As a result, the hydrogenation of dimethyl oxalate (DMO) to ethylene glycol, an important reaction well known for deactivation from copper nanoparticle sintering, can show greatly enhanced activity and stability with the CuZn alloy catalysts due to no noticeable sintering. HRTEM, nano-diffraction and EXAFS characterization reveal the presence of a small beta-brass CuZn alloy phase  which appears to greatly stabilise Cu atoms from aggregation in accelerated deactivation tests. DFT calculations also indicate that the small bcc CuZn phase is more stable against Cu adatom migration than the fcc CuZn phase with the ability to maintain a higher Cu dispersion on its surface.Incorporation of Zn atoms into a nanosize Cu lattice is known to alter the electronic properties of Cu, improving catalytic performance in a number of industrially important reactions. However the structural influence of Zn on the Cu phase is not well studied. Here, we show that Cu nano-clusters modified with increasing concentration of Zn, derived from ZnO support doped with Ga It is long believed that Zn atoms are essential in catalysts for textural dispersion of the copper phase181920262728Cu-based catalysts are widely used for many important reactions in the chemical industry, including methanol synthesis124578101112131415163+ doping into Cu-ZnO by controlling chemical composition and calcination temperature. We were particularly interested to develop these composite materials as effective catalysts for the hydrogenation of DMO to EG reaction due to the recent incentive to develop this non-oil based reaction route for chemicals synthesis. EG is an important raw material in the manufacture of polyester fibers and fabrics, and polyethylene terephthalate, widely used in our society11121314111215162O3 have been documented27282930In this work, we have systematically investigated Ga0 when Ga3+ is added to the Cu/ZnO precursors under H2, which gives rise to CuZn nanoclusters. A clear inverse correlation between Zn0 concentration and Cu particle size in the catalysts is, for the first time, revealed. From high resolution transmission electron microscopy (HRTEM), nano-diffraction, and extended X-ray absorption fine structure (EXAFS) analyses, a bcc CuZn phase can be identified. According to our density functional theory (DFT) calculations the bcc CuZn is more stable against metal sintering than the corresponding fcc Cu or fcc CuZn. Thus, this new type of catalyst shows remarkable activity and stability for the hydrogenation of DMO to EG. This conclusion, although demonstrated for the hydrogenation of DMO reaction, is expected to be applicable to other Cu catalyzed reactions that are susceptible to Cu sintering. It is thus believed that this finding can offer a new way to stabilize surface Cu clusters for high activity and stability.Here, we report the progressive formation of Zn3+ ions into the Cu and Zn containing precursor system a series of Zn modified Cu catalysts have been prepared by co-precipitation with control of precursor injection rate, pH value and precipitation temperature 5(CO3)2(OH)16 were identified by powder XRD  and Ga3+ (no reduction) exclusively. XPS also indicated partial reduction of Zn2+ to form Zn0 after the H2 treatment of the co-precipitated precursors. 2+ and Zn0, respectively. Thus, the surface composition of catalysts analysed by XPS is presented in 0 varies with chemical composition and calcination temperature. The Zn0/(Zn0 + Zn2+) ratio increased as the content of Ga3+ increased to a maximum of 0.32 at a chemical composition of 43%Cu, 52%Zn, and 5%Ga. The ratio then decreased as the Ga3+ concentration was increased further. When using the same chemical composition but varying different calcination temperatures, the Zn0/(Zn0 + Zn2+) ratio was found to reach a maximum value when the sample was calcined at 330 °C. The CZG29 sample gave the highest Zn0/(Zn0 + Zn2+) ratio among all the studied catalysts, which means it has the highest ratio of Zn atoms decorated on Cu nanoparticles, and has the highest Zn0/Cu ratio than the other samples. Although XPS is a useful technique to obtain near surface composition, it cannot provide the topmost atomic layer analysis because the photoelectron escaping depth can reach to few nano-meters. Therefore, by using low energy ion, the penetration depth to the solid sample can be greatly restricted. As a result, apart from the XPS analysis, we also used the more surface-sensitive technique of high-sensitive low-energy ion scattering (HS-LEIS), which provided the elemental composition of the outermost surface (refer to SI for the details of the experiment of HS-LEIS), to characterise two selected samples, CZG28 and CZG29. As seen from Through incorporation of Gawder XRD . Under H3+ into the Cu and Zn containing precursor system can clearly create a ZnGa2O4 phase, which depends on calcination temperature (>330 °C) and composition (2O4 and ZnO phases (both identified in XRD) suggests the formation of a material interface between these two phases. It has been reported that the formation of heterojunction interface with two semiconducting solid phases (ZnGa2O4 and ZnO) with different band energies could lead to charge separation of the phases. In this case, the reducibility of the refractory ZnO support phase is greatly enhanced0 indeed shows an optimum response to the presence of ZnGa2O4 and ZnO phases.Also, in the catalyst synthesis, an addition of Gaposition . The app3+ concentration increases, the activity increases, reaching a maximum value at the CZG29 sample. The activity then decreases as the Ga3+ concentration further increases. The EG selectivity has a similar trend to the activity. It can be seen that the calcination temperature of the CuZnGa precursors also affects the catalytic performance with a calcination temperature of 330 °C showing the best performance amongst the CZG28 series. Comparing the change in Ga3+ concentration with Zn0/(Zn0 + Zn2+) in 0/(Zn0 + Zn2+) or Zn0/Cu ratio closely, see in-situ treatment (by raising the temperature to 400 °C for 24 h under N2) shows very different behaviours than that without the high temperature treatment  and CZG28-450 (Zn0/Cu ratio of 0.54) with intermediate Zn0/Cu ratios deactivated by 5 and 11% of EG yields accordingly. Thus, there is a clear correlation of stability with Zn0/Cu ratio, which suggests the active Cu particles might have severe sintering after heat treatment but the presence of Zn0 minimized the extent of sintering. 2 indeed suffered from serious particle aggregation (average diameter increased from 3.1 nm to 66.7 nm), whereas CZG29 maintained the small particle size  is seen when the Zn concentration is higher than 62%, and finally to hcp if Zn ratio is higher than 75%. These structural changes are thought to be attributed to the increasing electron contribution from Zn into anti-bonding bands of CuZn, hence making the primary structures unstable0/Cu ratio, by microscopy clearly indicates a mixture of phases. The most prevalent phase in this reduced sample is the bulk ZnO (observable by XRD). There is also a clear evidence of the formation of the other phases: fcc Cu (may contain Zn as fcc CuZn with same lattice parameter), bcc CuZn and trace complex cubic Cu5Zn8 phases (at about 2 nm) (5Zn8 (Red), Cu (Green) and CuZn (Blue) represent the characteristic d-spacings of each structure: Cu (00–001–1241), CuZn (04–003–4270) and Cu5Zn8 (00–025–1128). The d-spacings of crystals were measured from the lattice fringes and the corresponding fast-Fourier Transform (FFT) images of selected HR-STEM images  when there is a large quantity of Zn0 in contact with Cu nanoparticles. This was predicted from the Cu-Zn phase diagram. On the other hand, the CZ catalyst without Ga (Cu/ZnO sample) contained exclusively the fcc Cu phase.According to the phase diagram of Cu-Znut 2 nm) . AlthougM images . The thr0 into the nano Cu lattice, extended X-ray absorption fine structure (EXAFS) of Cu was studied. This technique explores the local structural information of the Cu atom. The experimental data for the reduced CuZnGa samples was recorded and satisfactory R fittings were achieved, as can be seen in fcc Cu. Therefore, a mixed-structure model is introduced and it is found that the combination of both fcc metallic Cu and bcc CuZn alloy structures give significantly improved fits to the experimental spectra. We did not attempt to fit the complex cubic Cu5Zn8 since there was only a small quantity present in the sample. The EXAFS were fitted using scattering paths of Cu-Cu (2.56 Å) from metallic fcc Cu model, Cu-Zn (2.56 Å) and Cu-Cu (2.99 Å) from bcc Cu-Zn alloy model. Notice that the longer distance of Cu-Cu (2.99 Å) in the bcc Cu-Zn structure is distinctive from the shorter Cu-Cu (2.56 Å) of the fcc Cu model and Cu-Zn (2.56 Å) of the bcc Cu-Zn model. As shown in bcc Cu-Cu bond, thus indicates the highest concentration of bcc Cu-Zn alloy. We also found the bcc CuZn concentration is consistent with the concentration of Zn0/Cu observed from the XPS result  and bcc CuZn can be clearly observed after the reduction procedure. It is intriguing to find that the sample containing a higher quantity of bcc CuZn shows better stability against particle sintering when high temperature treatment is applied. It has recently been reported that SiO2 supported Cu nanoparticles continue to grow, with an Ostwald ripening process, during DMO hydrogenation, causing deactivation of the Cu catalystsfcc (111) 4 × 4 model (the similar surface fcc 50%Cu50%Zn (111), not shown) and 50%Cu50%Zn bcc (110) 3 × 3 model we adopted for the calculations. Additional Cu atoms (yellow) are placed on their surfaces progressively, as shown in fcc (111) Cu, fcc 50%Cu50%Zn (111) alloy and bcc 50%Cu50%Zn (110), bcc CuZn surface reduces the Cu atoms migration, hence decreases the sintering extent. It is interesting to note, from fcc Cu and fcc CuZn show similar energy stabilizations upon addition of Cu atoms despite their difference in chemical composition. It can be clearly seen that the bcc CuZn surface, with the same Zn content as fcc CuZn, gives the highest energy stabilization to accommodate additional Cu atoms as compared to the other two surfaces. This clearly suggests that Cu atoms on bcc CuZn surface are more stable than the corresponding fcc Cu or fcc CuZn surfaces, which reduces the extent of sintering of Cu atoms due to the stronger geometric effects holding these atoms in position. Previous theoretical studies suggested that the phase change from fcc to bcc structure in CuZn when Zn ≥ 50% is attributed to the electronic energy stabilization of the systembcc arrangement can also exert stronger binding to Cu adatoms than that of fcc. This information also matches with known stronger binding properties of beta brass (bcc CuZn) in metallurgy, which can only be worked with hot, are harder, stronger and more suitable for casting. In contrast, alpha brass (fcc CuZn) is more malleable with weaker binding. It can be worked with cold and is used in pressing, forging or similar applicationsbcc CuZn structure is shown to be more stable and can be used in Cu catalysts maintaining small CuZn clusters so as to keep their superior catalytic performance.In the present work, bcc Cu-Zn catalysts may provide a good starting point for improving this reaction.Nano alloys play a crucial role in many heterogeneous catalytic processes and their applications are expected to rise rapidly. This is due to the vast number of configurations and type of surface sites that multi-component materials can present. However, characterization of small alloy clusters of different compositions and temperature pre-treatments with respect to catalytic performance is technically challenging. In this case, using a range of surface sensitive, diffraction and microscopic techniques, we report for the first time that a clear structural role of Zn inclusion into the Cu nano-clusters by forming stable nano-beta phase CuZn. The structure can offer a good dispersion of active surface Cu atoms for catalysis and more importantly, stabilize them from aggregation. Regarding the hydrogenation of DMO the key challenge is to identify a new method to stabilize the Cu surface. A method to maintain higher Cu dispersion and thermal stability against sintering would provide an excellent direction to improve catalytic performance. We show that using highly dispersed 3)2·3 H2O (Aldrich), Zn(NO3)2·6 H2O (Aldrich), and Ga(NO3)3·9 H2O (Aldrich). For a typical preparation, the metal nitrates  were dissolved completely in 100 mL deionized water. A Na2CO3 aqueous solution was prepared by dissolving 3.50 g of Na2CO3 in 100 mL of DI water. The solutions were added simultaneously into a plastic reactor containing 250 mL of preheated DI water. A delivery pump with two 50 mL syringes was used to inject the precursor metal nitrate solution at a constant rate of 0.42 mL/min in an automatic and reproducible manner. An HPLC pump was used to deliver the Na2CO3 solution at a rate of 0.35–0.70 mL/min. The mixture was stirred at 1000 rpm, with pH of the precipitating solution carefully maintained at 6.5. The precipitation process took place at around 80 °C. The pH of the liquid was measured using a temperature-dependent pH meter and was controlled at pH 6.5, with an error range of ± 0.1. Once the addition of the precursor metal nitrate solution was completed, the first aging was carried out under atmospheric conditions to let the pH value to become stable. After 30 min, the pH was measured again to ensure that the target pH had been reached before putting the lid onto the reactor. The resulting precipitate was continued to the second aging process in solution at 100 °C for 15 h. After aging, the precipitate was extracted by centrifugation at 5000 rpm. The centrifuged precipitate was washed with DI water five times at 5000 rpm to remove residual Na+ ions. The resulting wet solid was dried in air at 80 °C overnight and then calcined in static air, at a ramp of 5 °C/min up to desired temperature  for 3 h to produce the final catalyst. For the Cu/SiO2 catalyst, the synthesis details could be found in the literatureCuZnGa catalysts were synthesized using a pH-controlled co-precipitation method. The metal precursors were hydrated metal nitrate salts: Cu using a Series III digital HPLC pump . Under a given condition, the outlet stream was sampled using an automatic Valco six-port sampling valve at 30 min intervals after the reaction reached a steady state. The products were analyzed using an online gas chromatograph (Shimadzu GC-2010) equipped with a DB-Wax capillary column (30 m × 0.32 mm × 0.25 μm) and a flame ionization detector with a relative standard deviation (RSD) of less than 2%. The RSD of analysis data for each sampling was less than 3%. The products were also collected and confirmed using a 7890GC–5975 MS system.Catalytic reactions were conducted using continuous flow in a stainless steel tubular reactor equipped with a computer-controlled auto-sampling system. 100 mg of as-calcined catalyst with the particle diameter of 0.25 mm to 0.42 mm (40–60 meshes) was placed in the center of the reactor, and both sides of the catalyst bed were packed with quartz powders (40–60 meshes). Prior to the evaluation of the catalytic performance, the catalyst precursor was pre-reduced in a 5%HHow to cite this article: Li, M. M.-J. et al. The remarkable activity and stability of a highly dispersive beta-brass Cu-Zn catalyst for the production of ethylene glycol. Sci. Rep.6, 20527; doi: 10.1038/srep20527 (2016).


U–Pb analyses of zircon from ten samples of augen gneisses, eight mafic and intermediate metaigneous rocks and six metasediments from some tectonic domains along the Calabria–Peloritani Terrane  contribute to knowledge of peri-Gondwanan evolution from Late-Proterozoic to Paleozoic times. All samples were equilibrated under amphibolite to granulite facies metamorphism during the Variscan orogeny. The zircon grains of all considered samples preserve a Proterozoic memory suggestive of detrital, metamorphic and igneous origin. The available data fit a frame involving: (1) Neoproterozoic detrital input from cratonic areas of Gondwana; (2) Pan-African/Cadomian assemblage of blocks derived from East and West African Craton; (3) metamorphism and bimodal magmatism between 535 and 579 Ma, within an active margin setting; (4) rifting and opening of Ordovician basins fed by detrital input from the assembled Cadomian blocks. The Paleozoic basins evolved through sedimentation, metamorphism and magmatism during the Variscan orogeny involving Palaeozoic and pre-Paleozoic blocks. The Proterozoic zircon records decidedly decrease in the high grade metamorphic rocks affected by Variscan pervasive partial melting.The online version of this article (doi:10.1186/s40064-016-1839-8) contains supplementary material, which is available to authorized users. Amalgamation and break up of supercontinents and superterranes  characterize the history of the Earth between Neoproteozoic and Palaeozoic times. All geological processes known today, starting with Rodinia fragmentation and culminating with the assemblage of Pangea, have been the focus of research in recent decades , together with previous data collected through traditional methods (ID-Tims) are today available on different rock types exposed in this area  to granulite facies (high-grade metamorphic complex).The Calabria–Peloritani Terrane (CPT) is an “exotic terrane”  pre-Triassic basements with metamorphic and igneous rocks pertaining to the south-European Variscan belt; (2) fragments of Jurassic to lower Cretaceous Tethyan oceanic crust affected by Alpine HP/LT metamorphism; and (3) Mesozoic to Cenozoic sedimentary rocks at the bottom  felIn the Aspromonte Massif , this Unit is sandwiched between the lower “Madonna di Polsi” Unit . Details of procedures are in Fornelli et al. .The zircon domains from twenty-four samples here considered, show various spectra of ages having different geological significance Table . The majVariscan zircon domains occur and are decidedly abundant only in the granulite facies metasediments and metabasites of the lower crust of the Serre and in garnet–biotite gneiss from Pollino massif, whereas they are absent in the augen gneisses and metasediments of the Aspromonte–Peloritani Unit and Mandatoriccio Complex Table .Ordovician–Silurian domains characterize the zircons from Calabria (Aspromonte and Castagna) augen gneisses, deep crustal rocks of the Serre, garnet–biotite gneiss of Pollino and metasediments of Mandatoriccio complex. However, in the Mandatoriccio complex and in garnet–biotite gneiss from Pollino, the Ordovician–Silurian ages are related to detritic grains  includes rounded or fractured cores appearing as detrital domains Fig. a. AccordThe augen gneisses from Peloritani contain zircon grains giving ages ranging from 3200 to 520 Ma as clusters or single spots , some tens of million years earlier than the felsic magmatic precursor of the augen gneisses at 543–545 Ma  from the granulite facies metasediments of the Serre, and many ages in the range 325–270 Ma  values (Table The Nd model ages (at 540 Ma) of the Calabria augen gneisses range from 1700 to 1530 Ma  both calculated at 565 and 545 Ma  and ε0 Ma and Nd (550 Ma) values (−7.5 and −14.7) are lower than in the augen gneisses (Table Nd (540 Ma) value of −6.6  and the magmatic crystallization ages  and basic 579 ± 15 Ma) magmas  record an older history with respect to Mandatoriccio complex, sliver of garnet–biotite gneiss from Pollino and very low-grade Variscan metasediments. In fact the very low-grade metasediments in southern Calabria and Sicily contain porphyroids and meta-andesites having Ordovician ages  age of inheritance  Precambrian ages have been mostly erased in the granulite facies deep crustal rocks of the Serre Fig. ; (2) zirThe lack of Ordovician–Silurian records in the Peloritani metasediments Fig. b can be Neoproterozoic sediments derived from both West and East African cratonic sources, formed the protoliths of high-grade metasediments of the Serre, those of the Aspromonte–Peloritani Unit and probably even those of Castagna unit, all these metasediments were intruded by Neoproterozoic–Cambrian magmas so they are older.Panafrican orogenesis and consequent assemblage of blocks (having East and West gondwana affinities) involved these terranes as evidenced by metamorphism between 573 ± 3 Ma (this paper) and 535 Ma (Williams et al. Uplift and extensional tectonic in the Ordovician times with rifting and opening of Ordovician basins Fig. . This teVariscan orogenesis involved all described rocks recording zircon ages in the range 370–270 Ma only in high-grade metamorphites Fig.  in whichFrom the synthesis of the available data it appears that the Peri-Gondwana Calabria–Peloritani Terrane, affected by Variscan tectono-thermal events, preserves records of previous geological processes Fig. :Neoprote


The study of developmental processes in the mouse and other vertebrates includes the understanding of patterning along the anterior–posterior, dorsal–ventral and medial– lateral axis. Specifically, neural development is also of great clinical relevance because several human neuropsychiatric disorders such as schizophrenia, autism disorders or drug addiction and also brain malformations are thought to have neurodevelopmental origins, i.e. pathogenesis initiates during childhood and adolescence. Impacts during early neurodevelopment might also predispose to late-onset neurodegenerative disorders, such as Parkinson’s disease. The neural tube develops from its precursor tissue, the neural plate, in a patterning process that is determined by compartmentalization into morphogenetic units, the action of local signaling centers and a well-defined and locally restricted expression of genes and their interactions. While public databases provide gene expression data with spatio-temporal resolution, they usually neglect the genetic interactions that govern neural development. Here, we introduce Mouse IDGenes, a reference database for genetic interactions in the developing mouse brain. The database is highly curated and offers detailed information about gene expressions and the genetic interactions at the developing mid-/hindbrain boundary. To showcase the predictive power of interaction data, we infer new Wnt/β-catenin target genes by machine learning and validate one of them experimentally. The database is updated regularly. Moreover, it can easily be extended by the research community. Mouse IDGenes will contribute as an important resource to the research on mouse brain development, not exclusively by offering data retrieval, but also by allowing data input.Database URL:http://mouseidgenes.helmholtz-muenchen.de. Brain formation during vertebrate development is a complex process that has been studied for decades. The understanding of neuronal development is a prerequisite for the fight not only against neurodegenerative diseases, e.g. Parkinson’s disease, but also toward neuropsychiatric disorders in particular schizophrenia, autism disorders and drug addiction.The emergence of the neural tube from the neural plate and the patterning of these structures along their anterior– posterior, dorsal–ventral and medial–lateral axes are fundamental processes during vertebrate neural development. The formation of forebrain, midbrain, hindbrain and spinal cord is determined by well-defined and locally restricted expression of genes and their gene regulatory networks . WhereasPositioning of the future MHB is almost exclusively achieved by the cross-inhibitory interaction of orthodenticle homolog 2 (Otx2) and gastrulation brain homeobox 2 (Gbx2), two transcription factors initially expressed in the anterior and posterior part of the developing embryo, respectively. The inductive mechanism for these two and other factors of the IsO in the neural plate are still unknown. Wingless-type MMTV integration site family member 1 (Wnt1) and fibroblast growth factor 8 (Fgf8) are two factors secreted from the anterior and posterior region of the MHB, respectively. Wnt1 is required for the maintenance of the MHB, and Fgf8 is necessary for the patterning of the midbrain and rostral hindbrain. The engrailed genes En1 and En2 as well as the paired box transcription factors Pax2 and Pax5 act up- and downstream of Wnt1 and Fgf8, mediating their maintenance as well as patterning function at the MHB , the main disadvantage of immunohistochemistry often represents the lack of a functional antibody. Notably, for many of the genes expressed at the MHB suitable antibodies are not available.The gene expression in neural development has been subject to many large-scale studies, and the results were stored in publically available databases. The most important of these resources were recently reviewed  and in tin Atlas ], GenePain Atlas  and the in Atlas . Furtherin Atlas . SAGE isTo facilitate, for example, dynamic modeling approaches, which necessitate a priori knowledge, i.e. highly curated data, a comprehensive collection of known genetic interactions containing spatial and temporal information is essential . These dhttp://mouseidgenes.helmholtz-muenchen.de. The Web interface offers detailed information about the expression of genes and their genetic interactions in the developing mid-/hindbrain region. Stored data were already used in part to understand regulatory gene interactions on the systems level  are available from the database.We thus developed Mouse IDGenes, which represents a manually curated reference database for genetic interactions in the developing mouse brain focusing on the MHB, but with the possibility to add gene expression and interaction data of the central nervous system (CNS) with the help of a graphical user interface. The freely available database can be accessed via a Web interface through the URL ms level , 27. TheSupplementary Table S1). The development of the MHB and of the mid-/hindbrain region initiates after gastrulation is finished. At E10.5, the establishment of the IsO at the MHB is completed, and its function at this stage is well characterized, meaning that most of the known interactions are taking place at this time point. The neural tube already exists at this stage, whereas specific neuronal populations have not developed yet. These neuronal populations, however, are first identifiable at around E12.5. Our model about the mouse anatomy is based on data reviewed from literature (http://www.informatics.jax.org). To comply with the Edinburgh Mouse Atlas Project (EMAP), ontologies of mouse developmental anatomy, which provides a standard nomenclature for the description of normal and mutant mouse anatomy (Supplementary Table S1). Because EMAP ontologies were recently updated to the EMAPA ontology (Supplementary Table S1). The regionalization model was kept as general as possible, but as exact as necessary and covers initially the anterior–posterior compartmentalization of the neural tube into the brain vesicles and spinal cord. These brain vesicles correspond to the regions of the brain, which have already developed at a given developmental stage. Within these compartments, we further divided the regions on the anterior–posterior  axis into lateral and medial or dorsal and ventral parts depending on the corresponding developmental stage. Therefore, we developed a tripartition of a respective CNS region, which has the general structure of vesicle→anterior–posterior localization→medial–lateral or dorsal–ventral localization . Whenever no further division was made, the description ‘all’ was used.We introduced a CNS regionalization model, which covers developmental stages E8.5, E10.5 and E12.5  representing three crucial stages in the development of the murine MHB and mid-/hindbrain region  axis. For all CNS regions, the new regionalization on the dorso–ventral axis is roof plate (RP), alar plate (AP), alar basal boundary (ABB), basal plate (BP) and floor plate (FP). The prosencephalon separates along the anterior–posterior axis into the telencephalon and the diencephalon, the developing mesencephalon separates into anterior and posterior, the rhombencephalon splits into met- and myelencephalon and further into eight rhombomeres. The metencephalon was defined as consisting only of rhombomere one (r1).Because these subdivisions are not defined as sharply as before and distinct neuronal populations have already arisen or are arising in the mid-/hindbrain region of the E12.5 mouse embryo, we additionally defined individual neuronal populations and new subdivisions of the regions (compartments) tel-, di-, mesencephalon, MHB, met- and myelencephalon and also spinal cord. At this stage, the telencephalon as well as the diencephalon show additional subdivisions along the dorsal–ventral axis, and the mesencephalon is subdivided into regions or neuronal populations (or both) also along the dorsal–ventral axis. While at E12.5 the MHB is still subdivided along the anterior– posterior and dorsal–ventral axis, in the met- and myelencephalon as well as spinal cord, dorsal and ventral regions or neuronal populations (or both) are defined along the dorsal–ventral axis. Along the anterior–posterior axis, the diencephalon is now subdivided into anterior hypothalamus, posterior hypothalamus, prethalamus and thalamus. In addition, at E12.5, the developing spinal cord is subdivided into five anterior–posterior regions, namely cervical, thoracic, lumbar, sacral and caudal.http://www.postgresql.org). Gene expression and interaction data were manually extracted from literature and stored in the database including references. Currently, the database contains 89 expression data sets and 145 genetic interactions. Genetic interactions as well as expression data sets are assigned to different anatomical regions at the mouse embryonic developmental stages E8.5, E10.5 and E12.5, as described before. To assess the quality of our data, we compared all interactions with the STRING database (v 9.1), a comprehensive resource of protein–protein interactions  can be selected with the help of combo boxes. Having set a specific developmental stage and an anatomical structure, users are further able to analyze whether a specific gene of interest is expressed during that specific developmental stage in the selected anatomical structure.Navigation on the Mouse IDGenes Web page by the ‘Detailed Search’ tab allows users to search for gene expression and interaction data in specific regions of the embryonic mouse CNS at mouse embryonic developmental stages E8.5, E10.5 and E12.5 (radio button: ‘display genes according to the chosen region’). CNS regions and developmental stages . Information about the region and at which stage the specific interaction takes place in the embryonic mouse CNS can be retrieved.Users can also search whether two genes of interest display an overlapping gene expression (by the ‘Search for overlapping gene expressions’ button).By selecting ‘all’ in either both or only one of the gene selection boxes, users can search for all stored interactions either of the whole database or in which a specific gene of interest is involved.– direct: We define a direct interaction in case interaction partner 1 binds directly to the promoter of interaction partner 2.– direct signaling: In case of a ligand that activates a signaling cascade or any other component of a signaling cascade that does not directly interact with (or binds to) the promoter of a target gene of this signaling pathway, a direct signaling interaction refers to the activation/repression of a direct target gene of this signaling pathway.– indirect: Interaction partner 1 does not bind directly to the promoter of interaction partner 2, and signaling or genetic interaction cascades have to occur between the two interaction partners.– indirect signaling: In case of a ligand that activates a signaling cascade or any other component of a signaling cascade that does not directly interact with (or binds to) the promoter of a target gene of this signaling pathway, an indirect signaling interaction refers to the activation/repression of an indirect target gene by a direct target gene of this signaling pathway.– maintenance: Interaction partner 1 is not required to activate (i.e. turn on) or to repress (i.e. turn off) the promoter (or expression) of interaction partner 2, but to keep this promoter (or expression) activated (‘on’) or repressed (‘off’) over (a longer period of) time.– IEXP: Inferred from experiment (e.g. loss of function/gain of function) and/or expression patternThe displayed interactions follow an overall scheme consisting of the attributes ‘effect’, ‘type’ and ‘name’ for a genetic interaction. The attribute ‘effect’ can be either activation, i.e. turning on gene expression, or repression, which is defined as shutting down gene expression. The attribute ‘type’ of an interaction is defined by the following six values:Currently, the database contains these general interaction schemes: direct activation, direct signaling activation, IEXP activation, maintenance activation, indirect activation, indirect signaling activation, direct repression, direct signaling repression, maintenance repression, IEXP repression, indirect repression and indirect signaling repression. The attribute ‘name’ of an interaction, which is displayed on the Mouse IDGenes Web page, is composed of the official gene symbol according to MGI of interaction partner 1, followed by an arrow symbol from the third column of As pointed out before, the maintenance activation occurs over a longer period to cause a downstream effect. Time-wise, such an interaction can occur over several developmental stages as, for example, is the case of the development of midbrain dopaminergic (mDA) neurons. There, initially a Wnt1-regulated network together with a Shh-controlled genetic cascade establishes the mDA progenitor domain, by maintaining Otx2 expression in the ventral midbrain . AnotherFurthermore, cooperative interactions, i.e. interactions with more than two interaction partners, can be stored in the database, for example, if two transcription factors or one transcription factor and another cofactor will bind on the promoter of a target gene, i.e. interaction partner 2.Supplementary Table S1), by choosing and subsequently storing specifications given by combo boxes, which follow our CNS model  with ‘direct signaling’ targets (class 1) and ‘indirect signaling’ targets (class 2) using both parameters C. ClassiTo further elucidate the Wnt1-controlled gene regulatory network at the MHB, we applied SVM prediction by using the number of conserved Lef1/Tcf binding sites and the average matrix similarity in the promoters of four interesting genes, Fgf8, Lef1, Islet1 and Dkk3, representing the test set from B to predin vitro.To show the predictive power of our approach, we experimentally validated the so far unknown Wnt1 target gene Dkk3 as a direct target gene of the Lef1/Tcf-mediated Wnt1/β-catenin signaling cascade in vitro context. Additional in vivo evidence indicates that the murine Dkk3 gene is also a direct target of Lef1-mediated Wnt1/β-catenin signaling in the mouse ventral midbrain .To identify conserved Lef1/Tcf binding sites in ∼700 bp extended promoter region (as used for the SVM prediction) of the Dkk3 gene of five different mammalian species , we applied the DiAlign TF program in the Genomatix software suite GEMS Launcher to evaluate the overall promoter similarity and to identify conserved Lef1/Tcf binding sites in these regions. For the alignment, we chose the five most conserved Dkk3 promoter sequences among 14 organisms from Genomatix homology group Hg3927. Four Lef1/Tcf binding sites were predicted in the putative mouse Dkk3 promoter, of which one  was highly conserved among all five species A. To det– luciferace activity was significantly decreased relative to the wild-type mDkk3 promoter after co-transfection of Lef1 cDNA H,– the activation of the mDkk3 promoter/reporter construct carrying a mutagenized Lef1/Tcf binding site ‘c’ by Lef1 was completely abolished relative to the pcDNA3.1 control, in contrast to a still significant activation of the mDkk3 promoter/reporter constructs carrying a mutagenized Lef1/Tcf binding site ‘a’ or Lef1/Tcf binding site ‘b’ I.To evaluate whether the predicted Lef1/Tcf binding sites in the murine Dkk3 promoter are functional, we mutagenized each of these binding sites either individually (mutation of a single Lef1/Tcf binding site) or altogether  within the mDkk3 promoter fragment such that they cannot be recognized by Lef1/Tcf transcription factors anymore B–E 46)..46). SitThis result strongly suggests that the most conserved  and proximal (relative to the TSS) Lef1/Tcf binding site ‘c’ is the functionally most important of the three Lef1/Tcf binding sites for Lef1-mediated activation of the murine Dkk3 gene by Wnt1/β-catenin signaling.Lef1/Tcf transcription factors were predicted to directly activate the mouse Dkk3 gene in the context of Wnt1/β-catenin signaling by our SVM analyses. Therefore, the experimental validation of Lef1/Tcf binding site ‘a’ and Lef1/Tcf binding site ‘c’ with a matrix similarity of 0.867 and 0.961, respectively, confirm the result of our SVM classification, indicating that direct Lef1/Tcf targets have in general more conserved binding sites than indirect targets B. Altogein silico analyses related to gene expression and interaction data in the developing murine CNS. Additionally, a broader data set will lead to improvement of dynamic modeling projects and to more precise prediction methods  was applied. The data matrix of the SVM is composed of two variables (columns) for each object (row), i.e. the frequency as well as the averaged matrix similarity of conserved TFBSs for each promoter sequence of a specific gene. The classification of the interaction type in training data set for each object (gene), meaning whether Lef1/Tcf transcription factor binds directly or indirectly to the promoter sequence, was derived from the Mouse IDGenes database. Promoter sequences for Wnt/β-catenin target genes were derived from the ElDorado genome database (Genomatix/Germany), versions 12-2010 and 08-2011. Orthologous promoter sequences from different mammalian species of the Genomatix homology group  were analyzed using the MatInspector program (with the Matrix Family Library Version 8.4) from Genomatix to identify potential Lef1/Tcf binding sites and to extract matrix similarities. Conserved binding sites were determined by using the DiAlign TF program (Genomatix) and by searching for common Lef1/Tcf sites occurring at the same position in  orthologous promoter sequences of each Wnt/β-catenin target gene. The length of the promoter regions used for the detection of Lef1/Tcf sites to derive the total number and the average matrix similarity of binding sites both included in the SVM algorithm were generally in the range of 600–1400 bp. In addition, a longer promoter region of mouse Dkk3 mRNA  was defined as 1800 bp upstream, including the proximal region, and 200 bp downstream of the TSS. Dkk3 promoter sequences of 2000 bp length from five different mammalian species  were analyzed with the MatInspector to predict Lef1/Tcf binding sites.ctcgag TGACCA G A T C C A G C T TGCA-3′ and reverse primer 5′-aagctt CCTC CTGA GG GTAG TTGAGA-3′ that included an XhoI and HindIII restriction site , respectively. The amplified fragment was cloned into the pCR®II TOPO TA vector  and sequenced throughout its entire length (Sequiserve/Germany). The mDkk3 promoter fragment was excised from the pCR®II TOPO TA vector by XhoI/HindIII digestion and subcloned into an XhoI/HindIII-digested pGL3-Basic Vector (Promega/USA).A 744-bp fragment of the putative mDkk3 promoter  was amplified from C57BL/6 mouse genomic DNA by PCR using the forward primer 5′-ctgcgttcattc gaa ttgggtg-3′; Dkk3-mB: 5′-gtccaagagatcccagtaatagaacggatgg ggaaa tagta aaggaa-3′; Dkk3-mC: 5′-ggtggtcctgcagtctgcga tagctttccgggac-3′ . Mutated promoter fragments were confirmed by sequencing (Sequiserve).Site-directed mutagenesis of the most conserved and proximal (relative to the TSS) Lef1/Tcf binding sites predicted in the 744-bp mDkk3 promoter fragment was done using the QuickChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies/USA) according to the manufacturer’s instructions. Mutagenic primers were the following: Dkk3-mA: 5′-ccagcttgcagctcag2 in DMEM medium + 10% fetal calf serum/glutamine (Life Technologies). HEK-293T cells (1.25 × 105 cells/well of a 24-well plate) were co-transfected with 300 ng/well pGL3-mDkk3 promoter/reporter vectors (wild-type and mutagenized sequences), 30 ng/well pRL-SV40  and 150–225 ng/well pcDNA3.1 (Life Technologies) ‘empty’ vector, alone or together with 75 ng/well, 150 ng/well or 300 ng/well constitutively active ΔN-β-catenin (HEK-293T cells were kept at 37°C and 5% CO-catenin  or rat L-catenin  using Lit tests using the SPSS 18.0 software (SPSS Inc./USA). A value of P < 0.05 was considered significant.All values shown are mean ± SEM. Statistical significance between groups was assessed by two-tailed independent-samples Supplementary data are available at Database Online.


Rhodopseudomonas palustris TIE-1 and the Fe(III)-reducing bacteria Geobacter sulfurreducens, comparing magnetite nanoparticles (d ≈ 12 nm) against microparticles (d ≈ 100–200 nm). By integrating surface-sensitive and bulk-sensitive measurement techniques we observed a particle surface that was enriched in Fe(II) with respect to a more oxidized core. This enables microbial Fe(II) oxidation to occur relatively easily at the surface of the mineral suggesting that the electron transfer is dependent upon particle size. However, microbial Fe(III) reduction proceeds via conduction of electrons into the particle interior, i.e. it can be considered as more of a bulk electron transfer process that is independent of particle size. The finding has potential implications on the ability of magnetite to be used for long range electron transport in soils and sediments.The ability for magnetite to act as a recyclable electron donor and acceptor for Fe-metabolizing bacteria has recently been shown. However, it remains poorly understood whether microbe-mineral interfacial electron transfer processes are limited by the redox capacity of the magnetite surface or that of whole particles. Here we examine this issue for the phototrophic Fe(II)-oxidizing bacteria  Geobacter sulfurreducens or Shewanella oneidensis), which combine the oxidation of organic compounds or hydrogen with the reduction of minerals with short range order, such as ferrihydrite, often leading to the release of Fe2+aq and the precipitation of siderite (FeCO3) and magnetite (Fe3O4), depending on conditions present during formation Acidovorax sp BoFeN1Gallionella ferrugineaRhodopseudomonas palustris TIE-1), which fix CO2 and oxidize Fe(II) in the presence of lightThe ubiquity of Fe-bearing minerals in the environment has led to a number of bacteria developing pathways through which they can use Fe in different oxidation states as either electron donors or terminal electron acceptorsrite FeCO and magntite Fe3O, dependi2+ R. palustris can use a solid electrode as an electron donorc-type cytochrome MtoA purified from a microaerophilic Fe(II)-oxidizing bacteria (Sideroxydans lithotrophicus ES-1) can oxidize the surface of magnetite and significantly change its stoichiometry10111416To date, much of the Fe which is bioavailable to Fe(II)-oxidizing bacteria has commonly been considered to originate from aqueous or complexed Fe2+ and Fe3+ ions in octahedral (Oh) and tetrahedral (Td) coordination according to the formula Fe2+OhFe3+TdFe3+OhO42−. Fe2+-Fe3+ valence alternation electron exchange between adjacent Oh sites is fast at room temperature (~1012 s−1) enabling low bulk resistivity. The spin imbalance between the antiparallel Oh and Td iron sublattices leads to the ferrimagnetic properties of the mineral, as the magnetic moments of the octahedral site ions (Fe2+ and Fe3+) are aligned anti-ferromagnetically with the tetrahedral site ions (Fe3+), resulting in the moments of the ferric ions effectively cancelling each other out and leaving the net magnetic moment to be due to the Fe2+Oh ions. Consequently, the magnetic properties of magnetite are highly sensitive to changes in stoichiometry. This is exemplified in the topotactically isomorphic mineral maghemite ((Fe3+Td[Fe3+5/3□1/3]OhO42−), which contains no Fe(II) and where □ represents a vacancy. Maghemite is known to have a lower bulk saturation magnetization (Ms) of ~75 A m2 kg−1 at room temperature in comparison to the Ms = 92 A m2 kg−1 for stoichiometric bulk magnetiteTV) which occurs at ~121 K and is a result of magnetite undergoing a first order phase transition from cubic crystal symmetry to a lower symmetry (likely monoclinic) structureTV is known to be exceptionally sensitive to the presence of additional elements, e.g. Ti4+ 3+ Magnetite is a mixed-valence material, based upon high-spin FeR. palustris) and Fe(III)-reducing bacteria (G. sulfurreducens) with two distinct types of magnetite, nanoparticles (d ≈ 12 nm) and microparticles (d ≈ 100–200 nm) in order to test the effect that surface area to volume ratio has on the microbe-mineral electron transfer processes. We combine synchrotron-based, surface-sensitive x-ray magnetic circular dichroism (XMCD) with bulk sensitive 57Fe Mössbauer spectroscopy to probe the impact of both Fe(II) oxidation and Fe(III) reduction on the stoichiometry of the magnetite, with ancillary applied magnetic measurements and transmission electron microscopy (TEM) based imaging and spectroscopic information to further inform our findings.Many questions remain about how Fe-metabolizing bacteria interact with magnetite, especially in their ability to use it as an electron donor or electron acceptor . In particular, it is unclear to what extent the bacteria are able to access Fe in the whole magnetite particle, or whether such biogenic redox processes are only confined to the surface of the magnetite particle. To answer this, we have investigated the ability for separate cultures of Fe(II)-oxidizing and Fe(III)-reducing bacteria to use magnetite with significantly different particle sizes. Here we have incubated Fe(II)-oxidizing bacteria  to 634.1 ± 1.7 × 10−6 SI), consistent with previous observations−6 SI to 718.3 ± 10.7 × 10−6 SI). Application of the T-test indicated that differences between the relative changes of the MS of Nano-ox and Micro-ox were statistically different (i.e. P < 0.05) at all time points. This result was expected due to the much higher surface area to volume ratio of the Nanomag compared to Micromag magnetite. However, changes under Fe(III)-reducing conditions are less clear (−6 SI to 816.7 ± 2.6 × 10−6 SI) compared to 8.0% for Micromag relative to the starting value (from 757.9 ± 21.3 × 10−6 SI to 818.6 ± 11.8 × 10−6 SI). Furthermore, the differences between the two are not statistically significant with overlapping error bars until the final time-point measurement (24 h). The T-test showed that the relative changes of the MS of Nano-red and Micro-red were not statistically different from each other (i.e. P > 0.05) at all time points until the final measurement. These data suggest that the surface area to volume ratio did not play a major role in the differences between Nanomag and Micromag reduction by Geobacter sulfurreducens. In all experiments, control groups showed some minor changes in MS, however these were determined to be statistically insignificant from setups containing bacteria.Two types of magnetite were synthesized for these experiments, denoted Nanomag and Micromag in reference to their respective sizes, i.e. Nanomag corresponds to magnetite nanoparticles and Micromag corresponds to magnetite microparticles. Magnetic susceptibility (MS) measurements enable direct measurements of the magnetic changes to the microbe-magnetite cultures y sample . From thy sample  occurredss clear . The MS ex situ mineralogical analysis using XMCD and Mössbauer spectroscopy to more effectively understand the changes induced by the bacteria. XMCD spectra  and Fe(III) oxidation states and their coordination environment, with the lowest energy negative peak matching Fe2+Oh, the positive peak matching Fe3+Td and the second negative peak matching Fe3+Oh 3+Td site because it is generally the most stable iron site in the magnetite structure, with each displaying the characteristic signature expected for magnetite2+Oh peak (708 eV), in which the intensity of Nano-ctrl is greater than that of Nano-ox. However, in the case of Micromag it appears as though the intensity of the Fe3+Oh and Fe2+Oh peaks have increased after oxidation (Micro-ox) in comparison to the starting material (Micro-ctrl). There were no obvious differences between the spectra of Nano-ctrl and Nano-red which suggests that no reduction of the Nanomag took place despite the evidence to the contrary shown by MS and Mössbauer (see below) data. The relative intensity of both octahedral Fe sites appears to increase for the microparticles, relative to Fe3+Td.Following the completion of MS experiments, samples were dried for  spectra  were col2+Oh is far greater than would be expected of stoichiometric magnetite (Fe(II)/Fe(III) = 0.5), with the Fe(II)/Fe(III) ratio ranging from 0.64 to 0.75 across the samples. This suggests that all of the magnetite nanoparticles investigated were highly reduced at their surfaces. Such a result has previously been observed for magnetite particles by Liu et al.R. palustris can be observed, with Fe(II)/Fe(III) decreasing from 0.75 to 0.64 for Nanomag, and decreasing from 0.75 to 0.70 for Micromag. There was little difference observed between the Nano-ctrl and Nano-red XMCD spectra, though a decrease in Fe(II)/Fe(III) from 0.75 to 0.73 respectively was calculated which in fact suggests some oxidation. The Fe(II)/Fe(III) ratios of Micro-ctrl and Micro-red were calculated to be 0.75 and 0.71 which would also suggest some oxidation.Based on fitting theoretical component spectra from atomic multiplet calculations, the relative intensity of each lattice site was determined . In all 57Fe Mössbauer spectroscopy was performed on all six samples at 140 K /Fe(III) ratio of 0.40 and 0.41 for Nanomag and Micromag, respectively, consistent with the observed Fe(II) enrichment at surfaces deduced from XMCD spectroscopy. Microbial Fe(II) oxidation of the nanoparticles leads to a notable decrease in the bulk Fe(II)/Fe(III) ratio, whilst reduction leads to a slight increase, following the expected trends. For Micromag, however, Fe(II)/Fe(III) ratio of the oxidized material remains largely unchanged (within error). The reduced microparticles show a small but significant increase, which is as expected. Attempts to calculate the extent of Fe(II) oxidation based on Mössbauer Fe(II)/Fe(III) ratios have been made see . These cFurther analysis of the changing properties of the magnetite induced by microbial interaction was carried out using low temperature superconducting quantum interference device (SQUID) magnetometry in conjunction with field-cooled and zero-field-cooled saturation magnetisation isothermal remanence magnetisation (FC-ZFC-SIRM) and room temperature SIRM (RT-SIRM) experimental protocols. Furthermore, Alternating Current (AC) field dependent measurements were also collected .TV)TV . This behaviour is not observed in either Nano-ctrl or Nano-red and is likely related to the effect of oxidationTV, which is present in the other samples. χ″ of the microparticles do not show clear differences.RT-SIRM curves show differences due to oxidation/reduction . This isHc) in comparison to the larger microparticles. Given the mean diameter of the Nanomag particles (12 nm), one would expect to see room temperature hysteresis loops dominated by superparamagnetic behaviour, whereas the larger mean grain sizes in the Micromag sample are expected to show more elevated coercivities and discernable hysteresis. The saturation magnetisation values (Ms) of the starting materials are close to the theoretical value of 92 A m2 kg−1 expected of magnetite at room temperature. The slightly smaller value observed for Nano-ctrl could again indicate that the sample is slightly oxidized, or due to the effect of its small particle sizeThe hysteresis loops  are charMs were observed for Nano-ox (−23%) and Micro-ox (−9%) compared to their respective starting materials. For the reduction experiments, the Ms for Nano-red decreased (−13%) which is suggestive of an oxidation process, whereas Micro-red Ms increased by (+6%). Very small differences were observed between the coercivities of the nanoparticle and microparticle series.Decreases in d = 12.3 nm (σ = 0.3 nm) and Nano-ox having d = 12.0 nm (σ = 0.3 nm), however, Nano-red appeared to show a slight increase in particle size in comparison to the other samples with d = 13.3 nm (σ = 0.3 nm). This is somewhat contradictory to previous studies, which suggest that Geobacter sulfurreducens can lead to the dissolution of magnetite11Both Nanomag and Micromag were imaged with TEM and SEM respectively. In each of the Nanomag series, an amorphous layer appeared to be present around all samples  and thoux ≈ 0.64–0.75) was far in excess of the expected stoichiometric value of 0.5. In comparison, the Mössbauer data indicated that both nanoparticles and microparticles were slightly oxidized (x ≈ 0.38–0.43). The XMCD signal is surface sensitive with an exponentially decreasing probing depth of 4.5 nm, whilst Mössbauer works via transmission and will probe the entire crystal. This suggests that the surfaces of the particles are highly enriched in Fe(II) compared to their cores. Scanning transmission electron microscopy–high angle annular dark field (STEM-HAADF) mapping was applied on Micro-ox, because their larger crystal sizes were more accessible to this technique, and confirmed such a surface layer to be present (3 buffer (pH 7) produces an oxidized interior (as shown by XRD data) while the surface region remains enriched in Fe(II) relative to Fe(III) (as shown by XMCD data)One of the most striking results of this study is the difference in stoichiometry of both the Nanomag and Micromag starting materials observed by XMCD compared to Mössbauer spectroscopy. The amount of Fe(II)/Fe(III) ratio determined by XMCD ; this simultaneous accumulation of an Fe(III) product at the surface results in a larger net decrease in the surface Fe(II)/Fe(III) ratio, whilst the core remains relatively intact. The surface saturation with Fe(II), however, may also hinder the electron transfer by Fe(III)-reducing bacteria as the surfaces of the magnetite particles are already so highly reduced that any further reduction of the surface is not or hardly possible. Instead, we suggest that an electron transport process must be enlisted to enable reduction to proceed, transferring charge through the surface via an electron hopping mechanisms to the core of the magnetite and leading to the overall reduction of the core of the particles, a process that could manifest the observed changes to the magnetic susceptibility.The high abundance of surface Fe(II) in these synthetic magnetites is likely one of the reasons why the Fe(II)-oxidizing and Fe(III)-reducing bacteria behave so differently with respect to the surface and the bulk. As the ratio of Fe(II)/Fe(III) determined using XMCD  appears to be high, it is possible that the magnetite surfaces are saturated with a supply of Fe(II), which the Fe(II)-oxidizer et al. (2016)41The low temperature magnetic properties of the samples reveal broad similarities between the reduced and starting materials as compared to the oxidized samples. The most notable changes occur in the RT-SIRM data for Nano-ox. Whilst the initial change in remanence magnetization is the smallest for Nano-ox samples on cooling, the warming curve shows dramatic differences to the other samples. These data suggest that low-temperature magnetic methods can distinguish magnetite particles which have undergone oxidation  from those that have not been oxidized. However, the reduction of both magnetite nanoparticles and microparticles does not yield significant differences in low temperature magnetic properties, which potentially rules it out as a diagnostic tool for such processes. Notwithstanding, it is evident that reduction does lead to significant changes to the room temperature volume dependent magnetic susceptibility , which cThe results presented here have several implications in terms of the interactions of bacteria with magnetite and other redox active Fe minerals in the environment. Perhaps most important is the relevance to the processes by which long range electron transport is mediated by extracellular electron transfer. For instance, the different methods through which Fe(III)-reducing bacteria are able to use solid electron acceptors, such as ferrihydrite, require either direct contact (operating over distances <1.8 nm), the production of chelators (nm-μm), use of electron shuttles (nm-μm), or the formation of nanowires, which are appendages that appear to facilitate electron transport over several micrometers. However, recently a number of different studies have attempted to learn more about these types of long range electron transport2), making it a suitable candidate for environmental remediation14154716Finally, these findings also have implications for the use of magnetite, predominantly in the form of nanoparticles, for environmental remediation strategies of waste water and contaminated soils and sedimentsi) the identification of biomarkers and changes to redox conditions in the rock record using magnetic signatures; (ii) the suitability of magnetite in mediating long range intercellular electron exchange within soils and sediments; (iii) use of magnetite as a remediation agent for treating wastewater or contaminated soils and sediments.We have shown that magnetite can undergo microbial redox processes as both nanoparticles and microparticles. Whilst microbial Fe(II) oxidation appears to be highly sensitive to the surface area to volume ratio of the magnetite, this relationship does not appear to be valid for microbial Fe(III) reduction. Instead we suggest that microbial Fe(III)-reducers drive an electron hopping mechanism through the particles, thus rendering it a bulk dependent effect. Such differences between surface/bulk processes could potentially have significant impact on  and FeCl3 (2 M) in HCl (0.3 M) was added dropwise into an anoxic solution of NH4OH (25%) under continuous stirring at 800 rpm in an anoxic glovebox (100% N2). This process led to the instantaneous precipitation of magnetite nanoparticles (d ≈ 12 nm), which were washed twice in ultrapure deionized water to remove chloride ions. The point of zero charge was determined to be pH 6-7 for the starting material and the long term stability of stoichiometry was determined via micro x-ray diffraction (μ-XRD) and X-ray magnetic circular dichroism (XMCD)Nanomag was synthesised at room temperature using the protocol outlined in Pearce 4 solution was stored in the presence of zerovalent Fe for one month prior to reaction to ensure no Fe(III) was present. Subsequent oxidation of the FeSO4 in an alkaline solution of KNO3 at 90 °C under a N2 atmosphere yielded the magnetite particles, which were dried and stored under N2 until analysis. Analysis following new hybrid oxidi-colorimetric method Amonette and MatyášTotal and 21.84 ± 0.23 wt% Fe(II).Micromagnetite particles were prepared following the modified method of Schwertmann and CornellRhodopseudomonas palustris strain TIE-1 was grown on modified basal mediumGeobacter sulfurreducens was cultivated on modified fresh water mediumR. palustris and G. sulfurreducens were grown without Fe until the late log phase after which they were washed by repeated cycles of centrifugation and resuspension in anoxic NaHCO3 buffer . After final centrifugation dense cell suspensions were prepared for each strain in 7.5 ml and 7.0 ml for R. palustris and G. sulfurreducens, respectively.3 buffer. Aliquots of magnetite (10 mg) were added to bottles from stock solutions and allowed to equilibrate for 3 days. Acetate (1 mM) was added to all reduction experiment bottles. Dense cell suspensions of either R. palustris or G. sulfurreducens were added into the bottles, equivalent to 100% (v/v) inoculum. Fe(II) oxidation experiments were incubated in the light at 20 °C for 48 h until changes in magnetic susceptibility no longer appeared to decrease. Fe(III) reduction experiments were incubated in the dark at 30 °C for 24 h, at which point magnetic susceptibility data showed no significant increase and the experiments were terminated to prevent potential mineral dissolution11Experiments were prepared in 50 ml headspace vials containing 25 ml of modified basal medium, pH 7, NaHCO2) to prevent abiotic oxidation. The samples were loaded into the Portable Octupole Magnet System (POMS) on beamline I10 at the Diamond Light SourceL2,3 edge (690–740 eV) with spectra recorded using total electron yield (TEY) detection in energy steps of 0.2 eV with an effective probing depth of ~45 Å 2+Oh, Fe3+Td and Fe3+Oh, where Oh and Td denote octahedral and tetrahedral coordination, respectively, fitted against calculated spectra54Dq crystal-field parameters taken as 1.4 and −0.7 eV for Fe Oh and Td sites, respectively. The results were convoluted by a Lorentzian with a half-width at half-maximum of Γ = 0.3 (0.5) eV for the L3 (L2) edge to account for intrinsic core-hole lifetime broadening and by a Gaussian of σ = 0.2 eV to account for instrumental broadening.For synchrotron-based XMCD measurements, dried magnetite samples were loaded onto carbon tape, which was fixed to a sample holder. All sample preparation was carried out in an anoxic glovebag (100% N2) by mixing the dried powder (~10 mg) with glucose (80 mg) and ground using a pestle and mortar to ensure a homogenous sample. Magnetite-glucose mixtures were loaded into Plexiglas holders (area 1 cm2) and then loaded into a closed-cycle exchange gas cryostat (Janis cryogenics) under a backflow of He. Spectra were collected at 140 K with a constant acceleration drive system (WissEL) in transmission mode with a 57Co/Rh source and calibrated against a 7 μm thick α-57Fe foil measured at room temperature. Spectral fitting was carried out using an Voigt based fitting (VBF) routine in the Recoil software (University of Ottawa)Mössbauer spectroscopy was carried out at the University of Tübingen, with samples prepared in a glovebox (100% Nn ≈ 150 for each sample.Scanning transmission electron microscopy images (STEM) and electron energy loss spectroscopy (EELS) were carried out using aberration corrected electron microscope, including JEOL JEM-ARM200CF microscope operated at 200 kV and FEI Titan 80–300 operated at 300 kV. Both microscopes are equipped with a probe spherical aberration corrector and post column attached with a Gantan Quantum EELS system for spectroscopic imaging. For the STEM-high angle annular dark field (HAADF) imaging, the inner and outer collection angles of annular dark field detector were set at 68 and 280 mrad, respectively. For EELS spectroscopic imaging, the energy resolution is typically in the range of 0.8 eV. The high resolution TEM image were collected using FEI Titan 80–300 operated at 300 kV and fitted with an aberractor for objective lens. The average particle size was determined by measuring the longest length of individually resolvable particles and a population size of Scanning electron microscopy (SEM) analysis was performed to analyse particle morphology amongst control and reacted, reduced and oxidized, magnetite samples. Samples were prepared for imaging by fixation of magnetite powders to carbon tape and carbon coating with a thickness of ~10 nm. SEM imaging was performed on Quanta 3D FEG, equipped with an energy-dispersive X-ray detector for qualitative analysis at acceleration voltage of 5 to 10 keV.In-situ magnetic susceptibility was measured on triplicate experiments at room temperature with a KLY-3 Kappabridge , at a peak magnetic field of 300 A m−1 and a frequency of 875 Hz. Bottles  containing the magnetite, media and bacteria were loaded directly into the instrument for each measurement. The susceptibility of the input vessel was subtracted from the final value to account for diamagnetic signal of the glass and media. The T-test function was applied to magnetic susceptibility data using two tails and two-sample unequal variance . The test was applied to experiments carried out in triplicate, with direct comparisons made between Nano-ox:Micro-ox, Nano-red:Micro-red, Nano-ox:Nano-ctrl, Micro-ox:Micro-ctrl, Nano-red:Nano-ctrl and Micro-red:Micro-ctrl. A value of P < 0.05 is considered to be statistically significant.2, 5% H2). Powdered sample were put between two layers of quartz wool packing material and sealed inside pharmaceutical gelatine capsules, which were then sealed with Kapton tape to maintain anoxic conditions. The capsules were transported to the instrument within N2 filled Eppendorf vials and only removed immediately prior to loading into an instrument. Several low temperature magnetic measurements were performed using a Quantum Design MPMPS-2 superconducting quantum interference device (SQUID) magnetometer, including (i) field-cooled (FC) and zero-field-cooled (ZFC) low temperature saturation isothermal remanence magnetization (LTSIRM) sweep curves which were obtained by applying 2.5 T during cooling from 300 K to 20 K with FC remanence magnetization measured during warming from 20 K to 300 K in the absence of an applied field. The samples were then cooled to 20 K in the absence of a field, magnetized by a field of 2.5 T, which was switched off before warming the sample to 300 K during which ZFC remanence magnetization measurements were recorded in steps of 5 K. First order differentiation was performed on FC and ZFC curves for all samples to more effectively identify sudden changes in the remanence magnetization curves. (ii) Room temperature saturation isothermal remanence magnetization (RT-SIRM) curves were obtained by first applying a magnetic field of 2.5 T at 300 K, which was switched off immediately prior to measuring the magnetization during thermal cycling to 20 K and back to 300 K. (iii) In-phase (χ′) and quadrature phase (χ″) frequency dependent magnetic susceptibility measurements were performed at 1, 10 and 100 Hz.Low temperature magnetic measurements were carried out on all of the dried magnetite samples at the Institute for Rock Magnetism, University of Minnesota, USA. Samples were transported in individual anoxic bags, which were only opened within a glovebox  with applied magnetic fields between −1 T and 1 T.How to cite this article: Byrne, J. M. et al. Size dependent microbial oxidation and reduction of magnetite nano- and micro-particles. Sci. Rep.6, 30969; doi: 10.1038/srep30969 (2016).


To determine the effect of intravenous iron supplementation on performance, fatigue and overall mood in runners without clinical iron deficiency.−1 were randomly assigned to receive three blinded injections of intravenous ferric-carboxymaltose  or normal saline (PLACEBO) over four weeks . Athletes performed a 3,000 m time trial and 10×400 m monitored training session on consecutive days at week 0 and again following each injection. Hemoglobin mass (Hbmass) was assessed via carbon monoxide rebreathing at weeks 0 and 6. Fatigue and mood were determined bi-weekly until week 6 via Total Fatigue Score (TFS) and Total Mood Disturbance (TMD) using the Brief Fatigue Inventory and Brunel Mood Scale. Data were analyzed using magnitude-based inferences, based on the unequal variances t-statistic and Cohen's Effect sizes (ES).Fourteen distance runners with serum ferritin 30–100 µg·L−1; p = 0.002) and remained elevated two weeks after the final injection , without significant changes in Hbmass. Supplementation had a moderate effect on TMD of IRON (ES -0.77) with scores at week 6 lower than PLACEBO . Similarly, at week 6, TFS was significantly improved in IRON vs. PLACEBO . There were no significant improvements in 3,000 m time in either group ; but IRON reduced their average time for the 10×400 m training session at week 2 .Serum ferritin increased in IRON only (Week 0: 62.8±21.9, Week 4: 128.1±46.6 µg·LDuring 6 weeks of training, intravenous iron supplementation improved perceived fatigue and mood of trained athletes with no clinical iron deficiency, without concurrent improvements in oxygen transport capacity or performance. Iron is an essential nutrient for the optimal functioning of the human body −1, with iron deficiency anemia diagnosed when iron stores are exhausted (serum ferritin <15 µg·L−1), and consequently hemoglobin (Hb) levels fall below 12 g·L−1Iron status is influenced by both intake from dietary sources and iron loss through sweat and urine −1 using a placebo-controlled blinded design. Reduced feelings of perceived fatigue were observed following treatment but without concomitant changes in hemoglobin concentration ([Hb]), leading the authors to speculate that the non-hematological functions of iron, namely its involvement in the formation of key enzymes involved in oxidative metabolism Iron supplementation, typically via the oral route, is commonly used for the treatment of iron deficiency. Recently, carbohydrate-encased intravenous (IV) compounds such as ferric carboxymaltose have proven successful in treating iron-deficiency anemia −1 and transferrin saturation <20%, or serum ferritin <15 µg.L−1) or “suboptimal” (serum ferritin <65 µg.L−1) iron status. Whilst both forms of supplementation improved iron status, the increase in serum ferritin was greater in the IV treated group and improvements in hemoglobin mass (Hbmass), maximal aerobic power (VO2max) and run time to exhaustion were evident only in the IV treated athletes with initial “low” iron status. Increases in Hbmass were not observed in the “suboptimal” group suggesting that their iron stores did not limit erythropoiesis or aerobic capacity. Unfortunately, neither subjective measures nor performance per se were formally assessed by Garvican et al. Indeed, the chosen delivery route , and subsequent bioavailability of the supplemented iron may also be a contributing factor to relatively novel findings of the Krayenbuehl study At present, the optimal iron status for overall wellbeing and training ability  in athletic populations is unclear. Previous investigations of the benefit of oral supplementation for endurance performance in athletes without a clinical iron deficiency, i.e. “normal” iron status, have been equivocal Given the efficacy of the newer formulations of iron  at improving fatigue in clinical settings The study was approved by both the Australian Institute of Sport Human Ethics Committee and the University of Canberra Human Research Ethics Committee. All athletes provided written informed consent prior to participation.−1) were randomly assigned to receive either 2 ml (100 mg) of ferric carboxymaltose (IRON) or normal saline (PLACEBO) intravenously every fortnight for four weeks. Each IV injection was closely followed by a 3000 m time trial run and a monitored training session consisting of an all-out 400 m time trial and 10×400 m training session, performed on consecutive evenings during the months of June and July in Canberra, Australia, with mean environmental temperature of 10.4°C. Iron status, mood and fatigue were examined throughout, with Hbmass assessed pre and post the intervention period  with serum ferritin between 30–100 µg·L−1 (determined in duplicate prior to inclusion in the study) were screened by a medical doctor to determine their suitability for participation. Athletes excluded from participation were those currently taking oral supplementation or presenting with iron deficiency anemia, anemia not related to simple iron deficiency, serum ferritin <30 µg.L−1 or> 100 µg.L−1, previously documented hypersensitivity to iron, iron overload, acute illness, renal and endocrine disorders, pregnancy, or those having completed altitude training within one month prior.Non-anemic athletes  were recruited from local distance running groups . All ath−1 bolus of CO was rebreathed through a closed-system spirometer for two minutes. A CO-oximeter  measured the percent carboxyhemoglobin of capillary blood at 0 and 7 minutes post-rebreathing, with five replicates of each sample performed where possible. Typical error, calculated previously from duplicate measures on a separate group of distance runners was 1.6 % (90% CL 1.3 to 2.2%) Hemoglobin mass was measured prior to the study, and two weeks following the final injection (week 6) using the optimized carbon monoxide (CO) rebreathing method of Schmidt and Prommer One to two weeks before the study commenced, two 3000 m time trials and one 400 m time trial were performed within one week on a synthetic 400 m running track for familiarization.2max, assessed at the start of the study as described previously Participants were pair-matched by an independent researcher based on their initial serum ferritin level, Hbmass, personal best time for 3000 m and aerobic fitness  was obtained via venepuncture by a trained phlebotomist to assess [Hb], hematocrit (Hct), and iron status . Whole blood was analyzed within four hours of collection , whilst iron status was assessed from serum . If Hct values exceeded 50%, or serum ferritin values exceeded 200 µg·LParticipants were assessed fortnightly for six weeks for their perceptions of fatigue and mood disturbance using the Brief Fatigue Inventory (BFI) Mood was assessed using the Brunel Mood Scale (BRUMS) 2max, and has been shown previously to be a reliable indicator of running performance in trained runners The day following each injection , and approximately two weeks after the completion of the supplementation period (week 6 follow-up), participants completed a maximal effort 3000 m time trial individually on a synthetic outdoor 400 m (7.5 laps) running track  to assesIn order to assess the athletes' ability to recover from the time trial, and their subsequent readiness to train, a monitored training session was completed 22–24 hours after the 3000 m time trial at week 0, 2 and 4. The timeframe between the time trial and monitored training session was ″24 h shorter than typically experienced by the athletes, but is reflective of competition schedules at major championship meets where athletes may be required to complete heats and finals on consecutive days. After a standardized self-selected warm-up, participants completed an all-out 400 m time trial individually. BLa and RPE were recorded three minutes post-trial. After 10 minutes of active recovery, athletes undertook as a group a training session consisting of 10×400 m, on a two minute time cycle , with inHow ready do you feel to train today? How easy was it to get out of bed this morning? How energized do you feel today? How much did you give in your training today? How sore are you feeling?” on a scale of 1–5. Athletes were also instructed not to change their normal dietary practices, and to refrain from taking any oral iron supplements.Participants were asked to record their training daily using an electronic training diary for the duration of the study period. In addition to recording total distance and training time, they were asked to rate their feelings related to “within each group over the duration of the study, as well as between the groups for the differences in their respective changes from baseline measures. Prior to analysis, raw data were log transformed to reduce non-uniformity of errors. The magnitude of an effect and the associated P-value was presented as percent change and standardized mean differences (ES); calculated as the difference in mean divided by the between-subject standard deviation (SD); where a small effect is> 0.2, moderate> 0.6 and large> 1.2 unclear if the confidence interval overlapped the thresholds for the smallest positive and negative effects. Data are reported as mean ± SD unless otherwise stated.Potential differences between groups at baseline were assessed using a Student's t-test, with significance set to p≤0.05. Otherwise, magnitude-based inferences were used in order to define the practical significance of the results using the unequal variances t-statistic and Cohen's Effect Sizes (ES) The overall change in the subjective parameters of the training diaries was assessed with within-individual linear regressions  as described by others There were no significant differences between the IRON and PLACEBO groups at the start of the study in terms of baseline characteristics , hematol−1; p = 0.002) and remained elevated two weeks after the final injection  effect on Total Mood Disturbance , with vaWithin the IRON group, supplementation resulted in a trend to decrease the Total Fatigue Score at week 6 , but the latter large effect size failed to attain statistical significance . BetweenMean 3000 m run time did not change significantly within IRON during the study but PLACEBO were slower than week 0 at week 4 (p = 0.04). Therefore, at week 4 there was a small effect on 3000 m time between the groups, with IRON faster than PLACEBO ; All-out 400 m time did not change significantly within or between either group over the course of the study . DiffereThe average time for the 10×400 m session of the IRON group was faster at week 2  compared with week 0. There were no significant differences within PLACEBO over time , nor werDay-to-day mean training diary scores improved in the following areas within the IRON group only: “How easy was it to get out of bed this morning?”, “How ready do you feel to train today?”, and “How energized do you feel today?” . The obs−1, without concomitant changes in Hbmass or [Hb]. Supplementation had no acute effects on 3000 m running performance; however iron supplemented athletes decreased their 10×400 m training set time at week 2, and recorded improvements in their perceptions about their preparedness to train, which collectively may indicate a potential training benefit of IV iron for the treatment group.The main finding of the present study was that, compared with PLACEBO, three fortnightly IV injections of ferric carboxymaltose over 4 weeks improved self-reported perceptions of fatigue and reduced mood disturbance in trained distance runners with “clinically normal”serum ferritin levels between 30 and 100 µg·L−1−1, functional compounds become compromised, including but not limited to hemoglobin, myoglobin and cytochromes −1), whose Hbmass was therefore unlikely to be compromised The most widely accepted clinical threshold for iron deficiency is 22 µg·LIntravenous ferric carboxymaltose supplementation increased serum ferritin throughout the supplementation period. Further, serum ferritin remained elevated by approximately double the pre-treatment levels two weeks following the final injection. However, Hbmass and [Hb] did not change, confirming previous research −1, erythropoiesis and thereby oxygen transport, are unlikely to be affected When storage iron is> 35 µg·LThe presence of blood lactate, a gross indirect measure of anaerobic metabolism, reflects the contribution of each of the body's energy systems with which an exercise effort is undertaken. A decrease in BLa within the IRON group was observed following the week 2 and week 6, 3000 m time trials, which is similar to previous findings Iron deficiency without anemia can result in a greater energy expenditure during a simulated time trial There is evidence that iron insufficiencies impact mental function and emotional health as a result of reduced neurotransmitter activity in the brain and central nervous system Despite careful pair matching of subjects based on initial ferritin, Hbmass and performance characteristics, the PLACEBO group reported lower fatigue scores on the one of the two questionnaire scales (BFI) at the start of the study. The BFI is a tool typically used to assess cancer-related fatigue but has been used previously in a non-athletic population to assess iron related fatigue No clear acute or chronic benefits of iron supplementation on 3000 m running performance were observed although consistently similar performances were evident throughout which was not the case in the PLACEBO group. Measuring a true maximal effort of an individual as well as obtaining optimal performance on a particular day, remain inherently difficult This study demonstrated improvements in fatigue and mood disturbance in the IRON group during a 6 week training period. Whether or not such improvements in fatigue and mood  may lead to better training quality and subsequently improved performance over a longer period of time cannot be determined from this study. However, the long term or repeated use of iron supplementation in athletes who are not iron deficient should not be advocated. In the short-term, the efficacy of IV Iron supplementation for athletes with moderate ferritin levels also requires careful consideration, because a performance improvement remains unclear. For this reason the data in this study do not provide strong evidence for IV iron supplementation in athletes with clinically normal ferritin levels, especially when taking into account the ethical and potential health implications of sustained iron supplementation for iron replete individuals. Specifically, the potential risk of toxicity and iron overload following IV administration must be acknowledged, particularly since the primary route for regulating whole body iron stores is bypassed using this method, allowing ferritin levels to be elevated rapidly. Therefore, IV injections should not be administered without first assessing existing iron stores, and careful consideration of the total dose to be given, and its likely impact on whole body iron stores, is required. IV iron should only be administered under medical supervision, with patients closely monitored for adverse reactions. Follow-up tests should also be conducted to ensure ferritin levels do not exceed healthy limits. Lastly, researchers need to be acutely up to date with any changes to the World Anti-Doping Agency code when planning future research with elite athletes −1 without associated changes in Hbmass. Running performance was not acutely affected by iron supplementation, but there was some evidence from self-reported training diaries that participants felt more able to train. Together, these results indicate a potential psychobiological influence of iron supplementation on feelings of mood and fatigue in athletic populations with clinically normal serum ferritin.Four weeks of IV ferric carboxymaltose supplementation doubled serum ferritin, and decreased feelings of fatigue and mood disturbance in non-anemic athletes with initial ferritin levels in the clinically normal range of 30–100 µg·L


The cells developed into distinct populations of neuroglial stem/progenitor cells, mature neurons or epithelial-mesenchymal cells. Importantly, the synchronized electrical activity in the newly developed cortical assemblies could be recorded as local field potential (LFP) measurements. This study therefore describes the first example of the development of a biologically relevant cortical plate assembly outside of the body. This model provides i) a preclinical basis for engineering cerebral cortex tissue autografts and ii) a biofidelic 3D culture model for investigating biologically relevant processes during the functional development of cerebral cortical cellular systems.Little is known about how cells assemble as systems during corticogenesis to generate collective functions. We built a neurobiology platform that consists of fetal rat cerebral cortical cells grown within 3D silk scaffolds (SF). Ivermectin (Ivm), a glycine receptor (GLR) agonist, was used to modulate cell resting membrane potential (V The selection of experimental models with which to study the biology of development and disease requires researchers to search for components that are specifically targeted to the organism and characteristic of the disease. Some insight into conserved cell biological functions has been provided by 2D tissue cultures, including spheroid cultures grown in a 2D environment, organ-on-chip microfluidic/multi electrode array technologies and cells  grown in fabricated 3D SFs. The latter of these three are typically referred to as 3D tissue culture models, and they can add more complex cell biological and anatomical relevance to a study123in vivo-like environment to investigate the behavior of brain cellular systems outside of the body while maintaining the highest possible relevance to the system’s natural environment. The model consists of cells that are freshly isolated from rat brain tissue. The model not only keeps the cells alive and functional but it also demonstrates the fundamental biological/electrical/anatomical features of a developing brain at a multicellular systems level over a long period of time. Rat brain cells were chosen because of their high degree of similarity to human brain cells5The biofidelic 3D model described in this paper presents a unique design and arrangement of biological, biomaterial and environmental components that can be used to nurture functional self-assembly and maintain the intrinsic functions of brain cellular systems in long term cultures. The purpose of this model is to provide an m) were monitored in three different culture systems: neurons (N), astrocytes (A), and co-cultures (NA). All three of the cellular systems grown in silk-based SFs showed long-term viability. The cells exhibited three distinct distribution patterns: some cells grouped as spherical free aggregates, others as laminar connected aggregates and some grouped as dispersed single cells. These results confirm the anatomical heterogeneity of cerebral cortex cellular systems89We evaluated biological parameters that are common across mammalian cellular systems in E18 fetal rat cerebral cortical cells that were grown for up to 3 wks in SF, and spontaneous cortical spike peaks were recorded after 11 wks. Mitochondrial activity, cell population-specific markers, cell resting membrane potentials, action potentials, and mitochondrial transmembrane potentials . Each presented with a different density. Cells were homogenously distributed within or along the walls of the 3D SF cultures -2-naphthalenyl]ethenyl]-1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS) after 1 wk. The phenotypic relevance of these electrophysiological changes is demonstrated in the following sections. Data reflecting the immediate changes observed in Vmem in response to the applied Ivm are shown in the Real-time measurements performed using cortical cultures after 1 wk and 3 wks revealed that the cultures displayed long-term changes in V at 1 wk  and at 3 at 1 wk . The neund 3 wks , whereaswith Ivm . Co-cultectively , and a mnditions . At 3 wkf −45 mV , indicatf −45 mV  and moref −45 mV . Figure Cultures were assessed to determine changes in ΔΨm, which is a hallmark of tumor cells. Although the neuronal cultures had a significantly more hyperpolarized ΔΨm at 1 wk than all of the other cells and conditions, the overall ΔΨm of the cells was similar at 3 wks (mem (Ivm)-mediated regulation of ΔΨm via gap junctions that has been observed in 2D cell cultures, cells grown under the same conditions in 3D SF did not display changes in ΔΨm when they were exposed to Ivm. In addition, ΔΨm was not affected by the gap junctional blocker Octanol, whereas under similar conditions, treatment with Ivm resulted in significant changes in ΔΨm in 2D cultures  cultures of E18 fetal rat cerebral cortical neurons grown in SF in the presence of Ivm contained spheres that were 400 μm in diameter. These spheres consisted of Beta-III tubulin- and MAP2-labeled cells that grew in an inside-out fashion  and werein vivo-like, gradual state of development between stemness and terminal differentiation because they displayed a more depolarized Vmem at 3 wks than at 1 wk  cells increased after 3 wks of treatment with Ivm  that resembled a cortical plate and the ability to generate synchronized population spikes. They therefore presented anatomical and physiological similarities to cortical microcircuit assembly42Self-assembled cerebral cortex cells grown in SF showed anatomical structures that are similar to those observed during mem) enabled the long-term retention of the neural stem/progenitor niche and a functionally and biologically relevant model of mammalian cerebral cortex development.Tissue elasticity can direct stem cell lineage specificationmem in the formation of cerebral cortical assemblies and connectivity in vitroXenopus laevis neural tissue developmentex vivo niches of different populations of neonatal cerebral cortical cells, and our findings may therefore serve as a biologically and biomedically relevant platform for developing novel cellular therapies.Given recent findings that have implicated endogenous VRattus norvegicus, Sprague-Dawley, Charles River) cerebral cortical tissues were generously provided every week by Dr. Steven Moss’s laboratory at Tufts Neuroscience Center. The brain tissue isolation protocol was approved by the Tufts University Institutional Animal Care and Use Committee and complied with the NIH Guide for the Care and Use of Laboratory Animals (IACUC # B2011–45). The isolation and dissociation of the rat cortical neurons and astrocytes and the preparation of the cell cultures were performed as previously described52E18 rat . Stocks of 10 mM ivermectin were dissolved in dimethyl sulfoxide and stored at −20 °C. Ivm was used at a final concentration of 1 μM. Stocks of 127 μM octanol (8-OH) were prepared by diluting 10 μl of 1-octanol  in 500 mL of distilled water. Octanol was used at a final concentration of 2.54 nM. For safety the information for Ivermectin and octanol, please refer to their product websites.B. mori cocoons as previously describedA silk solution was prepared from Ivm adsorption data were obtained during the monitoring of dynamic drug adsorption on the silk films using a quartz crystal microbalance, and these data can be found in the online 5 cells per scaffold in phenol-free DMEM supplemented with 1% penicillin/streptomycin, 2% GlutaMAX, and 2% FBS . Neurons were added at 3 × 105 cells per scaffold to the other scaffolds in phenol-free neurobasal medium supplemented with B27, 1% penicillin/streptomycin, and 1% GlutaMAX. The cells were incubated at 37 °C, and the cell culture media were changed once per week. The same number of cells was used to co-culture the astrocytes and neurons.The E18 fetal rat cerebral cortical cells were dissociated as previously describedmem of neuron cultures (N), astrocyte cultures (A) and cocultures (NA) for 0–4 weeks after plating. The cells were washed once with Hank’s Balanced Salt Solution (HBSS) prior to staining. Di-8-ANEPPS dye was diluted in HBSS to a final concentration of 2 μM and then added to the cells (100 μL/well). The cells were incubated at 37 °C for 30 min in the dark. Dye uptake was confirmed under a microscope using a red filter. The cells were washed once with HBSS to remove excess dye prior to measurement. Fluorescent intensities were measured using a top-read fluorescence multimode multiplate reader . The relative Vmem of the cells was measured as the ratio of readings at two excitation sites of the dye . The plots were generated using Excel.We used ratiometric di-8-ANEPPS dye  to measure the Vm of neuron cultures (N), astrocyte cultures (A) and cocultures (NA) at 0–4 wks after plating. The cells were washed once with HBSS prior to staining. The JC1 dye was diluted in HBSS to a final concentration of 5 μM and then added to the cells (100 μL/well). The cells were incubated at 37 °C for 30 min in the dark. The incubation time and the final JC1 concentration used for the primary cultures of rat cortical neurons and astrocytes were chosen based on previous studies56m of the cells was measured as the ratio of readings at two emission sites of the dye . The plots were generated using Excel. To assess the mitochondrial shapes shown in m.The dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide (JC1)  was used to measure the ΔΨA basic immunocytochemistry protocol was performed as previously describedMitochondria-based tetrazolium salt WST-1 assays were used to assess mitochondrial activity according to the manufacturer’s instructions . Cultures were plated for 0–4 weeks and then incubated for 2 hours at 37 °C with the WST-1 assay reagent (1:10 in cell culture medium). Mitochondrial dehydrogenase activity was measured as a function of mitochondrial activity at 440 nm using a multimode multiplate reader  with SoftMax Pro software.2, 2 mM MgCl2, 10 mM HEPES, and 10 mM D-glucose, with pH adjusted to 7.4 using NaOH). Sharp glass microelectrodes were pulled using Sutter Borosilicate Glass (BF150-86-10) with a Sutter Micropipette Puller (P-87). The microelectrodes were filled with extracellular solution and had a resistance of 60–80 Mohm. The cell cultures and 3D scaffolds were visualized using a Wild M3C Dissecting Microscope (Switzerland). The recording electrodes were positioned close to the cultured tissues. Fast local field potential changes (spikes) were recorded using the NPI amplifier at a bandwidth of 0.3–10 kHz and then further amplified using an A-M Systems Differential AC amplifier (Model 1700) to a combined total gain of 10,000×. The signals were digitized at 10 KHz using a Molecular Devices digitizer (DigiData 1550) using a Dell OptiPlex GX620 computer with pClamp 10 software (Molecular Devices). The electrical stimulations of the cultured tissues were generated using a Grass S44 Stimulator, passed through a Grass Stimulus Isolation Unit (SIU5), and delivered using a platinum parallel bipolar electrode (FHC PBSB0875). There was a distance of 800 μm between the two tips, which were positioned near the recording electrode. Images showing the measurement setup, which consisted of scaffolds and electrodes, were captured using an AmScope microscope digital camera (MU1000-CK).Dissociated E18 rat embryonic cortical neurons were seeded on poly-D-lysine-coated 35-mm Corning Culture Dishes or in 3D silk protein-based scaffolds. After a period of incubation (10 days to 3 months), the electrical spiking activities of the neurons grown in cultures and on 3D scaffolds were recorded extracellularly at room temperature using sharp glass microelectrodes and an NPI Bridge Amplifier . The cells grown in cultures and on 3D scaffolds were immersed in extracellular solution  or p < 0.01 (**). The data are presented as the mean ± SEM.How to cite this article: Ren, M. et al. A biofidelic 3D culture model to study the development of brain cellular systems. Sci. Rep.6, 24953; doi: 10.1038/srep24953 (2016).