Auxetics comprise a rare family of materials that manifest negative Poisson’s ratio, which causes an expansion instead of contraction under tension. Most known homogeneously auxetic materials are porous foams or artificial macrostructures and there are few examples of inorganic materials that exhibit this behavior as polycrystalline solids. It is now possible to accelerate the discovery of materials with target properties, such as auxetics, using high-throughput computations, open databases, and efficient search algorithms. Candidates exhibiting features correlating with auxetic behavior were chosen from the set of more than 67 000 materials in the Materials Project database. Poisson’s ratios were derived from the calculated elastic tensor of each material in this reduced set of compounds. We report that this strategy results in the prediction of three previously unidentified homogeneously auxetic materials as well as a number of compounds with a near-zero homogeneous Poisson’s ratio, which are here denoted “anepirretic materials”. There are very few inorganic materials with auxetic homogenous Poisson’s ratio in polycrystalline form. Here authors develop an approach to screening materials databases for target properties such as negative Poisson’s ratio by using stability and structural motifs to predict new instances of homogenous auxetic behavior as well as a number of materials with near-zero Poisson’s ratio. These materials have been shown to possess enhanced hardness and toughness, as well as absorb vibrations and sound better than their non-auxetic counterparts3. As a result, the atypical elastic behavior of auxetic materials is enabling advancements in a broad range of technologies such as impact-resistant composites, extremely precise sensors, tougher ceramics, and high-performance armor5.A material’s Poisson’s ratio reflects the deformation of its cross-section in response to an orthogonal tensile strain. The term “auxetic” defines a class of exotic materials that exhibit a negative Poisson’s ratio, which causes a counter-intuitive expansion under tension rather than contractionνij (where i and j are the directions of the applied tensile strain and the resultant transverse strain). For polycrystalline solids, which are macroscopically isotropic, these directional values average to a “homogeneous” value, μ2. A number of crystalline materials have been found to exhibit negative Poisson’s ratios in certain directions associated with specific features of their crystal structures9. However, nearly all of the currently known homogeneously auxetic materials are porous foams or purposefully designed hinged meta-materials with open, re-entrant structures11. The most well-known example of a crystalline material with a negative homogeneous Poisson’s ratio, α-cristobalite (SiO2), was discovered via laser Brillouin spectroscopy in 1992 by Yeganeh-Haeri et al.13. They hypothesized that the material’s unusual elastic behavior originates from the rotation of rigid SiO4 tetrahedra. Indeed, the prevailing theory for auxetic behavior in α-cristobalite and other inorganic materials relies on rigid unit modes (RUM’s) in which connected polyhedra rotate without deforming  calculations of their full elastic tensors. Of the 38 candidates, 7 new compounds are identified to exhibit negative homogeneous Poisson’s ratios in addition to the known auxetic α-cristobalite. The most notable of these predictions is a high temperature aluminum orthophosphate polymorph, HT-AlPO4. The other new materials predicted to exhibit auxetic behavior are four hypothetical SiO2 polymorphs and two hypothetical metal vanadate structures. Additionally, we report near-zero homogeneous Poisson’s ratios for a number of materials in various chemical systems, which we denote “anepirretic materials” after the Greek word for “unaffected.”In this work, we utilize a screening process for auxetic materials that employs open-source tools and algorithms to efficiently and systematically find promising candidates. Several open materials databases with structural information are available today31 contains data for over 67,000 distinct inorganic crystalline materials where of over 4000 have an associated calculated elastic tensor. Benchmarks of the Materials Project elastic tensor workflow suggest that it reproduces elastic-tensor derived bulk and shear moduli well; within 15% of experimentally measured values. However, the workflow requires computationally demanding input parameters to ensure this level of accuracy25. It is currently projected to take years to calculate the elastic tensor for all known crystalline compounds using available supercomputing resources. Hence, approaches which prioritize certain compounds based on structure-property descriptors are useful to accelerate the rate of discovery. For example, predictive statistical learning methods have been used to estimate bulk and shear moduli of materials in the MP database, illustrating that existing DFT-based data on elasticity and structure can be leveraged to guide the search for specific elastic behavior33. In this work, to expedite the discovery process with respect to Poisson’s ratio, materials were screened though a set of criteria designed to reduce the number of elasticity calculations required to identify novel auxetics. In Tier I  of materials display meiotic behavior, the current search identified a statistically significant number of calculated negative or near-zero homogeneous Poisson’s ratios. Of the 30 materials that passed Tier I and II, 1 known auxetic, 3 new auxetic, 9 new anepirretic, and 17 meiotic Poisson’s ratios were predicted. This has greatly increased the total number of predicted auxetic and anepirretic materials in the MP database, as shown in the inset of Fig. Instead of classifying the materials solely by the sign of the Poisson’s ratio, we introduce a new criterion based on both sign and magnitude of 2, a meiotic compound exhibiting a structure identical to α-cristobalite.The results of the elastic analysis for each of the auxetic and anepirretic materials identified by the screening process are included in Table 2(a) and (b), were identified as auxetic by the screening process. We note that SiO2(a) is the enantiomer of α-cristobalite SiO2, hence it is not surprising that its elastic properties are close to those of its chiral partner. Coh and Vanderbilt investigated the structural stability of cristobalite and described how the α and β structures may be considered higher-symmetry special cases of a three-dimensional manifold with general P212121 symmetry accessed by RUMs19. Both SiO2(a) and (b) appear on this manifold along with α and β cristobalite. Intuitively, the homogeneous Poisson’s ratios of these structures fall between those of α and β-cristobalite SiO2.Two novel silica polymorphs, denoted SiO2(c) and (d), as well as β-cristobalite, denoted SiO2(βC), were calculated as anepirretic. Furthermore, two ternary metal vanadates; FeV3O8 and CoV3O8 were identified as anepirretic with predicted Poisson’s ratios of −0.05 and −0.04, respectively. The four silica polymorphs SiO2 (a)–(d) and the two ternary vandates are part of the growing number of materials in the Materials Project which are ‘hypothetical’ new compounds35, e.g., they have been generated using various transformation schemes, such as substitutions on existing materials, to explore novel chemistries and structures for different applications. Hence, there is no existing literature on synthesis and testing of these materials; however, they exhibit favorable formation energies and may be amenable for synthesis34.Two other silica polymorphs, denoted SiO4. This known polymorph exhibits a structure very similar to α-cristobalite but has a lower crystal symmetry (C2221) due to its stoichiometry. Our calculations predict it has a Poisson’s ratio of −0.28, rivaling that of α-cristobalite SiO2. Berlinite AlPO4 spontaneously transforms to HT-AlPO4 at elevated temperatures, which remains metastable at room temperature36. Although NAT-type aluminophosphate zeolites have been investigated for negative Poisson’s ratio38, to our knowledge HT-AlPO4 has not been experimentally investigated for auxetic behavior39.The potentially most interesting of the novel identified auxetic materials is a high-temperature phase of aluminum orthophosphate: HT-AlPOνij, for the materials identified in Tier 3, we calculate ν32 over the entire range of possible orientations and compare to the known α-cristobalite SiO2. The elastic tensors were rotated around the Eulerian z, xʹ, and zʹʹ directions between 0 and 2π radians at a resolution of π/30 and for each orientation, ν32 was recorded as a function of the rotation angles. Figure ν32 for α-cristobalite and HT-AlPO4 over the subspace of orientations spanned by rotations about the xʹ and zʹʹ axes. In order to more directly compare the Poisson response of the two materials, the elastic tensor of α-cristobalite was first rotated by π/4 around z to align its orientation with that of HT-AlPO4’s elastic tensor with respect to its conventional standard unit cell.In order to study the extremal values of the anisotropic directional Poisson’s ratio, νmax is predicted as negative for both α-cristobalite and HT-AlPO4, implying that, similar to α-cristobalite SiO2, HT-AlPO4 will behave auxetically when strained in any direction. We note that these results are consistent with other DFT calculations of the elastic properties of α-cristobalite, which slightly overestimate the auxetic response such that νmax < 0, whereas Brillioun spectroscopy indicates νmax = +0.0840. Nevertheless, α-cristobalite SiO2 still exhibits overall auxetic behavior in experiment12 and the same is expected for HT-AlPO4. We find that νmin appears on the xʹ–zʹʹ rotation surfaces of α-cristobalite and HT-AlPO4 at coinciding rotations, hence the character of their Poisson behavior is highly similar. Figures α-cristobalite41. Moreover, these soft rotational modes are also activated in the α–β transformation of cristobalite SiO218. SiO2 (a) also exhibits a negative νmax, and hence global auxetic behavior, and SiO2 (b) manifests a νmax very close to 0.0, indicating auxetic behavior in a majority of directions.Indeed, νij22. While completely auxetic materials will always be homogeneously auxetic, partially auxetic materials can average to auxetic, anepirretic, or meiotic depending on the distribution of νij. Anepirretic materials typically exhibit directional Poisson’s ratios that are distributed across both positive and negative values resulting in a homogeneous Poisson’s ratio close to zero. As indicated in Table 2 (c) polymorph as well as the ternary vandates display weakly negative Poisson’s ratios while positive νmax. Uniformly small directional Poisson’s ratios as well as combinations of positive and negative directions can minimize the magnitude of the homogeneous Poisson’s ratio in anepirretic materials, which may be a useful materials design feature for applications where transverse strain is undesirable.Auxetic materials have conventionally been classified as “partially auxetic” if they possessed one or more negative directional Poisson’s ratios or “completely auxetic” if they exhibited no positive values for Accelerated discovery of novel materials with target properties is desirable to meet current and future material needs. In this study, we show how a tiered search, targeting specific structural motifs, can greatly accelerate the discovery of materials with unusual elastic behavior.α-cristobalite SiO2 are known to exhibit homogeneous auxetic behavior. Prior to this study, through the Materials Project calculations of elastic tensors, a tungsten oxide (WO3) with weakly negative Poisson’s ratio, μ = −0.08, was serendipitously found among the first 2000 materials calculated25. This result represents an ~0.02% chance to discover a material with a negative homogeneous Poisson’s ratio. Using the target screening described here, we find that almost one in five of the materials identified by the homologous structure search exhibit negative homogeneous Poisson’s ratios and 3/30 are proposed new homogeneous auxetics. Hence, the approach delivers an improvement of three orders of magnitude.To date, very few inorganic crystalline materials other than 4, β-cristobalite SiO2, and GaPO4. Both phosphates are metastable at room temperature and can be flux-grown or synthesized through annealing of quartz-structured AlPO4 and GaPO4 above the cristobalite transition temperature with subsequent quenching42. However, β-cristobalite SiO2 spontaneously transforms to α-cristobalite upon cooling and must be chemically stabilized to exist at room temperature43. The remaining seven materials identified by the search as auxetic or anepirretic are hypothetical compounds with low formation energies which indicate a fair chance of successful synthesis34. The polymorphs SiO2 (a), (c), and (d) are frameworks generated by simulated annealing35 while SiO2 (b) represents an early suggestion for the α-cristobalite structure proposed in 1932. The four vanadates  originate from a variety of structure manipulations of known compounds, such as orderings of disordered structures, and cation substitution in such frameworks44. We stress that the fact that these materials are hypothetical does not necessarily preclude the possibility that they can be made, and identifying them as auxetic may inspire design and discovery of similar compounds. Additionally, we note that the restriction on energy above the convex hull in Tier I of the screening process does not reflect an absolute limit for stability. As such, this screening method may exclude materials that can be synthesized and exploring higher energies may lead to the discovery of additional homogeneously auxetic materials of interest.It is important to note that the Materials Project database does not discern between materials that have been synthesized and those that remain hypothetical. Previously synthesized or known materials, identified by the tiered search as either auxetic or anepirretic, are HT-AlPOα-cristobalite GeO2 which exhibits the exact same crystal structure, symmetry, and stoichiometry as α-cristobalite SiO2. Indeed, GeO2 has previously been proposed as an auxetic material9. However, while auxetic directions are found in this material , S, as:νij represents the ratio of deformation in the principal direction j to a uniaxial strain applied along the orthogonal principal direction i in the basis frame of the elastic tensor26. The global minimum and maximum directional Poisson’s ratios were found by rotating the original elastic tensor through the range of possible orientations via the Euler–Rodriguez and Cayley method and Euler angle rotations around the z, xʹ, and zʹʹ axes at a resolution of π/30 radians49.The homogeneous approximation of the Poisson’s ratio was calculated from the elements of the calculated elastic tensors as:50, in which the equilibrium bulk structures are deformed in a single direction and optimized isotropically in the perpendicular directions. This allowed for an estimate of the structural response to a uniaxial load, and further reinforces the prediction of auxetic behavior in elastic tensor results.The tetrahedral rotations displayed in Figs. 32. Structure-matching proceeds by: 1. reducing to unit cells and comparing the total number of sites, 2. further reducing to Niggli cells and scaling by volume, 3. removing oxidation states, and 4. comparing, within specified tolerances, the lattice angles and atomic positions of all permutations of valid lattices. A full description of the algorithm is provided. (The structure-comparison algorithm described in this study was implemented using the structure matcher in Pymatgenrovided.  The struhttp://pymatgen.org and https://materialsproject.org. Any other information supporting the conclusions presented is available from the authors upon request.The code and data used to develop the screening process in this study are available at Supplementary InformationPeer Review File


Pa consisting of an endoribonuclease toxin and RNA antitoxin. ToxINPa is a heterohexameric quaternary complex in which pseudoknotted RNA inhibits the toxicity of the toxin until infection by certain phages causes destabilization of ToxINPa, leading to bacteriostasis and, eventually, lethality. However, it is still unknown why only certain phages are able to activate ToxINPa. To try to address this issue we first introduced ToxINPa into the Gram-negative enterobacterium, Serratia sp. ATCC 39006 (S 39006) and then isolated new environmental S 39006 phages that were scored for activation of ToxINPa and abortive infection capacity. We isolated three T4-like phages from a sewage treatment outflow point into the River Cam, each phage being isolated at least a year apart. These phages were susceptible to ToxINPa-mediated abortive infection but produced spontaneous “escape” mutants that were insensitive to ToxINPa. Analysis of these resistant mutants revealed three different routes of escaping ToxINPa, namely by mutating asiA ; by mutating a conserved, yet functionally unknown, orf84; or by deleting a 6.5–10 kb region of the phage genome. Analysis of these evolved escape mutants may help uncover the nature of the corresponding phage product(s) involved in activation of ToxINPa.Abortive infection is an anti-phage mechanism employed by a bacterium to initiate its own death upon phage infection. This reduces, or eliminates, production of viral progeny and protects clonal siblings in the bacterial population by an act akin to an “altruistic suicide.” Abortive infection can be mediated by a Type III toxin-antitoxin system called ToxIN Bacteria are susceptible to viral (bacteriophage) predation but have evolved several strategies to resist viral infection. One strategy is abortive infection (Abi) in which an infected bacterial cell dies precociously and thereby concomitantly blocks the production of mature phage progeny  was encoded by a cryptic plasmid (pECA1039) in Pectobacterium atrosepticum. ToxINPa was originally identified because ToxN shared 31% amino acid sequence identity with the AbiQ protein, which was involved in an abortive infection system in Lactococcus lactis , the escape locus might provide insight into how phage infection leads to activation of the ToxINPa complex.To date, ΦTE remains the only phage whose ToxINSerratia sp. ATCC 39006 (S 39006)-specific phages, ΦCHI14, ΦX20, and ΦCBH8. Comparison of the genomic sequences of spontaneous ToxINPa-escape mutants and their wild type progenitors revealed three different routes of escape: (1) mutation of asiA, encoding a predicted phage transcriptional co-activator in ΦCHI14 and ΦCBH8; (2) mutation of an unknown gene (orf84) in a ΦCHI14 mutant; (3) deletion of a large region (6.5–10 kb) of the phage genome in most mutants of all three T4-family phages.In this study, we isolated and characterized S 39006. Bacteria were cultured at 30°C in Luria-Broth (LB)  or on LB agar (LBA) containing 1.5% w v−1 or 0.35% w v−1 agar to make LBA or top-LBA plates respectively. Bacterial growth was monitored by measuring optical density at 600 nm (OD600) using a Thermo Scientific Helios Zeta spectrophotometer. Where required, media were supplemented with ampicillin at 100 μg mL−1. Bacteriophages ΦCHI14, ΦX20, and ΦCBH8 were isolated from treated effluent collected from a sewage treatment plant in Cambridge, United Kingdom. The bacteriophages were selected from a library of S 39006-specific phages isolated using an enrichment procedure detailed previously  was calculated after incubating serial dilutions of phage lysates overnight on bacterial lawns on LBA and dividing the titer of the phage on the test host by the titer of the phage on the control host  mL−1 was obtained.Plaques of rare spontaneous phage mutants were isolated on lawns of 8 p.f.u. mL−1) onto a charged copper grid for 3 min. The grids were then washed with water twice before being stained with 2% phosphotungstic acid (PTA) neutralized with potassium hydroxide (KOH). The accelerating voltage was 120.0 kV and the direct magnification used to image phages was 25,000x.Transmission Electron Micrograph (TEM) images of phages were taken at the Multi-Imaging Center, University of Cambridge using a Tecnai G2 series transmission electron microscope. Samples were prepared by adsorbing 10 μl of phage lysate  or SPAdes. The wild type sequences had coverage ranging from 57x to 100x of the full genome while the escape phage sequences had coverage ranging from 45x to 150x.The genomes of wild type phages and selected escape mutants were sequenced using the Junior Roche 454 Genome Sequencer FLX pyrosequencer at the Department of Biochemistry, University of Cambridge or using the Illumina MiSeq, and HiSeq 2500 platforms at MicrobesNG. The resulting contigs were assembled using Genomic Sequencer Phage genome open reading frames (ORFs) were defined using the gene prediction tools GeneMark.hmm  and oBH4 (5′- CGCTATATGTCAACAGGCCG-3′). Subsequent amplicons were subjected to Sanger sequencing.The S 39006 culture was used to inoculate LB in a 250 mL conical flask and incubated at 30°C to OD600 = 0.5. Phage samples were then added at a multiplicity of infection (M.O.I.) of 0.001 and the culture incubated with shaking at 150 rpm at 30°C. Samples were taken at different time points and chloroform-treated before titrating to determine the number of p.f.u. The one-step growth curve describes phages per initial infection center, over time.Phage burst size assays were performed as described previously  . None of the phages showed sensitivity to other Type III TA systems tested.Since 2013, three phages that could be aborted by ToxINMF036690), ΦCBH8 (MF036691), and ΦX20 (MF036692).TEM images revealed that all three phages had isometric, icosahedral heads, contractile tails and tail fibers Figure . This clΦCHI14 was used as a representative of the new isolates to compare with related phages Figure : ΦCHI14 Pa aborted T4-like S 39006 phages efficiently, rare phage plaques appeared at low frequencies. We presumed that these rare plaques arose due to viral mutations enabling phages to “escape” or circumvent the effects of ToxINPa. Therefore, we expected that definition of the corresponding mutations would enable identification of the phage genes encoding products responsible for “activation” of the ToxINPa system.Although ToxINS 39006 phages were subject to whole genome sequencing; 5 of ΦCHI14 mutants, 11 of ΦX20 mutants and 8 of ΦCBH8 mutants. Mutations were mapped to the corresponding wild type genomes to identify the putative ToxINPa resistance loci. Interestingly, the mutations did not all map to single locations in the corresponding genomes. Instead, three distinct mutational types were identified in the genomes of the 24 escape phages  had mutations in the ne Table . After dorf84. The A to C substitution caused an E66D change in the encoded ORF84 protein. Homologs of ORF84 have been found in 11 other phages, the most similar homolog belonging to enterobacterial phage, CC31. However, no functional information exists currently for any of the homologs from both sequenced-based predictions and structure-based predictions.One mutant (ΦCHI14e) had a single nucleotide substitution in the gene, S 39006 phages became insensitive to ToxINPa by a similar type of mutation—through a large deletion of a specific viral genome locus  to 10,094 bp (ΦCHI14c). The largest deleted region contains 19 ORFs and 13 tRNA genes, whereas the smallest contains 13 ORFs and 10 tRNA genes  of the sequenced “escape” mutants of s Figure .Pa infers that the 6.5–10 kb deleted region contains genetic elements directly or indirectly responsible for activating ToxINPa. In our limited pool of spontaneous “large deletion” mutants, 6.5 kb was the most compact deletion and therefore detailed inspection of the ORFs and tRNAs in this region was undertaken. Every individual predicted ORF was investigated by searching for DNA homologies using nucleotide BLAST; amino acid sequence homology using protein BLAST; and finally i-TASSER for predicted structural homologs.The ability of “large deletion” mutants to escape ToxINOf the 13 predicted ORFs within the smallest deletion region found in ΦCBH8o, 6 encode hypothetical proteins with no sequence, or structural, homologs, while 7 of the ORFs encode homologs in other T4-like phages. However, no functional information is available about any of these homologs—except for one hypothetical protein encoded by the 3′ end of the deletion region. This hypothetical protein, (ORF145), shows some similarity to the membrane anchor domain of an agglutinating adhesin (YadA) in both protein BLAST and i-TASSER structural predictions.S 39006 phages reported in this study. Therefore, the 6.5 kb smallest deletion region from ΦCBH8o was aligned with the genomes of the related phages that show high homology with the entire genomes of these environmental T4-like S 39006 phages. ACT alignment showed only very limited identity of the 6.5 kb region with phages T4, CC31, ΦR1-RT, ΦS16, PG7, and PEi20  and Leu (TAA) in the host genome suggests the possibility that their deletion from the phage genome might lead to inefficient translation of some viral transcripts encoding products that activate ToxINPa.The presence of multiple tRNA genes within the deleted regions may also suggest that these tRNAs play a role in susceptibility to ToxINS 39006 with an M.O.I. of 0.001 was measured by one-step growth (n = 5). Both wild type ΦCBH8 and ΦCBH8o showed a latent period of 25 min and a rise period of about 35 min. The average burst size was approximately 22 phage particles per initial infection center for wild type ΦCBH8 and 28 for ΦCBH8o  using stationary phase S 39006 instead of exponential phase host but retaining the same M.O.I. As shown in Figure Given the extent of the viral genome deletions and absence of any functional information on most of the deleted gene products, we decided to investigate the impact of the deletions on phage fitness, represented by adsorption efficiency and burst size. The burst size of wild type ΦCBH8 and ΦCBH8o infecting exponential phase o Figure . Howevero Figure , suggestS 39006 that are sensitive to ToxINPa-mediated Abi. Rare spontaneous mutants of these phages were able to circumvent ToxINPa via multiple escape routes. The independent isolation of ΦCHI14, ΦX20, and ΦCBH8 is interesting since pairwise alignment of their genomes with other T4-like phages showed that they are mosaics of one another—whole-genome alignments consist of varying-length stretches of high homology interspersed with stretches of no homology  suggests a relatively rare incidence of ToxINPa-sensitive viruses among environmental phages of S 39006. However, this rarity may not be surprising because wild type S 39006 does not carry ToxINPa naturally. Furthermore, it is possible that these new phages arose in the environment by propagation on alternative native hosts taxonomically unrelated to S 39006.In this study we isolated three highly homologous T4-family environmental phages of Pa system in the natural host, P. atrosepticum  to recognize and transcribe host genes but reconfigures the enzyme to transcribe phage middle-expressed genes  attempts to align the large deletion locus with several other T4-like phages  showed very little homology (Figure The most frequently found escape locus among the y Figure  and therYersinia species and serves as a virulence factor that mediates Yersinia adherence to epithelial tissue (Hoiczyk et al., The only clue to possible functions of ORFs in the deleted regions comes from ORF145, which is a YadA homolog. YadA was first found in S 39006 phages that could activate the ToxINPa-mediated abortive infection system. The data on the “escape” mutants of these environmental phages suggest that either ToxINPa can be activated by more than one phage product, or that production of the ToxINPa- activating product involves multiple biological processes and that defects in several could enable phages to circumvent Abi. Future investigations will involve further characterization of the products from the escape loci of these phages, and experiments to try to dissect their physiological roles during the process of abortive infection—and its circumvention.In summary, this study has discovered a distinct group of T4-family GS, BC, CA, and XF conceived and designed the experiments. BC, CA, and XF performed the experiments. BC and CA prepared figures and graphs. BC and GS wrote the manuscript. All the authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A central effort of today’s neuroscience is to study the brain’s ’wiring diagram’. The nervous system is believed to be a network of neurons interacting with each other through synaptic connection between axons and dendrites, therefore the neuronal connectivity map not only depicts the underlying anatomy, but also has important behavioral implications. Different approaches have been utilized to decipher neuronal circuits, including electron microscopy (EM) and light microscopy (LM). However, these approaches typically demand extensive sectioning and reconstruction for a brain sample. Recently, tissue clearing methods have enabled the investigation of a fully assembled biological system with greatly improved light penetration. Yet, most of these implementations, still require either genetic or exogenous contrast labeling for light microscopy. Here we demonstrate a high-speed approach, termed as Clearing Assisted Scattering Tomography (CAST), where intact brains can be imaged at optical resolution without labeling by leveraging tissue clearing and the scattering contrast of optical frequency domain imaging (OFDI). Clusters of neurons with diverse structural and functional traits comprise an astonishingly intricate network with interconnections spanning across the entire brain at both local and global scales. This network reflects not only the topological links among neurons but also their aggregate behavior leading to cognitive functionsmm to 100 μm are prevailingin vivo, the current resolution of one-cubic-millimeter makes wiring diagrams obtained through dMRI too coarse to disclose the complete neuronal network explicitly. At the opposite extreme, scanning electron microscopy (SEM) has a resolution of around 30 nm when applied to ultra thin, serially-sectioned specimens and is able to visualize the finest structure of neurons, such as synaptic connectionμm 8986In spite of its significance, mapping the comprehensive wiring diagram across the whole brain has been a daunting challenge for many years and our understanding on neuronal connectivity still remains staggeringly fragmented45Another approach for deep penetration optical imaging without sectioning, tissue clearing, originated decades ago14151618192015162021222414152226To address this, here we introduce a high-speed label-free methodology, termed as Clearing Assisted Scattering Tomography (CAST), for imaging intact brains. Rather than sensing the incoherent photons emitted by fluorophores or absorbed by chromophores, optical frequency domain imaging (OFDI) and optical coherence tomography (OCT) coherently detect photons scattered back from tissue3132μm3 (X × Y × Z) in air to demonstrate that CAST can work efficiently at the resolution gap between conventional LM (1 μm) and MRI (100 μm). This configuration also yielded the best balance between sufficient structural visualization and scanning time. As will be shown later, a mouse brain hemisphere can be scanned in around 22 minutes. While clearing indeed extends light penetration in tissue, it inevitably decreases OFDI signal due to scattering reduction. By adjusting the degree of lipid removal, we found that it was possible for CAST to image through very thick tissue while maintaining sufficient contrast. To introduce OFDI imaging on cleared samples, we first constructed an OFDI imaging system (Method). This platform was designed to have a nearly isotropic optical resolution volume of 10 × 10 × 9 mm thick coronal section from a mouse brain was used to examine the biological origins of OFDI contrast. To discover how the clearing process affects the scattering signal, we first compared the OFDI images of the same coronal section before and after a controlled clearing process. As shown in Next, we further investigated which biological structures within the cleared samples gave rise to OFDI signal. A 1 mm thick coronal section and a right hemisphere of mouse brain were cleared and then scanned. μm were projected to form the two-dimensional enface images. Various regions with high content of white matter tracts were clearly visualized, such as corpus callosum (CC)  , caudopusum (CC) , thalamusum (CC) , and glosum (CC) . One cansum (CC)  was discsum (CC)  were alsmm × 7.5 mm × 6.2 mm. Due to the limited depth of focus, an axial focus stepping , similar to the A three-dimensional rendering of the hemisphere is shown in The resolution of our current system is not sufficient for identifying individual cells as the system was intentionally designed to balance speed and resolution. OFDI or optical coherence microscope (OCM) platforms with cellular resolution have already been demonstrated and are evolving into mature research toolsBy detecting light coherently, CAST is fundamentally distinct from other existing methods based on incoherent detection. This allows the measurement of not only the intensity but also subtle characteristics of light such as phase and polarization, opening the possibility of a wide spectrum of new contrast mechanisms in cleared tissue. At the same time, the speed of all fluorescence methods will be ultimately limited by the fluorescence throughput while the speed of CAST is theoretically limited by shot noise during detection because there is no similar ‘photo-bleaching’ effect on light scattering.While individual neurons are the basic building blocks of the nervous system, brain function relies more on the interaction among the massive number of neurons. These connections exist across a broad range of scales. The scattering based imaging methodology described in this work offers a high-speed, label-free imaging alternative with a mesoscale resolution that potentially links connectomes across broad spatial scales.OFDI is particularly advantageous for volumetric imaging in that it offers a unique depth scan (A-scan) along the probing beam. Together with transverse sampling, three-dimensional image stacks can be obtained.Covega Corp., BOA-4379) as the gain medium and a polygon scanner  as the tunable filter to rapidly tune the wavelength at a rate of 54 kHz. This source had a center wavelength of 1300 nm and a sweeping range of 110 nm, resulting an axial resolution of 9 μm in air. It eventually delivered 10 mW optical power at sample. By employing an acousto-optic frequency shifter , the imaging depth of the system was extended to 6 mm.A customized OFDI imaging system was developed for this work. The system was based on an optical interferometer, where light from a wavelength swept laser was first branched into a sample arm and a reference arm, and then the returned light from tissue sample and a reference mirror was combined, interfered, and detected. The homemade wavelength swept laser utilized a semiconductor optical amplifier  were used to measure the interference fringes from two orthogonal polarization channels. The interference signals were captured by a two-channel 14 bit PCIe digitizer board  at 85 MHz, resulting in 1024 pixels per axial scan and an axial sampling pitch of 5.86 μm. A customized application based on C++ was developed to continuously stream the sampled data to a RAID hard-drive array. The application also processed and rendered the OFDI images at approximately 40% of the acquisition rate, allowing for real-time monitoring on image acquisition sessions.The resulting interference fringes, as a function of wavelength, were detected by a polarization diversity demodulator to avoid polarization artifacts. A pair of balanced detectors  at the sample arm was first expanded by a fiber collimator  to a diameter about 4 mm and then focused by a 18 mm scan lens , resulting a working distance of 7.5 mm and a lateral resolution of 10 μm. Transverse sampling was achieved by two orthogonal scan axes . The fast axis, X, was implemented by scanning a galvo mirror  at the back focal point of the lens. In this work, a 7.5 mm transverse field along X was scanned at a sampling pitch of 3.67 μm. This resulted in 26.4 frames per second with each frame comprised 2048 axial scan. A motorized translation stage  was used to translate samples along the slow axis, Y. A translation speed of 96 μm/sec was chosen to maintain an isotropic sampling. The scanned field along this direction was determined interactively based on tissue dimension. The axial scan and the transverse scans  were all synchronized with the acquisition by the custom application.The probing beam from a single mode fiber , and 1% glutaraldehyde (GA)). Upon the completion of the perfusion, brain tissues were removed from the skulls and incubated into 20 mL of perfusion solution with gentle shaking for 3 days at 4 °C. Then samples were washed twice in PBST with 6 hrs each at room temperature with gentle shaking. After the initial wash, samples were washed overnight in inactivation solution  acetamide, and 4% (w/v) glycine) at 37 °C. After another two washes with 6 hrs each in the clearing solution (200 mM sodium dodecyl sulfate (SDS) and 20 mM sodium sulfite), inactivated samples were incubated in the clearing solution with 70 °C water bath for lipid removal. Various clearing time and temperature were applied to achieve different levels of optical transparency on samples with different sizes.Mouse brain tissues from young adult wild type (C57BL/6) mice aged 6–8 weeks, were first preserved via a cardiac perfusion. The procedure started with perfusing 20 ThermoFisher, D7757) and two stains, DyLight 488-conjugated tomato lectin  for vessels, SMI-312 antibody  and Alexa Fluor 594-conjugated donkey anti-mouse IgG antibody  for neurofilaments. Samples were first incubated in label-OFF solution  to reach equilibrium before transferred to a fresh buffer of label-OFF solution with antibodies or other bio-markers added. The samples were incubated in the buffer at 37 °C with gentle shaking for 12 hrs to 7 days depending on their sizes. Finally, samples were incubated in PBST at 37 °C for 2 days.Cleared samples were labeled with DiD (w/v) n-methyl-d-glucamine, 29.4% (w/v) diatrizoic acid, and 32.4% (w/v) iodixanol) over-night at room temperature.Before imaging, samples were incubated in a refractive index matching solution  was used in this study. The system was configured with a 10× objective  and three channels at 488, 559, and 635 nm were employed. Transverse sampling pitch was 1.59 μm and axial pitch was 20 μm. The overall acquisition time for each 1 mm thick sample was about 2 hrs.A confocal fluorescence microscope  with a galvo-scanner  and then imaged with a second objective . Fluorescence signals were recorded by a sCMOS camera . Its laser unit covered a broad spectrum , matching various emission filters. The dual illumination arms on both sides of the sample illuminated the sample simultaneously. Rolling shutter mode of the camera was utilized for dynamic confocal detection. Images were acquired by synchronizing the focal plane of the detection objective and the position of the illumination light sheet. Multiple image stacks were stitched with Terastitcher  and visualized with Imaris (Bitplane). Transverse sampling pitch was 2.32 μm and axial pitch was 5 μm. The total acquisition time was about 1 hrs.A DiD-stained mouse brain hemisphere was imaged by a custom-built light sheet microscope. The sample was illuminated with a sheet of light generated by scanning a focused beam from an objective .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Thus, acoustophoresis can be easily integrated with further down-stream applications such as flow cytometry, making it a superior alternative to existing MNC isolation techniques.Effective separation methods for fractionating blood components are needed for numerous diagnostic and research applications. This paper presents the use of acoustophoresis, an ultrasound based microfluidic separation technology, for label-free, gentle and continuous separation of mononuclear cells (MNCs) from diluted whole blood. Red blood cells (RBCs) and MNCs behave similar in an acoustic standing wave field, compromising acoustic separation of MNC from RBC in standard buffer systems. However, by optimizing the buffer conditions and thereby changing the acoustophoretic mobility of the cells, we were able to enrich MNCs relative to RBCs by a factor of 2,800 with MNC recoveries up to 88%. The acoustophoretic microchip can perform cell separation at a processing rate of more than 1 × 10 Working with MNCs often requires enrichment or isolation of the cells which can be challenging due to their low abundance of less than 0.06% of all blood cells2. The deterministic behavior of fluids governed by the laminar flow conditions in microscale devices has driven the development of a wide range of modalities for separating cells, in which separation performances take advantages of physical scaling laws, but many times at the expense of system throughput9.Separation of MNCs is commonly performed in batch processes either by density gradient centrifugation or red blood cell (RBC) lysis and centrifugation, both involving several manual handling steps. Alternatives to replace these labor-intensive methods are intensely researched within the microfluidics field. Miniaturized and automated fluid handling holds great promise of reducing several of the shortcomings encountered in macroscale handling of cell samples2. The benefits of these microfluidic concepts can then be fully realized by integration with downstream unit operations or other lab-on-a chip devices11.A key area of interest has been to develop methods that can differentiate cells solely based on their intrinsic parameters, which would enable a label free separation of the target cells and thereby reduce manual handling steps and costs. To achieve this goal, the microfluidic systems have to be designed to utilize differences in physical properties of the cells such as size, shape, density, electrical charge and deformability12, hydrodynamic filtration14, hydrophoretic filtration15, deterministic lateral displacement9, inertial focusing19, dielectrophoresis21, leukocyte margination22, and erythrocyte lysis24. However, most of the microfluidic devices reported thus far are either not sufficiently efficient in terms of separation performance or operate at low throughput rates ranging from 0.018 µl/min to 2 µl/min of undiluted blood13 35, which has so far prevented the use of acoustophoresis to generate pure MNC populations from blood for bioanalytical and diagnostic purposes.There is a clear unmet need for a rapid, simple and efficient method to separate MNC from blood, with at least a 3-log reduction of the RBC fraction and high MNC recovery above 80%, as an alternative to current labor-intensive density gradient centrifugation or RBC lysis and centrifugation37 with a 2-dimensional pre-alignment zone preceding the separation zone but was modified by increasing the channel length  in order to enable an increased sample flow rate. Separation was realized by optimizing the acoustic properties of the suspending medium, thereby changing the acoustophoretic mobility of MNCs versus RBCs. This method has a much higher separation efficiency combined with higher throughput than previous reports on the label-free microfluidic separation of WBC components from blood21. The gentle, label-free and continuous acoustophoresis separation protocol developed herein presents an important step towards integration of downstream diagnostic applications such as direct analysis of MNCs in flow cytometry.In this paper, we report the development of an acoustophoresis protocol that enables the efficient and rapid separation of MNCs from diluted whole blood. The acoustophoresis chip was based on the earlier reported design by Augustsson3, density 1.055–1.070 g/cm3) as compared to red blood cells 47. Calculations of the acoustophoretic mobility showed an overlap between WBCs and RBCs in PBS . In human whole blood, the cell volumes and densities differ between various populations. Mononuclear cells are larger but less dense  solution, a standard buffer density modifier, we anticipated that the mobility of WBC should approach zero at a concentration of 70% SIP while the corresponding average mobility value for RBC should be about 4000 µmWe therefore investigated the acoustophoretic mobility of MNC and RBC, as measured by the obtained separation efficiency, in buffer compositions with different SIP concentrations. Dilution of SIP decreases the density of the medium as well as the speed of sound and increases the compressibility  of 390.6 ± 169.8-fold enrichment. Under these conditions, increasing the voltage to 30Vpp resulted in a higher fraction of MNCs that was translocated to the center, thus resulting in a decreased relative enrichment. In comparison, for higher SIP concentrations, more RBCs contaminated the side outlet fraction leading to a lower relative enrichment.The acoustic separation outcome can be expressed as the relative enrichment of MNC to RBC, calculated by MNC to RBC cell ratio after the separation divided by the corresponding input ratio. Figure pp actuation  which is clearly above the threshold for hydrodynamic coupling. Therefore, the influence of different starting blood concentrations diluted in 80% SIP on the acoustophoretic separation outcome was investigated. A 200 µl sample was separated at 25Vpp and the separation efficiencies of MNCs and RBCs for the side outlet fraction, as well as the relative enrichment of MNCs to RBCs as compared to the input sample were recorded. Dilution of blood decreased the average cell concentration to about 5 × 108 cells/ml  and 5 × 107 cells/ml . As shown in Fig. 33. Overall, the best results with a median relative enrichment of MNC to RBCs of 2806-fold (range 1318–5398) were achieved for a starting blood concentration of 5%  was achieved when using a 5% blood concentration.Acoustophoretic separation of blood cells outperforms other previously reported continuous and label-free microfluidic separation devices, both in relative enrichment as well as sample throughput. The published literature reports sample flow rates between 0.018 µl/min and 3.6 µl/min undiluted whole blood equivalent, however, relative enrichment rates of WBCs versus RBCs are often as low as 15–110 foldWhen calculating the overall enrichment of WBC (accounting for the depletion of granulocytes of 82.1 ± 11.5%) a median 1,013-fold enrichment of WBCs to RBCs was accomplished, which is considerably higher than most other reported microfluidic systems.The total MNC recovery, as calculated by the percentage of mononuclear cells in the side outlet compared to the input cell amount, was lower than the measured separation efficiency Fig. . Howeverpp which gave the highest relative enrichment of MNCs to RBCs We have shown that acoustophoresis can be used to enrich mononuclear cells from red blood cells with high efficiency and recovery. In agreement with theoretical calculations we demonstrate experimentally that the acoustophoretic mobilities of MNC and RBC can be differentially affected by modifying the acoustophysical properties of the buffer, thereby enabling efficient separation of the two cell types with otherwise overlapping acoustophoretic mobilities.The collection of blood samples from healthy volunteers was approved by the Regional Ethical Review Board at Lund University. All experiments were performed in accordance with relevant guidelines and regulations.Blood samples were obtained from healthy volunteers after informed consent at Lund University Hospital, Lund, Sweden and collected in vacutainer tubes , containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant.Percoll density medium  was used as central inlet buffer as well as to prepare dilutions of blood for the acoustophoretic separation. To adjust the osmolality of undiluted Percoll, 1 part (v/v) of 1.5 M NaCl (Sigma-Aldrich) was added to 9 parts (v/v) of Percoll to make a stock isotonic percoll (SIP) solution. Adjustments of the density of SIP were done by further dilution of SIP in 0.15 M NaCl.Blood samples were processed within 10 hours after collection, stained with monoclonal antibodies for 15 min at room temperature in the dark and further diluted in central inlet buffer. The following directly conjugated monoclonal antibodies were used in this study: CD3-APC (clone HIT3a), CD14-PE (clone MφP9), CD19 FITC (clone HIB19), CD45-PerCP (clone 2D1), CD61-PE (clone VI-PL2), CD66b FITC (clone G10F5) and CD235a APC (clone GA-R2), as well as matched isotype controls . Stained samples were analyzed before and after separation on a FACSCanto II flow cytometer (BD Bioscience) and acquired data was further analyzed using the FlowJo software .Mononuclear cells (MNC) were isolated by Ficoll density gradient centrifugation using Ficoll-Paque Premium . In brief, 5 mL blood was diluted four times and layered over 15 mL of Ficoll-Paque Premium. After 40 min of centrifugation at 400 × g the mononuclear cell layer was removed and washed twice at 200 × g, for 10 min.For comparison, selective lysis of red blood cells (RBCs) to enrich the MNC fractions was performed using BD Pharm Lyse . A total of 200 µL blood was lysed by adding 2 mL of 1x lysing solution, incubated for 15 min at room temperature in the dark and washed twice at 200 × g for 5 minutes.The acoustophoretic chip was manufactured by Micronit  using Deep Reactive Ion Etching and sealed with a glass lid. The channel structure was etched into silicon and consists of a sample inlet, a pre-focusing zone (26 mm × 300 µm × 150 µm), a v-shaped flow splitter around the central buffer inlet, a main separation channel (43 mm × 375 µm × 150 µm) and a trifurcation with a central outlet and a common side outlet for the two lateral branches , connected to signal amplifiers (in-house build) was used to drive both transducers while the voltage over each transducer was measured via a two-channel digital oscilloscope . Temperature regulation was achieved using a Peltier element and a PT100 resistance temperature detector attached to the acoustophoretic system.The standing wave field was created using piezoceramic transducers glued underneath the pre-focusing channel as well as underneath the main separation channel. Frequencies were set to 4.831 MHz for the pre-focusing channel with 5 V59. A total volume of 200–500 µl diluted blood was infused through the sample inlet at 100 µl/min and pre-aligned in width and height into two parallel bands in the pre-focusing channel. Pre-aligned blood entered the main-separation channel, with a central inlet buffer flow rate set to 300 µl/min, and cells were separated in the acoustic standing wave field, based on their acoustophysical properties into the center outlet fraction (100 µl/min) or the side outlet fraction (300 µl/min). Samples of both outlet fractions were collected in 5 ml tubes and analyzed for their cell content.An in-house designed pressure driven system with feedback regulation was used for controlling the fluids to and from the chip monitored by SLI-1000 Liquid Flow Meters The authors declare that all data supporting the findings of this study are provided in the paper and it’s Supplementary Information. Raw data is available on request.Supplementary Information


Although, Pangea B avoids any continental overlap marring classical Pangea A configuration (Wegener’s type), it requires a Carboniferous-Permian megashear of up to 1500 km to achieve the pre-Jurassic configuration. The existence of this megashear is controversial and has led to a wide range of hypotheses, in order to avoid Pangea A continental overlaps and consequently the need for major intra-Pangea movements and to accommodate the paleomagnetic database within a Pangea A reconstruction. We present paleomagnetic results from Permian volcanic rocks of the El Centinela, La Pampa, Argentina. Undeformed volcanic rocks are not affected by any inclination bias and are, therefore, ideal to test different paleogeographic models. The presence of two different paleopole positions, at the base and the top of the same stratigraphic sequence, makes this location optimal to constrain the track of the Gondwana’s path during the Late Paleozoic, which shows the transition from Pangea B during the Carboniferous-Permian, to Pangea A at the Permian – Triassic boundary.A long-standing debate regarding the configuration of Pangea during the Late Paleozoic has been going on among the paleomagnetic community concerning the validity of one of two significantly different Pangea reconstructions (Pangea A  They consist of a continuous volcanic sequence of lava flows that degrade to volcanic breccias, interbedded with pyroclastic rocks : N = 25, Lat.: 060.8°S; Long.: 356.6°E, A95 = 4.5° and El Centinela II PP: N = 15, Lat.: 69.2°S; Long.: 048.2°E, A95 = 3.5°  and the early Late Permian , Rio Curaco11 and San Roberto11 PPs, respectively, Sierra Chica (a)13 PP and Punta Sierra PP14. El Centinela I and II PPs have been calculated in volcanic rocks, and furthermore these poles are not the only PPs based on volcanic rocks of South America. The Sierra Chica (a) PP12 was also determined in volcanic rocks belonging to the Choiyoi volcanic province1, which fully coincides with the age and the position of El Centinela I. Some years later, a different paleomagnetic pole has been published for Sierra Chica (b) PP15. Although when it was performed on the same outcrops, the application of an erroneous structural correction and age interpretation of this data15 dislocated this PP position13.Both PPs have good consistency with coeval paleomagnetic poles from others regions of the Southwest Gondwana margin6 Figs  and 3 wi8 and El Centinela II PPs is about 15 Ma, the declination difference cannot be attributed to secular variation. Instead the difference in the declinations might be attributed to apparent polar wander  were accomplished using a DC squid cryogenic magnetometer (2 G model 750 R) at the “Daniel Valencio” Paleomagnetic laboratory of the Universidad de Buenos Aires IGEBA-CONICET. Thermal demagnetization was the only successful demagnetization method due to the high Curie temperatures of the magnetic carriers, and was applied in at least 15 steps, with maximum temperatures of 680 °C, using ASC ovens with a dual or single chamber. Bulk susceptibility was measured in all specimens after each step to monitor possible chemical changes during heating, with a Bartington MS2 susceptibility meter. Demagnetization results were analyzed using orthogonal vector plots19. Instrumental errors20 were corrected using the reference zircon GJ-1.The magmatic origin of the zircons was identified through BSE imaging with an electron microscope (JEOL JSM 5800), helped to identify its magmatic origin. Later, they were dated by the U-Pb method using a Laser Ablation Microprobe coupled to a MC-ICP-MS (Neptune), belonging to the Isotope Geology Laboratory of the Federal University of Rio Grande do Sul (Brazil). Isotopic data were acquired using a static mode analysis area of 25 μm in diameter. Calculations were carried out using the Isoplot/Ex 4.10


The first year after adoption constitutes a sensitive period for both strengthening the new emotional bond in the family and checking its appropriate development by adoption services. A key variable for children’s catch-up are adoptive parents’ socioemotional and individual features. The aim of this study is to investigate links between adoptive mothers’ individual features and behavioral problems in their children in the first year after adoption placement, by testing the moderating role of both age at adoption and maternal genetic polymorphisms. Seventy-eight adoptive mothers completed temperament and genetic measures. Mothers showed a specific pattern of interaction between basic temperament traits and genetic markers in their assessment of children’s behavioral problems; dopamine D4 receptor gene and children’s age at adoption are two moderators in the association in which mothers’ temperament was affecting the evaluation of their children’s behavioral problems. Findings highlight a still undervalued area of parenting resources in the process of post-institutionalized children’s catch-up after adoption placement, by showing how individual features count in the commonly measured variable of children’s behavioral and emotional problems. This could help in orienting identification and choice of key variables for family assessment after adoption placement, thus contributing in fostering children’s healthy development. Institutionalization is the more common background of internationally adopted children, with consistent research showing how institutional care leads to a number of developmental shortcomings ,2, with A key variable for children’s catch-up are adoptive parents’ socioemotional  and indiA growing body of literature focuses on susceptibility effects for several genes, including serotonin- and dopamine-related gene polymorphisms, in a “for better or for worse” manner: individuals who are more vulnerable to adverse social environments also benefit more from positive environments ,14,15. HThe first year after adoption placement represents a key period for acknowledging possible strengths and vulnerabilities of all adopted children and it usually serves as a point of reference for the monitoring work of adoption services that are in charge of checking the new family’s socioemotional adjustment. Children who were adopted before 12 months of age, named early adoptees, were almost as securely attached as their non-adopted peers , with reIn view of all mentioned so far, the objective of this research is to investigate links between adoptive mothers’ individual features, including basic temperament traits (negative affectivity and effortful control) and genetic markers , and maternal report of emotional and behavioral problems in their children in the first year after adoption placement, by testing the moderating role of both age at adoption and maternal genetic polymorphisms . We expect that maternal higher effortful control will be associated with less emotional and behavioral problems in children; and conversely that lower maternal negative affect will be associated with less behavioral problems in the children. Moreover, we hypothesize that children’s age at adoption and maternal genetic polymorphisms could play a moderator role in these relationships, regardless of children’s temperament traits.r = 0.96, p < 0.001), we choose to consider the first one as key variable [Eighty-one mothers with their first adopted children have been recruited through adoption services. Due to the incomplete data of 3 families, 78 adoptive families were considered. variable . All chiAll subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Department of Psychology  of the University of Pavia (Project identification code: 1-2013).The Child Behavior Checklist for Ages 1.5–5 (CBCL/1.5–5) [L/1.5–5) ,21. MothThe Adult Temperament Questionnaire-short form (ATQ) [rm (ATQ) . MothersThe Child Behavior Questionnaire (CBQ) [re (CBQ) . MothersNeurobiological markers. Genotyping was conducted at Padua, BMR Genomics lab, using well-established methods. DNA samples were extracted from buccal swabs using the kit IQ form Promega following manufacturer’s instructions. For genotyping the VNTR in exon 3 of the DRD4 gene, PCR was performed using the primers 5′-AGGACCCTCATGGCCTTG HEX-conjugated and 5′GCTCATGCTGCTGCTCTACT were used. PCR was performed as follows: 1 cycle of 5 min at 94 °C, followed by 10 cycles of 45 s at 94 °C, 30 s at 62 °C, 1 min 68 °C, 25 cycles of 30 s 94 °C, 30 s 52 °C, 1 min 68 °C and a final extension step of 7 min at 68 °C. The PCR-amplified DNA fragments were analyzed by loading the PCR product on an ABI PRISM 3130xl Genetic Analyzer.For amplification of the 5′ regulatory region of Serotonin Transporter gene (Locus Symbol SLC6A4), which contains a 43 bp insertion/deletion polymorphism (5HTTLPR), primers 5′-TCCTCCGCTTTGGCGCCTCTTCC 6FAM-conjugated and 5′-TGGGGGTTGCAGGGGAGATCCTG were used. PCR was carried out as follows: 1 cycle of 5 min at 94 °C, followed by 10 cycles of 45 s at 94 °C, 1 min 68 °C, 1 min 68 °C, 25 cycles of 45 s 94 °C, 45 s 55 °C, 1 min 68 °C and a final extension step of 7 min at 68 °C. The presence of the Short (S) and Long (L) allele for each sample was determined by loading the PCR product on an ABI PRISM 3130xl Genetic Analyzer. All the PCR were carried out in presence of 0.05% DMSO and 1 N betaine starting from 2 µL l of genomic DNA.Mothers were recruited by consecutive admission to adoption services in Italy and were asked, after agreement and signature of informed consent, for completing a demographic information form and the three questionnaires mentioned above. Regarding DNA collection, buccal swabs were collected at the same time of self-report administration and stored at −20 °C until DNA was extracted. The research was part of a more comprehensive intervention trial and was approved by the ethical committee of Pavia University Department of Brain and Behavioral Sciences.p > 0.05).As all variables were normally distributed, the study hypotheses were tested via parametric analyses. T-scores of CBCL-Total showed that mothers reported 11.5% of children as in the clinical range, 9% of them as in the borderline range, confirming previous findings showing a higher percentage of emotional and behavioral problems among these children, if compared to those reared in biological families. Moreover, there were no significant differences between girls and boys on any of outcome measures  = 4.06, p < 0.001) and the interaction were significant : Adoptive mothers with temperamental NA and being carrier of DRD4-7 repeat allele, significantly assessed their children as showing more emotional and behavioral problems . Conversely, adoptive mothers with temperamental EC and being carriers of DRD4-7 repeat allele significantly assessed their children as showing less emotional and behavioral problems  = 2.57, p = 0.02; interaction: b = −0.85, t = −2.11, p = 0.03; age at adoption as high: b = −13.72, t = −2.08, p = 0.04). The models to test the moderation effect of 5HTTLPR was not significant  = 2.01, p = 0.07; EC: R2 = 0.14, F = 1.57, p = 0.15)  .N = 53). We did not observe any moderator role of children’s temperamental features neither for the model with mothers’ NA  nor for the model with mothers’ EC .Lastly, in order to check if even children’s temperament features could count in the findings obtained, we examined whether both children’s negative affect and effortful control moderated the relationship between mothers’ temperament and children’s behavioral problems  on late adopted children’s emotional and behavioral problems and second to inquire how children’s age at adoption placement and maternal genetic polymorphisms influenced this relationship. Lastly, we also checked if children’s temperament features could count. The findings showed that adoptive mothers tended to report more behavioral problems of their children according to specific individual features; mothers who showed more temperamental features of negative affectivity-NA , and who were also carriers of dopamine 7r allele of DRD4, showed the highest evaluation of the presence of behavioral problems in their just adopted children. This outcome applies to a specific group of children, i.e., to children whose age at adoption was in the older range considered in our sample—that is about 51 months of age. Conversely, mothers who showed more temperamental effortful control-EC  and who were also carriers of the 7r allele of DRD4 tended to report less behavioral problems, especially for children who arrived in their family older. This result revealed how effective it could be to carefully analyze possible moderators of the association between mothers’ individual features and children’s behavioral problems in order to detect effects that otherwise would remain masked and, thus, undervalued. The interaction between temperamental features and neurobiological markers appears a key concept for identifying the “more affected” group of mothers’ perceptions, “for better and for worse”; the same neurobiological marker, i.e., the dopamine 7r allele of DRD4, appears to play a role in decreasing their children’s behavioral problems report if in interaction with the temperamental feature of mother’s EC and, conversely, in increasing the same perception if in interaction with mother’s NA . Worthy Outcomes found seem also to enlarge the long history of research on the interaction between parenting and temperament by considering whether, how, and how much parents’ perception of their children’s problem behaviors is influenced in the very demanding situation of the first year after adoption placement . Even ifN = 53). Future studies with a bigger sample size could contribute in confirming or rejecting this first finding.In our additional analysis, we did not find any significant role of children’s temperament on how maternal temperament influenced children’s behavioral problems. The absence of an effect is a first pilot indication and it needs to take into account even the size of the sub-sample of older children considered  and genetic markers in their assessment of children’s emotional and behavioral problems. In particular, dopamine D4 receptor gene and children’s age at adoption are two moderators in the association in which mothers’ temperament was affecting their evaluation of their children’s emotional and behavioral problems. Beyond the acknowledged data on the importance of age at adoption for children’s likelihood of showing behavioral problems , our res


Intercalation of alkali metal cations, like Li+ or Na+, follows the same three‐stage mechanism of the interfacial charge and mass transfer irrespective of the nature of the electrolyte, electrolyte composition or electrode material. It is nowadays a nonsurprising fact that the solar and wind power noticeably contribute to the energy supply in a number of countries.The performance of the majority of modern batteries is largely determined by the status of the electrode/electrolyte interfaces. Interfaces at battery electrodes have been studied for a long time; however, they are not fully understood yet. For instance, the so‐called solid electrolyte interface (SEI) formed at the negative electrode of common Li‐ion batteries has attracted a special attention since it drastically influences the key parameters in the performance of a battery, e.g., cycle‐life and safety.4Ti5O12 and TiO2) were selected, which operate at more positive potentials (1.5–1.8 V vs. Li/Li+) without the formation of thick organic films.4 operating at low potentials (≈3.55 V vs. Li/Li+) and preventing the presence of cathode electrolyte interphase was chosen. For the aqueous systems, state‐of‐the‐art Prussian Blue Analogues (PBAs)In the following, we give a brief rationale for the particular selection of battery systems used in this work. First, the voltage of the state of the art Li‐ion batteries (>3.7 V) requires the use of organic electrolyte solutions with larger electrochemical stability window compared to aqueous systems. However, the state‐of‐the‐art carbonate‐based solutions decompose at the very cathodic potentials necessary to lithiate graphite. The products of the decomposition precipitate onto the electrode and form a protecting film, which is electrically insulating, thus preventing further electrolyte decomposition. Therefore, for this work, two Li intercalation anode materials Li4Ti5O and TiO2Figure2O‐based). As a result, a sort of “total uncompensated charge” can likely appear , M′—an “optional” transition metal cation which does not participate in the redox reactions , Ly is a “ligand” , A− is an anion .In order to elucidate the interfacial processes during quasi‐reversible (de‐)intercalation, it is worth to notice that the process of (de‐)intercalation in many state‐of‐the‐art and perspective electrode materials starts with the change of the oxidation state of the host metal cation. For instance, in the case of numerous PBAs, the oxidation state of Fe is changed between 2+ and 3+. This process is very fast due to a relatively good electronic conductivity of intercalation electrodes. This is also schematically shown in r Figure B provokir Figure D, all exIt should be noted that there is unfortunately a very limited number of techniques capable to prove the above mentioned scheme experimentally. However, there is a relatively simple nondestructive mean to do so, namely electrochemical impedance spectroscopy (EIS). This is one of only few suitable approaches, when the system intercalates and de‐intercalates cations during the probing, which likely enables the most adequate characterization.As related to the EIS‐characterization, the general scheme involving three interconnected quasi‐reversible electrochemical stages where the last stage does not involve the net charge transfer often leads to a number of equations, which can be equivalently represented using a circuit shown in Figure Figure2O/CH3CN, and organic electrolytes.2Ni[Fe(CN)6] electrodes in aqueous Na2SO4 electrolytes. The curves show only a small hysteresis revealing generally high reversibility of the Na+ (de‐)intercalation. As can be seen in Figure 2Ni[Fe(CN)6] film stability but not the mechanism itself. The introduction of K+ instead of Na+ and changing the nature of the anions from SO42− to NO3− 6] and Na2Cu[Fe(CN)6] 6]  hexafluorophosphate (PF6−) anions, (iv) porous paste electrodes  and (v) battery‐type cell intercalation of alkali metal cations. In the following system, a number of parameters was changed with respect to the PBA‐based systems: (i) solely organic electrolyte solutions, (ii) solely Liphate PF6− anions,s Figure B,D,F aga+, Na+, and K+ intercalation compounds, SO42−, NO3−, and PF6− anions, thin films and porous paste electrodes. This indicates that this mechanism is rather common in batteries, surprisingly for both cathodes and anodes. Therefore, the interpretation of the battery test data as well as the design of the new battery systems should involve the above‐described considerations, especially in the case of fast (de‐)intercalation kinetics. In particular, the commonly observed in battery research “impedance loops” should in many cases be an indication of the specific mechanism of the interfacial charge and mass transfer, rather than an experimental error. However, further investigations including more experimental techniques, for instance other potentiodynamicIn conclusion, we have demonstrated that the three‐stage mechanismElectrochemical Measurements in Aqueous Media: Electrochemical experiments in aqueous media were carried out in different glass cells using a three‐electrode configuration, consisting of the PBA working electrodes, a platinum wire as a counter electrode and an Ag/AgCl  reference electrode, to which all the electrode potentials for the aqueous systems were referred. As the substrates for the PBA thin film working electrodes, either a sputtered gold on glass arrandee, an Au electrochemical quartz crystal microbalance (EQCM) electrode  or an Au(111) single crystal  were employed. The PBA thin films were electrochemically deposited from aqueous solutions and characterized as described in Supporting Information, Section 1. Electrochemical impedance spectroscopy measurements were performed as described in detail elsewhereMaterials and Electrochemical Characterization of Electrodes in Organic Media: Commercially available Li4Ti5O12 (2 m2 g−1), anatase TiO2 (100 m2 g−1) and carbon‐coated LiFePO4 (2–5 wt% content of carbon) materials were received from MTI Corp (USA), Sachtleben Chemie (Germany) and Phostech Lithium, Clariant Canada Inc. (Canada), respectively. The porous paste electrodes were prepared following the standard procedure . A current density of 80, 87, and 168 mA g−1 (C‐rate of 0.5 C) was applied to C‐LiFePO4, Li4T5O12, and TiO2 electrodes, respectively, within the potential window 4.0–3.0 V and 3.0–1.0 V for cathode (C‐LiFePO4) and anode materials (Li4T5O12 and TiO2). All the electrode potentials in organic electrolytes are reported versus Li/Li+ (in 1 m Li+). All electrochemical experiments were performed using a three‐electrode coaxial cell specially designed for EIS measurements  should be addressed to the authors.SupplementaryClick here for additional data file.


Toombak is a form of smokeless tobacco (SLT) that is locally made and consumed in Sudan and contains several carcinogenic elements. Use of Toombak has been etiologically linked to various oral diseases including oral cancer. This study aimed to obtain baseline information about the Toombak use among Sudanese school workers, as well as their knowledge about Toombak related health hazards and attitude towards their role in Toombak control. In addition, this study assessed the availability and effectiveness of control policies and preventive practices in the schools.A cross-sectional school-based study using one-stage stratified random sampling procedure; four schools were selected randomly from each of seven localities. A total of 239 school workers’ were recruited (census) from the selected schools in Khartoum State, Sudan.p < 0.001). Most of the school workers reported good knowledge, positive attitude towards their role in Toombak control and good preventive practice. Female school workers were more likely to report positive attitude towards their role in Toombak control (p < 05), and to report good knowledge. Those reporting good preventive practice in schools reported good knowledge more than two times than their counterpart (p < 0.001). Age was the strongest predictor of ever Toombak use among school workers (p < .001).Of the school workers, 63% (147) were ≤40 years, half were females and 79.2% (187) were teachers. A total of 9.6% (22) school workers confirmed ever use of Toombak and the percentage of daily users amounted to 64.7% (11). Moreover, 76.2% (16) of ever Toombak users were ≥40 years and all of them were males (The use of Toombak among school workers was associated with poor knowledge, negative attitude towards their role in Toombak control and poor preventive practice, Therefore, school workers use of Toombak may reduce their motivation and use of their potential in the prevention of a major health problem caused by Toombak use and affects their role model behaviour. On the other hand, school workers engagement with preventive practices in schools’ was associated with good knowledge which in turn empowers their positive attitude towards their role in Toombak control. The tobacco epidemic is a major public health problem and one of the main causes of death and disability worldwide . More thUse of Toombak has been etiologically linked to various oral diseases, such as periodontal diseases, mucosal lesions and may eventually lead to tooth loss , 8. MoreSchool workers  represent important role models for secondary school students. The daily interactions between students and school workers combined with their authority, make them a potentially influential group with respect to Toombak use control , 17. TheKhartoum is the capital of the Sudan and consists of three major cities; Khartoum, Omdurman and Khartoum North (Bahry). The total population of the Republic of the Sudan is 36 million people, where Khartoum state has a population of 6 million people .In Sudan, estimates of the prevalence and correlates of Toombak use are scarce. Research on Toombak is important and relevant regarding the magnitude and impact of the problem. This study aimed to assess baseline information about Toombak use among Sudanese school workers, their knowledge about Toombak related health hazards and their attitude towards their role in Toombak control. In addition, the study aimed to assess the availability and effectiveness of control policies and the preventive practices in the schools. Such data are necessary for the planning and implementation of intervention programs to prevent Toombak use among school workers.This cross-sectional school-based study was carried out during the period (2013–2014) as a part of a larger research project focusing on the use of Toombak among secondary school students in Khartoum State, Sudan.The sampling procedure was made for a larger-scale study of secondary schools students’ use of Toombak .The three cities and their respective localities were all represented in the sample a total of 28 schools; four schools from each locality  were randomly selected with the substitution of the schools which did not agree to participate. All school workers (census) in the 28 selected schools were invited to participate in the study. Eligibility criteria required was the presence of the school workers at the time of data collection. A total of 239 school workers  were invited and accepted to participate in the study from the 28 previously selected secondary schools.School workers completed self-administered questionnaires at their offices. The data collection was supervised by trained personnel (the main researcher and two assistants). After a brief explanation of the objectives of the study, written informed consent was obtained from the participants.The questionnaire, used in this study was based on the Global Schools’ Personnel Survey (GSPS) questionnaire; GSPS is an integral part of the Global Tobacco Surveillance System (GTSS), (SLT section questions) started by the WHO in 1999 . The queEver use of Toombak was measured by the question; “have you ever used Toombak” using response options (1) “yes”; (2) “no”. The original categories recoded into (0) non- user ; (1) ever user . The frequency of Toombak use was measured by the question; “how frequently do you use Toombak at present” using response options (1)” daily”; (2) “occasionally”.Age group was measured by the question; “how old are you” using response options (1) “19 years or younger”; (2) “20 to 29 years”; (3) “30 to 39 years”; (4) “40 to 49 years”; (5) 50 to 59 years; (6) “60 years old or older”. The original categories recoded into (0) < 40 years ; (1) ≥ 40 years .Knowledge about Toombak related health hazards was measured by four questions; “is Toombak use addictive”; “does Toombak use cause oral cancer”; “does Toombak use cause heart disease”; using response options (1) “yes”; (2) “no”; (3) “I don’t know”. The original categories recoded into (0) poor knowledge ; (1) good knowledge ; “does Toombak use cause malaria” using response options (1) “yes”; (2) “no”; (3) “I don’t know”. The original categories recoded into (0) poor knowledge ; (1) good knowledge . A sum score was constructed, labelled “knowledge about Toombak related health hazards” from the four questions (0–4) Cronbach’s α =0.45). For analysis, this sum score was dichotomized on the median split with values (0) poor knowledge ; (1) good knowledge .Attitude of school workers towards their role in Toombak control was measured by four questions; “school workers’ use of Toombak influences students use”; “schools should have a policy or rule specifically prohibiting Toombak use among students on school premises/property”; “Have you ever advised a student to stop using or quit use of Toombak”; using the response options (1)” yes”; (2) “no”; (3) “I don’t know”; the original categories were recoded into (0) negative attitude ; (1) positive attitude . “How concerned are you about Toombak use among youth in your community”; using response (1) “very concerned”; (2) “Somewhat concerned”; (3) “Not at all concerned”. Original categories were recoded into (0) negative attitude ; (1) positive attitude . A sum variable was constructed ranging from (0–4), labelled “Attitude of school workers towards their role in Toombak control” (Cronbach’s α = 0.54) and dichotomized into (0) negative attitude  (1) positive attitude .Availability of a policy to prevent use of Toombak in schools was measured by the question; “does school has policy that prohibits Toombak use within school premises” using the response options (1)” yes”; (2) “no”; (3) “I don’t know”; response options were recoded (0) no policy ; (1) yes, there is policy .Effectiveness of schools’ policy was measured by the question “does school policy prohibit Toombak for visitors, students, and school workers’” using response options (1) “yes”; (2) “no”; (3) “I don’t know”; response options recoded (0) no effective policy ; (1) effective policy .Preventive practices in school were measured by five questions; “did you participate in campaigns against Toombak use”; “is prevention of Toombak use included in the school curriculum”; “do you have access to teaching materials for Toombak prevention”; “have you received training to prevent Toombak use among students” ; “do you use non-classroom activities to teach Toombak prevention to students” using response options (1) “yes”; (2) “no”; (3) “I don’t know”; response options were recoded into (0) poor practice ; (1) good practice . A sum score was created from five questions (0–5) and labelled “preventive practices in schools” (Cronbach’s α = 0.61). This sum score was dichotomized on the median split  into (0) poor practice ; (1) good practice .p < 0.05. Multiple variable analysis was conducted using multiple logistic regression. Estimates were presented as odds ratios (OR) and 95% Confidence Intervals (CI). Nagelkerkes R2 was calculated for each multiple variable logistic regression model. Nagelkerkes R2 is a pseudo R square that generalize the coefficient of determination with values between 0and 1 where 0 denotes that the model for not explain anything about the variation in the dependent variable and 1 that the model completely explain the variance.Data were recorded and analysed using the Statistical Package for the Social Science, version 20 (IBM SPSS Statistics). Descriptive analyses were performed using frequencies and percentages. For the bivariate analysis, chi-square tests were performed to evaluate the categorical variables and the level of significance was set at Most of the school workers 63.1% (147) were <40 years. Females represented 50.2% (114) of the participants. The majority 79.2% (187) were teachers, 7.2% (17) were headmasters, 1.7% cleric, 6.4% service labourers and 5.5% others. Almost half of the participating school workers 50% (116) confirmed that they teach health issues, while 64.3% (148) confirmed the availability of schools’ policy that prohibits Toombak use. Only 17% (38) reported that policies were completely enforced among school workers, whereas 35.1% (78) reported complete enforcement among students.Almost half of the school workers, 55.3% (125), reported good knowledge with respect to health hazards related to using of Toombak, whereas 86.1% (192) had a positive attitude towards their role in Toombak control. About, 57.2% (127) confirmed good preventive practices in schools.p < 0.001). As depicted in Table Prevalence of ever use of Toombak among school workers was 9.6% (22) among them 64.7% 11) were daily users and 35.3% (6) were occasional users. A 55% (11) reported using Toombak inside the school (1 were dap < 0.05) and preventive practices in schools as those engaged in these practices tend to report good knowledge (p < 0.001).Whereas the larger proportion of female school workers reported positive attitude towards their role in Toombak control than males (p < 0.05). A significantly smaller proportion of ever Toombak users reported positive attitudes than did non-users (88.1% versus 63.6%) . School workers who confirmed preventive practices in schools were almost two times more likely than their counterparts to report good knowledge .As shown in Table  in the m2 .23). Only age and attitude of school workers towards their role in Toombak control were significant predictors of ever Toombak use. The older group of school workers had almost five times greater likelihood to be Toombak users than their younger counterparts, OR 4.43 . Those with negative attitude had almost four times the likelihood to be Toombak users compared to those with positive attitudes OR 0.26 .Ever use of Toombak was regressed upon age, enforcement of the policy among students and attitude towards their role in Toombak control and the model explained 23% of the variance  use, while this study focuses only on SLT (Toombak) use , 23.According to the present study, the prevalence of ever use of Toombak was low among the participating school workers, amounting to 9.6%. A previous GSPS study carried out in Sudan in 2009, reported that only 5% of the investigated school workers were using tobacco products other than cigarette (including Toombak and Shisha) . We coulThis study has clearly demonstrated that Toombak use is a male dominant habit among school workers in Khartoum State, as all of the users were males. This is different when compared to a study from India. Where, 13.7% of the female versus 35% of the male school workers were SLT users . Male doAge has been identified as a strong predictor of Toombak use as it is most prevalent among school workers ≥40 years. This is consistent with previous studies in Sudan. Idris et al.  found thToombak use inside school’s premises on a daily basis was high and this might have a direct influence on students’ use of tobacco and may cause a detrimental effect to the school workers’ function as a role model for students as was shown by other studies –35. In SSchools expected to be a favourable place to promote health and also to protect the students from tobacco exposure by effective school tobacco control policies . Low effIn this study, reduced knowledge of the health hazards of Toombak use among school workers is a common problem, especially in Africa as most of the school workers demonstrate low awareness of the health consequences of tobacco products use , 41. BeiKnowledge in this study is consistently significant among those who reported participation in preventive practices in schools. This is in accordance with Agaku et al.  who repoThe majority of the participants, more females than males, showed a positive attitude towards their role in Toombak control Table . This miHowever; this study has its limitations, as the findings are based on self-reporting that might be subjected to information bias , thus school workers may either have under- or over-reported their responses to the questionnaire. Also, the participation was voluntary; it may not be subjected to selection bias as all school workers accepted to participate in the study. In addition, the study sample of school workers might not be representative of all school workers in Khartoum state.The Alpha values (Cronbach’s α) reported in this study were lower than the expected rule of thumb (0.7), this may affect the reliability of some questions. However, some factors may affect the test as the number of items, the type of variables . Thus thAlthough the prevalence of Toombak use among the school workers investigated was low, Toombak use was associated with poor knowledge, negative attitude towards their role in Toombak control and poor preventive practice. Thus, school workers who use Toombak may be less motivated for the prevention of the major health problems caused by Toombak use and less suitable as role models for their students in tobacco prevention. On the other hand, school workers who were engaged in preventive practices in schools presented with good knowledge which, in turn, may empower their positive attitude towards their role in Toombak control.