To overcome these problems, an effective encapsulation for the sulfur cathode is necessary. By means of particle swarm optimization (PSO) and density functional theory (DFT), we have predicted a stable metallic two-dimensional sp2-hybridized carbon allotrope (DHP-graphene). This carbon sheet can prevent S atoms from cathode entering electrolyte. However, Li-ions can shuttle freely due to the increasing difference in Li-ions concentration between electrolyte and cathode along with the potential difference between cathode and anode during charge-discharge cycles. In addition, versatile electronic band structures and linear dispersion are found in DHP-graphene nanoribbons but only metallic band structure occurs for DHP-graphene nanotubes.Sulfur cathodes in lithium-sulfur (Li-S) batteries still suffer from their low electronic conductivity, undesired dissolution of lithium polysulfide (Li Li-ion rechargeable batteries have played a key role in commercial applications such as portable electronic devices and electric vehicles due to their superior energy density6. However, with growing energy demand for business, existing Li-ion rechargeable batteries have approached their bottlenecks and can not meet the critical demand for batteries energy density. Thus, a new high-energy storage system is urgently needed9. Li-S rechargeable batteries are widely considered to be the next-generation high-energy storage system, due to their high theoretical capacity (1675 mAh/g) and energy density (2600 Wh/kg)11. In addition, sulfur element is abundant and environmental-friendly for long-term use12. Despite their many advantages, Li-S batteries still exist some problems to be solved. Firstly, the low electronic conductivity of sulfur and its discharge products (Li2S and Li2S2) limits the actual utilization of active materials10. Secondly, polysulfides , the by-products during the charge-discharge, can highly solubilize in the electrolyte, and freely shuttle between cathode and anode, which is called “shuttle phenomenon”. It will lead to low coulomb efficiency, loss of active materials and short cycle life13. Lastly, large degree expansion and shrinkage of cathode volume during the lithiation/delithiation result in the stability damage and capacity decay of cathode14.The electrical storage with battery is one of the most significant requirements nowadays18, carbon-sulfur composites22, and carbon-polymer-sulfur composites25, have been investigated. Among the available carbon-based materials, graphene inherently shows excellent electrical conductivity, mechanical flexibility and high specific surface area29. However, in the cathode of sulfur-graphene composites, graphene acts as a substrate loading sulfur failed to immobilize the polysulfides in the cathode to alleviate the shuttle phenomenon30. To overcome this drawback, both replacing the graphene with graphene oxide and encapsulating graphene-sulfur composites with polymers are effective approaches to alleviating shuttle phenomenon, but suffer from the electrical conductivity decreasing instead30. Moreover, some metal oxides such as Ti4O732 and MnO233 can alleviate the shuttle phenomenon by converting soluble polysulfides to insoluble low order sulfides, but their electronic conductivity is also much lower than carbon materials just like graphene oxide and polymers. Archer’s group has successfully alleviated the shuttle phenomenon and meanwhile enhanced cathode electrical conductivity to some extent by encapsulating sulfur with porous hollow carbon34. However, the effective encapsulation of sulfur cathode with high electrical conductivity is still an open problem urgent to be solved for the practical application of Li-S batteries.In order to solve the above problems, a cathode material that not only has good electrical conductivity but also can immobilize the polysulfides is required. Various sulfur-based composites including polymer-sulfur compositessp2-hybridized porous carbon allotrope named as DHP-graphene and evaluate its dynamic and thermodynamic stability. Secondly, The electronic band structures of DHP-graphene and its derivatives, including nanotubes and nanoribbons, are investigated. Finally, the potential application of DHP-graphene in the cathode encapsulation of Li-S is evaluated. The main purpose of this study is to demonstrate the feasibility of alleviating the shuttle phenomenon and maintaining high electrical conductivity at the same time by using this novel carbon allotrope as the cathode of Li-S batteries.Herein, we firstly predict a new two-dimensional (2D) 35 predicted by Feng et al. is found again in this structure research. In addition, a few carbon allotropes, such as HHPG-L, HHPG-Z and OHPG, also have lower enthalpies but they are dynamic unstable (see the supporting information). Fortunately, a dynamic stable 2D sp2 hybridized carbon allotrope with low enthalpy attracts our attentions. Its structure is shown in Fig. decagonal, hexagonal and pentagonal rings and thus is named as DHP-graphene. Its unit cell is emphasized by black dashed lines, and the lattice constants a and b are 7.08 Å and 9.92 Å, respectively. DHP-graphene belongs to the 2D space group of Cmm. The primitive cell of DHP-graphene is highlighted by a red parallelogram. Six independent bond lengths of DHP-graphene are 1.505 Å, 1.440 Å, 1.431 Å, 1.409 Å, 1.412 Å, and 1.460 Å for b1, b2, b3, b4, b5, and b6, respectively. The average bond length (1.443 Å) of DHP-graphene is a little longer than that of graphene (1.42 Å). The bond angle is 108.95°, 107.91°, 104.26°, 113.83°, 132.33° and 105.05° for α12, α14, α25, α35, α55 and α46, respectively. The deviation of bond length and angle from ideal sp2 carbon (graphene) results in the increasing of total energy. However, DHP-graphene is still more thermodynamic stable than graphdiyne which has been experimentally synthesized by Li et al.36 Similar to other carbon sheets, the DHP-graphene is also a flexible structure, and many potential applications need to be investigated. The first Brillouin zone is also shown in Fig. x,y) of K point are calculated by the formulas of β is the angle between The enthalpy evolution for a 12-atom carbon system is shown in Fig. f). The projected DOS shows that the DOS near Ef is mainly ascribed to the contribution of pz orbital electron and slightly from px and py orbital electron. Thus the C-C bonding type is dominant π bonding below Ef and π* antibonding above Ef. It implies high electronic conductivity of DHP-graphene. In contrast with graphene oxide, polymers or metal oxide, if the DHP-graphene is used as the cathode of Li-S batteries, the present disadvantage of low electronic conductivity could be improved significantly.For a material to be applied in the cathode of Li-S barriers, high electronic conductivity is necessary. Therefore, we investigate the electronic band structure and density of state (DOS) of DHP-graphene as shown in Fig. Na and Nb, where the Na and Nb are the ratios of the width of DHP-GNR to lattice constant of Na and Nb are integers, DHP-GNRs have inversion symmetry center (ISC). Otherwise, Na and Nb are half-integers and no ISC exists. Strikingly, DHP-GNRs exhibit diversified electronic properties as shown in Fig. Na  and conduction band minimum (CBM) near Fermi energy level. In contrast, the DHP-GNRs are direct bandgap semiconductors as Na = 1.5, 2 and 2.5. And the bandgap is 150, 60, and 190 meV and occurs at Γ, X point and along Γ-X, respectively. When the Na is 3, DHP-GNR is a semiconductor too, but has a 42 meV indirect bandgap. Figure Nb = 0.5. It is worth noting that the dispersion is linear for both VBM and CBM. Thus, the electron and hole carriers are massless, and their mobility are very high. In contrast with zero bandgap graphene, this DHP-GNR exhibits distinct advantages using for semiconductor devices including field-effect transistor due to its open bandgap and linear dispersion.Similar to graphene or other 2D materials, DHP-graphene also has two kinds of derivatives, nanoribbons and nanotubes. In this study, zigzag and armchair DHP-graphene nanoribbons (DHP-GNRs) are obtained by tailoring the DHP-graphene along lattice vectors see Fig. . When thn, 0) zigzag DHP-GNTs and  armchair DHP-GNTs can be achieved. Two typical structures of DHP-GNTs are shown in Fig. n) DHP-GNT is more stable than the corresponding  DHP-GNT.The other kind of derivatives of DHP-graphene are DHP-graphene nanotubes (DHP-GNTs). By rolling DHP-graphene along vectors 37. Therefore, alleviating the shuttle phenomenon is necessary for the commercial application of Li-S batteries. The natural decagonal rings provide the possibility for DHP-graphene used as a selective filter for Li, S and soluble polysulfides  as illustrated in Fig. n−2 to penetrate through a decagon ring due to its small size (4.43–4.72 Å). A parameter, Δd, is defined as the vertical distance between Li (or S) atom and DHP-graphene as shown in Fig. d = 0 as shown in Fig. 5, S atoms will be confined into nanotubes for S and DHP-GNTs based cathode as illustrated in Fig. The main issues faced in Li-S batteries including low coulomb efficiency, loss of active materials and short cycle life, are arisen from the shuttle phenomenonIn summary, we have proposed a new 2D porous carbon allotrope (DHP-graphene) based on particle swarm optimization and density functional theory. This 2D carbon structure is flexible and belongs to Cmm space group. By phonon dispersion calculations, DHP-graphene is proved to be dynamic stable. As far as thermodynamic stability is concerned, it is more stable than graphdiyne which is also a 2D carbon allotrope experimentally synthesized. Electronic band calculations show that both DHP-graphene and its derivative (DHP-GNTs) are metallic carbon allotropes. However, the other derivative, DHP-GNR can be metallic or semiconducting, which depends on the width of nanoribbons and edge structure. Versatile electronic structures make DHP-graphene and its derivatives have potentials to be used for all carbon-based nano devices. Our DFT calculations show DHP-graphene could be used as a filter to encapsulate the cathode of Li-S batteries due to its decagon carbon rings. A 3.6 eV energy barrier prevent S atoms entering electrolyte. However, Li-ions can shuttle across DHP-graphene with the help of Li-ions concentration and potential differences during charge-discharge cycles. In addition, if DHP-GNTs is used for the cathode of Li-S batteries, the volume expansion of cathode can be alleviated because S atoms are confined the inside of DHP-GNT but not adsorb its outside surface.38. The population size and the number of generation were set to be 26, and there were 12 carbon atoms in the crystal cell. Density functional theory (DFT) calculations were carried out using general gradient approximation (GGA)39 as implemented in the Vienna ab initio simulation package (VASP)41. The interactions between the nucleus and valence electrons of carbon were described by the projector augmented wave (PAW) method42. A plane-wave basis with a cutoff energy of 400 eV was used to expand the wave functions for all carbon structures investigated in this work. The geometries of carbon structures including the atomic position and lattice parameters were fully relaxed until the residual forces on atoms were less than 0.0001 eV·Å−1. The Brillouin Zone sample meshes were 5 × 5 × 1 for DHP-graphene, 1 × 1 × 5 for one-dimensional carbon nanotube and 1 × 1 × 5 for nanoribbons, in our calculations. The phonon band structure for this new two-dimensional carbon allotrope was calculated using the direct supercell method implemented in the Phonopy program43.Crystal structure predictions were performed using particle-swarm optimization (PSO) algorithm within the evolutionary scheme as implemented in the CALYPSO codeSupporting Information


The system is characterized by a topological index, the mirror winding number, with its root lying in the Su-Schrieffer-Heeger model for polymer. Taking this point of view, one is rewarded by several ways for decorating graphene edge which result in similar mobile electronic states with topological protection. This work demonstrates that celebrated nanotechnology can be used to derive topological states.Localized electrons appear at the zigzag-shaped edge of graphene due to quantum interference. Here we propose a way for harnessing the edge electronic states to make them mobile, by incorporating a topological view point. The manipulation required is to introduce a pattern of strong-weak bonds between neighboring carbon atoms, and to put side by side two graphene sheets with strong-weak alternation conjugating to each other. The electrons with up and down  For device applications, however, it is desired to turn graphene into a semiconductor by opening a controllable energy gap in the linear dispersion, which in a physics language is equivalent to attaching a mass to electron governed by the Dirac equation4. Noticeably, the emergent mass term has also played a key role in the development of the topological phases of matter. Haldane was the first to recognize that, in terms of complex next-nearest-neighbor (nnn) hopping integral, a valley-dependent mass can be induced in the massless Dirac-like dispersion in honeycomb structure, resulting in a quantum anomalous Hall effect5. Kane and Mele then noticed that, taking into account the spin degree of freedom of electron, the spin-orbit coupling (SOC) yields naturally two copies of the Haldane system in the spin-up and -down channels related to each other by time-reversal (TR) operation7, which is known as a quantum spin Hall effect (QSHE).Graphene exhibits linear dispersions in its energy spectrum originated from the honeycomb structure8, the Dirac cones at K and K’ points at the corners of Brillouin zone of ambient graphene are folded to Γ point  hopping integral only can also open a gap in the Dirac-like linear dispersions in honeycomb lattice: due to the nesting effect caused by hopping texture over hexagonssee Fig.  and meansee Fig. .Figure 19  as / for m > 0/m < 0 respectively. Bringing the two insulators side by side  can be manipulated accurately by the advanced STM technique. Or, simply putting a graphene sample on an appropriate substrate possibly causes the hopping patterns12. Turning to metamaterials which are analogs to materials with ions replaced by periodic arrangements of dielectric constant and/or magnetic permeability, there are more spaces for precise real-space manipulation for achieving topological photonic and phononic states15. The SOC which are required for realization of QSHE is either very small or hardly conceived in these systems, whereas the topological energy (or frequency) gap induced by the texture of hopping integral in a tight-bind picture is considerably large.Real-space textures in the hopping energy can be realized in systems of honeycomb structure in terms of cutting-edge technologies of material preparation. In the molecular graphene achieved on the Cu [111] surface, a pattern in hopping integrals was realized by putting extra CO molecules, which induces an energy gap of ~100 meV in the otherwise Dirac dispersion16 and Rice-Mele model17 as the hallmark of 1D topology. This link is quite natural in the present 2D system, where the nontrivial topology arises from the bond alternating the same as the 1D counterpart. As a merit of this approach, we show that topologically protected edge states can be realized in terms of a variety of edge decorations at the graphene-vacuum interface in accordance to the bulk texture of hopping energy , and the Su-Schrieffer-Heeger (SSH) modelsee Fig. .ci) is the creation (annihilation) operator at site i, and 〈ij〉 denotes summation over the nn sites, with the hopping integral tij modulated spatially . To be specific, we introduce two values of hopping energy, intra- and inter-hexagon as t0 and t1 /t0, which is inherited from the zigzag-edged graphene without hopping texture9. That is, the edge mode is nothing like helical ones, although the bulk state acquires a gap due to the hopping modulation. Remarkably, putting two regions with δ > 0 and δ < 0 side by side, instead of the exposing to vacuum as in the ribbon geometry, the situation is drastically altered. Figure δ = ±0.1 with a graphene-zigzag interface. .With the clear edge-shape dependence of the edge mode observed in Fig. δ > 0 and δ < 0 regions does not. In the following, we provide a unified way of understanding this variety in terms of a topological invariant. Because the present system exhibits a bipartite nature, the Hamiltonian in k-space, which is obtained by the Fourier transformation from ci to ck, can be written asQk is a 3 × 3 matrix. The Hamiltonian (1) anticommutes with the chiral operator γ = diag, manifesting the chiral symmetry (or sublattice symmetry) of the present system. Then, regarding the momentum k parallel to the unit vector a1 defined in Fig. 21,So far, we have established that the topological modes at the ribbon edge depends on the edge shape, while the one at the interface between k|| = 0, we can do further. That is, the mirror symmetry with the mirror plane perpendicular to a1 enables us to decompose the Hamiltonian (1) into even and odd sectors k is replaced by k⊥, since our focus is now on k|| = 0. In this case, the mirror operation commutes with γ, and thus, Qk can be decomposed into even and odd sectors 22 , we can assign winding numbers for the even and odd sectors separately by plugging mirror winding number . Note that even though we also have a mirror plane parallel to a1, which is related to armchair type edge, another important class of graphene edge, that mirror operation does not commute with γ, leading to no mirror winding number for armchair edge. Note also that in a general context, the approach intertwining topology and crystal symmetry was first considered in the work on topological crystalline insulator28.For entclass1pt{minima30. Although the choice of unit cell is merely a unitary transformation of Hk, it does not necessarily correspond to a unitary transformation of Qk. Consequently, the unit cell choice can modify the winding number through equation , Qk is decomposed inton+, n−) =  for δ > 0 and  for δ < 0. On the other hand, for molecule-zigzag edge, we haven+, n−) =  for δ > 0 and  for δ < 0. The same prescription applies to partially-bearded edge, giving usn+, n−) =  for δ > 0 and  for δ < 0. The results are summarized in Table ntot = n+ + n−. While the mirror winding number is only defined at k|| = 0, the total winding number ntot is well defined for any momentum, and is conserved as far as the bulk gap is not closed. It is worthy noticing that, as can be read from Table δ > 0 and δ < 0 can be distinguished topologically in terms of the mirror winding numbers, but indistinguishable by the total winding number.Equipped with the appropriate unit cell, mentclass2pt{minimmentclass2pt{minimgraphene-zigzag edge, ntot = 1 for any k|| regardless of the sign of δ. Because vacuum is topologically trivial with zero mirror winding number, we expect the flat-band edge mode associated with the the nonzero total winding number29 for the ribbon geometry, which is in accordance with Fig. molecule-zigzag and partially-bearded edges, we observe the counterpropagating edge modes with n± ≠ 0 and ntot = 0. This can be understood as follows: for k|| = 0, one has two zero-energy edge modes, one from the even sector and the other from the odd sector; for k|| ≠ 0, there is no reason to have any zero-energy edge mode since ntot = 0. As the consequence, we end up with two edge modes with finite dispersions and crossing linearly at k|| = 0. Remarkably, the counterpropagating modes at the graphene-zigzag interface between two domains with opposite values of δ  and (d) to the molecule-zigzag edge, and (e-h) to the partially-bearded edge, respectively. The chiral symmetry permits us to adopt the following ansatz for the wave functions of zero-energy edge modes ΨA =  or ΨB = .A more intuitive and physical understanding of this unified picture is available by checking analytic solutions of the wave functions for the topological edge states. Here, we briefly describe the derivation of the wave functions for the zero-energy topological edge modes at see Fig. . We can graphene-zigzag edge t0 + t1 = 032 for a nontrivial zero-energy mode. From the fourth site on the central column, one has 2t0 + αst1 = 0. These two equations are satisfied by β ≡ (1 + δ)/2. The solution is physically relevant only when it decays from the edge into the bulk, namely when α < 1, which is achieved by δ > 0. On the other hand, for the odd-parity solution  =  for δ > 0 while  =  for δ < 0 for the graphene-zigzag edge.Let us begin with the even-parity solution for the dge Fig. . From thion Fig. , the ansmolecule-zigzag edge , which is physical for δ > 0. That is, the even- and odd-parity solutions are both physical when and only when δ > 0, in agreement with  in Table Next, we move on to the dge Fig. . Compariion Fig. , the anspartially-bearded edge, the top and the bottom edges should be considered separately since the ribbon becomes asymmetric with respect to the middle line of the ribbon  edge yields gapless counterpropagating edge modes. The present work provides a new designing guideline for topological edge states adaptive to the bulk hopping texture, which may pave a way to tailoring graphene and related materials in the topological point of view.To summarize, topological phases in honeycomb lattice induced by texture in hopping energy between nearest-neighboring sites are characterized in terms of the mirror winding number. Analytic wave functions are provided for zero-energy edge modes at the Γ point, which evolve into the dispersive counterpropagating edge states at finite momenta. Explicitly, when the intra-hexagon hopping energy is stronger (weaker) than the inter-hexagon one, The datasets generated during the current study are available from the corresponding author on reasonable request.


The effect of initial evenness on the temporal trajectory of synthetic communities in comprehensive, low-volume microcosm studies remains unknown. We used flow cytometric fingerprinting and 16S rRNA gene amplicon sequencing to assess the impact of time on community structure in one hundred synthetic ecosystems of fixed richness but varying initial evenness. Both methodologies uncovered a similar reduction in diversity within synthetic communities of medium and high initial evenness classes. However, the results of amplicon sequencing showed that there were no significant differences between and within the communities in all evenness groups at the end of the experiment. Nevertheless, initial evenness significantly impacted the cell density of the community after five medium transfers. Highly even communities retained the highest cell densities at the end of the experiment. The relative abundances of individual species could be associated to particular evenness groups, suggesting that their presence was dependent on the initial evenness of the synthetic community. Our results reveal that using synthetic communities for testing ecological hypotheses requires prior assessment of initial evenness, as it impacts temporal dynamics. Comprehensive understanding of community properties such as diversity and structure is currently insufficient and, consequently, the description of natural communities remains challenging4. Uncovering functional and/or active members of natural microbial communities is a complex task. Hence, model systems have been developed. Synthetic communities are simplified representations of natural ecosystems, with improved reproducibility5 in a controlled environment7. These have been applied to study microbial interactions and biodiversity-production relationships9. For instance, for bioethanol production11, bioremediation of contaminated areas12, recycling of waste products during long distance space exploration13, and as an alternative for human faecal transplants14.Microbial communities, where cells interact and communicate with one another and influence each other’s behaviour, are dynamic change agents in numerous ecosystems17 has been reported. Although evenness influences community dynamics19, the stability of synthetic communities with different initial evenness over time is yet to be elucidated. In this study, we monitored the progress of communities with different initial evenness, and we hypothesized that these communities evolve in a similar and simultaneous fashion. A previous report showed that initial community evenness is a key factor for preserving the stability of an ecosystem17. Hence, extensive characterization of the evolving community structure is essential for a comprehensive overview of this process.The impact of diversity, richness, and evenness on ecosystem functions such as stress resistance, invasion, and predation interactions20. FCM is a fast, high throughput method with a wide variety of potential applications, particularly in medical research and microbial ecology. Nonetheless, it is mostly limited to liquid samples and only partial information on community structure can be obtained from this analysis21. Flow cytometry fingerprinting (FCFP) is a promising approach to monitor complex microbial communities and to detect changes in the structure of communities. FCFP was applied to estimate the biodiversity of microbial communities, using a phenotypic diversity index based on single cell phenotypic characteristics, such as morphology and nucleic acid content20, but has not been used in synthetic communities previously. Additionally, FCM and sequencing techniques have been applied together to gain insight into community structure and dynamics over time28. In contrast with these studies, which surveyed natural communities, we monitored the temporal trajectory of synthetic communities. For these reasons, we employed complementary techniques to survey the community evolution of synthetic communities.Currently, microbial community structure and dynamics are often determined based on 16S rRNA gene amplicon sequencing, which remains the standard method for culture-independent surveys of microbial diversity. On the other hand, flow cytometry (FCM) couples high accuracy with sensitivity, ranging from a single cell level to the community levelIn this study, we assembled 100 different synthetic communities with the same richness but different initial evenness. We then combined flow cytometric fingerprinting and amplicon sequencing of the 16S rRNA gene, to monitor how initial evenness variations induced different temporal dynamics in community structure at the taxonomic and physiological level. Understanding these variations can assist us in recognising critical time points for community stability and resilience, which may be potentially modulated in synthetic microbial communities.Shifts in community structure were monitored in one hundred microcosms with same richness but varying initial evenness . Each synthetic community was sampled before a medium transfer that occurred at five discrete time points . A transfer consisted of inoculating five percent of the liquid microcosm into fresh medium, (48 h). A total of 1913 observations were recorded and a significant decrease in the phenotypic diversity (Hill order 2) (P < 0.001) in high and medium evenness groups was observed, following flow cytometric fingerprinting. Conversely, there was no significant difference (P = 0.18) in the low evenness group Fig. . Cell nu16S rRNA gene amplicon sequencing was performed to validate the phenotypic diversity trends revealed by flow cytometry, and to quantify each member of the synthetic communities at the start of the experiment (transfer 0) and after transfer 5.P < 0.05, Table P < 0.005) in communities with high initial evenness. The results of different diversity indices were confirmed by measuring three Hill orders . These observations confirmed that alpha and beta diversity were not significantly different within and between the communities of different initial evenness.Several diversity indices were utilized to describe community composition, structure, and dynamics from the sequencing data. We applied the Fisher’s alpha diversity, Shannon–Wiener index, Pielou’s Evenness, Simpson’s, and inverse Simpson indices in this experiment. All diversity indices excepting for Simpson index were significantly different between transfer 0 and 5. The decrease in diversity was significant in all evenness groups across transfers  further explained how the diversity and evenness metrics were associated with the relative abundance of each member of the synthetic community , and both were negatively associated with the relative abundances of Rhizobium and Burkholderia (P < 0.01). Fisher’s alpha diversity is an indicator for logarithmic changes in relative abundances. The positive association between richness and Fisher’s index indicated that high evenness communities tended to have high richness, but the distribution of these species was skewed. Enterococcus, Aeromonas and Clostridium were the main genera associated with dimension 4, potentially indicating that their relative abundances were associated with communities of high evenness. Delftia and Tissierella were associated with dimension 5, and therefore, with communities of low evenness.Dimension 1 explained the 28% of the variance, while dimension 2 described 24% of the variance and dimension 3, nearly 14%  is reached. When biodiversity attains saturation, interactions can steer elimination of some taxa31.Environmental filtering occurs when species are unable to tolerate environmental conditions. Based on this concept, the environment acts as a selective force which has effects on the distribution of biodiversity over the world33. These previously reported results were based on natural communities, but in our experiment, we worked with small synthetic communities. Thus, our communities may not follow the same underlying ecological principles of natural communities, as a result of the synthetic nature of the experiment34.As previously described, FCFP combined with multidimensional and statistical analysis have been used to detect changes in community composition of drinking water37. The phenotypic diversity estimation in our synthetic communities was based on optical features detected by flow cytometry, enabling us to describe the diversity of the synthetic communities across phenotypic characteristics (i.e. morphology and nucleic acid content), which cannot be inferred from molecular techniques. Recent research has shown strong correlation between the phenotypic and taxonomic diversity of natural freshwater communities in both low and high diversity environments38. In contrast to the sequencing-based approach, FCFP is fast (<1 h), cheap (<€1 sample−1) and requires only minute sample volumes (<1 mL) thereby allowing high-frequency and non-invasive tracking of sensitive biological processes, such as the feeding of invasive mussel species on bacterioplankton communities38.Dissimilarities observed between the results of FCFP and amplicon sequencing may be as well due to inherent differences among the techniques: FCFP is based on staining nucleic acids of cells in suspension, whereas amplicon sequencing applications focus on rRNA gene hypervariable regions, and some limitations have been found in both techniques. Sequencing errors, difficulties in assessing operational taxonomic units (OTUs) and numerous 16S rRNA gene copies in some species are limitations of 16S rRNA techniques20. In contrast to previous studies, we did not find a strong correlation between D0 and D2 of the phenotypic and taxonomic diversity for synthetic microbial communities  decreases with increasing population size41. Zhang et al. implied that the probability of ecological drifts associated with birth and death, and fluctuations in relative population abundance decrease when there are smaller populations and lower growth rates, which may be the case in our synthetic communities. Thus, it was expected that diversity and bacterial abundance decreased concurrently42.Moreover, FCFP showed that the phenotypic diversity tended to be low when bacterial cell numbers were high in all our evenness groups. Thus, communities with low and medium initial evenness were more diverse and had lower total cell counts than communities with high initial evenness. Communities with high initial evenness showed higher cells numbers but low diversity. Milici 43. For instance, fast growth may be inefficient, thereby impacting heterotrophic cell metabolism and ultimately entire population density44. In contrast, higher yields optimize limited environmental resources, and support the survival of the entire population45. Indeed, shifts between mixed and segregated cell lineages affect the evolution of cooperative and antagonistic phenotypes46. In natural settings, community members may differ in the level of cooperation, which makes social interaction open to exploitation43. For instance, P. aeruginosa delays investment into cooperation when its impact on fitness becomes negligible47.We artificially altered the fitness of certain strains by initially mixing the strains at different ratios. In this way, the differences in the evenness among communities may have triggered competitive interactions that ultimately drove the community to a low cell density. In addition, Zha et al. reported that evenness increased with increasing dispersal rates. It is possible that the repeated inoculation in our experiment may have had a similar effect48.Based upon individual growth curves (data not shown), all strains should have reached stationary phase in between each transfer (i.e. <48 h). Single cells possess traits that increase group performance but that may be detrimental for individual cellsBacillus, Rhizobium and Burkholderia had very low abundances in the Low evenness group, whereas Rhizobium and Burkholderia were almost absent in the Medium group. Therefore, higher cell numbers at the final transfer in the High evenness group may be due to the presence of all species, as indicated by the sequencing results.Our results showed a fluctuation of cell density over time in all communities. Thus, the effect of initial evenness on cell density after five subsequent transfers for the Low and Medium initial evenness categories was uncovered. Cell density decreased in all groups but communities with low initial evenness showed the fastest decrease over time. Communities with high initial evenness retained a higher cell density over time in comparison with other groups. The results at the final transfer in all evenness groups indicated that et al. proposed that communities with high initial evenness may present improved resistance during environmental oscillations, by creating specialized microcosms with varying evenness without changing richness. Communities with high initial evenness showed higher functional stability even under non-stressed conditions in comparison with those with low initial evenness17. Importantly, most synthetic community studies did not fully control for potentially evenness-dependent temporal effects on community diversity, function, and cell density. Understanding cooperative and competitive interactions assists to clarify community function, assembly, and stability. Collaborations within and among bacterial communities can arise when one or a group of species provide nutrition or protection to another, whereas competition occurs when some species compete for a shared metabolite49. In the context of this experiment, Bacillus, Enterococcus, Aeromonas, and Clostridium were associated with low evenness whereas Delftia and Tissierella were correlated with high evenness, suggesting that they are more abundant on either group. Therefore, Delftia and Tissierella thrived in our communities with High evenness, while Aeromonas and Enterococcus succeed in the communities designed with Low evenness. Aeromonas abundance was negatively correlated with Delftia, Serratia and Enterococcus abundance but positively correlated with Bacillus abundance. Thus, Serratia and Enterococcus were less present in communities with high relative abundance of Aeromonas. Burkholderia and Rhizobium were correlated with each other positively, which may explain their consistent low relative abundance in all the evenness groups.Knowledge of differences between communities is essential for identifying ecosystem dynamics. For instance, Wittebolle 50. Based on our results, the combined use of FCM and sequencing provided an overview of community structure dynamics, allowing for efficient monitoring of natural microbial communities. Synthetic communities are of special interest, because description of their characteristics has not been performed yet. Knowledge of these traits will assist us to elucidate the effect of time on community stability and resilience, which are key functional characteristics of microbial communities. Using synthetic and controlled microbial communities may also further our mechanistic understanding and support the development of models with increased predictive power. In the context of our study, deterministic selection may have impacted the population size over time. This finding may be relevant for the development of engineered communities with targeted functionality, such as those in wastewater treatment or environmental bioremediation. By monitoring diversity, structure, resilience and stability of communities, bioremediation efficiency51 may be predicted, for instance, and successful in situ bioremediation techniques can be implemented52.Understanding how community structure changes over time is a fundamental question in ecology. The structure and composition of natural microbial communities remains to be elucidated because of the presence of non-cultivable species. The complete description of their functions and microbial interactions remains challenging. Unlike temporal dynamics of animal and plant communities, the temporal dynamics exhibited by microbial communities have not been fully described54. In this way, the synthetic communities were grouped in Low, Medium, and High evenness , based on their initial Pielou evenness. One hundred synthetic communities were sampled from an experimental design of one million in silico simulated synthetic communities  was performed at transfer 0 and 5 to supplement the results of FCFP.A total of 10 environmental bacterial species from 4 phyla Table  were mix55. Four technical replicates of each sample were prepared and analysed to obtain robust estimates of the phenotypic characteristics. The data points belonging to the microbial community were isolated from (in-)organic and instrument noise using a gating strategy applied on the FL1-H and FL3-H channels  in 145 mM of NaCl and stained with SYBR Green I to determine total cell count20. Nucleic acid content and scatter signals were employed to calculate the phenotypic diversity metrics (i.e. Hill numbers) from these distributions. We utilized the flowBasis and Diversity functions with default settings on the denoised data, per the protocol available at https://github.com/rprops/Phenoflow_package . Samples with less than 1,000 cells were excluded from the analysis, resulting in a minimum sample size of 5,000 cells. We utilized the Hill diversity of order 2, also known as the inverse Simpson index, for inference on community diversity metrics.Microbial phenotypic diversity was estimated for all samples (n = 1913) using a novel computational method, which calculates various diversity estimators based on single-cell phenotypic features56. Cells were lysed with 1 mL of lysis buffer  polyvinylpyrrolidone and 2% (m/v) sodium dodecyl sulphate) and 200 mg of glass beads , in a FastPrep®− 96 instrument , two times for 40 s at 1600 rpm. After removing glass beads by centrifugation (5 min at 13000 g), DNA was extracted following a phenol–chloroform extraction. DNA was precipitated with 1 volume ice-cold isopropyl alcohol and 0.1 volume 3 M sodium acetate for 1 h at −20 °C. DNA pellet was dried and resuspended in 100 μL 1 × TE  buffer. Samples were immediately stored at −20 °C until further analysis. Quality of DNA was analysed by gel electrophoresis.One ml from each community was centrifuged 10 min at 13000 × g, supernatant was removed and the pellet was stored immediately at −20 °C until further analysiset al.57. Sequencing was performed using Illumina MiSeq sequencer and reads were clustered allowing for two mismatches as previously defined58. The dataset was then filtered to consider only those phylotypes that were present in at least one sample at a relative abundance >0.1% or were present in all samples at a relative abundance >0.001%57. A total of 200 samples were analysed and a total of 6 million reads was obtained. Sequence composition was compared using the RDP Classifier tool59 and SILVA database60. After examining read counts, data were rarefied to a chosen maximum depth of 3317 sequences, using the phyloseq package from R61 and rarefaction curves were plotted using the vegan package in R62.The V1-V2 hypervariable regions of the 16 S rRNA gene were amplified as described by Camarinha-Silva 62. Pielou’s index was used as indicator of evenness in the community. Differences in alpha diversity and evenness measures among transfers and evenness categories were compared using a repeated measures mixed model in SAS , and comparing multiple means using Tukey test. In this way, the differences in the diversity measures among communities could be attributed to either evenness category, transfer or to the interaction transfer*evenness category. Hill numbers or effective number of species63 were calculated from amplicon sequencing, as well as from FCM data, using the Phenoflow package. A generalized mixed model was constructed to evaluate the interaction effect between transfer and initial evenness on the phenotypic diversity (nlme package)64. Random intercepts were included for each biological replicate and autocorrelation was addressed by an AR1 structure. Parameters were estimated using the restricted maximum likelihood approach. Tukey’s all pair comparison was conducted for pairwise comparisons between initial evenness groups at each transfer. Normality and homoscedasticity of the model residuals were visually checked using diagnostic plots  and for transfer, using the vegan software package in Radonis was used to perform a one-way ANOVA, to determine the impact of evenness in community diversity. Differences in relative abundances of the bacterial genera composing each community were compared using a repeated measures mixed model in SAS, with the least squares means adjustment and Bonferroni correction for multiple comparisons65. Multiple factor analysis (MFA) was used to reveal whether any of the diversity metrics and/or evenness were associated with each member of the synthetic communities (FactoMineR package)29.Permutational multivariate analysis of variance (PERMANOVA) with 999 permutations was conducted to explore the percentage of variance that could be explained by the differences in beta diversity, based on Bray–Curtis distance matrices. Tukey’s test for pairwise comparisons of group mean dispersions was performed using the vegan package in R. Further, the function http://flowrepository.org/id/FR-FCM-ZYBR). The OTU table (97% identity) also has been added to supplementary information (Supplementary Table The authors declare that the main data supporting the findings of this study are available within the article and its Supplementary Information files. All FCS files of flow cytometry data, accompanying metadata have been deposited to the Flow Repository website with the repository ID FR-FCM-ZYBR (Supplementary information


Reports of exocrine-to-endocrine reprogramming through expression or stabilization of the transcription factor neurogenin 3 (NGN3) have generated renewed interest in harnessing pancreatic plasticity for therapeutic applications. NGN3 is expressed by a population of endocrine progenitor cells that give rise exclusively to hormone-secreting cells within pancreatic islets and is necessary and sufficient for endocrine differentiation during development. In the adult human pancreas, NGN3 is expressed by dedifferentiating exocrine cells with a phenotype resembling endocrine progenitor cells and the capacity for endocrine differentiation in vitro. Neurotrophic tyrosine kinase receptor type 2 (TRKB), which regulates neuronal cell survival, differentiation and plasticity, was identified as highly overexpressed in the NGN3 positive cell transcriptome compared to NGN3 negative exocrine cells. This study was designed to determine if NGN3 is regulated by TRKB signaling in the adult human exocrine pancreas.t-test and ANOVA.Transcriptome analysis, quantitative reverse transcriptase polymerase chain reaction (RTPCR) and immunochemistry were used to identify TRKB isoform expression in primary cultures of human islet-depleted exocrine tissue and human cadaveric pancreas biopsies. The effects of pharmacological modulation of TRKB signaling on the expression of NGN3 were assessed by Student’s Approximately 30 % of cultured exocrine cells and 95 % of NGN3+ cells express TRKB on their cell surface. Transcriptome-based exon splicing analyses, isoform-specific quantitative RTPCR and immunochemical staining demonstrate that TRKB-T1, which lacks a tyrosine kinase domain, is the predominant isoform expressed in cultured exocrine tissue and is expressed in histologically normal cadaveric pancreas biopsies. Pharmacological inhibition of TRKB significantly decreased the percentage of NGN3+ cells, while a TRKB agonist significantly increased this percentage. Inhibition of protein kinase B (AKT) blocked the effect of the TRKB agonist, while inhibition of tyrosine kinase had no effect. Modulation of TRKB and AKT signaling did not significantly affect the level of NGN3 mRNA.In the adult human exocrine pancreas, TRKB-T1 positively regulates NGN3 independent of effects on NGN3 transcription. Targeting mechanisms controlling the NGN3+ cell population size and endocrine cell fate commitment represent a potential new approach to understand pancreas pathobiology and means whereby cell populations could be expanded for therapeutic purposes.The online version of this article (doi:10.1186/s12964-016-0146-x) contains supplementary material, which is available to authorized users. Neurogenin 3 (NGN3) is a transcription factor that is essential for development of endocrine hormone-producing pancreatic islet cells. NGN3 also is expressed by dedifferentiated cells in the adult human exocrine pancreas that have begun to take on characteristics of endocrine progenitor cells during development. Neurotrophic tyrosine kinase receptor type 2 (TRKB), a cytokine receptor that promotes functional plasticity in cells of the developing and adult nervous system, was identified as being overexpressed by the NGN3+ cell population compared to NGN3- exocrine cells. In this report we demonstrate that NGN3+ cells in the adult human exocrine pancreas express TRKB-T1, an isoform that lacks a tyrosine kinase domain. Positive regulation of NGN3 protein by TRKB involves the cell-signaling molecule protein kinase B (AKT). Blocking tyrosine kinase activity had no effect on the percentage of cells expressing NGN3 protein. Pharmacological methods that target the NGN3+ cell population size and endocrine cell fate commitment may be an approach to treat diabetes.The transcription factor neurogenin 3 (NGN3) is necessary and sufficient for islet specification during development –4. NGN3 Here, we demonstrate that TRKB-T1 is the predominant isoform expressed in cultured adult human exocrine tissue and is coexpressed with NGN3 in cadaveric pancreas biopsies. In primary cultures of human exocrine tissue, the percentage of NGN3+ cells is positively regulated by TRKB signaling. Inhibition of AKT, but not tyrosine kinase, blocks the regulation of NGN3 by TRKB. TRKB-T1 signaling in NGN3+ cells may contribute to cellular dedifferentiation indicative of exocrine cell plasticity. As such, the mechanisms regulating NGN3 may provide insights into pancreas pathobiology and the therapeutic potential of this cell population.-1 reduced glutathione. Tissue was plated in low-adhesion plastic dishes and maintained in suspension culture at 37 °C, 5 % CO2 for 4–6 days with media replacement every 2 days. Drugs were administered initially, at media replenishment and 1–2 h before harvest. Drugs and final dosages were: 7,8-dihydroxyflavone ; N-[2-[[(Hexahydro-2-oxo-1H-azepin-3-yl)amino]carbonyl]phenyl]benzo[b]thiophene-2-carboxamide ; 4-Amino-5,8-dihydro-5-oxo-8-β-D-ribofuranosyl-pyridopyrimidine-6-carboxamide ; -2,3,9,10,11,12-Hexahydro-10-hydroxy-10-(hydroxymethyl)-9-methyl-9,12-epoxy-1H-diindolopyrrolo [Islet-depleted cadaveric exocrine tissue and histologically normal biopsies from human pancreata of non-diabetic adult cadaver donors was obtained from ICR Basic Science Islet Distribution Program (IIDP) under the auspices of the National Institutes of Health National Center for Research Resources (NCRR). Written informed consent for research use was obtained by the institutions that collected the tissue. Tissues were received without personal identifiers. Exocrine tissue was received within 48 h post mortem and cultured at a density of 100 ml media per ml of pelleted tissue in serum-free Miami Media 1A  supplemented with freshly prepared 0.01 g LTo measure expression of TRKB, live single cell suspensions of cultured exocrine tissue were stained with mouse anti-human TRKB  or mouse IgG1 isotype negative control  in phosphate-buffered saline (PBS), pH 7.2, 0.5 % bovine serum albumin , and 2 mM ethylenediaminetetraacetic acid (EDTA) for 30 min at 4 °C. Cells were then washed and stained with anti-mouse Alexa Fluor-647  for 30 min at 4 °C.To measure coexpression of TRKB and NGN3, cells were then fixed, blocked and permeabilized using Transcription Factor Buffer Set reagents  and stained with rabbit anti-human NGN3 , or an equivalent concentration of rabbit Ig negative control, overnight at 4 °C. NGN3 staining was detected with anti-rabbit Alexa Fluor-488  secondary antibody for 30 min at 4 °C. Cell populations were gated on FL1/SSC-A to identify the NGN3+ cell population and on FL4/SSC-A to identify the TRKB+ population. To quantify percentage of NGN3+ cells that are TRKB+, NGN3 gated cells were analyzed for TRKB expression in FL4. To quantify the percentage of TRKB+ cells that are NGN3+, TRKB gated cells were analyzed for NGN3 expression in FL1.To measure coexpression of TRKB and CD133, cells were then stained with mouse anti-CD133-phycoerythrin (PE)  or mouse IgG2b-PE isotype negative control  for 10 min at 4 °C. Staining for TRKB and anti-mouse secondary antibody prior to staining for CD133 with an antibody conjugated to PE was used to avoid cross-reactivity between the two mouse monoclonal primary antibodies. Cell populations were gated on FL2/SSC-A to identify the CD133+ population and on FL4/SSC-A to identify the TRKB+ population. To quantify the percentage of CD133+ cells that are TRKB+, CD133 gated cells were analyzed for TRKB expression in FL4. To quantify the percentage of TRKB+ cells that are CD133+, TRKB gated cells were analyzed for CD133 expression in FL2. Gating results shown in Additional file Cadaveric pancreas wedge biopsies frozen at -80 °C 1–5 h post mortem and cultured exocrine tissue were embedded in OCT mounting media  then snap frozen in a dry ice/2-methylbutane bath. Tissues were stored at -80 °C and sectioned as soon as possible after embedding. Eight micron frozen sections were fixed for 5 min in 4 % paraformaldehyde in PBS, quenched for 5 min in 50 mM glycine in PBS then blocked in 5 % donkey serum , 1 % BSA, 0.1 % Triton-X100 in PBS for 30 min at room temperature. Detection of NGN3 was carried out with a mouse monoclonal antibody  or rabbit polyclonal antibody . TRKB-T1 isoform was detected using a rabbit polyclonal antibody . Secondary antibodies were donkey antisera to the primary antibody conjugated to Alexa Fluor-488 or -546  and counterstained with Hoechst 33342  to visualize nuclei.http://imagej.nih.gov) blinded to treatment. Some drug treatment studies were carried out independently but are shown together. Significance was determined using a two-tailed homoscedastic Student’s t-test between drug and carrier control for single drug studies and using a one-way ANOVA with Bonferroni post hoc testing for multiple drug studies. Sample size (n) refers to biological replicate exocrine cultures. Results were reported as mean ± SEM (standard error of the mean) of technical and biological replicates.Quantitative immunohistochemistry was carried out by imaging 10 random fields  of >200 nuclei per field spanning >100 microns of tissue depth for each treatment group. Fields were captured with Metamorph morphometry software  and nuclei were counted using ImageJ image analysis software (t-test (n = 3) was performed on band intensities following staining with anti-p(S473)AKT divided by band intensities following staining with total AKT.Western blots were incubated in Li-cor blocking solution  1 h at room temperature then stained with primary antibodies in 5 % BSA, PBS, 0.1 % Triton-X100 overnight at 4 °C. Antibodies and dilutions were: total AKT , p(S473)AKT , total ERK1/2 , p(T202/Y204) ERK1/2 , GAPDH , pan-TRKB , TRKB-T1 . Bound proteins were detected with goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to Dylite-680 or Dylite-800  diluted 1:10,000 in Li-cor blocking buffer  with 0.1 % Tween-20, 0.05 % sodium dodecyl sulfate at 25 °C for 1 h then imaged on an Odyssey infrared imager . For quantification of western blot results, a Student’s -ΔΔCt of 5 technical replica readings for each biological replicate exocrine culture. Each technical replica reaction had approximately 50 ng cDNA template. TRKB expression was normalized to PPIA and expressed as percent of total TRKB level, which was determined separately for exocrine tissue and isolated cells. A mean threshold cycle (Ct) of >35 was treated as no expression.RNA from cultured exocrine tissue and isolated cells were prepared using the RNeasy miniprep kit . cDNA was synthesized using oligo(dT) primers in a standard reverse transcriptase reaction with 5 μg RNA. mRNA levels were determined by using TaqMan gene expression assays : NGN3 (Hs01875204_s1), Pan-TRKB (Hs00178811_m1), TRKB-FL (Hs01093096_m1), TRKB-T1 (Hs01093110_m1), HES1 (Hs00172878_m1), KI-67 (Hs01032443_m1) and primer-attenuated cyclophillin A . Expression was calculated as mean ± SEM 2n = 3 biological replicate exocrine cultures) were subjected to >20 million DNA sequencing reads per sample on an HiSeq2000  sequencing system then analyzed using the TopHat-Cufflinks-Cuffdiff workflow [t-test against 10 scores from randomly selected and equally sized gene lists matched to the expression dataset.Random-primed cDNA from CD133+ and CD133-depleted populations   .Isoform-specific quantitative RTPCR was used to quantify the relative transcription levels of total TRKB, TRKB-FL and TRKB-T1. TRKB-T1 expression was significantly higher than TRKB-FL in three biological replicate exocrine cultures (01) Fig.  and in t03) Fig.  in agreeL in Fig. S in Fig. Multiple TRKB isoforms also were observed in western blot analyses of protein lysates from three biological replicate exocrine cultures as well as lysates from paired NGN3+/CD133+ and NGN3D/CD133D cell populations. Immunostaining with a pan TRKB-specific antibody detected a weak band at approximately 140 kDa, which corresponds to the predicted size of TRKB-FL , in exocS size variant was enriched in the NGN3+/CD133+ cell population, detection of TRKB-T1L in both NGN3+/CD133+ and NGN3D/CD133D cell extracts conflicts with low or undetectable levels of TRKB detected in the NGN3D/CD133D population by FACS, transcriptome, and quantitative RTPCR. As it is detected in NGN3D/CD133D extract  following treatment of exocrine cultures with 7,8-dihydroxyflavone, an agonist that binds the TRKB extracellular domain . ANA-12 and 7,8-dihydroxyflavone had no significant effect on NGN3 mRNA expression . These significant and appropriate responses to TRKB-selective drugs of opposing action strongly suggest NGN3 protein is regulated by TRKB signaling.Drugs that selectively modulate receptor activity were used to determine if NGN3 is regulated by TRKB signaling. The percentage of NGN3+ cells significantly increased to a mean ± SEM of 170.5 ± 7.2 % of carrier control  and decreased AKT activation   7,8-dihydroxyflavone-dependent increase in AKT activation AKT), whereas 7,8-dihydroxyflavone increased p(S473)AKT Fig. . To deteion Fig. . In comb18) Fig. . Similarn = 3, p > 0.05) and in combination with 7,8-dihydroxyflavone, did not block the increase in NGN3 observed following treatment with 7,8-dihydroxyflavone alone   Fig. . CEP-701t >35, n = 4). This data, along with coexpression of with NGN3 in histologically normal pancreas biopsies, suggest TRKB signaling results in de novo NGN3 protein accumulation rather than NGN3+ cell proliferation.TRKB signaling can lead to proliferation and invasiveness of pancreatic adenocarcinoma cell lines –15. Whilp > 0.05, n = 4, Additional file NGN3 transcription and protein half-life are regulated by Notch signaling through the transcriptional regulation of repressor hairy and enhancer of split-1 (HES1) , 37–39. n = 10, p = 0.0001). In addition to TRKB itself, this analysis identified 41 genes upregulated and 54 genes down regulated by more than 2-fold in the NGN3+/CD133+ population (Table The expression level of 320 genes involved in, or regulated by, neurotrophin signaling were analyzed in NGN3+/CD133+ and NGN3/CD133D cell transcriptomes (genes listed in Additional file TRKB-FL activation results in intrinsic tyrosine kinase activity, adapter protein docking and downstream signal transduction. Low levels of TRKB-FL expression and failure of tyrosine kinase inhibition to block regulation of NGN3 suggest involvement of isoforms TRKB-T1, TRKB-T-Shc or TRKB-N-T1. TRKB-T1 is the predominant isoform expressed in the adult rodent central nervous system  and is eWhile NGN3 plays a critical role in endocrine lineage fate commitment during pancreas development, its role in the adult pancreas is largely unknown. Although it cannot be detected in the rodent pancreas, targeted disruption of murine NGN3 has a negative impact on islet function . UpregulAlthough beta cells within preexisting islets –60 have Approximately 30 % of total and 95 % of NGN3+ cells in primary cultures of adult human exocrine pancreas tissue expresses TRKB. TRKB-T1, which lacks a tyrosine kinase domain, is the predominant isoform expressed in cultured human exocrine tissue and is expressed in histologically normal cadaveric pancreas biopsies. TRKB signaling positively regulates NGN3 independent of tyrosine kinase activity in cultured adult human exocrine tissue without significantly affecting NGN3 mRNA levels. Identification of VAV3 overexpression in the NGN3+ cell transcriptome suggests TRKB signaling may also involve Rho/Rac GTPases.


Graphene, which is a two-dimensional crystal of carbon atoms arranged in a hexagonal lattice, has attracted a great amount of attention due to its outstanding mechanical, thermal and electronic properties. Moreover, graphene shows an exceptionally strong tunable light-matter interaction that depends on the Fermi level - a function of chemical doping and external gate voltage - and the electromagnetic resonance provided by intentionally engineered structures. In the optical regime, the nonlinearities of graphene originated from the Pauli blocking have already been exploited for mode-locking device applications in ultrafast laser technology, whereas nonlinearities in the terahertz regime, which arise from a reduction in conductivity due to carrier heating, have only recently been confirmed experimentally. Here, we investigated two key factors for controlling nonlinear interactions of graphene with an intense terahertz field. The induced transparencies of graphene can be controlled effectively by engineering meta-atoms and/or changing the number of charge carriers through electrical gating. Additionally, nonlinear phase changes of the transmitted terahertz field can be observed by introducing the resonances of the meta-atoms. Due to the difficulties of generating high fluence THz pulses, the experimental demonstrations of induced transparencies of bare graphene under high THz fluences were reported only recently131416et al. supported this prediction by fitting their experimentally measured induced transparency data to calculated results with different momentum scattering timesTunable light-matter interaction is one of most interesting properties of graphene124678971078controllable nonlinear light-matter interaction of graphene metadevices under illumination of high THz fluences. It is shown that the modulation range of the induced transparencies is strongly influenced by the resonance of metallic meta-atoms patterned on graphene, i.e., whether they are resonant with the exciting light field. Resonant meta-atoms are also shown to introduce nonlinear phase changes of the transmitted light. Moreover, the induced transparencies of graphene metadevices under high THz fluences can be controlled by shifting the Fermi level of graphene, for example, via electrical gating. These schemes of control over nonlinear light-matter interaction in graphene/meta-atom hybrid structures are promising for the realisation of ultra-thin THz devices with controllable nonlinear optical response.Here, we provide experimental proofs of Nonlinear light-matter interaction can be manifested when the light field is strong enough to change the state of the material and its optical response. Therefore, to understand the induced transmittance changes in graphene, we first need to contemplate how the strong light field modifies the carrier distribution and how, in turn, this change affects the transmission of the light field. Above a certain doping level, THz light does not have enough photon energy to induce interband transitions , so the conductivity is mostly determined by the intraband contribution. The intraband conductivity in the Kubo formula isτ is the momentum scattering time, T is the carrier temperature, EF is the Fermi level of the carrier distribution, and e, kB, and ω are the elementary charge, the reduced Plank constant, the Boltzmann constant and the angular frequency, respectivelyτ (or the increase of the collision rate) decreases the real part of conductivity and induces the nonlinear THz response.where The nonlinear response of materials can be amplified by local field enhancements0 and ΔΓ are the initial momentum scattering rate and the change in the momentum scattering rate, respectively21Graphene itself allows for a second way to improve the nonlinear response; namely, a higher carrier concentration leads to a higher conductivity, which in turn allows for stronger nonlinear interaction. According to the hot-carrier generation process explained above, a strong THz field increases the momentum scattering rate of the charge carriers. Conductivity variation due to the change in the momentum scattering rate can be approximately expressed as 3 crystal capable of generating electric field strengths of ~350 kV/cm. The induced transparency comes from graphene and not from other metallic structure by measurements performed on a sample fabricated without graphene resonant meta-atoms and measured the response of these devices to high THz fluences. The spectral transmittance was obtained via THz time-domain spectroscopy (THz-TDS) and the power-dependent transmittance was measured by a THz power meter. The structures depicted in FTHz = 0.06 μJ/cm2 to FTHz = 85.5 μJ/cm2, the changes in amplitude transmittance at 0.6 THz were measured to be ΔtGD@0.6THz = 2.9% and ΔtNRGD@0.6THz = 7.8%, respectively. The transmittance dropped because of the increase in reflection and absorption due to the incorporation of metallic meta-atoms, but the change in transmittance was enhanced. It is worth noting that larger nonlinear transmittance change for NRGD is achieved without substantial change in the spectral response.First, we investigated the role of field enhancement in the induced transmittance change of graphene devices by comparing the nonlinear response of a bare graphene device (GD) with a non-resonant meta-atom/graphene device (NRGD). Both devices were fabricated on the same wafer using chemical vapour deposition (CVD) grown graphene that is transferred from the same Cu foil. The THz transmittance data shown in tNRGD, was 2.95 times larger than that for the GD, ΔtGD. Therefore, our data indicate that the field enhancement by the meta-atoms contributes to the increase in nonlinear transmittance change.To confirm that the field enhancement effect leads to increased nonlinearity, we first calculated the average field enhancement factor in the capacitive gap between the meta-atoms by numerical simulations. The average field enhancement factor is defined here as ϕ = 0.25 rad near 0.5 THz, as opposed to the negligible phase change of NRGDs. The large nonlinear phase change is a result of the blue-shift of the resonance under higher THz fluences. The phase response of a meta-atom is highly dispersive in the vicinity of its resonance frequency; hence, a large nonlinear phase change can be observed for a slight resonance blue-shift with increasing fluence. However, for the case of NRGDs, their resonance is designed to be positioned far from the frequency of interest. Therefore, the fluence-dependent phase difference is almost negligible in NRGDs.In subsequent experiments, we used resonant meta-atom/graphene devices (RGDs) to investigate the additional nonlinear effects, such as the nonlinear phase change or a reversal of the nonlinear transmittance change  near the resonance frequency. RGD samples were fabricated to have a resonant response at 0.6 THz, as shown in 10EF = −226 meV, whereas the momentum scattering time varied from τ = 13 fs at FTHz = 0.06 μJ/cm2 to τ = 7 fs at FTHz = 85.5 μJ/cm2, as shown in τ = 13 fs at FTHz = 0.06 μJ/cm2 to τ = 7 fs at FTHz = 85.5 μJ/cm2. Although the scattering parameters are the same with GD for the lowest and the highest fluences, NRGD has lower scattering parameters for other fluences. Note that the Fermi level also altered with 184 meV by the interaction between graphene and goldThe decrease in momentum scattering time is confirmed by the comparison between the simulations and the experimental results for NRGDs . The expp-doped, and the corresponding Fermi level was estimated to be EF = −169 meV. EF = −230 meV to −56 meV. The power transmittance change ΔTEF increases as the Femi level is shifted away from the charge neutral point (CNP); for example, the change was ΔT−230 meV ~16% and ΔT−56 meV <1%. In another plot, 10Finally, the nonlinear power transmittance change for a GD was investigated as a function of the Fermi level. Without electrical gating, the graphene in the GD was slightly We have explored different mechanisms to control the nonlinear transmittance change of graphene metadevices at THz frequencies. Metamaterials concentrates the incident electric field in their gaps between meta-atoms, and the enhanced field can be exploited to control the nonlinear response of graphene. The metadevices can be designed to show either broadband induced transparency or resonant induced opacity by controlling the geometry of the meta-atoms. In addition, dynamic control over the nonlinear response has been demonstrated with a gated graphene sheet. The free carrier concentration of graphene can be controlled by the gate voltage, and a higher free carrier concentration produces a higher nonlinear transmittance change. This work constitutes a crucial early step towards the employment of ultra-thin and controllable graphene devices in nonlinear THz technology.TM) was used for the fabrication of protective dielectric layers and a spacer. The PI solution was spin-coated on a silicon wafer to have a final thickness of 1 μm and was then baked. The baking process consisted of two steps: first, the solvent was volatilised in a convection oven, and second, PI was cured in a furnace. All of the metal parts were made of 100-nm-thick gold with a 10-nm-thick adhesive chromium layer. The bottom terahertz transparent electrode was patterned by photolithography and electron beam evaporation deposition followed by metal lift-off. The PI layer as a spacer was stacked using the same procedure described above. Then, the array of meta-atoms was patterned on top of the PI spacer. Single-layer graphene (GRAPHENE SQUARE Inc.) was grown on a copper foil by chemical vapour deposition and was transferred to cover the whole metamaterial area by the thermal release tape method. The top (ground) electrode was fabricated on the graphene layer using a shadow mask. All layers were capped with another PI layer and were delaminated from the silicon wafer.The graphene metadevices were fabricated on a sacrificial silicon wafer substrate. Polyimide solution  crystal with a 1-kHz Ti:sapphire regenerative amplifier . The system was capable of generating near-infrared (NIR) pulses with an average power of 4 Watts. The pulse duration was approximately 100 fs, and the centre wavelength was 800 nm. The maximum power of the generated THz pulses and the peak-to-peak electric field amplitude that can be generated by the setup were 3.3 mW and 350 kV/cm, respectively. The spectral response of the graphene metadevices was recorded by the THz-TDS. The time-domain signal of the transmitted THz pulses was obtained by electro-optic sampling with gallium phosphide (GaP) crystal. The total power transmittance was measured by a calibrated pyroelectric detector , and the corresponding amplitude transmittance was calculated by taking the square root of the power transmittance. The fluence of the incident THz wave was varied by changing the relative angle between two THz wire-grid polarisers. The THz fluence could be varied from 0.008 μJ/cm2 to 85.5 μJ/cm2.To obtain the spectral response in the THz regime, THz time-domain spectroscopy (THz-TDS) was employed. High-power THz pulses were generated with a lithium niobate .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


These results suggest that a misfolding-specific intrabody prone to synergistic proteolysis by proteasomal and autophagic pathways is a promising strategy for mitigation of TDP-43 proteinopathy in ALS.Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is implicated in the pathogenesis of sporadic and certain familial forms of amyotrophic lateral sclerosis (ALS), suggesting elimination of TDP-43 aggregates as a possible therapeutic strategy. Here we generated and investigated a single-chain variable fragment (scFv) derived from the 3B12A monoclonal antibody (MAb) that recognises D247 of the TDP-43 nuclear export signal, an epitope masked in the physiological state. In transfected HEK293A cells, 3B12A scFv recapitulated the affinity of the full-length MAb to mislocalised TDP-43 with a defective nuclear localising signal and to a TDP-43 inclusion mimic with cysteine-to-serine substitution at RRM1. Moreover, 3B12A scFv accelerated proteasome-mediated degradation of aggregated TDP-43, likely due to an endogenous PEST-like proteolytic signal sequence in the VH domain CDR2 region. Addition of the chaperone-mediated autophagy (CMA)-related signal to 3B12A scFv induced HSP70 transcription, further enhancing TDP-43 aggregate clearance and cell viability. The 3B12A scFv also reduced TDP-43 aggregates in embryonic mouse brain following  Emerging evidence suggests that the accumulation of various disease-related misfolded proteins may underlie these pathogenic processes. Despite advances in understanding ALS pathogenesis, however, there is no curative or effective control therapy.Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterised by progressive muscle wasting and weakness, leading ultimately to lethal respiratory failure. Multiple pathogenic pathways have been implicated in the motor neuron degeneration of ALS, such as excitotoxicity, neuroinflammation, proteolysis impairment, oligodendrocyte dysfunction, mitochondrial dysfunction and disruption of RNA homeostasis9. TDP-43 contains a nuclear localising signal (NLS) as well as a nuclear export signal (NES)10, which enables the physiological shuttling of TDP-43 between nucleus and cytosol. Under stress-free conditions, TDP-43 interacts with mRNAs on which ribosomes are located separately, forming polysomes. Various stresses induce clustering of ribosomes into a ‘stalled’ state, resulting in the formation of stress granules (SG) containing TIA-1, G3BP, ataxin-2 and eIF4G1/2. In the stalled state, transcription is inhibited as a homeostatic response. However, sustained stress and ensuing TDP-43 misfolding creates aberrant SGs and pathogenic TDP-43 aggregates12. Moreover, membrane-less organelles in the cytosol formed by the liquid−liquid phase separation of RNPs4 and RNA13 are implicated in TDP-43 proteinopathy. Misfolding and cytosolic mislocalisation also lead directly to a loss of normal TDP-43 function, and the resultant disruption of protein and RNA homeostasis is considered another likely pathogenic mechanism14 in addition to the toxicity of inclusions. TDP-43 inclusions can be identified in the majority of ALS patients with the exception of patients with superoxide dismutase 1 (SOD1) mutations15, suggesting that these aggregates are a core pathology of sporadic ALS. Aberrant TDP-43 inclusions are also observed in familial ALS (FALS) cases associated with various mutations such as C9orf7216, MATR317, hnRNP118, UBQLN219, SQSTM120, VCP21 and OPTN22, as well as in cases with mutations of the TDP-43 gene (TARDBP) itself, indicating that misfolded TDP-43 is a common pathogenic molecule in familial as well as sporadic ALS. These lines of evidence suggest that targeting pathogenic TDP-43 species is a possible therapeutic strategy against ALS.The TAR DNA-binding protein of 43 kDa (TDP-43) is a DNA/RNA-binding protein predominantly located in the nucleus under physiological conditions. However, hyperphosphorylated, fragmented and ubiquitinated forms of TDP-43 were identified as core components of cytosolic inclusions in sporadic ALS and frontotemporal lobar degeneration (FTLD)23. Unexpectedly, the suppression of cytoplasmic human TDP-43 expression after disease onset (DOX-ON) reversed TDP-43 pathology and rescued the motor deficits23. These results indicate that mislocalised TDP-43 is directly toxic and further suggest that removal of misfolded TDP-43 is a promising therapeutic strategy for TDP-43-linked ALS, even after formation of TDP-43 inclusions. Ubiquitination and phosphorylation of TDP-43 are pathological hallmarks of sporadic ALS. However, they are relatively late phenomena in ALS progression26, and thus targeting the early conformational changes in pathogenic TDP-43 is required for pre-emptive therapy. We previously identified E246 and D247 as molecular epitopes of the mislocalised/misfolded TDP-4327. These residues are located at the assembly interface of the RNA Recognition Motif 2 (RRM2) domain formed upon interaction with single-stranded DNA28. Using these residues as epitopes, we successfully generated a misfolding-specific monoclonal antibody (MAb), 3B12A, which recognises mislocalised or misfolded TDP-43 in cultured cells and spinal cord sections from ALS patients, but not wild-type (WT) TDP-43 in the nucleus27.A transgenic mouse model with doxycycline (DOX)-dependent suppression of mutant human TDP-43 harbouring a defective nuclear localising signal (NLS) exhibited accumulation of cytoplasmic TDP-43 aggregates, loss of endogenous nuclear TDP-43 and progressive motor impairments in the DOX-OFF statein vitro and in vivo. This effect was mediated by dual proteolytic signals, an endogenous PEST-like signal and a chaperone-mediated autophagy (CMA)-localising signal. Moreover, aggregate binding to CMA-fused 3B12A scFv unexpectedly induced heat shock protein (HSP) 70, which further enhanced TDP-43 clearance by promoting protein refolding.Here we report the development of self-degradative intrabodies derived from 3B12A MAb, and demonstrate effective elimination of misfolded TDP-43 both 29. To apply this antibody inside cells as a potential scavenger of intracellular TDP-43 aggregates, we generated a single-chain variable fragment (scFv) to be used as an intrabody. The cDNAs encoding variable fragments of the heavy chain (VH) and light chain (VL) were obtained from hybridoma 3B12A mRNA, and domain profiles were investigated. Unexpectedly, DNA sequence analysis of these 3B12A intrabodies showed that the VH domain contains a RIDPEDGETK sequence with PEST score as high as 9.02 at the complementarity determining region 2  and VL-linker-VH (VL_VH) as scFv forms and VH and VL as nanobodies tagged with Myc at the C-terminus  as a model for misfolded and aggregated TDP-43, and TDP-43 with an NLS mutation (TDP-43mNLS)34 as a TDP-43 cytosolic aggregate mimic in the present study , but not with TDP-43WT. Moreover, VH_VL preferentially colocalised with TDP-43 aggregates over TDP-43WT whether in the nucleus or cytosol  leads to the formation of TDP-43 aggregates sharing pathological and functional features of the inclusions observed in sporadic ALSsol Fig. . Likewissol Fig. . These rsol Fig. . These 3mNLS, TDP-43C173S/C175S and TDP-43mNLS,C173S/C175S over TDP-43WT  chase study. For this purpose, we constructed a VH_VL intrabody containing two protein disassembling signals, CL1 for proteasome localisationy39 Fig. , based oy39 Fig. . The inty39 Fig. . Furthery39 Fig. . Consisty39 Fig. . We confy39 Fig. .Figure 3WT or misfolded TDP-43 species  but did not significantly accelerate clearance of the cytoplasmic TDP-43 mutant (TDP-43mNLS) , and cell lysates were analysed at several time points post-labelling. The HaloTag pulse chase assay demonstrated that 3B12A scFv co-expression markedly enhanced the degradation of the cytoplasmic aggregation-prone TDP-43 mutant TDP-433WT Fig. . Moreove3WT Fig. . Co-exprLS) Fig. , confirmLS) Fig. , indicatmNLS,C173S/C175S demonstrated a gradual increase in the number and size of cytoplasmic aggregates over 48-h post-transfection. Conversely, Hoechst counterstaining demonstrated a concomitant decline in live cells, indicating that these cytoplasmic TDP-43 aggregates are cytotoxic  cells at 48-h post-transfection, indicating reduced aggregation and thus rescued from cell death  induced the highest cytotoxicity, consistent with our previous studies in the NSC-34 cell line33. This cytotoxicity was significantly reduced in the presence of 3B12A scFv (VH_VL), and the inclusion of the CMA signal significantly enhanced this protective effect  TDP-43 by the distribution of GFP fluorescence45. Coronal brain sections exhibited GFP-TDP-43 mutant protein in both the lateral and medial neocortices, and the cellular distribution was grossly similar to that reported previously43 , a marker of mature neurons  . Similar to mice electroporated with the empty vector, those expressing 3B12A scFv-CMA were safely born at term, grew normally and exhibited age-appropriate behaviours until P21  was not degraded by 3B12A scFv in cultured cells /cullin-2 ubiquitin ligase complex for misfolded TDP-4329. This explains why 3B12A scFv specifically interacted with mislocalised/misfolded TDP-43 but not with TDP-43WT in cells  in RRM133. The sustained mislocalisation of TDP-43 in the cytosol by the NLS mutation induces cell death and occasionally forms inclusions both in vitro and in vivo54. However, these aggregates are relatively small in number and rarely phosphorylated or ubiquitinated54; thus TDP-43mNLS is an insufficient model for TDP-43 proteinopathy. Transgenic mice overexpressing human TDP-43WT and several known FALS mutants such as A315T and Q331K under native promoters develop similar mild phenotypes for FTLD-motor neuron diseases, but nuclear exclusion and cytosolic aggregates are prominent only in the FALS mutant models55. We reported that both WT and NLS mutant TDP-43 induce cell toxicity in immortalised cultured cells34. It should be noted that in the current work and our previous study33, aggregate-prone TDP-43mNLS,C173S/C175S induced greater cell toxicity than TDP-43WT or TDP-43mNLS, irrespective of mislocalisation. The pathologies induced by this TDP-43 mutant share several crucial properties with TDP-43 proteinopathy in addition to the strong trend for aggregation, including the involvement of prion-like domains, impaired RNA splicing, ubiquitination and phosphorylation, as well as motor neuron toxicity33. Based on the extent of aggregate toxicity, the TDP-43mNLS,C173S/C175S mutant may be the most robust form for modelling ALS-associated TDP-43 proteinopathy, especially for studies on therapeutic strategies and efficacy.There is yet no model TDP-43 species replicating all pathological features of sporadic ALS. In this study, we used the C173S/C175S artificial mutant as a TDP-43 inclusion mimic based on evidence of structural similarity to aggregated TDP-43 inclusions in ALSin utero electroporation did not show aberrant/ectopic neuronal cells, gliosis or microglial activation compared with vector controls. Of course, the restricted distributions of exogenous human TDP-43 and 3B12A scFv-CMA protein in mice following in utero electroporation limit future experiments on mitigation of neurological behaviour phenotypes, and the environment of the pup brain is distinct from the aged human brain. However, the successful elimination of TDP-43 aggregates in vivo validates the basic principle and justifies further analyses in older disease model animals. In the clinical setting, our intrabody may be effectively delivered by a virus vector, such as an adeno-associated virus (AAV). Indeed, AAV9-mediated intrathecal delivery of a scFv derived from the D3H5 monoclonal antibody against misfolded SOD1 reduced misfolded SOD1 in the spinal cord, delayed disease onset and enhanced the survival of SOD1G93A transgenic mice56. A recent study demonstrated that suppression of human TDP-43 mutant expression after disease onset in a mouse model decreased TDP-43 pathology, rescued motor deficits and prolonged survival23. These results provide strong support for our approach of pathological TDP-43 removal using a 3B12A intrabody, even after disease onset and formation of pathological TDP-43 aggregates. Further investigations evaluating the potential of our degradative 3B12A intrabody against pathogenic TDP-43 using appropriate animal models are required to advance this novel molecular targeting therapy for ALS.Most promising for clinical applicability, 3B12A scFv-CMA reduced cytoplasmic TDP-43 aggregates in the lateral CP of murine embryos Fig.  without 34. The TDP-43 substitution mutant with serine at Cys173 and Cys175 , familial ALS-linked mutations (A315T and Q331K) and mNLS (R82L/K83Q) were generated by site-specific mutagenesis34. A plasmid for Halo-tagged TDP-43  was generated by subcloning TDP-43 cDNA into pFC14A HaloTag® CMV Flexi® Vector . cDNA for variable fragments of the heavy chain (VH) and light chain (VL) were obtained using mRNA from a hybridoma producing 3B12A monoclonal antibody, which recognises mislocalised/misfolded TDP-4327. For in utero electroporation studies, target protein expression was driven by the CAGGS promoter57. Detailed protocols regarding construction and domain maps of Halo-tagged TDP-43 , CAGGS promoter-driven TDP-43, and single-chain variable fragment (scFv) for VH-VL-Myc, VL-VH-Myc, VH-Myc, VL-Myc, VH-VL-CL1-Myc and VH-VL-CMA-Myc are described in Supplementary Materials and Methods and Figure In cell culture studies, mammalian expression plasmids for TDP-43 tagged with FLAG (pcDNA3-TDP-43-FLAG) or EGFP (pEGFP-N3-TDP-43) were constructed using a conventional PCR technique as described previouslyAntibodies used in this work are described in the Supplementary Table.2 and 100% humidity. HEK293A  and N2a cells  were maintained in Dulbecco’s modified Eagle’s medium  containing 10% foetal bovine serum (FBS) and penicillin/streptomycin . The FuGene HD Transfection Reagent (Promega) was used for plasmid transfection. At 48 h after transfection, cells were treated for various analyses as indicated.All cultured cells were maintained at 37 °C under 5% CO58.Cultured cells were lysed in sodium dodecyl sulfate (SDS) buffer containing 2-mercaptoethanol for Western blotting or in immunoprecipitation assay (RIPA) buffer  containing protease inhibitor cocktail  for immunoprecipitation. Ten percent of each cell lysate volume was analysed as the total cell lysate, and the remaining 90% was incubated with anti-FLAG M2 affinity gel  or anti-Myc affinity beads  at 4 °C overnight. The affinity beads were subsequently washed with RIPA buffer five times, and the immunoprecipitates were eluted in 2% SDS sampling buffer for 5 min at 95 °C. The eluates were separated on polyacrylamide gels  and proteins transferred onto PVDF membranes . Proteins were detected using an enhanced chemiluminescence system . Densitometric analysis of protein bands was performed using ImageJ softwareCultured cells were fixed in 4% paraformaldehyde (PFA)/PBS (pH 7.2) and permeabilised with 0.1% Triton-X100/PBS containing 5% normal goat serum as a blocking agent. Cells were reacted with primary antibody  and subsequently with a fluorophore-tagged secondary antibody  for 1 h at room temperature. Cells were counterstained with 4′-6 diamidino-2-phenylindole (DAPI). Fluorescence images were obtained using a confocal laser microscope .Target FLAG antibody in coating buffer (Roche) was coated onto ELISA plates  at a 1:100 dilution. Coated FLAG antibody was then reacted with cell lysates from transfected HEK293A cells (prepared in RIPA buffer) for 1 h at room temperature and subsequently with anti-Myc antibody at a 1:500 dilution . Then, the coated antibody was reacted with the peroxidase-conjugated secondary antibody . Finally, reaction buffer containing 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonate (Roche) was applied, and the absorbance was measured by a spectrometer at 405 nm with a reference at 490 nm.At 48 h after cotransfection of HEK293A cells, the HaloTag-fused protein was labelled with 1 μM diAcFAM ligand (Promega) in FBS-free culture medium for 15 min in a cell culture incubator. Cells were then washed three times with warm PBS and cell lysates prepared in SDS sample buffer at 0, 12 and 24 h after ligand treatment. All lysate samples were separated on polyacrylamide gels (Wako) by SDS-PAGE, and gels were subsequently analysed on a fluorescence scanner .2 as aggregates. Intensity is expressed in arbitrary units.At 24 h after cotransfection, HEK293A cells were counterstained with Hoechst 33342 . Stained cells were observed at 30-min intervals over 48 h using a fluorescent microscope . For quantification of GFP-positive TDP-43 aggregates with ImageJ software, we removed background noise and set fluorescence thresholds of GFP fluorescence at 2.5% to distinguish TDP-43 aggregates from diffusely expressing cytoplasmic non-aggregated species and subsequently counted GFP-positive areas over 10 µmProtein half-life in HEK293A cells was estimated starting 48 h after transfection by chronological chase analysis. Cells were treated with CHX (100 μg/ml) to inhibit protein synthesis. In several experiments, lactacystin (10 μM) or bafilomycin (0.1 μM) was added with CHX to inhibit proteasome or autophagy–lysosome activity, respectively. Dimethyl sulfoxide (DMSO) was used as a vehicle control.We estimated the cytotoxicity and viability of transfected N2a cells using the MultiTox-Fluor multiplex cytotoxicity assay kit (Promega). Briefly, N2a cells were seeded onto 96-well black culture plates and plasmids encoding FLAG-tagged WT or mutant TDP-43 and 3B12A scFv were co-transfected at 0.1 µg each per well. At 48 h after transfection, cell-permeant glycyl-phenylalanylamino fluorocoumarin (GF-AFC) and cell-impermeant bisalanyl-alanyl-phenylalanyl-rhodamine 110 (bis-AAF-R110), fluorescent indicators for live cells and dead cells, respectively, were applied. After a 2-h incubation, relative fluorescence emission was measured on a multi-plate reader (Perkin Elmer) with excitation/emission of 400/505 nm for live cells and 485/520 nm for dead cells.HSP70 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were analysed using a real-time PCR Detection Systems  and SYBER quantitative PCR kit  with the following primer pairs: HSP70, 5′-CAA GAT CAC CAT CAC CAA CG-3′ and 5′-TCG TCC TCC GCT TTG TAC TT-3′; GAPDH, 5′-GCA CCG TCA AGG CTG AGA AC-3′; and 5′-TGG TGG TGA AGA CGC CAG TGG A-3′. GAPDH was used as an internal standard, and the relative mRNA expression levels were calculated by the ΔΔCT method according to the manufacturer’s protocol using the included software (BIO-RAD).Total RNA samples were purified from HEK293A cells using a commercially available kit (Invitrogen) and converted to cDNA with reverse transcriptase (Invitrogen). The mRNA expression levels of WT-GFP or pCAGGS-TDP-43mNLS,C173S/C175S-GFP], 1 μg/μl of effector plasmids [pCAGGS-3B12A-VH_VL-Myc-CMA or empty plasmid], and 0.5 μg/μl of normalisation plasmid [pCAGGS-mCherry]) were mixed and diluted in PBS (−) containing 0.05% Fast Green, and 1–2 μl of the solution was injected into the lateral ventricles of embryos. After plasmid injection, embryo heads were held using a tweezer-type electrode  with the anode on the forebrain side in which the plasmid was injected, and five cycles of electric pulses  were delivered by an electroporator . The uterus was repositioned in the abdominal cavity, the skin sutured and pregnancy allowed to continue.Timed-pregnant ICR mice (E13.5) were anaesthetised by inhalation of isoflurane  and intraperitoneal injection of sodium pentobarbital . The uterine horns were then exposed in the abdominal cavity. The expression vectors . The slides were washed with 0.1 M PBS/0.1% Triton X-100 (PBS-T) and blocked with 3% bovine serum albumin  in PBS-T at room temperature for 30 min. After subsequent incubation with primary antibody at 4 °C overnight, slides were incubated in Alexa Fluor-conjugated antibody (Invitrogen) at room temperature for 1 h, washed with PBS-T, and placed onto cover glass (Matsunami) with mounting medium containing DAPI for nuclear counterstaining . For quantification of GFP-positive TDP-43 aggregates in the lateral cortical plates with ImageJ software, we removed background noise and set fluorescence thresholds of GFP fluorescence at 1% to distinguish TDP-43 aggregates from diffusely expressing cytoplasmic non-aggregates, and subsequently counted GFP-positive areas over 20 µm2 as aggregates. Intensity is expressed in arbitrary units.Embryos were anaesthetised on ice and postnatal mice by intraperitoneal injection of sodium pentobarbital and isoflurane inhalation. Brains were fixed by transcardial perfusion with 4% paraformaldehyde (PFA) in 0.1 M PBS. The brains were immersed in 4% PFA at 4 °C overnight, incubated in 20% sucrose/0.1 M PBS at 4 °C overnight, embedded in OCT compound , and frozen in liquid nitrogen. The restricted distribution of the plasmids in the dorsolateral cortex of the pup mice by p < 0.05 was considered statistically significant.Multiple group means were compared by one-way ANOVA with post hoc Tukey’s multiple comparison tests for pair-wise comparisons. Factor estimation in the two chronological data groups was evaluated by two-way ANOVA using Prism software . A The protocols for both genetic transformation experiments and animal experiments were approved by and performed under the guidelines of the Kyoto University Graduate School of Medicine ethics committee (#130181 for the genetic transformation experiments) and the Shiga University of Medical Science .Supplementary Movie 1aSupplementary Movie 1bSupplementary Movie 2Supplementary information


Azadirachta indica) in India suggests its potential efficacy in periodontal diseases. The objective of this study is to explore the antibacterial effects of Neem leaf extract on the periodontophatic bacteria Porphyromonas gingivalis and Fusobacterium nucleatum, and its antioxidant capacities alone and in combination with bacteria and polycationic peptides that may be at the site of inflammation.The common usage of chewing sticks prepared from Neem tree  of Neem leaf extract against each bacterial strain. The effect of Neem leaf extract on the coaggregation of the bacteria was assessed by a visual semi-quantitative assay. The antioxidant capacities of Neem leaf extract alone and in combination with bacteria, with the addition of red blood cells or the polycationic peptides chlorhexidine and lisozyme, were determined using a chemiluminescence assay.Neem leaf extract was prepared by ethanol extraction. The growth kinetics of P. gingivalis, however, had no effect on the growth of F. nucleatum nor on the coaggregation of the two bacteria. Yet, it showed intense antioxidant activity, which was amplified following adherence to bacteria and with the addition of red blood cells or the polycationic peptides.Neem leaf extract showed prominent dose-dependent antibacterial activity against Neem leaf extract, containing polyphenols that adhere to oral surfaces, have the potential to provide long-lasting antibacterial as well as synergic antioxidant activities when in complex with bacteria, red blood cells and lisozyme. Thus, it might be especially effective in periodontal diseases. Porphyromonas gingivalis and Fusobacterium nucleatum. The adherence of bacteria to mucosal cells is a critical step in the development of periodontal infection. P. gingivalis is able to adhere to cellular and acellular surfaces  (100 μM) and 10 μM Co [II] , 36. TheNeem leaf extract may bind to mammalian and microbial surfaces , 37–39, p < 0.05.The results were expressed as the mean ± SEM. One or two-way ANOVA with the Newman-Keuls test for post-hoc comparisons were applied. Differences were considered statistically significant at P. gingivalis growth. Whereas approximately 50% growth inhibition was induced by 5 μM GAE of Neem leaf extract, higher concentrations (10 and 20 μM GAE) markedly inhibited P. gingivalis growth . Significant growth inhibition was noted already after 18 h . In contrast, Neem leaf extract had no effect on the growth of F. nucleatum even at the highest concentrations  [P. gingivalis and F. nucleatum.Coaggregation of t shown) . This inP. gingivalis pre-coated with Neem leaf extract. It should be noted that P. gingivalis alone did not possess any OSA.As shown in Fig. P. gingivalis coated with Neem leaf extract was further markedly enhanced by small amounts of RBC.As shown in Fig. F. nucleatum-Neem-lysozyme complex exerts greater OSA, with about 85% reduction of luminescence, as compared to F. nucleatum-Neem complex. However, the addition of the polycationic peptide chlorhexidene to F. nucleatum-Neem complex did not intensify the antioxidant effect of F. nucleatum-Neem complex, alone.Similarly, as shown in Fig. Azadirachta indica) leaves has antibacterial properties against the periodontophatic bacterium P. gingivalis and potent antioxidant abilities. Our findings also show that small numbers of RBC further enhance the OSA of P. gingivalis pre-coated with agents present in Neem leaf extract, and that lysozyme also amplifies the OSA of bacteria pre-coated with Neem leaf extract.Our results show that an ethanol extract derived from Neem tree (P. gingivalis and F. nucleatum. Also, surprisingly, it has no effect on the growth of F. nucleatum, as opposed to its antibacterial effect on P. gingivalis, when tested under similar experimental conditions (Fig. P. gingivalis and F. nucleatum exhibited diverse effects, such as antibacterial activities against both bacteria by propolis extracts [Neem leaf extract has no effect on the coaggregation of ons Fig. , maybe dextracts , and no extracts . On the extracts , 44. Theextracts , 46.F. nucleatum-Neem complex, as compared to the antioxidant capacity of this complex alone deserves further evaluation.Neem leaves are extremely rich in a large variety of polyphenol agents, possessing antioxidant properties that can modulate inflammation –18. FurtTaken together, Neem extract, adsorbed by bacteria, in addition to its antibacterial properties, provides profound antioxidant activity, intensified in the presence of RBC or lysozyme. However, the potent antioxidant activity in Neem extracts might also act as a double-edged sword. On one hand scavenging ROS might lower inflammatory damage to the gum, but on the other hand it may also protect periopathogenic catalase-negative bacteria against oxidants generated by inflammation or cariogenic streptococci.In addition, our report has two major limitations: First, the tested species in this study were evaluated in a planktonic state, whereas, in vivo they are part of a multi-specious biofilm, which has a profound impact in the effectiveness of antimicrobial agents. Therefore, further clinical studies using periodontal parameters are needed to shed more light on the potential clinical implication of Neem extracts as a modulator of periodontal diseases. Second, we studied fresh leaves obtained from Neem trees grown in Israel, applying a specific extraction protocol. Further experiments are needed to extend our findings to Neem extracts obtained from other parts of this plant and to explore comparable extracts produced from trees grown abroad. Furthermore, we did not label and quantify the numerous compounds plausibly extracted from the plant, and we did not identify components responsible for the antimicrobial properties of the extract. Nor did we compare the characteristics of our product with extracts obtained by other methods. Additional studies are needed to address these important limitations, and safety evaluation is required preceding clinical trials.P. gingivalis but not against F. nucleatum. It possesses strong anti-oxidant activity, which is amplified following adherence to bacteria, to red blood cells and to the polycationic peptide lysozyme. The clinical relevance of these findings is based on the co-existence of bacteria, RBCs and polycationic compounds in the oral cavity. Conceivably, Neem extract could adhere to these compounds, and provide a long lasting bacteriostatic as well as anti-oxidant activity at the site of inflammation in periodontal diseases. Prospective randomized trials in-vivo are needed to strengthen these in-vitro findings, suggesting potential clinical implication of the use of Neem leaf extract in periodontal diseases.We report that Neem extracts possess prominent dose-dependent bacteriostatic activity against