81–1.00 as excellent. The correlation between attenuation and percentages of RBCs, platelets and fibrin was calculated using Pearson's correlation with a coefficient . The mean NIHSS was 14, with a minimum of 5 and a maximum of 35. In 17 patients (77%) IV-rtPA was administrated with a median time of 88 minutes from onset. Only 2 patients (9%) were on chronic anticoagulation medication and 3 patients (14%) received IA-rtPA prior to mechanical thrombectomy. The cause of stroke was classified as LAA in eight (36%), cardioembolism in six (27%), dissection in three (14%), and as unknown in five (23%) patients.A total of 22 thrombi were retrieved from 22 patients with an average age of 60 years (±13) and 50% female Thrombi were fresh in 73% (n = 16), as lytic in 18% (n = 4) and as organized in 9% (n = 2). No significant differences were found in the age of thrombi between the different subtypes (p = 0.54). All lytic and organized thrombi also revealed a component of the thrombus that was fresh. All thrombi that were fresh did not have any organized parts and only a very few fresh thrombi showed minimal lytic parts..The median (range) percentages of components across all thrombi were 40% (2–90%) RBCs, 40% (5–80%) platelets, and 20% (5–65%) fibrin ( and ). Comparing the individual stroke subtypes; thrombi originating from LAA showed the highest percentage of RBCs (median 50% (35–90%)), which was not significantly different from the dissection subtype (median 35% (20–40%), p = 0.05) but was significantly higher than thrombi from cardioembolisms (median 35% (5–45%), p = 0.013) and unknown subtype (median 25% (2–40%), p = 0.006). The median percentages of RBCs in thrombi from dissection and cardioembolisms (p = 0.82), dissection and unknown subtype (p = 0.33) and cardioembolism and unknown (p = 0.39) were comparable . White blood cells were present in most thrombi but in very small amounts only, in one thrombus a small volume of atheromatous material was seen and calcification was not visible in any thrombus.The median percentages of RBCs differed significantly between the different stroke subtypes as a group (p = 0.010) while there were no significant differences in the median percentages of platelets (p = 0.16) and fibrin (p = 0.52) between the different subtypes  and . C. Thrombi from LAA were classified as red in half of the cases (n = 4) and as mixed in the other half. Cardioembolic thrombi were defined 5 times (83%) as mixed and once as white (17%). All 3 dissection thrombi were classified as mixed, while the unknown subtype contained 3 white (60%) and 2 mixed thrombi (40%). No white thrombi were found in LAA and none of the cardioembolic, dissection, or unknown subtype thrombi were classified as red. Thrombi in patients with LAA had a significantly higher proportion of red thrombi than in cardioembolisms (p = 0.04), and significantly less white thrombi than the unknown subtype (p = 0.012) while differences between all other subtypes in number of red and white thrombi were insignificant (p>0.06).Thrombi were defined as red (RBC-rich) in 4 (18%), as white (platelet-rich) in 4 (18%) and as mixed in 14 (64%) cases The interobserver reliability for the HU-measurements was excellent (ICC = 0.98). There was a moderate linear correlation between the attenuation and percentage of RBCs , a weak negative linear correlation between attenuation and platelets  and a negligible negative linear correlation for fibrin  or by removal of embolized cardioembolic thrombi from ilio-femoral and subclavian-brachial arteries during vascular surgery (11 patients) Our findings are not in line with the traditional assumption. We observed that thrombus from LAA contain the highest amount of RBCs and that all red thrombi originate from LAA and none from cardioembolisms.Our findings are, however, to a great extent in line with the previous histopathologic studies investigating cerebral thrombi, which also found that the traditional assumption is not valid for stroke patients. To our knowledge only three histopathologic studies were performed on thrombi retrieved from cerebral arteries of patients having acute stroke Other than only one brief report on a thrombus from a single patient with a dissection, in which the histological examination was described as “serpentine” (fibrin:platelet bands interspersed with accumulations of nucleated cells and RBC-rich accumulations) To our knowledge this is the first study investigating the age of thrombi in acute cerebral stroke patients. While the traditional assumption emphasizes that fresh thrombi originate mainly from cardioembolisms, we found no differences in age of thrombi between the stroke subtypes. Furthermore, we found a higher percentage of fresh thrombi compared to studies of myocardial infarction patients, in which at least half of the cardiac thrombi were days or weeks old As mentioned, thrombus attenuation could be related to the constituents and composition of the thrombus. The concentration of hemoglobin is one of the determining factors as it has a linear correlation with the attenuation while platelets and atheromatous or cellular debris are known to decrease the attenuation of the thrombus on CT There are some aspects that merit consideration. First, only a relative small number of thrombi were investigated, consequently making it hard to draw strong conclusions. Second, no clear definition of “red” and “white” thrombi exists which is probably due to the fact that just a few studies have pathologically assessed cerebral thrombi in vivo. Most histopathologic studies based the division in red or white thrombi on “macroscopic evaluation” without any thresholds In our study with relatively small numbers important findings were seen: we found that the majority of cerebral thrombi is fresh and that there are no differences in age of thrombi between the stroke subtypes. Thrombi originating from large artery atherosclerosis have the highest percentages of red blood cells followed by thrombi due to dissection, while cardioembolic and unknown stroke subtype thrombi have the least red blood cells. A moderate correlation was found between the red blood cell-component and attenuation on non-contrast CT, improving the validation of thrombus attenuation on non-contrast CT as an imaging biomarker for acute ischemic stroke management. These findings will have to be validated in a larger study.


The standard use of a single universal broad-range PCR in direct 16S rDNA sequencing from polybacterial samples leaves the minor constituents at risk of remaining undetected because all bacterial DNA will be competing for the same reagents. In this article we introduce a set of three broad-range group-specific 16S rDNA PCRs that together cover the clinically relevant bacteria and apply them in the investigation of 25 polybacterial clinical samples. Mixed DNA chromatograms from samples containing more than one species per primer group were analysed using RipSeq Mixed , a web-based application for the interpretation of chromatograms containing up to three different species. The group-specific PCRs reduced complexity in the resulting DNA chromatograms and made the assay more sensitive in situations with unequal species concentrations. Together this allowed for identification of a significantly higher number of bacterial species than did standard direct sequencing with a single universal primer pair and RipSeq analysis (95 vs 51). The method could improve microbiological diagnostics for important groups of patients and can be established in any laboratory with experience in direct 16S rDNA sequencing. Detection and identification of bacteria directly from clinical samples by broad-range PCR targeting the 16S rRNA gene followed by DNA sequencing (direct 16S rDNA sequencing) is a valuable tool in clinical microbiology  each matching one of the three groups. All primer pairs amplified approximately the first 500 bp of the gene. In addition to reducing competition for reagents they also increased the theoretical number of species possible to detect in a sample using RipSeq from three to three times three. The group-specific primers were used to reinvestigate 50 clinical samples, of which 25 were known to be polymicrobial and 25 assumed to be sterile based on previous investigations and the clinical information available.Based on careful examination of the 16S rRNA gene we identified a semi-conserved area splitting a wide selection of human bacterial pathogens into three variant groups: group A, Gram-positive cocci in chains and non-anaerobic Gram-positive rods; group B, non-anaerobic Gram-negative bacteria; group C, Escherichia coli DNA from the cloning process. Cross-reactivity with human DNA was tested for each primer pair by amplifying 1000 pg, 100 pg, 10 pg, 1 pg and 0 pg of target bacterial DNA together with a constant amount of 100 ng human DNA (Promega).For each primer pair , following the manufacturer’s protocol. E. coli containing verified clones were cultured aerobically overnight. Plasmids were isolated from 2 ml E. coli culture using PureLink Quick Plasmid Miniprep kit (Invitrogen). The isolated plasmids were diluted to 1010 µl−1 and frozen at −20 °C until they were used.The 16S rRNA genes from relevant bacterial strains were amplified using primers 4F+pH/1542r . The PCRn = 5), bile (n = 2), empyema (n = 6), liver abscess (n = 3), ovarian abscess (n = 1), pancreas abscess (n = 2), para-aortic abscess (n = 1), retro-peritoneal abscess (n = 2), soft tissue abscess (n = 2) and spleen abscess (n = 1). In addition 25 presumptive negative specimens and their controls were included: various abscess or abscess-like specimens (n = 5), bone biopsy (n = 1), pericardial fluid (n = 3), periprosthetic tissue (n = 3), peritoneal fluid (n = 2), pleural fluid (n = 6), subdural haematoma (n = 1) and synovial fluid (n = 4). All samples were investigated using the three separate group-specific real-time PCRs with SYBR-green detection followed by DNA sequencing. In parallel all samples were analysed using a single universal primer pair , 400 µl supernatant was transferred to a MagNa Pure Compact automated extractor (Roche) and DNA was extracted and purified using the ‘Total_NA_plasma_100_400’ programme according to the manufacturer’s instructions. The resulting 50 µl of eluate was stored at −80 °C until used.The samples had been extracted using the following protocol. Between 200 and 800 µl sample material was added to a tube containing a mixture of glass and ceramic beads , together with 400 µl Bacterial Lysis Buffer . Eight hundred microlitres was the maximum capacity of the bead-tube, and this volume was used for liquid samples with low viscosity. For other samples 400 µl was used if available. Two hundred microlitres was the lowest volume that would still provide 400 µl supernatant for the subsequent DNA purification and was the lowest volume accepted for all specimens. A negative control containing lysis buffer and 400 µl PCR-grade water (Qiagen) was included in every batch of samples. The samples were run for 2×45 s in a FastPrep machine (Cepheid) at speed 6.5. After a short spin (15 800 PCR primers are listed in Ct) was reached three or more cycles ahead of the negative control. For the group-specific PCRs the negative control that became positive first for a given sample defined the cut-off for positivity for all three group-specific PCRs for that sample (i.e. three or more cycles before the earliest negative control).For all PCRs every sample was run in parallel with its negative extraction and purification control. For the universal PCR a sample was defined as positive if the fluorescence threshold value (The amplicons from the positive PCRs were collected by centrifugation from the SmartCycler reaction tubes into a 1.5 ml Eppendorf tube and cleaned up using the ExoSAP-IT enzymic degradation kit (Affymetrix). Primers used in the cycle sequencing reactions are listed in http://www.ripseq.com/login/login.aspx). The RipSeq Mixed algorithm searches against the ‘16S human pathogen iSentio’ database currently containing about 1500 reference sequences. The definition of a positive identification with the RipSeq program has been described previously (blast search against the GenBank database (http://blast.ncbi.nlm.nih.gov). For the blast searches interpretation criteria given by the Clinical and Laboratory Standards Institute were followed ]; primer pair B, 46 genera (non-anaerobic Gram-negative bacteria); primer pair C, 41 genera (Staphylococcus spp. and anaerobic bacteria except those covered for by primer A). This distribution is to be viewed as a rule of thumb, and exceptions exist in particular for groups A and C. A more detailed overview is provided in Supplementary Table S1, available with the online version of this paper.The distribution of genera among the different primer pairs was as follows: primer pair A, 42 genera [Gram-positive cocci in chains (including some anaerobic species) and non-anaerobic Gram-positive rods (including the aerotolerant genera The highest tendency for cross-reactivity was between primer pair A (PCR ‘A’) and bacterial group C and between primer pair C (PCR ‘C’) and bacterial group A. For these combinations a 1000-fold higher copy number of non-target DNA was needed to reach equally high peaks as the target DNA in the resulting chromatograms. For primer pair B (PCR ‘B’) co-amplification of group ‘A’ and ‘C’ DNA was not detectable even when present at a 1000-fold higher concentration than group ‘B’ target DNA (Supplementary Table S2). No cross-reactivity with human DNA was observed with any of the primer pairs. In the solutions with 100 ng human DNA and no added bacterial DNA, only background bacterial DNA from the reagents was amplified.Ct distance between a sample and the corresponding negative control ranged from −20 to −8 cycles. For the specimens positive by PCR ‘A’ the Ct distance to the negative control ranged from −17 to −4 cycles. For the specimens negative by PCR ‘A’ the Ct distance ranged from −2.4 to +3.9. The corresponding intervals for PCR ‘B’ were −16 to −7 and −2.8 to +0.9 and for PCR ‘C’ −17 to −6 and −2.0 to +4.4. (− indicates before the negative control and+indicates after.)The polybacterial specimens contained high levels of bacterial DNA, and with the universal PCR the Staphylococcus haemolyticus isolated from sample 7, scarce growth of a coagulase-negative Staphylococcus together with a diphtheroid in sample 23 and two colonies of Streptococcus parasanguinis in sample 24. In addition an isolate of Klebsiella sp. from sample 6 was not possible to read from chromatogram 6B. Some of these isolates might represent sample contamination, but others are probably true findings that have not been detected by sequencing either because they had to compete for reagents with a more dominant species belonging to the same primer group or because direct sequencing can have lower sensitivity than culture in samples with living bacteria.Eighty per cent of the samples were affected by antibiotics administrated prior to specimen collection, and both standard direct 16S rDNA sequencing and direct sequencing using group-specific primers detected a higher number of bacteria than did culture. In total 37 species were found by culture, 51 by standard direct sequencing and 95 by the group-specific primers. A detailed comparison between these results is given in Enterococcus faecium and Finegoldia magna were amplified by both PCR ‘A’ and ‘C’, but PCR ‘A’ became positive 8.6 cycles prior to PCR ‘C’. Three of the group-specific chromatograms were found to be too complex to be completely resolved, indicating that these samples still might contain one or more unidentifiable species.The detailed results from the group-specific PCRs for the polybacterial specimens are given in Ct at cycle 31.5, exactly three cycles before the negative control. Melt-point analysis showed no distinct peak and it was negative by the group-specific PCRs. Sequencing was unsuccessful and the PCR result was defined as a false positive. Another sample (ID 50) was positive by PCR ‘B’. This reached Ct at cycle 30.6, 3.1 cycles before the closest negative control. Melt-point analysis gave an irregular peak. Sequencing produced a complex chromatogram dominated by a Pseudomonas sp. and an Acidovorax sp. This corresponds to what we typically find in our negative controls  and the PCR was judged to be a false positive. The remaining 98 PCRs were negative as expected.Among the 25 clinical specimens assumed to be negative  one sampDetection and identification of bacteria directly from polymicrobial clinical samples by broad-range PCR followed by DNA sequencing can be accomplished using RipSeq mixed chromatogram analysis. In this study we replaced the standard universal 16S rRNA gene PCR with a set of three separate group-specific PCRs, reducing the risk of species present at lower concentrations becoming outcompeted in the amplification step. The importance of this factor is illustrated by the fact that only eight of the chromatograms obtained with the universal primers were judged to contain more than three bacterial sequences .P<0.05 by Wilcoxon signed-rank test; The group-specific PCRs significantly increased the number of identified bacteria as compared to amplification with a universal PCR . Consequently, in reactions where the negative control did not contain any target DNA a poor amplification would lead to a very high Prevotella histicola and P. melaninogenica in the forward direction allowed for an ‘and’ instead of an ‘and/or’ identification with the universal protocol. A rational approach will be to use a single universal primer pair for specimens expected to be monobacterial  and reserve the group-specific primers for specimens known to frequently contain multiple organisms .By using group-specific primers the cost per analysis will see some increase. We kept the increase in both price and workload as low as possible by sequencing in one direction only, and the average number of sequencing reactions in our material was 2.55 per positive sample. There are several publications based on mono-directional sequencing, and it is also used in a commercially available 16S rDNA sequencing kit . Failure to detect these organisms can lead to inadequate antimicrobial coverage, in particular in situations where first-line empirical treatment cannot be used or when patients are to be transferred to oral treatment. For the brain abscesses our findings correlate well with what was described in a study by It is clear from this and other studies that the results achieved by routine culture-based diagnostics alone are not sufficient when it comes to patients who have received one or more doses of antibiotics prior to sample collection, or the investigation of anaerobe infections  have relatively unproblematic susceptibility patterns.The purposes of this study were to demonstrate the potential of the group-specific PCRs and to establish a robust protocol. The approach can contribute to better characterization of polybacterial clinical samples and has the advantage that it can be readily implemented in any diagnostic laboratory with experience in standard direct sequencing.